Vol 1 No 1 May 1998 IDrugs The Investigational Drugs Journal Editor Ian Tarr Deputy Editors Barbara Chan, Tara Dyson London Office IDrugs Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Tel: +44 (0)171 580 8393 Fax: +44 (0)171 580 5646 e-mail: [email protected] Product Manager Jennifer Garratt Production Editor Kate Marston Production Manager Wendy Palmer Subscriptions Hannah Lawrence Designer Juan Mico Editorial Board Sudhir Agrawal (USA) John F Barrett (USA) Robert Beamer (USA) Nigel Beeley (USA) James Black (UK) David P Bloxham (UK) Jean-Jacques Body (Belgium) Mike Briley (France) James A Bristol (USA) Leslie Brown (USA) Andre Bryskier (France) Simon Campbell (UK) Daniel TW Chu (USA) T Colatsky (USA) Stanley T Crooke (USA) Erik de Clercq (Belgium) Annette Doherty (France) Jürgen Drews (USA) Jay Gibbs (USA) William E Heydorn (USA) Ray Hill (UK) Richard Houghten (USA) David Howat (UK) Victor J Hruby (USA) Ian Hunneyball (UK) Paul AJ Janssen (Belgium) Graham Johnson (USA) Taff Jones (UK) Tim Kogan (USA) Hugo Kubinyi (Germany) Gerard Le Fur (France) William M Mackin (USA) Ronald Magolda (USA) John P Mallamo (USA) John M McCall (USA) Walter H Moos (USA) A Nistri (Italy) Stella O'Donnell (Australia) Stephen J Peroutka (USA) Petpiboon Peppi Prasit (Canada) George Poste (UK) Daniel H Rich (USA) Robert D Sindelar (USA) Grace Tang (Hong Kong) John B Taylor (UK) Bernard Testa (Switzerland) David J Triggle (USA) Marc Vasseur (France) Michael D Varney (USA) Camille G Wermuth (France) George Whitesides (USA) Wendell Wierenga (USA) Ruth R Wexler (USA) IDrugs The Investigational Drugs Journal IDrugs (ISSN 1369-7056) is published monthly by: Current Drugs Ltd Middlesex House 34-42 Cleveland Street London, W1P 6LB, UK Copyright © 1998 by Current Drugs Ltd No part of this publication may be reproduced, stored in a retrieval system, or transmitted by any form, or by any means, electronic or otherwise, without prior permission of the copyright owner. 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Subscription information Subscription rates including airspeed delivery Corporate IDdb subscribers second copy Personal Academic Rate Orders All orders (except for Japan) should be placed with Current Drugs Ltd Middlesex House 34-42 Cleveland Street London W1P 6LB UK Tel +44 (0)171 580 8393 Fax +44 (0)171 580 5646 North America $2380 $1190 $1190 $1190 Japan ¥350,000 ¥175,000 ¥175,000 ¥175,000 Rest of World £1400 £700 £700 £700 Orders for Japan Technomics Inc Nihonbashi TM Bldg 1-8-11 Nihonbashi Horidome-cho Chuo-ku Tokyo 103 Japan Subscription queries All enquiries should be addressed to Current Drugs Ltd Middlesex House 34-42 Cleveland Street London W1P 6LB UK Tel +81 3 3666 2952 Fax +81 3 3666 2730 Tel +44 (0)171 580 8393 Fax +44 (0)171 580 5646 In case of particular difficulty please contact Hannah Lawrence: [email protected] IDrugs The Investigational Drugs Journal Contents 1 Editorial 42 42 Cancer gene therapy D Banerjee 45 Cell-based gene therapy and late-breaking news E Hutchinson 47 Highlights of selected symposia AF Chambers Meeting Reports 3 American Chemical Society - 215th National Meeting 3 PET and Radionuclides P Robins 4 Adenosine receptors as targets for drug discovery JE Gomez-Galeno 6 Protein farnesyltransferase and adenosine receptors P Robins American Association for Cancer Research 89th Annual Meeting 49 Medicinal Chemistry in Eastern England Ninth Symposium P Norman 55 8 Protein prenylation D Leonard 10 ATP site-directed kinase inhibitors WA Denny 63 13 Anticoagulation strategies P Robins 67 17 Amyloid aggregation inhibitors MG Zagorski 19 Medicinal Chemistry Awards Symposium P Robins 21 Cytochrome P450 and drug design C Wu 22 Angiogenesis MS Wolfe 26 Aromatics, heterocycles and porphyrins KG Pinney 28 Total syntheses of alkaloids, antibiotics, anticancers and antivirals D Pleynet 30 Glucosidase and fucosidase inhibitors & Advances in nucleosides and nucleotides ZJ Witczak 32 Ernest Guenther award in the chemistry of natural products KG Pinney 35 Combinatorial carbohydrate chemistry - where are we now? M Panigot T Wright Selected state of the art topics - organic chemistry poster session E Juaristi 40 Interview with the Chair of the ACS MEDI division, Annette Doherty P Robins Prostate Cancer - CHI Conference PB Fisher American Society for Clinical Pharmacology & Therapeutics - 99th Annual Meeting D Morgan Reviews 73 Neurokinin antagonists as potential therapies for inflammation and rheumatoid arthritis Andreas von Sprecher et al Is there a clinical potential for the use of neurokinin antagonists in inflammatory diseases? 92 38 Japanese Pharmacological Society - 71st Annual Meeting Plant-derived anticancer agents currently in clinical use or in clinical trials Hui-Kang Wang Throughout history, plant products and their modified analogs have been rich sources of clinically useful drugs, including anticancer agents. Cancer chemotherapeutic drugs, including vinblastine, vincristine, Navelbine, etoposide, Teniposide, Taxol and, most recently, Taxotere, topotecan, and irinotecan are reviewed. 103 Mucosal immunity elicited by DNA vaccines Taff Jones Are there prospects for the clinical use of DNA vaccines? This review charts the progress towards the challenge of eliciting mucosal immunity through the delivery of DNA vaccines. 109 Involvement of 5-HT receptors in learning and memory François Roman & Evelyne Marchetti 141 Tranilast (Kissei) M Konneh Research has suggested that the 5-HT system plays a modulatory role in cognitive processes, particularly in learning and memory. However, few data are currently available for the role of the 5-ht5, 5-ht6 and 5-HT7 receptors. This article presents an overview of their brain locations and possible involvement in these highly cognitive processes. Tranilast, a known anti-allergic agent, is now being developed as a prophylactic agent for restenosis. Clinical trials have so far demonstrated a reduced rate of restenosis in patients. O OH O O N H Drug Evaluations O 122 GS-4104 (Gilead) A Billich 147 Iralukast (Novartis) A Bramley A neuraminidase inhibitor currently in phase III clinical trials for influenza virus infection. Current therapies for this indication are suboptimal and novel therapies, especially in elderly patients and infants, are required. This LTD4 / LTE4 antagonist is currently in phase II trials for the treatment of asthma. Animal studies have shown it to be more potent than other LT antagonists. O O S O O OH O O O OH O N H O OH F F F NH 2 152 Epristeride (SmithKline Beecham) SS Hedge 129 Cystemustine (INSERM) B Palmer A non-competitive steroid 5-α-reductase inhibitor in phase II trials for the treatment of benign prostatic hypertrophy. Analysts estimate that epristeride will be launched in Japan between 1999 and 2000 and is predicted to attract over 10 billion Yen in sales. This chloroethyl-nitrosourea is being developed as an anticancer drug. It is currently in phase II trials for several cancers. O O O S O Cl N H N H N N O H 136 KA-672 (Schwabe) H Mucke An α1 and D2 antagonist currently in phase II clinical trials for the treatment of Alzheimer’s disease. O O N O N H Cl H O O 158 O H HO Patent News Peter Steele Highlights of the patent literature over the past month from Schering-Plough, IDUN, NIH, SmithKline Beecham, Rhone-Poulenc Rorer, Sanofi, Merck, Eisai, Nutrimax, Eli Lilly, Meiji Seika Kaisha, Affymetrix, Shionogi, Columbia University, Johns Hopkins University, Harvard College, Millennium, Hokuriku Seiyaku, Kissei, Takeda, Du Pont Merck, Demeter, Guilford, Pherin, Hoechst Marion Roussel and ImmuLogic. Editorial 1 Editorial Ian Tarr Welcome to the first issue of a new journal, IDrugs – the Investigational Drugs journal. Each month, IDrugs will bring to its readers' attention, in a lively manner, the most noteworthy information reported on investigational drugs in the preceding weeks. In doing so, we will place particular emphasis on the following: Although there are now several excellent general magazines and review publications covering various aspects of pharmaceutical R&D, few assess the scientific aspects of drug R&D in a commercial context. In contrast, articles in IDrugs will emphasize the latter. • • • Neurokinin antagonists in the clinic? Rapid publication of meeting reports Timely reviews and analysis of R&D programs Expert evaluations of the literature on new drugs Much of the most valuable information on both the discovery and subsequent development of investigational drugs is first disclosed at scientific meetings – long before full papers appear in the primary literature. For this reason, IDrugs will provide unparalleled coverage of such meetings via its team of scientific reporters and editors. For example, the article by von Sprecher et al in this issue reviews the basic pharmacology of neurokinin antagonists in the context of drugs in active development and their prospective indications. The aim is to assess whether there is sufficient evidence to suggest a place for these novel compounds in the clinic. Reviews in future issues will place an even greater emphasis on the commercial implications of drug R&D, adding, for example, comments and forecasts from market analysts. Meetings in this issue – focus on the ACS Drug evaluations: a core review service In this first issue of IDrugs, we cover the following meetings in depth: • • • • the 215th National Meeting of the American Chemical Society, the 71st Annual Japanese Pharmacological Society meeting, the 89th Annual Meeting of the American Association for Cancer Research, the 99th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics. All these meetings took place less than 6 weeks prior to publication of this issue and we will strive to maintain this rapid publication process for meeting reports in future issues of IDrugs. In many cases, we will have several reports from the same conference, reflecting the multidisciplinary nature of the larger meetings and the number of parallel sessions in their programs. For example, the most recent ACS meeting is covered in this issue via no less than 14 separate reports; we also carry an interview with Dr Annette Doherty, who organised the program for the ACS’ Medicinal Chemistry Division. The interview provides a valuable insight into the original selection of symposium topics and reflects on which sessions proved to be most successful and the probable reasons for their success or otherwise). Here at Current Drugs Ltd, we have systematically collected all published references to investigational drugs over a period of several years. The resulting drug bibliographies are a core component of our Investigational Drugs database (IDdb). As part of this service, we periodically commission experts to review each of these bibliographies, so providing users of the IDdb with a guide to the literature cited in the database. This whole literature collation, organization and evaluation process results in a rich seam of value-added information on investigational drugs – one that will be mined regularly by IDrugs. The choice of which drug evaluations to include in IDrugs will be governed by the strength of commercial or scientific interest in the drug (or drug class) concerned. In this first issue, we have selected six drugs currently in clinical trials for a wide range of indications: this choice is intended to give our readers a general impression of the scope and quality of the literature evaluations that will be included in future issues of the journal. We hope that this unique blend of timely meeting reports, reviews and drug-literature evaluations will make IDrugs an indispensable read for anyone with an interest in investigational drugs. We also hope that you enjoy it. Meeting Report 215th ACS 3 American Chemical Society 215th National Meeting PET and radionucleotides 29 March - 1 April 1998, Dallas, Texas, USA Peter Robins Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):3 © Current Drugs Ltd ISSN 1369-7056 Over 10,000 scientists attended over the five days of the 215th National Meeting of the American Chemical Society (ACS). The ACS is the largest scientific society in the world with more than 155,000 members and 29 March - 2 April was duly designated ’American Chemical Society Week’ by the Mayor, Ronald Kirk. This is the first of a series of ACS reports beginning with the medicinal chemistry program followed by reports from the organic chemistry symposia. Diagnosis with PET Of the presentations on the first day, particularly striking were several that considered the growing use of diagnostic PET. When normal cells start to become cancerous, they develop an enormous appetite for glucose. Therefore, after injection 18 with an [ F]glucose analog, a whole body PET scan can then be used to determine the presence of a tumor and moreover, whether it is malignant, or if it has metastasized. Michael Phelps (UCLA School of Medicine, USA) described how such studies may be used in patients with lung, breast, head and neck, colorectal, ovarian and prostate cancers. These techniques may also identify the early metabolic alterations associated with neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). Further studies of HD, and the genetic form of AD have indicated that PET is able to detect metabolic abnormalities associated with these diseases prior to the appearance of symptoms. Serial scans may be used to monitor the progression of the disease through the brain. A 18 similar approach is taken with PD, except [ F] L-DOPA is used as the radiotracer. On January 1, 1998, the US Federal Government began reimbursing physicians who use PET to diagnose and identify the stage of lung cancer. The government has also agreed to perform expedited reviews of 12 other clinical indications for PET. In addition, the Food, Drug and Cosmetic Act was amended this year to direct the US FDA to take account of the special diagnostic capability of PET, and whilst this takes place, the US Pharmacopoeia (USP) will be used as the legal standard of FDA approval. The USP has approved the above mentioned radiotracers for numerous diagnostic indications in cancer, heart disease and neurological diseases such as AD, PD, cerebrovascular ischemia and epilepsy. Dr Phelps stated that advances in radiotracer chemistry are now allowing PET to take its place in every day medical practice. Radionuclides and bone cancer Researchers from Brookhaven National Laboratory described advances in the use of radionuclides for a variety of medical uses, but particularly bone cancer. This debilitating condition causes excruciating pain to more than 200,000 Americans each year, and is characterized by painful lesions which are often spread throughout the bony tissue. Conventional treatments are far from satisfactory, causing decreases in quality of life, or being of only limited effectiveness. Suresh Srivastava described how Brookhaven, in conjunction with SUNY at Stony Brook, has developed a new agent, tin-117m DTPA, which appears to provide pain relief with virtually no side-effects, including the bone marrow damage seen with other nuclear medicines. In a phase I/II clinical trial, tin-117m DTPA completely or substantially relieved the pain in 75% of 47 enrolled patients, with relief lasting up to one year. Bone marrow toxicity was also much less than comparable therapies. Two multi-center, double-blind, randomized clinical trials are currently in progress in 100 prostate cancer patients and 75 patients with osteolytic bone metastases (including breast and lung). This therapy may also hold promise for the slowing of metastatic cancer and prolonging patient survival time, and even for treating primary bone cancer and rheumatoid arthritis. Ritalin For many years, synthetic drugs have routinely been marketed as racemates. However, it has only recently become clear that individual enantiomers may possess quite distinct pharmacokinetic and side-effect profiles. The failure to take this into account has occasionally been catastrophic, as in the case of thalidomide. Advances in chemical technology have facilitated the routine synthesis of pure enantiomers, permitting the refinement of the previously ’dirty’ profiles of a number of drugs, such as methadone and local anesthetics. The latest such candidate is Ritalin (methylphenidate), widely prescribed in the US (an estimated 2 million recipients, with sales of $350 million) for the treatment of attention deficit disorder. Yu-Shin Ding (Brook-haven National Laboratory, NY, USA) described how evidence suggests that Ritalin may only be beneficial in its d-threo form, and that furthermore, the presence of the l-threo isomer may in fact compromise its effectiveness. PET scans following individual administration of the enantio13 13 mers of [ C]Ritalin revealed that d-threo-[ C]Ritalin binds exclusively to dopamine receptors, whilst the binding of its enantiomer was mostly non-specific. The high specificity of d13 threo-[ C]Ritalin for dopaminergic neurons suggests that it might be used as a radiotracer to probe the neuronal loss that accompanies neurodegenerative diseases, echoing the work of Dr Ding’s colleague, Dr Srivastava. 4 IDrugs 1998 Vol 1 No 1 Adenosine receptors as targets for drug discovery Jorge E Gomez-Galeno receptor antagonist. This xanthine derivative was licensed to Biogen from CV Therapeutics and has entered phase II clinical trials as a diuretic for the treatment of the edema associated with congestive heart failure (CHF). Address Metabasis Therapeutics 4575 Eastgate Mall San Diego CA 92121-1911 USA Email: [email protected] IDrugs 1998 1(1):4-5 © Current Drugs Ltd ISSN 1369-7056 This symposium was held on March 30, 1998 as part of the 215th American Chemical Society National Meeting. It was presided by PR McGuirk of Pfizer and was particularly aimed at medicinal chemists. There were more than 200 attendees at the symposium. Introduction The initial lecture by Professor RA Olsson (University of South Florida, USA), covered the subject of adenosine receptors (A1, A2A, A2B and A3) and their implication for drug development. Activation of these cell surface, G-protein coupled receptors causes cell-specific physiological responses with potential utility for the treatment of a variety of disease states. Intervention at the adenosine receptor level can be achieved in several ways: (i) through direct action on the receptor by either agonists or antagonists; (ii) by enhancement of tissue adenosine concentration by compounds which interfere with its metabolism; (iii) by treatment with adenosine precursors, such as ribose or AICAR (5-amino-4 carboxamido1-ribosylimidazole; Figure 1). Benefits and drawbacks to each of the approaches were reviewed. BG-9719 Russell C Peter of Biogen (Cambridge, MA, USA) presented some of the pharmacology of BG-9719 (1,3-dipropyl-8-[2-(5,6 epoxynorbornyl)]xanthine (Figure 1), previously known as CVT-124, a highly potent and selective adenosine A1 Typical diuretics act mainly by inhibiting reabsorption in the kidney distal tubule but inhibition of reabsorption at the proximal tubule appears to be more clinically relevant in CHF. The distribution of adenosine A1 receptors means that BG9719 is expected to inhibit reabsorption of sodium and chloride at the proximal and distal tubules, causing increased diuresis without an increase of potassium excretion. In saline loaded rats, oral dosing of BG-9719 at 1 mg/kg increased urine output to the same extent as furosemide at 30 mg/kg. There was an increase in potassium excretion which was statistically significant but was not believed to be clinically relevant. In humans, phase I clinical trials showed that sodium excretion was increased by a factor of about 2.3 in iv drug-treated patients versus a placebo-treated group. Potassium excretion was also increased, but not to a clinically relevant level. These effects peaked about 1 h after infusion. The effect on sodium excretion for BG-9719 (0.3 mg/kg, iv) was similar to the effect observed with furosemide (20 mg/kg, po). The compound has entered phase II clinical trials. A3-selective antagonists The therapeutic potential of, and challenges associated with, intervention at the A3 adenosine receptor were discussed by Marlene Jacobson (Merck Research Labs, PA, USA). There are unique differences in tissue distribution and affinity to xanthine antagonists between the rat and human adenosine A3 receptors. This has presented a challenge for the development of animals model relevant to clinical situations. Levels of A3 adenosine receptors are increased in individuals with airway inflammation. It has been proposed Figure 1. Drugs targeting adenosine receptors F O NH NH2 N NH2 N O O H3C N H HO OH AICAR (Gensia) O N N N N O HO N H N H O H3C H H3C HO BG-9719 (University of Florida) GP-3269 (Gensia) OH Meeting Report 215th ACS that selective A3 antagonists may be useful as anti-asthmatic, anti-inflammatory, and anticancer agents. Dr Jacobson described two approaches for the study of the role of the adenosine A3 receptor. The first, library screening and lead optimization, led to the identification of L-249313 (6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]triazolo[5,1-a][2,7]naphthyridine) which binds to the human A3 receptor with Ki = 5.4 nM, and shows high selectivity over A1 and A2A receptors. L-249313 increases cAMP levels and to decreases GTP-γS binding, which is not due to covalent modification. Jacobson’s second approach entailed development of an A3 knockout mouse, in collaboration with S Tilley (University of North Carolina, USA). This model simulates the effect of chronic antagonism at the A3 receptor. Amongst the most relevant preliminary observations were the lack of histological differences with wild-type mice, the absence of an effect on fertility and growth (which was interesting, given the high density of A3 receptor in testis) and the absence of any effect on baseline mean arterial pressure and heart rate. There was no apparent effect on the density of A1 125 and A2A receptors. As expected, [ I]ABA did not bind to bone marrow mast cell membranes derived from the transgenic mice. There was no observed increase of hexosaminidase release induced by adenosine or 2-chloroIB-MECA (a selective A3 agonist) in mast cells derived from the transgenic mice. This last result was relevant because it indicates that the A3 receptor, which has also been found in human lung eosinophils, plays an important role in mast cell degranulation, a key step in allergic reactions and asthma. Adenosine regulating agents The use of adenosine regulating agents (ARAs) to modulate pathways responsible for the production or metabolism of adenosine were described by Mark Erion (Metabasis Therapeutics, San Diego, CA, USA). The approach is based on the observation that adenosine is produced primarily at sites undergoing ATP breakdown; ARAs are therefore expected to enhance endogenous adenosine production in a site- and event-specific manner. Two enzymes were targeted as ARAs: adenosine kinase (AK) and AMP deaminase (AMPDA). GP-3269 (Gensia Sicor; 4-N-(4-fluorophenylamino)-5phenyl-7-(5-deoxy-1-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine; Figure 1) inhibits AK with IC50 = 5 nM. It also inhibits maximal electroshock-induced seizures in rats with ED50 = 5 mg/kg after oral administration. This inhibition occurs in the absence of hemodynamic effects, and its antagonism by theophylline indicates that adenosine is the key mediator of the antiseizure activity. GP-3269 has been tested in phase I clinical trials. In vitro studies reveal that inhibition of AK 5 results in decreased neutrophil adhesion and TNFα production. This suggests a further potential use of AK inhibitors for the treatment of inflammation. Coformycin monophosphate-based transition state inhibitors of AMPDA were designed and synthesized which were expected to have high binding affinity and enzyme specificity. The high affinity would allow for removal of the phosphate group from the molecule, and therefore enhance the cell penetration of these compounds. Highly potent AMPDA inhibitors were described (Ki = 2 nM) that also showed high selectivity versus adenosine deaminase. In rat hepatocytes subjected to anoxia, there is a decrease of about 30% in ATP levels. Treatment with AMPDA inhibitors increased the levels of ADP and ATP, and decreased IMP levels. AMPDA inhibitors were also shown to have good event specificity in these cells. Purinergic ligands The history and therapeutic potential of purinoceptors were reviewed by Michael Williams (Abbott Laboratories, IL, USA). In addition to a discussion on P1 (adenosine) receptors (A1, A2A, A2B and A3) and AK, he also reviewed the therapeutic potential and challenges associated with the eleven P2 (ATP/UTP) purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7). It was commented that 5’-deoxy-5’-iodotubercidin, an adenosine kinase inhibitor, had an analgesic effect in the hot plate and carrageenan hyperalgesia models of pain in rats, without a significant effect on their heart rate or blood pressure. P2 purinoceptors offer potential for the treatment of a variety of conditions, such as cystic fibrosis (P2Y2) and pain (P2X3). Many of these potential applications have been recently reviewed (Trends Pharmacol Sci (1997) 18(3):83; Curr Opin Neurobiol (1997) 7(3):346). The challenges associated with P2 purinoceptor intervention include the lack of selective pharmacological tools and of reliable binding assays. For example, the pharmacology of P2 purinoceptors is defined by the functional effect of agonists. Also, the known antagonists are relatively weak and have poor selectivity and high molecular weight. Recently, the P2X7 purinoceptor was found to have a role in apoptosis (Neuropharmacol (1997) 36(9):1149). It has also been implicated in the killing of bacteria by human macrophages, and in the mediation of host immunity to tuberculosis. These results open up a wide range of potential applications to drugs that interact with this receptor. Conclusion This symposium highlighted the importance of purinergic receptors as viable, novel therapeutic targets. 6 IDrugs 1998 Vol 1 No 1 Protein farnesyltransferase and adenosine receptors Peter Robins Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):6-7 © Current Drugs Ltd ISSN 1369-7056 Many exciting advances in the treatment of cancer were presented at the symposium entitled ’Protein prenylation’ which was chaired by Professor Richard A Gibbs (Wayne State University, Detroit, MI, USA). Prenylation, or the covalent attachment of isoprenoid lipids, is now recognized as an important component in the post-translational localization and functionalization of many cellular proteins. The majority of prenylated proteins are modified by one of two specific enzymes: protein farnesyl-transferase (FTase) and geranylgeranyltransferase type I (GGTase-I). The finding that farnesylation of oncogenic forms of Ras proteins is required for expression of their transforming activity has led to FTase becoming an important target for anticancer drug design. Farnesyltransferase inhibitors About one quarter of all human cancers are caused by genetic malfunctions in the Ras biochemical pathway, including up to 90% of pancreatic cancers, half of all colon cancers, and a quarter of all lung cancers. DM Leonard (Parke-Davis, MI, USA) explained how, as such, this is one of the most common types of mutation responsible for cancer, and how mutation of as little as one amino acid (eg, Gln61 to Leu61) may cause oncogenicity. She then discussed a series of histidine-N-(benzylglycinamides), including PD169451 (Figure 1), which have been developed as FTase inhibitors, and presented structure-activity relationship studies of this class. Parke-Davis uses three assays to assess activity against FTase: an enzyme scintillation proximity assay using rat affinity FTase purified from SF9 cells; a cellular mobility shift assay in NIH 3T3 cells which express H-ras (used if activity is detected in the above); and a twolayer soft agar system in which NIH 3T3 cells are continuously exposed to drug for several days. One compound, PD-161956, exhibited IC50 values of 0.062, 0.1 and 4.4 mM, respectively, in these assays, and served as a template for further SAR studies, which are ongoing. RJ Doll (Schering-Plough, NJ, USA) presented a series of novel trihalobenzocycloheptapyridine heterocycles with which his company claims to have overcome the problems of in-cell potency and oral bioavailability that have beset FTase inhibitors in the past. Again, several assays have been employed, including an in-cell H-ras FTase inhibition assay, and an anchorage-independent colony forming assay. In addition, pharmacokinetic studies have been carried out in mouse and cynomologus monkey, following both iv and po administration. Early leads showed only mediocre potency, but were selective for FTase over GGTase. The compounds that resulted from modification of these leads, including Sch-54419, Sch-56620 and Sch-44342 (Figure 1), suffered from poor pharmacokinetics, but provided useful clues for further SAR because of their characteristic metabolism. One of the compounds that has resulted, Sch-66336, entered the clinic in the third quarter of 1997. It is not clear which cancers the drug will be developed for if results from phase I studies are satisfactory, although it seems likely that these will be pancreatic in origin. Structure-activity studies of early FTase inhibitors were presented by SL Graham (Merck Research Laboratories, PA, USA). These compounds, which included L-744832 (Figure 1), all contained a thiol group which, because of the potential for immune-mediated toxicities, led to the abandonment of the series at the preclinical stage. SAR studies, some of which have been reported in J Med Chem (1996) 39:1345, led to a non-thiol series of cyanobenzylimidazole derivatives of piperazinones. An illustrated compound produced 100% inhibition of tumor growth at a dose of 80 mg/kg in h-ras positive mice, and 79% at a dose of 160 mg/kg in k-ras positive mice. These experiments in transgenic animals support the premise that FTase inhibitors will find utility in clinical oncology. Dr Gibbs, the symposium chair later commented: "The field of protein prenylation is a tremendously exciting one, not only because of its intrinsic scientific interest, but also because of the potential of prenylation inhibitors as anticancer drugs". Adenosine receptors Earlier in the day, CR McGuirk chaired a symposium on the use of adenosine receptors as drug targets. Perhaps the most useful talk for the novice was, somewhat perversely, reserved until last. M Williams (Abbott, IL, USA) began with an overview of currently known members of the class, then gave a short history of the molecular biology of the group, and, perhaps most usefully, clarified the IUPHAR position on the naming of its members. There are currently four P1 receptors (A1, A2A, A2B and A3) and eleven P2 receptors which form the focus of an intensive research interest. In contrast to the preceding lectures which focused on adenosine receptors, Dr Williams gave an overview of the latter class of purinoreceptors by describing the pharmacology, therapeutic potential and clinical status of modulators of each member receptor. Of the other presentations, that by Marlene Jacobson was particularly interesting as it dealt with the A3 receptor. This is mysteriously different from other adenosine receptors in that it shows unique species differences between rodents and humans, both in the pattern of distribution of expression and sensitivity to classical xanthine antagonists. Pharmacological characterization was hampered until recently by a lack of selective modulators, although many claims have been made in the literature in this regard. Merck are interested in the pulmonary applications of A3 modulators, since, in humans, the receptor is most strongly expressed in the lung. Meeting Report 215th ACS 7 Figure 1. Farnesyl transferase inibitors O O H N H N O H3C CH3 N N N O H O N N N N H O Cl PD-169451 (Parke-Davis) Sch-54419 (Schering-Plough) O N N HS O O O H2N H H3C O S CH3 O Sch-44342 (Schering-Plough) The company has used a classical SAR approach, but has also created an A3-knockout mouse in order to study the loss of function phenotype. A compound has emerged from these studies, L-249313, which binds to cloned human A3 receptors with Ki = 5.4 ± 0.34 nM (compared to 32.7 ± 12.9 mM for rat). Buckminsterfullerenes Elsewhere in the conference, an interesting presentation by JM Alford (TDA Research, CO, USA) described a new process for the manufacture of buckminsterfullerenes that has decimated their market cost. Commercial, much less medical applications of buckminsterfullerene and its derivatives have been slow to present themselves, hampered partly by the limited availability of the raw materials. CH3 O N Cl CH3 H N H N CH3 L-744832 (Merck & Co) However, the new method employs a continuous combustion process, in contrast to the labor-intensive and wasteful carbon-arc process that is currently used. TDA Research is currently capable of synthesizing up to one pound of the material per day and claims that it could easily be carried out on a multi-ton scale. The most promising clinical application of the fullerenes relies on their ability to act as free-radical traps; as antioxidants, they are much more potent that vitamin E. There is preclinical work underway at Washington University Medical School in St Louis for amyotrophic lateral sclerosis, and Lou Gehrig’s disease, but clinical trials are at least a year away. 8 IDrugs 1998 Vol 1 No 1 Protein prenylation session Daniele Leonard Address Parke-Davis Pharmaceutical Research 2800 Plymouth Road Ann Arbor MI 48105 USA Email: [email protected] IDrugs 1998 1(1):8-9 © Current Drugs Ltd ISSN 1369-7056 This session, which took place on Monday, 30 March 30 1998, was organized by Richard A Gibbs and Kim D Janda, and presided by Richard A Gibbs (Wayne State University, Detroit, MI, USA). The first two speakers discussed the mechanism of protein prenylation in yeast and mammalian species, while the next three lectures were about inhibitors of one of the prenylation enzyme, farnesyl transferase. As more research is carried out in identifying farnesyl transferase inhibitors, with companies starting clinical studies, it was of interest to have a session on this topic. The meeting was well attended, with about 300 people in the audience. Prenylation Prenylation is a post-translational modification which allows the attachment of a lipophilic isoprenoid group onto a protein. There are two types of prenylation, namely, farnesylation and geranylgeranylation. The enzymes for each of the respective prenylations have been isolated and characterized: farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase-I). Their substrates are proteins with the C-terminal motif CAAX (C is cysteine, A is any aliphatic amino acid, and X is either Ser or Met for proteins undergoing farnesylation, or Leu or Phe, for proteins undergoing geranylgeranylation). The Ras proteins undergo farnesylation at the cysteine residue in the presence of FTase that uses farnesyl pyrophosphate as a donor, forming a thioether linkage. The three terminal amino acids (AAX) are then proteolytically cleaved, followed by methyl esterification at the new Cterminal cysteine residue. The Ras protein then associates with the cell membrane, which enables signaling events leading to cell replication and transformations. Oncogenic versions of these proteins are also farnesylated and subsequently localized to the cell membrane, leading to uncontrolled growth. The ras oncogenes are implicated in a wide variety of human cancers. Since the farnesylation of the protein is the necessary step which allows for the membrane localization, inhibition of this modification via the inhibition of FTase could prove to be useful, and may have potential as antitumor therapy. Mechanistic studies of protein farnesyltransferase Professor C Dale Poulter (University of Utah, USA) was the first speaker in this session. His talk focused on the mechanism of prenylation by the yeast FTase enzyme, with regards to substrate binding for a fluorescent labeled peptide, difluorofluorescein-Arg-Thr-Arg-Cys-Val-Ile-Ala. Farnesylation can occur via a 1,4 condensation mechanism, involving the nucleophilic attack of the protein onto carbon1 on farnesyl pyrophosphate to give the farnesylated protein. Fluorescence anisotropy was used to determine the kinetic constants. For the substrate difluorofluorescein-ArgThr-Arg-Cys-Val-Ile-Ala, the Kcat was determined to be 0.6/s and the KM 2 µM. The Kd value was 2.5 µM (at pH 7) and independent of pH (Kd = 2.0 µM at pH 5.75, and 1.9 µM at pH 9). It was suggested that the binding of the protein to the yeast enzyme is independent of the cysteine side chain being either the thiol or the thiolate. In the yeast enzyme, it was also shown that the residues Asp-307, Cys-309 and His-363 2+ are involved in the binding of Zn . The mechanism of protein prenylation of human FTase was discussed by Professor Patrick J Casey (Duke University Medical Center, USA). Interestingly, for the human enzyme, the Kd value is pH-dependent, unlike the yeast enzyme. The human enzyme was shown to preferentially bind the thiolate form of the cysteine residue of the protein. The crystal structure of FTase was also discussed. A structure of FTase and farnesyl pyrophosphate (FPP) (3.5 Å) was presented. The binding pocket of the FPP moiety is sizespecific, causing the C-1 of the farnesyl moiety to interact 2+ with Zn . Protein farnesyltransferase inhibitors In vivo studies of L-744832 (Merck & Co; Figure 1), in four new transgenic mice models, ie, H-ras/p53 knockout mice, Hras/myc overexpression mice, N-ras mice and K-ras mice, were discussed by Samuel L Graham (Merck Research Laboratories, West Point, PA, USA). Although this compound was active in vivo, it did present liabilities: the presence of the thiol group, the prodrug efficacy, and the pharmacokinetic and pharmacodynamic profiles in rodent were not optimal. A new class of FTase inhibitors was then developed, and the results from SAR studies were presented. IL-744832 inhibited tumor growth inhibition by 100% at a concentration of 80 mg/kg/day, in H-ras (Rat 1 cells), and by 79% at 160 mg/kg/day against the K-ras tumor cell line (oral administration). The SAR studies around PD-169451 (Figure 1) and the results of in vivo evaluation in a panel of human tumor cell lines were presented by Daniele M Leonard (Parke- Davis Pharmaceutical Research, Ann Arbor, MI, USA). PD-169451 inhibited FTase with an IC50 value of 0.004 µM, and was effective at inhibiting H-ras processing at the concen-tration of 0.05 µM. Further evaluation of this compound in a panel of human tumor xenografts showed that it inhibited tumor growth for the following tumor cell lines: MCF-7 (breast): > 120% (200 mg/kg); HT-29 (colon): 108% (125 mg/kg); H460 (lung): 100% (125 mg/kg) and A549 (lung): 140% (125 mg/kg). Ronald J Doll (Schering-Plough Research Institute, Kenilworth, NJ, USA) reported on a tricyclic FTase inhibitor, Meeting Report 215th ACS 9 Figure 1. Protein farnesyltransferase inhibitors O HS O O CH3 O H2N O H H3C CH3 N H N H N H N H N H O CH3 O N O H3C N H O H3C S O CH3 O L-744832 (Merck & Co) PD-169451 (Parke-Davis) N C Br Cl CH3 N H N Br O F N F N N N N NH2 F O O SCH-66336 (Schering-Plough) trihalobenzocycloheptapyridine, Sch-66336 (Figure 1). This compound was developed from structure-activity relationships studies of a original tricyclic FTase inhibitors which had moderate potencies against FTase. Sch-66336 was active against FTase with an IC50 value of 1.9 nM, and inhibited Hras processing with an IC50 value of 10 nM. In an anchorageindependent soft agar assay it was active at 72 nM for H-ras and was 7-fold less active for K-ras (500 nM). The pharmacokinetic profile of this compound was reported: it is 76% and 50% bioavailable in mouse and in monkey, respectively; its half-life (iv) is 1.4 and 3 h in mouse and monkey, respectively. Sch-66336 inhibited tumor growth by Compound A (Merck & Co) 100%, in both EJ (bladder) and NIH 3T3–CVLS cell lines (nude mice xenograft sc qid, 40 mg/kg po), and is not cytotoxic. It is currently in phase I clinical trials. Conclusion The prenylation session was interesting and demonstrated the high level of activity in this field, from academia to industry. A better understanding of the enzyme and its interaction with the various classes of inhibitors will help to elucidate how these compound are useful as antitumor agents. 10 IDrugs 1998 Vol 1 No 1 ATP site-directed kinase inhibitors William A Denny Address Auckland Cancer Society Research Centre Faculty of Medicine and Health Science The University of Auckland New Zealand Email: [email protected] IDrugs 1998 1(1):10-12 © Current Drugs Ltd ISSN 1369-7056 This symposium was organised by Dr Michael South (Monsanto/Searle Discovery Research, USA), and Dr Alex Bridges (Parke-Davis, MI USA), as a half-day symposium within the Medicinal Chemistry Division of the ACS. It was intended to provide an update to the chemical community of recent developments in signal transduction enzymes, especially the progress towards clinical evaluation of a number of classes of ATP sitedirected inhibitors. This is a rapidly expanding field, as judged by both the near capacity attendance and the number of additional posters on this subject at the meeting. Michael South opened the symposium with the comment that there were a large number of kinase enzymes now known. These show considerable homology at the ATP site, and it was thus originally expected that inhibitors targeting this site were unlikely to be selective. However, the discovery of potent and selective ATP site inhibitors has been a major step forward in the development of clinical agents. ras/ERK pathways Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, USA) began the meeting with an overview of recent structural and mechanistic findings among kinases in the ras/ERK pathways. ERK1 and 2 are terminal enzymes in three-kinase cascades, activated by MAP/ERK kinases 1 and 2, and controlling gene transcription and cytoskeletal changes in cells. Phosphorylation at both tyrosine and threonine residues is required for full activation. A comparison of crystal structures of ERK2 in both its low-activity (non-phosphorylated) and high-activity (twice-phosphorylated) states showed that phosphorylation induces major conformational changes in the activation loop. These changes drive head-totail homodimerization by interaction between these loop regions. Crystal structures The literature on crystal structures of tyrosine kinase enzymes was reviewed by Ravi Kurumbail (Searle, IL, USA). About ten different structures have now been reported, including both apo-enzymes and complexes with ATP and/or inhibitors. These are typically bilobal structures, with a wide variability in interdomain angles. The Nterminal domains are comprised largely of β-sheets, and the C-terminal domains of α-helices, with the ATP site situated at the base of the N-terminal domain. ATP binds quite similarly to the different enzymes, with the adenosine accepting hydrogen (H)-bonds, the sugar donating H-bonds, and the triphosphate bonding to a conserved lysine-rich loop via metal interactions. Unlike the situation in many other ATP-accepting enzymes, the adenosine is deeply buried. A comparison of several enzymes complexed with diverse classes of inhibitors (isoquinolinesulfonamides, staurosporines, benzylindoles and isoflavones) showed that, in all cases, the inhibitors form both H-bonds and lipophilic interactions at the buried adenosine site, but have little common effects at the sugar and triphosphate sites. The high selectivity shown by ATP site binding inhibitors is thus surprising from a structural point of view, and depends critically on small inter-enzyme changes in the adenosine binding region. p38 MAP kinase p38 MAP kinase is part of a stress-induced signal transduction pathway downstream of MKK, leading to the synthesis of several pro-inflammatory cytokines (eg, IL-1 and TNF), and Tim Gallagher (SmithKline Beecham, PA, USA) reviewed the development of aminopyrimidinylimidazoles as inhibitors of this enzyme. The series of compounds were originally pursued as anti-inflammatory cyclooxygenase/THF inhibitors, but radiolabeling studies showed p38 MAP kinase as the sole molecular target. The 4-pyridyl analogs were very selective inhibitors, but also inhibited cytochrome P450 due to high-affinity binding of the 4-pyridyl group to the heme iron. Analogs where this was replaced by a 2-aminopyrimidine retained potent (IC50 = 20 nM) and selective p38 MAP kinase inhibition without P450 suppression, and downregulated TNF in vivo by approximately 80% at 5 mg/kg/day. Structureactivity studies showed that the 4-fluorophenyl ring was important for selectivity, fitting into a small hydrophobic pocket. The synthesis of these compounds has been adapted to a solid-phase combinatorial approach, to enable rapid further study of structure-activity relationships. trkA A staurosporine-based inhibitor of trkA, a receptor for NGF, as a potential drug for androgen-independent pros-tate cancer was discussed by Bob Hudkins (Cephalon, PA, USA). At least 90% of human prostate cancers overexpress one or more of the trk receptors, and the disease is driven by a neurotrophin/ NGF endocrine loop. Prostate cancers are low growth fraction tumors, poorly responsive to standard cell cycle-dependent drugs. Staurosporine analogs are known inhibitors of trks, and an extensive structure-activity study on more than 500 compounds showed that few changes could be made in the lactam or indole rings. The modified sugar derivative, CEP751 (Figure 1), was selected for development. This compound is a potent (IC50 = 3 nM) and selective (1000-fold over PKC, VEGF, PDGF) inhibitor of trkA. It is active in the refractory Dunning H rat prostate model in vivo, producing 65% tumor inhibition at 5 mg/kg/day po, and causing substantial tumor cell apoptosis. Because of low solubility, a series of prodrugs was evaluated, and a lysine/β-alanine analog was selected for phase I trials on the basis of high solubility and adequate stability. Meeting Report 215th ACS isolated EGFR enzyme, and showed that both N1 and N3 quinazoline nitrogens were required. Small lipophilic substituents at the 3’-anilino position and electron-donor substituents at the 6- and/or 7-quinazoline/ pyridopyrimidine positions greatly enhanced activity. These data are consistent with a model in which the drug binds in the ATP site, with the 3’-substituted anilino ring fitting into a 751 hydrophobic pocket (partly defined by unique Cys and 742 Met residues) adjacent to the ATP site. The N1 and N3 769 766 quinazoline atoms accept H-bonds from Met and Thr residues, and the drug 6- and 7-positions point towards the entrance of the ATP binding pocket. The latter feature has been exploited by attaching weakly basic side chains at these positions to improve aqueous solubility, and two such reversible inhibitors, ZD-1839 (Zeneca; Figure 2) and CP358774 (Pfizer; Figure 2) are currently undergoing clinical evaluation. Figure 1. CEP-751 H N O N N O CH3 H OH H3C O CEP-751 (Cephalon) Despite this success, there are potential advantages in irreversible inhibitors, since the compounds must compete with high levels of ATP, and even the tightest-binding reversible inhibitors shut down the enzyme for only a few hours. Irreversible inhibitors should provide longer enzyme shutdown, overcome only by synthesis of new receptors (turnover approximately 22 h). Modeling studies showed that 6-acrylamide analogs positioned the acrylamide directly 773 over a unique cysteine (Cys ) not far from the mouth of the ATP binding pocket, and kinetic studies showed that these 773 compounds rapidly and selectively alkylated the Cys of EGFR, but not other kinases. Structure-activity studies Epidermal growth factor receptor A joint program between Auckland University and ParkeDavis on the development of 4-anilinoquinazoline/ pyridopyrimidine acrylamides as irreversible inhibitors of the epidermal growth factor receptor (EGFR) was outlined by Bill Denny (Cancer Research Centre at the University of Auckland, New Zealand). This is a transmembrane receptor tyrosine kinase which is overexpressed in a significant proportion of tumors. Early work established 4-anilinoquinazolines as spectacularly potent reversible inhibitors of the Figure 2. Inhibitors of EGFR O N H3C H3C O O O N H3C O N O N N 11 HN O CH Cl HN H Cl F ZD-1839 (Zeneca) CP-358774 (OSI) O N O N N HN H2C HN O PD-169540 (Parke-Davis) Br C 12 IDrugs 1998 Vol 1 No 1 showed that a 6-acrylamide is superior to a 7-analog for irreversible inhibition, and that an additional weakly basic solubilizing substituent at the 7-position improved in vivo activity. The 7-propyloxymorpholide, PD-169540 (Figure 2), showed good in vivo activity against a range of human tumor xenografts in mice, with growth delays of 10 to 30 days using oral dosing at 6 to 25 mg/kg/day. This compound, and a small number of analogs, are in advanced development. Conclusion This was a well-planned program, with the first two talks providing the audience with an update on the functions and structures of many signal transduction kinase enzymes. The three concluding talks then gave disparate examples of the structure-based design of new classes of inhibitors directed at the ATP sites of different kinase targets. This field seems set to grow explosively, and to provide many new classes of drugs. Meeting Report 215th ACS 13 Anticoagulation strategies Peter Robins Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):13-16 © Current Drugs Ltd ISSN 1369-7056 A number of individual presentations at this meeting dealt with the burgeoning field of anticoagulation strategies, and the fourth day (Wednesday) provided one of the most interesting symposia of the meeting, entitled ’Anticoagulation Strategies: Thrombin and Factor Xa Inhibition’. Chair RR Wexler (DuPont Merck, PA, USA) pointed out that classical anticoagulants, such as warfarin, suffer from poor oral bioavailability, lack of selectivity and short plasma half-life. In addition, there are safety issues, for instance, an increased risk of bleeding, and a large peak-to-trough ratio. Hirudin is a recently-approved thrombin inhibitor, but is suitable for parenteral administration only. New therapies are needed and many advances in this field were presented in the ensuing sessions. Thrombin inhibitors 3DP-4147 JC Spurlino (3-Dimensional Pharmaceuticals, PA, USA) began the session with a description of the coagulation cascade and the role that thrombin plays. Scaffold selection for the lead compound in the program was achieved using a DirectedDiversity library. 3-Dimensional then employed a combination of structure-based design and combinatorial chemistry to generate a series of thrombin inhibitors. These include 3DP-4147 which has greater than 90% oral bioavailability and a duration of action in excess of 6 h in dogs. In addition, it is highly potent (ED 50 less than 10 nM) and, being non-peptidic, is easily synthesized. An unnamed compound from this series has been advanced for clinical trials. L-375378 Thrombin is a serine protease which has several procoagulant actions. For instance, it cleaves fibrinogen to generate fibrin, activates Factors V, VIII and XIII, and activates platelets via the thrombin receptor. Therapeutic indications for antagonists include deep vein thrombosis, pulmonary embolism, and the prophylaxis of cardiogenic thromboembolism. PEJ Sanderson (Merck Research Laboratories, PA, USA) explained how peptide based inhibitors have a relatively long history, but are far from satisfactory. Early compounds in Merck’s research program, were based on an efagatran template and included L-370518 and L371912. However, none of these compounds was significantly orally bioavailable, or particularly selective. The next generation of compounds, including L-373890, were based on a pyridinone acetamide template, but the basic guanidine group that they contained meant that, although the compounds were potent, poor oral bioavailability was still a problem. Replacing this group with aminopyridine led to a compound with good selectivity. The most potent of these, L-374087, exhibited Ki values of 0.5 and 3200 nM against thrombin and trypsin, respectively. However, the pharmacokinetic data were still inadequate to support further development, since the company’s aim was to provide a therapy suitable for once- or twice-daily dosing. The researchers found that by introducing an electron withdrawing group in to the central pyridine ring, the compound could be made more metabolically stable. L375378 (Figure 1), which showed the superior biological profile of the compounds that resulted, has Ki values of 0.8 and 1800 nM against thrombin and trypsin, respectively, a plasma t1/2 following iv injection of 185 min, and is 92% orally bioavailable. This compound has been selected for development in the clinic. UK-156406 The structure-activity studies that led to the development of UK-156406 (Figure 1), a potent, orally-active thrombin inhibitor that was disclosed towards the end of last year, were presented by David Fox (Pfizer, CT, USA). As with all such research programs, Pfizer’s main aim was to produce a potent, orally bioavailable inhibitor which was highly selective for thrombin over other serine proteases, such as plasmin and trypsin. Early benzamides in the program had moderate selectivity but suffered from a short duration of action due to biliary elimination. Incorporation of a tetrahydroiso-quinoline moeity into the structure produced a series of compounds that were well-tolerated, with good activity, but these compounds, which included UK-141364 and UK-141366, showed lethal hemodynamic toxicities. Introduction of a carboxyl group produced UK-142773, which prolonged thrombin time in rat at 10 mg/kg intraduodenally and was well-tolerated in mice at 50 mg/kg iv. Finally, the introduction of unsaturation at the piperidine moiety resulted in UK-156406, which shows increased potency and decreased hepatic clearance as a result of improved lipophilicity. In dog, UK-156406 demonstrates moderate clearance (11 ml/min/kg), a good volume of distribution (0.9 l/kg), a plasma half-life of 0.8 h, oral bioavailability of 45%, and a dose-related prolongation of clotting times following oral administration. The compound has progressed to phase I clinical trials, and the full pharmacokinetic profile in humans will be presented at a later date. Factor Xa inhibitors ZX-807191 Berlex maintained a high profile in the Medicinal Chemistry section this year as a result of its Factor Xa (FXa) inhibitor program. At the poster session on Wednesday morning, some 13 posters relating to structure activity studies had been displayed, and KJ Shaw from the company presented an outline of this program. FXa plays a central role in the coagulation cascade and affects coagulation without affecting platelet function. When Berlex began its program 14 IDrugs 1998 Vol 1 No 1 Figure 1. Thrombin inhibitors HN CH3 N CH3 CH3 O O O N N S NH NH NH2 N O O NH2 O L-375378 (Merck Research Laboratories) N H3C approximately five years ago, only a few low molecular weight inhibitors were known. One of these was E,E-2,7-bis(4-amidinobenzilidene)-cycloheptan-1-one (BABCH). Berlex quickly identified the Z,Z isomer of this compound, which was designated ZK-805412, as a highly-active lead. It was also selective (Ki = 0.66 and 530 nM against FXa and FIIa, respectively), but was photochemically unstable, and, being a bisaryl-amidine, belonged to a family for which there were little known toxicological data. With ZK-805412 as a lead, the company used an indolebased template and undertook an extensive series of SAR studies to optimize selectivity and potency whilst enhancing photochemical stability (structural alterations at each part of the molecule were reported in individual posters). The resulting compound, ZK-807191 (Figure 2), showed Ki values of 0.10 and 2000 nM against FXa and thrombin, respectively, and was efficacious in several in vivo models, eg, the rat model of venous stenosis. The compound is currently in phase I clinical trials and a detailed presentation of its pharma-cology will be presented at the forthcoming 1998 FASEB meeting. SK-549 DuPont Merck is another company which has been investigating the use of FXa antagonists. FXa was selected as a target because the thrombin cascade involves signal amplification with one molecule of FXa activating many molecules of prothrombin to thrombin. It was therefore reasoned that inhibition of FXa would be a more economical way of inhibiting coagulation. Mimi Quan, described how a lead was generated by screening the company’s IIb/IIIa library, since a similarity was detected between the binding site of the two enzymes based on inspection of the Arg-GlyAsp sequence of the former. Early compounds from the FXa program, including SF-303 and SF-324 were effective antithrombotics, but possessed an ester side chain at the chiral center. Replacement of the ester group with a nonhydrolyzable moiety led to SM-084 and SK-549. The latter shows an ID50 of 0.035 µmol/kg/h (23 mg/kg/h) in the arteriovenous shunt thrombosis model in anesthetized rabbits, and a Ki of 0.49 nM against FXa. OH UK-156406 (Pfizer) RPR-130737 WR Ewing (Rhone-Poulenc Rorer (RPR), PA, USA) stated how FXa is uniquely situated at the junction of the intrinsic and extrinsic branches of the coagulation cascade, and is the sole enzyme capable of generating thrombin. These attractive characteristics led RPR to begin an FXa program three years ago. The researchers began with a benzamidine ligand and used SKELGEN (see Toderov NPJ: Comput Aided Des (1997) 11:175) to generate a lead with an IC50 of 1.1 µM. However, this compound suffered from slow binding kinetics. Optimization via the use of a pyrrolidinone linker led to RPR-120844, and ultimately to RPR-130737 (Figure 2), which is highly potent (Ki = 2 nM) with more than 1000-fold selectivity against other serine proteases. It is a competitive inhibitor of FXa and is effective against the prothrombinase complex. In addition, it has no effect on heart rate or blood pressure at therapeutic doses. However, no indication was given regarding progression of this compound to the clinic. Poster session Both the medicinal chemistry poster sessions at this year’s conference were relatively modest in size, but some interesting disclosures were nonetheless made. RWJ-64777 R W Johnson Pharmaceutical Research Institute revealed details in MEDI 170 of its program to identify novel and selective inhibitors of p56 Lck tyrosine kinase. This enzyme is expressed primarily in T-cells and natural killer cells, and is critical for their proliferative and effector functions. One of the series of indandiones that the company has developed, RWJ-64777 (Figure 3), shows an IC50 against Lck of 32 nM, compared to 460 and 8000 nM for c-Src and PKA, respectively. H3 antagonists In a series of three posters (MEDI 145, 146, 147), Gliatech described SAR studies for a series of histamine H3 receptor antagonists. One of these compounds, GT-2227 (Figure 3), Meeting Report 215th ACS Figure 2. Factor Xa inhibitors N NH2 N N HN N CH3 OH N O O N N O O S O H2N NH O N O F F F OH N H3C HO F NH2 F HN O ZK-807191 (Berlex) SK-549 (DuPont Merck) NH S O NH2 O S O N N HN OH RPR-130737 (Rhône-Poulenc Rorer) Figure 3. Structures from Medicinal Chemistry Poster Session H O H H H O O HO OH H2N Br O Br HN OH GT-2227 (Gliatech) RWJ-64777 (R W Johnson Pharmaceutical Research Institute) O CH3 H3C N O H3C O N N N O H3C O H3C CH3 N N H N N N N CH3 N N H3C N N Br H3C N H N Br NH H3 C CH3 H3C CH3 H3C CH3 CH3 SJ-948 (DuPont Merck) SC-241 (DuPont Merck) O Cl N SG-058 (DuPont Merck) H 15 16 IDrugs 1998 Vol 1 No 1 shows Ki values of 13407, 4469 and 3.2 nM against human cloned H1, H2 and H3 receptors, respectively, and has been "chosen for additional characterization". It was also described as "…a possible clinical candidate". CRH-R1 antagonists In a series of five posters (MEDI 140-144), DuPont Merck described SAR studies on a series of triazolopyridines and pyrimidines which are corticotropin releasing hormone (CRH-R1) antagonists. Compounds named on the posters included SC-241, SG-058 and SJ-948, (Figure 3). The last compound is in late preclinical development, and extensive in vivo (male beagles) and in vitro data were presented. SJ948 was not in fact the most active of the compounds presented (Ki against hCRH-R1 is 14.2 nM, compared to 3.7 and 4.1 nM for SC-241 and SG-058, respectively), but was selected because of its superior pharmacokinetic profile. Meeting Report 215th ACS 17 Amyloid aggregation inhibitors Michael G Zagorski Address Department of Chemistry Case Western Reserve University Millis 414SA Cleveland OH 44106-7078 USA Email: [email protected] IDrugs 1998 1(1):17-18 © Current Drugs Ltd ISSN 1369-7056 Amongst the 4,700 presentations at the 215th National Meeting of the American Chemical Society (ACS), were fourteen research papers on Alzheimer’s disease (AD) and related issues. The dementia associated with AD is a progressive and common neurodegenerative disorder producing widespread brain destruction, with no curative therapies. The brains of AD patients have an abundance of amyloid plaques and neurofibrillary tangles. The major protein component of the amyloid plaques is the β-peptide that exists in two predominant forms: the shorter, 40-residue β1-40, and the longer, 42-residue β1-42. Recent genetic studies have established that amyloid deposition, particularly by the longer β1-42, is directly linked to early onset cases of AD. As a result, major research efforts are focused on uncovering effective therapeutic strategies to prevent or slow down the aggregation and the associated precipitation of the β-peptide into amyloid. In the amyloid deposits, the β-peptide adopts a β-sheet structure which is proposed to be neurotoxic. Introduction Of the fourteen related papers, seven dealt with β-amyloid and these are summarized in this report. In addition, a press briefing was held at the Adolphus Hotel with four invited speakers: Mony J DeLeon, EdD (Professor of Psychiatry, NYU Medical Center, New York, USA); Regina M Murphy, PhD (Associate Professor, Department of Chemical Engineering, University of Wisconsin, USA); Donald F Weaver, PhD, MD (Professor, Departments of Chemistry and Medicine, Queen’s University, Kingston, Canada); and Michael G Zagorski, PhD (Associate Professor, Department of Chemistry, Case Western Reserve University, OH, USA). Fibrillogenesis of the β -peptide On Wednesday, Girija Krishnamurthy (Wyeth Ayerst, NJ, USA), presented a poster summarizing measurements of fibrillogenesis of the β-peptide. This study is the first to correlate increased β-sheet production (as detected by circular dichroism), with both thioflavin-T fluorescence and neurotoxicity in rat hippocampal E18 cells. These results are in addition to earlier findings with neuroblastoma and PC12 cells, in which the aggregated β-sheet structure is indeed toxic, and support the idea that β-amyloid deposition is a primary pathological event in AD. Amyloid aggregation inhibitors Biophysical studies of β -amyloid The symposium entitled ’Amyloid aggregation inhibitors’, chaired by Corinne Augelli-Szafran (Parke-Davis, MI, USA), began with a presentation by Jeffrey Kelly (Scripps Institute, CA, USA). His seminar focused on the protein transthyretin (TTR), which is a constitutively-secreted protein that transports vitamin A and thyroxin retinol binding protein in both plasma and CSF. The three-dimensional structure of soluble TTR was resolved several years ago by X-ray crystallography, revealing it to be a highly β-sheet-rich homotetramer. This is presumably the species of TTR that is involved in transport of vitamin A and thyroxin retinol binding protein. However, under certain diseased states, including familial amyloid polyneuropathy (FAP), the TTR changes structure to an insoluble amyloid fibril, similar to the amyloid deposits produced by the β-peptide of AD. The proteins in the amyloid deposits have antiparallel cross-β-pleated sheet structures. Dr Kelly presented a mechanism of amyloidosis for TTR involving several alternative, misfolded intermediate structures. TTR has a greater propensity to produce amyloid at acidic pH; therefore, the notion that the low pH environment of the lysosome may be a site for the early formation of amyloid was proposed. The idea all amyloid-associated diseases may involve a common mechanism of misfolded intermediates was also suggested. Dr Kelly also described several small organic compounds that prevent amyloid formation of TTR. These compounds were non-steroidal anti-inflammatory agents and mostly derivatives of anthranilic acid. The X-ray crystal structure of TTR complexed with one such inhibitor was presented; this established that the inhibitors bind specifically to the soluble tetrameric structure. Problems associated with inhibitor binding to other proteins, such as albumin, and approaches to the solution of these problems, were discussed. Analysis of amyloid structure The second speaker, Paul E Fraser (University of Toronto, Canada), described improvements of fibril diffraction methods for analyzing the structure of amyloid deposits. In general, fibril diffraction studies have established that all amyloid-forming proteins produce anti-parallel cross-βpleated sheet structures in the plaques. The topology of these β-sheets appears as super-helical twists of protofilaments. To improve the resolution, the diffraction of the βpeptide was carried out in a magnetic field, since the βpeptide has a high magnetic anisotropy and therefore will orient in a discrete fashion with an external magnetic field. Additional high resolution pictures using atomic force microscopy (AFM) were presented. Analysis of the fibril diffraction and AFM images of the β-peptide complexed with inositol derivatives (putative amyloid inhibitors) revealed that some specific inhibition may result from interference with the characteristic super-helical twisting patterns. It is hoped that future studies will uncover more site-specific interactions that will facilitate the design of more therapeutically-useful β-amyloid inhibitors. NMR studies Michael G Zagorski spoke primarily about high-resolution, solution NMR studies of the β1-40 and β1-42 peptides. In many instances, the NMR approach can provide three-dimensional 18 IDrugs 1998 Vol 1 No 1 structures that are comparable to those obtained by X-ray crystallographic techniques. The three-dimensional, micellebound structure for both β-peptides was described. The conditions under which these were obtained adequately mimic the environment found at charged lipid surfaces such as possibly with lipoproteins (Apo-E and Apo-J). Both peptides have nearly identical and largely β-helical tertiary structures when bound to micelles, indicating that the greater propensity of the β1-42 to produce amyloid is not related to lipid-bound state. More recent NMR studies of the β1-40 and β1-42 peptides in aqueous solution at pH 7.4 demonstrated mostly extended chain structures. The peptides are relatively soluble at concentrations up to 0.8 mM, and aging over several days showed that the extendedchain structures can fold back on themselves to produce an intramolecular β-sheet structure. This is the first real evidence for an early folded intermediate that forms during the β-aggregation. The potential for studying the binding interactions with β-amyloid inhibitors was illustrated with melatonin (J Biol Chem (1998) 273:7185-7188). The NMR data showed that melatonin binds to the β1-40 His and Asp sidechains and this interferes with the salt bridge formation that is critical to β-aggregation. Azo-dye analogs A thorough examination of the inhibition of β-amyloidosis with a series of azo-dye analogs was presented by Warren J Porter (Eli Lilly, IN, USA). Both chrysamine-G and Congo Red show complete neuroprotection in primal hippocampal E18 cells, as measured by a lactase dehydrogenase assay. A critical structural requirement for neuroprotection is the biphenyl ring structure. Problems related to the azo-dyes quenching thioflavin-T fluorescence were briefly described, as this prevented measurements of amyloid formation using the standard thioflavin-T assay. Electron microscopy was used to establish the inhibitory effects of the different compounds. Peptide inhibitors Regina M Murphy described work to establish the specific recognition sequence of the β-peptide that is important to aggregation and toxicity. Both static and dynamic light scattering, together with analytical ultra-centrifugation were employed to measure β-amyloidosis, whilst toxicity studies used PC12 cells. Many synthetic recognition peptide segments were prepared and analyzed, and the greatest inhibition was observed with a Lys16-Leu17-Val18-Phe19Phe20 peptide with a Lys hexamer unit attached to the Cterminal end. The Lys segment functioned as a disrupting element for β-aggregation, which affected the fibril morphology as shown by light scattering. Without the Lys hexameric unit, the Lys16-Leu17-Val18-Phe19-Phe20 peptide inhibited toxicity but did not significantly slow down βaggregation. Design of anti-aggregants About 100 small organic molecules that bind to the putative β-peptide glycosaminoglycan binding motif (His13-His14Gln15-Lys16) were described by Donald F Weaver. The idea for such inhibitors was to mimic, and therefore prevent, the proteoglycan and/or glycosaminoglycan induced amyloid formation. The proteoglycans are normal cellular components of the basement membranes of blood vessels and other biological tissues. The majority of the compounds described by Dr Weaver were sulfonates which inhibit amyloid formation in mice with amyloid-infested spleens. Following molecular modeling simulations and extensive structure-activity relationship determinations, the Canadian group has found a lead molecule that looks very promising. Two pharmacophore models were ascertained, one in which two sulfonates were positioned 5.3 Å apart, and the second with them positioned 7 Å apart. However, Dr Weaver cautioned that any application of this research to humans is at least two years away, due to problems with passage of these compounds through the blood-brain barrier. Conclusions Following the conclusion of the symposium, the conference chair, Corinne Augelli-Szafran, reiterated the several new concepts introduced at this symposium, particularly the improvements in fibril diffraction and the use of solution NMR. These new structural methods should provide important new information that will facilitate the design of more effective amyloid inhibitors. Meeting Report 215th ACS 19 Medicinal Chemistry Awards Symposium Peter Robins Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] drugs which are receiving considerable attention in animal in vivo tests. Figure 1. Irinotecan N CH3 IDrugs 1998 1(1):19-20 © Current Drugs Ltd ISSN 1369-7056 Although of perhaps limited interest to scavengers of hard biological information, one of the undoubted highlights of the third day of the conference was the Medicinal Chemistry Awards Symposium. Two venerable chemists gave resumés of their life's work, and updated the audience on their current research. This was followed by a new award for team innovation. N O N O OH Dr Karle is interested in the variety of unusual conformations that have been observed in peptides (such as kinks, bulges and severely bent helices) and the ways in which these may be induced in synthetic or unnatural peptides. Several hybrid peptides containing three adamantyl linkers were shown which exhibited 2-fold symmetry instead of the predicted 3-fold. These insert themselves into artificial lipid bilayers and act as nonselective cationic pores. Following her presentation, Dr Karle was presented with a bound selection of her papers by the Symposium Chair, AM Doherty. H Cl O CH3 Irinotecan (Honsha) X-rays The first of the awardees was Isabella A Karle, currently at the Laboratory for the structure of matter at the Naval Research Laboratory in Washington DC. Dr Karle was introduced by Dr Ralph H Hirschmann, who praised her "seminal contribution to X-ray crystal structure determi-nation, and to peptide conformational analyses in particular". In addition, he drew attention to her "precision with great attention to detail, combined with a wise and informed overview of the implication of the results". N O O Team innovation Finally, a new award for team innovation was presented to the team at Lilly in Indianapolis responsible for the development of the insulin prodrug Humalog. Drs JH Andeson, RE Chance and BH Frank delivered a threehanded presentation of the development history of the compound which will go a long way to solving problems of patient compliance. Firstly, sc delivery of insulin is far from satisfactory since it must be carried out 45 min before eating to allow time for the self-associated hexameric insulin complex to dissociate into absorbable units. This is a problem if, for instance, a small child refuses to eat following an insulin injection. Humalog, on the other hand, is created by the simple expedient of reversing the ProB28LysB29 sequence, which weakens its tendency to selfassociate into dimers. The stabilized hexameric complex in which it exists thus dissociates immediately upon sc injection, improving the physiologic profile tremendously. HIV combination therapy Anticancers The second awardee was introduced in glowing terms by Dr F Ivy Carroll. Dr Monroe E Wall, a natural product chemist, made his name with the discovery of camptothecin, an extract of the Chinese tree Camptotheca acuminata. This has formed the basis of a whole family of anticancer compounds, such as topotecan (SmithKline Beecham) and irinotecan (Yakult Honsha; Figure 1), both derivatives of 10hydroxycamptothecin. However, Dr Wall is in the unusual position of having discovered two such seminal compounds, having added taxol to the list several years ago. In this presentation, Dr Wall concentrated on current work with third generation camptothecin analogs, which is being carried out with the support of Bristol-Myers Squibb. These new compounds, which include 7-chloromethyl-, 7-ethyland 9-amino-analogs, are water-soluble, low-toxicity pro- Elsewhere, an interesting combination therapeutic strategy using HIV protease inhibitors was described by HL Sham (Abbott, Illinois, USA). Combination therapies exist that include one of the four HIV protease inhibitors currently approved by the US FDA and these can suppress HIV to undetectable levels. However, the pharmacokinetics of these compounds are not entirely satisfactory because of the low trough concentration that is observed. If the plasma concentration falls below the EC50 (for instance, if a dose is missed), this creates a window of opportunity for the virus; five to ten billion virus particles are produced/patient /day, which, combined with the rapid rate of mutation that is characteristic of HIV, allows the proliferation of proteaseinhibitor-resistant strains within an individual. The results may be lethal. 20 IDrugs 1998 Vol 1 No 1 ritonavir would provide a double protease inhibition, whilst preventing rapid clearance, and Dr Sham showed that this was borne out in preclinical studies. ABT-378 is also highly active against many of the most common ritonavir-resistant mutants, so will likely be important in dealing with new mutants as they arise. The compound is now in early phase II trials. The protease inhibitor, ritonavir (Figure 2), is a potent and reversible inhibitor of the 3A subfamily of the cytochrome P450 enzymes, so inhibits its own metabolism. Nonetheless, its efficacy is limited by the fact that trough plasma concentrations are close to the EC50, requiring vigilance with dosing. On the other hand, ABT-378 (Figure 2), is very potent, but rapidly metabolized. Scientists at Abbot reasoned that coadministration of this compound with Figure 2. Ritonavir and ABT-378 S CH3 OH N O H N O N H N N H S CH3 O H3C Ritonavir (Abbott) N O CH3 OH CH3 CH3 O H N O NH N N H O O H3C CH3 ABT-378 (Abbott) CH3 Meeting Report 215th ACS 21 Cytochrome P450 and drug design Chengde Wu Address Texas Biotechnology Corp Houston TX 77630 USA Email: [email protected] IDrugs 1998 1(1):21 © Current Drugs Ltd ISSN 1369-7056 This session, devoted to the consideration of drug interactions with the cytochrome P450 family of enzymes, was presided by Professor Patrick Woster of Wayne State University. SAR considerations The first speaker, Paul Ortiz de Montellano (University of California at San Francisco, CA, USA), described work to determine the substrate specificity of cytochrome P450 enzymes through measurement of the oxidation by P450cam of a series of benzene substrates bearing alkyl groups of up to four carbon atoms. By examining the three uncoupling pathways of a complex of the aromatic substrates with ironO , the spin state shift of the complex was not found to correlate with any other parameter, such as coupled or uncoupled turnover. Similar studies were carried out with the T185L and T185F mutants, in which the active site volume was decreased. Coupled turnover varies widely, but is maximized when the phenyl ring bears a single large alkyl substituent. Decreasing the size of the active site cavity increases coupled turnover without altering the alkyl group size dependence. Finally, substrate oxidation occurred preferentially at secondary or tertiary C-H bonds, and aromatic oxidation was only observed when the alternative was a primary C-H bond. P450 versus target receptor A mere six or so of the more than 40 known human P450 enzymes are responsible for most of the metabolic oxidations of drugs. FP Guengerich (Vanderbilt University, TN, USA) explained how it is advantageous to investigate P450 interactions, in vitro, early in the drug discovery process, since the recognition of a drug as a substrate by P450 enzymes is a major impediment to bioavailabilty. He illustrated his talk using the metabolism of terfenadine as an example (Drug Metab Dispos (1993) 21:403-409), and described some of the procedures that have been employed to investigate the molecular determinants of P450-ligand interaction, such as classical SAR, and amino acid substitutions. Ronald White (Bristol-Myer Squibb, NJ, USA) gave an industrial perspective on P450 -related considerations in drug design. As pointed out by Dr Guengrich beforehand, the recognition of a drug by P450 enzymes can be as important in drug design as the structure-activity relationships for the target receptor. After giving a short introduction to enzyme kinetics, Cmax , t½ , and other basic pharmacological concepts, Dr White described the various biochemical tools that may be used to determine the affinity of a drug for P450. At Bristol-Myers Squibb, P450 recognition data are obtained at various stages in the drug development process, depending on need. For instance, during the initial screening phase, preparations of liver microsomes are sufficient to screen out highly metabolized compounds, with no consideration of structure, whereas more structure-related data may be required at a later stage in order to optimize the metabolic profile of a series of active compounds. Substrate specificity for P450 3-D Homology models of cytochromes P4502B1 and P4503A4 have been utilized by Professor JR Halper (University of Arizona), together with site-directed mutagenesis, to elucidate the molecular determinants of substrate specificity of these two enzymes. Most of the key residues identified in 2B enzymes fall within five substrate recognition sites (SRS) and have counterparts in bacterial P450 residues that regulate substrate binding or access. Analysis of the interaction of inhibitors with models of 2B enzymes has provided a plausible explanation for changes in susceptibility to mechanismbased inactivation that accompany particular amino acid side chain replacements. Based on the 3A4 model and single-site mutants, a double mutant in SRS-2 has been constructed that exhibits normal Michaelis-Menten kinetics. A further SRS, SRS-6, is thought to be the activating site for P4503A4 and Professor Halper presented the results of modeling and mutagenesis studies which suggest that the substrate and effector binding sites in P4503A4 are partially overlapping. ABT-378 Finally, Dr Hing L Sham (Abbott Laboratories, IL, USA) discussed the synergistic co-administration of Abbott’s ritonavir and ABT-378. Ritonavir is an approved HIV protease inhibitor but is also a 30 nM inhibitor of CYP3A4. ABT-378, on the other hand, is a potent second generation compound in phase II clinical trials, but with low oral availability. Dr Sham showed that by coadministering ritonavir and ABT-378, a dramatic, prolonged improvement in the AUC of ABT-378 was achieved in rat, dog, monkey and human. Abbott has recently published a patent (WO09701349) on such application of ritonavir with, for example, cyclosporin. 22 IDrugs 1998 Vol 1 No 1 Angiogenesis Michael S Wolfe Address Department of Pharmaceutical Sciences University of Tennessee 327 The Faculty Building 847 Monroe Street Memphis TN 38163 USA Email: [email protected] IDrugs 1998 1(1):22-25 © Current Drugs Ltd ISSN 1369-7056 This symposium, along with one on amyloid inhibition, was scheduled for the final day of the meeting, undoubtedly in an attempt to keep participants in attendance. The plan worked well: the number of symposia attendees varied from 150 to 300 on a day typically noted for sparse attendance. The organizers guessed correctly that the importance of this topic to drug discovery and development would generate plenty of interest. Michael Moyer (Pfizer) presided over the session. Inhibition of angiogenesis as a therapeutic target The process of angiogenesis and possible therapeutic targets were introduced by Jean S Beebe (Pfizer, CT, USA). Angiogenesis, the growth or sprouting of capillaries from existing blood vessels, is involved in a number of normal physiological processes, including embryonic growth and development, wound healing, ovulation and menstruation. Therapeutic indications for inhibitors include cancer, ocular diseases and chronic inflammation. There is a critical need for new therapies for these conditions, and the limited role of angiogenesis in adult physiology suggests that inhibitors may lead to new agents with reduced toxicities. The application of angiogenesis inhibitors to cancer chemotherapy also offers the hope of circumventing one of the most vexing problems in this arena: the development of resistance. Cytotoxic therapy targets cancer cells, which are genetically unstable, thus resistance develops frequently. Tumor growth and metastasis are dependent on angiogenesis, and since angiogenesis inhibitors target normal cells (eg, vascular endothelial cells), these inhibitors may be less susceptible to the development of resistance. As an illustration of the potential to avoid resistance, repeated cycles of therapy with endostatin, an endogenous protein inhibitor of angiogenesis, resulted in tumor regression in mice. No resistance was observed and the tumors eventually became dormant (see Boehm et al: Nature (1997) 390:404). One strategy for inhibiting angiogenesis is blocking the activation and initiation of endothelial cell replication. Tumors release growth factors, such as vascular endothelial growth factor (VEGF), that are responsible for this first step in angiogenesis, so developing antibodies that bind these growth factors, or small molecules that block the receptors or downstream signaling pathways for these growth factors are two key strategies in preventing neovascularization. Another strategy is to target processes involved in inhibition of angiogenesis; however, the mechanisms of action of negative regulators, such as endostatin and angiostatin, are ill-defined, and small molecule design will be contingent upon determining the signaling pathways involved. Blocking targets involved in vascular remodeling is also a key strategy in preventing angiogenesis. The matrix metalloproteinases (MMPs) are zinc-dependent enzymes that digest the endothelial cell matrix, thereby mediating invasion, metastasis and angiogenesis. Considerable effort has been directed toward inhibiting MMPs. Also, the integrin receptors (particularly αvβ3), involved in cell adhesion, are particularly ‘hot’ targets for antagonist design. Several angiogenesis inhibitors are in clinical trials. IFN-α, which decreases the production of fibroblast growth factor, has already been approved, and those in various phases of clinical trials include thalidomide, the fumagillin analog TNP-470 (Takeda; Figure 1), SU-5416 (Sugen; Figure 1) which is a KDR (a distinct tyrosine kinase receptor) inhibitor, the MMP inhibitor, Marimastat (British Biotech; Figure 1), and αvβ3 antibody, vitaxin (Scripps Research Institute). Dr Beebe ended her talk by raising issues that need to be addressed in order to develop effective agents. Since it is likely that these agents will need to be administered chronically, they must be safe and orally active. There is also a need for validating markers for angiogenesis in patients to determine whether the compounds are working. Although these new agents target normal cells, resistance may still develop if tumors can activate alternative growth factor pathways. Finally, combination therapies with cytotoxic agents, an idea not yet tested clinically, may give optimal responses through synergistic action. Blocking the receptor tyrosine kinase Flk1/KDR The development of novel 3-substituted indolin-2-ones as potent and selective inhibitors of Flk-1/KDR for inhibition of tumor angiogenesis was described by Connie Sun (Sugen, CA, USA). Flk-1/KDR, a receptor tyrosine kinase, dimerizes upon VEGF-binding, resulting in autophosphorylation and initializing a signal tranduction process leading to endothelial cell replication. Sugen has targeted the tyrosine kinase domain of this receptor. 3-Substituted indolin-2-ones (Figure 2) were initially identified as potent inhibitors, although compound 2 was non-selective with respect to other related receptor tyrosine kinases. A number of structural modifications of these 1 compounds led to the conclusions that: (i) N -methylation reduces activity; (ii) placing electron-donating groups in the phenyl 4-position provides high potency and selectivity (in contrast, installing electron-withdrawing substituents in this same position results in inactive compounds); and (iii) methylation of the vinyl group gives inactive analogs. Meeting Report 215th ACS 23 Figure 1. Angiogenesis inhibitors CH3 H3C O CH3 CH3 O H N O CH3 H N O H H O CH3 HO O O N H N H OH Cl O H N CH3 H N CH3 O H3C CH3 O CH3 H3C SU-5416 (Sugen) Marimastat (British Biotech) TNP-470 (Takeda) Figure 2. Flk-1/KDR inhibitors CH3 H3C N CH3 H3C N H O O N H 1 Co-crystallization of an inhibitor with Flk-1 provided insight into the importance of the double bond stereochemistry. In the bound structure of SU-4984, an analog of compound 2 where the isopropyl substituent is replaced by 4methylpiperidine, the oxindole moiety occupies the ATP binding site. Interestingly, the compound 2 analog is bound as the Z isomer, even though it is the E isomer in solution. Thus the E form must be converted to the Z form before binding. Apparently, this conversion can be mediated by acid, base or light. The pyrrole moiety of compound 3 was replaced with other heterocycles (thiophene, furan, pyrazole), but the pyrrole ring was the superior compound. In this series, all the E isomers were inactive, and the vinyl proton was again determined to be essential for activity. In the HUVEC assay, the 3,5-dimethyl pyrrole-analog (SU-5416) was the superior compound, with an IC50 of 40 nM. The compound was also highly selective for Flk-1, being essentially inactive at other related tyrosine kinase receptors, such as PDGF (plateletderived growth factor), EGF (epidermal growth factor), and IGF-1 (insulin-like growth factor). N H 2 O N H 3 SU-5416 was chosen for further evaluation. Cocrystallization revealed that this compound binds at the ATP domain of Flk-1 in essentially the same manner as SU-4984. SU-5416 displayed dose-dependent inhibition of subcutaneous growth of A375 human melanoma in mice, and in preclinical studies, no toxic effects were noted at efficacious doses. This compound dramatically inhibited tumor size and tumor neovascularization, suggesting that tumor growth is not affected directly. SU-5416 displayed a broad antitumor spectrum: ip administration inhibited eight of ten different cell lines grown subcutaneously in mice. Although the plasma half-life was short (30 min), the half-life for activity against Flk-1 was about 20 h, perhaps due to the lipophilicity of the compound. SU-5416 is currently in phase I clinical trials. Orally active vitronectin receptor antagonists The efforts to develop orally active angiogenesis inhibitors by targeting the vitronectin receptor (αvβ3) were described by James Samanen (SmithKline Beecham). This receptor, a member of the integrin family, is involved in cell adhesion, and the potential applications of antagonists include the 24 IDrugs 1998 Vol 1 No 1 treatment of osteoporosis, restinosis and proliferative disorders. The integrin receptors all recognize the RGD (Arg-Gly-Asp) sequence of their respective ligands, and selectivity of a given ligand for its integrin is thought to be primarily due to conformational differences in the RGD region. Thus conformationally restrained RGD-containing peptides possess selectivity for certain integrins. possessed no ability to inhibit migration of human dermal micro-vascular endothelial cell (HMVEC) migration induced by bFGF, while kringles 1-4 and 1-3 were active. Kringle 5 was an outstanding antichemotactic agent, with an IC50 of 200 pM. In comparison, the IC50 for kringle 1-4 (angiostatin) was > 100 nM. Kringle 5 also prevented cell adhesion onto gelatin-coated plates, whilst kringle 4 did not. Last year, the SmithKline Beecham groups reported an advanced, constrained RGD mimetic, SB-223245 (Keenan et al: J Med Chem (1997) 40:2289-92; Figure 3). Although SB223245 was αvβ3 selective and very effective in a cell adhesion assay (IC50 = 120 nM), this compound possessed a short plasma half-life and low oral bioavailability. One strategy employed to improve the bioavailability was to increase the lipophilicity: altering the benzodiazapine moiety by replacing NH with CH2 and fusing a phenyl in place of the amide, resulting in a carbocylic version of SB223245. Although this compound was 10-fold less active at αvβ3, it was racemic and retained selectivity for αvβ3 over αIIbβ3 receptors. Kringle 5 blocked HMVEC migration stimulated by a variety of inducers (VEGF, IL-8, HGF (hepatocyte growth factor), PDGF (platelet derived growth factor), aFGF (acidic fibroblast growth factor) or TGF-β1). Evidence for a receptor for this portion of plasminogen was obtained using 125 [ I]kringle 5; the binding was saturable, with little nonspecific binding and angiostatin competed with kringle 5 for binding. Figure 3. SB-223245 OH H N H N N O H3C O N N O CH3 SB-223245 (SmithKline Beecham) Further analog development resulted in the highly active 2aminopyridine-containing compounds (5 and 6). Resolution of compound 5 revealed that the S-isomer was the more active, and this isomer was still selective (ie, not active against αIIbβ3). The 4’-methyl analog, compound 6, possessed low clearance and astonishingly high oral bioavailability. This compound was effective in a mouse air pouch model, where angiogenesis is involved in the inflammatory response. It also inhibited tumor growth in mice injected sc with MOPC 315 plasmacytoma, a particularly virulent tumor cell line. In a mouse model using the B16F10 melanoma cell line, the compound decreased tumor size, but did not increase survival time. Kringle 5 domain of plasminogen inhibits endothelial cell migration The deconstruction of human plasminogen was described by Don Davidson (Abbott Laboratories, IL, USA). Plasminogen possesses five triple disulfide-containing domains called kringles, and proteolytic cleavage of plasminogen between kringles 4 and 5 by elastase results in kringle 1-4 (angiostatin), an endogenous angiogenesis inhibitor. Elastase was used to isolate other plasminogen fragments in the hope of defining the region responsible for angiogenesis inhibition. Kringle 2-3 Kringle 5 was also effective in two in vivo model systems. Systemic (sc) administration of kringle 5 inhibited bFGFinduced angiogenesis in mouse cornea completely in five of eight mice and partially in another two. 20 µM of protein also completely inhibited the growth of prostate tumor cells in rats. No blood vessels appeared to be growing in tumors isolated from treated animals, suggesting that the inhibition of tumor growth resulted from inhibition of angiogenesis. The group also detected a kringle 5-like protein in human urine: many breast cancer patients expressed this protein, whereas control patients did not. One control did test positive for kringle 5, but it was later discovered that she had previously had cancer. Thus kringle 5 is a candidate marker for disease diagnosis and progression. Natural products as molecular probes of angiogenesis The ability of natural product chemistry to reveal new protein players involved in angiogenesis was illustrated by Craig Crews (Yale University, CT, USA). Fumagillin, isolated from microbial sources, is an anti-angiogenic agent, and a related analog, AGM-1470 (Takeda), is currently in clinical trials. These compounds arrest proliferating cells in the late G1 phase with an IC50 in the pM range and display some selectivity for endothelial cells. Although it is appreciated that fumagillin blocks the phosphorylation of the RB protooncoprotein, its molecular mechanism of action remains largely unresolved. The presence of the epoxides indicates that the compounds might become covalently attached to the target. The Yale group synthesized an analog of fumagillin with an extra hydroxyl group (fumagillol), which could serve as a functional group handle for conjugation to biotin. In a HUVEC proliferation assay, this fumagillol-biotin conjugate was still active at nM concentrations, suggesting that the compound might serve as a molecular hook to fish out the immediate protein target. Visualizing the Western blot using avidin-horseradish peroxidase conjugate resulted in a single new band at 67 kDa; competition with non-biotinylated fumagillol showed that this binding was specific. Isolation, microsequencing, and cloning led to the identification of the target protein as eukaryotic methionine Meeting Report 215th ACS aminopeptidase-2 (MetAP-2) (Sin et al: Proc Natl Acad Sci USA (1997) 94:6099), a known enzyme responsible for cleaving amino terminal methionine after translation. The two forms of this peptidase, MetAP-1 and -2, are highly conserved, and the microbe Saccharomyces cerevisiae survives the loss of either enzyme, but not both. Fumagillin potently inhibits the growth of yeast with deleted MetAP-1 (IC50 < 1 nM), suggesting that the mechanism involves the inhibition of MetAP-2. Yeast cells with deleted MetAP-2 are not sensitive to fumagillin except at much higher concentrations, illustrating the selectivity of the compound for MetAP-2 over MetAP-1. Using numerous fumagillin analogs, Crews and colleagues found a correlation between potency against MetAP-2 and HUVEC proliferation. Currently, the group is developing a strategy for synthesizing and testing new fumagillin analogs by releasing analogs from beads using ammonia vapor. Thus beads with active compounds prevent the growth of yeast with deleted MetAP-1. The Yale group is also trying to identify the targets of two related compounds, eponemycin and epoxomicin. Synthesis of dihydroeponemycin and its biotinylated counterpart has led to the detection of a putative target protein which is apparently different from MetAP-2. Attempts to sequence this new protein are currently underway. The group has completed the first synthesis of epoxomicin, which should facilitate the identification of the epoxide stereocenter. 25 Conclusion Angiogenesis is an fascinating new area of basic science, and controlling this process holds considerable promise for drug development, particularly in the area of cancer chemotherapy. The identity and role of some molecular players (eg, vitronectin receptor, Flk-1) are well defined, and considerable effort has been spent in the development of compounds that interact with these targets. These agents are likely be approved for clinical use in the near future. Meanwhile, there are undoubtedly many unknown proteins and pathways involved in angiogenesis. Determining the mechanism of action of negative regulators of angiogenesis, as well as natural product inhibitors, will greatly increase the number of viable targets for drug development. In a conversation after the session, presiding officer Michael Moyer emphasized that the issue of resistance to antiangiogenic drugs is not yet resolved. He also believes, like Jean Beebe, that combination therapies with the cytotoxic agents are certainly worth testing. It is possible that such combinations would be synergistic and that lower (and therefore safer) doses of the cytotoxic drugs would be needed. 26 IDrugs 1998 Vol 1 No 1 Aromatics, heterocycles and porphyrins Kevin G Pinney Address Department of Chemistry Baylor University Waco TX 76798 USA Email: [email protected] IDrugs 1998 1(1):26-27 © Current Drugs Ltd ISSN 1369-7056 This session covered a variety of interesting synthetic methodologies and target directed syntheses with far-reaching applications towards the preparation of both natural and unnatural products, as well as analogs with potentially improved biological activity. Two presentations focused on molecular recognition of the colchicine binding site on tubulin, and included the presentation of molecular probes as well as lead candidates for new drug development, whilst five sessions centered on interesting new developments in porphyrin chemistry. The overall attendance at the session was very good and at some points reached approximately 250 attendees. Directed Remote Metalation Stephen Houldsworth from the laboratories of Professor Victor Snieckus at the University of Waterloo, Canada presented an interesting discussion on the further development of their popular methodology for the Directed Remote Metalation (DreM) of aromatic systems. This methodology has a wide range of applications including the preparation of dibenzo[b,d]pyranones, fluorenones, and other classes of aromatic systems. An eloquent application of this DreM methodology was presented which described the synthesis of several aromatic kinamycin antibiotics such as prekinamycin and kinobscurinone. In addition, a remote anionic Fries rearrangement was described with application towards the synthesis of the defucogilvocarcin family of natural products which have antitumor activity. The synthesis of piperolactam C, a representative benzylisoquinoline alkaloid, also with antitumor activity, was described. New classes of tubulin polymerization inhibitors Vani P Mocharla working at Baylor University in Dr Kevin G Pinney’s research group presented the develop-ment of several new types of inhibitors of tubulin polymerization which were designed to interact at the colchicine binding site on b-tubulin. Several interesting design features were discussed, as well as details of the chemical synthesis of each of the new inhibitors. The most prominent compounds presented included benzo[b]thiophenes, benzofurans, diarylethers, and a dihydronaphthalene derivative. The dihydronaphthalene ligand is especially promising since it demonstrates excellent inhibition of tubulin polymerization (IC50 = 1.7 µM) as well as significant activity against human cancer cell lines (for example, GI50 = 0.0038 µg/ml against a prostate cancer cell line (DU-145)). Additional design and synthetic work towards the development of ligands with improved bioavailability was also presented. A patent application was initially filed in March 1997 (patent pending), and a PCT application was filed on March 6, 1998. Molecular probes and chemotherapeutic agents based on combretastatin A-4 A second presentation from Dr Pinney’s group at Baylor University was given by Ma del Pilar Mejia. This talk focused on the development of new molecular probes for tubulin based on combretastatin A-4, which are designed to interact with the colchicine binding site on b-tubulin. Amongst the new probes, the most promising appears to be a 3-azido combretastatin analog which is being developed as a photoaffinity labeling agent for the colchicine site. This new compound demonstrates excellent inhibition of tubulin polymerization (IC50 = 1.3 µM) and displays significant activity against human cancer cell lines (for example, GI 50 = 0.0033 µg/ml against a neuroblast cancer cell line (SK-NSH)). This superb biological activity suggests a strong interaction at the colchicine binding site which is, of course, imperative if these ligands are to be employed as molecular probes for binding site elucidation. A new compound which is intriguing for potential development as an anticancer drug was also described. This 3-amino combretastatin analog is a potent inhibitor of tubulin polymerization (IC 50 = 1.7 µM) and is extremely active against human cancer cell lines (GI50 ≤ 0.001 µg/ml for numerous human cancer cell lines). Synthetic details of these ligands were presented, as well as efforts which are underway to develop prodrugs based on the 3-amino combretastatin derivative. A patent application has been filed on these combretastatin derivatives. Heterocyclic enediynes An interesting presentation by Dr KC Russell from the University of Miami highlighted an ongoing project aimed at exploring the synthetic utility of heterocyclic enediynes specifically as substrates in the Bergman cyclization. This work is especially relevant, since a variety of antitumor antibiotics (such as dynemicin) are known to effect their potent biological activity through an induced Bergman cyclization. Several new heterocyclic enediynes (diethynylpyridine and diethynylpyrimidine, for example) were prepared and their kinetics studied in this reaction. The heteroarenediynes react faster than the analogous arenediynes. For example, the diethynylpyrimidine derivative shows t1/2 = 7 min at 154 °C, whereas the figure for diethynylbenzene was 52 min at 152 °C). These results are encouraging for new drug development utilizing heterocyclic enediynes. Novel porphyrin ligands Three very informative presentations were given in sequence by members of Dr Kevin M Smith’s research group at the University of California, Davis, USA. The first talk, by Meeting Report 215th ACS Cinzia M Muzzi, centered on efforts to prepare a chiral dodecasubstituted porphyrin via a Suzuki coupling reaction. Since nonplanar distortions are thought to play a role in the biological activity of certain tetrapyrroles, it is of interest to prepare these sterically-congested porphyrins to investigate their physical and electronic properties. An ultimate goal might be to learn more about why cytochrome P450, for example, is able to carry out complex regiospecific and stereospecific oxidations. The second presentation was given by Kalyn M Shea and focused on dodecasubstituted chlorins (dihydroporphyrins). Numerous synthetic details were presented, as well as a discussion of the non-planarity induced by the dodecasubstitution pattern. The unique nature of these porphyrins may have far-reaching applications in material science and also in the development of interesting models of biological systems. The third and final presentation in this series by Richard G Khoury centered on calixarenes and specifically addressed the preparation of calix[4]arenoporphyrin. The X-ray structure of this compound has been determined, and it is noteworthy that the four porphyrins are all located on the same face of the calix system. Potential applications for these new compounds include the development of new molecular sensors and unique molecular receptors, to name just a few. Cobalt-precorrin-4 Patricio J Santander, working in Professor A Ian Scott’s laboratory (Texas A&M University, College Station, TX, USA), presented work to discover the intermediates and enzymes beyond precorrin-3 (an intermediate on the 27 Vitamin B12 anaerobic path). He makes use of the known 13 early enzymes to prepare [ C]isotopomers of precorrin-3. Details of the enzymes involved, as well as structural features of a cobalt-precorrin-4 (a tetramethylated intermediate) were presented. These results have far-reaching implications, not only because of their significance in advancing our understanding of the biosynthetic pathway of Vitamin B12, but also because they herald significant advances in the use of modern NMR techniques to elucidate complex solution structures of biomolecules. Porphyrins and phosphorescence quenching Two back-to-back presentations concerning porphyrin chemistry and its applications to biological systems were then presented by SA Vinogradov, working in conjunction with DF Wilson (University of Pennsylvania, PA, USA). In the first lecture, tetrabenzoporphyrins of palladium were described as near infrared phosphors, useful for oxygen determination by phosphorescence quenching. New synthetic approaches resulting in the preparation of bromine-substituted tetrabenzoporphyrins which are poised for further functionalization were described. In the second presentation, dendrimer-porphyrins were described, which have strong oxygen-dependent phosphorescence. Studies were described which address the accessibility of the core of the dendrimer-porphyrin, especially in terms of the size of the dendrimer constituents. Oxygen-dependent phosphorescence quenching was presented as a means of studying and analyzing structural features of the core of these porphyrins in terms of dendrimer branching. 28 IDrugs 1998 Vol 1 No 1 Total syntheses of alkaloids, antibiotics, anticancers and antivirals David Pleynet Address Amylin Pharmaceuticals Inc 9373 Towne Centre Drive San Diego CA 92121 USA Email: [email protected] IDrugs 1998 1(1):28-29 © Current Drugs Ltd ISSN 1369-7056 This session, attended by approximately 120 delegates was part of the Organic Chemistry Division. It offered relevant extensive and interesting approaches towards the synthesis of new anticancers, antivirals and antibiotics. The four-hour session was presided by Patrick Harran and comprised a succession of 12 speakers, each giving a twenty-minute delivery. which is used in the production of a variety of blue cheeses. The unique conformation of Roquefortine C, was investigated by computer modeling. Its rigid and stable structure, resulting from hydrogen bonding interactions, was highlighted. The key steps in the synthesis of Roquefortine C were the preparation of the prenyl pyrrolo [2,3b] indole from tryptophan and the subsequent synthesis of the diketopiperazine moiety by cyclodipeptide coupling. The final step consisted of the coupling of N-trityl protected imidazole-4-carboxalde-hyde with the previously described diketopiperazine intermediate. Stemfoline A synthetic approach to stemfoline, a hexacyclic insecticidal alkaloid was described by T Smalley (University of Rochester, NY, USA). A key step in the ongoing synthesis is the formation of three rings by means of a cascade reaction. Altohyrtin Wesley Trotter (Harvard University, USA), opened the meeting by describing the asymmetric total synthesis of Altohyrtin C (Spongistatin 2) in 31 steps. He described the coupling of the various fragments of the molecules. The most notable of which were the A-B and C-D rings coupling, which is effected by a double stereo-differentiating aldol reaction and, the subsequent coupling of the A-B-C-D fragment with the E-F portion, which is achieved by means of a Wittig reaction. This late introduction of the side chain was achieved by diastereoselective β-C-glycosidation. Gallotannin The first total synthesis of a dimeric gallotannin, which is an analog of the naturally-occurring ellagitannin Coriariin A, was reported by Kiran Sahasrabudhe (Pennsylvania State University, PA, USA). She highlighted that a possible biomimetic dimerization of an orthoquinone advanced intermediate by a Diels-Alder reaction led to the formation of a key diaryl ether linkage. It was also shown that the immunostimulatory activity of the dimer was comparable with that of Coriariin A and had a maximal activity after 24 h. Saponin OSW-1 The first synthesis of the aglycone of the potent antitumor steroid, OSW-1, was presented by Chuangxing Guo (Purdue University, IN, USA). This occurs in nine steps, from 5androsten-3b-ol-17-one, and in an overall yield of 15%. The key reactions involved the ene installation of the side chain, a regio- and stereoselective dihydroxylation and the diastereoselective reduction of the ketone function at C-16. Roquefortine C The conformational analysis and studies towards the total synthesis of Roquefortine C and related compounds, were discussed by Wei-Chuan Chen (University of Pennsylvania, PA, USA). Roquefortine C is an alkaloid isolated from culture filtrates of the common fungus, Penicillium roqueforti, Fluorine-containing camptothecin analogs The synthesis of fluorine-containing camptothecin analogs using N-fluoro bis(trifluoromethylsulphonyl) imide, prepared from bis(trifluoromethylsulfonyl) amine and F2 was discussed by Weiwen Ying (Clemson University, SC, USA). The major product, 14-fluoro-(20S)-camptothecin, was synthesized when the reaction was carried out in 2,2,2-trifluoroethanol at o 23 C. However two diastereomers 5-(2’-2’-2’-trifluoroethoxy)14-fluoro-(20S)-camptothecin were obtained by raising the o temperature to 80 C and employing 4 equivalents of N-fluoro bis(trifluoromethylsulfonyl) imide. The fluorinated camptothecin analogs showed enhanced biological activity. Sibirine Masato Koreeda (University of Michigan, USA), explained the radical cyclization of (alkoxycarbonyl-amino)methyl radicals and its application to the total synthesis of sibirine, which is effected in 11 steps and 13% overall yield from 3thiophenyl-cyclohexenone. He highlighted the key step, which involved the highly stereo- and regiocontrolled 6-exotrig cyclization of the radical which was generated from an advanced phenyl selenide intermediate and provided the spiro-bicyclic sibirine in a yield of 60%. A novel efficient method for opening spiroketals The first total syntheses of cephalostatin 1, North G and the hybrid analog, ritterostatin GN1N, were reported by Tom LaCour (Purdue University, IN, USA). Cephalostatin 1 is an extremely potent antineoplastic and ritterostatin GN1N is highly active in the NC160 panel. North ritterazine G was efficiently constructed from hecogenin acetate (30% yield, 10 steps) via a novel method for opening spiroketals and extension of a key photolysis/Prins sequence. Installation of the D14 moiety in the North G and South 1 units and experimental evidence for the biosynthesis of North A were presented. Unsymmetrical coupling provided the natural and analog pyrazines from cephalostatin and ritterazine components. Meeting Report 215th ACS 29 Epothilone analogs QSAR The design, synthesis and biological evaluation of epothilone analogs were described by Joanne Holland (University of Pennsylvania, PA, USA). The synthesis of constrained analogs, focusing on the eastern and western hemispheres, were presented. Molecular modeling established that a 2carbon tether between C-2 and C-10 of epothilone, renders the structure more rigid without changing the conformation. The key step for the generation of the cyclic structure of these analogs was an olefin metathesis reaction in the presence of a Lewis acid. These analogs were tested by Susan Horwitz and were found inactive despite a good overlay with the X-ray structure of epothilone. This reinforces the importance of the entire structure of epothilone for biological activity. QSAR and distribution studies of CADA analogs having activity against the human cytomegalovirus (HCMV) were presented by Meinrado Samala (University of Nevada, USA). He performed a comparative molecular field analysis (CoMFA) on a set of 23 compounds with EC50 values ranging from 2 to 5 mM and is currently trying to use the predictive abilities of the program to identify potential lead compounds. Finally, the remaining presentations illustrated the increasing importance of computer modeling and QSAR programs in the generation and development of new drugs. 30 IDrugs 1998 Vol 1 No 1 Glucosidase and fucosidase inhibitors & Advances in nucleosides and nucleotides Zbigniew J Witczak Address Department of Pharmaceutical Science School of Pharmacy University of Connecticut Storrs CT 06269 USA Email: [email protected] evaluation of the hitherto accepted mechanism of retaining b-glucosidases, particularly of hydrogen bonding and electrostatic interactions. These new aspects of the inhibition of retaining glycosidases are clearly supported by analysis of the crystal structure of the enzyme and the synthesis of novel, conformationally-defined neutral inhibitors from the glyconolactone versatile synthetic intermediates. IDrugs 1998 1(1):30-31 © Current Drugs Ltd ISSN 1369-7056 Professor Spencer Knapp of Rutgers University in New Jersey also presented observations on retaining glyco-sidases, in this case, neighboring group participation. New evidence suggests that certain N-acetyl-β-hexa-minidases and chitinases cleave their substrates with overall retention of configuration through participation at C-1 by the substrate acetamido carbonyl oxygen to form an oxazoline intermediate. For various retaining α- and β-glucosidases, it is strongly suggested that the enzyme active site carboxylate attack occurs at C-1, leading to a substrate-enzyme covalent intermediate. This leads to the question: is enzyme carboxylate attack considered as universal or might there be retaining glycosidases with substrate participating groups other than 2acetamido? The answer is that this is quite possible, but new, and existing, data need to be proven and generalized. The American Chemical Society Symposium "Glucosidase and fucosidase inhibitors" took place on 1 April 1998 and was organized by Professors Zbigniew J Witczak (UConn, School of Pharmacy, CT, USA), Kuniaki Tatsuta (Waseda University, Tokyo, Japan) and Waldemar Priebe, MD (Anderson Cancer Center, University of Texas, USA). Professor Witczak provided introductory remarks including the status of existing glucosidase inhibitors, and chaired the morning session, which consisted of six lectures. The symposium was well received, and was particularly attractive for those interested in networking, as attendance was about sixty. In addition, some participants and attendees presented posters on the subject during the regular poster session organized by the Division of Carbohydrate Chemistry. Glucosidase and fucosidase inhibitors Professor Steve Withers of the of University of British Columbia, set the proceedings in motion with an excellent overview of glucosidase inhibitors; this included new developments in the synthesis and use of 5-fluoro-5-deoxyand 2-fluoro-2-deoxy- derivatives of glucose to obtain new experimental evidence of transition state inhibitors via increased interaction at the anomeric center. Kuniaki Tatsuta gave a wonderful overview of the modern synthetic approaches to the various classes of existing natural glucosidase inhibitors including cyclophellitol, nagastatin and KD16-U1. Professor Tatsuta communicated that the analogs have different configurations and functionalities, confirming that they serve as antagonists of the corresponding α- or β-glycosidases. Linkage and configuration-specific inhibitors of glycosidase may be useful in treating diabetes and metastatic cancer, as well as lysozomal storage diseases. Professor Bruce Ganem (Cornell University, NY, USA) discussed enantioselective synthetic studies on novel glycosidase inhibitors, including trehazolin, and new functionalized analogs of isofagomine. Another extremely important aspect is their involvement in regulating the biosynthesis of N-linked glycoproteins and glycolipids, which play prominent roles in immune recognition phenomena such as lymphocyte recirculation and trafficking, and cell-cell or cell-matrix adhesion. Retaining glycosidases Professor Andrea Vasella, from the Swiss Federal Institute of Technology in Zurich, discussed retaining glycosidase inhibitors. Comparative inhibition tests have led to the re- The morning session ended with a lecture on new developments in the synthesis of C-glycosides as potential inhibitors of carbohydrate processing enzymes, by Professor Francesco Nicotra (Milano University, Italy). Two derivatives of glycosyl phosphates as spacer-connected disaccharides were selected as potential inhibitors of glycosyltransferases and glycosidases. The glucosidase inhibition data, however, suggest only weak activity. Fucosidase inhibitors Kuniaki Tatsuta chaired the afternoon session, which focused on fucosidase inhibitors and new synthetic approaches to azasugars as potential fucosidase inhibitors. The first lecture of the afternoon was presented by Yoshiyuki Kobayashi from the Medicinal Chemistry Research Laboratories of Sankyo in Tokyo, Japan. He reviewed the current status of the chemistry and biology of trehazolins, including comparative data on both stereoisomers of trehazolin and other analogs. The company is working on new approaches to analogs with the same or similar type of biological activity. Professor Olivier R Martin of SUNY, Binghampton, NY and University of Orléans, France, then summarized novel series of azasugars, ie, four- to seven-membered iminoheptitols and their derivatives. These included: azetidine derivatives; C-vinyl pyrrolidines; C-glycosyl compounds related to nojirimycin; aza-C-glycosyl amino acids; and, substituted azepanes derived from 7-amino-2-heptulose. Iminosugars The use of OH substituents to direct a chair conformation of 1-N-iminosugars and the relationship of this conformation Meeting Report 215th ACS to inhibition of β-glycosidases was discussed by Professor Yoshitaka Ichikawa (John Hopkins University School of Medicine, Baltimore, MD, USA). New families of 1-Niminosugars (nitrogen at anomeric position) are extremely potent inhibitors of the corresponding β-glycosidases, including glucosidase, galactosidase and glucuronidase. Inge Lundt (Technical University of Denmark, Lyngby, Denmark) described the regioselective functionalization of aldonolactones as a general strategy for the synthesis of iminosugars without the use of protecting groups. 6Deoxyisofagomin, and its L-ribo analog, were prepared and tested for enzyme inhibition. Both proved to be strong inhibitors of almond β-D-glucosidase, with Ki = 3 µM and Ki = 2 µM, respectively. The L-ribo analog also inhibits α-Lfucosidase, with Ki = 80 µM. Professor George Fleet, from Dyson Perrins Laboratory at Oxford University, began with an initial overview of natural and unnatural fucose and rhamnose mimics. General synthetic strategies were presented for the synthesis of bicyclic oxa- and aza-L-rhamnose mimics. These new analogs are commonly found in the cell walls of bacteria, such as Mycobacterium tuberculosis, and may function as effective inhibitors of α-L-rhamnosidases. Such new analogs have potential as selective therapeutics for the treatment of mycobacterial infections, such as tuberculosis. α-L-fucosidase inhibitors Zbigniew J Witczak presented developments from his laboratory in the synthesis of novel (1-4)-S-thiodisaccharides as α-L-fucosidase inhibitors. New stereoselective approaches utilize the Michael addition of protected 1-thiosugars to a bicyclic enone (levoglucosenone), producing target compounds in high yield. (1-4)-S-Thiodisaccharides containing the L-fucose moiety are, however, weak α-L-fucosidase inhibitors (Ki = 2.4 µM) with significant differences of inhibitory activity on α-L-fucosidase from a bovine kidney and a bovine epididymis. They are about 10-fold more potent on α-L-fucosidase from human placenta (Ki = 0.2 µM) than from bovine kidneys. Finally, Khushi L Matta (Roswell Park Cancer Institute, Buffalo, NY, USA) presented some aspects of the chemistry and biochemistry of α-fucosylated saccharides, and the substrate specificity of α-L-fucosidases and α-L-fucosyltransferases. Kuniaki Tatsuta and Zbigniew J Witczak then concluded the symposium with a summary of all lectures and highlighted some of the new developments in this field (eg, new iminosugars, trehazolins and azasugars) that have produced potential leads for the development of new pharmaceuticals. Advances in nucleosides and nucleotides The only morning session of the last day of the meeting was chaired by Professor Waldemar Priebe, MD, and consisted of five lectures. First, L Ma, an associate from Professor 31 Raymond F Schinazi’s laboratory at Emory University School of Medicine in Athens, GA, presented data from a collaborative project with Professor CK Chu of the University of Georgia in Athens on the separation and biological activity of enantiomers of 2’,3’-dideoxy-5-fluoro3’-thiacytidine (FTC) and its selenium analog (Se-FDDC). In primary human lymphocytes infected with HIV-1, (-)-β-SeFddC (mean EC50 = 0.21 µM) was 200-fold more potent than the (+)-β-counterpart and, interestingly, demonstrated no cytotoxicity in various cells. Fluorinated nucleosides and oligonucleotides Two presentations followed from the CRC Centre for Cancer Therapeutics, Sutton, UK, on the preparation of fluorinated analogs of nucleosides and oligonucleotides. Catherine A Brown presented a new method for the preparation of 2’and 3’-trifluoromethyl nucleosides via SN2’ substitution of the corresponding 2’- and 3’-difluoromethylene derivatives. ’ Interestingly, removal of the 2 - and 3’-O-trimethyl-silylethoxymethyl groups with tetrabutylammonium fluoride resulted in unexpected SN2’ substitution at the unsaturated difluoromethylene carbon with the formation of new analogs, mainly 2’,3-didehydro-2’,3’-dideoxy-5’-O-dimethoxytrityl-2’-trifluoromethyl uridine. Dr Pawel Serafinowski then discussed new studies on the synthesis of (2’-5’)-linked oligonucleotides modified with 3’-difluoromethylene groups. The protected dimers were synthesized by condensation of 3’-deoxy-3’-difluoromethylene-5’-O-dimethoxytrityluridine-2’-O-phosphor-amidite with 3’-O-acetyl or 3’-Olevulinylthy-midine. The synthesis of the target 2’-5’-linked oligonucleo-tides was performed by solid phase synthetic cycle using an oxalyl linker and t-butylhydroxyperoxide in the oxidation step. The final two presentations in this symposium related to work carried out at the Howard Hughes Medical Institute at the University of Chicago. Xiangmin Liao reported Tetrahymena ribozyme cleavage of 5’-methylene phos-phonate and the effect of replacing the 5’-bridging oxygen of the phosphate at the cleavage site with a methylene group. The ribozyme unexpectedly catalyzes the cleavage of this methylene phosphonate linkage 1000-fold faster than the unmodified linkage in the substrates containing all deoxynucleotides. This particular effect is attributed to a 10fold effect on docking of the substrate into the catalytic core and a 100-fold effect on the chemical step. Xiao-Quing Tang then discussed the synthesis and incorporation into oligonucleotides of phosphoroamidate derivatives of 2’-C-β-methylcytidine, which are potentially useful for antisense oligonucleotides and for probing the structure and function of RNA. The strategy developed for the synthesis of the phosphoroamidate derivatives of 2’-C-βmethylcytidine, ie, protection of the 2’-hydroxy group with either t-butyldimethyl silyl or tetrahydropyranyl groups, permitted their incorporation into oligonucleotides by solid phase synthesis. 32 IDrugs 1998 Vol 1 No 1 Ernest Guenther award in the chemistry of natural products Kevin G Pinney Address Department of Chemistry Baylor University Waco TX 76798 USA Email: [email protected] IDrugs 1998 1(1):32-34 © Current Drugs Ltd ISSN 1369-7056 A marvelous symposium was held to honor Professor GR Pettit (Arizona State University, Tempe, AZ, USA) as the 1998 recipient of the Ernest Guenther Award in the chemistry of natural products. Attendance at the symposium was fantastic, numbering well into the hundreds, and at various times there was standing-room only. Three outstanding lectures given by MR Boyd (National Cancer Institute, Frederick, MD, USA), David GI Kingston (Virginia Polytechnic Institute and State University, Blacksburg, VA, USA), and JP Kutney (University of British Columbia, Vancouver, USA) preceding the superb award address by GR Pettit. Professor Pettit was warmly acknowledged by each lecturer who preceded him. MR Boyd gave an especially touching tribute to Bob Pettit commenting on his professional excellence, scientific and personal integrity, dedication to public service, and declaring that he truly was a "National Treasure". On a personal note, I find Bob Pettit to be a brilliant scientist and tenacious researcher (in a sense, a true pioneer and statesman for science) who is driven by the humanitarian purpose of discovering and developing the most effective antitumor drugs possible and getting them to the people who need them the most, the cancer patients themselves. It is a further delight that Bob Pettit is a most humble and gracious man, as well as a true gentleman, who is a world leader in the battle against cancer. Discovery of naturally-occurring inhibitors of HIV Dr MR Boyd presented a fascinating lecture describing the current status of his laboratory’s HIV research as it pertains to new drug development from natural sources. With 40 million people affected worldwide, there is truly an escalating pandemic of HIV infection. This fact, coupled with the frequent and inevitable emergence of resistance to available drugs, mandates the need for the development of new therapeutic agents with non-overlapping resistance profiles and diverse mechanisms of biological activity. It was also mentioned that an effective vaccine or vaccines has thus far remained elusive. New natural product drug development leads have been discovered from both terrestrial and aquatic sources. The discovery process employs bioassay-guided chemistry. A cell-based bioassay using infectious HIV is utilized and the principal source of extracts for study come from the NCI Natural Products Repository, which is the world’s largest collection of samples with biodiversity for drug discovery. The lead compounds discovered from terrestrial plants have been mostly non-peptide in nature. For example, (+)- calanolide A, derived from the Malaysian tree, Calophyllum lanigerumunder, is a non-nucleoside HIV-1-specific reverse transcriptase inhibitor (NNRTI) which is active against diverse drug-resistant strains. This drug is being developed through a joint venture between MediChem Research (licensee to WO09320082, owned by the US government) and the government of Sarawak in Malaysia. Calanolide seems to have favorable bioavailability in initial clinical trials. In addition, prostratin, a non tumor-promoting phorbol ester, was described as a novel subclass of PKC agonist. A variety of new drugs have also been discovered from aquatic sources. These compounds seem especially useful for microbicide (anti-HIV) development, and as templates for new small-molecule inhibitors of HIV. Cyanovirin-N (CV-N), a protein initially isolated from a blue-green alga, Nostoc ellipsosporum, is a protective agent which targets viral gp120. Recombinant cyanovirin is now also available, and it has the same activity as the natural material. Low nanomolar concentrations of CV-N irreversibly inactivate diverse M-tropic, T-tropic, and dual-tropic primary isolates of HIV. Possible applications of cyanovirin include virucidal treatment of blood and topical use to avoid sexual transmission of HIV. Natural products from the tropical rainforest of Suriname Professor David GI Kingston gave an inspiring presentation of his group’s ongoing collection, isolation, structure determination, and synthetic analog preparation efforts in the tropical rainforest of Suriname. Prefacing his presentation with a factual reminder that a large number of clinically-significant drugs have resulted directly from discoveries in natural products chemistry, and that one-half of all prescriptions dispensed in the United States contain substances of natural origin, Professor Kingston challenged the audience with the escalating problem of biodiversity loss due, in part, to the harvesting of tropical rainforests for timber and other industrial concerns. These natural repositories of terrestrial life are disappearing rapidly and currently occupy only 7 to 8% of the earth’s surface, which is down from approximately 16% of the earth’s land surface in the 1950s. One effort to slow down this trend centers around the concept of biodiversity conservation in which a host country is rewarded financially for preserving its biodiversity. Newly-discovered drugs would be licensed by major pharmaceutical companies and certain royalty payments would be returned to the host country in compensation of their productive use of their biodiversity resources. One mechanism for such compensation is through the "Forest Peoples Fund" for distribution of upfront payments from Bristol-Myers Squibb in anticipation of future discoveries. Professor Kingston and his group, along with Bristol-Myers Squibb, their industrial collaborator, Conservation International, Missouri Botanical Garden, and their in-country partner BGVS, working closely with the shamans (tribal Meeting Report 215th ACS healers) have already made significant discoveries in Suriname. To date, approximately 1000 plant samples have been collected, 3000 extracts have been screened for biological activity, and 15 to 20 active compounds have been isolated from the 80 positive hits. The remainder of Professor Kingston’s interesting lecture focused on the structure and activity of these new compounds, including a new ellagic acid derivative, as well as other diterpenoids, quinones, alkaloids, and polyketides. The emerging role of biotechnology in natural product drug discovery The application of biotechnology to the field of natural product synthesis was eloquently presented by Professor JP Kutney, rightfully regarded as a true pioneer in the field. In the first part of his presentation, Professor Kutney illustrated the use of biotechnology in a synthetic application toward vincristine and vinblastine. These two potent anticancer drugs have been known for some time, but their cost is prohibitive, since they are isolated from plant sources. An intriguing biotechnological approach to this problem involves using waste by-products from the biosynthetic pathway as new starting materials in enzymatically-driven reactions. Using this method, vinblastine can now be produced in a one-pot process from catharanthine and vindoline with an overall yield of 42%. This translates into the ability to produce gram quantities of vinblastine in one day. In a move that may someday replace the current isolation of vinblastine from plants as the source of the drug, IGT (a Vancouver company) has licensed this biotechnological process for the production of vinblastine. A new drug, anhydrovinblastine (AVLB), has resulted from this work and is currently in preclinical development. In the second part of the presentation, Professor Kutney described the useful combination of synthetic organic chemistry with biotechnology. Synthesizing various precursors from biosynthetic pathways paves the way for the use of these compounds in combination with enzymes for the economical production of drugs. Various applications were discussed including the antimitotic agent podophyllotoxin and chiral analogs of podophyllotoxin, as well as etoposide. These processes can be carried out in "biological factories" which are large reactors operating in a semicontinuous fashion. In one particular example, the same cell line remained active for four months, allowing 22 batches of the product to be produced. Relatively inexpensive reagents can be employed to generate kilogram amounts of material. Aspects of this biotechnology have also been transferred to IGT for further development and commercialization. In the final portion of the lecture, an informative discussion of the use of plant tissue cultures for the production of high levels of products (when compared to living plants) was presented. A Chinese herbal plant, Tripterygium wilfordii, has been known to have medicinal value for over 2000 years and is used to treat various skin disorders, arthritis, and chronic hepatitis. Over the years, a variety of compounds, especially diterpenes and diterpene epoxides, have been isolated from the plant. Since some of these compounds have pronounced antitumor, immunosuppressive, and/or antispermatogenic activity, the development of high-yield production processes 33 is warranted. The remainder of the lecture centered on the application of plant cell cultures as a means not only of addressing this availability problem but also to discover new and novel analogs. Adventures in the discovery of naturally occurring anticancer drugs The afternoon concluded with a brilliant award presentation by Professor GR Pettit. Professor Pettit began his address by stating that he was deeply honored by the award and that he wished to dedicate his lecture to the memory of Dr Kenneth Paull (National Cancer Institute) and Sir Derek Barton (Texas A&M University) who both passed away recently and were especially good friends of his. Continuing with a comment that summarizes his tenacity as a researcher, Professor Pettit stated, "when I get up each morning, the chemistry of natural products is so exciting that I hardly know which way to run first!" With the cancer problem continuing to escalate (nearly 600,000 people will lose their lives to cancer in the United States this year), it is paramount that new second, third, and fourth generation drugs be developed to treat the over 200 types of human cancer. Beginning with a discussion of natural products isolated from terrestrial sources and amphibians in the early days of his academic career, Professor Pettit discussed work which has so far spanned more than four decades and has involved terrestrial and marine sources throughout the entire world. Pettit and coworkers collected the spongistatins from a red sponge off east coast of South Africa in the early 1970s. Extracts of these sponges more than doubled the lifetime of certain laboratory animals afflicted with the leukemia P388 cancer cell line. Often forced to collect several tons of source material to yield very small amounts of purified drugs, Professor Pettit and his group have remained steadfast in their dedication to the cancer problem. In 1982, for example, the first of the spongistatins had been purified resulting in approximately 800 mg! This corresponds to isolation yields -6 -8 in the range of 10 to 10 % for certain natural products from these sponges. The spongistatins have been shown to have spectacular cytotoxicity and antitumor activity, and the recent total synthesis of spongistatin 1 (Professor Kishi) and spongistatin 2 (Professor Evans) herald the future development of new and biologically significant analogs of these spongistatins. Professor Pettit continued his world tour with a description of the isolation and characterization of many natural products including halichondrin B, cribrostatin 1, and cephalostatin 1. The lecture shifted to a fascinating account of the isolation, characterization, and synthesis of dolastatin 10, and related compounds, isolated from Dolabella auricularia, a sea hare. These natural products are superb antineoplastic substances. When in vivo studies at the NCI in the 1980s confirmed that extremely low doses of dolastatin 10 could essentially cure certain cancer cell lines, it became essential to be able to quickly provide enough of the compound for clinical trials. This was not feasible due to low isolation yields of the material from the sea hare, as well as the conservation questions associated with removing 34 IDrugs 1998 Vol 1 No 1 many tons of this particular sea hare from the ocean. Professor Pettit and coworkers therefore set out to prepare the compound by total synthesis. Being unsure of the absolute configuration of the natural product, they were prepared to carry out the synthesis of all 512 possible stereoisomers; fortunately, they arrived at the correct analog on the fifteenth total synthesis (still an impressive feat, considering that their synthetic route encompassed approximately 28 steps). The lecture continued with the discussion of myriad other interesting and biologically valuable natural products, including bryostatin 1, a fascinating compound with superb antitumor activity. Professor Pettit concluded his lecture with an intriguing account of combretastatin A-4 which he originally isolated from the South African tree, Combretum caffrum, in 1972. This compound (as a prodrug) will enter phase I clinical trials this summer. In addition to being a potent inhibitor of tubulin polymerization, it has the remarkable ability to selectively inhibit the blood supply to a tumor; in an in vivo blood flow study, the blood supply to the tumor in a mouse was essentially completely stopped, approximately 4 h after administration of a combretastatin A-4 prodrug. Professor Pettit has now established the Cancer Research Institute at Arizona State University, which promises many more similar, exciting discoveries. Meeting Report 215th ACS 35 Combinatorial carbohydrate chemistry - where are we now? Mike Panigot Address Department of Chemistry P O Box 419 State University AR 72467-0419 USA Email: [email protected] IDrugs 1998 1(1):35-37 © Current Drugs Ltd ISSN 1369-7056 This symposium to highlighted the uses of combinatorial chemistry in the synthesis of complex polysaccharides possessing biological activity. Approximately 50 people attended, and most were involved in carbohydrate chemistry using either classical synthetic approaches or combinatorial methods. The invited speakers were from both academia and industry and the session was aimed both at academic researchers and those who employ combinatorial syntheses in the industrial production of biologically relevant polysaccharides. Progress in solid phase synthesis of oligosaccharides: impact in combinatorial synthesis The first address on the use of solid phase synthesis in the preparation of complex carbohydrates was presented by Professor Richard R Schmidt (Universitat Konstanz, Germany). The advantages of solid phase synthesis include the ability to modify the functionality of the carbon scaffolding of the sugar bonded to the resin, the highly functionalized carbohydrate structure (which may be viewed as a polyhydroxylated aldehyde or ketone and selectively protected or functionalized), and the defined chirality at each of the carbon atoms of these molecules. In the examples that Professor Schmidt discussed, D-glucosamine was bonded to the resin as the carbon scaffold. As a preliminary example, a polysaccharide found in human milk containing several galactose, fucose, and N-acetylglucosamine residues was prepared. This molecule was found to be an antigen determinant of the lacto- and lactono-series. This glycoform is linked to asparagine derivatives as part of a glycoprotein. Professor Schmidt then discussed the various types of polysaccharides and carbohydrate derivatives that exist, as well as the advantages and difficulties found in the chemical synthesis of each. Naturally-occurring compounds consist of homoglycans, heteroglycans, and glycoconjugates, such as glycolipids, glycosphingolipids, and glycoproteins. Efficient preparation of these molecules by combinatorial methods on a solid support requires that versatile chemical building blocks be available in the form of selectively-blocked or selectively-reactive carbohydrates, and that the desired reaction takes place in high yield for a resin-bound carbohydrate using chemistry developed for reactions in solution. Trichloroacetimidates were used as glycosyl donors, which Professor Schmidt has also used in the classical solution phase synthesis of glycosides. This compounds are easily prepared, near neutral in their pH and in the pH of the by products formed, exhibit a great deal of stereoselectivity in the glycosidic bond formed, and form no reactive intermediates or water during the glycosidation. The oligosaccharides which were synthesized were analyzed by MALDI-TOF MS, TLC and HPLC. As the length of the polysaccharide chain increased, the yield of each step decreased. Therefore, this method may be best suited for the synthesis of small oligosaccharides. As a demonstration of the feasibility of these methods, a β-1,3-linked homoglucan was prepared in an overall yield of 28% over 12 steps or 90%/step in the reaction sequence. Similarly, more complex N-glycan structures could be prepared in 20% overall yield or 82%/step. Strategy for the convergent assembly of oligosaccharide libraries Matthew Johnston (University of Birmingham, UK) discussed chemistry which was performed in the solution phase on protected carbohydrates that could act either as glycosyl acceptors or as glycosyl donors. These sugars were derived from a 3-buten-2-yl glycoside bearing benzyl protecting groups at the 2, 3 and 4 positions, and an acetate-protecting group at the 6 position. This, in turn, could be prepared from acetobromoglucose by Koenigs-Knorr glycosylation with 3buten-2-ol. It could also be prepared in a more environmentally friendly manner by reaction of D-glucose and 3buten-2-ol with almond β-glucosidase immobilized on Amberlite XAD-4 resin. The enzymatic process also provides some stereoselectivity in the side chain, leading to a 3:2 ratio with the R isomer predominating. Four glycosyl acceptors were reacted with four glycosyl donors to form 16 disaccharides, which were reacted with four glycosyl donors to form 64 trisaccharides, and so on. Initial compounds were partially protected glucose and galactose derivatives that could be reacted at the 4-position or at the 6-position. These were reacted with each other to form 16 possible disaccharides. Once the 16 disaccharides were prepared, they were mixed, deacetylated, split, and reacted with a fucosyl donor. After hydrolysis with trifluoroacetic acid and analysis by HPLC, the percentages of the different trisaccharides formed were in agreement with what was theoretically anticipated, indicating that this combinatorial method was successful. Emphasis then shifted towards the synthesis of sialyl Lewis X analogs utilizing acetate and 4-methoxybenzyl as orthogonal protecting groups. A series of trivalent carbohydrate ligands were prepared and screened. This process is currently being expanded to build libraries of oligosaccharides. Solid phase combinatorial synthesis of "carbohybrids" The biological importance of carbohydrates in molecular recognition and cell signaling events and the interactions of cell surfaces and carbohydrates in recognizing viruses, 36 IDrugs 1998 Vol 1 No 1 bacteria, enzymes, antibodies, and toxins, was discussed by Gerd Hummel (University of Alberta, Canada). The concept of what they have termed a "carbohybrid" is based on the observation that oligosaccharides are very complex structures - too complex to be adapted by the pharmaceutical industry on a large scale. The "carbohybrid" would contain a part of the oligosaccharide that is recognized and necessary for efficient binding and biological activity. The remainder of the oligosaccharide would be replaced by a small organic molecule, simpler in structure, which would provide some binding as well. A number of different leaving groups in glycosyl donors have been investigated including thioglycosides, sulfoxides, glycosyl halides, phosphates, and trichloro-acetimidates. From determination of the rates of glycosidation of a number of glycosyl donors and the chemical shifts of the 1 anomeric proton in the [ H]NMR spectra of these donors, it was found that the smaller the chemical shift, the more reactive the glycosyl donor. This strategy was used to make a tetrasaccharide by combining the glycosyl donors and acceptors initially and allowing them to react based on their rate of reactivity. The desired product was obtained in 35% yield. GM1 ganglioside As an example of this combinatorial approach, the GM1 ganglioside was investigated. This molecule binds cholera toxin and heat labile toxin found in Escherichia coli. The key structural feature required for the binding of heat labile toxin is a galactose residue. An anomeric thioacetate was prepared, converted to the thiogalactose, and reacted at the sulfur atom with a series of Michael acceptors, particularly cyclic unsaturated ketones. Once addition had occurred, the ketone could be reduced to the alcohol or converted to an amine by reductive amination. Conversion to the amine permitted the introduction of groups that could bind by interaction of opposite charges. An extensive series of Michael acceptors were reacted and processed, and their activity against heat labile protein was measured. In the combinatorial approach, the thiosugar was reacted with ten different Michael acceptor aldehydes and ketones. Each of these was processed to give 10 compounds for a total of 100 compounds. Screening of these compounds was carried out with ELISA and chemical analysis was performed by MS. The compounds were tested as a mixture of four diastereomers. Similar chemistry could be performed in the solid phase on a polymer-bound trityl chloride. Initially, the tetraacetate of thiogalactose was converted to the ethyl disulfide, deacetylated, bonded to the trityl chloride resin, and treated with dithiothreitol to liberate the thioglycoside. The chemistry described above worked equally well in the solid phase and the final products could be removed from the resin using TFA/TIS in dichloromethane. Programmable one pot synthesis of oligosaccharides Dr Chi-Huey Wong (Scripps Research Institute, CA, USA) began his presentation with a brief description of the classical glycosidation reaction in which a glycosyl acceptor is reacted with a glycosyl donor in the presence of an activating agent to form a disaccharide. The limiting step in this sequence is activation of the glycosyl donor to form a glycosidic bond with the glycosyl acceptor. Structural features that affect the rate of glycosidation were discussed for both the donor and acceptor carbohydrates. For the glycosyl donor, these features are stereochemistry of the carbohydrate, the structure(s) of protecting groups present, the leaving group to be displaced by the glycosyl acceptor, and the nature of the promoter being used. For the glycosyl acceptor, the structural features that affect glycosidation are the stereochemistry of the acceptor and the protecting groups present. Next, the use of orthogonal protection strategy in the synthesis of complex polysaccharides was discussed. By using four different protecting groups (4-methoxybenzyl, TBDMS, chloroacetyl and levulinate) that could be removed individually, a carbohydrate library was prepared containing a number of different carbohydrate branches attached to a common carbohydrate core. This was performed on a solid support and glycoside incorporation was determined by gated 13 decoupled [ C]NMR using Cr(acac)2 as relaxation agent. Generation of carbohydrate libraries for drug discovery The synthesis and uses of carbohydrate libraries in drug screening were discussed by Dr Michael J Sofia (Transcell Technologies, NJ, USA). It has been known for some time that combinatorial chemistry to form oligopeptides and small molecules greatly increases the number of biologicallyactive compounds that may be screened in a given time, but the use of such techniques in carbohydrate research is much less common. Moenomycin A is an inhibitor of cell wall biosynthesis with a pharmacophore that requires three structural units: a carbamate portion; an amide; and a phospho-glycerate. A lipid moiety and an N-acetylglucosamine group may also be necessary for biological activity. In the chemistry discussed by Dr Sofia, the carbamate was replaced by a dialkylurea and an orthogonally protected carbohydrate was prepared and bonded to a solid support as an amide through a uronic acid group. The required carbohydrate with the necessary functionality was prepared on a solid support, then glycosidated using the sulfoxide donor approach developed by Kahne. Quality control and analysis was through 1 [ H]NMR spectroscopy after cleavage of the oligosaccharide from the resin. Additional analysis of these compounds was obtained by electrospray MS. Next, primary screening libraries were investigated. These were broken down into cases in which there is an interaction of a small molecule with a protein and the molecular recognition sites are close and instances in which proteinprotein interactions predominate and the recognition sites are farther apart. The rationale for the use of carbohydrates in this particular area arises from their rigid structure, enantiomeric purity, defined stereochemistry, and high combinatorial density. The molecules also have the drug characteristics of small molecules. As a result of the three-point pharmacophore, the carbohydrate molecule was trifunctionalized as a uronic acid Meeting Report 215th ACS containing a free hydroxyl at C-4 and an Fmoc-NH group at C-3 in one series and at C-2 in another. The uronic acid was bound to a solid support as an amide and the nitrogen functionalized by reacting it with amino acids, isocyanates, acids and aldehydes. An IRORI Accutag 100 system was employed with a microcan system which allowed the library to be built by a mix and split technique. This permitted the incorporation of unnatural sugars to investigate nonstandard binding sites. The overall purity of the compounds being tested was greater than 80%. Lastly, a comparison was made in diversity space of several trisaccharides that had been prepared with several trifunctional small molecules and several tripeptides. These particular carbohydrates were found in a different diversity space from the small molecules and tripeptides. It is hoped 37 that this will lead to different biological activity for this particular class of molecules Conclusion The use of combinatorial chemistry in academic and industrial research laboratories has been widespread in the syntheses of small molecules and oligopeptides for quite some time. Similar methodology is now encompassing the field of carbohydrate chemistry. This should permit the rapid discovery and testing of many new carbohydratebased materials. Given the importance of cell signaling and molecular recognition by carbohydrates, this should allow more carbohydrate-containing pharmaceuticals to enter the marketplace more rapidly and at lower cost than by classical solution phase synthesis of individual compounds. 38 IDrugs 1998 Vol 1 No 1 Selected state of the art topics - organic chemistry poster session Eusebio Juaristi Address Cinvestav Department of Chemistry PO Box 14-1470 07360 Cd Mexico Email: [email protected] IDrugs 1998 1(1):38-39 © Current Drugs Ltd ISSN 1369-7056 Nearly 400 papers were accepted for presentation in the Division of Organic Chemistry during the 1998 Spring ACS National Meeting. From these, 38 poster papers were selected by Program Chairperson, SS Hall, as most representative of the state of the art, and ten of these selected papers related to drugs or drugs discovery. A brief report on these posters, which attracted a large crowd of interested attendees, is presented here. Design and synthesis of enediyne-based ADEPT prodrugs Samuel A Brown and Carmelo J Rizzo (Vanderbilt University, TN, USA) showed that the biological toxicity of enediynes is generally dependent upon conformational changes. As an example, the bioreduction of the trisulfide unit of calicheamicin, followed by addition of the resulting free thiol to the enone, afforded a cytotoxic agent presenting 1000-fold greater activity than adriamycin in murine tumor models. Synthesis of enediyne-cephalosporin hybrids using ADEPT (antibody-directed enzyme prodrug therapy) technology was also presented [ORGN 053]. Solid phase syntheses of turn structures The finding that the synthesis of cyclic peptides and peptidomimetics by means of disulfide, amide, or ester bond formation is usually complicated by racemization and other side reactions was discussed by Yangbo Feng, Zigi Wang and Kevin Burgess (Texas A&M University, USA) [ORGN 054]. These difficulties are a serious drawback for combinatorial chemistry applications. The goal of the presented work was the development of new methodologies for the synthesis of cyclic peptido-mimetic libraries on solid supports. In particular, SN2 and SNAr reactions were successful in the cyclization step leading to C-O, C-S or C-N single bonds. These reactions were sufficiently clean for library syntheses on solid supports, affording macrocyclic structures (20-membered rings or smaller), with purities above 90%. Conjugate addition of nitrogen nucleophiles to an unsaturated pyrrolidinone Functionalized pyrrolidines have a wide range of biological activities. Philip Chan, Ian Cottrell and Mark Moloney (University of Oxford, UK) [ORGN 058] revealed how a bicyclic lactam derived from pyroglutamic acid was a useful template for conjugate additions of nitrogen nucleophiles, in good yield and with high diastereoselectivity [for a preliminary communication, see Chan P et al: Tetrahedron Lett (1997) 38:5891]. The above protocol provides a route to β-amino- and α,β-diamino-acids, compounds which are recognized to be of substantial biological importance, and whose synthesis has recently attracted considerable attention [see Juaristi E: Enantioselective Synthesis of β-Amino Acids, Wiley-VCH, New York, 1997]. A novel approach to the synthesis of taxolchain analogs The continuing need for the development of efficient largescale syntheses of taxol was discussed by Marko Mihovilovic, Margaret Kayser, Sonia Rodríguez and Jon Stewart (University of New Brunswick, NJ, USA and University of Florida, USA) [ORGN 064]. The group has employed enantioselective reduction of αketo-β-lactams, which was accomplished with engineered strains of the yeast Saccharomyces cerevisiae. The main advantages are their low cost and easy access, and the fact that yeast are environmentally compatible and pathogenically benign. However, the authors report solubility problems and possible toxicity of the substrate for yeast. The applicability of the method was demonstrated with the preparation of a precursor to the side chain of taxol. Total synthesis of a potent proinflammatory 5-oxo-ETE Subhash Khanapure, Xiao-Xin Shi, William Powell and Joshua Rokach (Florida Institute of Technology, USA and McGill University, Montreal, Canada) showed that 5-oxoETE, a novel arachidonate metabolite, is a potent chemotactic agent for human neutrophils, being about 100-fold more potent than its precursor, 5(S)-HETE [ORGN 065]. In order to study the biochemical pathway for the oxidation of 5-oxo-ETE, the authors undertook the total synthesis of a potent proinflammatory 5-oxo-ETE and its radiolabeled derivatives [Rokach J et al: J Org Chem (1998) 63:337]. A convergent synthesis was accomplished via a bisdienyl phosphorium bromide and a dithiolane aldehyde. The acetylenic precursor made the incorporation of four tritium atoms possible. Synthesis of constrained cyclic peptide helices by ring-closing metathesis Helen Blackwell and Robert Grubbs (California Institute of Technology, USA) reported that ring-closing metathesis (RCM) [Grubbs R et al: J Am Chem Soc (1996) 118:9606] has recently been applied to the synthesis of cyclic peptides exhibiting a β-turn secondary structure. Indeed, ruthenium complexes catalyzed ring-closing metathesis reactions to form five-, six-, seven-, and eight-membered carbocycles and heterocycles. Meeting Report 215th ACS The aim of this research is to establish whether the acyclic precursors access a helical conformation in solution. Heptapeptide analogs of a known helical peptide were synthesized and found to adopt helical conformations in chloroform. The ruthenium olefin metathesis catalyst was highly active in these media, affording the macrocyclic peptides of interest [ORGN 072]. Biomimetic mineralization The widespread interest in the preparation of organized nanoscale, microscopic and bulk inorganic materials under mild conditions was discussed by Haimanot Bekele and Jeffrey Kelly (Scripps Research Institute, CA, USA). Inorganic composites of uniform size, shape and morphology may have applications as catalysts, as well as magnetic, optical and electrical materials. In order to achieve controlled crystallization of inorganic minerals, the authors employed an amphiphilic selfassembling peptidomimetic that adopts a two-dimensional β-sheet structure at an air-water interface to nucleate calcite into crystals of uniform size that are distinct from nonnucleated crystals [ORGN 075]. Synthesis of γ-lactams Lactams are known to be essential constituents in a diverse range of natural and synthetic products of therapeutic interest. For example, the β-lactam family has acquired a status of unparalleled importance in the treatment of infectious diseases caused by bacteria. Although γ- and δlactams have been investigated less extensively, they are of considerable interest as antitumor agents. Diana Danca and James Rigby (Wayne State University, MI, USA) reported a 39 general method for the synthesis of γ-lactams, which is based on the reaction of selenium-phenylselenocarbamates with [(CH3)3Si]3SiH and subsequent addition of the incipient free radical onto an appropriate double bond. This radical cyclization reaction displays high regioselectivity, with a preference for exo addition [ORGN 079]. Synthesis and application of γ,δ-unsaturated amino acids Unsaturated amino acids are an important class of natural products which display an array of interesting biological properties. Mark Burk, John Allen and William Kiesman (Duke University, NC, USA) reported a procedure for the highly regio- and enantio-selective catalytic hydro-genation of enamides in conjugated diene systems (α,γ-dienamide esters) to afford γ,δ-unsaturated amino acids [ORGN 085]. This new methodology was applied to the synthesis of the natural product, bulgecinine, a component of β-lactam synergists bulgecin A-C [see also Burk M et al: J Am Chem Soc (1998) 120:657]. Progress toward total syntheses of epothilones A and B Epothilones are 16-membered ring macrolides which have exhibited excellent antimitotic activity. Indeed, epothilones operate by the same mechanism as paclitaxel. Emily Reiff, Sajiv Nair, Ashok Tunoori, Sam Victory and Gunda Georg (University of Kansas, USA) presented pathways for the synthesis of the C1-C6 segment of the epothilones [ORGN 086]. These synthetic approaches are amenable to the synthesis of attractive analogs [for related work, see Tetrahedron Lett (1997) 38:1703]. 40 IDrugs 1998 Vol 1 No 1 An interview with the Chair of the ACS MEDI division Peter Robins Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] followed by three talks that summarized inhibitors of this enzyme, which are potential anticancer agents. There have been significant advances in this field in the last two or three years, now that pharmacokinetic and potency problems are beginning to be solved. In the next few years we should obtain information on the potential of the newer compounds in human cancer. IDrugs 1998 1(1):40-41 © Current Drugs Ltd ISSN 1369-7056 The schedule for Tuesday morning was very full, with the Awards Symposium and further general oral sessions. Following the conclusion of the 215th National Meeting of the American Chemical Society Meeting in Dallas, Current Drugs Editor, Peter Robins, spoke to the Chair of the ACS Division of Medicinal Chemistry, Annette Doherty, of Parke-Davis/ Jouveinal in Fresnes, France. The awards session took place on Tuesday morning and due to a large number of oral submissions we scheduled additional presentations for the Tuesday morning sessions. How did you come to be the Organizer of the Medicinal Chemistry Section? Medical chemists who are members of the division vote for Vice-Chair and Councillor positions each year. The members of the division voted for my appointment in 1996. This is a four-year commitment to the medicinal chemistry division. During the middle two years as Secretary of the Division, the responsibility involves organization of the medicinal chemistry schedule for ACS National Meetings. The conference began for the Medicinal Chemistry section with the two General oral sessions on Sunday. Could you tell me a little about those, and the poster session that followed. On the first day of the conference, we always have general oral papers. We usually receive between 30 and 40 of these submissions on a wide range of subjects. These particular sessions have become increasingly popular in the last two or three years, so we schedule two on Sunday in general. This year, both general sessions were very well attended. A poster session is always held on Sunday evening, again covering a wide range of medicinal chemistry topics. The adenosine symposium was remarkably popular as well. We decided to focus on this area because in recent years, with advances in the molecular biology of the adenosine receptors, a number of subtypes have emerged and the clinical indications for selective antagonists and agonists of the various adenosine receptors are becoming clearer. A number of different indications were discussed for the A1, A2A, A2B and A3 receptors and there were many important compounds disclosed. Selectivity has been a major issue in this area and significant advances have occurred in recent years. The session on Monday afternoon with Dr Gibbs presiding, was focused on protein prenylation and in particular, on Ras farnesyltransferase inhibitors. The first two talks were biochemical in nature, focusing on yeast, as well as the human Ras farnesyltransferase enzymes. These were The first presentation was for the Ralph Hirschmann Award, presented to Dr Isabella Karle. Her fascinating talk focused on her contributions to the field of peptide/ protein crystallography. Next was Monroe Wall, who spoke on the subject of camptothecins. He discussed the mechanism of action of these compounds as topoiso-merase inhibitors, and highlighted a number of other, newer camptothecin compounds with potential for anticancer therapy. What about the final award, the ACS Award for Team Innovation? This is a new award presented to teams of people who have developed a product in the laboratory and taken it to market, intended to highlight the value of multi-disciplinary teamwork in the development of a product in commercial use. The recipients this year were the group from Eli Lilly responsible for the development of the rapid-acting insulin analog, Humalog. Three members of the team gave a very well integrated presentation, illustrating how, in industry, the various departments work together to advance a successful new product. Humalog has the ability to normalize levels over a prolonged period of time and will likely reduce the long-term complications of diabetes. Overall, there seemed to be a large number of presentations that discussed angiogenesis, cancer or oncology in general. Well certainly, protein prenylation, ATP-directed kinase inhibitors, angiogenesis are all proliferative-type strategies, but these were not the sole focus of the program. For example, the anticoagulation strategies symposium was interesting, because the Factor Xa inhibitor area is one which has advanced significantly in recent years and there are many new, non-peptide inhibitors being reported. This strategy may have advantages over the thrombin inhibitors in terms of bleeding tendency. Also, we had a session relating to the application of amyloid aggregation inhibition to cognition. Thrombosis was covered quite recently at the ACS, I understand. The ACS planning committee for the Medicinal Chemistry division was unsure whether to have a thrombin/ Factor Xa Meeting Report 215th ACS inhibition symposium, because of the antithrombotic symposium in Orlando in 1996, but there have been many recent advances in this particular field. We decided, therefore, to schedule a late-breaking session to cover new developments in anticoagulation. In particular, the field of factor Xa inhibitors is attracting interest for either deep vein thrombosis or acute thrombotic events. As a result, there were many new compounds revealed at this symposium, particularly in the factor Xa area. What was the rationale behind the Cytochrome P450 symposium? This is a really interesting area because we don’t fully understand how to design drugs that do not inhibit P450. This is an important consideration because drug interactions are likely, if P450 inhibition occurs in vivo at plasma levels close to efficacious doses. In this session, there were several talks showing recent advances in our understanding of design features associated with avoidance of P450 inhibition. 41 What about ’Amyloid aggregation inhibitors’? Amyloid inhibition is an important area of research receiving increased attention and we felt that this would be a topic of interest to the medicinal chemistry community. Given my earlier point about antiproliferative strategies, would you say that there is a common theme to these symposia? Not really, because topics are voted for national meetings based on suggestions from division members. We try to provide a wide range of topics including a balance of new technologies and therapeutic areas of broad interest. Clearly, there are sessions devoted to cardiovascular medicine (Thrombosis), CNS diseases (Amyloid aggregation), design issues (P450 inhibition) and proliferative strategies. There were a number of sessions focusing on growth factormediated strategies at the meeting, including ATP-site directed kinase inhibitors, protein prenylation and angiogenesis, but we did not select this as a specific theme. 42 IDrugs 1998 Vol 1 No 1 American Association For Cancer Research 89th Annual Meeting Cancer gene therapy 28 March - 1 April 1998, New Orleans, LA, USA Debabrata Banerjee Address Molecular Pharmacology and Therapeutics Program Sloan-Kettering Institute For Cancer Research 1275 York Avenue New York NY 10021 USA Email: [email protected] IDrugs 1998 1(1):42-44 © Current Drugs Ltd ISSN 1369-7056 The annual meeting of the AACR, the largest meeting devoted to cancer research, covered all aspects of cancer research currently being conducted all over the world. The present report attempts to highlight some of the more interesting presentations dealing with cancer gene therapy. The field of cancer gene therapy has made tremendous progress in the last few years, as witnessed by the burgeoning numbers of proffered papers on the subject. The variety of presentations, from novel forms of gene therapy in vitro to clinical trials, attest to the maturity of the field from concept to practice. This is the first of three AACR reports. Gene transfer to hematopoietic progenitors The ’meet the expert at sunrise’ sessions provided an opportunity to learn about recent progress in the particular fields thought to be important by the scientific program committee. An authoritative lecture on the state of gene transfer to hematopoietic progenitors was delivered by Dr Mavilio (San Raffaele Institute, Milan, Italy). Gene transfer to hematopoietic stem cells (HSCs) will be useful for gene therapy of immune deficiencies, thalassemias, leukemias, lymphomas and AIDS, and for protecting the bone marrow from chemotherapy-induced toxicity during high dose treatment of solid tumors. Although many viral and nonviral vectors have been developed for successful gene transfer, HSCs remain particularly difficult cells to transduce. HSCs have been transduced with modest success using retroviral vectors but not with other vectors such as adenoviral and adeno-associated viral vectors. Improvements in retroviral vectors are constantly being made in order to increase the transduction efficiency of HSCs. These improvements include better pseudotyping of retrovirus particles with receptors that will possibly result in better transduction, such as the gibbon ape leukemia virus (GALV) receptor. Most of the retroviral vectors being used presently are derived from the Moloney murine leukemia virus whose genome has been modified to serve as a replication incompetent, but infective, particle carrying the gene of interest into the target cell genome. Dr Mavilio explained that retrovirus infection and integration requires that the HSCs be in cycle and many improvements have been made in culture conditions, including addition of growth factors to stimulate division of the HSCs ex vivo. On the other hand, it is becoming evident that certain types of growth factors such as interleukin 3 (IL3), while being able stimulate cell division, somehow adversely affect the ’stemness’ of the HSCs, ie, the IL-3stimulated cells are compromised in their ability to repopulate the hematopoietic compart-ments when reintroduced into suitable recipients. Research has indicated that stimulation with certain growth factors, such as thrombopoietin and the Flk3/Flt2 ligand, in place of IL-3, results in equal or better transduction efficiencies with no loss of ’stemness’ of the HSCs. Improvements in infection conditions such as the use of fibronectin-coated plates during the infection process improve the transduction efficiency. Evaluation of successful gene transfer has traditionally relied on the use of the neomycin phosphotransferase gene (neo) to confer resistance to the antibiotic, G-418, in culture. More recently, a cell surface marker, the mutated nerve growth factor receptor (mNGFR), is being increasingly used as it permits enrichment of successfully transduced cells by fluorescence activated cell sorter (FACS) technique. One can thus overcome the low transduction efficiency by sorting successfully transduced cells. Since the purpose of gene transfer is to introduce a therapeutically useful gene, the retroviral vector has to deliver a second gene besides the selectable marker (neo or mNGFR), explained Dr Mavilio. The second gene can be placed in the vector under the control of either a promoter separate from the one governing transcription of the marker gene, or the internal ribosomal entry site (IRES) element, to generate a bicistronic message, using the same promoter as the marker gene. Success has been reported using both systems although no systematic comparison has been made. Several clinical trials involving retrovirus-mediated gene transfer into HSCs have been conducted. Marking studies with the neo, as well as the mNGFR gene, have reported long-term expression in marked cells although the transduction efficiencies have been low. Two groups have reported on transduction and long-term expression of the adenosine deaminase (ADA) gene in severe combined immunodeficiency (SCID) children transplanted with bone marrow stem cells transduced with the ADA cDNA. Although these children received the ADA gene, and it was proven to function in the transduced cells these children have not been taken off the polyethylene glycol-ADA enzyme therapy which is the standard of care for SCID children. Transfer of the multidrug resistance gene, mdr-1, into HSCs has also been carried out. Although expression of the mdr-1 gene in retrovirally transduced cells has been documented in vivo, protection against myelotoxicity of chemotherapeutic drugs, such as taxol, is yet to be demonstrated. There appear Meeting Report 89th AACR to be several major hurdles that need to be overcome before HSC transduction for the purposes of clinical gene therapy can become a routine treatment. These hurdles include: efficient transduction; maintenance of self-renewal and longterm stem cell potential, when reintroduced into the recipient; and regulated and sustained expression of the introduced gene. Dr Mavilio also summarized the ongoing efforts of many laboratories to develop superior retroviral vectors based on lentiviruses, such as the HIV virus. These viruses are being re-engineered so that they are not pathogenic and yet retain their ability to infect hematopoietic cells easily. This theme was further developed by Dr Luigi Naldini (Cell Genesys Inc, Foster City, CA, USA) who presented two lectures on development of lentiviral vectors for in vivo gene delivery. Third generation lentiviral vectors, derived from the HIV virus, have recently been developed that are entirely safe and are efficient for in vivo gene delivery. The current generation of HIV-based vectors are self-inactivating, lack almost all viral sequences and yet retain good viral titers. These vectors have proven to be useful for in vivo gene delivery, especially by the intraportal vein and have shown long-term integration and expression of the transferred gene with no abnormal pathology. These viruses have greatly reduced chances of generating replication competent recombinants, as the number of events required to generate such a recombinant have been increased to an unlikely number. A major advantage of these vectors over the conventional Moloney-based vectors is that these HIV-based vectors can infect non-dividing cells thus eliminating the need to bring the target HSCs into cycle. Non-viral gene delivery into hematopoietic cells Retroviral vectors have been used for hematopoietic progenitor cell gene transfer as other methods, including non-viral methods, have not yet proven useful for such gene transfer work. This has not prevented laboratories from exploring non-viral means for transducing HSCs, both murine and human, for gene therapy purposes. Two posters described the use of liposome-based delivery systems for transduction of murine (Dr S-C Zhao, Memorial Sloan-Kettering Cancer Center, New York, USA) and human (Dr KA Winters, City of Hope National Medical Center, Duarte, CA, USA) HSCs. Interestingly both groups used liposome-based delivery systems to transfer drug resistance genes into HSCs. Dr Zhao et al compared the efficiencies of retroviral vectors and liposome-based systems to transfer a mutated human dihydrofolate reductase gene into murine bone marrow progenitor cells. They further compared the myeloprotection, imparted by bone marrow cells transduced retrovirally, with the myeloprotection imparted by the liposomally transduced cells in vivo. Their results suggest that liposome-based (DOTAP formulation; BoehringerMannheim) delivery systems are capable of transducing the murine HSCs as efficiently as the retroviral vector system. The second paper presented evidence for successful gene transfer of a bicistronic cDNA encoding the green fluorescence protein (GFP) and the human Mdr-1 P-glycoprotein 43 into murine bone marrow cells, human KB31 cells and + human CD34 cells. In this case the liposome used was the polycationic liposome DOSPER (Boehringer-Mannheim). Dr Winters showed that PCR analyses revealed presence of the GFP and the mdr-1 genes in the transduced cells. Approximately 50% of the transduced colonies were found to contain the exogenously delivered genes. More-over, these colonies were resistant to docetaxel, providing further evidence of Mdr-1 expression. Immunohistochemical staining of the colonies growing in presence of drug also indicated that the transduced cells expressed the P-glycoprotein. Both these papers suggested that liposome-based delivery systems need to be re-evaluated for their ability to transduce bone marrow progenitor cells. Cancer gene therapy The papers in this category could be subdivided into the following: direct antitumor effects as well use in bone marrow purging to eradicate contaminating tumor cells; gene therapy by restoring wtp53 activity, direct effect and chemosensitization; gene therapy by targeting angiogenesis. These papers were presented in two minisymposia entitled ’Human cancer gene therapy’ and ’Cytoreductive gene therapy of cancer’. Gene transfer of the suicide gene cytosine deaminase Papers given by Dr SQ Fu (Yale University, New Haven, CT, USA), Dr M Hirai (University of Nebraska Medical Center, Omaha, NE, USA) and Dr T Nanakorn (Yale University School of Medicine, New Haven , CT, USA) all dealt with purging of tumor cells from bone marrow by using adenoviral vectors (carrying either cytosine deaminase (CD) or wt p53 genes) which theoretically should only infect the tumor cells and not the bone marrow cells. Tumor cells transduced with, and expressing, the CD gene are then eliminated by treatment with the prodrug 5-fluorocytosine (5-FC) which is converted to the cytotoxic drug, 5fluorouracil (5-FU), by enzymatic action of CD. All groups reported success using either wt p53 or the CD gene for purging studies. Interestingly, Dr Nanakorn also demonstrated that bone marrow that has been purged of tumor cells by the CD-5-FC treatment maintain their ability to repopulate recipient mice for periods ranging from 4 days in vitro to 14 days after in vivo purging. This is important information for investigators wishing to use these systems for eradicating contaminating tumor cells from bone marrow samples prior to transplantation. Gene therapy with wt p53 Two interesting papers presented by Dr J Horwitz and Dr J Dell (Schering-Plough Research Institute, Kenilworth, NJ, USA) dealt with preliminary results of a phase I clinical trial where 64 patients with various cancers including hepatic metastases of colon cancer, head and neck cancer, ovarian cancer and melanoma were evaluated for transgene expression after administration of Ad-p53 (Sch-58500) by either intrahepatic, intraperitoneal or intratumoral routes. Transgene expression was documented by reverse transcriptase-PCR analyses and found to be dose- 44 IDrugs 1998 Vol 1 No 1 dependent. With increasing doses of Ad-p53, increasing numbers of patients were found to express the transgene and pre-existent antibodies to adenovirus did not affect such transgene expression. Dr Dell reported on a different aspect of the same work. It was observed that introduction of Ad-p53 into tumor cells increased the chemosensitivity of the tumors to standard chemotherapy agents including, 5-FU, cisplatin, etoposide, methotrexate, doxorubicin and paclitaxel. Tumor-bearing animal models showed enhanced tumor cell kill when the combination of Ad-p53 and paclitaxel was used, rather than either agent alone in equivalent doses. True synergism was demonstrated by means of statistical analyses using isobolograms for two drug combinations. An explanation for the observed synergism was the demonstration that paclitaxel enhanced the uptake of Ad-p53 into tumor cells, thereby increasing the effective p53 concentration in tumor cells. Gene therapy targeting angiogenesis A rather novel means of using angiogenesis inhibitors for cancer gene therapy was reported by Dr R Pawliuk (Harvard Medical School, MA, USA). Murine bone marrow cells were transduced with retroviral vectors containing a bicistronic construct encoding either endostatin-IRES-GFP or angiostatin-IRES-GFP and were transplanted into recipients. Donor and recipient cells could be distinguished by the CD45.1 marker on the recipient cells and CD45.2 on the donor cells. Transduced cells were analyzed by FACS sorting for CD45.2 and GFP expression. Engraftment of the recipient cells was demonstrated by detection of dual positive cells for CD45.2 and GFP for up to 12 weeks post transplant, with the earliest time of angiostatin/endostatin production being 5 weeks post transplant. These mice were then challenged with B16 melanoma cells. No untoward toxicities were observed and wound healing in the recipients was not compromised. Mice that received bone marrow cells transduced with the construct bearing the endostatin cDNA did not show tumor growth as compared to control animals or animals receiving cells transduced with the angiostatin gene. This raised the possibility that these angiogenesis inhibitors may have different tumor specificities. The authors went on to discuss future plans regarding transduction of bone marrow cells with a construct bearing a fusion protein that produced both angiostatin and endostatin in the same recipient. In the discussion following the paper it was pointed out that systemic bone marrow transduction for the in vivo production of angiogenesis inhibitors may not be entirely safe in the long run and a better alternative would be to implant depot cells producing these inhibitors within hollow fibers or capsules, so that they can be removed once the need for production of these angiogenesis inhibitors in vivo is over. Targeting tumor angiogenesis directly by introducing the suicide gene, HSV-TK, into the tumor-associated endothelial cells and killing them by ganciclovir (GCV) treatment, was discussed by Dr T Tanaka (Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA). Tumor-associated endothelial cell kill was found to be dose- and MOI- (multiplicity of infection) dependent. The HSV-TK transduced cells continued to grow in culture, as indicated by positive PCNA (proliferating cell nuclear antigen) staining, thus ensuring sensitivity to GCV which can kill only dividing cells. HUVEC (human vascular endothelial cells), a popular cell system with investigators in the angiogenesis field, were shown to be particularly sensitive to the HSV-TK/GCV combination therapy. Cancer gene therapy has come of age. Meeting Report 89th AACR 45 Cell-based gene therapy and late-breaking news Ezzie Hutchinson Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):45-46 © Current Drugs Ltd ISSN 1369-7056 New concepts in cell-based gene therapy Dendritic cells pulsed with prostate tumor antigens A phase II trial with prostate cancer patients, using dendritic cells pulsed with prostate tumor antigens, was described by Dr Michael Salgaller (Northwest Therapeutics, WA, USA). Firstly, dendritic cells, a type of antigenpresenting cell, were isolated from blood samples taken from patients with very advanced prostate cancer. These cells were then exposed to prostate tumor antigens (prostate-specific mem-brane antigens-P1 and P2) in cell culture, and injected back into the patients in six infusions given every six weeks. Half of the patients also received granulocyte-macrophage colony stimulating factor (75 µg/m2/day), to help boost the immune response. So far, 104 patients have been enrolled and 33 have completed therapy. Significant acute or chronic toxicity was not observed in any of the patients. Of the evaluable patients 27% achieved partial response, while 33% had stable disease. Some of these responders have lived to two years, which is impressive considering that the average survival time for these patients is only 6 to 12 months. SC-1 antibody Preliminary results of a phase I/II trial using a human monoclonal IgM antibody, SC-1, as an adjuvant therapy to treat gastric cancer, was reported by Professor H Peter Vollmers (Institute for Pathology, University of Wuerzburg, Germany). The SC-1 antibody was originally isolated by extracting B-cells from the spleen of a patient with a diffuse type adenocarcinoma of the stomach and testing the antibodies produced by these cells for antitumor activity. SC-1 was demonstrated to induce apoptosis of tumor cells. In the phase I/II clinical trial, 40 patients with poorly differentiated adenocarcinoma have been injected iv with 20 - 30 mg purified SC-1 two days prior to gastrectomy. Of these patients, 90% showed apoptosis in their tumor, 60% showed tumor regression and 80% had tumor-free lymph nodes, as opposed to only 10% in the control group. Adenovirus wt p53 A phase I study of replication-deficient, E1 deleted adenovirus re-engineered to deliver normal p53 (Sch-58500) into patients with various types of cancer, was reported by Dr M Rybak (Schering-Plough, NJ, USA). The rationale is that injection of functional p53 into cancer cells expressing mutant p53 will induce the cells to undergo apoptosis. Sch58500 was administered intratumorally in brain cancer and head and neck cancer, intraperitoneally in ovarian cancer, and via the hepatic artery in colon cancer. 74 Patients have had a single administration of Sch-58500, ranging from 7.5 x 109 to 7.5 x 1012 particles/dose. Transgene expression was evaluated in 69 tumors using a reverse transcriptase-PCRbased assay. Results showed that 33 tumors expressed the transgene and expression increased with increasing doses received. Her2/neu inhibition by E1A Dr Naoto Ueno (MD Anderson Cancer Center, TX, USA) described exciting work carried out with colleagues from Targeted Genetics and the University of Pittsburgh. Her2/neu is an oncogene overexpressed in a number of cancers, in particular, 20 to 30% of breast and ovarian cancers overexpress this oncogene. Expression of Her-2/neu is correlated with poor prognosis, increased tumor formation, increased metastasis and resistance to chemotherapeutic agents. The adenoviral tumor suppressor gene, E1A, is able to inhibit the promoter activity of Her-2/neu, thus downregulating expression. Delivery and expression of an E1A gene in human cancer and normal cells using a propri-etary liposomal delivery mechanism has been demonstrated. Dr Ueno outlined the phase I dose-escalation study which is underway with 12 patients with metastatic or recurrent breast and ovarian cancers. Patients are given weekly intra-abdominal injections of the E1A gene cationic liposome complex over 4 to 6 weeks. The first three patients have been evaluated and show E1A gene expression in both cancer and normal cells. These patients also exhibited decreased levels of Her2/neu and surrogate tumor markers. The maximum tolerated dose has been identified and phase II trials are planned which are hoped to confirm the antitumor activity of the E1A gene. Late-breaking news This session consisted of papers submitted after the closing date of October 1997, but were included at the conference due to their significant interest. The abstracts for these papers, therefore, are not to be found in the AACR abstract book (AACR Proceedings (1998) 39). Arsenic trioxide A presentation on the use of arsenic trioxide (As2O3) for treating cancer patients was given by Dr S Soignet (Memorial Sloan-Kettering Cancer Center, NY, USA). Arsenic was originally discovered to be of use as a drug in the 1800s, but the promise of efficacy in treatment of leukemias is a very recent finding. Arsenic trioxide homes in on sulfahydrols present in cancer, and induces apoptosis in these cells. In preclinical studies, Dr Soignet explained that As2O3 exhibited unexpectedly broad anticancer activity against a variety of acute and chronic myeloid and lymphoid cell lines. This drug was able to induce apoptosis, without the presence of the promyelocytic leukemia (PML) 46 IDrugs 1998 Vol 1 No 1 gene, in a variety of myeloid cell lines. In a phase I study of patients with relapsed and/or refractory acute promyelocytic leukemia (APL), all of whom had received extensive prior treatment with all-trans retinoic acid or cytotoxic chemotherapy, were treated with daily infusions of 0.07 to 0.21 mg/kg As2O3 until a bone marrow complete remission was achieved, up to a maximum of 60 days. Responding patients then received an additional 25 days of therapy after a rest of 4 to 6 weeks. So far, of seven patients evaluable for response, all have achieved complete remissions ranging from 14 to 39 days. Four of these patients no longer showed the characteristic markers for APL as demonstrated by reverse transcriptase-PCR. The drug was also very well tolerated. Following these striking results, an international multicenter study will be initiated using a daily dose of 0.15 mg/kg. CGP-57148B CGP-57148B (Novartis), a competitive inhibitor of a protein kinase, was the subject of a paper by Dr P le Coutre (Instituto Nazionale Tumori, Milan, Italy). This compound specifically inhibits the kinase which blocks proliferation of human bcr/abl+ leukemic cells and induces apoptosis in vitro. Nude mice received CGP-57148B, 50 mg/kg ip or 160 mg/kg po, every 8 h for 11 days. 87.5% control animals developed leukemia within 10 days, whereas 87.5% ip treated animals and 100% po treated animals remained free of cancer for more than 90 days. The toxicity of this treatment was low. Designer T-cells A new therapy to target tumor cells expressing carcinoembryonic antigen (CEA), a tumor-associated antigen, was discussed by Dr RP Junghans (Harvard Medical School, MA, USA). Preclinical evaluation has produced convincing data which has led to an IND approval by the FDA. This is the first anticancer gene therapy to be supported by the National Gene Vector Laboratory at NIH. In the phase I trial, which has recently begun, patients' T-cells are collected and retrovirally transduced with an immunoglobulin T-cell receptor construct which has fragments of a humanized anti-CEA antibody attached to it. The T-cells are then expanded in culture and re-infused into the patients. The hypothesis is that on contact with CEA+ tumor cells, the anti-CEA designer T-cells will be activated, leading to IL-2 production and lysing of tumor cells by cytotoxic T-lymphocytes. Recombinant Listeria monocytogenes expressing E6 and E7 proteins Cervical cancer is often associated with human papilloma virus (HPV) infection. More than 90% of HPV-16 transformed cervical cells express the viral proteins, E6 and E7, constitutively, so these proteins make ideal targets for a cervical cancer therapeutic. Dr A Zubair (University of Pennsylvania, PA, USA) presented in vitro data to show that a recombinant Listeria monocytogenes, which expresses and secretes E6 and E7 proteins, can elicit a potent E7 restricted cytotoxic T-cell response against murine tumor cells. Study of the ability of the vaccines to protect mice from transplanted E6 and E7 expressing tumors is ongoing. Telomerase inhibitors This year at the AACR, as last year, there was much excitement about telomerase as a target for anticancer drug development. A recent paper in Science (Bodnar AG: Science (1998) 279(5349):349-52) showed that elongated telomeres cause a huge increase in the life-span of cells in culture. In cancer, activation of telomerase causes elongation of telomeres which enables a cell to by-pass cellular Meeting Report 89th AACR senescence. Telomere shortening is thus a powerful tumor suppressor mechanism. Now that the controversy over the role of telomerase has been largely solved, researchers should be able to move forward in the quest to find telomerase inhibitors. A paper presented by Dr D Corey (University of Texas Southwestern Medical Center, TX, USA) in the late-breaking news session, discussed the development of a new class of agents to inhibit telomerase. The 2'-O-methyl RNA oligonucleotides have IC50 values as low as 1 nM, explained by the fact that these inhibitors have very slow rates of dissociation from the telomerase molecules. These potent sequence-specific inhibitors are promising lead compounds for drug development. Final news briefing Professor Donald Coffey (President AACR 1998, Johns Hopkins University School of Medicine, MD, USA) concluded the meeting by selecting the papers from the conference which he thought were the most exciting. The first paper he discussed involved research on dendritic cells pulsed with prostate tumor antigens by Dr M Salgaller, mentioned above under 'New concepts in cell-based therapy'. He also found the paper on arsenic trioxide by Dr S Soignet, outlined in the late-breaking news section, of particular merit. One of the major goals of current cancer research is to develop targeted chemotherapy strategies. A method for targeting endothelial cells in prostate cancer was described by Dr E Ruoslahti (The Burnham Institute, CA, USA) in this final news briefing. Endothelial cells in blood vessels within tumors are a promising target as they do not mutate like the cancer cells themselves. In vivo selection of phage display libraries was used to isolate peptides which bound specifically to endothelial cells. The anticancer drug, adriamycin, when coupled to these peptides, demonstrated increased efficacy against human prostate cancer xenografts in nude mice, as well as reduced toxicity. Results of a study of the role of insulin-like growth factor-I (IGF-I) in prostate cancer were presented by Dr JM Chan (Harvard School of Public Health, MA, USA) and published earlier this year (Chan JM: Science (1998) 279(5350):563-6). Dr Coffey summarized this interesting paper. A nested case-control study was conducted on prospectively collected plasma from cancer patients and healthy volunteers. It was found that the risk of developing prostate cancer increases 4-fold with high IGF-1 levels. This early marker is possibly as good as prostate-specific antigen (PSA) as a detector of prostate cancer cells. Antibodies to IGF-1 are already available, so possible use of this factor as therapy could be studied. Conclusion Donald Coffey ended the news briefing by noting that this AACR, organized by Frank Rauscher III, had been a particularly exciting one. The number of new drugs which are reaching the clinic for cancer treatment is expanding rapidly: a few years ago 30 to 50 new drugs would enter the clinic per year, whilst this year the number will be 320. 47 Meeting Report 89th AACR 47 Highlights of selected symposia Ann F Chambers Address Department of Oncology University of Western Ontario 790 Commissioners Road East London Ontario N6A 4L6 Canada Email: [email protected] IDrugs 1998 1(1):47-48 © Current Drugs Ltd ISSN 1369-7056 trials in establishing optimal treatments in breast cancer. Ernst Wynder (American Health Foundation, New York, NY, USA) was the recipient of the American Cancer Society Award for Cancer Epidemiology and Prevention. Dr Wynder presented a summary of his research, beginning in the 1950s, identifying smoking as a significant risk factor in lung cancer, and his systematic and persistent efforts in establishing smoking as a primary cause of lung cancer. Tumor angiogenesis and microcirculation The AACR annual meeting was attended by approximately 8000 international scientists, and consisted of more than 200 invited presentations, a plenary session, 28 symposia, two controversy sessions, multiple "meet-the-expert" sessions, two methods workshops and several educational sessions. In addition, more than 4500 submitted abstracts were accepted for presentation in 34 minisymposia, 30 poster discussion sessions and 109 poster sessions. The submitted abstracts are published in the 1998 AACR Proceedings (1998) 39, and are also available online at the AACR website (http://www.aacr.org). More than 30 organizations and foundations provided support for a variety of the sessions. In addition, over 250 commercial firms and other organizations exhibited this year. This report is thus, by necessity, only a small snapshot of selected portions of this large meeting. Major lectures and awards The meeting was opened with the presidential address, given by the outgoing AACR President, Donald Coffey (Johns Hopkins University School of Medicine, Baltimore, MD, USA) and was entitled "Cancer cell structure: aberrations in the decoder of DNA". Dr Coffey reviewed his and others’ work on the role of nuclear matrix structure in regulating gene expression in a tissue-specific fashion, with examples drawn from changes that occur during progression from benign hyperplasia to cancer of the prostate, and described the importance of nuclear morphology functionally, in regulating gene expression, as well as a prognostic indicator. Speakers in the plenary session (sponsored by Novartis) included Erkki Ruoslahti (The Burnham Institute, La Jolla, CA, USA), who described a strategy of using in vivo phage display to identify av integrin-binding peptides, for use in specific targeting of cytotoxic drugs, such as doxorubicin, to endothelial cells in newly formed vessels within tumors. Also in the Plenary Session, Waun Ki Hong (MD Anderson Cancer Center, Houston, TX, USA) summarized studies on strategies for chemoprevention using retinoids in patients at high risk of developing initial or second tumors of the aerodigestive system, including studies showing phenotypic reversion accompanied by persistence of "genetic scars" (eg, chromosomal changes such as loss of heterozygosity) in the tissue. Bernard Fisher (Allegheny University of the Health Sciences, Pittsburgh, PA, USA) was the recipient of the Joseph Burchenal Clinical Research Award (sponsored by a grant from Bristol-Myers Squibb Oncology). Dr Fisher summarized the history of the development of current concepts on breast cancer as a systemic disease, and the crucial role of clinical This symposium, chaired by Rakesh Jain (Massachusetts General Hospital, Boston, MA, USA) and sponsored by Janssen Pharmaceutica and Janssen Research Foundation, presented new information on the role of angiogenesis in cancer and described possible therapeutic approaches. George Yancopoulos (Regeneron, Tarrytown, NY, USA) summarized the cloning of angiopoietin genes, Ang-1 and Ang-2, which can function as activators and antagonists, respectively, of the Tie2 receptor gene in regulating vessel development, as well as Ang-3 (in mouse) and Ang-4 (human). Dr Jain described results from transgenic mice which overexpress Ang-1 in skin (giving rise to hyper-vascularity), Ang-2 knockout mice (which have fragile and leaky vessels), and Ang-3 knockout mice (currently being characterized). The relationship between angio-genesis and oncogenes, such as ras, was summarized by Robert Kerbel (Sunnybrook Health Science Centre, Toronto, Canada). He stressed the importance of cell growth in threedimensional versus monolayer culture. Studies on the microvasculature within tumors, and the roles of both the target organ and the tumor type in determining vessel properties, such as permeability to macromolecules, were presented by Dr Jain. The role of interferons in regulation of angiogenesis and tumor metastasis was discussed by Isaiah J Fidler (MD Anderson Cancer Center, Houston, TX, USA). He described how angiogenesis can be affected by a balance between factors that promote angiogenesis and those that inhibit it, and stressed that while acute diseases may be appropriately treated with acute therapies, metastasis, as a chronic disease, may be more appropriately treated with chronic therapies, such as those limiting angiogenesis. Finally, James Pluda (National Cancer Institute, Rockville, MD, USA) summarized the status of a series of anti-angiogenic compounds and strategies that are in, or will soon enter, clinical trials. He also described an evolving concept of how anti-angiogenic strategies may be used, from the initial hope that they would be useful as adjuncts to stabilize tumor growth after chemotherapy, to their potential use as inducers of tumor dormancy, to the current belief that anti-angiogenic strategies may have a possible role as front-line therapies with the potential for complete tumor eradication. Extracellular matrix proteases and invasion This minisymposium was chaired by Lynn Matrisian (Vanderbilt University School of Medicine, Nashville, TN, USA). Ruth Muschel (University of Pennsylvania School of Medicine, Philadelphia, PA, USA) summarized much of the current research on the role of proteases in cancer growth, 48 IDrugs 1998 Vol 1 No 1 invasion and metastasis. She presented an overview which focused particularly on the matrix metalloproteinases (MMPs), their association with malignancy, and data suggesting that they may play functional roles in tumor invasion and metastasis. Results indicating that neutrophil-derived serine prote-inases can activate proMMP-2 produced by endothelial cells, and that this process requires the membrane-type MT-MMP, were described by J Schwartz (New York University Medical Center, NY, USA). A Strongin (La Jolla Institute for Experimental Medicine, La Jolla, CA, USA) described how MT1MMP and the αvβ3 integrin cooperate in the activation of MMP-2. Studies suggesting that the propeptide region of MT1-MMP is important in the activation of proMMP-2, and that this region also is involved in tissue inhibitor of metalloproteinase-2 (TIMP-2) binding were presented by J Cao (SUNY Stony Brook, NY, USA). Using stromelysin-1 (Str1) null mice, Howard Crawford (Vanderbilt University School of Medicine, Nashville, TN, USA) assessed the role of Str-1 in tumorigenesis, and reported an increase in tumor formation and metastasis following chemical induction of tumors, supporting the idea that host stromal cell expression of Str-1 can restrict tumor growth. Results from in vivo videomicroscopy and histology, showing that the MMP inhibitor, batimastat (BB-94; British Biotech), inhibited growth of liver metastases of murine melanoma cells by inhibiting angiogenesis and not by inhibition of extravasation, were presented by Ann Chambers (London Regional Cancer Centre, Ontario, Canada). Finally, JM Müller (Novartis, Basel, Switzerland) presented results using the MMP inhibitor, CGS27023A, which showed that this compound had antitumor and anti-angiogenic activity in vivo and anti-angiogenic activity in vitro; however, development of this compound has been discontinued. Stromal-epithelial, cell-cell, and cellextracellular matrix interactions in normal development and cancer This symposium, chaired by Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA, USA), examined the interactions between different cell types in the context of three-dimensional tissues. Dr Bissell summarized her studies on the importance of the microenvironment in gene expression and behavior of mammary epithelial cells, and proposed the idea that 'form is the ultimate regulator of gene expression'. Keld Dano (Finsen Laboratory, Copenhagen, Denmark) reviewed his research on components of the plasminogen activator (PA) cascade (urokinase-type PA, urokinase-type PA receptor and PA inhibitor-1), including a description of clinical studies, where patterns of expression in tumor versus stromal tissue of various components of the cascade are characteristic of specific tumor types. Lynn Matrisian (Vanderbilt University School of Medicine, Nashville, TN, USA) summarized research from her laboratory on the role of various MMPs in cancer, and also described studies with the multiple intestinal neoplasia (min) adenomatous polyposis coli (APC)-mutated mouse, in which colocalization of matrilysin and increased β-catenin was found in adenomas, raising the possibility that matrilysin is a downstream gene in the APC signal pathway. Models and methods for invasion and metastasis This poster discussion session was chaired by Meenhard Herlyn (The Wistar Institute, Philadelphia, PA, USA) and Mary Hendrix (University of Iowa College of Medicine, IO, USA). A quantitative study on metastatic inefficiency of melanoma cells in mouse liver, using in vivo videomicroscopy, showed that the rate-limiting steps in this model were not cell survival in the circulation or failure to extravasate, but failure of most extravasated cells to begin and continue growth, was presented by Vincent Morris and Ann Chambers (University of Western Ontario, London, Canada). The generation and characterization of a series of cancer cell lines transfected to stably express green fluorescent protein, for use in studying tumor metastasis was described by Robert Hoffman (AntiCancer, San Diego, CA, USA). In two presentations, T Tachikawa, Y Tamura and colleagues (Fuji, Tokyo, Japan) described procedures they are developing to assess MMP activity using film-based in situ zymography. Finally, Claus Kristensen/Rakesh Jain (Massachusetts General Hospital, Boston, MA, USA) described an in vivo study in mice, in which cells shed from a primary tumor were collected and assessed for in vitro clonogenic ability; the shed cells were found to be less clonogenic in soft agar than the parental tumor cells. Alternative research models for cancer: case studies for consumer involvement This unique session explored various aspects of the changing role of cancer patients, advocates and survivors in research and public policy. The session was introduced by Donald Coffey, outgoing AACR President, who stressed the crucial and growing role played by such advocates in many aspects of academic and corporate cancer research, and cancer research funding. Frances Visco (National Breast Cancer Coalition, Washington DC, USA) chaired the session. The role played by Frances Visco and the coalition in lobbying for significant funding for breast cancer research was described by Anna Barker (Oxis, OR, USA). The positive role played by patients and advocates in Genentech's development and clinical trials of the company's anti-Her2 antibody for breast cancer treatment was described by Susan Hellman (Genentech, CA, USA). Finally, Carol Hochberg (SHARE/SelfHelp for Women with Breast or Ovarian Cancer, NY, USA), a breast cancer survivor, shared her own personal experiences in dealing with cancer. Conclusion This report can give only a small suggestion of the presentations available to the participants. The AACR website can be consulted for details on any of the submitted abstracts. The presentations spanned basic and clinical aspects of oncology, and provided information for researchers interested in nearly any aspect of current oncology research. Meeting Report 9th Med Chem East England 49 Medicinal Chemistry in Eastern England - Ninth Symposium 20 April 1998, University of Hertfordshire, Hatfield, UK Peter Norman Address Norman Consulting 18 Pink Lane Burnham Bucks SL1 8JW UK Email: [email protected] IDrugs 1998 1(1):49-54 © Current Drugs Ltd ISSN 1369-7056 This meeting is sponsored by the Royal Society of Chemistry’s (RSC) Biological and Medicinal chemistry sector and is now an annual event. Around 100 delegates representing a broad spectrum of the British pharmaceutical industry attended to hear lectures from seven invited speakers. The meeting included the presentation of the RSC’s Biological and Medicinal Chemistry Sector award to a team from SmithKline Beecham, UK, for their work on 5-HT4 antagonists, especially SB-207266. This was presented by the group’s chairman, Dr Derek Buckle, to Dr Paul Wyman. Transition state analogs as cytosolic phospholipase A2 inhibitors Cytosolic PLA2 is believed to the primary form of phospholipase (PLA2) involved in the pathogenesis of inflammatory disease. It selectively liberates arachidonic acid from phospholipids, and recent studies in knockout mice have implicated a role in the induction of airway hyperreactivity. Dr Tony Meete (Astra Charnwood, UK) described progress towards the identification of transition state analogs of PLA2 as inhibitors. High-throughput screening (HTS) has so far failed to identify any potential lead structures, but a designed inhibitor - AR-C69611 (1-(4-decyloxyphenoxy)-3methoxypropan-2-one; Figure 1) - was described (IC50 = 5 µM). Replacement of the methoxy group with substituted phenyl rings, especially p-benzoic acid (Figure 1), led to compounds with IC50 values in the 100 nM range, and activity in HL60 cells at 2 µM, but very poor aqueous solubility. Replacement of the decyloxy group by phenylalkoxy or phenylalkylthio groups yielded comp-ounds with better solubility and comparable potency. Enzyme activity was found to be a function of drug lipophilicity. AR-C73346 (4-(2-oxo-3-[4-(5phenylpentyl-thio)phenoxy]-propoxy)benzoic acid; Figure 1) was the most potent compound described (IC50 = 77 nM). The presence of both the proximal phenyl ring and the ketone is critical to activity, and replacement of either of the ether linkages reduced, or abolished, activity. Enhanced potency was achieved by oxidation of the thia linkage to a sulfone, to give a compound, AR-C71089 (Figure 1), with an 13 IC50 of 38 nM. Studies with [ C]-labeled inhibitor confirmed that the bound form is the hemiketal and that the binding is reversible. It was shown that inhibition is not timedependent, and that both on and off rates are rapid. This class has no effect against 5-lipoxygenase or cyclooxygenase, but the compounds inhibit the release of both prostaglandins and leukotrienes in whole cell assays, with potencies slightly lower than against isolated enzyme. Data were presented on one compound demonstrating inhibition of leukotriene B4 production in rat whole blood ex vivo at 2 µM, and inhibition of TPA-induced inflammation in the mouse ear. More potent compounds than those described are apparently under development for rheumatoid arthritis and asthma, and are active in rat paw edema models of inflammation. Orally active carbapenems Carbapenems offer a potential means of addressing the problems of increasing antibiotic resistance in communityacquired infections. The currently available carbapenems (iminipenem and meropenem) are unsuitable for oral administration, partly because of their susceptibility to cleavage by renal dihydropeptidase-1 (DHP-1). Dr Steve Coulton (SmithKline Beecham, UK) described the outcome of a program to identify orally active carbapenem derivatives. SB’s approach was first to identify a potent carbapenem with good stability against DHP-1 and second to prepare suitable crystalline prodrugs which were well absorbed. 2-Phenylcarbapenems are known to be stable to DHP-1, so a range of heterocycles as the 2-substituents was investigated. The synthesis of such penems has recently been published in the Journal of Antibiotics (1998) 51:210. From a range of 5-membered rings, only 2,3-disubstituted pyrazole-5-yl derivatives were found to combine the desired potency with sufficient stability against DHP-1 cleavage. Five compounds, substituted with methyl, ethyl or hydroxyethyl groups, were evaluated in vivo. SB-221691 (6(1-hydroxyethyl)3-([2-(2-methyl-3-ethylpyrazol-5-yl)-7-oxo1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; Figure 2) showed the best duration of action and superior in vivo activity to tribactam in a rat model of respiratory tract infection. The compound had good potency against resistant strains of Streptococcus pneumoniae and Haemophilus influenzae (MIC90 values of 1 and 0.25 µg/ml) and good potency against Staphylococcus aureus (S aureus), Streptococcus pyogenes and Escherichia coli; oral absorption was, however, poor (8%). Two crystalline ester derivatives of the hydroxymethoxy ester of SB-221691 were described. The bioavailability of SB223328 (the cyclohexyloxycarbonyl ester; Figure 2) and SB224617 (the isopropoxycarbonyl ester; Figure 2) in the rat was 45 and 41%, respectively. Both compounds were as potent as augmentin in a rat model of respiratory tract infection, at 5 and 50 mg/kg, respectively. Like meropenem 50 IDrugs 1998 Vol 1 No 1 Figure 1. Astra PLA2 inhibitors O O O R1 R2 X AR-C70484 = R1 CH3(I) R2 CH3(CH2)9CH3(CH2)9- AR-C73346 = (I) AR-C71089 = (I) Ph(CH2)5CH3(CH2)9- AR-C69611 = X O O OH S SO2 (I) O Figure 2. Carbapenems OH OH H H H CH3 H3C N N O HO H CH3 H3C N N CH3 N O O O O SB-221691 (SmithKline Beecham) O N CH3 O O SB-223328 (SmithKline Beecham) OH H H CH3 H3C N N O CH3 H3C O O O N CH3 O O SB-224617 (SmithKline Beecham) they are inactive against methicillin-resistant S aureus. Both compounds are currently under clinical evaluation. ZD-1839 - an EGF receptor-tyrosine kinase inhibitor The EGF receptor-tyrosine kinase is heavily expressed in a variety of cancer cells. Dr Keith Gibson (Zeneca, UK) described the identification of a novel, reversible, inhibitor of this kinase which is now in phase I trials. 4-Arylamino- quinazolines were initially identified as lead structures by 3D structural searching based on a transition state model, which was later found to be erroneous. Simple derivatives such as 4-(3-methyl-phenyl)aminoquinazoline (Figure 3) had reasonable potency in both enzyme and cellular assays (IC50 = 0.18 and 2.4 µM, respectively). Such compounds were readily prepared in four steps, and robotic synthesis was used to generate a compound library to delineate the SAR of the series. The presence of the secondary amine was critical, and 6- and/or 7- substitution with electron-donating groups Meeting Report 9th Med Chem East England 51 Figure 3. Zeneca tyrosine kinase inhibitors F HN CH3 O HN N O N N screening lead from HTS (Zeneca) Cl N H3C O N ZD-1839 (Zeneca) enhanced the activity. The 6,7-dimethoxy derivative was the most potent (IC50 = 5 nM), but was rapidly metabolized in vivo. Replacement of the toluyl group by a 3-chloro-4fluorophenyl ring improved the pharmacokinetic properties but compounds were only moderately effective in a tumor xenograft model. action. Re-introduction of the carboxyl group gave UK142773 ([3-(3-aminoiminoph-enyl)-2-[2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)sulf-onyl]amino-1-oxopropyl]-4-methyl-2-pipe-ridinecarboxylic acid; Figure 4). This compound was well-tolerated, orally active and 18-fold selective for thrombin (Ki = 3.1 nM) compared to trypsin. Introduction of an amino functionality in the 6-substituent dramatically improved absorption. Data were presented on a variety of substituents, and whilst potency was greatest with methylamino or dimethylamino substituents, drug absorption was better with 6-aminopropoxy substituents. ZD-1839 (4-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)-quinazoline; Figure 3) has IC50 values of 23 and 80 nM in enzymatic and cellular assays, respectively, and has little activity against a range of other kinases (eg, IC50 = 1.2 µM against erbB2). It displays 30-fold selectivity for EGF relative to other growth factors in human umbilical vein endothelial cell assays. UK-156406 (Figure 4) employs a flattened piperidine ring - 4methyl-1,2,5,6-tetrahydropyridine-6-carboxylic acid - and is a more potent compound (Ki values for thrombin and trypsin = 0.26 and 26 nM, respectively). The compound had an improved duration of action, due to reduced hepatic clearance, by both iv and id routes. It has a bioavailability of 45% in the dog and 15% in the rat; plasma half life = 0.8 h in the dog. In man there was a dose-dependent inhibition of ex vivo thrombin time at doses of 25 to 200 mg. Doses of 10 mg were ineffective, but higher doses had a duration of action of 4 to 5 h, and were well-tolerated. The compound is now in clinical trials. ZD-1839 produces sustained plasma levels (5.7 µM 24 h after a dose of 200 mg/kg). In mice it is orally effective at doses as low as 12.5 mg/kg and will inhibit tumor growth or cause regression of existing tumors (eg, AS49) over a sustained period. On cessation of drug treatment, tumor regrowth occurs. A back-up compound was prepared by designing compounds to interact with the lipophilic cleft between Ala-190 and Val213. Replacement of the benzamidine group by a 1-aminoimino-1,2,5,6-tetrahydropyridin-3-yl group gave a compound (UK-239326; Figure 4) with dramatically improved potency and selectivity and a similar profile of action in vivo. UK-156406 - an orally active thrombin inhibitor Large scale synthesis of chiral reagents Dr Alan Brown (Pfizer, UK) described the development of an orally active thrombin inhibitor which is now in clinical evaluation. Compounds were derived from the structures of the thrombin inhibitors argatroban and MD-205, and X-ray data of the argatroban-thrombin complex. The X-ray data pointed to an interaction with the Trp-60 portion of the enzyme and to a hydrophobic collapse of the quinoline ring onto the piperidine ring. These initial approaches led to the introduction of benzamidine rings, and to the selection of a 2-methyl-1,2,3,4tetrahydroisoquinolin-7-yl ring in place of the tetrahydroquinoline ring of argatroban. Early compounds were poorly selective and short-acting. Introduction of a 4-methyl group in the piperidine ring gave a compound (UK-141364) which was poorly tolerated but had a reasonable duration of Dr Jon Cummins (ChiRex) addressed some of the problems of obtaining large quantities of chiral reagents without using long and/or expensive synthetic routes. In particular, he illustrated how the catalysts recently described by Jacobsen may be applied to prepare, or resolve, chiral epoxides (eg, epichlorohydrin) and diols (eg, propanediol) in multikilogram quantities in high yield (> 98%). These catalysts are reusable and he predicted that the processes can be applied on the tonne scale. A novel serine protease pharmacophore [5,5]trans-fused lactones and lactams Dr Harry Kelly (Glaxo Wellcome, UK) described studies on [5,5]transfused lactones as inhibitors of the serine protease, thrombin. The program has generated orally active inhibitors of elastase and potent, orally active thrombin inhibitors with encouraging pharmacokinetics. 52 IDrugs 1998 Vol 1 No 1 Figure 4. Pfizer thrombin inhibitors HN HN NH2 NH2 H N N O CH3 N S H3C O H N N O HO CH3 N S H3C O O O HO O O UK-142773 (Pfizer) UK-156406 (Pfizer) HN NH2 N H N N O CH3 N S H3C O O HO O UK-239326 (Pfizer) An HTS program identified a terpenoid (GW-133686), found in wild sage, as a potent inhibitor of thrombin (IC50 = 4 nM) and the ability to double activated partial thromboplastin time at 40 µM. This compound was less potent as a trypsin inhibitor (IC50 = 70 nM), and had a sustained duration of action in both rat and human plasma. The compound contains the unusual [5,5]trans lactone system substituted in the 3-position by a 2-methyl-4-oxo-pent-2-en-5-yl substituent. The presence of the enone is critical to activity, and ring opening of the lactone reduces activity by 400-fold. X-ray crystallographic studies on the interaction of GW-133686 with thrombin have shown that Ser-195 opens the lactone ring (Biochemistry (1998), in press). The structural complexity of this lead led to the exploration of the activity of 3-substituted-[5,5]trans lactones. The parent compound had little activity against thrombin but was active against trypsin, whilst 3-m-thiophenyl or 3-enone substituted compounds had IC 50 values of 20 µM (Figure 5). The introduction of amidine groups into the side-chain improved the potency, with the 5-amidinopentyl derivative having an IC50 of 130 nM (Figure 5). Potency was further improved by preparing [5,5]trans lactones of indanes with 4- or 5-diethylcarbamoyl derivatives. The resulting compounds had IC50 values of 8 to 11 nM (Figure 5), but these compounds had poor stability in plasma. Replacement of the ring oxygen by nitrogen provided lactams with better stability but poorer potency. Potency was restored by introduction of N-substituents, with the N-carbomethoxy derivative having an IC50 value of 16 nM (Figure 5) and high stability in human plasma. Further efforts, which were not described, have concentrated on varying the S-2 and S-3 regions of the inhibitors. This has resulted in significant potency improvements and permitted the identification of potent inhibitors of other serine proteases. Thus the [5,5]trans lactone/lactam group is a novel pharmacophore for serine proteases in general. Meeting Report 9th Med Chem East England 53 Figure 5. Glaxo Wellcome serine protease inhibitors NH H3C CH3 O NH2 O S O O IC50 = 250 µM O O O lead pharmacophore IC50 = 21 µM O O IC50 = 20µM IC50 = 130 nM NH NH NH2 N H3C CH3 O O NH2 N H3C CH3 O O O N O CH3 O IC50 = 8 nM IC50 = 16 nM Figure 6. Knoll antidepressants O O N N CH3 N N N N N H2N N O H2N CH3 O O O O BTS-78554 (Knoll) BTS-79524 - a potential antidepressant with 5-HT1A agonist and α2A antagonist activity Dr Frank Kerrigan (Knoll, UK) outlined the need for novel antidepressants, and briefly described how sibutramine, a monoamine uptake inhibitor, had been identified and originally developed as an antidepresssant. However, when evaluated clinically it caused significant weight loss and has subsequently been developed, and approved in the US, as an anti-obesity drug. BTS-79524 (Knoll) The company’s alternative strategy for the identification of novel antidepressants has focused on drugs that combine 5-HT1A agonist and α2A antagonist activity. Such compounds synergistically inhibit synaptic neurotransmission. The original lead structures were based on the pyrimidinyl-piperazine portion of buspirone (BristolMyers Squibb), which is a potent antidepressant. 54 IDrugs 1998 Vol 1 No 1 This led to a series of 6-amino-5-(4-arylpiperazinyl)methyl-uracil derivatives. When both ring nitrogens were substituted, the compounds had nanomolar affinities for the desired receptors but were found to be mutagenic. Increasing the size of the substituents reduced the effects, but did not reduce mutagenicity. The use of 4-(4arylpiperazinyl)-(5-amino-3-cycloalkyl-1-phenyl-pyrazole) derivatives provided more effective compounds (although an injudicious choice of substituents led to enhanced α1 activity and the class retained genotoxicity). The use of an dihydropyrazolone ring led to BTS-79524 (5-amino-4-[4-(benzodioxan-5-yl)piperazin-1-yl]methyl-1methyl -4-oxo-3-phenyl-2,3-dihydropyrazole; Figure 6). This comp-ound has high affinity at 5-HT 1A (Ki = 26 nM) and α2A (Ki = 29 nM) receptors with only moderate affinity for α1 receptors (Ki = 401 nM) and was free of genotoxic effects. Some details of the synthetic approaches to this compound have recently been described in Tetrahedron Letters (1998) 39:2219. In in vivo models of depression, BTS-79524 produced 66% downregulation of 5-HT1 receptors (cf 32% for amitryptiline) and was more potent in the rat Porsholt test (minimum effective dose = 3 mg/kg). This compound is not under active development, as a consequence of a strategic decision, but has a similar profile to the structurally distinct CP-93393 (Pfizer), that is now in early clinical trials. Conclusion The organizers provided a well chosen program at a good, easily accessible venue. The meeting covered a broad range of therapeutic areas. One deficiency, pointed out in the closing remarks of Paul Leeson, was that the conference had not encompassed aspects of combinatorial chemistry, and he suggested that next year’s meeting should remedy this omission. Meeting Report 71st Japan Pharmacol Soc 55 The Japanese Pharmacological Society - 71st Annual Meeting 23 - 26 March 1998, Kyoto International Conference Center, Kyoto, Japan Trevor Wright Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):55-62 © Current Drugs Ltd ISSN 1369-7056 Angiotensin receptor subtype functions In a presentation concerning angiotensin receptor signaling mechanisms, T Inagami (Vanderbilt University School of Medicine, USA) discussed the mechanisms of function of individual angiotensin receptor subtypes and showed that AT2 gene deletion causes blood pressure elevation. He went on to discuss the possible mechanism of AT2 function. Other notable oral presentations Y-27632 This meeting was attended by over 2,700 pharmacologists and physiologists, and hosted nearly 350 oral presentations and close to 700 posters. The topics covered a wide range of contemporary pharmacological and physiological themes and provided an interesting update of the development of many novel compounds for a wide range of therapeutic indications. Antisense oligonucleotide pharmacology made an appearance this year and many presentations concerned the safety of medicines. Data on the pharmacological profile of a novel pyridine derivative, Y-27632 (Yoshitomi), were presented by T Ishizaki (Faculty of Medicine, University of Kyoto). This compound inhibits smooth muscle contraction induced by various agonists of the calcium sensitization pathway. It interacts with p160ROCK, with an affinity which is about 200-fold its affinity for PKC and it is thought to directly inhibit this Rho-associated protein kinase. CP-99994 Clinical pharmacy and pharmacology The six opening symposia concentrated on various aspects of clinical pharmacy and pharmacology. Clinical pharmacy education was not introduced into Japanese universities until the 1970s, despite the early establishment of this discipline in other countries, such as the US. There are currently only six departments of clinical pharmacology amongst the 80 medical schools throughout the country. The general consensus of the speakers was that the role of clinical pharmacy and pharmacology should be strengthened and that cooperation between them extended. The role of nitric oxide Special lectures focused on cardio- and cerebrovascular pharmacology. S Moncada (University of London, UK), in a slight departure from the scheduled lecture theme, presented an overview on the discovery of nitric oxide together with its function in health and disease. He highlighted the broad range of functions of this simple molecule, including its effects on immunological host defense and its role in the pathophysiology of septic shock, inflammation and cerebral ischemia. Professor Moncada discussed the potential effects that selective nitric oxide synthase inhibitors may have on protecting neurons from damage. Neuronal circuits Current understanding of how neurons within the CNS interact through both excitatory and inhibitory synapses and how multilayered neuronal circuits within the cortical tissues show remarkable computational functions was discussed by M Ito (Riken, Japan). He suggested that our knowledge of gene regulatory mechanisms will expand in future to shed light on mechanisms of cellular function and development which in turn will lead to the production of brain-like computers. A Nagahisa (Pfizer, Japan) described findings for CP-99994 (Pfizer; Figure 1), which is a centrally acting NK1 antagonist, showing that it is a broad spectrum anti-emetic agent. It inhibits vomiting and retching induced by morphine, cisplatin and ipecac in ferrets and highlights the potential advantages of neurokinin antagonists in the treatment of chemotherapy-induced emesis. GR-205171 The pharmacological profile of GR-205171 (Figure 1) was reported by S Liou (Nippon Glaxo, Japan). This too is an NK1 antagonist with potent anti-emetic effects and at 25 or 50 µg/kg iv was shown to abolish the gradual and rhythmic bursts of firings of the central pattern generator (CPG) concomitantly with retching discharges of both phrenic and abdominal muscle nerves. It is thought that the CPG neurons within the reticular area are the site of action of this anti-emetic. Mitragynine S Horie (Faculty of Pharmaceutical Sciences, University of Chiba, Japan) presented results for mitragynine, an alkaloid extract of a Thai medicinal plant. This monoterpenoid indole alkaloid is known to have a potent analgesic effect and is currently being investigated for its opioid agonist effects despite its lack of chemical similarity to morphine. It appears to act on µ- and δ-opioid receptors and causes antinociception. FR-146801 The effects of FR-146801 (Fujisawa), a spontaneous NO donor, were reported by Y Hirasawa (Fujisawa Pharmaceutical Co Ltd, Japan). This compound is a stable analog of FK-409 (Fujisawa), and was shown to suppress intimal thickening after balloon injury in a rat model. When given orally, FR-146801, showed a more selective antiproliferative effect than hypotensive effect. 56 IDrugs 1998 Vol 1 No 1 ATZ-1993 ATZ-1993 is an orally active non-peptide endothelin receptor antagonist and, according to H Azuma (Tokyo Medical and Dental Engineering, Japan), it significantly inhibits intimal hyperplasia following endothelial denudation in the rat, after dosing at 30 mg/200 ml/kg/day for 7 weeks. This compound did not affect the mean blood pressure and heart rate and its effects on intimal hyperplasia were at least partly due to its antagonist effects on endothelin receptors. Ad.CMV-cHK K Yayama (Kobe Gakuin University, Japan) reported on the use of an adenovirus-mediated delivery system for a human tissue kallikrein gene. This gene delivery system was shown to reduce blood pressure in a two-kidney, one-clip Goldblatt hypertensive rat model. The adenovirus (Ad.CMV-cHK) carrying this gene was under the control of a cytomegalovirus promoter and was administered iv. significantly decreased heart rate, rate-pressure product, left ventricular contraction and cardiac output and was 6- to 8fold more potent than esmolol on some parameters. It is thought to be a more potent β-blocker than esmolol. OPC-6535 OPC-6535 (Otsuka; Figure 1) is an inhibitor of leukocyte activation, including decreased superoxide production, lower neutrophil adhesion and decreased chemotaxis. G Miyakoda (Otsuka Pharmaceutical Co Ltd, Japan) reported on findings for the effect of this compound on left ventricular function in a pig myocardial model of 30 min ischemia and 4 week reperfusion. It was shown to significantly improve left ventricular function and reduce the extent of myocardial fibrosis after long-term reperfusion when given post-ischemically. EMD-57033 Results of using Bay-k-8644 (University of Alberta) were presented by T Nasu (Yamaguchi University, Japan). This dihydropyridine calcium agonist evoked strong rhythmic contraction in normal calcium medium in taenia coli. These results suggest that this compound allows entry of manganese ions into the cytoplasm via calcium channels while voltage-dependent calcium channels are activated by Bay-k-8644. Calcium sensitizers, such as EMD-57033 (Merck KGaA; Figure 1), are being developed for the treatment of congenital heart failure. K Sakurai (Yamagata University School of Medicine, Japan) reported results for this compound in a volume-loaded rabbit model 12 weeks or more after arteriovenous shunt formation. The cardiotonic effects of this compound do not appear to be affected in volume-overloaded cardiomyocytes although the response by these cells to β-blockers was markedly decreased. It is suggested that this compound may be of benefit in the treatment of contractile dysfunction in congestive heart failure where β-antagonists are ineffective. MKC-242 FR-171113 The involvement of presynaptic 5-HT1A receptor antagonist action in the MKC-242-induced potentiation of photic entrainment in circadian rhythms was reported by Y Yoshinobu (Waseda University, Japan). The results obtained indicate that MKC-242 (Mitsubishi; Figure 1) potentiates the photic entrainment of the circadian clock via activation of presynaptic 5-HT1A receptors, which is followed by a reduction in serotoninergic neuron activity. Y Kato (Fujisawa Pharmaceutical Co Ltd, Japan) presented findings for FR-171113 (Fujisawa), a novel non-peptide thrombin receptor antagonist, on platelet aggre-gation in guinea pigs. Dose-dependent inhibition was observed over a concentration range of 1 to 32 µM when aggregation was induced by TRP-6, but not when induced by ADP or collagen, even at 100 mM. FR-171113 inhibits thrombinreceptor mediated platelet aggregation both in vivo and in vitro. Bay-k-8644 OPC-14523 Data on the pharmacological effects of OPC-14523 (Otsuka) were presented by K Tottori (Otsuka Pharmaceutical Co Ltd, Japan). This novel antidepressant is a potent antagonist of σ receptors and 5-HT1A receptors as well as inhibiting 5-HT reuptake. Single doses of 3 mg/kg po, or higher, significantly and dose-dependently antagonized reserpineinduced ptosis in mice. These results indicate that OPC14523 may be a potent antidepressant drug. FK-409 S-8921 The hypocholesterolemic and anti-atherogenic effects of S8921 (Shionogi) were reported by S Hara (Shionogi & Co + Ltd). This compound is an ileal Na /bile acid cotransporter inhibitor and was shown to produce dose-dependent decreases in serum cholesterol levels when administered for 6 weeks in cholesterol-fed hamsters (the final concentration of S-8921 was 0.6 to 57 mg/kg). This compound was also shown to be > 50-fold more effective than cholestyramine. The protective effect of FK-409 (Fujisawa; Figure 1) on ischemic acute renal failure in rats was discussed by M Nishiura (Osaka University of Pharmaceutical Sciences, Japan). This compound, a spontaneous NO donor, was shown to attenuate a decrease in renal function due to occlusion following contralateral nephrectomy and indicates that it may be useful in the treatment of acute renal failure due to formation of NO. (+)-TAN 67 (Toray; Figure 1) has antinociceptive effects in mice according to J Kamei (Hoshi University, Japan). Subcutaneous pretreatment at doses from 3 to 30 µg/kg for 30 min, dose-dependently attenuated the antinociception induced by intrathecal administration of nociceptin (1 nmol). This compound may therefore act as an antagonist for opioid receptor-like-1 receptors. ONO-1101 S-0509 The cardiovascular effects of ONO-1101 (Ono; Figure 1) were presented by A Sugiyama (Yamanashi Medical University, Japan). This ultra-short acting β-blocker Findings for a new potent and selective gastrin/CCK-B receptor antagonist, S-0509, were presented by K Ikeda (Kyoto Pharmaceutical University). When 1 to 60 mg/kg of TAN 67 Meeting Report 71st Japan Pharmacol Soc 57 Figure 1. Structures of drugs from oral presentations H N CH3 O H N O N H N H H3C O F F O N H3C Cl N GR-205171 (Glaxo Wellcome) MKC-242 (Mitsubishi) OH HO N NH2 N O H N O H3C O H N F CP-99994 (Pfizer) O N O O N H H3C H3C O N H O N O O O O O ONO-1101 (Fujisawa) FK-409 (Fujisawa) O CH3 O O S O N N H3C N OH CH3 O S H3C O N O N H OPC-6535 (Otsuka) EMD-57033 (Merck KGaA) H N N OH TAN-67 (Toray) CH3 CH3 58 IDrugs 1998 Vol 1 No 1 this compound was administered orally 1 h before pyloric ligation, it significantly suppressed basal gastric acid secretion in rats without ulcers. It also appeared to be as potent as famotidine (Yamanouchi) in inhibiting gastric secretion and may be a useful anti-ulcer drug. Highlights of poster presentations Cardiovascular and circulatory NTE-122 Y Azuma et al (Nissin Food Products, Japan) presented findings for the effects of the ACAT inhibitor, NTE-122 (Nissin; Figure 2), on the metabolism and secretion of lipids in HepG2 and CaCo-2 cells. This is a potent and selective inhibitor of this lipid-metabolizing enzyme. It appears to lower cholesterol levels in these cells by the inhibition of VLDL secretion and the stimulation of bile acid secretion in the liver, and the inhibition of lipid incorporation via the intestinal mucosa. JTV-605 JTV-605 is a non-peptide vasopressin antagonist and results of its effects on diuresis were presented by K Wakitani et al (Japan Tobacco). It shows high selectivity for V1 receptors from rat liver and human platelets (IC50 values of 15 and 16 nM, respectively) and for V2 receptors from rat kidney and CHO cells stably expressing human V2 receptors (IC50 values of 22 and 19 nM, respectively). JTV-605 may be of potential use in the treatment of patients with edema. NS-7 The cerebroprotective effects of NS-7 (Nippon Shinyaku; Figure 2), a novel calcium channel blocker, on ischemia induced by transient occlusion of the middle cerebral artery were presented by Y Aoki et al (Nippon Shinyaku, Japan). The protective effects of this pyrimidine deriv-ative were obvious even when it was administered 6 to 12 h after onset of ischemia, indicating that it may be of use as a cerebroprotectant with a wide time window after ischemic insult. SR-33805 GR-144053 (Glaxo Wellcome; Figure 2), a fibrinogen receptor antagonist, enhances the suppression of neoin-timal formation by losartan (DuPont Merck) according to H Matsuno et al (Gifu University School of Medicine, Japan). This compound was coadministered with losartan, an angiotensin II receptor antagonist, to hamsters with intimal injury caused by the tip of a catheter and was found to suppress neointimal formation 2 weeks after injury. S Ieiri et al (Kyushu University, Japan) reported their findings for SR-33805 (Sanofi Recherche; Figure 2) on the cytosolic calcium concentrations and contractions of porcine muscle. This indole derivative, a novel calcium channel blocker, differs chemically from other known calcium antagonists such as 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. The results showed that it inhibited contraction of porcine artery to a lesser extent than would be predicted from its inhibition of calcium influx. Its mechanism of action has yet to be elucidated. KB-R9032 CP-060S In a comparative study of KB-R9032 (Kanebo), K Harada et al + + (Kanebo, Japan) showed that this novel Na /H ion-exchange inhibitor was a selective NHE1 inhibitor with weak inhibitory effects on NHE2, which were expressed in a fibroblast cell line. Species differences were observed for the actions of KBR9032 on human and rabbit platelets according to investigations on the attenuation of sodium proprionateinduced swelling caused by this compound. It may be of use in the treatment of ischemic reperfusion injury. CP-060S (Chugai) is a novel cardioprotective agent which prevents overloading of both sodium and calcium ions, and its effects on Arg-vasopressin-induced myocardial ischemia in rats were presented by K Tamura et al (Chugai Pharmaceutical Co Ltd, Japan). Orally administered CP060S, at doses of 3 and 10 mg/kg, significantly inhibited AVP-induced ST depression and this effect was sustained for 12 h at the higher dose. CP-060S was shown to have a slower onset of its vasodilatory effect due to its calcium antagonist effect than that seen for diltiazem. It may be of use in the treatment of angina. GR-144053 KRG-594 Y Inada et al (Kissei Pharmaceutical, Japan) presented their findings on KRG-594 (Figure 2), a novel AT1 receptor antagonist. This cyclopentenecarboxylate was shown to cause a sustained hypotensive effect in SHR and renal hypertensive rats and dogs, and was also shown to dosedependently improve glomerulosclerosis. The results suggest that KRG-594 ameliorates diabetic nephropathy accompanying hypertension. E-4010 E-4010 is a novel PDE V inhibitor according to H Ishihara (Eisai, Japan). It competitively inhibited the activity of PDE V purified from porcine and human platelets and was shown to be highly selective for this form compared to other isozymes. At 100 nM, E-4010 significantly potentiated increases in cGMP levels induced by SNP (1 µM) and ANP (10 nM) in isolated porcine pulmonary arteries without affecting cAMP levels. AT-1015 The antithrombotic effects of AT-1015 and its associated bleeding risks were presented by H Koganei (Ajinomoto, Japan). This is a novel 5-HT2 receptor antagonist which inhibits platelet aggregation and arterial contraction induced by 5-HT. It did not prolong bleeding time in rat tail at a dose of 3 mg/kg po, and it is suggested that it could be used as an antithrombotic agent with low bleeding tendency. OPC-33509 OPC-33509 (Otsuka) is a novel antithrombotic derivative of cilostazol (Otsuka) and shows more potent antithrom-botic and antihyperplastic effects than cilostazol. Y Inoue et al (Nagoya University School of Medicine, Japan) report that this compound has a dual mechanism of action by inhibiting vascular intimal thickening and inhibiting thrombogenesis, indicating that it may be of use in the treatment of arteriosclerosis-induced ischemic diseases. Meeting Report 71st Japan Pharmacol Soc 59 Figure 2. Cardiovascular and circulatory drugs H3 C OH N N CH3 O O N N H N H3 C N H N N H2 N O N NH CH3 GR-144053 (Glaxo Wellcome) NTE-122 (Nissin Food Products) O S CH3 O N CH3 N N N N N N N OH HN N O H K Cl K F KRG-594 (Wakunaga) NS-7 (Nippon Shinyaku) O CH3 H3C CH3 O CH3 S N O N O O CH3 CH3 SR-33805 (Sanofi Recherche) KIN-493 KIN-493 (Novartis; Figure 3), an analog of carbama-zepine, produces marked antinociceptive actions in STZ-induced diabetic rats according to S Kiguchi et al (Kissei Pharmaceutical, Japan). The antinociceptive effects of this compound, at 100 mg/kg po, were significantly reversed by pretreatment with theophylline, bicuculline and yohimbine, although naloxone, naltrindole, bromocriptine and sulpiride had no effect on this compound. These results indicate that the antinociceptive effects of KIN-493 are not mediated by opioid or dopamine receptors. MKC-242 Findings for the mechanism of action of MKC-242 (Mitsubishi; Figure 3) were presented by P Somboonthum et al (Department of Pharmacology, University of Osaka, Japan). It is a potent and selective 5-HT1A receptor agonist which has anxiolytic- and antidepressant-like effects in animal models. Results suggest that systemic injection of 60 IDrugs 1998 Vol 1 No 1 Figure 3. Central and peripheral nervous system drugs O S NH2 O OH N OH N Cl N N H3C HO O Cl H R-84760 (Sankyo) CP-101606 (Pfizer) KIN-493 (Novartis) O O O O H NH2 OH N H3C O O O N H Cl N H F O Cl F F MKC-242 (Mitsubishi) KMD-3213 (Kissei) Figure 4. Gastrointestinal drugs O O HO N H OH H3C N N H N O S N H N H3C HO O HO OH S O O T-593 (Toyama) BX-661A (Nippon Hoechst Marion Roussel) O N N NH C C N H N KW-5092 (Kyowa) N Meeting Report 71st Japan Pharmacol Soc this compound facilitates the release of ACh via activation of somadendritic 5-HT1A autoreceptors and that this mechanism differs from that of 8-OH-DPAT. KMD-3213 The potential use of a novel α1-adrenoceptor antagonist, KMD-3213 (Kissei; Figure 3), were outlined by S Murata et al (Kissei Pharmaceutical, Japan). This selective α1 adrenoceptor blocker has been developed for the treatment of urinary outlet obstruction associated with benign prostatic hyperplasia. KMD-3213 was found to be highly selective for α1A and α1L subtypes, rather than for α1B and α1D. This pharmacological profile differed from that of prazocin and tamsulosin (Yamanouchi) which show high affinity for the latter receptors. 61 mg/kg po in dogs chronically implanted with force transducers. Z-388 improves delayed gastric emptying to the normal level and may be useful in the treatment of abdominal symptoms due to hypomotility and delayed gastric emptying in patients with functional dyspepsia. T-593 The effects of T-593 (Toyama; Figure 4) on gastric ulcer healing were presented by Y Doi (Toyama Chemical, Japan). This is a potent H2 receptor antagonist with gastroprotective effects, and was shown to facilitate mucosal regeneration and collagen fiber proliferation when administered at 30 mg/kg po for 8 weeks to a rat model of cryoprobe-induced gastric tissue damage. It is effective in the re-epithelialization of gastric tissue and promotes the maturation of granulation tissue. KW-5092 Gastrointestinal BX-661A S Furuta et al (Nippon Hoechst Marion Roussel, Japan) presented findings for BX-661A (Salix; Figure 4), a compound being investigated for the treatment of ulcerative colitis. They observed the effects of this salicylate on PMNL functions following treatment with FMLP. BX-661A is suggested to inhibit reactive oxygen metabolite production and chemotaxis by inhibiting FMLP-binding to its receptor on PMNL, both activities possibly contributing to its effects in ulcerative colitis. YM-905 YM-905 (Yamaouchi) is a novel muscarinic receptor antagonist. Its effects on gastrointestinal motility and gastrocolic reflex were reported by M Suzuki et al (Yamanouchi Pharmaceutical, Japan). It potently and competitively inhibited carbachol-induced contractions of guinea pig colon, with a pA2 value of 7.5 ± 0.05. It was also shown to inhibit restraint stress-induced defecation and diarrhea over a dose range of 1 to 30 mg/kg. It is suggested that this compound may be useful in the treatment of irritable bowel syndrome. Z-338 Y Matsunaga et al (Zeria Pharmaceuticals, Japan) reported on the gastroprokinetic activity of Z-338. This thiazole-4carboxamide derivative showed a dose-dependent increase in fed gastric motor activity at 0.3 to 3 mg/kg iv and 3 to 30 N Kishibayashi et al (Kyowa Hakko, Japan) presented findings for KW-5092 (Kyowa Hakko; Figure 4), a novel gastroprokinetic agent which causes the release of ACh and also inhibits acetylcholinesterase. The EC50 value for this compound in the potentiation of ACh release was 570 nM and suggests that the stimulation of gastric emptying is due to its ACh releasing effect. Anti-inflammatory and anti-allergy T-164 A novel anti-inflammatory compound, T-164 (Toyama), suppresses NFκB activation from evidence presented by Y Konishi et al (Toyama Chemical, Japan). It is known to suppress immunoglobulin and cytokine production and to have anti-inflammatory actions as a consequence. It was shown that the mechanism of action of T-164 was due to the inhibition of NFκB production. HSR-609 The pharmacological characterization of a novel amphoteric anti-allergic compound, HSR-609 (Hokuriku Seiyaku; Figure 5), was presented by Y Hiraoka (Hokuriku Seiyaku, Japan). This selective H1 receptor antagonist was shown in saturation binding experiments to significantly reduce Bmax values when the membrane fraction from guinea pig cerebral cortex was pre-incubated with HSR-609, before Figure 5. Anti-inflammatory and anti-allergy drugs OH N N O O H N N N F N F N O HSR-609 (Hokuriku Seiyaku) FR-167653 (Fujisawa) O 62 IDrugs 1998 Vol 1 No 1 addition of tritiated pyrilamine. HSR-609 was shown to be an insurmountable antagonist due to its slow dissociation from H1 receptors. FR-167653 M Kawamura et al (Kitasato University School of Medicine, Japan) presented their results for the suppression of COX-2 expression by FR-167653 (Fujisawa; Figure 5) in a rat model of carrageenin-induced pleurisy. This IL-1 and TNF synthesis inhibitor at a dose of 30 mg/kg was shown to suppress the expression of COX-2 and the accumulation of pleural exudate 5 h after injection of carrageenin. KB-R7785 KB-R7785 (Kanebo) is a novel matrix metalloproteinase inhibitor and its antidiabetic effects were presented by N Nishimura et al (Kanebo KK, Japan). It was shown to inhibit TNF-α release from LPS-stimulated THP-1 cells in a dosedependent manner, with an IC50 of 2 µM. Subcutaneous administration for 5 weeks at 100 and 200 mg/kg/day, resulted in a significant reduction in plasma levels of glucose, insulin, triglycerides, total cholesterol and creatinine. These data suggest that inhibition of TNF-α release caused by KB-R7785 may be useful in the therapy of noninsulin dependent diabetes mellitus. XT-611 The effects of a novel PDE IV inhibitor, XT-611, on osteoclast and osteoblast formation were reported by K Miyamoto et al (Kanzawa University, Japan). This is a selective isozyme inhibitor, which relaxed tracheal smooth muscle without affecting the heart in guinea pigs, did not cause emesis in Suncus murinus at doses of 10 to 100 mg/kg po, unlike other known PDE IV inhibitors such as rolipram (Schering) and denbufylline (SmithKline Beecham) at doses of 10 to 30 mg/kg. XT-611 may be a good therapeutic candidate for the treatment of osteoporosis due to its side-effect profile. Antisense therapy PA-as and PB2-as T Mizuta et al (Rational Drug Design Laboratories, Japan) reported their findings for two antisense phosphorothioate oligonucleotides, PA-as and PB2-as. These were shown to be potent inhibitors of influenza A virus replication in vitro and were tested in a mouse model of respiratory tract infection with this virus. Treatment of mice with a liposomally encapsulated PB2-as 1 day before infection and 1 and 3 days post-infection significantly prolonged the mean survival time and increased the mean survival rate in a dosedependent manner. Meeting Report Prostate Cancer Conference 63 Prostate Cancer - CHI Conference Advances in Understanding Diagnostics and Therapy 23-24 March 1998, McLean, Virginia, USA Paul B Fisher Address Departments of Pathology and Urology Columbia University College of Physicians and Surgeons New York NY 10032 USA IDrugs 1998 1(1):63-66 © Current Drugs Ltd ISSN 1369-7056 As the title indicates, the purpose of this meeting was to highlight new information relative to the etiology, diagnosis and potential therapy of prostate cancer. Prostate cancer is the most prevalent internal cancer affecting men in the USA and the second most frequent cause of cancer-related deaths. Ongoing studies are identifying potentially important genes and target molecules involved in prostate cancer. These reagents will permit improved diagnostics and offer the promise of enhanced therapeutic targets for this cancer. The meeting was divided into four general areas. Approximately 70 scientists representing academic, governmental and industrial sectors attended this meeting. Keynote address Mr S Leslie Misrock (Pennie & Edmonds, New York, USA) discussed his perspective as a survivor of prostate cancer. He also presented an overview of Prostagen, a company designed to understand prostate cancer and to improve the diagnosis and therapy of prostate cancer. An additional aim of Prostagen is to define strategies to prevent the development of prostate cancer. Genetics of prostate cancer The chairman of this session was Dr Paul B Fisher (Columbia University, College of Physicians and Surgeons, New York, USA). Studies relating to familial prostate cancer in Utah kindreds were discussed by Dr Sean V Tavtigian (Myriad Genetics Inc, Utah, USA) and Dr Lisa Cannon-Albright (University of Utah, USA). Evidence was presented documenting an association between specific regions of chromosome 1q and familial prostate cancer susceptibility. Linkage to HPC1, a region of chromosome 1 previously shown to contain a prostate cancer susceptibility locus, as well as additional regions of chromosome 1q, was found to correlate with increased incidence of prostate cancer in specific Utah kindreds. Evidence indicating an additional region of chromosome 1q (42.2-43) that predisposes to early onset of prostate cancer in specific families was presented by Dr Philippe Berthon (UroGene, France). These studies suggest that multiple regions in addition to HPC1 may contain potential susceptibility loci for prostate cancer, and these regions may differ for specific subset populations. The overall strategy being used by Human Genome Sciences to identify genes was described by Dr Kenneth C Carter (Human Genome Sciences, Maryland, USA). This includes the construction of cDNA libraries, followed by highthroughput screening to identify and clone candi-date genes, followed by labor intensive screening for biological activity. Sequence information has now been generated for 1.4 million human cDNAs, expressed sequence tags (ESTs), including 40% known human genes, 20% genes similar to previously identified genes (or gene families), 10% common sequences and approxi-mately 30% novel ESTs. A detailed discussion was presented on one original EST, identified by cDNA library screening that is differentially expressed in prostate cancer, prostatic acid phosphatase. A modified differential RNA display approach, restriction enzyme analysis of differentially expressed sequences (READS), for identifying differentially expressed genes was reviewed by Dr William Munger (Gene Logic Inc, Maryland, USA). Although not presently documented, it was suggested that this approach could be applied to prostate cancer permitting the quantification of active genes (known and unknown) in a prostate tissue sample. Functional approaches for identifying and cloning human oncogenes and tumor-associated genes with specific application to prostate cancer were described by Dr Fisher. Using a rapid expression cloning strategy (RExCS) combined with differential RNA display, a novel oncogene, prostate tumor inducing gene-1 (PTI-1) was identified. PTI-1 is able to detect one prostate cancer cell in 100 million normal cells and has successfully identified patients with metastatic prostate cancer. In addition, inhibition of PTI-1 expression induces a reversion in cancer-related properties. A second approach, surface-epitope masking (SEM), has resulted in the production of monoclonal antibodies reacting with prostate cancer and the identification of prostate carcinoma tumor antigen gene-1 (PCTA-1). PCTA-1 is expressed in prostate cancer and high grade prostatic intraepithelial neoplasia (PIN) but not in lowgrade PIN, benign prostatic hypertrophy (BPH) or normal prostate. PCTA-1 expression is also a sensitive indicator of potential metastatic prostate cancer cells in the circulation of patients. These two genes provide useful diagnostic markers for staging prostate cancer and potential targets for prostate cancer therapy. Moreover, the approaches of RExCS and SEM represent valuable methodologies for identifying genes relevant to human cancer. Studies designed to develop suitable animal model systems that replicate distinct steps in prostate cancer progression were described by Dr Charles L Sawyers (UCLA School of Medicine, USA). Xenografting approaches have been used to develop models for prostate cancer metastasis and progression to androgen independence. Cells displaying specific stages of diseases are being investigated in an attempt to 64 IDrugs 1998 Vol 1 No 1 identify candidate genes involved in prostate cancer progression. One candidate gene associated with prostate cancer progression, ie, androgen-independent growth, was identified using a cDNA library (retrovirus) transfer approach. This gene encodes the cathepsin D protease and its potential role in prostate cancer is currently under investigation. Diagnostic and prognostic markers of prostate cancer The chairman of this session was Dr Jeffrey Ross (Albany Medical College, New York, USA). Novel amplification methods for immuno- and gene-based diagnostics were described by Dr David Ward (Yale University, Connecticut, USA). By using Padlock probes of defined sequence and the rolling circle amplification (RCA) technique, a sensitive and selective procedure has been developed for detecting molecules present at exceedingly low levels in a complex mixture. These approaches can be performed in solution and with immobilized targets and DNA arrays. Applications of Padlock probes and the RCA strategy include potential mutation detection, allele discrimination analyses, single molecule detection of antigens immobilized on a glass surface and in situ analyses with cytological sections. This scheme offers significant promise for the detection of specific target sequences and proteins that may contribute to prostate and other cancers. Evidence that human glandular kallikrein (hK2) may be a valuable clinical diagnostic marker for prostate cancer was provided by Dr Donald J Tindall (Mayo Clinic, Minnesota, USA). hK2 shares important properties of organ localization and hormonal regulation with prostate-specific antigen (PSA), a major diagnostic protein used in staging prostate cancer. Monoclonal antibodies have been developed that show less than 0.5% cross-reactivity with PSA. Analysis of 257 radical prostatectomy samples indicated a higher percentage of cells staining positive for hK2 in more advanced stages of cancer. In this context, its staining pattern was opposite to that of PSA and indicates that hK2 may provide useful diagnostic applications for staging prostate cancer. An approach to prostate cancer diagnosis and prognosis testing, called Biostage, was described by Dr Stephen Strum (Cancer Research Institute, Healing Touch Oncology, California, USA). This scheme involves the use of multiple parameters, including measurements of angiogenesis, to predict pathological stage in prostate cancer patients. Although further studies are required, this information could prove useful as part of a systematic approach, including CT scans, monitoring PSA levels and in more accurately staging prostate cancer. Emerging genetic markers, her-2/neu and glutathione Stransferase-pi (GST-pi), that may prove useful in prostate cancer detection and prediction of disease outcome were discussed by Dr Jeffrey S Ross (Albany College of Medicine, New York, USA). A fluorescence in situ hybridi-zation (FISH) approach was used to detect her-2/neu gene amplification indicating an adverse relationship between amplification and prognosis. In approximately 75% of nodal metastases gene amplification or protein over-expression was present. Amplification of her-2/neu by FISH independently predicts disease recurrence after primary radiation therapy. Using a second marker, GST-pi, hypermethylation of this gene was apparent in 52 of 57 prostate cancers. Immune-based approaches in prostate cancer Dr Michael I Sherman (Prostagen Inc, New Jersey, USA) chaired this session on immune-based approaches to the therapy of prostate cancer. An antibody targeting approach for solid tumors was described by Dr Paul G Abrams (NeoRx Corporation, Washington, USA). Avicidin (an antibody containing a receptor) delivers radiation to primary and metastatic disease by means of a Pretarget technology. Using this approach, early phase I trials indicate efficacy against metastasis in a subset of patients with prostate cancers who have failed standard therapies. Further studies with larger patient samplings are planned. An overview of cancer vaccines was provided by Dr Lynn E Spitler (Jenner Biotherapies Inc, California, USA). Dr Spitler also presented the results of phase I/II trials using OncovaxP containing a recombinant PSA formulated in liposomes, with lipid A as adjuvant. The results of four phase I/II trials have demonstrated safety and the ability to induce immune responses. On the basis of these encouraging results, expanded phase II trials and a phase III trial are anticipated. Recent studies using immunotherapy for prostate cancer based upon prostate-specific membrane antigen (PSMA) was discussed by Dr Michael L Salgaller (Northwest Biotherapeutics, Washington, USA). Dendritic cells were isolated from patients, pulsed with PMSA and readministered to patients (with and without granulocyte-macrophage colony stimulating factor). Of the patients (n = 107) with hormone refractory metastatic prostate cancer, 27% were classified as partial responders. In contrast, 33% of patients showed no change in disease status, 39% underwent disease progression and 21% of the test patients died. Dendritic cell immunotherapy for prostate cancer was the subject of a talk by Dr Frank H Valone (Dendreon Corp, California, USA). As outlined by Dr Salgaller, dendritic precursor cells can be isolated from patients, induced to mature with antigen in vivo (in this case prostatic acid phosphatase) and then infused back into patients 48 h later to stimulate an antigen-specific T-cell response in vivo. A phase I/II trial of immunotherapy with APC-8015 in patients with advanced, hormone-refractory prostate cancer was promising. This study demonstrated that APC-8015 is safe, immunologically active (T-cell responses in 12 of 12 patients, T-helper 1 response and evidence of a dose response) and signs of biological efficacy on the tumors. However, the studies are not suficiently advanced to assess clinical benefit. Studies using a recombinant replication competent adenovirus containing a PSA response element for treatment of prostate cancer were described by Dr Daniel R Henderson (Calydon Inc, California, USA). Virus replication was demonstrated in cells expressing PSA. A single injection Meeting Report Prostate Cancer Conference discussed by Dr Robert G Croy (MIT, USA). Compounds have been developed that exploit molecular features in prostate cancers, such as the expression of the androgen receptor to disable cellular defenses against DNA damage. By blocking repair of genotoxic damage, lethality in tumor cells is increased while sparing normal tissues. It is predicted that these new compounds should produce greater therapeutic benefit and fewer side-effects than conventional cytotoxic chemotherapy. Although further research is clearly necessary, including studies in animals, if successful, this strategy might prove of value for prostate therapy. of CN-706 (adenovirus containing the PSA response element driving the adenovirus E1A and E1B genes resulting in viral replication) into the human prostate cancer cell line LNCaP (which is PSA positive) inhibited tumor growth in vivo in nude mice. Phase I trials with patients with recurrent, locally advanced prostate cancer will be initiated shortly. Dr Lily Wu (UCLA School of Medicine, USA) provided additional documentation of the potential use of adenovirus vectors containing prostate-specific promoter sequences to target viral replication and cell lysis. Specific viral constructs have been engineered, using the prostate-specific PSA enhancer and promoter region, to produce specific therapeutic gene products, including IL-2/TNF-α, p53, Rb, thymidine kinase and antisense TGF-β, in PSA expressing prostate cancer cells. Efficacy of engineered viruses for targeting gene expression in cells expressing PSA was documented. Studies of the apoptosis-inducing drug, FGN-1 (sulindac sulfone), that is being developed as a pharmaceutical for the primary prevention of cancers was described by Dr Gary A Piazza (Cell Pathways Inc, Pennsylvania, USA). Studies have been conducted to evaluate the effect of FGN-1 in a number of in vitro and in vivo preclinical models to assess the extent to which it induces apoptosis and prevents the development of neoplasia, as well as to elucidate mechanism of action. Preliminary evidence suggests potential efficacy of FGN-1 in experimental model systems. Clinical trials are planned for determining the effect of FGN-1 on premalignant lesions and malignancy recurrence. Small molecule and other therapies for prostate cancer The chair of this session was Dr William D Figg (National Cancer Institute, Washington DC, USA). A novel technology for improving the utility of chemotherapy, with potential application to prostate cancer, was Figure 1. Angiogenesis inhibitors O O O HO S O OH S O O O O S S OH HO H3C O CH3 O O S HO S NH N H N H O O H N H N HN O O O Suramin (Warner-Lambert) H3C O OH H3C O CH3 H OH O N H NH O O S O S O TNP-470 (Takeda) NH O O O H N H2N CH3 Cl H N H2N H O O H N O O H N NH2 O NH N H H O N O HO CH3 H3C Somatuline (Ipsen-Beaufour) CH3 65 NH O O Thalidomide (Entremed) 66 IDrugs 1998 Vol 1 No 1 promoter region has been isolated, ligated to a luciferase reporter gene and stable cell constructs constitutively expressing luciferase have been generated. Cloned cells with the PEG-3 promoter/luciferase reporter construct are being used to screen several combinatorial small molecule libraries. This assay offers significant promise as a general screening method for the identification of compounds that inhibit both oncogene function and the progression phenotype. The activity of several angiogenesis inhibitors (suramin, Warner-Lambert Co; somatuline, Ipsen-Beaufour; pentosan, Ivax; TNP-470, Takeda Chemical Industry Ltd; carboxyamido-triazole, Merck and thalidomide, Entremed) in patients with androgen-independent prostate cancer, was evaluated by Dr William D Figg (National Cancer Institute, Maryland, USA). Activity was apparent with several of these agents; however, the duration of response was minimal in the heavily pretreated metastatic population. The present studies suggest that there may be greater potential if the angiogenesis inhibitors are evaluated in patients at earlier stages of disease progression. Conclusion Approaches for screening small molecule libraries for antagonists of prostate oncogenes and downstream pathway events were described by Dr Neil I Goldstein (GenQuest Inc, USA). A gene mediating cancer aggressiveness, progression elevated gene-3 (PEG-3), has been identified by subtraction hybridization. The biological effects of PEG-3 are downstream of multiple oncogenes, including Ha-ras, src, human papilloma virus type 18 and PTI-1. The PEG-3 This conference provided an updated and insightful overview of prostate cancer. The talks, in general, were of high quality and they were well received by the attendees. The use of a panel discussion format involving all of the speakers in each section permitted an active dialog between presenters and the audience. The organization of the meeting, including breaks that permitted interaction between the speakers and the attendees, resulted in very interactive and successful symposia. Meeting Report 99th Am Soc Clin Pharmacol Ther 67 American Society for Clinical Pharmacology and Therapeutics 99th Annual Meeting 30 March - 1 April 1998, New Orleans, Louisiana, USA Denise Morgan Address Current Drugs Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):67-72 © Current Drugs Ltd ISSN 1369-7056 This meeting was attended by over 1000 clinicians, and clinical pharmacologists from academia and industry. The three-day conference consisted of poster sessions, state-of-the-art lectures, plenary sessions, oral presentations, symposia and workshops. The Human Genome Project Genomic research and gene therapy are becoming increasingly popular strategies for the development of novel therapeutics. Based on the relationship between an individual’s genotype and phenotype, knowledge of the pathophysiology of disease at the gene level would enable one to ascertain differences in an individual’s symptoms, as well as differences in the pharmacokinetic and pharmacodynamic response to a drug (eg, changes in efficacy and increasing incidence of adverse events). Professor Richard Myers (Stanford Human Genome Center, CA, USA) opened the meeting by presenting a lecture on the progress of the Human Genome Project. Established in 1990, the Stanford Human Genome Center has built maps of human chromosome 4, to a 500 kb resolution, using a panel of 83 radiation hybrids and more than 10,000 sequencetagged sites. The center is actively involved in the Human Genome Project, which will determine the complete sequence of at least 17.5 million base pairs of human DNA using a directed sequencing strategy, in the hope of identifying genes and obtaining information on gene expression and function, gene-gene interaction and genomic regulation, in order to use this knowledge to advance and improve the drug discovery pharmaceutical process. As Chairman of the Human Genetics and Drug Therapy symposium, Dr B Michael Silber (Groton, CT, USA) suggested that mapping of the human genome will be completed by 1999, with complete sequences available by 2000/2001. Utilization of genomic research and the candidate gene approach to pharmaceutical development has already begun in select pharmaceutical companies and routine use of this approach is anticipated by 2000. The consensus within the pharmaceutical industry is that the use of genomics in drug development is a great step forward, leading to an optimization of the drug discovery process through retrospective analysis of discovery targets, a change in clinical trial design and interpretation, an impact on product labeling, faster regulatory approval, early diagnosis and estimation of disease risk, and quicker decisions regarding patient care. Dr Silber emphasized that problems do exist with this approach to drug development and such major concerns include: the notion that not all patients with the same genetic indiscrepancy will have the same disease (eg, differences may occur due to environmental factors, such as the duration of disease); misinterpretation of such information by the regulatory authorities and improper use by industry; the lack of regulation over validation procedures and the maintenance of confidentiality of DNA samples held in databases and DNA banks, which are already being developed by many pharmaceutical companies. Novel approaches to the prevention and treatment of acute renal failure Acute renal failure (ARF) is a complication of 10% of hospital administrations, 30% of renal allografts and 20% of abdominal aneurysm repairs. As a result, the pharmaceu-tical industry has begun looking for novel ways of treating ARF and improving renal function in these patients. Growth factors An overview of the use of growth factors, in particular, insulin-like growth factor-1 (IGF-1), for the treatment of ARF was presented by Dr Steven Miller (Washington University School of Medicine, St Louis, MO, USA). The mechanism of action of the IGFs is yet to be fully elucidated but mitogenic, hemodynamic, cytoprotective, apoptotic and cytokine modulatory actions are known to play an important role. Various companies are involved in the development of IGF-1 products for the potential treatment of ARF, plus a number of other conditions including osteoporosis, cachexia and metabolic disorders, neuropathies, neurodegenerative disease and diabetes; several clinical trials have been successfully completed with IGF-1 for the prevention of renal dysfunction. Other growth factors being investigated for the potential treatment of ARF include HGF (hepatocyte growth factor), EGF (epidermal growth factor), OP-1 (osteogenic protein-1, also termed BMP-7), HB-EGF (heparin binding epidermal growth factor) and α-MSH (a tridecapeptide derived from the post-tranlational processing). Dr Miller presented data from studies conducted in a rodent model of ARF involving 36 animals treated with IGF-1 and 7 treated with placebo. Results showed that IGF-1 reduces serum creatine levels and causes an increase in body weight via an anabolic mechanism. Mortality was decreased to 7.3% in those animals treated with IGF-1 compared to 36.7% in the placebo group. One other potential use of IGF-1 seems to be as a reversal agent for the ischemia caused by ARF. This was demonstrated in a randomized, blinded, placebo- 68 IDrugs 1998 Vol 1 No 1 Figure 1. Adenosine receptor antagonists H O O H3C O H N N H3C H N N NH O N N O N N H H N N H3C H3C N N CH3 KW-3902 (Kyowa Hakko Kogyo) CVT-124 (University of Florida) controlled study of ischemia in a canine model, where ischemia resulted from removal of both kidneys, one of which was subsequently stored in IGF-1 (n=8) or placebo (n=8), followed by re-implantation of either an IGF-1- or placebo-stored kidney. In this model, serum creatine levels were reduced by approximately 50% over a 6-day period. Adenosine receptor antagonists The merits of adenosine A1 receptor antagonism for the treatment of ARF were discussed by Dr George F Schreiner (Scios, Sunnyvale, CA, USA). Selective adenosine A1 receptor blockade reverses cAMP inhibition in the renal vasculature resulting in an increase in renal blood flow and GFR and a reduction in ischemia induced by secondary vasocon-striction. The resulting vasodilation enables the kidney to remove nephrotoxins and reduces the possibility of precipitation and reabsorption of these toxins in the proximal tubule. Dr Schreiner referred to four adenosine A1 receptor antagonists currently in development; FR-166124 (Fujisawa) and KW-3902 (Kyowa Hakko Kogyo; Figure 1), both for the treatment of ARF, CVT-124 (University of Florida; Figure 1) for the treatment of congestive heart failure (CHF), ARF and edema, and N-0861 (Discovery Therapeutics; Figure 1) being developed for the treatment of CHF and arrhythmia. All these compounds are in phase II clinical trials. The endothelin pathway, ARF and cardiovascular disease Drs David Brooks (SmithKline Beecham, PA, USA) and Michael R Goldberg (Merck, PA, USA) discussed endothelin (ET) receptor antagonism and ET converting enzyme (ECE) inhibition. Both of these points in the ET pathway are amenable to drug intervention and are under investigation for the potential treatment of ARF, pulmonary and essential hypertension, CHF and benign prostatic hyperplasia. Endothelin receptor antagonists Stimulation of ETA and ETB receptor subtypes, located in the kidney, is responsible for the regulation of renal blood flow, naturesis (via a reduction in GFR mediated by the ETA receptor), renal secretion and downregulation of the renin- N-0861 (UCB) angiotensin-aldosterone system. Stimulation of ET receptors is also responsible for ET-1 mediated vasodilation and nitric oxide release and ET-3 mediated vasoconstriction (via ETB receptors located in the blood vessels). BQ-123 (Banyu, Figure 2), a selective peptidic ETA antagonist, causes an increase in forearm blood flow and a vasodilatory response in CHF when given as an iv infusion, starting with a loading dose of 1 µg/kg/min. Chronic dosing of BQ-123 in a rat model also blocks the dilation and hypertrophy which results in left ventricular remodeling, a prognostic marker of CHF. Takeda Chemical Industries Ltd is developing TAK-044 (Figure 2), a mixed ETA and ETB antagonist, which lowers diastolic blood pressure and plasma ET at 0.15 to 10 µg/kg over a 15 min iv infusion. Roche is conducting phase III CHF clinical trials with bosentan (Figure 2) which has Ki values for ETA and ETB of 15 and 102 nm, respectively. SB-209670 (SmithKline Beecham, Figure 2), a mixed, non-peptidic antagonist with Ki values for ETA and ETB of 0.4 and 18 nM, respectively, reduces both systolic and diastolic blood pressure and causes a reflex tachycardia in patients with mild to moderate hypertension. Endothelin converting enzyme inhibitors Novartis AG is developing CGS-26303 (Figure 3), the first non-peptidic inhibitor of ECE and neutral endopeptidase 24.11, for the potential treatment of hypertension, cerebral vasospasm and renal failure. Three analogs of CGS-26303 (CGS-24592, CGS-31447 (Figure 3) and CGS-36112) are in the discovery phase of development. All four compounds have differential selectivity for ECE and neutral endopeptidase. In the spontaneous hypertensive rat model, CGS-26303 reduces blood pressure by 15 to 20 mmHg (an effect which remains for approximately 2 weeks after the initial dose). In a rabbit model of cerebral vasospasm, treatment with CGS26303 significantly increases vasodilation of the basilar artery and reduces arterial hyperplasia. CGS-26303 also causes a significant reduction (32%) in neointimal formation following angioplasty. Furthermore, in a puromycininduced model of nephropathy, sc injection of CGS-26303, 1 Meeting Report 99th Am Soc Clin Pharmacol Ther Figure 2. Endothelin receptor antagonists CH3 O H3C O Na Na O H3C O O CH3 O H N N NH O N H N H N H HN N OH O N H N H O O O O OH N O HN H N N H HN O H3C CH3 O S OH TAK-044 (Takeda) BQ-123 (Banyu) O CH3 HO O H3C OH O O H3C O O O N OH N S N N H H3C H3C O O O N O CH3 O Bosentan (Roche) SB-209670 (SmithKline Beecham) Figure 3. Endothelin converting enzyme inhibitors HO P HO N O N H N N N H CGS-26303 (Novartis) HO P HO N O N H N N N H CGS-31447 (Novartis) 69 70 IDrugs 1998 Vol 1 No 1 h after an iv bolus of puromycin, resulted in a modest reversal of puromycin aminoglycoside-induced injury. Chemoprevention - NSAIDs and colon cancer Dr Randall W Burt (University of Utah School of Medicine, Salt Lake City, USA) conducted a workshop on colon cancer and chemoprevention. The incidence of colon cancer is rapidly increasing, with approximately 131,000 new cases and 55,000 deaths reported in the US in 1997. Colon cancer arises due to either a genetic predisposition or a series of acquired mutations and, therefore, is an excellent candidate for chemoprevention. Dr Burt discussed the use of NSAIDs to aid in the regression and prevention of colon cancer in those patients with a genetic predisposition, mediated via the presence of the APC gene on the long arm of chromosome five. This phenomenon was first discovered in 1980, when animal studies in a rat tumor model showed that indocin inhibited carcinogenesis in a dose- and duration-dependent fashion via the blockade of cyclooxygenase and cell proliferation, and stimulation of apoptosis and immune surveillance. Dr Burt further discussed the use of, and current clinical trials in, FGN-1 (sulindac sulfone, Cell Pathways), celecoxib (Searle; Figure 4) and acetyl salicylic acid for the prevention and regression of familial adenomatous polyposis. Figure 4. Celecoxib O S NH2 O H3C N N F F F Celecoxib (Searle) Public policy forum - science and policy issues of tobacco addiction Nicotine addiction is a widespread problem throughout the Western world with approximately 3000 people a day becoming regular smokers, of which, 70% will express the desire to quit. The industry conceives cigarettes as a whole package, with nicotine being the product, the cigarette pack acting as the storage container for a day’s supply, the puff being the vehicle for nicotine delivery and the cigarette as an optimized dispenser for drug delivery. The American tobacco industry has declared that nicotine is used in cigarettes purely for taste, and not for pharmacological effect, but patents are available for the use of organic acids which have been developed to mask the taste of nicotine. In 1982, a study completed by the Federal Trade Commission demonstrated that nicotine levels in cigarettes had increased while tar levels had dropped, fueling suspicion that the tobacco industry recognized that nicotine exerted an intended pharma-cological effect. This study prompted the FDA to consider its position concerning the industry and initiate an investigation into the tobacco industry. Evidence compiled during the FDA’s investigation proved that the tobacco industry paid close attention to the quality and quantity of nicotine in the cigarette blend at every stage of manufacture. Adequate delivery of nicotine and the requirement of a high nicotine content when choosing the tobacco leaf have been, and still are, primary objectives in the development of the final product. Chemical manipulation of tobacco delivery has been used by the industry to liberate and increase the levels of free nicotine in the blend, thereby increasing the tobacco content and increasing the level of addiction. Other evidence produced during this investigation included internal memos and studies, and in particular, one project conducted by a company that was designed to find a non-nicotine product that would exert the same psychomotor effects in the advent that nicotine was removed from the market. In April 1997, after the investigation had produced sufficient evidence, the Federal District Court in Greensboro, NC, ruled that the FDA had jurisdiction under the Federal Food, Drug and Cosmetic Act, to regulate nicotine-containing cigarettes and smokeless tobacco products. Since gaining jurisdiction over the tobacco industry, the FDA has begun to implement a strategic plan, focused on the younger generation, which will reduce access to tobacco products, restrict the availability of vending machines selling these products, reduce the appeal of smoking to children via the toning down of advertising and banning of advertising around schools and by enforcing age and photographic identification restrictions on people under the age of 27 years. The FDA is currently contracting with state officials in order to police this plan. To date, approximately 12 states have joined the campaign and by the end of 1998, the FDA hopes to have implemented its plan in all US states. Only the age restrictions are being enforced at this time and compliance checks have already been initiated in participating states. With a budget of $34 million, and a planned budget of $134 million for 1999, a total of 7000 inspections have already taken place and this is hoped to increase to approximately 100,000 by the end of this year. The FDA has set up an internet site at http://www.fda.gov to increase awareness of its policies concerning this issue. Poster highlights Clinical studies Eli Lilly & Co presented a parallel, randomized, doubleblind study of LY-303870 (lanepitant; Figure 5), a selective NK-1 antagonist, conducted in 214 outpatients with moderate to severe lower limb osteoarthritis pain. Patients were randomized to an initial loading dose of 20, 60, 200 or 600 mg LY-303870, placebo, or 375 mg naproxen, followed by 10 (n=37), 30 (n=35), 100 (n=34) or 300 (n=36) mg LY303870 bid or naproxen 375 mg bid. The study compared Meeting Report 99th Am Soc Clin Pharmacol Ther 71 Figure 5. Drugs from poster presentations H N CH3 O H3C O O O CH3 H3C N N S CH3 NH N O CH3 N H O S N HBY-097 (Hoechst Marion Roussel) LY-303870 (Eli Lilly) Cl N H3C OH N O Br N O H N N N N CI-1007 (Parke-Davis) GR-117289 (Glaxo Wellcome) H3C H3C O N O O N NH2 LY-300164 (Eli Lilly) pain intensity and relief, patient global impression scores, adjunctive analgesic use and safety assessment between the three groups. There were no statistically significant differences for the initial dose assessment, however, naproxentreated patients had significantly less pain and used less adjunctive analgesics than the other two groups during the multiple-dose period. Although safely administered, both active drugs were associated with gastrointestinal sideeffects including diarrhea and gastric discomfort. Pharmacokinetic studies HBY-097 (talviraline, Hoechst-Roussel; Figure 5), a nonnucleoside reverse transcriptase inhibitor, and indinavir (Merck & Co) are metabolized via the same metabolic path- 72 IDrugs 1998 Vol 1 No 1 way (CYP3A4 metabolism) and may be used in combination therapy with zidovudine. Hoechst AG, Bayer AG and the UCSF presented a multiple-dose pharmacokinetic (PK) study of HBY-097, when given in combination with indinavir and zidovudine, in order to evaluate any potential drug interactions. Results showed that HBY-097 induces CYP3A4 metabolism of indinavir thus indicating a need for dose adjustments when both these drugs are used concomitantly. UCSF also presented a second poster, in collaboration with Aronex, of a PK study of AR-177 (Zintevir). AR-177, a synthetic oligonucleotide HIV integrase inhibitor, is more effective than reverse transcriptase inhibitors in the postinfected, acute phase of HIV, but less effective in the chronic or latent phase (as it does not affect gene transcription, protein synthesis, viral assembly or the HIV protease enzyme). The PK study investigated the single-dose kinetics of AR-177 in 16 asymptomatic HIV positive subjects, who received one of four doses (0.75, 1.5, 3 or 6 mg/kg) administered via a 2-h iv infusion. Results of the study indicated that AR-177 exhibits complex pharma-cokinetics with a trend towards decreasing plasma clearance with increasing dose. A multiple-dose study is ongoing to clarify the disposition of AR-177. The Center for Human Drug Research, supported by Glaxo Wellcome plc, reported on a completed PK study of GR117289 (Figure 5), an angiotensin II receptor antagonist which was being developed by Glaxo Wellcome for the treatment of hypertension (but has now been discontinued). The placebo-controlled, rising-dose trial was conducted in 19 male patients with a diastolic blood pressure (DBP) between 100-115 mmHg. Following an iv dose of up to 30 mg, regular measurement of BP, plasma renin and aldosterone took place and treatments were compared using the time-averaged values over a 12-h (for DBP) or 8-h period (renin activity and aldosterone). The study concluded that GR-117289 did not alter DBP in hypertensive patients at any given dose, while dose-dependent increases in renin activity and decreases in aldosterone were noted. The main adverse event recorded during the study was headache, with two patients withdrawning from the study for this reason. Parke-Davis & Co displayed a report of three PK studies conducted with CI-1007 (Figure 5), a dopamine D2 and D3 agonist with mild extrapyrimidal side-effects, under development for the potential treatment of psychosis and schizophrenia. The first study was a placebo-controlled, cross-over trial designed to evaluate the effects of CI-1007 as a single, oral dose of 0.5, 1, 2.5, 5, 10 or 15 mg (four subjects/dose group) plus placebo. The second study was a parallel group, double-blind, dose-escalation trial conducted in six healthy volunteers and the third study evaluated CI- 1007 (5, 10 and 15 mg doses) in 16 schizophrenic patients. The first study was terminated at the 15 mg dose due to adverse events; an increase in growth hormone and decrease in serum prolactin levels was observed in all three trials. A safety and tolerability study in CYP2D6 poor metabolizers is ongoing. Results of two single- and multiple-dosing PK studies of LY300164 (Eli Lilly), an orally available, potent, selective AMPA receptor antagonist for the treatment of epilepsy were also presented at the meeting. The two studies represent the first experience of LY-300164 in healthy subjects. A total of 24 healthy males received a single dose of up to 100 mg and 16 male subjects received multiple doses of 20 mg tid, or escalating doses of 30 to 60 mg tid for ten days. Adverse side-effects occurred at doses above 50 mg and included mild drowsiness (subjects showed a decrease in awareness 2.5 h after dosing, as measured by the Bond Lader Mood Rating Scale), dizziness, ataxia and paresthesiae. However, LY-300164 was shown to be safe with respect to all of the biochemical indices measured. Bristol-Myers Squibb unveiled the new potent, metabolically-stable, direct thrombin inhibitor, BMS-189664, which is being investigated for the prevention of deep venous thrombosis, venous thrombosis following hip and knee surgery and prevention of stroke in patients with atrial fibrillation. Intravenous infusion of BMS-189664 has been shown to be effective in rat and monkey models of venous and arterial thrombosis and platelet-dependent arterial thrombosis, respectively. Doses sufficient to inhibit arterial thrombosis can enable dissolution of preformed venous clots in rats without increasing bleeding time. In the randomized, double-blind, placebo-controlled clinical study conducted in 63 healthy subjects, clinically relevant prolongations in aPTT were observed after iv administration and oral doses above 1000 mg. The drug was well-tolerated and adverse events were seen in only four patients; these included headache, diarrhea, gum bleeding, an upper respiratory tract infection and forearm blister. Conclusion The diverse array of topics presented at the lectures, plenary sessions and symposium resulted in a meeting that was both informative and stimulating for those who attended. The organizers successfully managed to bring together representatives from academia and industry to present both new and established research which encompassed subjects from the entire discipline of clinical pharmacology. The 100th Annual meeting will be held in March 1999 in San Antonio, Texas. Information regarding ASCPT can be found on the world wide web at http://www.ascpt.org. Review Neurokinin antagonists 73 Neurokinin antagonists as potential therapies for inflammation and rheumatoid arthritis Andreas von Sprecher1, Marc Gerspacher2 and Gary P Anderson3 Addresses 1 Novartis Pharma AG K-136.3.93 CH-4002 Basel Switzerland pain, migraine, emesis, cancer and psychiatric disorders such as anxiety and schizophrenia [8-10]. This review focuses on the clinical potential of NK antagonists in inflammatory diseases, especially rheumatoid arthritis. 2 Rheumatoid arthritis Novartis Pharma AG K-136.3.25 CH-4002 Basel Switzerland 3 Department of Pharmacology University of Melbourne Parkville, 3052 VIC Australia IDrugs 1998 1(1):73-91 Current Drugs Ltd ISSN 1369-7056 Introduction Inflammatory diseases are major global causes of morbidity and mortality [1]. The importance of inflammatory disease has stimulated intense research into their pathogenic mechanisms and better treatment strategies [2]. Considerable evidence suggests that the very common inflammatory diseases, rheumatoid arthritis and asthma, are the consequence of inappropriate T-cell-mediated immunological responses that trigger secondary effector mechanisms, such as induction of growth factors or proteases that permanently alter tissue [3,4]. An emerging but controversial body of evidence suggests that neurokinins may play an important role in disease induction and progression. Neurokinins (NKs or tachykinins) are the most important of the peptides released from neuronal sensory afferents. They form a family of peptides which share the common C-terminal sequence Phe-X-Gly-Leu-Met-NH2 [5]. In mammals, three different NKs, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), act as neurotransmitters and neuromodulators [5,6]. SP and NKA are products of the same gene (preprotachykinin I), which is expressed both in the central (CNS) and peripheral nervous systems [5], whilst NKB is produced by a distinct gene (preprotachykinin II), which is selectively expressed in the CNS [5]. Based on the different rank order of potency of natural NKs, three distinct NK receptor types have been identified, which have been termed NK1 (SP-preferring), NK2 (NKA-preferring), and NK3 (NKB-preferring) [7]. The existence of these three receptors has been supported by the development of selective receptor agonists (synthetic ligands), the development of receptorselective competitive antagonists, and, ultimately, the isolation and cloning of the three receptor proteins [6]. With few exceptions, the expression of the NK3 receptor is confined to the CNS, whilst NK1 and NK2 receptors are widely distributed throughout the CNS and peripheral nervous systems [7]. NK antagonists have been proposed to have potential utility in a wide range of pathological conditions, including inflammatory processes such as asthma, rheumatoid arthritis and inflammatory bowel disease (colitis), The synovium is richly innervated in all species, including humans, by sensory nerves [11-13]. A subpopulation of the sensory nerves, the vagal C-fibers, and a minor population of spinal Aδ fibers [14], contain a number of neuropeptides including NKs and calcitonin gene-related peptide (CGRP). The physiological function of neuropeptides in the sensory nerve fiber endings is unknown, but it is established that activation of the sensory nerves leads to both afferent signals to the CNS and to the local release of neuropeptides within the synovium. This local release of neuropeptides causes an acute inflammatory response, termed neurogenic inflammation, characterized by vasodilation, microvascular leakage, recruitment of inflammatory leukocytes, promotion of histamine release from mast cells and the release of cytokines from the invading leukocytes [15]. Growth factors, such as leukemia inhibitor factor (LIF) and nerve growth factor (NGF) [16] are implicated in the proliferation of NKs-containing nerves and in the induction of NK genes [11]. It is possible that these growth factors may also directly stimulate non-neuronal tissues to produce NKs [16]. Indeed, in chronic disease, non-neuronal cells, such as tissue macrophages and epidermal cells, may be the major sources of NKs that drive disease [16]. These effects of neuropeptides released during neurogenic inflammation are mediated through NK1 and NK2 receptors expressed by target cells in the joint and mimic several of the typical features of rheumatoid arthritis [14,17,18]. Whilst neuronal versus non-neuronal sources of neurokinins may vary in their contribution to disease, it is likely that neurogenic mechanisms may be involved in the development of symmetrical rheumatoid arthritis, where inflammation in one joint is often followed by inflammation and hyperalgesia in the contralateral joint [19]. Contralateral joint inflammation has been observed in a strict monoarthritis model where latex spheres were injected intraarticularly to induce disease: neuronal processes may be one form of communication between the joints in this model. This response was accompanied by a concurrent increase in SP-like immunoreactivity (SPLIR) content of both the ipsilateral and the contralateral dorsal root ganglia, and contralateral joint inflammation was attenuated by inhibition of small unmyelinated nerve activity. Similarly several groups [20-24] have demonstrated increased SP-ergic innervation of the inflamed synovium in experimental arthritis. Ahmed et al also demonstrated that whilst surgical denervation attenuated ipsilateral joint inflammation, it did not prevent it [25]. Interestingly denervation reduced, but did not prevent, increased levels of SP in the inflamed joint [25], strongly suggesting local non-neuronal production. In experimental arthritis, spinal SP content appears to peak early, perhaps reflecting acute hyperalgesia: Garret and colleagues [26] found 74 IDrugs 1998 Vol 1 No 1 È-preprotachykinin mRNA levels to be increased one day after induction of experimental joint inflammation (but not at day 10) and Malcangio and Bowery [27] found no increase in release of SP from spinal cord sections one week after induction of inflammation. These data suggest that increases in NK mRNA levels may be transient or subject to rapid modulation during exacerbations of disease. Conversely, Hanesch et al [28] used in situ hybridization to demonstrate that the proportion of dorsal horn cells expressing preprotachykinin A mRNA was increased, both acutely and chronically, in experimental monoarthritis. Few studies have addressed the role of neurokinins in human disease. Elevated levels of SP have been detected in human rheumatoid synovial fluid [29,30], at levels correlating with the proinflammatory cytokine interleukin (IL)-6 [31]. Anichini et al [32] found significantly higher serum SP levels in rheumatoid arthritis patients in comparison to healthy controls. Sacerdote et al [33] found that the elevated levels of SP in their patients decreased following therapy with non-steroidal antiinflammatory drugs (NSAIDs). Gold salt therapy has also been shown to reduce synovial SPLIR in human rheumatoid arthritis patients [34]. Pain NKs may prove to be important mediators of pain in inflamed joints. Early studies demonstrated that peptide concentrations, particularly of SP, but also of CGRP, NKA and NPY, increased in the regions of the dorsal horn processing afferent projections from affected joints in experimental models of rheumatoid- and osteoarthritis [3538]. More recent research suggests that hyperalgesia is accompanied not only by increases in SP content, but also by compensatory changes in NK receptor function and a possible failure of GABA-ergic inhibitory pathways to adequately suppress SP-ergic nociception [39]. The receptor subtype(s) mediating nociception remain uncertain, with evidence for the contibution of both NK1 [40] and NK2 [41] to hyperexcitability of dorsal horn neurons and pain. Effector mechanisms The acute effects of NKs are consistent with direct promotion of inflammation, causing at least short-term increases in leukocyte adhesion and diapedesis, plasma exudation, alterations in blood flow, and, probably, some contribution to hyperalgesia through release of secondary mediators. Despite the indications from animal models, it is not known if NKs contribute to more chronic inflammation. The proliferation of SP-ergic fibers is consistent with a role in long-term inflammation. SP promotes tissue-destroying superoxide and hypochlorous anions via an NK1 receptor-mediated mechanism in synoviocytes and tissue macrophages [42]. Macrophage induction of coagulation products and protease induction have also been demonstrated, at least in vitro. It seems possible that autocrine/paracrine production of SP by macrophages may mediate at least some of the disregulation of matrix metalloproteinase/tissue inhibitor of metalloproteinase imbalance that contributes substantially to pannus formation and joint degradation. Although SP is a weak coangiogenic factor in vitro, there is little compelling evidence to suggest that SP or NKA have an important role in altered microcirculation, endothelial cell phenotype or proliferation. SP is, however, almost certainly involved in reciprocal induction of the cytokines IL-1 and IL-6, which exert potent proinflammatory effects in joints [31,43]. The role of NKs in driving the underlying T-lymphocytemediated immune responses that cause chronic joint disease is uncertain. SP is a weak co-mitogen for human and animal T-cells, but there are, however, no firm reasons to believe that NKs fundamentally affect either primary T-cell response or committed T-cell effector function to a great extent. Furthermore, it is possible that SP may mediate at least part of its effects on lymphocytes by non-receptormediated direct G protein activation in a manner analogous to that occurring in human mast cells. One possibility that has important implications for therapy is that NKs may exert both pathogenic and protective effects. Bileviciute et al [44] used a model of immunoprophylaxis of adjuvant-induced arthritis to demonstrate that reduced disease severity was actually accompanied by increased SPLIR and NKA-like immunoreactivity at 24 h after injection of Freund’s complete adjuvant. Similarly, Garrett and coworkers [26] found that dorsal horn α-preprotachykinin increased acutely in experimental arthritis (24 h) and in the joint at 10 days following challenge. However, Carleson et al [45] have consistently demonstrated increased SPLIR in the perfused joint during sustained inflammation, whereas Jeanjean et al [46] showed that dexamethasone, a highly effective anti-inflammatory drug, decreased IL-1β-induced long-term increases in axonal SP transport suggesting, on balance, a proinflammatory effect. Conclusions Although the role of NKs in inflammatory disease conditions is not fully elucidated, there is growing evidence that neurokinins may play a pivotal role both in disease induction and in the establishment of chronic inflammatory disease. The advent of safe effective antagonists of NK receptors is likely to contribute important new knowledge of disease pathogenesis. Hopefully, these advances may lead to better therapies which are more specific and with fewer side-effects than the treatment with currently prescribed anti-arthritis drugs (NSAIDs and steroids). With the exception of Lilly’s lanepitant, which had no effect in clinical trials for the treatment of ostheoarthritic pain, there are no reports of NKs antagonists in clinical trials for the treatment of rheumatoid arthritis. It is too early to draw any conclusions; however, it can be speculated that non-selective (dual NK1/NK2) antagonists will be more suitable to treat inflammatory diseases than selective NK1 antagonists like lanepitant. Further work is needed to clarify whether additional blockade of the NK3 receptor and penetration of the blood-brain barrier can improve the antiinflammatory profile of NKs antagonists, or whether peripheral blockade of NK1 and NK2 receptors is preferable. With the emergence of dual NK1/NK2 antagonists and compounds able to unselectively block all three receptors these questions can be addressed. Neurokinin antagonists (Tables 1-8) (Comments on the compounds in the tables) Tables 1-8 depict the neurokinin antagonists currently under investigation. With the exception of some important compounds, this is restricted to non-peptide compounds, as Review Neurokinin antagonists peptides often lack oral bioavailability and metabolic stability, limiting their clinical usefulness. The list of compounds is by no means complete but, every effort was made to include representative compounds of each important structural class. The neurokinin antagonists are sorted firstly according to receptor selectivity and secondly according to the chemical structure. A tabular format has been chosen in order to provide quick access to the relevant in vitro (receptor binding) and in vivo (emphasis on inflammation and pain) data. The tables also contain available clinical data, state of development and the most likely indications. The comments section below does not repeat the references given in the tables. Selective NK1 antagonists (Tables 1-4) Quinuclidine- and piperidine- derivatives (Table 1) The first non-peptide NK1 antagonist CP-96345, a (2S,3S)-cis- 3(2-methoxybenzyl)-amino-2-benzhydryl-quinuclidine, was described by Pfizer in 1991. The first lead structure that was eventually developed into CP-96345 was identified by a random screen. CP-96345 is a highly active and selective, competitive, NK1 antagonist. Further modifications of the structure of CP-96345 led to the discovery of CP-99994, where the quinuclidine ring system has been replaced by a more simple piperidine ring and a phenyl substituent acts as a replacement for the benzhydryl residue. CP-99994 is highly active in vitro, but the compound suffers from poor oral bioavailability, short duration of action and L-type calcium channel interaction. Nevertheless, CP-99994 underwent clinical trials for asthma and pain. Whereas in the asthma trial CP-99994 showed no beneficial effect, a small effect could be observed in the pain clinical trial, but not significant enough to justify further development. After these disappointing experiences, Pfizer turned its interest to another structure, CP-122721, which is also a piperidine derivative, bearing an additional trifluoromethoxy substituent. This structural modification led to an increased binding affinity to the NK1 receptor, in comparison with CP-99994. In a protein plasma extravasation (PPE) model in vivo, CP-122721 exhibited high oral activities (ED50 values in the 10 to 50 µg range). Despite its attractive pharmacological profile in an emesis clinical trial, CP-122721 showed activity only when applied in combination with a 5-HT3 antagonist. Like the parent compounds, CP-122721 also displayed significant Ltype calcium channel interaction, which could potentially limit its clinical usefulness. However, no side-effects were reported in this clinical trial. Merck has also synthesized piperidine NK1 antagonists based on the structure of CP-99994. Replacement of the 3amino group by an oxygen and a modified benzyl substituent (3,5-bis-trifluoro-methyl) led to the development of L-733060, a highly potent NK1 antagonist with reduced L- Selective NK1 antagonists Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives Compound O Stage of development Indication Phase I (pain) Suspended NH Tool: First non-peptidic SP antagonist Inflammation H3C In vitro pharmacology and clinical trials hNK1: IC50 = 0.77 nM (IM-9 cells) [47]. Pain L-type calcium channel interaction (27 nM) N CP-96345 (Pfizer) O H3C Phase II (asthma) Phase II (pain) Suspended Phase II (emesis) NH Poor oral bioavailability and duration of action N H L-type calcium channel interaction (3 µM) CP-99994 (Pfizer) 75 hNK1: Ki = 0.17 nM (IM-9 cells) [51]. Asthma: no effect on bronchoconstriction and cough induced by hypertonic saline in asthmatic subjects (250 µg/kg, iv) Pain: peripheral neuropathy: not better than placebo (0.2 mg/kg, iv). Dental surgery: better than placebo less effective than ibuprofen. Short duration of action (90min, 0.75 mg/kg, iv) [10] In vivo pharmacology: emphasis on inflammation, pain, arthritis Arthritis model: blockade of painsensitization response in the spinal cord induced by injection of kaolin and carrageenin into the rat knee joint. Inhibition of IL-2 production by lymphocytes and IL-3 production by bone marrow cells. Inhibition of SP-, capsaicin-, or antidromic nerve stimulation-induced PPE in different species and tissues. Inhibition of SPinduced decrease in blood pressure and cigarette smoke-induced airway edema in rats. Inhibition of nociception: acetic acid writhing in the mouse, hot plate test in the mouse or the reaction time after pressure of the rat paw inflamed with carrageenin, or with formalin [48-50]. Inhibition of capsaicin-induced PPE in the guinea pig airway with an ID50 value of 4 mg/kg, po. Penetration of blood-brain barrier. Anti-emetic activity [48]. 76 IDrugs 1998 Vol 1 No 1 Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives (continued) Compound O F H3C F F O Stage of development Indication Phase II (emesis) Inflammation, Pain Migraine Asthma Good oral bioavailability NH L-type calcium channel interaction (390 nM) N H CP-122721 (Pfizer) Preclinical Emesis Migraine Asthma F F F In vitro pharmacology and clinical trials hNK1: pIC50 = 9.8 nM (IM-9 cells) [52]. Emesis: Improvement of acute cisplatin-induced vomiting in tumor patients in combination with a 5-HT3 antagonist and almost complete suppression of delayed nemesis. No effect of 200 mg, po, of CP122721 alone on acute vomiting [10]. hNK1: IC50 = 0.87 nM (CHO cells) [53]. F O In vivo pharmacology: emphasis on inflammation, pain, arthritis PPE model: ED50 = 0.01 to 0.05 mg/kg, po. Inhibition of capsaicin-induced PPE in the guinea pig lung with ID50 = 0.01 mg/kg, po [52]. Potent anti-emetic activity [10]. Inhibition of dural PPE induced by electrical stimulation of the trigeminal ganglion in rats with an ID50 value of 212 µ g/kg, complete inhibition with 1 mg/kg, iv [54]. F F N H L-733060 (Merck) Preclinical Emesis Migraine, pain Inflammation, asthma F F F F F N H N HN hNK1: IC50 = 0.09 nM (CHO cells) [56]. Inhibition of SP-induced dermal PPE in guinea pigs: ED50 = 0.009 mg/kg, po (CP-99994: ED50 = 1.6 mg/kg, po [56]. L-758298: hNK1: IC50 = 2.8 nM (CHO cells). L-754030: hNK1: IC50 = 0.1 nM (CHO cells) [57]. Potent oral activity against a variety of emetic stimuli. Rapid conversion of L-758298 to L-754030 in vivo [57]. Good oral bioavailability in rats and monkeys Weak L-type calcium channel interaction N L-741671 (Merck) Preclinical Emesis Migraine, pain Inflammation, asthma F F O Inhibition of SP-induced dermal PPE in the guinea pig (ED50 = 0.037 mg/kg, po), PPE produced by stimulation of the trigeminal nerve in the dura mater of the rat (ED50 = 0.028 mg/kg, iv) and cisplatin-induced retching and vomiting in ferrets (ED50 = 1.0 mg/kg, iv) [55]. F O O hNK1: IC50 = 0.05 nM (CHO cells) [55]. F F O F F Good oral bioavailability N H N O HN N L-742694 (Merck) Phase I/II Emesis F F F H3C O F F O F N H N O F N Water-soluble prodrug (L-758298) N R L-758298 (R = -PO3H2), Prodrug L-754030 (R = H) (Merck) Good CNS penetration (L-754030) Clinical trials: Antagonism of SP-induced forearm vasodilatation in eight healthy male volunteers (0.25, 1, or 5 mg L-758298, iv) [58] Review Neurokinin antagonists 77 Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives (continued) Compound Stage of development Indication Preclinical Emesis Migraine O H3C N N NH N N N H In vitro pharmacology and clinical trials hNK1: pKi = 10.3 nM (CHO cells) hNK1: pKi = 10.5 nM (U373 MG astrocytoma cells) [59]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Dose-dependent inhibition (3 and 10 mg/kg, iv) of PPE in dura mater, cojunctiva, eyelid and lip, induced by electrical stimulation of the trigeminal ganglion in rats. Anti-emetic activity [60]. hNK1: pKi = 10.6 nM (CHO cells) [61]. Inhibition of cyclophosphamide-induced PPE in the bladder of pretreated ferrets (0.3 mg/kg, sc). Synergistic inhibition in combination with sc dexamethasone. The combined treatment also inhibited X-rayinduced PPE in the small intestine [62]. Potent oral anti-emetic activity [61]. Selected for clinical evaluation, but replaced by GR-205171 Good oral bioavailability Weak L-type calcium channel interaction GR-203040 (Glaxo Wellcome) F O F F H3C N NH N Phase II Emesis Migraine, pain Inflammation N N Good oral bioavailability Reduced L-type calcium channel activity (pKi = 5.6) N H GR-205171 (Glaxo Wellcome) PPE hNK1,2,3 Protein plasma extravasation Human neurokinin receptor binding affinity type calcium channel activity. Further modifications of L733060, namely the introduction of substituents to the piperidine nitrogen, led to even more potent NK1 antagonists exemplified by L-741671, a 3-oxo-1,2,4-triazolylmethylsubstituted derivative of L-733060. With the aim of further reducing the L-type calcium channel activity and thus reducing the potential of side-effects, the piperidine ring was replaced by the less basic morpholine acetal ring system leading to the 3-oxo-1,2,3-triazol-5-yl substituted morpholine derivative L-742694. This compound is one of the most potent NK1 antagonists known. When compared to CP99994, L-742694 was found to exhibit a 100-fold increase in oral activity in a plasma extravasation model. Further refinement of L-742694 led to the metabolically more stable L-754030 and the water-soluble and clinically used prodrug L-758298. Another company actively pursuing the piperidine structures originally discovered by Pfizer is Glaxo Wellcome. GR-203040, one of Glaxo’s earlier compounds, was selected for clinical trials but was replaced later by its close analog GR-205171. This last compound is reported to be under clinical evaluation for emesis and/or migraine. The structure of GR-205171 can be regarded as CP-99994 with an additional 5-trifluoro-1,2,3,4tetrazole substituent attached to the 2-methoxybenzyl residue. This apparently small structural modification led to a compound with good oral bioavailability as well as low Ltype calcium channel activity. Perhydroisoindole derivatives (Table 2) Screening of compound libraries followed by lead optimization led to the discovery of Rhône-Poulenc Rorer's early NK1 antagonist, RP-67580. This diarylperhydroisoindole derivative displayed potent affinity for the rat NK1 receptor but, binding to the human NK1 receptor was rather moderate. RP-67580 also suffered from poor bioavailability (accounted to the amidine structural element), as well as considerable L-type calcium channel interaction. On the other hand, RP-67580 showed activities in several animal models of inflammation. Replacement of the amidine by an amide together with the attachment of an additional phenyl substituent at position 4 of the perhydroisoindole ring system resulted in the discovery of RPR-100893 (dapitant). Dapitant was highly active in binding to the human NK1 receptor with little binding affinity for the rat receptor. Although dapitant displayed highly attractive in vivo (iv, po) activities in several models of inflammation and migraine, it proved to be ineffective in a migraine clinical trial and was therefore suspended. The follow up compounds RPR-107880 and RPR-111905 are close structural analogs of dapitant. The 7,7-diphenyl substitution pattern is replaced by either a 6-methyl (RPR-107880) or a 6-cyanomethyl (RPR111905) substitutent. Both compounds show improved binding affinities for the human NK1 receptor and a more attractive profile in in vivo inflammation models in comparison with dapitant. Analogs of FK-888, modified peptides (Table 3) The pseudopeptide compound FK-888 was identified as a potent NK1 antagonist through rational drug design starting from a tripeptide lead structure. Molecular modeling studies helped to define the essential structural elements (the indole, the two aryl substituents) and connecting structural elements necessary for good NK1 binding affinity. FK-888 was tested for anti-inflammatory activities in several in vivo 78 IDrugs 1998 Vol 1 No 1 Selective NK1 antagonists Table 2. Analogs of RP-67580, perhydroisoindole derivatives Compound H3C O H Stage of development Indication Preclinical Inflammation, pain Discontinued Selectivity for rat NK 1 receptor N In vitro pharmacology and clinical trials hNK1: IC50 = 39.8 nM (U373MG astrocytoma cells) ratNK1: IC50 = 7 nM (rat brain) [63,5]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Antinociceptive activity in the mouse phenylbenzoquinone writhing, ED50 = 0.7 mg/kg, sc, and formalin test, ED 50 = 3.7 mg/kg locally. Anti-inflammatory activity in the rat by inhibition of PPE induced by SP in the urinary bladder (0.04 mg/kg, iv), or by electrical stimulation of the saphenous nerve in the hindpaw (ID50 = 0.15 mg/kg, iv). PPE induced by sc SP or septide in the hindpaw was also inhibited [48]. hNK1: IC50 = 13 nM (IM-9 cells) ratNK1: IC50 = >1000 nM (rat brain) [64]. Analgesic in the formalin test in guinea pigs (ED50 = 3.1 mg/kg sc and 11 mg/kg po). Inhibition of septide-induced PPE in peripheral tissues (guinea pig trachea): ED50 = 0.1 mg/kg, iv and 0.33 mg/kg, po. Inhibition of capsaicin-induced PPE, guinea pig dura mater: ED50 = 8 µg/kg, po [65]. NH H Poor oral bioavailability O RP-67580 (Rhône-Poulenc Rorer) L-type calcium channel interaction (200nM) Phase II (migraine) Suspended H3C H N O O H OH CH3 Migraine: Ineffective in clinical trials for the treatment of migraine at doses up to 20 mg [8]. O CH3 RPR-100893, dapitant (Rhône-Poulenc Rorer) H3C H H3C Preclinical Inflammation, pain Migraine hNK1: Ki = 4 nM (IM-9 cells) [66]. Inhibition of septide-induced PPE in guinea pig peripheral tissues (ED50 = 0.02 mg/kg, iv) [66]. Preclinical Inflammation, pain Migraine hNK1: IC50 = 0.3 nM (IM9 cells) [67]. Analgesic in the formalin test in guinea pigs, ED50 = 17 mg, po. Inhibition of septide-induced PPE in guinea pig peripheral tissues (ureter), ED50 = 0.004 mg/kg, po. Inhibition of capsaicin-induced PPE, guinea pig dura mater: ED50 = 0.093 mg/kg, po [67]. N O H OH O O CH3 CH3 RPR-107880 (Rhône-Poulenc Rorer) H3C H C N N H OH O O O CH3 CH3 RPR-111905 (Rhône-Poulenc Rorer) models in guinea pigs, where it inhibited SP-induced PPE in the lower trachea and main bronchi. FK-888 also inhibited cigarette smoke- and vagus nerve stimulation-induced PPE in the airways of guinea pigs. In an asthma clinical trial (2.5 mg of inhaled FK-888), however, no beneficial effect on exercise-induced broncho-constriction was observed. The Sandoz group (now Novartis), starting from a novel proline thiourea lead, modified the structure to incorporate aromatic amino acid amides of proline with improved NK1 affinity. Highly potent antagonists, exemplified by SDZNKT-343 contain the same N-methyl-N-benzylamide residue found in FK-888. This compound exhibited good oral activity in guinea pig pain models. Replacement of the hydroxyproline part of FK-888 by 1aminocyclohexoyl led to Menarini’s MEN-10930. This compound had a comparable affinity to the human NK1 receptor as the parent compound. Another structurally-related derivative, MEN-11149, showed iv and po activities in anti- Review Neurokinin antagonists asthma models in guinea pigs. Furthermore, MEN-11149 did not interact with L-type calcium channels. Servier also actively pursued the FK-888 lead structure. Its efforts resulted in the discovery of S-18523, and S-19752 where the simple methyl substituent in the case of FK-888 is replaced by an alkyl tetrazolyl residue. Additionally, in S19752, the naphthyl group of FK-888 has been replaced by an indole residue. Both compounds were active in several analgesia assays in mice and rats, and also inhibited SPinduced PPE in the bladder and bronchi of guinea pigs after iv application. 79 A completely different peptide-based structure was discovered in the labs of Parke-Davis. The tryptophane derivative PD-154075 was discovered after systematic modification of a screening lead (Z-TrpPhe-NH2). The structure of this dipeptide lead was simplified, leading to conformationally flexible NK1 antagonists displaying micromolar binding affinities. In the next step, conformational restraints, in this case methyl-groups, were introduced into the backbone in order to enhance receptor affinity and selectivity until a sufficiently selective and potent compound was found. Selective NK1 antagonists Table 3. Analogs of FK-888, modified peptides Compound H3C N H N O O O Stage of development Indication Phase II (asthma) Suspended Asthma, bronchitis, migraine In vitro pharmacology and clinical trials hNK1: IC50 = 0.72 nM [68]. Phase I Inflammatory pain Arthritis Migraine hNK1: IC50 = 0.62 nM [70]. Preclinical Asthma Inflammation Neurological diseases hNK1: Ki = 1 nM and 2.5 nM (IM-9 cells and U373MG astrocytoma cells) [72,73]. Preclinical Asthma Inflammation Neurological diseases hNK1: Ki = 2.8 nM (IM-9 cells) No L-type calcium channel interaction [74]. N N OH H3C FK-888 (Fujisawa) H3C N H N O O O N H Asthma: 2.5 mg inhaled FK-888 had no effect on the acute exerciseinduced bronchoconstriction in asthmatic patients, but the recovery times were shortened [10]. N N O O SDZ-NKT-343 (Novartis) H N O H N O NH In vivo pharmacology: emphasis on inflammation, pain, arthritis Inhibition of SP-induced PPE in the lower trachea and main bronchi of guinea pigs (ID50 = 0.1 µmol/kg, iv; complete inhibition with 2 µmol/kg, iv). Inhibition of PPE in the airways induced by vagus nerve stimulation or by cigarette smoke [69]. Withdrawal thresholds to mechanical and thermal stimuli in a guinea pig hyperalgesia model in both inflamed and non-inflamed paws. ED50 values of 0.73 mg/kg, po for the mechanical hyperalgesia and 1.75 mg/kg, po for the thermal hyperalgesia [71]. Inhibition of PPE in guinea pig knee joint induced by Freund’s adjuvant [70]. N O CH3 MEN-10930 (Menarini) O H N NH O O N H MEN-11149 (Menarini) CH3 Inhibition of [Sar9,Met(O2)11]SP-induced bronchoconstriction in guinea pigs with an ED50 value of 83 nmol/kg, iv (duration of action > 3h) and PPE in guinea pig bronchi with ED50 values of 0.2 µmol/kg, iv, and 0.97µmol/kg, po [74]. 80 IDrugs 1998 Vol 1 No 1 Table 3. Analogs of FK-888, modified peptides (continued) Compound O H N O N CH3 O Stage of development Indication Preclinical Inflammation, pain Asthma In vitro pharmacology and clinical trials hNK1: Ki = 1.5 nM (IM-9 cells) NK1: Ki = 9.6 nM (rabbit vena cava) [75]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Potent analgesic activity (iv and po) in the hot plate test in mice, the phenylbenzoquinone-induced writhing response in mice and the formalin test in rats with ED50 values of 0.018 and 0.56 mg/kg, iv, and 48% inhibition at 2.5 mg/kg, iv, respectively. Inhibition of SP-induced PPE in the bladder and bronchi of guinea pigs with ED50 values of 0.18 and 0.14 mg/kg, iv [75]. Preclinical Inflammation, pain Asthma hNK1: Ki = 2.4 nM (IM-9 cells) NK1: Ki = 9.0 nM (rabbit vena cava) [76]. Inhibition of SP-induced bronchoconstriction in guinea pigs with an oral ID50 value of 50 nmol/kg at 3 min and 70% inhibition with 0.5 mM aerosol at 3 min. Duration of action at least 40 min. Reduction of capsaicin-induced PPE in the bronchi of guinea pigs with an IC50 value of 26 µg/kg, iv [76]. Preclinical Asthma, inflammation hNK1: Ki = 0.35 nM (IM-9 cells) [77]. Inhibition of SPOMe-induced PPE in the guinea pig bladder (ED50 = 0.02 mg/kg, iv) [77]. N N OH N N + N K N S-18523 (Servier) F O F H N O F O O NH N N F OH F F N N N K + N S-19752 (Servier) H N O HC 3 O O H N N H O CH 3 PD-154075 (CAM-4261) (Parke-Davis) Selective NK1 antagonists, miscellaneous structures (Table 4) Sanofi is also involved in the search for NK antagonists. Starting from a common lead structure that was identified by random screen, SR-140333 (and also the NK2 antagonist SR48968) was developed. In terms of binding affinity SR-140333 is one of the most potent NK1 antagonists described in the literature. SR-140333 showed long-lasting activity in antiinflammatory models. Thus it inhibited PPE in the rat paw (iv and sc), but its anti-arthritic potential may be limited, due to the fact that SR-140333 had no analgesic activity in rats. The compound is reported to be under clinical phase I evaluation (possible indications: migraine, pain, inflammation). Another important NK1 antagonist, which reached clinical trials (pain, migraine, suspended because of lack of efficacy) is lanepitant from Lilly. From a series of tryptophane-based nonpeptide structures, this compound was found to be the most suitable candidate for further development. Lanepitant exhibited an attractive profile (potent, long-acting) in several animal models of inflammation, pain and migraine. However, in the clinic, lanepitant had no beneficial effects on pain in acute migraine or chronic osteoarthritis. The absence of any clinical activity might be attributed to low bioavailability. Takeda reported the pyridopyridine structural class of compounds exemplified by N-[3,5-(bistrifluoromethyl)benzyl]-7,8-dihydro-N,7-dimethyl-8-oxo-5-(4-fluoro-phenyl)-6-py- rido[3,4b]-pyridinecarboxamide as potent NK1 antagonists. Combination of structural elements from a cholecystokinin (CCK) antagonist and from already known NK1 antagonists led to the design of this class of highly potent NK1 antagonists. The specific compound mentioned had very good oral activity in capsaicin-induced PPE from guinea pig trachea, thus demonstrating its potential as anti-inflammatory agent. Merck also produced a series of tryptophane-based NK1 antagonists exemplified by L-737488. Lead compounds (tryptophane esters) which were eventually developed into L-737488 were again identified by random screening of available compound pools. L-737488 exhibited good oral activity in an anti-inflammatory model (inhibition of SPinduced dermal PPE) in guinea pigs, without having sideeffects on heart rate or blood pressure. With the discovery of CGP-49823, Ciba-Geigy (now Novartis) established its own structural class: 1-benzoyl-2-benzyl-4aminopiperidines were developed from a piperazine lead structure, which was selected from a variety of other selected compounds for the NK1 screen as a conformationally constrained analog of the Phe-Phe-structural element of SP (previously shown to be essential for reasonable affinity to the NK1 receptor). Because of its ability to penetrate the bloodbrain barrier CGP-49823 was under development as potential anxiolytic agent. Review Neurokinin antagonists 81 Selective NK1 antagonists Table 4. Miscellaneous structures Compound Cl Cl O N + CH3 N Stage of development Indication Phase I Migraine Pain Inflammation In vitro pharmacology and clinical trials hNK1: IC50 = 0.01 nM (IM-9 cells) [78]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Complete inhibition of mustard oil-induced PPE in the dorsal skin of the hind paw of rats at 0.1 mg/kg, iv and 1.0 mg/kg, sc lasting for 24 h. No analgesic activity after ocular application of capsaicin in rats. No change of thermal nociceptive thresholds, or thermal hyperesthesia induced by intraplantar PGE2 [79]. Phase II (Migraine, pain) Suspended Migraine, pain, asthma hNK1: IC50 = 0.15 nM (IM9 cells) [80]. Inhibition of intrathecal (it) NK1 agonistdriven nociceptive behavior in mice with ED50 of 0.21 nM, it, and 1.73 mg/kg, ip. Inhibition of neurogenic dural inflammation following trigeminal ganglion stimulation in guinea pigs with ED50 = 15 ng/kg, iv, and 91 ng/kg, po [80]. Active in a model of persistent nociceptive activation by tissue injury (formalin test) with ED50 values of 4 mg/kg, ip, and 10 mg/kg, po, 24 h blockade of pain-related behavior by 10 mg/kg, po [82]. CH3 O Cl SR-140333 (Sanofi) N O O N CH3 NH N O CH3 N H LY-303870, lanepitant (Eli Lilly) Preclinical Inflammation, pain Asthma Migraine F F F O N N CH3 CH3 Migraine, arthritic pain: The ability of lanepitant (LY-303870 dihydrochloride trihydrate) to attenuate acute migraine pain and chronic osteoarthritis pain was assessed in double blind, placebo-controlled studies. The compound was well-tolerated with no serious adverse effects. In both studies, lanepitant had no effect on pain intensity and provided no pain relief when compared to placebo. In conclusion, lanepitant was found to be very safe but to have no effect on pain in acute migraine or osteoarthritis [81]. hNK1: IC50 = 0.21 nM (IM-9 cells) [83]. Capsaicin-induced PPE: guinea pig trachea: ED50 = 0.068 mg/kg, po, and 0.017 mg/kg, iv [83]. F N F F O F EP-00652218 EP-00585913 (Takeda) F F Preclinical Emesis Migraine Asthma F NH O F N H N F O L-737488 (Merck) F hNK1: IC50 = 0.9 nM (CHO cells) [84]. Inhibition of SP-induced dermal PPE in the guinea pig with an ID50 of 1.8 mg/kg, po [84]. 82 IDrugs 1998 Vol 1 No 1 Table 4. Miscellaneous structures (continued) Compound N CH3 H N Stage of development Indication Phase I Anxiety In vitro pharmacology and clinical trials NK1: IC50 = 12 nM (bovine retina) [85]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Anxiolytic activity in gerbils (social interaction) [85]. N H3C O CGP-49823 (Novartis) Selective NK2 antagonists (Tables 5-6) Analogs of SR-48968 (Table 5) SR-48968 (saredutant) was described as the first selective and highly potent non-peptide competitive NK2 antagonist in 1992. SR-48968 and the selective NK1 antagonist SR-140333 were developed from a common lead structure, which was identified by a random screen. In several in vivo models SR48968 inhibits a number of NK2-induced effects. SR-48968 inhibits NKA-induced bronchoconstriction (long-acting), citric acid or capsaicin-induced cough and prevents citric acid- and ovalbumin-induced airway hyperreactivity in guinea pigs. In a recently-published clinical trial, SR-48968, at an oral dose of 100 mg inhibited NKA-induced bronchoconstriction in asthmatic patients. SR-144190, a morpholine analog of SR-48968 demonstrated a similar pharmacological profile as the parent compound, with a greatly improved ability to cross the blood-brain barrier. Yamanouchi discovered another potent NK2 antagonist of the SR-48968 type by modification of the piperidine residue (introduction of a spiro-benzothiophene residue instead of the N-acetylphenyl substitutuent at position 4 of the piperidine ring). Besides the nanomolar affinity at the NK2 receptor YM-38336 also exhibits some affinity (~ 700nM) for the NK1 receptor. Zeneca obtained a series of highly potent NK2 antagonists also by modifying the SR-48968 lead structure through the introduction of different substituents at position 4 of the piperidine ring. ZD-7944 incorporates the benzamide residue of SR-48968 into a ring system. Selective NK2 antagonists, miscellaneous structures (Table 6) Menarini identified MEN-10627, a cyclopeptide, as a selective and potent NK2 antagonist. In vivo (iv and po) this compound inhibited a variety of NKA-induced effects. Due to the rigid structure, Menarini claims MEN-10627 to be resistant to degradation by peptidases and thus the compound is expected to exhibit a long duration of action. Selective NK2 antagonists Table 5. Analogs of SR-48968 Compound O HN N N H3C O CH3 Stage of development Indication Phase II Asthma Incontinence Pain Inflammatory bowel disorders In vitro pharmacology and clinical trials Rat, Hamster, guinea pig, human NK2 receptors Kd = 0.4 - 2.9 nM [86]. Asthma: 100 mg, po, SR48968, inhibited inhaled NKA-induced bronchoconstriction in asthmatic patients [87]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Anxiolytic activity (rodents, marmosets) Inhibition of NKA-induced bronchoconstriction in guinea pigs, iv and po. Inhibition of citric acid- or capsaicininduced cough in guinea pigs. Prevention of airway hyperreactivity induced by citric acid and ovalbumin challenges in guinea pigs [86]. Preclinical Asthma Incontinence Pain Inflammatory bowel disorders Rat, hamster, guinea pig, human NK2 receptors pA2 = 9.08 to 10.10 [88]. Similar pharmacological profile as SR48968, with increased bioavailability in the CNS [88]. Cl Cl SR-48968, saredutant (Sanofi) HN O N H3C N N O CH3 O F F SR-144190 (Sanofi) Good CNS penetration Review Neurokinin antagonists 83 Table 5. Analogs of SR-48968 (continued) Compound O S O N Stage of development Indication Preclinical Asthma Urinary incontinence Irritable bowel disease In vitro pharmacology and clinical trials NK2 (hamster urinary bladder): IC50 = 8.9 nM NK1 (hamster urinary bladder): IC50 = 680 nM [89]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Inhibition of NKA agonist-induced bronchoconstriction in guinea pigs: IC50 = 20 µg/kg, iv; ID 50 = 405 µg/kg, id [89]. Preclinical Asthma Suspended hNK2: Ki = 0.14 nM (mouse erythroleukemia (MEL) cells) [90]. Inhibition of NKA- or [β-ala8]-NKA(4-10)induced guinea pig trachea contractions: pKB = 9.4 [90]. Inhibition of [β-ala8]-NKA(4-10)-induced bronchoconstriction in guinea pigs: 100% protection at 5 µmol/kg po, (- 2 h) (similar compound disclosed in patent EP-00630887 [91]). N CH 3 Cl Cl YM-38336 (Yamanouchi) H3C S O N N H O CH3 Cl Cl Cl ZD-7944 (Zeneca) Glaxo developed its selective NK2 antagonist GR-159897, a fluoroindole linked via an ethyl spacer to a 4-(phenylsulfinylmethyl)-4-methoxypiperidine residue, starting from a structurally-related screening lead. GR-159897 showed high oral activity in a NK2 bronchoconstriction model in guinea pigs, and in addition, anxiolytic activity has been demonstrated in rodents and marmosets. The structure of Rhône-Poulenc Rorer’s selective NK2 antagonist, RPR-106145, is closely related to the structure of RPR-100893 (selective NK1 antagonist). In comparison with RPR-100893, the perhydroisoindole backbone of RPR-106145 possesses the enantiomeric configuration at the chiral centers. Besides an additional hydroxy group at C-5 and an indole acetic acid residue instead of the 2-methoxypropionic acid substituent, RPR-106145 can be viewed as the mirror image of RPR-100893. The selective NK2 antagonist PD-147714 (Parke-Davis) was obtained using the same strategy as described above for the discovery of the company’s selective NK1 antagonist, PD154075. Zeneca discovered a novel class of non-peptide NK2 antagonists. The pyrrolopyrimidine compound, ZM-253270, exhibited high affinity to hamster NK2 receptors; however, binding to human receptors was much weaker. Dual NK1/NK2 antagonists (Table 7) FK-224, a natural product from microbial cultures, showed moderate activity at human NK1 and NK2 receptors. The affinity to rat receptors was only weak. Nevertheless, FK-224 inhibited carrageenin-induced PPE into rat knee joints as well as carrageenin- and antigen-induced cell accumulation in the synovial cavity. Due to its activity in asthma models in guinea pigs, FK-224 was chosen for clinical evaluation. In clinical trials, FK-224 proved to be ineffective in mild to moderate asthmatics (no effects on lung function or on NKA-induced bronchoconstriction). On the other hand, FK224 inhibited bradykinin-induced bronchoconstriction and cough in asthmatics. This beneficial effect of FK-224 can, however, be explained by the known bradykinin antagonist activity of the compound. The credit for the first really potent dual NK1/NK2 antagonist can be given to Hoechst Marion Roussel. MDL-105212A, which is structurally related to SR-48968, exhibits nanomolar affinities to both human receptors, and it also had good binding affinity to the guinea pig NK3 receptor. In in vivo asthma models, the compound showed a variety of beneficial effects when administered intravenously; however, oral activity remained at a rather moderate level. Introduction of a morpholine group into the unsubstituted amide residue of MDL-105212A led to MDL-105172A. This compound had more balanced binding affinities to all three neurokinin receptors and improved activity in guinea pig asthma models, when compared with MDL-105212A. For the design of its dual NK1/NK2 antagonists, Merck also used Sanofi’s NK1 (SR-140333) and NK2 (SR-48968) antagonists, respectively, as lead structures. Modification of the piperidine residue, as well as the introduction of trifluoromethyl groups into the benzoyl substituent led to potent (at cloned human receptors) dual NK1/NK2 antagonists (L-743986 and analogs). Potent oral activity was demonstrated in an anti-inflammatory model of asthma in guinea pigs. Servier’s S-16476, a modified peptide structure, exhibited only moderate dual NK1/NK2 binding affinities, but was active in vivo, when administered iv. 84 IDrugs 1998 Vol 1 No 1 Selective NK2 antagonists Table 6. Miscellaneous structures Compound Stage of development Indication Preclinical Asthma Urinary incontinence Irritable bowel disease HN H N HN O O O O NH NH H MEN-11420: hNK2: Ki = 2.5 nM [110] O HN O O H3C In vitro pharmacology and clinical trials MEN-10627: Rabbit pulmonary artery, hamster trachea NK2 receptors: pKB = 8.110.1 [91]. In vivo pharmacology: emphasis on inflammation, pain, arthritis MEN-10627: iv and po: inhibition of NKA agonist-induced urinary bladder contractions in rats. Inhibition of NKA agonist- and antigeninduced bronchoconstriction in guinea-pigs. Reduction of PAF-induced bronchial hyperresponsiveness to histamine in guineapigs [91-92]. NH S N H MEN-11420: Improved in vivo potency (about 10-fold) and duration of action compared to MEN-10627 [110]. CH3 CH3 MEN-10627 (Menarini) H N OH O HN OH OH H3C O MEN-11420 (Nepadutant; Menarini) N F S+ Preclinical Anxiety Asthma Inhibition of [3H]GR100679 (NK2 agonist) binding:hNK2: pKi = 9.5 (CHO cells) and rat colon membranes: pKi = 10.0 [94]. Preclinical Asthma hNK2: Ki = 16 nM [66]. Preclinical NK2: Ki = 1.4 nM (hamster urinary bladder) [95,96]. Preclinical NK2: Ki = 2.0, 2.2, and 105 nM (hamster urinary bladder, cloned hamster and cloned human NK2 receptors) [97]. O N H CH3 O- GR-159897 (Glaxo) H Inhibition of NK2 agonist (GR-64349)induced bronchoconstriction in guinea pigs: 10 µmol/kg, po. Anxiolytic activity (rodents and marmosets). Bioavailability in dogs: 87% [94]. NH N HO O H HO O CH3 RPR-106145 (Rhône-Poulenc Rorer) H3C O NH O O H N O H3C O N H O NH2 N H H3C O PD-147714 (CAM-2291) (Parke-Davis) Cl HN H3C N N H3C N O N H3C OH N N O ZM-253270 (Zeneca) Inhibition of NKA-induced contractions of hamster trachea: -logKB = 7.5. 90-fold less potent as a competitive antagonist of NKA in human bronchus [97]. Review Neurokinin antagonists 85 Dual NK1/NK2 antagonists Table 7. Miscellaneous structures Compound CH3 HO H3C CH3 O H H N HN H N NH2 N H O O Stage of development Indication Phase II (asthma) Suspended Asthma, bronchitis Inflammation, arthritis O OH H3C CH3 O O N H H N O O CH3 NH O O OH FK-224 (Fujisawa) O Preclinical Asthma Suspended CH3 O H2N CH3 N N O O CH3 O Cl H Cl Cl MDL-105212A (Hoechst Marion Roussel) O CH3 Preclinical Asthma hNK1: IC50 = 4.3 nM (IM9 cells) hNK2: IC50 = 2.1 nM (HSKR-1 cells) NK3: IC50 = 2.5 nM (guinea pig cerebral cortex) [102]. Preclinical Asthma hNK1: IC50 = 0.2 nM (CHO cells) hNK2: IC50 = 1.5 nM (CHO cells) [103]. Preclinical Asthma [104] O O CH3 N N N O CH3 O O Cl H Cl In vitro pharmacology and clinical trials hNK1: IC50 = 190 nM hNK2: IC50 = 190 nM ratNK1: IC50 = 1.7 µM ratNK2: IC50 = 1.9 µM [68]. Asthma: Inhibition of bradykinin- induced bronchoconstriction and cough in 9 asthmatics (4 mg, aerosol). No effect on baseline lung function and no protection against NKAinduced bronchoconstriction in 10 mild asthmatic patients (4 mg, aerosol) [10]. No beneficial effects on symptoms and lung function in patients with mild to moderate asthma (4-week treatment, 4 mg, aerosol, qid) [98]. hNK1: IC50 = 3.1 nM (IM9 cells) hNK2: IC50 = 8.4 nM (HSKR-1 cells) NK3: IC50 = 21 nM (guinea pig cerebral cortex) [100]. Cl MDL-105172A (Hoechst Marion Roussel) O H3C N F O F N F N CH3 Cl F F F Cl L-743986 analog (Merck) F O F N F N CH3 Cl F F F Cl L-743986 (Merck) In vivo pharmacology: emphasis on inflammation, pain, arthritis Inhibition of PPE induced by carrageenin and SP into the knee joint of rats with an ED50 of 10 µg/knee, as well as carrageeninand antigen-induced cell accumulation in the synovial cavity [99]. Inhibition of SP-induced PPE in the lower trachea and main bronchi of guinea pigs (ID50 = 1.1 µmol/kg, iv; complete inhibition with 10 µmol/kg, iv). Inhibition of [β-Ala]NKA- and [Sar]SPinduced bronchoconstriction in guinea pigs. Inhibition of PPE in the airways induced by vagus nerve stimulation or by cigarette smoke [69]. Inhibition of SP-induced PPE in guinea pig trachea and primary bronchi: ED50 = 0.20 mg/kg, iv. Inhibition of NKA (aerosol)induced respiratory collapse in guinea pigs: ED50 = 5 mg/kg, iv and capsaicin-induced increases in pulmonary insufflation pressure: ED50 = 0.5 mg/kg, iv. Inhibition of capsaicin (aerosol)-induced effects in guinea pigs : ED50 = 5 mg/kg, iv and 50 mg/kg, po [101]. Inhibition of SP-induced PPE in guinea pig trachea and primary bronchi: ED50 = 1 mg/kg, iv. 8 Inhibition of [β-Ala ]NKA 4-10 (aerosol)induced respiratory collapse in guinea pigs: ED50 = 0.5 mg/kg, iv. Inhibition of capsaicin (aerosol)-induced respiratory effects in guinea pigs : ED 50 = 1 mg/kg, iv and 20 mg/kg, po. Lack of CNS penetration [102]. Inhibition of resiniferatoxin-induced PPE in guinea pigs (ED50 = 0.3 mg/kg, po) [103]. 86 IDrugs 1998 Vol 1 No 1 Table 7. Miscellaneous structures (continued) Compound Stage of development Indication Preclinical Asthma In vitro pharmacology and clinical trials hNK1: IC50 = 85 nM hNK2: IC50 = 129 nM Poor affinity for the rat NK1 receptor [105]. In vivo pharmacology: emphasis on inflammation, pain, arthritis Inhibition of SP-, NKA- and capsaicininduced bronchoconstriction in guinea pigs with ED50 values of approx 4 µmol/kg, iv, 7µ mol/kg, iv; and 30% inhibition at 20 µmol/kg, iv. Abolition of PPE in guinea pig bronchi evoked by endogenously released neurokinins under vagus nerve stimulation at 10 µmol/kg, iv [105]. Stage of development Indication In vitro pharmacology and clinical trials hNK3: Ki = 0.21 nM (CHO cells) NK3: Ki = 0.11 nM (Guinea pig brain cortex) NK3: Ki = 0.42 nM (Gerbil brain cortex) NK3: Ki = 15 nM (Rat brain cortex) h NK1: Ki = 60 nM (IM-9 cells) NK2: Ki = 73 nM (Guinea pig ileum) [106]. hNK3: IC50 = 7.3 nM (CHO cells) NK3: IC50 = 3.7 nM (Guinea pig brain cortex) hNK1: IC50 = 3000 nM (IM-9 cells) NK2: IC50 = 790 nM (hamster urinary bladder) [107]. In vivo pharmacology: emphasis on inflammation, pain, arthritis H O N HN O O H N CH3 N N H O O O N Na OH O S-16474 (Servier) Selective NK3 antagonists Table 8. Miscellaneous structures Compound H3C Preclinical Psychosis, anxiety O N N N H3C O Cl Cl SR-142801, osanetant (Sanofi) Preclinical Asthma F H3C F CH3 O H3C O H N N H O H N H2N O PD-161182 (Parke-Davis) Preclinical CNS indications CH3 O NH R N SB-223412 (R = -OH) SB-222200 (R = -CH3) (SmithKline Beecham) SB-223412: hNK3: IC50 = 1.2 nM, Ki= 1.0 nM (CHO cells) NK3: IC50 = 1.3 nM (Guinea pig brain cortex) NK3: IC50 = 31 nM (Rat brain cortex) hNK1: IC50 = >100,000 nM (CHO cells) hNK2: IC50 = 180 nM (CHO cells) [108]. SB-222200: hNK3: Ki= 4.4 nM (CHO cells) hNK1: Ki= >100,000 nM (CHO cells) hNK2: Ki= 250 nM (CHO cells) [109]. Inhibition of intrastriatally-injected senktide (NK3 agonist)-induced turning behavior in gerbils at 3 mg/kg, po. Oral activity, long duration of action (8 h). Penetration of blood-brain barrier [106]. Inhibition of senktide-evoked increases in intracellular calcium levels in CHO cells with a Ke of 0.88 nM and inhibition of senktideinduced increases in spontaneous firing of guinea pig habenula neurons with a Ke of 5.8 nM [107]. SB-223412: Activity against senktideinduced miosis in rabbits and behavioral responses in mice with ED50 values of 0.44 mg/kg, iv, and 12.2 mg/kg, po, respectively. Good oral bioavailability in rats and dogs [108]. SB-222200: Good CNS penetration; brain concentrations of twice the plasma concentrations were obtained in rats [109]. Review Neurokinin antagonists Selective NK3 antagonists (Table 8) Combination of structural elements from Sanofi’s NK1 and NK2 receptor antagonists led to the discovery of SR-142801 (osanetant), an NK3 antagonist. Besides high affinity to the human and to animal (brain) NK3 receptors, osanetant also displayed considerable affinity to the NK1 and NK2 receptors. In in vivo models, osanetant was shown to penetrate the blood-brain barrier and to exhibit a long duration of action. Osanetant is of particular interest for the treatment of central nervous system disorders such as psychosis or anxiety. PD-161182 (Parke-Davis), which has a peptoid structure, is a potent and selective NK3 antagonist showing only weak affinities for the NK1 and NK2 receptors. SmithKline Beecham is pursuing a series of 2,3,4-trisubstituted quinolines as NK3 antagonists. SB-223412 and SB222200 are highly potent and selective NK3 antagonists with some weak NK2 activity, but have no affinity for other receptors and ion channels. With potent and selective NK3 antagonists like the compounds mentioned above, it will be possible to elucidate the role of the NK3 receptor in physiological or pathophysiological processes in the central nervous system, and to define their utility as potential drugs. References 1. Schiff M. Emerging treatments for rheumatoid arthritis Am J Med (1997) 102(suppl 1A), 11S-15S. 2. Choy EHS, Scott DL. Drug treatment of rheumatic diseases in the 1990s; achievements and future developments Drugs (1997) 23(3), 337-348. 3. Krug N, Madden J, Redington AE, Lackie P, Djukanovic R, Schauer U, Holgate ST, Frew AJ, Howarth PH. T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am J Respir Cell Mol Biol (1996) 14, 319-326. 4. Larbre JP, Moore AR, Da-Silva JA, Iwamura H, Ioannou Y, Willoughby DA. Direct degradation of articular cartilage by rheumatoid synovial fluid: contribution of proteolytic enzymes. J Rheumatol (1994) 21, 1796-1801. 5. Maggi CA, Patacchini R, Rovero P, Giachetti A. Tachykinin receptors and tachikyinin receptor antagonists. J Auton Pharmacol (1993) 13, 23-93. •• Comprehensive review covering all aspects of tachykinin receptors and tachykinin antagonists. 6. Nakanishi S. Mammalian tachykinin receptors. Ann Rev Neurosci (1991) 14, 123-136. 7. Burcher E, Mussap CJ, Stephenson JA. Autoradiographic localization of receptors in peripheral tissues In Tachykinin Receptors. Ed: Buck SH, Humana Press, Totowa, NJ (1994) 125-163. 8. Longmore J, Swain CJ, Hill RG. Neurokinin receptors. Drug News Perspec (1995) 8(1), 5-23. •• Excellent discussion of the potential therapeutic uses for neurokinin receptor antagonists. 9. 87 Kucharczyk N. Tachykinin antagonists in development. Exp Opin Invest Drugs (1995) 4(4), 299-311. •• Excellent review of chemical structures and the clinical potential of neurokinin receptor antagonists. 10. Elliott J, Seward EM. Neurokinin receptor antagonists. Exp Opin Ther Patents (1997) 7(1), 43-54. •• Excellent review of recent clinical data of neurokinin receptor antagonists. 11. Schwab W, Bilgicyildirim A, Funk RH. Microtopography of the autonomic nerves in the rat knee: a fluroescence microscopic study. Anat Rec (1997) 247, 109-118. 12. Mapp PI. Innervation of the synovium. Ann Rheum Dis (1995) 54, 398-403. 13. Isasaki A, Ioue K, Hukuda S. Distribution of neuro-peptidecontaining nerve fibers in the synovium and adjacent bone of the rat knee joint. Clin Exp Rheumatol (1995) 13, 173-178. 14. Garrett NE, Mapp PI, Cruwys SC, Kidd BL, Blake DR. Role of substance P in inflammatory arthritis. Ann Rheum Dis (1992) 51, 1014-1018. 15. Geppetti P, Bertrand C, Ricciardolo FML, Nadel JA. New aspects on the role of kinins in neurogenic inflammation. Can J Physiol Pharmacol (1995) 73, 843-847. 16. Aloe L, Probert L, Kollias G, Micera A, Tirassa P. Effect of NGF antibodies on mast-cell distribution, histamine and substance P levels in the knee joint of TNF-arthritic transgenic mice. Rheumatol Internat (1995) 14, 249-252. 17. Sluka KA, Milton MA, Willis WD, Westlund KN. Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation. Br J Pharmacol (1997) 120, 1263-1273. 18. Lam FY, Wong MCS. Characterization of tachykinin receptors mediating plasma extravasation and vasodilatation in normal and acutely inflamed knee joints of the rat. Br J Pharmacol (1996) 118, 2107-2114. 19. Kidd BL, Cruwys SC, Garrett NE, Mapp PI, Jolliffe VA, Blake DR. Neurogenic influences on contralateral responses during experimental rat monoarthritis. Brain Res (1995) 688, 72-76. 20. Ahmed M, Bjurholm A, Schultzberg M, Theodorsson E, Kreicbergs A. Increased levels of substance P and calcitonin gene-related peptide in rat adjuvant arthritis. A combined immunohistochemical and radioimmuno-assay analysis. Arthritis Rheum (1995) 38, 699-709. 21. Mapp PI, Terenghi G, Walsh DA, Chen ST, Cruwys SC, Garrett N, Kidd BL, Polak JM, Blake DR. Monoarthritis in the rat knee induces bilateral and time-dependent changes in substance P and calcitonin gene-related peptide immunoreactivity in the spinal chord. Neuroscience (1993) 57, 1091-1096. 22. Bileviciute T, Lundeberg T, Ekblom A, Theodorsson E. Bilateral changes of substance P-, neurokinin A-, calcitonin gene-related peptide and neuropeptide Y-like immunoreactivity in rat knee joint synovial fluid during acute monoarthritis. Neurosci Lett (1993) 153, 37-40. 88 IDrugs 1998 Vol 1 No 1 23. Sluka KA, Westlund KN. Behavioral and immunohistochemical changes in an experimental arthritis model in rats. Pain (1993) 55, 367. 24. Stucky CL, Galeazza MT, Seybold VS. Time-dependent changes in Bolton-Hunter-labeled 125I-substance P binding in rat spinal cord following unilateral adjuvantinduced peripheral inflammation. Neuroscience (1993) 57, 397-409. 25. Ahmed M, Scrinivasan GR, Theodorsson E, Schultzberg M, Kreicbergs A. Effects of surgical de-innervation on substance P and calcitonin gene-related peptide in adjuvant arthritis. Peptides (1995) 16, 569-579. 26. Garrett NE, Kidd BL, Cruwys SC, Tomlinson DR. Changes in preprotachykinin mRNA expression and substance P levels in dorsal root ganglia of monoarthritic rats: comparison with changes in synovial substance P levels. Brain Res (1995) 675, 203-207. 27. Malcangio M, Bowery NG. Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of the GABAB receptor system. Br J Pharmacol (1994) 113, 15611566. 28. Hanesch U, Blecher F, Stiller VU, Emson PC, Schaible HG, Heppelmann B. The effect of a unilateral inflammation at the rat’s ankle joint on the expression of preprotachykinin-A mRNA and preprosomatostatis mRNA in dorsal root ganglion cells - a study using non-radioactive in situ hybridization. Brain Res (1995) 700, 279-284. 29. Menkes CJ, Renoux M, Laoussadi S, Mauborgne A, Bruxelle J, Cesselin F. Substance P levels in the synovium and synovial fluid from patients with rheumatoid arthritis and osteoparthritis. J Rheumatol (1993) 20, 714-717. 30. Hernanz A, De Miguel E, Romera N, Perez-Ayala C, Gijon J, Arnalich F. Calcitonin gene-related peptide II, substance P and vasoactive intestinal peptide in plasma and synovial fluid from patients with inflammatory joint disease. Br J Rheumatol (1993) 32, 31-35. 31. Arnalich F, de Miguel E, Perez-Ayala C, Martinez M, Vazquez JJ, Gijon-Banos J, Hernanz A. Neuropeptides and interleukin-6 in human joint inflammation relationship between intraarticular substance P and interleukin-6 concentrations. Neurosci Lett (1994) 170, 251-254. 32. Anichini M, Cesaretti S, Lepori M, Maddali Bongi S, Maresca M, Zoppi M. La substance P dans la serum des patients atteints de polyarthrite rheumatoide. Rev Rheum [Ed Fr] (1997) 64(1), 19-22. 33. Sacerdote P, Carrabba M, Galante A, Pisati R, Manfredi B, Panerai AE. Plasma and synovial fluid interleukin-1, interleukin-6 and substance P concentrations in rheumatoid arthritis patients effect of the nonsteroidal anti-inflammatory drugs idomethacin, diclofenac and naproxen. Infl Res (1995) 44, 486-490. 34. De Miguel E, Arnalich F, Tato E, Vazquez JJ, Gijon-Banos J, Hernanz A. The effect of gold salts on substance P level in rheumatoid arthritis. Neurosci Lett (1994) 174, 185-187. 35. Bileviciute I, Lundeberg T, Ekblom A, Theodorsson E. Substance P-, neurokinin R-, calcitonin gene-related peptide and neuropeptide Y-like immunoreactivity (-lI) in rat knee joint synovial fluid during acute monoarthritis is not correlated with concentrations of neuropeptide-LI in cerebrospinal fluid and plasma. Neurosci Lett (1994) 167, 145-148. 36. Kar S, Rees RG, Quirion R. Altered calcitonin gene-related peptide, substance P and enkephalin immuno-reactivities and receptor binding sites in the dorsal spinal cord of the polyarthritic rat. Eur J Neurosci (1994) 6, 345-354. 37. Calvino B, Couraud JY, Besson JM. Prevaccination with diluted Freund adjuvant prevents the development of chronic pain and transient release of cerebrospinal fluid substance P in adjuvant-induced arthritis in rats. Pain (1994) 58, 211-217. 38. Krause JE, DiMaggio DA, McCarson KE. Alternations in neurokinin 1 receptor gene expression in models of pain and inflammation. Can J Physiol Pharmacol (1995) 73, 854-859. 39. Malcangio M, Bowery NG. Calcitonin gene-related peptide content, basal outflow and electrically-evoked release from monoarthritic rat spinal cord in vitro. Pain (1996) 66, 351-358. 40. Neugebauer V, Weiretter F, Schaible HG. Involvement of substance P and neurokinin-1 receptors in the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat’s knee joint. J Neurophysiol (1995) 73, 1574-1583. 41. Neugebauer V, Rumenapp P, Schaible HG. Mechanisms of oxyradical production in substance P stimulated rheumatoid synovial cells. Rheumatol Int (1996) 16, 159-167. 42. Tanabe T, Otani H, Mishima K, Ogawa R, Inagaki C. The role of spinal neurokinin-2 receptors in the processing of nociceptive information from the joint and in the generation and maintenance of inflammation-evoked hyperexcitability of dorsal horn neurons in the rat. Eur J Neurosci (1996) 8, 249-60. 43. Rameshwar P, Ganea D, Gascon P. Induction of IL-3 and granulocyte-macrophage colony-stimulating factor by substance P in bone marrow cells is partially mediated through the release of IL-1 and IL-6. J Immunol (1994) 152, 4044-4054. 44. Bileviciute I, Rheodorsson E, Lundberg T. Is the pretreatment effect of low dose Freund’s adjuvant on adjuvant arthritis due to an activation of the nervous system? Neuropeptides (1996) 30, 405-411. 45. a) Carleson J, Alstergren P, Appelgren A, Applegren B, Kopp S, Theodorsson E, Lundeberg T. A model for the study of experimentally induced temporamandibular arthritis in rats: the effect of human recombinant interleukin-1α on neuropeptide-like immunoreactivity. J Orofacial Pain (1996) 10, 9-14. b) Carleson J, Alstergren P, Appelgren A, Applegren B, Kopp S, Srinivasan GR, Theodorsson E, Lundeberg T. Effects of adjuvant on neuropeptide-like immunoreactivity in experimentally induced temporomandibular arthritis in rats. Arch Oral-Biol (1996) 41, 705-712. Review Neurokinin antagonists 46. Jeanjean AP, Moussaoui SM, Maloteaux JM, Laduron PM. Interleukin-1 beta induces long-term increase of axonally transported opiate receptors and substance P. Neuroscience (1995) 68, 151-157. 47. Lowe,III JA, Drozda SE, Snider RM, Longo KP, Zorn SH, Morrone J, Jackson ER, McLean S, Bryce DK, Bordner J, Nagahisa A, Kanai Y, Suga O, Tsuchiya M. The discovery of (2S,3S)-cis-2-(Diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]-octan-3-amine as novel, nonpeptide Substance P antagonist. J Med Chem (1992) 35, 2591-2600. • Disclosure of the first non-peptide NK1 receptor antagonist, CP-96345. 48. Regoli D, Boudon A, Fauchére JL Receptors and antagonists for Substance P and related peptides. Pharmacol Rev (1994) 46(4), 551-599. 49. McLean S. Nonpeptide antagonists of the NK1 tachykinin receptor. Med Res Rev (1996) 16(4), 297-317. 50. Lowe,III JA. Nonpeptide tachykinin antagonists: medicinal chemistry and molecular biology. Med Res Rev (1996) 16(6), 527-545. 51. Desai MC, Lefkowitz SL, Thadeio PF, Longo KP, Snider RM. Discovery of a potent Substance P antagonist: recognition of the key molecular determinant. J Med Chem (1992) 35, 4911-4913. 52. McLean S, Ganong A, Seymour PA, Bryce DK, Crawford RT, Morrone J, Reynolds LS, Schmidt AW, Zorn S, Watson J, Fossa A, DePasquale M, Rosen T, Nagahisa A, Tsuchiya M, Heym J. Characterization of CP-122721; a nonpeptide antagonist of the neurokinin NK1 receptor. J Pharmacol Exp Ther (1996) 277, 900-908. 53. Harrison T, Owens AP, Williams BJ, Swain CJ, Baker R, Hutson PH, Sadowski S, Cascieri MA. Piperidine-ether based hNK1 antagonists 2: investigation of the effect of N-substitution. Bioorg Med Chem Lett (1995) 5(3), 209-212. 54. Seabrook GR, Shepheard SL, Williamson DJ, Tyrer P, Rigby M, Cascieri MA, Harrison T, Hargreaves RJ, Hill RG. L733060, a novel tachykinin NK1 receptor antagonist; 2+ effects in (Ca ) immobilisation, cardiovascular and dural extravasation assays. Eur J Pharmacol (1996) 317(1), 129135. 55. Ladduwahetty T, Baker R, Cascieri MA, Chambers MS, Haworth K, Keown LE, MacIntyre DE, Metzger JM, Owen S, Rycroft W, Sadowski S, Seward EM. N-Heteroaryl-2-phenyl3-(benzyloxy)piperidines: a novel class of potent orally active human NK1 antagonists. J Med Chem (1992) 39(15), 2907-2914. 56. Hale JJ, Mills SG, MacCoss M, Shah SK, Qi H, Mathre DJ, Cascieri MA, Sadowski S, Strader CD, MacIntyre DE, Metzger JM. 2(S)-((3,5-Bis(trifluormethyl)benzyl)-oxy-3(S)-phenyl-4((3-oxo-1,2,4-triazol-5-yl)methylmorpholine (1): A potent orally active, morpholine-based human neuro-kinin-1 receptor antagonist. J Med Chem (1996) 39(9), 1760-1762. 57. Swain CJ. Design and biological evaluation of novel NK1 antagonists. Tachykinins in Health and Disease (September 711, 1997), Cairns, Australia, Abstract book p 4. 89 58. Newby DE, Sciberras DG, Ferro CJ, Mendel CM, Gertz BJ, Lowry RC, Webb DJ. Antagonism of Substance P-induced forearm vasodilatation by the neurokinin type 1 receptor antagonist, L-758,298. Proc Br Pharmacol Soc (September 24, 1997), Edinburgh, UK, Abstract C110. 59. Beattie DT, Beresford IJM, Connor HE, Marshall FH, Hawcock AB, Hagan RM, Bowers J, Birch PJ, Ward P. The pharmacology of GR203040, a novel, potent and selective nonpeptide tachykinin NK1 receptor antagonist. Br J Pharmacol (1995) 116(8), 3149-3157. 60. Ward P, Armour DR, Bays DE, Evans B, Giblin GMP, Heron N, Hubbard T, Liang K, Middlemiss D, Mordaunt J, Naylor A, Pegg NA, Vinander MV, Watson SP, Bountra C, Evans DC. Discovery of an orally bioavailable NK1 receptor antagonist, (2S,3S)-(2-methoxy-5-tetrazol-1-ylbenzyl) (2-phenylpiperidin3-yl)amine (GR203040), with potent antiemetic activity. J Med Chem (1996) 38(26), 4985-4992. 61. Armour DR, Chung KML, Congreve M, Evans B, Guntrip S, Hubbard T, Kay C, Middlemiss D, Mordaunt J, Pegg NA, Vinander MV, Ward P, Watson SP. Tetrazole NK1 receptor antagonists: the identification of an exceptionally potent orally active antiemetic compound. Bioorg Med Chem Lett (1996) 6(9), 1015-1020. 62. Alfieri AB, Gardner CJ, Twissell DJ. Dexamethasone enhances GR205171 inhibition of cytotoxic-induced plasma protein extravasation in ferrets. Br J Pharmacol (1996) 118 (Proc Suppl)), 151P. 63. Fardin V, Foucault F, Bock MD, Garret C. Binding profile of RP-67580, a new non-peptide SP antagonist: comparison with CP 96,345. Br J Pharmacol (1992) 105, 80. 64. Tabart M, Peyronel J-F. Synthesis of RPR 100893, prototype of a new series of potent and selective NK1 antagonists: the triarylperhydroisoindolols. Bioorg Med Chem Lett (1994) 4(5), 673-676. 65. Moussaoui SM, Carruette A, Montier F, Fardin V, Garret C. In vivo pharmacological profile of RPR 100893, a novel and selective non-peptide antagonist of the human NK1 receptor. Can J Physiol Pharmacol (1994) 72(Suppl.1), 459. 66. Fardin V, Pradier L, Morgat A, Moussaoui SM, Menager J, Carruette A. Species and NK1 versus NK2 tachykinin receptor specificities of perhydroisoindoles. 25th Ann Meeting Soc Neurosci, San Diego CA, November 11-16, 1995, 402.6. 67. Achard D,Peyronel J-F, Carruette A, Collemine P, Fardin V, Garret C. Synthesis and structure-activity relationships in cyanomethylperhydroisoindole potent NK1 antagonists. 25th Nat Med Chem Symp, Ann Arbor, Michigan USA, June 18-22, 1996, 84. 68. Fujii T. Discovery and pharmacological properties of selective neurokinin-receptor antagonists, FK-224 and FK888. Folia Pharmacol Jpn (1995) 106(3), 193-204. 69. Hirayama Y, Lei Y-H, Barnes PJ, Rogers DF. Effects of two novel tachykinin antagonists, FK-224 and FK-888, on neurogenic airway plasma exudation, bronchoconstriction and systemic hypotension in guinea pigs in vivo. Br J Pharmacol (1993) 108, 844-851. 90 IDrugs 1998 Vol 1 No 1 70. Walpole CSJ, Ko SY, Brown M, Beattie D, Campbell E, Dickenson F, Ewan S, Gentry C, Hughes GA, Lerpiniere J, Patel S, Urban L. SDZ NKT 343- A potent human NK-1 receptor antagonist with good oral analgesic activity in chronic pain models. Tachykinins in Health and Disease (September 7-11, 1997), Cairns, Australia, Abstract book p 4. 71. Walpole C, Ko SY. New pyrrolidine derivatives are tachykinin antagonists- useful as analgesics and as antiinflammatory and antiemetic agents. World (PCT) Patent WO-9618643 (1996). 72. Astolfi M, Parlani M, Lopez G, Conte B, Cirillo R, Fincham CI, Terracciano R, Manzini S. MEN 11149, a potent antagonist of the tachykinin NK1 receptor. Br J Pharmacol (1997) 120(Proc. Suppl.), abstract 202P. 81. Goldstein D. Clinical trials with lanepitant. William Harvey Res Inst Conf Tachykinins and their antagonists (October 1011, 1996), London. 82. Iyengar S, Hipskind PA, Gehlert DR, Schober D, Gitter BD, McMillian C, Couture R, Li D, Simmons RMA. LY303870: a centrally active NK-1 antagonist with a long duration of action. Tachykinins ’95 Florence (October 16-18, 1995), Abstract book, 122. 83. Natsugari H, Ikeura Y, Kiyota Y, Ishichi Y, Ishimaru T, Saga O, Shirafuji H, Tanaka T, Kamo I, Doi T, Otsuka M. Novel, potent, and orally active Substance P antagonists: synthesis and antagonist activity of N-benzylcarboxamide derivatives of pyrido[3,4-b]pyridine. J Med Chem (1995) 38(16), 3106-3120. 73. Swain, CJ. Neurokinin receptor antagonists. Exp Opin Ther Patents (1996) 6(4), 367-378. • Patent-orientated review on neurokinin receptor antagonists in the years 1990 - 1995. 84. MacLeod AM, Cascieri MA, Merchant KJ, Sadowski S, Hardwicke S, Lewis RT, MacIntyre DE, Metzger JM, Fong TM, Shepheard S, Tattersall FD, Hargreaves R, Baker R. Synthesis and biological evaluation of NK1 antagonists derived from L-tryptophan. J Med Chem (1995) 38(6), 934-941. 74. Astolfi M, Goso C, Maggi M, Manzini S. Binding profile of a potent and selective antagonist of NK-1 tachykinin receptors: MEN 10930. Tachykinins ’95 Florence (October 1618, 1995), Abstract book, 118. 85. Ofner S, Hauser K, Schilling W, Vassout A, Veenstra SJ. SAR of 2-benzyl-4-amino-piperidines: CGP-49823, an orally and centrally active non-peptide NK1 antagonist. Bioorg Med Chem Lett (1996) 6(14), 1623-1628. 75. Bonnet J, Kucharczyk N, Robinau P, Lonchampt M, Dacquet C, Regoli D, Fauchere J-L, Canet E. A water-soluble, stable dipeptide NK1 receptor-selective neurokinin receptor antagonist with potent in vivo pharmacological effects: S18523. Eur J Pharmacol (1996) 310(1), 37-46. 86. Emonds-Alt X, Advenier C, Soubrié P, Le Fur G, Breliere J-C. SR 48968: nonpeptide antagonist of the tachykinin NK2 receptor. Drugs Future (1995) 20(7), 701-707. • Review of the first non-peptide NK2 receptor antagonists. 76. Fauchere J-L, Kucharczyk N, Jacoby E, Lonchampt M, Robinau P, Dacquet C, Regoli D, Canet E. The dipeptide neurokinin-1 receptor antagonist S19752 is a potent and long-acting inhibitor of bronchoconstriction when administered by aerosol to the guinea pig in vivo. Bioorg Med Chem Lett (1997) 7(2), 203-208. 77. Boyle S, Guard S, Higginbottom M, Horwell DC, Howson W, McKnight AT, Martin K, Pritchard MC, O’Toole J, Raphy J, Rees DC, Roberts E, Watling KJ, Woodruff N, Hughes J. Rational design of high affinity tachykinin NK1 receptor antagonists. Bioorg Med Chem (1994) 2(5), 357-370. 78. Emonds-Alt X, Doutremepuich JD, Heaulme M, Neliat G, Santucci V, Steinberg R, Vilain P, Bichon D, Ducoux JP, Proietto V, van Broeck D, Soubrié P, Le Fur G, Brelière J-C. In vitro and in vivo biological activities of SR 140,333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol (1993) 250, 403-413. 79. Amann R, Schuligoi R, Holzer P, Donnerer J. The nonpeptide NK1 receptor antagonist SR 140,333 produces long-lasting inhibition of neurogenic inflammation, but does not influence acute chemo- or thermonociception in rats. Arch Pharmacol (1995) 352(2), 201-205. 80. Hipskind PA, Howbert JJ, Bruns RF, Cho SSY, Crowell TA, Foreman MM, Gehlert DR, Iyengar S, Johnson KW, Krushinski JH, Li DL, Lobb, KL, Mason NR, Muehl BS, Nixon JA, Phebus LA, Regoli D, Simmons RM, Threlkeld PG, Waters DC, Gitter BD. 3-Aryl-1,2-diacetamidopro-pane derivatives as novel and potent NK-1 receptor antagonists. J Med Chem (1996) 39(3), 736-748. 87. van Schoor J, Joos G, Chasson B, Brouard R, Pauwels R. The effect of the oral nonpeptide NK2 receptor antagonist SR 48968 on neurokinin A-induced broncho-constriction in asthmatics. Eur Resp J (1996) 9(Suppl. 23), 289S. 88. Emonds-Alt X, Advenier C, Cognon C, Croci T, Daoui S, Ducoux JP, Landi M, Naline E, Neliat G, Poncelet M, Proietto V, Van Broeck D, Vilain P, Soubrié P, Le Fur G, Maffrand JP, Brelière J-C. SR 144190, A new potent non-peptide tachykinin NK2 receptor antagonist. Biochemical and pharmacological activities Tachykinins in Health and Disease (September 7-11, 1997), Cairns, Australia, 5. 89. Kubota H, Kakefuda A, Nagaoka H, Yamamoto O, Ikeda K, Shibanuma T, Isomura Y. Spiro-substituted piperidines: potent and selective neurokinin A antagonists. AFMC Int Med Chem Symp / AIMECS 95, Tokyo, Japan (September 3-8, 1995) M139. 90. Shenyi AB, Aharony D, Brown FJ, Buckner CK, Campbell JB, Dedinas RF, Gero TW, Green RC, Jacobs RT, Kusner EJ, Miller SC, Ohnmacht CJ, Palmer WE, Smith RW, Steelman GB, Ulatowski TG, Veale CA, Walsh SA. The design and synthesis of a potent, selective and orally active NK2 antagonist ZD-7944. 214th ACS National Meeting, Las Vegas, NV September 7-11 (1997), MEDI 264. 91. Jacobs RT. 4-(Aryl-substituted)-piperidines as neurokinin receptor antagonists. Eur Pat Appl EP630887 (1994). 92. Maggi CA, Astolfi M, Giuliani S, Goso C, Manzini S, Meini S, Patacchini R, Pavone V, Pedone C, Quartara L, Renzetti AR, Giachetti A. MEN 10,627, a novel polycyclic peptide antagonist of tachykinin NK2 receptors. J Pharmacol Exp Ther (1994) 271, 1489-1500. Review Neurokinin antagonists 93. Perretti F, Ballati L, Manzini S, Maggi CA, Evangelista S. Antibronchospastic activity of MEN 10,627, a novel tachykinin NK2 receptor antagonist, in guinea pig airways. Eur J Pharmacol (1995) 273, 129-135. 94. Cooper AWJ, Adams HS, Bell R, Gore PM, McElroy AB, Pritchard JM, Smith PW, Ward P. GR159897 and related analogues as highly potent, orally active non-peptide neurokinin NK2 receptor antagonists. Bioorg Med Chem Lett (1994) 4(16), 1951-1956. 95. Howson W. Rational design of tachykinin receptor antagonists. Drug News and Perspectives (1995) 8(2), 97-103. 96. Guard S, Horwell DC, Howson W, Hughes J, Pritchard MC, Roberts E. Rational design of high affinity NK1 and NK2 tachykinin receptor ligands. Br J Pharmacol (1993) 110 (Suppl.), 56P. 97. Aharony D, Buckner CK, Ellis JL, Ghanekar SV, Graham A, Kays JS, Little J, Meeker S, Miller SC, Undem BJ, Waldron IR. Pharmacological characterization of a new class of nonpeptide neurokinin A antagonists that demonstrate species selectivity. J Pharmacol Exp Ther (1995) 274, 12161221. 98. Lunde H, Hedner J, Svedmyr N. Lack of efficacy of 4 weeks treatment with the neurokinin receptor antagonist FK224 in mild to moderate asthma. Eur Resp J (1994) 7(Suppl. 18), 151S. 99. Fujii T, Kawamura A, Yasumitsu R, Hirayama Y. Effects of FK224 (NK1 + NK2 dual antagonist) on the knee joint inflammation induced by carrageenin, substance P, serotonin and antigen. Regul Pep (1992), Suppl 1, Abst. S59. • Disclosure of the first non-peptide dual NK1/NK2 receptor antagonist. 100. Burkholder TP, Kudlacz EM, Le T-B, Knippenberg RW, shatzer SA, Maynard GD, Webster ME, Horgan SW. Identification and chemical synthesis of MDL-105212, a non-petide tachykinin antagonist with high affinity for NK1 and NK2 receptors. Bioorg Med Chem Lett (1996) 6(8), 951-956. 101. Kudlacz EM, Scott A, Shatzer SA, Knippenberg RW, Logan DE, Poirot M, van Giersbergen PLM, Burkholder TP. In vitro and in vivo characterization of MDL 105,212A, a nonpeptide NK-1/NK-2 tachykinin receptor antagonist. J Pharmacol Exp Ther (1996) 277, 840-851. 102. Kudlacz EM, Knippenberg RW, Shatzer SA, Kehne JH, McCloskey TC, Burkholder TP. The peripheral NK-1/NK-2 receptor antagonist MDL-105172A inhibits tachykininmediated respiratory effects in guinea pigs. J Auton Pharmacol (1997) 17(2), 109-119. 91 103. Shah SK, Hale JJ, Qi H, MillerDJ, Dorn Jr CP, Mills SG. Discovery of substituted spiroindolinopiperidines as orally active dual antagonists of NK1 and NK2 receptors. 212th ACS National Meeting, Orlando, Florida, USA, August 25-29, (1996), MEDI 136. 104. Mills, SG et al. William Harvey Res Inst Conf Tachykinins and their antagonists (October 10-11, 1996), London, P5. 105. Robineau P, Lonchampt M, Kucharczyk N, Krause JE, Regoli D, Fauchère JL, Prost JF, Canet E. In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist. Eur J Pharmacol (1995) 294, 677684. • Disclosure of the first potent non-peptide NK3 receptor antagonist. 106. Emonds-Alt X, Bichon D, Ducoux JP, Heaulme M, Miloux B, Poncelet M, Proietto V, van Broeck D, Vilain P, Neliat G, Soubrié P, Le Fur G, Brelière JC. SR 142801, the first nonpeptide antagonist of the tachykinin NK3 receptor. Life Sci (1995) 56(1), PL27-32. 107. Boden P, Eden JM, Hodgson J, Horwell DC, Pritchard MC, Raphy J, Suman-Chauhan N. The development of a novel series of non-peptide tachykinin NK3 receptor selective antagonists. Bioorg Med Chem Lett (1995) 5(16), 1773-1778. 108. Giardina GAM, Sarau HM, Farina C, Medhurst AD, Grugni M, Foley JJ, Raveglia LF, Schmidt DB, Rigolio R, Vassallo M, Vecchietti V, Hay DWP. 2-Phenyl-4-quinoline-carboxamides: a novel class of potent and selective non-peptide competitive antagonists for the human neurokinin-3 receptor. J Med Chem (1996) 39(12), 2281-2284. 109. Sarau HM, Griswold DE, Potts W, Foley JJ, Schmidt DB, Martin LD, Medhurst AD, Giardina GAM, Hay DWP. Pharmacology of SB-222200, a selective high affinity orally active nonpeptide NK-3 receptor antagonist. Tachykinins in Health and Disease (September 7-11, 1997), Cairns, Australia, Abstract book p 5. 110. Catalioto RM, Criscuoli M, Cucchi P, Giachetti A, Giannotti D, Giuliani S, Lecci A, Lippi A, Patacchini R, Quartara L, Renzetti AR, Tramontana M, Arcamone F, Maggi CA. MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist. Br J Pharmacol (1998) 123, 81-91. 92 IDrugs 1998 Vol 1 No 1 Plant-derived anticancer agents currently in clinical use or in clinical trials Hui-Kang Wang Address Natural Products Laboratory Division of Medicinal Chemistry and Natural Products School of Pharmacy University of North Carolina Chapel Hill North Carolina 27599 USA Email: [email protected] IDrugs 1998 1(1):92-102 Current Drugs Ltd ISSN 1369-7056 Throughout history, plant products and their modified analogs have been rich sources of clinically useful drugs, including anticancer agents. This review covers those agents that are currently in clinical use or in clinical trials as cancer chemotherapeutic drugs, including vinblastine, vincristine, Navelbine, etoposide, Teniposide, Taxol and, most recently, Taxotere, topotecan, and irinotecan. Introduction Current estimates indicate that there are about 250,000 species of flowering plants on earth, and of these, approximately 155,000 are found in the tropics [1]. Using plants as resources for drug discovery can be traced back to prehistoric time, and has been very efficient. Medicinal plants used in Chinese traditional medicine and other ethnic medicine have been employed for centuries in many countries for treating cancers, and are great treasures for new drug discovery. However, only recently have systematic efforts been made to isolate and characterize the active principles from antitumor-active plant extracts. In the past three decades, numerous cytotoxic antitumor agents and their analogs have been discovered and developed in many laboratories, and these have been reviewed [2-7]. This review covers only those agents that are currently in clinical use or in clinical trials as cancer chemotherapeutic drugs. Alkaloids Camptothecin and its analogs Camptothecin (CPT, 1) and 10-hydroxycamptothecin (OPT, 2; Chiba) were first isolated from the wood, bark, and fruits of the Chinese tree Camptotheca acuminata (Nyssaceae) [8]. Sodium CPT was tested for treatment of gastric carcinoma and other malignancies in the US in the 1970s, but its use was halted due to the severe side-effects. However, in China, an effort was made to decrease its toxicity and increase its activity. The monoammonium glycyrrhizinate of CPT was found to be effective in decreasing the toxicity of CPT. Also, a CPT suspension with a particle size of approximately 1 mm was made for intravenous use. Clinical trials involving 450 patients showed that this CPT suspension had a definite therapeutic effect on liver carcinoma, increasing the percentage of successful surgical operations from 18% to 49%. The one year survival rate was raised from 39% to 54%. Clinical studies on 250 cancer patients revealed that OPT was effective against primary liver carcinoma, cancer of the head and neck, leukemia, and gastric carcinoma, with less adverse effects than those caused by sodium CPT [9]. Camptothecin has drawn great attention worldwide as a potent inhibitor of DNA topoisomerase I. (Topoisomerases are responsible for the winding and unwinding of the supercoiled DNA composing the chromosomes. If the chromosomes cannot be unwound, transcription of the DNA message cannot occur and the protein cannot be synthesized.) Structure-activity studies have revealed a direct relationship between the ability of CPT analogs to inhibit topoisomerase I catalytic activity and their potency as cytotoxic agents [10-13]. These studies support the view that the interaction between CPT and the topoisomerase I-DNA complex is responsible to the cytotoxicity of CPT and its analogs. Among the numerous camptothecin derivatives, topotecan (SKF-104864, TPT, 3; SmithKline Beecham), CPT11 (4; Yakult Honsha), and 9-aminocamptothecin (5; Research Triangle Institute) are being tested clinically as anticancer drugs against colon, ovarian, and other cancers in Europe, Japan, and the US, respectively. These drugs have shown significant activity in advanced malignancies. On May 29, 1996, the US Food and Drug Administration (FDA) approved topotecan for use in patients whose ovarian cancer had progressed after the failure of first-line chemotherapy. Topotecan is marketed by SmithKline Beecham under the name of Hycamtin®. CPT-11 received accelerated approval in June 1996, one day after the FDA Oncologic Drugs Advisory Committee recommended approval. The drug is ® marketed by Daiichi (Irinotecan ), Pharmacia & Upjohn ® ® (Camptosar ), and Rhone-Poulenc Rorer (Campto ) [14]. Vinca alkaloids Vinca alkaloids, including vinblastine (6; Gedeon Richter) and vincristine (7), were first isolated from Cantharanthus rosea in the 1960s. Since then, more than 90 unique bisindole alkaloids have been isolated from the genus Cantharanthus [15]. Vinblastine and vincristine are currently used in the treatment of various cancers in the US and other countries, while semisynthetic vindesine (8; Eli Lilly) is now in a phase II trial for the treatment of leukemia, hepatocellular cancer [16], and non-small cell lung cancer [17] in South Africa, Canada, and Europe, respectively. S-12363 (Vinfosiltine, 9; Servier), another semisynthetic vinca alkaloid derivative, which has an a-aminophosphate moiety at the C-23 position of O-deacetylvinblastine, has entered a phase II study for the treatment of advanced malignant melanoma [18] and advanced breast cancer [19]. Vinorelbine (5’-noranhydrovinblastine, 10) is a semisynthetic analog that was initially developed by Pierre Potier of France; it gained approval in France in April 1989 and April 1991, for the treatment of non-small cell lung cancer and advanced breast cancer, respectively [20]. In 1989, Burroughs Wellcome acquired the license to develop and market vinorelbine in North America. Since 1990, Burroughs Wellcome has been conducting studies of vinorelbine to meet US FDA requirements for approval and to investigate its potential for treating other cancers. To date, these have Review Plant-derived anticancer agents 93 Figure 1. Structures of camptothecin and analogs CH3 N CH3 HO N HO O N N O N N H3C O H3C HO O H3C HO O O 3 Topotecan (SmithKline Beecham) 2 10-Hydroxycamptothecin (Chiba) N NH2 CH3 N O HO O 1 Camptothecin O N O N O N O N O N H3C H3C O O HO HO O O 4 CPT-11 (Yakult) confirmed the earier results obtained from the European studies. More than 3,000 patients worldwide have received vinorelbine in clinical studies. The US FDA approved a ® semisynthetic vinorelbine (Navelbine , Pierre Fabre) for the treatment of advanced non-small cell lung cancer in 1995 [21]. 5 9-Aminocamptothecin (Research Triangle Institute) breast cancer [25], acute myelogenous leukemia [26], acute myelogenous leukemia [27], and in patients with myelodysplastic syndrome (MDS) and MDS evolving to acute myeloid leukemia [28]. Colchicine Homoharringtonine Cephalotaxus alkaloids, including harringtonine, isoharringtonine, and homoharringtonine (11; Chinese Academy of Medical Sciences) were isolated from Cephalotaxus harringtonia [22]. Cephalotaxus alkaloids are inhibitors of protein synthesis. The effects of harringtonine and homoharringtonine on protein synthesis are: degradation of polyribosomes; release of completed protein chains; and, delayed inhibition of initiation of protein synthesis (compared to cycloheximide) without affecting chain elongation [23]. Research progress on Cephalotaxus alkaloids has been summarized, including their discovery, nature of antitumor activity, isolation, structural characterization, synthesis, biogenesis, and mechanism of physiological action [24]. Homoharringtonine has been clinically tested in advanced Colchicine (12), an antimitotic alkaloid isolated from Colchicum autumnale and is a classical drug used in the treatment of gout and familial Mediterranean fever (FMF) [29]. Recent research has revealed that colchicine and its analogs also show anti-flammatory [30], antitumor [31], and anti-HIV activities [32]. It has been suggested that most of the biological effects of colchicine are related to its tubulin binding action. High toxicity has limited the clinical usage of colchicine. However, colchicinamide (13), a semisynthetic derivative of colchicine in which the C-14 methoxy group is replaced by an amino group, showed a therapeutic index which was 1.75-fold greater than that of colchicine against various solid tumors. Colchicinamide has been used in China for the treatment of breast cancer and other solid tumors [8]. 94 IDrugs 1998 Vol 1 No 1 Figure 2. Structures of vinca alkaloids OH OH N HN N HN CH3 CH3 O O H3C H3C N O O H3C CH3 O H N H3C N O O O H OH O O H3C CH3 O H N CH3 H CH3 6 Vinblastine O H OH O O O CH3 CH3 7 Vincristine OH OH N HN H3C O H3C N O O N CH3 H3C CH3 H3C H H N N H3C H O CH3 O CH3 O O N HN H3C OH OH H H OH O NH2 NH H3C H3C 8 Vindesine 9 Vinfosiltine N HN CH3 O H3C N O O H3C CH3 O H N H3C O H OH O O 10 CH3 CH3 O CH3 O CH3 P O Review Plant-derived anticancer agents Indirubin Figure 3. Structure of homoharringtonine Indirubin (19) is an antileukemic compound isolated from the Chinese medicine Indigo naturalis (Qing-Dai), a blue pigment made from the leaves of Baphicacanthus cusia, Indigofera tinctoria, Polygonum tingctorium, or Isatis tingctoris [40]. Qing-Dai has been used in a traditional Chinese prescription "Dang Gui Lu Hui Wan", which is used in the treatment of chronic myelocytic leukemia. Indirubin and its more potent water-soluble synthetic derivative, N-methylindirubin oxime (20), have been used for treatment of chronic myelocytic leukemia in China [41]. Interestingly, indirubin and indigo have been identified from the urine of an acute myelomonocytic leukemia patient after admin-istration of mitoxantrone (Immunex) and etoposide [42]. O N O O H3C H O H3C OH H3C OH O CH3 O 95 O 11 Homoharringtonine (Chinese Academy of Medical Science) Terpenes Taxol Deacetylcolchicine (14), which showed effectiveness against Hodgkin’s lymphoma, chronic granulocytic leukemia, and melanoma, has been in phase II clinical trials [33]. Ellipticine Ellipticine (15) is an antitumor alkaloid isolated from Ochrosia elliptica [34] and subsequently from several other species of Ochrosia, including O acuminata [35], and Bleekeria vitiensis [36]. Ellipticine is a topoisomerase II inhibitor which has demonstrated anticancer activity in several animal and human tumor systems. Three analogs of ellipticine have been tested clinically. DHE (N-2-(Diethylaminoethyl)-9hydroxy-ellipticinium chloride, 16) is an intercalating agent, which is currently in a phase II trial in Europe for the treatment of breast cancer using a weekly regimen [37]. A phase II trial of NMHE (2-N-methyl 9-hydroxy-ellipticine, 17; Sanofi) was conducted in 57 patients with advanced 2 metastatic breast cancer given as 100 mg/m weekly, and two complete regressions (of 3 and 12 months) and seven regressions of over 50% were observed. A total regression rate of 19% regression was mainly observed in cutaneous or subcutaneous metastases [38]. The compound has since been launched. SR-95325-B (retelliptine dihydrochloride, NSC-D626717-W, 18; Sanofi) is another ellipticine derivative having a very high level of antitumor activity in resistant murine solid tumor models and was in a phase I trial [39], before being discontinued. Taxol (21; NIH), a highly promising chemotherapeutic diterpene which is active against ovarian and breast cancers, was isolated from the bark of the Pacific yew tree, Taxus brevifolia [43]. Since yields of taxol are very low and collection of the bark destroys the tree, which is an endangered species, worldwide attempts have been made to identify other species as sources of taxol or related compounds for semisynthesis. A recent review has covered all aspects of taxane diterpenoids, including their isolation, total synthesis, and biological and SAR studies [44]. With a new synthetic process [101], baccatin III (22), extracted from a renewable source, Taxus baccata, is converted into taxol, and then formulated. The FDA approved the natural form of taxol (paclitaxel®) in December 1992 for treatment of metastatic ovarian cancer after failure of first-line or subsequent chemotherapy. The FDA has further determined that the semisynthetic paclitaxel® is bioequivalent to that produced from Pacific yew bark and has granted marketing approval for a semisynthetic form of paclitaxel® (BristolMyers Squibb) for treating of certain cancers of the breast and ovary. The use of taxol for treating metastatic breast cancer received marketing approval in April 1994. A shorter course of infusion for metastatic ovarian cancer (3 h instead of 24 h) was approved in June 1994 to reduce the incidence of neutropenia. Figure 4. Structures of colchicine and derivatives O O H3C H3C CH3 O O H3C NH H3C NH CH3 O O O H3C O H3C 12 Colchicine H3C NH2 O O O H3C H3C O H3C NH2 O H3C 13 Colchicinamide 14 Deacetylcolchicine 96 IDrugs 1998 Vol 1 No 1 Figure 5. Structures of ellipticine derivatives CH3 CH3 CH3 HO N N N H N H H CH3 CH3 N + Cl CH3 15 Ellipticine 16 DHE CH3 N + CH3 O H3C CH3 O CH3 N H3C N H N HN CH3 HO O N H CH3 H Cl CH3 17 NMHE 18 SR-95325B (Elf Sanofi) Figure 6. Structures of indirubin and derivatives O N H N OH H N N H N H3C O O 19 Indirubin In a separate action, an FDA advisory panel recom-mended against immediate approval for a semisynthetic compound, docetaxel (23, Taxotere®; Rhone-Poulenc Rorer). Docetaxel has greater cytotoxic effects than taxol on human tumor cells [45]. Although docetaxel is effective against advanced breast cancer and certain types of lung cancer, the FDA committee stated that further studies of side-effects, especially effects on the immune system, are required. Docetaxel is apparently more toxic to patients than taxol. However, Rhone-Poulenc Rorer officials have said that side-effects could be controlled through additional medication. Taxol possesses a 6-8-6 tri-ring taxane skeleton, with nine chiral centers; it was immediately chosen as a total synthesis target by competent organic chemists after its discovery. After a challenge of more than 20 years, the goal was finally 20 achieved independently in 1994 by Holton et al and Nicolaou et al [46-47]. Taxol exhibits an unique mechanism of action. It promotes polymerization of tubulin and stabilizes the structure of intracellular microtubules. This process effectively inhibits the normal dynamic reorganization of the microtubules that is necessary for interphase and mitotic functions [48]. Lignans Podophyllotoxins An important anticancer lignan is podophyllotoxin (24; Hafslund Nycomed), isolated from Podophyllum peltatum or Podophyllum emodii (Berberidaceae). The aqueous extracts of the roots or rhizomes of Podophyllum peltatum were called podophyllin and were included in the first US Review Plant-derived anticancer agents 97 Figure 7. Taxol derivatives O O O O N H H3C H3C O H3C H3C CH3 O O CH3 OH CH3 HO HO HO O O O CH3 OH CH3 CH3 OH O H O O O O H O O O O H3C H3C 21 Taxol 22 Baccatin III O O O O CH3 H3C H3C H3C H3C N H CH3 O CH3 OH O CH3 OH HO O O H O O O H3C 23 Docetaxel (Rhone-Poulenc Rorer) Pharmacopoeia (USP 1820). It was used hundreds of years ago as a cathartic and anthelmintic by the American Indians and by natives of the Himalayan mountain region. The American colonists subsequently used podophyllin as an emetic. The antimitotic properties of podophyllin were first discovered in 1946, and chemical analysis of podophyllin revealed a number of cytotoxic lignan compounds, including podophyllotoxin [49]. Due to the high toxicities of these natural products, Sandoz initiated a semisynthetic podophyllotoxin derivative program. The two most successful anticancer analogs from this program were etoposide (VP-16 or VP-16-213, 25) and teniposide (VM-26, 26). VM-26 was synthesized in late 1965 and VP-16 about nine months later. VM-26 was first tested in man in 1967, and VP-16 in 1971. Sandoz commercialized VM-26 in some countries in 1976 under the names of Vumon® and Vehem®. Etoposide, which was approved by the US FDA for the treatment of testicular cancer in 1983 and was introduced to the US market as VePesid® by Bristol-Myers Squibb, is one of the most active anticancer agents in the treatment of testicular teratoma, Hodgkin's and nonHodgkin's lymphomas, small-cell lung cancer, and a variety of other malignancies. Both VP-16 and VM-26 appear to be useful compounds in the treatment of certain tumors. Although they differ somewhat in their pharmacokinetic profiles, there is no evidence that one drug is superior to the other in a specific tumor type; their clinical toxicities are also similar [50]. A number of reviews have been published and cover all aspects of VM-16 and VM-26, including their chemistry, biology, pharmacokinetics, and clinical applications. Among these papers, two excellent reviews have detailed the discovery of etoposide and the development of podophyllotoxin derivatives [51-52]. Two recent reviews have summarized hundreds of newly synthesized VP-16 analogs, their biological evaluation and their bioanalysis [53-54]. Due to problems encountered with VP-16 or VM-26, such as 98 IDrugs 1998 Vol 1 No 1 Figure 8. Structures of lignans O H H 3C O O H O OH H H OH HO O O O O O O H O H3C O O CH3 O H3C O OH 24 Podophyllotoxin (Hafslund Nycomed) O H O O N H O O O CH3 26 Teniposide H H3C O OH 25 Etoposide H O O CH3 H3C H3C H O O H O O O H H 3C H O H O OH HO O O O H H O H S H H OH HO O O N CH3 HO H NH O H 3C O H H O O O O O O H H 3C O H O O HO H CH3 H3C O O O O CH3 H3C O O CH3 OH O OH P HO O O 27 Etoposide phosphate (Bristol-Myers Squibb) 28 NK-611 (Nippon Kayaku) toxicity, poor water solubility, and the development of resistance by tumor cells, investigators are exploring new synthetic analogs for new lead compounds with an enhanced therapeutic index, extensive therapeutic scope and higher water-solubility. From these endeavors, three analogs, etoposide phosphate (27; Bristol-Myers Squibb), NK-611 (28; Nippon), and GL-331 (29; University of North Carolina), have entered clinical trials. Etoposide phosphate Etoposide phosphate (BMY-40481, 27; Bristol-Myers Squibb) is a new water-soluble analog of VP-16. It acts as a prodrug, probably through activation by plasma phosphatase. In vitro, 27 was less potent than VP-16; however, in animals, 27 is converted to VP-16 within a few minutes, regardless of mode of administration [55]. The phase I clinical and 29 GL-331 (University of North Carolina) pharmacokinetic study of oral etoposide phosphate has been reported [56-59]. The recommended oral dose of 27 is 25 2 mg/m /day for 5 days, every 3 weeks in high-risk patients [59]. NK-611 NK-611 (4’-demethylepipodophyllotoxin-9(2-deoxy-2-dimethylamino-4,6-O-ethylidene)-b-D-glucopyranoside, 28) was developed by Nippon Kayaku (Tokyo, Japan). It is a new water-soluble derivative of VP-16 with potent antineoplastic activity [60]. A dimethylamino group was introduced into the sugar moiety to make the water-soluble amine salt. The simultaneous determination of 28 and its metabolite (DeNK611) in dog plasma, by column-switching HPLC, has been reported [60] and the clinical pharmacokinetic studies of 28 have been performed [61,62]. The maximum tolerated dose Review Plant-derived anticancer agents of NK-611 administered daily for 21 consecutive days was 12.5 mg/day. The dose-limiting toxicity is granulocytopenia. The recommended doses for further phase II clinical trials 2 are 10 mg/day [61] or 20 mg/m for 5 consecutive days every 4 weeks [62] for patients who had never been treated, or have undergone previous chemotherapy only once. GL-331 Recently, GL-331 (29), a new derivative of epipodophyllotoxin, discovered and developed in Dr Lee’s laboratory and licensed to Genelabs, CA, USA, completed phase I clinical trials as an anticancer drug at MD Anderson Cancer Center, TX, USA in May 1993, and entered phase IIa trials in Taiwan in January 1997. GL-331, a novel topoisomerase II inhibitor of the epipodophyllotoxin family, has been evaluated in a variety of in vitro and in vivo systems, and has shown activity in both VP-16-sensitive and resistant cell lines and in animal models. The selectivity and potency of GL-331 against tumors were evaluated by the National Cancer Institute (NCI) using the in vitro screening of drug cytotoxicity against a human tumor panel, consisting of 60 tumor cell lines derived from nine histological types. Results indicated that leukemia, non-small cell lung cancer, colon cancer and renal cancer exhibited preferential susceptibility to GL-331. GL-331 achieved optimal therapeutic effect against L1210/0 (13.5 mg/kg) in terms of ILS, long-term survival, and tumor burden reduction at approximately half the dosage of VP-16. GL-331 also outperformed VP-16 in terms of ILS, long-term survival, and tumor burden. The final publication of data from phase I clinical trials of GL-331 is not available yet, however, some details have been discussed in a review article [54]. Flavones Flavone-8-acetic acid Numerous flavonoids have been isolated as cytotoxic antitumor agents. Flavone-8-acetic acid (FAA, NSC-347512, 30; Merck & Co) is a synthetic derivative of the basic flavonoid skeleton with a unique form of preclinical antitumor activity, but its mechanism of action is still not known. The phase I clinical trials of FAA were conducted in the US [63], France [64], UK [65], and New Zealand [66] for the treatment of advanced malignant melanoma. One result of these trials is evidence that treatment of patients with FAA and rIL-2 induces the synthesis of nitric oxide, a physiological mediator and potential cytotoxic agent [66]. Development of FAA has now been discontinued. 99 Proteins Ricin Ricin is a plant protein isolated from Ricinus communis [67]. This widely used toxin consists of two 30,000 to 32,000 molecular weight polypeptide chains, A and B, linked by a disulfide bond. After binding to the cell via the B-chain, the A-chain subunit crosses a cell membrane by an ill-defined mechanism to reach the cytosol where it inactivates the 60S ribosomal subunit and, thus, terminates protein synthesis. A single A-chain molecule can inactivate 1500 ribosomes/min and, thus, very few A-chain molecules, perhaps as few as one, are sufficient to kill a cell. This extreme potency makes this protein an attractive candidate for monoclonal antibody targeting. The majority of recent research has concentrated on the use of A-chain immunotoxins in which the monoclonal antibody is attached to purified A-Chain via a disulfide bond [68]. To date, phase I/II clinical trials of some monoclonal antibody-ricin A chains have been conducted by Xoma for the treatment of metastatic melanoma [69], colon cancer [70], and B-cell lymphoma [71] in the US. Conclusion The new plant-derived anticancer agents and their analogs that are currently in clinical use or in clinical trials as cancer chemotherapeutic drugs have been briefly reviewed. To develop these anticancer agents, the cooperation between botanists, chemists, and biologists is necessary and should be further encouraged and strengthened. However, compared to the huge number of plants which exist on earth, only a small number of species have been explored, and even then incompletely. In most cases, the polar or water-soluble constituents, or the macro molecules of the plant extracts have not been thoroughly investigated and are worthy of further study. Recent significant advances in biological assay methodology, isolation technology and spectroscopic structural determination, will be helpful in solving these problems. Nine anticancer drugs isolated or developed from plant sources have been approved for clinical use in the US; these include vinblastine, vincristine, Navelbine, etoposide, Teniposide, Taxol and, most recently, Taxotere, topotecan, and irinotecan (Yakult). This continuing success guarantees an optimistic future. New anticancer agents, especially anti-solid tumor agents, will be continuously discovered. Continuing searches among medicinal plants and the semi-synthesis of analogs of lead active principles will undoubtedly lead to further examples of novel plant-derived anticancer agents. Figure 9. Structure of flavone-8-acetic acid References O OH 1. Prance GT: Floristic inventory of the tropics: where do we stand? Ann Missouri Bot Gard (1977) 64:659-684. O 2. Lee KH: Chinese plant antitumor agents In: Advances in Chinese Medicinal Materials Research, Chang HM, Yeung HW, Tso WW, Koo A (Eds), World Scientific Publishing Co, Singapore (1985):353-367. O 3. Lee KH: Antitumor Agents 91. Recent progress in the discovery and development of plant antitumor agents. Kaohsiung J Med Sci (1987) 3:234-250. 30 NSC-347512 (Merck) 100 IDrugs 1998 Vol 1 No 1 4. Lee KH, Yamagishi T: Antitumor Agents 95. Antineoplastic terpenoids from Chinese medicinal plants. Abstr of Chin Med (1987) 1:606-625. 5. Lee KH: Antineoplastic agents and their analogs from Chinese traditional medicine In: Am Chem Soc Symp Series 534 Human Medicinal Agents from Plants. Kinghorn AD, Balandrin M (Eds.) ACS, Washington DC (1993):170-190. 6. Lee KH, Sun L, Wang HK: Antineoplastic alkaloids from Chinese medical plants and their analogs. J Chin Chem Soc (Taiwan) (1994) 41:371-384. 7. Lee KH, Wang HK: Recent advances in the chemical and antitumor structure-activity correlation studies of camptothecin. Chemistry (Taiwan) (1995) 53:65-75. 8. Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA: Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc (1966) 88:3888-3890. •• The first report of the discovery of camptothecin. 18. Stanley AJ, Kaprielian R, Retsas S: Evaluation of S-12363, a novel vinca alkaloid drug in the treatment of advanced malignant melanoma. A phase II study. Anticancer Res (1993) 13:31-32. 19. Adenis AJ, Pion M, Fumoleau P, Pouillart P, Marty M, Giroux B, Bonneterre J: Phase II study of a new vinca alkaloid derivative, S12363, in advanced breast cancer. Cancer Chemother Pharmacol (1995) 35:527-528. 20. Goa KL, Faulds D: Vinorelbine, a review of its pharmacological propertities and clinical use in cancer chemotherapy. Drug Evaluation (1994) 5:200-234. 21. Crawford J: Update: vinorelbine (navelbine) in non-small cell lung cancer. Sem Oncol (1996):23 (2 Suppl 5) 2-7. 22. Powell RG, Weisleder D, Smith CR Jr, Rohwedder WK: Structures of harringtonine, isoharringtonine, and homoharringtonine. Tetrahedron Lett (1970) 11:815-818. 23. Huang MT: Harringtonine, an inhibitor of initiation of protein biosynthesis. Mol Pharmacol (1975) 11:511-519. 9. Hsu B: The use of herbs as anticancer agents. Am J Chin Med (1980) 8:301-306. • A comprehensive review of anticancer agents developed from Chinese herbal medicines. 24. Smith CR Jr, Powell RG, Mikolajczak KL: The genus Cephalotaxus: source of homoharringtonine and related anticancer alkaloids. Cancer Treat Rep (1976) 60:1157-1170. •• A comprehensive review of Cephalotaxus anticancer alkaloids. 10. Hertzberg RP, Caranfa MJ, Holden KG, Jakas DR, Gallagher G, Mattern MR, Mong SM, Bartus JO, Johnson RK, Kingsbury WD: Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity. J Med Chem (1989) 32:715-720. 25. Zhao TP, Ding GX, Gao HY, Shen ZZ, Li KY: A clinical trial of homoharringtonine in the treatment of advanced breast cancer. Tumori (1986) 72:395-398. 11. Hsiang YH, Liu LF, Wall ME, Wani MC, Nicholas AW, Manikumar G, Kirschenbaum S, Silber R, Potmesil M: DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res (1989) 49:43854389. 12. Jaxel C, Kohn KW, Wani MC, Wall ME, Pommier Y: Structureactivity study of the actions of camptothecinderivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity. Cancer Res (1989) 49:1465-1469. 13. Gallagher G, Caranfa MJ, McCabe FL, Faucette LF, Johnson RK, Hertzberg RP: Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity. J Med Chem (1991) 34:98-107. 14. Cruzan SM: FDA approves ovarian cancer drug. FDA Press Releases. May 1996. 15. Blasko G, Cordell GA: Isolation, structure elucidation, and biosynthesis of the bisindole alkaloids of Catharanthus In: The alkaloids Vol. 37. Brossi A (Ed). Academic Press, New York (1990):1-76. 16. Falkson G, Burger W: A phase II trial of vindesine in hepatocellular cancer. Oncology (1995) 52:86-87. 17. Bretti SG, Bonardi M. Celano A, Comandone A, Casadio C, Marchisio U, Forconi G, Manzoni SC: Non small cell lung cancer treatment with carboplatin and vindesine: a phase II study. Anticancer Res (1992) 12:1459-1461. 26. Feldman E, Arlin Z, Ahmed T, Mittelman A, Puccio C, Chun H, Cook P, Baskind P: Homoharringtonine is safe and effective for patients with acute myelogenous leukemia patients. Leukemia (1992) 6:1185-1188. 27. Feldman E, Arlin Z, Ahmed T, Mittelman A, Puccio C, Chun H, Cook P, Baskind P: Homoharringtonine in combination with cytarabine for patients with acute myelogenous leukemia. Leukemia (1992) 6:1189-1191. 28. Feldman EJ, Seiter KP, Ahmed T, Baskind P, Arlin ZA: Homoharringtonine in patients with myelodysplastic syndrome (MDS) and MDS evolving to acute myeloid leukemia. Leukemia (1996) 10:40-42. 29. Zemer D, Revach M, Pras M, Modan B, Schor S, Sohar E, Gafni J: A controlled trial of colchicine in preventing attacks of familial mediterranean fever. New Engl J Med (1974) 291: 932-934. 30. Sugio K, Maruyama M, Tsurufuji S, Sharma PN, Brossi A: Separation of tubulin-binding and anti-inflammatory activity in colchicine analogs and congeners. Life Sci (1987) 40:35-39. 31. Zweig MH, Chignell CF: Interaction of some colchicine analogs, vinblastine and podophyllotoxin, with rat brain microtubule protein. Biochem Pharmacol (1973) 22:2141-2150. 32. Tatematsu H, Kilkuskie RE, Corrigan AJ, Bodner AJ,. Lee KH: Anti-AIDS agents, 3. Inhibitory effects of colchicine derivatives on HIV replication in H9 lymphocyte cells. J Nat Prod (1991) 54:632-637. 33. Yoshikuni I, Brossi A: Colchicine models: Synthesis and binding to tubulin of tetramethoxybiphenyls. Helv Chim Acta (1988) 71:1199-1209. Review Plant-derived anticancer agents 101 34. Goodwin S, Smith AF, Horning EC: Alkaloids of Ochrosia elliptica Labill. J Am Chem Soc (1959) 81:1903-1908. 48. Horwitz SB: Taxol (paclitaxel): mechanisms of action. Ann Oncol (1994) 5 (Suppl 6):S3-6. 35. Lin YM, Juichi M, Wu RY, Lee KH: Antitumor Agents 69. Alkaloids of Ochrosia acuminata. Planta Med (1985) 6:545546. 49. Hartwell JL, Schrecker AW: Chemistry of Podpphyllum. Fortschr Chem Org Naturst (1959) 15:83-166. 36. Suffness M, Douros JD: Miscellaneous natural products with antitumor activity In: Anticancer agents based on natural product models. Cassedy JM, John D, Douros JD (Eds), Academic Press, New York (1980) 466-469. 37. Rouesse J, Spielmann M, Turpin F, Le Chevalier T, Azab M, Mondesir JM: Phase II study of elliptinium acetate salvage treatment of advanced breast cancer. Eur J Cancer (1993) 29A:856-859. 38. Rouesse J, Huertas D, Sancho-Garnier H, Le Chevalier T, Amiel JL, Brule G, Tursz T, Mondesir JM: 2-N-methyl-9hydroxy-ellipticine in treatment of metastatic breast cancers. Bull du Cancer (1981) 68:437-441. 39. Kattan J, Durand M, Droz JP, Mahjoubi M, Azab M: Phase I study of retelliptine dihydrochloride (SR 95325 B) using a single two-hour intravenous infusion schedule. Am J Clin Oncol (1994) 17:242-245. 40. Xiao PG: Traditional experience of Chinese herb medicine, its application in drug research and new drug searching In Natural Products as Medicinal Agents. Beal JL, Reinhard E (Eds). Hippokrates Verlag, Stuttgart (1981) 351-368. • A plenary lecture of Chinese herbal medicine as sources of new drug discovery (63 references). 41. Ma MZ, Yao BY: Progress in indirubin treatment of chronic myelocytic leukemia. J Trad Chin Med (1983) 3:245-248. 42. Blanz J, Ehninger G, Zeller KP: The isolation and identification of indigo and indirubin from urine of a patient with leukemia. Res Commun Chem Pathol Pharmacol (1989) 64:145-156. 43. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT: Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc (1971) 93:2325-2327. •• An original report of the discovery of Taxol. 44. Kingston DGI, Molinero AA, Rimoldi JM: The taxane diterpenoids In: Progress in the Chemistry of Organic Natural Products. Herz W, Kirby GW, Tamm C (Eds), Springer-Verlag, New York (1993) 61:1-206. •• A comprehansive review of taxol and its analogs (371 references) 45. Hanauske AR, Degen D, Hilsenbeck SG: Effects of Taxotere and taxol on in vitro colony formation of freshly explanted human tumor cells. Anticancer Drugs (1992) 3:121-124. 46. Holton RA, Somoza C, Kim HB, Liang F, Biediger RJ, Boatman PD, Shindo M, Smith CC, Kim S, Nadizadeh H, Suzuki Y, Tao C, Vu P, Tang S, Zhang P, Murthi, KK, Gentile LN, Liu JH: First total synthesis of taxol. J Am Chem Soc (1994) 116:15971600. •• One of two recently achieved total synthesis of Taxol. 47. Nicolaou KC, Yang Z, Liu JJ, Ueno H, Nantermet PG, Guy RK, Claiborne CF, Renaud J, Couladouros EA, Paulvannan K, Sorensen EJ: Total synthesis of taxol. Nature (1994) 367:630634. •• A further recently achieved total synthesis of Taxol. 50. Jardine I: Podophyllotoxins In: Anticancer Agents Based on Natural Product Models. Cassady JM, Douros JD (Eds), Academic Press, New York (1980):310-347. 51. Stahelin HF, von Wartburg A: From podophyllotoxin glucoside to etoposide. Prog Drug Res (1989) 33:168-266. • A comprehensive review on the discovery of etoposide. 52. Stahelin HF, von Wartburg A: The chemical and biological route from podophyllotoxin glucoside to etoposide: Ninth Cain memorial award lecture. Cancer Res (1991) 51:5-15 •• Further comprehensive review of etoposide and its analogs. 53. Zhang YL, Lee KH: Recent progress in the development of novel antitumor etoposide analogs. Chin Pharm J (1994) 46:319-369. •• A comprehensive review reflecting recent development of etoposide analogs (117 references). 54. Lee KH, Wang HK: Current status of bioanalysis of etoposide and related compounds. J Food Drug Anal (Taiwan) (1995) 3:209-232. 55. Schacter LP, Igwemezie LN, Seyedsadr M, Morgenthien E, Randolph J, Albert E, Santabarbara P: Clinical and pharmacokinetic overview of parenteral etoposide phosphate. Cancer Chemother Pharmacol (1994) 34:58-63. 56. Sessa C, Zucchetti M, Cerny T, Pagani O, Cavalli F, De Fusco M, De Jong J, Gentili D, McDaniel C, Prins C, Schacter L, Winograd B, D'incalci M: Phase I clinical and pharmacokinetic study of oral etoposide phosphate. J Clin Oncol (1995) 13:200-209. 57. Chabot GG, Armand JP, Terret C, de Forni M, Abigerges D, Winograd B, Igwemezie L, Schacter L, Kaul S, Ropers J, Bonnay M: Etoposide bioavailability after oral administration of the prodrug etoposide phosphate in cancer patients during a phase I study. J Clin Oncol (1996) 14:20202030. 58. Millward MJ, Newell DR, Mummaneni V, Igwemezie LN, Balmanno K, Charlton CJ, Gumbrell L, Lind MJ, Chapman F, Proctor M: Phase I and pharmacokinetic study of a watersoluble etoposide prodrug, etoposide phosphate (BMY40481). Eur J Cancer (1995) 31A:2409-2411. 59. Igwemezie LN, Kaul S, Barbhaiya RH: Assessment of toxicokinetics and toxicodynamics following intravenous administration of etoposide phosphate in beagle dogs. Pharm Res (1995) 12:117-123. 60. Machida M, Tanaka S, Nakamori K: Simultaneous determination of NK611, a novel water-soluble derivative of etoposide, and its metabolite (DeNK611) in dog plasma by column-switching high-performance liquid chromatography. Biomed Chromatogr (1993) 7:82-85. 61. Rassmann I, Schrodel H, Schilling T, Zucchetti M, KaeserFrohlich A, Rastetter J, Hanauske AR: Clinical and pharmacokinetic phase I trial of oral dimethylaminoetoposide (NK611) administered for 21 days every 35 days. Invest New Drugs (1996) 14:379-386. 102 IDrugs 1998 Vol 1 No 1 62. Pagani O, Zucchetti M, Sessa C, de Jong J, D’Incalci M, De Fusco M, Kaeser-Frohlich A, Hanauske A, Cavalli F: Clinical and pharmacokinetic study of oral NK611, a new podophyllotoxin derivative. Cancer Chemother Pharmacol (1996) 38: 541-547. 63. Holmund JT, Kopp WC, Wiltrout RH, Longo DL, Urba WJ, Janik JE, Sznol M, Conlon KC, Fenton RG, Hornung R: A phase I clinical trial of flavone-8-acetic acid in combination with interleukin 2. J Natl Cancer Inst (1995) 87:134-136. 64. de Forni M, Chabot GG, Armand JP, Gouyette A, Klink-Alak M, Recondo G: Phase I and pharmacology study of flavone acetic acid administered two or three times weekly without alkalinization. Cancer Chemother Pharmacol (1995) 35:219224. 65. O’Reilly SM, Rustin GJ, Farmer K, Burke M, Hill S, Denekamp J: Flavone acetic acid (FAA) with recombinant interleukin-2 (rIL-2) in advanced malignant melanoma: I. Clinical and vascular studies. Br J Cancer (1993) 67:1342-1345. 66. Thomsen LL, Baguley BC, Rustin GJ, O’Reilly SM: Flavone acetic acid (FAA) with recombinant interleukin-2 (rIL-2) in advanced malignant melanoma. II: Induction of nitric oxide production. Br J Cancer (1992) 66:723-727. 67. Tavasolian B, Kharrazy P: Extraction and partial purification of ricin from Ricinus communis L. Pahlavi Med J (1978) 9: 21-26. 68. Blakey D C, Thorpe PE: Treatment of malignant disease and rheumatoid arthritis using ricin A-chain immunotoxins. Scand J Rheumatol - Supplement (1988) 76:279-287. 69. Selvaggi K, Saria EA, Schwartz R, Vlock DR, Ackerman S, Wedel N, Kirkwood JM, Jones H, Ernstoff MS: Phase I/II study of murine monoclonal antibody-ricin A chain (XOMAZYMEMel) immunoconjugate plus cyclosporine A in patients with metastatic melanoma. J Immunother (1993) 13:201-207. 70. LoRusso PM, Lomen PL, Redman BG, Poplin E, Bander JJ, Valdivieso M: Phase I study of monoclonal antibody-ricin A chain immunoconjugate Xomazyme-791 in patients with metastatic colon cancer. Am J Clin Oncol (1995) 18:307-312. 71. Sausville EA, Headlee D, Stetler-Stevenson M, Jaffe ES, Solomon D, Figg WD, Herdt J, Kopp WC, Rager H, Steinberg SM: Continuous infusion of the anti-CD22 immunotoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: a phase I study. Blood (1995) 85:3457-3465. Reference to Patent Literature 101.Florida State University (Tallahassee, FL), US-5015744 (1991). •• Conversion of taxol from baccatin III. Review Mucosal immunity 103 Mucosal immunity elicited by DNA vaccines Taff Jones Address Experimental Vaccines Centre for Applied Microbiology & Research Porton Down Salisbury Wilts SP4 0JG UK Tel: +44 198 061 2615 Fax: +44 198 061 1096 E.mail: [email protected] IDrugs 1998 1(1):103-108 Current Drugs Ltd ISSN 1369-7056 The induction of mucosal immunity could improve vaccine prophylaxis by preventing pathogens from colonizing their host. As a consequence, the delivery of vaccines to mucosal surfaces has become a crucial issue. DNA vaccines, administered by intramuscular injection or by particle bombardment of the epidermis, have revolutionized vaccine research in the last few years, but these routes of delivery are unlikely to elicit mucosal immunity efficiently. This article reviews the progress being made towards meeting the vital challenge of efficiently eliciting mucosal immunity through the appropriate delivery of DNA vaccines. Introduction Over the last two decades, several factors have given fresh impetus to the development of novel vaccines. Drugresistant strains of an increasing number of pathogens continue to give worldwide cause for concern. In both the developed and developing world, large populations continue to die from vaccine-preventable diseases, new diseases continue to emerge and ’old’ diseases are reemerging. Vaccine research has flourished as a consequence, and in these stimulating times, there have been some significant developments. It is now generally accepted that systemic humoral immune responses are both insufficient and inadequate to combat the range of different pathogens which we are exposed to. For example, it is thought that over 95% of human and animal pathogens gain access to their host cells across a mucosal surface. It has been argued that the induction of immune responses at the appropriate mucosal surface could at least limit, if not abolish, the transmucosal passage of pathogens, minimizing colonization and access to host cells. Although most vaccines are currently delivered by intramuscular (im) injection, the most efficient way of inducing mucosal responses is by immunizing the mucosal surfaces. Thus there is now an emphasis on finding alternatives to im injection as a means of administering vaccines [1]. Further, it is known that humoral (circulating antibody) responses are ineffective against viruses and other intracellular pathogens, which require cell-mediated immunity (CMI) to eradicate the pathogen and infected host cells [2]. Whilst live, attenuated organism vaccines are most efficient at stimulating CMI, they carry their own risks of eliciting sideeffects in a small number of cases and occasionally, a worrying reversion to virulence. The current subunit vaccines, adsorbed onto aluminum salts, induce only weak CMI, if at all [3]. Newer adjuvants have demonstrated an ability to induce vigorous CMI and these will ultimately improve subunit vaccines. Perhaps the most promising development in vaccine research in recent years, has been the astonishing finding that im injection of ’naked DNA’ induces long-lasting, potent CMI, as well as humoral immunity. This finding has opened up a plethora of possibilities for vaccines, as well as raising some novel safety and regulatory issues [4]. These latter issues have been dealt with in a previous review [5]. This review addresses the induction of mucosal immunity by appropriate delivery of DNA vaccines. DNA immunization DNA vaccines are plasmid DNAs, comprised of a gene which encodes a defined protein, and expression vectors which contain the necessary components to allow the gene of interest to be expressed in eukaryotic cells. Compared with inactivated pathogens or subunit vaccines, which do not endogenously synthesize proteins, there are several advantages in expressing antigens within a cell. The loss of antigenicity through chemical inactivation or adjuvanting is avoided; intracellular synthesis of the protein allows for conformational and post-translational integrity; and intracellular processing of the antigen permits the presentation + by MHC class I molecules, which elicit CD8 cytotoxic Tlymphocytes (CTL), which are a vital component of CMI. In these respects, DNA vaccines resemble live, attenuated virus vaccines, but with the major safety feature that they cannot revert to virulence. For these, and for the more practical reasons that plasmid purification is a generic process and plasmids are generally more stable than proteins or whole organisms (which usually require a cold chain for storage and transport), research into DNA vaccines has increased rapidly since the first seminal publication in 1990 [6]. There is now a large amount of published data reporting the induction of immunity and protection in a variety of animal models, all of which support the efficacy of DNA vaccines [4]. Plasmid DNA ‘vaccines’ are also finding application in the treatment of autoimmune disorders, allergies and cancers [7,8]. Most of the literature has reported the administration of DNA vaccines by im, iv or intradermal injection, or by genegun delivery of DNA-coated gold particles to the epidermis. As demonstrated with subunit or whole-organism vaccines, the most efficient way of inducing mucosal immune responses is by direct immunization of a mucosal surface [1]. There are now a few studies which report the successful induction of mucosal immunity with DNA vaccines; these are discussed in this review. 104 IDrugs 1998 Vol 1 No 1 Induction of mucosal immunity by the genegun Herrmann and colleagues [9] compared the protection elicited by gene-gun immunization of a non-mucosal (abdominal epidermis) and a mucosal (perineal/anal mucosa) route in mice, using a plasmid encoding a rotavirus antigen. The animals were subsequently challenged with homologous virus and protection was assessed by determining the levels of fecally shed virus. Complete protection was achieved by immunizing mice perineally with a fifth of the dose used to immunize epidermally, suggesting that the mucosal route of immunization elicited immunity against a mucosal pathogen more efficiently than the non-mucosal route. Intestinal IgA was not detected prior to challenge in either case. Mucosal responses were also elicited in the female genital tract using the gene-gun [10]. A plasmid containing a reporter gene was used to transfect vaginal mucosa in rats. Control animals were immunized via abdominal epidermis and in both cases, a boost was administered by the same route. A third group received a primary dose epidermally followed by a vaginal boost. All immunization regimes resulted in significant titers of specific IgG and IgA in serum which persisted for 14 weeks post-boosting. All immunization regimes also elicited substantial titers of IgA and IgG in vaginal secretions at six weeks post boosting, but IgG and IgA antibodies were only detected thereafter in animals primed and boosted vaginally. Thus epidermal immunization with the gene-gun can elicit mucosal immunity and protection against a mucosal pathogen, though the mechanisms of protection remain unresolved. Epidermal immunization may directly elicit immune responses at mucosal surfaces distant from the site of immunization, or transudation of serum antibodies may occur either spontaneously or from disruption of epithelial barriers as a result of infection. However it is clear from both these studies that direct immunization of mucosal tissue is the more efficient means of inducing mucosal immunity. Direct application of DNA to mucosal tissues A number of studies have attempted to induce mucosal immune responses through the direct application of DNA to mucosal surfaces. Although not as effective in inducing protective efficacy against lethal challenge as im immunization (which resulted in 95% survival in immunized animals), the intranasal instillation of a flu hemagglutininencoding DNA vaccine to mice resulted in 76% survival, although the dose used was half that of the im dose [11]. Both routes of immunization elicited detectable but low serum antibody titers. The gene-gun immunization was as effective as im injection in protecting epidermallyimmunized mice, and elicited serum antibody titers which were 3- to 5-fold higher. Similar studies, though with a different outcome, have been reported. The intranasal instillation of mice with a plasmid encoding flu hemagglutinin, with or without coadministered cholera toxin (CT), a mucosal adjuvant, failed to elicit detectable antibodies in serum, saliva or nasal washes, although antibody forming cells (AFCs) were detected in both spleen and lung cell suspensions when CT was included in the instillate [12]. Although im injection of the same plasmid elicited full protection against challenge with live virus, mice immunized by intranasal instillation were not protected. The ability to induce mucosal immune responses by intranasal instillation of a plasmid encoding Herpes Simplex Virus glycoprotein B, with or without CT, was compared with im injection of the same plasmid [13]. The former regime induced a specific IgA response in vaginal secretions, which was enhanced by co-administration of CT, whereas im injection induced specific IgG in serum, and specific IgG but not IgA in vaginal secretions. Although the intranasal route of DNA administration elicited CMI in vivo, as assessed by delayed-type hypersensitivity (DTH), it was less efficient than the im route in protecting immunized mice against vaginal challenge [13]. Furthermore, and contrary to current dogma, the intranasal administration of DNA failed to prevent the transmucosal passage of HSV, despite successfully eliciting mucosal immunity. Inoculation of plasmids by the intranasal route has been directly compared with other mucosal routes, namely po, intrajejunally and via the buccal mucosa, [14]. Using a measles virus hemagglutinin, CTL activity in restimulated spleen cells was assessed after inoculating mice with a single dose of unformulated plasmid or plasmid plus CT. Unformulated plasmid given by the intranasal or buccal routes elicited the most vigorous CTL responses, with the po and intrajejunal routes eliciting CTL responses which were approximately 50% lower. The formulation of the plasmid with CT slightly diminished the CTL response following administration intranasally or via the buccal mucosa, but in contrast, more than doubled the responses to plasmid given po and significantly enhanced the response to plasmid given by the intrajejunal route. Other studies have demonstrated that intravaginal inoculation of mice with a plasmid encoding the HIV envelope glycoprotein, gp160, yielded detectable anti-gp160 IgA and IgG antibodies in vaginal washes which were capable of neutralizing HIV in syncytia-formation neutralization assays [15]. Thus direct application of DNA vaccines to mucosal tissues is feasible and elicits systemic and mucosal immunity and protection. Although DNA is taken up relatively efficiently by myocytes following im injection, the transfection of epithelial cells may be less efficient in the absence of a transfectant or facilitator, and the variability of some of the data emerging from the studies described are consistent with this. Lipid formulated DNA Lipids can facilitate the entry of macromolecules into cells, presumably through perturbation of the cell membrane. Lipids have been widely used to transfect cells with DNA, either as classical liposomes using phospholipids or as complexes with novel cationic lipids. Both formulations have proved successful in the immunization of mucosal epithelia. Review Mucosal immunity 105 Most applications of lipid-complexed DNA have used the intranasal route of immunization to elicit mucosal immune responses. The formulation of a plasmid DNA-lipid complex containing the reporter gene, luciferase, and the cationic lipids, DMRIE/DOPE, when administered intranasally to mice, showed a 30-fold higher expression of the enzyme in nasal mucosa compared to that achieved by naked DNA, and elicited a vigorous, broad spectrum, immune response [16]. Anti-luciferase IgA and IgG were detected in serum, as were specific IgA and IgG in vaginal fluids, indicating that intranasal immunization with a lipid-complexed plasmid induced systemic and mucosal antibody responses. An assay with a monoclonal antibody to the secretory component confirmed that the vaginal IgA was mucosally derived. Lymphoproliferation of in vitro stimulated spleen cells was observed in the immunized animals, while spleen and iliac lymph node effector cells also showed a substantial, specific CTL activity against MHC class I restricted target cells expressing luciferase. Thus this study demonstrated that the intranasal instillation of lipid-DNA complexes was a simple and potent mechanism of stimulating humoral and cellular, systemic and mucosal immunity, and these studies have now been extended to non-human primates with similar results [T Jones, personal communication]. In addition to assessing the induction of CTL activity following inoculation of nasal or gut mucosae with a plasmid encoding the measles virus hemagglutinin, the effects of adding the cationic lipid, DOTAP, to the plasmid prior to administration were also investigated [14]. The authors had previously observed a decrease in the CTL activity following nasal administration, and a substantial increase in CTL activity following po or intrajejunal administration, of CT- adjuvanted plasmid. Using DOTAP as the adjuvant instead of CT, these effects were enhanced. DOTAP complexed plasmid diminished the CTL activity even more than CT when administered intranasally, but enhanced the CTL activity nearly 3-fold over unformulated DNA when given po or intrajejunally. In another study, the induction of humoral and cellular, systemic and mucosal immune responses were compared by im or intranasal administration of a plasmid encoding an HIV-1 protein [17]. An im injection elicited serum IgG and fecal IgA titers, which were enhanced by co-administration of the plasmid with the lipid, monophosphoryl lipid A (MPL). The intranasal administration resulted in serum IgG and fecal IgA at titers comparable to those induced by im injection, and again, co-administration of MPL enhanced both titers but enhanced the fecal IgA titers to sigificantly higher levels. Both im and intranasal routes of administration elicited cellular responses. DTH activity, which was enhanced by co-administration of MPL, was observed in all immunized animals, as was specific CTL activity. Induction of DTH activity was comparable by both routes, but CTL activity induced by intranasal instillation of plasmid was higher than that induced by im injection. There is no doubt that complexing with lipids facilitates the transfection of DNA and the subsequent induction of mucosal immunity, though the issue of toxicity of many of the cationic lipids needs addressing, as does the limited stability of lipid and liposomal preparations. The successful induction of mucosal immunity by co-administration of MPL, a stabilized lipid adjuvant, suggests that the intranasal immunization with lipid-formulated DNA vaccines may be a realistic prospect. PLG entrapped DNA The po route is a convenient and non-invasive means of delivering vaccines, and allows access to the largest mucosal surface in mammals, the gastrointestinal tract (GIT). The GIT is rich in immune inductive tissues, comprising lymph nodes, lymphoid follicles and aggregations of lymphoid follicles known as Peyer’s patches. Collectively these tissues are called the gut-associated lymphoid tissue (GALT) and as part of the common mucosal immune system, immunization of the GALT elicits mucosal immune responses both locally in the gut as well as in the respiratory and genitourinary tracts [18]. However, to protect against hydrolysis and proteolysis, orally delivered antigens should be protected by a barrier. This is thought to be particularly true for DNA which has a half-life of minutes when injected im [19] and would be expected to be even shorter in the gut. This has commonly been achieved by encapsulation of antigens in poly(lactideco-glycolide) (PLG) microparticles [20]. PLG degrades by non-enzymic hydrolysis to the normal body metabolites, lactic and glycolic acids and is therefore biodegradable and biocompatible. As internal sutures and as human or veterinary implants, PLG has acquired a documented history of safe use [20]. Orally administered PLG microparticles ≤10 µm in diameter are readily phagocytosed from the lumen of the gut by M cells, the specialized epithelial cells which overlie Peyer’s patches, and whose function it is to transport particulate material to antigen presenting cells underlying the dome of Peyer’s patches. The presentation by dendritic cells within Peyer’s patches leads to T- and B-cell interactions and the subsequent initiation of the IgA cell cycle, ie, the induction of mucosal immunity. In contrast, the uptake of soluble antigens by Peyer’s patches is less efficient and occurs across villi with subsequent processing by macrophages in the lamina propria which may have a suppressive effect on the subsequent immune responses, ie, induction of tolerance. Systemic immune responses result from a concomitant redistribution of microparticles from Peyer’s patches to the spleen. Thus, po administration of microencapsulated antigens can elicit both systemic and mucosal immunity [2124]. To pursue the interest in the induction of mucosal immunity, The Centre for Applied Microbiology and Research have investigated the oral delivery of PLGmicroencapsulated plasmid DNA vaccines. The process of microencapsulation involves emulsifying an aqueous solution of antigen in a solution of PLG in organic solvent [25]. Shear, which is detrimental to the physical and biological integrity of DNA, is generated during emulsification, but conditions have been established in our laboratory which allows plasmid DNA to be encapsulated in microparticles ≤10 µm diameter with the retention of biological activity [26]. 106 IDrugs 1998 Vol 1 No 1 Figure 1. Serum anti-luciferase IgG Serum anti-luc IgG Enc po Non po Non im Enc ip Non ip 0 500 1000 1500 2000 2500 3000 End point titer The figure shows the serum anti-luciferase IgG titers 6 weeks after immunizing mice with a single 50 µg dose of encapsulated DNA by ip injection or gavage (Enc ip and Enc po). Control animals received 50 µg of DNA by ip or im injection (Non ip and Non im) or by gavage (Non po). To determine whether encapsulated plasmid DNA would elicit an immune response, mice were immunized with a single dose of naked or encapsulated plasmid DNA containing the gene encoding the insect enzyme, luciferase. Comparison of the serum IgG responses elicited in mice by the administration of non-encapsulated plasmid DNA by im or ip injection or gavage, with that of encapsulated plasmid, showed that at six weeks post-immunization, mice immunized with encapsulated DNA, given by both po and ip routes, showed enhanced titers of luciferase-specific serum IgG compared to mice given ’naked’ DNA by the same or im routes (Figure 1). These responses confirmed that plasmid DNA formulated in microparticles allowed the expression of the encoded protein and appropriate presentation of the expressed antigen to lymphoid tissues. The time course of induction of the immune responses elicited by a single 50 µg dose of DNA, following ip injection or gavage of encapsulated DNA, was investigated. Serum and fecal pellets were collected at various times postimmunization and assayed for elicited antibodies. The ip injection of PLG-encapsulated DNA elicited good antiluciferase IgG and IgM and only modest IgA titers. The po administered encapsulated DNA elicited good responses in all three antibody classes. Over the course of the study, the immune responses tended to increase with time although the IgG response following ip injection, peaked at six weeks post-immunization [27]. The administration of PLG encapsulated DNA also elicited a local mucosal antibody response. Very high titers of specific IgA were detected in fecal pellets from animals immunized po with encapsulated plasmid [27]. These titers peaked at nine weeks post-immunization but remained high, 16 weeks postimmunization (Figure 2). The lymphoproliferative responses of pulsed splenocytes from these mice were also measured and were found to be 50% higher than those measured in splenocytes from control animals given naked plasmid im. Thus orally administered encapsulated DNA is capable of both enhancing the responses elicited by im injection of naked DNA, and eliciting cellular immune responses. Preliminary protection experiments have been performed in a challenge model for measles. Following gavage of infant mice with an encapsulated plasmid encoding the measles virus nucleoprotein, immunized mice were challenged with live virus. At the time when all control animals had died, half the immunized animals had survived. Thus the vaccine induced a substantial degree of protection which dose escalation experiments will hopefully improve. The Centre for Applied Microbiology and Research’s collaboration with John Herrmann’s group at the University of Massachusetts investigated the immune responses and protection elicited by encapsulated rotavirus DNA vaccines. Mice were immunized, po, with a single dose of microencapsulated plasmid DNA encoding VP6, which elicited anti-VP6 serum antibodies and fecal IgA. Immunized animals were challenged with live homologous virus and fecal antigen was reduced to approximately 80% that of controls. Earlier and higher fecal VP6-specific IgA responses were observed during the period of viral shedding, suggesting that the protection was mediated by specific mucosal immune responses [28]. Although gene-gun immunization of abdominal epidermis or perineal mucosa Review Mucosal immunity 107 Figure 2. Fecal anti-luciferase IgA Fecal anti-luc IgA 3 weeks 6 weeks 9 weeks 12 weeks 16 weeks 0 500 1000 1500 2000 2500 3000 End point titer The figure shows the change with time of fecal anti-luciferase IgA titers elicited by gavage of a single 50 µg dose of encapsulated luciferase pDNA. with the VP6 plasmid did not elicit fecal IgA, gene-gun immunized animals were fully protected against challenge. Conclusions Over the last few years, DNA vaccines have made a huge impact on vaccine research and development and the prospects for human application of DNA vaccines are bright, with the results of current human trials being eagerly awaited. Most of the studies to date have delivered DNA vaccines to animals by im injection, or epidermally with the gene-gun. The studies discussed in this review bring another dimension to DNA vaccines, in offering both alternative means of delivery which may be more acceptable to patients, and potential for improving the immune responses elicited. Injections through the im route elicit vigorous immunity and protection, even against some mucosally-acquired pathogens and largely in the absence of detectable mucosal immune responses. In these cases, the mechanisms of protection need to be resolved but notwithstanding, the precision required in the injection of DNA, the availability of alternative, non-invasive means of delivery, and the lack of induction of mucosal immunity will probably argue against this method of delivery in favor of another. There is a strong case for using the gene-gun in preference to im injection. With much lower doses of DNA, vigorous immunity and protection against challenge are elicited, though in the few instances where protection elicited by epidermal or mucosal immunization have been directly compared [9,10], protection was higher in the perineal/anal or vaginal immunized groups, indicating a more efficient induction of protection by mucosal routes. These routes, however, are unlikely to be acceptable to patients. At present the gene-gun is somewhat cumbersome but disposable single-shot units are being developed which will allow, at higher cost per unit, more flexibility and access to the buccal mucosae. Perhaps the biggest drawback of this technology concerns the coating of the gold particles. DNA is precipitated onto the particles which must subsequently be stored completely dry otherwise DNA disassociates from the particles. Since the technique is entirely dependent on cell transfection achieved by the ‘shooting’ of high-density, DNA-coated particles directly into cells, this will inevitably limit the stability of gene-gun vaccines. Direct application of DNA vaccines is feasible, certainly by the intranasal route, although the efficiency of uptake would be greatly enhanced by complexing with lipids. It is possible that, as with subunit vaccines, comparable responses can be elicited by the administration of small doses of the vaccine combined with adjuvant (or in the case of a DNA vaccine, a transfectant) or larger doses of the unformulated vaccine. This, combined with concerns over possible degradation of unformulated DNA, suggests that formulation with a transfectant might offer both some degree of protection to the DNA and accelerate its internalization. Vigorous systemic and mucosal immunity can be elicited by lipidcomplexed DNA, especially when given intranasally, but such formulations tend to be unstable and until this issue is resolved, such vaccines will remain experimental. Furthermore, many of the cationic lipids possess a degree of toxicity. Perhaps the most promising and cost effective approach to inducing mucosal immunity with DNA vaccines is oral 108 IDrugs 1998 Vol 1 No 1 delivery. The simplicity and convenience of administration will be widely accepted by vaccinees, whilst the induction of broad spectrum immunity and protection demonstrates the efficacy of DNA vaccines delivered by this route. The encapsulation of DNA in PLG microparticles protects the DNA, ensures uptake by lymphoid tissues, and as a lyophilized preparation, adds an extra dimension of stability to the vaccines. These are compelling reasons to favour this means of delivering DNA vaccines for use in both the developed and the developing world. References 1. Walker RI: New strategies for using mucosal vaccination to achieve more effective immunization. Vaccine (1994) 12: 387-400. 2. Liu MA: The immunologist’s grail: Vaccines that generate cellular immunity. Proc Natl Acad Sci USA (1997) 94:1049610498. 3. Warren HS, Chedid LA: Future prospects for vaccine adjuvants. CRC Crit Rev Immunol (1988) 8:83-101. 4. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA: DNA vaccines. Annu Rev Immunol (1997) 15:617-648. 5. Meager A, Robertson JS: Regulatory and standardization issues for DNA and vectored vaccines. Current Research in Molecular Therapeutics (1998) In press. 6. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL: Direct gene transfer into mouse muscle in vivo. Science (1990) 247:1465-1468. 7. Manickan E, Karem KL, Rouse BT: DNA vaccines - a modern gimmick or a boon to vaccinology? Crit Rev Immunol (1997) 17:139-154. 8. Robinson HL, Torres CAT: DNA vaccines. Seminars Immunol (1998) In press. 9. Chen SC, Fynan EF, Robinson HL, Greenberg HB, Herrmann JE: Mucosal immunity induced by a rotavirus VP6 DNA vaccine. Abstr Amer Soc Virol (1996) W27-6:131. 10. Livingston JB, Lu S, Robinson H, Anderson DJ: Immunization of the female genital tract with a DNA-based vaccine. Infect Immun (1998) 66:322-329. 11. Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro, JC, Robinson HL: DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA (1993) 90:11478-11482. 12. Ban EM, van Ginkel FW, Simecka JW, Kiyono H, Robinson HL, McGhee, JR: Mucosal immunization with DNA encoding influenza hemagglutinin. Vaccine (1997) 15:811-813. 13. Kuklin N, Daheshia M, Karem K, Manickan E, Rouse BT: Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization. J Virol (1997) 7:31383145. 14. Etchart N, Buckland R, Liu MA, Wild TF, Kaiserlian D: Class Irestricted CTL induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin. J Gen Virol (1997) 78:1577-1580. 15. Wang B, Dang K, Agadjanyan MG, Srikantan V, Li F, Ugen KE, Boyer J, Merva M, Williams WV, Weiner DB: Mucosal immunization with a DNA vaccine induces immune responses against HIV-1 at a mucosal site. Vaccine (1997) 15:821-825. 16. Klavinskis LS, Gao L, Barnfield C, Lehner T, Parker S: Mucosal immunization with DNA-liposome complexes. Vaccine (1997) 15:818-820. 17. Sasaki S, Hamajima K, Fukushima J, Ihata A, Ishii N, Gorai I, Hirahara F, Mohri H, Okuda K: Comparison of intranasal and intramuscular immunization against Human Immunodeficiency Virus type 1 with a DNA-monophosphoryl lipid A adjuvant vaccine. Infect Immun (1998) 66: 823-826. 18. Mestecky J: The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol (1987) 7:265-276. 19. Lew D, Parker SE, Latimer T, Abai AM, Kuwahara-Rundell A, Doh SG, Yang ZY, Laface D, Gromowski SH, Nabel GJ, Manthorpe M, Norman J: Cancer gene therapy using plasmid DNA: pharmacokinetic study of DNA following injection in mice. Hum Gene Ther (1995) 6:553-564. 20. Morris W, Steinhoff MC, Russell PK: Potential of polymer microencapsulation technology for vaccine innovation. Vaccine (1994) 12:5-11. 21. Mestecky J, Moldoveanu Z, Novak M, Huang W-Q, Gilley RM, Staas JK, Scafer D, Compans RW: Biodegradable microspheres for the delivery of oral vaccines. J Controlled Release (1994) 28:131-141. 22. McGhee JR, Mestecky J, Dertzbaugh MT, Eldridge JH, Hirasawa M, Kiyono H: The mucosal immune system: from fundamental concepts to vaccine development. Vaccine (1992) 10:75-88. 23. Eldridge JH, Meulbroek JA, Staas JA, Tice TR, Gilley RM: Biodegradable and biocompatible poly(DL-lactide-coglycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxinneutralising antibodies. Infect Immun (1991) 59:2978-2986. 24. Jones DH, McBride BW, Thornton C, O’Hagan DT, Robinson A, Farrar GH: Protection of mice from Bordetella pertussis respiratory infection using orally administered microencapsulated pertussis fimbriae. Infect Immun (1996) 64:489-494. 25. Jones DH: Micro-encapsulation of vaccines. In: Methods in Molecular Medicine: Vaccine Protocols. Robinson A, Farrar GH, Wiblin CN (Eds) Humana Press Inc., Totowa, NJ, (1996):157-166. 26. Microbiological Res Authority & Centre For Applied Microbiology And Res (Clegg JCS, Farrar GH, Jones DH): Microencapsulation of DNA for vaccination & gene therapy. WO-09717063-A1 (1997). 27. Jones DH, Corris S, McDonald S, Clegg JCS, Farrar GH: Poly (DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine (1997) 15:814-817. 28. Chen SC, Jones DH, Fynan EF, Farrar GH, Clegg JCS, Greenberg HB, Herrmann JE: Mucosal immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. Submitted for publication. Review 5-HT receptors in learning and memory 109 Involvement of 5-HT receptors in learning and memory François S Roman & Evelyne Marchetti Address Laboratoire de Neurobiologie des Comportements UMR 6562 CNRS Université de Provence IBHOP Traverse Charles Susini 13388 Marseille Cedex 13 France Tel: +33 0491 28 8724 Fax: +33 0491 98 2697 Email: [email protected] IDrugs 1998 1(1):109-121 Current Drugs Ltd ISSN 1369-7056 Cumulative evidence indicates that the serotonin (5-HT) system plays a modulatory role in cognitive processes, particularly in learning and memory. The present review focuses on the effects of agonists and antagonists on different 5-HT receptors, administered during pre-learning, post-learning or pre-retention, in different behavioral tasks. The effects of these pharmacological compounds were either facilitative or disruptive, or allowed for recovery from impaired cognitive performance following the activation or blockade of 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptors. Since little or no data on learning and memory are currently available for the 5-ht5, 5-ht6 and 5-HT7 receptors, this article presents an overview of their brain locations and possible involvement in these highly cognitive processes. Introduction Since Twarog and Page [1] detected the presence of 5-HT in the cerebral tissues in 1953, intense research has been directed towards understanding the effect of this neurotransmitter on CNS functions. Anatomically, the serotonergic systems are amongst the most diffusely organized of the brain, and complex axonal systems project into almost all regions of the CNS, with particularly dense innervation of the cerebral cortex and limbic structures. Consequently, it has been suspected that this neurotransmitter is involved in learning and memory processes. However, the initial studies on behavior using pharmacological tools modified the activity of all serotonergic pathways at the same time, so the effects on the now well-characterized 5-HT receptors [2] could not be distinguished. To review these studies of the effect on learning and memory of the compounds and doses used for these different receptor classes (ie, mainly agonist or antagonist), the injection protocol must be taken into account. The pre-learning phase allows the study of the effects on acquisition, while postlearning administration can be used to determine the effects on consolidation. When administration is performed during pre-retention, the effect is ascribed to the retrieval stage of learning. This approach was recently proposed in a study of 5-HT1A receptors in learning and memory [3]. This review focuses on the effects of serotonergic compounds, with particular reference to compounds with selective affinity for a given receptor subtype, in different tasks in animal models of mnesic processes. Table 1. Effects of pre-training administration of 5-HT1A receptor agonists and antagonists (modified from Meneses and Hong, 1997) BEHAVIORAL TASK DRUGS EFFECTS 8-OH-DPAT buspirone ipsapirone MED (mg/kg) 0.125 16 8 Isolation-induced social behavioral deficit Open-field 8-OH-DPAT buspirone ipsapirone 0.25 8 8 - Passive avoidance 8-OH-DPAT buspirone ipsapirone gepirone tandospirone BMY-7378 NAN-190 0.125 1 -1.2 2.5 2.5 1 2 0 - Active avoidance 8-OH-DPAT buspirone ipsapirone tandospirone gepirone 0.1 1 20 1 10 0 - Repeated acquisition procedure 8-OH-DPAT buspirone 0.032 0.032 - Extinction 8-OH-DPAT 0.015 - Delayed matching to position 8-OH-DPAT 0.003 0.3 1 1 0.01 2 0.03 0 + + 0 0 0 ipsapirone buspirone NAN-190 WAY100635 - Delayed nonmatch to sample task 8-OH-DPAT 0.2 0 Autoshaping 8-OH-DPAT 0.015 + Water maze 8-OH-DPAT 0.003 0.25 1 0 - buspirone Radial maze 8-OH-DPAT 0.1 0.3 + - Delayed conditional flesinoxan NAN-190 1 0.1 1 3 0 - + enhanced; - impaired; 0 no parametric effect; MED Minimum effective dose 110 IDrugs 1998 Vol 1 No 1 Effects on 5-HT receptor subtypes Presently, 14 different types of 5-HT receptors have been cloned in vertebrates. They are divided into seven distinct receptor classes [4,5], which are discussed below. Effects on 5-HT1A receptors 5-HT1A receptor agonists are active in certain behavioral tests in animals [3,4] (Tables 1 - 3). In mice, the pre-learning injection (Table 1) of the 5-HT1A receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone (Bristol-Myers Squibb), and ipsapirone (Bayer), was found to accentuate the deficit on the isolation-induced social behavioral deficit test [6]. In addition, the compounds decreased exploratory activity in the open-field test. Since the minimum active doses were very similar in the social behavioral deficit and open-field tests, it was suggested that a common phenomenon of increased emotionality or reactivity accounted for both of these reductions in activity. In the rat, 8-OH-DPAT decreases the acquisition of learning in behavioral tasks involving passive and active avoidance, repeated-acquisition procedures, delayed conditional discrimination, and extinction [7-13]. At doses ranging from 0.003 to 0.3 mg/kg, this agonist has either no effect or enhances learning in delayed matching to position and autoshaping tasks [4,14-16], whereas at doses of 0.2 to 0.3 mg/kg, it decreases learning in water and radial maze behavioral tasks [17-20]. On delayed nonmatch to sample tasks, 8-OH-DPAT at 0.2 mg/kg has no effect [21]. Buspirone and ipsapirone decrease learning in passive and active avoidance, autoshaping, and water maze tasks; buspirone also decreases learning in repeated-acquisition procedures [1113,22-26]. In delayed matching to position task, buspirone has no effect while ipsapirone enhances learning [27,28]. Tandospirone (Sumitomo) at 1 mg/kg improves learning in active avoidance task, but at a higher dose (2.5 mg/kg) decreases learning of passive avoidance [9,29]. Gepirone (Bristol-Myers Squibb) decreases passive and active avoidance at 10.0 mg/kg, and flesinoxan (Solvay), at 1.0 mg/kg, disrupts the acquisition of delayed conditional discrimination [3,12]. Concerning the effects of 5-HT1A receptor antagonists, it has been reported that the 5-HT1A receptor partial agonists, BMY-7378 (Bristol-Myers Squibb) and 1-(2-methoxyphenyl)-4-[4-(2-phthalamido)butyl]-piperazine (NAN-190), either have no effect or decrease learning in passive avoidance, delayed conditional discrimination, and delayed matching to position tasks [3,9,11,27], whereas the silent 5-HT1A receptor antagonist , WAY-100635 (WyethAyerst), has no effect on the delayed matching to position task [30]. With post-learning injections (Table 2), 8-OH-DPAT enhances the consolidation of learning in autoshaping and extinction tasks [7,31], and either impairs or has no effect on passive avoidance and water maze tasks [10,28], respectively. Buspirone and tandospirone decrease learning in autoshaping and active avoidance tasks, respectively [32]. Tandospirone at 1.0 mg/kg impairs consolidation in active avoidance task, but at 2.5 mg/kg has no effect on passive avoidance [9,29]. In contrast, the partial 5-HT1A receptor agonist, NAN-190, and the silent 5- Table 2. Effects of post-training administration of 5-HT1A receptor agonists and antagonists on learning (modified from Meneses and Hong, 1997) BEHAVIORAL TASK Autoshaping DRUGS 8-OH-DPAT buspirone NAN-190 S-UH-301 WAY-100135 WAY-100635 MED (MG/KG) 0.015 1 0.5 0.3 5 0.001 EFFECTS + 0 0 0 0 Extinction 8-OH-DPAT 0.015 + Passive avoidance 8-OH-DPAT tandospirone 0.09 2.5 - Active avoidance tandospirone 1 - Water maze 8-OH-DPAT 0.1 0 + enhanced; - impaired; 0 no parametric effect; MED Minimum effective dose Table 3. Effects of pre-retention administration of 5-HT1A receptor agonists on learning (modified from Meneses and Hong, 1997) BEHAVIORAL TASK Delayed nonmatch to sample task DRUGS AGONISTS 8-OH-DPAT ipsapirone MED (mg/kg) 0.2 0.5 2.5 EFFECTS 0 0 Water maze 8-OH-DPAT buspirone 0.003 0.01 0 Plus maze 8-OH-DPAT 0.01 0 Passive avoidance 8-OH-DPAT buspirone 0.01 0.01 0 - Delayed matching to position ipsapirone 2.5 0 + enhanced; - impaired; 0 no parametric effect; MED Minimum effective dose HT1A receptor antagonists, 1-(1-naphthyl) piperazine hydrochloride (1-NP), WAY-100135 (Wyeth-Ayerst), WAY100635, and S-UH-301 (Solvay Duphar), do not alter the consolidation of learning in autoshaping tasks [3,33-35]. The effects of the preretention administration of 5-HT1A agonists (Table 3) reveal that 8-OH-DPAT, at 0.01 and 0.3 mg/kg, and buspirone, at 0.1 and 1.0 mg/kg, impair the retrieval of learning in passive avoidance [10,23,36]. In contrast, at 0.1 and 0.2 mg/kg, 8-OH-DPAT has no effect in the water maze, plus maze, or delayed nonmatching to sample tasks [18,21,36]. Both 8-OH-DPAT (0.5 mg/kg) and Review 5-HT receptors in learning and memory 111 agonist, CGS-12066B (Novartis), does not modify the escape deficit in the LH paradigm, but it reduces the ability of the 5HT reuptake blocker citalopram (Lundbeck) and fluvoxamine (Solvay) to reverse the behavioral deficit resulting from uncontrollable shocks [41], suggesting that 5HT1B receptor agonists, by their inhibition of 5-HT release, might reduce the stimulation of 5-HT transmission induced by 5-HT reuptake blockers. In addition, the mixed 5-HT1B/A agonist, RU-24969, (Roussel-Uclaf) dose-dependently increases the spontaneous locomotor activity of rats and mice [42,43], and the selective 5-HT1B agonist, CP-94253 (Pfizer), also produces hyperlocomotion in rats [44], whilst 5-HT1A receptor agonists [45] reduce locomotor activity in ipsapirone (2.5 mg/kg) decrease learning in the delayed matching to position, and delayed nonmatching to sample tasks [21,37]. Effects on 5-HT1B receptors In learned helplessness (LH) paradigm rats, 5-HT1B receptors are upregulated in the cortex, hippocampus, and septum, and downregulated in the hypothalamus [38,39]. These results suggest that a change in 5-HT1B receptor responsiveness might be related to the escape deficit. 5-HT release measured in vivo by microdialysis in the cortex of LH rats decreases [40], which is compatible with 5-HT1B autoreceptors being upregulated. The 5-HT1B receptor Figure 1. Drugs acting at 5-HT1A receptors N O N N N O N N N H O S Cl O H O Buspirone (Bristol-Myers Squibb) Cl Ipsapirone (Bayer) N N O H N N O N N N N N H3C H 3C N N N H O H Cl Gepirone (Bristol-Myers Squibb) O Tandospirone (Sumitomo) N N N O N O CH3 H Cl H Cl WAY-100635 (Wyeth-Ayerst) H Cl N 112 IDrugs 1998 Vol 1 No 1 enedioxyamphetamine (MDA); and dl-methylenedioxymethamphetamine (MDMA) also enhanced the acquisition of CRS [50,53,54]. Quipazine has a similar effect on the conditioned avoidance response (CAR) [55]. On the other hand, in studies that have examined the effects of various 5HT2A/2C receptor antagonists on learning, ritanserin (Janssen), glemanserin (MDL-11939, Hoechst Marion Roussel), pizotifen, and cyproheptadine have all been found to have a negative action in that they produced significant retardation of learning, whether measured by the conditioned NM response or the CAR [48,56,57]. In contrast, the 5-HT2A/2C antagonists, ketanserin (Janssen), mianserin, d-2-bromolysergic acid diethylamide (BOL) and LY-053857 (Eli Lilly), had no effects on learning [48,55]. However, other authors have reported that the 5-HT2A/2C antagonist receptors, 2,5dimethoxy-4-iodoamphetamine (DOI), ritanserin, and ketanserin improve learning in aging or animals with induced learning deficits [3,58]. For instance, ketanserin enhances the consolidation of learning, attenuates the learning deficits induced by aging or hypoxia, and improves the facilitative effects on memory brought about by the acetylcholinesterase inhibitor, physostigmine, in aged animals. These findings are contradictory to, but not incompatible with, the facilitating effects observed with agonists of these receptors [48]. mg/kg), also reduce locomotor activity in rats [46]. Finally, recent data indicate that the 5-HT1B/1D receptor antagonist, GR-127935T (Glaxo Wellcome), enhances the consolidation of learning [47]. 5-HT2 receptor family Few studies have concentrated on the effects of 5-HT2 receptors on learning and memory processes, and little information is available (Table 2 and Table 4) [48]. Initial reports indicated that d-lysergic acid diethylamide (LSD), an agonist of the 5-HT2A,2C,1A receptors, produced significant enhancement in the acquisition of the conditioned nictitating membrane (NM) response in the rabbit [49,50]. LSD (12.9 µg/kg) was shown to enhance acquisition of the conditioned NM response. More importantly, LSD enhanced the acquisition of conditioned responses (CRS) during classical appetitive conditioning of the jaw movement response as effectively as it enhances the acquisition of the defensive NM response [51]. The LSD-induced enhancement of acquisition was found to be due to the enhancement of associative learning, with no effect on such non-associative factors as sensitization, pseudoconditioning, baseline rate of responding, or to changes in the unconditioned responses [49,51,52]. Later studies employing the conditioned NM response indicated that other 5-HT2A/2C agonists, including dl-2,5 dimethoxy-4-methylamphetamine (DOM); dl-methyl- Figure 2. Drugs acting at 5-HT1B receptors H N H N N H3C F N H3C O O N N F F N CH3 RU-24969 (Hoechst Marion Roussel) CGS-12066B (Novartis) O H3C N H N H CP-94253 (Pfizer) N N N H N CH3 N O O GR-127935T (Glaxo Wellcome) CH3 CH3 Review 5-HT receptors in learning and memory 113 Table 4. Effects of pre-training administration of 5-HT2A/2C receptor agonists and antagonists (modified from Harvey, 1996) BEHAVIORAL TASK Conditioning of rabbits’ nictitating membrane response DRUGS AGONISTS LSD DOM MDA MDMA MED (mg/kg) 0.001 0.074 0.54 0.95 0.32 0.2 2.5 > 0.166 > 1.0 1.25 1.0 > 2.0 > 1.0 0.001 ANTAGONISTS Ritanserin MDL-11939 Pizotifen BOL LY-53857 Quipazine Active avoidance Cyproheptadine Ketanserin Mianserin LSD Classical appetitive conditioning EFFECTS + + + + 0 0 + 0 0 + + enhanced; - impaired; 0 no parametric effect; MED Minimum effective dose Figure 3. Drugs acting at 5-HT2 receptors OH O O CH3 H3C CH3 N F N N H S N H O CH3 N H3C CH3 F Ritanserin (Janssen) LY-53857 (Eli Lilly) N OH MDL-11939 (Hoechst Marion Roussel) Involvement of 5-HT3 receptor family Involvement of the 5-HT3 receptors in learning and memory was first studied using the antagonist, ondansetron (Glaxo Wellcome) [59]. With pre-training injections in mice on a habituation test, ondansetron (10 ng/kg) facilitated performance in young and aged animals, and inhibited impairment in habituation induced by scopolamine, electrolesions, or ibotenic acid lesions of the nucleus basalis magnocellularis [60]. Similarly, in the T-maze reinforced alternation task in rats, ondansetron (100 ng/kg) antagonized a scopolamine-induced impairment [60]. In an object discrimination and reversal learning task in the 114 IDrugs 1998 Vol 1 No 1 marmoset, pre-training administration of ondansetron, in doses ranging from of 1 to 10 ng/kg, was shown to improve learning during the initial discrimination and reversal tests [59-63]. In aversive experiments using other 5-HT3 antagonist receptors, granisetron (SmithKline Beecham; 1-10 mg/kg) given as a pre-training injection increased the stepdown latency when mice were tested 24 h after footshock on a passive avoidance task [64]; tropisetron (Novartis) improved the retrieval of a previously learned aversive habit in mice at doses of 1, 10, and 100 mg/kg; and pretreatment with itasetron (DAU-6215, Boehringer Ingelheim;1, 10, 30, and 100 mg/kg) antagonized a deficit in the acquisition of a passive-avoidance response caused by scopolamine administration in rats [65]. In aged animals, itasetron, at 10 mg/kg injected for 3 weeks, significantly improved task performance compared to that displayed by the old rats treated with the vehicle in the Morris water maze task [66]. In the water maze also, the effects of the 5-HT3 receptor antagonists mirisetron maleate (WAY-100579, WyethAyerst) and ondansetron at doses of 0.001, 0.01, and 0.1 mg/kg injected subcutaneously led to recovery of the deficit created by combined excitotaxic lesions of the nucleus basalis and medial septal brain regions. Linear dose-related improvement trends have been found with both 5-HT3 antagonists in acquisition as well as in retention during spatial learning in the water maze [67]. Given the importance of the hippocampus in learning and memory, and the fact that LTP may provide a substrate for certain forms of memory, ondansetron in the rat induces a reliable and dose-dependent (100 and 500 µg/kg) increase in hippocampal theta rhythm, a significant increase in the magnitude and duration of LTP, and improved retention in an odor matching problem and a spatial learning task [68]. The action of the 5-HT3 receptor antagonists may be taskdependent, since ondansetron, over a large dose range (1 ng/kg to 1 mg/kg) injected during the pre-session for 10 to 15 days, failed to attenuate a scopolamine-induced impairment in the stone maze, which is considered to be a complex spatial memory task [69]. Also, repeated treatments may be required, as suggested by the finding that itasetron was only effective with chronic treatment [65]. Moreover, post-training intraperitoneal injection of the 5-HT3 agonist, 1-(m-chlorophenyl)-biguanide hydrochloride (mCPBG; 1 to 10 mg/kg), has been shown to impair consolidation in the learning of a lever-press response in autoshaping, tropisetron (0.01 to 0.1 mg/kg) and ondansetron (0.1 to 1 mg/kg) improved it, and the effect induced by mCPBG (1.0 mg/kg) was prevented by each of these 5-HT3 antagonists at the lower dose. Interestingly, the individual effects of the 5-HT3 receptor agonists and antagonists are significantly decreased by p-chloroamphetamine (PCA) pre-treatment during the first and second autoshaping sessions (10 mg/kg x 2), implying that activation and blockade of presynaptic 5-HT3 receptors may be involved in the impairment and enhancement of learning, respectively [70]. Effects on 5-HT4 receptors Data suggest that the stimulation of 5-HT4 receptors plays a role in different aspects of the cognitive processes, eg, improving the acquisition of learning [4,71-73] but impairing its consolidation [74]. For instance, the 5-HT 4 receptor agonists BIMU-1 and BIMU-8 (Boehringer Ingelheim) improve acquisition on various behavioral learning tasks. Pre-training stimulation of 5-HT4 receptors improve social learning, prevent amnesia, and reverse deficits in mice performing a passive avoidance task following exposure to hypercapnia and hypoxia at 10 to 20 mg/kg [71-73]. When administered intraperitoneally immediately after the first presentation in social olfactory recognition tasks in rats, BIMU-1 (10.0 mg/kg) enhanced short-term memory (ie, recognition 2 h later). The effect of BIMU-1 was antagonized by intraperitoneal injection of 1 and 10 mg/kg GR-125487 (Glaxo Wellcome), a specific 5-HT4 antagonist [75]. Similarly, pre-training injection of BIMU-1 (5 to 20 mg/kg) or BIMU-8 (20 mg/kg) enhanced acquisition in the autoshaping task, but post-training administration of these drugs impaired consolidation at 10 to 20 mg/kg and 5 and 20 mg/kg, respectively [74]. Injection of the 5-HT4 receptor agonist, RS67333 (Roche Bioscience) (0.1, 10 and 1000 µg/kg) reverses atropine-induced performance deficits in the rat Morris water maze assay (30 mg/kg); such an effect is blocked by 5HT4 receptor antagonists [58]. Post-training administration of the 5-HT4 receptor, SDZ205557 (Novartis; 10.0 mg/kg) and GR-125487 (0.78 mg/kg), or PCA (10.0 mg/kg) pretreatment, does not appear to affect learning itself, but is able to reverse the effect of the 5-HT4 receptor agonist. This observation suggests the participation of postsynaptic 5-HT4 receptors [74]. It should be noted that BIMU-1 and BIMU-8 are mixed 5-HT4 agonists/5-HT3 antagonists. Nevertheless, recent data, using an olfactory association learning task with systemic pre-training administration of BIMU-1 at 1.0, 5.0, and 10.0 mg/kg, revealed a substantial improvement in associative memory [76]. One month later, difficulty rapidly reversing behavioral responses to previously learned associations, indicated that the BIMU-1 effect at 10.0 mg/kg was not transient but correlated to long-term memory. The effects of BIMU-1 are most likely mediated by 5-HT4 receptors, since they were blocked by GR-125487. These data suggest that activation of 5-HT4 receptors may modulate cognitive processes like learning and memory [76]. Such a view could be explained by the distribution of 5-HT4 receptors in adult rat brain in various regions belonging to the limbic system, especially in the hippocampus, olfactory tubercules, and Islands of Calleja [77-79]. Suspected roles of 5-ht5, 5-ht6 and 5-HT7 receptors 5-ht5 receptors At the present time there are no selective ligands to evaluate 5-HT5 function [80]. The possible functions of the 5-ht5 receptors can be predicted based on localization studies. For example, the limbic distribution in the mouse brain indicates a possible role in learning and in mood [81], while the distribution of 5-ht5A mRNA in the cerebellum suggests a role in the coordination of fine motor skills. The distribution of 5-ht5 messangers in the habenula is interesting. This region is a relay station between the limbic system and the midbrain, and it projects into the interpeduncular nucleus, raphe nuclei, substantia nigra, and ventral tegmental areas. Review 5-HT receptors in learning and memory 115 Figure 4. Drugs acting at 5-HT3 receptors H3C N H3C H3C N N O O N N N N H N CH3 HN N O O H3C Ondansetron (Glaxo Wellcome) Granisetron (SmithKline Beecham) Mirisetron (American Home Products) OH H N HO OH HN CH3 O N O O OH HO N O O HN O HO OH N H CH3 CH3 OH HN CH3 O Itasetron (Hoechst) The stimulation of GABAergic neurons in the lateral habenula inhibits both 5-HT neurons in the raphe and dopamine neurons in the substantia nigra and ventral tegmentum. Therefore, it is possible that modulation of 5HT and dopamine release through 5-ht5 receptors has behavioral consequences. Habenular lesions have been shown to lead to enhanced exploratory behavior. Together, these data indicate a possible role for the 5-ht5 receptors in the inhibition of behavior related to emotional states [80]. The first behavioral studies on possible 5-ht6-mediated functions were conducted using antisense oligonucleotides in male rats targeted to the 5-ht6 receptor subtype [83]. In these studies, the rats exhibited a behavioral phenotype consisting of an increased number of yawns and stretches. This behavior was blocked by atropine, suggesting the role of the 5-ht6 receptors in the control of cholinergic neuro-transmission. If so, then a 5-ht6 antagonist might be useful in the treatment of learning and memory disorders [82,83]. 5-ht6 receptors 5-HT7 receptors The distribution of 5-ht6 mRNA in limbic pathways, particularly in the dentate gyrus and CA1, CA2 and CA3 of the hippocampus [82], also suggests that 5-ht6 receptors may modulate learning and memory processes. The mRNA localization of the 5-ht6 receptor in the nucleus accumbens may also be indicative of a role in reinforcement/reward [82]. There are no selective ligands currently available to study the 5-HT7 receptors. Predictions of possible receptor function based on studies of the localization of 5-HT7 receptor mRNA and protein in the septum, hypothalamus, centromedial amygdala, and anterior hippocampal rudiment also include a role in the so-called limbic processes [84]. Moreover, studies of the role of 5-HT7 receptors require the development of selective subtype compounds. 116 IDrugs 1998 Vol 1 No 1 Figure 5. Drugs acting at 5-HT4 receptors N CH3 N H O H NH O N NH N O H Cl O N H H3C BIMU-1 (Boehringer Ingelheim) CH3 S N O O CH3 BIMU-9 (Boehringer Ingelheim) H N O O Cl F H2N O N H Cl N CH3 O CH3 CH3 GR-125487 (Glaxo Wellcome) Conclusions As a whole, it appears that the 5-HT1A, 5-HT2A/2C, 5-HT3, and 5-HT4 receptors are the main subtypes involved in learning and memory, whilst the role of the 5-ht5, 5-ht6, and 5-HT7 receptors in these cognitive processes can only be speculated. The role of 5-HT1A receptors in learning and memory processes is controversial. There are studies reporting that 5HT1A receptor agonists enhance learning, while some have failed to show such improvement, and others have observed impairments (Tables 1 - 3). It should be noted that the use of different experimental conditions and different behavioral tasks can help distinguish true cognitive effects from locomotor, motivational and emotional effects [24,85]. These inconsistencies may, in fact, be ascribed to differences in the behavioral tasks, doses, and drug administration schedules. This is true for all of the effects reported for the different 5HT receptor families in this review. Although the pre- or post-training and pre-retention stimulation of 5-HT1A receptors impairs learning in passive and active avoidance O CH3 H N CH3 Cl RS-67333 (Roche) tasks, the passive avoidance task cannot distinguish memory from motivational and sensory factors [eg, 24,48,86]. The observed difference between 8-OH-DPAT and buspirone has not yet been explained, but it is noteworthy that early studies found that buspirone does not affect learning and memory in humans [60,86], while more recent work has reported that it disrupts such processes [26]. The effects induced by the pre-training administration of 8-OH-DPAT seem to be non-specific, whereas those produced by the post-training injection of the drug are attributed to changes in learning [16,30,87,88]; however, 8-OH-DPAT enhances learning at doses of 0.1 mg/kg [21] or 0.015 to 0.062 mg/kg [15,87]. When administered 15 [87], 30 [14] or 20, but not 40 or 120 min [21,87] before the training session, 8-OH-DPAT does not alter visual learning or discriminability [16,18]. The changes observed in these tasks cannot be attributed to increments in feeding [15,87], as rats trained with the food magazine and treated 24 h later with 8-OH-DPAT displayed normal or enhanced learning [15]. In addition, the silent 5- Review 5-HT receptors in learning and memory 117 HT1A receptor antagonist, WAY-100635, blocks the nonspecific effects produced by 8-OH-DPAT in the delayed matching to position learning task [14,16]. Hence, it is reasonable to conclude that pre- or post-learning administration of 8-OH-DPAT enhances learning regardless of feeding, and that once learning is consolidated, the stimulation of 5-HT1A receptors does not alter it [3,15]. The learning and memory effects produced by pre- or posttraining injection of 8-OH-DPAT are eliminated by PCA and parachlorophenylalanine (PCPA) [15,33] and reversed by the silent 5-HT1A receptor antagonists, WAY-100135, WAY100635 and S-UH-301 [16,30,33]. These findings suggest that pre- and postsynaptic 5-HT1A receptors participate in the acquisition and consolidation of learning. To conclude, it should be noted that 5-HT1A receptors are: (i) coupled with inhibition of adenylate cyclase, directly + activating a K channel [89]; (ii) interact with other 5-HT receptor classes and subtypes; (iii) are co-expressed in single neurons with 5-HT2A/2C and 5-HT4 receptors in the hippocampus and cortex, and other neuronal populations [90,91]; and (iv) affect diverse neurotransmission systems [92,93]. Furthermore, from electrophysiological studies, it has been shown that 5-HT1A receptors in subregions of the hippocampus, and those located pre- and postsynaptically, exhibit different physiological responses [91,94,95]. Only a few studies have focused on the behavior of the 5HT2A/2C receptors [48]. Some behavioral paradigms (rabbit nictitating membrane conditioned response, rat conditioned avoidance response) have demonstrated the enhancement of learning 5-HT2A/2C receptor agonists, and that this enhancement is only blocked by drugs that are antagonists of these receptors [48,49,50]. However, it should be noted that the available 5-HT2A/2C compounds are not selective for these receptors [96,97], and some of these display negative intrinsic activity [48]. Various effects, including improvement of learning and anxiolysis, are observed with 5-HT3 receptor antagonists administered systemically and centrally [59,67,98-100]. For instance, granisetron, tropisetron, itasetron and mirisetron have precognitive effects, and counteract deficits in learning associated with dysfunction of central cholinergic neurons [67,100]. Moreover, the 5-HT3 agonist, 1-(m-chlorophenyl)biguanide (mCPBG), impairs the consolidation of learning, whereas tropisetron and ondansetron improve it; the effect induced by mCPBG is prevented by both 5-HT3 antagonists [70]. Interestingly, the individual effect of the 5-HT3 receptor agonists and antagonists is significantly reduced by PCA treatment, implying that activation and blockade of presynaptic 5-HT3 receptors may be involved in the impairment and enhancement of learning, respectively [70]. Presynaptic 5-HT3 receptors located in the amygdala and hippocampus may be involved in learning. In addition, 5HT3 receptors may be located presynaptically (in non-5-HT neurons, but postsynaptically in 5-HT neurons), possibly on the soma, axon, and/or nerve terminals of the GABAergic interneurons [67,100-102]. Electrophysiological studies have shown that 5-HT3 heteroreceptors modulate the activity of several neurotransmitters, including cholinergic and gluta- matergic neurotransmitters [92,93,102]. Previous findings suggest that postsynaptic 5-HT3 receptors are involved in learning and memory processes [68]. The pre-training stimulation of 5-HT4 receptors improves social learning and associative olfactory discrimination, prevents amnesia, and reverses defects in learning and memory resulting from hypercapnia and hypoxia [7173,75,76]. Injection of the 5-HT4 receptor agonist, RS- 67333, reverses the performance deficit produced by atropine; the effect is blocked by 5-HT4 receptor antagonists [103]. The post-training administration of the 5-HT4 receptor antagonists, SDZ-205557 and GR-125487, or pretreatment with PCA does not affect learning itself, but is able to reverse the effect of 5-HT4 receptor agonists. This observation suggests the participation of postsynaptic 5-HT4 receptors [74]. As autoradiographic studies have revealed that 5-HT4 receptors are located in the habenula, hippocampus, and amygdala [27], and electrophysiological studies have shown that 5-HT4 receptors mediate a slow but long-lasting excitatory response in the hippocampus [77,104], stimulation of the 5-HT4 receptors could modulate learning and memory processes. The development of new and more selective agonists will determine future studies of this receptor. Anatomical data confirm the location of 5-ht5, 5-ht6, and 5-HT7 receptors on cognitive pathways, especially in the hippocampus. A better understanding of the role played in cognition by these, and other 5-HT receptors, will be gained through the discovery of new specific ligands and new molecular tools, such as gene knockout and transgenic mice [4,80]. In conclusion, it is clear that 5-HT receptor families modulate cognitive processes, such as learning and memory. Nevertheless, more specific ligands with greater selectivity for a specific subtype of receptor and more elaborate behavioral tasks are required to enable the discrimination of the respective effects of these compounds on the different subcategories of learning and long-term memory. Such new compounds would be useful as therapeutic agents for patients with mnesic deficits, such as those observed in Alzheimer’s disease. Acknowledgements We wish to thank Joël Bockaert and Aline Dumuis for the preparation of the manuscript. We wish to acknowledge the expert secretarial assistance of Michelle Bauget. References 1. Twarog BM, Page JH: Serotonin content of some mammalian tissues and urine and a method of its determination. Am J Physiol (1953) 175:157-161. 2. Keane PE, Soubrié P: Animal models of integrated serotonergic functions: their predictive value for the clinical applicability of drugs interfering with serotonergic transmission. In: Handbook of experimental pharmacology. Serotonergic neurons and 5-HT receptors in the CNS. Baumgarten HG, Göthert M (Eds), Springer (1997) 27:707-719. 118 IDrugs 1998 Vol 1 No 1 3. Meneses A, Hong E: Role of 5-HT1A receptors in acquisition, consolidation and retrieval of learning. CNS Drug Rev (1997) 3(1):68-82. 4. Buhot MC: Serotonin receptors in cognitive behaviors. Curr Opin Neurobiol (1997) 7:243-254. 5. Peroutka SJ: 5-Hydroxytryptamine receptor subtypes. In: Serotonin receptors and their ligands. Olivier B, Van Wijngaarden I, Soudijn W (Eds), Elsevier Science BV (1997):3-13. 6. Francès H, Khidichian F, Monier C: Increase in the isolationinduced social behavioral deficit by agonists at 5-HT1A receptors. Neuropharmacology (1990) 29:103-107. 7. Balleine BW, Fletcher N, Dickinson A: Effect of the 5-HT1A agonist, 8-OH-DPAT on instrumental performance in rats. Psychopharmacology (1996) 125:79-88. 19. Kant JG, Meininger GR, Maughan KR, Wright WL, Robinson TN, Neely TM: Effects of the serotonin receptor agonists 8OH-DPAT and TFMPP on learning as assessed using a novel water maze. Pharmacol Biochem Behav (1996) 53:385390. 20. Winter JC, Petti DT: The effects of 8-hydroxy-2-(di-npropylamino)tetralin and other serotonergic agonists on performance in a radial maze: A possible role for 5-HT1A receptors in memory. Pharmacol Biochem Behav (1987) 27:625-628. 21. Deacon RMJ: Pharmacological studies of a rat spatial delayed nonmatch-to-sample task as an animal model of dementia. Drug Dev Res (1991) 24:67-79. 22. Bass EW, Means LW, Mcmillen BA: Buspirone impairs performance of a three-choice working memory water escape task in rats. Brain Res Bull (1992) 28:455-461. 8. Evenden JL: Effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) after repeated administration on a conditioned avoidance response (CAR) in the rat. Psychopharmacology (1992) 1-2:134-144. 23. Rowan MJ, Cullen WK, Moulton B: Buspirone impairment of performance of passive avoidance and spatial learning tasks in the rat. Psychopharmacology (1990) 100:393-398. 9. Mendelson SD, Quartermain D, Francisco T, Sherrer A: 5-HT1A receptor agonists induce anterograde amnesia in mice. Eur J Pharmacol (1993) 236:177-182. 24. Sarter M, Hagan J, Dudchenko P: Behavioral screening for cognition enhancers: from indiscriminate to valid testing. Part II. Psychopharmacology (1992) 107:461-473. 10. Riekkinen P: 5-HT1A and muscarinic acetylcholine receptors jointly regulate passive avoidance behavior. Eur J Pharmacol (1994) 262:77-90. 25. Torres C, Morales A, Candido A, Maldonado A: Differential effect of buspirone and diazepam on negative contrast in one-way avoidance learning. Eur J Pharmacol (1995) 280:277-284. 11. Sanger GJ, Joly D: Performance of a passive avoidance response is disrupted by compounds acting at 5-HT1A receptors. Behav Pharmacol (1989) 1:235-240. 12. Sanger GJ, Joly D, LePichon M: Buspirone, gepirone and ipsapirone disrupt both active and passive avoidance responding in rats. Behav Pharmacol (1989) 1:153-160. 13. Winsauer PJ, Bixler MA, Mele PC: Comparison of the effects of typical and atypical anxiolytics on learning in monkeys and rats. J Pharmacol Exp Ther (1996) 276:1111-1127. 14. Fletcher A, Cliffe IA, Dourish CT: Silent 5-HT1A receptor antagonists: Utility as research tools and therapeutic agents. TIPS (1993) 14:441-448. 15. Meneses A, Hong E: Mechanisms of action of 8-OH-DPAT on learning and memory. Pharmacol Biochem Behav (1994) 49:1083-1086. 16. Stanhope KJ, McLenachan AP, Dourish CT: Dissociation between cognitive and motor/motivational deficits in the delayed matching to position test: Effects of scopolamine, 8-OH-DPAT and EAA antagonists. Psychopharmacology (1995) 122:268-280. 17. Carli M, Samanin R: 8-Hydroxy-2-(di-n-propylamino)tetralin impairs spatial learning in a water maze: Role of postsynaptic 5-HT1A. Br J Pharmacol (1992) 105:720-726. 18. Carli M, Luschi R, Garofalo P, Samanin R: 8-O-DPAT impairs spatial but not visual learning in a water maze by stimulating 5-HT1A receptors in the hippocampus. Behav Brain Res (1995) 67:67-74. 26. Unrug-Neervoort A, VanLuijtelaar G, Coenen A: Cognition and vigilance: differential effects of diazepam and buspirone on memory and psychomotor performance. Neuropsychobiology (1992) 26:146-150. 27. Cole BJ, Jones GH, Turner JD: 5-HT1A receptor agonists improve the performance of normal and scopolamineimpaired rats in an operant delayed matching to position task. Psychopharmacology (1994) 116:135-142. 28. Riekkinen M, Sirviö J, Toiven T, Riekkinen P: Combined treatment with a 5-HT1A receptor agonist and a muscarinic acetylcholine receptor antagonist disrupts water maze navigation behavior. Psychopharmacology (1995) 122:137146. 29. Quatermain D, Clemente J, Shemer A: The 5-HT1A agonist, tandospirone, disrupts retention but not acquisition of active avoidance learning. Pharmacol Biochem Behav (1994) 48:805-807. 30. Fletcher A, Forster EA, Bill DJ et al.: Electrophysiological, biochemical, neurohormonal and behavioral studies with WAY-100,635, a potent, selective and silent 5-HT1A receptor antagonist. Behav Brain Res (1996) 73:337-353. 31. Meneses A, Hong E: Modification of the anxiolytic effects of 5-HT1A agonists by shock intensity. Pharmacol Biochem Behav (1993) 6:569-573. 32. Meneses A, Hong E: Effects of serotonergic compounds on associative learning. Proc West Pharmacol Soc (1991) 4:461464. Review 5-HT receptors in learning and memory 119 33. Hong E, Meneses A: The activation of serotonergic 5-HT1A presynaptic receptors or an enhancement of 5-HT postsynaptic activity increase learning. Proc West Pharmacol Soc (1995) 38:86-86. 49. Gimpl MP, Gormezano I, Harvey JA: Effects of LSD on learning as measured by classical conditioning of the rabbit nictitating membrane response. J Pharmacol Exp Ther (1979) 208:330-334. 34. Hong E, Meneses A: The increase in learning induced by indorenate and 8-OH-DPAT was blocked by silent 5-HT1A antagonists. Soc Neurosci Abstr (1995) 21:1228. 50. Harvey JA, Gormezano I, Cool VA: Effects of d-lysergic acid diethylamide, d-2-bromolysergic acid diethylamine, dl-2,5dimethoxy-4-methylamphetamine and d-amphetamine on classical conditioning of the rabbit nictitating membrane response. J Pharmacol Exp Ther (1982) 221:289-294. 35. Meneses A, Hong E: Effect of fluoxetine on learning and memory involves multiple 5-HT systems. Pharmacol. Biochem. Behav. (1995) 52:341-346. 36. Klint T: Effects of 8-OH-DPAT and buspirone in a passive avoidance test and in the elevated plus-maze test in rats. Behav J Pharmacol (1991) 2:481-489. 37. Jansen JHM, Andrews JS: The effects of serotonergic drugs on short-term spatial memory in rats. J Psychopharmacol (1994) 8:157-163. 38. Briley M, Chopin P, Marien M, Moret C: Functional neuropharmacology of compounds acting at 5-HT1B/D receptors. In: Handbook of experimental pharmacology. Serotonergic neurons and 5-HT receptors in the CNS. Baumgarten HG, Göthert M (Eds), Springer (1997) 27:269-284. 39. Edwards E, Harkins K, Wright G, Henn FA: 5-HT1B receptors in an animal model of depression. Neuropharmacology (1991) 30:101-105. 40. Petty F, Kramer G, Wilson L: Prevention of learned helplessness - in vivo correlation with cortical serotonin. Phamacol Biochem Behav (1992) 43:361-367. 41. Martin P, Puech AJ: Is there a relationship between 5-HT1B receptors and the mechanisms of action of antidepressant drugs in the learned helplessness paradigm in rats? Eur J Pharmacol (1991) 192: 193-196. 42. Green AR, Guy AP, Gardner CR: The behavioural effects of RU-24969, a suggested 5-HT1 receptor agonist in rodents and the effect on the behaviour of treatments with antidepressants. Neuropharmacology (1984) 23:655-661. 43. Noda Y, Ochi Y, Shimada E, Oka M: Involvement of central cholinergic mechanism in RU-24969-induced behavioral deficits. Pharmacol Biochem Behav (1991) 38:441-446. 44. Koe BK, Nielsen JA, Macor JE, Heym J: Biochemical and behavioral studies of the 5-HT1B receptor agonist, CP94,253. Drug Dev Res (1992) 26:241-250. 45. Mittman SM, Geyer MA: Effects of 5-HT1A agonists on locomotor and investigatory behaviors in rats differ from those of hallucinogens. Psychopharmacology (1989) 98:321329. 46. Lucki I, Frazer A: Behavioural effects of indole and piperazine-type serotonin receptor agonists. Soc Neurosci Abstr (1982) 8:101. 47. Meneses A, Hong E: Effects of 5-HT receptor antagonists GR-127935T (5-HT1B/1D) and MDL-100907 (5-HT2A) on learning. Soc Neurosci Abstr (1996) 22:1586. 48. Harvey JA: Serotonergic regulation of associate learning. Behav Brain Res (1996) 73:47-50. 51. Gormezano I, Harvey JA, Aycock E: Sensory and associative effects of LSD on classical appetitive conditioning of the rabbit jaw movement response. Psychopharmacology (1980) 70:137-143. 52. Schindler CW, Gormezano I, Harvey JA: Effects of morphine and LSD on the classically conditioned nictitating membrane response. Pharmacol Biochem Behav (1985):41-46. 53. Romano AG, Harvey JA: MDMA enhances associative and nonassociative learning in the rabbit. Pharmacol Biochem Behav (1994) 47:289-293. 54. Romano AG, Bormann NM, Harvey JA: A unique enhancement of associative learning produced by methylene dioxyamphetamine. Behav Pharmacol (1991) 2:225-231. 55. Alhaider AA, Ageel AM, Ginawi OT: The quipazine-andTFMPP-increased conditioned avoidance response in rats: Role of 5-HT1C/5-HT2 receptors. Neuropharmacology (1993) 32:1427-1432. 56. Ginn SR, Powell DA: Pizotifen attenuates classical eye-blink and heart rate conditioning in rabbits. Physiol Psychol (1986) 14:36-41. 57. Welsh S, Romano AG, Simansky KJ, Harvey JA: Evidence for an inverse agonist action of ritanserin on associative learning in the rabbit. Soc Neurosci Abstr (1994) 20:557.5. 58. Altman HJ, Normile HJ: What is the nature of the role of serotonergic nervous system in learning and memory: Prospects for development of an effective treatment strategy for senile dementia. Neurobiol Aging (1988) 9:627638. 59. Costall B, Naylor RJ: The psychopharmacology of 5-HT3 receptors. Pharmacol Toxicol (1992) 71:401-415. 60. Barnes JM, Costall B, Coughlan J, Domeney AM, Gerrard PA, Kelly ME, Naylor RJ, Onaivi ES, Tomkins DM, Tyers MB: The effects of ondansetron, a 5-HT3 receptor antagonist, on cognition in rodents and primates. Pharmacol Biochem Behav (1990) 35:955-962. 61. Carey GJ, Costall B, Domeney AM, Gerrard PA, Jones DNC, Naylor RJ, Tyers MB: Ondansetron and arecoline prevent scopolamine-induced cognitive deficits in the marmoset. Pharmacol Biochem Behav (1992) 42:75-83. 62. Costall B, Naylor RJ: Neuropharmacology of 5-HT3 receptor ligands. In: Handbook of experimental pharmacology. Serotonergic neurons and 5-HT receptors in the CNS. Baumgarten HG, Göthert M (Eds), Springer (1997) 27:409-427. 120 IDrugs 1998 Vol 1 No 1 63. Domeney AM, Costall B, Gerrard PA, Jones DN, Naylor RJ, Tyers MD: The effect of ondansetron on cognitive performance in the marmoset. Pharmacol Biochem Behav (1991) 38:169-175. 64. Chugh Y, Saha N, Sankaranarayanan A, Sharma PL: Memory enhancing effects of granisetron (BRL 43694) in a passive avoidance task. Eur J Pharmacol (1991) 203:121-123. 65. Brambilla A, Ghiorzi A, Pitsikas N, Borsini F: DAU-6215, a novel 5-HT3 receptor antagonist, selectively antagonizes scopolamine-induced deficit in a passive-avoidance task, but not scopolamine-induced hypermotility in rats. J Pharm Pharmacol (1993) 45(9):841-843. 66. Pitsikas N, Brambilla A, Borsini F: DAU-6215, a novel 5-HT3 receptor antagonist, improves performance in the aged rat in the Morris water maze task. Neurobiol Aging (1993) 14(6):561-564. 67. Hodges H, Sowinski P, Turner JJ, Fletcher A: Comparison of the effects of the 5-HT3 antagonists WAY- 100579 and ondansetron on spatial learning in the water maze in rats with excitotoxic lesions of the forebrain cholinergic projection system. Psychopharmacology (1996) 25:146-161. 68. Stäubli U, Bo Xu F: Effects of 5-HT3 receptor antagonism on hippocampal theta rhythm, memory, and LTP induction in the freely moving rat. J Neurosci (1995) 15(3):2445-2452. 69. Bratt AM, Kelly ME, Domeney AM, Naylor RJ, Costall B: Failure of ondansetron to attenuate a scopolamine-induced deficit in a stone maze task. Neuroreport (1994) 5:1921-1924. 70. Hong E, Meneses A: Systemic injection of p-chloroamphetamine eliminates the effect of the 5-HT3 compounds on learning. Pharmacol Biochem Behav (1996) 53:765-769. 71. Ghelardini C, Galeotti N, Meoni P, Giotti A, Rizzi CA, Bartolini A: Effect of 5-HT4 agonists: BIMU1 and BIMU8 on the social learning test in rats. CINP Congress Washington, DC, USA (1993). 72. Ghelardini C, Meoni P, Galeotti N, Malmberg-Aiello O, Rizzi CA, Bartolini A: Effect of the two benzimidazolone derivates: BIMU-1 and BIMU-8 on a model of hypoxia-induced amnesia in the mouse. 5-HT Third IUPHAR Satellite Meeting on Serotonin, Chicago, Illinois, USA (1994). 77. Eglen RM, Wong EH, Dumuis A, Bockaert J: Central 5-HT4 receptors. TIPS (1995) 16:391-398. 78. Grossman CJ, Kilpatrick GJ, Bunce KT: Development of a radioligand binding assay for 5-HT4 receptors in guinea-pig and rat brain. Br J Pharmacol (1993) 109:618-624. 79. Waeber C, Sebben M, Nieoullon A, Bockaert J, Dumuis A: Regional distribution and ontogeny of 5-HT4 binding sites in rodent brain. Neuropharmacology (1994) 33(3-4):527-541. 80. Branchek TA, Zgombick JM: Molecular biology and potential functional role of 5-ht5, 5-ht6, and 5-HT7 receptors. In: Handbook of experimental pharmacology. Serotonergic neurons and 5-HT receptors in the CNS. Baumgarten HG, Göthert M (Eds), Springer, 27:475-793. 81. Plassat JL, Boschert U, Amlaiky N, Hen R: The mouse 5-ht5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family. EMBO J (1992) 11:4779-4786. 82. Ward RP, Hamblin MW, Lachowicz JE, Hoffman BJ, Sibley DR, Dorsa DM: Localization of serotonin sybtype 6 receptor messenger RNA in the rat brain by in situ hybridization histochemistry. Neuroscience (1995) 64:1105-1111. 83. Bourson A, Boroni E, Austin RH, Monsma FJ Jr, Sleight AJ: Determination of the role of the 5-HT6 receptor in rat brain: a study using antisense oligonucleotides. J Pharmacol Exp Ther (1995) 274:173-180. 84. Gustafson EL, Durkin MM, Bard JA, Zgombick JM, Branchek TA: A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5-HT7 receptor in rat brain. Br J Pharmacol (1996) 117:657-666. 85. Decker MW, McGaugh JL: The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory. Synapse (1991) 7:151-168. 86. Dolmella A, Bandoli G, Nicolini M: Alzheimer’s disease: A pharmacological challenge. In: Advances in Drug Research. Testa B., Meyer UA (Eds), Academic Press, London (1994):203-294. 87. Meneses A, Hong E: Modification of 8-OH-DPAT effect on learning by manipulation of the assay conditions. Behav Neural Bull (1994) 61:29-35. 73. Ghelardini C, Malmberg-Aiello O, Bartolini A, Rizzi CA: Protective effect of BIMU-1 and BIMU-8 on amnesia induced by hypercapnia. 10th Congress of Eur Soc for Neurochem, Jerusalem (1994). 88. Meneses A, Hong E: A pharmacological analysis of serotonergic receptors: effects of their activation or blockade in learning. Prog Neuropsychopharmacol Biol Psychiat (1997) 21:273-296. 74. Meneses A, Hong E: Effects of 5-HT4 receptor agonists and antagonists in learning. Pharmacol Biochem Behav (1997) 56:347-351. 89. Sanders-Bush E, Canton H: Serotonin receptors: signal transduction pathways. In: Psychopharmacology: The Fourth Generation of Progress. Bloom EF, Kupfer DJ (Eds), Raven Press, New York (1995):431-441. 75. Letty S, Child R, Dumuis A, Pantaloni A, Bockaert J, Rondouin G: 5-HT4 receptors improve social olfactory memory in the rat. Neuropharmacology (1997) 36(4-5):681-687. 90. Andrade R: Electrophysiology of 5-HT1A receptors in the rat hippocampus and cortex. Drug Dev Res (1992) 26:275-286. 76. Marchetti-Gauthier E, Roman FS, Dumuis A, Bockaert J, Soumireu-Mourat B: BIMU1 increases associative memory in rats by activating 5-HT4 receptors. Neuropharmacology (1997) 36(4-5):697-706. 91. Blier P, Lista A, De Montigny C: Differential properties of preand postsynaptic 5-hydroxytryptamine1A receptors in the dorsal raphe and hippocampus: 1. Effect of spiperone. J Pharmacol Exp Ther (1993) 265:7-15. Review 5-HT receptors in learning and memory 121 92. Jacobs BL, Azmitia EC: Structure and function of the brain serotonin system. Physiol Rev (1992) 72:165-229. 93. Zifa E, Filion G: 5-Hydroxytryptamine receptors. Pharmacol Rev (1992) 44:401-458. 94. Francis PT, Pangalos MN, Pearson RCA, Middlemiss DN, Stratmann GC, Bowen DM: 5-Hydroxyiryptamine1A but not 5Hydroxyiryptamine2 receptors are enriched on neocortical pyramidal neurons destroyed by intrastriatal volkensin. J Pharmacol Exp Ther (1992) 261:1273-1281. 95. Handley SL: 5-Hydroxytryptamine pathways in anxiety and its treatment. Pharmacol Ther (1995) 66:103-148. 96. Hoyer D, Hartig PR, Humphrey PPA: International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rec (1994) 46:157-203. 97. Hoyer D, Martin GR: Classification and nomenclature of 5HT receptors: A comment on current issues. Behav Brain Res (1996) 3:263-268. 98. Gower AJ: 5-HT receptors and cognitive function. In: Central serotonin receptors and psychotropic drugs. Marsden CA, Heal DJ (Eds), Blackwell Scientific Publications, Oxford (1992):415429. 99. Lister RG: The effects of benzodiazepines and 5-HT1A agonists on learning and memory. In: 5-HT1A agonists, 5-HT3 antagonists and benzodiazepines: their comparative behavioral pharmacology. Rodgers RJ, Cooper SJ (Eds), John Wiley, Chichester (1991):267-280. 100.Passani MB, Corradetti R: Therapeutic potentials of itasetron (DAU-6215), a novel 5-HT3 receptor antagonist, in the treatment of central nervous system disorders. CNS Drug Rev (1996) 2:195-213. 101.Hjorth S: (-)-Pindolol, but not buspirone, potentiates the citalopram-induced rise in extracellular 5-hydroxytryptamine. Eur J Pharmacol (1996) 303:183-186. 102.Ramirez MJ, Cenarruzabeitia E, Lasheras B, del Rio J: Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex. Brain Res (1996) 12:274-280. 103.Fontana DJ, Daniels SE, Wong EHF, Clark RD, Eglen RM: The effects of novel, selective 5-hydroxytryptamine (5-HT4) receptor ligands in rat spatial navigation. Neuropharmacology (1997) 36(4-5):689-696. 104.Aghajanian GK: Electrophysiology of serotonin receptor subtypes and signal transduction pathways. In: Psychopharmacology: the fourth generation of progress. Bloom EF, Kupfer KJ (Eds), Raven Press, New York (1995):451-460. 122 IDrugs 1998 Vol 1 No 1 GS-4104 Gilead Sciences Inc Andreas Billich Address Novartis Research Institute General Dermatology PO Box 80 Brunner Strasse 59 A-1235 Vienna Austria Email: [email protected] IDrugs 1998 1(1):122-128 Current Drugs Ltd ISSN 1369-7056 Gilead and Roche are codeveloping GS-4104, a neuraminidase inhibitor, for the potential treatment and prevention of influenza virus infections, type A and type B. The companies began international phase II/III trials in December 1997 [272638]. The phase II/III double-blind, placebo-controlled study involves centers in the US, Canada, Europe and Hong Kong [272638]. The program will consist of two treatment studies and two prophylaxis studies, each of which will recruit 750 patients [273331]. The trials involve over 1500 patients, who will receive either GS-4104 or placebo for 6 weeks, as a once- or twice-daily dosing schedule [276650], [276895]. The company has also registered 73 centers for the two treatment studies, and 55 of these had been activated by the end of January 1998. Patients in the treatment arm will receive either GS-4104 or placebo for 5 days. Roche is conducting similar trials in Europe, Canada and Hong Kong [276895]. In September 1997, results were announced from two phase II studies, initiated in May 1997, in which volunteers were exposed intranasally to influenza A. The patients received oral capsules of GS-4104, or placebo, once-daily or twicedaily. In the first study, GS-4104 decreased the duration of symptoms by nearly 50%. In the second study, all patients who were given GS-4104 prophylactically did not have detectable levels of virus, as compared to 50% in the placebo group [264362]. In phase I trials, GS-4104 was welltolerated with good oral absorption and distribution in blood and tissue [248180]. Introduction Influenza viruses cause respiratory illness with significant morbidity and mortality. At present, influenza is managed mainly by preventative vaccination or by symptomatic treatment. The success of vaccinations is limited by the continuous mutation of the influenza virus. Symptomatic treatment with amantadine and rimantadine (Du Pont Merck) shortens the duration of symptoms caused by type A virus strains, but is inactive against type B strains, which account for about one third of infections. Also, side-effects have been associated with these drugs and the emergence of resistant influenza strains has been observed. The viral enzyme, neuraminidase (sialidase), is a surface glycoprotein that cleaves sialic acids from glycoproteins and Originator Gilead Sciences Inc Status Phase III clinical Indication Influenza virus infection Action Viral replication inhibitor, Sialidase inhibitor Biotechnology Oral formulation CAS 1-Cyclohexene-1-carboxylic acid, 4-(acetylamino)-5amino-3-(1-ethylpropoxy)-, [3R-(3.α.,4.β.,5.α.)]Registry No: 187227-45-8 Note: GS-4071 - free acid of GS-4104. Synonyms GS-4109, Ro-64-0796, GS-4071, GS-4116, GS4104 analogs, Gilead O O O O N H NH 2 glycolipids. The enzyme is responsible for the release of new particles from infected cells and may also assist in the spreading of the virus through the mucus within the respiratory tract. Therefore, neuraminidase is thought to represent a suitable target for antiviral therapy. Based on the initial discovery of 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en) as a transition state analog inhibitor of neuraminidases, a large range of more specific and more potent derivatives have been synthesized. The present evaluation decribes the status of one of these, GS-4104, which, based on data from recent clinical trials [266214], holds promise for future therapy of influenza infections. Synthesis and SAR Derivatives of the transition state inhibitor, Neu5Ac2en, featuring an amino or guanidino function in the 4-position have been identified as more potent inhibitors of influenza A and B virus sialidase than the original lead compound [254841,158324]. 4-Guanidino-Neu5Ac2en (zanamivir, Biota/ Glaxo) is a highly effective inhibitor of both the sialidase and the growth of a wide range of influenza A and B strains in vitro [158324]. Zanamivir has shown efficacy in phase II challenge studies for the prophylaxis and treatment of influenza virus infection [280306]. The drug has to be administered by either intranasal or inhaled routes since it exhibits poor oral absorption. In addition, the compound is rapidly excreted [165582]. The search for a molecule with similar potency, but improved pharmacokinetic properties lead researchers at Gilead to three major changes of the zanamivir structure [280310]: (i) the labile dihydropyran Drug evaluation GS-4104 123 ring was converted to a cyclohexene ring; (ii) the polar side chain in the 6-position of zanamivir was replaced by a more lipophilic ether moiety which is accommodated in a hydrophobic pocket in the active site of the enzyme; (iii) the 4-guanidino group was changed to an amino group. The resulting compound, GS-4071, was equipotent with zanamivir in an in vitro plaque reduction assay; the ester prodrug of GS-4071, designated GS-4104, showed good oral bioavailability in several species [280310]. The synthesis of multi-kilogram amounts of GS-4104 has been achieved by a lengthy linear sequence (about 16 steps, with no chromatography needed) starting from (-)-quinic acid [280310]. It is reasonable to expect that a more direct route can be achieved for process development. Clinical Development Phase I The phase I program demonstrated that GS-4104 was well tolerated with good oral absorption and distribution in blood and tissue [248180]. Phase II Eighty volunteers received doses (20 mg, bid, to 200 mg, qid) of GS-4104 for 5 days beginning 28 h after intranasal exposure to influenza A. The viral load was reduced by more than 100-fold in those receiving GS-4104 compared with placebo; the median time to cessation of symptoms in GS-4104-treated patients was 53 h versus 95 h in the placebo group [266214]. Synthesis of several series of derivatives has not yet led to further improvement of the activity of GS-4071, with the exception of the guanidino analog, GS-4116. However, this compound and its ethyl ester showed poor oral bioavailability [258727,258728,258052]. In a separate prophylaxis study, 37 patients received 100 mg GS-4104 once or twice daily, or placebo, for 5 days, beginning 26 h before intranasal exposure to influenza A. None of the treated patients had detectable virus in nasal passages compared with 50% of those receiving placebo [266214]. Pharmacology Although GS-4104 was generally well tolerated in both studies, transient, mild to moderate post-dosing nausea was reported in some individuals [266214]. GS-4071 is a potent inhibitor of influenza neuraminidase (Ki < 1 nM) [280313]. In a plaque reduction assay in cell culture, the compound was active against an influenza A strain (H1N1) (ED50 = 16 nM) [5]. In a more detailed study, GS-4071 showed potent inhibitory activity against a range of different isolates of influenza H1N1, H3N2, H2N2 and B strains (ED50 from 0.2 to 14 nM) [271744]. In addition, it was active against clinical isolates grown on cells as well as in eggs and in adenoid explants; GS-4071 had a potency and antiviral spectrum similar to zanamivir and was also active against certain viruses with reduced susceptibility to zanamivir [247132]. In a mouse infection model, on oral dosing at 1 mg/kg/day, the prodrug, GS-4104, exhibited excellent efficacy (87% survival versus 6% in the placebo group) [271744]. Delaying po administration with GS-4104 as late as 60 h post-virus exposure was still inhibitory to a lethal influenza A virus infection [254764]. In ferrets infected with influenza A virus, GS-4104 showed efficacy in reducing flu symptoms, such as pyrexia, nasal cell infiltrate, nasal signs, at an oral dose of 5 mg/kg [256493]. Metabolism GS-4071 has low (< 5%) oral bioavailability in animals, but the ethyl ester prodrug, GS-4104, has exhibited good oral bioavailability in rats (36%), mice and dogs (65 to 100%) [254763,247131,280313]. Following absorption from the gastrointestinal tract, GS-4104 undergoes rapid enzymatic conversion to the active form, GS-4071 [280314]. Furthermore, the parent drug, GS-4071, was delivered into rat bronchoalveolar fluid following oral administration of the prodrug [280313]. Toxicity In a 5-day repeat dose toxicology study in rats, GS-4104 did not demonstrate any toxic effects with a dose of 300 mg/kg/day, po [271744]. Current Opinion Currently available modalities for therapy of influenza infection are suboptimal. Therefore, novel drugs, such as the neuraminidase inhibitors, are needed, especially for therapy in elderly patients and in small children. GS-4104 is an orally bioavailable prodrug which is readily converted in the gastrointestinal tract to GS-4071, a potent and highly selective inhibitor of the viral sialidase. Following absorption, GS-4071 is distributed to the bronchoalveolar lavage fluid where, at least in rats, it is encountered at substantial and sustained concentrations. GS-4071 is approximately equipotent to zanamivir against influenza A and B strains. However, GS-4071 can be delivered orally by its prodrug, whilst zanamivir can only be used topically. Although arguments in favor of both routes of application can be sought, such as the convenience of oral dosage versus more direct and fast interference at the target site by the topical route, only extended phase III trials and finally, acceptance by the market, will determine which of the two approaches is superior with respect to efficacy, side-effects, and patient compliance. A general concern in antiviral therapy is the occurrence of resistance. Currently, it is not known how important an issue this will be in the case of the neuraminidase inhibitors. Any speculations about the superiority of this class of compound over the M2 protein inhibitors with respect to development of resistance, appear not to be based on experimental data. Combination therapy might be the future strategy for the treatment of influenza infection to potentiate the efficacy of the drugs and to minimize the risk for spreading of resistant virus. A concern voiced by epidemiologists, is the possible reluctance of people to be vaccinated, once effective drugs for treatment and prophylaxis are available [256487]. This 124 IDrugs 1998 Vol 1 No 1 argument cannot be used against the introduction of neuraminidase inhibitors, especially for use in high risk groups; rather, the issue should be considered as a challenge for the medical community and public health authorities to ensure thoughtful use of these drugs. To conclude from the currently available data, GS-4104 appears to be an effective drug that is based on a scientifically sound concept and is likely to be successful on the market. Licensing Hoffmann-La Roche AG Exclusive worldwide rights to Gilead’s proprietary neuraminidase inhibitors, including GS 4104. Roche will make an initial cash payment to Gilead of $10 million and up to an additional $40 million in cash upon achievement of developmental and regulatory milestones. In addition, Roche will fund all research and development costs and pay Gilead undisclosed royalties on the net sales of any products developed under the collaboration [220572]. Development history DEVELOPER COUNTRY STATUS INDICATION DATE REF F Hoffmann-La Roche Ltd UK C2 Influenza virus infection 30-MAY-97 248180 Gilead Sciences Inc US C3 Influenza virus infection 17-DEC-97 272638 Hoffmann-La Roche Inc US C3 Influenza virus infection 17-DEC-97 272638 Gilead Sciences Inc Western Europe C3 Influenza virus infection 17-DEC-97 272638 Gilead Sciences Inc Canada C3 Influenza virus infection 17-DEC-97 272638 Gilead Sciences Inc Hong Kong C3 Influenza virus infection 17-DEC-97 272638 Hoffmann-La Roche Inc Canada C3 Influenza virus infection 17-DEC-97 272638 Hoffmann-La Roche Inc Western Europe C3 Influenza virus infection 17-DEC-97 272638 Hoffmann-La Roche Inc Hong Kong C3 Influenza virus infection 17-DEC-97 272638 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Chemistry STUDY TYPE RESULT REF Synthesis and SAR Design, synthesis and structural analysis of GS-4104. 280310 SAR Synthesis of C3-thia and C3-carba analogs of GS-4071. 258727 SAR Synthesis of C2-substituted analogs of GS-4071. 258728 SAR Systemic variation in positions C3, C4 and C5 of GS-4071. 258052 Biology STUDY TYPE EFFECT STUDIED EXPERIMENTAL MODEL RESULT REF In vitro Activity against influenza virus. Plaque reduction assay. GS-4071 is active against a series of A and B strains (ED 50 = 0.1 to 14 nM). 271744 In vitro Activity against influenza virus. Assays using cell-, egg-, or tissue-grown viruses. GS-4071 shows similar potency and antiviral spectrum as zanamivir. 247132 In vivo Survival of mice after influenza infection. Oral dosing at 1 mg/kg/day after infection. 87% survival versus 6% in placebo group. 271744 In vivo Survival of mice after influenza infection. Oral dosing at 10 and 1 mg/kg/day after infection. GS-4104 protects from lethal infection with strain H1N1, even when administered 60 h exposure to the virus. 254764 Drug evaluation GS-4104 125 Biology (continued) STUDY TYPE EFFECT STUDIED EXPERIMENTAL MODEL RESULT REF In vivo Flu symptoms in ferrets. Oral dosing to ferrets infected with influenza A virus. Efficacy in reducing pyrexia, nasal cell infiltrate and nasal signs at a dose of 5 mg/kg, bid. 256493 STUDY TYPE EFFECT STUDIED MODEL USED RESULT REF In vivo Penetration of GS-4071 into rat bronchoalveolar lavage fluid. Oral administration of prodrug to rats. Measurement of parent drug in plasma and bronchoalveolar lavage fluid. Oral bioavailability based on plasma level is 36%. Significant delivery of GS-4071 to bronchoalveolar lavage fluid, longer persistence than in plasma. 280313 In vivo Oral bioavailability. Oral dosage to rats. GS-4104 undergoes rapid cleavage to GS-4071 after absorption from the GI tract; bioavailability in rats is 36%. 254763 In vivo Oral bioavailability. Oral dosage to dogs. Oral bioavailability in dogs is 100%. 247131 Metabolism Clinical EFFECT STUDIED MODEL USED RESULT REF Safety, tolerability and absorption in phase I study. Oral dosing in healthy volunteers. GS-4104 was well tolerated with good oral absorption and distribution. 248180 Phase II study examining the effect on flu symptoms and virus load. Exposure of volunteers to influenza A; dosing of GS-4104 either postexposure or prophylactically. GS-4104 showed efficacy by reducing the time to cessation of symptoms (therapeutic treatment) and preventing virus replication (prophylaxis). 266214 Bibliography 158324 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro. Woods JM, Bethell RC, Coates JAV, Healy N, Hiscox SA, Pearson BA, Ryan DM, Ticehurst J, tilling J, Walcott SM, Penn CR ANTIMICROB AGENTS CHEMOTHER 1993 37 7 1473 1479 220002 Gilead sciences announces discovery of orally active influenza inhibitors. Gilead Sciences Inc PRESS RELEASE 1996 September 17 220572 Roche and Gilead Sciences announce worldwide influenza collaboration. Hoffmann-La Roche AG PRESS RELEASE 1996 September 30 220573 Hoffmann-La Roche, Gilead Sciences announce collaboration to co-promote potential hepatitis C treatment in the U.S. Gilead Sciences Inc PRESS RELEASE 1996 September 30 221067 Gilead to copromote Roche’s roferon-A for hepatitis C. FDC REPORTS PINK SHEET 1996 58 41 T&G-9 - T&G-10 221313 Gilead, Roche sign potential $50 M deal. BIOWORLD WEEK 1996 4 41 1 221842 Roche teams up with Gilead and ArQule. SCRIP 1996 2170 7 221857 Nasal ’flu vaccine proves effective. 27 SCRIP 1996 2170 222094 Gilead claims preclinical success with influenza neuraminidase inhibitor. EMERGING PHARMACEUTICALS 1996 5 10 P8 - P9 222233 36th Interscience Conference on Antimicrobial Agents and Chemotherapy: Antivirals. IDDB MEETING REPORT 1996 15 - 18 September 226699 Roche joins Gilead on flu treatments ProScript on cancer cachexia. EMERGING PHARMACEUTICALS 1996 5 11 P7 - P8 226824 Re: Vistide, GS-4104, GS-840. 3RD QUARTER REPORT 1996 October 25 227642 Drug development. DRUG DEV PIPELINE 1996 1 7 1 - 4 231659 Human tests to start on flu pill. RELEASE 1997 January 28 Reuters PRESS 231741 Preliminary tests indicate potential influenza cure, PRESS RELEASE 1997 preventative. ACS News Service January 29 237533 Hoffman-La Roche and Gilead Sciences initiate human testing of oral influenza drug GS-4104. F Hoffmann La Roche Ltd PRESS RELEASE 1997 March 11 126 IDrugs 1998 Vol 1 No 1 237812 Vistide boosts Gilead. SCRIP 1997 2207 13 238976 Xoma presents data from meningococcemia trial at London symposium. PRESS RELEASE 1996 March 14 239780 Drug development digest. CEUTICALS 1997 6 3 15 254766 GS-4071 is a potent and selective inhibitor of the growth and neuraminidase activity of influenza A and B virus in vitro. Mendel DB, Tai CY, Escarpe PA, Li WX, Kim CU, Williams MA, Lew W, Zhang L, Bischofberger N, Huffman JH, Sidwell RW, Chen MS ANTIVIRAL RES 1997 34 2 A73 EMERGING PHARMA- 243409 10th International Conference on Antiviral Research (Part II). IDDB MEETING REPORT 1997 April 6-11 IDdb meeting report. Effect of combination therapy (HBY-097 + 3TC) on HIV-1 infected CEM cells 245844 10th International Conference on Antiviral Research (Part IV). IDDB MEETING REPORT 1997 April 6-11 247129 A new class of carbocyclic influenza neuraminidase inhibitors: structure-activity relationships. Williams MA, Kim CU, Lew W, Liu H, Chen MS, Mendel D, Li W, Tai L, Escarle P, Swaminathan S, Bischofberger N, Stevens et al RC ANTIVIRAL RES 1997 34 2 A86 247131 GS-4104: A highly potent orally bioavailable influenza neuraminidase inhibitor. Williams MA, Kim CU, Lew W, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel D, Li W, Tai L, Escarpe P, Cundy et al KC ANTIVIRAL RES 1997 34 2 A86 247132 In vitro activity of the neuraminidase inhibitor, GS-4071, against influenza viruses. Hayden FD, Rollins BS ANTIVIRAL RES 1997 34 2 A86 248059 The regio and stereospecific synthesis of potent carbocyclic influenza neuraminidase inhibitors: GS4071 and analogs. Zhang L, Williams MA, Lew W, Liu H, Kim CU ACS 1997 213th San Francisco ORGN 106 248180 Hoffmann-La Roche and Gilead Sciences start phase II human testing of oral influenza drug GS 4104. Hoffmann La Roche PRESS RELEASE 1997 May 29 250791 Gilead Sciences Annual Report. Gilead Sciences Inc ANNUAL REPORT 1996 256286 International Conference on Acute Respiratory Infections (Part II) Canberra, Australia. IDDB MEETING REPORT 1997 July 7-10 256487 Researchers seek new weapon against the flu [news]. Service RF SCIENCE 1997 275 5301 756 - 757 256493 Efficacy of GS-4104 in ferrets infected with influenza A virus. Kim CU, Bischofberger N, Williams MA, Lew W, Merson J, Sweet C ANTIVIRAL RES 1997 34 2 A74 256533 GS 4104: A new orally administered anti-influenza drug candidate. Hitchcock MJM INT ANTIVIRAL NEWS 1996 4 9 175 256538 New potent, orally active neuraminidase inhibitors as anti-influenza agents: In vitro and in vivo activity of GS-4071 and analogs. Kim CU, Lew W, Williams M, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel D, Li W, Tai L, et al ICAAC 1996 36th New Orleans Abs 171 258052 Structure-activity relationships of carbocyclic influenza neuraminidase inhibitors. Williams MA, Lew W, Mendel DB, Tai CY, Escarpe PA, Laver WG, Stevens RC, Choung UK BIOORGANIC MED CHEM LETT 1997 7 14 1837 - 1842 258727 C3-thia and C3-carba isosteres of a carbocyclic influenza neuraminidase inhibitor, (3R, 4R, 5S)-4-acetamido-5amino-3-propoxy-1-cyclohexene-1-carboxylic acid. Lew W, Williams MA, Mendel DB, Escarpe PA, Kim CU BIOORGANIC MED CHEM LETT 1997 7 14 1843 - 1846 258728 Synthesis and Activity of C2-substituted analogs of influenza neuraminidase inhibitor GS-4071. Zhang L, Williams MA, Mendel DB, Escarpe PA, Kim CU BIOORGANIC MED CHEM LETT 1997 7 14 1847 - 1850 259524 Half-year report 1997. F Hoffmann La Roche Ltd PRESS RELEASE 1997 August 15 251002 Clinical trials update. SCRIP 1997 2241 27 253232 GS-4104. CLIN TRIALS MONITOR 1997 6 7 7 261712 Rational drug design of orally active influenza inhibitors: from discovery to the clinical candidate. Kim CU AFMC INT MED CHEM SYMP 1997 Jul-Aug 6 254751 Current antiviral agents factfile. 3rd edition: Part I herpesviruses, hepatitis viruses and respiratory viruses. Balzarini J, Schinazi RF, Kinchington D INT ANTIVIRAL NEWS 1997 5 5 74 - 81 264362 Oral influenza compound reduced symptoms and prevented illness in humans. Gilead Sciences Inc PRESS RELEASE 1997 September 29 254752 From crystal structures to resistant clinical isolates Landmarks in the lives of antiviral agents. Goldthorpe Stein S INT ANTIVIRAL NEWS 1997 5 5 70 264437 214th American Chemical Society (ACS) National Meeting (Part XI) Influenza inhibitors symposium, Las Vegas, Nevada, USA. IDDB MEETING REPORT 1997 September 7-11 254763 GS-4104 is a highly bioavailable prodrug of the novel, potent influenza neuraminidase inhibitor GS-4071. Li W X, Escarpe PA, Chen MS, Lew W, Williams M, Zhang L, Kim CU, Bischofberger N, Cundy KC, Eisenberg EJ, Mendel DB ANTIVIRAL RES 1997 34 2 A74 265214 214th American Chemical Society (ACS) National Meeting (Part XIV), Highlights from the Medicinal Chemistry Division, Las Vegas, Nevada, USA. IDDB MEETING REPORT 1997 September 7-11 254764 In vivo influenza virus-inhibitory activity of an orally administered neuraminidase inhibitor. Sidwell RW, Huffman JH, Bailey KW, Wong MH, Kim CU ANTIVIRAL RES 1997 34 2 A74 265491 GW’s ’flu drug passes phase II trials. SCRIP 1997 2272 19 Drug evaluation GS-4104 127 265562 Interscience Conference on Antimicrobial Agents and Chemotherapy: 37th annual meeting (Part III), treatment resistant viruses, Toronto, Ontario, Canada. IDDB MEETING REPORT 1997 Sept 28 - Oct 1 265880 Novel inhibitors of influenza sialidases related to zanamivir. Heterocyclic replacements of the glycerol side chain. Smith PW, Whittington AR, Sollis SL, Howes PD, Taylor NR BIOORGANIC MED CHEM LETT 1997 7 17 2239 - 2242 266214 Roche gaining on Glaxo Wellcome ’flu drug. 1997 2275 27 SCRIP 267203 Efficacy of GS-4104: A potent influenza neuraminidase inhibitor. Choung KU ACS 1997 214th Las Vegas MEDI 137 267207 A new carbocyclic neuraminidase inhibitor related to GS-4071: (3R, 4R, 5S)4-acetamido-5-amino-3-(1-(S)-(2-phenethyl)propoxy)-1-cyclohexene-1-carboxylic acid. Willard L, Williams MA, Mendal DB, Escarpe P, Stevens RC, Laver GW, Choung KU ACS 1997 214th Las Vegas MEDI 185 267445 AIMECS 97, AFMC International Medicinal Chemistry Symposium (Part II) Seoul, Korea. IDDB MEETING REPORT 1997 July 27 - August 1 269518 10th International Conference on Antiviral Research April 6-11, 1997, Atlanta, Georgia, USA. Brideau RJ EXP OPIN INVEST DRUGS 1997 6 11 1753 - 1756 • This meeting report indicates consensus opinion on AIDS treatment modalities as from early 1997. 271739 Inhibitors of influenza virus neuraminidase and endonuclease. Sham HL, Chen X CURR PHARM DESIGN 1997 3 2 159 - 168 271744 New, potent, orally active neuraminidase inhibitors as anti-influenza agents: In vitro and in vivo activity of GS-4071 and analogs. Kim CU, Lew W, Williams M, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel D, Li W, Tai L, Escarpe P, et al ICAAC 1996 36 New Orleans 171 271748 Analyst Report 1997. ANALYST REPORT 1997 276895 Gilead Sciences - 4Q report January 1997. Gilead Sciences Inc 4TH QUARTER REPORT 1998 January 277125 Analyst report - Roche - January 1998. Colletta A, Brewer M, Beadle D, Brindle S, Black J, ABN AMRO Hoare Govett ANALYST REPORT 1998 January 277127 Development of antivirals against influenza. Cianci C, Krystal M EXP OPIN INVEST DRUGS 1998 7 2 149 - 165 277501 DRUG DEV PIPELINE 1998 3 1 1 - 4 277572 Flu patients in US may now have access to experimental oral treatment for influenza. Gilead Sciences Inc PRESS RELEASE 1998 February 12 279997 Clinical trials update. SCRIP 1998 2313 21 280306 Safety and efficacy of the neuraminidase inhibitor, GG167, in experimental human influenza. Hayden FG, Treanor JJ, Betts RF, Lobo M, Esinhart J, Hussey EK J AM MED ASSOC 1996 275 295 - 299 280310 Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogs with potent anti-influenza activity. Kim CU, Lew W, Williams MA, Liu H, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel DB, Tai CY, Laver WG, Stevens RC J AM CHEM SOC 1997 119 681 - 690 • Design, synthesis and structural analysis of carbocyclic sialic acid analogs, culminating GS-4071 and its prodrug, GS-4104. 280313 Penetration of GS-4071, a novel influenza neuraminidase inhibitor, into rat bronchoalveolar lining fluid, following oral administration of the prodrug, GS-4104. Eisenberg EJ, Bidgood A, Cundy KC ANTIMICROB AGENTS CHEMOTHER 1997 41 1949 - 1952 • Demonstration of bioavailability and delivery into the bronchoalveolar lavage of GS-4071, after oral aministration of the prodrug, GS-4104, to rats. 272638 Gilead and Roche begin phase II/III studies of an experimental product to treat or prevent influenza. Gilead Sciences Inc PRESS RELEASE 1997 December 16 280314 New potent, orally active neuraminidase inhibitors as anti-influenza agent: in vitro and in vivo activity of GS-4071 and analogs. Kim CU, Lew W, Williams M, Zhang L, Swaminathan S, Bischofberger N, Chen MS, Mendel D, Li W, Tai L, Escarpe P, et al ICAAC 1996 36 New Orleans Abs H44 273228 Gilead/Roche ’flu drug enters phase III. SCRIP 1997 2295 19 281963 Pharmaceutical and Biotechnology Bulletin. Merrill Lynch ANALYST REPORT 1998 February 18 273331 Trials underway. CLIN TRIALS MONITOR 1998 7 1 5 - 12 282057 Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2enderived inhibitors. McKimm BRESCHKINJL, Sahasrabudhe A, Blick TJ, McDonald M, Colman PM, Hart GJ, Bethell RC, Varghese JN J VIROL 1998 72 3 2456 - 2462 273508 Gilead begins oral flu drug phase II/III. BIOWORLD WEEK 1997 5 51 3 275686 Gilead’s oral drug blocks, relieves flu. BIOWORLD WEEK 1997 5 40 3 282059 ’Flu’ and structure-based drug design. Wade RC STRUCTURE 1997 5 9 1139 - 1145 275707 DRUG DEV PIPELINE 1997 2 11 1 - 4 276077 Gilead announces fourth quarter and 1997 year-end financial results. Gilead Sciences Inc PRESS RELEASE 1998 January 29 276650 Gilead/Roche flu vaccine went from phase I to phase III in nine months. FDC REPORTS PINK SHEET 1998 60 4 T&G-13 - T&G-14 282061 Efficacy of GS-4104: A potent influenza neuraminidase inhibitor. Kim CU ACS 1997 214 1-2 MEDI 137 282074 Rational drug design approach to an orally bioavailable neuraminidase inhibitor. Kim CU, Lew W, Williams MA, Zhang L, Swaminathan S, Mendel DB, Tai CY, Escarpe PA, Laver WG, Stevens RC, Bischofberger N ICAAC 1997 37 Toronto 388 128 IDrugs 1998 Vol 1 No 1 282075 The novel influenza neuraminidase inhibitor prodrug, GS-4104, is highly bioavailable in animals. Cundy KC, Eisenberg G, Bidgood A, Li W, Mendel D, Francis RJ, Angus D, Sharp SR, Lee WA ICAAC 1997 37 Toronto 237 282080 [Influenza viruses: Clinical testing of neuraminidase blocker]. GRIPPEVIREN. NEURAMINIDASE-BLOCKER IN KLINISCHER PRUFUNG. Jungmayr P DTSCH APOTH ZTG 1997 137 21 27 282084 Tolerability and pharmacokinetics of the influenza neuraminidase inhibitor, Ro-64-0802 (GS-4071), following oral administration of the prodrug, Ro-64-0796 (GS-4104) to healthy male volunteers. Wood ND, Aitken M, Sharp S, Evison H ICAAC 1997 37 Toronto 25 282087 Enhanced delivery of the novel influenza neuraminidase inhibitor, GS-4071, to rat lung after oral administration of the prodrug, GS-4104. Cundy KC, Eisenberg G, Bidgood A, Lynch G, Lee WA ICAAC 1997 37 Toronto 25 Drug evaluation Cystemustine 129 Cystemustine INSERM Brian Palmer Address Auckland University Cancer Research Laboratory School of Medicine Private Bag 92019 Auckland New Zealand Email: [email protected] IDrugs 1998 1(1):129-135 Current Drugs Ltd ISSN 1369-7056 Cystemustine is a chloroethyl-nitrosourea under development by INSERM as a potential anticancer drug. It is in phase II trials in France for malignant melanoma [207163], advanced neck and head cancer [207164], advanced renal cancer [207165], and colorectal cancer [207187]. Cystemustine appears to have moderate activity against 2 malignant gliomas using a 60 mg/m dosing regimen as a second line therapy, following surgery, chemotherapy or radiotherapy. Cystemustine has potent antitumor activity in mice. It has a short plasma half-life which makes it potentially amenable to circadian scheduling in order to enhance tolerability and dose intensity. The influence of circadian dosing time on toxicity was first investigated in a total of 368 synchronized male B6D21F1 mice. Leukopenia was the 6 main hematological effect encountered. The activity of O alkylguanine-DNA alkyltransferase (AGT) was studied in the liver of 31 additional mice obtained at six different circadian times. A large amplitude circadian rhythm characterizes both cystemustine toxicity and liver AGT activity in mice. This suggests that the AGT rhythm is an important mechanism of the chronopharmacology of cystemustine [203827]. Introduction The [2-chloroethyl]nitrosoureas, such as BCNU (carmustine), CCNU (lomustine) and MeCCNU, are a wellestablished class of DNA-alkylating antitumor agents, which are particularly useful for the treatment of gliomas and lymphomas [273753]. As a result of their lipophilicity, they readily cross the blood-brain barrier and display good bioavailability. Under physiological conditions, they are extensively decomposed, liberating small electrophilic species which are capable of alkylating DNA at nucleotide base and phosphate sites. Alkylation at O-6 of guanine has been suggested as an important cytotoxic event for this class of agent. Recently, a series of second generation nitrosoureabased agents, such as fotemustine (Servier), nimustine (ACNU; Sankyo) and chlorozotocin (DCNU; NIH), have been developed, which display an improved spectrum of activity, as well as better activity against drug-resistant tumors. The development of clinical resistance to the nitrosourea class of agents is frequently associated with 6 overexpression of the enzyme, O -alkylguanine DNA alkyltransferase (AGT), which acts to repair the cytotoxic Originator INSERM Status Phase II clinical Indication Neoplasm, Melanoma, Head & neck tumor, Renal tumor, Colorectal tumor, Glioma, Carcinoma, Sarcoma Action Anticancer, Alkylating agent CAS Urea, N-(2-chloroethyl)-N’-[2-(methylsulfonyl)ethyl]-NnitrosoRegistry No: 79955-36-5 Synonyms CENU, chloroethylnitrosourea, CYST, CMSO2EN2 O O O S Cl N H N N O lesion formed upon DNA alkylation. The co-administration of inhibitors of this enzyme improves the antitumor effect of nitrosoureas in experimental systems, and many of the newer agents of this class are under evaluation in conjunction with newly synthesized alkyltransferase inhibitors. Cystemustine is a second generation nitrosourea which is derived from the amino thiol cysteamine, and which has recently completed a series of phase II clinical trials against a variety of tumor types. Synthesis and SAR Cystemustine (N-[2-chloroethyl]-N’-[(2-methylsulfonyl)ethyl]-N-nitrosourea, CENU, CMSO2EN2, CYST) was originally identified as a metabolite of the experimental agent CNCC [N,N’-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cystamine], where it occurred with the corresponding sulfoxide [273755]. Both metabolites result from the in vivo reduction of the disulfide linkage in CNCC, followed by S-methylation and oxidation. Subsequent studies established that both cystemustine and the sulfoxide, perrimustine, were as active as CNCC against the L1210 leukemia in mice, and led to the development of cystemustine as an experimental antitumor agent in its own right [273755], [207185]. Cystemustine is prepared in four steps from 4-nitrophenol and 2-chloroethyl isocyanate and is obtained as light yellow crystals (melting ° point 94 to 96 C). It has good water solubility, enabling its direct formulation in aqueous solutions for in vivo studies. 14 [ C]cystemustine has been synthesized for pharmacokinetic studies with the label in complementary portions of the 130 IDrugs 1998 Vol 1 No 1 14 14 molecule, from either [ C]ethanolamine [273757] or [ C]2chloroethyl isocyanate [273762]. Pharmacology The in vivo activity of cystemustine was evaluated in a panel of 12 murine tumors, and was compared with that of CNCC [273765]. It was at least as active as CNCC against eight lymphomas and solid tumors, and gave a higher number of long term survivors. Cystemustine was clearly better than CNCC against B16 melanoma, 26 glioma and grafted L1210 leukemia, with an optimum iv dose of 15 mg/kg, half that of CNCC. The activity of cystemustine was significantly improved following the co-administration of an experimental, water soluble inhibitor of AGT to nude mice bearing the M4Beu melanoma xenograft [207169]. Metabolism 14 The disposition of [ C]cystemustine was evaluated in nontumor bearing rats following an iv dose of 60 µmol/kg [273766]. Elimination of cystemustine and its metabolites was predominantly via the urine, with 70% of the total radioactivity collected in this fraction, 72 h following drug administration. At this time, the feces contained only 5% of the radioactivity, with 10% excreted as carbon dioxide in the lung. An analysis of the tissue distribution of unchanged cystemustine at 5 min and 30 min, following iv administration, showed a particularly high level of drug in the kidney, together with significant levels of the drug in the brain, lung and liver. In this study, cystemustine was cleared with first order kinetics, with a plasma half-life of 30.5 min, and a volume of distribution of 1.05 l/kg. The AUC was 42.8 nmol/h/ml. The human pharmacokinetics of cystemustine have been reported as part of a phase II clinical trial [207184]. HPLC analysis was used to determine plasma levels up to 3 h following iv administration of cystemustine, 2 at doses in the range 40 to 90 mg/m . The drug was eliminated with a half-life of 50 min, and had a distribution half-life of less than 10 min. The rapid metabolism and clearance of cystemustine is similar to that observed for the clinically used nitrosoureas, CCNU and BCNU. Cystemustine is extensively metabolized in the rat, with the main urinary metabolites being thiodiacetic acid and its sulfoxide (sulfonylthiodiacetic acid), which together comprise about half of the urinary radioactivity. N-acetyl-Shydroxyethylcysteine and N-acetyl-S-carboxymethylcysteine together account for 7% and 2-(methylsulfonyl)ethylamine 16% of the urinary radioactivity. About 10% of urinary radioactivity is associated with four unidentified metabolites. The main plasma metabolite is 2-chloroethanol [273766, 273768]. Toxicity As with other nitrosourea-based antitumor agents, cystemustine showed mainly hematological toxicity in preclinical murine studies [273765]. Cystemustine caused dose-related leukopenia in mice, peaking on days 8 to 12 following administration of multiple iv doses of the drug up to day 5, which recovered to control levels by day 19. Cystemustine had little effect on platelets or red blood cells, in contrast to CNCC. In murine studies, the toxicity of cystemustine displayed a surprising dependence on the point in the circadian cycle of the mice at which drug administration occurred [268219]. This effect is pronounced for cystemustine due to its short plasma half-life. Following administration of a single dose of cystemustine, the nadir of leukocyte count (day 7) was lower for those mice injected with drug 7 h after light onset, as compared with 13 or 19 h afterwards, and bone marrow necrotic lesions were also more pronounced in the former case. Furthermore, recovery from leukopenia was faster, following administration of cystemustine 19 h after light onset. The 60-day survival rate following a dose of 35 mg/kg was 4% when injected 7 h after light onset, rising to 90% when given at 19 h. Overall, cystemustine displayed the lowest toxicity when administered in the middle of the active phase of the rest-active circadian cycle. Further studies established a significant 5-fold circadian variation in the liver AGT activity of mice which correlated with cystemustine toxicity, suggesting a basis for the observed chronotoxicity of cystemustine [203827]. The dose-limiting toxicity of cystemustine in the phase I clinical trial was dose-dependent and hematological. Mild 2 toxicity began, following a cumulative dose of 55 mg/m of cystemustine, and the maximum tolerated dose was 2 established as 90 mg/m [207188]. In the phase II studies, toxicity per cycle of treatment was mild and well-tolerated 2 2 at the 60 mg/m dose level, while at 90 mg/m , the toxicity was also hematological, grade 3 to 4 anemia (19%), grade 3 to 4 neutropenia (23%), grade 3 to 4 thrombopenia (27%) and grade 3 to 4 nausea and vomiting (11%) [156265]. All toxicities were reversible. Clinical Development A phase I study of cystemustine was carried out in 34 patients with a variety of advanced metastatic cancers [207188]. The drug was administered iv in seven or eight cycles over a period of up to 190 days, in escalating doses 2 ranging from 0.9 to 180 mg/m . The recommended dose for 2 2 phase II evaluation was 60 mg/m , increasing to 90 mg/m . Three partial responses were reported in this phase I study. The phase II evaluation of cystemustine began in 1990 in a range of tumor types, under the auspices of the EORTC. More than 160 patients with advanced or recurrent tumors were given cystemustine q2 weeks as a 15 min iv infusion at 2 2 a dose of either 60 mg/m or a dose of 90 mg/m for the first 2 three cycles, with subsequent doses of 60 mg/m [156265], [207165]. Although the drug had been observed to concentrate to some extent in the kidney in preclinical rat studies, when cystemustine was evaluated in 56 patients with advanced renal cancer, it had only minimal activity [207165]. Cystemustine also displayed only marginal activity against metastatic colorectal carcinoma (27 patients) [207187], soft tissue sarcoma (32 patients) [273774], head and neck cancer (28 patients) [207164] and non-small cell lung cancer (19 patients) [156265]. Against advanced malignant melanoma in 25 patients, cystemustine displayed limited activity at the lower dose level [268218], but a smaller study in 17 pre-treated patients using the higher dosing regimen of 2 90 mg/m gave an overall response rate of 23% [273770]; this is significant for this tumor type, but remains to be verified Drug evaluation Cystemustine 131 in a larger number of patients. Cystemustine was evaluated 2 at the 60 mg/m dose in 38 patients with malignant gliomas, comprising 14 glioblastomas, 20 grade 3 to 4 astrocytomas and four grade 3 to 4 oligodendrogliomas [156265], [273771]. The majority of these patients had received prior radiotherapy. Of the 32 evaluable patients, three had partial remissions (giving an overall response rate of 9.4%), while 18 patients had stable disease for at least 8 weeks. Several of the responding patients had received high doses of BCNU in previous adjuvant chemotherapy, suggesting that cystemustine might be cross-resistant to conventional nitrosourea-based agents. There have been no specific reports of the clinical evaluation of cystemustine against either Hodgkin’s or non-Hodgkin’s lymphoma. Current Opinion The clinical utility of the first generation of nitrosoureas is limited mainly to the treatment of lymphomas and the palliative treatment of gliomas (the latter usually in combination with radiotherapy and/or surgery). As expected, the second generation nitrosourea-based agents are also active against lymphoproliferative diseases and gliomas, but in addition, have low to moderate activity against small cell lung carcinoma, gastrointestinal cancers, head and neck cancer and melanoma [273753]. The published phase II clinical data for cystemustine suggest that it is no more active than other second generation agents against these latter tumor types, and cystemustine is unlikely to be developed further for these indications. Cystemustine appears to have moderate activity against 2 malignant gliomas, using the 60 mg/m dosing regimen, when used as a second line therapy following surgery, chemotherapy or radiotherapy. The response rate observed with cystemustine against this tumor type appears to be broadly comparable with response rates obtained with currently used nitrosoureas, such as CCNU and BCNU [23593], [273776], although strict comparisons await a detailed analysis of the median survival times (rather than response rates) obtained with cystemustine, in conjunction with the adjuvant therapy used and tumor staging at the start of treatment. Any further advancement of cystemustine for gliomas will depend on a clear demonstration of its superior activity to currently used agents at the higher 2 dosing regimen of 90 mg/m , and/or a demonstration of clinical activity in tumors which have become resistant to conventional nitrosourea-based antitumor agents. Development History DEVELOPER COUNTRY STATUS INDICATION DATE REF INSERM France C2 Melanoma 08-MAY-96 207163 INSERM France C2 Head & neck tumor 08-MAY-96 207164 INSERM France C2 Renal tumor 08-MAY-96 207165 INSERM France C2 Neoplasm 08-MAY-96 207184 INSERM France C2 Colorectal tumor 08-MAY-96 207187 INSERM France C2 Glioma 21-JAN-98 273771 INSERM France C2 Carcinoma 21-JAN-98 156265 INSERM France C2 Sarcoma 21-JAN-98 273774 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Chemistry STUDY TYPE RESULT REF Synthesis and antitumor activity Synthesis of four metabolites of the experimental agent, CNCC, one of which is cystemustine. 273755 Synthesis Synthesis of [ 14C]cystemustine from labeled ethanolamine. 273757 Synthesis Synthesis of [ 14C]cystemustine from labeled 2-chloroethyl isocyanate. 273762 132 IDrugs 1998 Vol 1 No 1 Biology STUDY TYPE In vivo EFFECT STUDIED Toxicity. EXPERIMENTAL MODEL Cystemustine was administered at 7, 13 or 19 h after light onset (HALO) and toxicity was evaluated in B6D2F1 mice. RESULT Leukopenia recovery was faster and bone marrow necrotic lesion was not severe after cystemustine at 19 HALO. This was dependant on the rhythm of AGT activity. REF 203827 In vivo Antitumor activity. L1210 tumors grown ip in mice. Activity of cystemustine against murine L1210 tumors was found to be greater than that of CNCC. 273755 In vivo Antitumor activity. 12 murine and xenograft tumors in mice. Cystemustine demonstrated greater overall activity than CNCC, at half the dose. 273765 In vivo Antitumor activity in the presence of AGT inhibitors. M4Beu melanoma xenografts in mice. In vitro and in vivo activity of cystemustine evaluated in the presence of a series of inhibitors of the enzyme, AGT. Enzyme inhibitors containing benzyl groups augmented the antitumor activity. 207169 In vivo Circadian toxicity. 368 synchronized B6D2F1 mice. Cystemustine hematological toxicity markedly lower when administered in the middle of the active portion of the activerest cycle. 268219 In vivo Circadian toxicity. Radiochemical detection of liver AGT activity at six circadian timepoints in 31 mice. 5-fold variation in AGT activity found, correlating with the chronotoxicity of cystemustine. 203827 Metabolism STUDY TYPE EFFECT STUDIED MODEL USED RESULT REF In vivo Drug disposition and pharmacokinetics. Non-tumor bearing rats, dose of 60 mmol/kg of [ 14C]cystemustine. 70% of radioactivity in the urine after 72 h, with 5% in feces, 10% in lung. Plasma halflife 30.5 min, first order. Volume of distribution = 1.05 l/kg, AUC = 42.8 nmol/h/ml. 273766 In vivo Pharmacokinetics. Phase II trial. HPLC analysis of plasma levels. Distribution half-life less than 10 min. Plasma half-life = 50 min. 207184 In vivo Metabolism. [14C]cystemustine in the rat. Extensive metabolism, with metabolites appearing mainly in the urine. Major metabolites identified. 273768 Clinical EFFECT STUDIED MODEL USED RESULT REF 2 Phase I trial. Phase I evaluation in 34 pretreated cancer patients. Dose for phase II trials set at 60 mg/m , increasing to 90 mg/m2. Three responses seen. 207188 Antitumor activity. Phase II evaluation in 27 patients with metastatic colorectal carcinoma. No activity. 207187 Antitumor activity. Phase II evaluation in 56 patients with advanced renal cancer. Minimal activity. 207165 Antitumor activity. Phase II evaluation in 32 patients with soft tissue sarcoma. Minimal activity. 273774 Antitumor activity. Phase II evaluation in 28 patients with advanced head and neck cancer. Minimal activity. 207164 Antitumor activity. Phase II evaluation in 19 patients with non small cell lung carcinoma. Minimal activity. 156265 Drug evaluation Cystemustine 133 Clinical (continued) EFFECT STUDIED MODEL USED RESULT REF Antitumor activity. Phase II evaluation in 25 patients with advanced malignant melanoma. Limited activity at a dose of 60 mg/m 2. 268218 Antitumor activity. Phase II evaluation in 25 patients with advanced malignant melanoma. Limited activity at a dose of 60 mg/m 2. 268218 Antitumor activity. Phase II evaluation in 17 patients with advanced malignant melanoma. Overall response rate of 23% at a dose of 90 mg/m2. 273770 Antitumor activity. Phase II evaluation in 38 patients with mostly preirradiated gliomas, comprising 14 glioblastomas, 20 high-grade astrocytomas and four oligodendrogliomas. Three partial remissions, and a total of 18 patients with stable disease for at least 8 weeks. An observation that cystemustine could have activity against BCNU-refractory gliomas. 156265 Antitumor activity. Phase II evaluation in 38 patients with mostly preirradiated gliomas, comprising 14 glioblastomas, 20 high-grade astrocytomas and four oligodendrogliomas. Three partial remissions, and a total of 18 patients with stable disease for at least eight weeks. An observation that cystemustine could have activity against BCNU-refractory gliomas. 273771 Bibliography 23593 The role of chemotherapy in the treatment of gliomas in adults. Stewart DJ CANCER TREAT REV 1989 16 129 - 160 • A review and analysis of the use of chemotherapy in the treatment of gliomas. 156265 Results of two phase II trials with cystemustine in five different tumoral localizations. Chollet Ph, Adenis A, Chauvergne J, Fargeot P, Roche H, Kerbrat P, Lentz MA, van Glabbeke M, Urosevic V, Fumoleau P, Chevallier B NCI EORTC SYMP NEW DRUGS CANCER THER 1994 Abs 289 • A conference abstract summarizing the results of two phase II trials of cystemustine (differing in dose level) in five different tumor types. The overall toxicity profile of cystemustine is reported. 203827 Circadian rhythm in toxic effects of cystemustine in mice and its relationship with alkyltransferase activity. Martineau Pivoteau N, Levi F, Cussac C, Rolhion C, Debiton E, Rapp M, Kwiatkowski F, Lemaigre G, Filipski E, Chollet P NCI EORTC SYMP NEW DRUGS CANCER THER 1996 9th Amsterdam Abs 406 • A study of the circadian cycle dependence of alkyltranferase activity in the livers of mice, which established a correlation between lower systemic cystemustine toxicity and high levels of enzyme activity at the midpoint of the active phase of the rest-active cycle of the mice. 207163 Results of a phase II trial with cystemustine in advanced malignant melanoma. A trial of the EORTC clinical screening group [7]. Urosevic V, Chollet PH, Adenis A, Chauvergne J, Fargeot P, Roche H, Kerbrat P, Lentz MA, Fumoleaus P, Chevallier B EUR J CANCER PART A GEN TOP 1996 32 1 181 - 182 207164 Phase II study of cystemustine in advanced head and neck cancer. A trial of the EORTC Clinical Screening Group [letter]. Cappelaere P, Lentz MA, Degardin M, Chauvergne J, Mayer F, Chollet P, Chevallier B EUR J CANCER PART B ORAL ONCOL 1995 31B 2 151 - 152 • The report of a phase II evaluation of cystemustine in advanced head and neck cancer, establishing only low activity. 207165 Phase II study of cystemustine in advanced renal cancer [letter]. Chauvergne J, Kerbrat P, Adenis A, Chollet P, Fargeot P, Pujade Lauraine E, Dieras V, Madelmont JC, Lentz MA, Toulouse C, et al EUR J CANCER PART A GEN TOP. 1995 31A 1 130 - 131 • The report of a phase II evaluation of cystemustine in advanced renal cancer, establishing only marginal activity. 207167 Phase II study of cystemustine in advanced head and neck cancer. A trial of the EORTC Clinical Screening Group [1]. Cappelaere P, Lentz MA, Degardin M, Chauvergne J, Mayer F, Chollet PH, Chevallier B EUR J CANCER PART B ORAL ONCOL 1995 31 2 151 - 152 207168 Phase II study of cystemustine in advanced renal cancer [4]. Chauvergne J, Kerbrat P, Adenis A, Chollet P, Fargeot P, Pujade Lauraine E, Dieras V, Madelmont JC, Lentz MA, Toulouse C, Chevallier B EUR J CANCER PART A GEN TOP 1995 31 1 130 - 131 207169 Enhancement by O6-benzyl-N-acetylguanosine derivatives of chloroethylnitrosourea antitumor action in chloroethylnitrosourea- resistant human malignant melanocytes. Cussac C, Rapp M, Mounetou E, Madelmont JC, Maurizis JC, Godeneche D, Dupuy JM, Sauzieres J, Baudry JP, Veyre A J PHARMACOL EXP THER 1994 271 3 1353 - 1358 • A study investigating the enhancement of antitumor activity of cystemustine by a series of experimental inhibitors of the enzyme, AGT. Both in vitro and in vivo studies in nude mice bearing human leukemia xenografts established that some of the AGT inhibitors were able to augment the activity of cystemustine. 207182 Disposition and metabolism of O6-alkylguanine-DNA alkyltransferase inhibitor in nude mice bearing human melanoma. Cussac C, Mounetou E, Rapp M, Madelmont JC, Maurizis JC, Labarre P, Chollet P, Chabard JL, Godeneche D, Baudry JP, et al DRUG METAB DISPOS 1994 22 4 637 - 642 207183 Diagnosis and treatment of soft tissue sarcomas in adults. Dirix LY, Van Oosterom AT CURR OPIN ONCOL 1994 6 4 372 - 383 134 IDrugs 1998 Vol 1 No 1 207184 Pharmacokinetics of two new 2-chloroethylnitrosoureas in cancer patients submitted to phase II clinical trials. Godeneche D, Labarre P, Cussac C, Madelmont JC, Dupuy JM, Fontanon C, Tisserant A, Chollet P, Baudry JP, Veyre A DRUG INVEST 1994 7 5 234 - 243 • A description of the pharmacokinetics of cystemustine in humans, measured during a phase II evaluation of the drug. An HPLC method is described for measurement of drug levels in plasma at least until 3 h following drug infusion. 207185 Cystemustine. Chloroethyl nitrosourea antineoplastic alkylating agent. Madelmont JC DRUGS FUTURE 1994 19 1 27 30 • A useful review of the preclinical and clinical development of cystemustine up until 1994, written by one of the original investigators of the drug. 207187 Phase II study of cystemustine in metastatic colorectal carcinoma. A trial of the EORTC Clinical Screening Group. Kerbrat P, Adenis A, Rebattu P, Roche H, Chevallier B, Chollet P, Krakowski I, Lentz MA, Fumoleau P EUR J CANCER PART A GEN TOP. 1993 29A 11 1597 - 1599 • The report of a phase II evaluation of cystemustine in metastatic colorectal carcinoma, establishing no activity for the drug at that dose and schedule. 207188 Phase I trial of cystemustine, a new cysteamine (2chloroethyl) nitrosourea: an intrapatient escalation scheme. Mathe G, Misset JL, Triana BK, Goden‘ eche D, Madelmont JC, Meyniel G DRUGS EXP CLIN RES 1992 18 4 155 - 158 • A report of the phase I evaluation of cystemustine in 34 patients in a variety of tumor types, which established a recommended dose level for phase II trials. 268218 Results of a phase II trial with cystemustine in advanced malignant melanoma. A trial of the EORTC Clinical Screening Group [letter]. Urosevic V, Chollet P, Adenis A, Chauvergne J, Fargeot P, Roche H, Kerbrat P, Lentz MA, Fumoleau P, Chevallier B EUR J CANCER PART A GEN TOP. 1996 32A 1 181 - 182 • The report of a phase II evaluation of cystemustine in advanced malignant melanoma, establishing only marginal activity at the dose level used, but noting that a further trial at a higher dose is underway. 268219 Circadian rhythm in toxic effects of cystemustine in mice: relevance for chronomodulated delivery. Martineau PIVOTEAUN, Levi F, Rolhion C, Kwiatkowski F, Lemaigre G, Filipski E, Chollet P INT J CANCER 1996 68 5 669 - 674 • A study of the effect on cystemustine toxicity of the timing of drug administration within the circadian cycle of the mouse. The toxicity profile of cystemustine in the mouse was compre-hensively described. 268240 Cystemustine. DRUGS FUTURE 1996 21 1 76 268242 Cystemustine. DRUGS FUTURE 1995 20 1 75 268246 Coordination of clinical research in Europe: The EORTC data center. Meunier F, McVie JG BIOMEDICAL HEALTH RESEARCH (ED: VERMORKEN AJM) 1994 5 151 - 159 268251 Chronotoxicity of cystemustine CYST a new nitrosourea in mice relevance for chronomodulated drug delivery. Martineau N, Levi F, Deloche C, Chollet P, Madelmont JC PROC AM ASSOC CANCER RES 1993 34 Abs 271 273753 A critical appraisal of the evolution of N-nitrosoureas as anticancer drugs. Gnewuch CT, Sosnovsky GA CHEM REV 1997 829 - 1013 • A useful recent review of the nitrosourea class of antitumor agents. 273755 New cysteamine (2-chloroethyl)nitrosoureas. Synthesis and preliminary antitumor results. Madelmont JC, Godeneche D, Parry D, Duprat JL, Chabard JL, Plagne R, Mathe G, Meyniel G J MED CHEM 1985 28 1346 - 1350 • A report detailing the identification of cystemustine as a metabolite of CNCC. Its synthesis and preliminary biological evaluation in mice are described. 273757 Use of ethanolamine 14C as a precursor. 1. 14C labeling of 1-(2-chloroethyl)-3-[2-(methylsulfinyl)ethyl]-1-nitrosourea and 1-(2-chloroethyl)-3-[2-(methylsulfonyl)ethyl]-1-nitrosourea. Madelmont JC, Parry D, Godeneche D, Duprat J J LABEL COMPOUNDS RADIOPHARM 1985 15 851 - 862 • A report on the use of 14C labeled ethanolamine for the preparation of labeled cystemustine (and perrimustine) for use in pharmacokinetic and metabolism studies. 273762 14C Labeling of 2-chloroethyl isocyanate. Application in labeling of chloroethyl tetrazinone and of chloroethyl nitrosoureas. Madelmont JC, Moreau MF, Godeneche D, Labarre P, Veyre A J LABEL COMPOUNDS RADIOPHARM 1988 25 1135 1142 • A report of the use of 14C labeled 2-chloroethyl isocyanate for the preparation of labeled cystemustine for use in pharmacokinetic and metabolism studies. 273765 Cytostatic action of two nitrosoureas derived from cysteamine. Bourut C, Chenu E, Godeneche D, Madelmont JC, Maral R, Mathe G, Meyniel G BR J PHARMACOL 1986 89 539 546 • An in vivo evaluation of the antitumor activity of cystemustine (and perrimustine) in 12 murine tumor models, in comparison with another nitrosourea, CNCC. Hematological toxicity studies are reported. 273766 Disposition of new sulfur-containing 2-(chloroethyl)nitrosoureas in rats. Godeneche D, Madelmont JC, Labarre P, Plagne R, Meyniel G XENOBIOTICA 1987 17 59 - 70 • A distrubution study of 14C radiolabeled cystemustine in rats. 273768 Main urinary metabolites of two cysteamine containing 2-(chloroethyl)nitrosoureas in rats. Godeneche D, Labarre P, Moreau MF, Madelmont JC, Rapp M, Veyre A DRUG METAB DISPOS 1993 21 93 - 99 • A study of the urinary metabolites of 14C radiolabeled cystemustine in the rat. 273770 Results of two phase II trials with cystemustine in advanced malignant melanoma. Chollet PH, Adenis A, Chauvergne J, Fargeot P, Roche H, Kerbrat P, Lentz MA, van Glabbeke M, Urosevic V, Chevallier B, Fumoleau P NCI EORTC SYMP NEW DRUGS CANCER THER 1994 Abs 290 • The preliminary report of a phase II evaluation of cystemustine in advanced metastatic melanoma, establishing only low activity at a dose of 60 mg/m2, but more significant activity at the higher dose of 90 mg/m2. 273771 Phase II trial of cystemustine, a new nitrosourea, as a second line treatment of malignant gliomas. Roche M, Adenis L, Cure H, Faregot P, Guiochet N, Ouabdessalam R, Houyan P, Lentz MA, Fumoleau P, Chollet P ANN ONCOL 1996 7 Suppl 5 Abs 130 • A report from the early studies group of the EORTC on the activity of cystemustine in the second line treatment of malignant glioma. Drug evaluation Cystemustine 135 273774 Cystemustine (a new nitrosourea) phase II trial in advanced soft tissue sarcoma. Krakowsky Y, Tubiana N, Cappelaere P, Rebattu P, Schneider M, Lentz MA, van Glabbeke M, Chollet PH, Chevallier B, Fumoleau P ANN ONCOL 1992 3 Suppl 5 Abs 181 • A report of a phase II trial of cystemustine against advanced soft tissue sarcoma, establishing only marginal activity. 273776 An overview of published results from randomised studies of nitrosoureas in primary high grade malignant glioma. Stenning SP, Freedman LS, Bleehen NM BR J CANCER 1987 56 89 - 90 • A short note evaluating the usefulness of nitrosoureas as adjuvant therapy for gliomas. 282039 O6-methylguanine-DNA methyltransferase (MGMT) transfectants of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)sensitive colon cancer cell line selectively repopulate heterogenous MGMT+/MGMT- xenografts after BCNU and O6benzylguanine plus BCNU. Phillips WPJR, Willson JK, Markowitz SD, Zborowska E, Zaidi NH, Liu L, Gordon NH, Gerson SL CANCER RES 1997 57 21 4817 - 4823 282043 Pretreatment prediction of the chemotherapeutic response of human glioma cell cultures using nuclear magnetic resonance spectroscopy and artificial neural networks. El Dered YW, Ashmore SM, Branston NM, Darling JL, Williams SR, Thomas DGT CANCER RES 1997 57 19 4196 - 4199 282045 O6-(alkyl-aralkyl)guanosine and 2’-deoxyguanosine derivatives: Synthesis and ability to enhance chloroethylnitrosourea antitumor action. Mounetou E, Debiton E, Buchdahl C, Gardette D, Gramain J C, Maurizis J C, Veyre A, Madelmont J C J MED CHEM 1997 40 18 2902 - 2909 282047 Enhancement by O6-benzyl-N2-acetylguanosine of N’-[2chloroethyl]-N- [2-(methylsulphonyl)ethyl]-N’-nitrosourea therapeutic index on nude mice bearing resistant human melanoma. Debiton E, Cussac Buchdhal C, Mounetou E, Rapp M, Dupuy JM, Maurizis JC, Veyre A, Madelmont JC BR J CANCER 1997 76 9 1157 - 1162 282049 Evidence for nucleotide excision repair as a modifying factor of O6-methylguanine-DNA methyltrans-ferase-mediated innate chloroethylnitrosourea resistance in human tumor cell lines. Chen ZP, Malapetsa A, McQuillan A, Marcantonio D, Bello V, Mohr G, Remack J, Brent TP, Panasci LC MOL PHARMACOL 1997 52 5 815 - 820 282053 Application of antisense ribonucleic acid complementary to O6- methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas. Nagane M, Asai A, Shibui S, Nomura K, Kuchino Y, Ali Osman F, Greenberg HS, Raffel C, Rutka JT NEUROSURGERY 1997 41 2 434 - 441 136 IDrugs 1998 Vol 1 No 1 KA-672 Dr Wilmar Schwabe GmbH & Co Hermann Mucke Address Sanochemia Enenkelstrasse 28/32 A-1160 Wien Austria Email: [email protected] IDrugs 1998 1(1):136-140 Current Drugs Ltd ISSN 1369-7056 KA-672 is a multifunctional antidementia agent under development by Schwabe as a potential agent to combat cognitive and other symptoms of Alzheimer’s disease (AD), via modulation of the interplay between serotonergic, adrenergic, dopaminergic and cholinergic systems [260590]. It has also been categorized as a neuronal activator. Various studies support the notion that this compound could indeed have a broad range of nootropic properties. The compound is in phase II clinical trials [260592]. KA-672 selectively protects against NMDA-induced neurotoxicity but not against other chemoconvulsants or electroshock [260590]. At 0.3 mg/kg it inhibits the reduction in 5-HT brain levels following NMDA administration, and exacerbates the reduction in dopamine levels [260593]. It has been shown to bind to 5-HT1A, 5-HT7, α1, D2 and D3 receptors with nanomolar affinities, acting as an antagonist at α1 and D2 receptors [260592]. It also has nerve growth factor (NGF)-like activity [260591]. Introduction Most drugs that are being developed for the symptomatic treatment of AD are directed exclusively towards one of the several neurotransmitter deficits that are known to occur in primary degenerative dementia; in most cases the target is the cholinergic system. A more balanced approach would be desirable, especially with respect to the treatment of noncognitive symptoms which are not directly mediated through acetylcholine (ACh) deficits. Whether such an approach requires combination therapy, or can be achieved in a single molecule, remains an open question. Originator Dr Wilmar Schwabe GmbH & Co Status Phase 2 Clinical Indication Alzheimer’s disease Action Nootropic agent CAS 2H-Benzopyran-2-one, 7-methoxy-6-[3-[4-(2methoxyphenyl)-1-piperazinyl]propoxy]-3,4-dimethylRegistry No: 155773-59-4 O N O O O N H Cl O with 2-methyl-3-oxobutanoic acid ethyl ester in 75% sulfuric acid. This intermediate is reacted with 1-[3-chloropropyl]-4[2-methoxyphenyl]piperazine in the presence of K2CO3 and KI in ethanol, resulting in the target compound, KA-672, as free base [281905], [258435]. Pharmacology KA-672 binds to 5-HT1a, 5-HT7, α1, D2 and D3 receptors in nanomolar concentrations, but does not interact with other subtypes of monoaminergic receptors or ion channels. Functional studies suggest that KA-672 is an α1 and D2 antagonist, but the nature of receptor interaction remains to be defined [260592]. Given to rats at a dose of 0.3 mg/kg, po, the compound elevated serotonin levels and decreased the levels of dopamine and its metabolite, 3-methoxytyramine. The parent structure for KA-672 is scoparone (6,7dimethoxycoumarin), a naturally-occuring compound with vasodilatory, anti-atherogenic, antiproliferative and immunosuppressive properties. It is isolated from the flower, Artemisia scoparia, an agent in Chinese herbal medicine. In rat brain homogenates and in commercial Torpedo preparations, the potency of KA-672 as an acetylcholinesterase inhibitor (AChEI) is reported to be as equal to that of tacrine and 10-fold higher than that of galanthamine, corresponding to an IC50 value in the submicromolar range [281915], [258435]. However, no such effect was observed in vivo, and Schwabe no longer classifies KA-672 as an AChEI [281908]. The compound does not inhibit plasma butyrylcholinesterase, and is also without effect on rat brain arylacylamidase. At moderately high doses of 3 to 5 mg/kg po it does not produce tremor, salivation or other peripheral cholinergic effects. Furthermore, KA-672 does not bind to nicotinic or muscarinic ACh receptors [281915]. KA-672 is synthesized via 6-hydroxy-7-methoxy-3,4dimethyl-2H-1-benzopyran-2-one, which is accessible through a Pechmann reaction of 2-methoxy-1,4-benzenediol Most importantly, however, KA-672 appears to be a functional NMDA antagonist. In doses that are effective in cognitive rodent models [177791], it protects mice against Schwabe has dropped its provisional synonym for KA-672, anseculin [281908]. No international non-proprietary name has been assigned so far. Synthesis and SAR Drug evaluation KA-672 137 convulsions and mortality induced by NMDA or homocystein, but protection against chemoconvulsants or electroshock is not achieved even at doses several-fold higher. The compound does not compete with glutamic or kainic acid, glycine, AMPA or MK-801 for binding sites at the NMDA receptor complex. In in vitro electrophysiological studies, KA-672 abrogated currents triggered by NMDA, but not those activated by AMPA or GABA. If administered prior to an NMDA challenge, it acted synergistically with NMDA in lowering brain dopamine by enhancing its metabolism along the homovanillic acid pathway, antagonized the serotonin depletion induced by NMDA, and also increased the brain norepinephrine content. [260593]. Finally, in an in vitro system of rat embryonic septal cells, KA-672 has been shown to have NGF-like activity (comparable to that of NGF in terms of potency and quality). No synergy between the compound and NGF was observed, suggesting that both might stimulate neurite outgrowth using essentially the same mechanisms [260591]. In neuropsychological standard screening tests, KA-672 is either inactive or behaves as a weak antidepressant. In cognitive paradigms, such as passive or conditioned avoidance, oral doses of 0.1 to 1.0 mg/kg strongly facilitate acquisition and retention in rats and mice, and antagonize the amnestic effect of scopolamine [177791], [258435]. Metabolism No data have yet been reported concerning the metabolic fate of KA-672; however, educated guesses can be made based on what is known from coumarin derivatives used as anticoagulants. These compounds show almost complete plasma protein binding, and a metabolic rate that might in part be genetically determined. The aromatic ring system is extensively hydroxylated. This results in glucuronidation or sulfatation products being excreted in urine, although hardly any unchanged parent compound is lost in this way. The metabolism of scoparone, the parent compound of KA672, has been thoroughly investigated because it can be used as a substrate to differentiate cytochrome P450 monooxygenase induction stages. Scoparone is regio-selectively demethylated at different rates for the methyl groups at positions 6 and 7, yielding scopoletin (7-hydroxy-6methoxycoumarin) and isoscopoletin (6-hydroxy-7methoxycoumarin). Rat hepatocytes metabolize scoparone 7to 10-fold slower than those of hamsters and monkeys. Human hepatocytes do not secrete scopoletin in detectable amounts, suggesting that scoparone metabolism in humans may be qualitatively different from that in other mammals [281907]. Toxicity In mice and rats, the only dose-dependent toxicological symptoms observed after oral doses of 10 mg/kg or more were sedation, hypothermia and anorexia, with doses up to 50 mg/kg not causing mortality. In 26-week chronic exposure studies, daily oral doses of up to 13 mg/kg in rats and 1.5 mg/kg in dogs were well-tolerated, and side-effects were reversible. Oral doses up to 40 mg/kg were not clastogenic in the mouse bone marrow micronucleus test [258435]. Clinical Development Phase I Single and repeat oral dose tolerability of KA-672 was determined in adult and elderly healthy male and female subjects in the non-fasting state. The compound was welltolerated and linear pharmacokinetics, in terms of Cmax and AUC, were observed after repeat doses. Half-life was in excess of 10 h, suggesting that therapeutic trials with 10 or 20 mg once-daily dosing could be feasible [260594]. Phase II KA-672 has been reported to have entered phase II in 1997 [260592], [258423]; however, no data are currently available. Side-effects and Contraindications In phase I studies, the side-effect profile of 40 mg, po, single doses was indistinguishable from placebo; at 60 mg, about half the subjects experienced a moderate drug-related orthostatic syndrome. Repeated dosing of up to 20 mg po/per day, for 14 days, resulted in only mild adrenergic side-effects (decrease in semisupine and standing blood pressure, but no orthostasis) without a systematic pattern [260594]. Current Opinion The current lack of peer-reviewed original papers on KA-672, and the multitude of pharmacological activities reported in patents and congress abstracts, makes it difficult to form a solid opinion on this compound. The all-in-one concept of a single molecule combining several activities that are relevant to the therapy of AD is certainly intriguing, and would indeed put KA-672 in a class of its own if these claims can be fully substantiated. If this were case, it would add dramatic support to the notion that a strategy consisting of targeted synthesis and pharmacological screening could, at this stage of our knowledge, still be superior to the onslaught of combinatorial chemistry, or purely rational drug design through molecular modeling. It is, however, difficult to imagine that so many different functional elements should actually reside in a single chemical structure. In particular, it should be investigated whether some of the claimed effects might be conferred by metabolites of KA-672. Regardless of what explanation can be found for this broad activity spectrum, an inseparable and fixed combination of an NMDA receptor antagonist with an α-adrenergic, dopaminergic and NGF agonist might not be without problems in clinical practice. While functional NMDA antagonism could be invaluable in combating neurodegeneration, and a measure of stimulatory activity in the aminergic systems might allow better therapeutic access to the non-cognitive disturbances of AD, NGF-like- and D2antagonistic-activity might not be desirable for all patients, or in all stages of the syndrome. It is also conceivable that interactions with other drugs could be difficult to control. 138 IDrugs 1998 Vol 1 No 1 There could be significant additional potential for KA-672 as an anticonvulsant, in ischemic stroke, and in traumatic brain injury. Patent Commentary Based on the German patent, DE-04111861 (April 11, 1991), Schwabe has secured a family of patents (Benzopyranones, a method for producing them and uses thereof) covering the synthesis of the series of coumarin derivatives to which KA672 belongs, and their use as neuroprotectants and antiallergics. This family includes, among others, WO-09218493 (October 29, 1992), US-05428038 (June 27, 1995), and JP94506922. This family is extended by another series under the same title (DE-04233963, October 8, 1992; WO-09408985, April 28, 1995; US 05550129, August 27, 1996). Development History DEVELOPER COUNTRY STATUS INDICATION DATE REF Dr Wilmar Schwabe GmbH & Co Germany C2 Alzheimer’s disease 08-AUG-97 258423 Dr Wilmar Schwabe GmbH & Co Germany C1 Alzheimer’s disease 23-MAY-95 177791 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Chemistry STUDY TYPE RESULT REF Synthesis and SAR 110 novel 2H-1-benzopyran-2-ones (coumarin derivatives) prepared. Neuroprotective and analgesic properties described. 281905 Biology STUDY TYPE In vitro EFFECT STUDIED NGF-like activity. EXPERIMENTAL MODEL Embryonic rat septal cells. RESULT Addition of 100 ng/ml KA-672 to the culture medium induced outgrowth of predominantly short (5 to 25 mm) neurites, comparable to NGF at the same concentration. REF 260591 In vitro Spasmogenic activity. Isolate guinea pig ileum. No interference with the effects of acetylcholine, bradykinin, serotonin, substance P and nicotine. Dose-dependent inhibition of histamine effects. 177791 In vitro Cholinesterase inhibition. Rat brain homogenate. Electric eel acetylcholinesterase. KA-672 inhibited acetylcholinesterase as potently as tacrine and was approximately 10-fold stronger than galanthamine. Butyrylcholinesterase and arylacylamidase were not inhibited. 281915 In vivo Cognitive effect (passive and conditioned avoidance). Rat and mouse. KA-672 (0.1 to 1.0 mg/kg, po) dose-dependently enhanced memory acquisition and retrieval. 0.01 to 0.3 mg/kg, po, shortened conditioned avoidance latencies from day 1 to 2 of training, and increased the number of correct responses up to 15 days after discontinuation of treatment. 177791 In vivo Cholinergic activity. Rat and mouse. KA-672 (0.1 to 0.3 mg/kg, po) protected against scopolamine-induced amnesia. No tremor or salivation at 3 to 5 mg/kg. 177791 In vivo Adrenergic activity. Rat and dog (anesthetized). KA-672 (10 mg/kg, iv) inhibited the cardiovascular actions of phenylephrine, and lowered systolic and diastolic blood pressure 15 to 20%, without altering heart rate. 258435 Drug evaluation KA-672 139 Biology (continued) STUDY TYPE In vivo EFFECT STUDIED Serotonergic activity. EXPERIMENTAL MODEL Rat and mouse. RESULT KA-672 dose-dependently inhibited 5-HT-induced head twitch in mice, and potentiated the 8-OHDPAT-induced inhibition of spontaneous cageleaving behavior in rats. REF 258435 In vivo Functional NMDA receptor antagonism. Mouse. KA-672 (0.1 to 0.5 mg/kg, po) protects against convulsions induced by NMDA, but not against those triggered by pentetrazol, picrotoxin, strychnine, or electroshock. No binding interference with glutamic or kainic acid, AMPA, glycine, MK-801 or TCP. Electrophysiological currents activated by NMDA (but not those activated by AMPA or GABA) were inhibited. 260590 In vivo Receptor binding. Rat. KA-672 drastically reduced 5-HT in the striatum, and elevated its levels in the hippocampus. Binds to 5- 260592 HT1a, 5-HT7, α1 and D2 receptors in nanomolar concentrations. No binding to other monoaminergic receptor subtypes or ion channels. In vivo Serotonin and dopamine metabolism. Mouse. KA-672 (0.3 mg, po, 1 h prior to injection of 50 mg/kg NMDA) potentiated the reduction of dopamine levels, and antagonized the reduction of 5-HT levels. 260593 In vivo Nootropic activity. Passive avoidance test in rats and mice. KA-672 (0.1 to 1 mg/kg po) dose-dependently enhanced learning ability and improved consolidation and retrieval of memory. 177791 In vivo Onset and duration of action. Onset and duration of action in conditioned avoidance response tests in rats. Rapid onset of action following administration of 0.01 to 0.3 mg/kg, po, resulting in correct avoidance response from first to second training day; long duration with nootropic activity 15 days after discontinuation of treatment. 177791 RESULT Maximal side-effect (free single) dose is 40 mg, po; 50% of individuals experienced orthostatic syndrome after single doses of 60 mg, po. Mild decrease in supine and standing blood pressure seen as the only side-effect with repeat dosing of 10 or 20 mg, po. Linear pharmacokinetics; terminal-phase half-life values after 20 mg repeat doses and single doses were 11.1 h and 13.7 h, respectively REF 260594 Clinical EFFECT STUDIED Tolerability, safety and pharmacokinetics. EXPERIMENTAL MODEL Double-blind, randomized, placebocontrolled study in healthy males and females aged 52 to 74 years. Associated patent WO-09218493 (Dr Willmar Schwabe GmbH Co) Abstract Heterocyclic substituted coumarins have been synthesized and are claimed as specific NMDA agonists. They act as anti-allergic and neuroprotective agents and may be useful in the treatment of hayfever, asthma and urticaria. Compounds were tested in vivo in mice for NMDA agonist activity, the most potent having efficiency at doses of 5 to 25 mg/kg, iv. Sixty-six compounds are exemplified by synthesis via 44 intermediates, and using standard techniques. Yields and melting points are provided. Twenty-six compounds are specifically claimed, one of which is 6-ethoxy-7-(3-(4-phenyl-1piperazinyl)propoxy)-2H-1-benzopyran-2-one. Priority DE-00111861 11-APR-91 Inventors Chatterjee,S; Noeldner,M; Hauer,H; Koch,E. Designated States AT AU BE CA CH DE DK ES FR GB GR IT JP LU MC NL SE US Title Benzopyranones, methods of manufacture and use thereof. Action NMDA agonist Indication Allergy, Neurodegenerative disease, Urticaria Publication WO-09218493-A1 29-OCT-92 Title Benzopyranones, methods of manufacture and use thereof. Action NMDA agonist Indication Allergy, Neurodegenerative disease, Urticaria Publication WO-09218493-A1 29-OCT-92 Priority DE-00111861 11-APR-91 Inventors Chatterjee,S; Noeldner,M; Hauer,H; Koch,E. Designated States AT AU BE CA CH DE DK ES FR GB GR IT JP LU MC NL SE US 140 IDrugs 1998 Vol 1 No 1 Bibliography 177482 Neurologic Drugs. DRUG NEWS PERSPECT 1995 8 1 47-48 177791 KA-672.HCL: A new orally active antidementia agent. Klusa V, Germane S, Chatterjee SS, Noeldner M EUR J PHARM SCI 1994 2 1-2 • The seminal presentation on KA-672, describing cognitive effects in mice and rats. 258423 Willmar Schwabe GmbH COMPANY COMMUNICATION 1997 August 08 • Confirmation of KA-672 being in phase II trials for Alzheimer’s disease. 258435 Anseculin hydrochloride. DRUGS FUTURE 1996 21 8 779-781 • A review on KA-672. 260590 Effects of anseculin on some excitatory amino acidinduced phenomena. Chatterjee SS, Noldner M PHARMACOL TOXICOL 1997 80 Suppl 1 abs16 • The interactions of KA-672 with the NMDA receptor complex and its neuroprotective and anticonvulsant effect is discussed. 260591 Neurotrophic effects of KA-672 HCl on cultured embryonic septal cells. Klimaviciusa L, Chatterjee SS, Noldner M, Svirskis S, Klusa V PHARMACOL TOXICOL 1997 80 suppl 1 abs28 • KA-672 and nerve growth factor act in a comparable way in vitro. 260592 Modulation of neurotransmitter by Anseculin. Noldner M, Chatterjee SS PHARMACOL TOXICOL 1997 80 suppl 1 abs38 • An overview of the interaction of KA-672 with neurotransmitter receptors, and its influence on serotonin and dopamine levels and metabolism. 260593 Influence of KA-672.HCl on the NMDA-induced neurotoxicity in mice: the contents of the brain monoamines. Skujins A, Svirskis M, Noldner M, Chatterjee SS, Klusa V PHARMACOL TOXICOL 1997 80 suppl 1 abs48 • This abstract describes the separate and combined actions of KA-672 and NMDA, as reflected in 5-HT and dopamine levels. The effect of KA-672 on NMDA-induced convulsions and mortality might be mediated through central monoaminergic mechanisms. 260594 Anseculin, a neuronal activator against dementia: tolerability, safety, preliminary pharmacokinetics in male and female volunteers. Sourgens H, Horr R, Steinbrede H, Derendorf H PHARMACOL TOXICOL 1997 80 suppl 1 abs50 • A presentation of the phase I studies of KA-672. 270790 Therapeutic approach in dementia. Trabucchi M, Bianchetti A RIV PSICHIATR 1997 32 4 SUPPL. 20-26 275467 [Treatment of dementia with Tacrine]. THERAPIE DEMENTIELLER ERKRANKUNGEN MIT TACRIN. Gastpar M FORTSCHR FORTBILD MED 1997 21 - 451-458+495 281905 Benzopyranones, a method for producing them and uses thereof. Chatterjee SS, Noeldner M, Hauer H, Kock E US PATENT 1995, June 27 Dr W Schwabe GmbH & Co, Germany. 281907 Biotransformation of scoparone used to monitor changes in cytochrome P450 activities in primary hepatocyte cultures derived from rats, hamsters and monkeys. Mennes WC, van Holsteijn CW, Timmerman A, Noordhoek J, Blaauboer BJ BIOCHEM PHARMACOL 1991 41 8 1203-1208 • The demethylation of scoparone (the parent structure of KA-672) in hepatocytes of various species is described. 281908 Dr Wilmar Schwabe GmbH & Co COMPANY COMMUNICATION 1998 March 6 •Schwabe has dropped its provisional synonym for KA-672 HCl, anseculin. No international non-proprietary name has been assigned to date. 281915 KA-672 HCl: A novel anticholinesterase inhibitor with no effects on arylacylamidase or butyrylcholinesterase. Noeldner M, Chatterjee SS 9TH INT SYMPOSIUM CHOLINERGIC MECHANISMS 19957-10 June • The cholinesterase inhibitor properties of KA-672 are discussed. Drug evaluation Tranilast 141 Tranilast Kissei Pharmaceutical Matthew Konneh Address Institut des Vaisseaux et du Sang University VII of Paris Paris France IDrugs 1998 1(1):141-146 Current Drugs Ltd ISSN 1369-7056 Kissei, in collaboration with SmithKline Beecham, is developing tranilast (Rizaben) as a prophylactic agent for restenosis, following percutaneous transluminal coronary angioplasty (PTCA). As of May 1997, tranilast was awaiting approval in Japan as an oral formulation given for three months following PTCA [246273], [248281]. The agent has been launched for use in allergic rhinitis, asthma and atopic dermatitis. Analysts had expected the early approval of Rizaben for PTCA-associated restenosis. However, due to delays by the Ministry of Health and Welfare, it is unlikely that Rizaben will be approved until the end of 1998. Sales of Rizaben for the treatment of its launched indications did not reach company expectations. The companies are also developing tranilast, in a nasal spray formulation, as a potential treatment for allergic rhinitis. The drug is in phase II trials for this indication [262810]. EP-00588518 from Kissei discloses the use of tranilast for the treatment of restenosis. Kissei has also filed patents disclosing the use of tranilast for arteriosclerosis (JP09227371) and diabetic retinopathy (WO-09729744). Introduction Tranilast (N-3,4-dimethoxycinnamoyl anthranilic acid) has been in clinical use in Japan since 1982 for the treatment of allergic diseases, such as allergic rhinitis, bronchial asthma and atopic dermatitis. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils [275344]. Tranilast is also effective against keloids, the formation of which is thought to be associated with excessive proliferation of, and excessive collagen synthesis by, fibroblasts. From these lines of evidence, researchers have investigated the therapeutic benefit of tranilast against the development of fibroproliferative restenotic lesions, following PTCA. Coronary heart disease (CHD) may be treated surgically by PTCA, coronary artery bypass graft or cardiac transplantation. The method of intervention depends on the severity of the disease. These invasive surgical interventions are associated with an early complication of increased vascular smooth muscle cell growth and extracellular matrix expression. Coronary angioplasty uses a balloon catheter to dilate coronary stenotic lesions. Restenosis at the site of dilation of these lesions occurs in 30 to 40% of cases [275349], usually over a time course of three to six months. Acute failure of angioplasty in 30% of cases is due to ’elastic recoil’ and can be prevented, to a certain extent, by stenting. The Originator Kissei Pharmaceutical Co Ltd Status/Indication Launched (Allergic rhinitis, Asthma, Atopic dermatitis); Pre-registration (Restenosis) Synonyms MK-341, Tramelas, Rizaben O OH O O N H O late failure of PTCA is normally due to fibrous intimal proliferation; the excessive overgrowth of vascular smooth muscle cells (VSMCs) together with the excess production of extracellular matrix (ECM), including collagen, elastin and glycoproteins (fibronectin, vitronectin and proteoglycans) lead to the narrowing of the vessel lumen. Pharmacology The pharmacological approaches under consideration for reducing restenosis include anti-inflammatory drugs, antiplatelet agents, antithrombotics, anticoagulants, antiproliferatives, vasodilators, antioxidants, lipid-lowering drugs, chimeric toxins, antisense oligonucleotides and gene therapies. Several medical devices, such as atherectomy systems that use a rapidly rotating device to reduce plaques, intravascular stents and laser technology have come into clinical testing and are used for the types of lesions for which they are suited. Although some show benefit with regard to acute complications, most do not seem to offer distinct advantages in avoiding restenosis. Using VSMC in culture, Tanaka et al [167010] showed that tranilast could inhibit DNA synthesis, and hence cell growth, as well as their migration and their ability to synthesize collagen. These inhibitory effects occurred in a dose-dependent manner; tranilast is also able to modulate the effects of contractile agents. In isolated rat aortic rings, which were made to contract in an organ bath by the addition of endothelin (ET-1), exposure of these rings to tranilast (0 to 500 µM) dose-dependently inhibited ET-1induced increases in tension and intracellular calcium [258570]. Chronic recoil, following PTCA, may account for more than 60% of the late lumen loss in restenosis. This recoil may be due to a chronic vasoconstrictor component such as endothelin release [275350] or loss of nitric oxide (NO) vasodilatory activity. The impairment of NO release by platelet-derived growth factor (PDGF) production can be abrogated in the presence of tranilast [258582]. Therefore, 142 IDrugs 1998 Vol 1 No 1 the ability of tranilast to inhibit vasoconstrictor activity may be an important mechanism in its anti-restenotic profile. Mechanical injury causes endothelial denudation and platelet accumulation, which results in the release of several growth factors [275350]. These growth factors may stimulate the migration and proliferation of smooth muscle cells (SMCs) in the dilated artery and some of the growth factors may be acting through intracellular calcium release, [Ca2+]i. Nie et al [266805] have shown that in rat VSMCs, 1 nM of PDGF induced a biphasic elevation of cytoplasmic free calcium concentration, however, tranilast abolished the sustained phase of [Ca2+]i in a dose-dependent manner. Tranilast also abolished the concomitant DNA synthesis of these cells with a similar IC50 to that of the inhibition of calcium flux. In a model of rat-vessel denudation, in which insult to the femoral artery is induced by a photochemical reaction between light and a photosensitive dye (Rose Bengal), the generation of singlet molecular oxygen leads to endothelial injury with subsequent platelet adhesion and aggregation, and growth factor release. This initiates the cascade responsible for recruiting SMCs and eventually neointimal lesion formation. In rats which received tranilast (10, 100, 300 mg/kg) two days before surgery and continued for three weeks after, lesional changes, measured by morphometric analysis, showed that there was a dose-dependent decrease in the cross-sectional area of the intimal layer (28.8, 62.4, 86.9%, respectively), a trend which was also observed in intimal/medial ratios. In SMCs cultured from the same rat species, incubation with tranilast (3 to 300 µM) 24 h previously caused a dose-dependent inhibition in [3H]thymidine uptake [266806]. Although investigations employing injury to animal carotid artery have provided invaluable insight into neointimal development, the response to injury in these models may not be entirely similar to the situation following PTCA of atherosclerotic lesions in man. Leukocytes, which are minimal in rat vessels, comprise a major but variable cellular component of human and hypercholesterolemic rabbit experimental models [275351], [275358], [275359]. In white male rabbits fed 1% cholesterol and 5% lard, followed by balloon catheter injury in the right common carotid artery, there was increased medial thickening, intimal hyperplasia, and lipid deposits, compared to rabbits on a normal diet. Tranilast was given orally (100 to 300 mg/kg/day) one day after the balloon injury. At 28 days after injury, morphometric analysis showed that treatment with tranilast (300 mg/kg) decreased the intimal thickening as measured by intimal/medial ratios, and also increased the lumenal/total area ratio, which was decreased in the hypercholesterolemic rabbits compared with normocholesterolemic rabbits [258580]. Clinical Development Tranilast (600 mg/day) given to patients post-angioplasty for three months, demonstrated a reduced rate of post-PTCA restenosis [275420]. In an enlarged multicenter, double-blind, placebo-controlled study of patients with native coronary lesions and other predisposing factors, tranilast (300 mg/day or 600 mg/day) or placebo was given for three months following PTCA. Patients receiving tranilast (600 mg/day) had a reduced rate of restenosis; 14.7% in comparison to 46.5 % for the placebo group (p < 0.001) [266811]. In another study, the efficacy of tranilast was investigated in patients with restenosis undergoing directional atherectomy of coronary lesion. Patients were randomized into two groups; 40 patients receiving tranilast (600 mg/kg) and 152 patients received placebo. In comparing angiographic and clinical variables, the minimal lumen diameter was significantly larger in the tranilast group at both the threeand six-month follow-up times than in the placebo group. The restenosis rates (loss of > 50% of the initial gain) were lower in the tranilast treated patients (11% versus 26%, p = 0.03). The number of clinical events over the 12-month period after the procedure was significantly reduced by tranilast administration (p = 0.0013) [275361]. Current Opinion Hospital registries show that acute complications, including hospital deaths, myocardial infarctions, and emergency bypass surgery, have greatly declined. Much of this improvement has been attributed to the aggressive use of agents to prevent elastic recoil (antispasmodics), anticoagulants, thrombolysis and new mechanical devices including perfusion balloons and intravascular stents. There are 300,000 procedures performed annually in the US. Although the acute occurrence of complications has been greatly reduced, the marked prevalence of restenosis clearly compromises the long-term benefits associated with PTCA. The advantage of the lower cost of performing PTCA relative to coronary artery bypass surgery has been greatly diminished by the expense of repeat diagnostic and therapeutic catheterization as a result of restenosis [275362], currently estimated to be US$2 billion dollars per year in the United States alone [275423]. Tranilast has a potent inhibitory effect on smooth muscle cell proliferation, migration and extracellular matrix production. It also reduces the neointimal response to balloon injury in the various animal models used to simulate this condition. Of the few trials in which tranilast has been used in the clinical setting, it is encouraging to see that all have shown a beneficial outcome with respect to tranilast-induced decreases in restenosis rates, following PTCA and coronary atherectomy procedures. It remains for tranilast to undergo the ’gold standard’ in clinical trials in which a large cohort of patients (2000 to 3000) are enrolled to evaluate clinical events following PTCA with a further subanalysis of patients using angiography to determine the restenosis rate in doubleblind randomized, placebo-controlled conditions. With the huge increase in healthcare costs as a result of restenosis and its resultant complications (angina and repeat procedures), a drug which limits these events would provide a powerful pharmacological therapy for interventional cardiologist and reduce the cost of coronary heart disease management. Drug evaluation Tranilast 143 Licensing SmithKline Beecham plc In May 1997, SmithKline Beecham acquired exclusive development and marketing rights for tranilast in areas outside of Japan, Taiwan, South Korea, China and Russia [248281]. Development History DEVELOPER COUNTRY STATUS INDICATION DATE REF Kissei Pharmaceutical Co Ltd Japan C1 Restenosis 12-MAR-97 275361 Kissei Pharmaceutical Co Ltd Japan PR Restenosis 14-MAY-97 246273 SmithKline Beecham plc UK PR Restenosis 14-MAY-97 246273 SmithKline Beecham plc UK C2 Allergic rhinitis 15-SEP-97 262810 Kissei Pharmaceutical Co Ltd Japan L Allergic rhinitis 18-MAR-98 Kissei Pharmaceutical Co Ltd Japan L Asthma 18-MAR-98 Kissei Pharmaceutical Co Ltd Japan L Atopic dermatitis 18-MAR-98 Kissei Pharmaceutical Co Ltd South Korea L Allergic rhinitis 18-MAR-98 Kissei Pharmaceutical Co Ltd South Korea L Asthma 18-MAR-98 Kissei Pharmaceutical Co Ltd South Korea L Atopic dermatitis 18-MAR-98 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Biology STUDY TYPE In vitro EFFECT STUDIED Vasorelaxation. EXPERIMENTAL MODEL Isolated rat aortic strips in an organ bath were induced to contract by endothelium ET-1 and high potassium + (K ) addition. Changes in calcium 2+ (Ca ) levels were measured by fura-2 loading. RESULT Prior exposure of these rings to tranilast (0 to 500 µM), dose-dependently inhibited ET-1-induced increases in tension and 2+ Ca elevation in the muscle strips. REF 258570 In vitro Vasorelaxation. Transverse strips of porcine coronary artery in an organ bath prior to incubation with tranilast for 30 min, followed by challenge with histamine and KCl. Histamine-induced contractions were decreased by 25% and 65% on incubation with 100 µM and 500 µM tranilast, respectively. Tranilast inhibited the + contraction induced by K (30 mM) in a dose-dependent manner. 258569 In vitro Smooth muscle cell (SMC) proliferation. Extracellular matrix production and migration. Stimulated [ H]thymidine and proline uptake by SMC. Induced SMC migration on culture plates. Tranilast inhibited thymidine uptake dosedependently, with maximal inhibitory activity being reached at 50 µg/ml. The calcium antagonist, nilvadipine and the PDE antagonists, E-1020, and elastase, all showed concentration-dependent inhibitory effects on vascular smooth muscle cell proliferation, with maximal effects that were approximately half that of tranilast. 167010 In vivo Neointima formation. Intimal thickening in femoral artery of rats initiated by photochemical reaction between green light directed at the artery by an optic fiber and iv infusion of Rose Bengal (10 mg/kg). Tranilast at doses of 30, 100, 300 mg/kg, po, given two days prior to endothelial injury, decreased intimal area by 29%, 62% and 87%, respectively, in comparison to vehicle control-treated animals. 266806 3 144 IDrugs 1998 Vol 1 No 1 Biology (continued) STUDY TYPE In vivo EFFECT STUDIED Neointima formation. EXPERIMENTAL MODEL Intimal thickening in common carotid artery induced by balloon catheterization in rabbits which had been fed a 1% cholesterol diet. RESULT 28 Days after balloon angioplasty, marked intimal thickening was observed in control rabbits. Treatment with tranilast (300 mg/kg, po) decreased the intimal thickening as measured by intimal/medial ratios. REF 258580 Clinical EFFECT STUDIED Restenosis after percutaneous transluminal coronary angioplasty (PTCA). MODEL USED Double-blind, placebo-controlled study of patients with native coronary lesions and other predisposing factors. Patients were given either 600 mg/day, 300 mg/day, or placebo for three months following PTCA. RESULT Patients receiving tranilast 600 mg/day showed a restenosis rate of 14.7% in comparison to 46.5% for the placebo group (p < 0.001). REF 266811 Restenosis following successful directional atherectomy. Patients who underwent directional atherectomy of coronary lesions were randomized into groups. 40 Patients received tranilast (600 mg/kg) and 152 patients received placebo. Comparing angiographic and clinical variables; the minimal lumen diameter was significantly larger in the tranilast group at both the threemonth and six-month follow-up times. The restenosis rates (loss of > 50% of the initial gain) were lower in the tranilast treated patients than those on placebo (11% versus 26%; p = 0.03). The number of clinical events over the 12month period after procedure was significantly reduced by tranilast administration (p = 0.0013). 275361 Bibliography 167010 Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by vascular smooth muscle cells. Tanaka K; Honda M; Kuramochi T; Morioka S ATHEROSCLEROSIS 1994 107 2 179-185 258558 Inhibitory effects of tranilast on proliferative reactions, angiogenesis and contraction of fibrotic tissue. Isaji M, Miyata H, Ajisawa Y, Yoshimura N INVEST OPHTHAL-MOL VISUAL SCI 1997 38 4 PART 1-2 S753 199768 The pathogenesis of atherosclerosis : a perspective for the 1990s. Ross R NATURE 1993 362 801-809 244866 New Drugs in Kissei R&D Pipeline. PHARMA JPN 1997 1540 19 258560 Tranilast prevents restenosis after directional coronary atherectomy more strongly in larger sized vessels. Kosuga K, Tamai H, Ueda K, Hsu Y S, Ono S, Tanaka S, Matsui S, Minami M, Motohara S, Uehata H J AM COLL CARDIOL 1997 29 2 Suppl A 419A 246273 SmithKline Beecham and Kissei Pharmaceuticals in agreement for new cardiovascular compound. SmithKline Beecham plc PRESS RELEASE 1997May 14 258562 Tranilast inhibits the growth of rat mesangial cells. Ikeda M, Ikeda U, Shimada K, Fujita N, Okada K, Saito T, Minota S, Kano S EUR J PHARMACOL 1997 324 2-3 283-287 248281 Kissei licenses-out Rizaben preventing restenosis postPTCA to SB. PHARMA JPN 1997 1550 8 258563 Effects of tranilast on proliferation and protein tyrosine phosphorylation stimulated by PDGF and EGF in cultured human-derived coronary artery smooth muscle cells. Watanabe S, Matsuda A, Umemura K, Kondo K, Suzuki Y, Hashimoto H, Nakashima M JPN J PHARMACOL 1997 73 Suppl 1 202P 258566 Antiallergic effects of tranilast in rats and guinea pigs. Jiayu C, Shaohui C, Songbai Y, Qiangmin X, Fei Y J WEST CHINA UNIV MED SCI 1997 28 2 179-183 250724 Tranilast suppresses intimal hyperplasia in the balloon injury model and cuff treatment model rabbits. Fukuyama J, Ichikawa K, Miyazawa K, Hamano S, Shibata N, Ujiie A JPN J PHARMACOL 1996 70 4 321-327 257399 Pathophysiology and pharmacological approaches for prevention of coronary artery restenosis following coronary artery balloon angioplasty and related procedures. Landzberg BR, Frishman WH, Lerrick K PROG CARDIOVASC DIS 1997 39 4 361-398 258556 Successful treatment of Pemphigus vulgaris with prednisolone and tranilast [letter]. Miyamoto H, Takahashi I ACTA DERM VENEREOL 1997 77 1 87-88 258557 Tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) down-regulates the growth of scirrhous gastric cancer. Yashiro M, Chung YS, Sowa M ANTICANCER RES 1997 17 2A 895-900 258567 Treatment of keloid and hypertrophic scars by iontophoretic transdermal delivery of tranilast. Shigeki S, Murakami T, Yata N, Ikuta Y SCAND J PLAST RECONSTR SURG HAND SURG 1997 31 2 151-158 258568 Suppression of atherosclerotic development in Watanabe Heritable Hyperlipidemic (WHHL) rabbits treated with an oral anti-allergic drug, tranilast. Matsumura T, Kugiyama K, Ohgushi M, Sugiyama S, Ohta Y, Doi H, Yasue H J AM COLL CARDIOL 1997 29 2 Suppl A 151A Drug evaluation Tranilast 145 2+ 258569 Tranilast inhibits contraction and C movement of porcine coronary arteries. Ishibashi S, Ikeda U, Ihara T, Shimada K ATHEROSCLEROSIS 1997 130 1-2 113-119 258570 Tranilast inhibits contraction of rat aortic smooth muscle. Ihara T, Ikeda U, Ishibashi S, Shimada K EUR J PHARMACOL 1997 329 1 43-48 258571 Tranilast-induced cystitis: A case report. Takada T, Kondoh N, Nakamura Y, Kitamura M, Takeyama M, Kiyohara H NISHINIHON J UROL 1997 59 1 35-38 258573 Antiproliferative and c-myc mRNA suppressive effect of tranilast on newborn human vascular smooth muscle cells in culture. Miyazawa K, Hamano S, Ujiie A BR J PHARMACOL 1996 118 4 915-922 258575 Pharmacokinetics and relative bioavailability of tranilast in healthy volunteers. Wang D, Yu H, Hu C CHIN PHARM J 1996 31 11 670-672 258577 Efficacy of tranilast on restenosis after coronary stenting. Hsu Y S, Tamai H, Ueda K, Ono S, Kosuga K, Tanaka S, Matsui S, Motohara S, Uehata H CIRCULATION 1996 94 8 Suppl I620 258578 The impact of tranilast on restenosis following coronary angioplasty: the second tranilast restenosis following angioplasty trial (TREAT-2). Tamai H, Katou K, Hayakawa H, Yamaguchi T, Kanmatsuse K, Haze K, Aizawa T, Nakanishi N, Suzuki S, Suzuki T, Takase S, Nishikawa H, TREAT study investigators CIRCULATION 1996 94 8 Suppl I620 258579 Tranilast inhibits expression of TGF-b isoforms and receptors after balloon injury. Ward MR, Sasahara T, Agrotis A, Dilley RJ, Jennings GL, Bobik A CIRCULATION 1996 94 8 Suppl I466 258580 Tranilast suppresses the vascular intimal hyperplasia after balloon injury in rabbits fed on a high-cholesterol diet. Fukuyama J, Ichikawa K, Hamano S, Shibata N EUR J PHARMACOL 1996 318 2-3 327-332 258581 Inhibitory effect of tranilast on activation and transforming growth factor-b1 expression in cultured rat stellate cells. Ikeda H, Inao M, Fujiwara K BIOCHEM BIOPHYS RES COMMUN 1996 227 2 322-327 258582 Tranilast restores cytokine-induced nitric oxide production against platelet-derived growth factor in vascular smooth muscle cells. Hishikawa K, Nakaki T, Hirahashi J, Marumo T, Saruta T J CARDIOVASC PHARMACOL 1996 28 2 200-207 258584 Tranilast inhibits smooth muscle cell proliferation by induction of c-myc and c-fos proto-oncogenes. Taniguchi T, Oda A, Yoshida H, Shimizu H, Ishikawa Y, Yokoyama M, Takahashi A CIRCULATION 1996 94 8 Suppl I107 258585 Inhibitory effects of tranilast on proliferation, migration, and collagen synthesis of human vascular smooth muscle cells. Fukuyama J, Miyazawa K, Hamano S, Ujiie A CAN J PHYSIOL PHARMACOL 1996 74 1 80-84 262810 New drugs in the R&D pipeline at Kissei Pharmaceutical. PHARMA JPN 1997 1564 20 266803 Clinical trials to prevent restenosis after percutaneous coronary revascularization. Mak K. H, Topol EJ, Clowes A, Berk B, Libby P, Moncada S, Nabel G ANN NY ACAD SCI 1997 811 255-288 266804 Effects of pemirolast and tranilast on intimal thickening after arterial injury in the rat. Miyazawa N, Umemura K, Kondo K, Nakashima M J CARDIOVASC PHARMACOL 1997 30 2 157-162 266805 Blockade of DNA synthesis induced by platelet-derived growth factor by tranilast, an inhibitor of calcium entry, in vascular smooth muscle cells. Nie L, Mogami H, Kanzaki M, Shibata H, Kojima I MOL PHARMACOL 1996 50 4 763-769 266806 Tranilast suppresses intimal hyperplasia after photochemically-induced endothelial injury in the rat. Kikuchi S, Umemura K, Kondo K, Nakashima M EUR J PHARMACOL 1996 295 2-3 221-227 266807 Efficacy of tranilast on restenosis after directional coronary atherectomy (DCA). Kosuga K, Tamai H, Ueda K, Hsu Y S, Ono S, Tanaka S, Doi T, Wang M U, Motohara S, Uehata H CIRCULATION 1995 92 8 Suppl I346 266808 Inhibition of PDGF- and TGF-b 1-induced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats. Miyazawa K, Kikuchi S, Fukuyama J, Hamano S, Ujiie A ATHEROSCLEROSIS 1995 118 2 213-221 266809 Suppressive effect of an anti-allergic drug, tranilast, on the vascular intimal thickening induced by balloon catheter. Ichikawa K, Fukuyama J, Miyazawa K, Hamano S, Shibata N, Ujiie A PHARMACOMETRICS 1995 50 5 539-548 266810 Effect of tranilast on the intimal thickening after arterial injury in rabbits. Ichikawa K, Miyazawa K, Tazawa S, Shibata N, Hamano S, Ujiie A JPN J PHARMACOL 1995 67 Suppl 1 186P 266811 The impact of tranilast on restenosis following coronary angioplasty: the tranilast restenosis following angioplasty trial (TREAT). The TREAT study investigators CIRCULATION 1994 90 4 Pt 2 I652 267352 Making efforts to conduct simultaneous develop-ment in Japan, US and EU: Mr T Usuda of Kissei. PHARMA JPN 1997 1570 4 267747 Kissei establishes US subsidiary. SCRIP 19972279 269877 Suppressive effects of tranilast on the expression of inducible cyclooxygenase (COX2) in interleukin-1b-stimu-lated fibroblasts. Inoue H, Ohshima H, Kono H, Yamanaka M, Kubota T, Aihara M, Hiroi T, Yago N, Ishida H BIOCHEM PHARMACOL 1997 53 12 1941-1944 269878 Effect of topical tranilast and corticosteroids on subepithelial haze after photorefractive keratectomy in rabbits. Furukawa H, Nakayasu K, Gotoh T, Watanabe Y, Takano T, Ishikawa T, Kanai A J REFRACT SURG 1997 13 5 Suppl S457S458 269879 Effects of insulin and tranilast on cytosolic free Ca2+ concentration and DNA synthesis of aortic smooth muscle cells. Asakura Y, Okuda Y, Asano M, Tachi Y, Suzuki S, Kawakami Y, Yamashita K DIABETOLOGIA 1997 40 SUPPL. 1 A436 146 IDrugs 1998 Vol 1 No 1 269880 Protective effect and its mechanism of tranilast on paracetamol-induced hepatotoxicity in mice. Fu YP, Wang J, Wu R. S CHIN J PHARMACOL TOXICOL 1997 11 3 239-240 275420 Effects of tranilast on prevention of chronic restenosis after PTCA. Ueda K, Tamai H, Kyo E, et al JPN J INTERVENTIONAL CARDIOLOGY 1993 8 Suppl 104 271492 Kissei: profits decline by double digits due to price cuts, transfer of marketing rights of Spiropent. PHARMA JPN 19971576 25 275423 Restenosis: The clinical issues. Hillegrass WB, Ohman EM, Califf RM TEXTBOOK INTERVENTIONAL CARDIOLOGY 1994 415-435 275344 The inhibition mechanisms of histamine release by N(3,4,-dimethoxycinnamoyl) anthranilic acid. Koda A, Kurashina Y INT ARCH ALLERGY APPL IMMUNOL 1985 77 244-245 280214 Major M&As and tie-ups in 1997. PHARMA JPN 19981588 8-12 275349 Long-term results of coronary balloon angioplasty. Meier B ANNU REV MED 1991 42 47-59 275350 Circulating immunoreactive endothelin in patients undergoing percutaneous transluminal coronary angio-plasty. Tahara A, Kohno M, Yanagi S, Itagane H, Toda I, Akioka K, Tergaki M, Yasuda M, Takeuchi K, Takeda T METABOLISM 1991 40 12 1235-1237 275351 Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. Austin GE, Ratliff NB, Hollman J, Tabei S, Phillips DF J AM COLL CARDIOL 1985 6 369-375 275358 Morphogenesis and clinicopathologic charac-teristics of recurrent carotid disease. Clagett GP, Robinowitz M, Youkey JR, Fisher DF, Fry RE, Myers SI, Lee EI, Collins GJ, Virmani R J VASC SURG 1986 3 10-23 275359 Morphological observations late (greater than 30 days) after clinically successful balloon coronary angioplasty. Waller BF, Pinkerton CA, Slack JD, VanTassel JW, Peters T CIRCULATION 1991 83 Suppl 2 I28-I41 275361 Effectiveness of tranilast on restenosis after directional coronary atherectomy. Kosuga K, Tamai H, Ueda K, Hsu YS, Ono S, Tanaka S, Doi T, Myou UW, Motohara S, Uehata H AM HEART J 1997 275362 Is percutaneous coronary angioplasty less expensive than bypass surgery? Reeder GS, Krishnan I, Nobrega FT , et al NEW ENGL J MED 1984 311 1157-1162 280803 Topical delivery of keloid therapeutic drug, tranilast, by combined use of oleic acid and propylene glycol as a penetration enhancer: evaluation by skin microdialysis in rats. Murakami T, Yoshioka M, Yumoto R, Higashi Y, Shigeki S, Ikuta Y, Yata N J PHARM PHARMACOL 1998 50 1 49-54 280804 The effect of tranilast on subepithelial corneal opacity after excimer laser keratectomy. Sakai T, Okamoto S, Iwaki Y ACTA SOC OPHTHALMOL JPN 1997 101 10 783-787 281057 Pharmaceutical Industry: Global Model Book Highlights: Models for the US, Europe, Emerging Europe and Japan. Merrill Lynch ANALYST REPORT 1998 282064 Suppression by tranilast of fetal myosin heavy chains and intimal hyperplasia in rabbits. Ohkawa H, Ito M, Shigeno K, Gupta PC, Matsushita M, Nishikimi N, Sakurai T, Nimura Y CURR THER RES CLIN EXP 1997 58 10 764-772 282065 Effectiveness of tranilast on restenosis after directional coronary atherectomy. Kosuga K, Tamai H, Ueda K, Hsu YS, Ono S, Tanaka S, Doi T, Myou UW, Motohara S, Uehata H AM HEART J 1997 134 4 712-718 282070 Overview of in-stent restinosis: from benchtop to bedside. Ward M ASIA PACIFIC HEART J 1997 6 1 70 Drug evaluation Iralukast 147 Iralukast Novartis AG Andrew Bramley Address Pulmonary Research Laboratory 123-3440 West Broadway Vancouver V6R 4R2 Canada Email: [email protected] IDrugs 1998 1(1):147-151 Current Drugs Ltd ISSN 1369-7056 Iralukast is an LTD4 and LTE4 antagonist under development by Novartis and in phase II clinical trials as a potential treatment for asthma [244117], [177071]. In a double-blind, placebo-controlled trial in 16 patients with mild to moderate asthma, a single 1.5 mg inhaled dose of iralukast reduced the incidence of exercise-induced bronchiospasm and was well-tolerated [272161]. Originator Novartis AG Status Phase II clinical Indication Asthma Action LTD4 antagonist, LTE 4 antagonist CAS 4H-1-Benzopyran-2-carboxylic acid, 7-[[9-(4-acetyl-3hydroxy-2-propylphenoxy)-1-[hydroxy[3(trifluoromethyl)phenyl]methyl]-2,4-nonadienyl]thio]-4-oxo-, monosodium salt, [S-[R*,S*-(E,Z)]]Registry No: 125617-94-9 Note: CGP-45715A - Irakulast sodium salt. CAS 4H-1-Benzopyran-2-carboxylic acid, 7-[[9-(4-acetyl-3hydroxy-2-propylphenoxy)-1-[hydroxy[3(trifluoromethyl)phenyl]methyl]-2,4-nonadienyl]thio]-4-oxo-, [S-[R*,S*-(E,Z)]]Registry No: 151581-24-7 Synonyms CGP-45715A Novartis has an agreement with Rhone-Poulenc Rorer to use its Ultrahaler delivery system for iralukast and phase II trials are underway [220836]. The agreement also involves the development of further related compounds and delivery systems. O S O OH O OH O Introduction Leukotrienes (LT) are potent LTD4 antagonists which are synthesized from arachidonic acid and play a major role in the inflammatory response in asthma. LT C4, D4 and E4 are some of the most potent bronchoconstrictors known and have recently been shown to play an important role in the complex interactions of inflammatory cells in asthma. Iralukast (CGP-45715A) is an LTD4/LTE4 antagonist which is under development by Novartis (formerly Ciba-Giegy) and is currently in phase II trials for the treatment of asthma [244117], [177071]. Synthesis and SAR The structure of iralukast is based on LTD4 [194318]. The carboxylic acid group in the eicosanoid chain of LTD4 is replaced by a trifluoromethyl group that is important for the antagonist properties [274603], and the dipeptide sulfur side chain is replaced by a chromone carboxylic acid [274604]. The polar region of the eicosanoid backbone is stabilized by phenyl binding [274605]. In addition, the lipophilic region of the molecule is stabilized by a substituted acetophenone which serves as a lipophilic anchor [274605]. Iralukast has the same chain length as LTD4, ie, 20 carbon atoms [194318]. During the development of LT antagonists, the introduction of a methyl group at carbon 1 led to the LTD4 antagonist activity with no sign of agonist activity [274603]. The development towards a highly stable and potent LTD4/E4 antagonist involved the introduction of a chromone group to replace the cysteinyl-glycine residue, which gave the compound better chemical stability. This was then enhanced O OH F F F by the integration of the double bonds into a phenyl group, or as in the case of other LT antagonists, by reducing the number of double bonds [274604]. Introduction of a phenyl group at carbons 2, 3 and 4 was found to stabilize the polar region of the eicosanoid structure [274605], and further stability was confirmed by replacing a methyl group at carbon 1 with a trifluoromethyl group [194318]. The 1R,2S configuration of the iralukast structure is absolutely crucial for LTD4 antagonist potency. Various other stereoisomers were at least 20-fold less potent. The number and geometry of double bonds in the eicosanoid backbone are also essential for the activity of the agonist both in vitro and in vivo. Analogs having the same number and geometry of double bonds as synthetic LTD4 were less active in vitro [194318]. Pharmacology 3 Iralukast displaces [ H]LTD4 from its receptor in guinea pig lung membrane preparations; it also displaces 1 nM LTD4 in a dose-dependent fashion (IC50 = 60 nM, Ki = 26 nM) when added at the same time as the ligand. In studies where the preparation was pre-incubated with the iralukast at 15, 30 and 45 min prior to the ligand, the IC50 values were 7.35, 10 and 15.3 nM, respectively (Ki values = 2.2, 2.9 and 4.0 nM) [194318]. The increased potency when pre-incubated with the antagonist is also reflected in the increased potency of the compound when pre-incubated in bioassays. 148 IDrugs 1998 Vol 1 No 1 Iralukast is an extremely potent antagonist of both LTD4 and LTE4 receptors on guinea pig ileum, with IC50 values of 2.7 nM and 0.4 nM at 2 min and 15 min incubation, respectively, and PA2 of 10.1. For LTE4 antagonism, the IC50 values were 7.9 nM and 0.36 nM at 2 min and 15 min incubation, respectively. Although the IC50 values are similar for both LTD4 and LTE4, there were differences in the agonist concentration used in this assay, ie, for LTD4, 1.8 nM and LTE4, 22.7 nM. This suggests that iralukast is more potent as an LTE4 antagonist on guinea pig ileum [194318]. Iralukast also inhibits histamine, bradykinin, PGE1, ACh, SP and barium chloride, with IC50 values in the µM range when added 2 min prior to the agonist. For all of these agonists, other than BK and SP, the potency of iralukast was increased when incubation was increased to 15 min. and 240 min and 24 h pre-treatment. The iv ED50 value in this model was 0.11 mg/kg at 60 min prior to challenge. Iralukast also has a non-specific action against LTC4 and LTD4-induced responses in the guinea pig isolated heart [26731]. Aerosolized iralukast was found to be a potent LTD4 antagonist on spontaneously breathing guinea pigs stimulated with LTD4. IC50 values of 0.09, 0.03, 0.2, 0.1 and 10 µg/kg for 1 min aerosol at incubation times of 15, 60, 120, 240 and 480 min. Iralukast is also a potent antagonist of LTE4 receptors in the same model. For animals treated with iralukast 15 min prior to LTE4 challenge, the IC50 value was 0.3 µg/kg. When administered 60 min prior to challenge, iralukast was even more potent, with an IC50 value of 0.01 µg/kg. Iralukast has also been shown to be orally active against LT-induced bronchospasm in this in vivo model. ED50 values range from 4.3 mg/kg for a 1 h pre-treatment to 0.32 mg/kg after a pre-treatment period of 2 h. In addition, activity was well maintained at a pre-treatment period of 4 h (ED50 = 1.1 mg/kg). This declined at 8 and 24 h pretreatment to 2.9 and 10.9 mg/kg, respectively. Toxicity Iralukast administered iv, was also active, with ED50 values of 0.16, 0.046 and 0.024 mg/kg at 1, 15 and 60 min prior to aerosol LTD4 challenge. Similar activity has been shown at 120, 180 and 240 min pre-treatment, with ED50 values less than 0.1 mg/kg. In addition, iralukast showed potent antagonism of antigeninduced leukotriene-mediated bronchospasm in sensitized guinea pigs [194318]. IC50 values were approximately 2, 3, 2.5 and 0.8 µg/kg at 15, 60 and 480 min, respectively. Oral and iv activities have also been demonstrated in this antigen model, with oral ED50 values of 2.8, 3.8 and 3.9 mg/kg at 120 In comparison with the other more established and clinically advanced LT antagonist, such as zafirlukast (Zeneca) and montelukast (Merck), iralukast has been shown to be considerably more potent in inhibiting LTD 4-induced bronchospasm in the guinea pig model. However, both zafirlukast and montelukast are far more advanced in clinical trials than iralukast, and it is not yet known whether iralukast is more potent than its rivals in clinical trials. No toxic effects have been reported. Clinical Development Phase I No data are currently available. Phase II To date, results of only one phase II trial on iralukast have been published [272161]. In this double blind, placebocontrolled crossover trial, the ability of 1500 µg/kg of inhaled iralukast, administered 60 min prior to evaluation, was assessed for its ability to prevent exercise-induced bronchospasm in asthmatics. In 16 patients with mild to moderate asthma, iralukast significantly prevented exerciseinduced bronchospasm as assessed by a fall in FEV1 and was shown to be well tolerated. Current Opinion Iralukast is a potent LTD4/LTE4 antagonist which blocks both leukotriene and antigen-mediated bronchoconstriction in guinea pigs. In animal studies, its activity compares well with the other competing LT antagonists and, in fact, was shown to be more potent than pranlukast (Ono), montelukast and zafirlukast. However, as yet there are very little data demonstrating its efficacy and toxicity in human subjects. Licensing Rhone-Poulenc Rorer SA Co-development and co-marketing agreement with Ultrahaler technology. Development History DEVELOPER COUNTRY STATUS INDICATION DATE Ciba-Geigy AG Switzerland C1 Asthma 01-OCT-92 Ciba-Geigy AG Switzerland C2 Asthma 01-NOV-94 Drug evaluation Iralukast 149 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Chemistry STUDY TYPE SAR RESULT Evaluation and structure-activity relationships of various synthetic analogs of LTD 4 found to be potent leukotriene antagonists. REF 194318 Biology STUDY TYPE In vitro EFFECT STUDIED Leukotriene antagonistic effects. EXPERIMENTAL MODEL Guinea pig; isolated heart; leukotriene-induced vascular contraction; inhibitory effects. RESULT Leukotriene-induced vasculature contraction inhibited non-specifically. REF 26731 In vitro LTD4 binding. Ability of iralukast to displace LTD 4 from its receptor, using guinea pig lung membrane preparations. Iralukast displaced 1 nM LTD4 with IC50 of 60 nM (Ki = 26 nM). 194318 In vitro Smooth muscle relaxant. Antagonist properties of iralukast on LT contractions of guinea pig ileum. IC50 value for iralukast on LTD4 (2.7 nM) LTE4 (7.9 nM) at 2 min incubation and at 15 min incubation LTD4 (0.4 nM) LTE4 (0.36 nM). 194318 In vivo LTD4-induced bronchoconstriction. Effect of aerosol administration of iralukast on LTD4 challenge. Iralukast is an effective and potent LTD 4 antagonist. 194318 Associated patent EP-00419411 (Ciba-Geigy AG) Abstract Novel alkanophenone derivatives possessing leukotriene antagonistic and phospholipase A2 inhibitory activities are disclosed. The compounds are useful for treating various inflammatory diseases, including contact dermatitis, asthma and allergies. Protocols for the in vitro evaluation of the compound's leukotriene D4 antagonist activity and phospholipase A2 and phospholipase C inhibitory activity are disclosed. Thirty two compounds are specifically claimed, but no specific data are given. Title Additional alkanophenones. Indication Allergy, Asthma, Dermatitis, Inflammation Action LT antagonist, LTD4 antagonist, PLA2 inhibitor Publication EP-00419411-A2 27-MAR-91 Priority CH-00003402 19-SEP-89 Inventors Von Sprecher,A; Schaub,B; Lang,RW. Designated States AT BE CH DE DK ES FR GB GR IT LI LU NL SE Bibliography 11985 CGP 45715 A: a leukotriene D4 analogue with potent peptido-LT antagonist activity. Bray MA; Anderson WH; Subramanian N; Niederhauser U; Kuhn M; Erard M; von Sprecher A ADV PROSTAGLANDIN THROMBOXANE LEUKOT RES 1991 21B 503-507 148117 A leukotriene (LT) analogue with potent, long lasting, peptido-LT (pLT) antagonist activity in vivo. Bray MA; Anderson GP; Erard E; Kuhn M; Niederhauser U; Reisbrodt P; Rordorf C; Sills M; Stalder R; et al AM REV RESPIR DIS 1992 145 A284 26731 Effect of ICI 198,615, SK+F 104,353, MK-571 and CGP45715A on cysteinyl leukotriene-induced responses in guinea-pig heart. McLeod JD, Piper PJ PROSTAGLANDINS 1991 41 395-406 • Reports on effects of iralukasts non-specific actions on LTC4 and LTD4-induced responses in guinea pig isolated heart. 148119 The effects of a leukotriene (LT) D4 receptor antagonist (CGP 45715A) on LTD4 and antigen-induced responses in allergic sheep. Ahmed A; Cortes A; Sielczak MW; Abraham WM AM REV RESPIR DIS 1992 145 A288 150 IDrugs 1998 Vol 1 No 1 162584 Peptidoleukotriene antagonists; state of development with special emphasis on the ciba development compounds. Gerspacher M, Von Sprecher A, Beck A EUR MED CHEM CONF 1993 2 L4 168509 CGP-45715A. Iralukast Sodium. DRUGS FUTURE 1994 19 9 872 177071 Ciba targets new products. SCRIP 1995 2023 13 • Report on Ciba (Novartis) pipeline as of 1995. 194180 Leukotrienes as therapeutic target in asthma. Pauwels A, Joos F, Kips JC ALLERGY EUR J ALLERGY CLIN IMMUNOL 1995 50 8 615-622 194318 Peptidoleukotriene antagonists: structural analogs of leukotriene D4 with special emphasis on CGP 45715A. Sprecher A Von, Beck A, Sallmann A, Breitenstein W, Wiestner H, Kimmel S, Anderson GP, Subramanian N, Bray MA DRUGS FUTURE 1991 16 9 827-843 • Report on synthesis and structure-activity relationships of synthetic leukotriene antagonist drugs based on LTD4. 203429 Novartis - Backgrounder. BROCHURE 1996 March 27 Novartis COMPANY 268059 Leukotrienes as therapeutic target in asthma Pauwels A, Joos F, Kips JC ALLERGY 1995 50 8 615-622 272161 Information update DRUGS FUTURE 1997 22 9 10271061 • Reports on the only phase II trial using iralukast. 272174 [Leukotrienes antagonists: their interest in asthma]. Les anti-leucotrienes: leur positionnement dans l’asthme. Devillier P, Millart H, Advenier C REV MED BRUX 1997 18 4 279-285 274603 Samuelsson B, Paoletti R, Ramwell PW ADV PROSTAGLANDIN THROMBOXANE LEUKOT RES 1987 17 519-525 • Reports on the importance of methyl substitution of the carboxylic group in the eicosanoid backbone of LTD4 to produce leukotriene antagonist properties. 274604 Von Sprecher et al ANN NY ACAD SCI 1988 524 438-441 • Shows the importance of replacing the cysteinyl-glycine dipeptide sulfur side chain with a chromone carboxylic acid to confer chemical stability. 274605 Samuelsson B, Wong PYK, Sun FF ADV PROSTAGLANDIN THROMBOXANE LEUKOT RES 1988 19 647-650 • Shows the importance of stabilizing the polar region of the eicosanoid backbone of an LTD4 antagonist by phenyl binding. 207193 Novartis’ "new skills". SCRIP 1996 2111 7 275902 Zeneca ANALYST REPORT 19974 November 212858 Agents for the treatment of asthma: patent analysis 1990-1995. Mlodzik H EXP OPIN THER PAT 1996 6 1 57-60 • Reports on possible commercial viability and market potential of agents used for the treatment of asthma. 215839 Modulators of leukotriene biosynthesis and receptor activation. Brooks CDW, Summers JB J MED CHEM 1996 39 14 2629-2654 220836 Rhone-Poulenc Rorer to launch 18 new products before 2000. SCRIP 1996 2168 6 244117 Update of selected products in clinical trials. Novartis AG COMPANY COMMUNICATION 1997 April 29 251162 Pharmacological characteristics of leukotriene antagonists. Nicosia S MONALDI ARCH CHEST DIS 1996 51 6 556 257897 The action of the peptidoleukotriene, LTD4, on intracellular calcium in rat mesangial cells. Ochsner M EXPERIENTIA 1996 52 9 856-864 257899 Protective effect of inhaled iralukast (a new LTD4 antagonist) on exercise-induced bronchospasms. Djaballah K, Dessanges JF, Patalano F, Lockhart A EUR RESPIR J SUPPL 1996 9 23 272S 257900 CGP-45715A. Iralukast sodium. DRUGS FUTURE 1994 19 9 872 257901 45715a, A leukotriene LT analogue with potent long lasting peptido-LT PLT antagonist activity in vivo. Bray MA, Anderson GP, Erard E, Kuhn M, Niederhauser U, Reisbrodt P, Rordorf C, Sills M, Stalder R, et al AM REV RESPIR DIS 1992 145 4 PART 2 A284 257904 Iralukast sodium, CGP-45715A. DRUGS FUTURE 1995 20 9 957 278113 LY-191704 inhibits type I steroid 5-α-reductase in human scalp. Neubauer BL, Gray HM, Hanke CW, Hirsch KS, Hsiao KC, Jones CD, Kumar MV, Lawhorn DE, Lindzey J, McQuaid L, Tindall DJ, Toomey RE, Yao RC, Audia JE J CLIN ENDOCRINOL METAB 1996 81 6 2055-2060 281980 Pharmacological characterization of the cysteinylleukotriene antagonists, CGP- 45715A (iralukast) and CGP57698 in human airways in vitro. Capra V, Bolla M, Belloni PA, Mezzetti M, Folco MC, Nicosia S, Rovati GE BR J PHARMACOL 1998 123 3 590-598 281982 Montelukast sodium. Antiallergic/antiasthmatic leukotriene CysLT1 antagonist. Graul A, Martin L, Castaner J DRUGS FUTURE 1997 22 10 1103-1111 281983 Quantitative determination of iralukast in human plasma using LC-MS-MS. Linberg L, Melamed D PHARM RES 1997 14 (11 SUPPL) S677 281984 Leukotrienes: pathophysiologic role and therapeutic potentials in asthma. Sanico AM, Togias A ALLERGY ASTHMA PROC 1996 17 6 331-334 281985 CGP-45715A. Iralukast sodium. DRUGS FUTURE 1994 19 9 872. 281986 CGP-45715A. A leukotriene LT analogue with potent long lasting peptido-LT PLT antagonist activity in vivo. Bray MA, Anderson GP, Erard E, Kuhn M, Niederhauser U, Reisbrodt P, Rordorf C, Sills M, Stalder R, et al AM REV RESPIR DIS 1992 145 4 PART 2 A284 281987 Peptidoleukotrine antagonists: structural analogs of leukotriene D4 with special emphasis on CGP-45715A. Von Sprecher A, Beck A, Sallmann A, Breitenstein W, Wiestner H, Kimmel S, Anderson GP, Subramanian N, Bray MA DRUGS FUTURE 1991 16 9 827-843 Drug evaluation Iralukast 151 281989 Pharmacological and molecular evidence for the expression of the two steroid 5-α-reductase isozymes in normal and hyperplastic human prostatic cells in culture. Berthaut I, Mestayer C, Portois MC, Cussenot O, Mowszowicz I PROSTATE 1997 32 3 155-163 152 IDrugs 1998 Vol 1 No 1 Epristeride SmithKline Beecham Sharath S Hegde Address Roche Bioscience Center for Biological Research Department of Urogenital Pharmacology 3401 Hillview Avenue Palo Alto CA 94304 USA IDrugs 1998 1(1):152-157 Current Drugs Ltd ISSN 1369-7056 Epristeride is a transition-state, noncompetitive steroid 5-αreductase inhibitor under development by SmithKline Beecham for the treatment of benign prostatic hyperplasia and acne. Phase III trials for prostatic hypertrophy with an oral formulation, have been initiated in the UK, the US and Japan [188478], [219166]. Drug name Epristeride Originator SmithKline Beecham plc Status Phase III clinical Indication Acne, Prostatic hypertrophy, Prostate disease Action α-reductase inhibitor CAS Androsta-3,5-diene-3-carboxylic acid, 17-[[(1,1dimethylethyl)amino]carbonyl]-, (17β)Registry No: 119169-78-7 Synonyms SKF-105657, Zariflo, ONO-9302, SKB-105657 O In healthy male volunteers, epristeride caused a reduction in serum dihydrotestosterone levels but serum testos-terone levels remained stable. In animal studies, it showed a similar potency to finasteride for the inhibition of 5-αreductase [164200]. Epristeride is licensed to Ono which has exclusive rights in Japan, South Korea and Taiwan. Recordati has comarketing rights for epristeride [162547], [162519]. Analysts at Yamaichi estimate that epristeride will be launched in Japan between 1999 and 2000 and peak annual sales have been predicted to be over ten billion Yen [216018]. Introduction Epristeride is a potent and selective non-competitive inhibitor of the Type 2 steroid, 5-α-reductase, the enzyme responsible for the conversion of testosterone to dihydrotestosterone. By suppressing the biosynthesis of dihydrotestosterone, epristeride has the ability to retard prostate growth and relieve bladder outlet obstruction in patients with benign prostatic hyperplasia. Other potential therapeutic uses of epristeride include the treatment of acne, prostate cancer and male pattern baldness. Synthesis and SAR Epristeride (17-β-(N-tert-butylcarbomyl)androsta-3,5-diene3-carboxylic acid) was identified from a series of steroid 5-αreductase inhibitors that belong to the 3-androstene-3carboxylic acid class. Structure-activity studies showed that 2 the high affinity is dependent on sp hybridization at C-2, C3 and C-4 [275886]. Furthermore, the negative charge supplied by the carboxylate function at C-3 is essential for activity in as much as reduction to the aldehyde or alcohol leads to a pronounced drop in inhibitory potency. Two synthetic routes of epristeride have been reported [199657]. The first route starts from methyl 3-oxoandrost-4ene-17-β-carboxylate, which is converted to epristeride in H N H H HO H O four steps, with a 44% overall yield. The second route starts from the commercially available 3-oxoandrost-4-en-17-betacarboxylic acid, which is converted to epristeride in two synthetic steps, with a 63% overall yield. Both routes have been employed to produce kilogram quantities of epristeride of high purity. Pharmacology Epristeride is a potent inhibitor of the human Type 2 steroid 5-α-reductase (Ki = 0.7 to 2 nM) but is a much weaker inhibitor of the human Type 1 isoform of steroid 5-αreductase (Ki = 400 to 450 nM) [164200]. Epristeride also inhibits the native form of the enzyme in human prostate (Ki = 2 to 15 nM), rat prostate (Ki = 10 to 20 nM), rat liver (Ki = 5 to 10 nM), rat epididymis (Ki = 2 to 4 nM) and monkey (cynomologus) prostate (Ki < 1 nM) [164200], [275888]. Epristeride is a non-competitive inhibitor of both isoforms of steroid 5-α-reductase by the formation of a ternary complex with NADP+ and the enzyme [164200]. It is a highly specific inhibitor of steroid 5-α-reductase since it has at least a 1000fold lower affinity for seven other steroid processing enzymes and five steroid hormone receptors [164200]. In rats and cynomologus monkeys, orally-administered single doses of epristeride produce significant and longlasting (>24 h after a single dose) reductions in plasma 5-αdihydrotestosterone levels and also prostatic 5-αdihydrotestosterone content, consistent with inhibition of Drug evaluation Epristeride 153 steroid 5-α-reductase [164198]. Chronic oral adminis-tration of epristeride (0.1, 1.0 or 10 mg/kg, bid, for six weeks) to mature rats resulted in significant decreases in prostatic 5-α-dihydrotestosterone content, which were associated with dose- and time-dependent decreases in mass of ventral prostate [164198]. Toxicology In castrated rats, epristeride (25 mg/kg, bid, po) reduced testosterone, but not 5-α-dihydrotestosterone. It induced increases in ventral prostate weight, prostatic secretion and glandular proliferation, which is consistent with the notion that the compound operates by inhibiting the conversion of testosterone to 5-α-dihydrotestosterone and is not an androgen receptor antagonist [199669]. Epristeride (25 or 50 mg/kg, bid, po) produced significant decreases in 5-αdihydrotestosterone content and inhibited tumor growth in rodents transplanted with androgen-responsive prostatic cancer cells (Dunning R-3327 G and PC-82) [199674]. Phase I In cultured stromal cells from benign, hyperplastic adult prostates, epristeride dose-dependently inhibited the proliferative response to testosterone, but had no effect on 5-adihydrotestosterone-induced growth or growth of androgenunresponsive, testosterone-treated cells [275889]. In these studies, upregulation of prostate-specific antigen secretion from epithelial cells by androgens was downregulated by epristeride in testosterone-treated cells. Furthermore, transforming growth factor-β-1 secretion was downregulated by testosterone treatment and this was reversed by epristeride. Metabolism The phamacokinetics of epristeride have been investigated in young and elderly healthy male subjects [275890], [275891]. Following oral dosing, peak plasma levels were usually attained around 1.5 to 4 h. Epristeride plasma concentrations decline in a biexponential fashion with secondary peaks usually evident around 24 h, following intravenous and oral dosing. The mean apparent termination half-life estimates were similar after intravenous and oral dosing and in the range of 24 to 27 h, which allows once-daily dosing. The mean plasma clearance and steadystate volume of distribution were 0.33 ml/min/kg and 0.54 l/kg, respectively. The mean absolute bioavailability was 93%. Epristeride was highly bound to plasma proteins (98.9%) and binding was concentration-dependent over the therapeutic range and was reduced in patients with liver disease [206422]. The pharmacokinetics of epristeride have been studied in benign prostatic hyperplasia patients after daily dosing (5, 20 and 80 mg) for 8 weeks [199659]. Mean trough concentrations were dose-proportional and steady-state levels were attained at the end of the first week. No evidence of drug accumulation were noted over the 8-week period. A good correlation was observed between steady-state drug levels and the reduction of 5-α-dihydro-testosterone levels in serum and prostate. 14 Preclinical studies in rats and dogs with [ C]epristeride have shown that the majority of radioactivity can be recovered in the bile, predominantly as the acyl glucoronide conjugate, whereas less than 0.5% was excreted in the urine [164198]. Enterohepatic circulation of the drug has also been demonstrated in animals. The data from toxicological studies with epristeride have not yet been reported. Clinical Development In single dose phase I studies, epristeride (median dose of 0.25 mg/kg), decreased serum 5-α-dihydrotestosterone levels by more than 50% in 86% of the patients. This effect persisted for more than 24 h [276064]. Two subsequent multi-dose phase I studies in healthy male subjects confirmed the biochemical efficacy of epristeride [275897]. In the first open label study, 40 subjects received ascending doses of 0.4, 2.0, 20, 80 or 160 mg/day. In the second placebo-controlled and double-blind study, 51 subjects were randomized to receive doses of 4, 10, 20 or 40 mg/day of epristeride or placebo. In both studies, epristeride produced suppression of serum 5-α-dihydrotestosterone levels at all doses, with the maximal effect being noted at about 12 h post-dose. Phase II Two double-blind, placebo-controlled phase II trials with a total of 96 men have evaluated the effects of epristeride (0.4 to 80 mg once daily for 10 to 14 days) in patients with benign prostatic hyperplasia prior to undergoing trans-urethral resection of the prostate [276075], [275898]. Epristeride at 80 mg daily produced significant lowering (74 to 78%) of prostatic 5-α-dihydrotestosterone levels when compared to placebo. In another study, 56 patients with benign prostatic hyperplasia were treated with placebo or epristeride (2, 10 or 80 mg) for 10 days. Levels of 5-α-dihydrotestosterone in serum and prostate decreased with increasing serum epristeride levels. Phase III No phase III data are currently available. Side-effects and Contraindications No side-effects and contraindications have yet been reported. Current Opinion Previous clinical studies with finasteride (Merck), the first selective steroid 5-α-reductase inhibitor, have shown that this therapeutic approach can confer symptomatic benefit, albeit modestly, to patients with benign prostatic hyperplasia. Like finasteride, epristeride is also a selective inhibitor of steroid 5α-reductase and reduces serum and prostatic levels of dihydrotestosterone in benign prostatic hyperplasia patients. Epristeride differs from finasteride in its mode of inhibition of steroid 5-α-reductase, ie, non-competitive as opposed to competitive. From a theoretical standpoint, the noncompetitive behavior of epristeride may be advantageous, since this would diminish the potential of excessive substrate (testosterone) surmounting the inhibitory effects of epristeride. From the clinical view, it is unclear whether the non-competitive effects of epristeride would augment the therapeutic efficacy of the drug. The data from large ongoing phase III trials are awaited to ascertain the therapeutic value and superiority of epristeride in comparison to other inhibitors of steroid 5-α-reductase. 154 IDrugs 1998 Vol 1 No 1 Licensing Ono Pharmaceutical Co Ltd A cross-licensing agreement exists between SmithKline Beecham and Ono Pharmaceuticals for the products, epristeride and pranlukast, respectively [167442]. Ono has the rights in Japan, South Korea and Taiwan. Recordati SpA Recordati has co-marketing rights for SmithKline Beecham’s epristeride and ropinirole in Italy and Spain. This is in exchange for rights given to SmithKline Beecham to develop and market Recordati’s highly-selective α-receptor antagonist compounds indicated for benign prostatic hypertrophy. This includes the lead compound, REC-15-2739 [162547], [162519]. Development history DEVELOPER COUNTRY STATUS INDICATION Ono Pharmaceutical Co Ltd Taiwan C2 Prostatic hypertrophy DATE REFERENCE 167442 Ono Pharmaceutical Co Ltd South Korea C2 Prostatic hypertrophy 167442 Recordati SpA Italy C3 Prostatic hypertrophy 162519 Recordati SpA Spain C3 Prostatic hypertrophy 162519 SmithKline Beecham plc UK C3 Prostatic hypertrophy 188421 SmithKline Beecham plc UK C? Acne SmithKline Beecham plc UK C3 Prostate disease SmithKline Beecham plc US C3 Prostate disease Ono Pharmaceutical Co Ltd Japan C3 Prostatic hypertrophy 203772 203772 11-SEP-96 219166 Literature classifications Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the bibliography. These headings are as follows: Chemistry: References which discuss synthesis and structure-activity relationships. Biology: References which disclose aspects of the drug’s pharmacology in animal models. Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether the experiment is placebo-controlled or double or single blind; number of patients; dosage. Metabolism: References that discuss metabolism, pharmacokinetics and toxicity. Chemistry STUDY TYPE RESULT REF SAR Studies provide data for identification of key pharmacophores for activity. 275886 Synthesis Describes two commercially-viable synthetic routes for epristeride. 199657 Biology STUDY TYPE EFFECT STUDIED EXPERIMENTAL MODEL RESULT REF In vitro Inhibitory potency against steroid 5-α-reductase. Recombinant human type I and II steroid 5-α-reductase; native steroid 5-α-reductase in human, monkey and rat tissues. Ki (Type I) = 0.7 to 15 nM; K i (Type II) = 400 to 450 nM. 164200 In vitro Proliferative response and prostate-specific antigen secretion and TGFβ-1 secretion. Cultured stromal and epithelial cells from benign hyperplastic adult prostates; 1 nM to 0.3 mM epristeride. 275889 In vivo Prostatic 5-α- dihydrotestosterone content, ventral prostate weight, prostate secretion and glandular proliferation. Castrated rats 25 mg/kg bid, po Inhibition of proliferative response to testosterone and upregulation of prostate-specific antigen secretion and TGFβ-1 secretion. Reduces testosterone, but not 5α- dihydrotestosterone-induced increase in ventral prostate weight, prostatic secretion and glandular proliferation. In vivo 5-α-dihydrotestosterone content and tumor growth. Rats transplanted with androgenresponsive prostate cancer cells (Dunning R-3327G and PC-82); epristeride 25 or 50 mg/kg bid, po. Decreases in 5-αdihydrotestosterone content and inhibition of tumor growth. 199669 199669 Drug evaluation Epristeride 155 Metabolism STUDY TYPE EFFECT STUDIED MODEL USED RESULT REF In vivo Clearance of [14C]epristeride. Rats and dogs. Majority of radioactivity recovered in bile, principally as acyl glucoronide, < 0.5% excreted in urine. 164198 Clinical EFFECT STUDIED Pharmacokinetics. MODEL USED Young and elderly male subjects. RESULT Pharmacokinetics. Patients with benign prostatic hyperplasia; 5, 20, 80 mg, qid, for 8 weeks. Dose-dependent increase in mean trough concentrations. Steady-state achieved in 1 week. No evidence of drug accumulation. 199659 Biochemical efficacy. Healthy male subjects (n = 40) in open label study. Ascending doses of 0.4, 2.0, 20, 80 or 160 mg/day. Decreases in serum 5-α-dihydrotestosterone levels. 275897 Biochemical efficacy. Healthy male subjects (n = 51) in double-blind, placebo-controlled study. 4, 10, 20 or 40 mg/day. Decreases in serum 5-α-dihydrotestosterone levels. 275897 Biochemical efficacy. Benign prostatic hyperplasia patients (n = 56) in double-blind, placebo-controlled study; 2, 10 or 80 mg/day for 10 days. Reduction in prostatic and serum 5-α-dihydrotestosterone levels. 275898 Biochemical efficacy. Benign prostatic hyperplasia patients (n = 96) in double-blind, placebo-controlled study; 0.4 to 80 mg/day for 10 to 14 days. Reduction in prostatic 5-α-dihydrotestosterone levels. 276075 Termination t1/2 = 24 to 27 h; plasma clearance = 0.33 ml/min/kg; steady state volume of distribution = 0.54 l/kg; bioavailability = 93%. REF 275890 Associated patent EP-00427434 (SmithKline Beecham) Abstract 11-Keto or 11-hydroxy 3,5-diene steroids are claimed as inhibitors of steroid 5-α-reductase. The compounds are useful in the treatment of androgenrelated diseases. The treatment of benign prostatic hypertrophy is indicated in one claim. Inhibition of human prostatic steroid 5-α-reductase is described for two compounds. 17β-(N,N-diisopropylcarboxamide)-11-oxoandrosta-3,5-diene-3-carboxylic acid had a Ki value of 30 nM while the corresponding 11β-hydroxy compound had a Ki value of 20 nM. Eight examples with details are given. Four compounds are claimed, including N,N-diisopropylcarboxamide-11-oxo-acid. Title 11-Keto or hydroxy 3,5-diene steroids as inhibitors of steroid 5-αreductase. Indication Prostatic hypertrophy Action Testosterone 5-α-reductase inhibitor, Androgen synthesis inhibition Publication EP-00427434-A2 15-MAY-91 Priority US-00430152 01-NOV-89 Inventors Holt,DA; Metcalf,BW; Levy,MA. Designated states AT BE CH DE DK ES FR GB GR IT LI LU NL SE Bibliography 158362 Cancer drug development: current research and patents 1992 - part 1. Bair KW CURR OPIN THER PAT 1993 3 6 695 - 742 162689 Steroid 5-α-reductase: Two genes/two enzymes. Russell DW, Wilson JD ANNU REV BIOCHEM 1994 63 25 - 61 162519 Licensing confirmation. SmithKline Beecham Pharmaceuticals COMPANY COMMUNICATION 1994 September 7 163327 5-α-Reductase inhibitors and prostatic Schroder FH CLIN ENDOCRINOL 1994 41 2 139 - 147 162547 SmithKline Beecham and Recordati announce codevelopment agreement. SmithKline Beecham Pharmaceuticals PRESS RELEASE 1993 November 23 164198 Steroid 5-α-reductase inhibitor treatment for benign prostatic hyperplasia. Audet PR; Baine NH; Benincosa LJ; Holt DA; Wier PJ; Rappaport EB; Metcalf BW; Levy MA DRUGS FUTURE 1994 19 7 646 - 650 162562 Licensing confirmation. Ono Pharmaceuticals COMPANY COMMUNICATION 1994 September 8 disease. 156 IDrugs 1998 Vol 1 No 1 164199 Synthesis of carbon-14 and tritiated steroidal 5-αreductase inhibitors. Shu AYL; Heys JR J LABEL COMPOUNDS RADIOPHARM 1994 34 7 587 - 596 199646 Treatment of prostatic hyperplasia with 4-α-reductase inhibitors. Stoner E, Guess H ENDOCRINOLOGIST 1995 5 2 140 - 146 164200 Epristeride is a selective and specific uncompetitive inhibitor of human steroid 5-α-reductase isoform 2. Levy MA, Brandt M, Sheedy KM, Dinh JT, Holt DA, Garrison LM, Bergsma DJ, Metcalf BW J STEROID BIOCHEM MOL BIOL 1994 48 2-3 197 - 206 • Provides definitive evidence for the ability of epristeride to produce potent and selective inhibition of Type 2 steroid 5-α-reductase in vitro. 199651 Some aspects of the biology and endocrinology of prostate cancer. Griffiths K, Harper ME, Peeling WB SCAND J CLIN LAB INVEST 1995 55 221 23 - 31 164201 5-α-reductase inhibitors and the treatment of benign prostatic hyperplasia. Isaacs JT DRUGS TODAY 1993 29 5 335 342 199657 Improved synthesis of epristide, a potent human 5-αreductase inhibitor. Baine NH, Owings FF, Kline DN, Resnick T, Ping L J, Fox M, Mewshaw RE, Tickner AM, Kowalski CJ J ORG CHEM 1994 59 20 5987 - 5989 • Describes the synthetic route for epristeride. 167442 Ono Pharmaceutical Co Ltd annual report 1993 171208 SB claims top R&D performance. SCRIP 1995 1992 12 13 • Comments by SB management on better cost/toxicity profile of SKF 107647 compared with CSFs. 172957 SmithKline Beecham plc. PHARMA BUSINESS 1995 1 123 - 129 176815 Sanofi information BROCHURE 1995 Spring meeting. SANOFI 199655 Benzophenone- and indolecarboxylic acids: Potent type-2 specific inhibitors of human steroid 5-α-reductase. Holt DA, Yamashita DS, Konialian-Beck AL, Luengo JI, Abell AD, Bergsma DJ, Brandt M, Levy MA J MED CHEM 1995 38 1 13 - 15 199658 Pharmacotherapy for benign prostatic hyperplasia. Narayan P, Indudhara R WEST J MED 1994 161 5 495 - 506 199659 Pharmacodynamic analysis of plasma epristeride concentrations and dihydrotestosterone levels in patients with benign prostatic hyperplasia. Benincosa LJ, Miller A, Knox S, Rappoport E, Morris R, Lamb Y PHARM RES 1993 10 10 Suppl S362 COMPANY 188421 Research and development review - SB Pharmaceuticals advanced development portfolio: Products in phase III clinical trials. SMITHKLINE BEECHAM COMPANY BROCHURE 1995 October 02 188478 Research and development review - SB Pharmaceuticals: Development portfolio by therapeutic area. SMITHKLINE BEECHAM COMPANY BROCHURE 1995 October 02 189127 SmithKline Beecham to file 13 new chemical entities and 8 vaccines over the next 3 years. SmithKline Beecham plc PRESS RELEASE 1995 October 06 189136 New drugs in the R&D Pipeline : Ono. PHARMA JPN 1995 1467 22 • Disclosure of ONO-1603 having reached phase II. 189187 Smithkline Beecham upbeat on R&D. SCRIP 1995 2066 14 199660 Normal-phase high performance liquid chromatographic determination of epristeride, a prostatic steroid 5-αreductase enzyme inhibitor, in human plasma. Boppana VK, Miller-Stein C, Rhodes GR J CHROMATOGR 1993 631 1-2 251 254 199667 Steroidal 5-α-reductase inhibitors: Patent activity July 1991 to September 1992. Combs DW CURR OPIN THER PAT 1992 2 11 1803 - 1805 199669 Prostatic involution in rats induced by a novel 5-αreductase inhibitor, SK&F-105657: role for testosterone in the androgenic response. Lamb JC, English H, Levandoski PL, Rhodes GR, Johnson RK, Isaacs JT ENDOCRINOLOGY 1992 130 2 685 - 694 • Provides convincing evidence that epristeride operates by inhibiting the conversion of testosterone to 5-α-dihydrotestosterone and not by antagonism of androgen receptors. 199671 Hormonal balance and the risk of prostatic cancer. Isaacs JT J CELL BIOCHEM 1992 50 Suppl H 107 - 108 189207 Ono Pharmaceutical Co Ltd Annual report 1995 March 192905 Recent progress in the pharmacotherapy of diseases of the lower urinary tract. Hieble JP, McCafferty GP, Naselsky DP, Bergsma DJ, Ruffolo RR Jr EUR J MED CHEM 1995 30 Suppl 269s - 298s 199625 Chemoprevention for prostate cancer. Brawer MK, Ellis WJ CANCER 1995 75 7 Suppl 1783 - 1789 199629 Epristeride. SKandF-105657. DRUGS FUTURE 1995 20 7 724 199638 Cloning, expressing and functional characterization of type 1 and type 2 steroid 5-α-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes. levy MA, Brandt M, Sheedy KM, Holt DA, Heaslip JI, Trill JJ, Ryan PJ, Morris RA, Gasrrison LM, Bergsma DJ J STEROID BIOCHEM MOL BIOL 1995 52 4 307 - 319 199674 Response of rat and human prostatic cancers to the novel 5-α-reductase inhibitor, SK&F 105657. lamb JC, Levy MA, Johnson RK, Isaacs JT PROSTATE 1992 21 1 15 - 34 199675 Finasteride. Testosterone-5-α-reductase inhibitor agent for treatment of BPH. DRUGS FUTURE 1991 16 11 996 - 1000 203772 Striving to make peoples lives better. SmithKline Beecham plc ANNUAL REPORT 1995 March 206422 Pharmacokinetic (PK) and protein binding (PB) of epristeride in liver disease patients LDP and healthy male subjects. Benincosa LJ, Thompson KA, Everitt DE, Oldham H, Patterson S, Jorkasky D, Bay MK, Schenkler S AM SOCIETY CLIN PHARMACOL THER 1996 97 Annu Meet March 20 - 22 PIII-52 206442 Recordati: A healthcare company. Recordati SA Chem & Pharm Co COMPANY BROCHURE 1996 March 30 Drug evaluation Epristeride 157 219166 New drugs in the R&D pipeline. 1515 18 PHARMA JPN 1996 255734 5-α-Reductase (5α-R) and androgenic responses in HOS-TE85 (TE85) human osteosarcoma cells. Leibovitch IY, Gygi CM, Goode RL, Dixon EP, Little SP, Sutkowski DM, Neubauer BL PROC AM ASSOC CANCER RES 1997 38 Abs 3863 261150 Effects of steroid 5-α-reductase inhibitor, ONO-9302, and anti-androgen allylestrenol on the prostatic growth, and plasma and prostatic hormone levels in rats. Yasuda N, Fujin K, Shiraji T, Nambu F, Kondo K JPN J PHARMACOL 1997 74 3 243 252 263452 Epristeride. DRUGS FUTURE 1997 22 7 789 275886 Potent inhibition of human steroid 5-α-reductase (EC 1.3..30) by 3-androstene-3-carboxylic acids. Metcalf BW, Holt DA, Levy MA, Erb JM, Heaslip JI, Brandt M, Oh HJ BIOORG CHEM 1989 17 372 - 376 275888 3-Phosphonic acid and 3-phosphonic acid steroids as inhibitors of steroid 5-α-reductase: Species comparison and mechanistic studies. Levy MA, Metcalf BW, Brandt M, Erb JM, Oh HJ, Heaslip JI, Yen HK, Razamus LW, Holt DA BIOORG CHEM 1991 19 245 - 260 275889 Effects of a new 5-α-reductase inhibitor (epristeride) on human prostate cell cultures. Robinson EJ, Collins AT, Robson CN, Neal DE PROSTATE 1997 32 4 259 - 265 275890 Pharmacokinetics and pharmacodynamics of SK&F 105657 in healthy elderly male subjects. Chapelsky MC, Nichols A, Jorkasky DK, Pue MA, Lundberg BS, Knox MS, Audet PR CLIN PHARMACOL THER 1992 51 154 275891 Pharmacokinetics and absolute bioavailability of epristeride in healthy male subjects. Benincosa LJ, Audet PR, Lundberg D, Zariffa N, Jorkasky DK BIOPHARM DRUG DISPOS 1996 17 3 249 - 258 275897 Effect of multiple doses of epristeride, a steroid 5-αreductase inhibitor on serum dihydrotestosterone (DHT) in older male subjects. Audet P, Nurcombe H, Lamb D, Jorkasky K, Lloyd-Davies A, Morris R J CLIN PHARMACOL 1993 53 231 275898 Effect of fourteen days treatment with epristeride, an uncompetitive 5-α-reductase inhibitor on serum and prostatic testosterone and dihydrotestosterone in men with benign prostate hyperplasia. Johnsonbaugh RE, Cohen BR, McCormack EM, George FW, Wilson JD J UROL 1993 149 432A • Provides preliminary evidence for the biochemical efficacy of epristeride in patients with benign prostatic hyperplasia. 276064 Hormonal effect of SK&F 105657, a 5-α-reductase inhibitor in normal healthy male subjects. Audet P, Ilson B, Jorkasky D 9TH INT CONGRESS ENDOCRINOLOGY 1992 30 Aug - 5 Sept Nice Abst 13.03.033 276075 Double-blind, placebo-controlled study to evaluate the pharmacodynamic effect of SK&F 105657 in patients with benign prostatic hypertrophy. Peeling WB, Abrams P, Ramsey JWA, et al 10TH CONGRESS EUROPEAN ASSOCIATION UROLOGY 1992 240 22 July - 25 July, Genoa 148 • Provides preliminary evidence for the biochemical efficacy of epristeride in patients with benign prostatic hyperplasia. 158 IDrugs 1998 Vol 1 No 1 Patent News Peter Steele Address Current Patents Limited Middlesex House 34-42 Cleveland Street London W1P 6LB UK Email: [email protected] IDrugs 1998 1(1):158-161 © Current Drugs Ltd ISSN 1369-7056 Schering-Plough If there was a patent award, then one of the winners might well be the nine-part invention from Schering-Plough Corp, published as WO-09811091 to WO-09811093, WO-09811096 to WO-09811100 and WO-09811106. Dr Karl Thomae GmbH, however, is also a strong contender, with 75 pages of claims in WO-09811128 to libraries of neurotransmitter modulators, the complete specification extending to almost 500 pages. Schering-Plough’s invention relates to farnesyl protein transferase (FPTase) inhibitors, and the tricyclic compounds belong to a series which the company has been investigating for some time. Several code-numbered compounds of this type, including Sch-54419, have already been the subject of preclinical studies. FPTase inhibitors have become a very popular target for the industry, and more than 20 such agents are currently undergoing preclinical investigations. Schering-Plough’s major commit-ment to this field is clearly yielding a rich supply of promising compounds, although both Merck & Co Inc and Warner-Lambert Co are also known to have located several interesting leads. ICE/CED-3 from IDUN Three substantial applications from IDUN Pharmaceuticals Inc, WO-09810778, WO-09811109 and WO-09811129, relate to inhibitors of the interleukin-1β-converting enzyme CED-3 family. There are two distinct strands to this work, involving indoles on the one hand and peri-fused tricyclics on the other. The indoles are shown to act as apoptosis inhibitors, with potential in transplantation as a result of their effectiveness in expanding and prolonging survival of hematopoietic cell populations. IDUN’s work on apoptosis inhibition has attracted long-term support from Novartis AG, and these compounds may well fall within the terms of that agreement. The tricyclics too are linked to the work of Novartis, since there is a very close structural similarity with CGS-28106, an azepino[3,2,1-hi]indole in which dual ACE/NEP inhibitory activity is being studied. NIH NIH is commonly understood to denote the National Institutes of Health, but in management-speak stands for "not invented here", a syndrome afflicting chauvinistic R&D personnel. There are several examples this month of inventions which appear to build on other companies’ earlier discoveries, which is precisely the way in which the patent system is intended to work. Servier, for example, patenting as ADIR & Co, claims PDE-IV inhibitors in EP00831090 which are clearly derived from rolipram, Schering AG’s phase III antidepressant. Likewise Astra AB, a company with no background in protein kinase C inhibitors, is now seen to be following the lead of Eli Lilly & Co, a company with several such compounds in active development. The bis-indolyl compounds claimed by Astra in WO-09811102, WO-09811103 and WO-09811105 are structurally similar to LY-333351; the latter, though still only at the preclinical stage, has been the subject of several Lilly process and use cases in recent months, which seems to indicate that intensive development activity is in progress. The antihyperproliferatives claimed by Celltech Group plc in WO-09811095 also act on PKC, but in addition are described as inhibitors of the p56lck and ZAP-70 kinases. The 2-pyrimidineamine template adopted by Celltech for this project has already been used by Novartis as the basis for such candidates as CGS-53716 and CGS-57148, which act as inhibitors of tyrosine kinase. Cross-site developments A sure sign of precommercial activity is the patenting of secondary inventions by inventors located at two or more corporate sites. An example this month is WO-09810762, in which workers at SmithKline Beecham plc’s (SB) Collegeville, Pennsylvania site join forces with those in the UK to claim a controlled release form of the azabicyclooctyl acetonitrile SB-202026; this muscarinic agonist, in phase III trials in Alzheimer’s disease, has now been assigned the approved name sabcomeline, and seems destined to become an important new product for SB within a year or two. Another Pennsylvania-based team, this time Rhone-Poulenc Rorer Inc (RPR), is collaborating with colleagues in France on the development of chiral syntheses of ethylamine derivatives. From an inspection of RPR’s pipeline, it becomes clear that the optically active intermediates claimed in WO-09811064 are intended for adenosine receptor modulators such as RPR-100579, a cardiovascular agent which is already progressing well in clinical trials. Sanofi Recherche SA’s WO-09811053 is also concerned with chiral synthesis, though without input from abroad; the claimed benzyl alcohol derivatives could be used in the synthesis of SR-58611A, a spasmolytic atypical β3 agonist which is in phase II trials. Another SB invention, WO-09811067, relates to the use of SB-207266A in irritable bowel syndrome; the 5HT4 antagonist is in phase III trials for this indication. In WO-09810782, Merck & Co claims various combinations of pneumocandins with other antifungals, naming such compounds as ER-30346, Sch-56592, CAN-296 and XMP-97. Most of Merck’s antifungal development effort seems to be devoted to echinocandins rather than pneumocandins, and it is not clear which, if any, of the latter are in active development. Several of the other antifungals, on the other hand, are progressing well; Eisai Co Ltd’s ER-30346, for example, is reported to be licensed to Bristol Myers Squibb Co, and the Schering-Plough compound, already the subject of prodrug patenting, has reached phase II trials. There can be no doubt that secondary patenting of this type, though Patent News 159 not necessarily of great scientific interest, gives advanced warning of future commercial activity, if placed in context. It is no coincidence that several of the candidates mentioned above are regarded as leading compounds in their respective classes. York, describes how as many as 108 compounds might be prepared by light-directed synthesis on the surface of a single 5½" disc, and then assayed in a similar fashion. Not the least interesting aspect of this specification is the prior art review, covering more than a dozen seminal references in this emerging field. Se and SAM The rise of selenium as a therapeutic element continues with the publication of WO-09811122, in which Nutramax Laboratories Inc of Maryland claims nutritive compositions consisting of selenium and S-adenosylmethionine; optional additional ingredients in this concoction include vitamin B 12 and, rather improbably, cyanide. The efficacious properties of this nutritional supplement are associated with leukotriene B4 antagonist activity. Crystal forms Eli Lilly & Co’s antipsychotic, olanzapine, is the subject of WO-09811893, in which a novel physical form known as dihydrate D is claimed. Patenting of this type is now common, but the timing of this particular invention is interesting. Registration submissions were deposited towards the end of 1995, and were followed a year later by the first product launches. The present application was filed initially in September 1996, which seems to indicate that this work on crystal forms was taking place as registration was pending, and that possibly the active ingredient was switched to the dihydrate D form immediately prior to launch. Olanzapine, acting by a combination of 5-HT2 and dopamine antagonist mechanisms, is regarded as a leading product in its class, and would normally enjoy patent protection until the year 2011, based on claims to the novel chemical entity. These claims to the new physical form should in effect extend that protection until 2017. Meiji Seika Kaisha Ltd is seen to be adopting a similar tactic in WO09812200, although in this instance the need for extended protection is more pressing, The subject is a crystalline form of the antibacterial cefditoren pivoxil, which was launched in Japan in 1994 as Meiact, but which is still in clinical trials elsewhere with licensees such as Grüenenthal GmbH and Abbott Laboratories. Basic patent protection for cefditoren is due to begin expiring in 2005, so that these licensees will be heavily dependent on the reinforced protection which Meiji Seika is now securing. CD-ROM chemistry and signatures One of the more intriguing aspects of pharmaceutical patenting is the occurrence of interdisciplinary inventions, relating to subjects which cannot be fitted neatly into a single location in the International Patent Classification (IPC). One such case is WO-09812354, classed under nucleic acids (C07h), microbiological processes (C12q) and computers (G06f). The invention claimed by Affymetrix Inc is a technique for identifying molecular sequence signatures, clearly of relevance to the discovery of gene therapy products. In contrast, WO-09812559 is assigned to only a single IPC, G01n-33, which is concerned with analysis of biological materials. However, the invention is unmistakably multidisciplinary, since it relates to the use of a CDROM format for spatially addressable combinatorial chemical arrays. The inventor, James P Demers of New Alcohol manufacture Two companies are claiming processes leading to alcohols, and though the inventions are themselves relatively unspectacular, they indicate that vigorous development activity is under way. Merck & Co Inc's WO-09812340 claims the reduction of bisaryl ketones to the corresponding alcohols, the aim being a chiral product useful in the synthesis of PDE-IV inhibitors. This is a class of compound not strongly represented in the company's extensive development portfolio, although some previous patenting of bisphenyl ethanes has been noted. This lack of correlation is explained by the fact that Merck had licensed-in CDP-840 from Celltech Group plc; that collaboration ceased early in 1996, when the candidate was in phase II trials, but the present process patenting seems to indicate that Merck retains an interest in this class of compound. Further intelligence on this subject appears in WO-09812178, in which a different Merck team claims bisarylethanes bearing a 4-pyridyl substituent, as in CDP-840. The accumulated evidence suggests that Merck is now actively developing an antiasthmatic acting by PDE-IV inhibition, either CDP-840 itself or a close analog. Chiral alcohols are also the subject of WO09812155, in which Shionogi & Co Ltd claims a reduction process employing asymmetric boranes. This process seems to have broad applicability, although one of the suggested target structures is saphingol, Lilly's phase II PKC inhibitor. A similar ambiguity exists in WO-09812197, since the target molecule, duocarmycin SA, is associated with Kyowa Hakko Co Ltd, rather than with the joint applicants, Kyorin Pharmaceutical Co Ltd and the Sagami Institute. HIP hedgehog and tub genes Hedgehog proteins are known to have therapeutic potential in a range of CNS and metabolic diseases, and are being studied in several institutions. Proteins from the Indian, Desert and Sonic hedgehogs, for example, form part of a project at Ontogeny Inc, under which both Biogen Inc and Boehringer Mannheim GmbH are now licensees. Much of the work which underpins this progress has however been carried out in academic institutions, including Columbia University and Johns Hopkins University. Harvard College has been involved too, and now in WO-09812326 claims HIP-1, the hedgehog-interacting protein-1; HIPs show high affinity for hedgehog binding protein. This again is a conspicuously eclectic invention in terms of the technologies upon which it draws, being placed in no fewer than six distinct subclasses of the IPC. A similarly broadly-based invention, Millennium Pharmaceuticals Inc's WO-09812302, is concerned with tub and fat, loci on the ob gene implicated in the development of obesity. Genetic approaches to obesity control are now being widely studied, by companies such as Lilly, Amgen Inc, Amylin Pharmaceuticals Inc and Ligand Pharmaceuticals Inc. Millennium's own involvement has attracted the attention of Roche Holding AG, now 160 IDrugs 1998 Vol 1 No 1 collaborating on the development of targeted gene products. Another company to have studied the tub gene is AxyS Pharmaceuticals Inc, newly formed by the merger of Arris Pharmaceutical Corp and Sequana Therapeutics. β2-Agonist in labor β2-Agonists might be thought of as a rather unfashionable research field, since the pioneering products with this selectivity, salbutamol and ritodrine, were first synthesized more than 30 years ago. Much of the development work currently being carried out on these compounds is concerned with formulations and compositions, principally in the context of asthma therapy. However, the primary indication for ritodrine has always been control of premature labor, and from WO-09813035 it is clear that, in Japan at least, improved agents for this indication are still being sought. The applicant, Hokuriku Seiyaku Co Ltd, has enlisted the help of Nitto Denko Corp, a specialist manu-facturer of transdermal tape, and is claiming a percutaneous formulation of HSR-81. This compound, a close analog of the anti-asthma agent tulobuterol, is in phase III trials for control of labor, and has been licensed to Tokyo Tanabe Co Ltd. Another Japanese company, Kissei Pharmaceutical Co Ltd, is also reported to be developing a novel β2-agonist for this indication, although it is apparently still at the preclinical stage. Kissei is one of several companies to have licensed ritodrine from the originator, Solvay SA (originally Philips). Subepithelial turbidity Kissei attracts attention also in the context of subepithelial turbidity, a condition associated with corneal trauma. The active agent in WO-09813038 is an anthranilic acid derivative identifiable as tranilast, an anti-allergic agent first synthesized in the early 1970s, which has now begun to lose its basic patent protection. An ophthalmic solution for use in allergic conjunctivitis is already marketed, but Kissei clearly needs the strongest possible protection for any additional indications. SmithKline Beecham plc is reportedly studying the use of tranilast in stenosis, but for the ophthalmic indication Kissei’s partner is Nihon Tengan-yaku, a company with no previous history of international patenting, presumably an ophthalmics specialist; one of the inventors is known to have worked in this field previously, apparently with Fujisawa Pharmaceutical Co Ltd. Beecham, collaborating with the National Institutes of Health and the Virus Research Institute Inc, respectively. Now, according to WO-09813065, Merck & Co Inc has also adopted this target. One of the workers assigned to this project was previously working on fibroblast growth factor formulations, a topic now apparently abandoned in favor of rotavirus. NNRTIs from Du Pont Merck The antiviral research at Du Pont Merck Pharm Co’s Wilmington site is directed mainly towards HIV protease inhibitors. However, from WO-09814436, it is clear that there is now also interest in non-nucleoside type reverse transcriptase inhibitors, or NNRTIs. The company’s own research into such agents in the late 1980s yielded a lead which was designated DuP-925, but which seems not to have progressed beyond the preclinical stage. The present benzoxazinones resemble DMP-266, now known as efavirenz, in clinical development jointly with the originator, Merck & Co Inc. This candidate has progressed rapidly through the early stages of development, having first appeared in June 1994 when WO-09403440 was published. Demeter’s MIMs A rather younger company, Demeter Biotechnologies Ltd, had two applications published early in 1996 describing membrane interacting molecules (MIMs), WO-09603519 and WO-09603522. Now, in WO-09814201, further work on these MIMs is described, in the context of HIV therapy. Demeter’s entire product development pipeline is oriented around the concept of membrane permeability enhancement, but there is no evidence to date that even this ultra-specialized approach has attracted any licensing partners to support product development. The relatively small scale of Demeter’s operations is further emphasized by the naming of the company’s R&D VP as the sole inventor. From the previous inventions of this individual, however, it is possible to trace Demeter’s lytic peptides back to work on plant pathogens carried out in the mid 1980s in the Agricultural and Mechanical College of Louisiana State University. This rather unconventional background is confirmed by the naming of the US Department of Agriculture as joint applicant on one of the earlier Demeter cases mentioned previously. PGI2 agonists Oddly, although several dozen PGI2 agonists have been under development during the past decade or so, and a handful have matured into successful products, there seem to be virtually no candidates under investigation currently. Nevertheless, Takeda Chemical Industries Ltd is now entering the field, with claims in WO-09813356 to agents with high affinity for the receptor and useful platelet aggregation inhibitory action. The tricyclic templates on which these compounds are based include naphtho[1,2d]thiazole, a fragment seen previously in certain naturallyoccurring rifamycins. Merck’s rotavirus vaccine Rotavirus has attracted the attention of several companies, including American Home Products Corp and SmithKline NAALADase inhibitors from Guilford Baltimore-based Guilford Pharmaceuticals Inc filed no fewer than nine patent applications at the US Patent Office between September 1996 and June 1997 relating to inhibitors of NAALADase. This enzyme, N-acetylated-α-linked acid dipeptidase, is involved in the conversion of N-acetylaspartyl glutamate into glutamate, which results in presynaptic glutamate release. Compositions useful in the treatment of neurological disease, including compulsive disorders, are claimed in WO-09813044 and WO-09813046, following on from the publication of two related international applications at the end of 1997. This complex family of patents began to appear, however, in September 1997, when US-05672592 was granted with claims to novel phosphonomethylpentanedioic acid derivatives. At about the same time, Guilford announced that these NAALADase Patent News 161 inhibitors were showing promise in neurodegenerative disease and amyotrophic lateral sclerosis, naming GPI-5000 as the lead compound. Guilford has until now filed very few patent applications, and has focused on the enzyme rotamase; the inventor team working on NAALADase is relatively inexperienced too, the only possible exception being a chemist who may previously have been associated with a neurological project at Zeneca Group plc’s US site. A puzzling aspect of Guilford’s earlier patenting was the use of an agent in Turin; for the current substantial project, however, the services of a Washington-based attorney are being used. Merrell NMDA antagonists With only a brief diversion into IL-1 antagonists, a small team at the Merrell Pharmaceuticals Inc site in Cincinnati has been steadily researching NMDA antagonists for more than a decade. The latest compounds, claimed in WO09814427, are indole derivatives, and from the sheer consistency of Hoechst Marion Roussel Inc’s effort in this particular field, it seems likely that by now some promising compounds must have been identified. Astra AB’s remacemide, now in phase III trials, is among the more advanced NMDA modulators in development. ImmuLogic collaborations Vomeronasal organ targeted Pherin Pharmaceuticals of California is a small company specializing in vomeropherins, synthetic compounds acting on the vomeronasal organ, or VNO. Such agents are described in WO-09814194, a document of well over 500 pages, almost half of which are devoted to figures and diagrams. Vomeropherins have potential in the therapy of a range of conditions, including anxiety and prostate cancer; there is a suggestion that two distinct classes of compound are involved. Although the present patenting forms part of a series of cases going back to the early 1990s, it is only within the past year that a licensee, Organon NV, has been identified to support Pherin in the development of these pheromone-related compounds. A key inventor, associated with the company throughout this period, has also appeared on inventions from companies such as BioSource International Inc and Erox Corp. Unlike some of the smaller biotech companies, ImmuLogic Pharmaceutical Corp has always adopted a policy of vigorous patenting. Several dozen inventions have accumulated during the decade since the Massachusettsbased company was founded, and the high level of joint patenting suggests a very outgoing attitude towards collaboration, especially with non-industrial partners. Within the US there have been joint filings with North Carolina University, Leland Stanford University, and the New England Medical Center. However, there has also been collaboration with academics elsewhere, including teams based in Perth, Western Australia, and London. The latest addition to this interesting portfolio is WO-09814216, in which hapten-carrier conjugates for drug-abuse therapy are claimed. On this occasion there is no joint applicant, and the company seems not to have identified a development partner so far. Schering AG is known to be cooperating in the development of a potential multiple sclerosis therapy, and at one time Merck, ImmuLogic’s only industrial copatentee, was reported to have an interest in a candidate for use in transplant rejection. IDrugs – REQUEST FOR INFORMATION Fax back to Paul Baird on + 44 (0) 171 580 5646 IDrugs - the Investigational Drugs journal - is a new monthly review journal from Current Drugs. IDrugs brings you the most noteworthy information reported on investigational drugs in the preceding weeks. Particular emphasis will be placed on the rapid publication of information disclosed at scientific meetings, as well as a selection of expert evaluations of investigational drugs, reviews highlighting important issues in drug research and analysis taken from the Investigational Drugs database (IDdb). 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