Salute ed ambiente in Italia Istituto Superiore di Sanità 5 - 6 dicembre 2011 I NANOMATERIALI AL SERVIZIO DELLA SALUTE UMANA Agnese Molinari Dipartimento di Tecnologie e salute Nanomateriali al servizio della salute umana: “needs and challenges” Attività svolte nel Dipartimento di Tecnologie e salute LIMITATIONS HINDERING DRUG CLINICAL TRANSLATIONS AND SUCCESS -  Physico-chemical characteristic of the drugs (low water solubility: paclitaxel 0,0015 mg/ml; dexamethasone 0,1 mg/ml); - Biodistribution (1/10,000-1/100,000 drug molecules reach target site, high doses needed); - Narrow therapeutic window. THUS THERE IS THE NEED OF: -  Increasing drug stability; -  Increasing drug solubility; -  Targeting the drug to the site of action. Nanotechnology allows creation of platforms with: •  superior drug carrier capabilities •  selective responsiveness to the environment •  unique contrast enhancement profiles •  improved accumulation at the disease site. Nano-scaled materials and devices Imaging !  Nanoporous silica chips !  Smart hydrogel particles Diagnosis !  Nano-biosensors: !  Nanowires !  Micro- and nano-cantilever systems !  Superparamagnetic agents !  Metal nanoparticles !  Synthetic carbon-based nanoparticles !  Others: (Liposomes, Dendrimers, !  Polymer conjugates, bacteriophage, etc.) Therapy 1-100 nm Regenerative medicine !  Nanofibrous scaffolds !  Amphiphilic peptides !  Nanoparticles (spheres, capsules, liposomes, micelles, densrimers) !  Biodegradable polymers (PLA, PEG, PGA) !  Nano-sized drug crystals !  Cross-linked nanogels !  Carbon nanoparticles !  Gold nanoparticles !  Nanoshell !  Albumin nanoparticles !  Polymer. drug conjugates !  Liposomes !  Polymer micelles !  Nanoshelles !  Denrimers !  Immunopolymers, immunotoxins IMAGING Nanoparticle-based contrast agents. I Superparamagnetic agents Advantages: Metal nanoparticles !  high contrast !  tunable size and shape !  surface properties !  multiple functionalities !  long circulation times Synthetic carbon-based nanoparticles Others: liposomes, dendrimers, polymer conjugates Magnetic Resonance Imaging Positron emission tomography Single-photon emission computed tomography IMAGING Nanoparticle-based contrast agents. III (1) Preclinical development Superparamagnetic metal nanoparticles Composition Contrast source Target Indication Poly-L-lysine coated IO IO Mammalian cells Tracking of transplanted cells Antibody-targeted IO IO Her-2 Breast cancer Peptide/protein-targeted SPIO IO Clotted plasma proteins, MMP-2 Various tumors Radiolabeled antibody-targeted SPIO 111In, Membrane glycoproteins, EGFR-2 Various cancer Aptamer-doxorubicin SPIO conjugate IO, doxorubicin PSMA Prostate cancer Peptide-targeted USPIO IO αvβ3, E-selectin Various tumors, inflammation Antibody-targeted USPIO IO CD20 antigen, E-selectin Non-Hodkin’s lynphoma, inflammation Baculovirus-targeted USPIO IO, LacZ Mammalian cells Gene therapy Micelle-encapsulated MnSPIO IO Macrophages Liver lesions Antibody-targeted MnMEIO IO Her-2 Breast cancer Radiolabeled passive-targeted MnMEIO 124I, Lymph nodes Lymph node mapping Fluorescent CLIO IO, Cy5.5 Macrophages Macrophage infiltration Radiolabeled fluorescent CLIO 64Cu, Macrophages Macrophage infiltration Fluorescent peptide-targeted CLIO IO, Cy5.5/FITC Proteases, bombesin receptor, plectin, uMUC-1, hepsin, αvβ3, H-2Kd, VCAM-1, phosphatidylserine Various tumors, autoreactive Tcells, inflammation, apoptosis Fluorescent siRNA-CLIO conjugate IO, Cy5.5 Birc5 gene Various cancer IO, IRDye 800CW IO IO, Cy5.5 Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89 IMAGING Nanoparticle-based contrast agents. IV other metal nanoparticles (2) Preclinical development Composition Contrast source Target Indication Polymer-coated gold nanoshells Au Tumor accumulation Solid tumors Fluorescent passive-targeted gold Au, Hilyte 647 Tumor accumulation Solid Tumors Fluorescent antibody-targeted gold Au, ICG EGFR Epithelial cancer Antibody-targeted QD QD Her-2, PSMA, VEGFR Various tumors Growth factor-targeted QD QD EGFR Epithelial cancers Radiolabeled peptide-targeted QD 64Cu, Αvβ3, VEGFR Various cancer Protein-targeted paramagnetic QD Gd, QD Phosphatidylserine Apoptosis QD liposome-based nanoparticles (3) Preclinical development Composition Contrast source Target Indication Radiolabeled peptide-targeted liposomes 18F Macrophages Inflammation Gd, Texas red ICAM-1 Inflammation and neuroinflammatory disease 99mTc, Lymph nodes Lymph node identification, inflammation Antibody-targeted paramagnetic liposomes Radiolabeled, dye-filled liposomes blue dye Fluorescent protein-targeted paramagnetic liposomes Gd, AF680 Transferrin receptor, Eselectin Various cancers Electron dense liposomes Gd, AF680 - Blood pooling Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89 IMAGING - Nanoparticle-based contrast agents. V (4) Preclinical development synthetic carbon-based nanoparticles Composition Contrast source Target Indication Peptide-targeted SWNT SWNT Integrin αvβ3 Various cancers Gd-filled fullerenes, fullerenols, and SWNT Gd Macrophages Macrophage infiltration, blood pooling Radiolabeled antibody-targeted SWNT 111In, CD20 Lymphoma Radiolabeled peptide-targeted SWNT 64Cu, 111In, Integrin αvβ3, EGFR Various cancers Radiolabeled MWNT 99mTc, 125I - TBD SWNT SWNT Nanoparticle-based contrast agents in preclinical development (4): other platforms Composition Contrast source Target Indication Bismuth sulfide polyvinylpyrrolidone nanoparticles Bi - Blood pooling Radiolabeled hormone-targeted bacteriophage 111In MC-1 receptor Melanoma Ioxilan carbonate particles Iodine Macrophages Liver lesions Antibody-targeted paramagnetic perfluorocarbon emulsions Gd, 19F Fibrin, Integrin αvβ3, collagen III Atheroslerosis Radiolabeled amphiphillic block copolymers 64C Folate receptor Various cancer Iodinated amphiphillic block copolymers Iodine Macrophages Lymph lesions Fluorescent paramagnetic dendrimers Gd, Cy5.5 - Sentinal lymph node identification Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89 IMAGING Nanoparticle-based contrast agents. II Clinically approved nanoparticle-based contrast agents Composition Trade name Company Indication Administration Destran coated SPION (ferumoxides) Feridex I.V./Endorem Bayer Healthcare Pharmaceuticals, Inc. Detection and evaluation of liver lesions i.v. Carboxydextran-coated SPION (ferucarbotran) Resovist/Cliavist (EU, AUS, JPN only) Bayer Schering Pharma AG Detection and evaluation of liver lesions i.v. Silicon-coated SPION (ferumoxsil) GastroMARK Covidien, Ltd. Bowel marking Oral Nanoparticle-based contrast agents in clinical trials Trade name Company Indication Administration Combidex/Sinerem AMAG Pharmaceuticals, Inc. Differentiation of cancerous from noncancerous lymph nodes i.v. Carboxy dextran-coated USPIO (ferucarbotran) Supravist Bayer Schering Pharm AG Detection of blood pooling using MRA i.v. Polyglucose sorbitol carboxymethyl ethercoated SPIO (ferumoxytol) - AMAG Pharmaceuticals, Inc. Nervous system disease, brain neoplasms, peripheral artery disease i.v. Citrate-coated very small SPIO VSOP-C184 Charité-Universitätsmedizin Berlin Detection of blood pooling using MRA i.v. Radiolabeled-Her-2Affibody® ABY-025 Affibody Holding AB Breast cancer i.v. Composition Dextran-coated USPIO (ferumoxtran-10) Sakamoto et al., Enabling individualized therapy through nanotechnologyPharmacol Res 2010, 62:57-89 Theranostic SPIONs SuperParamagnetic Iron Oxide Nanoparticles Surface Engineering of Iron Oxide Nanoparticles for Targeted Cancer Therapy" FORREST M. KIEVIT AND MIQIN ZHANG*" Biological barriers. I Biological barriers. II ACCOUNTS OF CHEMICAL RESEARCH Vol. 44, No. 10 ’ 2011 ’ Enhanced permeability and retention effect EPR ACCOUNTS OF CHEMICAL RESEARCH Vol. 44, No. 10 ’ 2011 ’ Nano-based Injectable drug-delivery devices Functional taxonomy First generation: passive mechanisms (e.g. liposomes - EPR mechanism) Second generation: active mechanisms (e.g. m-Ab conjugated liposomes magnetic liposomes) Third generation: active mechanisms (multistage delivery system) Sakamoto et al. 2010 Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH MULTISTAGE NANOVECTORS (MSVs). I Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH MULTISTAGE NANOVECTORS (MSVs). II Multistage Nanovectors: From Concept to Novel Imaging Contrast Agents and Therapeutics Vol. 44, No. 10 ’ 2011 ’ 979–989 ’ ACCOUNTS OF CHEMICAL RESEARCH Nanomaterials for Drug Delivery EMEA approved Manomaterial Trade name Company Indication Current Status Caelyx Janssen Pharmaceutica Metastatic breast cancer Commercialized" (doxorubicin) Pegylated Liposomes Ovarian cancer Multiple myeloma anticancer AIDS-related Kaposis Mepact Pegylated Liposomes (mifamurtide) Mitsubishi Pharmaceutical, Japan immunomodulator Pegylated Liposomes High grade non metastatic osteosarcoma Commercialized" Myocet Cephalon Europe Metastatic breast Commercialized" Sistemi di drug delivery attualmente approvati cancer (doxorubicin) anticancer Nano-scale particles of the active substance Abraxane Nano-scale particles of the active substance Emend Nano-scale particles of the active substance Rapamune Celgene Europe Limited Metastatic breast cancer Commercialized" Merck Sharp & Dome Ltd Cancer Commercialized" Wyeth Lederle Rejection of transplanted kidney Commercialized" (paclitaxel) (aprepitant) Anti-emetic (sirolimus) FROM THE BENCH TO THE BED Design, characterization, production PHASE 1: Preclinical studies: Healthy subjects: Effects on body functions, dose definition, pharmacokinetics •  in vitro studies •  in vivo studies In vitro testing In vivo testing • Cytoxicity • Haematocompatibility • Drug release • Therapeutic efficacy Approval from FDA or EMEA General use Long-term benefit-risk evaluation • Organ specific toxicity • Immunogenicity • Intracellular fate PHASE 3: • Body distribution PHASE 2: Selected patients • Pharmacological activityEffect on disease: PHASE 2: Patient groups: Comparison with shandon therapy Safety efficacy dose pharmacokinetics NEEDS FOR SAFETY AND EFFICACY DEFINITION. I GENERALLY, IMPROVEMENT OF NON-CLINICAL METHODOLOGY TO AID DEFINITION OF LIKELY CLINICAL EFFICACY AND TOXICITY IS NEEDED Models should be developed to more closely correlates with the appropriate pathophysiology scenario present in the specifc, target clinical situation. Important considerations must be: • disease localization • disease progression • likely access to target tissues and cells • impact of angiogenesis (vascular permeability) • immune status (Gaspar and Duncan, Adv Drug Del Rev. 61: 1220-1231 - 2009) NEEDS FOR SAFETY AND EFFICACY DEFINITION. II SELECTION OF OPTIMAL PATIENT POPULATION TO ENTER CLINICAL TRIALS There is a need to identify those patients who are most likely to benefit from a novel therapy and to select the optimal patient population to enter clinical trials. INDIVIDUALIZED NANO-THERAPY Patient-specific molecular profiling allow the individuation of specific biomarkers useful to identify: • target specific site of disease • follow up pharmacological response • identify potential adverse reactions. (Gaspar and Duncan, Adv Drug Del Rev. 61: 1220-1231 - 2009) CONGRESS TOPIC DEVELOPING A NANOPARTICLE !Design, !Modelling, !Characterization FROM THE ADMINISTRATION TO THE TARGET SITE !Administration routes !Biological barrier !Immunological respons CLINICAL TRANSLATION OF NANODRUGS !Laboratory optimization !Pre-clinical safety evaluation !I/II/III phase clinical studies !Clinical successes !Industry reports Chairperson: Dr.ssa Giovanna Mancni CNR, Roma Dr.ssa Agnese Molinari Istituto Superiore di Sanità PROSSIMO EVENTO" Department of Technology and Health UNITS involved in Nanomaterials and Human Health Research Biomaterials and Contaminants Ultrastructural Infectious Pathology Ultrastructural Methods for Innovative Anticancer Therapies Risk Assessment Studies Department of Technology and Health CTR! ZnO 10 µg/cm2! ZnO 5 µg/cm2! TiO2 5 µg/cm2! Nanoparticles cultured cells toxicity on Relation between nanoparticles cytotoxicity and their physicochemical characteristics Effect of pre- and post-exposure to nanoparticles on viral and bacterial infections Biomedical applications Department of Technology and Health Nanoparticles-plasma membrane interaction: uptake and transport EXTRACELLULAR SPACE! LIPOSOME! Employment of Nanoparticles as selective drug carriers at the site of disease PROTOPLASMIC FRACTURE FACE! LIPOSOMES! CYTOPLASM! Possible use of Nanoparticles as antimicrobial agents Studies on implantable devices made or covered with nanomaterials: characterization of biomechanical alterations and potential surface performance Impiego di nanomateriali per terapie innovative antitumorali Reparto Metodi ultrastrutturali per terapie innovative antitumorali Dipartimento di Tecnologie e Salute Tumor markers Natural products ! BAF Device INNOVATIVE ANTICANCER THERAPIES Nanotechnology Collaborations Transmission electron microscopy 2 µ m ! Laser scanning confocal microscopy Scanning electron microscopy Impiego di liposomi cationici per la terapia fotodinamica del glioblastoma Reparto Metodi ultrastrutturali per terapie innovative antitumorali MECCANISMO DI AZIONE DELLA PDT SUI TUMORI REAZIONE DI TIPO I OH OH REAZIONE DI TIPO II * F STATO ECCITATO SINGOLETTO F H H N H N N H Radicali liberi SUBSTRATO N OH HH OH Meso-tetraidrossifenilclorina m-THPC, Foscan® (650-700 nm) * STATO FONDAMENTALE DI TRIPLETTO STATO ECCITATO TRIPLETTO 3O 2 NECROSI E APOPTOSI STATO FONDAMENTALE DI SINGOLETTO F STATO ECCITATO DI SINGOLETTO * 1O 2 CHIUSURA DEL MICROCIRCOLO INFIAMMAZIONE Reparto Metodi ultrastrutturali per terapie innovative antitumorali Impiego di liposomi cationici per la terapia fotodinamica del glioblastoma Dipartimento di Tecnologie e Salute Reparto Metodi ultrastrutturali per terapie innovative antitumorali L’EFFICACIA DELLA PTD PUO’ ESSERE MIGLIORATA USANDO DELLE FORMULAZIONI LIPOSOMICHE : "  Migliorano l’accumulo del fotosensibilizzante nei tumori "  Limitano la formazione di aggregati in soluzione acquosa dei aumentando la popolazione fotoattiva fotosensibilizzanti idrofobici, "  Possono influenzare in modo positivo la farmacocinetica ed il destino subcellulare del fotosensibilizzante I LIPOSOMI SONO VESCICOLE CHIUSE COSTITUITE DA UNO O PIU’ DOPPI STRATI DI FOSFOLIPIDI SEPARATI DA COMPARTIMENTI ACQUOSI. LA MICROGRAFIA ELETTRONICA RAPPRESENTA LIPOSOMI UNILAMELLARI OOSSERVATI 100 nm MEDIANTE LA TECNICA DEL FREEZE-FRACTURING. Dipartimento Tecnologie e salute - Istituto Superiore di Sanità - Roma DMPC/G1 m-THPC/DMPC m-THPC/DMPC/G1 Formulazioni! DMPC! G1! (DMPC + G1 =12,5 mM)! (%mol)! (%mol)! DMPC/G1! 60! 40! ---! m-THPC/DMPC! 100! ---! 50 µM! m-THPC/DMPC/G1(8:2)! 80! 20! 50 µM! m-THPC/DMPC/G1(7:3)! 70! 30! 50 µM! m-THPC/DMPC/G1(6:4)! 60! 40! 50 µM! m-THPC! Reparto Metodi ultrastrutturali per terapie innovative antitumorali ACCUMULO DI m-THPC CELLULE DI GLIOBLASTOMA DI RATTO (C6): confronto con il FOSCAN® 120 Canale medio di fluorescenza 100 80 DMPC/G1 6:4 m-THPC/DMPC Foscan 60 m-THPC/DMPC/G1 8:2 m-THPC/DMPC/G1 7:3 40 m-THPC/DMPC/G1 6:4 20 0 4 h! 1 h! 30 min! 30 min! 1 h! 4 h! DMPC/G1! 4.85 ± 0.5 5.61 ± 0.6 m-THPC/DMPC! 4.04 ± 0.66 4.01 ± 0.25 6.54 ± 0.67 Foscan! 3.21 ± 0.95 5.79 ± 0.39 14.29 ± 3.25 76.98 ± 6.38 4.79 ± 0.41 m-THPC/DMPC/G1 8:2! 35.66 ± 3.9 42.27 ± 4.53 m-THPC/DMPC/G1 7:3! 42.44 ± 3.87 58.24 ± 3.11 80.82 ± 3.47 m-THPC/DMPC/G1 6:4! 73.32 ± 4.83 90.13 ± 3.91! 105.53 ± 5.56 Foscan m-THPC/DMPC m-THPC/DMPC/G1 7:3 Liposomi+ LASER INTERSTIZIALE CITOTOSSICITA’ confronto con il FOSCAN® Test di clonogenicità CTR DMPC/G1 6:4 Foscan m-THPC/DMPC m-THPC/DMPC/G1 8:2 m-THPC/DMPC/G1 7:3 LN229 % 100! 90! 80! m-THPC/DMPC/G1 6:4 70! Frazione di sopravvivenza 60! 50! 40! FOSCAN + LASER INTERSTIZIALE 30! 20! 10! 0! CTR! DMPC/G1!FOSCAN! m-THPC/DMPC! 8:2! 7:3! 6:4! LN229+drugs+laser! 100! 100 ± 0.0 73.66 ± 7.243.33 ± 9.63.66 ± 1.180.02 ± 0.020.34 ± 0.5 LN229+drugs! 100! 100 ± 10 100 ± 20 100 ± 1.21 100 ± 12 100 ± 3 94.0 ± 4.6 LIPOSOMI/m-THPC+ LASER INTERSTIZIALE Lysozyme Microbubbles for magnetic resonance imaging and ultrasound triggered drug delivery " Reparto Metodi ultrastrutturali per terapie innovative antitumorali Ferritin Coating In vitro cytotoxicity test (MTT) In vitro cellular uptake studies in breast cancer cells (4 hrs) SKB3 Partecipanti all’attività Nanomateriali Dipartimento di Tecnologie e salute Ing. Velio Macellari Rossella Bedini Giuseppina Bozzuto Annarica Calcabrini Marisa Colone Maria Condello Giuseppe Formisano Magda Marchetti Stefania Meschini Agnese Molinari Annarita Stringaro Fabiana Superti Laura Toccacieli Collaboratori esterni Istituto di Neurochirurgia, Università Cattolica, Roma Giulio Maira Annunziato Mangiola Stefano Mannino Istituto di Metodologie Chimiche, CNR, Roma Cecilia Bombelli Paola Luciani Giovanna Mancini!