INVESTIGATOR’S BROCHURE BUTHIONINE SULFOXIMINE NSC 326231

INVESTIGATOR’S BROCHURE
BUTHIONINE SULFOXIMINE
NSC 326231
IND # 69,112
Initial Version for IND 32,981 June 1994
(Revised June 1996)
Division of Cancer Treatment
National Cancer Institute
Diagnosis and Centers
Bethesda, Maryland
Revised December 2003 Version 2.0
Developmental Therapeutics Program
USC-CHLA Institute for Pediatric Clinical Research
Childrens Hospital Los Angeles
Los Angeles, CA
This document contains both published and unpublished data describing the results of
preclinical studies with this agent. The unpublished data are preliminary and should not be
quoted or used for any purpose other than as guidance in developing clinical trials.
Table of Contents
Page
Summary
3
Introduction
5
Pharmaceutical Information
10
Mechanism of Action
12
Preclinical Activity
18
GSH Depletion and Effect on L-PAM Cytotoxicity
18
Tissue Selectivity of BSO-Mediated GSH Depletion
21
Tumor Cell Selectivity
23
Effect of BSO and L-PAM on Survival
27
BSO Sensitization of Agents Other Than L-PAM
31
Preclinical Toxicology and Pharmacology
36
Experience with BSO in Humans
50
Summary of Data and Guidance for the Investigator
56
References
58
Appendix
Attached
2
SUMMARY
Glutathione (GSH) is a ubiquitous intracellular tri-peptide that protects cells from
oxidative stress and plays an important role in detoxifying alkylating agents such as melphalan
(L-PAM) via dechlorination, quenching of DNA adducts, and conjugation reactions catalyzed by
glutathione S-transferase.1-3
L-S,R-buthionine sulfoximine (BSO), a specific inhibitor of γ-
glutamyl cysteine synthetase (the rate-limiting enzyme in GSH synthesis), depletes GSH in vitro
and in tumors in vivo, and BSO has been shown in preclinical
studies, to reverse alkylator
resistance.2; 4-7
BSO, as a single agent, is highly cytotoxic for neuroblastoma cell lines in vitro and
results in apoptosis due to increased generation of reactive oxygen species (ROS).8 ;
9
ROS that
are elaborated during catecholamine synthesis are the hypothesized etiology of this unique single
agent BSO activity which has not been reported for other pediatric tumors such as
rhabdomyosarcoma, Ewing’s Family Tumors, or retinoblastoma.
The in vitro combination of
BSO with clinically achievable (non-myeloablative) concentrations of L-PAM was highly
synergistic for neuroblastoma cell lines established at diagnosis or following non-myeloablative
therapy and led to the a recently completed pediatric pilot study (see appendix).10;
11
Preclinical
toxicology studies in animals showed no major toxicity at doses of BSO used in human clinical
trials.12; 13
Clinically, BSO has been given to adults, alone and in combination with L-PAM.
The
most typically used dosing schedule has been a loading dose of 3 g/m2 bolus over 30 minutes i.v.
followed by a 72 hour continuous infusion of 0.75 g/m2 /hr and L-PAM (15 mg/m2 bolus at hour
3
48 of BSO infusion).
A pilot study in pediatrics tested the same schedule and also tested
increasing the c.i.v. for BSO to 1.0 g/m2 /hr.10; 11
The reported systemic toxicities of BSO and L-PAM in adults were unremarkable with
reversible myelosuppression being the most common.14-16
Documented responses have occurred
in patients with melanoma (complete response 18 months), ovarian cancer, breast cancer, and
small cell carcinoma of the lung.14-16 A pilot study of BSO combined with 15 mg/m2 of L-PAM
in children with recurrent/refractory high-risk neuroblastoma achieved a 29% response rate
(duration 1-3 months), with 7 partial (6 of 7 had previous myeloablative therapy) and 2 minor
response (Preprint in Appendix).10;
11
Toxicity in the pediatric study was similar to that in adults,
with most patients showing grade 3-4 leukopenia and thrombocytopenia.
The degree of
hematopoietic toxicity seen with BSO + L-PAM appears to be increased over what would be
expected for L-PAM alone at the same dose. In contrast to the adult studies, there were 2 deaths
due to CNS toxicity in the first pediatric study of BSO + L-PAM.
Both patients having a toxic
death presented with acute tubular necrosis; one had a large, intracranial mass.
No other
significant toxicities were seen. In addition to the possibility of the CNS mass pre-disposing to
toxicity from BSO + L-PAM, there is the possibility that a drug interaction between BSO and
cephalosporin antibiotics could lead to BSO toxicity (preprint in appendix).11
Thus, patients
receiving BSO should not be given cephalosporin antibiotics, or any agent known to deplete
GSH, such as acetaminophen.
The t½ of BSO is ~ 2.3 hours and BSO may decrease the
clearance of L-PAM, potentially increasing the L-PAM AUC.
Depletion of GSH by BSO in
blood mononuclear cells occurred to 30-40% of baseline in adults and ~46% in children, and
appeared to return to normal within 4 days of stopping the BSO.
4
INTRODUCTION
DL-Buthionine-S, R-sulfoximine (BSO) is an analog of methionine sulfoximine (MSO),
which was identified as the toxic agent in agonized grain.
synthetase and
MSO inhibits both glutamine
γ-glutamylcysteine synthetase (γ-GCS), enzymes which participate in the
synthesis of glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH).
GSH is a ubiquitous tripeptide
found in mammalian cells and in other animal and plant cells, as well as in prokaryotic cells.
GSH is the major intracellular nonprotein sulfhydryl and is involved in a variety of metabolic
and physiologic functions, such as catalysis of enzymatic reactions, amino acid transport,
detoxification of xenobiotics, and normal cellular reductive processes.
GSH exists in millimolar
concentrations (0.5-10 mM) under normal steady-state conditions in the cell, but can also exist in
interchanging states as glutathione disulfide (GSSG), as part of mixed disulfides, or as a thiol
ester. Biosynthesis of GSH proceeds through two successive reactions which require ATP.
Although MSO was effective in depleting intracellular thiols when tested in vitro using
isolated synthetases, it did not possess the specificity required for in vivo studies and was also
inherently toxic.
Consequently, analogs of MSO, such as BSO and prothionine sulfoximine
(PSO), were synthesized and evaluated for specificity of GSH inhibition and activity both in
vitro and in vivo. Among the sulfoximine derivatives examined, BSO was the most specific and
least toxic.
BSO was noted to selectively bind to the active site of γ-GCS, the enzyme that
catalyzes the first of two steps in the synthesis of GSH. The synthesis of GSH and the structure
and site of action for BSO is shown in Figure 1.
5
BSO Inhibits Synthesis of Glutathione
γ-glutamylcysteine
synthase
GSH synthase
cysteine
γ-glu-cys
glutamine
CH3
CH2
H C CH2
H
BSO
NH
S O
(CH2)2
H C NH3
COO-
γ-glu-cys-gly
Glutathione
glycine
(GSH)
Figure 1
Once γ-GCS is inactivated by BSO, de novo GSH synthesis is inhibited. This results in
depletion of intracellular GSH, especially under conditions that promote enhanced GSH
utilization.
BSO has demonstrated tissue selectivity with respect to GSH depletion, and this
observation may be of clinical importance.
In an in vivo study in mice, GSH was maximally
depleted (70-89%) in certain tissues such as the liver and kidney and minimally depleted (40%)
in the bone marrow. This reduced effect on bone marrow is thought to be responsible for BSO’s
lack of myelosuppression.
Intracellular levels of GSH have been shown to modulate the toxicity of various
antineoplastic agents, such as cytotoxics and radiation therapy.
action of GSH, in this regard, is not fully understood.
However, the mechanism of
The following mechanisms have been
proposed: 1) GSH may detoxify antineoplastics such as melphalan (L-PAM) via conjugation; 2)
GSH may promote repair cellular injury resulting from the action of antineoplastics or radiation,
through its role in oxidative-reductive or nucleophilic reactions;
formation of active species which cause cellular injury;
4)
3) GSH may mediate the
GSH is known to decrease
intracellular reactive oxygen species and the latter promote the toxic effects of radiation and
some antineoplastic drugs or, 5) GSH may interfere with delivery or uptake of antineoplastic
agents through its role in amino acid transport.
6
GSH and drug resistance.
The hypothesis that thiols may be involved in cellular
response to alkylating agents was explored. In one study, an inverse relationship was observed
between the presence of protein and nonprotein sulfhydryls and the cytotoxicity of the alkylating
agent, merophan. This prompted further studies to determine whether the major nonprotein thiol
(GSH) plays a role in the modulation of the cytotoxicity of melphalan (L-PAM), an alkylating
agent widely used in clinical oncology.
Results of these studies suggested that tumor cells
resistant to L-PAM contained higher levels of GSH than those sensitive to L-PAM. Data from
these studies also demonstrated that nutritional deprivation of L-cysteine could reduce
intracellular GSH to an equivalent extent in both L1210 murine leukemia cells sensitive to LPAM and L1210R cells resistant to L-PAM, which resulted in re-sensitization of L1210R cells to
L-PAM to the same extent as L1210 cells grown under normal conditions.
Some reports have indicated that the mechanism of cellular resistance to L-PAM is
related to decreased L-PAM uptake.
However, other reports have shown no difference in L-
PAM uptake between L1210R or human ovarian cancer cells (1847me), both resistant L-PAM,
and their respective L-PAM sensitive parent lines. Both resistant lines had approximately a 2fold higher intrinsic level of GSH.
Data from these studies indicated that L-PAM was more
readily converted to its noncytotoxic dechlorinated dihydroxy metabolite in the resistant than in
the sensitive cells. Furthermore, the interaction between L-PAM and GSH was postulated to be
mediated by GSH-S-transferase which normally is involved in the dehalogenation of several
electrophiles. However, it is possible that this interaction may vary with cell type.
Despite the role of extracellular L-cysteine as a modulator of intracellular GSH,
nutritional deprivation of cysteine ws not considered a feasible therapeutic method to reduce
GSH levels in vivo.
Consequently, BSO was explored as a thiol-depleting agent. Results
indicated the GSH could be effectively depleted in vitro in L1210R cells at a minimally toxic
dose of BSO (50 µM), but that long exposure time (24 hours) was required for maximum
depletion. These findings were confirmed in an in vitro study in which BSO effectively reduced
GSH levels in both L-PAM resistant (1847me) and L-PAM-sensitive (A1847) human ovarian
cancer cells.
Similar results were reported in another study of human ovarian cancer cells
(NIH:OVCAR-3), which were derived from patients clinically resistant to alkylataing agents.
7
The levels of GSH in the OVCAR-3 cells were intrinsically 4-fold higher than those in cells from
untreated patients.
Treatments with a nontoxic dose (50 µM) of BSO reduced intracellular GSH
levels by 93.4% and decreased the inhibitory concentration (IC)50 of L-PAM by 73%, resulting
in a dose modification factor (DMF) of 3.6.
Despite the effectiveness of BSO in reducing intracellular GSH and increasing the
cytotoxicity of L-PAM in vitro, two earlier in vivo studies did not indicate a significant increase
in survival in mice bearing L1210R treated with BSO and L-PAM when compared to mice
treated with L-PAM alone.
However, in a recent study, a significant increase in survival was
observed in mice bearing clinically resistant OVCAR-3 cells when the animals were treated with
L-BSO and L-PAM.
The median survival time (MST) for the untreated mice was 46 days,
which increased to 68 days when the mice received L-PAM alone, and further increased to ≤ 125
days when L-BSO was administered prior to L-PAM.
Although BSO is being evaluated only with L-PAM in the initial clinical trials, BSO has
been shown in preclinical studies to increase the cytotoxicity of other cytotoxics, such as
carmustine (BCNU), cisplatin (CDDP), doxorubicin (DOX), and cyclophosphamide (CTX), and
radiosensitizers, such as SR-2508, misonidazole (MISO), and tirapazamine. BSO has also been
shown to increase radiation response, although it is unclear as to the conditions (normoxic or
hypoxic) required.
Treatment with BSO has shown promising activity in thermo sensitization
and inhibition of thermo tolerance. BSO has also been shown to enhance the anti-tumor activity
anti-neoplastics acting via a variety of mechanisms, such as the retinoid fenretinide,
dexamethasone, and arsenic trioxide in leukemia cell lines, and diethylstilbesterol in a prostate
cancer model.
Although modulation of cytotoxicity by BSO may present a promising
therapeutic approach, the tissue selectivity of BSO-mediated GSH depletion may be a limiting
factor in developing combination regimens which include BSO.
that BSO increases the renal toxicity of MeCCNU.
Some reports have indicated
Consequently, for maximum therapeutic
benefit, BSO may be best combined with agents whose principal toxicity is myelosuppression.
Preclinical toxicology.
Studies were conducted to determine the toxicity and
pharmacokinetic behavior of BSO in mice and dogs and to explore the effect of BSO on L-PAM
–induced toxicity in mice.
For the toxicity studies of BSO alone, CD2F1 mice were given
8
multiple oral (PO) doses of BSO 100-800 mg/kg/dose or multiple intravenous (IV) doses of 4001500 mg/kg/dose.
Beagle dogs received a single IV dose of BSO 120 or 3200 mg/kg/dose,
multiple IV doses of 50,120, and 240 mg/kg/dose (every 12 hours x 6 doses, or multiple PO
doses of 100-800 mg/kg/dose).
BSO was toxic to the gastrointestinal, urinary, and central nervous systems of beagle
dogs.
Possible hepatotoxicity was indicated by clinical pathology, but no treatment-related
microscopic lesions were found in the liver.
BSO was lethal to dogs.
At the highest single PO or IV doses evaluated,
BSO produced severe GSH depletion in the dogs; however, a
relationship could not be established between the magnitude of GSH depletion and BSO-induced
toxicity. With the multiple IV dose schedule, a slight decrease in weight was observed in one
dog at the 240 mg/kg/dose level. Minor hematologic effects were noted among dogs receiving
the 120 and 2340 mg/kg/dose schedule. Toxicity was reversible and was not sex-related in dogs.
In contrast, mice tolerated large multiple PO or IV doses of BSO up to 800 mg/kg/dose without
apparent toxicity, except for slight weight loss, and despite marked depletion of GSH levels.
Transient depression in white blood cell count was observed in female mice at the highest IV
dose level (1600 mg/kg/dose), but no bone marrow lesions were observed at necropsy.
Pretreatment with BSO up to 800 mg/kg/dose PO or IV did not significantly alter the toxicity of
L-PAM 5 mg/kg IV in mice. At high single IV doses (1600 mg/kg/dose), BSO potentitated LPAM-induced renal, bone marrow, and lymphoid toxicity in mice.
However, this potentiation
did not appear to be related to magnitude of GSH depletion.
Preclinical pharmacology.
Elimination of drug from the plasma appeared to be
biexponential in either dogs or mice given BSO 2400 mg/m2 PO or IV. Although the initial and
terminal half-lives (T ½) were similar in both species, the area under the plasma concentrations
vs. time curve (AUC) in the initial phase accounted for 94% of the total AUC in mice and 20%
of the total AUC in dogs.
approximately 2% in mice.
Based on plasma drug levels, oral bio-availability of BSO was
Since GSH depletion was comparable following either p.o. or i.v.
BSO, however, it is likely that the actual oral bioavailability is higher than that observed,
possibly secondary to rapid removal of BSO from the plasma into tissue compartments.
9
INVESTIGATIONAL DRUG – PHARMACEUTICAL DATA
BSO INJECTION
(NSC-326231)
The following information applies to the investigational dosage form of BSO injection.
The Division of Cancer Treatment, Diagnosis, and Centers, National Cancer Institute provided
the product below to the current IND sponsor, The Developmental Therapeutics Program USCCHLA Institute for Pediatric Clinical Research, for clinical trials.
Chemical Name: Butanoic acid, 2-amino-4 (S-butylsulfonimidoyl), 2S
CH3
CH2
H C CH2
H
NH
S O
(CH 2)2
H C NH3
COO-
Other Names: buthionine sulfoximine
Molecular Formula: C8 H18 N2 O3 S
M.S.: 222.3
How Supplied:
Sterile injection, 100 mg/ml, 50 ml/vial:
The product is prepared as an aqueous solution with
sodium hydroxide for adjustment of pH to 7 to 8.
Storage: The intact vials should be stored at controlled room temperature (15 to 30° C)
10
Stability:
Shelf life surveillance of the intact vials is ongoing.
In aqueous solution, BSO is
most stable at neutral and basic pH values. At pH values of 6 to 9.9, little or no decomposition
occurs in 48 hours at 90ºC. Increasing rates of decomposition occur at lower pH values. At a
concentration of 0.1 mg/ml in 5% Dextrose Injection, USP, and 0.9% Sodium Chloride Injection,
USP, in both glass bottles and PVC plastic bags, no decomposition or loss of drug due to
sorption has been noted over a 14 day period.
Route of Administration: Intravenous
Distribution: The product has been supplied for authorized investigational studies to the USCCHLA Insittute for Pediatric Clinical Research by the Division of Cancer Treatment, Diagnosis,
and Centers, National Cancer Institute, Bethesda, Maryland 20892, USA, via the Developmental
Therapeutics Program, NCI.
Drug supplies for studies conducted under the current IND will be provided by:
Developmental Therapeutics Program
USC-CHLA Institute for Pediatric Clinical Research
Childrens Hospital Los Angeles
4650 Sunset Blvd
Los Angeles, CA 90027
11
MECHANISM OF ACTION
The mechanism through which BSO modulates the activity of cytotoxic agents is related
to two distinct actions; 1) the inhibition of GSH synthesis; and 2) the affect of intracellular GSH
depletion on the activity of the antineoplastic agent.17-19
Inhibition of GSH synthesis by BSO is related to the BSO-mediated activation of ?-GCS,
in the presence of ATP, catalyzes the formation of ?-glutamylcysteine (g-GC) from glutamate
and cysteine; second, GSH synthetase, also in the presence of ATP, catalyzes the formation of
GSH from ?GC and glycine.
?-Glutamyl phosphate is the enzyme-bound intermediate formed
from the action of ?-GCS on glutamate.20 BSO was anticipated to be a potent inhibitor of GSH
synthesis due to its close structural resemblance to ?-glutamyl phosophate-a-amniobutyrate,
which binds to ?-GCS,21 and its inhibition of de novo synthesis of GSH.
Griffith22;
23
first attempted to elucidate the mechanism of inhibition of ?-GCS by BSO.
Their results were consistent with the proposed mechanism for PSO that the S-alkyl moiety of
PSO binds at the site of ?-GCS to which the acceptor amino acid, cysteine or a-aminobutyrate,
would normally bind.
BSO is thought to initially bind to the enzyme and then undergo
phosphorylation in the presence of ATP to the ?-GCS-bound BSO-phosphate, which is the actual
?-GCS inhibitor.21
Compared to the other sulfoximine derivatives examined, such as MSO and PSO, BSO
was the most potent inhibitor of ?-GCS activity. BSO-induced inhibition of ?-GCS was at least
100-fold greater than that of MSO and approximately 20-fold greater than that of PSO.
Complete inhibition of ?-GCS was achieved after a 10-minute incubation with 0.02 mM BSO.
In contrast, complete inhibition of ?-GCS activity was not achieved after 10 minutes with 2 mM
MSO. This high specificity of BSO for ?-GCS may account for its low toxicity relative to other
thiol-depletors, such as MSO and PSO.23
12
The mechanism of ?-GCS inhibition by BSO was further examined by Griffith.24 In this
study, it was established that ?-GCS, in the presence of MgATP, catalyzes the phosphorylation
of BSO to form the BSO-phosphate adduct. The inhibition of ?-GCS by the enantiomers, L-BSO
and D-BSO, was also examined.
At a concentration of 10 mM, D-BSO did not significantly
inhibit ?-GCS, whereas 20µM L-BSO resulted in complete inhibition of ?-GCS activity after
only 10 minutes.
The rate of ?-GCS inactivation produced by incubation with 20 µM L-BSO
was equivalent to that produced by 40 µM DL-BSO (a mixture of the D and L enantiomers).
Furthermore, unlike the reversibility of inactivation previously demonstrated with MSO,22 a 200fold dilution of BSO-bound γ-GCS did not result in enzyme reactivation.
The inhibition kinetics of γ-GCS and BSO were also studied by Griffith. 24
Because
inhibition of γ-GCS by L-BSO occurred too rapidly (maximal inhibition within 1 minute) for
kinetic measurements by conventional methods, DL-BSO was used for kinetic studies. Enzyme
inhibition followed first order kinetics at any fixed concentration within the range (10-100 µM)
of BSO examined.
The rate-limiting constant was 3.7 min,20 which corresponds to a T1/2 of
approximately 11s at a saturating concentration of inhibitor.
The maximal estimate of the
apparent initial binding constant for BSO was 100 µM. However, since BSO has two centers of
asymmetry, one at the a-carbon and another at the sulfur, four isomers may possibly exist.21 In
this study, it was assumed that only one isomer was active, thus the actual initial binding
constant was extrapolated to be 25 µM.
Extensive binding studies using column chromatography showed that extent of binding γGCS to BSO in the presence of ATP was examined using buffers with or without Mg and/or
ATP to determine the role of these agents in the interaction of BSO and ?-GCS. Results of these
studies indicated that 1 M equivalent of the BSO-phosphate adduct was bound tightly, noncovalently, and irreversibly to the fully inhibited enzyme.
?-GCS (725 U., 0.84 nmol was
inhibited to > 98% at 15 minutes in the presence of 0.83 mM L-BSO. However, when the
enzyme was pre-incubated with cystamine, ?-GCS was not inactivated by subsequent exposure
to BSO.
A possible explanation is that cystamine may bind to an enzyme sulfhydryl near the
active site of G-GCS. If the buffer contained Mg without ATP, γ-GCS-BSO binding was slightly
13
diminished, but the enzyme retained substantial (approximately 30%) activity.
When the buffer
contained neither Mg nor ATP, binding did not occur, and the enzyme retained full activity. In
additional experiments using [?- 32 P]ATP or [8- 14 C]ATP, both radiolabels were bound to the
enzyme inhibited with BSO.5
When the BSO-bound enzyme was analyzed by chromatography,
35
radiolabeled L-[ S]BSO or [?- 32 P]ATP coeluted distinctly from either BSO, P?, or ATP. The
elution profile of the BSO-phosphate adduct indicated a net charge which was more negative
than that of P?.
These observations were consistent with the profile expected for the BSO-
phosphate adduct.24
Other studies have attempted to elucidate the effects of BSO-induced GSH depletion on
the activity of certain cytotoxic agents.
The mechanism through which GSH modulates
cytotoxicity is thought to be related to its role in cellular metabolism. Once γ-GCS is inactivated
by BSO, GSH synthesis is inhibited, resulting in depletion of intracellular GSH.
Decreased
levels of GSH have been shown to increase the toxicity of various antineoplastic agents, in
particular melphalan (L-PAM).6; 7; 25-27
Through
its
nucleophilic
and
oxidative-reductive properties, GSH may detoxify
potentially harmful reactive intermediates formed form oxidant injury, either by normal aerobic
metabolism or through the action of xenobiotics.
GSH can react with electrophiles to form
thioethers, or, through its oxidation to GSSG, GSH can donate hydrogen to radical compounds to
form the original molecule.
During normal steady-state conditions, GSH (0.5-10 mM) exists
primarily in its reduced form, but it may also exist in any of the following three interchanging
states: 1) GSH may be oxidized by GSH peroxidase or by nonenzymatic means to a disulfide,
GSGG (normally 5-50 µM), which may in turn be reduced to GSH by GSH reductase or by an
exchange reaction;
2) GSH may form mixed disulfides with both protein and nonprotein
sulfhydryls; or, 3) GSH may exist in the form a thiol ester.28;
29
Peristeris et al.,30 investigated the
role of the antioxidative function of GSH in regulating tumor necrosis factor (TNF) production in
vivo in lipopolysaccharide (LPS) treated mice.
TNF is a mediator in the pathogenesis of
endotoxic and septic shock and TNF inhibition has a protective effect in several animal models.
14
Pretreatment with GSH produced a significant inhibition of TNF production, whereas treatment
with BSO had the reverse effect.
Arrick and Nathan28 have classified the action of GSH on the therapeutic efficacy of
antineoplastic agents as follows: 1) GSH may detoxify active species produced by agents such
as L-PAM, CTX, nitrosoureas, radiation, and radiosensitizers or repair direct injury induced by
such species through the role of GSH in oxidation-reduction and nucleophilic reactions; 2) GSH
may increase the toxicity of certain agents such as bleomycin by participating in the formation of
active cytotoxic species;
3) GSH may enhance the delivery of certain cytotoxics such as
methrotrexate (MTX) to their target site through its role in amino acid transport; or,
4)
alterations of GSH levels may interfere with the effectiveness or toxicity of concomitant or
subsequent therapy.
Since the mechanism of the interaction between GSH and antineoplastics
has not been fully elucidated, the classification of certain drugs into these categories may be
arguable.
BSO has been effective both in vitro and in vivo in enhancing the cytotoxicity of L-PAM.
However, the data from some studies have been conflicting as to the mechanism of the
modulation of L-PAM cytotoxicity by GSH. Susukake et al, 31 studied the role of GSH in the
modulation of L-PAM toxicity in L1210 L-PAM-sensitive and L1210R L-PAM resistant murine
leukemia cell lines.
No differences in L-PAM uptake were noted between the resistant or
sensitive cell lines exposed to L-PAM 2.5 µg/ml for up to 45 minutes.
In addition, no
differences in L-PAM efflux were observed over 90 minutes after a 45-minute incubation of each
cell line with L-PAM. However, marked differences between the two cell lines were noted with
respect to the metabolism of L-PAM. In the L1210R cells exposed to 1.25 µg/ml of L-PAM for 3
hours, the ratio of unchanged L-PAM to its dechlorinated noncytotoxic derivative, dihydroxy LPAM
(4-[bis(2-hydroxyethyl)amino]-L-phenylalanine),
was
approximately
20:18.
The
corresponding ratio (approximately 24:7) in the L1210 cells was markedly different and
indicated that dechlorination occurred to a lesser extent in the L-PAM-sensitive cells. When the
L1210R cells were treated with the 0.1-0.25 mM N-ethylmaleimide (a sulfhydryl agent) 5
minutes prior to exposure to L-PAM, dechlorination of L-PAM was inhibited and occurred at the
15
same level as in the L1210 cells. Levels of the dihydroxy metabolite in either cell line correlated
positively with GSH levels, which in turn correlated inversely with sensitivity to L-PAM.
These observations led the authors to conclude that GSH-mediated resistance to L-PAM
was probably not associated with a classical transport system in either L1210 cell line
investigated, since neither accumulation nor retention of L-PAM correlated with drug resistance.
Because of another report32 which indicated changes in transport of L-PAM as the mechanism of
drug resistance, the authors conceded that such mechanisms may vary among cell lines
examined.
Although the precise mechanism of GSH-mediated resistance to L-PAM remains
undefined, the authors have postulated that the interaction between L-PAM and GSH may be
mediated by GSH-S-transferase, an enzyme which is involved in the dehalogenation of many
electrophiles.31
Results of the study of Green et al6 with human ovarian cancer cells confirmed the
findings of Suzukake, et al.31 No detectable difference was observed in the uptake of L-PAM
(3.5 µM) between cells (1847me) with 4-fold resistance to L-PAM acquired in vivo and the parent
cell line (1847) which was sensitive to L-PAM. There was a marked difference, however, in the
cellular content of dihydroxy L-PAM which was 2-fold higher in the 1847me than in the 1847
cells. These observations led the authors to conclude that GSH-conferred resistance to L-PAM
in this cell line may be related to increased catabolism of L-PAM to the noncytotoxic metabolite.
Hamilton et al7 were also unable to detect any difference in L-PAM uptake in another L-PAMresistant human ovarian cancer cell line (2780ME) following exposure to 25 µM BSO. It was
noted that the activity of GSH-transferase was higher in the 2780ME cells than in the L-PAMsensitive parent cells.
Koberle and Speit 33 studied the effect of GSH depletion of sister chromatid exchange
(SCE) induction by cytostatic drugs. V79 cells were treated with BSO for 24 hours either before
BrdU application or together with BrdU.
The results indicate that BSO itself did not induce
SCEs either before BrdU treatment or together with BrdU . However, cellular depletion of GSH
by BSO had a clear effect on SCE induction by doxorubicin and cyclophosphamide, with drug
induced SCE frequencies were significantly increased after BSO pretreatment
16
Kang et al,34 compared the effects of cadmium on epidermal growth factor (EGF)induced DNA synthesis and its effects on GSH metabolism in quiescent NRK-49F cells with
those of BSO.
Cadmium inhibited EFG-induced DNA synthesis in a dose-dependent manner
and increased cellular GSH content in growth arrested and EGF-stimulated NRK-49F cells.
BSO also inhibited EGF-induced DNA synthesis but depleted cellular GSH content in EGF
stimulated NRK-49F cells.
The BSO effects showed different dose dependencies.
The
combination of BSO and cadmium inhibited EGF induced DNA synthesis in NRK-49F cells in
an additive manner, suggesting different mechanisms of action for the two agents. The results of
this study demonstrate that cadmium inhibition of EGF-stimulated DNA synthesis is not
mediated by GSH depletion.
Possible mechanisms by which BSO inhibits cell proliferation were investigated using
normal rat kidney fibroblasts (NRK-49F). NRK-49F cells were cultured in Dulbecco’s modified
Eagle medium(DMEM) with and without glutamine.
glutamine-deficient DMEM.
the cells.
NRK-49F cells did not proliferate in the
Addition of glutamine into the medium stimulated proliferation of
Addition of BSO and glutamine inhibited the glutamine-stimulated cell proliferation in
a BSO dose-dependent fashion. Based on these results, glutamine is required for NRK-49F cell
proliferation.
BSO-induced cytostasis is most likely due to inhibition of cellular uptake of
glutamine as well as other amino acids.34
It is important to recognize that inhibition of GSH synthesis with BSO likely provides an
advantage in attacking multi-drug resistant tumor cells that extends beyond just the utilization of
GSH which occurs with a variety of drugs.
Upregulation of GSH and GSH synthetic and
utlization enzymes is associated with drug resistance in a variety of tumor types.18;
35-40
An
increased ability to synthesize GSH potentially allows tumor cells to overcome depletion of GSH
though increased utilization that would be overcome by blocking GSH synthesis with BSO.
Indeed, that has been observed in neuroblastoma cell lines, pointing toward the importance of
targeting γ-GSH with BSO.41
17
PRECLINICAL ACTIVITY
GSH Depletion and Effect on L-PAM Cytotoxicity
BSO will be evaluated in combination with L-PAM in the initial clinical trials. L-PAM is
a bifunctional alkylating agent active in several malignancies, including ovarian cancer.
The
response rate of intravenous (IV) L-PAM <65 mg/m2 as a single agent in ovarian cancer was
reported to be 78%.42 Despite such high activity, successful treatment of ovarian cancer remains
a clinical problem because of acquired drug resistance.
The idea that thiols may be involved in cellular response to alkylating agents was
explored by Calcutt et al. 1 In this study, tumor response to the alkylating agent, merophan, was
reported to be inversely related to the intracellular level of both protein and nonprotein
sulfhydryls. This result prompted in vitro studies by Suzukake et al. to determine the role of GSH
in the development of cellular resistance to L-PAM, another alkylating agent.27
Intracellular
levels of GSH were compared to three L1210 murine leukemia cell lines: 1) L1210 sensitive to
L-PAM; 2) L1210R cells in which resistance to L-PAM was developed in vivo in female DBA/2
mice; or, 3) L1210R variant cells which were isolated from one colony following clonal growth
of L1210R. The L1210R cells and the variant were 5-fold and 8-fold, respectively, more resistant
to L-PAM than the sensitive parent line and contained approximately 2-fold and 4-fold,
respectively, higher levels of GSH and GSSG than the sensitive cells.
To confirm the
relationship between increased levels of GSH and resistance to L-PAM, L1210R cells were
grown in the presence of reduced concentrations of glycine, L-glutamic acid, or L-cysteine. The
latter served as a source for GSH biosynthesis. L-cysteine was effective in reducing intracellular
GSH content, whereas either glycine or L-glutamic acid was not.
Following an 18-hour
exposure to L-cysteine at reduced concentrations, levels of both GSH and GSSG were reduced to
similar levels in L1210 and L1210R cells.
Consequently, L-PAM was equally cytotoxic to
L1210R cells grown in the presence of reduced L-cysteine for 24 housr and L1210 cells grown in
the presence of a normal concentration (100 mg/L) of L-cysteine.
18
Somfai-Relle et al. (1984) reported that, in L1210R cells which were sensitized to L-PAM
in the presence of reduced concentrations of L-cysteine, sensitization could be reversed by
subsequent exposure to normal concentrations (100 mg/L) of L-cysteine. However, despite the
successful modulation of L-PAM sensitivity by L-cysteine, nutritional deprivation of L-cysteine
was not considered a feasible method for L-PAM sensitization in vivo. Consequently, SomfaiRelle et al. (1984) employed BSO, a thiol-depleting agent, to sensitize L-PAM-resistant L1210R
cells.
BSO was previously demonstrated by Griffith and Meister to be an effective thiol
depleting agent because of its specificity for ?-GCS and its low toxicity.23
Somfai-Relle et al.
demonstrated that L-PAM cytotoxicity was increased din both L1210 and L1210R cells at all
concentrations (10-500 µM) of BSO was effective in sensitizing the L1210R cells but a long
exposure time (18-24 hours) was required prior to treatment with L-PAM for maximal
sensitization.43 Following a 24-hour exposure to 50 µM BSO, cellular GSH was reduced by 50%
of its pretreatment value in L1210R cells.
BSO-conferred sensitization was examined in vitro in human ovarian cancer cells by
Green et al. 6 An ovarian cancer cell line (1847ME) was developed in vitro with an acquired 4fold greater reisistance, as defined by the IC50, to L-PAM than the parent line, A1847, which
was sensitive to L-PAM. Three other cell lines, NIH:OVCAR-1,-2, and –3 were also studied.
These were derived from patients with ovarian cancer clinically resistant to alkylating agents.
Both the IC50 and levels of intracellular GSH were comparable for the NIH:OVCAR and
1847ME cells. These similarities led the authors to assume that studies of L-PAM resistance with
the 1847ME cell line would be clinically relevant. Both the level of GSH and the level of the
noncytotoxic dihydroxy L-PAM metabolite were twice as high in the 1847ME cells compared to
that of the parent line; however, no difference in uptake of 3.5 µM L-PAM was observed
between these two cell lines. Intracellular GSH content was reduced in both 1847ME and A1847
cells at all concentrations (10-100 µM) of BSO after an 18-hour exposure to the agent.
Following exposure to 50 µM BSO, GSH was reduced by 75% of control values in the A1847
cells and by 68% in 1847ME cells. Following exposure to 100 µM BSO, GSH was reduced by a
similar degree (82-85%) in both cell lines; however, the absolute GSH content in the 1847ME
cells remained twice that of the parent line. When both cell lines were treated with 3 µM L-PAM
19
for 2 hours following an 18-hour exposure to 100 µM BSO, L-PAM cytotoxicity, as defined by
the IC50, was increased in both 1847ME cells and A1847 cells. In the resistant cells, sensitivity
was increased by 3.5 fold, as evidenced by a reduction in the IC50 of L-PAM to 1.4 µM. This
concentration was comparable to the IC50 (1.7 µM) for the A1847 cells not treated with BSO.
Pretreatment with BSO caused a similar degree of reduction in the IC50 of L-PAM in the parent
cells.
Maximal enhancement of sensitivity to L-PAM was produced when 1847ME cells were
exposed to 50 µM BSO for 18 hours followed by an additional dose of 50 µM BSO in
combination with L-PAM for another 18 hours. Under these conditions, the cytotoxicity of LPAM was increased 10-fold. Dose-response curves were generated which indicated that BSO at
a concentration of 100 µM had a synergistic effect upon the cytotoxicity of 3 µM L-PAM.
However, it was also noted that 100 µM BSO was itself inherently toxic and caused a 30%
reduction in colony function.
Hamilton et al.7 and Ozols et al. 26 examined the effect of BSO on intracellular GSH
levels and on sensitivity to L-PAM, CDDP, or DOX in several human ovarian cancer cell lines,
which included 2780ME cells with a 10-fold resistance to L-PAM acquired in vitro and 2780CP
cells 14-fold resistant to CDDP. At doses (15-25 µM) that were minimally toxic to the cells,
BSO depleted GSH levels by 70-85% in both the resistant and sensitive parent cell lines. As
evidenced by the dose modification factors (DMF) for L-PAM or CDDP, BSO potentiated the
cytotoxicity of either agent in the resistant as well as the sensitive cells. The DMF for L-PAM in
A2780, 2780ME, and 2780CP cells was 6.8, 3.4 and 6.3, respectively. It was also observed that
these cell lines expressed a pleiotropic phenotype, as evidenced by cross-resistance to drugs not
used in the induction of primary resistance. Depletion of GSH levels by BSO also affected these
patterns of cross-resistance in these cells. As evidenced by the DMF, complete reversal of crossresistance to L-PAM occurred in the 2780CP cells following exposure to 25 µM BSO for 48
hours.
Ozols et al.26 examined the in vitro effects of L-BSO on the cytotoxicity of L-PAM in a
human ovarian cancer cell line, NIH:OVCAR-3 (OVCAR-), which was initially derived from a
patient refractory to therapy with DOX, CDDP, and CTX. The GSH levels in the OVCAR-3
20
cells were 4-fold higher than that of a cell line from a patient with no prior therapy.
At a
concentration of 50 µM, L-BSO reduced the cloning efficiency of the OVCAR-3 cells by < 10%,
did not significantly affect colony size, and caused a 93.4% reduction in levels of GSH.
This
reduction was accompanied by a decrease in the IC50 of L-PAM from 0.44 to 0.12 µM, resulting
in a DMF of 3.6.
Kramer et al. 25 conducted an in vivo study on the cytotoxic effect of BSO in combination
with L-PAM in BDF male mice bearing L1210R cells resistant to L-PAM.
In L1210R cells
harvested 18 hours after mice were treated with multiple intraperitoneal injections (IP) of BSO
450 mg/kg every 6 h x 5 followed 6 hours later by L-PAM 0.5-2.5 mg/kg, cytotoxicity of LPAM was increased.
As the highest dose (2.5 mg/kg) of L-PAM, pretreatment with BSO
resulted in 92% reduction in the clonogenicty of L1210R cells compared to a 78% reduction in
cells from mice treated with L-PAM alone.
BSO alone also was noted to produce a 10%
reduction in the clonogenicity of the L1210R cells. The total number of cells recovered from the
peritoneal cavity was lower in mice treated with L-PAM alone relative to untreated mice; this
recover was further reduced in mice that received BSO in combination with L-PAM.
Kramer et al. 25 also examined the in vivo effect of BSO pretreatment on DNA synthesis,
which was extrapolated from incorporation of [3 h]dTTP. At lower doses (0.5- 1 mg/kg), L-PAM
alone caused an increase in [3 h]dTTP incorporation.
Treatment with BSO prior to L-PAM
caused both an inhibition of the increased incorporation observed at lower doses of L-PAM and a
further reduction of DNA synthesis at higher doses of L-PAM. The ED50 (dose which produces
a 50% inhibition of dTTP incorporation in vivo) of L-PAM alone was 3.9 mg/kg. For L-PAM in
combination with BSO, the ED50 was decreased to 1.8 mg/kg. This represents an overall dose
reduction factor of 2.2 mg/m2 .
Tissue Selectivity of BSO-Mediated GSH Depletion
Kramer et al. 25 conducted in vivo studies on the effect of BSO on tissue levels of GSH
and BDF male mice.
BSO was administered at a dose of 450 mg/kg either as a single IP
21
injection or IP every 6 h x 5. BSO given as a single injection depleted the GSH levels of all
tissues examined, which included the liver, stomach, plasma, lung, kidney, and heart.
Maximal
GSH depletion occurred within 2-4 hours in the plasma, kidney and liver and within 5-8 hours in
the other tissues.
Differences in the initial rate of GSH depletion appeared to reflect the rate of
GSH turnover in a given tissue. However, there appeared to be no direct correlation between
inherent levels of GSH, degree of depletion, and degree of recovery of GSH levels 24 hours after
completion of the BSO dose. GSH levels were the lowest in bone marrow, which was the most
refractory to depletion by BSO among the tissues investigated. When BSO was given IP,
maximal depletion of GSH in the bone marrow was approximately 40%, in contrast to that of 7080% in the liver, kidney, heart, or stomach.
Also, in this study the efficacy of orally (PO) administration BSO compared to BSO IP
was examined with respect to selective GSH depletion in tissues. BSO administered either PO
(1mg/d x 3) or IP resulted in comparable GSH depletion in the heart. GSH depletion by BSO PO
was slightly lower in the kidney, lung, and stomach compared to BSO IP. However, in the liver,
GSH was depleted by approximately 36% with BSO PO and by 80% with BSO IP. Compared to
all tissues investigated, GSH depletion (90%) was greatest in L1210R cells harvested from the
peritoneum of tumor-bearing mice treated with BSO IP.
Furthermore, ni mice treated with L-
PAM following BSO, potentiation of L-PAM cytotoxicity was dependent on extent of GSH
depletion, which was more efficient overall with BSO IP compared to BSO PO.25
Lee et al. have compared the effect of BSO administration on GSH levels in normal and
tumor tissues.12
Mice bearing either the murine sarcomas KHT or RIF-1, or the mammary
carcinoma 16C were administered a single IP dose (2.5 mmol/kg) of BSO. The extent and rate
of GSH depletion varied among different tissues. GSH levels were lowest in the bone marrow
(17% of control), kidney (20%), and liver (26%). GSH nadirs in these tissues varied from 33%
of controls (in KHT) in approximately 41% (RIF-1 and 16C). Nadirs occurred in these within
1012 hrs.
GSH depletion was intermediate in the heart and lung (46% and 60% of controls,
respectively).
The rate of depletion (4-15 hrs to nadir) in the lung approximated that in other
tissues, but was much slower in the heart (24-72 hrs to nadir).
22
GSH depletion was minimal in
RBCs (GSH values were 8% of control). Recovery of tumor GSH levels required 48 hrs, which
was longer than that required in the liver (16 hrs), kidney (30 hrs), or lungs (32 hrs), but shorter
than the bone marrow (72 hrs) or heart (96 hrs). The investigators concluded that differences in
GSH depletion between normal and tumor tissues may possibly be exploited in order to
minimize the adverse effects of chemotherapy in normal tissues.
The effect of BSO administration on GSH depletion has also been compared in tumored
and normal brain tissue.44
Mice and intracranial human glioma-derived (D54 MG) xenografts
received BSO (5mmol/kg) IP on either a single or multiple (q 12 hr) dose schedule. Tumor GSH
levels were 47% of controls 12 hrs after a single dose of BSO, and declined to 23% after 24 hrs.
When BSO was administered q 12 hrs, tumor GSH levels at 24 hrs were the same as that seen on
the single dose schedule (22% versus 23% of controls, respectively). However, GSH levels at 36
hrs were lower on the multiple dose schedule than the single dose schedule (16.5% versus 26.8%
of controls, respectively).
Administration of a single, higher dose (20 mmol/kg) did not
significantly alter GSH depletion.
GSH levels in the contralateral (non-tumor) hemisphere were
minimally affected by a single dose of BSO (5 mmol/kg).
If a second BSO dose was
administered at 12 hrs, the GSH levels were depleted to 74.5% of control at 24 hrs. The data
suggest that BSO selectively depletes GSH in CNS tumors.
Tumor Cell Selectivity
Because of the potential therapeutic advantage from treatment with an agent which could
selectively target tumor rather than normal tissue, some investigators have examined the action
of BSO on tumor cells relative to normal cells. The results of some in vitro studies have
indicated no tumor selectivity in the action of BSO.12;
25
by Ozols et al. suggest that such selectivity may exist.45
However, data from an in vivo study
Although BSO improved survival in
tumor-bearing nude mice treated with L-PAM in this study, it did not increase lethality in nontumor-bearing mice.
Anderson et al. showed that unlike many cell types, BSO treatment of
neuroblastoma cells in vitro was highly cytotoxic, again providing evidence of tumor selectivity.8
23
Russo et al. examined both intracellular GSH content and BSO-modulated GSH depletion
in vitro in normal human lung fibroblasts (CCL-210) and human lung adenocarcinoma cells
(A549).4
Intracellular GSH levels in A549 cells were 7-fold higher than in normal CCL-210
cells. Following exposure to 10 mM BSO, GSH was depleted more rapidly in the normal cells
than in A549 cells. Within 7 hours after treatment with BSO, the level of GSH in the CCL-210
cells had declined by 95%, but had declined by only 60% in the similarly treated A549 cells.
Oxothiazolidine-4-carboxylate (OTZ) was used to stimulate GSH synthesis. When the cells were
treated with OTZ, GSH levels increased to 140-170% over baseline values in the normal cells,
but no response was observed in the cancer cells.
Melanoma.
The antitumor activity of BSO has been evaluated in melanoma and non-
melanoma cell lines.46
Prior to this study, GSH was shown to be indirectly involved in the
melanin biosynthetic pathway, and may also serve as a scavenger for radicals produced during
melanogenesis.46;
47
Kable et al. demonstrated that seven melanoma cell lines were more
sensitive to BSO alone than were six non-melanoma lines (fibroblasts, HeLA cells, ovarian cells,
and three ovarian tumor cell lines).46
The D37 (dose at which cell survival is 37%) of BSO
ranged from 0.73-8.5 µM in the non-melanoma cell lines. No correlation was found between
antitumor activity and levels of total soluble sulfhydryl, GSH, GSH reductase, GSH
transferase,or with the amount of GSH depletion induced by the drug.
Exposure of a human melanoma cell line (RPMI 8322) to 0.01 mM BSO for 24 hours
reduced the cellular GSH content to 14% of the control without affecting cellular growth rate,
clonogeneic capacity, or cell volume.48
BSO pretreatment also enhanced the cytotoxicity of
melphalan and nitrogen mustard by a dose-modification factor (DMF) of 3.4 and 3.3,
respectively (the DMF was calculated as the ratio of the slopes of the survival curves for BSO
pretreated and control cells), and an increase in DNA cross-linking was observed.
A slight
increase in DMF was seen with cisplatin and BCNU, but little or no increase in DNA crosslinking was observed.
Despite the increase in melphalan cytotoxicity after BSO pretreatment,
intracellular melphalan concentrations were not significantly affected.
24
The authors concluded
that BSO pretreatment reduced intracellular binding of melphalan with GSH, thereby increasing
drug binding to DNA targets.
Neuroblastoma. Anderson et al. demonstrated that BSO as a single agent can produce
up to 4 log cell kills in neuroblastoma cell lines and pre-incubation with glutathione, vitamin E,
or ascorbate partially reverses this effect.8 The cytotoxicity was variable, with most of the IC50 ’s
in the range of 08.79 µM. Five out of eighteen neuroblastoma cell lines had 1C50 ’s greater than
100 µM. BSO caused GSH to fall to less than 10% of control within 24 hours and maximal
cytotoxicity is seen by 48 hours.
The mechanisms of BSO-induced neuroblastoma cytotoxicity
may be related to tyrosine/catecholamine metabolism or unique alterations in baseline levels of
GSH related enzymes such as ?-GCS or GST isoenzymes. BSO toxicity for neuroblastoma cells
was markedly decreased in hypoxic conditions, but can be restored using the bioreductive agent
tirapazamine.41
Pre-treatment of neuroblastoma cell lines with 10 µM BSO for 24 hours enhanced the
cytotoxicity of 10 µM L-PAM by 1 to 2 logs of cell kill.9 In the eight cell lines tested, BSO + LPAM provided an average of 1.37 additional logs of cytotoxicity over that obtained by L-PAM
alone (P
= 0.0009).
Since 10 µM BSO is far lower than the peak (3000-8000 µM) or
continuous steady state (500 µM) plasma levels in humans, these studies suggested that BSO
may enhance tumor cell kill of neuroblastoma in vivo.
Studies of human neuroblastoma cell
lines established from patients with recurrent disease after myeloablative therapy or BSO/LPAM therapy (including lines that had lost p53 function) also demonstrated the ability of BSO to
increase the cytotoxic response to L-PAM, but only at high L-PAM levels that require
hematopoietic stem cell support.49-51
Figure 2 shows the ability of BSO to enhance L-PAM
cytotoxic activity in multi-drug resistant neuroblastoma cell lines.
25
BSO Enhances L-PAM Activity in
Neuroblastoma Lacking Functional p53
FRACTIONAL SURVIVAL
CHLA-90
CHLA-171
102
101
100
10-1
L-PAM alone
BSO alone
L-PAM + BSO
10-2
10-3
0
1
3
6
L-PAM alone
L-PAM + 500 µM BSO
9
0
12
L-PAM µg/ml or BSO µM (x 10)
2
4
6
8
10 12 14
L-PAM (µg/ml)
Figure 2
BSO and Tumor Hypoxia.
Since area of solid tumors may be hypoxic, antineoplastic
agents selectively toxic to hypoxic cells may have a therapeutic benefit over agents that are not
active in hypoxia.
Shrieve and Harris examined the effect of GSH depletion by BSO on the
cytotoxicity of a number of chemotherapeutic agents, such as L-PAM and CDDP, and on
radiation response in EMT6/SF cells under hypoxic or normoxic conditions.52 For measurement
of GSH depletion, the cells were incubated in air with 0-1000 µM BSO for up to 75 hours. After
75 hours, GSH in the untreated cells was decreased by approximately 30% of baseline values.
In the cells treated with BSO, maximum GSH depletion occurred at a concentration of 50 µM
BSO.
In cells exposed to BSO for longer than 24 hours, dose-dependent inhibition of cell
growth occurred. When the cells were treated with cytotoxic agents under normoxic or hypoxic
conditions following a 12-hour incubation with BSO, cytotoxicity was increased for CDDP,
mitomycin-C, L-PAM, and nitrogen mustard under oxic conditions.
The greatest sensitization,
either under hypoxic or normoxic conditions, occurred when BSO was used in combination with
L-PAM.
Although this was generally observed with other cytotoxic agents tested, sensitization
was never greater under hypoxic relative to normoxic conditions.
The enhancement ratio (ER)
for cells treated with L-PAM under hypoxic conditions was 3.1; when BSO was used prior to LPAM, the ratio increased to 4.3. However, when the cells were treated with BSO + L-PAM
under normoxic conditions, the ER was 6.5. The results of this study suggest that the degree of
sensitization by BSO may depend on the antineoplastic agent used and the availability of oxygen
in the environment of the tumor cell.
26
Cervical cancer xenografts in athymic mice were studied for the ability of BSO to deplete
GSH in hypoxic regions of the tumors (identified using nitroimidazole EF5). BSO was found to
deplete GSH more effectively in hypoxic regions of the tumor.53
A study with neuroblastoma cell lines showed hypoxia can antagonize BSO
cytotoxicity.41
However, the bioreductive agent tirapazamine caused highly syngergistic
cytotoxicity in hypoxia, suggesting that hypoxia-active cytotoxic agents may provide the best
combinations with BSO in any settings where tumor hypoxia is present.
Further pre-clinical
studies combining BSO with various potential drugs under hypoxic conditions are needed to
better define these issues.
Effect of BSO and L-PAM on Survival of Mice with Tumors
In an in vivo study by Kramer et al., GSH levels were examined in cells harvested from
the peritoneal cavity of mice bearing L1210 or L1210R cells.25
The intracellular GSH
concentration was 1.6-fold higher in the L1210R than in the L1210 cells. After a single injection
of BSO 450 mg/kg in the mice, GSH levels were depleted more rapidly in the harvested L1210R
cells relative to the L1210 cells. However, 24 hours after the BSO dose, GSH was depleted to
almost an equivalent extent in both cell lines. In contrast, Green et al. reported that the levels of
GSH in L-PAM-resistant human ovarian carcinoma 1847ME cells, treated with 100 uM BSO for
18 hours in vitro, remained about twice as high as that in the sensitive cell line; however, the
extent of GSH depletion was similar for both cell lines.6
It is difficult to assess the validity of
these differences, however, since one cannot compare an in vivo versus an in vitro system.
Mice bearing either EMT6 mammary or RIF fibrosarcoma tumors were given a single
subcutaneous (sc) dose of 4 mmol/kg or 40 µmol/kg BSO had complete tumor regression, and
appeared cured on day 91. In the RIF group, tumor growth was delayed for one day in animals
receiving 4 mmol/kg BSO. No effect was seen at the lower dose. Another set of mice was
administered BSO (4 mmol/kg) sc twice daily for nine days.
Drug administration was
discontinued at that time due to toxicity. In mice the EMT 6 tumors, tumor growth lagged 9 days
27
behind controls. Growth inhibition persisted for 11 days after the last BSO dose. No direct
correlation was found between tumor growth delay and GSH levels in either group (EMT6 or
RIF) on the single or multiple dose schedule. GSH values two hrs after a single injection were
similar in EMT6 and RIF tumors (1.3-1.7 versus 2.2-2.5 fold less than control values). Recovery
to pre-injection levels occurred within 28 hours in both tumor groups.
On the multiple dose
schedule, GSH levels ere three and four-fold less than controls in the EMT6 and RIF groups,
respectively. GSH levels returned to control values within 48 hours in both tumor systems.
Tsuji et al. examined aflatoxin B1 (AFB1 )-induced glutathione s-transferase placental
form (GST-P) positive single hepatocytes in male and female rats and male hamsters with and
without BSO pretreatment.54
Male rats formed significantly more AFB1 -induced GST-P
positive hepatocytes by approximately 120% above the controls.
Male hamsters formed
significantly less AFB1 -induced GST-P positive hepatocytes than male rats.
Pretreatment with
BSO did not increase AFB1 -induced GST-P positive hepatocytes in hamsters although it did
produce an increase in hepatic necrosis.
Ozols et al. studied the effect of BSO on the in vivo cytotoxicity of L-PAM in nude mice
bearing a clinically resistant human ovarian cancer cell line relative to mice without tumors.45
Although cytotxicity of normal versus tumor tissue was not directly compared, such selectivity
was inferred from the results of this study, which indicated that treatment with BSO and L-PAM
improved the survival of the tumor-bearing mice compared to those treated with L-PAM alone.
BSO did not enhance the lethality of L-PAM in mice without tumors and caused only slight
weight loss in tumor-bearing mice. BSO did not significantly increase cytotoxicity of L-PAM in
human bone marrow cells obtained from either cancer patients receiving chemotherapy or
normal volunteers. In addition, the levels of GSH in the ascitic tumor cells, bone marrow cells,
and cells from the gastrointestinal mucosa were reduced by 96%, 79%, and 86%, respectively, on
day 5 in mice treated with L-BSO 30 mM PO d x 5. These results indicated that BSO may
selectively increase the cytotoxicity of L-PAM in the tumor cell population compared to normal
tissue.
28
Somfai-Relle et al conducted in vivo studies of the effect of BSO on the survival of
L1210R –bearing mice treated with L-PAM.43
L-PAM 13 mg/kg was administered IP 2 hours
after the last dose of BSO given either as a bolus (4.5 mmole/kg d 1,2) or as a 48-hour
continuous infusion (02. mmole/kg/d). Two groups of control mice received either BSO alone as
a continuous infusion or L-PAM alone. Mice that received L-PAM and the continuous infusion
of L-PAM and the single bolus of BSO and 106% for those given L-PAM alone. Ascites were
remarkably absent at the time of death only in mice that received L-PAM and the continuous
infusion of BSO. The authors postulated that the highly metastatic (primarily hepatic) nature of
the L1210 leukemia may account for the discordance between the disappointing in vivo special
data and the results of the in vitro cytotoxicity studies, which indicated increased cell kill of
1210R cells by L-PAM in combination with BSO.
Differences in survival between mice treated with L-PAM or L-PAM and BSO were also
assessed in the in vivo study in Kramer et al.25 Despite the enhanced cytotoxicity, the effective
reduction of GSH levels, and the lack of increased myelosuprression which resulted from
pretreatment with BSO, the survival of mice bearing the 1210R leukemia treated with BSO and
L-PAM was not significantly increased compared to those treated with L-PAM alone. A 132 ±
6% increase in life span was observed in mice treated with BSO 450 mg/kg IP every 6 h x 5 and
L-PAM 10 mg/kg, compared to that of 120 ± 11% for those treated with L-PAM alone. Based
on the ratio of median cell survival to number of inoculated 1210R cells, this increase was
equivalent to a log-fold increase in tumor cell kill. At the highest dose (13 mg/kg) of L-PAM
evaluated, pretreatment with BSO resulted in increased L-PAM toxicity, as evidenced by 34%
decrease in median survival.
In this study and that of Somfai-Relle et al. above, the lack of
increased survival was attributed to the metastatic potential of the L1210 leukemia.43
In support
of this, Kramer et al.25 point to the results of a study by Ahmad et al.,55 which indicated that
L1210 cells metastatic to the liver had elevated GSH levels and were more resistant to L-PAM
relative to the ascitic cells derived from the peritoneal cavity of CDF1 mice.
Studies investigating the role of BSO pretreatment on melphalan activity have also been
done in mice bearing sc xenografts of the human rhabdomyosarcoma-derived cell line TE-671.56
29
Two doses (5 mmol/kg q 12 hr) given IP decreased tumor GSH levels to 26% of control 12 hrs
after BSO injection. When melphalan (36 mg/m2 ) ws given 12 hr after BSO, tumor growth delay
was 4.6 days longer than in mice receiving melphalan alone.
depleted GSH levels to 17% of control.
Oral BSO (30 mM for 96 hr)
Melphalan given immediately after the completion of
the last BSO dose delayed tumor growth for 6.3 days longer than in mice that received
melphalan alone.
Mice bearing P388 lymphocytic leukemia, MOPC-315 plasmacytoma, or colon 38
tumors were given BSO in drinking water for 24 hrs at dose of 50 or 500 mg/kg. 57
Tumor GSH
content declined in colon 38 tumors within 24 hrs after the drug was stopped. When mice with
P388 leukemia were pretreated with BSO (50 mg/kg for 24 hrs) followed by melphalan (15
mg/kg), median survival increased by 13 days compared to the control group which received
melphalan alone.
A high BSO dose pretreatment did not enhance the survival of melphalan-
treated mice bearing MOPC-315 plasmacytomas or colon-38 tumors.
In contrast to the lack of increased survival reported by Somfai-Relle et al. (1984) for
CDF1 mice bearing 1210R cells and by Kramer et al. 25 for BDF mice bearing 1210R cells treated
with L-PAM and BSO compared to those treated with L-PAM alone, Ozols et al. have reported
substantial increases n survival in their study of nude athymic mice bearing a human ovarian
cancer cell line (NIH:OVCAR-3), clinically resistant to DOX, CTX, and CDDP, treated with
BSO and L-PAM.45
In Experiment 1 of this study, mice received either 1) water alone (control)
d 3-9 post-inoculation or in combination with L-PAM 5 or 10 mg/kg IP d8, 04 2) BSO 30 mM
PO (in drinking water) alone d 3-9 post-inoculation or in combination with L-PAM 5 or 10
mg/kg IP d 8.
Part of this experiment was repeated (Experiment 2) with additional mice that
received either 1) water alone (control) or with L-PAM 5 mg/kg IP d 7, or 2) BSO 30 mM PO d
2-8 alone (control) or with 5 mg/kg of L-PAM IP d 7. Survival was monitored for up to 130
days. For mice treated in Experiment 1, the median survival time (MST) was 46 days for mice
receiving water alone, 75 days for those receiving water and L-PAM 5 mg/kg, and 102.5 days for
mice given water and 10 mg/kg of L-PAM. The latter group also had one long-term (>130 days)
survivor. Mice treated with BSO alone had a MST of 43 days. For the 15 mice given BSO and
30
10 mg/kg of L-PAM, MST was approximately 120 days, and there were 3 long-term survivors.
For mice treated in Experiment 2, the MST was 46 days for mice given water alone, 68 days for
mice given water and 5 mg/kg of L-PAM, and > 125 days for those treated with BSO and 5
mg/kg of L-PAM.
It is unclear why Ozols et al.45 observed an improved survival in tumor-bearing mice
treated with L-PAM and BSO, whereas Kramer et al. 25 and Somfai-Relle et al.43 did not.
One
possible explanation is that the extent of GSH depletion achieved in the study by Ozols et al.45
was greater than that in the study of Kramer et al. 25 In the former, nude mice received drinking
water with L-BSO 30 mM PO d x 5.
Five days following treatment with BSO, GSH was
depleted 79% in the bone marrow cells and 88% in the GI mucosa.45 In the study by Kramer et
al.,25 BDF mice were given BSO 20 M PO d x 3 following a loading dose (450 mg/kg IP) or
BSO 450 mg/kg IP every 6 h x 5. Six hours after the last dose of BSO, GSH was reduced by
approximately 40% (BSO IP) in the bone marrow. Another possible explanation is use of the LBSO isomer specified by Ozols et al. in their study.45
whether the L isomer was used.25;
43
The other investigators did not specify
Ozols et al. also believed their in vivo system closely
represented an in vivo model of human ovarian cancer,45 and indeed, that ovarian xenograft was
multi-drug resistant.
It is possible that multi-drug resistant tumors may show an advantage to
combining BSO with L-PAM that would not be seen when testing BSO and L-PAM in other
systems, such as the L1210 leukemia system previously investigated.25; 43
BSO-Mediated Sens itization of Agents Other Than L-PAM
Because GSH is potentially involved in modulating the activity of many antineoplastic
agents, pre-clinical studies of BSO in combination with a variety of antineoplastic drugs and
radiation have been conducted.
Cyclophosphamide. Increased GSH as been associated with resistance to 4hydroperoxycyclophosphamide
medulloblastoma
58
and leukemia
(4-HC,
59; 60
the
active
metabolite
of
cyclophosphamide)
in
cell lines, and in both of these systems, BSO enhanced 431
HC cytotoxicity. Similar results for BSO combined with other cyclophosphamide metabolites in
leukemia cells have also been reported.61 It has been postulated GSH can diminish metabolism
of 4-HC into metabolites that are more active at DNA alkylation. 62
BSO showed increased the
cytotoxicity of cyclophosphamide against pulmonary metastases in mice of the NFSa tumor,
without increasing cytotoxicity to bone marrow in the mice.63 In the KHT saracoma,64 the MBT2 murine bladder cancer cells,65 and EMT6/SF tumors in mice,
66; 67
BSO was also able to
enhance the cytotoxicity of cyclophosphamide against the tumor cells.
Dorr et al. studied the effect of BSO-mediated GSH depletion on the in vivo cytoxicity of
the sulfhydryl-dependent drugs, BCNU, CTX, DOX, and L-PAM in BALB/c mice bearing
MOPC-315 tumor cells.68
BSO at 50 mg/kg PO was administered 6 days after tumor
implantation, and the cytotoxics were administered at various doses one day following BSO.
Twenty-four hours after BSO, GSH levels in the kidney or liver were reduced to < 10% of
control values.
Pretreatment with BSO increased the % survival in mice by 20% at a BCNU
dose of 20 mg/kg and 25% at 35 mg/kg.
At low CTX doses (50 mg/kg), % survival as increased
by 37%; however, at higher doses (100-200 mg/kg) of CTX, % survival was decreased by 1621%. BSO produced a 15% increase in % survival at a DOX dose of 10 mg/kg, but there was no
increase at 15 mg/kg of DOX. These results indicate that BSO may increase antitumor activity
at lower doses of these agents, but alternatively, may increase adverse toxicity at higher doses.
Subsequent work studying the effect of BSO on busulphan cytotoxicity against hematopoietic
precursors in vitro did not show enhanced toxicity, again suggesting a tissue- or tumor-specific
effect of BSO.69
BSO has been studied in combination with 4-HC in a rat brain tumor model in which
both BSO and 4-HC were given by local installation in a polymer.70 Interestingly, local BSO
delivery enhanced animal survival, whereas systemic BSO therapy did not. However, BSO was
also
reported
to
diminish
the
protective
effect
of
challenging
cyclophosphamide (CTX) prior to higher-dose CTX treatment.71
mice
with
low-dose
The latter points toward the
possibility that sequence and timing of drug usage with BSO may effect the tolerability of the
agent.
32
The mixed results with combining cyclophosphamide with BSO are likely due to the
combination being active against tumors, but at the same time capable of systemic toxicity For
example, BSO has been reported to increase urotoxicity of cyclophosphamide in mice.72;
73
.
Rapid cardiac toxicity (3 hours) has been reported when BSO was combined with
cyclophosphamide in mice and rats.74 In vitro studies showed this to be a direct cytotoxic effect
against cardiac myocytes, and also showed the ability to diminish the toxicity of combining 4HC + BSO by addition of thiol anti-oxidants, such as N-acetylcysteine or MESNA. 75
Platinum compounds. Increased detoxification through GSH pathways is
thought to be a major mechanism of resistance to cisplatin (CDDP) and its analog carboplatin
(CBDCA), the first line agents in ovarian cancer.
It has been shown that BSO can increase
sensitivity to platinum drugs in tumor cells cultured directly from individual patients,76 and in a
similar study BSO enhanced cytotoxicity of cisplatin or doxorubicin.77
Lai et al. isolated lung cancer cells with acquired CDDP resistance by intermittent drug
exposure.78 Enhanced DNA repair activity was seen in three cisplatin resistant cell lines.
Pretreatment of the cells with BSO, followed by pentoxifyline (another resistance modulator)
and CDDP combined treatment, effectively overcame acquired cisplatin resistance in vivo.
The cytotoxicity of a 2 hr CDDP or tetraplatin (TP) exposure with or without a 24 hr
BSO pretreatment was evaluated against human ovarian A2780 and murine leukemia L1210/0
cells and their corresponding CDDP resistant (A2780/CP and L1210/DDP).79 BSO significantly
decreased the IC 50 values of CDDP and TP against both ovarian lines. BSO enhanced only the
cytotoxicity of TP, but not CDDP against the L1210/0 cells, whereas it enhanced only the effect
of CDDP against the L1210/DDP cells. BSO also increased platinum and L-PAM toxicity (but
not that of doxorubicin or camptothean) for the U-937 leukemia cell line.80
Interestingly, in
cisplatin-resistant K562 leukemia cells, BSO enhanced NK cell killing, suggesting a role for
GSH in the latter process.81
Kramer et al. suggested that the tissue selectivity of BSO-mediated GSH depletion may
potentially determine which cytotoxic agents would be compatible for use with BSO.25 The
33
authors point to reports by Litterst et al. 82 and Kramer et al.,83 which indicated increased renal
toxicity of CDDP at time of maximal GSH depletion by diethylmaleate, a thiol depleting agent,
when used in combination with BSO.
Since BSO has been shown to have little effect on the
myelosuppression of L-PAM, Kramer et al. suggested that BSO might be more successfully
combined with agents whose dose-limiting toxicity is myelosuppression.25
Radiation. Most of the data concerning the effect of BSO and radiation response have
been generated from in vitro studies.
Biaglow et al. (1986) demonstrated that prolonged
exposure (24-120 hours) to 0.1 mM BSO increased, in a time-dependent fashion, the radiation
response of A549 cells under normoxic conditions.84
An increase in single-strand breaks and
irreparable cross-linking of DNA was also noted. Following treatment with BSO and radiation,
the increase in radiosensitivity was greater under normoxic than hypoxic conditions. The DMF
was 2.4 under normoxic conditions. In contrast, Shrieve and Harris reported a small increase in
radiation response in EMT6/SF cells treated with 50 µM BSO under hypoxic conditions and no
increase in cells treated similarly under normoxic conditions.52
BSO has also been shown to increase the radiosensitizing effect of nitroimidazoles. Ono
et al. examined the effect of BSO on radiation response to MISO in C3H/He mice bearing the
NFSa tumor.85
Radiation response was assessed using tumor growth delay time.
BSO 5
mmole/kg IP produced a marked increase in the radiosensitizing effect of MISO 0.5 mmole/kg.
The ER for MISO plus radiation, BSO plus radiation, and for BSO and MISO in combination
with radiation was 1.44, 1.16, and 1.93, respectively.
BSO has been useful in determining the role of GSH in intrinsic cellular resistance to
heat and in the development of thermotolerance.
Shrieve et al. examined the effect of GSH-
depletion by BSO in heat-sensitive and heat-resistant HA-1 Chinese hamster fibroblast cell
lines.86
Intracellular levels of GSH did not correlate with sensitivity to heat, but depletion of
GSH by BSO increased the sensitivity of both heat-sensitive and resistant cells to thermal stress.
Furthermore, development of thermotolerance was inhibited in the heat-sensitive cells only when
low levels of GSH were maintained for extended periods.
34
Other agents. As described above, BSO synergistically enhanced the cytotoxicity of the
bioreductive agent tirapazamine in neuroblastoma cell lines.41 A number of other drugs, of
diverse mechanisms, have been shown to have enhanced cytotoxicity for various tumor cell types
when combined with BSO.
The benzocronycine deriviatve S29306-1, apparently an aklyating
agent, showed enhanced cytotoxicity when combined with BSO.87 Depletion of GSH by BSO in
a glucocorticoid-resistant B-lineage ALL cell line restored the sensitivity to dexamethasone,
apparently via a caspase-independent mechanism.88
The cytotoxic retinoid fenretinide, has been
shown to generate enhanced reactive oxygen species and apoptosis in leukemia cell lines when
combined with BSO.89 BSO was able to overcome resistance to arsenic trioxide in leukemia cell
lines,90 but the combination of arsenic trioxide and BSO was rapidly fatal to mice (Reynolds CP,
et al, unpublished).
Dethylstilbesterol cytotoxicity for a protstate cancer cell line was enhanced
by BSO.91
35
PRECLINCIAL TOXICITY OF BUTHIONINE SULFOXIMINE (BSO, NSC-326231)
ALONE AND IN COMBINATION WITH MELPHALAN (NSC-8806)
(note, the following section is NCI data as reproduced from the original BSO Investigator’s Brochure)
SUMMARY:
Buthionine sulfoximine (BSO NSC-326231) inhibits tissue glutathione (GSH) synthesis,
lowers intracellular GSH levels and increases the efficacy of alkylating agents used in cancer
chemotherapy.
Single intravenous and oral doses of BSO produced a reversible depletion of
GSH levels in plasma, lung, liver and kidney tissue in mice.
BSO treatment resulted in no
clinical signs of toxicity nor microscopic lesions in mice when given as single and multiple oral
and intravenous doses ranging from 100 to 800 mg/kg.
Multiple intravenous doses of 1500
mg/kg dose of BSO (q4h x 6) produced a transient decrease in peripheral WBC in female mice.
BSO produced no changes in other clinical pathology parameters and no gross or microscopic
lesions were found in mice treated with 1600 mg/kg/dose (q4h x 6). A drug combination study
was designed to address the effect of BSO on melphalan-induced toxicity. Multiple intravenous
doses of 1600 mg/kg/dose (q4h x 6) of BSO resulted in maximal GSH depletion and caused a
potentiation of melphalan-induced bone marrow, lymphoid and rental toxicity. Multiple oral and
intravenous doses of up to 800 mg/kg/dose of BSO resulted in maximal liver GSH depletion and
no potentiation of melphalan toxicity.
BSO was lethal to dogs following a single intravenous dose of 3200 mg/kg/or up to 10
oral doses of 800 mg/kg/dose (q8h).
Clinical signs included emesis, diarrhea, hyperactivity,
tonic/clonic convulsions and seizures in the 800 mg/kg/dose group given up to 10 doses and
emesis and diarrhea in the 400 and 100 mg/kg/dose groups given a total of 15 doses.
BSO
administration also resulted in a significant decrease of GSH concentration in whole blood, lung,
liver and kidney.
Significant increases in SGPT, SGOT and alkaline phosphatase occurred in
dogs receiving a single intravenous dose of 3200 mg/kg or 9-10 oral doses of 800 mg/kg dose of
BSO (q8h).
Dogs sacrificed in a moribund condition after a single intravenous dose of 3200
mg/kg of BSO had microscopic lesions in the urinary bladder and exocrine pancreas.
36
Fifteen
oral doses (q8h) of 100 mg/kg dose of BSO were well tolerated by beagle dogs. Multiple IV
doses (60, 120 and 240 mg/kg/dose q 12 hours x 6) were also well tolerated in beagle dogs,
although slight decreases in RBC, HGB, and HCT were noted.
EXPERIMENTAL PROCEDURES:
Drug Preparation
L-Buthionine-S, R-sulfoximine (BSO, NSC-326231) was formulated in sterile 0.9%
sodium chloride injection.
Melphalan (L-Pam, NSC-8806) was supplied as the clinical
formulation. Sterile sodium chloride (0.9%) for injection was used as the vehicle (VCTL) in all
studies.
Animals
Purebred beagle dogs used in these studies were supplied by the Developmental
Therapeutics Program (DTP), Division of Cancer Treatment (DCT), and National Cancer
Institute (NCI).
CD2F1 mice were supplied by the NCI through Charles River Breeding
Laboratories (Portage, MI).
Bioavailability Study in Mice
Male CD2F1 mice were administered either 0.9% sodium chloride for injection (VCTL)
or 800 mg/kg of BSO orally or intravenously. Liver and plasma samples were collected at 0, 1,
2, 4, 8, 12, and 24 hours after dosing and assayed for GSH concentration. Blood samples for
plasma BSO level analysis were collected at 0, 2, 5, 10, 20, 30, 60, 90 minutes and 2, 4, 8, 12,
16, 20 and 24 hours after dosing. In a second group of mice, lung, liver and kidney samples
were collected 4 hours after BSO of VCTL administration and analyzed for GSH concentration.
37
Dose Response Study in Mice
Male CD2F1 mice were assigned to 3 dose groups and a VCTL group (12 mice per
group). Mice received a single dose of 1600, 1200, 800, or 0 mg/kg of BSO and were sacrificed
8 hours or 24 hours after dosing.
Liver and plasma samples were collected and analyzed for
GSH concentration.
Multiple Dose Toxicity in Mice
Oral Dose Study – Mice and female CD2F1 mice were assigned to 3 dose groups with a
vehicle control (VCTL) group (21 male and 15 female per group). Mice received BSO or 0.9%
sodium chloride (VCTL) orally by gavage every 8 hours for a total of 15 doses. BSO doses
administered were 800,400,100 or 0 mg/kg/dose. Three mice of each sex in each dose group
were sacrificed after 5 and 10 doses of BSO and on study days 9, 13, and 30. The mice were
evaluated for adverse clinical signs, changes in clinical pathology parameters, gross and
microscopic lesions. Additionally, 6 male mice per dose group were sacrificed 4 hours after the
15th dose of BSO and plasma, lung, liver and kidney GSH concentrations were determined.
Intravenous Dose Study – Male and female CD2F1 mice were assigned to 3 dose groups and a
VCTL group (30 male and 25 female mice per group). Mice were treated intravenously with
BSO or 0.9% sodium chloride every 4 hours for a total of 6 doses. Doses were 1600, 800, 400
and 0 mg/kg/dose BSO. Tissues from five mice of each sex in each dose group were evaluated
histologically on study day 2 and 29. Clinical pathology samples were taken on study day 2, 8,
15, 22, and 29. Additionally, 5 male mice per dose group were sacrificed one hour after the 5th
dose of BSO and liver GSH concentration was determined.
38
Effect of BSO on Melphalan-Induced Toxicity in Mice
Oral Dose Study – Male and female CD2F1 mice were assigned to 5 dose groups (25 male and
25 female mice per dose group) as follows:
Dose
Pretreatment
Treatment
Group
BSO (mg/kg/dose)
Melphalan (mg/kg)
A
800
5
B
800
1
C
800
0
D
0
5
E
0
1
BSO or pretreatment VCTL (0.9% sodium chloride) was administered orally by gavage every 8
hours for a total of 6 doses. One hour after the 5th dose of BSO or pretreatment VCTL, mice were
treated with a single intravenous dose of melphalan or treatment VCTL.
parameters were determined on study days 4, 5, 11, 18, and 30.
Clinical pathology
Gross and histopathology
examinations were done on days 11 and 30. Additionally, 5 mice of each sex were treated with
800 mg/kg/dose BSO or pretreatment VCTL every 8 hours and sacrificed for plasma, lung, liver
and kidney GSH determinations one hour after the 5th dose of BSO.
Intravenous Dose Study – Male and female CD2F1 mice were assigned to 3 BSO dose groups
and once VCTL group (35 male and 30 female mice per group).
Mice were pretreated with
1600, 800, 400 mg/kg/dose of BSO or 0.9% sodium chloride (VCTL) intravenously every 4
hours for a total of 6 doses. One hour after the 5th dose of BSO, a single intravenous 5
mg/kg/dose of Melphalan was administered.
Clinical pathology parameters were determined on
39
days 2, 4, 6, 11, 17 and 30. Histopathological examinations was done on tissues collected on
days 2 and 30. Liver glutathione levels were determined in 5 male mice from each dose group
sacrificed one hour after the 5th dose of BSO.
Bioavailability Study in Dogs
Male beagle dogs were dosed with either 120 mg/kg or 0 mg/kg BSO intravenously (3
dogs per group). Blood samples were collected for plasma BSO and GSH analyses at 0, 25, 5,
10, 20, 30, 60, 90, 120 minutes and 4, 8, 10, 12, 16, 20 and 24 hours after dosing. Due to lack of
GSH depletion, the dogs in the BSO group were treated with 3200 mg/kg BSO intravenously on
study day 29 and blood was collected for plasma GSH analysis of 0, 1, 2, 4, 8, 12 and 24 hours
after BSO. Complete necropsy and gross and microscopic examinations were done on all dogs
sacrificed moribund on day 30.
In addition, liver, kidney and lung samples were collected for
tissue GSH analysis.
Multiple Dose Toxicity Study in Dogs
Male and female beagle dogs were assigned to 3 dose groups and VCTL group (4 dogs
per group).
BSO doses (800, 400, 100n and 0 mg/kg/dose) were administered orally
approximately every 8 hours for a total of 15 doses. Blood was collected for whole blood GSH
levels on study days 2, 4 and 8 and for clinical pathology determinations on days 2, 4, 8 and
weekly thereafter to study day 65.
Liver GSH was assayed in all dogs sacrificed in a stress
moribund condition and all dogs sacrificed on study day 8.
Complete gross and microscopic
examinations were done on dogs sacrificed moribund and at scheduled necropsies on study day 8
and 65.
RESULTS
Pharmacokinetics
BSO was rapidly cleared from the plasma of dogs and mice after single intravenous or
oral doses of 2400 mg/m2 (Table 1). The plasma BSO concentrations versus time data is best
40
described as a bi-exponential function in mice and in 2 out of 3 dogs. In mice, the initial phase
had t1/2 of approximately 5 minutes and accounted for 94% of the total AUC. The T1/2 of the
terminal phase was approximately 40 minutes and accounted for 6% of the total AUC in mice.
By contrast, dogs dosed with 2400 mg/m2 had an initial phase t1/2 of about 8 minutes and the
initial phase represented only 20% of the total AUC. The t1/2 of the terminal phase was 36.3
minutes and accounted for 80% of the total AUC in dogs.
Even with the difference in BSO
elimination, mice and dogs had a similar rate of plasma drug clearance. Clearance of BSO from
plasma was 84 ml/min/m2 and 104 ml/min/m2 in mice and dogs, respectively.
Bioavailability
Bioavailability of BSO given orally, based on plasma drug levels, appeared to be about
2% in mice, suggesting that BSO was either not absorbed from the gastrointestinal tract or that it
was rapidly removed from the plasma. There was no apparent correlation between plasma BSO
levels and tissue GSH depletion after oral or intravenous dosing.
Additionally, there was no
significant difference in the time course or magnitude of plasma and liver GSH depletion after
oral and intravenous dosing with BSO. A single oral or intravenous dose of 800 mg/kg (2400
mg/m2 ) of BSO resulted in 75% depletion of plasma GSH and a 70% depletion of liver GSH.
Maximal GSH depletion occurred in liver and plasma within 2-4 hours after intravenous and 4-8
hours after oral dosing. There is no significant difference between the GSH depletion after oral
and intravenous dosing, which suggests that BSO is well absorbed from the gastrointestinal trace
and is rapidly removed from the plasma into tissue compartments.
Studies in Mice
In the dose response study, mice were treated with a single oral dose of 1600, 1200 and
800 mg/kg of BSO. Eight hours after dosing, plasma GSH levels were depleted about 70% and
liver GSH levels were depleted about 73%.
At 24 hours after dosing, plasma GSH
concentrations were 37 and 53% depleted, even though liver GSH had returned to 85-100% of
control levels. This data suggests that maximum GSH depletion occurs in mice after a single 800
mg/kg oral dose of BSO.
41
Mice given BSO orally at doses of 800, 400, and 100 mg/kg every 8 hours for a total of
15 doses showed a significant decrease in plasma GSH at all dose levels although the depletion
did not appear dose-dependent. Four hours after the 15th dose of BSO, plasma GSH levels were
depleted by 61-72% compared to vehicle control values. A dose response was present in some
tissues; four hours after the 15th dose of BSO, kidney GSH was depleted by 89%, 82% and 41%
and lung GSH was depleted by 60%, 48% and 35% in the 800, 400, and 100 mg/kg/dose groups,
respectively. Approximately 35% GSH depletion occurred in the livers of mice in the 800 and
400 mg/kg/dose groups, but there was no apparent dose response presumably due to rapid
recovery of liver GSH levels after BSO administration. In spite of marked GSH depletion, mice
treated orally with 800, 400, and 100 mg/kg dose for a total of 15 doses of BSO showed no
adverse clinical signs, no changes in clinical pathology parameters, or gross or microscopic
lesions at any dose level (Table 2). The only adverse sign of toxicity was a slight loss in body
weight in male mice during the dosing period. Between study day 2 and 6, male mice lost an
average of 10.7% of their day – 1 body weight in the 800 mg/kg/dose group. By study day 10,
body weights of these mice were comparable with the VCTL group.
BSO administered intravenously at doses of 1600, 800, and 400 mg/kg/dose (q 4h x 5)
caused an 88% depletion of liver GSH in male mice. There was no apparent dose response in
liver GSH depletion at the administered BSO dose levels; all 3 BSO doses produced maximal
GSH depletion.
Toxicity of intravenously administered BSO was limited to a transient
depression (67%) in WBC levels and a slight 10% body weight loss in female mice in the 1600
mg/kg/dose group (Table 2).
There were no drug-related gross or microscopic lesions in any
mice treated with BSO intravenously (q 4h x 6) at doses up to 1600 mg/kg/dose.
BSO given to mice on a chronic basis caused cataracts,92 but this particular toxicity has
not seen in acute dosing of animals or humans.
The effect of BSO on melphalan-induced toxicity was assessed in CD2F1 mice. A single
intravenous dose of 5 mg/kg of Melphalan produced gastrointestinal toxicity and a 50%
depression of peripheral WBC counts with histopathological evidence of thymic atrophy, but no
evidence of bone marrow atrophy.
Pretreatment of mice with up to 800 mg/kg/dose of BSO
42
either intravenously (q 4h x 6) or orally (q 8h x 6) did not potentiate melphalan-induced
leukopenia, however, BSO pretreatment appeared to slightly delay the recovery of WBC to
control values.
In mice pretreated intravenously with 1600 mg/kg/dose of BSO (q 4h x 6),
melphalan produced a rise in blood urea nitrogen and the appearance of renal nephrosis in 5/5
male mice and 1/5 female mice.
In addition, BSO potentiated the bone marrow toxicity of
melphalan as evidenced by the appearance of histopathological evidence of bone marrow atrophy
in 2/5 male mice.
Accompanying bone marrow atrophy, mice pretreated intravenously with
1500 mg/kg/dose of BSO before melphalan administration had splenic myeloid tissue atrophy
and splenic lymphoid and mesenteric lymph node necrosis. All of these lesions were reversible
and were completely resolved by day 30.
Studies in Dogs
A single intravenous dose of 3200 mg/kg of BSO was lethal to beagle dogs producing
hyperactivity, repeated tonic/clonic convulsions, acute hemorrhage and ulceration of the urinary
bladder with hematuria and mild to marked depletion of exocrine pancreas.
Multiple intravenous doses (60, 120, and 240 mg/kg/dose) of BSO were administered
every 12 hours x 6. No apparent hematologic changes occurred at the 60 mg/kg/ dose level. An
incidental decrease in the neutorphil count of one dog occurred on days 5 and 36 (38% and 23%
below baseline, respectively).
Drug-related hematologic changes which included a slight
depression of the RBC, HGG, and HCT were observed in one dog at the 120 mg/kg/dose level.
Increases in neutrophil and lymphocyte were observed in three dogs at the same dose on several
days throughout the study. One female exhibited an incidental decrease in platelet count (56%
below baseline) in RBC, HGB, and HCT in two males on day 5. Two males and one female
exhibited an incidental decrease in neutrophils on day 5 (28% below baseline in one male, 2440% in the other, and 300-36% in the female). All parameters had returned to baseline by the
end of the study. There were no treatment-related changes in clinical chemistry parameters. In
addition, there were no apparent drug-related gross necropsy observations or histiopathological
lesions.
43
Multiple doses (800, 400 and 100 mg/kg/dose) of BSO were administered orally to dogs
approximately every 8 hours for up to 15 doses.
Whole blood GSH levels were maximally
depleted by 52-70%. These changes in whole blood GSH, however, did not occur in a doserelated manner. Liver GSH levels in the 800 mg/kg/dose group were 95% depleted in the 3 dogs
sacrificed moribund 6-24 hours after their 9th or 10th dose of BSO. Sixty hours after the 15th dose
of BSO, dogs in the 400 mg/kg/dose group had a 78% depletion of liver GSH.
In the 100
mg/kg/dose group, liver GSH levels were comparable to control values 60 hours after the 15th
dose of BSO. Dogs in the 800 mg/kg/dose group received only 9 or 10 doses group received
only 9 or 10 doses of BSO and displayed adverse clinical signs of emesis, diarrhea,
hyperactivity, and convulsions during the dosing period (Table 3).
Three out of four of these
dogs had multiple convulsions, so BSO dosing was discontinued and the dogs were sacrificed as
moribund on study day 4 or 5 for humane reasons.
scheduled sacrifice on study day 65.
The remaining dog survived until its
Increases in SGOT and alkaline phosphatase activities
occurred in all the dogs in the 800 mg/kg/dose group beginning on day 2. Emesis and diarrhea
occurred in the 400 mg/kg/dose group beginning on study day 2 and continued during the dosing
period. One out of four dogs in the 400 mg/kg/dose group was hyperactive after receiving the 8th
dose of BSO, but no convulsive activity was present. Increased SGPT activity occurred in ¾
dogs in the 400 mg/kg/dose group and elevated SGOT and alkaline phosphatase occurred in one
dog between study days 4 and 15. There were no drug-related microscopic lesions in any of the
dose groups.
DISCUSSION:
Buthionine sulfoximine (BSO, NSC-326231) is a potent inhibitor of t-glutamylcysteine
synthetase, an enzyme involved n the synthesis of glutathione (GSH).
tripeptide that acts to protect cells from a variety of cytotoxins.
GSH is an intracellular
Because of the chemically
reactive nature of most anti-neoplastic agents, GSH also protects tumor cells from the
cytotoxicity of chemotherapeutic drugs used in the treatment of cancer.
Numerous pre-clinical
studies have shown that GSH depletion produced by BSO administration increases the efficacy
44
of melphalan, a potent alkylating agent, in human tumor cell lines and human tumor xenografts
in nude mice. The present studies were designed to determine the toxicity of BSO in dogs and
mice and additionally to explore the effect of BSO on melphalan-induced toxicity in mice.
BSO
administration
caused
toxic
effects
gastrointestinal and urinary tracts of beagle dogs.
to
the
central
nervous
system
and
Clinical pathology changes indicated possible
hepatic toxicity (increases in SGOT, SGPT and alkaline phosphatase), however, no drug-related
microscopic lesions were present in the liver. BSO administration caused severe GSH depletion
in dogs, however, the condition of the dogs after multiple convulsions make quantitative
interpretation of the changes in GSH levels difficult, so no direct comparison in the magnitude of
GSH depletion and the toxicity of BSO in mice and dogs can be made. Toxicity was reversible
and there was no apparent sex difference in the response of dogs to BSO. Mice tolerated large
multiple oral and intravenous doses of up to 800 mg/kg/dose of BSO without apparent adverse
effects. The only adverse effect of BSO in male mice receiving multiple oral doses of BSO was
a significant but quantitatively small weight loss.
The administration of 1600 mg/kg/dose of
BSO (q4h x 6) intravenously caused a transient depression of WBC counts in female mice,
however, no bone marrow lesions were present at necropsy. Pretreatment of mice with multiple
oral or intravenous doses of up to 800 mg/kg/dose of BSO to induce GSH depletion did not
significantly alter the toxicity of melphalan.
However, melphalan administration produced renal,
bone marrow and lymphoid toxicity in mice pretreated with very high intravenous doses of BSO
(1600 mg/kg/dose q4h x 6).
This potentiation of melphalan toxicity by BSO pretreatment
doesn’t appear to be related to GSH depletion, since a quantitatively similar amount of GSH
depletion occurred at lower doses of BSO without any potentiation of melphalan toxicity.
The elimination of BSO from plasma fit a bi-exponential function in both dogs and mice.
The elimination half-life values for the initial and terminal phases were similar in dogs and mice.
However, in mice the AUC of the initial phase accounted for 94% of the total AUC while in
dogs it accounted for only 20% of the total AUC. In dogs, elimination was represented by the
terminal phase of the curve and in mice it was represented by the initial phase. This difference in
the pattern of BSO elimination may, in part, account for the absence of toxic effects of BSO in
45
mice.
Oral bio-availability of BSO based on plasma drug levels was approximately 2%.
However, comparable GSH depletion occurred after intravenous and oral dosing, suggesting
BSO is rapidly removed from plasma into tissue compartments and that the oral bio-availability
of BSO is most likely higher than 2%.
46
TABLE 1
PHARMACOKINETIC PARAMETERS OF BUTHIONINE SULFOXIMINE
SPECIES
BEAGLE DOGS
CD2F1 MICE
(mg/kg)
120
800
(mg/m2 )
2400
2400
(ml/min/kg)
5.18 ±0.53
28
(ml/min/m2)
104
84
t1/2
initial (min)
7.7
5
t1/2
terminal (min
36.3 ± 9.7
37
Vd term
(ml/kg)
266 ± 72
1490
Vd 55
(ml/kg)
233 ± 23
280
AUC Total
(µg/ml-min)
23800 ±2400
28500
Dose
C1
47
TABLE 2
CHARACTERISTICS OF BUTHIONINE SULFOXIMINE TOXICITY IN MICE
SCHEDULE
Doses
ORAL (q8h x 15)
I.V. (q4h x 6)
800 mg/kg/dose
1500 mg/kg/dose
400 mg/kg/dose
800 mg/kg/dose
100 mg/kg/dose
400/mg/kg/dose
Mortality
•
none
•
none
Clinical Signs
•
none
•
none
Clinical Pathology
•
none
Body Weights
Histopathology
•
•
1600 mg/kg/dose
•
day 2
•
1 WBC in females
1600 mg/kg/dose
none
no drug related lesions
48
•
day 2
•
10% decrease in females
•
no drug related lesions
TABLE 3
BUTHIONINE SULFOXIMINE TOXICITY IN DOGS
SCHEDULE
Dose(s)
SINGLE DOSE (IV)
MULTIPLE DOSES (Oral q8h)
X 9/10
800 mg/kg/dose
mg/kg/dose
3200 mg/kg
x 15
400
120 mg/kg
Mortality
Clinical Signs
100
mg/kg/dose
3 /4 800 mg/kg/dose x 9/10 dose
800 mg/kg/dose
3 /3 3200 mg/kg
3200 mg/kg
•
day 1
•
hyperactivity/aggressive
behavior
•
•
•
•
•
day 1 – 6
diarrhea/emesis
hyperactivity
convulsions
convulsions/seizures
400 and 100 mg/kg/dose
•
•
•
Clinical Pathology
hematuria
dilated pupils
hypersalivation
3200 mg/kg
•
•
day 2 – 15
diarrhea/emesis
800, 400 mg/kg/dose
•
•
day 1
increases in
•
•
day 4 – 15
increases in:
Alkaline phosphatase
Alkaline phosphatase
SGOT
SGOT
SGPT
SGPT
Serum Glucose
Chloride
Hematocrit
Histopathology
3200 mg/kg
•
no drug related lesions
urinary bladder:
hemorrhage
ulceration
•
pancreas:
depletion of
49
EXPERIENCE WITH BSO IN HUMANS
BSO has been tested in phase I trials in adults, alone and in combination with L-PAM.
The initial phase I studies tested 30 min infusions of BSO every 12 hours, alone (6 to 10 doses,
escalation of BSO dosing from 1.5 to 13 g/m2 ), or in combination with 15 mg/m2 of L-PAM
given as a bolus, alone or in combination with BSO.14;
16
alone to L-PAM given with BSO showed a
significant increase in leukopenia and
Comparsion of courses of L-PAM
thrombocytopenia when L-PAM was given with BSO compared to L-PAM alone.
To further
enhance the degree of GSH depletion at the time of L-PAM administration, another dose
schedule was tested, with a loading dose of 3 g/m2 bolus over 30 minutes i.v. followed by a 24,
48, or 72 hour continuous infusion of BSO at 0.75 g/m2 /hr.15 The preferred dosing appeared to
be the BSO 3 g/m2 bolus loading dose followed by 72 hours of 0.75 g/m2 /hr BSO with L-PAM
(15 mg/m2 bolus at hour 48 of BSO infusion).15
The latter schedule of BSO alone produced
minimal systemic toxicity, but when combined with L-PAM caused substantial, but tolerable,
myelosuppression. A pilot study in pediatrics tested the same schedule and also tested increasing
the c.i.v. for BSO to 1.0 g/m2 /hr.10; 11
Human Pharamacokinetics
The T ½ of BSO was < 2 hours when given as 30 min infusions every 12 hours,14;
~ 2.3 hours after continuous i.v. infusion.10;
L-PAM AUC.10;
11; 15
11; 15
16
and
BSO may decrease clearance and increase the
Significant differences in clearance and t1/2 have been observed between
the R and S stereoisomers that comprise the clinical formulation of BSO.93 The pediatric BSO
50
and L-PAM (given with BSO) pharmacokientic data and a summary of that from the adult
clinical trials is presented in Table 4.
TABLE 4 PHARMACOKINETCIS OF BSO AND L-PAM + BSO
Dose level
Total BSO
Css (µM)
1
2
1 vs. 2
Adult data
Dose level
(0.75 g/m2/hr x 72 hours) 94
1
2
1 vs. 2
Adult
data
2
BSO CLss
(ml/min/m2)
Total BSO
t 1/2 hr
346 +/- 176
524 +/- 207
P = 0.02
465 +/- 189
Total BSO
AUC (mM x
hr)
26 +/- 12
41 +/- 16
P = 0.01
31 +/- 8
193 +/- 77
177 +/- 72
P = 0.08
136 +/- 45
2.3 +/- 1.2
2.2 +/- 1.1
P = 0.60
3.7
L-PAM Peak
(µM)
3.02 +/- 0.3
3.32 +/- 1.2
P = 0.46
N/A
L-PAM AUC
(mM x hr)
66 +/- 24
82 +/- 27
P = 0.15
53 +/- 24
L-PAM CLss
(ml/min/m2)
252 +/- 88
204 +/- 83
P = 0.22
343 +/- 171
L-PAM
t 1/2 hr
1.5 +/- 0.5
1.9 +/- 0.5
P = 0.13
1.1 +/- 0.4
(15 mg/m L-PAM + q 12 hr BSO)
Pharmacodynamic studies showed both decreased GSH (40% of baseline) and inhibition
of the γ-GCS activity (the enzyme targeted by BSO) were demonstrated in blood mononuclear
cells.14; 16
A high degree of GSH depletion in tumor tissue (< 10% of baseline) during
continuous infusion BSO was demonstrated in a subsequent study.15 Depletion of GSH by BSO
in blood mononuclear cells occurred to 30-40% of baseline in adults and ~46% in children, and
appears to return to normal within 4 days of stopping the BSO. Pharmacodynamic modeling of
data from a phase I trial suggested a gradual depletion of GSH occurring over about 30 hours, 95
51
and RNA expression of γ-GCS in mononuclear cells, which showed a 3-fold variability from
patient to patient, also showed a compensatory upregulation in response to BSO treatment.80
Safety and Efficacy
The reported systemic toxicities of BSO and L-PAM in adults were unremarkable with
reversible myelosuppression being the most common.14-16
Documented responses have occurred
in patients with melanoma (complete response 18 months), ovarian cancer, breast cancer, and
small cell carcinoma of the lung.14-16
A pilot study of BSO combined with 15 mg/m2 of L-PAM in children with
recurrent/refractory high-risk neuroblastoma achieved a 29% response rate (duration 1-3
months), with 7 partial (6 of 7 had previous myeloablative therapy) and 2 minor responses
(Preprint in Appendix).10;
relapse therapy.
11
Of 9 responders, 5 were treated with BSO/L-PAM as their primary
One patient, whose pre-relapse therapy only included chemotherapy with no
myeloablative therapy or 13-cis-retinoic acid, had a marked response to BSO/L-PAM with 98%
reduction in size of left pelvic mass, improvement of liver nodules and resolution of bone
marrow disease following 2 courses of therapy. There were 7 patients that experienced a greater
than 25% improvement of their disease and most patients had a maximal response after 1 course
of BSO/L-PAM therapy.
Three patients had multi-log improvement in bone marrow
involvement by immunocytology that corresponded to a marked reduction of tumor observed by
routine microscopic examination of biopsy and aspirate. An additional patient showed resolution
of bone marrow (tumor undetectable by immunocytology) of a marrow showing 50% tumor
involvement by light microscopy prior to BSO/L-PAM therapy
52
(no immunocytology available
at baseline). Two patients had painful, subcutaneous scalp masses that completely resolved, one
of these patients required continuous morphine that was no longer required within days after the
1st course of BSO/L-PAM.
Except for two patients with severe toxicity (see below), toxicity in the pediatric study
was similar to that in adults, with most patients showing grade 3-4 leukopenia and
thrombocytopenia. The degree of hematopoietic toxicity seen with BSO + L-PAM appears to be
increased over what would be expected for L-PAM alone at the same dose. There appeared to be
cumulative hematopoietic toxicity for patients treated with more than 2 courses of BSO + LPAM, especially with respect to platelet recovery.
In contrast to the adult studies, there were 2 deaths due to CNS toxicity in the first
pediatric study of BSO + L-PAM.
Both patients having a toxic death presented with acute
tubular necrosis; one had a large, intracranial mass.
No other significant toxicities were seen.
The first patient experiencing a toxic death had received one course of BSO + L-PAM without
undue toxicity, and developed renal toxicity followed by neurotoxicity during the BSO infusion
in the 2nd course; L-PAM was not given during the course of therapy with toxicity. The second
patient (patient with the CNS mass) received both BSO and L-PAM before developing renal and
neurotoxcity.
The onset of toxicity in the first patient during BSO alone might suggest an
unusual sensitivity to GSH depletion, but the patient had received BSO + L-PAM one month
before without any remarkable toxicity.
Review of all data from the two toxic deaths suggests the possibility of a drug interaction
between BSO and cephalosporin antibiotics could have lead to BSO toxicity (preprint in
appendix).11
Clinical review of both cases found that cephalosporin antibiotics were
53
administered during the period of BSO infusion (treatment of cellulitis in first patient; fever in
second).
At least one other patient is known to have received cephalosporin (treatment of
cellulitis) during their first of three courses of BSO and had no renal or neurologic sequelae.
An analysis of the pediatric pilot trial found that 14/32 patients had a history of either
total body irradiation (TBI) (N=7), abdominal radiation (kidneys in field; N=6 not including TBI
patients), or cranial radiation (N=6; not including TBI patients) prior to BSO therapy. 11
Of the 2
toxic deaths, one (pt #6) had previous TBI, whereas the other (pt. #30) had radiation to a nonspecified pelvic site (no kidney or cranial radiation).
Assessment of renal function during the
first course of therapy revealed 19/32 patients with no abnormalities in urinalysis or serum
creatinine while receiving BSO, 4 patients had pre-existing 1-3+ proteinuria/glucosuria with
normal serum creatinine, and 4 patients developing 1-2+ proteinuria/glucosuria with normal
serum creatinine while on BSO.
The new onset during BSO therapy of > 3+ proteinuria and
glucosuria, combined with elevation of baseline serum creatinine, had a significant (P = 0.02 by
Fisher’s exact test) correlation with BSO toxicity leading to death.
The complete etiology of the 2 toxic deaths remains to be defined, as adult trials have had
no reports of significant neurologic or renal toxicity.
Pre-clinical studies of BSO in animals
demonstrated minimal GSH depletion in brain (> 70% control) with rapid recovery13 and no
neurologic toxicity except that when substantial doses of BSO were given to large animals,
reversible aggressiveness and convulsions were observed.12-14
It is speculated that the large
intracranial mass contributed to the second pediatric patient’s grade 5 toxicity, perhaps in
addition to the mass effect, by altering the blood-brain barrier and increasing intracranial drug
levels.
54
Although descriptive, the analysis of prior radiation therapy in the 32 children treated
with BSO suggested that there is no evidence of an association between a history of radiation
prior to BSO therapy and the subsequent development of neurologic or renal toxicity. 11
Despite
no difference in plasma creatinine, pre-clinical evaluation of BSO has shown sub-clinical
evidence of acute tubular necrosis by light microscopy in mice treated with BSO.96
Although early cephalosporins such as cephaloridine are known to cause acute tubular
necrosis by a GSH-dependent process involving free radicals made during proximal renal tubular
transport,97 the third generation cephalosporins used by the children experiencing grade 5
toxicity have a very low potential for proximal renal tubular membrane transport.97;
98
However,
even forms of cephalosporins considered safe for patients with renal failure have had anecdotal
reports of acute tubular necrosis mediated by hypersensitivity reactions.99
Importantly, in this group of children treated with BSO, the new onset of high-grade proteinuria
and glucosuria appears to foreshadow life-threatening complications. Although high-dose BSO
has been reported to cause substantial nigral changes in rodents,100 review of the literature
revealed no definite cause of these renal and neurologic toxicities. Neurotoxicity from BSO in
cell culture has also been associated in vitro with increased extracellular copper levels,101 though
there was no known reason why increased copper would have occurred in the patients with BSO
toxicity. Until the exact etiology of BSO toxicity is defined, the authors recommend patients
with intracranial disease be excluded and all cephalosporin antibiotics be avoided during BSO
administration. It is also important that drugs known to deplete GSH, such as acetaminophen,
not be given just prior to, together with, or soon after BSO administration.
55
SUMMARY OF DATA AND GUIDANCE FOR THE INVESTIGATOR
Data obtained with a variety of tumor systems have established a role for GSH in
mediating drug resistance to several types of drugs, in particular alkylating agents and platinum
compounds.
Increased glutathione (GSH) is common in drug-resistant cancer cells, and the
mechanism appears to be transcriptional up-regulation of RNA for, and consequently increased
production of γ-glutamylcysteine synthetase
(γ-GCS), the initial enzyme in the synthetic
pathway for GSH. BSO is selective inhibitor of γ-GCS that can decrease GSH levels in tumor
cells, prevent tumor-cell reactionary GSH increases in response to chemotherapy, and appears to
have preferential activity in depleting GSH from tumor rather than many normal cells (including
bone marrow).
BSO has been shown to be tolerable when given alone, and in combination with the
alkylating agent melphalan (L-PAM) to animals and to humans, including children.
BSO
increases the degree of hematopoietic toxicity seen with L-PAM, limiting the use of L-PAM to
low levels when combined with BSO. Because the dose-limiting systemic toxicity of BSO when
combined with L-PAM is hematopoietic, increased activity may be achieved if extra-medullary
toxicities allow dose escalation of L-PAM when given with BSO by using hematopoietic stem
cell support.
Two toxic deaths (renal and neurotoxicity) occurred in the pediatric BSO + L-PAM pilot
study, one associated with BSO treatment alone, the other with BSO + L-PAM. The etiology of
those two toxic deaths remains unclear, though careful review of the two toxic deaths suggests
56
that an intracranial mass in one patient, and the possibility of an interaction between BSO and
cephalosporin
antibiotics
pre-disposed,
or
perhaps
caused,
the
observed
toxicities.
Accordingly, investigators are cautioned that use of BSO in patients with intracranial mass
disease, or in patients being treated with any potentially nephrotoxic or neurotoxic drug
(including cephalosporin antibiotics) is contraindicated until further information on the potential
for BSO toxicity in those situations is obtained.
Other drugs known to interact with GSH,
especially acetaminophen, are also contraindicated during the use of BSO.
The clinical activity seen with BSO + low-dose L-PAM in recurrent neuroblastoma
suggests that BSO + L-PAM is a promising novel therapy for recurrent neuroblastoma,
especially if L-PAM levels can be dose-escalated using hematopoietic stem cell support.
While
clinical studies have only been conducted with BSO combined with melphalan, it is possible that
BSO may enhance the activity of other anti-neoplastic drugs.
However, there remains the
significant concerns for increased systemic toxicity, especially with agents such as the platinum
compounds.
Thus, further pre-clinical toxicology studies will be necessary before undertaking
any clinical trials with combinations other than BSO + L-PAM.
In the case of apparent BSO toxicity, N-acetylcysteine (NAC) is not currently
recommended, as the BSO will prevent NAC from enhancing GSH synthesis and it is unknown
if the oral formulation of NAC available in the USA can be safely given in such a setting. An
alternative would be the parenteral thiol agent sodium thiosulfate (STS), but again caution should
be employed as the effects of combining STS with BSO are not well understood.
57
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