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Phase II Trial of Carmustine Plus O⁶-Benzylguanine for Patients With Nitrosourea-Resistant Recurrent or Progressive Malignant Glioma

From the Departments of Surgery, Medicine, Pathology, Radiology, and Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC; Department of Medicine, University of Chicago, Chicago, IL; Department of Cellular and Molecular Physiology and Pharmacology, Pennsylvania State University School of Medicine, Milton S. Hershey Medical Center, Hershey, PA; Chemistry of Carcinogenesis Laboratory, Advanced Bioscience Laboratories, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick; and Investigational Drug Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Address reprint requests to Jennifer A. Quinn, MD, The Brain Tumor Center, Duke University Medical Center, Box 3624, Durham, NC 27710; email: quinn008{at}mc.duke.edu

	ABSTRACT

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PURPOSE: We conducted a phase II trial of carmustine (BCNU) plus the O⁶-alkylguanine-DNA alkyltransferase inhibitor O⁶-benzylguanine (O⁶-BG) to define the activity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resistant to nitrosoureas.

PATIENTS AND METHODS: Patients were treated with O⁶-BG at an intravenous dose of 120 mg/m² followed 1 hour later by 40 mg/m² of BCNU, with cycles repeated at 6-week intervals.

RESULTS: Eighteen patients were treated (15 with glioblastoma multiforme, two with anaplastic astrocytoma, and one with malignant glioma). None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity. Twelve patients experienced reversible grade 3 hematopoietic toxicity. There was no difference in half-lives (0.56 ± 0.21 hour v 0.54 ± 0.20 hour) or area under the curve values (4.8 ± 1.7 µg/mL/h v 5.0 ± 1.3 µg/mL/h) of O⁶-BG for patients receiving phenytoin and those not treated with this drug.

CONCLUSION: These results indicate that O⁶-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitrosourea-resistant malignant glioma, although stable disease was seen in five patients for 6, 12, 12, 12, and 18 weeks. Future use of this approach will require strategies to minimize dose-limiting toxicity of BCNU such as regional delivery or hematopoietic stem-cell protection.

	INTRODUCTION

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THE FAILURE TO cure patients with a diverse spectrum of malignancies, including malignant glioma, can be directly attributed to resistance to chemotherapy. Because virtually all malignant gliomas display marked de novo or acquired drug resistance, standard therapy, including alkylnitrosourea-based chemotherapy, cures the minority of patients with anaplastic astrocytoma and few, if any, patients with glioblastoma multiforme.^1-3 Because carmustine (BCNU) is associated with modest increase in median survival, it remains the community gold standard of care.^4-5 Unfortunately, resistance to this alkylnitrosourea invariably occurs with subsequent therapy failure and patient death.

Preclinical and clinical studies have demonstrated that the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) creates resistance to alkylnitrosoureas by removing groups damaged by chloroethylation or methylation from the O⁶-position of DNA guanines in time to prevent cell injury or death.^6-20

The high incidence of AGT activity in human CNS tumors suggests that AGT may play a role in the resistance of these tumors to alkylnitrosoureas.²¹ Recent clinical trials have shown an inverse relationship between survival and AGT levels in patients with malignant glioma who receive BCNU therapy,^22-25 thus providing a strong rationale for strategies designed to deplete tumor AGT levels before therapy with BCNU. O⁶-benzylguanine (O⁶-BG) is an AGT substrate that inactivates AGT and enhances alkylnitrosourea activity both in vitro and in vivo.^26-35 We previously reported a phase I trial of O⁶-BG that defined the dose of O⁶-BG effective in depleting AGT to be 100 mg/m².³⁶ This was followed by a phase I trial of BCNU plus O⁶-BG where we found the maximum-tolerated dose of BCNU to be 40 mg/m² when used in combination with O⁶-BG in patients with recurrent malignant glioma.³⁷ We now report a phase II trial of BCNU plus O⁶-BG in patients with recurrent or progressive, nitrosourea-resistant malignant gliomas that was designed to define the activity of this combination in patients who failed a nitrosourea regimen.

	PATIENTS AND METHODS

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Protocol Objectives
The objectives of the study were as follows: to define the activity of BCNU plus O⁶-BG in the treatment of adults with progressive or recurrent malignant glioma, and to further define the toxicity of this regimen.

Patient Eligibility Criteria
Patients were eligible if they had a recurrent or progressive glioblastoma multiforme, anaplastic astrocytoma, or gliosarcoma that was initially diagnosed by histologic examination and was resistant to nitrosoureas. Nitrosourea resistance was defined as progressive or recurrent disease within 8 weeks of receiving a nitrosourea. Patients with progressive disease after implantation of Gliadel (Guilford Pharmaceuticals, Baltimore, MD) wafers did not constitute resistance to nitrosourea. Patients with apparent tumor progression after stereotactic radiosurgery or brachytherapy required histologic confirmation of tumor by biopsy. For entry onto the study, there must have been measurable disease on a contrast-enhanced magnetic resonance imaging (MRI) or computed tomographic (CT) scan. The patient must have been at least 18 years of age, and have had a Karnofsky performance status 60%. Additional enrollment criteria included the following: adequate pretreatment bone marrow function (hemoglobin > 10 g/dL, absolute neutrophil count [ANC] > 1,500 cells/µL, platelet count > 100,000 cells/µL), renal function (serum creatinine < 1.5 mg/dL, blood urea nitrogen 25 mg/dL), hepatic function (serum AST 2.5 times the upper limits of normal, total bilirubin within normal limits), and pulmonary function (diffusing capacity [DLCO] > 80% predicted). For patients on corticosteroids, a stable dose was required at baseline neuroimaging and 2 weeks before entry. Patients must have recovered from toxicity of prior treatment of a nitrosourea, procarbazine, or mitomycin C within 6 weeks of protocol entry. Patients with reproductive potential were required to practice effective birth control measures for the duration of the study and 2 months after completing therapy. All patients were informed of the investigational nature of the study and were required to provide informed consent as approved by the institutional review board.

The following patients were excluded from the study: pregnant women, nursing women, potentially fertile women or men who were not using an effective contraceptive method, and patients who received prior nitrosourea exposure more than 1,200 mg/m². Patients who received prior chemotherapy or radiotherapy within 4 weeks of protocol entry were also excluded.

Treatment Design
Patients were treated with 120 mg/m² intravenous O⁶-BG followed 1 hour later by 40 mg/m² of intravenous BCNU. Both drugs were administered on the first day of each cycle. Cycles were repeated at 6-week intervals. Treatment was administered until unacceptable toxicity or tumor progression occurred.

O⁶-BG was supplied by the National Cancer Institute (NCI) (Bethesda, MD) in a dual pack with a diluent. The O⁶-BG was provided as a 100-mg vial of lyophilized powder with 670 mg mannitol (United States Pharmacopeia) and sodium hydroxide. The diluent was provided as a 30-mL vial containing a sterile solution of 40% polyethylene glycol 400 in pH 8 phosphate buffer (106 mg dibasic sodium phosphate, 102 mg monobasic potassium phosphate in sterile water for injection [United States Pharmacopeia]). The 30 mL of diluent was added to the O⁶-BG vial. Completely in solution, the O⁶-BG was further diluted to 0.04 mg/mL with 0.9% saline and given intravenously over 1 hour. BCNU was commercially available and administered intravenously in 0.9% saline over 1 hour starting 1 hour after the completion of the O⁶-BG infusion.

The dose of BCNU was reduced to 33 mg/m² for any patient experiencing the following: febrile neutropenia, ANC less than 500 cells/µL, grade 4 thrombocytopenia (under the NCI expanded common toxicity criteria), a platelet transfusion, or 14-day delay in the administration of a cycle of therapy. Patients were allowed a second dose reduction to 25 mg/m². Any patient requiring more than two dose reductions was removed from study.

The criteria for retreatment consisted of the following: ANC more than 1,500 cells/µL, platelets more than 100,000 cells/µL, hemoglobin more than 10 g/dL, AST 2.5 times upper limit of normal, blood urea nitrogen 25 mg/dL, creatinine less than 1.5 times upper limit of normal, total bilirubin within normal limits, and DLCO more than 75% of baseline DLCO. All other toxicities should have resolved to baseline or grade 1 for retreatment.

Therapy was discontinued and the patient was considered off study for the following reasons: if the patient showed evidence of progressive or recurrent disease as documented by MRI or CT scan anytime after the completion of at least one cycle of therapy, if the patient experienced grade 4 nonhematopoietic toxicity, if the patient failed to achieve the retreatment criteria within 8 weeks of the previous BCNU plus O⁶-BG dose, if the patient’s DLCO was 60%, if the patient required more than two dose reductions, if the patient was noncompliant with the protocol, and if the patient chose to voluntarily withdraw from the study. Also, therapy was discontinued and the patient was considered off study if the investigator thought it was in the patient’s best interest to stop therapy.

Supportive Care
Prophylactic antiemetics were permitted as needed. Neurologic stability was provided with the lowest corticosteroid dose when required. Hemopoietic growth factors were administered at the treating physician’s discretion for the management of grade 4 myelosuppression. However, these patients were removed from treatment but followed on the study. Blood products were administered at the treating physician’s discretion.

Evaluation Before and During Therapy
Patients underwent physical and neurologic examinations, hematology tests, chemistries, and MRI scans before every 6-week cycle including the first cycle. In addition to these examinations, a urinalysis and pulmonary function test were performed before the first cycle along with beta human chorionic gonadotropin in women with reproductive potential. Pulmonary function tests were also repeated before every odd-numbered cycle. Weekly hematology tests and every-other-week chemistries were also required. Pharmacokinetic analysis was performed only during the first cycle.

Toxicity Evaluation
Toxicity was graded according to the NCI expanded common toxicity criteria version 2. Patients were evaluated for acute toxicity by physical examination and quantitation of hematologic, renal, hepatic, pulmonary function, and serum electrolytes.

Response Evaluation
Response determination was derived from both of the following: comparison of baseline contrast-enhanced MRI scan with those obtained before every 6-week course of therapy and changes in physical findings on neurologic examination. Complete response was defined as the disappearance of all enhancing tumor on contrast-enhanced MRI or CT scans at least 1 month apart, with the patient receiving no corticosteroids and determined to be neurologically stable or improved. Partial response was defined as a greater than 50% reduction in the size (product of largest perpendicular diameters) of enhancing tumor maintained for at least 1 month in a patient who was neurologically stable or improved while on a stable or reduced dose of corticosteroids. No response was defined as either no change in tumor size after 1 month, or a change in tumor size after 1 month that did not qualify as a complete response, partial response, or progressive disease. Progressive disease was defined by the following: either a greater than 25% increase in size (product of largest perpendicular diameters) of enhancing tumor or any new tumor on MRI or CT scan, or deterioration of the patient’s neurologic status while on a stable or increased dose of corticosteroids, or a permanent increase in steroid dose while remaining neurologically stable.

Plasma Sampling
Whole-blood samples were collected in a sodium-heparinized Vacutainer (Becton Dickinson, Franklin Lakes, NJ) before treatment at 30 and 60 minutes into the infusion of O⁶-BG and at 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, and 24 hours after completion of the infusion. Plasma was obtained by centrifugation at 2,500 rpm for 10 minutes. Samples were stored at -70°C until analysis.

High-Performance Liquid Chromatography Analysis of O⁶-BG and Metabolites
Total plasma concentrations of O⁶-BG and 8-oxo-O⁶-BG were measured by high-performance liquid chromatography analysis with methods modified from those described previously.^36,38 For O⁶-BG and 8-oxo-O⁶-BG, aliquots of plasma (500 µL) were spiked with 150 µL of internal standard (O⁶-[p-fluorobenzyl]guanine) and extracted with 5 mL of ethyl acetate two times. After centrifugation at 1,500 x g for 20 minutes, the supernatant was evaporated to dryness under nitrogen. Samples were reconstituted in mobile phase and separated isocratically with 35% methanol 10 mmol/L potassium phosphate buffer, pH 7.5, using a Waters Novapak 4 µm phenyl column (3.9 x 300 mm; Waters, Milford, MA) at ambient temperature and a flow rate of 1.0 mL/min. Retention times were 16.3, 18.9, and 21.5 minutes for 8-oxo-O⁶-BG, O⁶-BG, and O⁶-(p-fluorobenzyl)guanine, respectively. Samples were monitored at 280 nm using a Hitachi variable-wavelength UV detector (Hitachi, Ltd, Tokyo, Japan). The limits of detection for O⁶-BG and 8-oxo-O⁶-BG were 30 ng/mL, respectively. Extraction efficiency was determined to be 92% for O⁶-BG, 85% for 8-oxoBG, and 97% for O⁶-(p-fluorobenzyl)guanine.

Pharmacokinetic Analysis
The pharmacokinetics of O⁶-BG and 8-oxo-O⁶-BG were analyzed using noncompartmental methods with WinNonlin (PharSight Corp, Apex, NC). The apparent elimination half-life of O⁶-BG was estimated from the slope of the terminal concentrations of the log concentration-time curve of individual patients. The area under the concentration-time curve (AUC) for the study period, AUC_last, for O⁶-BG and 8-oxo-O⁶-BG were calculated by the linear trapezoidal method. Because only a small group of patients had a value above the limit of detection at 24 hours, AUC to infinity was not calculated for 8-oxo-O⁶-BG. AUC was determined up to 3 hours for O⁶-BG and up to 7 hours for 8-oxo-O⁶-BG.

Statistical Considerations
The primary goal of this study was to evaluate the activity of BCNU plus O⁶-BG in the treatment of patients with recurrent or progressive malignant gliomas resistant to nitrosourea. A two-stage "minimax" phase II design that differentiates between a response rate of 5% and 20% was used to evaluate the activity of this regimen. Specifically, the hypothesis that was to be tested was as follows: H₀: P .05 versus H₁: P .2, where P is the proportion of patients who responded (complete response, partial response) to treatment.

The first stage of the study would treat 18 patients, but if none of the patients responded to treatment, the study would be terminated. If any of those patients responded to treatment, however, 14 patients would be added to the study. If four or more of the total 32 patients responded, it was to be decided whether the treatment regimen was worthy of further testing.

The following characteristics are true of this study design: (1) the probability of erroneously concluding a treatment is active (P .2) when it is actually ineffective (P .05) is less than .1 (ie, alpha = .1); and (2) the probability of erroneously concluding that the treatment is ineffective (P .05) when the treatment is actually active (P .2) is .1 (ie, beta = .1). Under the null hypothesis, the probability of early termination was .40.

	RESULTS

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Patient Data
Eighteen patients were enrolled onto the study. The patients’ age, diagnosis, prior therapy, total number of cycles of BCNU plus O⁶-BG received, and toxicity are detailed in Table 1. The patients were between the ages of 39 and 73 years and all had received between one and three prior chemotherapeutic regimens. All patients had received and failed a nitrosourea-containing regimen immediately before enrollment onto the BCNU plus O⁶-BG trial.

Antitumor Responses
None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity.

Pharmacologic Studies
Nine patients were evaluated for plasma concentrations of O⁶-BG and 8-oxo-O⁶-BG during and after the O⁶-BG infusion. O⁶-BG is oxidized to 8-oxo-O⁶-BG by human CYP1A1 (unpublished observations), CYP1A2, CYP3A4, and aldehyde oxidase.³⁹ Six of the nine patients evalu-ated used phenytoin and three of the patients used no obvious CYP3A4-inducing medication. Because phenytoin can increase the elimination of agents metabolized by CYP3A4,^40,41 we compared the plasma drug and metabolite concentrations as measured by high-performance liquid chromatography with ultraviolet detection. Figure 1 illustrates the mean plasma concentrations (± SD) of O⁶-BG and 8-oxo-O⁶-BG over time after a dose of 120 mg/m² O⁶-BG. The mean O⁶-BG AUC_{0 to 3 hours} values for six patients using phenytoin and three patients not on CYP3A4-inducing medication were 4.8 ± 1.7 µg/mL/h and 5.0 ± 1.3 µg/mL/h, and O⁶-BG half-lives were 0.56 ± 0.21 hour and 0.54 ± 0.2 hour, respectively. In addition, the AUC_{0 to 7 hours} values for 8-oxo-O⁶-BG in patients using phenytoin and those not on phenytoin were 52 ± 14 µg/mL/h and 44 ± 18 µg/mL/h and half-lives were 5.8 ± 2.6 hours and 6.3 ± 2.2 hours, respectively. The half-lives of O⁶-BG and 8-oxo-O⁶-BG are in agreement with previously reported values.³⁷ Thus, phenytoin did not alter the pharmacokinetics of O⁶-BG and 8-oxo-O⁶-BG.

View larger version (20K): [in this window] [in a new window]   Fig 1. Plasma concentrations of O6-GB (squares) and 8-oxo-O6-BG (circles) of patients treated with phenytoin (open symbols, n = 6) or not on CYP3A4-inducing drugs (closed symbols, n = 3) were determined at various time points after administration of 06-BG. Zero time refers to start of infusion. Each point of O6-BG and 8-oxo-O6-BG represents the mean ± SD.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

This current phase II trial of O⁶-BG plus BCNU was designed to define the therapeutic impact of reducing the AGT content of malignant gliomas confirmed to be resistant to alkylnitrosoureas. Previous studies have shown a high incidence of AGT in CNS tumors as well as compelling data demonstrating a correlation between the outcome of patients treated with BCNU for a malignant glioma and tumor AGT levels.^22-25 The results suggest that several obstacles may exist to using O⁶-BG to restore sensitivity to BCNU.

Our initial phase I trial showed that O⁶-BG at a dose of 100 mg/m² could deplete the AGT content of malignant gliomas when it was measured 18 hours later, a period adequate for conversion of a BCNU-induced adduct to an interstrand cross-link immune from AGT removal.⁴² We chose a dose of O⁶-BG for this phase II trial that was 20% higher so that we could be absolutely confident that optimal AGT depletion had been achieved. Therefore, it is unlikely that inadequate AGT depletion in the tumors would explain the failure to see tumor regressions. Our subsequent phase I trial of O⁶-BG plus BCNU demonstrated the need for a marked reduction in the dose of BCNU that could be administered, with a maximum-tolerated dose of only 40 mg/m² as compared with the usual dose of 200 mg/m² when BCNU is used alone. The myelosuppression seen in the phase I trial was also seen in the current phase II trial, with a magnitude and incidence suggesting that the dose of BCNU chosen for the phase II trial was appropriate. This profound reduction of BCNU may be the underlying factor in the failure of this drug combination to cause frank tumor regressions.

Another explanation for failure to see tumor regressions is the possibility that alternative mechanisms of resistance to BCNU are operational in human malignancies, in this case malignant glioma. Previous work has identified only one other mechanism mediated by glutathione and/or glutathione S-transferase.^43-45 However, we and presumably other investigators have demonstrated additional mechanisms of resistance whose precise pathways remain unidentified.⁴⁶ Nevertheless, the weight of prior laboratory and clinical studies suggests that AGT is the primary mechanism of resistance to nitrosoureas, which makes it unlikely that the 18 tumors in our trial had non-AGT mechanisms of BCNU resistance.

O⁶-BG is at least partly metabolized to 8-oxo-O⁶-BG by CYP3A4 and further metabolized to 8-oxoguanine by CYP1A1/2.³⁹ We studied the possible effect of the CYP3A4-inducing antiepileptic agent phenytoin on the pharmacokinetics of O⁶-BG. Phenytoin is known to accelerate the elimination of carbamazepine and vincristine, probably by stimulating cytochrome P-450 CYP3A4, thereby reducing plasma drug concentrations.^41,47 Phenytoin was prescribed to some patients to reduce seizures. Using noncompartmental analysis, we found no difference in the AUCs or half-lives of O⁶-BG or 8-oxo-O⁶-BG between patients treated with phenytoin and those not receiving CYP3A4-enhancing agents. Furthermore, the mean plasma concentration curve for both groups was superimposable. These results are consistent with a previous study comparing results from patients treated with 100 mg/m² receiving CYP3A4-inducing drugs with a separate phase I study of patients treated with doses between 10 and 120 mg/m² of O⁶-BG not on CYP3A4-inducing drugs.³⁷ Therefore, concurrent administration of agents that induce CYP3A4 does not affect the metabolism of O⁶-BG.

We believe that the current results indicate that the dose reduction of BCNU, mandated by the enhanced myelosuppression seen when O⁶-BG is also administered, explains the lack of activity of this regimen in the treatment of nitrosourea-resistant malignant glioma. Several approaches may provide a strategy for overcoming this obstacle. Regional administration of an alkylnitrosourea such as with the BCNU-releasing polymer Gliadel, coupled with administration of systemic O⁶-BG, may limit extraneural drug exposure and reduce myelosuppression.⁴⁸ Alternatively, protection of hematopoietic stem cells with a mutant AGT gene refractory to O⁶-BG may be effective in allowing a higher dose of alkylnitrosourea to be used in combination with systemic O⁶-BG.⁴⁹

In summary, our results demonstrate that the use of systemic O⁶-BG plus BCNU did not restore sensitivity to BCNU-resistant malignant glioma. Strategies will need to address the enhanced systemic (particularly hematopoietic) toxicity of BCNU when given with O⁶-BG to more effectively deal with AGT-mediated nitrosourea resistance in these tumors.

	ACKNOWLEDGMENTS

 
Supported by National Institute of Neurological Disorders and Stroke grant no. 1K23NS41737-01 and National Institutes of Health grant nos. NS30245 and NS20023.

	NOTES

 
H.S.F. had a consulting relationship with Procept, Boston, MA, which held the license for O⁶-benzylguanine at the time of this study and now has a consulting relationship with Access Oncology, New York, NY, which currently holds the license. M.E.D., A.E.P., and R.C.M. are coinventors of O⁶-benzylguanine.

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Submitted September 19, 2001; accepted January 24, 2002.

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