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

From the Departments of Surgery, Medicine, Pathology, Radiology, and Community and Family Medicine, Duke University Medical Center, Durham, NC; Departments of Medicine and Pediatrics, 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; Investigational Drug Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; and Pediatric Hematology-Oncology, Royal Children’s Hospital, Melbourne, Australia.

Address reprint requests to Henry S. Friedman, MD, DUMC-3624, Duke University Medical Center, Durham, NC 27710; email fried003{at}nc.duke.edu

	ABSTRACT

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PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O⁶ position of guanine. O⁶-benzylguanine (O⁶-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O⁶-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O⁶-BG with recurrent or progressive malignant glioma.

PATIENTS AND METHODS: Patients were treated with O⁶-BG at a dose of 100 mg/m² followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O⁶-BG, 8-oxo-O⁶-BG, and 8-oxoguanine concentration.

RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m²) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O⁶-BG rapidly disappeared from plasma (elimination half-life = 0.54 ± 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O⁶-BG (elimination half-life = 5.6 ± 2.7 hours) and further to 8-oxoguanine. There was no detectable O⁶-BG 5 hours after the start of the O⁶-BG infusion; however, 8-oxo-O⁶-BG and 8-oxoguanine concentrations were detected 25 hours after O⁶-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O⁶-BG was 17.5 times greater than the mean AUC for O⁶-BG.

CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O⁶-BG at a dose of 100 mg/m² is 40 mg/m² administered at 6-week intervals. This study provides the foundation for a phase II trial of O⁶-BG plus BCNU in nitrosourea-resistant malignant glioma.

	INTRODUCTION

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RESISTANCE TO chemotherapy remains the central reason for the failure to cure patients with a diverse spectrum of malignancies. Malignant glioma is a particularly dismal case, with virtually all tumors displaying marked de novo or acquired drug resistance and ultimate lethal growth. Conventional treatment with surgery, radiotherapy, and alkylnitrosourea-based chemotherapy cures the minority of patients with anaplastic astrocytoma but no patients with glioblastoma multiforme.^1-3 Although carmustine (BCNU) remains the community gold standard of care because of a modest increase in median survival,^4,5 resistance to this alkylnitrosourea invariably occurs with subsequent patient death.

An extensive series of preclinical and clinical studies has demonstrated that the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) is responsible for resistance to alkylnitrosoureas.^6-20 AGT removes chlorethylation or methylation damage from the O⁶ position of DNA guanines before cell injury and death.

The high incidence of AGT activity in human CNS tumors,²¹ as well as recent clinical trials that show an inverse relationship between survival and AGT levels in patients with malignant glioma who receive BCNU therapy,^22-24 provides the 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.^25-35 We now report a phase I trial of BCNU plus O⁶-BG in patients with recurrent or progressive malignant glioma that is designed to define the maximum-tolerated dose (MTD) of BCNU, the toxicity of this regimen, and the pharmacokinetic parameters at this dose of O⁶-BG.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Protocol Objectives
The objectives of the study were as follows: to define the MTD of BCNU when administered after O⁶-BG (100 mg/m²), to characterize any toxicity associated with the combination of O⁶-BG and BCNU, and to observe patients for clinical antitumor response when they were treated with O⁶-BG and BCNU.

Patient Eligibility Criteria
Patients were eligible if they were 18 years or older with a recurrent or progressive anaplastic astrocytoma or glioblastoma multiforme. Patients must have had a Karnofsky performance status 60% at study entry and have been treated previously with an alkylnitrosourea.

Treatment Design
Cohorts of three to six patients were treated with O⁶-BG at a dose of 100 mg/m² followed approximately 1 hour later by BCNU at an initial dose of 13.5 mg/m². Additional cohorts of three to six patients were treated with escalating doses of BCNU until dose-limiting toxicity was observed. The first three assessable patients at a dose level must have been observed for 6 weeks after the BCNU/O⁶-BG treatment without experiencing dose-limiting toxicity before patients could enter the next dose level.

O⁶-BG was supplied by the National Cancer Institute (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 of 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 of dibasic sodium phosphate and 102 mg of 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.

Dose Escalation and Statistical Consideration
Succeeding dose levels of BCNU were designed to use 100% dose escalations until two grade 2 or one grade 3 hematopoietic toxicities were seen. Additional dose escalations at that time were designed to use 33% increases, although this was modified by the Cancer Therapy Evaluation Program for dose levels 3 and 4.

The dose level was escalated in successive cohorts of three patients so long as no dose-limiting toxicity was observed. If one instance of dose-limiting toxicity was observed among the initial three assessable patients treated at a dose level, an additional three patients had to be treated at that dose level with no further dose-limiting toxicity for dose escalation to proceed. If two instances of dose-limiting toxicity were observed at a dose level, the MTD was surpassed and a total of six patients had to be treated at the previous level to assure its tolerability. The MTD was the highest dose to cause dose-limiting toxicity in no more than one of six patients at that dose level. Patients who had stable or responding disease who developed dose-limiting toxicities were allowed to continue to be treated at the next lower dose level, provided that their toxicities resolved to the appropriate levels for retreatments with no greater than a 2-week delay for recovery. However, if dose-limiting toxicity occurred again on the lower dose, then the patient was removed from the study.

Dose-limiting toxicity was defined as greater than or equal to grade 3 nonhematopoietic or grade 4 hematologic toxicity. A decrease in the carbon monoxide diffusing capacity of the lungs (DLCO) by 25% from baseline was considered a dose-limiting toxicity. Furthermore, failure to recover from any non–dose-limiting toxicity to less than or equal to grade 1 toxicity within 2 weeks of the end of the cycle (ie, 8 weeks from drug administration) was considered a dose-limiting toxicity.

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

Response Evaluation
Tumor response was evaluated using radiographic criteria with gadolinium-enhanced magnetic response imaging as follows: complete response was defined as the disappearance of all enhancing tumor on consecutive contrast-enhanced magnetic resonance imaging or computed tomography scans at least 1 month apart, with patient not receiving corticosteroids and neurologically stable or improved. Partial response was defined as an at least 50% reduction in the size (product of largest perpendicular diameters) of enhancing tumor maintained for at least 1 month, use of corticosteroids stable or reduced, and patient neurologically stable or improved. No response was defined as no changes in tumor size that qualified as complete response, partial response, or progressive disease. Progressive disease was defined by an at least 25% increase in size (product of largest perpendicular diameters) of enhancing tumor or any new tumor on magnetic resonance imaging or computed tomography scan, or the patient being neurologically worse, and the use of corticosteroids stable or increased.

Plasma Sampling
Whole-blood samples were collected in sodium-heparinized Vacutainer before treatment at 30 and 60 minutes into the infusion of 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-Pressure Liquid Chromatography (HPLC) Analysis of O⁶-BG and Metabolites
Total plasma concentrations of BG, 8-oxo-O⁶-BG, and 8-oxoguanine were measured by HPLC using methods modified from those described previously.^36,37 For O⁶-BG and 8-oxo-O⁶-BG, aliquots of plasma (500 µL) were spiked with 75 µL of internal standard (O⁶-(p-fluorobenzyl)guanine) and extracted with 5 mL of ethyl acetate. After centrifugation at 1,500 x g for 20 minutes, the supernatant was evaporated to dryness under N₂. 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 µ phenyl column (3.9 x 150 mm; Waters, Milford, MA) at ambient temperature and a flow rate of 1.5 mL/min. Retention times were 9.2, 10.5, and 12.8 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 of O⁶-BG and 8-oxo-O⁶-BG were 30 ng/mL and 20 ng/mL, respectively. Extraction efficiency was determined to be 92% for O⁶-BG, 85% for 8-oxoBG, and 97% for O⁶-(p-fluorobenzyl)guanine. For 8-oxoguanine, aliquots of plasma (500 µL) were spiked with 10 µL of internal standard (0.25 mmol/L 8-hydroxy-2'deoxyguanosine) and protein precipitated with 5 mL of methanol. Samples were centrifuged at 1,500 x g for 20 minutes, evaporated to dryness under N₂ and reconstituted in mobile phase. An HPLC method with electrochemical detection was used to quantitate 8-oxoguanine. Briefly, the chromatographic separation was achieved on a TosoHaas 5µm, ODS-80 column (TosoHaas, Montgomeryville, PA). The pump was set to deliver 2% of acetonitrile and 98% of 0.1 M sodium acetate, pH 5.2, at a flow rate of 1 mL/min for 10 minutes, then increase linearly to 50% acetonitrile in 10 minutes. CoulArray potentials (ESA, Inc, Chelmsford, MA) were set at +280 mV for detection of 8-oxoguanine and +300 mV for internal standard. The limit of detection of 8-oxoguanine was 35 ng/mL with 93% recovery.

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 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 BG and 8-oxoBG were calculated by the linear trapezoidal method. The AUC to infinity was calculated as the sum of AUC_last and C_last/_term, where C_last is the final measured concentration and _term is the terminal elimination rate constant. Time points used to derive the latter were based on visual inspection of the data. When C_last was below the limit of quantification, a concentration of O⁶-BG (15 ng/mL), 8-oxo-O⁶-BG (10 ng/mL), or 8-oxoguanine (17 ng/mL) equivalent to one half of the limit of quantification was used for the calculation of AUC.

	RESULTS

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Patient Data
Twenty-three patients were enrolled onto the study at one of four dose levels of BCNU: 13.5, 27.0, 40, or 55 mg/m². Twenty-two patients had glioblastoma multiforme, and one had an anaplastic astrocytoma. The patients’ age, prior therapy, total number of cycles of BCNU plus O⁶-BG received, and toxicity are detailed in Table 1.

Toxicity Evaluation
Table 1 details three categories of toxicity: dose-limiting toxicities as defined above, toxicities that precluded a patient from further treatment with BCNU plus O⁶-BG, and grade 3 toxicities that resolved allowing further treatment. The observed toxicities were almost exclusively hematopoietic, specifically neutropenia and thrombocytopenia. Two patients displayed pulmonary toxicity. Patient no. 7 had a greater than 25% decrease in the DLCO, which was a dose-limiting toxicity. Patient no. 13 demonstrated a DLCO less than 80% that prevented retreatment but did not have a greater than 25% decrease from baseline. Four of six patients treated with a BCNU dose of 55 mg/m² displayed dose-limiting neutropenia or thrombocytopenia. The MTD of BCNU was therefore 40 mg/m².

Antitumor Responses
Fifteen patients demonstrated progressive disease as their best response, with thirteen experiencing progression after one cycle of therapy and two after two cycles. Five patients demonstrated stable disease for one or two cycles but incurred toxicity that precluded additional therapy. Two patients with stable disease continued on treatment, having received seven plus and three plus cycles, respectively. This latter patient (no. 20), although having stable disease by definition, demonstrated a 15% decrease in tumor size after two cycles of treatment. One patient (no. 19) was nonassessable since she self-referred for additional radiotherapy 1 week after receiving BCNU plus O⁶-BG.

Pharmacologic Studies
Twelve patients were evaluated for plasma concentration of O⁶-BG and metabolites during and up to 24 hours after the O⁶-BG infusion. O⁶-BG was oxidized to 8-oxo-O⁶-BG similar to that demonstrated in rodents,³⁸ nonhuman primates,³⁹ and humans.^36,40 8-Oxoguanine was also found in plasma and urine using electrochemical detection,³⁷ presumably as a result of debenzylation of 8-oxo-O⁶-BG (Fig 1). We determined that 21.1% ± 10.7% of BG administered is excreted as 8-oxoguanine (range, 4.2% to 40.8%). The mean plasma concentrations (± SD) of O⁶-BG, 8-oxo-O⁶-BG, and 8-oxoguanine over time after a dose of 100 mg/m² are shown in Fig 2. The apparent plasma half-lives of O⁶-BG and 8-oxo-O⁶-BG were 0.54 ± 0.14 hours and 5.6 ± 2.7 hours, respectively. The plasma concentration of 8-oxoguanine increased up to 7 hours and decreased slightly at 25 hours after O⁶-BG infusion. The AUC_0-inf of 8-oxo-O⁶-BG (58.2 ± 23.8 µg/mL.h) was 17.5-fold higher than that of O⁶-BG (3.32 ± 0.93 µg/mL.h). The clearance of O⁶-BG was 32 ± 9 L/h/m².

View larger version (9K): [in this window] [in a new window]   Fig 1. Oxidation of O6-BG to 8-oxo-O6-BG and debenzylation to 8-oxoguanine.

View larger version (20K): [in this window] [in a new window]   Fig 2. Mean plasma profile of drug and metabolite concentration levels after administration of O6-BG. Plasma concentration of O6-BG (), 8-oxo-O6-BG (•), and 8-oxoguanine () were determined at various time points. Zero time refers to the start of infusion. Each point of O6-BG (n = 13), 8-oxo-O6-BG (n = 13), and 8-oxoguanine (n = 13) represents the mean ± SD.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Therapeutic strategies designed to combat resistance to chemotherapy in general take three approaches. The first is selection of alternative chemotherapeutic agents to which the tumor is not resistant. Unfortunately, despite large federal and corporate efforts to identify new and active chemotherapeutic agents, there are few new drugs with activity in malignant glioma. The second approach is to try to enhance activity of chemotherapeutic agents by use of concomitant additional modalities such as radiotherapy or hyperthermia. These strategies have proven modestly effective but do not seem to have benefited patients with malignant glioma. Finally, efforts are underway with a number of different chemotherapeutic agents to try to directly identify and reverse the mechanisms of resistance operational in the tumor. This approach includes efforts to deplete tumor AGT levels as a means of restoring sensitivity to chloroethylating nitrosoureas and methylating agents. The role of AGT in mediating tumor resistance to nitrosoureas and methylating agents has been precisely defined in both laboratory and clinical studies.^6-20,25,26 Accordingly, the question is not whether depletion of AGT will enhance alkylator and methylator activity in these tumors but rather whether it can be done in a fashion that does not increase normal organ toxicity and merely reduce the dose of chemotherapeutic agent administered without therapeutic gain.

We have previously demonstrated in a phase I trial the dose of O⁶-BG required to deplete the alkyltransferase activity in glioblastoma multiforme and anaplastic astrocytoma.³⁵ Tumor AGT levels were measured at 18 hours after treatment of O⁶-BG to prepare for a subsequent phase I trial in which BCNU would be administered immediately after O⁶-BG. BCNU-induced crosslinks seem to peak at 12 hours, and therefore, it was critical that the AGT depletion last throughout this time period.⁴¹ Once the crosslink is formed, AGT is ineffective in removing them. We successfully identified an O⁶-BG dose of 100 mg/m² and immediately initiated a phase I trial in which O⁶-BG was administered in combination with BCNU.

Although we have previously demonstrated oxidation of O⁶-BG to 8-oxo-O⁶-BG in humans,³⁶ we now report the presence of 8-oxoguanine in the plasma of patients. 8-Oxoguanine is most likely formed as a result of debenzylation of 8-oxo-O⁶-BG. O⁶-BG and 8-oxo-O⁶-BG inactivate AGT to a similar extent³⁸; however, loss of the benzyl group to form 8-oxoguanine abolishes AGT inactivating activity. The plasma concentration of 8-oxoguanine varies considerably and is noted in some patients within 30 minutes of the start of the infusion of O⁶-BG, whereas in others it is not detectable until 10 hours after the initiation of O⁶-BG infusion. We observed a steady increase in plasma 8-oxoguanine concentration that remained between 0.15 and 0.27 µg/mL from the end of the O⁶-BG infusion until 25 hours after infusion. It is not known whether this product is further metabolized or excreted unchanged in the urine.

The human enzymes responsible for oxidation of O⁶-BG to 8-oxo-O⁶-BG include cytochrome P450 isoforms, CYP1A2 and CYP3A4, and cytosolic aldehyde oxidase.⁴⁰ Preliminary data from our laboratory indicates that debenzylation of 8-oxo-O⁶-BG to 8-oxoguanine occurs via CYP1A2 (Dolan, unpublished observations). All of the patients evaluated for pharmacokinetic parameters in this study received concomitant corticosteroids that could affect drug metabolizing enzymes. Phenytoin and dexamethasone were prescribed to patients to reduce seizures and brain edema. Both of these drugs are known to induce CYP450 and UDPGT enzymes.^42-44 Dexamethasone is known to induce CYP3A,⁴⁴ one of the enzymes responsible for oxidation of O⁶-BG to 8-oxo-O⁶-BG. We compared pharmacokinetic parameters obtained on the phase I patients described herein with parameters obtained from patients on a previous phase I study who were not receiving corticosteroids.^36,45 Using noncompartmental analysis, we determined that the clearance values of O⁶-BG for patients receiving and not receiving concomitant corticosteroids were 32 ± 9 (n = 12) and 32 ± 10 (n = 23), respectively. In addition, the AUC of O⁶-BG and 8-oxo-O⁶-BG for patients who received corticosteroids at a dose of 100 mg/m² was directly proportional to the AUC calculated from patients not on corticosteroids who received doses between 10 and 120 mg/m² of O⁶-BG.^36,45 Thus, it is interesting that concurrent administration of corticosteroids/anticonvulsants does not seem to alter the rate of clearance or AUC of O⁶-BG or the AUC ratio of 8-oxo-O⁶-BG to O⁶-BG.

We now report the results of this phase I trial of O⁶-BG (100 mg/m²) plus BCNU, in which the MTD of BCNU was shown to be 40 mg/m². Dose-limiting toxicity was, as previously noted, myelosuppression. Severe but reversible neutropenia and thrombocytopenia was observed at a BCNU dose of 55 mg/m², which required reduction to 40 mg/m² as the suggested dose for phase II trials. Recent treatment with O⁶-BG plus BCNU (55 mg/m²) in two patients who had not previously been treated with a nitrosourea confirmed that the MTD of BCNU in this group was also 40 mg/m² because both patients demonstrated dose-limiting myelosuppression. All of the patients treated on this study had previously been treated with nitrosourea, which thus increased the likelihood that pulmonary toxicity would be a problem. Although we were concerned about dose-limiting pulmonary toxicity, particularly because of the pulmonary dysfunction seen with the use of high-dose BCNU,⁴⁶ only two patients were identified with significant alterations in their DLCO. The ubiquitous presence of AGT in all human organs and tissue predicted that BCNU toxicity would be enhanced, and the dose of BCNU defined as the MTD in this trial was far less than that when BCNU is administered alone. However, it is not clear if the BCNU dose has been reduced to a homeopathic level at which antitumor activity will not be seen. All patients had active progressing tumors at the time of their enrollment onto the study, and several patients may have benefited. Patient no. 11 demonstrated stable disease for 66+ weeks on therapy with BCNU and O⁶-BG. Patient no. 17 had a small reduction in tumor size but did not qualify for partial response. The observed antitumor activity was provocative, but the regimen was certainly not convincingly beneficial in patients with nitrosourea-resistant malignant glioma.

The current phase I trial of O⁶-BG plus BCNU has defined the therapeutic approach for a newly opened phase II trial of BCNU plus O⁶-BG. Patients with recurrent nitrosourea-resistant malignant glioma, defined as tumor growth within 8 weeks of receiving the nitrosourea, will be enrolled onto this trial. This will provide the first unequivocal evidence of whether BCNU plus O⁶-BG can restore nitrosourea sensitivity in patients for whom this agent has previously failed or whether the reduction of BCNU dose required by the O⁶-BG-mediated reduction of AGT levels has merely shifted the dose response curve so that BCNU activity will not be seen. Certainly there are other strategies, such as the use of an AGT mutant gene transfected into stem cells, that may allow an even higher dose of BCNU to be used in combination with O⁶-BG.⁴⁷ However, it is not clear whether that strategy will be required, and the use of BCNU plus O⁶-BG alone is medically easier and less financially cumbersome than the use of transfected stem-cell support. Future studies that use O⁶-BG–mediated depletion of AGT in combination with either alkylators or methylators such as temozolomide are warranted.⁴⁸ Alternatively, O⁶-BG could be used with regional chemotherapeutic approaches such as Gliadel wafers (Rhône-Poulenc Rorer, Collegeville, PA).⁴⁹ Successful use of this strategy for reversing alkylator/methylator resistance could be a major step forward in the treatment of human neoplasia.

	ACKNOWLEDGMENTS

 
Supported by National Institutes of Health grants no. NS30245, NS20023, and CA57725.

	NOTES

 
Drs Dolan, Pegg, and Moschel have a financial relationship with Procept, the company that is presently licensing O⁶-benzylguanine. Partial support for the trial was provided by Procept.

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Submitted March 6, 2000; accepted June 9, 2000.

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