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Phase I Trial of Polifeprosan 20 With Carmustine Implant Plus Continuous Infusion of Intravenous O⁶-Benzylguanine in Adults With Recurrent Malignant Glioma: New Approaches to Brain Tumor Therapy CNS Consortium Trial

Jon Weingart, Stuart A. Grossman, Kathryn A. Carson, Joy D. Fisher, Shannon M. Delaney, Mark L. Rosenblum, Alessandro Olivi, Kevin Judy, Stephen B. Tatter, M. Eileen Dolan

From the New Approaches to Brain Tumor Therapy CNS Consortium, Baltimore, MD; and the Department of Medicine, University of Chicago, Chicago, IL

Address reprint requests to Joy Fisher, Cancer Research Building 2, Suite 1M16, 1550 Orleans St, Baltimore, MD 21231; e-mail: jfisher{at}jhmi.edu

	ABSTRACT

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Purpose: This phase I trial was designed to (1) establish the dose of O⁶-benzylguanine (O⁶-BG) administered intravenously as a continuous infusion that suppresses O⁶-alkylguanine-DNA alkyltransferase (AGT) levels in brain tumors, (2) evaluate the safety of extending continuous-infusion O⁶-BG at the optimal dose with intracranially implanted carmustine wafers, and (3) measure the pharmacokinetics of O⁶-BG and its metabolite.

Patients and Methods: The first patient cohort (group A) received 120 mg/m² of O⁶-BG over 1 hour followed by a continuous infusion for 2 days at escalating doses presurgery. Tumor samples were evaluated for AGT levels. The continuous-infusion dose that resulted in undetectable AGT levels in 11 or more of 14 patients was used in the second patient cohort. Group B received the optimal dose of O⁶-BG for 2, 4, 7, or 14 days after surgical implantation of the carmustine wafers. The study end point was dose-limiting toxicity (DLT).

Results: Thirty-eight patients were accrued. In group A, 12 of 13 patients had AGT activity levels of less than 10 fmol/mg protein with a continuous-infusion O⁶-BG dose of 30 mg/m²/d. Group B patients were enrolled onto 2-, 4-, 7-, and 14-day continuous-infusion cohorts. One DLT of grade 3 elevation in ALT was seen. Other non-DLTs included ataxia and headache. For up to 14 days, steady-state levels of O⁶-BG were 0.1 to 0.4 µmol/L, and levels for O⁶-benzyl-8-oxoguanine were 0.7 to 1.3 µmol/L.

Conclusion: Systemically administered O⁶-BG can be coadministered with intracranially implanted carmustine wafers, without added toxicity. Future trials are required to determine if the inhibition of tumor AGT levels results in increased efficacy.

	INTRODUCTION

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Despite advances in tumor biology, there has been little impact on the outcome of patients with malignant glioma. The median survival of patients with malignant gliomas in the United States is less than 2 years.¹ As a result, novel therapeutic approaches are imperative. Most malignant gliomas recur within 2 cm of the original resection site and within the radiation field. Polifeprosan 20 with carmustine implant (Gliadel; Guilford Pharmaceuticals, Baltimore, MD) delivers carmustine (BCNU) locally to the tumor site without significant systemic or local adverse effects.^2,3 This biodegradable copolymer is implanted during surgery and releases carmustine locally for approximately 2 weeks.⁴ It has been evaluated in three phase III trials, and it prolongs survival when placed at the initial tumor resection or at recurrence.^5-7

O⁶-alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein known to remove and repair O⁶-alkylguanine lesions introduced by alkylating agents such as carmustine or temozolomide.⁸ AGT activity has been shown to be a major factor in the resistance of tumor cells to alkylating agents.^8-10 O⁶-benzylguanine (O⁶-BG) is a low molecular weight substrate that inactivates AGT, requiring de novo synthesis to replenish the protein.¹¹ In vitro and in vivo studies using tumor cell lines and intracranial and subcutaneous brain tumor xenografts demonstrate that O⁶-BG treatment before carmustine increases its therapeutic effectiveness.^10,12,13 In animals and humans, systemically administered O⁶-BG severely limits the amount of intravenous carmustine that can be given as the repair mechanisms of normal cells also are affected by exposure to O⁶-BG.^10,12-16

Prior studies demonstrated that 100 mg/m² of O⁶-BG given intravenously completely inhibits AGT activity in brain tumors.^17,18 Combining systemic O⁶-BG with locally delivered carmustine could provide a synergistic cytotoxic effect on the tumor without increased systemic toxicity. Since carmustine is released over several weeks from wafers, tumor AGT activity would need to be suppressed by O⁶-BG during this period to maximize potential therapeutic benefit.

This trial's objectives were to (1) establish the continuous-infusion dose of O⁶-BG that completely suppresses AGT levels (group A), (2) evaluate the safety of increasing the duration of continuous-infusion O⁶-BG for up to 2 weeks at a dose that suppresses tumor AGT activity when combined with intracranially implanted polifeprosan 20 wafers with carmustine (group B), and (3) measure plasma concentrations of O⁶-BG and its metabolite, O⁶-benzyl-8-oxoguanine (8-oxoBG; groups A and B).

	PATIENTS AND METHODS

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This study was approved by the Cancer Therapy Evaluation Program at the National Cancer Institute (NCI) and the institutional review boards of all participating institutions. Informed consent was obtained from each patient. Accrual took place between May 2000 and September 2002 at the following institutions of the New Approaches to Brain Tumor Therapy: Cleveland Clinic (Cleveland, OH), Emory University (Atlanta, GA), Henry Ford Hospital (Detroit, MI), Johns Hopkins University (Baltimore, MD), University of Pennsylvania (Philadelphia, PA), and Wake Forest University (Winston-Salem, NC). Pharmacokinetic analysis was performed at the University of Chicago (laboratory of M.E.D.; Chicago, IL).

Patients
Patients were eligible if they were 18 years or older and could provide informed consent. In addition, they had to have had a prior diagnosis of malignant glioma, a supratentorial ( 1 cm) recurrence, surgical treatment indicated, a Karnofsky performance status of 60% or higher, previous radiation therapy completed more than 3 months prior, and an intraoperative diagnosis of viable glioma. At least 4 weeks must have elapsed since the patients' last chemotherapy dose, and they could not receive additional chemotherapy during the first 56 days on this study. Normal hematologic, renal, and liver functions were required. Patients were ineligible if they had a bilateral or multifocal tumor, projected survival of less than 60 days, known hypersensitivity to nitrosoureas, prior malignancy, or if they were pregnant or breast-feeding.

Study Design
The objective for group A was to determine the continuous-infusion dose of O⁶-BG that resulted in undetectable AGT tumor levels in at least 11 of 14 patients. Continuous-infusion O⁶-BG was initiated 2 days presurgery with a bolus of 120 mg/m² over 1 hour followed by a continuous infusion of 30 mg/m²/d, which continued for the next 2 days. On the day of surgery, the patients underwent surgical debulking and implantation of up to eight polifeprosan 20 with carmustine wafers. During the surgical procedure, a sample of tumor was obtained, frozen, and stored for AGT analysis. The infusion of O⁶-BG stopped once the surgery was completed. Although a previous study demonstrated that a bolus of 100 mg/m² adequately depleted AGT levels for 18 hours, we chose a 20% higher dose to extend the time of depletion.¹⁸

Up to 14 patients were to be enrolled until 11 patients had unmeasurable tumor AGT activity. Tumor AGT depletion was defined as AGT levels of less than 10 fmol/mg protein. In the event that four patients had measurable AGT activity, the dose of continuous-infusion O⁶-BG was to be increased by 10 mg/m²/d. This decision rule was chosen to have a greater than 95% chance of concluding that O⁶-BG inhibited AGT when it was effective and a small chance (P < .00001) of concluding that it inhibited AGT when in reality it was not effective.

Group B was designed to evaluate the safety of increasing the duration of continuous-infusion O⁶-BG at a dose that suppresses tumor AGT activity when combined with intracranially implanted carmustine wafers. The bolus of O⁶-BG was administered at least 1 hour presurgery, and the continuous-infusion duration was increased in a stepwise fashion (2, 4, 7, and 14 days) postoperatively. Six patients were studied at each infusion's duration. At the time of wafer implantation, steady-state pharmacokinetics data were obtained. Toxicity was assessed during the initial 28 days, and at days 42 and 56. All patients were followed for 12 months unless they progressed or started another form of treatment. All were followed for survival status until death.

O⁶-BG was provided by NCI and prepared by the pharmacy staff according to the guidelines provided. After reconstitution of the drug in the 40% PEG-buffered diluent (20 mmol/L potassium phosphate, 5 mmol/L EDTA, and 20% volume-to-volume ratio glycerol), the appropriate dose was administered by continuous infusion for up to 2 weeks. O⁶-BG is stable for 24 hours after preparation, and thus the bag/cassette containing the drug was changed daily.

Polifeprosan 20 wafers with carmustine were provided by Guilford Pharmaceuticals (Baltimore, MD). The aim was to cover the surface of the resection cavity with eight wafers. The number of implanted wafers was recorded. Corticosteroid doses were based on an individual patient's needs and were tapered as indicated.

Dose Modification for Toxicity
For the purposes of this study, drug-related toxicity was arbitrarily defined as any of the following outcomes without other explanation in the 28 days after implantation: 40-point decline in the Karnofsky performance status (sustained for at least 2 weeks), two or more episodes of status epilepticus with therapeutic anticonvulsant levels, a brain abscess requiring surgical intervention, and WBC count of less than 2,000/mm³; ANC of less than 1,000/mm³; platelets of less than 50,000/mm³ or hemoglobin of less than 8 gm/dL; ALT, AST, alkaline phosphatase, or blood urea nitrogen of more than 5x the upper limit of normal (ULN); bilirubin of more than 1.5x the ULN; creatinine of more than 3x the ULN; and proteinuria (> 10 gm/L) or unexplained gross hematuria, dyspnea on exertion, or treatment-related death. Any death related to the use of O⁶-BG and/or carmustine wafers would stop enrollment until all data were formally reviewed.

AGT Activity
Extracts were prepared from tumors by homogenization in 50 mmol/L Tris (pH 7.5), 0.1 mmol/L EDTA, and 5 mmol/L dithiothreitol. AGT activity was determined as described previously.¹⁹ Briefly, cell extracts were incubated with [³H]-methylated DNA substrate (5.77 Ci/mmol). The DNA was precipitated by adding ice-cold prechloric acid at a final concentration of 0.25 N, which was then hydrolyzed in 0.1 N HCL at 70°C for 30 minutes. The modified bases were eluted on a C18 reverse-phase column using 10% methanol/0.05 M ammonium formate pH 4.5 at 37°C. Each assay was performed with a positive control cell line (DAOY or DuPro cell extract) and negative control (CHO cell extract). Protein concentration was determined by the Bradford method.²⁰ The results were expressed as femtomole of O⁶-methylguanine released from DNA per mg of protein.

Pharmacokinetic Measurements
Plasma concentrations of O⁶-BG and 8-oxoBG were measured before administration of O⁶-BG (120 mg/m² over 1 hour followed immediately by 30 mg/m²/d continuous infusion) and at 24, 48, 72, and 96 hours after administration of O⁶-BG bolus. Whole blood samples (7 mL) were collected in sodium-heparinized vacutainers and centrifuged for 10 minutes at 1,500 x g. Samples were stored at –70°C until analysis. O⁶-BG and 8-oxoBG were measured by high-performance liquid chromatography as described previously with slight modifications.²¹ Aliquots of plasma were spiked with an internal standard, O⁶-[p-fluorobenzyl]guanine, extracted with ethyl acetate, and centrifuged at 2,500 x g for 20 minutes, and the supernatant was evaporated to dryness under nitrogen. Samples were reconstituted in mobile phase (32% methanol/10 mmol/L potassium phosphate buffer; pH 7.5) and separated isocratically using a Waters Bondapak 125Å 4 µ phenyl column (3.9 x 300 mm) at 30°C and a flow rate of 1.0 mL/min. Retention times were 21.5, 24.6, and 28.9 minutes for 8-oxoBG, O⁶-BG, and O⁶-[p-fluorobenzyl]guanine, respectively. Samples were monitored at 280 nm using a UV detector and at 295 excitation and 360 emission on a fluorescence detector. The limit of detection for both O⁶-BG and 8-oxoBG was determined to be 10 ng/mL.

Statistical Considerations
Demographic and clinical characteristics were summarized using appropriate descriptive statistics. Categoric data were summarized with frequencies and percents and continuous data with medians and ranges. Toxicities were tallied by treatment cohort. Survival time was calculated from the start of therapy until death from any cause, and survival was estimated using the Kaplan-Meier method.²² CIs were calculated using standard methods. Analyses of demographic and clinical characteristics and toxicities were performed using SAS Version 9.1 (SAS Institute, Cary, NC).

	RESULTS

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Patient Characteristics
Forty-two patients were accrued to this study. Demographic and clinical characteristics by treatment group are presented in Table 1. Thirty-nine patients (93%) received eight polymer wafer implants. One patient (2%) each received four, five, and six wafers.

Treatment Administration for Group B
For treatment group B, continuous infusion was successfully increased to the 14-day time point. Six patients were enrolled at the 2-, 4-, and 7-day continuous-infusion cohorts, and no dose-limiting toxicities (DLTs) were observed. Six patients were initially enrolled at the 14-day time point. However, in four out of the first six patients treated, O⁶-BG began precipitating in the intravenous catheter after 10 days, so these infusions were temporarily stopped. With a new supply of O⁶-BG, four additional patients were accrued to this cohort without precipitation. All 10 patients in the cohort were evaluated for toxicity. One patient developed grade 3 elevation in ALT. Although O⁶-BG was not the likely cause, it prompted the investigator (K.J.) to stop the therapy. As a result, this was considered a DLT.

All significant toxicities related to carmustine polymer or O⁶-BG are presented in Table 2. The only grade 4 toxicity was one cerebrospinal fluid leak. Although an infection and CNS hemorrhage were noted in one patient in the 14-day infusion cohort, these occurred after the 28-day evaluation period for DLTs.

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Mean (± standard deviation) of O6-benzylguanine (O6-BG; ) and O6-benzyl-8-oxoguanine (8-oxoBG; •) plasma concentration in group A patients. A total of 14 patients were treated with 120 mg/m2 O6-BG over 1 hour followed immediately by a continuous infusion of 30 mg/m2/d O6-BG for 48 hours presurgery.

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. A total of 26 patients were treated with 120 mg/m2 of O6-benzylguanine (O6-BG) over 1 hour, administered at least 1 hour presurgery, and followed immediately by a 30 mg/m2/d of continuous-infusion O6-BG beginning on the day of surgery. The length of the infusion increased up to14 days. Concentrations of O6-benzyl-8-oxoguanine (8-oxoBG; •) and O6-BG () were evaluated before the O6-BG bolus, day of surgery, and various days postsurgery.

	DISCUSSION

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Alkylating drugs, such as carmustine and temozolomide, are the most effective agents in patients with primary brain tumors. Although systemic carmustine results in tumor responses in some patients, its benefit in adjuvant trials is modest. Placement of locally administered carmustine using sustained-release polymers prolonged survival for 8 to 10 weeks in randomized placebo-controlled trials in this patient population.^5-7 In addition, this is devoid of systemic toxicities. Thus, improving the efficacy of locally administered carmustine is a reasonable therapeutic strategy.

AGT, a DNA repair protein present in a significant percentage of high-grade gliomas, confers relative resistance to treatment with temozolomide and carmustine.⁸ In 167 patients treated with carmustine, patients with low tumor AGT levels lived longer than patients with high AGT levels.²⁵ Similarly, survival in patients treated with radiation and temozolomide was correlated with the absence of the AGT repair protein.²⁶ These studies suggest that the efficacy of carmustine could be enhanced by depletion of tumor AGT.

O⁶-BG is a low molecular weight substrate that inactivates AGT. Preclinical studies combining O⁶-BG with carmustine suggest that this results in improved tumor control and survival.^10,12,13 However, systemically administering the O⁶-BG and carmustine markedly enhances myelosuppression and reduces the maximum tolerated dose of systemic carmustine from 200 mg/m² to 40 mg/m².¹⁵ In contrast, local delivery of high doses of carmustine directly to the tumor results in minimal systemic exposure to carmustine.²⁷ Therefore, the combination of local carmustine delivered via polymer wafer and O⁶-BG should minimize systemic carmustine toxicity while maximizing the efficacy of the carmustine within the tumor.

This hypothesis was tested in a preclinical F98 rat brain tumor model. F98 expresses high levels of AGT and is resistant to treatment with alkylating agents. Rats treated with carmustine polymer alone showed no increased survival compared to controls, whereas combination treatment of O⁶-BG with carmustine polymer resulted in significant prolonged survival. This suggests that O⁶-BG potentiates the effects of locally delivered carmustine, and for tumors expressing AGT, it may be necessary for carmustine to provide a meaningful benefit.²⁸

This study aimed to identify a continuous-infusion dose of O⁶-BG that would suppress tumor AGT activity for 2 weeks and to assess the local and systemic toxicity of O⁶-BG combined with implanted carmustine wafers. The results demonstrate that 120 mg/m² of O⁶-BG followed by 30 mg/m²/d by continuous infusion reduces tumor AGT levels to an undetectable level for 48 hours. In group A patients, the plasma levels of O⁶-BG and 8-oxoBG, the active metabolite of O⁶-BG, at 48 hours was 0.4µm for O⁶-BG and 1.2µm for 8-oxoBG (Fig 1).

Patients in group B exhibited no local or systemic toxicity with the combination treatment. Plasma concentrations of O⁶-BG and 8-oxoBG in these patients were measured at various time points during the continuous infusion. Figure 2 demonstrates that the initial effect on plasma drug levels of the bolus followed by the continuous infusion continues for 48 to 72 hours. The steady-state plasma drug levels stabilize at 72 hours and were found to be 0.7 to 1.3 µm for 8-oxoBG and 0.1 to 0.4 µm for O⁶-BG. Previous in vitro results indicate that the effective doses for 50% depletion of AGT activity in 30 minutes in HT29 colon tumor cell extract are 0.2 µmol/L for O⁶-BG and 0.3 µmol/L for 8-oxoBG.²⁴ In group A, we found absolute suppression of AGT activity at 48 hours, and we expected this suppression to last for the entire 2-week infusion period. However, when the group B data were analyzed using later time points, the steady-state drug levels were lower. The effect of 8-oxoBG plasma levels of 0.7 to 1.3 µm on brain tumor AGT levels cannot be determined from this study.

This study demonstrates that AGT levels in brain tumors can be suppressed using systemically administered O⁶-BG. No added toxicity was noted when this was combined with locally administered carmustine. This is in contrast to combining O⁶-BG with systemic carmustine. These data support proceeding with clinical trials designed to test the hypothesis that O⁶-BG will increase the efficacy of local carmustine and improve patient survival. These trials should include newly diagnosed patients and a higher concentration of carmustine polymers.²⁷ The pharmacokinetics raise the possibility that the continuous-infusion dose required to completely suppress AGT levels for 2 weeks may need to be higher.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment: N/A Leadership: N/A Stock: N/A Honoraria: N/A Research Funds: N/A Testimony: N/A Other: M. Eileen Dolan, KERYX Biopharmaceuticals; Jon Weingart, MGI PHARMA

	AUTHOR CONTRIBUTIONS

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Conception and design: Stuart A. Grossman, Kathryn A. Carson, Kevin Judy, Stephen B. Tatter, M. Eileen Dolan

Financial support: M. Eileen Dolan

Administrative support: Joy D. Fisher

Provision of study materials or patients: Stuart A. Grossman, Joy D. Fisher, Mark L. Rosenblum, Alessandro Olivi, Kevin Judy, Stephen B. Tatter

Collection and assembly of data: Stuart A. Grossman, Joy D. Fisher, Shannon M. Delaney, Kevin Judy, Stephen B. Tatter, M. Eileen Dolan

Data analysis and interpretation: Stuart A. Grossman, Kathryn A. Carson, Joy D. Fisher, Shannon M. Delaney, Stephen B. Tatter, M. Eileen Dolan

Manuscript writing: Jon Weingart, Stuart A. Grossman, Kathryn A. Carson, Shannon M. Delaney, Alessandro Olivi, Stephen B. Tatter, M. Eileen Dolan

Final approval of manuscript: Jon Weingart, Stuart A. Grossman, Kathryn A. Carson, Shannon M. Delaney, Kevin Judy, Stephen B. Tatter, M. Eileen Dolan

	NOTES

 
Supported by New Approaches to Brain Tumor Therapy Grant No. CA62475, a supplement to Grant No. CA69852 (M.E.D.), and the University of Chicago Cancer Research Center Support Grant No. P30 CA14599.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted April 6, 2006; accepted November 2, 2006.

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