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Vol. 6, 3025–3031, August 2000 Clinical Cancer Research 3025

Phase I Clinical and Pharmacological Study of O6-Benzylguanine Followed by Carmustine in Patients with Advanced Cancer1

Richard L. Schilsky,2 M. Eileen Dolan,3 dose-limiting toxicity of BG combined with carmustine and Donna Bertucci, Reginald B. Ewesuedo, was cumulative in some patients. The neutrophil nadir oc- Nicholas J. Vogelzang, Sridhar Mani, curred at a median of day 27, with complete recovery in most patients by day 43. Nonhematological toxicity included Lynette R. Wilson, and Mark J. Ratain fatigue, anorexia, increased bilirubin, and transaminase el- Department of Medicine, Section of Hematology-Oncology, Cancer evation. Recommended doses for Phase II testing are 120 Research Center and Committee on Clinical Pharmacology, mg/m2 BG given with carmustine at 40 mg/m2. BG rapidly University of Chicago, Chicago, Illinois 60637 disappeared from plasma and was converted to a major metabolite, O6-benzyl-8-oxoguanine, which has a 2.4-fold ABSTRACT higher maximal concentration and 20-fold higher area un- O6-benzylguanine (BG) is a potent inactivator of the der the concentration versus time curve than BG. AGT DNA repair protein O6-alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells was rapidly (AGT) that enhances sensitivity to in tumor cell and completely suppressed at all of the BG doses. The rate lines and tumor-bearing animals. The major objectives of of AGT regeneration was more rapid for patients treated this study were to define the optimal modulatory dose and with the lowest dose of BG but was similar for BG doses 2 associated toxicities of benzylguanine administered alone ranging from 20–120 mg/m . In conclusion, coadministra- and in combination with carmustine; to define the maxi- tion of BG and carmustine is feasible in cancer patients, but mally tolerated dose and associated toxicities of carmustine the maximal dose of carmustine that can be safely adminis- administered with benzylguanine and to describe the phar- tered with BG is approximately one-third of the standard macokinetics of BG in humans and its effects on AGT clinical dose. Bone marrow suppression, which may be cu- depletion and recovery in peripheral blood mononuclear mulative, is the dose-limiting toxicity of the combination. cells. Patients with histologically confirmed advanced solid Prolonged AGT suppression is likely attributable primarily 6 tumors or that had failed to respond to standard to the effect of O -benzyl-8-oxoguanine. therapy or for which no standard therapy was available were eligible to participate in this study. Patients initially INTRODUCTION received BG as a 1-h i.v. infusion without carmustine. After The DNA repair protein AGT4 (1) plays an important role a 14-day washout (i.e., without therapy) period, patients in the protection of cells from the cytotoxic effects of alkylni- received BG as a 1-h i.v. infusion followed, 1 h later, by a trosoureas and methylating agents. AGT removes adducts from 15-min i.v. infusion of carmustine. Cycles of the O6 position of in DNA through covalent binding of were repeated every 6 weeks. Cohorts of patients received the alkyl group to a cysteine residue on the protein within the BG doses ranging from 10 to 120 mg/m2 and carmustine active site (1). During this process, irreversible inactivation of doses ranging from 13 to 50 mg/m2. Plasma and urine the protein occurs and the synthesis of new protein molecules is samples were collected and analyzed for BG, and O6-benzyl- required to regenerate AGT activity. 8-oxoguanine concentrations and AGT activity was deter- There is an inverse relationship between the level of AGT mined in peripheral blood mononuclear cells. and the sensitivity of tumor cells grown in culture and as There was no toxicity attributable to BG alone at any xenografts to the cytotoxic effects of alkylnitrosoureas (1). dose tested. Bone marrow suppression was the primary and Increased AGT activity has been found in many human solid tumors including colon cancer (2), malignant melanoma (3), lung cancer, (4, 5), and others (6), and may account for the relative ineffectiveness of therapy in these dis- Received 12/6/99; accepted 2/15/00. eases. Inactivation of AGT leads to an enhancement of the The costs of publication of this article were defrayed in part by the cytotoxic effects of chloroethylnitrosoureas (e.g., carmustine) payment of page charges. This article must therefore be hereby marked and methylating agents (e.g., , ) in advertisement in accordance with 18 U.S.C. Section 1734 solely to both cell culture and animal tumor xenograft models (7–9). indicate this fact. 1 Supported by USPHS Grants CA14599 (to R. L. S.), CA67098 (to M. E. D.), and CA69852 (to M. J. R.) from the National Cancer Insti- tute, NIH, Bethesda, MD, and by Grant MO1 RR00055 to The General Clinical Research Center, University of Chicago. 4 The abbreviations used are: AGT, O6-alkylguanine-DNA alkyltrans- 2 To whom requests for reprints should be addressed: Division of the ferase; BCNU, bischloronitrosourea, carmustine; BG, O6-benzylgua- Biological Sciences, University of Chicago, 5841 South Maryland nine; MTD, maximally tolerated dose; PBMC, peripheral blood mono- Avenue, MC1000, Chicago, IL 60637. Phone: (773) 834-3914; Fax: nuclear cell; BG max, maximal dose of BG; DLT, dose-limiting

(773) 834-3915; E-mail: [email protected]. toxicity; DLCO, carbon monoxide diffusion capacity; AUC, area under 3 Author has disclosed a financial interest in Procept, a company that has the concentration versus time curve; 8-oxo-BG, O6-benzyl-8-oxogua- licensed O6-benzylguanine. nine. 3026 Phase I Trial of Benzylguanine plus Carmustine

Therefore, various strategies have been attempted to deplete creatinine level less than 1.7 mg/dl or measured creatinine cells of AGT and thereby increase the sensitivity of tumor cells clearance of at least 60 ml/min. Patients must have been off of to carmustine and related agents. all previous anticancer therapy for at least 4 weeks (6 weeks if Agents that methylate DNA, such as and the previous therapy included or a nitrosourea) and dacarbazine, deplete AGT activity incompletely and produce must have recovered from the toxic effects of any prior therapy. significant clinical toxicity (10). BG, a low-molecular-weight Patients were excluded from the study if they had a significant AGT substrate, binds readily to the same cysteine residue on cardiac, pulmonary, neurological, endocrine, gastrointestinal, AGT that is used for alkyl group transfer and inactivates the rheumatological, dermatological, or allergic disorder that would protein stoichiometrically, requiring micromolar concentrations make administration of the therapy hazardous or would obscure and only minutes of exposure to completely inactivate AGT (11, the interpretation of adverse effects. Pregnant and lactating 12). Once AGT is inactivated, cells become vulnerable to killing women were also excluded, and all of the patients with repro- by nitrosoureas because lesions that are present at the O6 posi- ductive potential were required to use an effective contraceptive tion of guanine cannot be repaired until new AGT synthesis method during treatment and for 2 months after completion of occurs (13–16). There is a strong correlation between the degree treatment if they were sexually active. All of the patients gave of sensitization that can be achieved and the level of AGT written informed consent according to institutional and federal activity in cells, with little or no BG-induced enhancement of guidelines. BCNU cytotoxicity occurring in cells that express low levels of Study Design. The initial goal of the study was to define AGT and the greatest enhancement observed in cells with high the dose of BG that produced maximal AGT suppression in AGT activity (13). BCNU preceded by BG treatment results in human PBMCs without excessive toxicity and to define the time significantly greater growth inhibition of human brain and colon course of this inhibition. Once this dose of BG was defined, tumor xenografts in nude mice compared with that observed in cohorts of patients were to be treated at a fixed dose of BG and animals treated with BCNU alone (8, 17). at increasing doses of BCNU to determine the MTD of BCNU Preclinical toxicology studies in mice and dogs revealed given with BG. Thus, patients who were enrolled in the study BG alone to be nontoxic. When combined with BCNU, bone initially received BG as a 1-h i.v. infusion without BCNU. After marrow toxicity was dose-limiting, and the MTD of BCNU was a 14-day washout period, patients received the assigned dose of 2- to 3-fold lower in mice and 6-fold lower in dogs than in the BG as a 1-h i.v. infusion followed, 1 h later, by a 15-min i.v. absence of BG (18, 19). infusion of BCNU. Cycles of chemotherapy were repeated every On the basis of the strong preclinical evidence that BG 6 weeks as long as the patient’s tumor was stable or responding administration could potentially reverse resistance to alkylnitro- to treatment and the patient did not experience DLT. 2 soureas, we conducted a Phase I study of the combination of BG The starting dose of BG was 10 mg/m . Escalation of the and BCNU in patients with advanced cancer. The major objec- BG dose occurred in increments of 100% until maximal AGT tives of the study were to define the optimal modulatory dose suppression was observed in PBMCs 6 h after dosing or until the and associated acute and cumulative toxicities of BG adminis- appearance of grade 1–2 toxicity attributable to BG. Dose es- tered alone and in combination with BCNU; to define the MTD calation of BG was to continue until AGT suppression of at least and associated acute and cumulative toxicities of BCNU admin- 90% was observed in at least two of three patients in two istered with BG; to determine the time course of AGT depletion successive dose cohorts (BG max). During the period of BG and recovery in PBMCs after administration of BG; and to dose escalation, all of the patients received a fixed dose of 2 describe the pharmacokinetics of BG in humans. BCNU of 13 mg/m . Once BG max was defined, all of the subsequent patients were to receive BG max, and the BCNU dose was to be escalated in cohorts of at least three patients until PATIENTS AND METHODS the MTD of BCNU was defined. Planned BCNU dose levels Patient Selection. Patients with histologically confirmed were 25 mg/m2, 40 mg/m2, 60 mg/m2, 80 mg/m2, and 100 advanced solid tumors or lymphoma that had failed to respond mg/m2. No intrapatient dose escalation was permitted. Midway to standard therapy or for which no standard therapy was avail- through the study, we became aware of data from other trials able were eligible to participate in this study. Patients with that suggested that AGT suppression in PBMCs did not corre- malignant gliomas were eligible if they were on a stable steroid late with the extent of AGT suppression in solid tumor tissues; dose for at least 2 weeks before enrollment and met all of the that BG doses greater than 80 mg/m2 were required to com- other eligibility criteria. Patients with malignant melanoma were pletely suppress AGT activity in tumor tissue; and that doses of required to undergo a computed tomography head scan within 2 at least 100 mg/m2 were required to completely suppress AGT weeks of entry on-study to determine whether asymptomatic activity in brain tumors (20, 21). We, therefore, studied addi- brain metastases were present. Other eligibility criteria included tional cohorts of patients at BG doses higher than those deter- measurable or assessable disease by computed tomography, mined to be BG max in PBMCs. magnetic resonance imaging, radiograph or physical examina- Definition of Study End Points. BG max was defined as tion; age of at least 18 years; Karnofsky performance status of stated above. DLT of BCNU was defined as the dose of BCNU at least 60%; life expectancy of at least 8 weeks and adequate that produced at least grade 3 nonhematological toxicity (except organ function defined as: a WBC count of at least 3500/␮l, alopecia, nausea, or vomiting) or grade 4 hematological toxicity platelets of at least 100,000/␮l, and hemoglobin at least 10 g/dl; lasting more than 3 days, or fever in the presence of an absolute total bilirubin level less than 1.7 mg/dl; aspartate aminotrans- neutrophil count of less than 1000/␮l. The MTD of BCNU was ferase less than twice the upper limit of normal; and serum defined as the dose of BCNU that produced DLT in more than Clinical Cancer Research 3027

Table 1 Patient characteristics Table 2 Dose levels Characteristic Number of patients Benzylguanine BCNU No. of No. of 2 2 a Number of patients 78 (mg/m ) (mg/m ) patients cycles M/F 45/33 10 13 3 6 Median age, yr (range) 53 (23–82) 20 13 7 9 Karnofsky performance status 100 30 40 13 3 5 90 24 80 13 3 9 80 14 40 25 3 5 70 9 40 32 3 5 60 1 40 40 9 9 Prior therapy 40 50 8 9 Chemotherapy only 42 80 25 7 14 Chemotherapy and radiation 34 80 32 3 6 Radiation only 2 80 40 3 7 Diagnosis 120 25 6 8 Colorectal 45 120 32 7 11 Renal 9 120 40 10 16 Cervical 2 120 50 3 2 6 Total 78 121 Endometrial 2 Other 14 a Total enrolled.

two of six patients during the first cycle of chemotherapy. The than that required for a partial response. Disease progression recommended Phase II dose was one dose level below the MTD was defined as Ն25% increase in the sum of the products of the of BCNU. perpendicular diameters of all of the measured lesions over the Drug Supply. BG (NSC 637037) was supplied by the smallest sum observed or as the appearance of any new lesions National Cancer Institute, Division of Cancer Treatment and or the clear worsening of assessable disease. Diagnosis, in 100-mg vials containing white lyophilized powder Plasma Sampling and Pharmacokinetic Studies. Pa- with 670 mg mannitol, USP, and sodium hydroxide to adjust pH tients were admitted to the General Clinical Research Center at to 7–8.5. Once diluted, each ml of solution contained 3.3 mg of the University of Chicago for administration of the initial dose BG; 22 mg of mannitol, USP; 0.4 ml of polyethylene glycol of BG. Whole-blood samples were collected in sodium-hepa- 400; and approximately 0.6 ml (pH 7) phosphate buffer. BCNU rinized vacutainers before treatment, at 30 and 60 min during the was commercially available from Bristol-Myers Squibb and was infusion of BG and at 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and prepared for administration according to package labeling. 48 h after the completion of the infusion. Plasma was obtained Pretreatment and Follow-Up Studies. Before the initi- by centrifugation at 2500 rpm for 10 min. Urine was collected ation of therapy, all of the patients had a history and physical at various time periods up to 48 h after completion of the examination, assessment of Karnofsky performance status, chest infusion. Samples were stored at Ϫ70°C until analysis. Total radiograph, 12-lead electrocardiogram, determination of tumor plasma and urine concentrations of BG and 8-oxo-BG were measurements, dipstick urinalysis, and routine laboratory stud- measured by high-pressure liquid chromatography using meth- ies that included a complete blood count with differential WBC ods that have been described previously (22). For determination count, electrolytes, urea, creatinine, glucose, total protein, albu- of AGT activity in PBMCs, blood (40 ml) was collected twice min, calcium, phosphate, uric acid, alkaline phosphatase, total before the BG infusion was begun and at 1, 6, 10, 24, 48, 72, and direct bilirubin, and alanine aminotransferase and aspartate 168, and 336 h postinfusion. Within an hour of collection, an aminotransferase levels. Baseline pulmonary function studies equal volume of RPMI medium was added, and the diluted

including DLCO and arterial blood gases were performed on all blood was layered on Ficoll-Paque (Histopaque 1077). After of the patients. Complete blood counts were assessed weekly centrifugation at 400 ϫ g for 30 min, the lymphocyte layer was during therapy and daily if grade 4 neutropenia or thrombocy- removed and resuspended in 15 ml PBS and centrifuged at topenia was documented. History and physical examination, 250 ϫ g for 10 min at room temperature. RBCs were lysed by determination of performance status, toxicity evaluation, and the addition of 6 ml of deionized water for 15 s after which 2 ml serum chemistries were performed every 2 weeks and before of 3.6% NaCl and 15 ml of PBS were added. The samples were each cycle of therapy. Disease assessment and pulmonary func- centrifuged at 250 ϫ g for 10 min. Final pellets were resus- tion tests were performed after every two cycles of therapy. A pended in 50 mM Tris (pH7.5), 0.1 mM EDTA, and 5 mM DTT. complete response was defined as the disappearance of all The pharmacokinetics of BG were analyzed using noncom- clinical, biochemical, and radiographic evidence of the tumor partmental methods with WinNonlin (PharSight Corp., Apex,

for a minimum of 4 weeks and the absence of no disease-related NC). The terminal elimination t1/2 of BG was estimated from the symptoms. A partial response was defined as at least a 50% slope of the terminal concentration-time curve of individual

decrease from baseline in the sum of the products of the per- patients. The AUC for the study period, AUClast, for BG and pendicular diameters of all measurable lesions lasting at least 4 8-oxo-BG were calculated by the linear trapezoidal method. The

weeks during which time no new lesions had appeared. Minor AUC to infinity (AUC0-inf) was calculated as the sum of AUClast ␭ response was defined as objective decrease of disease but less and Clast/ , where Clast is the final measured concentration and 3028 Phase I Trial of Benzylguanine plus Carmustine

Table 3 Hematological toxicity—cycle 1 Table 4 Effect of benzylguanine dose on DLT of BCNU (cycle 1) No. of patients with Նgrade 3 BG dose BCNU dose BG BCNU No. of (mg/m2) (mg/m2) No. of patients with DLT (mg/m2) (mg/m2) patientsa Neutropenia Thrombocytopenia DLT 40 32 0/3 10 13 3 0 0 0 80 32 0/3 20 13 5 0 0 0 120 32 0/6 40 13 3 0 0 0 40 40 2/7 80 13 3 0 0 0 80 40 0/3 40 25 3 1 2 0 120 40 1/8 40 32 3 0 0 0 40 50 3/4 40 40 7 2 2 2 120 50 1/2 40 50 4 3 3 3 80 25 6 3 3 1 80 32 3 0 0 0 80 40 3 0 0 0 120 25 3 0 0 0 listed in Table 3. Grade 3 or higher toxicity was noted at BCNU 120 32 6 1 0 0 doses as low as 25 mg/m2 given with BG. As originally defined 120 40 8 2 2 1 2 120 50 2 1 1 1 in the protocol, a BG dose of 40 mg/m was determined to be a BG max based on suppression of AGT activity in PBMCs. At Evaluable for toxicity. this BG dose, two of seven patients treated with BCNU at 40 mg/m2 had dose-limiting hematological toxicity and three of four patients treated at a BCNU dose of 50 mg/m2 had dose- limiting hematological toxicity. Further escalation of the BG ␭ is the terminal elimination rate constant. The latter was based dose was not associated with increased BCNU toxicity (Table on visual inspection of the data. When Clast was below the limit 4), which suggests that BG doses of Ն40 mg/m2 produced of quantitation, a concentration of BG (15 ng/ml) or 8-oxo-BG maximal AGT suppression in bone marrow progenitor cells. (10 ng/ml) equivalent to one-half of the limit of quantitation was Table 5 summarizes cycle-one hematological toxicity data for used for the calculation of the AUC. BCNU doses of 32 mg/m2, 40 mg/m2, and 50 mg/m2 given with AGT Activity. The assay for alkyltransferase activity BG at doses of 40 mg/m2 or higher. Dose-limiting hematolog- was performed as described previously (22). Briefly, alkyltrans- ical toxicity occurred in 4 of 6 patients treated with BCNU at 50 6 3 ferase activity was measured as the removal of O -[ H]methyl- mg/m2; in 3 of 18 patients treated with BCNU at 40 mg/m2; and 3 guanine from a H-methylated DNA substrate (5.8 Ci/mmol) in 0 of 12 patients treated with BCNU at 32 mg/m2. Prior after incubation with extract at 37°C for 30 min. The DNA was therapy received by the patients with DLT is summarized in precipitated by adding ice-cold perchloric acid (0.25 N) and was Table 6. It should be noted that one of the patients with DLT at hydrolyzed by the addition of 0.1 N HCl at 70°C for 30 min. The a BCNU dose of 40 mg/m2 had previously had high-dose modified bases were separated by reverse-phase high-pressure chemotherapy consisting of 1-␤-D-arabinofuranosylcytosine, liquid chromatography with 0.05 M ammonium formate (pH 4.5) , , and carmustine and an auto- containing 10% methanol. Each assay was performed with a logous stem-cell transplant as therapy for refractory lymphoma. positive control cell line (DaOY cell extract) and lymphocytes On the basis of this information, we recommend doses of BG at from three normal volunteers. Protein was determined by the 120 mg/m2 with BCNU at 40 mg/m2 for subsequent Phase II method of Bradford (23), and the results were expressed as fmol studies. The higher BG dose is recommended based on data 6 of O -methylguanine released from the DNA substrate per mg reported by other investigators, which suggested that 120 mg/m2 of protein. is the optimal dose to completely suppress AGT in tumor tissue. Statistical Considerations. Comparisons of DLCO post- In patients with DLT, the neutrophil nadir occurred be- cycle 2 to baseline were performed using a paired t test. tween days 11 and 36 of the treatment cycle (median, day 27) with complete recovery in most patients by day 43. The platelet RESULTS nadir occurred between days 17 and 27 of the treatment cycle The characteristics of the 78 patients enrolled in the study (median, day 21) with complete recovery in most patients by are listed in Table 1. Sixteen patients did not complete the first day 37. Sepsis or febrile neutropenia were uncommon with only cycle of therapy and are, therefore, not assessable for toxicity or four episodes documented. Similarly, only three patients re- response; toxicity data on the remaining 62 patients form the quired transfusion of platelets at any time during their chemo- basis of this report. In most cases, failure to complete the first therapy treatment. Because nitrosoureas are known to cause cycle of therapy was attributable to rapid disease progression. cumulative bone marrow toxicity, we tried to determine whether Table 2 lists the dose levels evaluated in this study. A total of severe hematological toxicity was common during the second 121 cycles of chemotherapy were administered at BG doses cycle of chemotherapy. Table 7 summarizes episodes of grade 3 ranging from 10 to 120 mg/m2 and BCNU doses ranging from or higher bone marrow suppression occurring during cycle 2 in 13 to 50 mg/m2. patients treated with BCNU doses of 32 mg/m2, 40 mg/m2,or50 Toxicity. No clinically significant toxicity was observed mg/m2 regardless of the BG dose. Of note is that, although 1 of after administration of BG alone at any dose level. Bone marrow 12 patients treated at 32 mg/m2 experienced grade 3 or higher suppression was the primary toxicity and DLT of BG combined toxicity during cycle one (Table 3), 3 of 10 patients who with BCNU. Hematological toxicity observed during cycle 1 is received a second cycle of chemotherapy at this BCNU dose Clinical Cancer Research 3029

Table 5 Summary of hematological toxicity—cycle 1 Table 6 Prior therapy for patients with DLT during cycle 1 No. of patients with Նgrade 3 Dose (mg/m2) BG BCNU No. of (BG/BCNU) Prior chemotherapy (mg/m2) (mg/m2) patients Neutropenia Thrombocytopenia DLT 40/40 CHOPa/ABMT Ն40 32 12 1 0 0 40/40 IL-12/raltritrexed plus Ն40 40 18 4 4 3 80/25 5-FU/CPT-11/Rhizoxin/raltritrexed plus paclitaxel Ն40 50 6 4 4 4 40/50 Raltritrexed plus paclitaxel/tamoxifen 40/50 CPT-11/5FU-eniluracil 40/50 5-FU/CPT-11 120/40 plus //paclitaxel 120/50 CCNU// experienced grade 3 or 4 neutropenia or thrombocytopenia, a CHOP, cyclophosphamide, , vincristine, prednisone; which clearly indicated the potential for cumulative hematolog- AMBT, autologous bone marrow transplant; 5-FU, 5-; ical toxicity to occur. One patient, initially thought to have CCNU, cyclohexylchloroethylnitrosourea; CPT-11, ; IL-12, treatment-related grade 4 bone marrow suppression after his interleukin 12. second cycle of chemotherapy at BG and BCNU doses of 120 and 40 mg/m2, respectively, was diagnosed with acute promy- Table 7 Hematological toxicity (Նgrade 3) during cycle 2 elocytic leukemia. This patient had a hepatocellular carcinoma and had previously been treated with pyrazaloacridine. Cytoge- BG dose BCNU dose No. of patients with 2 2 Ն netic examination of the leukemic blasts revealed a t(15;17) (mg/m ) (mg/m ) grade 3 toxicity translocation. No other cytogenetic abnormalities were noted. Ն40 25 1/12 Ն Given the low cumulative dose of BCNU received (80 mg/m2) 40 32 3/10 Ն40 40 3/9 and the short interval from initiation of therapy until diagnosis Ն40 50 3/4 (10 weeks), this leukemia was unlikely to be related to the BG/BCNU therapy. Nonhematological toxicity was uncommon and not clearly dose-related in this study. The most common toxicities of grade Ϯ Ϯ 2 2 or higher were fatigue, anorexia, increased bilirubin, and 12.1, 34.7 12.9, and 31.6 10.6 liters/h/m , at BG doses of 2 transaminase elevation. None of these events occurred in more 40, 80, and 120 mg/m , respectively. than 15% of the patients. The relative AGT activity in PBMCs was determined after Because high cumulative doses of carmustine are known administration of BG. The rate of AGT regeneration was more 2 to be associated with , we monitored rapid for patients receiving the lowest dose of BG (10 mg/m ); D CO in these patients after every two cycles of therapy. however, the rate of regeneration was similar for doses ranging L 2 Pulmonary function tests were performed prestudy in every from 20 to 120 mg/m . AGT inactivation curves have been patient, but only 30 patients returned for testing after two published for the initial 25 patients (22). Fig. 2 provides data for cycles of therapy. Among patients with both baseline and a total of 50 patients. postcycle 2 measurements, mean DLCO uncorrected fell ap- ϭ proximately 9% (P 0.002) and mean DLCO corrected fell DISCUSSION approximately 10% (P ϭ 0.006). Twenty of the 30 patients Inactivation of AGT is a potentially important strategy to had a decrease in the percentage of predicted DLCO. Of improve the clinical utility of the alkylnitrosoureas. This study these, the median percent decrease in the percentage of and others (21) clearly demonstrate that BG completely sup- predicted DLCO was 10% (range, 2–46%). Only four patients presses AGT activity in PBMCs and tumor tissues at nontoxic had a percent decrease in percentage of predicted DLCO of doses. Coadministration of BG and BCNU is feasible in cancer Ն20%. These patients had been treated with BG/BCNU patients but, as expected based on pre-clinical data, the maximal doses of 40/32, 80/25, 120/25, and 120/32. No patient expe- dose of BCNU that can be safely administered in the presence of rienced clinical signs or symptoms of pulmonary toxicity. BG is approximately one-third of the standard clinical dose. Antitumor Response. No complete or partial responses Clinicians must monitor the BCNU dose particularly carefully were observed. in the presence of BG because even small increments in BCNU Pharmacological Studies. We have previously reported dosage can produce severe toxicity when AGT is inactivated. results of pharmacological studies performed in 25 patients Patients and clinicians must also be alert to the possibility of enrolled in this study and treated at BG doses ranging from 10 cumulative bone marrow suppression and the potential for se- 2 to 80 mg/m (22). BG is rapidly eliminated from plasma (t1/2 at vere hematological toxicity to occur in the second or later cycles 120 mg/m2 averaged 1.9 h) and is replaced by a major metab- of therapy, even in patients who do not experience significant olite, 8-oxo-BG. This biologically active metabolite of BG has toxicity during the initial treatment cycle. Although pulmonary 2 a longer t1/2 (averaging 4.0 h at 120 mg/m ) and a 12- to 29-fold toxicity is not clinically significant after two cycles of therapy, higher AUC than BG. we cannot rule out the possibility of pulmonary dysfunction Fig. 1 illustrates the relationship between BG dose and after higher cumulative doses of BCNU. AUC for patients treated at doses ranging from 40 to 120 mg/m2 The study design that we used clearly illustrates the advan- of BG. As suggested by Fig. 1, the BG clearance decreases as tages and disadvantages of using PBMCs as surrogates for the dose increases from 40 to 120 mg/m2, with values of 40.9 Ϯ tumor tissue in assessing biochemical events. PBMCs are easily 3030 Phase I Trial of Benzylguanine plus Carmustine

Fig. 2 AGT activity relative to pretreatment values in PBMCs of patients after a 1-h infusion of BG. Lymphocytes were isolated from blood at various times and AGT activity determined. This plot repre- sents data from 50 patients including data from the initial 25 patients that have been published previously (22).

Fig. 1 Plot of AUC for BG and 8-oxo-BG as a function of BG dose. AUC was determined as described in the text. AUC values for BG are that tumor responses would occur. A number of possible expla- 1.1 Ϯ 0.6, 2.7 Ϯ 1.2, and 4.2 Ϯ 1.4 ␮g⅐h/ml and for 8-oxo-BG are Ϯ Ϯ Ϯ ␮ ⅐ nations exist for the apparent lack of tumor efficacy. Only 24 19.4 9.7, 52.6 15.2, and 80.9 19.0 g h/ml at BG doses of 40,80, 2 and 120 mg/m2, respectively. patients received a BCNU dose of 40 mg/m or higher and, of these, only 10 received BG at a dose of 120 mg/m2. Treatment of a heterogeneous group of only 10 patients at the recom- mended Phase II doses or above is not an adequate test of accessible and can be sampled serially with little risk to the antitumor efficacy. It is also reasonable to consider whether this patient. PBMCs provide ample cellular material and enable schedule of BG administration is sufficient to produce sustained quantitative assays even with relatively insensitive techniques. AGT depletion throughout the period of time when BCNU- However, PBMCs cannot be presumed to be an adequate sur- induced DNA cross-links are most likely to occur, i.e., 12–18 h rogate for the assessment of biochemical events in tumor tissue. after BCNU administration (24). Synthesis of new AGT mole- Using the definition of BG max described previously, we ini- cules after BG depletion occurs at different rates in different 2 tially concluded that a BG dose of 40 mg/m was sufficient to tissues. Persistence of BG or 8-oxo-BG at sufficient concentra- completely suppress AGT activity. Although this is clearly the tion is necessary to insure that newly synthesized AGT is case with respect to PBMCs and bone marrow progenitors, inactivated or the removal of alkylnitrosourea mono- Spiro et al. (21) have convincingly demonstrated that AGT adducts from DNA is likely to occur, thereby limiting the suppression in PBMCs does not correlate with AGT suppression effectiveness of BCNU. Pharmacological data from this study in solid tumor tissue. Their study also demonstrated that BG demonstrates that plasma concentrations of 8-oxo-BG of at least 2 doses Ͼ80 mg/m are necessary to completely suppress AGT in 100ng/ml are detectable in most patients at 10 h post-BG dosing, tumors. and Spiro et al. (21) have demonstrated undetectable AGT We previously reported (22) linearity in the AUC of BG activity in tumor biopsies obtained 18 h after a BG dose of 120 and nonlinearity in the AUC of 8-oxo-BG with increasing BG mg/m2. Thus, it seems likely that the present schedule of ad- dosage based on an initial evaluation of 25 patients. Our updated ministering BG over a 1-h period prior to BCNU is adequate to analysis suggests some nonlinearity in BG pharmacokinetics suppress AGT throughout the period of BCNU-induced DNA over a wider dosage range, inasmuch as the mean clearance cross-link formation. decreased approximately 23% over a 3-fold increase in dosage. Phase II studies of the combination of BG and BCNU are In addition, the 8-oxo-BG AUC increased more than propor- ongoing or planned in patients with malignant gliomas, my- tionately, despite the apparent saturation of its formation, which eloma, colorectal cancer, melanoma, soft tissue sarcoma, and suggests that its catabolism is also saturable, as reported previ- pediatric brain tumors. Recommended dosage for these studies ously (22). The pharmacokinetics of BG are unlikely to require is BG 120 mg/m2 given over a period of 1 h followed 1 h later precise titration because the clinical toxicity remains the same by BCNU 40 mg/m2. when the BG dosage is increased from 40 to 120 mg/m2 at any given BCNU dosage. ACKNOWLEDGMENTS Although essentially all of the patients in this study had We gratefully acknowledge the assistance of Phillip Schumm in advanced cancer that was unresponsive to prior therapy, very analyzing pre- and posttreatment pulmonary function tests as well as the few patients had received alkylnitrosoureas or alkylating agents assistance of Wendy Miland in preparation of the manuscript. of any kind prior to enrollment on this trial. We were disap- pointed, therefore, that no objective tumor responses were ob- REFERENCES served among 62 evaluable patients. If AGT is the primary 1. Pegg, A. E., Dolan, M. E., and Moschel, R. C. Structure, function and mechanism of resistance to alkylnitrosoureas and if AGT activ- inhibition of O6-alkylguanine-DNA alkyltransferase. Prog. Nucleic ity is completely suppressed by BG, one might have anticipated Acid Res. Mol. Biol., 51: 167–223, 1995. Clinical Cancer Research 3031

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