O 6-Benzylguanine-mediated Enhancement of Chemotherapy 1

We have previously demonstrated (A. E. Pegg, Cancer Res., 50: 6119–6129, 1990) that O-benzylguanine (OBG) enhances nitrosourea, temozolomide, and cyclophosphamide activity in malignant glioma xenografts growing in athymic nude mice. More recently, we have demonstrated (V. J. Patel et al., Clin. Cancer Res., 6: 4154–4157, 2000; P. Pourquier et al., Cancer Res., 61: 53–58, 2001) that the combination of temozolomide plus irinotecan (CPT-11) displays a schedule-dependent enhancement of antitumor activity secondary to trapping of topoisomerase I by Omethylguanine residues in DNA. These studies suggested that there might be favorable therapeutic interactions between O-BG and combinations of 1,3bis(2-chloroethyl)-1-nitrosourea (BCNU) plus cyclophosphamide or temozolomide plus CPT-11, respectively. Our present results indicate that the combination of cyclophosphamide plus BCNU plus OBG produces growth delays modestly-to-markedlysuperior to combinations of cyclophosphamide with BCNU. Although the combination of temozolomide and CPT-11 reveals a marked increase in activity compared with either agent used alone, the addition of O-BG to this combination dramatically increased the growth delay of the O-alkylguanine-DNA alkyltransferase (AGT)-positive malignant glioma D-456 MG. These results suggest that a Phase I trial of CPT-11 plus temozolomide plus O-BG in AGT-positive tumors may be an important intervention to maximize the therapeutic benefits of the combination of CPT-11 and temozolomide. Introduction Several preclinical and clinical studies have demonstrated that the DNA repair protein AGT is responsible for resistance to chlorethylation or methylation damage at the O position of guanine in DNA (1–15), and several studies have shown that this enhances BCNU, temozolomide, and cyclophosphamide resistance. O-BG inactivates AGT activity both in vitro and in vivo (16–27). More recently, we have demonstrated that the combination of temozolomide plus CPT-11 displays a schedule-dependent enhancement of antitumor activity secondary to the trapping of topoisomerase I by O-methylguanine residues in DNA (28, 29). These studies suggested that there might be favorable therapeutic interactions between O-BG, and combinations of BCNU plus cyclophosphamide or temozolomide plus CPT-11, respectively. We now report the marked increase in antitumor activity produced by these combinations after O-BGmediated AGT inactivation. Materials and Methods Animals. Male and female athymic BALBc mice (nu nu genotype, 6 weeks of age or older) were used for all of the studies and were maintained as described previously (30). Athymic mice were housed in an isolation facility with highefficiency particulate air-filtration. All of the mice were maintained under a controlled 12-h light/12-h dark cycle and were provided food and water ad libitum. Xenografts. Human central nervous system tumorderived xenografts were used for in vivo studies. The xenografts were maintained as described previously (31). The derivation and AGT status of these xenografts is detailed in Table 1 from a previous publication (25). Drugs. Temozolomide was provided by Schering-Plough Research Institute (Kenilworth, NJ). CPT-11 was provided by Pharmacia and Upjohn Global Distribution Center (Kalamazoo, MI). Cyclophosphamide and BCNU were purchased from Sigma (St. Louis, MO). O-BG was synthesized as described previously (32). s.c. Xenograft Transplantation. s.c. tumor transplantation was performed into the right flank of the animals with an inoculation volume of 50 l using a brei prepared from xenografts (33). Tumor Measurements. Tumors were measured twice weekly with hand-held vernier calipers (Scientific Products, McGraw, IL). Tumor volume was calculated according to the following formula: V [(width) (length)]/2. Drug Toxicity. Mice were weighed twice weekly to assess weight loss and were checked daily for survival. Received 5/6/02; revised 7/23/02; accepted 7/31/02. 1 This work was supported by NIH Grants NS30245 (to H. S. F.), NS20023 (to H. S. F), CA57725 (to A. E. P., H. S. F., and M. E. D.), and CA81485 (to M. E. D.). Drs. Pegg, Moschel, and Dolan have a financial relationship with Access Oncology, the company that is presently licensing O-benzylguanine. Dr. Friedman is a paid consultant for Access Oncology. 2 To whom requests for reprints should be addressed, at Duke University Medical Center, Room 047, Baker House, Trent Drive, Durham, North Carolina 27710. 3 The abbreviations used are: AGT, O-alkylguanine-DNA alkyltransferase; O-BG, O-benzylguanine; CPT-11, irinotecan; LD10, lethal dose to 10% of animals; 4-HC, 4-hydroperoxycyclophosphamide; BCNU, 1,3bis(2-chloroethyl)-1-nitrosourea; CHO, Chinese hamster ovary. 943 Vol. 1, 943–948, September 2002 Molecular Cancer Therapeutics on May 2, 2017. © 2002 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Xenograft Therapy. Groups of 10 randomly selected mice were treated when the median tumor volume was in the range of 100–500 mm and were compared with control animals receiving drug vehicle. Cyclophosphamide was administered via i.p. injection at a single dose of 300 mg/m (100 mg/kg) in 0.9% saline, which represents 22% of the LD10. BCNU was administered via i.p. injection at a single dose of 24.0 mg/m (8 mg/kg) in 5% ethanol, which represents 23.8% of the LD10. Temozolomide was administered via i.p. injection at a single dose of 105 mg/m (35 mg/kg) in 0.9% saline, which represents 10% of the LD10. CPT-11 was administered via i.p. injection at a dose of 12 mg/m (4 mg/kg) in 0.9% saline on days 1–5 and 8–12, which represents 10% of the LD10. O -BG was administered at a dose of 240 mg/m (80 mg/kg) in polyethylene glycol-400/0.9% saline (4:6) 1 h before any of the other agents. At this dose, O-BG depletes tumor AGT levels by 90% within 1 h. In additional studies, groups of five non-tumor-bearing mice were treated with the combination of temozolomide at 25% of the LD10 plus CPT-11 at 25% of the LD10 given alone or with O-BG. Similar studies with five non-tumor-bearing mice/group using temozolomide at 50% of the LD10 plus CPT-11 at 50% of the LD10 given alone or with O -BG were also conducted. Assessment. The response of the s.c. xenografts was assessed by delay in tumor growth and by tumor regression. Growth delay, expressed as T C, is defined as the difference in days between the median time required for tumors in treated (T) and control (C) animals to reach a volume five times greater than that measured at the start of treatment. Tumor regression is defined as a decrease in tumor volume over two successive measurements. Statistical analyses were performed using a personalized SAS statistical analysis program, the Wilcoxon rank order test for growth delay, and Fisher’s exact test for tumor regression as described previously (34).