4110 Vol. 6, 4110–4118, October 2000 Clinical Cancer Research Antitumor Activity of Temozolomide Combined with Irinotecan Is Partly Independent of O6-Methylguanine-DNA Methyltransferase and Mismatch Repair Phenotypes in Xenograft Models1 Peter J. Houghton,2 Clinton F. Stewart, phases of clinical evaluation against other tumors. Phase II trials Pamela J. Cheshire, Lois B. Richmond, in Europe have also confirmed some activity against melanoma Mark N. Kirstein, Catherine A. Poquette, (1) and suggest activity against high-grade gliomas (2). Temo- zolomide is considered to exert its toxic effects primarily by Ming Tan, Henry S. Friedman, and generating O6-methylguanine in DNA (3, 4). This adduct is Thomas P. Brent subject to a single-step, error-free repair reaction that simply Departments of Molecular Pharmacology [P. J. H., T. P. B.], transfers the methyl group to a cysteine residue within the repair Pharmaceutical Science [C. F. S., P. J. C., L. B. R., M. N. K.], and protein MGMT,3 thus restoring the DNA to its intact state. Biostatistics and Epidemiology [C. A. P., M. T.], St. Jude Children’s Hence, MGMT is a major determinant of temozolomide cyto- Research Hospital, Memphis, Tennessee 38105, and Duke University Medical Center, Durham, North Carolina 27710 [H. S. F.] toxicity (4, 5). Furthermore, intact MMR function is critically required for the cytotoxicity of the methylating drugs. Recently, we reported the sensitivity of a series of pediatric tumor xe- ABSTRACT nografts to temozolomide. Sensitivity correlated with MGMT The activity of temozolomide combined with irinotecan deficiency and MMR proficiency (6). (CPT-11) was evaluated against eight independent xe- CPT-11 is a camptothecin prodrug activated by carbox- nografts (four neuroblastomas, three rhabdomyosarcomas, ylesterases to the active topoisomerase I poison SN-38. CPT-11 and one glioblastoma). In all studies, temozolomide was has demonstrated broad activity against both murine and human administered p.o. daily for 5 consecutive days/cycle, found in tumor xenograft models (reviewed in Ref. 7) and clinically preliminary studies to be the optimal schedule for adminis- significant activity against many types of cancer (reviewed in 8, tration. Irinotecan was administered i.v. for 5 days for 2 9). Camptothecins have been reported to synergize with ionizing consecutive weeks/cycle. Treatment cycles were repeated radiation (10, 11) and chemical agents that damage DNA, in- every 21 days for a total of three cycles over 8 weeks. In cluding platinum and alkylating agents (12–14). Potentially, combination, temozolomide and CPT-11 induced complete modification to DNA can lead to recruitment of topoisomerase responses in four neuroblastomas, two rhabdomyosarcomas, I, thus potentially increasing the probability of a camptothecin and the glioblastoma line. The activity of the combination drug stabilizing the DNA-enzyme covalent complex (15, 16). was significantly greater than the activity of either agent Because topoisomerase I preferentially cuts DNA between T administered alone in four tumor lines. Of interest, the and G residues, we speculated that methylation of O6-guanine interaction appeared independent of tumor MGMT or mis- would lead to recruitment of topoisomerase I and potentially match repair phenotype, suggesting that the mechanism of enhance the probability of inducing camptothecin-mediated synergy may be independent of O6-methylation by temozo- damage. This formed the biochemical rationale for combining lomide. Pharmacokinetic studies indicated no detectable in- temozolomide with a camptothecin. Recently, Pourquier et al. teraction between these two agents. Further, coadministra- (17) demonstrated that O6-alkylation of guanine induces topoi- tion of CPT-11 appeared to reduce the toxicity of somerase-I DNA covalent complexes in N-methyl-NЈ-nitro-N- temozolomide in tumor-bearing mice. nitrosoguanidine-treated cells. Conceptually, this would in- crease the probability of a collision with the advancing INTRODUCTION replication fork and generation of a double-strand DNA break, Temozolomide is a methylating agent that has been ap- considered the initiating event in inducing cell death (reviewed proved for treatment of astrocytoma and is entering various in Ref. 18). In addition to the biochemical rationale for the interaction between temozolomide and CPT-11, in clinical trials these agents have relatively nonoverlapping toxicities. The limiting Received 5/23/00; revised 7/14/00; accepted 7/14/00. toxicity of temozolomide is noncumulative, transient myelosup- The costs of publication of this article were defrayed in part by the pression (19), whereas when CPT-11 is given as protracted daily payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by USPHS Awards CA23099, CA71628, CA14799, and CA21765 (Cancer Center Support Grant) from the National Cancer 3 The abbreviations used are: MGMT, O6-methylguanine-DNA methyl- Institute and by American, Lebanese, Syrian Associated Charities. transferase; MMR, mismatch repair; CPT-11, irinotecan [7-ethyl-10-(4- 2 To whom requests for reprints should be addressed, at Molecular [1-piperidino)-1-piperidino]-carbonyloxy-camptothecin]; SN-38, 7- Pharmacology, St. Jude Children’s Research Hospital, 332 North Lau- ethyl-10-hydroxy-camptothecin; CR, complete response; MTIC, derdale Street, Memphis, TN 38105-2794. Phone: (901) 495-3440; Fax: 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide; AUC, area under the (901) 521-1668; E-mail: [email protected]. concentration-time curve. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2000 American Association for Cancer Research. Clinical Cancer Research 4111 Table 1 MGMT, MMR, and p53 phenotypes of xenografts because this was found optimal in initial studies. Temozolomide Tumor MGMTa MLH1a MSH-2a p53b was administered 1 h prior to administration of CPT-11. Cycles of therapy were repeated twice at 21-day intervals. CPT-11, at NB-SD ϮϮϩϩMut NB-1643 ϪϩϩWt doses listed for individual experiments, was administered i.v. NB-1771 ϩϩϩϩϩWt daily for 5 consecutive days for 2 consecutive weeks, shown SJ-GBM2 ϪϮϩϩMut previously to be an optimal schedule (days 1–5 and 8–12). ϩϩ ϩ ϩϩ Rh12 Wt Temozolomide and CPT-11 were generously supplied by Scher- Rh18 ϩϩ Ϫ Ϯ Wt/MDM2 Amp. Rh30 ϪϮϩϩMut/Wt ing-Plough and Upjohn-Pharmacia, respectively. a MGMT, MLH1 and MSH2 were determined by Western blot analysis. Data are from Middlemas et al. (6). Pharmacokinetic Studies b Mut, mutant p53; Wt, wild type. Wt/Mut, heterozygous for p53. MDM2 amp., amplified mdm2 with wild-type p53. Pharmacokinetics of Temozolomide and MTIC in Mice. We conducted pharmacokinetic studies of the combination of temozolomide and CPT-11 to determine whether a pharmaco- kinetic interaction existed between the two drugs. Temozolo- dosing, the limiting toxicity is primarily diarrhea (20). Here we mide was administered as a single oral dose (66 mg/kg), fol- report the significant activity of CPT-11 in combination with lowed by a single i.v. dose of irinotecan (10 mg/kg). To measure temozolomide. Of interest is the finding that the combination of each agent at dose levels that as monotherapy have minimal temozolomide and MTIC, blood samples were collected from antitumor activity resulted in complete regressions of several mice (three animals/point) at 0, 0.25, 0.5, 1, 1.5, 2, 3, and 6 h. ϫ tumors. This occurred in tumors that were MGMT proficient Samples were immediately centrifuged at 5.5 g for 2 min in and MMR deficient, suggesting that the interaction between a tabletop refrigerated centrifuge at 4°C. Plasma was then di- these agents may in part be independent of temozolomide- vided into aliquots for processing to assay either temozolomide induced O6-methylation of guanine. In the companion paper, or MTIC by isocratic high-performance liquid chromatography Patel et al. (21) have examined the sequence dependence of this as described previously in detail (6). Temozolomide and MTIC combination in brain tumor xenografts. were quantitated by UV detection at 325 and 318 nm, respec- tively. The lower levels of quantitation for temozolomide and MTIC were 0.25 and 0.5 ␮g/ml, respectively. All calibrators and MATERIALS AND METHODS controls were prepared in murine plasma (Hill Top Lab Ani- Tumor Models mals, Inc., Scottdale, PA). Each of the xenografts used has been described previously Pharmacokinetics of Irinotecan and SN-38 in Mice. (22–24). Studies used four lines of neuroblastoma, three rhab- The disposition of irinotecan and SN-38 was evaluated after domyosarcomas, and one glioblastoma. Tumors were grown in administration of a single oral dose of temozolomide (66 mg/ the s.c. space of immune-deprived, female CBA/CaJ mice, as kg), followed by a single i.v. dose of irinotecan (10 mg/kg). described (25). Previously, we have reported the MGMT, Heparinized blood samples (ϳ1 ml) were collected (three ani- MMR, and p53 phenotype of each tumor and related this to mals/time point) pre, 0.25, 0.5, 1, 2, 4, and 6 h after i.v. temozolomide sensitivity (6). The phenotype determined from irinotecan administration. Samples were immediately centri- tumor tissue for each tumor is summarized in Table 1. fuged at 5.5 ϫ g for 2 min. Plasma was separated, and proteins were precipitated by the addition of 200 ␮l of plasma to 800 ␮l Tumor Response and Tumor Failure Time of cold methanol (Ϫ30°C), followed by vigorous agitation with For individual tumors, partial response was defined as a a vortex mixer, and centrifuged again at 5.5 ϫ g for 2 min. The volume regression Ͼ50% but with measurable tumor (Ն0.10 supernatant was decanted and stored at Ϫ70°C until analysis. cm3) at all times. CR was defined as a disappearance of meas- Irinotecan and SN-38 lactone plasma concentrations were urable tumor mass (Ͻ0.10 cm3) at some point within 12 weeks determined by an isocratic high-performance liquid chromatog- after initiation of therapy.