Antitumor Activity of Temozolomide Combined with Irinotecan Is Partly Independent of O6-Methylguanine-DNA Methyltransferase

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.

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 pharmacokinetic 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, respectively. 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. A maintained complete response was raphy assay with fluorescence detection, as described previously CR without tumor regrowth within a 12-week study time frame. in detail (24, 26). The excitation and emission wavelengths were Methods for statistical analysis of data, and evaluation of tumor 375 and 520 nm, respectively. The lower level of quantitation responses have been reported previously (25). Animal care was was 1 ng/ml for irinotecan and SN-38. All calibrators and in accord with institution guidelines. controls were prepared in murine plasma (Hill Top Lab Ani- mals, Inc.). Drug Formulation and Administration Temozolomide and MTIC Pharmacokinetic Analysis. Initial studies were designed to determine the optimal Temozolomide and MTIC plasma concentration-time data from schedule for administration of temozolomide and to determine oral administration were modeled using maximum likelihood any schedule-dependent antitumor activity. Tumor-bearing mice estimation as implemented in ADAPT II (27). A first-order were treated with temozolomide by oral gavage for 5 days (days absorption, one-compartment linear model, which included 1–5) or two 5-day courses (1–5 and 8–12) per 21-day cycle. first-order MTIC formation and elimination, was used to simul- Alternatively, mice received three 5-day courses per 28-day taneously describe temozolomide and MTIC disposition (28). cycle. The cumulative dose in all groups was 630 mg/kg. In AUC was calculated from the model parameters. The apparent subsequent combination studies, temozolomide was adminis- time of maximum concentration (tmax) and maximum plasma tered daily for 5 consecutive days (days 1–5) of each cycle, concentration (Cmax) were also noted.

Fig. 1 Schedule-dependent antitumor activity of temozolomide against NB-1382 neuroblastoma xenografts. Tumor-bearing mice received either no treatment (control) or a cumulative dose of 210 mg/kg per cycle. Schedules used were daily for 5 days [(dx5)1], two 5-day courses on consecutive weeks [(dx5)2] with cycles repeated every 21 days over 8 weeks. Alternatively, mice received temozolomide for three 5-day courses on consecutive weeks [(dx5)3], and cycles were repeated every 28 days over 11 weeks. Each curve shows the growth of an individual tumor.

Irinotecan and SN-38 Pharmacokinetic Analysis. The ments, administration of temozolomide in the most intensive disposition of irinotecan and SN-38 was evaluated using a schedule (daily for 5 consecutive days/cycle) was either more three-compartment model with linear distribution and elimina- active or equally active compared with the other schedules. tion (29). Pharmacokinetic parameters for each set of data were Consequently, for combination studies we selected the daily ϫ initially fit by maximum likelihood estimation, as implemented 5 schedule for combination with CPT-11. in ADAPT II. Pharmacokinetic parameters calculated from Dose levels of CPT-11 and temozolomide were chosen so these estimates included systemic clearance (CL) and AUC. The that neither drug alone caused CR. For CPT-11 dose, levels maximum observed irinotecan and SN-38 plasma concentra- between 2.5 and 0.18 mg/kg daily were administered, deter- tions (Cmax) and time to maximum plasma concentration (Tmax) mined by the intrinsic sensitivity of any particular xenograft were determined. line. These dose levels give SN-38 systemic exposures consistent with those achieved in patients receiving CPT-11 using the RESULTS same schedule of administration (20). Temozolomide was ad- Previously, we and others have shown that the antitumor ministered at dose levels ranging from 66 to 19 mg/kg and are activity of camptothecins is highly schedule dependent. For consistent with doses that in patients give achievable levels of example, the same total dose of CPT-11 given over 10 days was parent drug and MTIC the active metabolite (6). Statistical significantly more active than when administered over 5 days or analysis of the antitumor activity of single agents and com- as a single administration. To determine whether there was bination treatments are summarized in Table 2. The activity similar schedule dependency for temozolomide, we examined of combinations was significantly better than the activity of the antitumor activity of this agent given daily for 5 days, 2 ϫ either single agent used at the same dose level, with a few 5 days per 21-day cycle or 3 ϫ 5 days per 28-day cycle. The exceptions. For example, in studies where monotherapy with total dose per cycle was constant. Results for NB-1382 xe- either CPT-11 or temozolomide at the doses used caused nografts are presented in Fig. 1 and demonstrate the most maintained CR, it was not possible to determine whether pronounced schedule-dependent activity of temozolomide. In combinations were superior to monotherapy (e.g., Rh30 and this experiment, mice received a cumulative dose of 210 mg/kg NB-1643). However, against several tumor lines temozolo- per cycle. Mice received three cycles of treatment (total cumu- mide combined with CPT-11 demonstrated significant activ- lative dose was 630 mg/kg). When temozolomide was given ity against tumors at dose levels that had little activity when over 5 days (42 mg/kg/dose), CRs were achieved in all mice and administered as monotherapy. For example, against NB-SD were maintained at week 12. Lower doses given over more neuroblastoma xenografts, temozolomide had little activity at protracted periods were progressively less effective. Far less tolerated dose levels, and similarly tumors progressed in mice schedule-dependent antitumor activity was observed in five treated with CPT-11 at 0.4 mg/kg (Fig. 2). In combination other xenograft lines (data not shown); however, in all experi- these agents induced CR that was maintained at week 12. The

Table 2 Time to tumor failure: results of log-rank and Gray’s tests Average time P of exact to 4ϫϮSD Growth delay log rank Gray’s test Tumor Group Treatment (weeks) (wk) (unadjusted) (unadjusted P) NB-SD A Control 2.75 Ϯ 0.5 B TMZa 66 6.5 Ϯ 3.5 3.75 0.033 0.022 C CPT 0.4 5. Ϯ 1.7 2.25 0.015 0.013 D TMZ 66 ϩ CPT 0.4 Ͼ12 Ͼ9.25 0.012 0.015 E TMZ 42 6.3 Ϯ 4.9 3.55 0.043 0.082 F TMZ 42 ϩ CPT 0.4 Ͼ12 Ͼ9.25 0.002 0.020 B vs. D 0.033 0.132 C vs. D 0.036 0.027 C vs. F 0.020 0.034 E vs. F 0.106 0.056 NB-SD A Control 2.7 Ϯ 0.8 B TMZ 33 6 Ϯ 2.6 3.3 0.004 0.001 C CPT 0.4 9 Ϯ 2.8 6.3 0.002 0.001 D TMZ 33 ϩ CPT 0.4 Ͼ12 Ͼ9.3 0.002 0.001 E CPT 0.26 8.3 Ϯ 3.8 5.6 0.004 0.001 F TMZ 33 ϩ CPT 0.26 Ͼ12 Ͼ9.3 0.002 0.001 G TMZ 22 4.9 Ϯ 2.0 Ͼ9.3 0.012 1.0 H TMZ 22 ϩ CPT 0.4 Ͼ12 Ͼ9.3 0.002 0.001 I TMZ 22 ϩ CPT 0.26 Ͼ12 Ͼ9.3 0.001 0.000 B vs. D 0.009 0.063 B vs. F 0.052 0.067 C vs. D 0.054 0.151 C vs. H 0.21 0.162 E vs. F 0.061 0.069 E vs. I 0.002 0.047 G vs. H 0.002 0.002 G vs. I 0.001 0.000 D vs. H 0.091 0.23 E vs. I 0.024 0.005 B vs. D 0.013 0.064 C vs. D 0.73 C vs. F 0.46 E vs. F 0.021 0.008 NB-1771 A Control 3.5 Ϯ 1.0 B TMZ 19 7.6 Ϯ 1.8 4.1 0.010 0.004 C CPT 1.25 11.7 Ϯ 0.6 8.2 0.004 0.004 D CPT 0.61 10.8 Ϯ 0.8 7.3 0.005 0.002 E TMZ 19 ϩ CPT 1.25 Ͼ12 Ͼ8.5 0.002 0.004 F TMZ 19 ϩ CPT 0.62 Ͼ12 Ͼ8.5 0.004 0.003 B vs. E 0.002 0.009 B vs. F 0.004 0.006 C vs. E 0.182 0.059 D vs. F 0.007 0.002 NB-1643 A Control 3.8 Ϯ 1.3 B TMZ 28 Ͼ12 Ͼ8.2 0.002 0.001 C CPT 0.61 Ͼ12 Ͼ8.2 0.002 0.001 D TMZ 28 ϩ CPT 0.61 Ͼ12 Ͼ8.2 0.002 0.001 E TMZ 28 ϩ CPT 0.4 Ͼ12 Ͼ8.2 0.002 0.001 B vs. D1 B vs. E1 C vs. D1 SJ-GBM2 A Control 2.25 Ϯ 0.5 B TMZ 66 Toxic 0.013 0.019 C TMZ 42 9.5 Ϯ 1.7 7.25 0.044 0.018 D CPT 2.5 4.5 Ϯ 2.1 2.25 0.074 0.088 E CPT 1.25 6 Ϯ 2.8 3.25 0.043 0.017 F TMZ 66 ϩ CPT 2.5 Ͼ12 Ͼ9.75 0.001 0.018 G TMZ 66 ϩ CPT 1.25 Ͼ12 Ͼ9.75 0.013 0.018 H TMZ 42 ϩ CPT 2.5 Ͼ12 Ͼ9.75 0.013 0.028 I TMZ 42 ϩ CPT 1.25 Ͼ12 Ͼ9.75 0.013 0.018 C vs. H 0.005 0.034 C vs. I 0.023 0.025 D vs. F 0.070 0.142 D vs. H 0.070 0.172

Table 2 Continued Average time P of exact to 4ϫϮSD Growth delay log rank Gray’s test Tumor Group Treatment (weeks) (wk) (unadjusted) (unadjusted P) E vs. G 0.039 0.151 E vs. I 0.056 0.153 C vs. D1 Rh12 A Control 5.7 Ϯ 0.6 B TMZ 66 Toxic 0.61 0.95 C TMZ 3 4.6 Ϯ 1.1 Ϫ1.1 0.49 0.133 D CPT 1.25 Ͼ12 Ͼ6.3 0.003 0.019 E TMZ 66 ϩ CPT 1.25 Ͼ12 Ͼ6.3 0.001 0.018 F TMZ 33 ϩ CPT 1.25 Ͼ12 Ͼ6.3 0.027 0.021 E vs. D 0.139 E vs. B 0.46 0.28 D vs. F 0.53 F vs. C 0.010 0.003 Rh18 A Control 3.4 Ϯ 1.5 B TMZ 42 6.5 Ϯ 2.2 3.1 0.039 0.138 C TMZ 28 6.3 Ϯ 2.3 2.9 0.031 0.033 D CPT 0.61 11.2 Ϯ 1.1 7.8 0.006 0.005 E CPT 0.4 6.8 Ϯ 2.8 3.4 0.016 0.052 F TMZ 42 ϩ CPT 0.61 12 8.6 0.002 0.009 G TMZ 42 ϩ CPT 0.4 Ͼ12 Ͼ8.5 0.001 0.009 H TMZ 28 ϩ CPT 0.61 8.1 0.004 0.005 11.5 Ϯ 0.7 B vs. F 0.001 0.003 B vs. G 0.001 0.003 F vs. D 0.28 0.28 D vs. H 0.181 0.143 H vs. C 0.029 0.058 E vs. G 0.015 0.005 Rh30 A Control 3.5 Ϯ 1 B TMZ 28 Ͼ12 Ͼ8.5 0.007 0.015 C CPT 0.61 Ͼ12 Ͼ8.5 0.002 0.014 D TMZ 28 ϩ CPT 0.61 Ͼ12 Ͼ8.5 0.002 0.014 E TMZ 28 ϩ CPT 0.4 Ͼ12 Ͼ8.5 0.014 0.044 B vs. D 0.182 C vs. D1 B vs. E 0.79 a TMZ, temozolomide; CPT, CPT-11.

glioblastoma line, SJ-GBM2, has a similar phenotype to tumor that regrew, whereas at the lower dose, four tumors NB-SD, except in this tumor MGMT is not detected (see regrew during the period of observation. Rh18 rhabdomyo- Table 1). Although temozolomide induced some partial re- sarcoma expresses high MGMT levels and is deficient in sponses and an occasional CR during the 8 weeks of treat- MLH1 expression. Consequently, this xenograft is poorly ment at 66 or 42 mg/kg, the higher dose level was lethal sensitive to temozolomide as a single agent (Fig. 5). Ineffec- during cycle 3 of treatment (Fig. 3). At 42 mg/kg, the overall tive doses of temozolomide combined with doses of CPT-11, effect of treatment was stasis, because at the end of treatment which alone induced few CRs, resulted in a high frequency tumor volumes were similar to that at initiation of temozo- of CRs. lomide. CPT-11 combined with temozolomide (66 or 42 To determine whether the antitumor activity observed mg/kg) resulted in CR of all tumors with no tumor regrowth with the drug combination could be a result of one agent during the period of observation (12 weeks). Furthermore, altering the systemic exposure to the other, detailed pharma- combination with CPT-11 decreased the toxicity of temozo- cokinetic studies were performed. Temozolomide and MTIC lomide (described below). NB-1771 tumors have detectable concentrations exceeded the limit of assay sensitivity for the

MGMT and appear to be MMR proficient. These tumors were duration of the study. After administration, the apparent tmax relatively sensitive to temozolomide as a single agent (6); was 30 min for temozolomide and 1 h for MTIC. The Cmax hence for this study the dose was reduced to 19 mg/kg. for temozolomide and MTIC were 36 and 0.8 ␮g/ml, respec-

CPT-11 induced some CRs at 1.25 mg/kg and an occasional tively. The plasma AUC03ϱ for temozolomide and MTIC CR at 0.61 mg/kg; however, at week 12 all tumors had were 47 and 1.3 mg/L-hr, respectively. Irinotecan and SN-38 progressed. As shown in Fig. 4, temozolomide combined concentrations exceeded the limit of assay sensitivity for the

with CPT-11 resulted in CRs of all tumors. In groups of mice duration of the study. After administration, the apparent tmax receiving the higher dose of CPT-11, there was a single was 15 min for both irinotecan and SN-38. The Cmax for

Fig. 2 Antitumor activity of temozolomide (TMZ) and CPT-11 as single agents and in combination against NB-SD neuroblastoma xenografts. Mice received TMZ p.o. for daily (days 1–5) and CPT-11 (days 1–5 and 8–12) i.v. of each treatment cycle. CPT-11 was administered 1 h after TMZ. Cycles of therapy were repeated every 21 days over 8 weeks. Each curve represents growth of an individual tumor.

irinotecan and SN-38 was 764 and 192 ␮g/ml, respectively. or days 1–5 ϩ 8–12 repeated for three cycles at 21-day intervals ϩ ϩ The plasma AUC03ϱ for irinotecan and SN-38 was 993 and or days 1–5 8–12 15–19 repeated at 28-day intervals). In 463 mg/L-hr, respectively. each schedule, the total cumulative dose/cycle was constant. Temozolomide demonstrated only moderate schedule-depen- DISCUSSION dent antitumor activity, but in all experiments the most intensive CPT-11 has demonstrated significant activity against var- schedule (daily for 5 days every 21 days) was either more ious human cancers including refractory pediatric neoplasms effective or equally effective with other schedules of drug (20). In vitro, camptothecins have demonstrated synergy with administration. Thus, for combination studies with CPT-11, various DNA-damaging agents, including alkylating agents, cis- temozolomide was given for 5 consecutive days at the start of platin, and ionizing radiation. In vivo, 9-aminocamptothecin acts each cycle of CPT-11 treatment. as a radiation sensitizer, and a single report indicates synergy Dose levels of CPT-11 were used that in mice give sys- between topotecan and temozolomide (30). Recently, we re- temic exposures to SN-38 consistent with achievable exposures ported the activity of temozolomide in xenografts where the in children receiving CPT-11 using the same schedule (20). The MGMT and MMR status had been determined (6). In the current highest dose of temozolomide (66 mg/kg) used also gives clin- study, we evaluated the antitumor activity of temozolomide, a ically relevant drug exposures (6). For most studies, the com- DNA methylating agent, combined with CPT-11, the prodrug binations had significantly greater activity than either agent form of SN-38, a topoisomerase I poison. In part, the rationale administered as monotherapy at the same dose level. Exceptions for this combination was the nonoverlapping toxicities of the were where either or both agents were effective at inducing two agents. Dose limiting toxicity of temozolomide is cumula- maintained CRs. Examples were Rh30 and NB-1643, where at tive myelosuppression, notably neutropenia, whereas in the the dose levels administered, monotherapy resulted in CRs schedule used here, that of CPT-11 is diarrhea with only mod- maintained at week 12. For NB-SD, NB-1771, SJ-GBM2, and erate myelosuppression in children (20). Rh18 xenografts, combinations evaluated were significantly Previously, we and others have shown that the antitumor more effective than monotherapy. Against tumors such as Rh18 activity of several camptothecin drugs is highly schedule de- that express relatively high levels of MGMT, temozolomide pendent (Ref. 22; reviewed in Ref. 7). We were interested, induced few regressions and caused relatively little growth therefore, in initially determining whether there was a similar inhibition. However the combination resulted in more CRs than schedule dependency for the antitumor activity of temozolo- either drug given as monotherapy. The glioblastoma, SJ-GBM2, mide. Tumor-bearing mice were administered temozolomide is also relatively refractory to temozolomide. Although this daily for 5 days for 1, 2, or 3 consecutive weeks (i.e., days 1–5 tumor is deficient in MGMT, it has barely detectable levels of

Fig. 3 Antitumor activity of temozolomide (TMZ) and CPT-11 as single agents and in combination against SJ-GBM2 glioblastoma xenografts deficient in MGMT expression. Drugs were administered as described in Fig. 2. Each curve represents growth of an individual tumor, and mice were observed for up to 12 weeks.

Fig. 4 Antitumor activity of temozolomide (TMZ) and CPT-11 as single agents and in combination against NB-1771 neuroblastoma xenografts proficient in MMR and expressing MGMT. Each curve represents growth of an individual tumor, and mice were observed for up to 12 weeks.

Fig. 5 Antitumor activity of temozolomide (TMZ) and CPT-11 as single agents and in combination against Rh18 rhabdomyosarcoma xenografts deficient in MMR and expressing high MGMT levels. Each curve represents growth of an individual tumor, and mice were observed for up to 12 weeks.

the MMR protein MLH1. At the highest tolerated dose of this study when temozolomide was coadministered with irino- temozolomide (42 mg/kg), there were tumor regressions, but at tecan. These results are not surprising, especially because temo- the end of treatment (week 8) tumors were similar in mass to the zolomide is metabolized through nonenzymatic, pH-dependent pretreatment values. Furthermore, there were no maintained hydrolysis (31). Likewise, the AUC03ϱ for irinotecan and CRs. In contrast, temozolomide combined with CPT-11 was less SN-38 was similar to those values reported by us previously toxic and resulted in maintained CRs for all animals in groups after single-agent irinotecan (26, 32). Thus, the enhanced anti- receiving 66 or 42 mg/kg temozolomide combined with an tumor activity of the combination is unlikely attributable to a ineffective dose level of CPT-11 (1.25 mg/kg). Similar results drug interaction between the two agents. were obtained against NB-SD neuroblastoma xenografts that are In summary, combination of temozolomide and CPT-11 resistant to temozolomide. Furthermore, all NB-1771 neuroblas- administered on optimal schedules is effective in inducing CR in toma xenografts in mice treated with the combination had CR, a series of xenografts derived from childhood solid malignan- although some tumors had regrown by week 12. Thus, for cies. Taken together with results from pediatric brain tumor several tumors the combination of CPT-11 with temozolomide xenografts in the companion paper by Patel et al. (21), clinical induced superior tumor responses than either agent alone against evaluation of this combination may be of interest. tumors that were either MGMT proficient or MMR deficient and irrespective of wild-type or mutant p53. These results sug- ACKNOWLEDGMENTS gest that the interaction between CPT-11 and temozolomide We thank Lorina Dudkin for technical assistance that contributed to may be, in part, independent of O6-methylation of guanine, the part of this study. primary mechanism considered to lead to cytotoxicity of temozolomide. In addition to methylation of O6-guanine, temozolo- REFERENCES mide methylates N7-guanine or N3-adenine. These are the pre- 1. Bleehen, N. M., Newlands, E. S., Lee, S. M., Thatcher, N., Selby, P., dominant sites of modification by temozolomide. Potentially, Calvert, A. H., Rustin, G. J., Brampton, M., and Stevens, M. F. Cancer recruitment of topoisomerase I to DNA may be facilitated research campaign Phase II trial of temozolomide in metastatic melanoma. J. Clin. Oncol., 13: 910–913, 1995. through such modifications, although this remains to be tested. 2. Bower, M., Newlands, E. S., Bleehen, N. M., Bada, M., Begent, R. J., Of interest also was the observation that toxicity-related death Calvert, A. H., Colquhoun, I., Lewis, P., and Brampton, M. H. Multi- was consistently lower in groups of mice treated with temozo- centre CRC Phase II trial of temozolomide in recurrent or progressive lomide combined with higher dose levels of CPT-11. high-grade glioma. Cancer Chemother. Pharmacol., 40: 484–488, 1997. The results of our pharmacokinetic analyses showed no 3. Tisdale, M. J. Antitumor imidazotetrazine. XV. Role of guanine O6 difference in temozolomide or irinotecan disposition when the alkylation in the mechanism of cytotoxicity of imidazotetrazines. Bio- two agents are coadministered. We have previously studied chem. Pharmacol., 36: 457–462, 1987. the disposition of single-agent temozolomide and MTIC in the 4. Wedge, S. R., Porteus, J. K., May, B. L., and Newlands, E. S. Potentiation of temozolomide and BCNU cytotoxicity by O6- benzyl- xenograft model after a single oral dose of 66 mg/kg (6). The guanine: a comparative study in vitro. Br. J. Cancer, 73: 482–490, 1996. ␮ ⅐ temozolomide and MTIC AUC03ϱ were 40 g/ml hr and 1.9 5. Graziani, G., Faraoni, I., Grohmann, U., Bianchi, R., Binaglia, L., ␮g/ml⅐hr, respectively, values very similar to those observed in Margison, G. P., Watson, A. J., Orlando, L., Bonmassar, E., and D’Atri,

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Peter J. Houghton, Clinton F. Stewart, Pamela J. Cheshire, et al.

Clin Cancer Res 2000;6:4110-4118.

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