Evaluation of Irinotecan in Combination with 5-Fluorouracil Or Etoposide in Xenograft Models of Colon Adenocarcinoma and Rhabdomyosarcoma

Vol. 2, 107-118, January 1996 Clinical Cancer Research 107

Evaluation of Irinotecan in Combination with 5-Fluorouracil or Etoposide in Xenograft Models of Colon Adenocarcinoma and Rhabdomyosarcoma

Janet A. Houghton, Pamela J. Cheshire, tecan and etoposide were combined using the (dx5)2 i.v. James D. Hailman II, Lois Lutz, Xialolong Luo, schedule for both drugs, in which irinotecan was given 2 h Yulan Li, and Peter J. Houghton 2 before or 2 h after the administration of etoposide. Each drug could be combined at only 38% of its respective max- Departments of Molecular Pharmacology [J. A. H., P. J. C., J. D. H., imum tolerated dose when administered as a single agent, L. L., P. J. H.] and Biostatistics [X. L., Y. L.], St. Jude Children's Research Hospital, Memphis, Tennessee 38101 indicating greater than additive toxicity. Toxicity was similar irrespective of the sequence of administration and was manifested by loss of weight (73% of the initial weight, nadir ABSTRACT day 7), myelosuppression, and prolonged thrombocytopenia. Irinotecan [7-ethyl-10-(4-[1-piperidino]- 1-piperidino)- The responses of colon carcinomas to the combination given carbonyloxy-camptothecin] administered i.v. in two courses, in either sequence were similar to that achieved with irino- each course consisting of administration every day for 5 tecan given alone at the same dose as used in the combina- days [(dx5)2] on days 1-5 and 8-12, has demonstrated tion. Similarly, when etoposide was given before irinotecan, significant activity against advanced human tumor xe- the responses of rhabdomyosarcomas were similar to those nografts derived from colon adenocarcinomas and several for irinotecan. However, in experiments in which etoposide childhood cancers. To build on this therapy, we have eval- was administered 2 h after each dose of irinotecan, there was uated the combination of irinotecan given on this schedule significant antagonism of the antitumor activity of irinote- with 5-fluorouracil given on days 1, 7, and 14 with or can. without leucovorin [(d×5)3 i.v.] against colon tumors, or combined with etoposide administered (d x 5)2 i.v. either 2 h before or 2 h after irinotecan for treatment of colon tumors INTRODUCTION and rhabdomyosarcomas. A combination of 5-fluorouracil Irinotecan [7-ethyl- 10-(4- [ 1-piperidino]- 1-piperidino)-car- at 75% and irinotecan at 50% of their respective maximum bonyloxy-camptothecin], a water-soluble prodrug, has demon- tolerated doses when administered as single agents on this strated broad-spectrum activity against experimental tumor schedule gave acceptable toxicity. Against colon adenocar- models (1-7) and against P-glycoprotein expressing multidrug cinoma xenografts, 5-fluorouracil did not enhance the re- resistant cell lines (8). The dose-limiting toxicity of irinotecan sponse rate compared with that obtained with the optimum given as a single i.v. administration to humans was severe dose of irinotecan given as a single agent. Against GC3/TK- myelosuppression (9). In this Phase I trial, objective responses xenografts, which lack thymidine kinase and cannot salvage were observed in previously treated patients with sarcoma of the thymidine to circumvent the inhibition of thymidylate syn- stomach, melanoma, colon adenocarcinoma, and non-SCLC) thase, the addition of leucovorin to the combination in- Irinotecan administered weekly by short infusion has demon- creased the complete response rate from 10 to >90%, strated activity against refractory or relapsed SCLC (10, 11) and whereas the response rates for the optimal doses of irinote- responses have been measured in non-Hodgkin's lymphoma, can or 5-fluorouracil, as single agents, were 30 and <10%, acute lymphocytic leukemia (12), non-SCLC (13, 14), and colon respectively. Etoposide d x5 i.v. for two or three courses or adenocarcinoma (15-19) with differing schedules. In most stud- (d x 5)3 p.o. did not cause objective regression of any colon ies, the dose-limiting toxicity has been neutropenia and severe tumors. In contrast, three of five rhabdomyosarcoma lines diarrhea (20). The latter toxicity may be related to low gluco- demonstrated a high frequency of partial regressions or ronidation and biliary elimination of the active metabolite complete regressions when treated (dx5)l i.v. Repetitive SN-38 (21). courses [e.g., (dx5)2 or (d×5)3] i.v. or p.o. or by 4-h infusion These data suggest quite broad-spectrum clinical activity d x 3 i.v. were either equally effective or less effective. Irino- for irinotecan, and combination Phase I trials with cisplatin (22, 23), vindesine (24), 5-fluorouracil (25), and etoposide (26, 27) have been reported. In previous studies (6, 7), we have demonstrated a therapeutic advantage of low-dose protracted schedules of irinotecan in the treatment of both colon adenocarcinoma and Received 6/29/95; revised 9/19/95; accepted 9/20/95. ~ Supported in part by Public Health Service Awards CA32613 and CA23099, Cancer Center Support CORE Grant CA21765, and the American Lebanese Syrian Associated Charities. 2 To whom requests for reprints should be addressed, at Department of 3 The abbreviations used are: SCLC, small cell lung cancer; (d×5)2, two Molecular Pharmacology, St. Jude Children's Research Hospital, 332 courses, every day for 5 days; PR, partial response; CR, complete North Lauderdale, Memphis, TN 38101. Phone: (901) 495-3440; Fax: response; MCR, maintained complete response; MTD, maximum toler- (901) 521-1668. ated dose; TK, thymidine kinase.

rhabdomyosarcoma models. Here, we report the evaluation of consecutive days. Blood (20 pA) was obtained from the orbital schedules of irinotecan in combination with 5-fluorouracil sinus of each animal before the start of treatment and on days 4, against a panel of colon adenocarcinoma xenografts and irino- 10, and 14 and analyzed using a Sysmex 180A Hematology tecan given prior to or subsequent to the topoisomerase II Analyzer. inhibitor etoposide in both colon and rhabdomyosarcoma mod- Formulation and Administration. Irinotecan (clinical els. The rationale for combination with 5-fluorouracil was that formulation) was diluted in sterile saline and administered i.v. this antimetabolite causes single-strand DNA breaks (28, 29), (0.1 ml/10 g body weight) (d×5)2 in 2 consecutive weeks (days which may involve direct incorporation into DNA (30, 31) or 1-5 and 8-12). Where appropriate, control mice received di- may occur as a consequence of accumulation and incorporation luted vehicle [0.26 ml sorbitol (70% w/w) and 0.9 mg lactic of dUTP with subsequent excision repair (32, 33). It has been acid/ml, (pH 3.9)]. 5-Fluorouracil and leucovorin were formu- postulated that topoisomerase I has an active role in repair (34, lated and administered i.v. as described previously (49). For 35), and, indeed, the basis for the reported synergy between combination studies, 5-fluorouracil was administered on days 1, ionizing radiation and inhibitors of this enzyme has been as- 7, and 14 (rapid i.v. push), and leucovorin was given on days cribed to drug inhibition of DNA repair (36-38). Combination 1-5, 8-12, and 15-19, whereas irinotecan was given (d×5)2 i.v. of irinotecan with an inhibitor of topoisomerase II was based on For the combination of irinotecan with etoposide, both agents several observations that indicate such combinations to be an- were given daily (d × 5)2 i.v. with administration separated by 2 tagonistic against cell lines in culture (39-41). We hypothe- h. All drugs were given i.v. by tail vein injection. sized that if such antagonism extended to normal tissues, par- Irinotecan was provided generously by Dr. K. Terada ticularly the hematopoeitic system, then this may offer (Yakult Co., Ltd., Tokyo, Japan) or Dr. A. Mathieu-Bou6 (Labo- protection, whereas tumors, such as colon adenocarcinomas that ratoire Roger Bellon, Paris, France). Etoposide was obtained may be intrinsically resistant to drugs such as etoposide as a through the pharmacy at this institution, and leucovorin and consequence of overexpression of P-glycoprotein (42), would 5-fluorouracil were purchased from Sigma Chemical Co. not be protected from the effects of irinotecan (or its active Tumor Response and Statistical Analysis. For compar- metabolite, SN-38), which seems either not to be a substrate for ison of different treatment regimens, tumor responses were P-glycoprotein-mediated efflux or, at best, to be a poor substrate analyzed for the time (days) individual tumors required to reach (8). four times their volume at initiation of therapy, tumor regression, and the area under the estimated relative tumor volume growth curve. (Relative tumor volume = volume day x after MATERIALS AND METHODS treatment/volume at initiation of treatment). For individual tu- Immune Deprivation of Mice. Female CBA/CaJ mice mors, PR was defined as a volume regression >50% but with a (Jackson Laboratories, Bar Harbor, ME), 4 weeks of age, were measurable tumor at all times. CR was defined as the disappear- immune deprived by thymectomy, followed 3 weeks later by ance of a measurable tumor mass at some point after initiating whole-body irradiation (1150 cGy) using a 137Cs source. Mice therapy. MCR was CR without tumor regrowth within the study received 3 × 106 nucleated bone marrow cells within 6-8 h of time frame (usually 12 weeks). Relative tumor growth was fitted irradiation (43). Tumor pieces of approximately 3 mm 3 were by a log linear model. Descriptive statistics of outcome mea- implanted in the s.c. space of the dorsolateral flanks of mice to sures were calculated. Growth between bilateral tumors in the initiate tumor growth. Tumor-bearing mice were randomized same mouse was examined, revealing a high correlation be- into groups of five to seven prior to initiating therapy. tween responses of tumors within the same host. Consequently, Tumor Lines. Each of the colon adenocarcinoma and comparisons among groups were based on one tumor per mouse, rhabdomyosarcoma xenografts have been described previously in mice in which only one tumor was present, or if two tumors (44-48). For chemotherapy studies, all tumors were used within were available in a mouse, one was chosen randomly. It was 27 passages of their engraftment in mice. Each tumor grows assumed that missing data would not change distributions of routinely in more than 90% of recipient mice, and all are human observed outcomes. The proportions of tumors that failed to as determined by karyotype and species-specific isoenzyme reach four times their volume at the start of therapy were given patterns. by Kaplan-Meier estimates (50) with the log-rank test (51) Growth Inhibition Studies, Mice bearing bilateral s.c. comparing differences among groups. The Kruskal-Wallis test tumors each received the agent when tumors were approxi- was used to compare tumor responses (PR and CR) among mately 0.20-1 cm in diameter. Procedures have been reported groups. Areas under the curve were compared by multiple t tests previously (6). Briefly, two perpendicular diameters were de- with adjusted P values (52). termined at 7-day intervals using digital Vernier calipers inter- faced with a Dell 486 66-MHz computer. Tumor volumes were calculated assuming tumors to be spherical using the formula RESULTS [(w/6) × d3], where d is the mean diameter (44). Toxicity of lrinotecan in Combination with 5-Fluorou- Toxicity Studies, Toxicity of individual agents and com- racil. When 5-fluorouracil was administered on days 1, 7, and binations was conducted using seven nontumored, immune- 14 in combination with irinotecan given (d×5)2 in 2 consecu- deprived mice per dose group. Body weight and death were tive weeks (days 1-5 and 8-12), it was necessary to reduce the monitored daily for 21 days after the last drug administration. dose of 5-fluorouracil by 25% to 56 mg/kg/dose and that of To assess hematological toxicity for irinotecan and etoposide, as irinotecan by 25 to 50% (dependent on the tumor line) compared single agents, or in combination, drugs were administered for 5 with the MTD of each agent administered alone on the same

schedule. A higher dose intensity was associated with higher caused only a minor (<5%) loss of body weight. Hematological frequencies of drug-related deaths. Combinations in which profiles for mice receiving either agent alone or in combination 5-fluorouracil was administered with irinotecan at 40 or 30 for a single 5-day course are shown in Fig. 1. There was greater mg/kg/dose resulted in deaths of 19 (56%) of 34 and 12 (29%) than additive myelosuppression when etoposide preceded or of 42 mice, respectively. The addition of leucovorin did not followed the administration of irinotecan, causing marked significantly alter the toxicity of this combination when irino- thrombocytopenia that reversed by day 14 after starting ther- tecan was given at 40 or 30 mg/kg/dose (deaths in 8 of 21 and apy. 3 of 21 mice, respectively). Responses of Colon Tumor and Rhabdomyosarcoma Responses of Colon Adenocarcinomas. Previous stud- Xenografts to Etoposide Treatment. Previously, we have ies demonstrated that irinotecan given (d×5)2 was more effec- reported the sensitivity of both colon tumor and rhabdomyosar- tive than a single administration (6). Consequently, we have coma models to agents that inhibit topoisomerase I (6, 7, 48). In evaluated the efficacy of this schedule of daily administration in contrast to the sensitivity of colon tumors to irinotecan, the combination with 5-fluorouracil. Tumor responses to irinotecan colon adenocarcinoma xenografts were considerably less sensi- or 5-fluorouracil as single agents or in combination are summa- tive to etoposide, irrespective of the schedule or route of ad- rized in Table 1. Given daily 5 days/week for 2 weeks, irinote- ministration evaluated (Table 2). As shown in Table 2, d×5 can caused a high frequency of partial and complete regressions administration either by i.v. or p.o. routes for two or three of HC1, GC3/TK-, and VRC 5 xenografts when administered at consecutive courses did not cause any objective responses in the MTD (40 mg/kg/dose). Irinotecan arrested the growth of any of the colon tumor lines examined, although the growth of ELC 2 xenografts completely but did not induce volume regres- VRC 5 and ELC 2 tumors was inhibited when etoposide was sions. 5-Fluorouracil also inhibited the growth of ELC2 and HC1 given (d×5)3 i.v. In contrast, etoposide caused PR and CR in tumors and alone or in combination with leucovorin caused a Rh28 rhabdomyosarcomas over a 5-fold dose range. There was partial regression of the GC3/TK- variant, which is deficient in also a high frequency of PR in both Rhl8 and IRS56 rhabdomy- thymidine salvage. Neither VRC 5 nor its derivative, VRCJcl, osarcomas when etoposide was given d× 5 by i.v. administration were sensitive to 5-fluorouracil. (Table 3). Alternative schedules of i.v. d×5 for two or three Against GC3/TK- xenografts, which demonstrate the consecutive courses or given by 4-h i.v. infusion on 3 consec- greatest sensitivity to 5-fluorouracil, the combination of 5-flu- utive days did not cause objective responses in Rhl2 or Rh30 orouracil at 56 mg/kg/dose with irinotecan at 30 mg/kg/dose rhabdomyosarcoma xenografts. Similarly, etoposide given by resulted in acceptable levels of toxicity in these experiments (0 p.o. gavage (d×5)3 did not increase the response rate and was deaths in 28 mice). However, the combination was not more less active than more intense treatment regimens in Rh28 xe- efficacious than irinotecan (P = 0.35) given as a single agent at the MTD (40 mg/kg/dose). In replicate experiments, there were nografts. PRs in 14 of 24 tumors and CRs in 6 of 24 tumors in the Efficacy of Irinotecan-Etoposide Combinations against combination groups compared with PRs in 7 of 10 tumors and Colon Adenocarcinoma Xenografts. Combinations of irino- CRs in 3 of 10 tumors in the group receiving only irinotecan (40 tecan given at 10 mg/kg/dose with etoposide administered at 3.1 mg/kg/dose). The addition of leucovorin enhanced the fre- mg/kg/dose were evaluated, when irinotecan preceded or fol- quency of CR with 22 of 26 tumors maintaining CR at the end lowed the administration of etoposide by 2 h, and were com- of the 12-week observation period (P = 0.021). The response pared with the activity of either agent given alone at the same rates for HC~, VRCs/cl, and ELC 2 colon tumors in mice re- dose and schedule (Table 4). Against HC 1 tumors, irinotecan ceiving the optimal combination of 5-fluorouracil and irinotecan produced a high proportion of objective regressions (PR + CR, (at 56 and 20 mg/kg/dose, respectively) were similar or slightly 93 and 100%). Similar activity was seen whether irinotecan less than could be achieved at the optimal dose of irinotecan preceded etoposide (PR + CR, 80 and 100%) or was adminis- given as a single agent. tered subsequent to etoposide (PR + CR, 85 and 100%) in Toxicity of Irinotecan in Combination with Etoposide. replicate experiments. The combination of etoposide with irino- Preliminary studies in both nontumored and tumor-bearing, tecan also gave no greater efficacy than irinotecan given as a immune-deprived mice showed, that given by i.v. administration single agent when evaluated against the VRC 5 xenograft model. on the (d×5)2 schedule, the MTDs were 40 mg/kg/dose for Efficacy of Irinotecan-Etoposide Combinations against irinotecan and 12.5 mg/kg/dose for etoposide. When irinotecan Rhabdomyosarcoma Xenografts. The activity of irinotecan was administered either 2 h prior to or 2 h after etoposide, given as a single agent at 15 and 10 mg/kg/dose on the (d×5)2 toxicity was greater than anticipated. Combinations of etoposide i.v. schedule is presented in Table 5. Each tumor line demon- and irinotecan each at 50% of the MTD (i.e., 20 and 6.25 strated considerable sensitivity to irinotecan given on this sched- mg/kg/dose, respectively) were not tolerated on the (dx5)2 ule. Combinations, in which etoposide at 4.2 or 3.1 mg/kg/dose schedule, but were tolerated when given on days 1-5 and was combined with 15 or 10 mg/kg/dose irinotecan, respec- 14-19. The maximum doses that produced acceptable host tively, were examined. In combinations in which etoposide toxicity using the (d×5)2 schedule were 15 and 4.7 mg/kg/dose preceded the administration of irinotecan (Table 6), there was a for irinotecan and etoposide, respectively, each representing slightly higher frequency of CR in Rhl8/VCR (a subline of 38% of the MTD when given as single agents. This combination Rhl8 selected in vivo for resistance to vincristine) than that resulted in a nadir in body weight of 73% irrespective of the given by irinotecan at 10 mg/kg/dose (100 versus 65% CR), order in which drugs were administered. In contrast, irinotecan with a higher frequency of maintained complete responses (15 mg/kg/dose) alone or etoposide (4.7 mg/kg/dose) alone (MCR at 12 weeks). For other tumors, the responses to this

Table 1 Efficacy of 5-fluorouracil with and without leucovorin in combination with irinotecan against colon

Growth Responses (%) Time to 4X delay Toxicity-related Tumor Dose (mg/kg) (days) _ SE (days) PRa CR MCR deaths (%) HC~ 0 28.0 ___ 1.1 FUra 56 67.9 _ 3.8 39 10 0 0 0 FUra 56 + CPT 20 >84 >56 43 50 50 0 FUra 56 + CPT 10 75.5 --- 1.6 47 79 14 14 0 FUra 56 + CPT 5 >84 >56 29 55 55 0 HC1 b 0 27.0 ___ 3.2 CPT 10 >84 >53 23 77 53 0 CPT 20 >84 >53 25 75 75 0 CPT 40 >84 >53 18 82 82 0 GC3/TK- 0 32.6 _+ 2.4 FUra 75 72.2 ± 3.3 39 45 0 0 0 FUra 75 + LV500 77.7 _+ 2.3 45 90 0 0 0 GC3/TK- 0 29.4 ± 1.8 FUra 75 72.8 + 2.9 43 55 18 9 0 FUra 56 68.6 --- 4.9 39 41 8 8 0 CPT 40 79.1 + 1.4 49 70 30 30 0 CPT 20 >84 >54 60 40 40 0 CPT 10 >84 >54 50 50 50 0 FUra 56 + CPT 30 >84 >54 90 10 10 0 FUra 56 + CPT 20 >84 >54 55 45 22 0 FUra 56 + CPT 10 >84 >54 88 12 12 0 GC3/TK- 0 30.9 --- 3.5 Fura 56 + CPT 40 >84 >51 55 33 33 14 FUra + CPT 40 + LV 500 >84 >51 67 33 33 28 FUra 56 + CPT 30 >84 >51 67 17 8 0 FUra 56 + CPT 30 + LV 500 >84 >51 8 92 84 0 FUra 56 + CPT 40 + LV 500 >84 >51 0 100 92 0 FUra 56 + CPT 30 + LV 500 >84 >51 0 100 88 0 VRCs/C1 0 15.1 ___ 3.3 CPT 40 49.7 + 1.2 34 30 0 0 17 CPT 20 44.4 ___ 1.1 29 23 0 0 0 FUra 75 18.5 ± 1.5 3 0 0 0 0 FUra 56 12.0 ___ 0.8 0 0 0 0 FUra 56 + LV 500 20.7 ___ 1.5 5 0 0 0 14 FUra 56 + CPT 40 54.8 __+ 3.8 39 25 17 17 14 FUra 56 + CPT 30 43.8 +__ 1.1 28 0 0 0 43 FUra 56 + CPT 40 + LV 500 Toxic 86 FUra 56 + CPT 30 + LV 500 33.3 ___ 1.9 18 0 0 0 43 VRC 5 0 13.5 ± 1.0 CPT 40 71.4 + 1.2 57 84 16 8 0 CPT 20 72.1 ± 3.2 58 36 43 29 0 FUra 75 27.4 ___ 2.7 13 0 0 0 14 FUra 56 19.6 ___ 2.2 6 0 0 0 0 FUra 56 + CPT 40 74.9 ___ 3.1 61 83 17 17 50 FUra 56 + CPT 30 69.6 ± 3.5 56 80 20 20 14 Fura 56 + CPT 20 70.0 ± 2.9 56 57 29 29 0 ELC2 0 50.0 + 4.9 FUra 75 71.2 _+ 2.4 21 0 0 0 0 FUra 56 60.5 ___ 3.1 10 0 0 0 0 CPT 40 75.7 ___ 2.4 25 15 0 0 0 FUra 56 + CPT 40 Toxic 100 FUra 56 + CPT 30 Toxic 71 ELC 2 0 55.5 _ 2.9 FUra 75 75.5 _ 2.5 20 0 0 0 0 FUra 56 68.3 ___ 3.7 13 0 0 0 0 CPT 40 75.5 ___ 2.1 20 7 0 0 0 FUra 56 + CPT 40 Toxic 100 Fura 56 + CPT 30 >84 >29 13 0 0 43 FUra 56 + CPT 20 >84 >29 14 0 0 0

a PR, -->50% regression; MCR, maintained CR at 12 weeks; CPT, irinotecan, mg/kg/dose (dx5)2; FUra, 5-fluorouracil, mg/kg/dose, days 1, 7, and 14; LV, leucovorin, mg/kg/dose (dx5)3. b From Houghton et al. (6), with additional data.

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3 600 I. L 2 . soo ,l,,a ~- 400 1 3OO

0 ' ' 200 0 2 4 6 8 10 "12 14 16 0 2 4 6 8 10 12 14 16 Days Days Fig. 1 Hematopoeticparameters in mice treated dx5 with irinotecan, 15 mg/kg/dose (O); etoposide, 4.7 mg/kg/dose ( O ); etoposide, 4.7 mg/kg/dose 2 h before irinotecan, 15 mg/kg/dose (I'); or the reverse schedule (A). Q, control. A, WBC; B, platelets. Each point is the mean + SD (n = 7).

combination were similar to that elicited by the administration time to a drug above some critical concentration, we have of irinotecan as a single agent. evaluated protracted schedules of drug administration. In mice The efficacy of combinations in which irinotecan preceded bearing xenografts of human cancers, protracted schedules of the administration of etoposide is shown in Table 7. Against relatively low dose intensity seem to offer greater efficacy than Rhl8/VCR, Rh30, and IRS68 xenografts, the activity of this more intense schedules of administration (7, 48). Because iri- combination and sequence was decreased significantly com- notecan at 10 mg/kg/dose given on the (dX5)2 i.v. schedule can pared with the activity of irinotecan given as a single agent. be repeated every 21 days for three cycles with acceptable Thus, against Rh 18/VCR xenografts, irinotecan as a single agent toxicity in the mouse (7), we have used this schedule of admin- given at 15 mg/kg/dose induced 100% objective regressions (PR istration as a platform for combining this agent with both and CR), which were reduced to 28% in the combination treat- 5-fluorouracil, for colon adenocarcinomas, and etoposide, for ment group (P = 0.002). Similarly, for Rh30 and IRS68, the both colon and rhabdomyosarcoma xenografts. overall objective response rate was reduced from 100% (CR) to 5-Fluorouracil (56 mg/kg/dose; 75% of the MTD as a 21 and 30% (CR and PR, respectively; P < 0.02). Representa- single agent) could be combined with irinotecan (20-30 mg/kg/ tive studies are shown for Rh30 and IRS68 rhabdomyosarcomas dose; 50-75% of the MTD), giving a cumulative toxicity index in mice receiving irinotecan at 15 or 10 mg/kg/dose as a single of 1.25-1.5, depending on the tumor line, indicating less than agent or in which etoposide was administered 2 h after each additive toxicity. Thus, when irinotecan was combined at 75% dose of irinotecan (Figs. 2 and 3). of its MTD (30 mg/kg/dose) with 75% of the 5-fluorouracil MTD, there were toxicity-related deaths in 12 (29%) of 42 mice. Of these, 8 were in mice bearing ELC 2 colon tumors. In con- DISCUSSION trast, there were 0 toxic deaths among 21 mice bearing GC3/ Irinotecan is one of several water-soluble camptothecin TK- tumors treated identically. When 75% of the 5-fluorouracil analogues that are in clinical development. This class of drug MTD was combined with 50% of the irinotecan MTD, there targets the nuclear enzyme topoisomerase I, which relaxes su- were 0 toxic deaths in 28 mice, representing a probably accept- percoiled DNA and seems important for semiconservative rep- able MTD for the combination. 5-Fluorouracil as a single agent, lication of double-helical DNA, transcription, and chromosomal administered at the MTD, inhibited the growth of GC3/TK- decondensation (53, 54) and represents a novel target for cyto- xenografts significantly, and in combination with leucovorin, it toxic agents. Initial results indicate that this agent may have a increased the objective response rate from 45 to 90% PR. broad spectrum of activity against tumors, such as colon ade- Irinotecan at 20 mg/kg/dose caused all tumors to regress (PR nocarcinomas, that are regarded generally as chemorefractory. and CR, 60 and 40%, respectively). The combination of 5-flu- Because inhibitors of topoisomerase I seem to be highly S-phase orouracil and irinotecan was not superior to irinotecan alone, specific (55), and in vitro, cytotoxicity is a function of exposure unless leucovorin was used, a combination for which the fre-

Table 2 Efficacy of etoposide against colon adenocarcinoma xenografts Growth Responses (%) Time to 4X delay Toxicity-related Tumor Dose (mg/kg) Schedule (days) .+ SE (days) PR CR MCR deaths (%) HC~ 0 9.3 -+ 3.3 10 (d X 5) 2 i.v. 12.6 + 1.4 3 0 o o 0 12.5 (d x 5) 2 i.v. 8.2 _+ 1.0 0 0 0 0 o GC3/cl 0 29.4 ___ 2.1 5 (d X 5) 2 i.v. 19.0 ___ 2.8 0 0 0 0 0 10 (d X 5) 2 i.v. 23.0 _ 3.0 0 0 0 0 0 12.5 (d x 5) 2 i.v. 51.3 _+ 3.6 21 0 0 0 0 VRC 5 0 12.4 ___ 1.8 5 (d x 5) 2 i.v. 23.6 -+ 2.1 11 0 0 0 0 10 (d x 5) 2 i.v. 17.5 + 0.7 5 0 0 0 o 12.5 (d x 5) 2 i.v. 23.5 _+ 3.4 11 0 0 0 0 ELC 2 0 28.5 .+ 2.7 5 (d X 5) 2 i.v. 35.0 + 3.2 6 0 0 0 0 10 (d X 5) 2 i.v. 37.5 --- 2.8 9 0 0 0 0 12.5 (d X 5) 2 i.v. 38.5 --- 3.2 10 0 o o o HC 1 0 13.3 .+ 1.2 7.5 (d x 5) 3 i.v. 17.5 .+ 0.8 4 0 o o o 10 (d X 5) 3 i.v. 18.1 ___ 1.0 5 0 0 0 0 GC3/cl 0 38.5 - 3.7 7.5 (d x 5) 3 i.v. 29.4 ,+ 5.9 0 7 0 0 0 10 (d x 5) 3 i.v. 38.0 --- 5.5 0 0 o o o VRC5 0 9.8 -+ 0.8 7.5 (d × 5) 3 i.v. 29.1 _+ 3.2 20 0 0 0 0 10 (d × 5) 3 i.v. 31.1 _+ 4.1 21 0 0 0 0 ELC z 0 33.0 ,+ 2.0 7.5 (d X 5) 3 i.v. 44.5 _+ 4.4 11 0 0 0 0 10 (d X 5) 3 i.v. 69.0 -+ 5.0 36 0 0 0 0 HC1 0 12.5 _+ 3.6 15 (d X 5) 3 p.o. 24.5 ,+ 2.7 12 0 0 0 0 20 (d x 5) 3 p.o. 23.0 _+ 3.7 10 0 o o o GC3/cl 0 33.6 + 3.1 15 (d × 5) 3 p.o. 47.0 .+ 5.3 13 0 0 0 0 20 (d X 5) 3 p.o. 45.5 _+ 5.7 12 0 o 0 0 VRC5 0 16.5 _+ 3.7 15 (d x 5) 3 p.o. 34.5 _+ 2.8 18 0 o 0 o 20 (d x 5) 3 p.o. 40.0 _+ 3.0 23 0 o o o ELC 2 0 36.0 .+ 5.3 15 (d X 5) 3 p.o. 41.0 .+ 3.5 5 0 o o 0 20 (d X 5) 3 p.o. 43.0 -+ 3.0 7 0 o 0 0

quency of CR increased to >90% (P = 0.02) with a high potentiation of cytotoxicity in several xenograft models when maintained CR rate at 12 weeks. In the limited studies reported, irinotecan was administered 24 h after doxorubicin but antago- leucovorin, given on this schedule, did not potentiate the toxic- nism when the agents were administered simultaneously. We ity of 5-fluorouracil as a single agent or in combination with hypothesized that if the data using cell lines could be extrapo- irinotecan. However, the sample size was relatively small, and lated to normal hematopoietic tissues, it may be possible to the organ-limiting toxicity was not determined. The GC3/TK- protect from toxicity, whereas in tumors that were intrinsically cell line was developed specifically to overcome the problem of resistant to etoposide, for example, by overexpressing P-glyco- thymidine salvage in xenograft models, in which plasma thymi- protein, there would be no antagonistic effect. Protection of dine levels in the mouse may be >10-fold higher than in normal tissues would allow for increased dose intensity and humans; therefore, the result from this line may be more pre- potentially a greater therapeutic effect. Although a systematic dictive of activity of this combination in man. In the other study of P-glycoprotein expression has not been undertaken tumors, HC1, VRCs, and ELC 2, the combination did not in- with this series of xenografts, GC3/cl colon tumors have levels crease the frequency of objective regressions, even in tumors in similar to KB-8-5 multidrug resistant cells, whereas Rh30 has which leucovorin supplementation was evaluated. undetectable levels by immunoblot analysis. 4 Although there is a conceptual basis for anticipating syn- Therefore, we examined etoposide as a single agent against ergistic activity between inhibitors of topoisomerase I and II, several studies have demonstrated antagonism in vitro in the hamster ovary (39), human promyelocytic leukemia (40), and HT-29 human colon cancer cells (41). Kim et al. (56) reported 4 L. Shu and P. J. Houghton, submitted for publication.

Table 3 Responsiveness of rhabdomyosarcoma xenografts to etoposide Growth Responses (%) Dose Time to 4X delay Tumor (mg/kg) Schedule (days) -+ SE (days) PRa CR MCR Rhl2 0 21.0 _ 1.0 25 (d × 5) i.v. 25.6 _+ 4.6 5 0 0 0 Rhl8 0 11.2 + 7.8 15 (d × 5) i.v. 8.2 - 4.5 41 0 0 20 (d × 5) i.v. 11.8 _+ 5.4 1 66 0 0 25 (d × 5) i.v. 13.2 - 7.3 2 41 8 8 Rh28 0 11.6 _+ 2.7 5 (d × 5) i.v. 30.1 + 1.5 19 100 40 40 15 (d × 5) i.v. 29.0 _+ 3.5 17 100 75 16 20 (d × 5)i.v. 31.0 18 100 83 83 25 (d × 5) i.v. 31.5 20 100 100 85 Rh30 0 9.1 +0.8 10 (d × 5) i.v. 7.8 _+ 3.2 0 0 0 15 (d × 5) i.v. 13.4 _+ 3.6 4 0 0 0 20 (d × 5) i.v. 14.5 _+ 2.7 5 30 50 0 IRS56 0 35.3 -+ 7.8 15 (d × 5) i.v. 58.0 + 11.1 23 35 0 0 2O (d × 5) i.v. 65.1 _+ 8.8 30 64 0 0 IRS68 0 17.8 - 3.2 15 (d × 5) i.v. 14.9 _+ 5.7 0 0 0 2O (d × 5) i.v. 11.9 _+ 2.1 0 0 0 Rhl2 0 19.5 12.5 (d × 5) 2 i.v. 44.2 _+ 6.1 25 0 0 0 10 (d × 5) 3 i.v. 39.2 -+ 3.9 20 0 0 0 45.5 inf (d × 3) i.v. 38.2 19 0 0 0 Rh30 0 7.4 _+ 1.2 12.5 (d × 5) 2 i.v 20.6 _+ 1.7 13 0 0 0 10 (d × 5) 3 i.v.. 25.1 -+ 6.3 18 0 0 0 45.5 inf (d × 3) i.v. 26.9 - 3.2 20 0 14 14 Rhl2 0 19.9 _+ 8.2 15 (d × 5) 3 p.o. 18.8 -+ 14.0 0 0 0 20 (d × 5) 3 p.o. 33.6 +_ 13.4 14 0 0 0 Rhl8 0 14.8 -+ 5.4 15 (d × 5) 3 p.o. 16.4 -+ 8.2 2 0 0 0 20 (d × 5) 3 p.o. 24.9 _+ 8.8 10 0 0 0 25 (d × 5) 3 p.o. 20.1 _+ 5.6 5 14 7 0 Rh28 0 9.1 -+3.8 15 (d × 5) 3 p.o. 41.2 +- 12.5 32 42 14 7 2O (d × 5) 3 p.o. 47.6 -+ 18.6 39 75 41 16 Rh30 0 20.0 + 4.7 2O (d × 5) 3 p.o. 36.6 _+ 17.2 16 0 0 0

both colon and rhabdomyosarcoma models using a variety of gavage d×5 for 3 courses seemed less effective than the more schedules and routes of drug administration. Etoposide was intense 5-day treatment schedule. administered d×5 each week for up to 3 weeks by i.v. injection Subsequently, we evaluated the combination of etoposide or p.o. gavage. Etoposide demonstrated only marginal activity with irinotecan, a combination in which both agents were ad- against the colon tumors, with no objective regressions using ministered i.v. using the (d×5)2 schedule. Using this schedule any dose or schedule. These results are in contrast to the of administration, in which i.v. injection of agents was separated significant activity of irinotecan given on similar schedules in by 2 h, the combination was synergistically toxic. In combina- the same tumor models (6). Etoposide given for 5 consecutive tion, irinotecan and etoposide could be administered at only days caused a high frequency of CR in Rh28 tumors and PRs in 38% of their respective MTDs when given as a single agent Rhl8 and IRS56 rhabdomyosarcoma xenografts. Repeated (toxicity index, 0.76). For a single 5-day course of therapy, courses of etoposide administered either i.v. or p.o. increased toxicity was associated with significant weight loss (27%), the growth inhibition in Rhl2 and Rh30 xenografts but did not reaching a nadir on day 7. In contrast, administration of either increase the frequency of tumor regressions. Similar results agent alone caused <5% loss of body weight. Hematopoietic were obtained when etoposide was administered by 4-h tail vein toxicity of the combination was marked by significant myelo- infusion on 3 consecutive days, conditions that approximate the suppression and prolonged thrombocytopenia. Toxicity was plasma concentration time profile in children receiving etopo- similar whether irinotecan was administered before or after side at 100 mg/mZ/day (57). Administration of etoposide by p.o. etoposide. These results are in marked contrast to the antago-

Table 4 Combinations of irinotecan and etoposide against colon adenocarcinomas

Growth Responses (%) Time to 4X delay Toxicity-related Tumor Dose (mg/kg) (days) _ SE (days) PR CR MCR deaths (%) HC~ 0 30.8 _+ 3.6 CPT 10a >84 >53 29 64 50 0 VP 3.1 36.4 _+ 2.8 5 0 0 0 0 CPT 10 ~ VP 3.10 >84 >53 10 70 40 0 VP 3.1 ~ CPT 10 >84 >53 39 46 38 0 HC~ 0 42 ± 6.2 CPT 10 >84 >42 8 92 16 14 VP 3.1 60.9 - 4.9 18 0 0 0 0 CPT 10 ~ VP 3.1 77.6 ± 1.4 35 82 18 9 0 VP 3.1 ~ CPT 10 75.1 ___ 2.2 33 54 46 8 0 VRC5 0 31.5 _+ 5.5 CPT 10 73.5 ± 2.8 42 23 54 54 0 CPT 10 ~ VP 3.1 72.1 ± 3.6 40 46 46 46 0 VP 3.1 ~ CPT 10 77.7 - 2.1 46 42 50 50 0

a CPT, irinotecan; VP, etoposide. o Drugs were administered 2 h apart. Both drugs were given on the (d × 5) 2 i.v. schedule.

Table 5 Rhabdomyosarcomas: efficacy of irinotecan

Growth Responses (%) Time to 4X delay Tumor Dose (mg/kg) (days) _+ SE (days) PR CR MCR Rhl2 0 29 ± 2.8 10 >84 >55 57 43 36 Rhl8 0 11.5 -+ 2.5 10 >84 >72 0 100 100 15 >84 >72 0 100 100 Rh 18/VCR 0 15.1 _+ 0.7 10 62.0 _ 5.2 47 35 65 35 15 83.5 ± 1.9 68 0 100 92 Rh30 0 13.2 + 1.3 10 >84 >71 0 100 92 15 >84 >71 0 100 100 IRS68 0 24.3 + 4.5 10 >84 >59 0 100 100 15 >84 >59 0 100 100

Table 6 Rhabdomyosarcomas: efficacy of etoposide administered before irinotecan

Growth Responses (%) Time to 4X delay Tumor Dose (mg/kg) (days) _ SE (days) PR CR MCR Rhl8 0 17.5 -+ 2.7 VP 3.1 ~ CPT 10a >84 63 92 92 VP 4.7 --* CPT 15 80 -+ 4.1 >66 100 100 Rhl8/VCR 0 16.9 -+ 1.9 VP 3.1 --~ CPT 10 >84 >67 100 100 VP 4.7 ~ CPT 15 >84 >67 100 100 Rh30 0 17.0 + O.9 VP 3.1 ~ CPT 10 70.3 + 3.0 53 100 43 VP 4.7 ~ CPT 15 77.5 + 2.3 61 100 86 IRS68 0 26.3 ± 1.6 VP 3.1 --* CPT 10 >84 >58 100 100 VP 4.7 --~ CPT 15 >84 >58 100 100 a VP, etoposide; CPT, irinotecan.

Table 7 Rhabdomyosarcomas: efficacy of irinotecan administered before etoposide

Growth Responses (%) Time to 4X delay Tumor Dose (mg/kg) (days) -+ SE (days) PR CR MCR Rhl2 0 29.0 _+ 2.8 CPT 10 ~ VP 3.1 a 71.5 - 2.7 41 0 7 7 CPT 15 ~ VP 4.7 69.5 --- 3.2 43 28 28 21 Rhl8 0 13.0 _+ 0.9 CPT 10 ~ VP 3.1 59.0 --- 5.0 46 14 50 29 CPT 15 ~ VP 4.7 68.5 + 3.7 56 35 57 43 Rh 18/VCR 0 15.5 _+ 4.9 CPT 10 ~ VP 3.1 37.5 _+ 3.5 22 14 14 0 CPT 15 --~ VP 4.7 39.3 + 5.5 24 28 28 7 Rh30 0 12.3 _+ 3.2 CPT 10 ~ VP 3.1 28.0 --+ 5.6 16 7 14 14 CPT 15 ~ VP 4.7 33.6 - 3.0 21 25 16 8 IRS68 0 18.5 _+ 2.5 CPT 10 ~ VP 3.1 32.4 _+ 4.4 14 20 10 0 CPT 15 ~ VP 4.7 56.0 + 2.8 38 10 10 0 a CPT, irinotecan; VP, etoposide.

10L 11L

0.1 ~ ~ ~ 0.1 , , , ~ OAt , , i 0.1 ~ k ~ i 0.1 L, 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 O0L 2 4 6 8 10 12 0 2 4 6 8 10 12 10 10 D E F ? 1 m 1 1

0.1 J ~ ~ ~ 0.1 i 0.1 I 0.1 0.11 ~ LI ~ L I 0.1 0 2 4 6 8 lO 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Weeks Weeks Weeks Weeks Weeks Weeks

Fig. 2 Responses of Rh30 rhabdomyosarcoma xenografts to irinotecan Fig. 3 Responses of IRS68 rhabdomyosarcoma xenografts to irinote- or irinotecan administered 2 h prior to etoposide. A and D, controls; B, can or irinotecan administered 2 h prior to etoposide. A and D, controls; irinotecan, 15 mg/kg/dose; C, irinotecan, 10 mg/kg/dose; E, irinotecan, B, irinotecan, 15 mg/kg/dose; C, irinotecan, 10 mg/kg/dose; E, irinote- 15 mg/kg/dose, followed by etoposide, 4.7 mg/kg/dose; F, irinotecan, 10 can, 15 mg/kg/dose, followed by etoposide, 4.7 mg/kg/dose; F, irinote- mg/kg/dose, followed by etoposide, 3.1 mg/kg/dose. Mice received i.v. can, 10 mg/kg/dose, followed by etoposide, 3.1 mg/kg/dose. Mice treatment on the (d×5)2 schedule. Each line demonstrates the growth of received i.v. treatment on the (d×5)2 schedule. Each line demonstrates an individual tumor. the growth of an individual tumor.

nism between the topoisomerase inhibitors predicted from the ated with a topotecan-induced increase in CD34 + bone marrow combination studies using malignant cells in culture (39-41). cells in the cycle (58). Clinically, the combination of irinotecan and etoposide was The combination then was compared with the activity of associated with leukopenia, although thrombocytopenia was irinotecan or etoposide given as single agents at the same dose mild (27). This may reflect a species difference or a difference levels used in the combination. Against the colon tumor models, in the scheduling of these agents. Of note, however, is that in the combination showed similar activity to that determined after patients, the sequential administration of topotecan (a topoi- irinotecan as a single agent administered at the dose used in the somerase I inhibitor) and adriamycin (a topoisomerase II inhib- combination. Thus, etoposide did not antagonize the activity of itor) resulted in unexpectedly severe myelosuppression, associ- irinotecan, but also demonstrated no synergism, irrespective of

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