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Mitomycin as a Modulator of Irinotecan Anticancer Activity

Mitomycin as a Modulator of Irinotecan Anticancer Activity

ABSTRACT: Irinotecan and mitomycin (Mutamycin) possess significant single-agent activity against several tumor types, and mitomycin activates topoisomerase I, the cellular target of irinotecan. We conducted a phase I dose-escalation study of irinotecan and mitomycin in 37 evaluable patients with solid tumors. Antitumor responses included 2 complete responses, 5 partial responses, 10 minor responses, and a CA 19-9 tumor marker response. Responders included 14 patients previously treated with chemotherapy for metastatic disease. No pharmacokinetic interaction between mitomycin and irinotecan was apparent when these agents were given 24 hours apart. Responders (complete and partial responses) demonstrated the largest topoisomerase I induction 24 hours following mitomycin infusion. In addition, since maximum topoisomerase I up-regulation was reached 24 hours after administration of mitomycin, a delay in the administration of irinotecan after mitomycin appeared justified. Based on these encouraging phase I data, phase II clinical trials in breast and esophageal/gastroesophageal junction adenocarcinomas at the recommended doses and schedule are under way. [ONCOLOGY 16(Suppl 7):21-25, 2002]

Irinotecan (CPT-11, Camptosar) is a semisynthetic analog of camptothecin, a compound originally isolated from the Chinese/Tibetan ornamental tree Camptotheca acuminata. In vivo, cellular carboxylesterases cleave the ester bond of irinotecan, which is inactive, thereby producing the active compound 7-ethyl-10-hydroxycamptothecin (SN-38). Irinotecan/SN-38 interacts with cellular topoisomerase I/DNA complexes and has S-phase-specific cytotoxicity.[1] Irinotecan binds to topoisomerase I single-strand DNA breaks, and this reversible topoisomerase I/irinotecan/DNA cleavable complex, though not in itself lethal to the cells, collides with the advancing replication forks, leading to the formation of a double-strand DNA break, irreversible arrest of the replication fork, and cell death.[1] The collision of the irinotecan-topoisomerase I complex with the replication fork also results in G2 arrest/delay by signaling the presence of DNA damage to an S-phase checkpoint mechanism.[2]

Because topoisomerase I is the cellular target of irinotecan, it is conceivable that the cellular level of topoisomerase I would be proportional to irinotecan cytotoxic effects. This notion is supported by experimental evidence from yeast systems and mammalian cell lines.[3,4] The total activity of topoisomerase I was reduced (by one-fourth) in irinotecan cell lines rendered resistant by stepwise, continuous treatment with the drug compared to the irinotecan-sensitive parental cell line.[4] Theoretically, the topoisomerase I expression in tumor specimens may serve as a predictor for sensitivity to irinotecan chemotherapy. Since tumor cells may escape irinotecan cytotoxic effects by down-regulating their levels of topoisomerase I, strategies to increase topoisomerase I expression might enhance the antitumor effect of this agent.

Mitomycin and Increased Topoisomerase I Activity in Preclinical Systems

Mitomycin (Mutamycin) is an antitumor antibiotic isolated from Streptomyces caespitosus. This agent is activated in vivo to an alkylating moiety that covalently crosslinks complementary DNA strands, resulting in the inhibition of DNA synthesis.[5] Metabolic activation of mitomycin is accomplished by reduction of the quinone moiety by the enzyme DT-diaphorase (NQO1), which releases a methanol residue from the molecule.

Pilot data from our laboratory demonstrated that irinotecan decreases NQO1 in vivo gene expression in peripheral blood lymphocytes by approximately 50%, suggesting that pretreatment with irinotecan may interfere with mitomycin activation.[6] These findings are consistent with the observed lack of potentiation of mitomycin activity by pretreatment (24 hours prior) with irinotecan in the EH-6, H-111, and CH-6 human tumor xenografts.[7]

Gobert et al studied topoisomerase I activity in MCF cells under conditions in which p53 expression was induced by mitomycin.[5] These investigators observed that mitomycin increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. This increase in catalytic activity occurred in conjunction with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 hour of drug treatment. Further studies demonstrated that the interaction between p53 and topoisomerase I is observed with both latent and activated wild-type p53, as well as with several mutant and truncated p53 proteins in vitro.[8]

Because both irinotecan and mitomycin possess significant single-agent activity against several tumor types, and mechanistically mitomycin may result in activation of topoisomerase I, we evaluated the combination of irinotecan and mitomycin in a phase I clinical trial. We hypothesized that administering mitomycin prior to irinotecan would permit mitomycin activation without interference and might result in increased topoisomerase I expression/activity, leading to increases in tumor cell sensitivity to irinotecan. Because of possible activation interference, irinotecan was administered 24 hours after mitomycin.

Patients and Methods

Patients with histologically confirmed advanced solid malignancies were candidates for this study. Eligibility criteria also included adequate hematopoietic (absolute neutrophil count [ANC] ³ 1,500/µL, platelet count ³ 100,000/µL, and hemoglobin ³ 9.0 g/dL), hepatic (total bilirubin level < 1.5 mg/dL; transaminases [AST, ALT] and alkaline phosphatase £ 3 times the upper limit of normal), and renal (serum creatinine £ 1.5 mg/dL) functions. No prior treatment with mitomycin, irinotecan, or nitrosourea, and no more than six courses of chemotherapy containing an alkylating agent (four courses for carboplatin [Paraplatin]) were permitted, as well as no prior irradiation to more than 20% of bone marrow reserve. Due to possible interference with activation of mitomycin, concomitant treatment with coumarin anticoagulants was not permitted. All patients gave informed written consent.

Treatment Plan and Dose Escalation

The dose schedule was mitomycin on day 1 and irinotecan on days 2 (24 hours after mitomycin), 8, 15 and 22, with cycles repeated every 6 weeks (actual recovery period of 21 days). Some patients experienced late diarrhea, which caused delays in late doses of irinotecan. As a result, the schedule duration was decreased to 4 weeks, with irinotecan administered on days 2 and 8 after mitomycin on day 1. The starting dose of irinotecan was 50 mg/m² administered IV over 90 minutes, whereas the dose of mitomycin was kept at 6 mg/m². The number of cycles of mitomycin was limited to a maximum of six (36 mg/m² total dose) to avoid potential cumulative toxicities of mitomycin.

Dose escalation of irinotecan was to proceed in 25-mg/m² increments in cohorts of at least three new patients until dose-limiting toxicity was observed in at least two patients. Dose reduction by one level was allowed for patients who experienced dose-limiting toxicity or grade 3/4 diarrhea. The recommended dose (maximum tolerated dose) was defined as the highest dose at which no more than one of six new patients developed dose-limiting toxicities during the first course. The dose-limiting toxicity was defined as: (1) ANC < 500/µL lasting at least 5 days, or associated with fever; (2) grade 4 thrombocytopenia; (3) nonhematologic toxicity  ³ grade 3, except diarrhea or nausea/vomiting; and (4) grade 4 diarrhea despite optimal antidiarrheals, or grade 4 vomiting despite optimal antiemetics.

Pharmacokinetic and Molecular Correlates Sampling and Analysis

Blood was sampled from a site contralateral to the drug infusion during the first course of treatment at 2, 4, and 24 hours postinfusion to evaluate plasma concentrations of irinotecan, SN-38, and SN-38G. Topoisomerase I, carboxylesterase 1, carboxylesterase 2 (CE1 and CE2), and NQO1 gene expression were analyzed in peripheral blood mononuclear cells. RNA was extracted from blood samples collected at the following times: (1) baseline, (2) 5 minutes into the mitomycin infusion, (3) at the end of mitomycin infusion, (4) 3 hours after the end of mitomycin infusion, (5) 24 hours after end of mitomycin, (6) at the end of irinotecan infusion, (7) 2 hours after the end of irinotecan, and (8) 24 hours after the end of irinotecan infusion. Gene expression samples were analyzed by reverse-transcription polymerase chain reaction with a reaction-specific internal standard. Detection was done by capillary electrophoresis with laser-induced fluorescence.

Results

Dose Escalation and Toxicities

A total of 38 patients (37 evaluable) from two institutions (Cancer Therapy and Research Center, San Antonio, Texas, and The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute at the Ohio State University, Columbus, Ohio) were treated with 119 courses of the irinotecan/mitomycin combination (pertinent demographic characteristics are displayed in Table 1). Five patients received 14 additional courses of irinotecan as a single agent after the maximum allowed number of courses with mitomycin (six) was exceeded. Thirty-two patients had received prior chemotherapy, including 21 with previous radiation treatment (patients with previous pelvic irradiation were ineligible).

Table 2 depicts the dose escalations and dose-limiting toxicities in the study. Initially, patients were treated in the 6-week schedule (mitomycin on day 1, irinotecan on days 2, 8, 15, and 22). Although no severe toxicities were observed among seven patients receiving irinotecan at 50 mg/m²/wk, two of six patients completing a full course (4 weeks) of irinotecan at 75 mg/m²/wk in this schedule developed grade 3/4 diarrhea. Since the diarrhea occurred after patients received the third or fourth dose of irinotecan, the study design was amended to shorten the courses to 4 weeks (mitomycin on day 1, irinotecan on days 2 and 8).

Higher weekly doses of irinotecan were tolerated in the amended schedule, with only 1 of 14 patients treated at irinotecan doses of 100 to 125 mg/m²/wk developing moderate to severe toxicity (grade 3 diarrhea) during the first course of treatment. However, dose-limiting toxicities (febrile neutropenia and grade 3/4 diarrhea) were observed in two of three patients treated with irinotecan at 150 mg/m²/wk. Therefore, the 125-mg/m²/wk dose level on the 4-week schedule is the dose and schedule of the combination recommended for phase II studies. Other mild to moderate toxicities included nausea/vomiting, asthenia/fatigue, and peripheral edema. No renal dysfunction, hemolysis, or pulmonary interstitial fibrosis occurred. Two patients died on study, one from congestive heart failure and another from pulmonary embolism (necropsy proven).

Antitumor Activity

Of 37 evaluable patients, 7 had major responses. This included two complete responses and five partial responses. An additional 10 patients had a minor response. Among patients with any degree of response, 14 had previous chemotherapy for metastatic cancer. The types of tumor in which activity was detected included refractory breast cancer (one complete response, one partial, one minor), chemoradiation-resistant esophageal cancer (one complete response [Figure 1], two partial, two minor), gastric carcinoma (one partial response, two minor), previously treated non-small-cell lung cancer (one partial response, two minor, and prolonged disease stabilization in one patient), pretreated pancreatic carcinoma (one minor response, one tumor marker response), cholangiocarcinoma (one minor response), and hepatocellular carcinoma (one minor response).

Pharmacokinetics

Pharmacokinetic evaluation was performed by Dr. L. Schaaf (Pharmacia Corporation, Peapack, New Jersey) and Dr. J. Kuhn (University of Texas Health Sciences Center, San Antonio, Texas). Irinotecan and SN-38 mean t1/2 were 5.7 ± 0.7 and 10.7 ± 4.4 hours, respectively, and clearance was 12.7 ± 3.86 L/h/m² for irinotecan. The SN-38/irinotecan AUC0-24 ratio was 0.03 ± 0.01 (n = 29). Irinotecan, SN-38, and SN-38G pharmacokinetic parameters were more similar at 100 and 125 mg/m² than at historical controls, which indicated that no pharmacokinetic interactions occurred between mitomycin and irinotecan on the schedule tested in this study.

Molecular Correlates

Topoisomerase I gene expression at baseline was 516 ng/µL ± 83 with maximal induction (1,863 ng/µL ± 310) by 24 hours after mitomycin infusion. Patients with major responses to therapy (complete and partial responses) had the greatest topoisomerase I induction, to 6,114 ng/µL ± 1,019 compared to the rest of the patients at 151 ng/µL ± 25.2 (P = .023). Mitomycin induced NQO1 gene expression from a baseline level of 4.0 ng/mL ± 0.67 to maximum induction of 18 ng/µL ± 3.12 by 3 hours after mitomycin infusion. Irinotecan induced the expression of CE1 from a baseline level of 0.23 ng/µL ± 0.04 to a maximum induction of 1.90 ng/µL ± 0.30 by 3 hours after irinotecan infusion, whereas neither irinotecan nor mitomycin induced CE2.

Discussion

Our clinical data suggest that the mitomycin/irinotecan combination is feasible and active in patients with refractory malignancies. No pharmacokinetic interactions between mitomycin and irinotecan are apparent when these agents are given 24 hours apart. As predicted from in vitro models, mitomycin induces topoisomerase I gene expression in human subjects, and responders (complete and partial responses) demonstrate the largest topoisomerase I induction 24 hours following mitomycin infusion. Mitomycin also induces NQO1, while irinotecan decreases NQO1 gene expression shortly after administration, which suggests that a serum irinotecan-free interval should follow mitomycin treatment.[9,10] In addition, since maximum topoisomerase I up-regulation is reached 24 hours after administration of mitomycin, a delay in the administration of irinotecan after mitomycin appears justified.

Due to the encouraging antitumor activity observed in this phase I study, phase II clinical trials in breast and esophageal/gastroesophageal junction adenocarcinomas at the doses and schedule recommended in this study are under way.

References

1. Liu LF, Desai SD, Li TK, et al: Mechanism of action of camptothecin. Ann NY Acad Sci 922:1-10, 2000.

2. Shao RG, Cao CX, Zhang H, et al: Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J 18:1397-1406, 1999.

3. Reid RJ, Benedetti P, Bjornsti MA: Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs. Biochim Biophys Acta 1400:289-300, 1998.

4. Kanzawa F, Sugimoto Y, Minato K, et al: Establishment of a camptothecin analogue (CPT-11) -resistant cell line of human non-small cell lung cancer: Characterization and mechanism of resistance. Cancer Res 50:5919-5924, 1990.

5. Gobert C, Bracco L, Rossi F, et al: Modulation of DNA topoisomerase I activity by p53. Biochemistry 35:5778-5786, 1996.

6. Kolesar J, Villalona-Calero M, Eckhardt G, et al: Detection of a point mutation and the alternative splice NQO1 gene in patients with colon cancer (abstract 402). Proc Am Soc Clin Oncol 15:189a, 1996.

7. Kim R, Hirabayashi N, Nishiyama M, et al: Experimental studies on biochemical modulation targeting topoisomerase I and II in human tumor xenografts in nude mice. Int J Cancer 50:760-766, 1992.

8. Gobert C, Skladanowski A, Larsen A: The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53. Proc Natl Acad Sci USA 96:10355-10360, 1999.

9. Traver RD, Horiskoshi T, Danenburg K, et al: NAD(P)H: Quinone oxidoreductase gene expression in human colon carcinoma cells: Characterization of a mutation which modulates DT-diaphorase activity and mitomycin C sensitivity. Cancer Res 52:797-802, 1992.

10. Kolesar JM, Burris HA, Kuhn JG: Detection of a point mutation in NQO1 (DT-diaphorase) in a patient with colon cancer. J Natl Cancer Inst 87:1022-1024, 1995.

 
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