1Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II, Napoli, Italy; 2Dipartimento di Medicina Sperimentale F. Magrassi, Seconda Universitá di Napoli, Napoli, Italy
Received 18 May 2001; revised 23 August 2001; accepted 6 September 2001.
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Abstract |
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DNA damage caused by platinum agents is frequently followed by induction of topoisomerase I, providing a rationale for use of platinum-based compounds with topoisomerase I inhibitors.
Materials and methods
We studied the effect of a sequential schedule of oxaliplatin on day 1 and topotecan on days 25, in human colon and ovarian cancer cells in vitro, in nude mice bearing human cancer xenografts and finally in cancer patients in a phase I trial.
Results
We demonstrated a supra-additive effect of this combination on inhibition of colony formation and induction of apoptosis in vitro. We then demonstrated that the two agents in combination markedly inhibit tumor growth in nude mice. We translated these results into a clinical setting, conducting a phase I study in cancer patients with oxaliplatin 85 mg/m2 on day 1 and topotecan at doses escalating from 0.5 to 1.5 mg/m2 on days 25. Sixty cycles of treatment were administered to 18 patients affected prevalently by ovarian and colorectal cancer. Combination with topotecan 1.5 mg/m2 caused a dose-limiting toxicity. Therefore the maximum tolerated dose of topotecan was 1.25 mg/m2, at which six patients experienced a mild hematological and gastrointestinal toxicity. We also obtained evidence of clinical activity, particularly in ovarian cancer.
Conclusions
Our results provide a solid biological and clinical rationale for a phase II trial at the recommended doses of oxaliplatin 85 mg/m2 and topotecan 1.25 mg/m2, possibly in ovarian cancer patients.
Key words: ovarian cancer, oxaliplatin, phase I, topotecan
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Introduction |
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Topotecan is a camptothecin derivative selective for topo-isomerase I [3], currently used in a variety of tumors at a standard dose of 1.5 mg/m2 for 5 days, including ovarian cancer patients who had received failed treatment with platinum derivatives and paclitaxel [4].
It has been shown that DNA damage produced by platinum agents is frequently followed by induction of topoisomerase I- dependent cleavage activity. In vitro studies have demonstrated that oxaliplatin induces topoisomerase I-mediated single strand breaks and that subsequent addition of topotecan increases DNA damage and cell death [5]. These data represent the biochemical rationale for the reported cooperative effect of platinum agents in combination with topoisomerase I inhibitors [6, 7]. A phase I clinical trial conducted on this basis (oxaliplatin 85 mg/m2 given on day 1 and topotecan 0.51 mg/m2 given for 5 days starting on day 1) has shown a severe and partly unexpected myelotoxicity resulting in the recommendation of careful selection of patients, based on previous treatment, for future studies [8]. However, the interesting biological rationale and the responses observed have led to the conclusion that further exploration of this combination of agents is worthwhile.
With the aim of reducing toxicity whilst preserving potential antitumor activity, we have studied the effect of oxaliplatin and topotecan in combination on a sequential schedule from cell culture to clinical settings. In the present study we first evaluated the activity of the combination on cancer cell proliferation and apoptosis in vitro. Then we confirmed these data in vivo, in nude mice bearing human tumor xenografts. Finally, we designed and conducted a phase I study in patients refractory to standard treatments.
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Materials and methods |
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Flowcytometric analysis of cell cycle
Cells were harvested, fixed in 70% ethanol, stained with a propidium iodide staining solution and the DNA content analyzed in duplicate by a FACScan flow-cytometer (Becton and Dickinson, Mountain View, CA, USA) as described previously [9]. Cell cycle data analysis was performed by a CELL-FIT program (Becton and Dickinson).
Apoptosis
The induction of apoptosis was determined by the Cell Death Detection ELISA Plus Kit (Roche Molecular Biochemicals, Mannheim, Germany) which detects cytosolic histone-associated DNA fragments. OVCAR-3 and GEO cells (5 x 104 cells/dish) were seeded into 35 mm dishes and treated with different concentrations of oxaliplatin (0.1 µg/ml on the day of seeding, day 0) and/or topotecan (1, 5 or 10 nM on days 14). On day 5, cells were washed once with PBS and lysed for 30 min then supernatant was recovered and assayed for DNA fragments as recommended by the manufacturer [10]. Each treatment was performed in quadruplicate. The total number of cells was measured with a hemocytometer in additional plates receiving an identical treatment. The optical density at 405 nm was normalized for cell number and the optical density ratio (treated:untreated cells) was considered an apoptotic index and expressed in arbitrary units [10].
In vivo studies in nude mice
Five- to six-week-old female Balb/c athymic (nu+/nu+) mice were purchased from Charles River Laboratories (Milan, Italy). Mice were maintained in accordance to the institutional guidelines of the University of Naples Animal Care and Use Committee. They were acclimatized to the University of Naples Medical School Animal Facility for 1 week prior to s.c. injection with 107 GEO cells resuspended in 200 µl of Matrigel (Collaborative Biomedical Products, Bedford, MA, USA) as described previously [11]. After 7 days, when well-established tumors of ~0.2 cm3 were detected, 10 mice per group were i.p. treated with the following doses and schedules: oxaliplatin alone (either 10 mg/kg on day 1 every week for 4 weeks or 15 mg/kg on day 1 every 2 weeks); topotecan alone (2 mg/kg on day 1 every week for 4 weeks, or 2 mg/kg on days 1 and 2 every 2 weeks, or 0.5 mg/kg on days 14 every 2 weeks); oxaliplatin (10 mg/kg on day 1) in combination with topotecan (0.5 mg/kg on days 25) repeated every 2 weeks for a total of three cycles of treatment. Tumor size was measured twice weekly using the formula: (/6) x larger diameter x (smaller diameter)2.
Phase I study
Patient selection. Patients enrolled had a histologically confirmed solid tumor refractory to conventional therapy, no treatment of the malignancy for 6 weeks before study entry, a minimum life expectancy of 3 months and were at least 18 years old. Minimum eligibility requirements included: Eastern Cooperative Oncology Group performance status 2; neutrophil count
1500/µl; platelet count
100 000/µl; hemoglobin
9.5 g/dl; serum creatinine
2.0 mg/dl; total bilirubin <1.5 mg/dl; and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities
2.5 x the upper limit of normal. Informed, signed consent was obtained as a condition of patient registration.
Study design. The study protocol was approved by the institutional Scientific Review Committee. Patients underwent a physical examination and performance status determination. Electrocardiogram and chest X-ray, complete blood count with platelet and differential counts, coagulation tests, serum chemistry profile and determination of serum tumor markers were performed within 14 days before initiating therapy and, at indicated times, during the entire period of study.
We combined oxaliplatin (Eloxatine) 85 mg/m2 administered in 2 h i.v. on day 1 with topotecan (Hycamtin) at doses ranging from 0.5 mg/m2 up to 1.5 mg/m2 in five different dose levels administered in 30 min i.v. on days 25. Each cycle of treatment was repeated every 3 weeks. Patients were scheduled to receive at least three courses of therapy at the same dose level. Cohorts of three patients were scheduled for entry at each dose level. Escalation of the dose to the next higher level proceeded after all three patients received the first cycle of therapy and had been observed for at least 21 days without evidence of a dose-limiting toxicity (DLT), as defined below. Drug-related toxicities were evaluated during each cycle of therapy and graded according to the NCI Common Toxicity Criteria version 1 [12]. A DLT was defined as any of the following events: grade 4 neutropenia, grade 4 thrombocytopenia, any drug-related non-hematological toxicity greater than or equal to grade 3 (except alopecia). At the occurrence of a DLT in three patients from any cohort, an additional three patients were enrolled at the preceding dose level which was considered the maximum tolerated dose (MTD). Patients experiencing toxicities that were not dose-limiting could be retreated at the same dose level upon full recovery. Treatment was discontinued upon occurrence of a DLT or tumor progression.
Evaluation of response. A baseline assessment of all measurable disease sites using appropriate radiological techniques was performed within 21 days before the first cycle of therapy. Tumor burden was calculated as the sum of the products of the longest perpendicular diameters of all measurable lesions and response was evaluated by common criteria [13]. Duration of response was measured from the date that the response was first recorded to the date of documented disease progression.
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Results |
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We studied the effect of oxaliplatin and topotecan, alone and in combination, on the induction of apoptosis in OVCAR-3 and GEO cells. As shown in Figure 1C, oxaliplatin or topotecan alone at the low doses of 0.1 µg/ml and 1 nM, respectively, did not induce apoptosis in ovarian OVCAR-3 cells, as compared with untreated cells. A 2-fold and a 2.3-fold induction were observed with topotecan alone at the higher doses of 5 and 10 nM, respectively. When oxaliplatin was used in combination with topotecan we observed at least an additive effect with topotecan 1 and 5 nM and a supra-additive effect with topotecan 10 nM (Figure 1C). The same doses of the two agents also caused at least an additive effect in GEO cells (data not shown).
Effect in vivo in nude mice bearing human cancer xenografts
We translated the results obtained in vitro into a model of nude mice xenografted with GEO cells. We first studied the antitumor effect of different doses and schedules of oxaliplatin and topotecan, selecting moderately effective doses of each single agent. We observed similar effects for oxaliplatin either 10 mg/kg once a week for 4 weeks or 15 mg/kg once a week every 2 weeks (data not shown). We also demonstrated similar growth inhibitory effects with three different doses and schedules of topotecan, including the low dose 0.5 mg/kg for 4 days every 2 weeks (Figure 2A). Therefore, to be consistent with the in vitro model, we randomized mice to receive the combination regimen of oxaliplatin 15 mg/kg on day 1 and topotecan 0.5 mg/kg on days 25. Each cycle was repeated every 2 weeks for a total of three cycles of treatment (Figure 2B). We have shown that oxaliplatin alone was ineffective in inhibiting tumor growth since by day 35 all oxaliplatin-treated mice, as well as mice in the control group, were dead. In the group treated with topotecan alone we observed a growth inhibitory effect accompanied by increased mice survival. A marked inhibitory effect was observed in mice treated with the two agents in combination. In fact, at 7 weeks following tumor injection, tumor volume was 90% smaller than that observed in mice treated with topotecan alone and tumors did not achieve a size incompatible with normal life until at least 5 weeks after treatment withdrawal (Figure 2B).
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We obtained evidence of the clinical activity of this regimen, particularly in ovarian cancer, although all patients had already received treatment with paclitaxel and a platinum agent. In fact, we observed a complete response maintained for >9 months in two stage IV ovarian cancer patients and stabilization of disease in two patients with ovarian cancer and two patients with metastatic colorectal cancer. Moreover, most ovarian cancer patients also obtained a reduction of serum CA 125 levels at the lower dose levels.
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Discussion |
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We demonstrated that oxaliplatin and topotecan used in sequential combination cause a supra-additive inhibition of growth in human GEO colon and OVCAR-3 ovarian cancer cells, expecially evident with lower doses of each agent. These effects were associated with enhancement of apoptosis and perturbation of cell cycle distribution with increased accumulation of cells in S and G2-M phases.
We confirmed these findings in vivo, in nude mice bearing GEO tumor xenografts, using different doses and schedules of the two agents alone, then selecting the doses to be used in combination with the same sequential schedule used in vitro. Animals received three cycles of treatment. All mice untreated or treated with oxaliplatin alone were dead 5 weeks after tumor injection. Treatment with topotecan alone caused ~60% inhibition of growth at the end of treatment as compared with the previous groups. However, 2 weeks following treatment withdrawal all topotecan-treated mice were dead. Conversely, at the same time point, animals receiving oxaliplatin and topotecan in combination were all alive and had a tumor volume 90% smaller than mice treated with topotecan alone. In addition, once the tumors recovered their growth they did not cause the death of the mice before 5 weeks following treatment withdrawal. No signs of acute or delayed toxicity were observed in treated mice.
On this basis, we designed and conducted a phase I study. We used oxaliplatin on day 1 at 85 mg/m2, a dose commonly used and well tolerated in combination regimens [2] and topotecan on days 25 at five different dose levels ranging from 0.5 to 1.5 mg/m2. We administered 60 cycles of treatment to 18 patients. The treatment regimen was generally well tolerated and toxic effects were mainly hematological (neutropenia and thrombocytopenia) and gastrointestinal (mucositis and diarrhea). The DLT was attained in the combination with topotecan at 1.5 mg/m2. Three patients affected by ovarian cancer and three by colon cancer received a total of 30 cycles of treatment at the MTD, with topotecan 1.25 mg/m2. Although patients at this dose level had been pretreated with drugs responsible for hematological toxicity and diarrhea, they experienced only manageable side effects during the study, with moderate bone marrow and gastrointestinal toxicity, except for one case of grade 4 neutropenia. Several patients received multiple cycles of treatment, up to six, without signs of cumulative toxicity. Interestingly, we have not observed neurotoxicity, a peculiar toxicity attributed to oxaliplatin, even in patients who had previously received platinum derivatives and taxanes.
Both topotecan and oxaliplatin have been widely studied in combination regimens with different agents. In the majority of studies the relative recommended doses have been between 85 and 100 mg/m2, on day 1, for oxaliplatin and up to 1 mg/m2 for 5 days for topotecan [2, 3]. The schedule used in our study allows the administration of approximately two-thirds of the standard dose of topotecan as a single agent.
Topotecan is used as a single agent to treat various types of cancer, including ovarian cancer [3, 4]. At the standard dose of 1.5 mg/m2 it induces an average response rate of 14% in ovarian cancer patients, varying between 12% and 20% in those refractory or sensitive to cisplatin, respectively [4, 15]. However, in other types of cancer, such as those of the gastrointestinal tract, the percentage of responses is relatively poor [16].
We obtained evidence of clinical activity of this combination regimen, particularly in ovarian cancer patients. In fact, we observed complete responses, maintained for >9 months, in two heavily pretreated stage IV ovarian cancer patients. Like all the other ovarian cancer patients enrolled, both had previously received paclitaxel and a platinum agent, also one of them had an early relapse after two different treatment regimens. Two other ovarian patients obtained a stabilization of the disease, while the majority of them experienced a reduction of serum CA 125 levels, even at the lower dose levels. We have also reported a stabilization of the disease in two patients with metastatic colorectal cancer.
For these reasons our results are encouraging and support further exploration of such a combination regimen in a phase II study at the recommended doses of oxaliplatin 85 mg/m2 on day 1 and topotecan 1.25 mg/m2 on days 25, in early relapsed or refractory ovarian cancer patients.
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Acknowledgements |
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Footnotes |
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References |
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2. Misset JL, Bleiberg H, Sutherland W et al. Oxaliplatin clinical activity: a review. Crit Rev Oncol Hematol 2000; 35: 7593.[ISI][Medline]
3. Burris HA 3rd, Fields SM. Topoisomerase I inhibitors. An overview of the camptothecins analogs. Hematol Oncol Clin North Am 1994; 8: 333355.[ISI][Medline]
4. Bookman MA, Malmstrom H, Bolis G et al. Topotecan for the treatment of advanced epithelial ovarian cancer: an open-label phase II study in patients treated after prior chemotherapy that contained cisplatin or carboplatin and paclitaxel. J Clin Oncol 1998; 16: 33453352.[Abstract]
5. Goldwasser F, Bozec L, Zeghari-Squalli N, Misset JL. Cellular pharmacology of the combination of oxaliplatin with topotecan in the IGROV-1 human ovarian cancer cell line. Anticancer Drugs 1999; 10: 195201.[ISI][Medline]
6. Ma J, Maliepaard M, Nooter K et al. Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro. Cancer Chemother Pharmacol 1998; 41: 307316.[ISI][Medline]
7. Romanelli S, Perego P, Pratesi G et al. In vitro and in vivo interaction between cisplatin and topotecan in ovarian carcinoma systems. Cancer Chemother Pharmacol 1998; 41: 385390.[ISI][Medline]
8. Misset JL, Goldwasser F, Riofrio M et al. Topotecan-oxaliplatin every three weeks: a phase I and pharmacological study. Proc Am Soc Clin Oncol 1999; 18: 845 (Abstr).
9.
Ciardiello F, Pepe S, Bianco C et al. Down-regulation of RI sub- unit of the cAMP-dependent protein kinase induces growth inhibition of human mammary epithelial cells transformed by c-Ha-ras and c-erbB-2 proto-oncogenes. Int J Cancer 1993; 53: 438443.[ISI][Medline]
10. Tortora G, Caputo R, Damiano V et al. A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer. Int J Cancer 2000; 88: 804809.[ISI][Medline]
11.
Ciardiello F, Damiano V, Bianco R et al. Anti-tumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. J Natl Cancer Inst 1996; 88: 17701776.
12. National Cancer Institute. Guidelines for reporting of adverse drug reactions. Bethesda, MD: Division of Cancer Treatment, National Cancer Institute, 1988.
13. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer 1981; 47: 207214.[ISI][Medline]
14.
Zeghari-Squalli N, Raymond E, Cvitkovic E, Goldwasser F. Cellular pharmacology of the combination of the DNA topoisomerase I inhibitor SN-38 and the diaminocyclohexane platinum derivative oxaliplatin. Clin Cancer Res 1999; 5: 11891196.
15. Coleman RL, Miller DS. Topotecan in the treatment of gynecologic cancer. Semin Oncol 1997; 24 (6 Suppl 20): 5563.
16. Creemers GJ. Topotecan in advanced colorectal cancer. Semin Oncol 1997; 24 (Suppl 20): 4248.