Synergistic cytotoxicity with 2'-deoxy-5-azacytidine and topotecan in vitro and in vivo.

Synergy, when it can be convincingly established, is an effective strategy for the development of novel drug combinations. We have evaluated the interaction between 2'-deoxy-5-azacytidine (DAC) and 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan) based on our hypothesis that DAC, through DNA hypomethylation, might increase the transcription of topoisomerase I (topo I) leading to increased sensitivity to topotecan. Five human tumor cell lines, A375 melanoma, DX-3 melanoma, DMS4C non-small cell lung carcinoma, UP-1 unknown primary adenocarcinoma, SN12C renal carcinoma, and the murine CT-26 tumor cell line, were studied. Drug interactions were assessed using the multiple drug effect analysis of Chou and Talalay (Chors, T-C, and Talalay, P. Adv. Enzyme Regul., 22:27-54, 1984.). A synergistic interaction was documented in four human cell lines and the murine CT-26 line. An antagonistic interaction was observed with the SN12C cell line. The toxicology and efficacy of this combination were analyzed using CT-26 in BALB/c mice. Various treatment schedules were studied, including: single doses of each agent; single sequential combination treatments where DAC was administered followed by topotecan 24 h later; and multiple sequential treatments where DAC and topotecan were administered on days 1, 2, 8, and 9. Efficacy studies showed that the single sequential combination of DAC (50 mg/kg) and topotecan (10 mg/kg) resulted in tumor growth delay as compared to single doses of DAC (50 mg/kg) or topotecan (10 mg/kg). When the multiple sequential combination schedule was used, the antitumor effect was more pronounced. In that experiment 50% of the control animals had tumors of 20 mm by day 28. For animals receiving a single sequential treatment with DAC and topotecan, the median time until the mean tumor size reached 20 mm was 38 days, and for the group with multiple sequential combination treatments the time was 51 days. Studies of the mechanism of the interaction showed that the activity of topotecan versus each cell line correlated with the topo I activity in nuclear extracts However, there was no correlation between topo I levels and synergy and no reproducible increase in topo I activity following exposure to DAC. Thus, while the exact mechanism of the interaction remains unclear, DAC can be effectively combined with topotecan to enhance antitumor activity.