A series of drug combination sequence studies was conducted in vitro using HEC-1A human endometrial carcinoma cells or 8226 myeloma cells. Four drugs were evaluated for schedule-dependent and sequence-dependent inhibition of human tumor colony formation in soft agar. Six different two-drug combinations were analyzed using the median dose effect method, and three different three-drug combinations were examined using the cumulative surviving fraction method. The results show that the specific sequence and method of drug exposure significantly influenced the production of antagonistic, additive, or synergistic cytotoxicity patterns. Drug combinations that were consistently synergistic included bleomycin or mitomycin C and cisplatin in 8226 cells, and etoposide plus bleomycin in human endometrial cancer (HEC-1A) cells. Most other two-drug combinations of bleomycin, etoposide, cisplatin, and mitomycin C were antagonistic in vitro, irrespective of the sequence of exposure. Among the three-drug combinations tested, consistent synergism was noted with cisplatin, etoposide, and bleomycin when either of the latter two agents was tested as a continuous exposure in vitro. Within individual two- and three-drug combinations, it was possible to observe synergism, additivity, or antagonism based on the particular exposure sequence tested. These results suggest that antitumor agent cytotoxicity in vitro can be radically influenced by the sequence of drug administration, a feature commonly overlooked in many clinical combination drug regimens.
