Characterisation of a synergistic interaction between a thymidylate synthase inhibitor, ZD1694, and a novel lipophilic topoisomerase I inhibitor karenitecin, BNP1100: Mechanisms and clinical implications

We developed a combination protocol for inhibitors of thymidylate synthase (TS) and DNA topoisomerase I (Topo I) that can exert highly lethal effects in vitro against HCT-8 human colorectal cancer cells. The specific schedule was constructed so that a TS inhibitor could induce not only primary DNA d amage but also cellular conditions optimal for the efficient action of a To po I inhibitor. The initial drug treatment consisted of a brief exposure to a quinazoline-based antifolate, ZD1694. After an interval of approximately one cell-doubling time, cells were exposed for 8-24h to BNP1100, a Karenit ecin-class 7-thiomethyl-camptothecin, in the presence of 1-10 mu M thymidin e; the latter acted as a crucial factor to promote the collision of moving replication forks with the drug-stabilised DNA-Topo I cleavable complexes e ven under continuous TS inhibition. Clonogenic analyses confirmed that thes e mechanistically distinct drugs at clinically achievable concentrations wo rked in a highly synergistic manner, with a maximum effect abolishing the v iability of virtually all cancer cells (> 99.9%). The pretreatment with ZD1 694 increased the amount of DNA-bound Topo I by up to 4-fold and the DNA-da maging capability of BNP1100 by up to 15-fold. The possibility of at least four DNA-damaging pathways is proposed which might have resulted from the i ndividual actions of TS and Topo I inhibitors as well as their concerted ac tions. Taken together, the present findings provided a logically permissibl e explanation as to why TS and Topo I inhibitors in concerted interactions induced a highly lethal effect which was more than a simple additive effect . Since these drugs are effective specifically on actively proliferating ca ncer cells, but not on non-cycling G(0)/G(1) cells, this mechanism-based pr otocol may warrant consideration for clinical verification. (C) 1999 Elsevi er Science Ltd. All rights reserved.