BIOCHEMICAL MODULATION OF IODODEOXYURIDINE BY INOSULFONYL)BENZYL]-N-6-METHYL-2,6-DIAMINOBENZ[CD] INDOLE GLUCURONATE (AG-331) LEADING TO ENHANCED CYTOTOXICITY

Inhibition of thymidylate synthase (TS) may increase incorporation of thymidine analogues into DNA, leading to increased inhibition of colon y formation in tumor cells. We have reported previously that TS inhibi tion by -6,-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ICI D 1694 or Tomudex), a folate-based TS inhibitor, increases the cytotoxic ity of iododeoxyuridine (IdUrd), a thymidine analogue, in MGH-U1 human bladder and HCT-8 human colon cancer cells. onyl)benzyl]-N-6-methyl-2 ,6-diaminobenz[cd]-indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not underg o intracellular polyglutamation. Exposure of MGH-U1 cells to 5 mu M AG -331 for 24 hr decreased clonogenic survival by 30%, but almost comple tely inhibited TS activity. IdUrd is a cytotoxic thymidine analogue, w ith IC50 and IC90 values after 24-hr exposures in MGH-U1 cells of 13 a nd 81 mu M, respectively. The combination of IdUrd and AG-331 resulted in an enhanced antitumor effect, as compared with the effect of eithe r agent alone. The cytotoxic IC50 of IdUrd decreased from 13 to 1.5 mu M, and the IC90 decreased from 81 to 5 mu M with the addition of 5 mu M AG-331. Biochemical studies of the combination revealed that pretre ating MGH-U1 cells with 5 mu M AG-331 increased IdUrd incorporation in to cellular DNA by 3.8-fold. This increased incorporation was associat ed with a greater proportion of DNA single-strand breaks than observed with either agent alone, and the combination of 5 mu M AG-331 plus Id Urd produced up to a 2.5-fold increase in DNA single-strand breaks as compared with IdUrd alone. The effects of AG-331, IdUrd, and the combi nation of IdUrd and AG-331 on the colony-forming ability of normal hum an bone marrow CFU-GM cells was determined as a measure of myelosuppre ssion. The combination of IdUrd and AG-331, at the same concentrations as those used in the MGH-U1 cells, produced a wider therapeutic index relative to that of IdUrd alone, and the therapeutic index for the co mbination was 6.5, as compared with 4.0 for IdUrd plus ICI D1694 in pr evious studies from this laboratory. These observations suggest that t he combination of IdUrd and AG-331 may enhance antitumor effects with minimal myelosuppression in vivo.