Antitumor Activity of Antifolate Inhibitors of Thymidylate and Purine Synthesis in Human Soft Tissue Sarcoma Cell Lines with Intrinsic Resistance to Methotrexate

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3,4-dihydro- 2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2- thenoyl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5,10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibidon studies showed that ED₅₀ values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. ln contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensidve and the three resistant sarcoma cell lines. Uptake of D1694,1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower K_m values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with V_max values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in padents with soft tissue sarcomas.