Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034) - Comparison across sensitive and resistant human tumor cell lines

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonp olyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamy latable inhibitors of other folate-requiring enzymes, such as glycinamide r ibonucleotide formyltransferase (GARFT) and thymidylate synthase. Since thi s phenomenon is potentially therapeutically useful, we explored its univers ality by examining the combined action of a DHFR inhibitor, trimetrexate (T MQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro 3H-pyri midino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic aci d (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, H CT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-FdUrd-resistant), and HCT- 8/50 (adapted to 50 nM Pte- Glu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung c arcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the p resence of 2.3 or 40 mu M PteGlu. Cell growth was measured with the sulforh odamine B assay at the end of this pried. Drug interactions were assessed b y fitting a 7-parameter model including a synergism parameter, alpha, to da ta with weighted nonlinear regression. Isobologam analysis was also applied . At 2.3 mu M PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TM(I + AG2034. Loewe synergy was abolished in HCT-8/ 50 cells cultured and studied in 50 nM PteGlu. At 40 mu M PteGlu, the inten sity of the combined action in all cell lines was increased. However, the m ost intense: Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253 , and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the p henomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline d eficient in folylpolyglutamate synthetase (FPGS). The universality of the P teGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activ ity and low-folate adaptation prior to treatment significantly lessen the d egree of PteGlu enhancement. BIOCHEM PHARMACOL 57;5:567-577, 1999. (C) 1999 Elsevier Science Inc.