During Fiscal Year 1985 we have continued our basic projects on antifolate pharmacology and pleiotropic drug resistance. In the antifolate area, studies have been completed on the direct inhibition of thymidylate synthase and AICAR transformylase by methotrexate polyglutamates and by dihydrofolate polyglutamates. These studies have shown that several hundred- to a thousandfold increases in inhibitory activity occur when methotrexate is metabolized to its higher polyglutamate forms. The consequences of this direct inhibition of purine and thymidylate synthesis by methotrexate polyglutamates are being examined in new studies that relate effects on these pathways to intracellular folate levels. Dr. Allegra has worked out an HPLC method for separating intracellular folate cofactors and has shown in the presence of methotrexate there is a rapid rise in dihydrofolate, a fall in 5-methyltetrahydro folate, but only minor changes in the intracelluar levels of 10-formyltetrahydrofolate (the cofactor required in purine biosynthesis). These findings indicate that folate depletion does not account for the inhibition of purine synthesis and point to the possibility of direct inhibition of this pathway by either methotrexate polyglutamates or dihydrofolate polyglutamates. Currently we favor the latter as being responsible, because the rise in dihydrofolate correlates most closely with the very rapid inhibition of this pathway in cultured cells. We are now undertaking careful studies of the methotrexate analog trimetrexate, which cannot be converted to a polyglutamate, and have shown a similar rapid increase in purine inhibition that correlates with dihydrofolate increases. In addition, Dr. Allegra has undertaken studies of leucovorin rescue. His preliminary evidence shows that cells recover the ability to synthesize purines and thymidylate only after restoration of competitive amounts of intracellular reduced folates. A simple return of the intracellular pool to pretreatment cofactor levels does not provide rescue, indicating that folate depletion is probably not the mechanism of inhibition of purine synthesis, but that direct inhibition by polyglutamates is a more likely explanation.
