Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer.

The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC50 < 50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria.

Comparison of the protection of cells from antifolates by transduced human dihydrofolate reductase mutants.

Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.

Leucovorin protection against repeated daily dose toxicity of trimetrexate in rats.

Repeated high doses of trimetrexate (TMX), a potent non-classical antifolate, have been administered as an experimental treatment for life-threatening Pneumocystis carinii infections in man. This therapy includes the coadministration of leucovorin, a reduced folate cofactor, to prevent antifolate toxicity in the host. The purpose of this investigation was to assess possible toxicologic sequelae of this combination regimen in an animal model. TMX at daily oral doses of 25, 35, and 45 mg/kg produced dose-related myelosuppression, thymic lymphoid depletion, seminiferous tubular atrophy, and degenerative lesions of the gastrointestinal tract. Mortality observed with TMX alone occurred earlier at higher doses and was specifically associated with severe degenerative enteropathy of the cecum. Oral leucovorin doses of 1, 5, 20, or 50 mg/kg administered twice daily, at the time of TMX administration and 6 hr later, protected against TMX lethality and target organ toxicity in a dose-related manner. Leucovorin was only partially protective against TMX-induced macrocytic anemia and the degree of protection was not dose-related. Leucovorin protection against cecal enteropathy was associated with increased DNA synthetic rates and higher mitotic activity of cecal epithelium than those in rats administered TMX alone. Importantly, the combination of daily administration of high dose TMX for 4 weeks with protective coadministration of leucovorin did not result in target organ toxicities that differed from TMX alone.