Leflunomide inhibits pyrimidine de Novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated, This study, using C-14-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 mu M), like Brequinar (0.5 and 1 mu M), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G(1) phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [C-14]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 mu M) restored expansion of all pools, but [C-14]bicarbonate incorporation into ATP and GTP only, not UTP, [C-14]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors, [C-14]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 mu M Leflunomide. 100 mu M Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 mu M and above.