102 Leflunomide (LFM) and its MNA analogs, HMR1279 and HMR1715, are effective in various experimental models of allo and xenotransplantation. Our study is the first to show that the in vitro and in vivo antiproliferative and immunosuppressive effects of MNAs (A77 1726 [active metabolite of LFM], HMR1279, and HMR1715) are caused by inhibition of de novo pyrimidine biosynthesis. Methods: The antiproliferative effects of MNAs were measured using an optimized whole blood, Con A-stimulated lymphocyte proliferation assay. DHODH activity was assessed histochemically in frozen tissue sections. The immunosuppressive effects of MNAs were assessed using the mouse ear heart transplant model (BALB/c to C3H/km). Results: MNAs concentration-dependently inhibited lymphocyte proliferation when added in vitro to whole blood samples (A77 1726, IC50%=7.2 mM; HMR1279, IC50%=60 mM; HMR1715, IC50%=100 mM) or when administered to Lewis rats (A77 1726, IC50%=6.7 mM; HMR1279, IC50%=37.1 mM; HMR1715, IC50%=91.2 mM). Uridine concentration-dependently reversed the antiproliferative effects of MNAs when added in vitro to whole blood samples(0-2 mM) or when administered to Lewis rats (0.5 to 1 g/kg/dose IP). DHODH and succinate dehydrogenase (SDH) activity were detected in kidney, liver, and intestinal tissue sections from normal C3H/km mice. DHODH, but not SDH activity, was inhibited by in vitro addition of 10 uM of A77 1726, HMR1279, or HMR1715. CsA (1 uM) had no effect on either enzyme. DHODH activity was also detected in lymphocytes infiltrating rejecting mouse heterotopic heart grafts(BALB/c to C57) removed on day 6 from untreated mice. Treatment with LFM (20 mg/kg), HMR1279 (40 mg/kg), or HMR1715 (20 mg/kg) from day 4 to 6 inhibited DHODH lymphocyte activity while CsA (25 mg/kg) had no effect. Administration of 20 mg/kg of LFM or HMR1715 starting at day 1 prolonged graft survival to 22.1±3.8 (n=8) and 23.3±3.9 (n=4) days, respectively, compared to nontreated mice (9.8±1, n=9, p<0.001). Coadministration of uridine (3 g/kg tid) with LFM (20 mg/kg) or HMR1715 (20 mg/kg) reduced graft survival significantly to 12±2.5 (n=12) and 14.2±1.4 (n=5) days, respectively, compared with LFM (20 mg/kg) or HMR1715 (20 mg/kg) monotherapy(p<0.001). Administration of FK506 alone (0.6 mg/kg, n=5) or in combination with uridine (n=5) produced similar prolongations of graft survival(19.6±2.6 vs. 18±1.4 days, p>0.05). Conclusions:(1) Uridine supplementation antagonized both the in vitro and in vivo antiproliferative effects of MNAs, (2) DHODH activity in tissues and graft-infiltrating lymphocytes was specifically inhibited by MNAs, (3) In vivo uridine treatment abrogated prolongation of heart allograft survival by LFM and HMR1715, but not by FK506. These are the first studies to define the in vivo mechanism of immunosuppressive action for xenobiotic drugs. Research funded by Hoechst-Marion-Roussel, Milton Keynes, UK.