Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase.

16 BACKGROUND: Leflunomide (Lef) previously has been shown to reverse the histologic features of chronic rejection in a rodent cardiac model. The purpose of this study is to explore the effect of Lef treatment on early chronic rejection of renal allografts (ECRRA), using a modified rodent model described by Tilney and his colleagues. METHODS: Fifteen Lewis rats with bilateral nephrectomy received renal allografts from Fisher-334 rats and were treated with cyclosporine (Cya) at 2.5 mg/kg/day for 10 days. On day 60, 5 grafts were harvested and the remaining 10 recipients were divided into two groups, treated with 0 mg/kg and 15 mg/kg of Lef daily for another 60 days respectively, and then, sacrificed on day 120. Ten untreated Lewis rats with bilateral nephrectomy received syngeneic renal transplants were harvested on day 60 (5 grafts) and 120 (5 grafts). All grafts were stained with H&E, PAS, and antibodies against α-smooth muscle actin, IgM, IgG, ED1, TCRαβ, CD4 and CD8β for pathological analysis. One hundred glomeruli from H&E, PAS and IgM stained sections were examined randomly and rates of chronic injury were recorded. RESULTS: In untreated allografts, the primary pathology was an increasing glomerular matrix deposition and progressive glomerulosclerosis (GS). The incidences of involved glomeruli were 36.0±5.9% and 53.2±17.9% days 60 and 120 post transplantation, respectively. Immunohistochemically, the predominant finding was intensive IgM deposition in 44.8±13.8% and 53.4±14.0% of the observed glomeruli at 60 and 120 days respectively; as well as diffuse IgG deposition in glomerular and cortical tubular basement membrane. Both IgM and IgG were also deposited in the cortical microvessels and a few injured arteries. The cellular infiltration was mainly interstitial, including multifocal, sparse T cells (TCRαβ+, CD4+/CD8β+), IgM+ and ED1+ cells. The proteinuria in untreated groups increased from 10.4±4.8mg/day at day 0 to 34.7±25.7 mg/day by day 60 and 63.5±31.2 mg/day by day 120 post transplantation. After 60 days of Lef therapy, the rate of injured glomeruli decreased to 16.0±5.5% (P<0.001), the percent of glomeruli with intensive IgM deposition decreased to 16.4±5.6% (P<0.001), and IgG deposition substantially was reduced. The elevated proteinuria was also reduced to 26.2±21.9 mg/day (P=0.02). The serum creatinine was not significantly different in all groups. There were no significant pathological or urinary changes observed in syngeneic grafts. CONCLUSION: 1. The central pathological feature of ECRRA in this rodent model is progressive GS with patterns of IgM and IgG deposition, indicating a humoral component in the immune mechanism. 2. Creatinine level was not significantly increased in ECRRA up to 120 days. 3. The ECRRA was stabilized and histologically reversed by Lef (15 mg/kg/day) therapy.

Leflunomide is a compound recently shown to reduce T and B cell-mediated responses in a number of experimental rat, mouse, and human systems. To explore its potential as an immunosuppressant, we studied leflunomide in 128 Brown-Norway/Lewis cardiac transplants and in 48 unoperated Lewis rats. At doses ranging from 0.63 mg/kg to 10 mg/kg given for 7 days, leflunomide significantly prolonged graft survival compared with controls. When cyclosporine or leflunomide was given for 21 days at a dose of 5 mg/kg, indefinite graft survival occurred in 3/6 animals receiving leflunomide but in none of the 21-day cyclosporine-treated animals. When acute rejection was allowed to develop for four days in untreated rats, leflunomide but not cyclosporine reversed the rejection, returning histology to a normal appearance by seven days. Alloantibody responses measured in microcytoxicity assays as well as total allospecific IgG and IgM in the rejecting animals also were returned to baseline levels by leflunomide but not cyclosporine. When both drugs were used together, a synergistic effect was observed at low doses of both drugs. Pharmacokinetics studies showed that their combined use for up to 28 days did not affect the trough levels of cyclosporine or cyclosporine elimination, suggesting that the synergistic effect was not caused by reduced elimination. The toxicity of each drug was negligible in a group of 32 rats receiving the drugs alone or in combination as measured by serial observation of general appearance, testing of serum ALT, AST, bilirubin, creatinine, white blood cell counts, hemoglobin, and gross necropsy appearance. Weight gain was slightly reduced by both drugs but combined drug use did not alter the pattern. The results of these experiments show leflunomide to be a potent, well-tolerated immunosuppressant, synergistic in its activity with cyclosporine, and would seem to encourage a closer look at this drug for potential use in man.