INHIBITION OF CYTOMEGALOVIRUS IN VITRO AND IN VIVO BY THE EXPERIMENTAL IMMUNOSUPPRESSIVE AGENT LEFLUNOMIDE

Abstract

825 Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) continues to complicate the clinical course of many allograft recipients. Enhancement of viral activity by requisite immune suppression frequently engages the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein tryrosine kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent with demonstrated effectiveness against acute and chronic rejection in animal models. We have recently discovered that the active metabolite of this agent, A77 1726, inhibits the production of multiple isolates of human CMV in both human fibroblasts and endothelial cells in vitro. Viral plaque assays demonstrated a consistent dose-dependent reduction of virus yield over a range of concentrations equivalent to those which inhibit in vitro immune activation. Unlike anti-CMV drugs currently in use, A77 1726 has no effect upon viral DNA synthesis, as demonstrated by CMV-specific DNA dot blot analysis and direct biochemical assay of viral DNA polymerase activity. Rather, as indicated by transmission electron microscopy, this agent appears to act at the level of virion assembly by interfering with nucleocapsid tegumentation, possibly by inhibition of tegument protein phosphorylation. Likely as a consequence of this unique mechanism of action, A77 1726 is equally effective against drug-resistant isolates. To determine the effectiveness of leflunomide in the control of viral load in vivo, groups of immunodeficient nude rats were inoculated with rat CMV (RCMV, Maastricht strain, 105 plaque-forming units/animal, IP) and treated with either leflunomide (15 mg/kg/day for 14 days), ganciclovir (10 mg/kg/day for 5 days), or drug-free vehicle. Experiments also included uninfected control animals. Following euthanization at 14 days post-inoculation, viral infection was confirmed by histologic observation of typical cytomegalic ductal epithelium in salivary glands, and further verified by immunohistochemical detection of RCMV antigens in multiple tissues. Plaque assay of tissue homogenates prepared from salivary gland, spleen, and lung demonstrated 75-99% reduction in virus yield in organs harvested from leflunomide-treated animals, and 85-99% reduction in those harvested from ganciclovir-treated animals. No virus was ever recovered from uninfected control rats. Thus in addition to its in vitro antiviral activity, leflunomide is capable of reducing viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.