Canine pharmacodynamic assays: applications in immune-mediated disease

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Immune-mediated diseases are commonly diagnosed in veterinary medicine and a high rate of mortality, expense and complications of immunosuppressive therapy in dogs underscores the need for optimization of drug dosing. Pharmacodynamic assays are utilized in human medicine to evaluate the efficacy of immunosuppressive therapy in individual patients. The purpose of this Master's project was to validate an in vitro flow-cytometric assay evaluating canine B and T lymphocyte proliferation. In doing so, this assay could then be used to evaluate the effects of a panel of immunosuppressants (dexamethasone, cyclosporine, and the active metabolites of azathioprine and leflunomide) on lymphocyte proliferation in individual dogs. The first-phase of this project focused on peripheral blood mononuclear cells to evaluate canine T lymphocyte proliferation and the concentration of all four immunosuppressants that would result in 50% inhibition of T lymphocyte proliferation (IC50) in five healthy dogs. The mean (±SEM) IC50 were: dexamethasone, -4.6±0.5 M; cyclosporine 16±2 ng/mL; 6-mercaptopurine 64±31 nM; and A77 1726, 24±3 μM. The second-phase of the project focused on a whole blood assay utilizing both 5-bromo-2'-deoxyuridine and Ki-67 as markers of proliferation and evaluated both B and T lymphocyte proliferation utilizing flow cytometry. The validation of these canine pharmacodynamic assays represents the first steps in veterinary medicine to tailor immunosuppressant protocols and evaluate lymphocyte proliferation in individual dogs with immune-mediated disease.