Vol. 289, Issue 2, 800-806, May 1999

Evaluation of Individual and Combined Neurotoxicity of the Immunosuppressants Cyclosporine and Sirolimus by In Vitro Multinuclear NMR Spectroscopy¹

Natalie Serkova, Lawrence Litt, Thomas L. James, Wolfgang Sadée, Dieter Leibfritz, Leslie Z. Benet and Uwe Christians

Departments of Biopharmaceutical Sciences (N.S., W.S., L.Z.B., U.C.), Anesthesia (L.L.), and Pharmaceutical Chemistry (T.L.J.), University of California at San Francisco, San Francisco, California; and Department of Chemistry and Biology, University of Bremen, Bremen, Germany (D.L.)

Neurotoxicity, a crucial side effect of immunosuppressive therapy with cyclosporine, also has been demonstrated in vitro for sirolimus, a novel macrolide immunosuppressant, which is under clinical investigation in combination with cyclosporine. NMR spectroscopy was used to study the separate and combined effects of cyclosporine and sirolimus on cerebral metabolism, both in brain cells and in perfused rat brain slices. The high-energy phosphate metabolism was already affected significantly at cyclosporine concentrations as low as 100 µg/liter: phosphocreatine was reduced by 10 ± 2% [half-maximal inhibition concentration (IC₅₀) = 1850 ± 600 µg/liter], and nucleoside triphosphate was reduced by 11 ± 5% (IC₅₀ = 1110 ± 420 µg/liter; n = 4, P < .05). At 500 µg/liter cyclosporine, N-acetylaspartate and glutamate were decreased by 13 ± 7% (IC₅₀ = 1100 ± 330 µg/liter) and 22 ± 9% (IC₅₀ = 360 ± 220 µg/liter; n = 4, P < .05), respectively. As evaluated using an algorithm based on Loewe isobolograms, combination of cyclosporine and sirolimus resulted in a synergetic reduction of high-energy phosphate metabolites. Addition of sirolimus to the perfusion medium increased brain slice concentrations of cyclosporine. It is concluded that cyclosporine significantly reduced high-energy phosphate metabolism in brain tissue at in vivo relevant concentrations. Combination with sirolimus resulted in synergism, which, in part, is explained by a greater distribution of cyclosporine into the brain tissue in the presence of sirolimus.