Abstract
An efficient drug-induced rabbit liver microsomal enzyme system, that may be easily scaled up, has been applied to produce milligram (mg) quantities of cyclosporin G metabolites (CMs). Using hepatic microsomal preparations from rabbits pretreated with rifampicin (RIF) and a NADPH regenerating system, we converted 60% of the cyclosporin G (CsG) to 12 CMs in 2 hr. CMs were recovered by solid-phase extraction. Separations performed on a semipreparative scale with two sequential 250 × 10 mm reversed-phase HPLC columns yielded mg quantities of CMs. The yield of GM1 and GM9 obtained by a single HPLC separation step was estimated to be 1585 ± 103 μg and 648 ± 33 μg, respectively. Other CMs produced by this microsomal enzyme system were GM19, GM4N, GM1c, the aldehydic isomers (GM1cAL and GM1AL), GM14N, GM4N9, GM1A, and GM1cA. The purity of CMs was confirmed by analytical HPLC and their molecular weights (M.W.) were determined by Fast Atomic Bombardment Mass Spectrometry (FABMS). Biotransformation of CsG and cyclosporine (CsA) was also compared using this in vitro model system. Significant difference was found between the rate of aldehydic metabolite formation from CsG and that from CsA. This translates to a faster rate of AM1cAL and AM1AL formation than that of GM1cAL and GM1AL. In addition, the rate of CsG conversion to GM9 appears to be faster than that of CsA conversion to AM9.
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