beta-Oxidation of simvastatin in mouse liver preparations

All current 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors [simvastatin (SV), lovastatin (LV), atorvastatin, pravastatin, fluvastatin, and cerivastatin] are believed to undergo an atypical beta-oxidation of the dihydroxy heptanoic or heptanoic acid side chain. Metabolites, which are shortened by two- and/or four-carbon units consistent with beta-oxidation products, have been reported exclusively in rodents following LV and SV administration and across species (rodents, dogs, and humans) following the other statins. In this study, in vitro formation of a beta-oxidation product of simvastatin hydroxy acid (SVA) and its intermediates in mouse livers is described. Incubation of SVA with mouse liver preparations fortified with CoASH and ATP led to formation of SV and two major products (P1 and P2). Based on mass spectrometry (MS), tandem mass spectrometry, and/or NMR spectral characteristics, P1 was an alpha,beta-unsaturated metabolite, formed by dehydration of the D,D-dihydroxy heptanoic acid side chain, whereas P2 was probably the L,D-dihydroxy acid isomer of SVA, formed by stereospecific hydration of P1. When NAD(+) was also included in the incubation mixture, there were two additional metabolites with the MS and/or NMR characteristics consistent with a two-carbon shortened product (P3) and its dehydrated derivative (P4). In a complete incubation system with all cofactors (ATP, CoASH, NAD(+), and NADPH) present, there was an additional product with MS spectra and liquid chromatography retention time identical to the beta-oxidized, unsubstituted pentanoic acid metabolite (P5) detected in rats and mice following simvastatin administration. The involvement of CoASH and NAD(+) and the presence of the four metabolic intermediates suggest that SVA (and presumably the other statins) is a substrate for the beta-oxidation enzyme complex in mice. Additionally, the present finding of CoASH-dependent formation of SV substantiates a mechanism proposed previously for the in vivo lactonization of statin hydroxy acids.