Short Communication -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 -oxidation of the dihydroxy heptanoic or heptanoic acid side chain. Metabolites, which are shortened by twoand/or four-carbon units consistent with -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 -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 , -unsaturated metabolite, formed by dehydration of the D,Ddihydroxy 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 -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 -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. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors, or “statins”, are used widely for the treatment of hypercholesterolemia and hypertriglyceridemia (Mauro, 1993). Except for simvastatin (SV) and lovastatin (LV), all current statins are administered as the pharmacologically active hydroxy acid form. SV and LV are inactive lactones, which upon conversion to their respective hydroxy acids (SVA and LVA), are potent competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (Duggan and Vickers, 1990). Most of the statins have been shown to undergo extensive metabolism in both animals and humans (Vickers et al., 1990a; Everett et al., 1991; Dain et al., 1993; Halpin et al., 1993; Cheng et al., 1994; Le Couteur et al., 1996; Prueksaritanont et al., 1997; Black et al., 1999). Biotransformation of the statins exhibits noticeable species differences and some qualitative similarities. In the case of SV and LV, esterase-dependent hydrolysis to SVA or LVA in plasma was very rapid in rodents but not in humans and dogs (Vickers et al. 1990b; Draganov et al., 2000). One of the biotransformation pathways reported for all statins is oxidation at the dihydroxy heptanoic or heptanoic acid side chain, a structural feature common to all statins. Pentanoic acid derivatives of SVA or LVA, corresponding to the loss of a two-carbon unit from the dihydroxy heptanoic acid side chain, have been reported to occur exclusively in rodents following SV or LV administration (Vickers et al., 1990b; Halpin et al., 1993). Metabolites shortened by twoor four-carbon units, resulting in pentanoic derivatives or propanoic products, respectively, have been observed in vivo for atorvastatin and pravastatin (both contain the same dihydroxy heptanoic side chain) primarily in rodents and minimally in dogs and in humans (Arai et al., 1988; Everett et al., 1991; Le Couteur et al., 1996; Black et al., 1998, 1999). Analogous metabolites also have been described in animals and humans for cerivastatin and fluvastatin, both of which contain the dihydroxy heptanoic acid moiety (Dain et al., 1993; Boberg et al., 1998). These metabolites have been referred to as -oxidation products because they resemble those observed in the -oxidation of fatty acids, which is characterized by stepwise oxidation of the carbon chain, two carbons for each cycle. Mechanistically, the -oxidation of fatty acids comprises CoASHdependent activation of the carboxyl group, dehydrogenation followed by stereospecific hydration to give a L-hydroxy derivative, NAD dependent dehydrogenation, and thiolytic cleavage of the , -bond (Nelson and Cox, 2000). Since all of the statins have a D-hydroxy configuration, an epimerization to the L-configuration is needed for the -oxidation cycle to occur. Statins that form propanoic or propanoic acid metabolites (loss of the four-carbon unit) are believed to undergo two cycles of -oxidation (Boberg et al., 1998). The mechanisms for the formation of unsubstituted pentanoic acid products of statins, including LVA or SVA, have been proposed to occur following a cycle of fatty acid oxidation yielding a D-hydroxy pentanoic acid derivative, followed by fatty acid biosynthetic processes (Halpin et al., 1993; Boberg et al., 1998). The biosynthesis requires the 1 Abbreviations used are: SV, simvastatin; LV, lovastatin; SVA, hydroxy acid form of simvastatin; LVA, hydroxy acid form of lovastatin; HPLC, high-pressure liquid chromatography; ACN, acetonitrile; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry. Address correspondence to: Dr. Thomayant Prueksaritanont, Department of Drug Metabolism, WP 75-100, Merck Research Laboratories, West Point, PA 19486. E-mail: [email protected] 0090-9556/01/2910-1251–1255$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 10 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 384/929778 DMD 29:1251–1255, 2001 Printed in U.S.A. 1251 at A PE T Jornals on Sptem er 1, 2017 dm d.aspurnals.org D ow nladed from D-configuration of the hydroxyl moiety and involves dehydration of the remaining D-hydroxyl group, followed by hydrogenation to form the unsubstituted pentanoic acid metabolites (Halpin et al., 1993). Although theoretically conceivable, evidence supporting these proposals is lacking. To date, there have been no reports concerning CoASH and NAD involvement or demonstrating the presence of key anticipated intermediates leading to the chain shortened products of these statins. Therefore, the present studies were undertaken, using an in vitro approach and SVA as a model substrate, to illustrate CoASH and NAD involvement in the -oxidation process of statins, and to provide evidence for -oxidation intermediates for the unsubstituted pentanoic acid derivative of SVA in mice. Experimental Procedures Materials. SV, SVA, and [C]SVA with specific activity of 50 Ci/ mol (Fig. 1) were synthesized at Merck Research Laboratories (Rahway, NJ). Triton X-100, CoASH, ATP, NAD , and NADPH were obtained from Sigma (St. Louis, MO). All other reagents were of analytical or HPLC grade. Animals. Ten male CF-1 mice ( 25–40 g) were obtained from Charles River Laboratories (Wilmington, MA). Following cervical dislocation, livers were quickly removed, weighed, and washed with 1.15% potassium chloride. The livers were homogenized in 4 volumes of ice-cold 0.05 M Tris buffer, pH 7.4, and 1.15% potassium chloride. Liver S2 was prepared following centrifugation of the homogenate at 2000g for 15 min at 8°C to remove cell debris. Protein concentrations were measured by the method of Lowry et al. (1951). In Vitro Metabolism of SVA. A typical incubation mixture, in a final volume of 0.2 ml, contained 0.6 mg of liver S2 preincubated with 30 l of 2% Triton X-100 for 15 min, 20 mM MgCl2, 6 mM ATP, 0.5 mM CoASH, and 0.05 M Tris buffer, pH 8.0. The preincubation step with Triton X-100 was found to be necessary for high-enzyme activity. Unless otherwise specified, the reaction was started by the addition of [C]SVA (prepared by mixing [C]SVA with nonradiolabeled SVA to achieve an isotopic ratio of C/C close to 1:1) following a 3-min preincubation at 37°C, and the reaction was incubated for up to 60 min. Experiments also were conducted in the presence of the cofactors NAD (2 mM) and/or NADPH (1 mM), in addition to CoASH and ATP. Control experiments included incubation mixtures with one or more components missing. The reaction was terminated at appropriate time intervals by the addition of 0.2 ml of acetonitrile (ACN). The samples were centrifuged, and the supernatants were analyzed immediately by HPLC or LC/MS, as