Use of the entero-test, a novel approach for the noninvasive capture of biliary metabolites in dogs.
The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding delta-lactone. By means of an LC-NMR technique, the glucuronide structure was established to be a 1-O-acyl-beta-D-glucuronide conjugate of the statin acid. The formation of statin glucuronide and statin lactone in human liver microsomes exhibited modest intersubject variability (3- to 6-fold; n = 10). Studies with expressed UDP glucuronosyltransferases (UGTs) revealed that both UGT1A1 and UGT1A3 were capable of forming the glucuronide conjugates and the corresponding lactones for all three statins. Kinetic studies of statin glucuronidation and lactonization in liver microsomes revealed marked species differences in intrinsic clearance (CL(int)) values for SVA (but not for AVA or CVA), with the highest CL(int) observed in dogs, followed by rats and humans. Of the statins studied, SVA underwent glucuronidation and lactonization in human liver microsomes, with the lowest CL(int) (0.4 microl/min/mg of protein for SVA versus approximately 3 microl/min/mg of protein for AVA and CVA). Consistent with the present in vitro findings, substantial levels of the glucuronide conjugate (approximately 20% of dose) and the lactone form of SVA [simvastatin (SV); approximately 10% of dose] were detected in bile following i.v. administration of [(14)C]SVA to dogs. The acyl glucuronide conjugate of SVA, upon isolation from an in vitro incubation, underwent spontaneous cyclization to SV. Since the rate of this lactonization was high under conditions of physiological pH, the present results suggest that the statin lactones detected previously in bile and/or plasma following administration of SVA to animals or of AVA or CVA to animals and humans, might originate, at least in part, from the corresponding acyl glucuronide conjugates. Thus, acyl glucuronide formation, which seems to be a common metabolic pathway for the hydroxy acid forms of statins, may play an important, albeit previously unrecognized, role in the conversion of active HMG-CoA reductase inhibitors to their latent delta-lactone forms.