ABSTRACT: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 ␦-lactone.

Dean L. Olson (first row, right) completed his Ph.D. degree in Analytical Chemistry from the University of Illinois in 1994. He then joined the academic research group of Professor Sweedler performing the first high-resolution and sensitivity studies with nanoliter-volume NMR microcoils. He also conducted the first experiments using high-resolution NMR as a detector for capillary electrophoresis. He is now employed at MRM Corp. (Savoy, IL) conducting research toward the commercial development and application of capillarybased NMR probes. Andrew G. Webb (second row, right) received his Ph.D. degree in Medicinal Chemistry from the University of Cambridge in 1990 under Professor Laurie Hall. He was a postdoctoral researcher in the Department of Radiology at the University of Florida before joining the faculty in the Department of Electrical and Computer Engineering at the University of Illinois at Urbana−Champaign.He is currently an Associate Professor, with a full appointment in The Beckman Institute for Advanced Science and Technology. His research areas include the design of microcoils for NMR of mass-limited samples, the use of MRI temperature mapping for hyperthermia, and human brain mapping using functional MRI. Jonathan V. Sweedler (second row, left) received his Ph.D. degree in Analytical Chemistry from the University of Arizona in 1989 under Professor M. Bonner Denton and then spent 3 years at Stanford with Professors Richard Zare and Richard Scheller developing new methods to study neurotransmitters in individual neurons. He is currently a Professor of Chemistry, Neuroscience, and of the Beckman Institute for Advanced Science and Technology at the University of Illinois. His current research interests are twofold: first, he is developing information-rich methods with improved mass sensitivity for nanoliter-volume samples, including microcoil NMR, mass spectrometric imaging, and capillary-scale separations. In addition, he applies these techniques to understanding the role of neurotransmitter and neuropeptide co-transmission and the regulation of behavior in well-defined neuronal networks of opisthobranch molluscs.

ABSTRACT:This study investigated the metabolic interaction between fibrates and statin hydroxy acids in human hepatocytes. Gemfibrozil (GFZ) modestly affected the formation of ␤-oxidative products and CYP3A4-mediated oxidative metabolites of simvastatin hydroxy acid (SVA) but markedly inhibited the glucuronidation-mediated lactonization of SVA and the glucuronidation of a ␤-oxidation product (IC 50 ϳ50 and 15 M, respectively). In contrast, fenofibrate had a minimal effect on all the metabolic pathways of SVA. GFZ also significantly inhibited (IC 50 ϳ50-60 M) the oxidation of cerivastatin (CVA) and rosuvastatin (RVA), but not of atorvastatin (AVA), while effectively decreasing (IC 50 ϳ30 to 60 M) the lactonization of all three statins. As was observed previously with other statin hydroxy acids, RVA underwent significant glucuronidation to form an acyl glucuronide conjugate and lactonization to form RVA lactone in human liver microsomes and by UGT 1A1 and 1A3. While GFZ is not an inhibitor of CYP3A4, it is a competitive inhibitor (K i ‫؍‬ 87 M) of CYP2C8, a major catalyzing enzyme for CVA oxidation. These results suggest that 1) the pharmacokinetic interaction observed between GFZ and statins was not likely mediated by the inhibitory effect of GFZ on the ␤-oxidation, but rather by its effect primarily on the glucuronidation and non-CYP3A-mediated oxidation of statin hydroxy acids, and 2) there is a potential difference between fibrates in their ability to affect the pharmacokinetics of statins, and among statins in their susceptibility to metabolic interactions with GFZ in humans.Fibrates, lipid-regulating agents, and hydroxymethylglutaryl-coenzyme A reductase inhibitors or so called "statins", cholesterol lowering agents, are frequently prescribed together to treat patients with mixed hyperlipidemia (Shek and Ferrill, 2001). There have been reports of increased risk of myopathy, including rhabdomyolysis with this coadministration (Murdock et al., 1999). Despite being generally accepted as a class effect for all fibrate-statin combinations, this increased risk has been observed at varied incidences with different fibrates and statins. More documented cases for myopathy have been reported with gemfibrozil (GFZ 1 )-statin combined therapy than with other fibrate-statin combinations (Shek and Ferrill, 2001). Recently, cerivastatin (CVA) was withdrawn from the market due to disproportionate numbers of fatal rhabdomyolysis cases (compared with other marketed statins), many of which occurred in patients receiving concomitant GFZ (Farmer, 2001).Although it has generally been accepted that the increased risk of myopathy is due primarily to a pharmacodynamic drug-drug interaction, recent studies have suggested that the increased risk might also have a pharmacokinetic origin. In recent clinical studies, increases in the exposure mainly to statin hydroxy acids, but minimally to the lactone form of statins, were observed following coadministration of GFZ and statins (Backman et al., 2000;Kyrklund et al., 2001). Subsequently, ...

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS(2) (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MS(All) (also called MS(E)) techniques. We compared these three techniques in their capabilities to produce comprehensive MS(2) data by assessing both metabolite MS(2) acquisition hit rate and the quality of MS(2) spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MS(All)) using an ultrahigh-performance liquid chromatography-qudrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS(2) acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS(2) acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS(2) acquisition with either the SWATH or MS(All) method. However, the quality of the acquired MS(2) spectra decreased in the order of IDA, SWATH, and MS(All) methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS(2) spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MS(All) methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS(2) spectra but with a lower MS(2) acquisition hit rate than the other two methods. SWATH outperformed the MS(All) method given its better quality of MS(2) spectra with an identical MS(2) acquisition hit rate.

Metabolic activation of a drug leading to reactive metabolite(s) that can covalently modify proteins is considered an initial step that may lead to drug-induced organ toxicities. Characterization of reactive metabolites is critical to designing new drug candidates with an improved toxicological profile. High performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) predominates over all analytical tools used for screening and characterization of reactive metabolites. In this review, a brief description of experimental approaches employed for assessing reactive metabolites is followed by a discussion on the reactivity of acyl glucuronides and acyl coenzyme A thioesters. Techniques for high-throughput screening and quantitation of reactive metabolite formation are also described, along with proteomic approaches used to identify protein targets and modification sites by reactive metabolites. Strategies for dealing with reactive metabolites are reviewed. In conclusion, we discuss the challenges and future needs in this field of research.