Human and Rat Liver UDP-Glucuronosyltransferases Are Targets of Ketoprofen Acylglucuronide

Abstract

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.

In Vitro–In Vivo Correlation for Low-Clearance Compounds Using Hepatocyte Relay Method

“…1). The exceptions had a high hepatic uptake component by transporters [e.g., fexofenadine (Poirier et al, 2009), fluvastatin (Watanabe et al, 2010a), ketoprofen (Morita et al, 2005), prazosin (Qin et al, 1994), and bosentan (Huang et al, 2012)] or potential contribution from extrahepatic metabolism [e.g., hydrolysis of acebutolol (Andresen and Davis, (Terrier et al, 1999)]. …”

“…In rats, significant contribution of transporter-mediated clearance was observed for ketoprofen (Poirier et al, 2009), leading to underprediction of in vivo clearance based on metabolic clearance from hepatocytes since transporter uptake also contributes to the systemic clearance (Yamazaki et al, 1996;Shitara et al, 2006;Watanabe et al, 2010b). In addition, potential extrahepatic contribution of UGT metabolism in rats can lead to underprediction of in vivo clearance 2022 Di et al at ASPET Journals on May 10, 2018 dmd.aspetjournals.org (Terrier et al, 1999). In dogs, the major elimination pathway for ketoprofen was through UGT metabolism, and the kidneys made a significant contribution to the metabolism (Granero and Amidon, 2008).…”

Gilbert's disease and atazanavir: From phenotype to UDP-glucuronosyltransferase haplotype

“…[39][40][41][42] Some of these are UGT inhibitory such as amitriptyline and acyl glucuronides of ketoprofen that form adducts with the UGT protein. 27,28 UGT1A7 expressed primarily in stomach, esophagus, 43 and pancreas 44 is characterized by the glucuronidation of mutagenic polyaromatic hydrocarbons, and the irinotecan metabolite SN-38. 13,43 It is additionally associated with a predisposition to cancer.…”

“…Commonly administered drugs with inhibitory potential include amitriptyline, 24 protease inhibitors, 25 ketoconazole, 26 and acyl glucuronide adducts of ketoprofen. 27,28 Therefore, the combination of genetic variants in addition to environmental or therapeutic xenobiotic exposure defines glucuronidation activity and thus the potential risk profile of an individual.…”

Interaction of the Electrophilic Ketoprofenyl-Glucuronide and Ketoprofenyl-Coenzyme A Conjugates with Cytosolic GlutathioneS-Transferases

“…KPF, among other drugs, has been used as a model compound to study the possible reactivity of acyl metabolites with cellular proteins. Thus, it was previously observed that both of these KPF acyl derivatives are chemically reactive species that can trigger the formation of adducts with various proteins such as albumin (Presle et al, 1996), UDP-glucuronosyltransferases (Terrier et al, 1999), the cyclooxygenase COX-2 (Levoin et al, 2004), and glucose-6-phosphate dehydrogenase (Asensio et al, 2007).…”