title: The role of pH in the glucuronidation of raloxifene, mycophenolic acid and ezetimibe.
abstract: The UDP-glucuronosyltransferase (UGT) active site faces the lumen of the endoplasmic reticulum and is enclosed behind a lipid bilayer. Consequently, observed UGT activity is latent in microsomal preparations, and thus, mechanical and/or chemical disruptions of the vesicle membrane are commonly employed to better expose the active site. The aim of the present investigation was to explore the impact of incubation pH on the glucuronidation of raloxifene, mycophenolic acid (MPA) and ezetimibe, which are basic, acidic and neutral compounds, respectively. Their glucuronidation was examined in human liver microsomal incubations by monitoring for the production of the glucuronide metabolites at pHs ranging between 5.4 and 9.4. Compared to physiological pH, unbound intrinsic clearance (CL(int,u)) was 11- and 12-fold higher at pH 9.4 for raloxifene 4'-glucuronide (R4G) and raloxifene 6-glucuronide (R6G), respectively; whereas a 10-fold increase was observed at pH 5.4 for MPA glucuronide (MPAG). In contrast, ezetimibe glucuronidation did not vary as the pH deviated from 7.4. Kinetic analysis revealed that increases in CL(int,u) were accompanied by less than a 2-fold change in V(max). Instead, K(m,u) decreased 8-, 13- and 5-fold for R4G, R6G and MPAG, respectively. Similar pH dependency on glucuronidation was observed in experiments utilizing recombinant UGT enzymes (recUGT). Particularly, recUGT1A9 was one of the major isoforms involved in the glucuronidation of raloxifene and MPA. While the highest rate of glucuronidation was found at pH 9.4 for raloxifene, the pH for optimal glucuronidation of MPA was between 5.4 and 7.4. In summary, these results suggest that microsomal glucuronidation may be enhanced for acidic and basic compounds by altering the incubation pH, perhaps by improving substrate membrane permeability.
authors.affiliation: Non-Clinical Safety, Department of Drug Metabolism and Pharmacokinetics, Roche Palo Alto, Palo Alto, California 94304, USA. jaechang@gmail.com
authors.forename: Jae H
authors.initials: JH
authors.lastname: Chang
authors.forename: Phillip
authors.initials: P
authors.lastname: Yoo
authors.forename: Theresa
authors.initials: T
authors.lastname: Lee
authors.forename: Wendy
authors.initials: W
authors.lastname: Klopf
authors.forename: Denise
authors.initials: D
authors.lastname: Takao
mesh_terms: D001384:Azetidines / Q000378:metabolism*; D002853:Chromatography, Liquid; D000069438:Ezetimibe; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000378:metabolism*; D009173:Mycophenolic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D020849:Raloxifene Hydrochloride / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001384:Azetidines; D020719:Glucuronides; D011994:Recombinant Proteins; D020849:Raloxifene Hydrochloride; C113145:mycophenolic acid glucuronide; D014453:Glucuronosyltransferase; D000069438:Ezetimibe; D009173:Mycophenolic Acid