TY - JOUR T1 - Structural Characterization of Anti-HIV Drug Candidate PA-457 [3-<em>O</em>-(3′,3′-Dimethylsuccinyl)-betulinic acid] and Its Acyl Glucuronides in Rat Bile and Evaluation of in Vitro Stability in Human and Animal Liver Microsomes and Plasma JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1436 LP - 1442 DO - 10.1124/dmd.106.009233 VL - 34 IS - 9 AU - Zhiming Wen AU - Stephan T. Stern AU - David E. Martin AU - Kuo-Hsiung Lee AU - Philip C. Smith Y1 - 2006/09/01 UR - http://dmd.aspetjournals.org/content/34/9/1436.abstract N2 - PA-457 [3-O-(3′,3′-dimethylsuccinyl)-betulinic acid] represents a new class of anti-HIV drug candidates termed maturation inhibitors. After oral administration to rats, PA-457 was metabolized to several glucuronide conjugates and mainly eliminated into rat bile. Liquid chromatography-electrospray ionization-mass spectrometry analysis showed that the glucuronidation products of PA-457 were acyl glucuronides including one di-glucuronide, di-PA-457G, and two mono-glucuronides, referred to as mono-PA-457G (I) and mono-PA-457G (II), respectively. In-source fragmentation of MS spectra supported the conclusion that mono-PA-457G (I) was glucuronidated at the C-28 carboxyl of PA-457, whereas mono-PA-457G (II) was conjugated at the dimethylsuccinic acid side chain of the C-3 position. Quantification demonstrated that the predominant glucuronide of PA-457 in rat bile was mono-PA-457G (I) with lower amounts of mono-PA-457G (II) and di-PA-457G. In vitro stability indicated that the mono-acyl glucuronides of PA-457 were not degraded after incubation with 0.1 M phosphate buffer (pH 4, 7.4 and 9), plasma (human, rat, and mouse), and UDP-glucuronosyltransferase reaction media (without uridine 5′-diphosphoglucuronic acid) with microsomes (human, rat, and mouse liver microsomes), respectively, whereas the minor diglucuronide was unstable in rodent liver microsomes. All glucuronides of PA-457 could be hydrolyzed both by β-glucuronidase and alkaline (1 M NaOH). Minor putative acyl migration products were slowly formed at pH 9, suggesting that the acyl glucuronides of PA-457 have relatively high in vitro stability. The American Society for Pharmacology and Experimental Therapeutics ER -