title: Glucuronidation of anticancer prodrug PR-104A: species differences, identification of human UDP-glucuronosyltransferases, and implications for therapy.
abstract: PR-104, the phosphate ester of a dinitrobenzamide mustard [PR-104A; 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino] carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate], is currently in clinical trial as a hypoxia- and aldo-keto reductase 1C3 (AKR1C3)-activated prodrug for cancer therapy. Here, we investigate species (human, dog, rat, mouse) differences in metabolism to the corresponding O-glucuronide, PR-104G, and identify the human UDP-glucuronosyltransferase (UGT) isoforms responsible. After intravenous PR-104, plasma area under the concentration-time curve ratios (PR-104G/PR-104A) decreased in the order of dog (2.3) > human (1.3) > mouse (0.03) > rat (0.005). The kinetics of uridine 5'-diphosphoglucuronic acid-dependent glucuronidation by liver microsomes in vitro fitted the single-enzyme Michaelis-Menten equation with similar K(m) (∼150 μM) but differing V(max) (472, 88, 37, and 14 nmol/h/mg for dog, human, rat, and mouse, respectively), suggesting that facile glucuronidation is responsible for the anomalously rapid clearance of PR-104A in dogs. In vitro-in vivo extrapolation of PR-104A glucuronidation kinetics is consistent with this also being a major clearance pathway in humans. Recombinant UGT screening identified UGT2B7 as the only commercially available human isoform able to conjugate PR-104A, and UGT2B7 protein concentrations were highly correlated (r = 0.93) with PR-104A glucuronidation by liver microsomes from 24 individuals. The active hydroxylamine metabolite of PR-104A, PR-104H, was also glucuronidated by UGT2B7, although with slightly lower specificity and much lower rates. UGT2B7 mRNA expression was highly variable in human tumor databases. Glucuronidation of PR-104A greatly suppressed nitroreduction by AKR1C3 and NADPH-supplemented anoxic human liver S9 (9000g postmitochondrial supernatant). In conclusion, PR-104A is glucuronidated by UGT2B7 with high specificity and seems to make a major contribution to clearance of PR-104A in humans, but it also has the potential to confer resistance in some human tumors.
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Gu
authors.forename: Malcolm D
authors.initials: MD
authors.lastname: Tingle
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D019540:Area Under Curve; D004285:Dogs; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008875:Middle Aged; D009588:Nitrogen Mustard Compounds / Q000097:blood / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology*; D011355:Prodrugs / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity
chemical_list: D018906:Antineoplastic Agents, Alkylating; D020719:Glucuronides; D009588:Nitrogen Mustard Compounds; C522381:PR-104; C524170:PR-104A; D011355:Prodrugs; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase