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 Km (∼150 μM) but differing Vmax (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.
Footnotes
This work was supported by the Health Research Council of New Zealand [Grant 08/103] and a Technology in Industry Fellowship from the Foundation for Research, Science, and Technology of New Zealand (to Y.G.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.180703.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- rUGT
- recombinant UGT
- rhUGT
- recombinant human UGT
- PR-104A
- 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate
- AKR1C3
- aldo-keto reductase 1C3
- AUC
- area under the concentration-time curve
- CL
- clearance
- d-SL
- d-saccharic acid 1,4-lactone
- HLM
- human liver microsome
- MS
- mass spectrometry
- MS/MS
- tandem MS
- S9
- 9000g postmitochondrial supernatant
- UDPGA
- uridine 5′-diphosphoglucuronic acid
- LC
- liquid chromatography
- UHPLC
- ultra high-performance LC
- SN25378
- 2-chloro-N-(2-chloropropyl)-N-methyl-N-(2-nitrobenzyl)propan-1-ammonium chloride
- MeCN
- acetonitrile
- Nrf2
- nuclear factor erythroid-2 related factor-2
- SN-38
- 7-ethyl-10-hydroxy-camptothecin.
- Received February 15, 2011.
- Accepted March 21, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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