DMD Large equally mixed donor pool

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

Drug Metabolism and Disposition Fast Forward
First published on December 17, 2004; DOI: 10.1124/dmd.104.002915

0090-9556/05/3303-359-364$20.00
DMD 33:359-364, 2005

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
dmd.104.002915v1
33/3/359    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Di Marco, A.
Right arrow Articles by Laufer, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Di Marco, A.
Right arrow Articles by Laufer, R.

DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP2C9 INHIBITION ASSAY USING TRITIATED DICLOFENAC

Annalise Di Marco, Isabella Marcucci, Ashok Chaudhary, Marina Taliani, and Ralph Laufer

Department of Pharmacology, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Merck Research Laboratories, Rome, Italy (A.D.M., I.M., M.T., R.L.); and Labeled Compound Synthesis, Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey (A.C.)

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4'-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4'-hydroxylation of diclofenac labeled with tritium in the 4' position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4'-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC50 values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4'-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4'-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.

Address correspondence to: Dr. Ralph Laufer, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Merck Research Laboratories, Via Pontina km 30,600, 00040 Pomezia (Roma), Italy. E-mail: ralph_laufer{at}merck.com




This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
A. Di Marco, A. Cellucci, A. Chaudhary, M. Fonsi, and R. Laufer
High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin
Drug Metab. Dispos., October 1, 2007; 35(10): 1737 - 1743.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
A. D. Marco, I. Marcucci, M. Verdirame, J. Perez, M. Sanchez, F. Pelaez, A. Chaudhary, and R. Laufer
DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP3A4/5 INHIBITION ASSAY USING TRITIATED TESTOSTERONE
Drug Metab. Dispos., March 1, 2005; 33(3): 349 - 358.
[Abstract] [Full Text] [PDF]


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
All ASPET Journals Molecular Pharmacology Pharmacological Reviews
Molecular Interventions Drug Metabolism and Disposition

Copyright © 2005 by the American Society for Pharmacology and Experimental Therapeutics.