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Inhibition of Cytochrome P450 3A4 by a Pyrimidineimidazole:  Evidence for Complex Heme Interactions

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Pharmacokinetics, Dynamics and Metabolism (PDM), Department of Medicinal Chemistry, Pfizer Global Research and Development, 700 Chesterfield Parkway West T3A, Chesterfield, Missouri 63017, and Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455
Cite this: Chem. Res. Toxicol. 2006, 19, 12, 1650–1659
Publication Date (Web):November 24, 2006
https://doi.org/10.1021/tx060198m
Copyright © 2006 American Chemical Society

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    Abstract

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    PH-302 inhibits the inducible form of nitric oxide synthase (iNOS) by coordinating with the heme of the monomeric form and preventing formation of the active dimer. Inherent with the mechanism of pharmacology for this compound was the inhibition of cytochrome P450 3A4 (P450 3A4), observed from early ADME screening. Further investigation showed that PH-302 inhibited P450 3A4 competitively with a Ki of ∼2.0 μM against both midazolam and testosterone hydroxylation in human liver microsomes. As expected, spectral binding analysis demonstrated that inhibition was a result of type II coordination to the P450 heme with the imidazole moiety of PH-302, although only 72% of the maximal absorbance difference was achievable with PH-302 compared to that of the smaller ligand imidazole. Time-dependent inhibition of P450 3A4 by PH-302 was also observed because of metabolite-inhibitory (MI) complex formation via metabolism of the methylenedioxyphenyl group. The profile for time-dependent inhibition in recombinant P450 3A4 was biphasic, and was kinetically characterized by a kinact of 0.08 min-1 and a Ki of 1.2 μM for the first phase (0−1.5 min) and a kinact of 0.06 min-1 and a Ki of 23.8 μM for the second phase (1.5−10 min). Spectral characterization of the PH-302 MI complex demonstrated that formation began to plateau within 3 min, consistent with the kinetic profile of inactivation by PH-302. Meanwhile, spectral evidence for the imidazole-derived type II difference spectrum of PH-302 was captured simultaneously with the formation of the MI complex. The presence of simultaneously operable type II coordination and rapidly saturable MI complex formation with heme by PH-302 indicates the presence of complex heme interactions with this unique molecule. Information from these mechanistic studies adds to our understanding of the nature of P450 3A4 inhibition by PH-302 and provides a potentially useful tool compound for future studies investigating binding interactions in this important drug-metabolizing enzyme.

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    *

     Corresponding author. Tel:  314-274-0261. Fax:  314-274-4426. E-mail:  [email protected].

     Pfizer Global Research and Development.

     University of Minnesota.

    §

     Current address:  Amgen, Inc., Pharmacokinetics & Drug Metabolism, AW2-D / 2381, 1201 Amgen Court West, Seattle, WA 98119-3105.

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    Initial P450 inhibition screening data showing potent inhibition of P450 3A4, IC50 data showing a shift in IC50 from 5.2 to 4.4 μM for inhibition of midazolam 1‘-hydroxylation following a 10 min preincubation of PH-302 in human liver microsomes, data demonstrating lack of significant depletion of PH-302 when incubated with P450 3A4 Supersomes under the same incubation conditions as those of the inactivation experiments, and verification of type II spectra with PH-302 with reduced Fe-II. This material is available free of charge via the Internet at http://pubs.acs.org.

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