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Characterization of CYP3A5 Selective Inhibitors for Reaction Phenotyping of Drug Candidates

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Abstract

CYP3A is one of the most important classes of enzymes and is involved in the metabolism of over 70% drugs. While several selective CYP3A4 inhibitors have been identified, the search for a selective CYP3A5 inhibitor has turned out to be rather challenging. Recently, several selective CYP3A5 inhibitors have been identified through high-throughput screening of ~ 11,000 compounds and hit expansion using human recombinant enzymes. We set forth to characterize the three most selective CYP3A5 inhibitors in a more physiologically relevant system of human liver microsomes to understand if these inhibitors can be used for reaction phenotyping studies in drug discovery settings. Gomisin A and T-5 were used as selective substrate reactions for CYP3A4 and CYP3A5 to determine IC50 values of the two enzymes. The results showed that clobetasol propionate and loteprednol etabonate were potent and selective CYP3A5 reversible inhibitors with selectivity of 24-fold against CYP3A4 and 39-fold or more against the other major CYPs. The selectivity of difluprednate in HLM is much weaker than that in the recombinant enzymes due to hydrolysis of the acetate group in HLM. Based on the selectivity data, loteprednol etabonate can be utilized as an orthogonal approach, when experimental fraction metabolized of CYP3A5 is greater than 0.5, to understand CYP3A5 contribution to drug metabolism and its clinical significance. Future endeavors to identify even more selective CYP3A5 inhibitors are warranted to enable accurate determination of CYP3A5 contribution to metabolism versus CYP3A4.

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Abbreviations

ACN:

Acetonitrile

CLint :

Intrinsic clearance

CYP:

Cytochrome P450

DDI:

Drug-drug interactions

FDA:

U.S. Food and Drug Administration

f m :

Fraction metabolized

HLM:

Human liver microsomes

IC50 :

Half-maximal inhibitory concentration

IS:

Internal standards

K m :

The concentration of substrate which permits the enzyme to achieve half Vmax

k obs :

Inactivation rate constant

LC–MS/MS:

Liquid chromatography with tandem mass spectrometry

MgCl2 :

Magnesium chloride

MRM:

Multiple-reaction monitoring

NADPH:

Nicotinamide adenine dinucleotide phosphate, reduced form

QSAR:

Quantitative structure–activity relationship

rCYP:

Recombinant cytochrome P450

RT:

Room temperature

TDI:

Time-dependent inhibition

V max :

The maximum rate of reaction

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Authors and Affiliations

  1. Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, MA, USA

    Jie Chen

  2. Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Groton, CT, USA

    Lloyd Wei Tat Tang, Samantha Jordan, Makayla Harrison, Gabrielle M. Gualtieri, Ethan DaSilva, Danial Morris & Li Di

  3. Discovery Sciences, Pfizer Worldwide Research and Development, Groton, CT, USA

    Gary Bora & Ye Che

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Contributions

Substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of data for the work: JC, LT, SJ, MH, GG, ED, DM, GB, YC, and LD.

Drafting the work or revising it critically for important intellectual content: JC, LT, SJ, ED, and LD.

Final approval of the version to be published: JC, LT, SJ, MH, GG, ED, DM, GB, YC, and LD.

Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: JC, LT, SJ, MH, GG, ED, DM, GB, YC, and LD.

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Correspondence to Li Di.

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The authors are employees of Pfizer Inc., New York, NY, USA, and may hold Pfizer stocks.

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Chen, J., Tang, L.W.T., Jordan, S. et al. Characterization of CYP3A5 Selective Inhibitors for Reaction Phenotyping of Drug Candidates. AAPS J 26, 26 (2024). https://doi.org/10.1208/s12248-024-00894-x

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