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Midazolam Hydroxylation Assay as a Marker for Endogenous CYP3A4‐mediated Metabolism: Metabolic Interaction with Natural Health Supplements
Author(s) -
Deb Subrata,
Clay Karen,
Zaleski Kimberly,
Alexander Lisa
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.833.8
Subject(s) - chemistry , midazolam , cyp3a4 , quercetin , gallic acid , pharmacology , biochemistry , hydroxylation , metabolism , chromatography , cytochrome p450 , enzyme , antioxidant , biology , sedation
Background Endogenous substances (endobiotics) such as vitamin D 3 and estrogen are metabolized by cytochrome P450 3A4 (CYP3A4). However, measurement of endobiotic metabolites is often technology‐limited and expensive. Because of structural similarity of midazolam with vitamin D 3 , midazolam metabolic profile could present valuable information in identifying potential interaction of drugs or natural health supplements (NHS) with endogenous substances. Polyphenols (quercetin, raspberry ketone, gallic acid, rutin), furanocoumarin (imperatorin) and terpenoids (ursolic acid) are the biologically active human dietary constituents from grapes, berry fruits, pomegranate, and olive oil, but are also widely ingested as over‐the‐counter natural health supplements (NHS) in United States. NHS have the ability to inhibit CYP3A4‐mediated metabolism, possibly causing the plasma concentrations of the midazolam as well as vitamin D 3 to increase. The objective of the present study was to develop a simple and effective metabolism assay to measure CYP3A4‐mediated midazolam hydroxylation and to screen certain NHS (polyphenols, furanocoumarin, terpenoids) for their potential to cause CYP3A4 related herb‐endobiotic interactions in humans. Methods The hydroxylation of midazolam to 1′‐hydroxymidazolam assay was developed and optimized. Reaction mixtures containing potassium phosphate buffer (pH 7.4), recombinant CYP3A4 protein (30 pmol/ml), NADPH‐regenerating system, and midazolam (5 uM or 2 uM) were incubated for 15 min. Substrate, metabolites and internal standard were analyzed with a reverse phase assay by a Shimadzu Prominence high‐performance liquid chromatography (HPLC) coupled to a photodiode array detector set at 220 nm using a C 18 column. SigmaPlot enzyme kinetics module (version 13; Systat Software, Inc., San Jose, CA) and GraphPad Prism software (GraphPad Software Inc., San Diego, CA) were used to determine the kinetics parameters and inhibitory constants, respectively. Results The Km and Vmax values for rCYP3A4‐mediated 1′‐hydroxymidazolam formation were 1.9±0.3 uM and 75.4±3.7 pmol/min/mg, respectively. Imperatorin inhibited 1′‐hydroxymidazolam formation to undetectable levels at both 2 uM and 5 uM concentrations of midazolam, whereas quercetin inhibited the formation by 31–44%. The IC 50 values of imperatorin for 1′‐hydroxymidazolam and 4‐hydroxymidazolam were 7.7 uM and 2.6 uM, respectively. Ketoconazole, a known potent inhibitor of CYP3A4, yielded IC 50 concentrations of 0.16–0.20 uM for midazolam hydroxylation. Conclusions A midazolam hydroxylation assay was developed to screen potential metabolic interactions. Imperatorin was identified as an inhibitor of rCYP3A4. Quercetin showed low rCYP3A4 inhibition potential. This research further shows that NHS studied here can potentially inhibit one of the most ubiquitous enzymes responsible for inactivation of endobiotics and may provide additional health benefits. Vitamin D 3 is metabolized by CYP3A4 and midazolam could potentially be used as a marker for interaction of commonly used NHS with vitamin D 3 . The cost‐effective HPLC method developed in this study provides a useful tool in predicting NHS‐endobiotic interactions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .