Interindividual Variability in CYP3A Activity: Influence on CYP3A‐related Herb‐Drug Interactions from Selected Natural Health Supplements in Human Liver
Authors: Deb Subrata , Bukowska Annetta , Heo Nakyo , Rajab Ibraheem 
Published: November 2023

Background

CYP3A proteins metabolize >50% of the currently available drugs including antihypertensive, antihyperlipidemic and anticancer agents. Interindividual variability in CYP3A activity may arise from diverse factors including genetic polymorphism (e.g. CYP3A5), xenobiotic‐mediated protein expression or pathophysiological conditions. Considering the ability of diverse chemical structures (e.g. drug, diet) to bind with the CYP3A4‐regualting nuclear receptors, it is plausible that CYP3A4 expression will widely vary in population. Polyphenols (quercetin, raspberry ketone, gallic acid, rutin), furanocoumarin (imperatorin) and terpenoids (ursolic acid) are active human dietary constituents and are also widely ingested as FDA‐unregulated over‐the‐counter natural health supplements (NHS) in United States. Due to the structural features NHS could inhibit CYP activity. Since the impact of drug interaction is depended on the level of intrinsic activity, differences in hepatic CYP3A activity at the time of herb‐drug interaction could determine the overall toxic outcome. The objectives of the present study were to examine interindividual variations in CYP3A‐mediated metabolism of midazolam (FDA‐recommended marker substrate) in human liver and to identify the impact of differences in CYP3A activities on the inhibition of midazolam hydroxylation by NHS.

Methods

Four individual human liver microsomes (HLM; Xenotech LLC, KS) were used to determine hydroxylation of midazolam to 1′‐hydroxymidazolam (1′‐OH MDZ) and 4‐hydroxymidazolam (4‐OH MDZ). The in vitro reaction mixtures (with or without NHS) containing potassium phosphate buffer (pH 7.4), HLM (0.1 mg/ml or 0.6 mg/ml), NADPH‐regenerating system, and midazolam (0.5–100 μM or 5 μM) was incubated for 30 min. Metabolites were analyzed with a reverse phase assay by a Shimadzu Prominence high‐performance liquid chromatography coupled to a photodiode array detector (220 nm) using a C18 column. The SigmaPlot Enzyme Kinetics Module and GraphPad Prism 5 were utilized to estimate the enzyme kinetics and IC50 values for NHS.

Results

Comparison of enzyme kinetic parameters for the formation of 1′‐OH MDZ and 4‐OH MDZ indicates that there is up to 20‐fold difference in the hepatic CYP3A activities in this study. High and low activity HLM phenotypes had a Vmax of 260.1 pmol/min/mg and 20.1 pmol/min/mg, respectively, for 1′‐OH MDZ formation, whereas for 4‐OH MDZ formation the Vmax were determined as 118.8 pmol/min/mg (high) and 8.4 pmol/min/mg (low). The Km values were comparable for different HLM samples for the same metabolite. Significant CYP3A inhibitory effect was observed from imperatorin, quercetin and gallic acid. Interestingly, for the high HLM phenotype there was up to 200 unit drop in CYP3A activity, compared to 20 unit drop for the low HLM phenotype following incubation of midazolam with NHS. Both high and low activity HLMs were completely inhibited by imperatorin in a concentration‐dependent manner with IC50 values of 4.1 μM and 5.3 μM for 1′‐OH MDZ formation, respectively. Similarly, ketoconazole (positive control) showed IC50 values of 0.03 μM and 0.19 μM for 1′‐OH MDZ formation in high and low HLM phenotypes, respectively.

Conclusions

Significant differences exist in CYP3A activities between individual human liver samples. CYP3A activity is differentially inhibited by imperatorin, quercetin and gallic acid in high and low activity HLM phenotypes. High activity HLM phenotype is inhibited more than the low activity HLM phenotype by select NHS. Thus, the toxic impact of herb‐drug interactions may depend on the hepatic CYP3A activity status of the individual.

Support or Funding Information

Roosevelt University Summer Research Grant (2016)