Effects of herbal components on cDNA-expressed cytochrome P450 enzyme catalytic activity

Abstract

We evaluated the effects of 25 purified components of commonly used herbal products on the catalytic activity of cDNA-expressed cytochrome P450 isoforms in in vitro experiments. Increasing concentrations of the compounds were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit metabolism of surrogate substrates by 50%) was estimated and compared with IC50's for the positive control inhibitory drugs furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole. Constituents of Ginkgo biloba (ginkgolic acids I and II), kava (desmethoxyyangonin, dihydromethysticin, and methysticin), garlic (allicin), evening primrose oil (cis-linoleic acid), and St. John's wort (hyperforin and quercetin) significantly inhibited one or more of the cDNA human P450 isoforms at concentrations of less than 10 uM. Some of the test compounds (components of Ginkgo biloba, kava, and St. John's wort) were more potent inhibitors of the isoforms 1A2, 2C19, and 2C19 than the positive controls used in each assay (furafylline, sulfaphenazole, and tranylcypromine, respectively), which are known to produce clinically significant drug interactions. The enzyme most sensitive to the inhibitory of effects of these compounds was CYP2C19, while the isoform least effected was CYP2D6. These data suggest that herbal products containing evening primrose oil, Ginkgo biloba, kava, and St. John's Wort could potentially inhibit the metabolism of co-administered medications whose primary route of elimination is via cytochrome P450.

Introduction

Because little is known about the metabolic interactions between herbal products and pharmaceuticals, we conducted a series of in vitro experiments to evaluate the effects of components of commonly used herbal products on the catalytic activity of cDNA-expressed cytochrome P450 isoforms in in vitro experiments. Increasing concentrations of the compounds were incubated with a panel of recombinant human CYP isoforms and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. We chose this methodology because inhibition of cytochrome P450 mediated metabolism is often the mechanism of drug-drug interactions and the use of recombinant human cytochrome P450 enzymes with specific surrogate substrates is recognized as a cost-effective technique for predicting such interactions [1]. Our laboratory and others have extended the use of these assays to evaluate metabolic interactions between herbal products and standard medications [2], [3]. We chose to test purified components of commonly used herbs rather than commercial herbal products or whole plant material because the IC50's of purified components can be expressed in uM concentration and, therefore, can be compared with known CYP inhibitors; because the purity and potency of herbal products have been shown to vary considerably [4]; and because some of these extracts exhibit native fluorescence or quenching which can interfere with these tests [5]. Many of the compounds tested are, however, “marker” compounds for herbal products, i.e., constituents of the plant thought to be associated with its biological activity or used to standardize herbal products for the purpose of quality control.

Twenty-five compounds, components of some of the most commonly used herbal products [6], were tested for their ability to inhibit the catalytic activity of cDNA-derived human P450 enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. These isoforms are involved in the majority of clinically important drug metabolizing reactions [7]. Results of these studies are reported herein.