Inhibition Of Human P450 Enzymes Research Articles

Inactivation of CYP3A4 by Benzbromarone in Human Liver Microsomes.

Benzbromarone is a uricosuric drug in current clinical use that can cause serious hepatotoxicity. Chemically reactive and/or cytotoxic metabolites of benzbromarone have been identified; however there is a lack of available information on their role in benzbromarone hepatotoxicity. The reactive metabolites of some hepatotoxic drugs are known to covalently bind, or alternatively are targeted, to specific cytochrome P450 (P450) enzymes, a process that is often described as mechanism-based inhibition. We examined whether benzbromarone causes a mechanism-based inhibition of human P450 enzymes. Microsomes from human livers were preincubated with benzbromarone and NADPH, followed by evaluation of CYP2C9 and CYP3A4 activities. Benzbromarone metabolism resulted in inhibition of CYP3A4 but not CYP2C9 in a time-dependent manner. Confirmation of pseudo-first order kinetics of inhibition, a requirement for NADPH, and a lack of protection by scavengers suggested that benzbromarone is a mechanism-based CYP3A4 inhibitor. Modification of the P450 enzyme by the reactive metabolite is a common trait of drugs that induce idiosyncratic hepatotoxicity, and might provide a speculative, mechanistic model for the rare occurrences of this type of drug toxicity.

Inhibition of human P450 enzymes by natural extracts used in traditional medicine

Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts, two natural polyphenol-rich extracts, diminished CYP1A1/2 and 3A4 activities, but not the CYP2D6 activity. These results suggest that these herbs inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions.

Inhibition of human P450 enzymes by compounds from Curcuma aromatica Salisb. rhizome extract

A growing number of naturally occurring phytochemicals in foods and herbal supplements have been shown to be substrates or inactivators of P450 enzymes [1]. Curcuma aromatica Salisb. is one of many distributed healthy supplements in Japan recently. We isolated and identified 10 sesquiterpenes and 2 diarylheptanoids from Curcuma aromatica. Above 12 compounds were evaluated on inhibition of human CYP3A4, CYP2D6, CYP2E1, and CYP1A2 using fluorescence assay in vitro. (4S, 5S) – (+) – germacrone 4, 5– expoxide (7), curcumin (11) and demethoxy-curcumin (12) showed inhibitory effects against CYP 3A4 and 2C9. These compounds in particular had inhibitory activity for three substrates (BFC, BQ, and DBF) in the case of CYP3A4. Although coumarins and flavonoids are known to inhibit the human P450s so far, there are few reports that sesquiterpene inhibit CYP3A4 and CYP2C9.

IN VITRO METABOLISM OF THE CALMODULIN ANTAGONIST DY-9760e (3-[2-[4-(3-CHLORO-2-METHYLPHENYL)-1-PIPERAZINYL]ETHYL]-5,6-DIMETHOXY-1-(4-IMIDAZOLYLMETHYL)-1H-INDAZOLE DIHYDROCHLORIDE 3.5 HYDRATE) BY HUMAN LIVER MICROSOMES: INVOLVEMENT OF CYTOCHROMES P450 IN ATYPICAL KINETICS AND POTENTIAL DRUG INTERACTIONS

Human cytochrome P450 (P450) isozyme(s) responsible for metabolism of the calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) and kinetic profiles for formation of its six primary metabolites [M3, M5, M6, M7, M8, and DY-9836 (3-[2-[4-(3-chloro-2-methylphenyl)piperazinyl]ethyl]-5,6-dimethoxyindazole)] were identified using human liver microsomes and recombinant P450 enzymes. In vitro experiments, including an immunoinhibition study, correlation analysis, and reactions with recombinant P450 enzymes, revealed that CYP3A4 is the primary P450 isozyme responsible for the formation of the DY-9760e metabolites, except for M5, which is metabolized by CYP2C9. Additionally, at clinically relevant concentrations, CYP2C8 and 2C19 make some contribution to the formation of M3 and M5, respectively. The formation rates of DY-9760e metabolites except for M8 by human liver microsomes are not consistent with a Michaelis-Menten kinetics model, but are better described by a substrate inhibition model. In contrast, the enzyme kinetics for all metabolites formed by recombinant CYP3A4 can be described by an autoactivation model or a mixed model of autoactivation and biphasic kinetics. Inhibition of human P450 enzymes by DY-9760e in human liver microsomes was also investigated. DY-9760e is a very potent competitive inhibitor of CYP2C8, 2C9 and 2D6 (Ki 0.25-1.7 microM), a mixed competitive and noncompetitive inhibitor of CYP2C19 (Ki 2.4 microM) and a moderate inhibitor of CYP1A2 and 3A4 (Ki 11.4-20.1 microM), suggesting a high possibility for human drug-drug interaction.

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 ( K i=14±4 μg/mL), and to a lesser extent, CYP1A2 ( K i=106±24 μg/mL), CYP2E1 ( K i=127±42 μg/mL), and CYP3A4 ( K i=155±43 μg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 ( K i=15 ± 6 μg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 ( K i’s between 4.9 and 55 μg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC 50<40 μg/mL).

Inhibition of human P450 enzymes by nicotinic acid and nicotinamide

Nicotinic acid has been used as a cholesterol-lowering agent for a few decades already, whereas the cytoprotective and antiviral properties of nicotinamide are slowly gaining attention. In both cases however, very high doses are needed to achieve a therapeutic effect, resulting in blood concentrations sometimes as high as 15 mM. Based on their common pyridine functionality, we hypothesized that these two molecules could inhibit human P450 enzymes. In vitro inhibition studies demonstrate that, at their therapeutic concentrations, both nicotinic acid and nicotinamide inhibit CYP2D6 ( K i =3.8±0.3 and 19 ± 4 mM, respectively). Nicotinamide also inhibits CYP3A4 ( K i =13±3 mM) and CYP2E1 ( K i =13±8 mM). As expected for nitrogen-containing heteroaromatic molecules, spectrophotometric analysis indicates that the inhibition occurs via coordination of the pyridine nitrogen atom to the heme iron.

In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus

1. Several unique flavonoid compounds have recently been isolated from hops, Humulus lupulus, and their presence has been detected in beer. Their chemical structures are similar to other plant-derived compounds, many present in the human diet, that have been shown to have cancer chemopreventive properties due, in part, to inhibition of cytochrome P450 enzymes that activate carcinogens. Additionally, preliminary studies have shown these flavonoids (at 100 muM) to be inhibitory of P450-mediated activation reactions in a variety of in vitro systems. Thus, the in vitro effects of these phytochemicals on cDNA-expressed human CYP1A1, CYP1B1, CYP1A2, CYP3A4 and CYP2E1 were currently examined by the use of diagnostic substrates and the carcinogen AFB1. 2. At 10 muM, the prenylated chalcone, xanthohumol (XN), almost completely inhibited the 7-ethoxyresorufin O-deethylase (EROD) activity of CYP1A1. At the same concentration, other hop flavonoids decreased the EROD activity by 90.8-27.0%. 3. At 10 muM, XN completely eliminated CYP1B1 EROD activity, whereas the other hop flavonoids showed varying degrees of inhibitory action ranging from 99.3 to 1.8%. 4. In contrast, the most effective inhibitors of CYP1A2 acetanilide 4-hydroxylase activitywere the two prenylated flavonoids, 8-prenylnaringenin (8PN) and isoxanthohumol (IX), which produced > 90% inhibition when added at concentrations of 10 mu M. 5. CYP1A2 metabolism of the carcinogen AFB1 was also inhibited by IX and 8PN as shown by decreased appearance of dihydrodiols and AFM1 as analysed by hplc. IX and 8PN also decreased covalent binding of radiolabelled AFB1 to microsomal protein in a concomitant manner. 6. XN, IX and 8PN, however, were poor inhibitors of CYP2E1 and CYP3A4 as measured by their effect on chorzoxazone hydroxylase and nifedipine oxidase activities respectively. 7. These results suggest that the hop flavonoids are potent and selective inhibitors of human cytochrome P450 and warrant further in vivo investigations.