Inhibitors Of Human Cytochrome P450 Research Articles

Inhibition of cytochrome P450 2A participating in coumarin 7‐hydroxylation in pig liver microsomes

Five commonly used human cytochrome P450 (CYP) inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A) activity in pig liver microsomes. The K(m) and V(max) values for coumarin 7-hydroxylation in pig liver microsomes were estimated to be 1 μm and 0.26 nmol·mg/min, respectively. The following human CYP inhibitors caused little or no inhibition of CYP2A as defined by a K(i) > 200 μm: quinidine (CYP2D6), troleandomycin (CYP3A4), and sulfaphenazole (CYP2C9). The other two human CYP inhibitors were classified as strong inhibitors of CYP2A: 8-methoxypsoralen (CYP2A6) and α-naphthoflavone (CYP1A1/2). In the absence of a preincubation period, 8-MOP inhibited the 7-hydroxylation of coumarin with a K(i) value of 1.1 μm, which decreased to 0.1 μm when 8-MOP was preincubated with pig liver microsomes for 3 min. α-Naphthoflavone inhibited the 7-hydroxylation of coumarin with a K(i) value of 32 μm, which did not increase ability to inhibitor CYP2A when α-naphthoflavone was preincubated with pig liver microsomes for 3 min. These results of this study suggest that 8-MOP is a potent, mechanism-based inhibitor of pig CYP2A activity in pig liver microsomes.

A Comparison of 2-Phenyl-2-(1-piperidinyl)propane (PPP), 1,1′,1″-Phosphinothioylidynetrisaziridine (ThioTEPA), Clopidogrel, and Ticlopidine as Selective Inactivators of Human Cytochrome P450 2B6

The use of selective chemical inhibitors of human cytochrome P450 (P450) enzymes represents a powerful method by which the relative contributions of various human P450 enzymes to the metabolism of drugs can be determined. However, the identification of CYP2B6 in the metabolism of drugs has been more challenging because of the lack of a well established inhibitor of this enzyme. In this report, we describe the selectivity of 2-phenyl-2-(1-piperidinyl)propane (PPP) as an inactivator of CYP2B6 and compare this selectivity versus other CYP2B6 inactivators: 1,1',1''-phosphinothioylidynetrisaziridine (thioTEPA), clopidogrel, and ticlopidine. Values of K(I) and k(inact) for PPP were 5.6 microM and 0.13/min for bupropion hydroxylase catalyzed by pooled human liver microsomes, and values for thioTEPA were similar (4.8 microM and 0.20/min, respectively). Intrinsic inactivation capability was considerably greater for clopidogrel because of a greater k(inact) value (1.9/min). Ticlopidine was potent with K(I) and k(inact) values of 0.32 microM and 0.43/min, respectively. The selectivity of these four agents for CYP2B6 was determined by testing their effects on other human P450 enzyme activities using conditions that yield approximately 90% inactivation of CYP2B6 activity. The results showed that preincubation of human liver microsomes with PPP at 30 microM for 30 min provided more selective inhibition for CYP2B6 than thioTEPA, clopidogrel, and ticlopidine. Furthermore, the use of clopidogrel is complicated by the observation that this agent is not stable in the presence of human liver microsomes, even without addition of NADPH. Therefore, PPP can serve as a selective chemical inactivator of CYP2B6 and be used to define the role of CYP2B6 in the metabolism of drugs.

Three dimensional pharmacophore modeling of human CYP17 inhibitors. Potential agents for prostate cancer therapy.

We report here a molecular modeling investigation of steroidal and nonsteroidal inhibitors of human cytochrome P450 17alpha-hydroxylase-17,20-lyase (CYP17). Using the pharmacophore perception technique, we have generated common-feature pharmacophore model(s) to explain the putative binding requirements for two classes of human CYP17 inhibitors. Common chemical features in the steroid and nonsteroid human CYP17 enzyme inhibitors, as deduced by the Catalyst/HipHop program, are one to two hydrogen bond acceptors (HBAs) and three hydrophobic groups. For azole-steroidal ligands, the 3beta-OH group of ring A and the N-3 of the azole ring attached to ring D at C-17 act as hydrogen bond acceptors. A model that permits hydrogen bond interaction between the azole functionality on ring D and the enzyme is consistent with experimental deductions for type II CYP17 inhibitors where a sixth ligating atom interacts with Fe(II) of heme. In general, pharmacophore models derived for steroid and nonsteroidal compounds bear striking similarities to all azole sites mapping the HBA functionality and to three hydrophobic features describing the hydrophobic interactions between the ligands and the enzyme. Using the pharmacophore model derived for azole-steroidal inhibitors as a 3D search query against several 3D multiconformational Catalyst formatted databases, we identified several steroidal compounds with potential inhibition of this enzyme. Biological testing of some of these compounds show low to high inhibitory potency against the human CYP17 enzyme. This shows the potential of our pharmacophore model in identifying new and potent CYP17 inhibitors. Further refinement of the model is in progress with a view to identifying and optimizing new leads.

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.