Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes

Abstract

Aims Chemical inhibitors of cytochrome P450 (CYP) are a useful tool in defining the role of individual CYPs involved in drug metabolism. The aim of the present study was to evaluate the selectivity and rank the order of potency of a range of isoform-selective CYP inhibitors and to compare directly the effects of these inhibitors in human and rat hepatic microsomes. Methods Four chemical inhibitors of human cytochrome P450 isoforms, furafylline (CYP1A2), sulphaphenazole (CYP2C9), diethyldithiocarbamate (CYP2E1), and ketoconazole (CYP3A4) were screened for their inhibitory specificity towards CYPmediated reactions in both human and rat liver microsomal preparations. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, chlorzoxazone 6-hydroxylation and testosterone 6b-hydroxylation were monitored for enzyme activity. Results Furafylline was a potent, selective inhibitor of phenacetin O-deethylation (CYP1A2-mediated) in human liver microsomes ( IC 50 =0.48 mm), but inhibited both phenacetin O-deethylation and tolbutamide 4-hydroxylation (CYP2C9-mediated) at equimolar concentrations in rat liver microsomes ( IC 50 =20.8 and 24.0 mm respectively). Sulphaphenazole demonstrated selective inhibition of tolbutamide hydroxylation in human liver microsomes but failed to inhibit this reaction in rat liver microsomes. DDC demonstrated a low level of selectivity as an inhibitory probe for chlorzoxazone 6-hydroxylation (CYP2E1-mediated). DDC also inhibited testosterone 6b-hydroxylation (CYP3A-mediated) in man and rat, and tolbutamide 4-hydroxylase activity in rat. Ketoconazole was a very potent, selective inhibitor of CYP3A4 activity in human liver ( IC 50 =0.04 mm). Although inhibiting CYP3A in rat liver it also inhibited all other reactions at concentrations ≤5 mm. Conclusions It is evident that CYP inhibitors do not exhibit the same selectivity in human and rat liver microsomes. This is due to differential selectivity of the inhibitors and/or differences in the CYP isoform responsible for metabolism in the different species.Keywords: cytochrome P450, cytochrome P450 inhibition, differential selectivity other species such as the rat are used and predictions made Introduction to man. Currently gene families 1, 2 and 3 are thought to be The correct assignment of individual cytochrome P450 (CYP) isoforms to specific metabolic pathways is an area of involved in the biotransformation of xenobiotics in both humans and rodents. However, isoforms are not conserved considerable importance; in particular in the rational prediction of drug-drug interactions. Many different between species and differences are known to occur in catalytic and regulatory specificities between human CYP strategies are currently employed in the unambiguous identification of CYP isoforms responsible for the biotransisoforms and their rat orthologues, although the CYP1A and CYP2E subfamilies show remarkable conservation formation of therapeutic agents. These include the use of selective chemical inhibitors of CYP isoforms, inhibitory between human and rat [3...

Oxidation of the Flavonoids Galangin and Kaempferide by Human Liver Microsomes and CYP1A1, CYP1A2, and CYP2C9

“…Furafylline (20 M), a selective CYP1A2 inhibitor (Sesardic et al, 1990;Clarke et al, 1994;Clement and Demesmaeker, 1997) was preincubated for 15 min with the enzyme mixture before the addition of substrate. Sulfaphenazole (25 M), a selective inhibitor of CYP2C9 (Eagling et al, 1998;Giancarlo et al, 2001), was added together with the substrates.…”

“…Chrysin was not a substrate for CYP2C9. We also examined the effect of the CYP2C9-selective inhibitor sulfaphenazole (Eagling et al, 1998;Giancarlo et al, 2001) on the oxidation of galangin and kaempferide by human liver microsomes. With 25 M sulfaphenazole, there was a small (35%) inhibition, which was statistically significant (P Ͻ 0.05).…”

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

“…A major advantage of using selective P450 inhibitors is that the fractional inhibition of a reaction in microsomes or another crude preparation indicates the extent to which a specific P450 is responsible for the metabolic reaction being studied (Halpert et al, 1994). It is important to note, however, that human P450 inhibitors do not necessarily exhibit the same selectivity when used with microsomes obtained from another species (Eagling et al, 1998). Thus caution needs to be taken when interpreting results obtained in animal studies.…”

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“…Thus, an inhibitor may lose isoform selectivity at high concentrations. For example, ketoconazole, a selective inhibitor of CYP3A4 at low concentrations, inhibited CYPl A2-mediated phenacetin 0-deethylation with an ICso value of 60.0 pM in human liver microsomes (Eagling et al 1998) and caused inhibition of CYP2C19 in another study (Rasmussen et al 1998b). Therefore, it is possible that inhibition of other enzymes than CYP3A4 contributed to the effects of ketoconazole on lidocaine metabolism observed in the present study.…”

“…Nevertheless, the possibility that the effects of fluvoxamine on the biotransformation of lidocaine were partly mediated by inhibition of other CYP enzymes than CYPlA2 cannot be excluded. In contrast to fluvoxamine, ketoconazole and erythromycin preferably inhibit CYP3A4 (Newton et al 1995;Eagling et al 1998;Pelkonen et a/. 1998).…”