Br J Clin Pharmacol 1998; 45: 107–114 Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes Victoria A. Eagling, John F. Tjia & David J. Back Department of Pharmacology & Therapeutics, University of Liverpool, P.O. Box 147, Liverpool L69 3GE, UK 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 eVects 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 CYP- mediated 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 (IC50 0.48 mm), but inhibited both phenacetin O-deethylation and tolbutamide 4-hydroxylation (CYP2C9- mediated) at equimolar concentrations in rat liver microsomes (IC50=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 (IC50 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 diVerential selectivity of the inhibitors and/or diVerences in the CYP isoform responsible for metabolism in the diVerent species. Keywords: cytochrome P450, cytochrome P450 inhibition, diVerential selectivity other species such as the rat are used and predictions made Introduction to man. The correct assignment of individual cytochrome P450 Currently gene families 1, 2 and 3 are thought to be (CYP) isoforms to specific metabolic pathways is an area of involved in the biotransformation of xenobiotics in both considerable importance; in particular in the rational humans and rodents. However, isoforms are not conserved prediction of drug-drug interactions. Many diVerent between species and diVerences are known to occur in strategies are currently employed in the unambiguous catalytic and regulatory specificities between human CYP identification of CYP isoforms responsible for the biotrans- isoforms and their rat orthologues, although the CYP1A formation of therapeutic agents. These include the use of and CYP2E subfamilies show remarkable conservation selective chemical inhibitors of CYP isoforms, inhibitory between human and rat [3]. The major human hepatic CYP CYP antibodies, studies with purified, reconstituted enzymes subfamilies are CYP2C and CYP3A which account for 20% and the correlation of immunoquantified CYP levels and and 30% of total CYP respectively [4], with CYP1A2, metabolic rates [1, 2]. Recent advances in the field of CYP2E1, CYP2A6, CYP2D6 and CYP2B6 comprising chemical inhibitors of CYP have greatly facilitated the 13%, 7%, 4%, 2% and <1% respectively. Levels of CYP characterization of the catalytic specificities of individual isoforms in male rats are very diVerent with CYP2C11 CYP isoforms involved in drug metabolism. The lack of accounting for 54% of total CYP content, CYP3A2 being availability of human liver samples for metabolism/inhibitor 17% abundant and CYP1A2 being expressed at much lower studies may mean that hepatic microsomal preparations from levels (2%) in untreated rat liver samples [5, 6]. DiVerences in the levels of individual CYP isoforms and Correspondence: Professor D. J. Back, Department of Pharmacology & Therapeutics, indeed the expression of distinct isoforms may lead to University of Liverpool, P.O. Box 147, Liverpool L69 3GE, UK. diVerences in the metabolism of alleged probe substrates © 1998 Blackwell Science Ltd 107 V. A. Eagling et al. between species. In addition, these diVerences have also and 2.0 mg protein for rat liver microsomes and an been shown to influence the selectivity of inhibitor probes. incubation time of 30 min in both species. A 500 ml reaction For example, the inhibition of tolbutamide 4-hydroxylation mixture typically containing 0.5 mg microsomal protein by sulphaphenazole (CYP2C9 inhibitor in human) diVers (human or rat) was incubated with phenacetin in the markedly when this reaction is catalysed by human, rat or presence of MgCl2 (10 mm) and NADPH (2.5 mm)in rabbit liver microsomes [7]. phosphate buVer (0.067 m; pH 7.4). Metacetamol was added In the present study, four chemical inhibitors of human as internal standard and the mixture was extracted with cytochrome P450 isoforms, namely, furafylline (CYP1A2, DCM (10 ml; 20 min) to remove unreacted phenacetin [1, 8–10]); sulphaphenazole (CYP2C9, [1, 2, 10]); diethyldi- followed by ethylacetate (10 ml; 20 min). Samples were thiocarbamate (DDC) (CYP2E1, [10, 11]) and ketoconazole reconstituted in mobile phase (200 ml) prior to h.p.l.c. (CYP3A4, [12–14]) were screened for their inhibitory analysis. Paracetamol and metacetamol were separated using − specificity towards CYP-mediated reactions in both human an isocratic mobile phase (flow rate 1 ml min 1) consisting and rat liver microsomal preparations. Phenacetin O- of AcN: sodium phosphate buVer (10590, v/v; 0.1 m; deethylation, tolbutamide 4-hydroxylation, chlorzoxazone pH 4.3) and a Spherex 5 mC18 column (25 cm x 4.6 mm; 6-hydroxylation and testosterone 6b-hydroxylation were Phenomenex, Macclesfield, UK) with u.v. detection at chosen as markers for human CYP1A2 [15], CYP2C9 [16], 245 nm. Formation of paracetamol was quantified by CYP2E1 [17] and CYP3A4 [18] activity respectively and interpolating peak height ratios of paracetamol and metaceta- incubations performed in human and rat liver microsomes. mol from a standard curve of known paracetamol concentrations. Inter- and intra- assay coeYcients of variation were 8.7% and 6.5% (determined at 100 pmol paracetamol) Methods respectively. The lower limit of determination was 25 pmol. Chemicals b) Tolbutamide 4-hydroxylation Initial linearity studies Phenacetin, paracetamol, metacetamol, tolbutamide, indicated that this reaction was linear up to 4 mg and 2 mg chlorzoxazone, zoxazolamine, testosterone, 6b-OH testos- microsomal protein for human and rat liver microsomes terone, 11b-OH testosterone, sulphaphenazole, diethyldithio- respectively and an incubation time of 16 min for human carbamate and b-NADPH (reduced form) were purchased liver microsomes and 10 min for rat liver microsomal from the Sigma Chemical Company (Poole, Dorset, UK). preparations. A 500 ml reaction mixture containing 0.5 mg Furafylline and 6-OH chlorzoxazone were obtained from microsomal protein (human or rat) was incubated with Ultrafine Chemicals (Manchester, UK). Chlorpropamide tolbutamide in the presence of MgCl2 (10 mm) and NADPH and 4-OH tolbutamide were gifts from Hoescht AG (1 mm) in phosphate buVer (0.067 m; pH 7.4) according to (Frankfurt, Germany); ketoconazole was a gift from Janssen the method of Back et al. [21]. Termination, extraction and (Beerse, Belgium). H.p.l.c. grade acetonitrile (AcN), h.p.l.c. analysis of samples were as previously described. The dichloromethane (DCM), methanol and ethyl acetate were inter- and intra- assay coeYcients of variation (determined purchased from Fisons Plc (Loughborough, UK). All other at 0.6 nmol hydroxytolbutamide) were 3% and 1.3% reagents were of the highest grade possible. respectively, with a lower limit of determination of 20 pmol. c) Chlorzoxazone 6-hydroxylation Initial linearity studies Human liver samples revealed that this reaction was linear upto 2 mg microsomal Histologically normal human livers were obtained from protein in both species and an incubation time of 40 min. kidney transplant donors. Liver samples were transferred on A 500 ml incubation volume containing 0.2 mg microsomal ice to the laboratory within 30 min where they were protein (human or rat) was incubated with chlorzoxazone sectioned into 10–20 g portions and frozen in liquid (CLZ) in the presence of MgCl2 (10 mm) and NADPH nitrogen. These were then stored at −80° C until required. (1 mm) in phosphate buVer (0.067 m; pH 7.4). The reaction Washed microsomes (105, 000g pellets) were prepared from was terminated by the addition of zoxazolamine as internal human liver samples by the diVerential centrifugation standard and the mixture was extracted with DCM (5 ml; technique [19] and microsomal protein yield was determined 10 min). The organic phase was evaporated to dryness and by the method of Lowry et al. [20] using bovine serum reconstituted into mobile phase (200 ml) prior to h.p.l.c. albumin as standard. analysis. Formation of 6-hydroxychlorzoxazone (6-OHCLZ) was measured by h.p.l.c. with u.v. detection at 295 nm and quantified by interpolating peak