The xenobiotic inhibitor profile of cytochrome P4502C8

Abstract

Aims: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A).

Methods: CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure.

Results: At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM.

Conclusions: Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.

Figure 2

Inhibition of CYP2C8 catalysed torsemide tolylmethylhydroxylation by prototypic CYP isoform selective probes. The torsemide concentration was 170 μm and concentrations of inhibitors are shown beside the abbreviation for each compound. Inhibition data represent the mean of duplicate measurements. Abbreviations: FUR, furafylline; COUM, coumarin; SPZ, sulphaphenazole; MEPH, mephenytoin; QUIN, quinidine; DDC, diethyldithiocarbamate; TAO, troleandomycin; KET, ketoconazole.

Figure 3

Inhibition of CYP2C8 catalysed torsemide tolylmethylhydroxylation by azole and triazole antifungal agents. The torsemide concentration was 170 μm. Each azole/triazole was screened at added concentrations of 10 and 100 μm. Inhibition data represent the mean of duplicate measurements. Abbreviations; FLU, fluconazole; ITPvA, itraconazole; BIF, bifonazole; MIC, miconazole; ECO, econazole; CLO, clotrimazole.

Figure 1

Eadie-Hofstee plots for paclitaxel 6α-hydroxylation by microsomes from a representative human liver (panel a) and Sf21-expressed CYP2C8 (panel b), and for torsemide tolylmethylhydroxylation by Sf21-expressed CYP2C8 (panel c). Points are experimentally derived values (means of duplicate measurements at each concentration) while the solid lines show computer-derived curves of best fit.

Figure 4

Dixon plots for inhibition of CYP2C8 catalysed torsemide tolylniethylhydroxylation by ketoconazole, clotrimazole, terfenadine, midazolam, triazolam and quinine. Concentrations of substrate (torsemide) are shown in the left hand panel of each plot. Each point represents the mean of duplicate measurements.