Evaluation of 6β-hydroxycortisol, 6β-hydroxycortisone, and a combination of the two as endogenous probes for inhibition of CYP3A4 in vivo

Abstract

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.

Figure 1. Inhibition of cortisol and cortisone 6β-hydroxylation by CYP isoform specific inhibitors and itraconazole in human liver microsomes

The percent activity remaining in comparison to control in human liver microsomes (HLMs) is shown when HLMs were incubated with 1.4μM cortisol (panel A) or cortisone (panel B) in the presence of the indicated specific CYP inhibitors. The error bars indicate the range of individual measurements. Control 1 refers to the control used for preincubation with mechanism-based inhibitors and Control 2 refers to the control for reversible inhibitors. The abbreviation are as follows: troleandomycin (TAO), furafylline (FURA), orphenadrine (ORPH), (+)-N-3-benzylnirvanol (BENZ), montelukast (MONT), sulfaphenazole (SULF), quinidine (QUIN) and ketoconazole (KETO). The IC₅₀–values for itraconazole (ITZ) towards cortisol (panel C) and cortisone (panel D) hydroxylation (1.4 μM) were determined in human liver microsomes and the obtained IC₅₀ value and the maximum % inhibition (E_max value) are shown in the insets of each panel.

Figure 2. Kinetic characterization of cortisol 6β-hydroxylation by CYP3A4 (A) and CYP2B6 (B), and cortisone 6β-hydroxylation by CYP3A4 (C)

Cortisol (A and B) or cortisone (C) was incubated with CYP3A4 (panel A and C) or CYP2B6 (panel B) supersomes and the formation of 6β-hydroxylation products was measured. No interconversion between cortisol and cortisone or the 6β -hydroxy-metabolites was observed. The Michaelis-Menten equation was fitted to the data to obtain the indicated kinetic parameters (inset in A and C). For CYP2B6 no saturation was observed and hence only the intrinsic clearance was calculated from the slope of the plot (panel B).

Figure 3. Plasma concentration-time curves of cortisol (A) and cortisone (B) in the presence and absence of itraconazole (ITZ)

The average plasma concentration (6 subjects) of cortisol (panel A) and cortisone (panel B) during the 24-hours after itraconazole administration are shown after control session and after 50, 200 and 400 mg administration of itraconazole. The error bars show the standard deviation of the concentration in the six subjects.

Figure 4. Determination of in vivo IC₅₀ of itraconazole towards the formation clearance of 6β-hydroxycortisone and 6β-hydroxycortisol and estimation of the maximum extent of inhibition

The CL_in/CL was calculated for each subject following escalating itraconazole (ITZ) doses using the formation clearance (CL_f) values for combined cortisol and cortisone hydroxylation according to equation 1. The plasma concentrations of ITZ were measured in each individual and the CL_in/CL –values were plotted as a function of ITZ concentration. The IC₅₀ and E_max values were determined by fitting equation 4 to the data.