Validation of 4β‐hydroxycholesterol and evaluation of other endogenous biomarkers for the assessment of CYP3A activity in healthy subjects

Aims This study aimed to assess changes in the plasma concentrationss of 4β‐hydroxycholesterol (4β HC ) against intravenous (i.v.) and oral midazolam ( MDZ ) pharmacokinetics ( PK ) after administration of a potent CYP3A inhibitor [ketoconazole ( KETO )] and inducer [rifampicin ( RIF )]. Methods Thirty‐two healthy subjects ( HS ) were allocated into three groups of 12 each in KETO and RIF and 10 in a placebo group ( PLB ). All HS were randomized to receive oral and i.v. MDZ on day 1 or 2 and on day 15 or 16 after receiving RIF (600 mg once daily ), KETO (400 mg once daily) or PLB for 2 weeks. Subjects were followed until day 30. The effect of treatments on 4β HC was assessed by analyzing % change from baseline using a linear spline mixed effects model. Results Compared with PLB , KETO decreased 4β HC mean values up to 13% ( P = 0.003) and RIF increased 4β HC mean values up to 220% ( P < 0.001). Within 14 days of stopping KETO and RIF , 4β HC had either returned to baseline ( KETO ) or was still returning to baseline ( RIF ). Compared with baseline, mean oral MDZ AUC increased by 11‐fold (90% CI ranging from 9‐fold to 13‐fold increase) and decreased by 92% (90% CI ranging from 90% to 95% decrease) after KETO and RIF , respectively. Similar trends were observed for 6β‐hydroxycortisol : cortisol (6β HCL : CL ) urinary ratios. Conclusions Changes in plasma 4β HC can be utilized as a surrogate for MDZ PK after multiple doses of potent CYP3A inducers. There is a more limited dynamic range for 4β HC for assessment of potential CYP3A inhibitors. 4β HC is a valuable tool for the assessment of potential CYP3A inducers in early drug development.