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

Br J Clin Pharmacol. 2014 Nov;78(5):1122-34. doi: 10.1111/bcp.12425.


Aims: This study aimed to assess changes in the plasma concentrationss of 4β-hydroxycholesterol (4βHC) 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 4βHC was assessed by analyzing % change from baseline using a linear spline mixed effects model.

Results: Compared with PLB, KETO decreased 4βHC mean values up to 13% (P = 0.003) and RIF increased 4βHC mean values up to 220% (P < 0.001). Within 14 days of stopping KETO and RIF, 4βHC 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 (6βHCL : CL) urinary ratios.

Conclusions: Changes in plasma 4βHC can be utilized as a surrogate for MDZ PK after multiple doses of potent CYP3A inducers. There is a more limited dynamic range for 4βHC for assessment of potential CYP3A inhibitors. 4βHC is a valuable tool for the assessment of potential CYP3A inducers in early drug development.

Keywords: 4β-hydroxycholesterol; CYP3A; biomarkers; drug-drug interactions; midazolam; saliva.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Biomarkers / blood
  • Cytochrome P-450 CYP3A / metabolism*
  • Cytochrome P-450 CYP3A Inducers / pharmacology
  • Cytochrome P-450 CYP3A Inhibitors / pharmacology
  • Dose-Response Relationship, Drug
  • Healthy Volunteers
  • Humans
  • Hydroxycholesterols / blood*
  • Injections, Intravenous
  • Ketoconazole / pharmacology
  • Limit of Detection
  • Midazolam / administration & dosage
  • Midazolam / blood
  • Midazolam / pharmacokinetics*
  • Middle Aged
  • Rifampin / pharmacology
  • Saliva / chemistry
  • Substrate Specificity
  • Time Factors
  • Tissue Distribution
  • Young Adult


  • Biomarkers
  • Cytochrome P-450 CYP3A Inducers
  • Cytochrome P-450 CYP3A Inhibitors
  • Hydroxycholesterols
  • cholest-5-ene-3,4-diol
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • Midazolam
  • Ketoconazole
  • Rifampin