We have proposed that 4b-hydroxycholesterol (4b-OHC) may be used as an endogenous marker of CYP3A activity. The cholesterol metabolite 4b-OHC is formed by CYP3A4. Treatment of patients with strong inducers of CYP3A enzymes, e.g. anti-epileptic drugs, resulted in 10-fold increased concentrations of plasma 4b-OHC, while treatment with CYP3A inhibitors such as ritonavir or itraconazole resulted in decreased plasma concentrations. There was a relationship between the 4b-OHC concentration and the number of active CYP3A5*1 alleles showing that 4b-OHC was not only formed by CYP3A4, but also by CYP3A5. The concentration of 4b-OHC was higher in women than in men, confirming previous studies indicating a gender difference in CYP3A4/5-activity. The rate of elimination of 4b-OHC is slow (half-life 17 days) which results in stable plasma concentrations within individuals, but limits its use to study rapid changes in CYP3A activity. In short-term studies exogenous markers such as midazolam or quinine may be superior, but in long-term studies 4b-OHC is a sensitive marker of CYP3A activity, especially to assess induction but also inhibition. Under conditions where the cholesterol concentration is changing, the ratio of 4b-OHC : cholesterol may be used as an alternative to 4b-OHC itself. The use of an endogenous CYP3A marker has obvious advantages and may be of value both during drug development and for monitoring CYP3A activity in patients.

CYP3A4, considered the most important enzyme in drug metabolism, is often involved in drug-drug interactions. When developing new drugs, appropriate markers for detecting CYP3A4 induction are needed. Our study compared endogenously formed 4β-hydroxycholesterol with the midazolam clearance in plasma and the 6β-hydroxycortisol/cortisol ratio in urine as markers for CYP3A4 induction. To this end, we performed a clinical trial in which 24 healthy subjects were randomized to 10, 20, or 100 mg daily doses of rifampicin for 14 days (n = 8 in each group) to achieve a low and moderate CYP3A4 induction. The CYP3A4 induction could be detected even at the lowest dose of rifampicin (10 mg) via the estimated midazolam clearance, the 4β-hydroxycholesterol ratio (both P < 0.01), and the 6β-hydroxycortisol ratio (P < 0.05). For the three dosing groups (10, 20, and 100 mg), the median fold induction from baseline was 2.0, 2.6, and 4.0 for the estimated midazolam clearance; 1.3, 1.6, and 2.5 for the 4β-hydroxycholesterol/cholesterol ratio; and 1.7, 2.9, and 3.1 for the 6β-hydroxycortisol/cortisol ratio. In conclusion, the 4β-hydroxycholesterol ratio is comparable to midazolam clearance as a marker of CYP3A4 induction, and each may be used to evaluate CYP3A4 induction in clinical trials evaluating drug-drug interactions for new drugs.

We investigated the effects of pharmacogenetic variations and efavirenz pharmacokinetics on inter-individual differences in the extent of CYP3A induction by efavirenz using 4β-hydroxycholesterol/cholesterol (4β-OHC/Chol) as a marker for CYP3A induction. Plasma 4β-hydroxycholesterol and cholesterol concentrations were determined at baseline, and at the 4th, 16th and 48th week of efavirenz-based highly active antiretroviral therapy in antiretroviral therapy-naive HIV patients (n=77). Efavirenz plasma concentrations were quantified at weeks 4 and 16. CYP2B6, CYP3A5, ABCB1, UGT2B7 genotyping were done. Compared with baseline, the median plasma 4β-OHC/Chol ratio increased at the 4th (257%), 16th (291%) and 48th (165%) week (P<0.0001). CYP2B6*6 genotype significantly influenced 4β-OHC/Chol ratio at weeks 16 (P=0.02) and 48 (P=0.04) being highest in CYP2B6*6/*6>*1/*6>*1/*1. There were positive correlations between plasma efavirenz and 4β-OHC/Chol ratios (week 4: P=0.02, week 16: P=0.001). CYP3A enzyme induction by efavirenz is pronounced in CYP2B6 slow metabolizers who have high efavirenz plasma exposure.