4β-hydroxycholesterol-to-cholesterol ratio as an endogenous biomarker in human plasma to determine treatment effects of induction and inhibition of CYP3A4 enzyme activity

Ryan, Yashara

4β-hydroxycholesterol-to-cholesterol ratio as an endogenous biomarker in human plasma to determine treatment effects of induction and inhibition of CYP3A4 enzyme activity

Thesis / Dissertation

2026

Publisher

University of Cape Town

Department

Department of Medicine

Faculty

Faculty of Health Sciences

Abstract

Stemming from the induction and/or inhibition of their enzymatic activity, the drug-metabolizing cytochrome P4503A (CYP3A) enzymes, specifically the enzyme CYP3A4, are implicated in drug-drug interactions in several therapeutic areas, including antiretroviral treatment. Whereas the inhibition of CYP3A4 could lead to less clearance and thus toxic buildup of the enzyme's substrates, CYP3A4 induction could compromise drug efficacy. To circumvent the potential danger of co-administered drugs, drug-drug interactions that involve CYP3A activity must be reliably predicted. To assess CYP3A activity and thus predict drug-drug interactions, probe drugs such as midazolam are mostly used. However, the challenges presented by probe drugs have prompted interest in alternative ways to predict drug-drug interactions, such as endogenous biomarkers. One such endogenous compound, 4β-hydroxycholesterol, formed by CYP3A4/5 induced cholesterol metabolism, has been proposed in literature as a biomarker for assessing CYP3A4/5 4β-hydroxycholesterol activity. In this project, an LC-MS/MS method was developed and validated according to the US Food and Drug Administration and the European Medicines Agency guidelines to quantify 4βhydroxycholesterol in human plasma. The method makes use of a surrogate analyte (4βhydroxycholesterol-d7) to mitigate the challenges of the endogenous presence of the 4βhydroxycholesterol and takes the difference in ionization efficiency of the surrogate analyte (4β-hydroxycholesterol-d7) and endogenous 4β-hydroxycholesterol into account. The LCMS/MS method was applied to measure the 4β-hydroxycholesterol concentrations in patient samples of three clinical studies. Cholesterol concentrations, for the calculation of 4βhydroxycholesterol/cholesterol ratios, were determined by the National Health Laboratory Services. Multilevel mixed-effects models were developed using unadjusted 4β-hydroxycholesterol concentrations and 4β-hydroxycholesterol/cholesterol ratios. These were used to describe the effects of sequential treatments and covariate effects on CYP3A4/5 activity in each study. Models using the 4β-hydroxycholesterol/cholesterol ratio were supportive, but not conclusive of the superiority of the ratio over the unadjusted 4β-hydroxycholesterol concentrations, in the prediction of drug-drug interactions, thus supporting prior suggestions of the use of the ratio, 4β-hydroxycholesterol/cholesterol as an endogenous biomarker of CYP3A4 activity.

Keywords

enzyme CYP3A4

biomarkers

Reference:

Collections

PhD / Doctoral

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