Identification and exploration of novel biomarkers for CYP3A activity using global metabolomics

Abstract

Introduction: The measurement of cytochrome P450 (CYP)3A activity has been quantitatively assessed using midazolam, which is a specific exogenous probe drug. To avoid unnecessary drug exposure and invasive sampling, evaluating an individuals CYP3A phenotype through measurement of sensitive endogenous biomarkers, especially urinary compounds, would be ideal. Quantitative models of endogenous metabolites are useful in predicting CYP3A-mediated drug–drug interactions. This study aimed to identify novel predictive markers for the magnitude of CYP3A induction and inhibition in male and female subjects. Methods: Twenty-four Korean male and 12 female volunteers aged 20–50 years were received intravenous midazolam during 3 study phases: phase 1, the control phase, in which we evaluated midazolam Pharmacokinetics (PK)

phase 2, the CYP3A-inhibition phase, in which we explored the changes in midazolam PK caused by pretreatment with ketoconazole

and phase 3, the CYP3A-induction phase, in which we assessed the effects of pretreatment with rifampicin on midazolam PK. Their 12-h interval urine samples in each phase were analyzed for global metabolomics using ultra-performance liquid chromatography time-of-flight mass spectrometry. To identify endogenous metabolic markers associated with CYP3A inhibition and induction, we selected metabolic features that satisfied the following conditions in male or female subjects: (1) fold-change of mean relative intensity in inhibition versus control phase < 0.5, (2) fold-change of mean relative intensity in induction versus control phase > 2.0, and (3) correlation between relative intensity of the compound and midazolam clearance (Pearsons r > 0.7). To assess the enzyme kinetics, recombinant enzymes of CYP3A4, CYP4A11, and CYP4F2 were used, and Km, Vmax, and Vmax/Km values were compared. Results: We report five ω- or (ω-1)-hydroxylated medium-chain acylcarnitines as novel CYP3A4 markers. Recombinant enzyme assays were used to determine the ω- and (ω-1)-hydroxylation activities of CYP3A4, CYP4A11, and CYP4F2. CYP3A4 catalyzed ω- and (ω-1)-hydroxylated MCFAs with the lowest Km and highest Vmax/Km values. Finally, we derived a model to predict midazolam clearance using these markers and demonstrated that the predictive model including three ω- or (ω-1)-hydroxylated medium-chain acylcarnitines, 6β-OH cortisol, and gender as covariates shows reliable predictability (r2 = 0.911). Ln(clearance) = 1.4443 – 0.5559 ∙ gender† + 0.1637 ∙ ln(Car C8:1-OH II/Cr + 1) + 0.09661 ∙ ln(Car C10:2-OH/Cr+1) + 0.1261 ∙ ln(Car C11:2-OH/Cr + 1) + 0.1191 ∙ ln(6β-OH cortisol/Cr + 1) †male = 0, female = 1 Conclusions: Using a global metabolomics approach, we identified urinary hydroxy acylcarnitines as novel CYP3A biomarkers and found that CYP3A4 efficiently catalyzes the ω- and (ω-1)-hydroxylation of MCFAs. The selected markers of hepatic CYP3A activity and the proposed model can be applied to predict DDI mediated by CYP3A.