Leveraging Human Plasma‐Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Cytochrome P450 3A4 by Modafinil

Preparations of plasma‐derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4β‐hydroxycholesterol‐to‐cholesterol (4βHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1‐fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8–2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold‐increase (90% CI)) of both liver (1.3 (1.1–1.5), P value = 0.014) and nonliver (1.9 (1.6–2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre‐dose) 4βHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil‐induced changes in plasma 4βHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α‐ethinylestradiol, and abemaciclib) area under the plasma concentration‐time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma‐derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.