Evaluation of cytochrome P450 (CYP) induction using RT‐qPCR in exosomes isolated from plasma samples: Method development and qualification

AIM: Induction of cytochrome P450 (CYP) enzymes is one of the major mechanisms for drug-drug interactions (DDIs), which requires accurate assessment for dose adjustments. This study aimed to develop a reliable method for isolating exosomes from rat and human plasma samples, quantifying several CYP enzymes in the isolated exosomes at the mRNA level, and further validating their utility for studying enzyme induction in both preclinical and clinical settings. METHODS: We evaluated and validated exosome isolation methods from human plasma and serum samples using nanoparticle tracking analysis (NTA) for physical characterization and RT-qPCR for exosomal mRNA quantification. CYP mRNA induction was quantified in plasma-derived exosomes from rats and humans following dexamethasone or modafinil treatment. RESULTS: Among six exosome isolation methods, the ExoQuick kit was selected and further optimized based on the high yield and purity of isolated exosomes. This workflow of exosome isolation and RNA extraction exhibited high precision and reproducibility, and demonstrated excellent assay linearity, with gene detectability and expression levels increasing in a volume-dependent manner from 0.5, 1 and 2 mL plasma samples. Following dexamethasone treatment in rats, significant induction of Cyp3a23/3a1 mRNA was observed in both plasma-derived exosomes and liver tissues. Additionally, CYP3A4, CYP3A5 and CYP1A2 mRNA in human plasma-derived exosomes were induced in clinical studies following 200 mg and 400 mg modafinil administration. CONCLUSION: We present a robust workflow for detecting several CYP mRNAs in plasma-derived exosomes and demonstrate proof-of-concept induction in preclinical and clinical samples. However, larger prospective studies are required to validate clinical utility.