Liver‐derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two‐step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N = 3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold‐increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5–4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1–6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22–0.34, P < 0.0001; and 0.17, 95% CI = 0.13–0.22, P < 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤ 1.5‐fold). It was also possible to measure liver sEV‐catalyzed dextromethorphan (DEX) O‐demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r = 0.917, P = 0.0001; N = 10) and 3‐hour plasma DXO‐to‐DEX concentration ratio (r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2‐fold, P = 0.003) and CYP2D6 (3.7‐fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

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Vemurafenib, a B rapidly accelerated fibrosarcoma inhibitor, is commonly used in combination of cobimetinib for the treatment of melanoma. In the current study, we evaluated the relationship between vemurafenib exposure, as measured by the estimated apparent clearance (CLB) at steady state and any grade ≥3 toxicity, grade ≥3 skin rash, or toxicity requiring dose modification using pooled data from 3 prospective clinical trials involving 898 patients. A total of 69% had any grade ≥3 toxicity; grade ≥3 skin rash in 15% and 47% had a dose reduction/interruption or cessation. The median vemurafenib CLB was 1.35 L/h (interquartile range, 1.15‐1.65 L/h). Lower vemurafenib CLB was significantly associated with an increased risk of grade ≥3 toxicity (hazard ratio [HR], 0.62; P < .001), grade ≥3 rash (HR, 0.29; P < .001), and adverse events requiring vemurafenib dose reduction/interruption or cessation (HR, 0.5; P < .001). When the patients were divided into 3 groups based on the vemurafenib CLB thresholds, those with low CLB (<1.22 L/h) had significantly increased incidence of any grade ≥3 toxicity or skin rash or dose adjustment, interruption, or cessation at 12 months and at day 28 when compared to those with medium (≥1.22 and <1.55 L/h) or high (>1.55 L/h) vemurafenib CLB. In conclusion, the estimated CLB of vemurafenib is associated with severe toxicities and dose adjustment or cessation, suggesting that an early estimation of vemurafenib exposure may be useful in identifying patients at risk of experiencing toxicity.