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.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, affecting approximately one-third of the global population. Most affected individuals experience only simple steatosis—an accumulation of fat in the liver—but a proportion of these patients will progress to the more severe form of the disease, non-alcoholic steatohepatitis (NASH), which enhances the risk of cirrhosis and hepatocellular carcinoma. Diagnostic approaches to NAFLD are currently limited in accuracy and efficiency; and liver biopsy remains the only reliable way to confirm NASH. This technique, however, is highly invasive and poses risks to patients. Hence, there is an increasing demand for improved minimally invasive diagnostic tools for screening at-risk individuals and identifying patients with more severe disease as well as those likely to progress to such stages. Recently, extracellular vesicles (EVs)—small membrane-bound particles released by virtually all cell types into circulation—have emerged as a rich potential source of biomarkers that can reflect liver function and pathological processes in NAFLD. Of particular interest to the diagnosis and tracking of NAFLD is the potential to extract microRNAs miR-122 and miR-192 from EVs circulating in blood, particularly when using an isolation technique that selectively captures hepatocyte-derived EVs.

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.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.

Extracellular vesicles (EVs) are small, non-replicating, lipid encapsulated particles that contain a myriad of protein and nucleic acid cargo derived from their tissue of origin. The potential role of EV derived biomarkers to the study of drug metabolism and disposition (DMD) has gained attention in recent years. The key trait that makes EVs an attractive biomarker source is their capacity to provide comparable insights to solid organ biopsy through an appreciably less invasive collection procedure.Blood derived EVs exist as a heterogenous milieu of biologically distinct particles originating from different sources through different biogenesis pathways. Furthermore, blood (plasma and serum) contains an array of vesicular and non-vesicular contaminants such as apoptotic bodies, plasma proteins and lipoproteins that are routinely co-isolated with EVs albeit to a different extent depending on the isolation technique. The following mini-review summarises current studies reporting DMD biomarkers and addresses elements of EV isolation and quantification relevant to the application of EV derived DMD biomarkers. Evidence based best practice guidance aligned to Minimum Information for the Study of Extracellular Vesicles (MISEV) and EV-TRACK reporting standards are summarised in the context of DMD studies. SIGNIFICANCE STATEMENTExtracellular vesicle (EV) derived protein and nucleic acid cargo represent a potentially game changing source of novel DMD biomarkers with the capacity to define within-and betweenindividual variability in drug exposure irrespective of aetiology. However, robust translation of EV derived biomarkers requires the generation of transparent reproducible evidence. This review outlines the critical elements of data generation and reporting relevant to achieving this evidence in a drug metabolism and disposition context.