Tolvaptan- and Tolvaptan-Metabolite-Responsive T Cells in Patients with Drug-Induced Liver Injury.

Tolvaptan is an effective drug for the treatment of autosomal dominant polycystic kidney disease, but its use is associated with a significant risk of liver injury in a small number of patients. Herein we describe the presence of tolvaptan- and tolvaptan-metabolite-responsive T cell clones within the peripheral circulation of patients with liver injury. Drug treatment of the clones resulted in a proliferative response and secretion of IFN-γ, IL-13, and the cytolytic molecule granzyme B. Future work should explore pathways of tolvaptan driven T cell activation and the role of T cells in the disease pathogenesis.

Mouse Population-Based Approaches to Investigate Adverse Drug Reactions.

Genetic variation is now recognized as a key factor in the toxicity of pharmaceutical agents. However, genetic diversity is not present in standard nonclinical toxicology models, and small clinical studies (phase I/II) may not include enough subjects to identify toxicity liabilities associated with less common susceptibility factors. As a result, many drugs pass through preclinical and early clinical studies before safety concerns are realized. Furthermore, when adverse drug reactions are idiosyncratic in nature, suggesting a role for rare genetic variants in the toxicity susceptibility, even large clinical studies (phase III) are often underpowered (due to low population frequency and/or small effect size of the risk factor) to identify associations that may be used for precision medicine risk mitigation strategies. Genetically diverse mouse populations can be used to help overcome the limitations of standard nonclinical and clinical studies and to model toxicity responses that require genetic susceptibility factors. Furthermore, mouse population-based approaches can be used to: 1) identify sensitive strains that can serve as a screening tool for next-in-class compounds, 2) identify genetic susceptibility factors that can be used for risk mitigation strategies, and 3) study mechanisms underlying drug toxicity. This review describes genetically diverse mouse populations and provides examples of their utility in investigating adverse drug response. It also explores recent efforts to adapt mouse population-based approaches to in vitro platforms, thereby enabling the incorporation of genetic diversity and the identification of genetic risk factors and mechanisms associated with drug toxicity susceptibility at all stages of drug development.

Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug.

A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug.

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance.

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

PPARγ coactivator-1α contributes to exercise-induced regulation of intramuscular lipid droplet programming in mice and humans.

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.

Neurexin-1α contributes to insulin-containing secretory granule docking.

Neurexins are a family of transmembrane, synaptic adhesion molecules. In neurons, neurexins bind to both sub-plasma membrane and synaptic vesicle-associated constituents of the secretory machinery, play a key role in the organization and stabilization of the presynaptic active zone, and help mediate docking of synaptic vesicles. We have previously shown that neurexins, like many other protein constituents of the neurotransmitter exocytotic machinery, are expressed in pancreatic ß cells. We hypothesized that the role of neurexins in ß cells parallels their role in neurons, with ß-cell neurexins helping to mediate insulin granule docking and secretion. Here we demonstrate that ß cells express a more restricted pattern of neurexin transcripts than neurons, with a clear predominance of neurexin-1α expressed in isolated islets. Using INS-1E ß cells, we found that neurexin-1α interacts with membrane-bound components of the secretory granule-docking machinery and with the granule-associated protein granuphilin. Decreased expression of neurexin-1α, like decreased expression of granuphilin, reduces granule docking at the ß-cell membrane and improves insulin secretion. Perifusion of neurexin-1α KO mouse islets revealed a significant increase in second-phase insulin secretion with a trend toward increased first-phase secretion. Upon glucose stimulation, neurexin-1α protein levels decrease. This glucose-induced down-regulation may enhance glucose-stimulated insulin secretion. We conclude that neurexin-1α is a component of the ß-cell secretory machinery and contributes to secretory granule docking, most likely through interactions with granuphilin. Neurexin-1α is the only transmembrane component of the docking machinery identified thus far. Our findings provide new insights into the mechanisms of insulin granule docking and exocytosis.

Activation of tolvaptan-responsive T-cell clones with the structurally-related mozavaptan.

Tolvaptan is an effective drug for the treatment of autosomal dominant polycystic kidney disease, but its use is associated with a significant risk of T-cell-mediated liver injury in a small number of patients. An important clinical conundrum following the contraindication of tolvaptan is whether administration of agents of similar pharmacological action and structure will be tolerated. Herein, we addressed this question through the exposure of tolvaptan-responsive T-cell clones to similar pharmaceutical agents. Whilst lixivaptan and conivaptan did not activate tolvaptan-responsive T-cells, mozavaptan evoked proliferative responses comparable with tolvaptan itself, indicating that there may be collateral immunological intolerance to this compound as a product of sensitization to tolvaptan.

Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.

The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.

Shedding Light on Drug-Induced Liver Injury: Activation of T Cells From Drug Naive Human Donors With Tolvaptan and a Hydroxybutyric Acid Metabolite.

Exposure to tolvaptan is associated with a significant risk of liver injury in a small fraction of patients with autosomal dominant polycystic kidney disease. The observed delayed onset of liver injury of between 3 and 18 months after commencing tolvaptan treatment, along with rapid recurrence of symptoms following re-challenge is indicative of an adaptive immune attack. This study set out to assess the intrinsic immunogenicity of tolvaptan and pathways of drug-specific T-cell activation using in vitro cell culture platforms. Tolvaptan (n = 7), as well as oxybutyric (DM-4103, n = 1) and hydroxybutyric acid (DM-4107, n = 18) metabolite-specific T-cell clones were generated from tolvaptan naive healthy donor peripheral blood mononuclear cells. Tolvaptan and DM-4103 T-cell clones could also be activated with DM-4107, whereas T-cell clones originally primed with DM-4107 were highly specific to this compound. A signature cytokine profile (IFN-γ, IL-13, granzyme B, and perforin) for almost all T-cell clones was identified. Mechanistically, compound-specific T-cell clone activation was dependent on the presence of soluble drug and could occur within 4 h of drug exposure, ruling out a classical hapten mechanism. However, antigen processing dependence drug presentation was indicated in many T-cell clones. Collectively these data show that tolvaptan-associated liver injury may be attributable to an adaptive immune attack upon the liver, with tolvaptan- and metabolite-specific T cells identified as candidate effector cells in such etiology.

Human-relevant mechanisms and risk factors for TAK-875-Induced liver injury identified via a gene pathway-based approach in Collaborative Cross mice.

Development of TAK-875 was discontinued when a small number of serious drug-induced liver injury (DILI) cases were observed in Phase 3 clinical trials. Subsequent studies have identified hepatocellular oxidative stress, mitochondrial dysfunction, altered bile acid homeostasis, and immune response as mechanisms of TAK-875 DILI and the contribution of genetic risk factors in oxidative response and mitochondrial pathways to the toxicity susceptibility observed in patients. We tested the hypothesis that a novel preclinical approach based on gene pathway analysis in the livers of Collaborative Cross mice could be used to identify human-relevant mechanisms of toxicity and genetic risk factors at the level of the hepatocyte as reported in a human genome-wide association study. Eight (8) male mice (4 matched pairs) from each of 45 Collaborative Cross lines were treated with a single oral (gavage) dose of either vehicle or 600 mg/kg TAK-875. As expected, liver injury was not detected histologically and few changes in plasma biomarkers of hepatotoxicity were observed. However, gene expression profiling in the liver identified hundreds of transcripts responsive to TAK-875 treatment across all strains reflecting alterations in immune response and bile acid homeostasis and the interaction of treatment and strain reflecting oxidative stress and mitochondrial dysfunction. Fold-change expression values were then used to develop pathway-based phenotypes for genetic mapping which identified candidate risk factor genes for TAK-875 toxicity susceptibility at the level of the hepatocyte. Taken together, these findings support our hypothesis that a gene pathway-based approach using Collaborative Cross mice could inform sensitive strains, human-relevant mechanisms of toxicity, and genetic risk factors for TAK-875 DILI. This novel preclinical approach may be helpful in understanding, predicting, and ultimately preventing clinical DILI for other drugs.

Characterization of primary mouse hepatocyte spheroids as a model system to support investigations of drug-induced liver injury.

Primary mouse hepatocytes isolated from genetically defined and/or diverse lines and disease models are a valuable resource for studying the impact of genetic and environmental factors on drug response and disease. However, standard monolayer cultures result in a rapid decline in mouse hepatocyte viability and functionality. Therefore, we evaluated 3D spheroid methodology for long-term culture of primary mouse hepatocytes, initially to support investigations of drug-induced liver injury (DILI). Primary hepatocytes isolated from male and female C57BL/6J mice were used to generate spheroids by spontaneous self-aggregation in ultra-low attachment plates. Spheroids with well-defined perimeters were observed within 5 days after seeding and retained morphology, ATP, and albumin levels for an additional 2 weeks in culture. Global microarray profiling and quantitative targeted proteomics assessing 10 important drug metabolizing enzymes and transporters demonstrated maintenance of mRNA and protein levels in spheroids over time. Activities for 5 major P450 enzymes were also stable and comparable to activities previously reported for human hepatocyte spheroids. Time- and concentration-dependent decreases in ATP and albumin were observed in response to the DILI-causing drugs acetaminophen, fialuridine, AMG-009, and tolvaptan. Collectively, our results demonstrate successful long-term culture of mouse hepatocytes as spheroids and their utility to support investigations of DILI.

Pregnancy-Related Hormones Increase Nifedipine Metabolism in Human Hepatocytes by Inducing CYP3A4 Expression.

Pregnancy-related hormones (PRH) have emerged as key regulators of hepatic cytochrome P450 (CYP) enzyme expression and function. The impact of PRH on protein levels of CYP3A4 and other key CYP enzymes, and the metabolism of nifedipine (a CYP3A4 substrate commonly prescribed during pregnancy), was evaluated in primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRH (estradiol, estriol, estetrol, progesterone, and cortisol), individually or in combination as a cocktail. Absolute protein concentrations of twelve CYP isoforms in SCHH membrane fractions were quantified by nanoLC-MS/MS, and metabolism of nifedipine to dehydronifedipine in SCHH was evaluated. PRH significantly increased CYP3A4 protein concentrations and nifedipine metabolism to dehydronifedipine in a concentration-dependent manner. CYP3A4 mRNA levels in hepatocyte-derived exosomes positively correlated with CYP3A4 protein levels and dehydronifedipine formation in SCHH. PRH also increased CYP2B6, CYP2C8 and CYP2A6 levels. Our findings demonstrate that PRH increase nifedipine metabolism in SCHH by inducing CYP3A4 expression and alter expression of other key CYP proteins in an isoform-specific manner, and suggest that hepatocyte-derived exosomes warrant further investigation as biomarkers of hepatic CYP3A4 metabolism. Together, these results offer mechanistic insight into the increases in nifedipine metabolism and clearance observed in pregnant women.

Understanding Idiosyncratic Toxicity: Lessons Learned from Drug-Induced Liver Injury.

Idiosyncratic adverse drug reactions (IADRs) encompass a diverse group of toxicities that can vary by drug and patient. The complex and unpredictable nature of IADRs combined with the fact that they are rare makes them particularly difficult to predict, diagnose, and treat. Common clinical characteristics, the identification of human leukocyte antigen risk alleles, and drug-induced proliferation of lymphocytes isolated from patients support a role for the adaptive immune system in the pathogenesis of IADRs. Significant evidence also suggests a requirement for direct, drug-induced stress, neoantigen formation, and stimulation of an innate response, which can be influenced by properties intrinsic to both the drug and the patient. This Perspective will provide an overview of the clinical profile, mechanisms, and risk factors underlying IADRs as well as new approaches to study these reactions, focusing on idiosyncratic drug-induced liver injury.

Identification of Candidate Risk Factor Genes for Human Idelalisib Toxicity Using a Collaborative Cross Approach.

Idelalisib is a phosphatidylinositol 3-kinase inhibitor highly selective for the delta isoform that has shown good efficacy in treating chronic lymphocytic leukemia and follicular lymphoma. In clinical trials, however, idelalisib was associated with rare, but potentially serious liver and lung toxicities. In this study, we used the Collaborative Cross (CC) mouse population to identify genetic factors associated with the drug response that may inform risk management strategies for idelalisib in humans. Eight male mice (4 matched pairs) from 50 CC lines were treated once daily for 14 days by oral gavage with either vehicle or idelalisib at a dose selected to achieve clinically relevant peak plasma concentrations (150 mg/kg/day). The drug was well tolerated across all CC lines, and there were no observations of overt liver injury. Differences across CC lines were seen in drug concentration in plasma samples collected at the approximate Tmax on study Days 1, 7, and 14. There were also small but statistically significant treatment-induced alterations in plasma total bile acids and microRNA-122, and these may indicate early hepatocellular stress required for immune-mediated hepatotoxicity in humans. Idelalisib treatment further induced significant elevations in the total cell count of terminal bronchoalveolar lavage fluid, which may be analogous to pneumonitis observed in the clinic. Genetic mapping identified loci associated with interim plasma idelalisib concentration and the other 3 treatment-related endpoints. Thirteen priority candidate quantitative trait genes identified in CC mice may now guide interrogation of risk factors for adverse drug responses associated with idelalisib in humans.

Bioprinted liver provides early insight into the role of Kupffer cells in TGF-ß1 and methotrexate-induced fibrogenesis.

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-ß1 and 0.209 µM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-ß1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.

Hepatocyte-Derived Exosomes Promote Liver Immune Tolerance: Possible Implications for Idiosyncratic Drug-Induced Liver Injury.

Most idiosyncratic drug-induced liver injury appears to result from an adaptive immune attack on the liver. Recent evidence suggests that the T-cell response may be facilitated by the loss of immune tolerance. In this study, we explored the hypothesis that constitutively released hepatocyte-derived exosomes (HDE) are important for maintaining normal liver immune tolerance. Exosomes were isolated from the conditioned medium of primary human hepatocytes via polymer precipitation. Mock controls were prepared by processing fresh medium that was not hepatocyte exposed with precipitation reagent. THP-1 monocytes were then treated with HDE or an equivalent volume of mock control for 24 h, followed by a 6-h stimulation with LPS. HDE exposure resulted in a significant decrease in the LPS-induced media levels of interleukin-1ß and interleukin-8. Gene expression profiling performed in THP-1 cells just prior to LPS-induced stimulation identified a significant decrease among genes associated with innate immune response. MicroRNA (miRNA) profiling was performed on the HDE to identify exosome contents that may drive immune suppression. Many of the predicted mRNA target genes for the most abundant microRNAs in HDE were among the differentially expressed genes in THP-1 cells. Taken together, our data suggest that HDE play a role in maintaining normal liver immune tolerance. Future experiments will explore the possibility that drugs causing idiosyncratic liver injury promote the loss of homeostatic HDE signaling.

Quantitative Systems Toxicology Analysis of In Vitro Mechanistic Assays Reveals Importance of Bile Acid Accumulation and Mitochondrial Dysfunction in TAK-875-Induced Liver Injury.

TAK-875 (fasiglifam), a GPR40 agonist in development for the treatment of type 2 diabetes (T2D), was voluntarily terminated in Phase III trials due to adverse liver effects. The potential mechanisms of TAK-875 toxicity were explored by combining in vitro experiments with quantitative systems toxicology (QST) using DILIsym, a mathematical representation of drug-induced liver injury. In vitro assays revealed that bile acid transporters were inhibited by both TAK-875 and its metabolite, TAK-875-Glu. Experimental data indicated that human bile salt export pump (BSEP) inhibition by TAK-875 was mixed whereas sodium taurocholate co-transporting polypeptide (NTCP) inhibition by TAK-875 was competitive. Furthermore, experimental data demonstrated that both TAK-875 and TAK-875-Glu inhibit mitochondrial electron transport chain (ETC) enzymes. These mechanistic data were combined with a physiologically based pharmacokinetic (PBPK) model constructed within DILIsym to estimate liver exposure of TAK-875 and TAK-875-Glu. In a simulated population (SimPops) constructed to reflect T2D patients, 16/245 (6.5%) simulated individuals developed alanine aminotransferase (ALT) elevations, an incidence similar to that observed with 200 mg daily dosing in clinical trials. Determining the mode of bile acid transporter inhibition (Ki) was critical to accurate predictions. In addition, simulations conducted on a sensitive subset of individuals (SimCohorts) revealed that when either BSEP or ETC inhibition was inactive, ALT elevations were not predicted to occur, suggesting that the two mechanisms operate synergistically to produce the observed clinical response. These results demonstrate how utilizing QST methods to interpret in vitro experimental results can lead to an improved understanding of the clinically relevant mechanisms underlying drug-induced toxicity.

Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion.

The composition of the beta-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of beta-cells and neurons substantially overlap. beta-Cells secrete gamma-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the beta-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in beta-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe beta-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 beta-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the beta-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.

Optimized Methods to Explore the Mechanistic and Biomarker Potential of Hepatocyte-Derived Exosomes in Drug-Induced Liver Injury.

Recent evidence supports that alterations in hepatocyte-derived exosomes (HDE) may play a role in the pathogenesis of drug-induced liver injury (DILI). HDE-based biomarkers also hold promise to improve the sensitivity of existing in vitro assays for predicting DILI liability. Primary human hepatocytes (PHH) provide a physiologically relevant in vitro model to explore the mechanistic and biomarker potential of HDE in DILI. However, optimal methods to study exosomes in this culture system have not been defined. Here we use HepG2 and HepaRG cells along with PHH to optimize methods for in vitro HDE research. We compared the quantity and purity of HDE enriched from HepG2 cell culture medium by 3 widely used methods: ultracentrifugation (UC), OptiPrep density gradient ultracentrifugation (ODG), and ExoQuick (EQ)-a commercially available exosome precipitation reagent. Although EQ resulted in the highest number of particles, UC resulted in more exosomes as indicated by the relative abundance of exosomal CD63 to cellular prohibitin-1 as well as the comparative absence of contaminating extravesicular material. To determine culture conditions that best supported exosome release, we also assessed the effect of Matrigel matrix overlay at concentrations ranging from 0 to 0.25 mg/ml in HepaRG cells and compared exosome release from fresh and cryopreserved PHH from same donor. Sandwich culture did not impair exosome release, and freshly prepared PHH yielded a higher number of HDE overall. Taken together, our data support the use of UC-based enrichment from fresh preparations of sandwich-cultured PHH for future studies of HDE in DILI.

Challenges and Solutions for Future Pharmacy Practice in the Era of Precision Medicine.

As precision medicine research and its clinical applications continue to advance, it is critical for pharmacists to be involved in these developments to deliver optimal, tailored drug therapies for patients. To ensure pharmacists remain leaders in the field, the annual Pharmaceutical Sciences Conference convened by the University of North Carolina at Chapel Hill Eshelman School of Pharmacy focused on the role of pharmacy within precision medicine. This is a summary of the conference, highlighting the major challenges and solutions that will help advance individualized pharmacological methods within practice and research.