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Abstract Non-alcoholic steatohepatitis (NASH) is a major health problem leading to liver fibrosis and hepatocellular carcinoma, among other diseases, and for which there is still no approved drug treatment. Previous studies in animal models and in LX-2 cells have indicated a role for serotonin (5-HT) and 5-HT receptors in stellate cell activation and the development of NASH. In the current study, we investigated the extent to which these findings are applicable to a human NASH in vitro model consisting of human liver spheroids containing hepatocytes and non-parenchymal cells. Treatment of the spheroids with 5-HT or free fatty acids (FFA) induced fibrosis, whereas treatment of the spheroids with the 5-HT receptor antagonists ketanserin, pimavanserin, sarpogrelate, and SB269970 inhibited FFA-induced fibrosis via a reduction in stellate cell activation as determined by the expression of vimentin, TGF-β1 and COL1A1 production. siRNA-based silencing of 5-HT2A receptor expression reduced the anti-fibrotic properties of ketanserin, suggesting a role for 5-HT receptors in general and 5-HT2A receptors in particular in the FFA-mediated increase in fibrosis in the human liver spheroid model. The results suggest a contribution of the 5-HT receptors in the development of FFA-induced human liver fibrosis with implications for further efforts in drug development.

Background:. The prevalence of NAFLD is rapidly increasing. NAFLD can progress to NASH, fibrosis, cirrhosis, and HCC, which will soon become the main causes of liver transplantation. To date, no effective drug for NASH has been approved by the Food and Drug Administration. This is partly due to the lack of reliable human in vitro models. Here, we present a novel human liver spheroid model that can be used to study the mechanisms underlying liver fibrosis formation and degradation. Methods and Results:. Such spheroids, which contain hepatocytes, stellate cells, KC, and LSECs, spontaneously develop fibrosis that is exacerbated by treatment with free fatty acids. Conditioned medium from activated LSECs caused similar activation of fibrosis in spheroids containing primary human hepatocyte and NPCs, indicating the action of soluble mediators from the LSECs. Spheroids containing LSECs treated with free fatty acids produced tissue inhibitor of metalloproteinases inhibitor 1, a matrix metalloproteinases inhibitor important for fibrosis progression. Tissue inhibitor of metalloproteinases inhibitor 1 knockdown using siRNA led to a reduction in collagen and procollagen accumulation, which could be partially rescued using a potent matrix metalloproteinases inhibitor. Interestingly, tissue inhibitor of metalloproteinases inhibitor 1 was found to be expressed at higher levels, specifically in a subtype of endothelial cells in the pericentral region of human fibrotic livers, than in control livers. Conclusion:. Potential anti-NASH drugs and compounds were evaluated for their efficacy in reducing collagen accumulation, and we found differences in specificity between spheroids with and without LSECs. This new human NASH model may reveal novel mechanisms for the regulation of liver fibrosis and provide a more appropriate model for screening drugs against NASH.

Abstract Cytochrome P450 2D6 (CYP2D6) is important for metabolism of 20%–25% of all clinically used drugs. Many known genetic variants contribute to the large interindividual variability in CYP2D6 metabolism, but much is still unexplained. We recently described that nuclear factor 1B (NFIB) regulates hepatic CYP2D6 expression with the minor allele of NFIB rs28379954 T>C significantly increasing CYP2D6‐mediated risperidone metabolism. In this study, we investigated the effect of NFIB T>C on metabolism of solanidine, a dietary CYP2D6 substrate. Analyses of solanidine and metabolites (M414, M416, and M444) were performed by ultra‐high performance liquid chromatography‐high‐resolution mass spectrometry in a cohort of 463 CYP2D6‐genotyped patients of which with 58 (12.5%) carried NFIB TC (n = 56) or CC (n = 2). Increased metabolism of solanidine was found in CYP2D6 normal metabolizers (NMs; n = 258, 55.7%) carrying the NFIB C variant (n = 27, 5.8%) with 2.83‐ and 3.38‐fold higher M416‐to‐solanidine (p = 0.039) and M444‐to‐solanidine (p = 0.046) ratios, respectively, whereas this effect was not significant among intermediate metabolizers (n = 166, 35.9%) (p ≥ 0.09). Importantly, no effect of the NFIB polymorphism on solanidine metabolism was seen in TC or CC carriers lacking CYP2D6 activity (poor metabolizers, n = 30, 6.5%, p ≥ 0.74). Furthermore, the NFIB polymorphism significantly explained variability in solanidine metabolism (M414 p = 0.013, M416 p = 0.020, and M416 and M444 p = 0.009) in multiple linear regression models for each metabolic ratio in the entire population, correcting for covariates (including CYP2D6 genotypes). Thus, the study confirms the effect of NFIB in regulating CYP2D6 activity, suggesting an about 200% increase in CYP2D6‐mediated clearance in NMs being NFIB CT or CC carriers, comprising around 6% of Europeans.

Abstract Clinical response of clozapine is closely associated with serum concentration. Although tobacco smoking is the key environmental factor underlying interindividual variability in clozapine metabolism, recent genome‐wide studies suggest that CYP1A and NFIB genetic variants may also be of significant importance, but their quantitative impact is unclear. We investigated the effects of the rs2472297 C>T (CYP1A) and rs28379954 T>C (NFIB) polymorphisms on serum concentrations in smokers and nonsmokers. The study retrospectively included 526 patients with known smoking habits (63.7% smokers) from a therapeutic drug monitoring service in Norway. Clozapine dose‐adjusted concentrations (C/D) and patient proportions with subtherapeutic levels (

Abstract Sertraline is a commonly used SSRI antidepressant drug, metabolized by CYP2C19 and CYP2B6, that exhibits a substantial interindividual variation in clinical response, of which only a part can be attributed to known genetic variants. In the current study we have examined the role of a newly discovered ultrarapid CYP2C:TG haplotype and CYP2B6 variants in order to identify the possible missing heritability for such variation in sertraline response in a large patient population (n = 840). Compared to the reference group (CYP2C19*1/*1, n = 160), sertraline exposure was increased by 128% in CYP2C19 PMs (n = 29, p

Connective tissue growth factor (CTGF) is involved in the regulation of extracellular matrix (ECM) production. Elevated levels of CTGF can be found in plasma from patients with liver fibrosis and in experimental animal models of liver fibrosis, but the exact role of CTGF in, e.g., diet-induced human liver fibrosis is not entirely known. To address this question, we utilized a 3D human liver co-culture spheroid model composed of hepatocytes and non-parenchymal cells, in which fibrosis is induced by TGF-β1, CTGF or free fatty acids (FFA). Treatment of the spheroids with TGF-β1 or FFA increased COL1A1 deposition as well as the expression of TGF-β1 and CTGF. Recombinant CTGF, as well as angiotensin II, caused increased expression and/or production of CTGF, TGF-β1, COL1A1, LOX, and IL-6. In addition, silencing of CTGF reduced both TGF-β1- and FFA-induced COL1A1 deposition. Furthermore, we found that IL-6 induced CTGF, COL1A1 and TGF-β1 production, suggesting that IL-6 is a mediator in the pathway of CTGF-induced fibrosis. Taken together, our data indicate a specific role for CTGF and CTGF downstream signaling pathways for the development of liver inflammation and fibrosis in the human 3D liver spheroid model.

The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in response to toxicity and that more focus should be placed on alterations of ECM-derived protein expression as biomarkers of liver disease and chronic drug-induced hepatotoxicity, changes that can be studied in stable in vivo-like hepatic cell systems, such as the spheroids.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Using pharmacogenetics in guiding drug therapy experiences a steady increase in uptake, although still leads to discussions as to its clinical use. Psychiatry constitutes a field where pharmacogenomic testing might help in guiding drug therapy. To address current challenges, this minireview provides an update regarding genotyping (SNP analysis/arrays/NGS), structural variant detection (star-alleles/CNVs/hybrid alleles), genotype-to-phenotype translations, cost-effectiveness, and actionability of results (FDA/CPIC/PharmGKB) regarding clinical importance of pre-emptive pharmacogenomic testing for prescription of antidepressants and antipsychotics.

Abstract The liver is a highly regenerative organ. While mature hepatocytes under homeostatic conditions are largely quiescent, upon injury, they rapidly enter the cell cycle to recover the damaged tissue. In rodents, a variety of injury models have provided important insights into the molecular underpinnings that govern the proliferative activation of quiescent hepatocytes. However, little is known about the molecular mechanisms of human hepatocyte regeneration and experimental methods to expand primary human hepatocytes (PHH). Here, a 3D spheroid model of PHH is established to study hepatocyte regeneration and integrative time‐lapse multi‐omics analyses show that upon isolation from the native liver PHH acquire a regenerative phenotype, as seen in vivo upon partial hepatectomy. However, proliferation is limited. By analyzing global promoter motif activities, it is predicted that activation of Wnt/β‐catenin and inhibition of p53 signaling are critical factors required for human hepatocyte proliferation. Functional validations reveal that activation of Wnt signaling through external cues alone is sufficient to inhibit p53 and its proliferative senescence‐inducing target PAI1 (SERPINE1) and drive proliferation of >50% of all PHH. A scalable 3D culture model is established to study the molecular and cellular biology of human hepatocyte regeneration. By using this model, an essential role of Wnt/β‐catenin signaling during human hepatocyte regeneration is identified.

In recent decades, very few new psychiatric drugs have entered the market. Thus, improvement in the use of antidepressant and antipsychotic therapy has to focus mainly on enhanced and more personalized treatment with the currently available drugs. One important aspect of such individualization is emphasizing interindividual differences in genes relevant to treatment, an area that can be termed neuropsychopharmacogenomics. Here, we review previous efforts to identify such critical genetic variants and summarize the results obtained to date. We conclude that most clinically relevant genetic variation is connected to phase I drug metabolism, in particular to genetic polymorphism of CYP2C19 and CYP2D6. To further improve individualized pharmacotherapy, there is a need to take both common and rare relevant mutations into consideration; we discuss the present and future possibilities of using whole genome sequencing to identify patient-specific genetic variation relevant to treatment in psychiatry. Translation of pharmacogenomic knowledge into clinical practice can be considered for specific drugs, but this requires education of clinicians, instructive guidelines, as well as full attention to polypharmacy and other clinically relevant factors. Recent large patient studies (n > 1,000) have replicated previous findings and produced robust evidence warranting the clinical utility of relevant genetic biomarkers. To further judge the clinical and financial benefits of preemptive genotyping in psychiatry, large prospective randomized trials are needed to quantify the value of genetic-based patient stratification in neuropsychopharmacotherapy and to demonstrate the cost-effectiveness of such interventions.

Abstract Background Variability in genes implicated in drug pharmacokinetics or drug response can modulate treatment efficacy or predispose to adverse drug reactions. Besides common genetic polymorphisms, recent sequencing projects revealed a plethora of rare genetic variants in genes encoding proteins involved in drug metabolism, transport, and response. Results To understand the global importance of rare pharmacogenetic gene variants, we mapped the variability in 208 pharmacogenes by analyzing exome sequencing data from 60,706 unrelated individuals and estimated the importance of rare and common genetic variants using a computational prediction framework optimized for pharmacogenetic assessments. Our analyses reveal that rare pharmacogenetic variants were strongly enriched in mutations predicted to cause functional alterations. For more than half of the pharmacogenes, rare variants account for the entire genetic variability. Each individual harbored on average a total of 40.6 putatively functional variants, rare variants accounting for 10.8% of these. Overall, the contribution of rare variants was found to be highly gene- and drug-specific. Using warfarin, simvastatin, voriconazole, olanzapine, and irinotecan as examples, we conclude that rare genetic variants likely account for a substantial part of the unexplained inter-individual differences in drug metabolism phenotypes. Conclusions Combined, our data reveal high gene and drug specificity in the contributions of rare variants. We provide a proof-of-concept on how this information can be utilized to pinpoint genes for which sequencing-based genotyping can add important information to predict drug response, which provides useful information for the design of clinical trials in drug development and the personalization of pharmacological treatment.

CYP2E1 is one of the fifty-seven cytochrome P450 genes in the human genome and is highly conserved. CYP2E1 is a unique P450 enzyme because its heme iron is constitutively in the high spin state, allowing direct reduction of, e.g., dioxygen, causing the formation of a variety of reactive oxygen species and reduction of xenobiotics to toxic products. The CYP2E1 enzyme has been the focus of scientific interest due to (i) its important endogenous function in liver homeostasis, (ii) its ability to activate procarcinogens and to convert certain drugs, e.g., paracetamol and anesthetics, to cytotoxic end products, (iii) its unique ability to effectively reduce dioxygen to radical species causing liver injury, (iv) its capability to reduce compounds, often generating radical intermediates of direct toxic or indirect immunotoxic properties and (v) its contribution to the development of alcoholic liver disease, steatosis and NASH. In this overview, we present the discovery of the enzyme and studies in humans, 3D liver systems and genetically modified mice to disclose its function and clinical relevance. Induction of the CYP2E1 enzyme either by alcohol or high-fat diet leads to increased severity of liver pathology and likelihood to develop ALD and NASH, with subsequent influence on the occurrence of hepatocellular cancer. Thus, fat-dependent induction of the enzyme might provide a link between steatosis and fibrosis in the liver. We conclude that CYP2E1 has many important physiological functions and is a key enzyme for hepatic carcinogenesis, drug toxicity and liver disease.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2–6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research.

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

Hepatotoxicity is one of the most cited reasons for withdrawal of approved drugs from the market. The use of nonclinically relevant in vitro and in vivo testing systems contributes to the high attrition rates. Recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into pure cultures of hepatocyte-like cells expressing functional drug metabolizing enzymes open up possibilities for novel, more relevant human cell based toxicity models. The present study aimed to investigate the use of hiPSC derived hepatocytes for conducting mechanistic toxicity testing by image based high content analysis (HCA). The hiPSC derived hepatocytes were exposed to drugs known to cause hepatotoxicity through steatosis and phospholipidosis, measuring several endpoints representing different mechanisms involved in drug induced hepatotoxicity. The hiPSC derived hepatocytes were benchmarked to the HepG2 cell line and generated robust HCA data with low imprecision between plates and batches. The different parameters measured were detected at subcytotoxic concentrations and the order of which the compounds were categorized (as severe, moderate, mild, or nontoxic) based on the degree of injury at isomolar concentration corresponded to previously published data. Taken together, the present study shows how hiPSC derived hepatocytes can be used as a platform for screening drug induced hepatotoxicity by HCA.

Human cytochome P450 2W1 (CYP2W1) enzyme is expressed in fetal colon and in colon tumors. The level of expression is higher in colon metastases than in the parent tumors and the enzyme is a possible drug target for treatment of colorectal cancer, as demonstrated in mouse xenograft studies. A previous study published in this journal reported that CYP2W1 is highly expressed in normal and transformed adrenal tissue. However, adrenal expression of CYP2W1 protein was not seen in previous studies in our research group. To clarify this inconsistency, we have used qRT-PCR and Western blotting with CYP2W1-specific antibodies to probe a panel of 27 adrenocortical carcinomas and 35 normal adrenal cortex samples. CYP2W1 mRNA expression is seen in all samples. However, significant CYP2W1 protein expression was found in only one tumor sample (a testosterone-producing adrenocortical carcinoma) and not in any normal tissue. Differences in the specificity of the CYP2W1 antibodies used in the two studies may explain the apparent discrepancy. We conclude that normal adrenal tissue lacks P450 2W1 enzyme expression; also, adrenocortical carcinomas generally do not express the enzyme. This information thus underline the colon cancer specificity of CYP2W1 enzyme expression and has implications for the development of anti-colon cancer therapies based on CYP2W1 as a drug target, since 2W1-dependent bioactivation of prodrugs for CYP2W1 will not take place in normal adrenal tissue or other non-transformed tissues.

Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of N-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown. Here we demonstrate a specific developmental pattern of expression of these enzymes. mARC1, but not mARC2, was found to be expressed in fetal human liver, whereas both, in particular mARC2, are abundant in adult liver and also expressed in omental and subcutaneous fat. Caloric diet restriction of obese patients caused a decreased expression of mARC2 in liver, similar to that seen in the livers of starved rats. Knock down of mARC2 expression by siRNA in murine adipocytes had statistically significant effect on the level of diglycerides and on the fatty acid composition of some triglycerides, concomitantly a clear trend toward the reduced formation of most of triglyceride and phospholipid species was observed. The involvement of mARC2 in the metabolism of the hepatotoxic drug ximelagatran was evaluated in hepatocytes and adipocytes. Ximelagatran was shown to cause oxidative stress and knock down of mARC2 in adipocytes prevented ximelagatran induced inhibition of mitochondrial respiration. In conclusion, our data indicate that mARC1 and mARC2 have different developmental expression profiles, and that mARC2 is involved in lipogenesis, is regulated by nutritional status and responsible for activation of ximelagatran into a mitotoxic metabolite(s).

Responses to drugs and pharmacological treatments differ considerably between individuals. Importantly, only 50%–75% of patients have been shown to react adequately to pharmacological interventions, whereas the others experience either a lack of efficacy or suffer from adverse events. The liver is of central importance in the metabolism of most drugs. Because of this exposed status, hepatotoxicity is amongst the most common adverse drug reactions and hepatic liabilities are the most prevalent reason for the termination of development programs of novel drug candidates. In recent years, more and more factors were unveiled that shape hepatic drug responses and thus underlie the observed inter-individual variability. In this review, we provide a comprehensive overview of different principle mechanisms of drug hepatotoxicity and illustrate how patient-specific factors, such as genetic, physiological and environmental factors, can shape drug responses. Furthermore, we highlight other parameters, such as concomitantly prescribed medications or liver diseases and how they modulate drug toxicity, pharmacokinetics and dynamics. Finally, we discuss recent progress in the field of in vitro toxicity models and evaluate their utility in reflecting patient-specific factors to study inter-individual differences in drug response and toxicity, as this understanding is necessary to pave the way for a patient-adjusted medicine.