Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations.

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model's functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12's robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10-14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development.

Application of Immunocompetent Microphysiological Systems in Drug Development: Current Perspective and Recommendations.

Immune responses are heavily involved in the regulation and pathogenesis of human diseases, including infectious diseases, inflammatory and autoimmune conditions, cancer, neurological disorders, and cardiometabolic syndromes. The immune system is considered a double-edged sword serving as a powerful host defense mechanism against infection and cancerous cells and causing detrimental tissue damage when the immune response is exaggerated or uncontrollable. One of the challenges in studying the efficacy and toxicity of drugs that target or modulate the immune system is the lack of suitable preclinical human models that are predictive of human response. Recent advancements in human microphysiological systems (MPS) have provided a promising in vitro platform to evaluate the response of immune organs ex vivo, to investigate the interaction of immune cells with non-lymphoid tissue cells, and to reduce the reliance on animals in preclinical studies. The development, regulation, trafficking, and responses of immune cells have been extensively studied in preclinical animal models and clinically, providing a wealth of knowledge by which to evaluate new in vitro models. Therefore, the application of immunocompetent MPS in drug discovery and development should first verify that the immune response in an MPS model recapitulates the complexity of the human immune physiology. This manuscript reviews biological functions of immune organ systems and tissue-resident immune cells and discusses contexts-of-use for commonly used immunocompetent and immune organ MPS models. Current perspective and recommendations are provided to guide the continued development of immune organ and immunocompetent MPS models and their application in drug discovery and development.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

Gut-Selective Design of Orally Administered Izencitinib (TD-1473) Limits Systemic Exposure and Effects of Janus Kinase Inhibition in Nonclinical Species.

Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2-6× plasma Cave:JAK IC50 ratio, 10-33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.

Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Challenging the pipeline.

This commentary presents a thought experiment seeking to answer the key question: "If you were to put aside all the traditional drug discovery processes and start a new drug discovery program that places the highest priority on human and disease-relevant models throughout the entire process, how could it be done?"

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague-Dawley Rats.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague-Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

Drug-induced skin toxicity: gaps in preclinical testing cascade as opportunities for complex in vitro models and assays.

Skin is the largest organ of the body and serves as the principle barrier to the environment. Composed of multiple cell types arranged in stratified layers with highly specialized appendages, it serves sensory and immune surveillance roles in addition to its primary mechanical function. Several complex in vitro models of skin (i.e. microphysiological systems (MPS) including but not limited to 3D tissues, organ-on-a-chip, organoids), have been developed and assays validated for regulatory purposes. As such, skin is arguably the most advanced organ with respect to model development and adoption across industries including chemical, cosmetic, and to a somewhat lesser extent, pharmaceutical. Early adoption of complex skin models and associated assays for assessment of irritation and corrosion spurred research into other areas such as sensitization, absorption, phototoxicity, and genotoxicity. Despite such considerable advancements, opportunities remain for immune capabilities, inclusion of appendages such as hair follicles, fluidics, and innervation, among others. Herein, we provide an overview of current complex skin model capabilities and limitations within the drug development scheme, and recommendations for future model development and assay qualification and/or validation with the intent to facilitate wider adoption of use within the pharmaceutical industry.

Folate receptor-beta expression as a diagnostic target in human & rodent nonalcoholic steatohepatitis.

INTRODUCTION: Nonalcoholic steatohepatitis (NASH) afflicts 20-36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-ß) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-ß protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-ß imaging agent to the liver of a rodent NASH model using FR-ß. METHODS: Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-ß protein. The FR-ß-targeted agent, Etarfolatide was injected into MCD and MC + -fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-ß expression across the stages of human NAFLD from normal to NASH was assessed. RESULTS: FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8 weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-ß in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-ß protein when compared to normal liver. FR-ß transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples. CONCLUSION: The findings in this study indicate that FR-ß expression in NASH may be harnessed to target agents directly to the liver.

Impaired N-linked glycosylation of uptake and efflux transporters in human non-alcoholic fatty liver disease.

BACKGROUND & AIMS: N-linked glycosylation of proteins is critical for proper protein folding and trafficking to the plasma membrane. Drug transporters are one class of proteins that have reduced function when glycosylation is impaired. N-linked glycosylation of plasma proteins has also been investigated as a biomarker for several liver diseases, including non-alcoholic fatty liver disease (NAFLD). The purpose of this study was to assess the transcriptomic expression of genes involved in protein processing and glycosylation, and to determine the glycosylation status of key drug transporters during human NAFLD progression. METHODS: Human liver samples diagnosed as healthy, steatosis, and non-alcoholic steatohepatitis (NASH) were analysed for gene expression of glycosylation-related genes and for protein glycosylation using immunoblot. RESULTS: Genes involved in protein processing in the ER and biosynthesis of N-glycans were significantly enriched for down-regulation in NAFLD progression. Included in the down regulated N-glycan biosynthesis category were genes involved in the oligosaccharyltransferase complex, N-glycan quality control, N-glycan precursor biosynthesis, N-glycan trimming to the core, and N-glycan extension from the core. N-glycan degradation genes were unaltered in the progression to NASH. Immunoblot analysis of the uptake transporters organic anion transporting polypeptide-1B1 (OATP1B1), OATP1B3, OATP2B1, and Sodium/Taurocholate Co-transporting Polypeptide (NTCP) and the efflux transporter multidrug resistance-associated protein 2 (MRP2) demonstrated a significant loss of glycosylation following the progression to NASH. CONCLUSIONS: These data suggest that the loss of glycosylation of key uptake and efflux transporters in humans NASH may influence transporter function and contribute to altered drug disposition observed in NASH.

Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.

Renal xenobiotic transporter expression is altered in multiple experimental models of nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease is the most common chronic liver disease, which can progress to nonalcoholic steatohepatitis (NASH). Previous investigations demonstrated alterations in the expression and activity of hepatic drug transporters in NASH. Moreover, studies using rodent models of cholestasis suggest that compensatory changes in kidney transporter expression occur to facilitate renal excretion during states of hepatic stress; however, little information is currently known regarding extrahepatic regulation of drug transporters in NASH. The purpose of the current study was to investigate the possibility of renal drug transporter regulation in NASH across multiple experimental rodent models. Both rat and mouse NASH models were used in this investigation and include: the methionine and choline-deficient (MCD) diet, atherogenic diet, fa/fa rat, ob/ob and db/db mice. Histologic and pathologic evaluations confirmed that the MCD and atherogenic rats as well as the ob/ob and db/db mice all developed NASH. In contrast, the fa/fa rats did not develop NASH but did develop extensive renal injury compared with the other models. Renal mRNA and protein analyses of xenobiotic transporters suggest that compensatory changes occur in NASH to favor increased xenobiotic secretion. Specifically, both apical efflux and basolateral uptake transporters are induced, whereas apical uptake transporter expression is repressed. These results suggest that NASH may alter the expression and potentially function of renal drug transporters, thereby impacting drug elimination mechanisms in the kidney.

Increased susceptibility to methotrexate-induced toxicity in nonalcoholic steatohepatitis.

Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1-4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40-100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.

Systems level metabolic phenotype of methotrexate administration in the context of non-alcoholic steatohepatitis in the rat.

Adverse drug reactions (ADRs) represent a significant clinical challenge with respect to patient morbidity and mortality. We investigated the hepatotoxicity and systems level metabolic phenotype of methotrexate (MTX) in the context of a prevalent liver disease; non-alcoholic steatohepatitis (NASH). A nuclear magnetic resonance spectroscopic-based metabonomic approach was employed to analyze the metabolic consequences of MTX (0, 10, 40, and 100 mg/kg) in the urine and liver of healthy rats (control diet) and in a model of NASH (methionine-choline deficient diet). Histopathological analysis confirmed baseline (0 mg/kg) liver necrosis, liver inflammation, and lipid accumulation in the NASH model. Administration of MTX (40 and 100 mg/kg) led to liver necrosis in the control cohort, whereas the NASH cohort also displayed biliary hyperplasia and liver fibrosis (100 mg/kg), providing evidence of the synergistic effect of MTX and NASH. The complementary hepatic and urinary metabolic phenotypes of the NASH model, at baseline, revealed perturbation of multiple metabolites associated with oxidative and energetic stress, and folate homeostasis. Administration of MTX in both diet cohorts showed dose-dependent metabolic consequences affecting gut microbial, energy, nucleobase, nucleoside, and folate metabolism. Furthermore, a unique panel of metabolic changes reflective of the synergistic effect of MTX and NASH was identified, including the elevation of hepatic phenylalanine, urocanate, acetate, and both urinary and hepatic formiminoglutamic acid. This systems level metabonomic analysis of the hepatotoxicity of MTX in the context of NASH provided novel mechanistic insight of potential wider clinical relevance for further understanding the role of liver pathology as a risk factor for ADRs.

Synergistic interaction between genetics and disease on pravastatin disposition.

BACKGROUND & AIMS: A genome wide association study and multiple pharmacogenetic studies have implicated the hepatic uptake transporter organic anion transporting polypeptide-1B1 (OATP1B1) in the pharmacokinetics and musculoskeletal toxicity of statin drugs. Other OATP uptake transporters can participate in the transport of pravastatin, partially compensating for the loss of OATP1B1 in patients carrying the polymorphism. Non-alcoholic steatohepatitis (NASH) in humans and in a diet-induced rodent model alter the expression of multiple OATP transporters. METHODS: To determine how genetic alteration in one Oatp transporter can interact with NASH-associated changes in Oatp expression we measured the disposition of intravenously administered pravastatin in Slco1b2 knockout (Slco1b2(-/-)) and wild-type (WT) mice fed either a control or a methionine and choline deficient (MCD) diet to induce NASH. RESULTS: Genetic loss of Oatp1b2, the rodent ortholog of human OATP1B transporters, caused a modest increase in pravastatin plasma concentrations in mice with healthy livers. Although a diet-induced model of NASH decreased the expression of multiple hepatic Oatp transporters, it did not alter the disposition of pravastatin compared to WT control mice. In contrast, the combination of NASH-associated decrease in compensatory Oatp transporters and Oatp1b2 genetic loss caused a synergistic increase in plasma area under the curve (AUC) and tissue concentrations in kidney and muscle. CONCLUSIONS: Our data show that NASH alters the expression of multiple hepatic uptake transporters which, due to overlapping substrate specificity among the OATP transporters, may combine with the pharmacogenetic loss of OATP1B1 to increase the risk of statin-induced adverse drug reactions.

An experimental approach to evaluate the impact of impaired transport function on hepatobiliary drug disposition using Mrp2-deficient TR- rat sandwich-cultured hepatocytes in combination with Bcrp knockdown.

Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR(-)) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1-10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR(-) status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR(-) rat SCH decreased by 28.6 (95% CI: 5.8-51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5-9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR(-) rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR(-) rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0-12.8) and 7.5 (3.7-11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.

Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models.

Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.

Experimental nonalcoholic steatohepatitis increases exposure to simvastatin hydroxy acid by decreasing hepatic organic anion transporting polypeptide expression.

Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy.