Recent advances in the exploration of the bile salt export pump (BSEP/ABCB11) function

ntroduction: The bile salt export pump (BSEP/ABCB11), residing in the apical membrane of hepatocyte, mediates the secretion of bile salts into the bile. A range of human diseases is associated with the malfunction of BSEP, including fatal hereditary liver disorders and mild cholestatic conditions. Manifestation of these diseases primarily depends on the mutation type; however, other factors such as hormonal changes and drug interactions can also trigger or influence the related diseases. Areas covered: Here, we summarize the recent knowledge on BSEP by covering its transport properties, cellular localization, regulation and major mutations/polymorphisms, as well as the hereditary and acquired diseases associated with BSEP dysfunction. We discuss the different model expression systems employed to understand the function of the BSEP variants, their drug interactions and the contemporary therapeutic interventions. Expert opinion: The limitations of the available model expression systems for BSEP result in controversial conclusions, and obstruct our deeper insight into BSEP deficiencies and BSEP-related drug interactions. The knowledge originating from different methodologies, such as clinical studies, molecular genetics, as well as in vitro and in silico modeling, should be integrated and harmonized. Increasing availability of robust molecular biological tools and our better understanding of the mechanism of BSEP deficiencies should make the personalized, mutation-based therapeutic interventions more attainable

Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction

Idiosyncratic drug-induced liver injury (iDILI) encompasses the unexpected harms that pre- scription and non-prescription drugs, herbal and dietary supplements can cause to the liver. iDILI remains a major public health problem and a major cause of drug attrition. Given the lack of biomarkers for iDILI prediction, diagnosis and prognosis, searching new models to predict and study mechanisms of iDILI is necessary. One of the major limitations of iDILI preclinical assessment has been the lack of correlation between the markers of hepatotoxicity in animal toxicological studies and clinically significant iDILI. Thus, major advances in the understanding of iDILI susceptibility and pathogenesis have come from the study of well-phenotyped iDILI patients. However, there are many gaps for explaining all the complexity of iDILI susceptibility and mechanisms. Therefore, there is a need to optimize preclinical hu- man in vitro models to reduce the risk of iDILI during drug development. Here, the current experimental models and the future directions in iDILI modelling are thoroughly discussed, focusing on the human cellular models available to study the pathophysiological mechanisms of the disease and the most used in vivo animal iDILI models. We also comment about in silico approaches and the increasing relevance of patient-derived cellular models.This work was supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional-FEDER (contract numbers: PI18/01804, PI19-00883, PT20/00127, UMA18-FEDERJA-194, PY18-3364, Spain) and grants of Consejería de Salud de Andalucía cofounded by FEDER (contract number: PEMP-0127-2020, Spain). M.V.P. holds a Sara Borrell (CD21/00198, Spain) research contract from ISCIII and Consejería de Salud de Andalucía. C.L.G. holds a Juan de la Cierva Incorporación (IJCI-2017-31466, Spain) research contract from Ministerio de Ciencia del Gobierno de España. SCReN and CIBERehd are funded by ISCIII (Spain). This publication is based upon work from COST Action “CA17112dProspective European Drug-Induced Liver Injury Network” supported by COST (European Cooperation in Science and Technology); www.cost.eu. The figures in this review were created with Biorender.com

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI

An updated review on drug-induced cholestasis: Mechanisms and investigation of physicochemical properties and pharmacokinetic parameters

Drug-induced cholestasis is an important form of acquired liver disease and is associated with significant morbidity and mortality. Bile acids are key signaling molecules, but they can exert toxic responses when they accumulate in hepatocytes. This review focuses on the physiological mechanisms of drug-induced cholestasis associated with altered bile acid homeostasis due to direct (e.g. bile acid transporter inhibition) or indirect (e.g. activation of nuclear receptors, altered function/expression of bile acid transporters) processes. Mechanistic information about the effects of a drug on bile acid homeostasis is important when evaluating the cholestatic potential of a compound, but experimental data often are not available. The relationship between physicochemical properties, pharmacokinetic parameters, and inhibition of the bile salt export pump (BSEP) among seventy-seven cholestatic drugs with different pathophysiological mechanisms of cholestasis (i.e. impaired formation of bile vs. physical obstruction of bile flow) was investigated. The utility of in silico models to obtain mechanistic information about the impact of compounds on bile acid homeostasis to aid in predicting the cholestatic potential of drugs is highlighted

Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction

Idiosyncratic drug-induced liver injury (iDILI) encompasses the unexpected harms that prescription and non-prescription drugs, herbal and dietary supplements can cause to the liver. iDILI remains a major public health problem and a major cause of drug attrition. Given the lack of biomarkers for iDILI prediction, diagnosis and prognosis, searching new models to predict and study mechanisms of iDILI is necessary. One of the major limitations of iDILI preclinical assessment has been the lack of correlation between the markers of hepatotoxicity in animal toxicological studies and clinically significant iDILI. Thus, major advances in the understanding of iDILI susceptibility and pathogenesis have come from the study of well-phenotyped iDILI patients. However, there are many gaps for explaining all the complexity of iDILI susceptibility and mechanisms. Therefore, there is a need to optimize preclinical human in vitro models to reduce the risk of iDILI during drug development. Here, the current experimental models and the future directions in iDILI modelling are thoroughly discussed, focusing on the human cellular models available to study the pathophysiological mechanisms of the disease and the most used in vivo animal iDILI models. We also comment about in silico approaches and the increasing relevance of patient-derived cellular models.This work was supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional-FEDER (contract numbers: PI18/01804, PI19-00883, PT20/00127, 3714 Antonio Segovia-Zafra et al. UMA18-FEDERJA-194, PY18-3364, Spain) and grants of Consejeríaa de Salud de Andalucı ́a cofounded by FEDER (contractnumber: PEMP-0127-2020, Spain). M.V.P. holds a Sara Borrell (CD21/00198, Spain) research contract from ISCIII and Consejerí a de Salud de Andalucía. C.L.G. holds a Juan de la Cierva Incorporación (IJCI-2017-31466, Spain) research contract from Ministerio de Ciencia del Gobierno de España. SCReN and CIBERehd are funded by ISCIII (Spain). This publication is based upon work from COST Action “CA17112dProspective European Drug-Induced Liver Injury Network” supported by COST (European Cooperation in Science and Technology)Ye

Mechanisms of drug-induced liver injury: the role of hepatic transport proteins

The objectives of this research were to investigate mechanisms of drug-induced liver injury (DILI) that involve drug-bile acid (BA) interactions at hepatic transporters, and develop a novel strategy to reliably predict human DILI. Troglitazone (TGZ), an antidiabetic withdrawn from the market due to severe DILI, was employed as a model hepatotoxic drug. Pharmacokinetic modeling of taurocholic acid (TCA, a model BA) disposition data from human and rat sandwich-cultured hepatocytes (SCH) revealed that species differences exist in TCA hepatocellular efflux pathways; in human SCH, TCA biliary excretion predominated, whereas biliary and basolateral excretion contributed equally to TCA efflux in rat SCH. This finding explains, in part, why rats are less susceptible to DILI compared to humans after administration of drugs that inhibit BA biliary excretion. The present study also revealed for the first time that TGZ sulfate (TS), a major TGZ metabolite, inhibits BA basolateral efflux in addition to biliary excretion. These findings support the hypothesis that TS is an important mediator of altered hepatic BA disposition; increased hepatic TS exposure due to impaired canalicular transport function might predispose a subset of patients to hepatotoxicity. A novel in vitro model system, rat SCH lacking selected canalicular transporters [breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein 2 (Mrp2)] was established to test this hypothesis; biliary excretion of hepatically-generated TS was not significantly altered, suggesting that alternate transporters can excrete TS into bile, and loss of Bcrp and/or Mrp2 function would not necessarily be risk factors for increased hepatocellular TS accumulation in rats. To translate experimental data to in vivo humans, a mechanistic model that incorporated TGZ/TS disposition, BA physiology/pathophysiology, hepatocyte life cycle, and liver injury biomarkers was developed; intracellular BA concentrations and toxicity measured in SCH were used to link BA homeostasis and hepatotoxicity. This mechanistic model adequately predicted the incidence, delayed presentation, and species differences in TGZ hepatotoxicity. This dissertation research revealed a number of important and novel findings that improve our understanding about mechanisms underlying BA-mediated DILI, and establish a framework to integrate biological information and experimental data to evaluate DILI mechanisms and predict hepatotoxic potential of chemical entities.Doctor of Philosoph

Computational Approaches for Drug-Induced Liver Injury (DILI) Prediction: State of the Art and Challenges

Drug-induced liver injury (DILI) is one of the prevailing causes of fulminant hepatic failure. It is estimated that three idiosyncratic drug reactions out of four result in liver transplantation or death. Additionally, DILI is the most common reason for withdrawal of an approved drug from the market. Therefore, the development of methods for the early identification of hepatotoxic drug candidates is of crucial importance. This review focuses on the current state of cheminformatics strategies being applied for the early in silico prediction of DILI. Herein, we discuss key issues associated with DILI modelling in terms of the data size, imbalance and quality, complexity of mechanisms, and the different levels of hepatotoxicity to model going from general hepatotoxicity to the molecular initiating events of DILI

Study of the cholestatic potential of relevant concentrations of cyclosporine A in primary human hepatocyte spheroids

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.O fígado é o principal órgão responsável pelo metabolismo e excreção de compostos endógenos e exógenos, sendo o órgão mais exposto a compostos tóxicos. A colestase induzida por fármacos é causada pela acumulação tóxica de ácidos biliares nos hepatócitos, podendo a sua manifestação ter um início retardado. A colestase é uma patologia onde a secreção da bílis para o duodeno está reduzida, quer por reduzida funcionalidade dos hepatócitos, quer por obstrução da via secretória da bílis. O principal objetivo desta investigação era o estudo dos efeitos da ciclosporina A a longo termo em culturas de esferóides de hepatócitos primários humanos. Foi efetuado um estudo de dose-resposta durante 28 dias, onde as células eram expostas a diversas concentrações de fármaco. A viabilidade celular era medida pela quantificação do conteúdo total de ATP. Foi observado que a ciclosporina A era tóxica de forma dependente da concentração e tempo. Para além disso, foi estudado os efeitos sinérgicos entre a ciclosporina A e os ácidos biliares. Os esferóides eram expostos a ciclosporina A na presença e na ausência de ácidos biliares durante 28 dias. Os resultados sugeriram que os esferóides ficavam sensibilizados pela ciclosporina A na presença de ácidos biliares, confirmando o seu sinergismo. O índice colestático foi calculado para determinar que concentrações de ciclosporina A tinham o potencial de causar colestase. O índice colestático correspondia ao rácio entre o conteúdo de ATP dos hepatócitos na presença de ácidos biliares e o conteúdo de ATP dos hepatócitos na ausência de ácidos biliares. Valores inferiores ou iguais a 0.8 indicam que o composto tem o potencial de causar colestase. Os resultados demonstraram valores de índice colestático inferiores a 0.8, quando os esferóides estavam expostos a ciclosporina A a 5 μM, após 14 dias de exposição. De acordo com os resultados, a ciclosporina A foi considerada como exibindo potencial para induzir colestase. Mais estudos podem ser realizados nesta área, de forma a descobrir por quais mecanismos a ciclosporina A induz colestase, dado que estes ainda não são totalmente compreendidos. Ensaios de RT-qPCR e Western Blot podem ser executados para estudar a expressão génica e proteica, respetivamente.The liver is the major organ responsible for the metabolism and excretion of endogenous and exogenous compounds, being more exposed to toxic compounds than any organ. Drug-induced cholestasis is attributed to the toxic accumulation of bile acids inside the hepatocytes and its manifestation can be delayed in onset. Cholestasis is a pathology where bile secretion to duodenum is reduced, whether by reduced functionality of the hepatocytes or obstruction of the excretory pathway of bile. The main objective of this research was to study the long-term effects of cyclosporine A in primary human hepatocytes spheroid cultures. A dose-response assay was conducted for 28 days, where the cells were exposed to several drug concentrations. Cell viability was measured by quantification of the total ATP content. It was assessed that cyclosporine A was toxic in a dose and time dependent manner. Furthermore, it was studied the synergistic effects between cyclosporine A and bile acids. Spheroids were exposed to cyclosporine A in the presence and in the absence of bile acids for 28 days. Findings suggested that spheroids were sensitized to cyclosporine A in the presence of bile acids, therefore confirming their synergism. Cholestatic index was calculated to determine which cyclosporine A concentrations had the potential to cause cholestasis. Cholestatic index was the ratio between the ATP content of the hepatocytes in the presence of bile acids and the ATP content of the hepatocytes in the absence of bile acids. Values lower than or equal to 0.8 indicated that the compound had the potential to cause cholestasis. Results showed cholestatic index values lower than 0.8, when spheroids were exposed to 5 μM cyclosporine A after 14 days of drug exposure. According to the results, cyclosporine A was suggested to demonstrate potential to develop cholestasis. More studies should be done in this area to discover by which mechanisms cyclosporine A induces cholestasis, since it is not yet fully understood. This can be achieved by performing RT-qPCR and Western Blot assays to study gene and protein expression, respectively.Com o patrocínio da Vrije Universiteit Brussel