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Here I detail the use of measures of permeability rate and solubility in predicting drug disposition characteristics through the utilization of the Biopharmaceutics Drug Disposition Classification System (BDDCS) and the Extended Clearance Classification System (ECCS) as well as the accuracy of the systems in predicting the major route of elimination and the extent of oral absorption of a new small molecule therapeutics. I compare the BDDCS and ECCS with the FDA Biopharmaceutics Classification System (BCS). I also detail the use of the BCS in predicting food effects and the BDDCS in predicting brain disposition of small molecule therapeutics and in validating DILI predictive metrics. This review provides an update of the current status of these classification systems and their uses in the drug development process.

The Biopharmaceutics Drug Disposition Classification system (BDDCS) is a four-class approach based on water solubility and extent of metabolism/permeability rate. Based on the BDDCS class to which a drug is assigned, it is possible to predict the role of metabolic enzymes and transporters on the drug disposition of a new molecular entity (NME) prior to its administration to animals or humans. Here, we report a total of 1475 drugs and active metabolites to which the BDDCS is applied. Of these, 379 are new entries, and 1096 are revisions of former classification studies with the addition of references for the approved maximum dose strength, extent of the systemically available drug excreted unchanged in the urine, and lowest solubility over the pH range 1.0-6.8 when such information is available in the literature. We detail revised class assignments of previously misclassified drugs and the literature analyses to classify new drugs. We review the process of solubility assessment for NMEs prior to drug dosing in humans and approved dose classification, as well as the comparison of Biopharmaceutics Classification System (BCS) versus BDDCS assignment. We detail the uses of BDDCS in predicting, prior to dosing animals or humans, disposition characteristics, potential brain penetration, food effect, and drug-induced liver injury (DILI) potential. This work provides an update on the current status of the BDDCS and its uses in the drug development process.

Introduction: Apart from cessation of the implicated agent leading to drug-induced liver injury (DILI), there is no standard therapy for DILI. Corticosteroids have been used in DILI, although their efficacy is unclear. Published data showed either beneficial effects or no improvement associated with steroid therapy. The aim of the current study was to perform a systematic review of the role of corticosteroids in the treatment of DILI. Methods: A search was performed in PubMed, searching for the terms: “corticosteroids” and “drug-induced liver injury”. Observation studies were included, but case reports excluded. Results: A total of 24 papers were retrieved. Most of these were observational studies on the effects of corticosteroids in moderate/severe DILI (n = 8), reports on the corticosteroid treatment in patients with drug-induced autoimmune hepatitis (DI-AIH) (n = 5), and effects of corticosteroids in drug-induced fulminant acute liver failure (ALF, n = 2). Furthermore, treatment of corticosteroids in patients with liver injury due to check point inhibitors (CPIs) was addressed in nine studies. In moderate/severe DILI, six out of eight studies suggested steroid treatment to be beneficial, whereas two studies showed negative results. All five observational studies on the effects of corticosteroids in DI-AIH showed good therapeutic response with rapid and long lasting effects after discontinuation of corticosteroids and without evidence of relapse. Steroid therapy was not associated with improved overall survival in patients with drug-induced fulminant ALF. CPIs-induced liver injury was found to improve spontaneously in 33–50% without corticosteroids, and the rate of patients who were treated responded to steroids in 33–100% (mean 72%). Conclusions: The majority of studies analyzing the effects of corticosteroids in moderate/severe DILI have demonstrated beneficial effects. However, this was not the case in drug-induced fulminant ALF. Patients with DI-AIH had an exc

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and pri

The Biopharmaceutics Drug Disposition Classification system (BDDCS) is a four-class approach based on water solubility and extent of metabolism/permeability rate. Based on the BDDCS class to which a drug is assigned, it is possible to predict the role of metabolic enzymes and transporters on the drug disposition of a new molecular entity (NME) prior to its administration to animals or humans. Here, we report a total of 1475 drugs and active metabolites to which the BDDCS is applied. Of these, 379 are new entries, and 1096 are revisions of former classification studies with the addition of references for the approved maximum dose strength, extent of the systemically available drug excreted unchanged in the urine, and lowest solubility over the pH range 1.0-6.8 when such information is available in the literature. We detail revised class assignments of previously misclassified drugs and the literature analyses to classify new drugs. We review the process of solubility assessment for NMEs prior to drug dosing in humans and approved dose classification, as well as the comparison of Biopharmaceutics Classification System (BCS) versus BDDCS assignment. We detail the uses of BDDCS in predicting, prior to dosing animals or humans, disposition characteristics, potential brain penetration, food effect, and drug-induced liver injury (DILI) potential. This work provides an update on the current status of the BDDCS and its uses in the drug development process.

The Biopharmaceutics Drug Disposition Classification system (BDDCS) is a four-class approach based on water solubility and extent of metabolism/permeability rate. Based on the BDDCS class to which a drug is assigned, it is possible to predict the role of metabolic enzymes and transporters on the drug disposition of a new molecular entity (NME) prior to its administration to animals or humans. Here, we report a total of 1475 drugs and active metabolites to which the BDDCS is applied. Of these, 379 are new entries, and 1096 are revisions of former classification studies with the addition of references for the approved maximum dose strength, extent of the systemically available drug excreted unchanged in the urine, and lowest solubility over the pH range 1.0–6.8 when such information is available in the literature. We detail revised class assignments of previously misclassified drugs and the literature analyses to classify new drugs. We review the process of solubility assessment for NMEs prior to drug dosing in humans and approved dose classification, as well as the comparison of Biopharmaceutics Classification System (BCS) versus BDDCS assignment. We detail the uses of BDDCS in predicting, prior to dosing animals or humans, disposition characteristics, potential brain penetration, food effect, and drug-induced liver injury (DILI) potential. This work provides an update on the current status of the BDDCS and its uses in the drug development process. Graphical abstract: [Figure not available: see fulltext.]

Introduction: Apart from cessation of the implicated agent leading to drug-induced liver injury (DILI), there is no standard therapy for DILI. Corticosteroids have been used in DILI, although their efficacy is unclear. Published data showed either beneficial effects or no improvement associated with steroid therapy. The aim of the current study was to perform a systematic review of the role of corticosteroids in the treatment of DILI. Methods: A search was performed in PubMed, searching for the terms: “corticosteroids” and “drug-induced liver injury”. Observation studies were included, but case reports excluded. Results: A total of 24 papers were retrieved. Most of these were observational studies on the effects of corticosteroids in moderate/severe DILI (n = 8), reports on the corticosteroid treatment in patients with drug-induced autoimmune hepatitis (DI-AIH) (n = 5), and effects of corticosteroids in drug-induced fulminant acute liver failure (ALF, n = 2). Furthermore, treatment of corticosteroids in patients with liver injury due to check point inhibitors (CPIs) was addressed in nine studies. In moderate/severe DILI, six out of eight studies suggested steroid treatment to be beneficial, whereas two studies showed negative results. All five observational studies on the effects of corticosteroids in DI-AIH showed good therapeutic response with rapid and long lasting effects after discontinuation of corticosteroids and without evidence of relapse. Steroid therapy was not associated with improved overall survival in patients with drug-induced fulminant ALF. CPIs-induced liver injury was found to improve spontaneously in 33–50% without corticosteroids, and the rate of patients who were treated responded to steroids in 33–100% (mean 72%). Conclusions: The majority of studies analyzing the effects of corticosteroids in moderate/severe DILI have demonstrated beneficial effects. However, this was not the case in drug-induced fulminant ALF. Patients with DI-AIH had an exc

The Biopharmaceutics Drug Disposition Classification system (BDDCS) is a four-class approach based on water solubility and extent of metabolism/permeability rate. Based on the BDDCS class to which a drug is assigned, it is possible to predict the role of metabolic enzymes and transporters on the drug disposition of a new molecular entity (NME) prior to its administration to animals or humans. Here, we report a total of 1475 drugs and active metabolites to which the BDDCS is applied. Of these, 379 are new entries, and 1096 are revisions of former classification studies with the addition of references for the approved maximum dose strength, extent of the systemically available drug excreted unchanged in the urine, and lowest solubility over the pH range 1.0–6.8 when such information is available in the literature. We detail revised class assignments of previously misclassified drugs and the literature analyses to classify new drugs. We review the process of solubility assessment for NMEs prior to drug dosing in humans and approved dose classification, as well as the comparison of Biopharmaceutics Classification System (BCS) versus BDDCS assignment. We detail the uses of BDDCS in predicting, prior to dosing animals or humans, disposition characteristics, potential brain penetration, food effect, and drug-induced liver injury (DILI) potential. This work provides an update on the current status of the BDDCS and its uses in the drug development process. Graphical abstract: [Figure not available: see fulltext.]

Background & Aims Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period. Methods Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected. Results A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%). Conclusions AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid phy

Many drugs are known to potentially cause liver injury; however, only a few reports investigate the association between levofloxacin and acute liver failure (ALF).  The case describes a 65-year-old man who was admitted with primary diagnoses of cerebrovascular accident (CVA) and acute coronary syndrome (ACS) who developed an upper respiratory tract infection for which he was started on levofloxacin. Following its administration, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased more than 100-fold above the upper limit of normal. Over the next 24 hours, AST peaked at 9334 U/L, ALT at 4525 U/L, prothrombin time to 24.6 seconds, international normalized ratio (INR) to 2.22, and serum ammonia to 157 µmol/L. The patient developed signs and symptoms of decompensated liver disease, namely hepatic encephalopathy (HE). Levofloxacin was discontinued immediately, and evidence-based treatment per society guidelines from The American Association for the Study of Liver Diseases consisting of IV n-acetylcysteine as well as lactulose and rifaximin was initiated. Such medical management resulted in clinical resolution of his ALF, but he had a poor overall prognosis and eventually succumbed to critical illness.

Background & Aims Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period. Methods Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected. Results A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%). Conclusions AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid phy

Background & Aims Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period. Methods Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected. Results A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%). Conclusions AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid phy

[Background & Aims] Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period., [Methods] Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected., [Results] A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%)., [Conclusions] AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid physicians in DILI characterisation and management., [Lay summary] Clinical information on drug-induced liver injury (DILI) collected from enrolled patients in the Spanish DILI Registry can guide physicians in the decision-making process. We have found that older patients with hepatocellular type liver injury and patients with additional liver conditions are at a higher risk of mortality. The type of liver injury, patient sex and analytical values of aspartate aminotransferase and total bilirubin can also help predict clinical outcomes.

microRNAs (miRNAs or miRs) are short non-coding RNA molecules which have been shown to be dysregulated and released into the extracellular milieu as a result of many drug and non-drug-induced pathologies in different organ systems. Consequently, circulating miRs have been proposed as useful biomarkers of many disease states, including drug-induced tissue injury. miRs have shown potential to support or even replace the existing traditional biomarkers of drug-induced toxicity in terms of sensitivity and specificity, and there is some evidence for their improved diagnostic and prognostic value. However, several pre-analytical and analytical challenges, mainly associated with assay standardization, require solutions before circulating miRs can be successfully translated into the clinic. This review will consider the value and potential for the use of circulating miRs in drug-safety assessment and describe a systems approach to the analysis of the miRNAome in the discovery setting, as well as highlighting standardization issues that at this stage prevent their clinical use as biomarkers. Highlighting these challenges will hopefully drive future research into finding appropriate solutions, and eventually circulating miRs may be translated to the clinic where their undoubted biomarker potential can be used to benefit patients in rapid, easy to use, point-of-care test systems.

[ES] El objetivo de este proyecto es el diseño experimental de la síntesis de hidrogeles como modelo 3D para el cultivo de células hepáticas de cara a su empleo como plataformas de hepatotoxicidad in vitro. Los materiales y procedimientos seleccionados están basados en literatura existente y consisten en una combinación de macromoléculas presentes en la matriz extracelular del hígado. Primeramente, se explica la síntesis de los hidrogeles combinados de colágeno tipo I y ácido hialurónico (HA) en forma de redes interpenetradas, detallando la química de las reacciones y la evaluación de las propiedades biomecánicas. Asimismo, se describe la obtención de induced pluripotent stem cells (iPSCs) a partir de células de sangre humana y la diferenciación hasta hepatocyte-like cells (HLCs). Además, se describen los métodos de caracterización de ambos tipos celulares. Por otro lado, se plantea la encapsulación de las HLCs en los hidrogeles para evaluar la capacidad de mantener la funcionalidad y el fenotipo celular. La comparación del cultivo 3D con el cultivo de HLCs en monocapa, permite valorar si el modelo 3D es más predictivo que el cultivo en monocapa. Se lleva a cabo el planteamiento de la idoneidad del modelo para la evaluación de la hepatotoxicidad inducida por fármacos, tras la exposición a 15 fármacos modelo y el estudio mediante un ensayo de high-content screening (HCS) en el que se evalúan simultáneamente diferentes endpoints para determinar la capacidad predictiva del modelo. Se plantea el cultivo de la línea HepG2 para estandarizar los protocolos y realizar una puesta a puesto en un modelo más sencillo, pudiendo determinar si el modelo propuesto es adecuado. Por último, tras la selección del hidrogel óptimo en base a las características descritas en la literatura, la descripción del desarrollo del modelo in vitro, y el planteamiento del ensayo de hepatotoxicidad, se realiza una justificación de la posible eficacia e idoneidad del mismo para su uso en el ensayo de, [EN] The main objective of this project is the experimental design of a hydrogel as a 3D model for the culture of hepatocytes and its use as an in vitro hepatotoxicity platform. The selection of materials and procedures is based on scientific literature and consists of a combination of macromolecules present in the extracellular matrix of the liver. First, the synthesis of combined hydrogels of collagen type I and hyaluronic acid, resulting in an interpenetrating network, is explained. Then, the chemistry of the reactions and how to evaluate the biomechanical properties is detailed. The reprogramming of human blood cells to obtain iPSCs and their differentiation to hepatocytes are described. In addition, methods for the characterization of both cell types are outlined. On one side, the encapsulation of iPSCs-derived hepatocytes in hydrogels, as well as the procedures for the hepatic differentiation of iPSCs to hepatocyte-like cells (HLCs) is detailed. Therefore, the in vitro model is based on the 3D culture of HLCs on collagen and HA hydrogels. On the other side, the encapsulation of HLCs in hydrogels is proposed to evaluate the capacity to maintain cell functionality and phenotype. The comparison of the 3D culture with monolayer HLC culture allows us to assess whether the 3D model is more predictive than monolayer culture. The ability of the model for the prediction of drug-induced hepatotoxicity is assessed after the exposure to 15 drugs and the analysis by a high-content screening (HCS) in which different endpoints are simultaneously evaluated. The culture of the HepG2 line is proposed to standardize the protocols and carry out a set up in a simpler model, being able to determine if the proposed model is adequate. Finally, after selecting the optimal hydrogel based on the bibliography, describing the development of the in vitro model, and planning the hepatotoxicity test, the efficacy and suitability for its use in the testing of hepatotoxic drugs is justified. T

[ES] El objetivo de este proyecto es el diseño experimental de la síntesis de hidrogeles como modelo 3D para el cultivo de células hepáticas de cara a su empleo como plataformas de hepatotoxicidad in vitro. Los materiales y procedimientos seleccionados están basados en literatura existente y consisten en una combinación de macromoléculas presentes en la matriz extracelular del hígado. Primeramente, se explica la síntesis de los hidrogeles combinados de colágeno tipo I y ácido hialurónico (HA) en forma de redes interpenetradas, detallando la química de las reacciones y la evaluación de las propiedades biomecánicas. Asimismo, se describe la obtención de induced pluripotent stem cells (iPSCs) a partir de células de sangre humana y la diferenciación hasta hepatocyte-like cells (HLCs). Además, se describen los métodos de caracterización de ambos tipos celulares. Por otro lado, se plantea la encapsulación de las HLCs en los hidrogeles para evaluar la capacidad de mantener la funcionalidad y el fenotipo celular. La comparación del cultivo 3D con el cultivo de HLCs en monocapa, permite valorar si el modelo 3D es más predictivo que el cultivo en monocapa. Se lleva a cabo el planteamiento de la idoneidad del modelo para la evaluación de la hepatotoxicidad inducida por fármacos, tras la exposición a 15 fármacos modelo y el estudio mediante un ensayo de high-content screening (HCS) en el que se evalúan simultáneamente diferentes endpoints para determinar la capacidad predictiva del modelo. Se plantea el cultivo de la línea HepG2 para estandarizar los protocolos y realizar una puesta a puesto en un modelo más sencillo, pudiendo determinar si el modelo propuesto es adecuado. Por último, tras la selección del hidrogel óptimo en base a las características descritas en la literatura, la descripción del desarrollo del modelo in vitro, y el planteamiento del ensayo de hepatotoxicidad, se realiza una justificación de la posible eficacia e idoneidad del mismo para su uso en el ensayo de, [EN] The main objective of this project is the experimental design of a hydrogel as a 3D model for the culture of hepatocytes and its use as an in vitro hepatotoxicity platform. The selection of materials and procedures is based on scientific literature and consists of a combination of macromolecules present in the extracellular matrix of the liver. First, the synthesis of combined hydrogels of collagen type I and hyaluronic acid, resulting in an interpenetrating network, is explained. Then, the chemistry of the reactions and how to evaluate the biomechanical properties is detailed. The reprogramming of human blood cells to obtain iPSCs and their differentiation to hepatocytes are described. In addition, methods for the characterization of both cell types are outlined. On one side, the encapsulation of iPSCs-derived hepatocytes in hydrogels, as well as the procedures for the hepatic differentiation of iPSCs to hepatocyte-like cells (HLCs) is detailed. Therefore, the in vitro model is based on the 3D culture of HLCs on collagen and HA hydrogels. On the other side, the encapsulation of HLCs in hydrogels is proposed to evaluate the capacity to maintain cell functionality and phenotype. The comparison of the 3D culture with monolayer HLC culture allows us to assess whether the 3D model is more predictive than monolayer culture. The ability of the model for the prediction of drug-induced hepatotoxicity is assessed after the exposure to 15 drugs and the analysis by a high-content screening (HCS) in which different endpoints are simultaneously evaluated. The culture of the HepG2 line is proposed to standardize the protocols and carry out a set up in a simpler model, being able to determine if the proposed model is adequate. Finally, after selecting the optimal hydrogel based on the bibliography, describing the development of the in vitro model, and planning the hepatotoxicity test, the efficacy and suitability for its use in the testing of hepatotoxic drugs is justified. T

[ES] El objetivo de este proyecto es el diseño experimental de la síntesis de hidrogeles como modelo 3D para el cultivo de células hepáticas de cara a su empleo como plataformas de hepatotoxicidad in vitro. Los materiales y procedimientos seleccionados están basados en literatura existente y consisten en una combinación de macromoléculas presentes en la matriz extracelular del hígado. Primeramente, se explica la síntesis de los hidrogeles combinados de colágeno tipo I y ácido hialurónico (HA) en forma de redes interpenetradas, detallando la química de las reacciones y la evaluación de las propiedades biomecánicas. Asimismo, se describe la obtención de induced pluripotent stem cells (iPSCs) a partir de células de sangre humana y la diferenciación hasta hepatocyte-like cells (HLCs). Además, se describen los métodos de caracterización de ambos tipos celulares. Por otro lado, se plantea la encapsulación de las HLCs en los hidrogeles para evaluar la capacidad de mantener la funcionalidad y el fenotipo celular. La comparación del cultivo 3D con el cultivo de HLCs en monocapa, permite valorar si el modelo 3D es más predictivo que el cultivo en monocapa. Se lleva a cabo el planteamiento de la idoneidad del modelo para la evaluación de la hepatotoxicidad inducida por fármacos, tras la exposición a 15 fármacos modelo y el estudio mediante un ensayo de high-content screening (HCS) en el que se evalúan simultáneamente diferentes endpoints para determinar la capacidad predictiva del modelo. Se plantea el cultivo de la línea HepG2 para estandarizar los protocolos y realizar una puesta a puesto en un modelo más sencillo, pudiendo determinar si el modelo propuesto es adecuado. Por último, tras la selección del hidrogel óptimo en base a las características descritas en la literatura, la descripción del desarrollo del modelo in vitro, y el planteamiento del ensayo de hepatotoxicidad, se realiza una justificación de la posible eficacia e idoneidad del mismo para su uso en el ensayo de, [EN] The main objective of this project is the experimental design of a hydrogel as a 3D model for the culture of hepatocytes and its use as an in vitro hepatotoxicity platform. The selection of materials and procedures is based on scientific literature and consists of a combination of macromolecules present in the extracellular matrix of the liver. First, the synthesis of combined hydrogels of collagen type I and hyaluronic acid, resulting in an interpenetrating network, is explained. Then, the chemistry of the reactions and how to evaluate the biomechanical properties is detailed. The reprogramming of human blood cells to obtain iPSCs and their differentiation to hepatocytes are described. In addition, methods for the characterization of both cell types are outlined. On one side, the encapsulation of iPSCs-derived hepatocytes in hydrogels, as well as the procedures for the hepatic differentiation of iPSCs to hepatocyte-like cells (HLCs) is detailed. Therefore, the in vitro model is based on the 3D culture of HLCs on collagen and HA hydrogels. On the other side, the encapsulation of HLCs in hydrogels is proposed to evaluate the capacity to maintain cell functionality and phenotype. The comparison of the 3D culture with monolayer HLC culture allows us to assess whether the 3D model is more predictive than monolayer culture. The ability of the model for the prediction of drug-induced hepatotoxicity is assessed after the exposure to 15 drugs and the analysis by a high-content screening (HCS) in which different endpoints are simultaneously evaluated. The culture of the HepG2 line is proposed to standardize the protocols and carry out a set up in a simpler model, being able to determine if the proposed model is adequate. Finally, after selecting the optimal hydrogel based on the bibliography, describing the development of the in vitro model, and planning the hepatotoxicity test, the efficacy and suitability for its use in the testing of hepatotoxic drugs is justified. T

[ES] El objetivo de este proyecto es el diseño experimental de la síntesis de hidrogeles como modelo 3D para el cultivo de células hepáticas de cara a su empleo como plataformas de hepatotoxicidad in vitro. Los materiales y procedimientos seleccionados están basados en literatura existente y consisten en una combinación de macromoléculas presentes en la matriz extracelular del hígado. Primeramente, se explica la síntesis de los hidrogeles combinados de colágeno tipo I y ácido hialurónico (HA) en forma de redes interpenetradas, detallando la química de las reacciones y la evaluación de las propiedades biomecánicas. Asimismo, se describe la obtención de induced pluripotent stem cells (iPSCs) a partir de células de sangre humana y la diferenciación hasta hepatocyte-like cells (HLCs). Además, se describen los métodos de caracterización de ambos tipos celulares. Por otro lado, se plantea la encapsulación de las HLCs en los hidrogeles para evaluar la capacidad de mantener la funcionalidad y el fenotipo celular. La comparación del cultivo 3D con el cultivo de HLCs en monocapa, permite valorar si el modelo 3D es más predictivo que el cultivo en monocapa. Se lleva a cabo el planteamiento de la idoneidad del modelo para la evaluación de la hepatotoxicidad inducida por fármacos, tras la exposición a 15 fármacos modelo y el estudio mediante un ensayo de high-content screening (HCS) en el que se evalúan simultáneamente diferentes endpoints para determinar la capacidad predictiva del modelo. Se plantea el cultivo de la línea HepG2 para estandarizar los protocolos y realizar una puesta a puesto en un modelo más sencillo, pudiendo determinar si el modelo propuesto es adecuado. Por último, tras la selección del hidrogel óptimo en base a las características descritas en la literatura, la descripción del desarrollo del modelo in vitro, y el planteamiento del ensayo de hepatotoxicidad, se realiza una justificación de la posible eficacia e idoneidad del mismo para su uso en el ensayo de, [EN] The main objective of this project is the experimental design of a hydrogel as a 3D model for the culture of hepatocytes and its use as an in vitro hepatotoxicity platform. The selection of materials and procedures is based on scientific literature and consists of a combination of macromolecules present in the extracellular matrix of the liver. First, the synthesis of combined hydrogels of collagen type I and hyaluronic acid, resulting in an interpenetrating network, is explained. Then, the chemistry of the reactions and how to evaluate the biomechanical properties is detailed. The reprogramming of human blood cells to obtain iPSCs and their differentiation to hepatocytes are described. In addition, methods for the characterization of both cell types are outlined. On one side, the encapsulation of iPSCs-derived hepatocytes in hydrogels, as well as the procedures for the hepatic differentiation of iPSCs to hepatocyte-like cells (HLCs) is detailed. Therefore, the in vitro model is based on the 3D culture of HLCs on collagen and HA hydrogels. On the other side, the encapsulation of HLCs in hydrogels is proposed to evaluate the capacity to maintain cell functionality and phenotype. The comparison of the 3D culture with monolayer HLC culture allows us to assess whether the 3D model is more predictive than monolayer culture. The ability of the model for the prediction of drug-induced hepatotoxicity is assessed after the exposure to 15 drugs and the analysis by a high-content screening (HCS) in which different endpoints are simultaneously evaluated. The culture of the HepG2 line is proposed to standardize the protocols and carry out a set up in a simpler model, being able to determine if the proposed model is adequate. Finally, after selecting the optimal hydrogel based on the bibliography, describing the development of the in vitro model, and planning the hepatotoxicity test, the efficacy and suitability for its use in the testing of hepatotoxic drugs is justified. T

Dili, the capital city of Timor-Leste has been faced with serious solid waste problems in recent years. Responding to this issue, the government has adopted various policies including setting up solid waste collection sites in community areas and outsourcing collection to the private sector to collect waste directly from homes in several areas. Despite, these efforts, waste is still found scattered on the roads and disposed of in rivers and open lands. A proper solid waste management strategy is necessary to transform the city into a clean city. In order to develop an effective solid waste management strategy, reliable data and public participation are required. This study, therefore, investigated whether crowdsourcing, in particular, Volunteered Geographic Information (VGI) can effectively be used to collect data about solid waste disposal and collection practices in Dili and raise awareness of the impact of waste disposal practices among the public. The study result demonstrated that crowdsourcing is a viable method for collecting solid waste data. Challenges such as collecting accurate location-specific data still remain, hence, the crowdsourced dataset may not entirely substitute for the usual traditional dataset. At this stage, however, the collected data can still be utilized as a supplementary data source. In the future, by improving data collection methodologies, such as using smaller rewards or providing necessary facilities, a crowdsourcing-based data collection method could be utilized as an adequate substitute for traditional data source because of its ability to collect data in real- time with lower operational costs. This approach is feasible for a developing country such as Timor-Leste where critical area such as waste management has less priority for funding.

N/A

The research has been conducted on the incident and analysis of risk factors drug liver injury (DILI) in a Surabaya Hospital. The aim of this study was to determine the incident of DILI, know which drugs cause DILI, and see the association of risk factors to DILI. The research method was descriptive and analytical observational (prospective cohort). Danan-Benichou scale is a tool used to ascertain drugs that cause DILI. Based on data collected for 3 months, the population was 1202 patients. Samples fulfilling the inclusion and exclusion criteria were 310 patients, the risk drug group of DILI were 285 patients (11 DILI, 274 Non-DILI), and the non-risk drug group 25 patients (11 DILI, 14 Non-DILI). The incident of DILI was 3.55%. Drugs that cause DILI are ranitidine (4 cases), omeprazole (1 case), rifampicin (2 cases), meropenem (1 case), ciprofloxacin (1 case), methotrexate (1 case), and dexamethasone (1 case). Characteristic of patients with DILI (11 patients) are average age of 59.27 ± 15.54 years (23-73 years), belonging to high risk group (54.55%), male gender (81,82%), have moderate comorbid disease (54.55%), and are not comsumsing alcohol (100%). This research use logistic regression analysis through SPSS 17.0 program to see the relation of risk factor to DILI incident. The p results were obtained from sex (0,156), age (0,534), and comorbid isease (0,213)> α (0,05) which means gender, age, and comorbid disease do not significantly affect the incident of DILI.

The research has been conducted on the incident and analysis of risk factors drug liver injury (DILI) in a Surabaya Hospital. The aim of this study was to determine the incident of DILI, know which drugs cause DILI, and see the association of risk factors to DILI. The research method was descriptive and analytical observational (prospective cohort). Danan-Benichou scale is a tool used to ascertain drugs that cause DILI. Based on data collected for 3 months, the population was 1202 patients. Samples fulfilling the inclusion and exclusion criteria were 310 patients, the risk drug group of DILI were 285 patients (11 DILI, 274 Non-DILI), and the non-risk drug group 25 patients (11 DILI, 14 Non-DILI). The incident of DILI was 3.55%. Drugs that cause DILI are ranitidine (4 cases), omeprazole (1 case), rifampicin (2 cases), meropenem (1 case), ciprofloxacin (1 case), methotrexate (1 case), and dexamethasone (1 case). Characteristic of patients with DILI (11 patients) are average age of 59.27 ± 15.54 years (23-73 years), belonging to high risk group (54.55%), male gender (81,82%), have moderate comorbid disease (54.55%), and are not comsumsing alcohol (100%). This research use logistic regression analysis through SPSS 17.0 program to see the relation of risk factor to DILI incident. The p results were obtained from sex (0,156), age (0,534), and comorbid isease (0,213)> α (0,05) which means gender, age, and comorbid disease do not significantly affect the incident of DILI.

The research has been conducted on the incident and analysis of risk factors drug liver injury (DILI) in a Surabaya Hospital. The aim of this study was to determine the incident of DILI, know which drugs cause DILI, and see the association of risk factors to DILI. The research method was descriptive and analytical observational (prospective cohort). Danan-Benichou scale is a tool used to ascertain drugs that cause DILI. Based on data collected for 3 months, the population was 1202 patients. Samples fulfilling the inclusion and exclusion criteria were 310 patients, the risk drug group of DILI were 285 patients (11 DILI, 274 Non-DILI), and the non-risk drug group 25 patients (11 DILI, 14 Non-DILI). The incident of DILI was 3.55%. Drugs that cause DILI are ranitidine (4 cases), omeprazole (1 case), rifampicin (2 cases), meropenem (1 case), ciprofloxacin (1 case), methotrexate (1 case), and dexamethasone (1 case). Characteristic of patients with DILI (11 patients) are average age of 59.27 ± 15.54 years (23-73 years), belonging to high risk group (54.55%), male gender (81,82%), have moderate comorbid disease (54.55%), and are not comsumsing alcohol (100%). This research use logistic regression analysis through SPSS 17.0 program to see the relation of risk factor to DILI incident. The p results were obtained from sex (0,156), age (0,534), and comorbid isease (0,213)> α (0,05) which means gender, age, and comorbid disease do not significantly affect the incident of DILI.

Nous rapportons le contenu de la PEAU’se dermatologique du 24 avril 2017 organisée par le service de Dermatologie des Cliniques universitaires Saint-Luc et consacrée au foie et à la peau. Le Professeur Y. Horsmans, Hépatologue et Chef du Département de Médecine Interne aux Cliniques a exposé la toxicité hépatique des médicaments prescrits en dermatologie. Le Professeur D. Tennstedt, Dermatologue du Service a ensuite rappelé les signes cutanés associés à l’infection par le virus de l’hépatite C., We report the « PEAUse dermatologique » meeting of the Dermatology department of the Cliniques universitaires Saint-Luc which took place on April 24, 2017 and dedicated to liver and skin . Professor Y . Horsmans, Hepatologist and Head of the Department of Internal Medicine, presented the liver toxicity of drugs . Professor D . Tennstedt, Dermatologist of the Department, then described the cutaneous signs associated with hepatitis C virus infection .

Non-alcoholic fatty liver disease (NAFLD) represents a wide spectrum of disease, ranging from simple fatty liver through steatosis with inflammation and necrosis to cirrhosis. One of the most challenging problems in biomedical research and within the chemical industry is to understand the underlying mechanisms of complex disease, and complex adverse outcome pathways (AOPs). Based on a set of 28 steatotic chemicals with gene expression data measured on primary hepatocytes at three times (2, 8, and 24 h) and three doses (low, medium, and high), we identified genes and pathways, defined as molecular initiating events (MIEs) and key events (KEs) of steatosis using a combination of a time series and pathway analyses. Among the genes deregulated by these compounds, the study highlighted OSBPL9, ALDH7A1, MYADM, SLC51B, PRDX6, GPAT3, TMEM135, DLGDA5, BCO2, APO10LA, TSPAN6, NEURL1B, and DUSP1. Furthermore, pathway analysis indicated deregulation of pathways related to lipid accumulation, such as fat digestion and absorption, linoleic and linolenic acid metabolism, calcium signaling pathway, fatty acid metabolism, peroxisome, retinol metabolism, and steroid metabolic pathways in a time dependent manner. Such transcription profile analysis can help in the understanding of the steatosis evolution over time generated by chemical exposure.

Background: Several side-effects related to prolonged benzodiazepines (BZD) use have been reported. Given the primary role of liver in BZD metabolism, toxicity related to prolonged high-dose BZD use could be conceivable. No data are available on the long-term impact of high-dose BZD use on liver. Research design and methods: A total of 201 BZD mono-abusers admitted to an Addiction Unit for detoxification were evaluated. Liver enzymes were evaluated at admission, before starting any treatment. An elevation of more than five times the upper limit of normal range (ULN) in serum ALT or conjugated bilirubin, or a combined elevation of AST, alkaline phosphatase and total bilirubin, one of which exceeding >2 the ULN, was considered diagnostic for drug-induced liver injury. Results: None of the evaluated subjects showed significant alterations of liver enzymes. Those with the highest transaminase levels were showing high body mass index. Twenty patients (10%) showed elevated gamma-glutamyl-transferase. No alteration of alkaline phosphatase, nor bilirubin was found in any patient. The average dosage of BZD was 307 mg of diazepam-equivalents for 7 years. Conclusions: Present data suggest that prolonged use of high-dose BZD, although very dangerous for several reasons, does not seem to produce a significant drug-induced liver injury.

Non-alcoholic fatty liver disease (NAFLD) represents a wide spectrum of disease, ranging from simple fatty liver through steatosis with inflammation and necrosis to cirrhosis. One of the most challenging problems in biomedical research and within the chemical industry is to understand the underlying mechanisms of complex disease, and complex adverse outcome pathways (AOPs). Based on a set of 28 steatotic chemicals with gene expression data measured on primary hepatocytes at three times (2, 8, and 24 h) and three doses (low, medium, and high), we identified genes and pathways, defined as molecular initiating events (MIEs) and key events (KEs) of steatosis using a combination of a time series and pathway analyses. Among the genes deregulated by these compounds, the study highlighted OSBPL9, ALDH7A1, MYADM, SLC51B, PRDX6, GPAT3, TMEM135, DLGDA5, BCO2, APO10LA, TSPAN6, NEURL1B, and DUSP1. Furthermore, pathway analysis indicated deregulation of pathways related to lipid accumulation, such as fat digestion and absorption, linoleic and linolenic acid metabolism, calcium signaling pathway, fatty acid metabolism, peroxisome, retinol metabolism, and steroid metabolic pathways in a time dependent manner. Such transcription profile analysis can help in the understanding of the steatosis evolution over time generated by chemical exposure.

NO ABSTRACT

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis.

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis.

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis.

Aims Acute drug overdose, especially with paracetamol, may cause acute liver failure leading to registration for transplantation (ALFT). Population statistics and between-country differences for ALFT related to overdose have been poorly described. The aim of the present study was to evaluate overdose ALFT in the multi-country Study of Acute Liver Transplantation (SALT). Methods All adult overdose-related ALFT, with or without suicidal intent, in France, Greece, Ireland, Italy, the Netherlands, Portugal and the UK between 2005 and 2007 were identified from liver transplant registries and hospital records. These were compared with whole-country and per capita use of paracetamol. Results Six hundred cases of ALFT were identified in 52 of 57 eligible transplant centres, of which 114 involved overdose (72 intentional, 10 non-intentional, 32 uncertain). Overdose represented 20% of all-cause ALFT: Ireland 52%, UK 28%, France 18%, the Netherlands 8%, and Italy 1%. Overdose ALFT were mostly females (61%), mean age 33.6 ± 10.9 years. A total of 111 (97%) of the overdoses involved paracetamol. Event rates ranged from one ALFT for 20.7 tons of paracetamol in Ireland, to one for 1074 tons in Italy and one case in 60 million inhabitants over 3 years in Italy to one case in 286 000 inhabitants per year in Ireland. Per-country event rates for non-overdose ALFT exposed to paracetamol were between 2.5 and 4.0 per million treatment-years sold. Conclusions Paracetamol overdose was found to represent one-sixth of all-cause ALFT. There was a 50-fold difference in Europe in the rates of paracetamol overdose ALFT, and a 200-fold difference per million inhabitants.

Current testing models for predicting drug-induced liver injury are inadequate, as they basically under-report human health risks. We present here an approach towards developing pathways based on hepatotoxicity-associated gene groups derived from two types of publicly accessible hepatotoxicity databases, in order to develop drug-induced liver injury biomarker profiles. One human liver omics-based and four text-mining-based databases were explored for hepatotoxicity-associated gene lists. Over-representation analysis of these gene lists with a hepatotoxicant-exposed primary human hepatocytes data set showed that human liver omics gene lists performed better than text-mining gene lists and the results of the latter differed strongly between databases. However, both types of databases contained gene lists demonstrating biomarker potential. Visualizing those in pathway format may aid in interpreting the biomolecular background. We conclude that exploiting existing and openly accessible databases in a dedicated manner seems promising in providing venues for translational research in toxicology and biomarker development.

Current testing models for predicting drug-induced liver injury are inadequate, as they basically under-report human health risks. We present here an approach towards developing pathways based on hepatotoxicity-associated gene groups derived from two types of publicly accessible hepatotoxicity databases, in order to develop drug-induced liver injury biomarker profiles. One human liver omics-based and four text-mining-based databases were explored for hepatotoxicity-associated gene lists. Over-representation analysis of these gene lists with a hepatotoxicant-exposed primary human hepatocytes data set showed that human liver omics gene lists performed better than text-mining gene lists and the results of the latter differed strongly between databases. However, both types of databases contained gene lists demonstrating biomarker potential. Visualizing those in pathway format may aid in interpreting the biomolecular background. We conclude that exploiting existing and openly accessible databases in a dedicated manner seems promising in providing venues for translational research in toxicology and biomarker development.

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a dataset of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, while positive molecular charge was associated with a lack of inhibition. All approved drugs in the dataset (n=182) were categorized according to DILI warnings in drug labels issued by the FDA and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including nine drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux while BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models we

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4 alpha, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4 alpha), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

‘Nation-building across Urban and Rural Timor-Leste’ provided an opportunity for East Timorese and people from around the world to reflect, discuss and debate the nation-building process in Timor-Leste since 1999. In this context, nation-building in Timor-Leste is taken to mean the many different attempts since 1999 to ensure the political, economic and cultural integration of the population to fulfil the ambition of self rule. Ten years after the 1999 vote for independence, this conference considered how nation-building is being experienced and responded to across urban and rural communities in Timor-Leste. Broadening the discussion beyond that of ‘state-building’, at the core of the conference was a consideration of the myriad ways the new republic has been ‘built’. Here ‘nation-building’ considers not only in terms of policy and programmatic initiatives but also grass roots experiences and perceptions of how Timor-Leste as a nation is seen and understood. For this reason, the majority of participants were East Timorese representatives from across the districts, allowing them top share their stories of nation-building. At this conference, nation-building was discussed in terms of what appears to be one of the most significant characteristics of contemporary Timor-Leste, namely the sharp distinction found between the urbanized capital and the rural communities where the majority of the population live. Dili has emerged as the centre for economic and political power in a way that is extraordinarily disproportionate with the remainder of the country, while rural areas often remain highly isolated and continue to be dominated by subsistence agriculture.