RESUMO
The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.
Assuntos
Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Masculino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Metionina/metabolismo , Ratos Sprague-Dawley , Purina-Núcleosídeo Fosforilase/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , MultiômicaRESUMO
The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.
Assuntos
Neoplasias , Humanos , Entropia , Metionina Adenosiltransferase/metabolismoRESUMO
An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.
Assuntos
Citotoxinas/toxicidade , Hepatócitos/efeitos dos fármacos , Testes de Toxicidade/métodos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas , Fluoresceínas/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla/antagonistas & inibidores , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismoRESUMO
Drug induced liver injury (DILI) can require significant risk management in drug development and on occasion can cause morbidity or mortality, leading to drug attrition. Optimizing candidates preclinically can minimize hepatotoxicity risk, but it is difficult to predict due to multiple etiologies encompassing DILI, often with multifactorial and overlapping mechanisms. In addition to epidemiological risk factors, physicochemical properties, dose, disposition, lipophilicity, and hepatic metabolic function are also relevant for DILI risk. Better human-relevant, predictive models are required to improve hepatotoxicity risk assessment in drug discovery. Our hypothesis is that integrating mechanistically relevant hepatic safety assays with Bayesian machine learning will improve hepatic safety risk prediction. We present a quantitative and mechanistic risk assessment for candidate nomination using data from in vitro assays (hepatic spheroids, BSEP, mitochondrial toxicity, and bioactivation), together with physicochemical (cLogP) and exposure (Cmaxtotal) variables from a chemically diverse compound set (33 no/low-, 40 medium-, and 23 high-severity DILI compounds). The Bayesian model predicts the continuous underlying DILI severity and uses a data-driven prior distribution over the parameters to prevent overfitting. The model quantifies the probability that a compound falls into either no/low-, medium-, or high-severity categories, with a balanced accuracy of 63% on held-out samples, and a continuous prediction of DILI severity along with uncertainty in the prediction. For a binary yes/no DILI prediction, the model has a balanced accuracy of 86%, a sensitivity of 87%, a specificity of 85%, a positive predictive value of 92%, and a negative predictive value of 78%. Combining physiologically relevant assays, improved alignment with FDA recommendations, and optimal statistical integration of assay data leads to improved DILI risk prediction.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Teorema de Bayes , Sobrevivência Celular , Desenvolvimento de Medicamentos/métodos , Células Hep G2 , Humanos , Aprendizado de Máquina , Mitocôndrias/efeitos dos fármacos , Medição de Risco/métodos , Células THP-1RESUMO
Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose- and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC50 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Modelos Biológicos , Esferoides Celulares/efeitos dos fármacos , Acetaminofen/toxicidade , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/toxicidade , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Esferoides Celulares/metabolismoRESUMO
Organ-Chips are micro-engineered systems that aim to recapitulate the organ microenvironment. Implementation of Organ-Chips within the pharmaceutical industry aims to improve the probability of success of drugs reaching late stage clinical trial by generating models for drug discovery that are of human origin and have disease relevance. We are adopting the use of Organ-Chips for enhancing pre-clinical efficacy and toxicity evaluation and prediction. Whilst capturing cellular phenotype via imaging in response to drug exposure is a useful readout in these models, application has been limited due to difficulties in imaging the chips at scale. Here we created an end-to-end, automated workflow to capture and analyse confocal images of multicellular Organ-Chips to assess detailed cellular phenotype across large batches of chips. By automating this process, we not only reduced acquisition time, but we also minimised process variability and user bias. This enabled us to establish, for the first time, a framework of statistical best practice for Organ-Chip imaging, creating the capability of using Organ-Chips and imaging for routine testing in drug discovery applications that rely on quantitative image data for decision making. We tested our approach using benzbromarone, whose mechanism of toxicity has been linked to mitochondrial damage with subsequent induction of apoptosis and necrosis, and staurosporine, a tool inducer of apoptosis. We also applied this workflow to assess the hepatotoxic effect of an active AstraZeneca drug candidate illustrating its applicability in drug safety assessment beyond testing tool compounds. Finally, we have demonstrated that this approach could be adapted to Organ-Chips of different shapes and sizes through application to a Kidney-Chip.
Assuntos
Dispositivos Lab-On-A-Chip , Imagem Óptica/instrumentação , Animais , Automação , Avaliação Pré-Clínica de Medicamentos , Humanos , Rim/diagnóstico por imagem , Rim/efeitos dos fármacos , Fígado/diagnóstico por imagem , Fígado/efeitos dos fármacos , RatosRESUMO
Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Esferoides Celulares/efeitos dos fármacos , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Criopreservação , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Valor Preditivo dos Testes , Cultura Primária de Células , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Tempo , Testes de Toxicidade/normasRESUMO
Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Desenho de Fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Hepatócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Descoberta de Drogas/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Hepatócitos/patologia , Humanos , Concentração Inibidora 50 , Fígado/efeitos dos fármacos , Fígado/patologia , Valor Preditivo dos Testes , Medição de Risco/métodos , Fatores de TempoRESUMO
Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit no functional expression of hepatic cytochrome P450 (P450) when compared to wild type (WT) mice, but have normal hepatic and extrahepatic expression of other biotransformation enzymes. We have assessed the utility of HRN™ mice for investigation of the role of metabolic bioactivation in liver toxicity caused by the nonsteroidal anti-inflammatory drug (NSAID) fenclozic acid. In vitro studies revealed significant NADPH-dependent (i.e. P450-mediated) covalent binding of [14C]-fenclozic acid to liver microsomes from WT mice and HRN™ mice, whereas no in vitro covalent binding was observed in the presence of the UDP-glucuronyltransferase cofactor UDPGA. Oral fenclozic acid administration did not alter the liver histopathology or elevate the plasma liver enzyme activities of WT mice, or affect their hepatic miRNA contents. Livers from HRN™ mice exhibited abnormal liver histopathology (enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration, necrosis, inflammatory cell infiltration) and plasma clinical chemistry (elevated alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities). Modest apparent improvements in these abnormalities were observed when HRN™ mice were dosed orally with fenclozic acid for 7 days at 100 mg kg-1 day-1. Previously we observed more marked effects on liver histopathology and integrity in HRN™ mice dosed orally with the NSAID diclofenac for 7 days at 30 mg kg-1 day-1. We conclude that HRN™ mice are valuable for assessing P450-related hepatic drug biotransformation, but not for drug toxicity studies due to underlying liver dysfunction. Nonetheless, HRN™ mice may provide novel insights into the role of inflammation in liver injury, thereby aiding its treatment.
RESUMO
Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.
Assuntos
Microambiente Celular/efeitos dos fármacos , Criopreservação , Avaliação Pré-Clínica de Medicamentos/métodos , Drogas em Investigação/efeitos adversos , Hepatócitos/efeitos dos fármacos , Células 3T3 , Animais , Técnicas de Cultura Celular por Lotes , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Técnicas de Cocultura , Drogas em Investigação/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Cinética , Células de Kupffer/citologia , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/imunologia , Lipopolissacarídeos/agonistas , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Camundongos , Modelos Moleculares , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologiaRESUMO
Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.
Assuntos
Diclofenaco/administração & dosagem , Diclofenaco/efeitos adversos , Fígado/efeitos dos fármacos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Administração Oral , Animais , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Diclofenaco/farmacocinética , Diclofenaco/urina , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/genéticaRESUMO
Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Antagonistas dos Receptores de Endotelina/toxicidade , Isoxazóis/toxicidade , Fenilpropionatos/toxicidade , Piridazinas/toxicidade , Sulfonamidas/toxicidade , Tiofenos/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Bosentana , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Antagonistas dos Receptores de Endotelina/farmacocinética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoxazóis/farmacocinética , Mitocôndrias/metabolismo , Estrutura Molecular , Consumo de Oxigênio/fisiologia , Fenilpropionatos/farmacocinética , Piridazinas/farmacocinética , Sulfonamidas/farmacocinética , Tiofenos/farmacocinética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Nanomedicine strategies have produced many commercial products. However, no orally dosed HIV nanomedicines are available clinically to patients. Although nanosuspensions of drug particles have demonstrated many benefits, experimentally achieving >25 wt% of drug relative to stabilizers is highly challenging. In this study, the emulsion-templated freeze-drying technique for nanoparticles formation is applied for the first time to optimize a nanodispersion of the leading non-nucleoside reverse transcriptase inhibitor efavirenz, using clinically acceptable polymers and surfactants. Dry monoliths containing solid drug nanoparticles with extremely high drug loading (70 wt% relative to polymer and surfactant stabilizers) are stable for several months and reconstitute in aqueous media to provide nanodispersions with z-average diameters of 300 nm. The solid drug nanoparticles exhibit reduced cytoxicity and increased in vitro transport through model gut epithelium. In vivo studies confirm bioavailability benefits with an approximately four-fold higher pharmacokinetic exposure after oral administration to rodents, and predictive modeling suggests dose reduction with the new formulation may be possible.
Assuntos
Nanopartículas/administração & dosagem , Nanopartículas/química , Administração Oral , Alcinos , Animais , Benzoxazinas/administração & dosagem , Benzoxazinas/química , Benzoxazinas/farmacocinética , Disponibilidade Biológica , Células CACO-2 , Ciclopropanos , Avaliação Pré-Clínica de Medicamentos , Emulsões/administração & dosagem , Emulsões/química , Emulsões/farmacocinética , Humanos , Masculino , Tamanho da Partícula , Polímeros/administração & dosagem , Polímeros/química , Polímeros/farmacocinética , Ratos , Ratos WistarRESUMO
Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC50 ≤ 200 µM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC50 values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Células Epiteliais/efeitos dos fármacos , Fígado/efeitos dos fármacos , Bioensaio , Biotransformação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Humanos , Concentração Inibidora 50 , Fígado/enzimologia , Fígado/patologia , Medição de Risco , Fatores de Risco , Testes de Toxicidade , TransfecçãoRESUMO
Drug-induced liver injury (DILI) is a major cause of failed drug development, withdrawal and restricted usage. Therefore screening assays which aid selection of candidate drugs with reduced propensity to cause DILI are required. We have investigated the toxicity of 144 drugs, 108 of which caused DILI, using assays identified in the literature as having some predictivity for hepatotoxicity. The validated assays utilised either HepG2 cells, HepG2 cells in the presence of rat S9 fraction or isolated human hepatocytes. All parameters were quantified by multiplexed and automated high content fluorescence microscopy, at appropriate time points after compound administration (4, 24 or 48h). The individual endpoint which identified drugs that caused DILI with greatest precision was maximal fold induction in CM-H2DFFDA staining in hepatocytes after 24h (41% sensitivity, 86% specificity). However, hierarchical clustering analysis of all endpoints provided the most sensitive identification of drugs which caused DILI (58% sensitivity, 75% specificity). We conclude that multi-parametric high content cell toxicity assays can enable in vitro detection of drugs that have high propensity to cause DILI in vivo but that many DILI compounds exhibit few in vitro signals when evaluated using these assays.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/patologia , Algoritmos , Animais , Caspase 3/metabolismo , Contagem de Células , Linhagem Celular , Sobrevivência Celular , Análise por Conglomerados , Criopreservação , Técnicas Citológicas/instrumentação , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Células Hep G2 , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Cultura Primária de Células , Ratos , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
BACKGROUND: Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). METHODS: Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. RESULTS: Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. CONCLUSION: A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. GENERAL SIGNIFICANCE: Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.
Assuntos
Biomarcadores/metabolismo , Glutationa/metabolismo , Fígado/efeitos dos fármacos , Modelos Biológicos , Acetaminofen/toxicidade , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Humanos , Fígado/metabolismo , Espectrometria de Massas em TandemRESUMO
The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.
Assuntos
Antagonistas de Receptores de Canabinoides/química , Iminas/química , Piperidinas/química , Pirazóis/química , Antagonistas de Receptores de Canabinoides/metabolismo , Antagonistas de Receptores de Canabinoides/toxicidade , Radioisótopos de Carbono/química , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/metabolismo , Humanos , Íons/química , Metaboloma/efeitos dos fármacos , Piperidinas/metabolismo , Piperidinas/farmacologia , Piperidinas/toxicidade , Cianeto de Potássio/química , Cianeto de Potássio/farmacologia , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Pirazóis/metabolismo , Pirazóis/toxicidade , Rimonabanto , Ritonavir/química , Ritonavir/farmacologiaRESUMO
The hepatic SV40 large T-antigen immortalized human liver epithelial (THLE) cell line and sublines transfected with cytochromes P450 (P450s) are increasingly being used for evaluation of potential drug-induced liver injury. So far, the available information on transporter and enzyme expression in these transfected cell systems is scattered. The purpose of this study was to characterize THLE cell lines with respect to transporter and enzyme expression. The mRNA expression of 96 typical drug absorption, distribution, metabolism and excretion genes, which encode a selection of transporters, phase I and II drug-metabolizing enzymes, and nuclear hormone receptors, was investigated in five THLE cell lines transfected with individual human P450s and in mock-transfected THLE-null cells using real-time polymerase chain reaction. The majority of the analyzed genes was either absent or expressed at low levels in the THLE-null and THLE-P450 cells, apart from housekeeping genes and the individual transfected P450s. Enzyme activity measurements provided confirmatory functional data for CYP2C9 and CYP3A4. Comparison with gene expression in human liver revealed an overall much lower gene expression in the THLE cell lines. The low levels of expression of a broad range of P450 genes in the THLE cell lines highlight the value of studies undertaken with P450-expressing cell lines for investigation of mechanisms of P450 metabolite-mediated hepatotoxicity. However, when attempting to translate between data obtained in THLE cell lines in vitro and functional consequences in vivo, it is important to take account of their limited expression of genes encoding many other drug-metabolizing enzymes and hepatic transporters.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/citologia , Fígado/enzimologia , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Expressão Gênica , Humanos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Idiosyncratic adverse drug reactions (IADRs) in humans can result in a broad range of clinically significant toxicities leading to attrition during drug development as well as postlicensing withdrawal or labeling. IADRs arise from both drug and patient related mechanisms and risk factors. Drug related risk factors, resulting from parent compound or metabolites, may involve multiple contributory mechanisms including organelle toxicity, effects related to compound disposition, and/or immune activation. In the current study, we evaluate an in vitro approach, which explored both cellular effects and covalent binding (CVB) to assess IADR risks for drug candidates using 36 drugs which caused different patterns and severities of IADRs in humans. The cellular effects were tested in an in vitro Panel of five assays which quantified (1) toxicity to THLE cells (SV40 T-antigen-immortalized human liver epithelial cells), which do not express P450s, (2) toxicity to a THLE cell line which selectively expresses P450 3A4, (3) cytotoxicity in HepG2 cells in glucose and galactose media, which is indicative of mitochondrial injury, (4) inhibition of the human bile salt export pump, BSEP, and (5) inhibition of the rat multidrug resistance associated protein 2, Mrp2. In addition, the CVB Burden was estimated by determining the CVB of radiolabeled compound to human hepatocytes and factoring in both the maximum prescribed daily dose and the fraction of metabolism leading to CVB. Combining the aggregated results from the in vitro Panel assays with the CVB Burden data discriminated, with high specificity (78%) and sensitivity (100%), between 27 drugs, which had severe or marked IADR concern, and 9 drugs, which had low IADR concern, we propose that this integrated approach has the potential to enable selection of drug candidates with reduced propensity to cause IADRs in humans.
Assuntos
Pró-Fármacos/efeitos adversos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Galactose/farmacologia , Glucose/farmacologia , Células Hep G2 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Ratos , Ratos Sprague-Dawley , Fatores de RiscoRESUMO
Water-insoluble organic compounds are often used in aqueous environments in various pharmaceutical and consumer products. To overcome insolubility, the particles are dispersed in a medium during product formation, but large particles that are formed may affect product performance and safety. Many techniques have been used to produce nanodispersions-dispersions with nanometre-scale dimensions-that have properties similar to solutions. However, making nanodispersions requires complex processing, and it is difficult to achieve stability over long periods. Here we report a generic method for producing organic nanoparticles with a combination of modified emulsion-templating and freeze-drying. The dry powder composites formed using this method are highly porous, stable and form nanodispersions upon simple addition of water. Aqueous nanodispersions of Triclosan (a commercial antimicrobial agent) produced with this approach show greater activity than organic/aqueous solutions of Triclosan.