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1.
Arch Toxicol ; 93(4): 1021-1037, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30915487

RESUMO

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/metabolismo
2.
J Pharmacol Exp Ther ; 350(2): 387-402, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24902585

RESUMO

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-: derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-: related hypersensitivities had either a single drug-: derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/metabolismo , Glucuronídeos/metabolismo , Espectrometria de Massas/métodos , Albumina Sérica/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica
3.
Med Res Rev ; 33(5): 985-1080, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23090860

RESUMO

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.


Assuntos
Desenho de Fármacos , Preparações Farmacêuticas/metabolismo , Animais , Pesquisa Biomédica , Sistema Enzimático do Citocromo P-450 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Glucuronídeos/metabolismo , Humanos
4.
Biopharm Drug Dispos ; 31(7): 367-95, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20830700

RESUMO

Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response.


Assuntos
Biotransformação , Ácidos Carboxílicos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Glucuronídeos/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas/metabolismo , Acilação , Animais , Ácidos Carboxílicos/efeitos adversos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Glucuronídeos/efeitos adversos , Glucuronídeos/farmacocinética , Glucuronídeos/farmacologia , Humanos , Ligação Proteica
5.
Toxicol Appl Pharmacol ; 239(3): 297-305, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19523481

RESUMO

UNLABELLED: Methapyrilene, [N,N-dimethyl-N'-pyridyl-N'(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. CONCLUSION: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.


Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Metapirileno/toxicidade , Tiofenos/metabolismo , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutamato-Cisteína Ligase/biossíntese , Glutationa/metabolismo , Heme Oxigenase-1/biossíntese , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Metapirileno/farmacocinética , Necrose , Oxirredução , Ratos , Ratos Wistar
6.
Toxicol Sci ; 162(2): 655-666, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29329425

RESUMO

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/normas
7.
J Med Chem ; 55(22): 9773-84, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-23088585

RESUMO

The anticonvulsant carbamazepine 1 is associated with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metabolism of 1 has been implicated in the pathogenesis of the ADRs. We report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogues of 1 that were intended to minimize reactive metabolite formation via arene oxidation and 10,11-epoxidation. Halo analogues were obtained either by rearrangement of halogenated N-arylindoles or from specifically halogenated iminodibenzyl derivatives. In rat hepatocytes, none of the analogues underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but aromatic hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of aromatic hydroxylation required at least monochlorination or difluorination of 1. In human liver microsomes, difluoro analogue 5b underwent 10,11-epoxidation but gave no arene oxidation.


Assuntos
Anticonvulsivantes/farmacologia , Carbamazepina/análogos & derivados , Halogênios/química , Hepatócitos/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Animais , Anticonvulsivantes/síntese química , Carbamazepina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Glutationa/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade
8.
Org Lett ; 13(20): 5592-5, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-21939196

RESUMO

Efficient procedures for the synthesis of benzo-fluorinated dibenz[b,f]azepines (iminostilbenes) from fluorinated isatins or indoles using a number of ring-expansion reactions are described. A range of mono- and difluorinated analogues is accessible, and the syntheses can deliver gram quantities of the final products, which are precursors of fluoro analogues of the important anticonvulsant carbamazepine.


Assuntos
Acridinas/química , Azepinas/síntese química , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/síntese química , Indóis/química , Isatina/análogos & derivados , Isatina/química , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Azepinas/química , Carbamazepina/análogos & derivados , Carbamazepina/síntese química , Carbamazepina/química , Catálise , Estrutura Molecular
9.
Toxicol Sci ; 112(2): 521-31, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19783637

RESUMO

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.


Assuntos
Acetaminofen/toxicidade , Apoptose/efeitos dos fármacos , Proteína HMGB1/sangue , Queratina-18/sangue , Acetilação , Sequência de Aminoácidos , Animais , Western Blotting , Inibidores de Caspase , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Proteína HMGB1/química , Queratina-18/química , Masculino , Camundongos , Dados de Sequência Molecular , Necrose , Espectrometria de Massas em Tandem
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