RESUMO
The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HµREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.
Assuntos
Hepatócitos , Humanos , Técnicas de Cocultura , Células Cultivadas , Carga Corporal (Radioterapia) , Hepatócitos/metabolismo , Medição de RiscoRESUMO
The NO-sGC-cGMP signaling pathway plays an important role in the cardiovascular system. Loss of nitric oxide tone or impaired signaling has been associated with cardiovascular diseases, such as hypertension, pulmonary hypertension and heart failure. Direct activation of sGC enzyme independent of NO represents a novel approach for modulating NO signaling with tremendous therapeutic potential. Herein, we describe the design of a structurally novel class of heme-dependent sGC stimulators containing the 3,3-dimethylpyrrolidin-2-one moiety which resulted in the identification of the potent, selective stimulator 30 (MK-2947) for the treatment of hypertension.
Assuntos
Anti-Hipertensivos/farmacologia , Descoberta de Drogas , Hipertensão/tratamento farmacológico , Guanilil Ciclase Solúvel/metabolismo , Anti-Hipertensivos/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.
Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Descoberta de Drogas/métodos , Ácido Taurocólico/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos , Humanos , Concentração Inibidora 50 , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismoRESUMO
MK-8666, a selective GPR40 agonist developed for the treatment of type 2 diabetes mellitus, was discontinued in phase I clinical trials due to liver safety concerns. To address whether chemically reactive metabolites played a causative role in the observed drug induced liver injury (DILI), we characterized the metabolism, covalent binding to proteins, and amino acid targets of MK-8666 in rat and human hepatocytes or cofactor-fortified liver microsomes. MK-8666 was primarily metabolized to an acyl glucuronide in hepatocytes of both species and a taurine conjugate in rat hepatocytes. Similar levels of covalent binding to proteins were observed in rat and human hepatocytes following incubation with [3H]MK-8666. After protease digestion of hepatocyte pellets, amino acid adducts A1, A2, and A3 were identified as transacylated products with lysine, serine, and cysteine residues, respectively. Amino acid adducts A4a-c were identified as glycation adducts resulting from rearrangement of MK-8666-1-O-ß-acyl glucuronide to ring-opened aldehydes which further condensed with lysine residues of proteins into imine adducts. Adducts A1-A3 and A4a-c were detected in rat and human liver microsomes fortified with UDPGA. Adducts A1-A3 were detected in rat and human liver microsomes fortified with CoA and ATP. Additionally, a trace amount of CoA thioester metabolite of MK-8666 and its transacylated GSH adduct were detected in human liver microsomes fortified with CoA, ATP, and GSH. Higher levels of covalent binding to protein were observed when [3H]MK-8666 was incubated in liver microsomes supplemented with CoA and ATP compared to UDPGA. Addition of GSH attenuated levels of CoA thioester-mediated covalent binding by 41-45%. Collectively, these studies indicated that metabolism of the -COOH moiety of MK-8666 can form a reactive acyl glucuronide and an acyl CoA thioester, which covalently modifies proteins and may represent one causative mechanism of the observed DILI.
Assuntos
Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Microssomos Hepáticos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Acilação , Aminoácidos/metabolismo , Animais , Ésteres/metabolismo , Glucuronídeos/metabolismo , Humanos , Ligação Proteica , RatosRESUMO
GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet ß-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet ß-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.
Assuntos
Indanos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Desenho de Fármacos , HumanosRESUMO
Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.
Assuntos
Benzimidazóis/química , Ácidos Carboxílicos/química , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Benzimidazóis/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografia Líquida de Alta Pressão , Cicloexanonas/química , Diacilglicerol O-Aciltransferase/metabolismo , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Isomerismo , Espectrometria de Massas , Camundongos , RatosRESUMO
A potent and selective Factor IXa (FIXa) inhibitor was subjected to a series of liver microsomal incubations, which generated a number of metabolites. Using automated ligand identification system-affinity selection (ALIS-AS) methodology, metabolites in the incubation mixture were prioritized by their binding affinities to the FIXa protein. Microgram quantities of the metabolites of interest were then isolated through microisolation analytical capabilities, and structurally characterized using MicroCryoProbe heteronuclear 2D NMR techniques. The isolated metabolites recovered from the NMR experiments were then submitted directly to an in vitro FIXa enzymatic assay. The order of the metabolites' binding affinity to the Factor IXa protein from the ALIS assay was completely consistent with the enzymatic assay results. This work showcases an innovative and efficient approach to uncover structure-activity relationships (SARs) and guide drug design via microisolation-structural characterization and ALIS capabilities.
Assuntos
Automação , Desenho de Fármacos , Fator IXa/antagonistas & inibidores , Fibrinolíticos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Animais , Relação Dose-Resposta a Droga , Fator IXa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Humanos , Ligantes , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened α,ß-unsaturated imine conjugated to the ε-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a ß-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.
Assuntos
Azetidinas/metabolismo , Proteínas/metabolismo , Receptores de Lisoesfingolipídeo/agonistas , Tiofenos/metabolismo , Administração Oral , Animais , Azetidinas/toxicidade , Biotransformação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Marcação por Isótopo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Peptídeos/análise , Proteínas/química , Ratos , Receptores de Lisoesfingolipídeo/metabolismo , Tiofenos/toxicidadeRESUMO
A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-ß-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2. Compound 9m blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucagon/metabolismo , Pirazóis/farmacologia , Receptores de Glucagon/antagonistas & inibidores , beta-Alanina/análogos & derivados , Animais , Área Sob a Curva , Células CHO , Cricetinae , Cricetulus , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Cães , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Concentração Inibidora 50 , Macaca mulatta , Camundongos , Camundongos Obesos , Microssomos Hepáticos/metabolismo , Pirazóis/química , Pirazóis/uso terapêutico , Ratos , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , beta-Alanina/química , beta-Alanina/farmacologia , beta-Alanina/uso terapêuticoRESUMO
A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes.
Assuntos
Glicemia/efeitos dos fármacos , Descoberta de Drogas , Hipoglicemiantes , Indóis/síntese química , Indóis/farmacologia , Receptores de Glucagon/antagonistas & inibidores , Administração Oral , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cães , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Indóis/química , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
In the course of the development of an aminobenzimidazole class of human glucagon receptor (hGCGR) antagonists, a novel class of cyclic guanidine hGCGR antagonists was discovered. Rapid N-dealkylation resulted in poor pharmacokinetic profiles for the benchmark compound in this series. A strategy aimed at blocking oxidative dealkylation led to a series of compounds with improved rodent pharmacokinetic profiles. One compound was orally efficacious in a murine glucagon challenge pharmacodynamic model and also significantly lowered glucose levels in a murine diabetes model.
Assuntos
Glicemia/efeitos dos fármacos , Descoberta de Drogas , Guanidinas/síntese química , Receptores de Glucagon/antagonistas & inibidores , Administração Oral , Animais , Ciclização , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Cães , Guanidinas/química , Guanidinas/farmacologia , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
The synthesis, selectivity, rat pharmacokinetic profile, and drug metabolism profiles of a series of potent fluoroolefin-derived DPP-4 inhibitors (4) are reported. A radiolabeled fluoroolefin 33 was shown to possess a high propensity to form reactive metabolites, thus revealing a potential liability for this class of DPP-4 inhibitors.
Assuntos
Alcenos/farmacocinética , Amidas/química , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Hidrocarbonetos Fluorados/farmacocinética , Hipoglicemiantes/farmacocinética , Microssomos Hepáticos/efeitos dos fármacos , Alcenos/síntese química , Animais , Inibidores da Dipeptidil Peptidase IV/síntese química , Hidrocarbonetos Fluorados/síntese química , Hipoglicemiantes/síntese química , Microssomos Hepáticos/patologia , Modelos Químicos , Mimetismo Molecular , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A series of beta-substituted biarylphenylalanine amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the metabolic profile of early analogues led to the discovery of (2S,3S)-3-amino-4-(3,3-difluoropyrrolidin-1-yl)-N,N-dimethyl-4-oxo-2-(4-[1,2,4]triazolo[1,5-a]pyridin-6-ylphenyl)butanamide (6), a potent, orally active DPP-4 inhibitor (IC(50) = 6.3 nM) with excellent selectivity, oral bioavailability in preclinical species, and in vivo efficacy in animal models. Compound 6 was selected for further characterization as a potential new treatment for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Hipoglicemiantes/síntese química , Fenilalanina/análogos & derivados , Inibidores de Proteases/síntese química , Triazóis/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , Cristalografia por Raios X , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Proteínas Musculares/antagonistas & inibidores , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Coelhos , Canais de Sódio , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologiaRESUMO
Interest in the parkinsonian-inducing proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine has prompted extensive studies into the oxidative pathways mediating its bioactivation to the corresponding pyridinium species, a potent inhibitor of the mitochondrial electron transport chain. The initial step in the overall reaction is the two-electron ring alpha-carbon oxidation to give the 1-methyl-4-phenyl-2,3-dihydropyridinium species, a reaction that is catalyzed by monoamine oxidase B. The same a-carbon oxidation is catalyzed by members of the cytochrome P-450 family of oxidases. This paper examines the impact that various structural features of 1,4-disubstituted-1,2,3,6-tetrahydropyridinyl derivatives have on the oxidative fate of this class of compound.