RESUMO
Diabetic nephropathy is a leading cause of end-stage renal disease, characterized by endothelial dysfunction and a compromised glomerular permeability barrier. Dysregulation of the angiopoietin 1 (ANGPT1)/angiopoietin 2 (ANGPT2) signaling axis is implicated in disease progression. We recently described the discovery of an IgG1 antibody, O010, with therapeutic potential to elevate circulating endogenous ANGPT1, a tyrosine kinase with Ig and epidermal growth factor (EGF) homology domains-2 (TIE2) agonist. Studies are described that detail the effect of various ANGPT1-elevating strategies to limit progression of renal dysfunction in diabetic-obese (db/db) mice. Results demonstrate that adeno-associated virus- or DNA minicircle-directed overexpression of ANGPT1 elicits a reduction in albuminuria (56%-73%) and an improvement in histopathology score (18% reduction in glomerulosclerosis). An improved acetylcholine response in isolated aortic rings was also observed indicative of a benefit on vascular function. In separate pharmacokinetic studies, an efficacious dose of the ANGPT1 DNA minicircle increased circulating levels of the protein by >80%, resulting in a concomitant suppression of ANGPT2. At a dose of O010-producing maximal elevation of circulating ANGPT1 achievable with the molecule (60% increase), no suppression of ANGPT2 was observed in db/db mice, suggesting insufficient pathway engagement; no reduction in albuminuria or improvement in histopathological outcomes were observed. To pinpoint the mechanism resulting in lack of efficacy, we demonstrate, using confocal microscopy, an interference with TIE2 translocation to adherens junctions, resulting in a loss of protection against vascular permeability normally conferred by ANGPT1. Results demonstrated the essential importance of ANGPT1 to maintain the glomerular permeability barrier, and, due to interference of O010 with this process, led to the discontinuation of the molecule for clinical development. SIGNIFICANCE STATEMENT: This body of original research demonstrates that elevation of systemic angiopoietin 1 (ANGPT1) is protective against diabetic nephropathy. However, using a novel biotherapeutic approach to elevate systemic ANGPT1 renoprotection was not observed; we demonstrate that protection was lost due to interference of the therapeutic with ANGPT1/ tyrosine kinase with Ig and EGF homology domains-2 translocation to adherens junctions. Thus, the clinical development of the antibody was terminated.
Assuntos
Angiopoietina-1 , Diabetes Mellitus , Nefropatias Diabéticas , Albuminúria , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Fator de Crescimento Epidérmico , Camundongos , Camundongos Obesos , Proteínas Tirosina QuinasesRESUMO
Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.
Assuntos
Ativadores de Enzimas/química , Glucoquinase/química , Hipoglicemiantes/química , Regulação Alostérica , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/uso terapêutico , Glucoquinase/metabolismo , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/uso terapêutico , Imidazóis/química , Injeções Intravenosas , Niacina/análogos & derivados , Niacina/química , Ratos , Distribuição TecidualRESUMO
Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.
Assuntos
Desenho de Fármacos , Glucoquinase/química , Hipoglicemiantes/síntese química , Indazóis/química , Pirazinas/síntese química , Pirazóis/química , Piridinas/química , Administração Oral , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucoquinase/metabolismo , Teste de Tolerância a Glucose , Meia-Vida , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Indazóis/síntese química , Indazóis/farmacocinética , Indazóis/uso terapêutico , Insulina/metabolismo , Cinética , Ligação Proteica , Pirazinas/farmacocinética , Pirazinas/uso terapêutico , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Piridinas/farmacocinética , Piridinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Glucagon is an important hormone in the regulation of glucose homeostasis, particularly in the maintenance of euglycemia and prevention of hypoglycemia. In type 2 Diabetes Mellitus (T2DM), glucagon levels are elevated in both the fasted and postprandial states, which contributes to inappropriate hyperglycemia through excessive hepatic glucose production. Efforts to discover and evaluate glucagon receptor antagonists for the treatment of T2DM have been ongoing for approximately two decades, with the challenge being to identify an agent with appropriate pharmaceutical properties and efficacy relative to potential side effects. We sought to determine the hepatic & systemic consequence of full glucagon receptor antagonism through the study of the glucagon receptor knock-out mouse (Gcgr-/-) compared to wild-type littermates. RESULTS: Liver transcriptomics was performed using Affymetric expression array profiling, and liver proteomics was performed by iTRAQ global protein analysis. To complement the transcriptomic and proteomic analyses, we also conducted metabolite profiling (~200 analytes) using mass spectrometry in plasma. Overall, there was excellent concordance (R = 0.88) for changes associated with receptor knock-out between the transcript and protein analysis. Pathway analysis tools were used to map the metabolic processes in liver altered by glucagon receptor ablation, the most notable being significant down-regulation of gluconeogenesis, amino acid catabolism, and fatty acid oxidation processes, with significant up-regulation of glycolysis, fatty acid synthesis, and cholesterol biosynthetic processes. These changes at the level of the liver were manifested through an altered plasma metabolite profile in the receptor knock-out mice, e.g. decreased glucose and glucose-derived metabolites, and increased amino acids, cholesterol, and bile acid levels. CONCLUSIONS: In sum, the results of this study suggest that the complete ablation of hepatic glucagon receptor function results in major metabolic alterations in the liver, which, while promoting improved glycemic control, may be associated with adverse lipid changes.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Fígado/metabolismo , Proteômica , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genética , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos/genética , Diabetes Mellitus/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucagon/deficiênciaRESUMO
CX3CR1 has been identified as a highly attractive target for several therapeutic interventions. Despite this potential, no potent antagonists, either small molecule or monoclonal antibody, have been identified. Here we describe the lead finding and engineering approach that lead to the identification of BI 655088, a potent biotherapeutic antagonist to CX3CR1. BI 655088 is a potent CX3CR1 antagonist that, upon therapeutic dosing, significantly inhibits plaque progression in the standard mouse model of atherosclerosis. BI 655088 represents a novel and highly selective biotherapeutic that could reduce inflammation in the atherosclerotic plaque when added to standard of care treatment including statins, which could result in a significant decrease in atherothrombotic events in patients with existing cardiovascular disease.
Assuntos
Aterosclerose/patologia , Receptor 1 de Quimiocina CX3C/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Animais , Progressão da Doença , Humanos , Macaca fascicularis , CamundongosRESUMO
Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies' pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies. Nevertheless, we showed that non-neutralizing antibodies may have therapeutic potential by increasing the level of a target molecule in the circulation.
Assuntos
Angiopoietina-1/imunologia , Anticorpos Monoclonais/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Angiopoietina-1/sangue , Angiopoietina-1/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Macaca fascicularis , Mutação , Ligação Proteica , Receptor TIE-2/genética , Receptor TIE-2/imunologia , Receptor TIE-2/metabolismoRESUMO
Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic ß-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativadores de Enzimas/síntese química , Glucoquinase/metabolismo , Hepatócitos/metabolismo , Hipoglicemiantes/síntese química , Imidazóis/síntese química , Ácidos Nicotínicos/síntese química , Sítio Alostérico , Animais , Glicemia/metabolismo , Cães , Ativadores de Enzimas/farmacocinética , Ativadores de Enzimas/farmacologia , Haplorrinos , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Imidazóis/farmacocinética , Imidazóis/farmacologia , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Masculino , Modelos Moleculares , Ácidos Nicotínicos/farmacocinética , Ácidos Nicotínicos/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
An IgG mouse monoclonal antibody (10F05) against polyethylene glycol has been generated. The antibody reacts with PEG regardless of the linker used for PEG attachment, and is able to recognize a PEGylated peptide in plasma at concentrations as low as 3 pg/mL. The antibody is readily purified in substantial quantities. The PEG IgG will find significant utility in the sensitive detection of PEG derivitives during the pharmacokinetic characterization of PEGylated compounds.
Assuntos
Anticorpos Monoclonais/biossíntese , Polietilenoglicóis/química , Animais , Antígenos/química , Ensaio de Imunoadsorção Enzimática , Hemocianinas/imunologia , Hibridomas/imunologia , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologiaRESUMO
Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Quinolinas/farmacologia , Desenho de Fármacos , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Diester Fosfórico Hidrolases/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
The closely related peptides glucagon-like peptide (GLP-1) and glucagon have opposing effects on blood glucose. GLP-1 induces glucose-dependent insulin secretion in the pancreas, whereas glucagon stimulates gluconeogenesis and glycogenolysis in the liver. The identification of a hybrid peptide acting as both a GLP-1 agonist and a glucagon antagonist would provide a novel approach for the treatment of type 2 diabetes. Toward this end a series of hybrid peptides made up of glucagon and either GLP-1 or exendin-4, a GLP-1 agonist, was engineered. Several peptides that bind to both the GLP-1 and glucagon receptors were identified. The presence of glucagon sequence at the N terminus removed the dipeptidylpeptidase IV cleavage site and increased plasma stability compared with GLP-1. Targeted mutations were incorporated into the optimal dual-receptor binding peptide to identify a peptide with the highly novel property of functioning as both a GLP-1 receptor agonist and a glucagon receptor antagonist. To overcome the short half-life of this mutant peptide in vivo, while retaining dual GLP-1 agonist and glucagon antagonist activities, site-specific attachment of long chained polyethylene glycol (PEGylation) was pursued. PEGylation at the C terminus retained the in vitro activities of the peptide while dramatically prolonging the duration of action in vivo. Thus, we have generated a novel dual-acting peptide with potential for development as a therapeutic for type 2 diabetes.
Assuntos
Peptídeos/química , Receptores de Glucagon/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Glucagon/agonistas , Homologia de Sequência de AminoácidosRESUMO
5-Hydroxyalkyl-4-phenylpyridines have been identified as a novel class of glucagon antagonists with potential utility for the treatment of diabetes. A lead structure with moderate activity was discovered through a high throughput screening assay. Structure-activity relationships led to the discovery of a potent antagonist, IC(50)=0.11 microM, more than 60-fold improvement over the lead structure.
Assuntos
Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Receptores de Glucagon/antagonistas & inibidores , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Relação Estrutura-AtividadeRESUMO
Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.
Assuntos
Neuropeptídeos/farmacologia , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Peptídeo Intestinal Vasoativo/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuropeptídeos/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Peptídeo Intestinal Vasoativo/químicaRESUMO
A narrow structure-activity relationship was established for the 4-aryl group in 4-aryl-pyridine glucagon antagonists, with only small substituents being well-tolerated, and only at the 3'- and 4'-positions. However, substitution with a 2'-hydroxy group gave a ca. 3-fold increase in activity (e.g., 4'-fluoro-2'-hydroxy analogue 33, IC50=190 nM). For efficient preparation of 2'-substituted phenylpyridines, a novel synthesis via pyrones and 4-methoxy-pyridines was developed.
Assuntos
Glucagon/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Piridinas/química , Relação Estrutura-AtividadeRESUMO
Optimized substituent patterns in 4-aryl-pyridine glucagon receptor antagonists were merged to produce highly potent derivatives containing both a 3-[(1R)-hydroxyethyl] and a 2'-hydroxy group. Due to restricted rotation of the phenyl-pyridine bond, these analogues exist as four isomers. A diastereoselective methylcopper reaction was developed to facilitate the synthesis, and single isomers were isolated with activities in the range IC(50)=10-25 nM.