RESUMEN
Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.
Asunto(s)
Péptidos , Proteínas Proto-Oncogénicas c-mdm2 , Péptidos/farmacología , Péptidos/químicaRESUMEN
The combination of insulin and incretin-based therapies has emerged as a potential promising tactic for the treatment of diabetes. Here we report the first example of a unimolecular triagonist to simultaneously target insulin, GLP-1, and glucagon receptors, aiming for better glycemic control and superior weight loss. The strategy for constructing such a unimolecular triagonist is the conjugation of the insulin moiety and GLP-1R/GCGR coagonist peptide via alkyne-azide click chemistry. Two tractable series differentiated by insulin conjugation sites, B1F and B29K, were identified. Triagonist 13 prepared through the conjugation at insulin B1F and position 24 of GLP-1R/GCGR coagonist exhibited insulin activity comparable to that of insulin degludec and potent and balanced GLP-1R and GCGR activities. Pharmacokinetic profiles of 13 in both rat and minipig were also discussed.
RESUMEN
We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of â¼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.
Asunto(s)
Diabetes Mellitus Tipo 2 , Hipoglucemia , Animales , Glucemia , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Perros , Hipoglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Receptor de Insulina , Porcinos , Porcinos Enanos , Índice TerapéuticoRESUMEN
Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/farmacología , Insulina/farmacología , Receptor de Insulina/agonistas , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Aloxano/administración & dosificación , Aloxano/toxicidad , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Células CHO , Cricetulus , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/metabolismo , Células HEK293 , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Lipólisis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratas , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Porcinos , Porcinos EnanosRESUMEN
Due to their relatively large molecular sizes and delicate nature, biologic drugs such as peptides, proteins, and antibodies often require high and repeated dosing, which can cause undesired side effects and physical discomfort in patients and render many therapies inordinately expensive. To enhance the efficacy of biologic drugs, they could be encapsulated into polymeric hydrogel formulations to preserve their stability and help tune their release in the body to their most favorable profile of action for a given therapy. In this study, a series of injectable, thermoresponsive hydrogel formulations were evaluated as controlled delivery systems for various peptides and proteins, including insulin, Merck proprietary peptides (glucagon-like peptide analogue and modified insulin analogue), bovine serum albumin, and immunoglobulin G. These hydrogels were prepared using concentrated solutions of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), which can undergo temperature-induced sol-gel transitions and spontaneously solidify into hydrogels near the body temperature, serving as an in situ depot for sustained drug release. The thermoresponsiveness and gelation properties of these triblock copolymers were characterized by dynamic light scattering (DLS) and oscillatory rheology, respectively. The impact of different hydrogel-forming polymers on release kinetics was systematically investigated based on their hydrophobicity (LA/GA ratios), polymer concentrations (20, 25, and 30%), and phase stability. These hydrogels were able to release active peptides and proteins in a controlled manner from 4 to 35 days, depending on the polymer concentration, solubility nature, and molecular sizes of the cargoes. Biophysical studies via size exclusion chromatography (SEC) and circular dichroism (CD) indicated that the encapsulation and release did not adversely affect the protein conformation and stability. Finally, a selected PLGA-PEG-PLGA hydrogel system was further investigated by the encapsulation of a therapeutic glucagon-like peptide analogue and a modified insulin peptide analogue in diabetic mouse and minipig models for studies of glucose-lowering efficacy and pharmacokinetics, where superior sustained peptide release profiles and long-lasting glucose-lowering effects were observed in vivo without any significant tolerability issues compared to peptide solution controls. These results suggest the promise of developing injectable thermoresponsive hydrogel formulations for the tunable release of protein therapeutics to improve patient's comfort, convenience, and compliance.
RESUMEN
NMR measurements of rotational and translational diffusion are used to characterize the solution behavior of a wide variety of therapeutic proteins and peptides. The timescales of motions sampled in these experiments reveal complicated intrinsic solution behavior such as flexibility, that is central to function, as well as self-interactions, stress-induced conformational changes and other critical attributes that can be discovery and development liabilities. Trends from proton transverse relaxation (R2 ) and hydrodynamic radius (Rh ) are correlated and used to identify and differentiate intermolecular from intramolecular interactions. In this study, peptide behavior is consistent with complicated multimer self-assembly, while multi-domain protein behavior is dominated by intramolecular interactions. These observations are supplemented by simulations that include effects from slow transient interactions and rapid internal motions. R2 -Rh correlations provide a means to profile protein motions as well as interactions. The approach is completely general and can be applied to therapeutic and target protein characterization.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Proteínas/químicaRESUMEN
Insulin oligosaccharide conjugates hold promise as potential glucose-responsive insulins (GRIs), which can improve the therapeutic index of insulins and mitigate the risk of hypoglycemia. A key challenge for the analytical development of such molecules is finding an efficient method to characterize the purity and impurities of conjugated insulins. Using the S-Matrix Fusion QbD-ultrahigh performance liquid chromatography (UHPLC) integrated system, we were able to quickly screen and develop two short UHPLC methods. These methods were used to support process development, clinical batch drug substance (DS) release, and stability studies of MK-2640, an insulin oligosaccharide conjugate. Both methods used a Waters CSH C18 column, with a shallow gradient of acetonitrile to aqueous mobile phase containing 25 mM sodium perchlorate and 0.05% perchloric acid. The 10-min run time method was well suited for process development and monitoring as it was able to separate the main product, MK-2640, six oligosaccharide-substituted recombinant human insulin (RHI) impurities, A21 deamidated MK-2640, and the starting material RHI. The 13-min run time method provided improved separation of the major impurities and demonstrated good chromatographic reproducibility on different instruments or using columns from different lots of stationary phase, which made it ideal for the final DS release. Validation of the 13-min method demonstrated great linearity for both the MK-2640 main peak and its related impurities, low limit of detection (0.02%), and limit of quantitation (0.05%). The high specificity of the method allowed the separation of the degradation products from main peak, thus makes it suitable for stability monitoring. The major impurities in the DS were characterized by two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glucosa/metabolismo , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Humanos , Límite de Detección , Espectrometría de Masas/métodos , Reproducibilidad de los ResultadosRESUMEN
A synthetic method to access novel azido-insulin analogs directly from recombinant human insulin (RHI) was developed via diazo-transfer chemistry using imidazole-1-sulfonyl azide. Systematic optimization of reaction conditions led to site-selective azidation of amino acids B1-phenylalanine and B29-lysine present in RHI. Subsequently, the azido-insulin analogs were used in azide-alkyne [3 + 2] cycloaddition reactions to synthesize a diverse array of triazole-based RHI bioconjugates that were found to be potent human insulin receptor binders. The utility of this method was further demonstrated by the concise and controlled synthesis of a heterotrisubstituted insulin conjugate.
Asunto(s)
Azidas/síntesis química , Insulina/química , Secuencia de Aminoácidos , Aminoácidos/química , Azidas/química , Reacción de Cicloadición , Humanos , Proteínas Recombinantes/química , Triazoles/químicaRESUMEN
We evaluated the hepatic and nonhepatic responses to glucose-responsive insulin (GRI). Eight dogs received GRI or regular human insulin (HI) in random order. A primed, continuous intravenous infusion of [3-3H]glucose began at -120 min. Basal sampling (-30 to 0 min) was followed by two study periods (150 min each), clamp period 1 (P1) and clamp period 2 (P2). At 0 min, somatostatin and GRI (36 ± 3 pmol/kg/min) or HI (1.8 pmol/kg/min) were infused intravenously; basal glucagon was replaced intraportally. Glucose was infused intravenously to clamp plasma glucose at 80 mg/dL (P1) and 240 mg/dL (P2). Whole-body insulin clearance and insulin concentrations were not different in P1 versus P2 with HI, but whole-body insulin clearance was 23% higher and arterial insulin 16% lower in P1 versus P2 with GRI. Net hepatic glucose output was similar between treatments in P1. In P2, both treatments induced net hepatic glucose uptake (HGU) (HI mean ± SEM 2.1 ± 0.5 vs. 3.3 ± 0.4 GRI mg/kg/min). Nonhepatic glucose uptake in P1 and P2, respectively, differed between treatments (2.6 ± 0.3 and 7.4 ± 0.6 mg/kg/min with HI vs. 2.0 ± 0.2 and 8.1 ± 0.8 mg/kg/min with GRI). Thus, glycemia affected GRI but not HI clearance, with resultant differential effects on HGU and nonHGU. GRI holds promise for decreasing hypoglycemia risk while enhancing glucose uptake under hyperglycemic conditions.
Asunto(s)
Evaluación Preclínica de Medicamentos , Drogas en Investigación/efectos adversos , Metabolismo Energético/efectos de los fármacos , Hipoglucemiantes/efectos adversos , Insulina Regular Humana/análogos & derivados , Hígado/efectos de los fármacos , Absorción Fisiológica/efectos de los fármacos , Animales , Glucemia/análisis , Glucemia/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Drogas en Investigación/administración & dosificación , Drogas en Investigación/farmacocinética , Gluconeogénesis/efectos de los fármacos , Técnica de Clampeo de la Glucosa , Glicosilación , Humanos , Hiperglucemia/metabolismo , Hiperglucemia/prevención & control , Hipoglucemia/inducido químicamente , Hipoglucemia/metabolismo , Hipoglucemia/prevención & control , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacocinética , Infusiones Intravenosas , Insulina Regular Humana/administración & dosificación , Insulina Regular Humana/efectos adversos , Insulina Regular Humana/farmacocinética , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica , Distribución Aleatoria , Somatostatina/administración & dosificación , Somatostatina/efectos adversosRESUMEN
Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.
Asunto(s)
Glucosa/metabolismo , Hipoglucemia/tratamiento farmacológico , Insulina/farmacología , Animales , Antígenos CD , Glucemia , Línea Celular , Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Hipoglucemiantes/farmacología , Lectinas Tipo C/efectos de los fármacos , Hígado/patología , Macrófagos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratas , Receptor de Insulina/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacosRESUMEN
Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by â¼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diseño de Fármacos , Hipoglucemiantes/uso terapéutico , Insulina Regular Humana/análogos & derivados , Lectinas Tipo C/agonistas , Lectinas de Unión a Manosa/agonistas , Receptor de Insulina/agonistas , Receptores de Superficie Celular/agonistas , Animales , Animales Endogámicos , Unión Competitiva , Células CHO , Cricetulus , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Hiperglucemia/prevención & control , Hipoglucemia/inducido químicamente , Hipoglucemia/prevención & control , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacocinética , Insulina Regular Humana/efectos adversos , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/uso terapéutico , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Tasa de Depuración Metabólica , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Porcinos , Porcinos EnanosRESUMEN
Glucokinase (GK, hexokinase IV) is a unique hexokinase that plays a central role in mammalian glucose homeostasis. Glucose phosphorylation by GK in the pancreatic ß-cell is the rate-limiting step that controls glucose-stimulated insulin secretion. Similarly, GK-mediated glucose phosphorylation in hepatocytes plays a major role in increasing hepatic glucose uptake and metabolism and possibly lowering hepatic glucose output. Small molecule GK activators (GKAs) have been identified that increase enzyme activity by binding to an allosteric site. GKAs offer a novel approach for the treatment of Type 2 Diabetes Mellitus (T2DM) and as such have garnered much attention. We now report the design, synthesis, and biological evaluation of a novel series of 2,5,6-trisubstituted indole derivatives that act as highly potent GKAs. Among them, Compound 1 was found to possess high in vitro potency, excellent physicochemical properties, and good pharmacokinetic profile in rodents. Oral administration of Compound 1 at doses as low as 0.03mg/kg led to robust blood glucose lowering efficacy in 3week high fat diet-fed mice.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Activadores de Enzimas/química , Activadores de Enzimas/uso terapéutico , Glucoquinasa/metabolismo , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Indoles/química , Indoles/uso terapéutico , Regulación Alostérica/efectos de los fármacos , Animales , Glucemia/análisis , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacocinética , Activadores de Enzimas/farmacología , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Indoles/farmacocinética , Indoles/farmacología , Insulina/sangre , Insulina/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Systemically acting glucokinase activators (GKA) have been demonstrated in clinical trials to effectively lower blood glucose in patients with type II diabetes. However, mechanism-based hypoglycemia is a major adverse effect that limits the therapeutic potential of these agents. We hypothesized that the predominant mechanism leading to hypoglycemia is GKA-induced excessive insulin secretion from pancreatic ß-cells at (sub-)euglycemic levels. We further hypothesized that restricting GK activation to hepatocytes would maintain glucose-lowering efficacy while significantly reducing hypoglycemic risk. Here we report the discovery of a novel series of carboxylic acid substituted GKAs based on pyridine-2-carboxamide. These GKAs exhibit preferential distribution to the liver versus the pancreas in mice. SAR studies led to the identification of a potent and orally active hepatoselective GKA, compound 6. GKA 6 demonstrated robust glucose lowering efficacy in high fat diet-fed mice at doses ⩾10mpk, with ⩾70-fold liver:pancreas distribution, minimal effects on plasma insulin levels, and significantly reduced risk of hypoglycemia.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Activadores de Enzimas/farmacología , Glucoquinasa/metabolismo , Hipoglucemiantes/farmacología , Piridinas/farmacología , Animales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Descubrimiento de Drogas , Activadores de Enzimas/química , Activadores de Enzimas/farmacocinética , Activadores de Enzimas/uso terapéutico , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Piridinas/química , Piridinas/farmacocinética , Piridinas/uso terapéuticoRESUMEN
Several series of novel non-thiourea-containing aminopyrazine derivatives were designed based on the MK-2 inhibitors 1-(2-aminopyrazin-3-yl)methyl-2-thioureas. These compounds were synthesized and evaluated for their inhibitory activity against MK-2 enzyme in vitro. Compounds with low micromolar to sub-micromolar IC50 values were identified, and several compounds were also found to be active in suppressing the lipopolysaccharide (LPS)-stimulated TNFα production in THP-1 cells with minimum shift compared to their enzyme activity.
Asunto(s)
Diseño de Fármacos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Pirazinas/síntesis química , Pirazinas/farmacología , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Pirazinas/químicaRESUMEN
Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.
Asunto(s)
Colesterol/sangre , Pirazoles/farmacología , Receptores de Glucagón/antagonistas & inhibidores , beta-Alanina/análogos & derivados , Animales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Humanos , Hipercolesterolemia/inducido químicamente , Concentración 50 Inhibidora , Absorción Intestinal , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirazoles/efectos adversos , beta-Alanina/efectos adversos , beta-Alanina/farmacologíaRESUMEN
A novel, potent series of glucagon receptor antagonists (GRAs) was discovered. These indazole- and indole-based compounds were designed on an earlier pyrazole-based GRA lead MK-0893. Structure-activity relationship (SAR) studies were focused on the C3 and C6 positions of the indazole core, as well as the benzylic position on the N-1 of indazole. Multiple potent GRAs were identified with excellent in vitro profiles and good pharmacokinetics in rat. Among them, GRA 16d was found to be orally active in blunting glucagon induced glucose excursion in an acute glucagon challenge model in glucagon receptor humanized (hGCGR) mice at 1, 3 and 10mg/kg (mpk), and significantly lowered acute glucose levels in hGCGR ob/ob mice at 3 mpk dose.
Asunto(s)
Indazoles/química , Indazoles/farmacología , Indoles/química , Indoles/farmacología , Receptores de Glucagón/antagonistas & inhibidores , Animales , Células CHO , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Obesos , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
Glucokinase activators (GKAs) are small-molecule agents that enhance glucose sensing by pancreatic ß cells and glucose metabolism by hepatocytes. There is strong interest in these agents as potential therapies for type 2 diabetes. Here, we report key pharmacokinetic and pharmacodynamic findings from preclinical studies of the GKA 3-[[6-(ethylsulfonyl)-3-pyridinyl]oxy]-5-[(1S)-2-hydroxy-1-methylethoxy]-N-(1-methyl-1H-pyrazol-3-yl)benzamide (MK-0941). Incubated in vitro with recombinant human glucokinase, 1 µM MK-0941 lowered the S(0.5) of this enzyme for glucose from 6.9 to 1.4 mM and increased the maximum velocity of glucose phosphorylation by 1.5-fold. In 2.5 and 10 mM glucose, the EC(50) values for activation of GK by MK-0941 were 0.240 and 0.065 µM, respectively. Treatment of isolated rat islets of Langerhans and hepatocytes with 10 µM MK-0941 increased insulin secretion by 17-fold and glucose uptake up to 18-fold, respectively. MK-0941 exhibited strong glucose-lowering activity in C57BL/6J mice maintained on a high-fat diet (HFD), db/db mice, HFD plus low-dose streptozotocin-treated mice, and nondiabetic dogs. In both mice and dogs, oral doses of MK-0941 were rapidly absorbed and rapidly cleared from the blood; plasma levels reached maximum within 1 h and fell thereafter with a half-life of ~2 h. During oral glucose tolerance testing in dogs, MK-0941 reduced total area-under-the-curve postchallenge (0-2 h) plasma glucose levels by up to 48% compared with vehicle-treated controls. When administered twice daily to mice for 16 days, and once daily to the dog for 4 days, MK-0941 remained efficacious on successive days. These findings support further investigation of MK-0941 as a potential therapeutic agent for treatment of type 2 diabetes.