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Robust methods are needed for preclinical evaluation of novel Alzheimer Disease (AD) therapies to accelerate drug discovery. Quantitative Gradient Recalled Echo (qGRE) MRI has shown promise to provide insight into neurodegeneration in AD prior to atrophy development in humans, highlighting areas of low neuronal density. In this study a novel qGRE method (20 echoes, TE=2-40ms) is shown to non-invasively measure the longitudinal neuronal loss in the hippocampus of a mouse model of AD tauopathy Tg4510. Tg4510 (n=10) and wild type (WT, n=6) mice underwent MRI (7T field strength) at 3-7 months old. 3D qGRE approach was used to generate brain-specific R2* maps free of magnetic field inhomogeneity artifacts. Light-sheet microscopy of the brains stained with NeuN and MBP served to visualize neuronal nuclei and myelin content respectively. Significant decrease in NeuN staining between 3mo and 5mo was observed in the hippocampus of Tg4510, validating the mouse AD model. Longitudinal analysis showed clear decreases in R2* metric of qGRE signal in the Tg4510 mice hippocampus undergoing neurodegeneration between 3 and 5 months old. Histogram analysis revealed an upward trend in patterns of low R2* value (Dark Matter, DM), and broadening of R2* distribution. These were quantified as significant increase in both DM Volume Fraction (DMVF) and R2* Standard Deviation (SD) in Tg4510 mice (p=0.004/p=0.016 DMVF/SD) but not in WT controls (p>0.25). Further monotonical increase was also observed in both metrics in time. A significant negative correlation was observed between the DMVF and myelin content (p=0.01, r=-0.76), suggesting sensitivity of the technique to the loss of myelinated axons. The presented qGRE technique, validated by histological measurements, can be readily applied as in vivo tool in preclinical models of neurodegeneration for pharmacodynamics and mechanism of action assessment.
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The neuronal tricarboxylic acid and glutamate/glutamine (Glu/Gln) cycles play important roles in brain function. These processes can be measured in vivo using dynamic 1H-[13C] MRS during administration of 13C-labeled glucose. Proton-observed carbon-edited (POCE) MRS enhances the signal-to-noise ratio (SNR) compared with direct 13C-MRS. Ultra-high field further boosts the SNR and increases spectral dispersion; however, even at 7 T, Glu and Gln 1H-resonances may overlap. Further gain can be obtained with selective POCE (selPOCE). Our aim was to create a setup for indirect dynamic 1H-[13C] MRS in the human brain at 7 T. A home-built non-shielded transmit-receive 13C-birdcage head coil with eight transmit-receive 1H-dipole antennas was used together with a 32-channel 1H-receive array. Electromagnetic simulations were carried out to ensure that acquisitions remained within local and global head SAR limits. POCE-MRS was performed using slice-selective excitation with semi-localization by adiabatic selective refocusing (sLASER) and stimulated echo acquisition mode (STEAM) localization, and selPOCE-MRS using STEAM. Sequences were tested in a phantom containing non-enriched Glu and Gln, and in three healthy volunteers during uniformly labeled 13C-glucose infusions. In one subject the voxel position was alternated between bi-frontal and bi-occipital placement within one session. [4-13C]Glu-H4 and [4-13C]Gln-H4 signals could be separately detected using both STEAM-POCE and STEAM-selPOCE in the phantom. In vivo, [4,5-13C]Glx could be detected using both sLASER-POCE and STEAM-POCE, with similar sensitivities, but [4,5-13C]Glu and [4,5-13C]Gln signals could not be completely resolved. STEAM-POCE was alternately performed bi-frontal and bi-occipital within a single session without repositioning of the subject, yielding similar results. With STEAM-selPOCE, [4,5-13C]Glu and [4,5-13C]Gln could be clearly separated. We have shown that with our setup indirect dynamic 1H-[13C] MRS at 7 T is feasible in different locations in the brain within one session, and by using STEAM-selPOCE it is possible to separate Glu from Gln in vivo while obtaining high quality spectra.
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Weight gain is a common harmful side effect of atypical antipsychotics used for schizophrenia treatment. Conversely, treatment with the novel phosphodiesterase-10A (PDE10A) inhibitor MK-8189 in clinical trials led to significant weight reduction, especially in patients with obesity. This study aimed to understand and describe the mechanism underlying this observation, which is essential to guide clinical decisions. We hypothesized that PDE10A inhibition causes beiging of white adipose tissue (WAT), leading to weight loss. Magnetic resonance imaging (MRI) methods were developed, validated, and applied in a diet-induced obesity mouse model treated with a PDE10A inhibitor THPP-6 or vehicle for measurement of fat content and vascularization of adipose tissue. Treated mice showed significantly lower fat fraction in white and brown adipose tissue, and increased perfusion and vascular density in WAT versus vehicle, confirming the hypothesis, and matching the effect of CL-316,243, a compound known to cause adipose tissue beiging. The in vivo findings were validated by qPCR revealing upregulation of Ucp1 and Pcg1-α genes, known markers of WAT beiging, and angiogenesis marker VegfA in the THPP-6 group. This work provides a detailed understanding of the mechanism of action of PDE10A inhibitor treatment on adipose tissue and body weight and will be valuable to guide both the use of MK-8189 in schizophrenia and the potential application of the target for weight loss indication.
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Tejido Adiposo Blanco , Inhibidores de Fosfodiesterasa , Ratones , Animales , Inhibidores de Fosfodiesterasa/farmacología , Obesidad/genética , Tejido Adiposo Pardo/patología , Pérdida de Peso , Imagen por Resonancia Magnética/efectos adversosRESUMEN
Administration of long-acting injectable suspensions is an increasingly common approach to increasing patient compliance and improving therapeutic efficacy through less frequent dosing. While several long-acting suspensions have recently been marketed, parameters modulating drug absorption from suspension-based formulations are not well understood. Further, methods for predicting clinical pharmacokinetic data from preclinical studies are not well established. Together, these limitations hamper compound selection, formulation design and formulation selection through heavy reliance on iterative optimization in preclinical and clinical studies. This article identifies key parameters influencing absorption from suspension-based formulations through compilation and analysis of preclinical and clinical pharmacokinetic data of seven compounds marketed as suspensions; achievable margins for predicting the clinical dose and input rate from preclinical studies as a function of the preclinical species, the clinical injection location and the intended therapeutic duration were also established.
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Suspensiones , Humanos , Estudios Retrospectivos , InyeccionesRESUMEN
BACKGROUND: The development of treatments for cognitive deficits associated with central nervous system disorders is currently a significant medical need. Despite the great need for such therapeutics, a significant challenge in the drug development process is the paucity of robust biomarkers to assess target modulation and guide clinical decisions. We developed a novel, translatable biomarker of neuronal glutamate metabolism, the 13C-glutamate+glutamine (Glx) H3:H4 labeling ratio, in nonhuman primates using localized 1H-magnetic resonance spectroscopy combined with 13C-glucose infusions. METHODS: We began with numerical simulations in an established model of brain glutamate metabolism, showing that the 13C-Glx H3:H4 ratio should be a sensitive biomarker of neuronal tricarboxylic acid cycle activity, a key measure of overall neuronal metabolism. We showed that this biomarker can be measured reliably using a standard 1H-magnetic resonance spectroscopy method (point-resolved spectroscopy sequence/echo time = 20 ms), obviating the need for specialized hardware and pulse sequences typically used with 13C-magnetic resonance spectroscopy, thus improving overall clinical translatability. Finally, we used this biomarker in 8 male rhesus macaques before and after administration of the compound BNC375, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor that enhances glutamate signaling ex vivo and elicits procognitive effects in preclinical species. RESULTS: The 13C-Glx H3:H4 ratios in the monkeys showed that BNC375 increases neuronal metabolism in nonhuman primates in vivo, detectable on an individual basis. CONCLUSIONS: This study demonstrates that the ratio of 13C-Glx H3:H4 labeling is a biomarker that may provide an objective readout of compounds affecting glutamatergic neurotransmission and could improve decision making for the development of therapeutic agents.
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Ácido Glutámico , Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Bencetonio , Biomarcadores , Clorobencenos , Ácido Glutámico/metabolismo , Humanos , Macaca mulatta , Espectroscopía de Resonancia Magnética , MasculinoRESUMEN
Despite being first published over 40 years ago, the combination of 13C nuclear magnetic resonance spectroscopy (NMR) and the isolated perfused liver preparation remains a unique and relevant approach in investigating the effects of pharmacological interventions on hepatic metabolism. The use of intact, perfused livers maintains many metabolic reactions at their respective rates in vivo, while the use of 13C-labelled substrates in combination with 13C NMR allows for a detailed study of specific pathways, as well as the design of robust assays which can be used to evaluate novel pharmacological agents. In this review article, we share some of the methods used to probe glucose metabolism, and highlight key findings and successes derived from the application of this specialized technique to the area of drug development for diabetes and related metabolic disorders.
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Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.
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Bencetonio/farmacología , Clorobencenos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Regulación Alostérica , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cognición/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Macaca mulatta , Masculino , Neurotransmisores/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Escopolamina/farmacologíaRESUMEN
Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.
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Dieta , Fructoquinasas/deficiencia , Errores Innatos del Metabolismo de la Fructosa/genética , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/genética , Dieta Alta en Grasa , Ingestión de Alimentos/genética , Fructoquinasas/genética , Fructoquinasas/metabolismo , Errores Innatos del Metabolismo de la Fructosa/metabolismo , Fructosafosfatos/sangre , Intolerancia a la Glucosa/genética , Resistencia a la Insulina , Metabolismo de los Lípidos/genética , Ratones NoqueadosRESUMEN
GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.
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Glucosa/metabolismo , Incretinas/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Línea Celular , Cricetulus , Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Ratones , RatasRESUMEN
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.
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Proteínas Quinasas Activadas por AMP/metabolismo , Cardiomegalia/inducido químicamente , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Imidazoles/farmacología , Piridinas/farmacología , Animales , Bencimidazoles , Glucemia/efectos de los fármacos , Ayuno , Glucógeno/metabolismo , Hipoglucemia/inducido químicamente , Imidazoles/efectos adversos , Imidazoles/química , Insulina/farmacología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Piridinas/efectos adversos , Piridinas/químicaRESUMEN
GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.
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Benzopiranos/farmacología , Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Receptores Acoplados a Proteínas G/agonistas , Tiazolidinedionas/farmacología , Animales , Benzopiranos/química , Glucemia/metabolismo , Células CHO , Cricetulus , Diabetes Mellitus Experimental/metabolismo , Evaluación Preclínica de Medicamentos , Ayuno/sangre , Células HEK293 , Humanos , Hipoglucemiantes/química , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones Noqueados , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tiazolidinedionas/química , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.
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Glucemia/metabolismo , Indicadores y Reactivos , Animales , Glucemia/efectos de los fármacos , Isótopos de Carbono , Dieta Alta en Grasa , Femenino , Técnica de Clampeo de la Glucosa , Hipoglucemiantes/farmacología , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Resistencia a la Insulina , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proyectos Piloto , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Rosiglitazona , Tiazolidinedionas/farmacologíaRESUMEN
Liver glycogen represents an important physiological form of energy storage. It plays a key role in the regulation of blood glucose concentrations, and dysregulations in hepatic glycogen metabolism are linked to many diseases including diabetes and insulin resistance. In this work, we develop, optimize, and validate a noninvasive protocol to measure glycogen levels in isolated perfused mouse livers using chemical exchange saturation transfer (CEST) NMR spectroscopy. Model glycogen solutions were used to determine optimal saturation pulse parameters which were then applied to intact perfused mouse livers of varying glycogen content. Glycogen measurements from serially acquired CEST Z-spectra of livers were compared with measurements from interleaved natural abundance (13)C NMR spectra. Experimental data revealed that CEST-based glycogen measurements were highly correlated with (13)C NMR glycogen spectra. Monte Carlo simulations were then used to investigate the inherent (i.e., signal-to-noise-based) errors in the quantification of glycogen with each technique. This revealed that CEST was intrinsically more precise than (13)C NMR, although in practice may be prone to other errors induced by variations in experimental conditions. We also observed that the CEST signal from glycogen in liver was significantly less than that observed from identical amounts in solution. Our results demonstrate that CEST provides an accurate, precise, and readily accessible method to noninvasively measure liver glycogen levels and their changes. Furthermore, this technique can be used to map glycogen distributions via conventional proton magnetic resonance imaging, a capability universally available on clinical and preclinical magnetic resonance imaging (MRI) scanners vs (13)C detection, which is limited to a small fraction of clinical-scale MRI scanners.
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Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Glucógeno/análisis , Hígado/química , Espectroscopía de Resonancia Magnética , Animales , RatonesRESUMEN
The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.
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Hígado/metabolismo , Receptores de Glucagón/antagonistas & inhibidores , Animales , Células CHO , Radioisótopos de Carbono , Cricetinae , Humanos , Glucógeno Hepático/biosíntesis , Espectroscopía de Resonancia Magnética/métodos , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Ácido Pirúvico/metabolismo , Receptores de Glucagón/metabolismo , Factores de TiempoRESUMEN
Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 +/- 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a KDB of 81 +/- 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon-induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo.