Discovery of Vibegron: A Potent and Selective ß3 Adrenergic Receptor Agonist for the Treatment of Overactive Bladder.

The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis.

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.

Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver.

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.

Type B Ventricular Preexcitation With Abnormal Contraction of the Ventricular Septum in a Dog.

A 14monthold female beagle had ventricular preexcitation (VP).The finding was characterized by a wide positive QRS complex with a tall notched R wave in leads I, II, III, and aVF, an inverted QRS complex in leads aVR and aVL, a Q wave in lead I, and a short PR interval. Compression of the carotid sinus caused an anticipated marked decrease in heart rate, but did not reveal latent electrocardiographic abnormalities. We did not detect evidence of ventricular hypertrophy during echocardiography. Examination of the M-mode image indicated abnormal movements of the septum. There were 2 sharp waves at each systole instead of a single wider wave that is seen for clinically normal dogs. To further characterize this ECG finding, the affected dog and 2 clinically normal female beagles (positive control dogs) were given atropine (0.025 mg/kg of body weight, i. v.) Increases in heart rate, relative to values obtained before atropine administration, were evident in all 3 dogs. Increase in heart rate in the dog with VP appeared sooner after injection than in the clinically normal dogs; it was evident at the conclusion of the atropine injection. When the increase in heart rate was maximal in the affected dog (3 min after atropine administration), notching of the R wave disappeared, and the QRS duration decreased to about 60 ms. Echocardiographically, atropine produced a decrease in end diastolic, end systolic and stroke volumes in all treated dogs, which was similar between clinically normal dogs and the dog with VP. Atropine administration also was associated with a decrease in the percentage of thickening of the septum in the dog with VP, but not in the clinically normal dogs. We did not detect histopathologic abnormalities in the heart of the dog with VP.