Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist, Vibegron: Evaluation of Antimuscarinic Receptor Selectivity for Combination Therapy for Overactive Bladder.

Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of ß3-adrenergic receptors (ß3ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel ß3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of ß3AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full ß3AR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a ß3AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel ß3AR agonist probe, [3H]MRL-037, that selectively labels ß3 receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of ß3AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.

Chronic treatment with a glucokinase activator delays the onset of hyperglycaemia and preserves beta cell mass in the Zucker diabetic fatty rat.

AIMS/HYPOTHESIS: Glucokinase activators (GKAs) are currently being developed as new therapies for type 2 diabetes and have been shown to enhance beta cell survival and proliferation in vitro. Here, we report the effects of chronic GKA treatment on the development of hyperglycaemia and beta cell loss in the male Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes with severe obesity. METHODS: Cell protection by GKA was studied in MIN6 and INS-1 cells exposed to hydrogen peroxide. Glucose homeostasis and beta cell mass were evaluated in ZDF rats dosed for 41 days with Cpd-C (a GKA) or glipizide (a sulfonylurea) as food admixtures at doses of approximately 3 and 10 mg kg(-1) day(-1). RESULTS: Incubation of MIN6 and INS-1 832/3 insulinoma cell cultures with GKA significantly reduced cell death and impairment of intracellular NADH production caused by exposure to hydrogen peroxide. Progression from prediabetes (normoglycaemia and hyperinsulinaemia) to overt diabetes (hyperglycaemia and hypoinsulinaemia) was significantly delayed in male ZDF rats by in-feed treatment with Cpd-C, but not glipizide. Glucose tolerance, tested in the fifth week of treatment, was also significantly improved by Cpd-C, as was pancreatic insulin content and beta cell area. In a limited immunohistochemical analysis, Cpd-C modestly and significantly enhanced the rate of beta cell proliferation, but not rates of beta cell apoptosis relative to untreated ZDF rats. CONCLUSIONS/INTERPRETATION: These findings suggest that chronic activation of glucokinase preserves beta cell mass and delays disease in the ZDF rat, a model of insulin resistance and progressive beta cell failure.

Chronic treatment with a glucagon receptor antagonist lowers glucose and moderately raises circulating glucagon and glucagon-like peptide 1 without severe alpha cell hypertrophy in diet-induced obese mice.

AIMS/HYPOTHESIS: Antagonism of the glucagon receptor (GCGR) represents a potential approach for treating diabetes. Cpd-A, a potent and selective GCGR antagonist (GRA) was studied in preclinical models to assess its effects on alpha cells. METHODS: Studies were conducted with Cpd-A to examine the effects on glucose-lowering efficacy, its effects in combination with a dipeptidyl peptidase-4 (DPP-4) inhibitor, and the extent and reversibility of alpha cell hypertrophy associated with GCGR antagonism in mouse models. RESULTS: Chronic treatment with Cpd-A resulted in effective and sustained glucose lowering in mouse models in which endogenous murine Gcgr was replaced with human GCGR (hGCGR). Treatment with Cpd-A also led to stable, moderate elevations in both glucagon and glucagon-like peptide 1 (GLP-1) levels, which were completely reversible and not associated with a hyperglycaemic overshoot following termination of treatment. When combined with a DPP-4 inhibitor, Cpd-A led to additional improvement of glycaemic control correlated with elevated active GLP-1 levels after glucose challenge. In contrast to Gcgr-knockout mice in which alpha cell hypertrophy was detected, chronic treatment with Cpd-A in obese hGCGR mice did not result in gross morphological changes in pancreatic tissue. CONCLUSIONS/INTERPRETATION: A GRA lowered glucose effectively in diabetic models without significant alpha cell hypertrophy during or following chronic treatment. Treatment with a GRA may represent an effective approach for glycaemic control in patients with type 2 diabetes, which could be further enhanced when combined with DPP-4 inhibitors.

Characterization of collagen fibril segments from chicken embryo cornea, dermis and tendon.

The cornea, dermis and tendon have extracellular matrix architectures with differences in fibril diameter, packing and organization. An early step in fibril assembly is the formation of a striated fibril of discrete length (segment). Fibril segments were isolated from developing chicken cornea, dermis and tendon by physical disruption and the structure characterized. In all three tissues, intact but relatively short fibril lengths were isolated. These segments were asymmetric, having long (alpha) and short (beta) tapered ends. They were also centrosymmetric with respect to molecular packing. Segments isolated from 12- to 16-day corneas, dermis and tendons had identical structures, but their lengths and diameters were distinct. We propose that the increase in length is, at least in part, the result of lateral associations of adjacent segments. In the developing tendon, there is a rapid increase in length and diameter between day 16 and 17, while in the dermis the increase is more linear with respect to time. In the cornea, the fibril segments grow longer, but their diameters remain constant. Disruption of corneas in phosphate-buffered saline yielded larger diameter segments than seen in situ, while tendon or dermis maintained tissue-specific diameters. When corneas were disrupted in buffers that stabilized the water layer associated with the collagen molecules or containing the corneal proteoglycans, then tissue-specific diameters were maintained. These data suggest differences in the stabilization of segments during growth in tissues where diameter increases versus those where diameter remains constant, and this may be related to collagen-proteoglycan interactions.

Potential role for epidermal Langerhans cells in nicotinic acid-induced vasodilatation in the mouse.

OBJECTIVE: Our objective is to study the role of cutaneous Langerhans cells on a mouse model of nicotinic acid-induced vasodilatation. METHODS: Nicotinic acid-induced vasodilatation was studied in the mouse ear by laser Doppler flowmetry prior to and at intervals after Langerhans cells depletion by treatment with hydrocortisone. RESULTS: Nicotinic acid evoked a dose-dependent increase in perfusion in the mouse ear. Treatment with 1 % hydrocortisone resulted in substantial depletion of Langerhans cells, accompanied by failure to show vasodilatation in response to nicotinic acid. Partial recovery of Langerhans cells on day 53 post-treatment was associated with a partial vasodilatation response. To exclude non-specific effects of hydrocortisone on arachidonic acid metabolism, the ability of the mice to mount an edema response to phorbol 12-myristate 13-acetate was evaluated. On day 9 post hydrocortisone, phorbol 12-myristate 13-acetate failed to evoke an edema response. However, on day 22 post hydrocortisone, the edema response in the hydrocortisone-treated animals was indistinguishable from that of control animals. CONCLUSIONS: These results suggest that Langerhans cells are responsible for nicotinic acid-induced vasodilatation.

Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia.

AIMS/HYPOTHESIS: Under normal physiological conditions, glucagon signalling is important in glucose homeostasis. Hyperglucagonaemia or altered insulin:glucagon ratio plays a role in maintaining hyperglycaemia in subjects with type 2 diabetes. It has been reported that glucagon receptor knockout (Gcgr (-/-)) mice develop normally and have lower plasma glucose on a normal diet. The goal of the current research was to further investigate the role of glucagon signalling in metabolic control and glucose homeostasis. METHODS: Gcgr (-/-) mice were challenged with a high-fat diet (HFD) and with streptozotocin, which induces beta cell damage. They were then analysed for whole-body and serum metabolic phenotypes as well as pancreatic islet morphology. RESULTS: In comparison with wild-type mice, Gcgr (-/-) mice exhibited decreased body weight and food intake, reduced plasma glucose levels, and improved oral and intraperitoneal glucose tolerance. Elevated glucagon-like peptide-1 levels and reduced gastric emptying were also observed in Gcgr (-/-) mice, which also had reduced HFD-induced hyperinsulinaemia and hyperleptinaemia, and were resistant to the development of hepatic steatosis. In addition, Gcgr (-/-) mice were resistant to STZ-induced hyperglycaemia and pancreatic beta cell destruction. CONCLUSIONS/INTERPRETATION: This study demonstrates that blocking glucagon signalling by targeted Gcgr gene deletion leads to an improvement in metabolic control in this mouse model.

Assembly of the tendon extracellular matrix during development.

The assembly of the collagenous extracellular matrix during tendon development was studied to determine the mechanisms involved in collagen fibril growth. Developing avian metatarsal tendons were studied using structural, immunochemical and biochemical approaches. Tendon fibroblasts were shown to establish a hierarchy of extracellular compartments associated with fibrils, bundles, and macroaggregates during development. These distinct domains provide a mechanism for the fibroblast to influence the extracellular steps in matrix assembly. A discontinuous fibrillar matrix was assembled and fibril segments approximately 10-20 microns long were deposited into bundles by the 14 d embryo fibroblasts. The fibril segment is a normal assembly intermediate, permitting orderly linear, lateral and intercalatory development and growth. A lateral and/or linear fusion of segments may be responsible for the formation of mature continuous fibrils. Fibril segments were isolated from 12-18 d chick embryo metatarsal tendons. Homogenisation almost completely disrupted the 12-15 d tendons. Transmission electron microscopy demonstrated intact segments. Between d 12 and 15 of development, mean segment length increased from approximately 22 to 33 microns. The incremental increase in length with development indicates a limited linear fusion of segments which is supported by morphological examination. At 16 d, there was a significant decrease in segment extractability and by 17-18 d, intact segments were unextractable. Mean segment lengths were 37 microns and > 70 microns for 16 and 17 d tendons respectively. During this period, fibril diameter also increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen fibrillogenesis in situ: fibril segments undergo post-depositional modifications resulting in linear and lateral growth during matrix development.

Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, stability, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post-depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14-day chicken embryo tendons had a mean length of 29 microns. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12- to 16-day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associations among adjacent segments. Analysis demonstrated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of development. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril-associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post-depositional fusion allows for appositional and intercalary growth and would be essential for normal development, growth, and repair.

Collagen fibril assembly and deposition in the developing dermis: segmental deposition in extracellular compartments.

The hierarchy of extracytoplasmic compartmentalization and fibrillar organization as well as the assembly and deposition of collagen fibrils was characterized in the 15-day chick embryo dermis using transmission electron microscopy. At least two levels of extracellular compartmentalization are recognizable at this stage of dermal development. The first compartment consists of a series of narrow channels containing single or small groups (less than 5) of collagen fibrils. These channels course deep within the cell and are open to the extracellular space. The second extracellular compartment consists of fibrils grouped as small bundles in close association with the cell surface and is most often defined by a single fibroblast. A third level of fibril organization and compartmentalization is sometimes apparent at this stage of dermal development consisting of laterally associated bundles, more characteristic of the mature dermis. This compartment is associated with the fibroblast surface, but is less well defined than the fibril channels or bundle-forming compartments. Dermal collagen fibrils within bundles are discontinuous. Numerous fibrils ends are identified from serial sections and the ends gradually taper. These data indicate that the dermal fibroblast compartmentalizes the extracellular space and deposits collagen fibril segments during dermal morphogenesis. A model for the genesis of the extracellular compartments and their role in collagen fibrillogenesis and development of regularly arranged connective tissues, tendon, and cornea has been proposed. Dermal development conforms to this model and we suggest that extracytoplasmic compartmentalization of the steps in matrix assembly and segmental deposition of collagen fibrils are important mechanisms in the development of a wide variety of connective tissues.

Collagen fibrillogenesis in situ: fibril segments are intermediates in matrix assembly.

The assembly of discontinuous fibril segments and bundles was studied in 14-day chicken embryo tendons by using serial sections, transmission electron microscopy, and computer-assisted image reconstruction. Fibril segments were first found in extracytoplasmic channels, the sites of their polymerization; they also were found within fibril bundles. Single fibril segments were followed over their entire length in consecutive sections, and their lengths ranged from 7 to 15 microns. Structural differences in the ends of the fibril segments were identified, suggesting that the amino/carboxyl polarity of the fibril segment is reflected in its architecture. Our data indicate that fibril segments are precursors in collagen fibril formation, and we suggest that postdepositional fusion of fibril segments may be an important process in tendon development and growth.

Collagen fibrillogenesis in situ: fibril segments become long fibrils as the developing tendon matures.

Tissue architecture, stability, and mechanical attributes are all determined by the structure and organization of collagen fibrils. Therefore, the characterization of fibril growth steps and determination of how this growth is regulated is essential to the elucidation of how tissues are assembled. We have proposed that fibril segments are intermediates in the formation of mature fibrils. The purpose of this study was to determine the length and structure of fibrils within a relatively mature tendon. The in situ determination of length performed here was only the second direct determination of fibril length in a vertebrate connective tissue and the first for a relatively mature tissue. The data demonstrate that the fibrils were discontinuous at 18 days of tendon development. However, both ends were not present in any of the analyzed fibrils within the 18-day tendon. Because the data set was 50-60 microm, this indicates a mean fibril length greater than 60 microm. These data are in contrast to data from the 14-day tendon, in which 80% of the fibrils had both ends in a 26-microm data set and the mean segment length was shown to be 10-30 microm. There were equal numbers of alpha and beta ends in the 18-day tendon. The structure of the ends was comparable to that in the less mature tendon. The data also indicate that fibril asymmetry and structure were maintained. The increase in fibril length is interpreted as being the result of a post-depositional, regulated assembly of segments via a lateral association/fusion to form mature fibrils. This hypothesis predicts an increase in diameter at this stage of development. The diameter increases have been documented, but this is the first demonstration of increases in length and maintenance of segment structure during this important stage of tendon development.

Collagen fibril bundles: a branching assembly unit in tendon morphogenesis.

The assembly, deposition and organization of collagen fibril bundles and their composite fibrils were studied during morphogenesis of the chick embryo tendon using electron microscopy, serial sections and computer-assisted three-dimensional reconstruction techniques. The 14-day chick embryo is a stage when tendon architecture is being established and rapid changes in the mechanical properties occur between days 14 and 17 of development. Tendon matrix structure develops from discrete subunits, bundles of collagen fibrils. The bundles branch; undergo a gradual rotation over several micrometers; are intimately associated with the cellular elements of the developing tendon; and form arborizing networks within and among fascicles. The organization of discrete fibril segments into bundles, during the establishment of tendon architecture and function, where the segmental fibrillar components could interact with the interfibrillar matrix as well as with adjacent fibrils would contribute to the stabilization of this structure. The observed gradual rotation of the bundles would serve to stabilize the immature bundle through the physical twining of the composite fibrils while the extensive branching of the bundles observed at 14-days of development and their intimate association with the cellular elements would provide a higher order of structure stabilization.