Β-adrenoceptor Agonist Research Articles

Effects of β3-adrenoceptor agonist on acute urinary retention in a rat model.

To investigate the protective effect of mirabegron on bladder dysfunction in an acute urinary retention rat model. Thirty-six 16-week Sprague-Dawley rats were assigned to the mirabegron and normal saline (N/S) groups. Each group of eighteen was divided into sub-groups of 6 for 30min, 2h, and 24h. They were administered mirabegron (10mg/kg) and N/S daily for 4weeks, respectively. Mirabegron and N/S groups were divided into sub-groups of 6 rats for 30min, 2h, and 24h. The changes in bladder blood flow were measured using laser Doppler (moorVMS-LDF2). Histopathological examination of the bladder and nitric oxide (NO) measurement were performed. During the urinary retention phase in the mirabegron group, it showed higher and rapider recovery of blood flow; the lowest at 19.5% ± 3.68% at 3min, a significant recovery from the lowest value as 23.7 ± 3.4% at 10min, than that in the N/S group; 15.1 ± 1.84% at 5min, 23.7 ± 3.4% at 20min, respectively (P < 0.05). At 30min, 120min, and 24h after reperfusion, the recovery of blood flow in the mirabegron group was significantly higher than that in the N/S group (mirabegron: 41.1 ± 1.7%, 59.9 ± 7.2%, and 89.7 ± 4.4%, N/S: 31.3 ± 2.1%, 47.3 ± 4.5%, 83.9 ± 3.6%, respectively (P < 0.05)). NO levels tended to be higher in the mirabegron group; however, the difference was not statistically significant. Histological examination revealed that the mirabegron group showed recovery close to normal tissue after 24h. In an acute urinary retention rat model, mirabegron maintained and restored higher bladder blood flow, resulting in protective and recovery effect after acute urinary retention.

Efficacy of vibegron, a novel β3-adrenoreceptor agonist, for lower urinary tract dysfunction in mice with spinal cord injury.

To investigate the effect of vibegron, a new clinically approved β3-adrenoceptor agonist in lower urinary tract dysfunction in mice with spinal cord injury. Investigators performed cystometry under awake conditions in 4-week spinal cord injury female mice. Two weeks after spinal cord injury, saline or vibegron (30mg/kg) was orally administered for 2weeks prior to the urodynamic study. Investigators removed L6-S1 dorsal root ganglia from the saline- or vibegron-treated spinal cord injury mice as well as from saline-treated normal (spinal intact) mice to evaluate the levels of transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, activating transcription factor 3, and inducible nitric oxide synthase transcripts using real-time polymerase chain reaction. In vibegron-treated spinal cord injury mice, nonvoiding contractions during bladder filling, which were increased in spinal cord injury compared to spinal intact mice, were significantly decreased. Micturition pressure or voiding efficiency was not significantly increased in comparison to measurements in saline-treated spinal cord injury mice. The expression of transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, activating transcription factor 3, and inducible nitric oxide synthase messenger RNA was increased in spinal cord injury mice compared to spinal intact mice, but significantly decreased after vibegron treatment. Vibegron improves spinal cord injury-induced detrusor overactivity in addition to significantly reducing C-fiber afferent receptors such as transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, and inflammatory cytokines/markers, such as activating transcription factor 3 and inducible nitric oxide synthase, in spinal cord injury mice. Thus, vibegron might be effective in the treatment of storage lower urinary tract dysfunction induced by C-fiber afferent activation after spinal cord injury.

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Diet-Induced Non-anemic Iron Deficiency Attenuates Adaptive Thermogenesis via Defective Iron Metabolism of Adipose Tissue in C57BL/6 Mice

ObjectivesIron deficiency is one of the most common nutrient deficiencies worldwide. Adaptive thermogenesis plays a critical role for maintaining energy homeostasis. Currently, the role of dietary iron deficiency on adaptive thermogenesis is unknown. We hypothesized that dietary iron deficiency attenuates adaptive thermogenesis via dysfunctional iron metabolism in adipose tissue. MethodsC57BL/6 male mice were fed a diet containing either adequate-iron (Cont: 35ppm) or deficient-iron (ID: 3ppm) throughout for 14 weeks and the last 10 weeks were fed a high-fat diet to induce obesity. Systemic iron status was evaluated by measuring serum ferritin, hemoglobin (Hb), and hematocrit (HCt). The iron content of adipose tissue was determined by ICP-MS spectrometer. The protein and gene expressions related to iron-handling and thermogenesis were measured in adipose tissue by Western blot analysis and qPCR. To assess adaptive thermogenic function, mice were exposed to cold temperature acutely (3 hour) or administrated with β3-adrenoceptor agonist (CL) for 5 days. The core body temperature and thermogenesis were determined by a rectal thermometer and infrared camera, respectively. ResultsThe ID mice displayed a non-anemic iron deficiency with reduced serum ferritin (p < 0.01), increased weight gain (p < 0.05) and decreased insulin sensitivity (p < 0.05) compared to Cont. The iron content was significantly reduced in the inguinal (iWAT, p < 0.05) of the ID mice, which was linked with reduced thermogenic heat release upon acute cold treatment. In addition, ID mice markedly reduced the adipose tissue browning and thermogenic heat release upon CL stimulation compared with Cont. In terms of iron metabolism, CL-induced coordinated iron uptake for mitochondrial biogenesis in the iWAT was impaired in the ID mice, which was comparable to inflammation-mediated defective adipose tissue browning and thermogenesis. ConclusionsIron deficiency induced visceral obesity and compromised thermogenic function due to defective iron metabolism in C57BL6 mice. Funding SourcesNational Institutes of Health Grant 1R21HD094273.

Effect of oxybutynin patch versus mirabegron on nocturia-related quality of life in female overactive bladder patients: A multicenter randomized trial.

To investigate the effect of oxybutynin patch versus β3-adrenoceptor agonist mirabegron on nocturia-related quality of life in female overactive bladder patients. In the present study, female overactive bladder patients were enrolled. The patients were randomly allocated into two groups: the oxybutynin patch group and the mirabegron group. Each of the drugs was given for 8weeks. The changes in the total Nocturia Quality of Life Questionnaire score were evaluated. Parameters on a frequency volume chart were also evaluated. In total, 100 patients (51 oxybutynin patch, 49 mirabegron) were treated with oxybutynin patch or mirabegron. The changes in the Nocturia Quality of Life Questionnaire score 4weeks after administration were 3.8±18.6 and 8.7±13.1 with the oxybutynin patch group and the mirabegron group, respectively, which were significantly higher than those at the baseline. Furthermore, the changes in the Nocturia Quality of Life Questionnaire score 8weeks after administration were 4.3±16.5 and 7.7±12.3, respectively. A statistical difference was seen only in the mirabegron group. Regarding the Nocturia Quality of Life Questionnaire subscores, oxybutynin patch and mirabegron significantly improved the Nocturia Quality of Life Questionnaire bother/concern subscore 4 and 8weeks after administration, whereas the Nocturia Quality of Life Questionnaire sleep/energy subscore was not significantly improved in each period. Eight weeks after administration, 24-h frequency, 24-h urinary urgency and mean voided urine volume were improved in both groups statistically. The oxybutynin patch improves quality of life, focusing mainly on nocturia by improving the bother/concern subscores of the Nocturia Quality of Life Questionnaire in the short term.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens.

Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a β2-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.

P2Y 14 receptor regulates adipocyte lipid mobilization and whole‐body glucose homeostasis

The incidence and prevalence of obesity and type 2 diabetes (T2D) is rapidly increasing worldwide. Obesity is associated with chronically elevated plasma free fatty acid (FFA) levels, causing peripheral insulin resistance and T2D. Drugs that are able to regulate adipocyte lipolytic processes, improving insulin sensitivity and whole-body glucose homeostasis, have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R), activated by UDP-glucose is abundantly expressed in mouse and human adipocytes. P2Y14R activation in primary mouse adipocytes decreased cAMP levels and CL-316,243 (β3-adrenoceptor agonist) induced lipolysis. To investigate the physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis in-vivo, we generated a mouse model lacking P2Y14R specifically in adipocytes. Lack of adipocyte P2Y14R increased lipolysis only in the fasting state, resulting in decreased body weight, improved glucose tolerance and insulin sensitivity. Accordingly, high fat diet increased, whereas fasting decreased P2Y14R expression in visceral white adipocytes. Mechanistic studies suggested that activation of P2Y14R reduced the activity of lipolytic enzymes including ATGL and HSL, thereby inhibiting lipolysis. Supporting these findings, administration of P2Y14R agonist in control mice decreased lipolysis. Coadministration of P2Y14R antagonist with the P2Y14R agonist rescued the decrease in lipolysis. In conclusion, we demonstrate that lack of adipocyte P2Y14R decreased diet-induced obesity by enhancing lipolysis in the fasting state, suggesting that P2Y14R antagonists may prove beneficial for the therapy of obesity and T2D.

Inhibition of cAMP‐Phosphodiesterase 4 (PDE4) Induces Salivation in Mice

While exploring potential anti-inflammatory benefits of PDE4 inhibition in a mouse model of bacterial lung infection, we noticed that treatment with the PDE4 inhibitor Piclamilast visibly induced salivation in the animals. Given the predominant role of autonomic nervous system regulation in the control of salivation, and the potential impact of altered autonomics triggered by PDE4 inhibition on our infection model, we have further explored this observation. InKetamine/Xylazine-anesthetized mice, in which saliva production was tracked by the weight increase of thin filter paper strips placed into the mouths of the animals, treatment with PDE4 inhibitors did not induce saliva production per se, but synergized with low doses of the β-adrenoceptor agonist Isoprenaline to induce substantial salivation that was ablated by treatment with the β-blocker Propranolol, but not the mACh-blocker Atropine, and was absent in CFTRΔF508/ΔF508 mice that lack functional CFTR. Conversely, in awake mice, visual scoring (elevated/abnormal salivation=1; no elevated/abnormal salivation=0) indicated that treatment with PDE4 inhibitors was by itself sufficient to induce substantial salivation, which was ablated by treatment with either Propranolol or Atropine, and was also absent in ΔF508-CFTR-mutant mice. Our findings suggest that in anesthetized mice, in which autonomic nervous system regulations are suppressed, inhibition of cAMP degradation by PDE4 within salivary gland cells potentiates the effects of increased cAMP production resulting from β-adrenoceptor activation, and thereby promote the cAMP/PKA-dependent activation of CFTR that promotes saliva secretion. However, in awake mice, PDE4 inhibition acts via an additional mechanism on the autonomic, muscarinic regulation of salivation, likely by affecting neurotransmitter release. In both awake and anesthetized mice, salivation was induced by several structurally distinct PDE4 inhibitors, but not by a PDE3 inhibitor, suggesting that salivation is a class effect of PAN-PDE4 inhibitors. The PDE4 family comprises four PDE4 subtypes, PDE4A to D. Genetic ablation of any individual PDE4 subtype in the respective KO mouse strains did not induce salivation in either awake or anesthetized mice, indicating that salivation must result from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data identify PDE4 as a critical regulator of CFTR function in an in vivo model and suggests a therapeutic potential of PDE4 inhibition to alleviate CFTR hypofunction in cystic fibrosis. In addition, our data reveal a therapeutic potential for PDE4 inhibition in the treatment of dry mouth, a condition frequently associated with drug-, radiation-, or ageing-induced salivary gland dysfunction.

Activation of the kidney sodium chloride cotransporter by the β2-adrenergic receptor agonist salbutamol increases blood pressure

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In exvivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

The Effect of the Clenbuterol-β2-Adrenergic Receptor Agonist on the Peripheral Blood Mononuclear Cells Proliferation, Phenotype, Functions, and Reactive Oxygen Species Production in Race Horses In Vitro.

Clenbuterol, the β2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reactive oxygen species (ROS) production, as well as cell proliferation, were evaluated by flow cytometry. In addition, IL-1β, IL-4, IL-6, IL-10, IL-17, INF-γ and TNF-α concentrations were evaluated by ELISA. β2-adrenoceptor stimulation leads to enhanced anti-inflammatory properties in well-trained horses, as do low doses in untrained animals. In contrast, higher clenbuterol doses create a pro-inflammatory environment in inexperienced horses. In conclusion, β2-adrenoceptor stimulation leads to a biphasic response. In addition, the immune cells are more sensitive to drug abuse in inexperienced individuals under physical training.

Attenuated β-adrenergic response in calcium/calmodulin-dependent protein kinase IV-knockout mice.

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the β-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to β-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the β-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac β-adrenergic signal transduction.

Pericardial effusion, cardiomegaly, oedema, and IgA deficiency in a child: coeliac disease

A 4-year old girl was admitted to our hospital after she collapsed at home while having dinner. She had a history of recurrent upper airway infections and wheezing, which were being treated with inhaled β2-adrenoceptor agonist, as an outpatient.

Bezpieczeństwo i skuteczność mirabegronu i solifenacyny w leczeniu nadaktywnego pęcherza moczowego u pacjentów w wieku podeszłym

Lower urinary tract symptoms, including urgency, urgency incontinence, pollakiuria and nocturia are common in the elderly people and has significant impact on quality of life. Pharmacological treatment is recommended when conservative management, such as fluid intake normalization or bladder training, does not alleviate symptoms. Antimuscarinics were often used as the I line treatment for OAB, as these drugs block the activity of the muscarinic M2/M3 receptors in the bladder and provide a significant clinical benefit for OAB symptoms in the elderly people, but their side effects are common, often leading to treatment discontinuation. Mirabegron, a β3-adrenoceptor agonist, shows similar efficacy to antimuscarinic drugs without the risk of anticholinergic effects.

Biomimetic in vitro respiratory system using smooth muscle cells on ECIS chips for anti-asthma TCMs screening

Airway smooth muscle (ASM) contraction is a major pathophysiological characteristic of asthma. Although β2-adrenoceptor (β2-AR) agonists are currently used as bronchodilators, they cause rapid effect and long-term agonist-induced desensitization. Thus, it is necessary to search for more effective and safer relaxant agents for ASM cells. In this work, bitter taste receptors (TAS2Rs) were demonstrated to be expressed in primary mouse ASM cells endogenously, and they were considered as new drug targets for asthma treatment. Traditional Chinese medicines (TCMs) contained a wide range of TAS2R agonists and some of them had the efficacy of relieving cough and asthma with less toxic side effects. Then the electronic cell-substrate impedance sensor (ECIS) was used for the first time to establish a method to detect the contraction/relaxation effects of ASM cells. Therefore, we introduced a biomimetic in vitro respiratory system using ASM cells on ECIS chips to screen for potential TCMs against asthma. Quinine, nobiletin, and picfeltarraenin IA screened in this study could effectively inhibit the ASM contraction in a concentration-dependent manner, showing potential value as novel anti-asthma drugs. Furthermore, the effective screening of anti-asthma drugs was realized based on 3D ASM cell arrays and gel imaging system. Consistent results were found and the reliability of the biomimetic in vitro respiratory system for the screening of TCMs against asthma was further verified. The biomimetic system designed in this study has the advantages of operation simplicity, high throughput, non-invasive, real-time, and high sensitivity, and therefore provides a promising drug screening platform for asthma disease.

Synthesis of the Lipophilic Amine Tail of Abediterol Enabled by Multiphase Flow Transformations

The development of a continuous-flow sequence for the synthesis of an important drug candidate precursor is reported. Abediterol is a β2-adrenoceptor agonist that has undergone phase IIa clinical trials for the treatment of respiratory disease. A flow sequence is developed for the preparation of the lipophilic amine tail portion of abediterol. The sequence comprises of a phase-transfer-catalyzed liquid/liquid O-alkylation, a rhodium-catalyzed hydroformylation, and a ruthenium-catalyzed reductive amination. The reactions were optimized separately within continuous-flow environments to identify important parameter effects. The strongly basic O-alkylation operates with greater than 90% conversion within a 23 min residence time. The hydroformylation uses 1 mol % Rh(acac)(CO)2 (acac = acetylacetone) as a catalyst and 6 mol % Xantphos as a ligand with 1.1 equiv of hydrogen and carbon monoxide. The optimized O-alkylation and hydroformylation telescoped flow process was successfully operated over 6 h. The protocol is shown to be high yielding for the desired linear aldehyde (75% gas chromatography yield, ∼2.5 g/h). The sequence requires a solvent switch prior to the reductive amination. The final step is a high-pressure (40 bar) and high-temperature (150 °C) Ru-catalyzed reductive amination using ammonia and hydrogen to afford the amine tail. The solution yield for the formation of the amine tail was 78%. The yield of the reductive amination with an unoptimized isolation was 50%, resulting in an overall isolated yield for the three-step sequence of 38%. This compares favorably against the batch yield of 26% using a different synthetic route.

Efficacy of the combination of KPR-5714, a novel transient receptor potential melastatin 8 (TRPM8) antagonist, and β3-adrenoceptor agonist or anticholinergic agent on bladder dysfunction in rats with bladder overactivity

Transient receptor potential melastatin 8 (TRPM8) channels may contribute to the pathophysiological bladder afferent hyperactivity, thus a TRPM8 antagonist would be a promising therapeutic target for the bladder hypersensitive disorders including urinary urgency in overactive bladder (OAB). We aimed to investigate a pharmacological effect of KPR-5714, a novel selective TRPM8 antagonist, on TRPM8 channels, M3 receptors and β3-adrenoceptors using the transfected cells of each gene at first. Then, combination effects of KPR-5714 and mirabegron, a β3-adrenoceptor agonist, or tolterodine tartrate, an anticholinergic agent, were studied on rhythmic bladder contractions (RBCs) in normal rats and bladder function in frequent-voiding rats. In vitro measurements showed that KPR-5714 acts on neither β3-adrenoceptor nor M3 receptor. In normal rats, KPR-5714 and mirabegron significantly reduced the frequency of RBCs, and a combined administration showed an additive effect. In rats with cerebral infarction, KPR-5714 and mirabegron significantly reduced the voiding frequency, and a combined administration showed an additive effect. In rats exposed to cold temperature, KPR-5714 and tolterodine tartrate significantly reduced the voiding frequency accompanied by the increased mean voided volume, and a combined administration showed additive effects. The present study demonstrated that the combined administration of KPR-5714 and mirabegron or tolterodine tartrate showed the additive effects on bladder dysfunction in different animal models, suggesting that the combination therapy of TRPM8 antagonist and β3-adrenoceptor agonist or anticholinergic agent can be the potential treatment option for obtaining additive effects in comparison with monotherapy for OAB.

Possible Involvement of Muscarinic Receptor Blockade in Mirabegron Therapy for Patients with Overactive Bladder.

The selective β 3-adrenoceptor agonist mirabegron, an established alternative to antimuscarinic therapy for patients with overactive bladder, induces additional effects against receptors, transporters, and hepatic enzymes. The present study aimed to elucidate the effects of mirabegron on muscarinic receptors in the rat bladder using radioligand binding and functional assays. Mirabegron (0.1-100 μM) inhibited specific [N-methyl-3H]scopolamine methyl chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. Binding affinity in the bladder was similar to that in the heart and significantly higher than those in the submaxillary gland and brain. Mirabegron induced the concentration-dependent relaxation of carbachol-induced contractions in the rat isolated bladder. Further analyses using a two-site model revealed that the relative quantities of high- and low-affinity components for mirabegron were 44.5% and 55.5%, respectively. Respective pEC50 values were 7.06 and 4.97. Based on the receptor binding affinity and pharmacokinetics of mirabegron, muscarinic receptor occupancy in the human bladder for 24 hours after the administration of a single oral dose of 50 mg mirabegron was 37%-76%. The present results demonstrate for the first time that mirabegron may relax the detrusor smooth muscle not only by β 3-adrenoceptor activation but also muscarinic receptor blockade. SIGNIFICANCE STATEMENT: Mirabegron, the first selective β 3-adrenoceptor agonist, represents an alternative to antimuscarinic agents for management of overactive bladder (OAB). The present study aimed to clarify whether mirabegron directly binds to muscarinic receptors and affects cholinergic agonist-induced contractions in rat urinary bladder and to predict muscarinic receptor occupancy in human bladder after oral administration of mirabegron. The results demonstrated that mirabegron therapy for patients with OAB may be due not only to β 3-adrenoceptor activation but also muscarinic receptor blockade.

089 Mirabegron for Erectile Dysfunction

PDE5 inhibitor is the only approved oral medication to treat erectile dysfunction (ED) in the US. However, there is a need for an alternative medication when PDE5 inhibitor treatment fails or there is a contraindication. The β3-adrenoceptor has been shown in human corpus cavernosum (CC). CC relaxation is induced by β3-adrenoceptor activation via cAMP-PKA dependent and/or cAMP-PKA independent pathway. Preclinical studies suggested mechanisms such as activation of K+ channels and closure of voltage-gated Ca2+ channels, cGMP accumulation by NO release and H2S pathway. However, the role of β3-adrenoceptor on penile erection in men is still unclear. Mirabegron is the first β3-adrenoceptor agonist that has been approved for the treatment of overactive bladder (OAB). The disorders of lower genitourinary tract such as OAB and ED share a common pathophysiologic mechanism. Mirabegron may act simultaneously in bladder and CC via β3-adrenoceptor activation and can offer a pharmacologic target for ED.

Effect of β1 /β2 -adrenoceptor blockade on β3 -adrenoceptor activity in the rat cremaster muscle artery.

The physiological role of vascular β3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β3 -adrenoceptors are functionally effective after down-regulation of β1 /β2 -adrenoceptors. The functional interaction between the β3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated. Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography. All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β3 -adrenoceptor agonists (in the presence of β1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β3 -adrenoceptor mediated vasodilation was only evident after blockade of β1/2 -adrenoceptors. This suggests constitutive β1/2 -adrenoceptor activity inhibits β3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.