Characteristics of AZD9708, a novel, selective β2-adrenoceptor agonist with rapid onset and long duration of action

Here we describe the pre-clinical pharmacological profile of AZD9708, a novel long-acting β₂-adrenoceptor agonist that has potential as a once-daily therapy for asthma and chronic obstructive pulmonary disease (COPD).AZD9708 is a potent and selective agonist at the human β₂-adrenoceptor, with selectivity over human β₁- and β₃-adrenoceptors of >500 and >24 fold, respectively. AZD9708 relaxes carbachol-induced contraction of human bronchial rings with a time to 90% of maximal relaxation of 13–20 min, similar to that seen with formoterol and quicker than salmeterol. In anesthetized guinea pigs, AZD9708 provides significant protection against histamine-induced airway constriction at 24 h after intratracheal and nebulized doses. This is longer than with intratracheal salmeterol, which is bronchoprotective for approximately 8 h, and formoterol, which is bronchoprotective for 8 and 12 h following nebulized and intratracheal dosing, respectively.AZD9708 also shows the potential for a greater therapeutic margin than widely used β₂-adrenoceptor agonists such as formoterol. At a defined efficacy dose that provides 80% bronchoprotection (ED₈₀), formoterol leads to a decrease in blood potassium levels in guinea pigs, whilst AZD9708 is not associated with significant reductions in potassium levels at doses up to 7 times the ED₈₀. [¹⁴C]AZD9708 is associated with extensive protein binding in both human (mean 1.0% free) and rat (mean 2.6% free) plasma.This pharmacological profile indicates the potential of AZD9708 to become an important addition to the range of bronchodilators available for the treatment of patients with obstructive airways disease.     

Effect of an oral β2-adrenoceptor agonist in a patient with idiopathic interstitial pneumonia

A 63-year-old man was referred to our hospital because of a dry cough. His chest roentgenogram revealed ground-glass opacities and honeycomb formations bilaterally in the lower lung fields. Pulmonary function tests showed a depleted lung volume and decreased arterial oxygen tension. He was clinically diagnosed as having idiopathic interstitial pneumonia (IIP). A β₂-adrenoceptor agonist was administrated because the patient's symptoms improved after its inhalation. Following treatment with an oral β₂-adrenoceptor agonist, the dry cough disappeared, lung function tests remained unchanged and an improvement in arterial oxygen tension was observed. Although β₂-adrenoceptor agonist therapy does not improve disease activity or progression in patients with IIP, its use may mitigate symptoms associated with the disease.     

Treatment of chronic heart failure with β-adrenergic receptor antagonists: a convergence of receptor pharmacology and clinical cardiology

Despite the absence of a systematic development plan, β-blockers have reached the top tier of medical therapies for chronic heart failure. The successful outcome was due to the many dedicated investigators who produced, over a 30-year period, increasing evidence that β-blocking agents should or actually did improve the natural history of dilated cardiomyopathies and heart failure. It took 20 years for supportive evidence to become undeniable, at which time in 1993 the formidable drug development resources of large pharmaceutical companies were deployed into Phase 3 trials. Success then came relatively quickly, and within 8 years multiple agents were on the market in the United States and Europe. Importantly, there is ample room to improve antiadrenergic therapy, through novel approaches exploiting the nuances of receptor biology and/or intracellular signaling, as well as through pharmacogenetic targeting.     

Early clinical investigation of Viozan™ (sibenadet HCI), a novel D2 dopamine receptor, β2-adrenoceptor agonist for the treatment of chronic obstructive pulmonary disease symptoms

Viozan™, (Sibenadet HCI, AR-C68397AA) is a dual D₂ dopamine receptor, β₂-adrenoceptor that combines bronchodilator activity with the sensory afferent modulating effects associated with D₂-receptor agonism. Investigation in animal models of key chronic obstructive pulmonary disease (COPD) symptoms has demonstrated that sibenadet effectively inhibits sensory nerve activity, thereby reducing reflex cough, mucus production and tachypnoea. The results of the early clinical evaluation of this novel agent are reported.An initial proof of concept study (Study I) aimed to determine the clinical potential of this novel agent by assessing the effects of three doses of sibenadet therapy relative to placebo, with two commonly used bronchodilators, intended to provide a benchmark against which sibenadet activity could be judged. In all, 701 patients were randomized to one of three sibenadet dose groups (400, 600 or 1000 μgex valve), salbutamol 200 μg, ipratropium bromide (IB) 40 μg or placebo, all three times daily via pressurized metered dose inhaler (pMDI) for 4 weeks. Once the results of Study I had been evaluated, a dose-ranging, study (Study 2) involving 872 patients randomized to receive sibenadet (45, 270, or 495 μgex actuator), or placebo all three times daily via pMDI, for 6 weeks commenced. Both studies were preceded by a 2-week baseline phase and followed by a 2-week follow up period. The primary efficacy variable identified changes in key COPD symptoms over the treatment period (compared with baseline data) as determined by the novel Breathlessness, Cough and Sputum Scale (BCSS©). In addition, data on lung function, health-related quality of life and adverse events were collected.Patients receiving sibenadet therapy three times daily exhibited statistically significantly greater improvements in BCSS total score than those receiving placebo or bronchodilator therapy alone. A clear dose-response was evident in Study 2. Symptom improvement in this study was also accompanied by improved lung function and healt-related quality of life. Sibenadet therapy was well tolerated with an adverse events profile comparable to current bronchodilator therapy. These data were viewed as extremely encouraging, warranting further, large-scale clinical investigation.     

Construction of a cell model of β2-adrenoceptor downregulation

Objective

To establish a cell model of β₂-adrenergic receptor (β₂AR) downregulation of murine airway smooth muscle induced by salbutamol to elucidate the molecular and biological mechanisms of β₂AR downregulation.

Methods

Airway smooth muscle cells (ASMCs) derived from Balb/c mice were primary cultured. Passage 2-5 cells were characterized by cell morphology and indirect immunofluorescence. More than 95% pure cells at passage 3 or 4 were randomly divided into two groups: control and salbutamol-treated groups. β₂AR mRNA and protein expression levels were then detected by RT-PCR and western blot analyses.

Results

Primary cultured cells demonstrated a typical “peak and valley”-like growth characteristic. Smooth muscle α-actin filaments paralleled the cell longitudinal axis in the cytoplasm. The origin of the ASMCs was validated and consistent with their morphology and biological characteristics. β₂AR mRNA expression in the salbutamol-treated group was lower than that in the control group (P<0.05), and β₂AR protein expression was also markedly lower than that in the control group (P<0.05).

Conclusions

We successfully established a cell model of β₂AR downregulation in ASMCs, which may provide the foundation for further study of the mechanism of β₂AR downregulation in asthmatic patients.KEY WORDS : Airway smooth muscle cells (ASMCs), β₂-adrenergic receptor (β₂AR), salbutamol, cell culture     

Conclusion lessons from the novel D2 dopamine receptor, β2-adrenoceptor agonist,Viozan™: chronic obstructive pulmonary disease and drug development implications

The development of novel drugs for the treatment of chronic obstructive pulmonary disease (COPD) poses significant challenges. The mechanisms through which the chronic symptoms of COPD arise are poorly understood, making identification of potential therapeutic targets andin vivo evaluation of potential therapies extremely difficult. Despite these challenges, a unique approach of combined D₂ dopamine, β₂-adrenoceptor agonism was identified as a valid potential target for the treatment of key COPD symptoms, the therapeutic potential of which was investigated in a series of pre-clinical evaluations. Subsequent clinical assessment has amassed a wealth of data from over 4000 patients, providing valuable insights into COPD, clinical trial design and the value of patient self-assessment tools.     

Differential expression of estrogen receptor α, β1, and β2 in lobular and ductal breast cancer

The role of estrogen receptor (ER) α as a target in treatment of breast cancer is clear, but those of ERβ1 and ERβ2 in the breast remain unclear. We have examined expression of all three receptors in surgically excised breast samples from two archives: (i): 187 invasive ductal breast cancer from a Japanese study; and (ii) 20 lobular and 24 ductal cancers from the Imperial College. Samples contained normal areas, areas of hyperplasia, and in situ and invasive cancer. In the normal areas, ERα was expressed in not more than 10% of epithelium, whereas approximately 80% of epithelial cells expressed ERβ. We found that whereas ductal cancer is a highly proliferative, ERα-positive, ERβ-negative disease, lobular cancer expresses both ERα and ERβ but with very few Ki67-positive cells. ERβ2 was expressed in 32% of the ductal cancers, of which 83% were postmenopausal. In all ERβ2-positive cancers the interductal space was filled with dense collagen, and cell nuclei expressed hypoxia-inducible factor 1α. ERβ2 expression was not confined to malignant cells but was strong in stromal, immune, and endothelial cells. In most of the high-grade invasive ductal cancers neither ERα nor ERβ was expressed, but in the high-grade lobular cancer ERβ was lost and ERα and Ki67 expression were abundant. The data show a clear difference in ER expression between lobular and ductal breast cancer and suggest (i) that tamoxifen may be more effective in late than in early lobular cancer and (ii) a potential role for ERβ agonists in preventing in situ ductal cancers from becoming invasive.Despite decades of research, the etiology of breast cancer remains unclear. It is currently thought that most breast cancers occur in the normal terminal duct lobular unit and progress in a stepwise fashion over time (1). Ductal carcinoma in situ (DCIS) means the cancer has not spread beyond the duct into any normal surrounding breast tissue and is thought by some to be the direct precursor of invasive ductal carcinoma (IDC).Estrogens play an important role in normal breast development as well as breast cancer progression (2). Most of the effects of estrogen are mediated through its two receptors: estrogen receptor α (ERα) and β (ERβ) (3). ERα is expressed in 50–80% of breast tumors, and its presence is the main indicator for antihormonal therapy (4). ERβ was first discovered in 1996, and its role in breast cancer is still being explored (5–7).The first step in understanding the role of ERβ in breast cancer was to define the expression pattern of ERβ in the normal human breast and in various stages of cancer. Since its discovery, several laboratories have reported ERβ expression in clinical samples (8–28). Most of these studies investigated the expression of ERβ in invasive breast cancer samples (12–15, 17, 19, 21–23). Some studies have reported ERβ expression in invasive breast cancer and normal breast tissue (11, 18, 26–28), but few have compared the expression of ERβ in the normal tissue, DCIS, and IDC within the same sample. Usually tumor samples are taken from one patient and normal tissue from another patient (8–10). Samples taken from different patients have intrinsic limitation (i.e., they cannot account for variations between different patients). In addition, because tumors are heterogeneous, core biopsies do not fully reflect the histological and biological diversity of breast tumors (29).The roles of ERβ1 and its splice variant ERβ2 in breast cancer are still unclear. As reviewed by Murphy and Leygue (30), some studies show a loss of ERβ1 as ductal cancer progresses, but others do not. Some studies show ERβ2 as a marker of bad prognosis (31), and others not (19). Some of these differences may be due to differences in antibody use and differences in tissue fixation and handling.When ERα and ERβ are coexpressed in breast cancer it is unclear whether tamoxifen treatment will be successful. This is because tamoxifen acts as an agonist of ERβ at activator protein 1 (AP-1) sites (32) and thus should oppose the antiproliferative effects of the tamoxifen–ERα complex. Yan et al. (33) have found that expression of ERβ predicts tamoxifen benefit in patients with ERα-negative early breast cancer, whereas Esslimani-Sahla et al (23) have found that low ERβ level is an independent marker, better than ERα level, to predict tamoxifen resistance. Although apparently saying different things, these two results actually agree with each other: in ERα-negative breast cancer, estrogen is not driving proliferation, so tamoxifen via ERβ may interfere with another growth signaling pathway. In ERα-positive cancers whose proliferation is driven by E2, tamoxifen with ERβ would oppose the antiproliferative effects of the ERα–tamoxifen complex.Investigation of the expression pattern of ERβ in normal tissue, DCIS, and IDC is important to understand the function of this receptor in the progression of breast cancer. We have a set of samples obtained from surgical excision of breast tumors from women before pharmacological intervention. The cohorts include lobular cancer, which has not yet been thoroughly studied for ERβ expression. Lobular cancer is an ERα-positive form of breast cancer characterized by loss of E-cadherin and relatively low proliferation rate. It is accompanied by a resistance to anoikis (34). It accounts for 10–15% of diagnosed breast cancer, and there are still many questions about the optimal therapeutic approach to this cancer. We have explored the changes in expression of the two ERs using identical protocols and reagents in different developmental stages of breast cancer within each patient.     

New therapeutic options for IBS: the role of the first in class mixed µ- opioid receptor agonist and δ-opioid receptor antagonist (mudelta) eluxadoline

Introduction: Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disorder which represents a major cost to healthcare services. IBS-D patients represent about one-third of the IBS population and are currently treated with antispasmodics, loperamide, bile acid sequestrants and antidepressants. Alosetron and rifaximin are also available in USA, ramosetron in Japan, Korea and Thailand and ondansetron as an off-label treatment.

Areas covered: This article focuses on eluxadoline, a novel pharmacological agent that has recently been approved by both the FDA and EMA for treatment of patients with IBS-D.

Expert commentary: The efficacy and safety of eluxadoline in treating bowel habit alterations and pain, both in the short and long-term, make the drug a welcome addition to our therapeutic alternatives in IBS-D. Its positioning in any IBS algorithm will depend on the ‘real world’ prevalence of the small risk of sphincter of Oddi spasm and mild pancreatitis.     

Effects of prazosin, an α1-adrenergic receptor antagonist, on the seeking and intake of alcohol and sucrose in alcohol-preferring (P) rats

Background: Previous studies show that prazosin, an α₁‐adrenergic receptor antagonist, decreases alcohol drinking in animal models of alcohol use and dependence [Rasmussen et al. (2009) Alcohol Clin Exp Res 3:264–272; Walker et al. (2008) Alcohol 42:91–97] and in alcohol‐dependent men [Simpson et al. (2009) Alcohol Clin Exp Res 33:255–263]. This study extended these findings by using a paradigm that allows for separate assessment of prazosin on motivation to seek versus consume alcohol or sucrose in selectively bred rats. Methods: Alcohol‐preferring (P) rats were trained to complete an operant response that resulted in access to either 2% sucrose or 10% alcohol. A 4‐week Seeking Test Phase examined responding in single, weekly extinction sessions when no reinforcer could be obtained. A 4‐week Drinking Test Phase consisted of rats lever‐pressing to “pay” a specified amount up front to gain access to unlimited alcohol (or sucrose) for a 20‐minute period. On Seeking and Drinking test days, prazosin (0.0, 0.5, 1.0, and 1.5 mg/kg) was administered intraperitoneally 30 minutes prior to behavioral sessions. Results: Rats were self‐administering an average of 0.9 (±0.09) g/kg alcohol on vehicle test day and had pharmacologically relevant blood ethanol concentrations. Prazosin significantly decreased alcohol seeking at all doses tested. The highest dose of prazosin also increased the latency to first response for alcohol and decreased alcohol intake. While sucrose‐seeking and intake were similarly affected by prazosin, the high dose of prazosin did not increase response latency. Conclusions: These findings are consistent with and extend previous research and suggest that prazosin decreases motivation to initiate and engage in alcohol consumption. The specificity of prazosin in attenuating the initiation of alcohol‐ but not sucrose‐seeking suggests that this effect is not because of prazosin‐induced motor‐impairment or malaise. Together with previous findings, these data suggest that prazosin may be an effective pharmacotherapy, with specific application in people that drink excessively or have a genetic predisposition to alcohol abuse.     

The β2-adrenoceptor agonist formoterol improves structural and functional regenerative capacity of skeletal muscles from aged rat at the early stages of postinjury

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of β2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of β2-adrenergic agonist formoterol (2 μg/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.     

Association of polymorphisms in the β2-adrenergic receptor gene with obesity, hypertriglyceridaemia, and diabetes mellitus (Short Communication)

Summary To assess the role of polymorphisms in the β2-adrenergic receptor gene in the development of obesity and obesity-related metabolic disorders, we analysed Arg16Gly, Gln27Glu, and Thr164Ile polymorphisms in 400 non-obese subjects (body mass index < 27 kg/m²) and 108 obese subjects (body mass index ≥ 27 kg/m²). The Gln27Glu substitution was twice as common in obese subjects as in non-obese subjects (0.14 vs 0.07, p = 0.001, odds ratio 2.14, 95 % confidence interval 1.35–3.41). The frequency of the Glu27 allele was also higher in patients with Type II (non-insulin-dependent) diabetes mellitus than non-diabetic subjects (0.14 vs 0.07, p = 0.001, odds ratio 2.13, 95 % confidence interval 1.34–3.41). Analysis of variance of multiple variables showed an association between 2-h post-load glucose concentrations and body mass index but not with the Glu27 variant, suggesting that the association with diabetes could be secondary to obesity. Obese subjects carrying the variant allele had higher concentrations of serum triglyceride than obese subjects homozygous for the wild type allele (2.68 ± 1.90 vs 1.18 ± 1.15 mmol/l, p = 0.02). Conversely, the frequency of Gly16 homozygotes was lower in obese women when compared with non-obese women (11 % vs 28 %, p = 0.01, odds ratio 0.30, 95 % confidence interval 0.12–0.75), although the association was not present in male subjects. Thr164Ile substitution was not detected in the subjects of this study. These observations suggest that the amino-terminal polymorphisms of the β2-adrenergic receptor gene could be involved in the molecular pathogenesis of obesity and hypertriglyceridaemia, and thereby the development of Type II diabetes mellitus. [Diabetologia (1999) 42: 98–101] Received: 18 May 1998 amd in final revised form: 24 August 1998     

Use of a mixed endothelin receptor antagonist in portopulmonary hypertension: a safe and effective therapy?

BACKGROUND & AIMS: Portopulmonary hypertension (PPHTN), a severe complication of portal hypertension is observed in 3%-6% of patients evaluated for liver transplantation. Endothelin-1, a potent vasoconstrictor, is likely to play a role in the pathogenesis of primary pulmonary hypertension, and, in 2 recent trials, the dual endothelin receptor antagonist bosentan has shown beneficial effects in this disease. A role for endothelins in the development of both pulmonary hypertension and cirrhosis has been suggested. We therefore hypothesized that endothelin receptor blockade may be beneficial in the treatment of PPHTN. METHODS: We report a case of a 42-year-old patient with PPHTN and alcoholic cirrhosis treated with the mixed endothelin receptor antagonist bosentan. RESULTS: The patient rapidly improved from NYHA IV to stage II, experienced a remarkable improvement of 6-minute walking distance from 0 to 590 m within 6 months, and resumed working full-time as a locksmith after 7 months of treatment. Improvement of cardiovascular parameters included a reduction of pulmonary vascular resistance by 60%, a decrease of mean pulmonary artery pressure (mPAP) from 55 to 44 mm Hg at 9 months, and a decline of plasma B-type natriuretic peptide (BNP) from 339 pg/mL to 19 pg/mL after 1 year. There were no adverse events except for a transient decrease in systemic blood pressure. CONCLUSIONS: To our knowledge, this is the first report of a patient with PPHTN treated with an endothelin receptor antagonist. The marked and sustained improvement supports the undertaking of controlled studies of the safety and efficacy of bosentan in PPHTN.     

Dual dopamine D2 receptor and β2-adrenoceptor agonists for the treatment of chronic obstructive pulmonary disease: the pre-clinical rationale

This paper describes the rationale for the development of dual dopamine D₂-receptor and β₂-adrenoceptor agonists as potential treatments for the symptoms of chronic obstructive pulmonary disease (COPD). The putative involvement of pulmonary sensory afferent nerves in mediating the key COPD symptoms of breathlessness, cough and excess sputum production is outlined and the hypothesis that activation of D₂-receptors on such nerves would modulate their activity is developed. This premise was tested, in a range of animal models, using the first of a novel class of dual dopamine D₂-receptor and β₂-adrenoceptor agonists, sibenadet HCI (Viozan™, AR-C68397AA). In the course of these studies it was demonstrated that sibenadet, through activation of D₂-receptors, inhibited discharge of rapidly adapting receptors and was effective in reducing reflex-induced tachypnoea, mucus production and cough in the dog. Sibenadet, through its activation of β₂-adrenoceptors, was also shown to be an effective bronchodilator with a prolonged duration of action following topical administration to the lungs. These studies also indicated that sibenadet had a wide therapeutic ratio with respect to expected undesirable side-effects such as emesis and cardiovascular disturbances. These results provided a compelling rationale for the initiation of a clinical development programme with sibenadet for the treatment of COPD.     

Effect of α2-adrenoceptor antagonist on platelet activation during insulin-induced hypoglycaemia in Type 2 (non-insulin-dependent) diabetes mellitus

Summary The role of epinephrine in platelet activation and the effect of an ₂-adrenoceptor antagonist, midaglizole, during insulin-induced hypoglycaemia in Type 2 (noninsulin-dependent) diabetes mellitus were examined. The action of midaglizole as a platelet ₂-antagonist was confirmed by in vitro studies using platelet-rich plasma and washed platelet suspension. Hypoglycaemia was induced by a bolus injection of short-acting insulin in 24 diabetic patients. They were divided into two groups, a control group (n=12) and an ₂-group (n=12), and midaglizole was administered orally 60 min before insulin injection in the latter. Blood glucose and plasma C-peptide levels were significantly decreased (p<0.005) by insulin injection in both groups. Counter-regulatory hormones, including epinephrine, and arginine vasopressin were similarly increased at the hypoglycaemic nadir compared with the levels at 0 min in both groups. Plasma -thromboglobulin was increased at the hypoglycaemic nadir (165.5±12.6ng/ml) compared with the level at 0 min (121.0±11.5, p<0.005) in the control group, whereas no significant increase was demonstrated in the ₂ group. These results suggest that plasma epinephrine plays an important role in platelet activation during hypoglycaemia in Type 2 diabetes mellitus, and that the platelet activation is prevented by ₂-adrenoceptor antagonist.     

Atypical β- and α(2)-adrenoceptor Activation, Dibutyryl cAMP and Iloprost Stimulate [(45)Ca(2+)] Uptake by Human Platelets

The effect of a range of α- and β-adrenoceptor agonists and antagonists on the in vitro uptake of [(45)Ca(2+)] by human platelets was investigated. Isoprenaline and adrenaline stimulated [(45)Ca(2+)] uptake. Isoprenaline-stimulated ([(45)Ca(2+)] uptake was inhibited by β-adrenoceptor antagonists (mabuterol [β(2)] > metoprolol [β(1)] > atenolol [β(1)] > pindolol [β(1)/β(2)]), but not by yohimbine [αz] or prazosin [αll. Adrenaline-stimulated [(45)Ca(2+)] uptake was inhibited (and in this order of potency) by yohimbine, mabuterol, metoprolol, prazosin, atenolol and pindolol. [(45)Ca(2+)] uptake was stimulated by β-adrenoceptor agonists (BRL37344 [β(3)] > terbutaline [β(2)] > xamoterol [β(1)] > salbutamol [β(2)]). Ca(2+) ionophore A23187-stimulated [(45)Ca(2+)] uptake was unaffected by pindolol, atenolol, metoprolol or mabuterol, indicating that these antagonists were not exerting nonspecitic inhibitory effects. [(45)Ca(2+)] uptake was also stimulated by dibutyryl cAMP and by iloprost (a stable prostacyclin analogue and stimulator of cAMP synthesis). It is concluded that: (1) β- adrenoceptor-linked Ca(2+) uptake is mediated by an atypical β-adrenoceptor, possibly of a β(3)-subtype; (2) the stimulatory action of β-adrenoceptor activation and prostacyclin may be mediated by adenylate cyclase, and (3) the paradoxical finding that both α- and β-adrenoceptor activation, cAMP and stimulators of CAMP elicit [(45)Ca(2+)] uptake suggests that the Ca(2+) mobilisation monitored by the present methodology is not associated with platelet aggregation but to adrenoceptor activation per se and possibly other signal transduction mechanisms that occur at the plasmalemma.