A re-evaluation of the use of glyceryl trinitrate to assess the degree of adrenergic beta receptor blockade

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor

Historically, calcitonin gene-related peptide (CGRP) receptors have been divided into two classes, CGRP(1) and CGRP(2).After the cloning of calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMPs), it became clear that the CGRP(1) receptor was a complex between CLR and RAMP1. It is now apparent that the CGRP(2) receptor phenotype is the result of CGRP acting at receptors for amylin and adrenomedullin. Accordingly, the term "CGRP(2)" receptor should no longer be used, and the "CGRP(1)" receptor should be known as the "CGRP" receptor.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

Modulation of glucagon receptor pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, whilst having clinical efficacy, have been associated with severe adverse side-effects and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems, to provide a more complete understanding of glucagon receptor signaling considering the effect of multiple ligands, association with the receptor-interacting protein, receptor activity modifying protein-2 (RAMP2) and individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Future glucose-lowering drugs for type 2 diabetes

The multivariable and progressive natural history of type 2 diabetes limits the effectiveness of available glucose-lowering drugs. Constraints imposed by comorbidities (notably cardiovascular disease and renal impairment) and the need to avoid hypoglycaemia, weight gain, and drug interactions further complicate the treatment process. These challenges have prompted the development of new formulations and delivery methods for existing drugs alongside research into novel pharmacological entities. Advances in incretin-based therapies include a miniature implantable osmotic pump to give continuous delivery of a glucagon-like peptide-1 receptor agonist for 6-12 months and once-weekly tablets of dipeptidyl peptidase-4 inhibitors. Hybrid molecules that combine the properties of selected incretins and other peptides are at early stages of development, and proof of concept has been shown for small non-peptide molecules to activate glucagon-like peptide-1 receptors. Additional sodium-glucose co-transporter inhibitors are progressing in development as well as possible new insulin-releasing biological agents and small-molecule inhibitors of glucagon action. Adiponectin receptor agonists, selective peroxisome proliferator-activated receptor modulators, cellular glucocorticoid inhibitors, and analogues of fibroblast growth factor 21 are being considered as potential new approaches to glucose lowering. Compounds that can enhance insulin receptor and post-receptor signalling cascades or directly promote selected pathways of glucose metabolism have suggested opportunities for future treatments. However, pharmacological interventions that are able to restore normal β-cell function and β-cell mass, normalise insulin action, and fully correct glucose homoeostasis are a distant vision.

Management of type 2 diabetes:new and future developments in treatment

The increasing prevalence, variable pathogenesis, progressive natural history, and complications of type 2 diabetes emphasise the urgent need for new treatment strategies. Longacting (eg, once weekly) agonists of the glucagon-like-peptide-1 receptor are advanced in development, and they improve prandial insulin secretion, reduce excess glucagon production, and promote satiety. Trials of inhibitors of dipeptidyl peptidase 4, which enhance the effect of endogenous incretin hormones, are also nearing completion. Novel approaches to glycaemic regulation include use of inhibitors of the sodium-glucose cotransporter 2, which increase renal glucose elimination, and inhibitors of 11ß-hydroxysteroid dehydrogenase 1, which reduce the glucocorticoid effects in liver and fat. Insulin-releasing glucokinase activators and pancreatic-G-protein-coupled fatty-acid-receptor agonists, glucagon-receptor antagonists, and metabolic inhibitors of hepatic glucose output are being assessed. Early proof of principle has been shown for compounds that enhance and partly mimic insulin action and replicate some effects of bariatric surgery.

The challenge of managing coexistent type 2 diabetes and obesity

The presence of obesity with type 2 diabetes increases morbidity and mortality from each condition. Excess adiposity accentuates insulin resistance and complicates the treatment of type 2 diabetes. Glucagon-like peptide 1 receptor agonists promote weight loss, whereas metformin, dipeptidyl peptidase 4 inhibitors, and a glucosidase inhibitors are typically weight neutral. The anabolic effects of increased insulin secretion and action restrict the benefits of treatment in obese patients. New treatments should ideally reduce hyperglycaemia and excess adiposity. Potential new treatments include analogues of intestinal and adipocyte hormones, inhibitors of renal glucose reabsorption and cellular glucocorticoid activation, and activators of cellular energy production.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

1 Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2 In the presence of indomethacin (3μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0 ± 0.1, n = 5). 3 Establishment of a rank order of potency using the EP4 > EP2 agonist 11-deoxy PGE1, and the EP2 > EP4 agonist PGE1-OH (mean pEC 50 of 7.6 ± 0.1 (n = 6) and 6.4 ± 0.1 (n = 4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations < 1 μM failed to relax the tissues. 4 Blockade of EP 4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration-response curves, exhibiting pA2 and pKB values of 5.7 ± 0.1, n = 3, and 8.4, n = 3, respectively. 5 The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3 ± 0.1 (n = 4) and 8.1 ± 0.1 (n = 9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGFα2 failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4 ± 0.3, n = 3). 6 These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

New drugs for type 2 diabetes mellitus:what is their place in therapy?

Oral therapy for type 2 diabetes mellitus, when used appropriately, can safely assist patients to achieve glycaemic targets in the short to medium term. However, the progressive nature of type 2 diabetes usually requires a combination of two or more oral agents in the longer term, often as a prelude to insulin therapy. Issues of safety and tolerability, notably weight gain, often limit the optimal application of anti-diabetic drugs such as sulforylureas and thiazolidinediones. Moreover, the impact of different drugs, even within a single class, on the risk of long-term vascular complications has come under scrutiny. For example, recent publication of evidence suggesting potential detrimental effects of rosiglitazone on myocardial events generated a heated debate and led to a reduction in use of this drug. In contrast, current evidence supports the view that pioglitazone has vasculoprotective properties. Both drugs are contraindicated in patients who are at risk of heart failure. An additional recently identified safety concern is an increased risk of fractures, especially in postmenopausal women. Several new drugs with glucose-lowering efficacy that may offer certain advantages have recently become available. These include (i) injectable glucagonlike peptide-1 (GLP-1) receptor agonists and oral dipeptidyl peptidase-4 (DPP-4) inhibitors; (ii) the amylin analogue pramlintide; and (iii) selective cannabinoid receptor-1 (CB1) antagonists. GLP-1 receptor agonists, such as exenatide, stimulate nutrient-induced insulin secretion and reduce inappropriate glucagon secretion while delaying gastric emptying and reducing appetite. These agents offer a low risk of hypoglycaemia combined with sustained weight loss. The DPP-4 inhibitors sitagliptin and vildagliptin are generally weight neutral, with less marked gastrointestinal adverse effects than the GLP-1 receptor agonists. Potential benefits of GLP-1 receptor stimulation on P cell neogenesis are under investigation. Pancreatitis has been reported in exenatide-treated patients. Pramlintide, an injected peptide used in combination with insulin, can reduce insulin dose and bodyweight. The CB1 receptor antagonist rimonabant promotes weight loss and has favourable effects on aspects of the metabolic syndrome, including the hyperglycaemia of type 2 diabetes. However, in 2007 the US FDA declined approval of rimonabant, requiring more data on adverse effects, notably depression. The future of dual peroxisome proliferator-activated receptor-alpha/gamma agonists, or glitazars, is presently uncertain following concerns about their safety. In conclusion, several new classes of drugs have recently become available in some countries that offer new options for treating type 2 diabetes. Beneficial or neutral effects on bodyweight are an attractive feature of the new drugs. However, the higher cost of these agents, coupled with an absence of long-term safety and clinical outcome data, need to be taken into consideration by clinicians and healthcare organizations.

Consistent reduction of postprandial glucagon and insulin by lixisenatide in the GetGoal clinical trial programme

Background and aims: Glucagon-like peptide-1 (GLP-1) receptor agonists improve islet function and delay gastric emptying in subjects with type 2 diabetes mellitus. We evaluated 2-hour glucose, glucagon and insulin changes following a standardized mixed-meal tolerance test before and after 24 weeks of treatment with the once-daily prandial GLP-1 receptor agonist lixisenatide (approved for a therapeutic dose of 20 μg once daily) in six randomized, placebo-controlled studies within the lixisenatide Phase III GetGoal programme. In the studies, the mixed-meal test was conducted before and after: (1) lixisenatide treatment in patients insufficiently controlled despite diet and exercise (GetGoal-Mono), (2) lixisenatide treatment in combination with oral antidiabetic drugs (OADs) (GetGoal-M and GetGoal-S), or (3) lixisenatide treatment in combination with basal insulin ± OAD (GetGoal-Duo 1, GetGoal-L and GetGoal-L-Asia).Materials and methods: A meta-analysis was performed (lixisenatide n=1124 vs placebo n=707) combining ANCOVA least squares (LS) mean values using an inverse variance weighted analysis. Results: Lixisenatide significantly reduced 2-hour postprandial glucose from baseline (LS mean difference vs placebo: -4.9 mmol/L, p<0.0001, Figure) and glucose excursions (LS mean difference vs placebo: -4.5 mmol/L, p<0.0001). As measured in two studies, lixisenatide also reduced postprandial glucagon (LS mean difference vs placebo: -19.0 ng/L, p<0.0001) and insulin (LS mean difference vs placebo: -64.8 pmol/L, p<0.0001), although the glucagon/insulin ratio was increased (LS mean difference vs placebo: 0.15, p=0.02) compared with placebo. Conclusion: The results show that lixisenatide potently reduces the glucose excursion after meal ingestion in subjects with type 2 diabetes, in association with marked reductions in glucagon and insulin levels. It is suggested that diminished glucagon secretion and slower gastric emptying contribute to reduced hepatic glucose production and delayed glucose absorption, enabling postprandial glycaemia to be controlled with less demand on beta-cell insulin secretion. Clinical Trial Registration Number: NCT00688701; NCT00712673; NCT00713830; NCT00975286; NCT00715624; NCT00866658 Supported by: Sanofi

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM(2) receptor, whereas when paired with the calcitonin receptor AMY(3) receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT((a))). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM(2) and AMY(3(a)) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

Effects of some 5-hydroxytryptamine and related ligands in anxiety models

Drugs acting at 5-HT receptors were evaluated on three animal models of anxiety. On the elevated X-maze test the majority of 5-HT1 agonists were found to be anxiogenic. However, ipsapirone was anxiolytic and buspirone and gepirone were inactive. The 5-HT2 agonist DOI and the 5-HT2 antagonist ritanserin were anxiolytic while ICI 169,369, a 5-HT2 antagonist was inactive. All 5-HT3 antagonists tested were inactive in this test, while the indirect serotomimetics zimeldine and fenfluramine were anxiogenic. Neither beta-adrenoceptor agonists nor antagonists had reproducible effects on anxiety in this model. Combined beta-1/beta-2 adrenoceptor antagonists reversed the anxiogenic effects of 8-OH-DPAT while selective beta-1 or beta-2 antagonists did not. On the social interaction model the 5-HT1 agonists 8-OH-DPAT, RU 24969 and 5-MeODMT were anxiogenic and ipsapirone was anxiolytic. The 5-HT2 agonist DOI and the beta-adrenoceptor- and 5-HT- antagonist pindolol were anxiolytic, while the 5-HT2 and 5-HT3 antagonists were inactive. In the marble burying test, the 5-HT upake inhibitors zimeldine, fluvoxamine, indalpine and citalopram, the 5-HT1B/5-HT1C agonists mCPP and TFMPP and the 5-HT2/5-HT1C agonist DOI reduced marble burying without affecting locomotor activity. 5-HT1A agonists and the 5-HT2 and 5-HT3 antagonists were without effect. Lesions of the dorsal raphe nucleus reversed the anxiogenic effects of 8-OH-DPAT in the X-maze model. The implication of these results for the understanding of the pharmacology of 5-HT in anxiety is discussed.

An investigation into the pharmacology of tics and tic-like movements

The study of tic-like movements in mice has demonstrated close parallels both in characteristics and in pharmacology with the tics which occur in TS. Head-shakes and/or other tic-like behaviours occurring spontaneously or induced by the selective 5-HT2/1C agonist DOI, alpha-melanocyte stimulating hormone, adrenocorticotrophic hormone (1-39), thyrotropin releasing hormone, or RX336-M were blocked when tested with neuroleptics such as haloperidol and/or the alpha-2 adrenoceptor agonist clonidine. The selective dopamine D1 antagonists SCH23390 and SCH39166 dose-dependently blocked spontaneous and DOI head-shakes but the selective dopamine D2 antagonists sulpiride and raclopride were ineffective. The 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone, gepirone, MDL 73005EF and buspirone (i.p) dose-dependently blocked DOI head-shakes, pindolol blocked the inhibitory effect of 8-OH-DPAT on DOI head-shakes. Parachlorophenylalanine blocked the inhibitory effect of 8-OH-DPAT and buspirone, suggesting that the 5-HT1A receptor involved is located presynaptically. The alpha-2 adrenoceptor antagonists yohimbine, idazoxan, 1-PP and RX811059 prevented the inhibitory effect of 8-OH-DPAT on DOI head-shakes suggesting that this 5-HT1A - 5-HT2 receptor interaction is under the modulatory control of adrenoceptors. Because kynurenine has previously been found to potentiate head-shaking, plasma kynurenine concentrations were measured in seven TS patients and were significantly higher than controls, but neopterin and biopterin were unchanged. The relationship between tic-like movements in rodents and their implications for understanding the aetiology and treatment of TS is discussed.