Synthesis of substituted 3-anilino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-ones and their evaluation as cholecystokinin-ligands

3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reacted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodiazepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepines 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzodiazepines 16-38 with CCK 1 receptor selectivity to CCK 2. The compounds 18, 24, 28 and 33 have shown affinities at the CCK 1 receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK 1 ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was identified in the tail suspension- and the Porsolt swimming-test. The ED 50 values for 24 and 28 were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in addition to the antidepressant effects anxiolytic properties. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analogue of GLP-1, N-pyroglutamyl-GLP-1 as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC50-37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16.3 and 27 nM respectively compared with GLP-1 (EC50 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P< 0.05 to P< 0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes. © 2004 Society for Endocrinology.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

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.

The pharmacology of CGRP-responsive receptors in cultured and transfected cells

Historically, CGRP receptors have been classified as CGRP(1) or CGRP(2) subtypes, chiefly depending on their affinity for the antagonist CGRP(8-37). It has been shown that the complex between calcitonin receptor-like receptor (CRLR or CL) and receptor activity modifying protein (RAMP) 1 provides a molecular correlate for the CGRP(1) receptor; however this does not explain the range of affinities seen for CGRP(8-37) in isolated tissues. It is suggested that these may largely be explained by a combination of methodological factors and CGRP-responsive receptors generated by CL and RAMP2 or RAMP3 and complexes of RAMPs with the calcitonin receptor.

Heterodimers and family-B GPCRs:RAMPs, CGRP and adrenomedullin

RAMPs (receptor activity-modifying proteins) are single-pass transmembrane proteins that associate with certain family-B GPCRs (G-protein-coupled receptors). Specifically for the CT (calcitonin) receptor-like receptor and the CT receptor, this results in profound changes in ligand binding and receptor pharmacology, allowing the generation of six distinct receptors with preferences for CGRP (CT gene-related peptide) adrenomedullin, amylin and CT. There are three RAMPs: RAMP1-RAMP3. The N-terminus appears to be the main determinant of receptor pharmacology whereas the transmembrane domain contributes to association of the RAMP with the GPCR. The N-terminus of all members of the RAMP family probably contains two disulphide bonds; a potential third disulphide is found in RAMP1 and RAMP3. The N-terminus appears to be in close proximity to the ligand and plays a key role in its binding, either directly or indirectly. BIBN4096BS, a CGRP antagonist, targets RAMP1 and this gives the compound very high selectivity for the human CGRP(1) receptor.

Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.

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.

Diabetes therapies in renal impairment

Depending on age, duration of diabetes and glycaemic control, 20-40% of patients with type 2 diabetes will incur a moderate or severe deterioration of renal function. This will impact the choice of blood glucose-lowering therapy and require more frequent monitoring of both renal function and glycaemic control. Moderate renal impairment (glomerular filtration rate 30-<60 ml/min) requires consideration of dose reduction or treatment cessation for metformin, glucagon-like peptide-1 receptor agonists, some sulphonylureas and some dipeptidyl peptidase-4 inhibitors. At lower rates of glomerular filtration down to about 15 ml/min it may be appropriate to use a meglitinide, pioglitazone or certain sulphonylureas with careful consideration of dose and co-morbidities. Dipeptidyl peptidase-4 inhibitors can be used at reduced dose in patients with very low rates of glomerular filtration, and linagliptin can be used without dose reduction, and has been used in patients on dialysis. Insulin can be used at any stage of renal impairment, but the regimen and the dose must be suitably adjusted and accompanied by adequate monitoring. © The Author(s), 2012.

Pronounced reduction of postprandial glucagon by lixisenatide:a meta-analysis of randomized clinical trials

Glucagon-like peptide-1 (GLP-1) receptor agonists improve islet function and delay gastric emptying in patients with type 2 diabetes mellitus (T2DM). This meta-analysis aimed to investigate the effects of the once-daily prandial GLP-1 receptor agonist lixisenatide on postprandial plasma glucose (PPG), glucagon and insulin levels. Methods: Six randomized, placebo-controlled studies of lixisenatide 20μg once daily were included in this analysis: lixisenatide as monotherapy (GetGoal-Mono), as add-on to oral antidiabetic drugs (OADs; GetGoal-M, GetGoal-S) or in combination with basal insulin (GetGoal-L, GetGoal-Duo-1 and GetGoal-L-Asia). Change in 2-h PPG and glucose excursion were evaluated across six studies. Change in 2-h glucagon and postprandial insulin were evaluated across two studies. A meta-analysis was performed on least square (LS) mean estimates obtained from analysis of covariance (ANCOVA)-based linear regression. Results: Lixisenatide significantly reduced 2-h PPG from baseline (LS mean difference vs. placebo: -4.9mmol/l, p<0.001) and glucose excursion (LS mean difference vs. placebo: -4.5mmol/l, p<0.001). As measured in two studies, lixisenatide also reduced postprandial glucagon (LS mean difference vs. placebo: -19.0ng/l, p<0.001) and insulin (LS mean difference vs. placebo: -64.8 pmol/l, p<0.001). There was a stronger correlation between 2-h postprandial glucagon and 2-h PPG with lixisenatide than with placebo. Conclusions: Lixisenatide significantly reduced 2-h PPG and glucose excursion together with a marked reduction in postprandial glucagon and insulin; thus, lixisenatide appears to have biological effects on blood glucose that are independent of increased insulin secretion. These effects may be, in part, attributed to reduced glucagon secretion. © 2014 John Wiley and Sons Ltd.

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.

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK 1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 CCK 1 for the CCK 1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg -1 was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine. © 2006 The Authors.