The apelinergic system as an alternative to catecholamines in low-output septic shock.

Catecholamines, in concert with fluid resuscitation, have long been recommended in the management of septic shock. However, not all patients respond positively and controversy surrounding the efficacy-to-safety profile of catecholamines has emerged, trending toward decatecholaminization. Contextually, it is time to re-examine the "maintaining blood pressure" paradigm by identifying safer and life-saving alternatives. We put in perspective the emerging and growing knowledge on a promising alternative avenue: the apelinergic system. This target exhibits invaluable pleiotropic properties, including inodilator activity, cardio-renal protection, and control of fluid homeostasis. Taken together, its effects are expected to be greatly beneficial for patients in septic shock.

Intermedin protects against myocardial ischemia-reperfusion injury in hyperlipidemia rats.

Hyperlipidemia is a well-established risk factor for the development of coronary atherosclerosis, while intermedin (IMD) has been identified as a novel calcitonin/calcitonin gene-related peptide family member involved in cardiovascular protection. However, whether IMD protects against hyperlipidemia-associated myocardial ischemia/reperfusion (MI/R) injury is unknown. We established a hyperlipidemia model using Sprague-Dawley rats, and created a MI/R condition by ligating the cardiac left circumflex artery. The possible pathophysiological role of IMD and its physiological function in MI/R was further studied. The level of IMD significantly decreased in hyperlipidemia rats (P < 0.05). After MI/R, the IMD level was increased both in the plasma and myocardial tissue of hyperlipidemia rats compared to the sham-operated rats (P < 0.001). As evaluated by the activity of LDH, CK-MB, MDA and SOD, additional IMD was revealed to alleviate MI/R heart injury in hyperlipidemia rats (P < 0.05). By regulating the process of cardiomyocyte apoptosis and inflammatory reaction, IMD could perform an important role in cardio-protection, especially against hyperlipidemia-associated MI/R injury. Additional IMD could protect cardiac myocytes against MI/R injury via reduction of apoptosis and inflammation in the hyperlipidemia rat model, and thus, it may play a potential role as a novel therapeutic target for cardiac ischemic injury in hyperlipidemic patients.

Effect of Fc-Elabela-21 on renal ischemia/reperfusion injury in mice: Mediation of anti-apoptotic effect via Akt phosphorylation.

INTRODUCTION: Renal ischemia/reperfusion injury (IRI) is the most common cause of acute kidney injury (AKI), and patients with AKI have a high rate of mortality. Apelin is a therapeutic candidate for treatment of IRI and Elabela (ELA) is a recently discovered hormone that also activates the apelin receptor (APJ). We examined the use of ELA as a preventive treatment for IRI using in vitro and in vivo models. METHODS: Male mice were subjected to renal IRI, with or without administration of a stabilized form of ELA (Fc-ELA-21) for 4 days. Renal tubular lesions were measured using H&E staining, reactive oxygen species (ROS) were measured using a dihydroethidium stain assay, and renal cell apoptosis was measured using the TUNEL assay and flow cytometry. Immortalized human proximal tubular epithelial (HK-2) cells were pretreated with or without LY294002 and/or ELA-32, maintained at normoxic or hypoxic conditions, and then returned to normal culture conditions to mimic IRI. Cell apoptosis was determined using the TUNEL assay and cell proliferation was determined using the MTT assay. The levels of Akt, p-Akt, ERK1/2, p- ERK1/2, Bcl-2, Bax, caspase-3 and cleaved caspase-3 were measured using western blotting. RESULTS: Fc-ELA-21 administration reduced renal tissue damage, ROS production, and apoptosis in mice that had renal IRI. ELA-32 reduced HK-2 cell apoptosis and restored the proliferation of cells subjected to IRI. Akt phosphorylation had a role in the anti-apoptotic effect of ELA. CONCLUSION: This study of in vitro and in vivo models of IRI indicated that the preventive and anti-apoptotic effects of ELA were mediated via the PI3K/Akt signaling pathway.

Glucagon-Like Peptide-1 (GLP-1)-Based Therapeutics: Current Status and Future Opportunities beyond Type†2 Diabetes.

Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells following food intake, and plays an important role in glucose homeostasis due to its stimulation of glucose-dependent insulin secretion. Further, GLP-1 is also associated with protective effects on pancreatic ß-cells and the cardiovascular system, decreased appetite, and weight loss, making GLP-1 derivatives an exciting treatment for type 2 diabetes and obesity. Despite these benefits, wild-type GLP-1 exhibits a short circulation time due to its poor metabolic stability and rapid renal clearance, and must be administered by injection, making it a poor therapeutic agent. Many strategies have been used to improve the circulation time of GLP-1 (e.g., mutations, unnatural amino acids, depot formulations, use of exendin-4 sequences, and fusions with high-molecular-weight proteins or polymers), with its therapeutic utility further improved by adding agonist activity for gastric inhibitory peptide and glucagon receptors. This minireview focuses on strategies that have been used to improve the pharmacokinetics of GLP-1 and provides an overview of GLP-1-based therapeutics in the pipeline.

Controlling the bioactivity of a peptide hormone in vivo by reversible self-assembly.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics.

Chitosan nanocapsules as carriers for oral peptide delivery: effect of chitosan molecular weight and type of salt on the in vitro behaviour and in vivo effectiveness.

We have recently reported preliminary data showing the efficacy of chitosan nanocapsules as carriers for oral peptide delivery. In the present work, our aim was to investigate the influence of some chitosan properties, such as the molecular weight and type of salt, on the interaction of these nanocapsules with the Caco-2 cells and also on their in vivo effectiveness. Chitosan nanocapsules were prepared by the solvent displacement technique using high (450 kDa) and medium (160 kDa) molecular weight chitosan glutamate as well as high molecular weight chitosan hydrochloride (270 kDa). The results indicated that the size of the nanocapsules was dependent on the chitosan molecular weight, whereas the zeta potential and the association efficiency of salmon calcitonin were not affected by the chitosan properties. Upon incubation with the Caco-2 cells, chitosan nanocapsules exhibited a dose-dependent cellular viability, which was hardly affected by, either the chitosan molecular weight or, the type of salt. In addition, it was observed that the transepithelial electrical resistance of the Caco-2 monolayer was not significantly modified upon their exposure to chitosan nanocapsules. The results of the in vivo studies, following oral administration to rats, indicated that chitosan nanocapsules were able to reduce significantly the serum calcium levels, and to prolong this reduction for at least 24 hours, irrespective of the type of chitosan salt and molecular weight of chitosan. Consequently, the performance of chitosan nanocapsules as oral carriers for salmon calcitonin was not affected by the characteristics of chitosan.

Sex, social status, and CRF receptor densities in naked mole-rats.

Naked mole-rats (Heterocephalus glaber) live in groups that are notable for their large size and caste structure, with breeding monopolized by a single female and a small number of males. Recent studies have demonstrated substantial differences between the brains of breeders and subordinates induced by changes in social standing. Corticotropin-releasing factor (CRF) receptors-which bind the hormone CRF as well as related peptides-are important regulators of stress and anxiety, and are emerging as factors affecting social behavior. We conducted autoradiographic analyses of CRF1 and CRF2 receptor binding densities in female and male naked mole-rats varying in breeding status. Both globally and in specific brain regions, CRF1 receptor densities varied with breeding status. CRF1 receptor densities were higher in subordinates across brain regions, and particularly in the piriform cortex and cortical amygdala. Sex differences were present in CRF2 receptor binding densities, as is the case in multiple vole species. CRF2 receptor densities were higher in females, both globally and in the cortical amygdala and lateral amygdalar nucleus. These results provide novel insights into the neurobiology of social hierarchy in naked mole-rats, and add to a growing body of work that links changes in the CRF system with social behavior.

Radiometallation of receptor-specific peptides for diagnosis and treatment of human cancer.

Radiolabeled, receptor-specific peptides are becoming increasingly popular as targeting vectors for the design and development of new diagnostic and therapeutic radiopharmaceuticals. The over-expression of functioning receptors on a variety of human cancers makes this method of drug development a viable tool for tumor targeting in vivo. This review describes some of the more recent efforts that are currently underway towards development of new receptor-specific radiopharmaceuticals. Diagnostic/therapeutic radionuclides, specific metal co-ordinating ligands/chelating systems, spacer technology, radiolabeling protocols, and specific peptides/peptide conjugates will be discussed in detail.

Annual banned-substance review: analytical approaches in human sports drug testing.

Within the mosaic display of international anti-doping efforts, analytical strategies based on up-to-date instrumentation as well as most recent information about physiology, pharmacology, metabolism, etc., of prohibited substances and methods of doping are indispensable. The continuous emergence of new chemical entities and the identification of arguably beneficial effects of established or even obsolete drugs on endurance, strength, and regeneration, necessitate frequent and adequate adaptations of sports drug testing procedures. These largely rely on exploiting new technologies, extending the substance coverage of existing test protocols, and generating new insights into metabolism, distribution, and elimination of compounds prohibited by the World Anti-Doping Agency (WADA). In reference of the content of the 2014 Prohibited List, literature concerning human sports drug testing that was published between October 2013 and September 2014 is summarized and reviewed in this annual banned-substance review, with particular emphasis on analytical approaches and their contribution to enhanced doping controls.

Effects of ghrelin on the insulin and glycemic responses to glucose, arginine, or free fatty acids load in humans.

Ghrelin possesses central and peripheral endocrine actions including influence on the endocrine pancreatic function. To clarify this latter ghrelin action, in seven normal young subjects [age (mean +/- SEM), 28.3 +/- 3.1 yr; body mass index, 21.9 +/- 0.9 kg/m(2)), we studied insulin and glucose levels after acute ghrelin administration (1.0 microg/kg i.v.) alone or combined with glucose [oral glucose tolerance test (OGTT), 100 g orally], arginine (ARG, 0.5 g/kg i.v.) or free fatty acid (FFA, Intralipid 10%, 250 ml). Ghrelin inhibited (P < 0.05) insulin and increased (P < 0.05) glucose levels. OGTT increased (P < 0.01) glucose and insulin levels. FFA increased (P < 0.05) glucose but did not modify insulin levels. ARG increased (P < 0.05) both insulin and glucose levels. Ghrelin did not modify both glucose and insulin responses to OGTT as well as the FFA-induced increase in glucose levels; however, ghrelin administration was followed by transient insulin decrease also during FFA. Ghrelin blunted (P < 0.05) the insulin response to ARG and enhanced (P < 0.05) the ARG-induced increase in glucose levels. In all, ghrelin induces transient decrease of spontaneous insulin secretion and selectively blunts the insulin response to ARG but not to oral glucose load. On the other hand, ghrelin raises basal glucose levels and enhances the hyperglycemic effect of ARG but not that of OGTT. These findings support the hypothesis that ghrelin exerts modulatory action of insulin secretion and glucose metabolism in humans.

Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects.

OBJECTIVE: It has been demonstrated that ghrelin plays a major role in the regulation of GH secretion and food intake. These actions make ghrelin a strong candidate for the treatment of GH deficiency, anorexia and cachexia. However, only preliminary studies have been performed to assess ghrelin administration in humans. In this study, we have conducted a double-blind, randomized, placebo-controlled trial to investigate the pharmacokinetics, safety, and endocrine and appetite effects of ghrelin in young healthy volunteers. DESIGN: Eighteen male volunteers were randomly assigned into three groups of six subjects: low- and high-dose ghrelin groups, who received intravenous injections of 1 and 5 microg/kg ghrelin (acylated form) respectively, and a placebo group who were injected with mannitol instead of ghrelin. RESULTS: Acylated ghrelin disappeared more rapidly from plasma than total ghrelin, with elimination half life (t(1/2)) of 9-13 and 27-31 min respectively. The number of subjects that experienced adverse effects did not significantly differ among the three groups, and all adverse effects were transient and well tolerated. Both the low and high doses of ghrelin strongly stimulated GH release (peak plasma concentration (C(max,0-90 min)): 124.2+/-63.9 and 153.2+/-52.2 ng/ml for 1 and 5 microg/kg ghrelin respectively). Slight alterations of blood glucose and insulin levels after the injection were observed. Although not statistically significant, ghrelin administration tended to increase hunger sensation in a dose-dependent manner. CONCLUSIONS: These results suggest that ghrelin is safe, and that clinical trials may be started to assess the usefulness of ghrelin for the treatment of disorders related to GH secretion and appetite.

Ghrelin clearance is reduced at the late stage of polymicrobial sepsis.

The cardiovascular response to sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor (i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide in addition to its effects on growth hormone release and energy homeostasis. We have shown that ghrelin (via its receptor) may play an important role in regulating cardiovascular responses in the progression of polymicrobial sepsis. However, it remains unknown whether the clearance of this peptide is altered in sepsis. To determine this, male adult rats were injected with 125I-ghrelin through the jugular vein at 5 or 20 h after cecal ligation and puncture (CLP, i.e., sepsis model) or sham operation. The blood sample was collected every 2 min for 30 min for determining half-life (t1/2). Tissue samples (i.e., kidneys, liver, brain, heart, lungs, spleen, stomach, small intestine, large intestine, skin and muscle) were then harvested. The radioactivities of samples were counted. The results indicate that 125I-ghrelin's t1/2 and its distribution were not significantly altered in early sepsis (5 h after CLP). However, the t1/2 increased significantly in late sepsis (20 h after CLP). Tissue distribution of 125I-ghrelin was far greater in the kidneys than in any other tissues tested in both sham and septic animals. Moreover, the kidneys and liver had significantly less radioactive uptake at 20 h after CLP, but the radioactivity in blood was much higher at the same time point. There were no significant changes in 125I-ghrelin distribution in other organs at the late stage of sepsis. These results indicate that the kidneys are the primary site of ghrelin clearance, which is significantly diminished in late sepsis. In addition, the liver also plays a role in the clearance of ghrelin, which was also reduced in late sepsis. The decreased clearance of ghrelin by the kidneys and liver may be due to renal and hepatic dysfunctions under such conditions.

Correlation of tetradecylmaltoside induced increases in nasal peptide drug delivery with morphological changes in nasal epithelial cells.

The effect of tetradecylmaltoside (TDM) on nasal peptide drug absorption was assessed with four peptides of distinct molecular size: insulin (5.7 kDa), leptin (16 kDa), somatropin (22.1 kDa), and epoetin alfa (30.4 kDa). The nasal uptake of the smallest peptides, insulin and leptin, was significantly increased at a TDM concentration of only 0.06%. The uptake of somatropin was significantly increased when concentrations of 0.125% or more were used. The uptake of the largest peptide, epoetin alfa, was not significantly increased, in the presence of 0.125-0.5% TDM. Light microscopy revealed that formulations containing 0.125% TDM caused moderate alterations in nasal epithelial cell morphology, while higher concentrations of TDM (0.5%), caused more extensive morphological changes. Following treatment with 0.125% TDM, the distribution of cilia was altered and the number of pinocytotic vesicles was increased, at a time that correlated with increased nasal absorption of insulin. Consistent with these findings, FITC-insulin applied nasally in the absence of TDM did not enter nasal epithelial cells, whereas FITC-insulin co-administered with 0.125% TDM was internalized into the cells, with a uniform distribution, consistent with transcellular movement of the peptide through the cells.

Ghrelin attenuates the development of acute pancreatitis in rat.

BACKGROUND: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. METHODS: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. RESULTS: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. CONCLUSIONS: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems Background: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. Methods: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. Results: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta conc; concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. Conclusions: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems to be related the inhibition in inflammatory process and the reduction in liberation of pro-inflammatory IL-1beta.

Three-peptide control of pulsatile and entropic feedback-sensitive modes of growth hormone secretion: modulation by estrogen and aromatizable androgen.

The present review highlights a simplified perspective of growth hormone (GH) secretory control, which incorporates the individual and joint effects of final-common signals that converge on somatotrope cells. Critical peptidyl effectors are GH-releasing hormone (GHRH), GH-releasing peptide (GHRP, ghrelin), and somatostatin. The latter three-peptide ensemble mediates stimulation, inhibition, and feedback suppression of GH secretion via homologous and heterologous interactions. Pubertal sex steroids putatively act via post-aromatized estrogen (e.g., testosterone converted to estradiol by aromatase) to augment sensitivity to GHRH, potentiate GHRP action, and mute somatostatin restraint. The dynamic interactions in this three-peptide network, rather than the activity of any single effector, subserve core adaptations in GH secretion across development.

Comparing monoclonal antibodies and small peptidic hormones for local targeting of malignant gliomas.

Monoclonal antibodies, F(ab')2 fragments and peptidic vectors have been clinically tested for systemic and locoregional treatment of malignant gliomas. Since these brain-intrinsic neoplasms are characterized by relentless tumor cell infiltration of normal brain parenchyma, targeting agents require diffusive properties in order to reach invading tumor cell clusters that migrate along vascular clefts and axonal pathways. Tumor uptake was significantly improved by using small peptidic hormone receptors, e.g. modified octreotide, following systemic injections as compared to macromolecules which only led to limited stabilization of the disease. More importantly, biodistribution was found to be superior following direct intratumoral injection by using these small drug-like radioconjugates. Rapid and extensive distribution within 30 minutes was observed in large tumors, even crossing the corpus callosum in bihemispheric lesions following injection of 2-3 ml of the radiopharmakon injected into the center of non-resected tumors. Distribution was far more extensive after direct intratumoral injection as compared to intracavitary injection after surgical debulking. Increased interstitial pressure gradients and the much larger and chaotic structure of the interstitial space of a tumor compared to the extremely tight architecture of normal brain tissue might explain this unexpected biodistribution pattern. Peptidic hormone vectors might become useful agents to deliver radiopharmaceuticals into human invasive gliomas.

Backbone modification of a polypeptide drug alters duration of action in vivo.

Systematic modification of the backbone of bioactive polypeptides through ß-amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein-coupled receptor in the B-family, we present an approach for αâ†’ß residue replacement that enables both high activity and improved pharmacokinetic properties in vivo.

Patents on enhancing the potency and longevity of highly valuable protein hormones.

One major issue regarding the clinical use of many peptides is their short half-life span in the body, due to the rapid clearance from the circulation. Thus, at the clinical level, there is a need for a regime of frequent injections of the peptides into the patients to overcome this low stability factor. The major strategies for overcoming this problem by pharmaceutical companies are based on chemical techniques and using specific peptidase inhibitors or cocktails. For this purpose, the cassette gene contains the sequence of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin ß subunit which was ligated to the coding sequence of follitropin (FSH), erythropoietin (EPO), growth hormone (GH) and thus to increase the longevity and bioactivity of these proteins in vivo. Interestingly, FSH-CTP and GH-CTP were found to be not immunogenic in humans. FSH-CTP was approved by The European Commission. In addition, GH-CTP is not toxic and it passed successfully clinical trials Phase II in adults. Thus, using this technology seems to be promising in designing long acting peptides. Development of long acting peptides will diminish the cost of these drugs and perhaps reduce the number of injections in the clinical protocols. The article also summarizes some relevant patents.

In vitro and in vivo stability and pharmacokinetic profile of unacylated ghrelin (UAG) analogues.

Ghrelin, an endocrine hormone predominantly produced by the stomach, exists in acylated and unacylated forms in the circulation. Unacylated ghrelin (UAG), the more abundant form in blood, possesses similar, independent or opposite physiological actions as acylated ghrelin (AG). AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG(6-13)), and its full (AZP531) and partially (AZP533) cyclised derivatives, exhibit the same pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We investigated the stability of these three fragments in vitro in human blood samples and in vivo after subcutaneous and intravenous injection in rats and dogs using liquid chromatography-mass spectrometry. In both species, AZP502 is rapidly degraded generating two major metabolites. Partial cyclisation of AZP502 and acylation at its N-terminus (AZP533 peptide) improves its stability in human plasma in vitro. Full cyclisation of AZP502 (AZP531 peptide) also completely protects the peptide from peptidase degradation in vitro in human blood samples. Moreover this cyclisation strongly improves the stability and the bioavailability of this peptide in vivo in both dogs and rats (mean bioavailability of 10-15% and 85-95% for AZP502 and AZP531 respectively). Taken together these results support the rationale for developing AZP531 as a long-acting UAG analogue for subcutaneous injection for the treatment of type 2 diabetes mellitus and other metabolic disorders.

Chemical modification of class II G protein-coupled receptor ligands: frontiers in the development of peptide analogs as neuroendocrine pharmacological therapies.

Recent research and clinical data have begun to demonstrate the huge potential therapeutic importance of ligands that modulate the activity of the secretin-like, Class II, G protein-coupled receptors (GPCRs). Ligands that can modulate the activity of these Class II GPCRs may have important clinical roles in the treatment of a wide variety of conditions such as osteoporosis, diabetes, amyotrophic lateral sclerosis and autism spectrum disorders. While these receptors present important new therapeutic targets, the large glycoprotein nature of their cognate ligands poses many problems with respect to therapeutic peptidergic drug design. These native peptides often exhibit poor bioavailability, metabolic instability, poor receptor selectivity and resultant low potencies in vivo. Recently, increased attention has been paid to the structural modification of these peptides to enhance their therapeutic efficacy. Successful modification strategies have included d-amino acid substitutions, selective truncation, and fatty acid acylation of the peptide. Through these and other processes, these novel peptide ligand analogs can demonstrate enhanced receptor subtype selectivity, directed signal transduction pathway activation, resistance to proteolytic degradation, and improved systemic bioavailability. In the future, it is likely, through additional modification strategies such as addition of circulation-stabilizing transferrin moieties, that the therapeutic pharmacopeia of drugs targeted towards Class II secretin-like receptors may rival that of the Class I rhodopsin-like receptors that currently provide the majority of clinically used GPCR-based therapeutics. Currently, Class II-based drugs include synthesized analogs of vasoactive intestinal peptide for type 2 diabetes or parathyroid hormone for osteoporosis.