Glepaglutide, a novel glucagon-like peptide-2 agonist, has anti-inflammatory and mucosal regenerative effects in an experimental model of inflammatory bowel disease in rats.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) enhances intestinal repair and attenuates inflammation in preclinical inflammatory bowel disease (IBD) models, making GLP-2 analogues attractive candidates for IBD therapy. Glepaglutide is a long-acting GLP-2 receptor agonist in clinical development for treatment of short bowel syndrome. Here, we investigated if glepaglutide is therapeutically beneficial in rats with small intestinal inflammation. METHODS: Small intestinal inflammation was induced with indomethacin in naive Wistar rats, followed by glepaglutide administration at different disease stages. Glepaglutide was administered in co-treatment and post-treatment regimens. Small intestinal length and concentrations of inflammatory markers α-1-acid glycoprotein and myeloperoxidase were used to assess anti-inflammatory effects. Small intestinal mass was evaluated to determine intestinotrophic effects. RESULTS: Glepaglutide co- and post-treatment significantly reduced severity of small intestinal inflammation, evidenced by reversed small intestinal shortening and decreased α-1-acid glycoprotein and/or myeloperoxidase concentration(s). Co- and post-treatment with glepaglutide also significantly increased small intestinal mass, indicating intestinal regenerative effects. Similar effects were observed in naive rats after glepaglutide treatment. CONCLUSION: Glepaglutide has anti-inflammatory and intestinotrophic effects without the need for pre-treatment in a rat model of small intestinal inflammation. Thus, glepaglutide is of potential clinical interest for patients with IBD.

Inhibition of Cx43 gap junction uncoupling prevents high glucose-induced apoptosis and reduces excess cell monolayer permeability in retinal vascular endothelial cells.

The aim of this study was to investigate whether inhibition of connexin 43 gap junction-uncoupling is sufficient to prevent retinal vascular cell loss under high glucose condition and reduce cell monolayer permeability. Rat retinal endothelial cells were grown for 3, 5, and 7 days in normal (5 mM) or high glucose (30 mM) medium; in parallel, cells grown in high glucose medium were exposed for 3, 5, and 7 days to 100 nM danegaptide, which stabilizes connexin 43-mediated cell coupling. Additionally, cells grown in normal medium were treated with a connexin 43 blocker as a negative control. To determine gap junction intercellular communication, scrape load dye transfer assay was performed at the three time points. Cells were assessed for apoptosis and cell monolayer permeability by differential dye staining and in vitro permeability assays, respectively. Cells treated with danegaptide preserved gap junction intercellular communication, decreased cell death, and reduced cell monolayer permeability. Scrape load dye transfer assay indicated that cells exposed to danegaptide for 3, 5, and 7 days under high glucose condition maintained gap junction intercellular communication. Importantly, danegaptide significantly prevented high glucose-induced apoptosis at all three time points, and inhibited cell monolayer permeability by day 5. Cells exposed to a connexin 43 blocker, which decreased cell coupling, showed excess apoptosis and cell monolayer permeability. These findings suggest that prevention of high glucose-induced compromised cell-cell coupling may be a useful strategy for inhibiting apoptosis and excess vascular permeability associated with diabetic retinopathy.

The design and discovery of lixisenatide for the treatment of type 2 diabetes mellitus.

INTRODUCTION: Lixisenatide is a once-daily short-acting glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1RA) used in the treatment of type 2 diabetes mellitus (T2DM). It is used in combination with oral antidiabetics and/or basal insulin in patients inadequately controlled on these medications and who are undergoing diet and lifestyle modification. GLP-1RAs glucose-dependently increase insulin secretion, decrease glucagon secretion, and slow gastric emptying, thereby improving glycemic control. GLP-1RAs are associated with body weight benefits and low rates of hypoglycemia which are welcome in patients with T2DM. AREAS COVERED: The authors describe the identification of GLP-1RAs as suitable targets for modification with structure-inducing probe technology to improve stability and resistance to proteolytic degradation. Clinical studies have assessed lixisenatide across > 5000 patients as a monotherapy or add-on to a variety of commonly used antidiabetic medications. These studies highlighted the effects of lixisenatide on gastric emptying, explaining its particular improvements in postprandial plasma glucose (PPG) excursions and underscoring its efficacy in combination with insulin glargine. Lixisenatide was well tolerated, with nausea and vomiting being the most frequently reported adverse events. EXPERT OPINION: The once-daily administration of lixisenatide as well as its substantial sustained effect on gastric emptying and, hence, PPG excursions are all important features compared with the other GLP-1RAs. The combination of two injectables, such as basal insulin to lower fasting plasma glucose and a GLP-1RA that curtails PPG excursions, is clinically valuable and could differentiate lixisenatide from other GLP-1RAs, especially from those continuously acting GLP-1RAs with little effect on gastric emptying and PPG excursions.

Osteoclastogenesis is influenced by modulation of gap junctional communication with antiarrhythmic peptides.

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.

Design and characterization of the first peptidomimetic molecule that prevents acidification-induced closure of cardiac gap junctions.

BACKGROUND: Gap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions. OBJECTIVE: The purpose of this study was to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels. METHODS: Experiments were based on a combination of biochemical, spectroscopic, and electrophysiologic techniques as well as molecular modeling of active pharmacophores with Cx43 activity. RESULTS: Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels and pH gating of cardiac gap junctions. CONCLUSION: Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacologic agents for the treatment of a selected subset of cardiac arrhythmias.

Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides.

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

Discovery of (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride (GAP-134)13, an orally active small molecule gap-junction modifier for the treatment of atrial fibrillation.

Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.

Identification of ischemia-regulated phosphorylation sites in connexin43: A possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123).

Previous studies suggest that dephosphorylation of connexin43 (Cx43) is related to uncoupling of gap junction communication, which plays an important role in the genesis of ischemia-induced ventricular tachycardia. We studied changes in Cx43 phosphorylation during global ischemia in the absence and presence of the antiarrhythmic peptide analogue rotigaptide (formerly known as ZP123). Phosphorylation analysis was performed on Cx43 purified from isolated perfused rat hearts using matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography electrospray ionization tandem mass spectrometry. Thirteen different serine phosphorylation sites were identified in Cx43 during non-ischemic conditions, three of which had not previously been described. Within the first 7 min of ischemia, Ser306 became fully dephosphorylated whereas Ser330 became phosphorylated. Between 15 and 30 min of ischemia, the critical time interval where gap junction uncoupling occurs, Ser297 and Ser368 also became fully dephosphorylated. During the same time period, all untreated hearts developed asystole. Treatment with rotigaptide significantly increased the time to ischemia-induced asystole and suppressed dephosphorylation of Ser297 and Ser368 at 30 min of ischemia. Our results suggest that phosphorylation of Ser297 and Ser368 may be involved in functional gating of Cx43 during ischemia and may be possible downstream targets for rotigaptide signaling.

Glucagon-like peptide 1 receptor agonist ZP10A increases insulin mRNA expression and prevents diabetic progression in db/db mice.

We characterized the novel, rationally designed peptide glucagon-like peptide 1 (GLP-1) receptor agonist H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSK KKKKK-NH2 (ZP10A). Receptor binding studies demonstrated that the affinity of ZP10A for the human GLP-1 receptor was 4-fold greater than the affinity of GLP-1 (7-36) amide. ZP10A demonstrated dose-dependent improvement of glucose tolerance with an ED50 value of 0.02 nmol/kg i.p. in an oral glucose tolerance test (OGTT) in diabetic db/db mice. After 42 days of treatment, ZP10A dose-dependently (0, 1, 10, or 100 nmol/kg b.i.d.; n = 10/group), decreased glycosylated hemoglobin (HbA1C) from 8.4 +/- 0.4% (vehicle) to a minimum of 6.2 +/- 0.3% (100 nmol/kg b.i.d.; p < 0.05 versus vehicle) in db/db mice. Fasting blood glucose (FBG), glucose tolerance after an OGTT, and HbA1C levels were significantly improved in mice treated with ZP10A for 90 days compared with vehicle-treated controls. Interestingly, these effects were preserved 40 days after drug cessation in db/db mice treated with ZP10A only during the first 50 days of the study. Real-time polymerase chain reaction measurements demonstrated that the antidiabetic effect of early therapy with ZP10A was associated with an increased pancreatic insulin mRNA expression relative to vehicle-treated mice. In conclusion, long-term treatment of diabetic db/db mice with ZP10A resulted in a dose-dependent improvement of FBG, glucose tolerance, and blood glucose control. Our data suggest that ZP10A preserves beta-cell function. ZP10A is considered one of the most promising new drug candidates for preventive and therapeutic intervention in type 2 diabetes.

Pharmacological characterization of the new stable antiarrhythmic peptide analog Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2 (ZP123): in vivo and in vitro studies.

Antiarrhythmic peptides (AAPs) are a group of compounds with antiarrhythmic properties; however, their use has been hampered by very low plasma stability. The aim of this study was to compare the in vitro and in vivo stability of our new stable AAP analog Ac-d-Tyr-d-Pro-d-Hyp-Gly-d-Ala-Gly-NH2 (ZP123) with the previously described AAP analog AAP10. Moreover, the effect of the two compounds was examined in a murine in vivo model of ouabain-induced second degree AV-block, and the effect on dispersion of action potential duration (APD dispersion) was studied during hypokalemic-ischemia in isolated perfused rabbit hearts. The in vitro t1/2 of ZP123 in rat and human plasma was about 1,700 times longer than t1/2 of AAP10. Due to rapid elimination, it was not possible to obtain an in vivo pharmacokinetic characterization of AAP10; however, calculations suggested that the clearance of ZP123 was at least 140 times slower than for AAP10. AAP10 and ZP123 produced a dose-dependent delay in onset of ouabain-induced AV-block in mice at doses of 10-11 to 10-7 mol/kg i.v. ZP123 and 10-11 to 10-6 mol/kg i.v. AAP10. Maximal efficacy of ZP123 was reached at a 10-fold lower dose (10-8 mol/kg i.v.) than with AAP10. In the isolated rabbit hearts, ZP123 and AAP10 had no effect on dispersion during control conditions. The increased APD dispersion during hypokalemic ischemia is considered a major arrhythmic substrate and only ZP123 prevented the increase in APD dispersion. In conclusion, ZP123 is a new potent AAP analog with improved stability.

Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice.

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.