Linking (Pyr)1apelin-13 pharmacokinetics to efficacy: Stabilization and measurement of a high clearance peptide in rodents.

Apelin, the endogenous ligand for the APJ receptor, has generated interest due to its beneficial effects on the cardiovascular system. Synthesized as a 77 amino acid preproprotein, apelin is post-translationally cleaved to a series of shorter peptides. Though (Pyr)1apelin-13 represents the major circulating form in plasma, it is highly susceptible to proteolytic degradation and has an extremely short half-life, making it challenging to quantify. Literature reports of apelin levels in rodents have historically been determined with commercial ELISA kits which suffer from a lack of selectivity, recognizing a range of active and inactive isoforms of apelin peptide. (Pyr)1apelin-13 has demonstrated beneficial hemodynamic effects in humans, and we wished to evaluate if similar effects could be measured in pre-clinical models. Despite development of a highly selective LC/MS/MS method, in rodent studies where (Pyr)1apelin-13 was administered exogenously the peptide was not detectable until a detailed stabilization protocol was implemented during blood collection. Further, the inherent high clearance of (Pyr)1apelin-13 required an extended release delivery system to enable chronic dosing. The ability to deliver sustained doses and stabilize (Pyr)1apelin-13 in plasma allowed us to demonstrate for the first time the link between systemic concentration of apelin and its pharmacological effects in animal models.

Discovery of Heteroaryl Urea Isosteres for Formyl Peptide Receptor 2 Agonists.

Formyl peptide receptor 2 (FPR2) agonists have shown efficacy in inflammatory-driven animal disease models and have the potential to treat a range of diseases. Many reported synthetic agonists contain a phenylurea, which appears to be necessary for activity in the reported chemotypes. We set out to find isosteres for the phenylurea and focused our efforts on heteroaryl rings. The wide range of potencies with heterocyclic isosteres demonstrates how electronic effects of the heteroatom placement impact molecular recognition. Herein, we report our discovery of benzimidazole and aminophenyloxadiazole FPR2 agonists with low nanomolar activity.

Selective FPR2 Agonism Promotes a Proresolution Macrophage Phenotype and Improves Cardiac Structure-Function Post Myocardial Infarction.

Dysregulated inflammation following myocardial infarction (MI) leads to maladaptive healing and remodeling. The study characterized and evaluated a selective formyl peptide receptor 2 (FPR2) agonist BMS-986235 in cellular assays and in rodents undergoing MI. BMS-986235 activated G proteins and promoted ß-arrestin recruitment, enhanced phagocytosis and neutrophil apoptosis, regulated chemotaxis, and stimulated interleukin-10 and monocyte chemoattractant protein-1 gene expression. Treatment with BMS-986235 improved mouse survival, reduced left ventricular area, reduced scar area, and preserved wall thickness. Treatment increased macrophage arginase-1 messenger RNA and CD206 receptor levels indicating a proresolution phenotype. In rats following MI, BMS-986235 preserved viable myocardium, attenuated left ventricular remodeling, and increased ejection fraction relative to control animals. Therefore, FPR2 agonism improves post-MI healing, limits remodeling and preserves function, and may offer an innovative therapeutic option to improve outcomes.

Discovery of BMS-986235/LAR-1219: A Potent Formyl Peptide Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure.

Formyl peptide receptor 2 (FPR2) agonists can stimulate resolution of inflammation and may have utility for treatment of diseases caused by chronic inflammation, including heart failure. We report the discovery of a potent and selective FPR2 agonist and its evaluation in a mouse heart failure model. A simple linear urea with moderate agonist activity served as the starting point for optimization. Introduction of a pyrrolidinone core accessed a rigid conformation that produced potent FPR2 and FPR1 agonists. Optimization of lactam substituents led to the discovery of the FPR2 selective agonist 13c, BMS-986235/LAR-1219. In cellular assays 13c inhibited neutrophil chemotaxis and stimulated macrophage phagocytosis, key end points to promote resolution of inflammation. Cardiac structure and functional improvements were observed in a mouse heart failure model following treatment with BMS-986235/LAR-1219.

Biphenyl Acid Derivatives as APJ Receptor Agonists.

The APJ receptor and its endogenous peptidic ligand apelin have been implicated as important modulators of cardiovascular function, and APJ receptor agonists may be beneficial in the treatment of heart failure. In this article, we describe the discovery of a series of biphenyl acid derivatives as potent APJ receptor agonists. Following the identification of initial high-throughput screen lead 2, successive optimization led to the discovery of lead compound 15a. Compound 15a demonstrated comparable in vitro potency to apelin-13, the endogenous peptidic ligand for the APJ receptor. In vivo, compound 15a demonstrated a dose-dependent improvement in the cardiac output in male Sprague Dawley rats with no significant changes in either mean arterial blood pressure or heart rate, consistent with the hemodynamic profile of apelin-13 in an acute pressure volume loop model.

Preservation of Post-Infarction Cardiac Structure and Function via Long-Term Oral Formyl Peptide Receptor Agonist Treatment.

Dysregulated inflammation following myocardial infarction (MI) promotes left ventricular (LV) remodeling and loss of function. Targeting inflammation resolution by activating formyl peptide receptors (FPRs) may limit adverse remodeling and progression towards heart failure. This study characterized the cellular and signaling properties of Compound 43 (Cmpd43), a dual FPR1/FPR2 agonist, and examined whether Cmpd43 treatment improves LV and infarct remodeling in rodent MI models. Cmpd43 stimulated FPR1/2-mediated signaling, enhanced proresolution cellular function, and modulated cytokines. Cmpd43 increased LV function and reduced chamber remodeling while increasing proresolution macrophage markers. The findings demonstrate that FPR agonism improves cardiac structure and function post-MI.

Quantification of platelet composition in experimental venous thrombosis by real-time polymerase chain reaction.

INTRODUCTION: Platelets play a key role in thrombus formation. Determination of the platelet component in a thrombus provides pathophysiological insights to the thrombotic event and aids in selecting an appropriate therapeutic intervention. In this study a sensitive and reliable method to characterize the cellular components of experimental thrombi was developed using real-time polymerase chain reaction (PCR). METHODS AND RESULTS: Vena cava thrombosis was induced by either oxidative injury to topical FeCl(2) (FeCl(2)-VT) or stenosis-limited blood flow and a hypotonic pressure stress (stasis-VT) in rats. High levels of platelets were identified in the thrombus containing vessels by real-time PCR analysis of target gene amplification using the 2(-DeltaDeltaCT) values by normalizing the data with gene expression in naive vessels and with a housekeeping gene, ribosomal protein L32. By this analysis, the levels of PF-4 (as a platelet marker) mRNA were significantly higher in FeCl(2)-VT (2(-DeltaDeltaCT)=7.8) than in stasis-VT (2(-DeltaDeltaCT)=4.2, p<0.05). Enhanced platelet enrichment in FeCl(2)-VT was also confirmed qualitatively by scanning electronic microscopic analysis. In addition, real-time PCR using a panel of genes representing vascular injury, inflammation and thrombosis showed marked induction (2(-DeltaDeltaCT)>5) in MCP-1, IL-1beta, iNOS and P-selectin mRNA expression in both models. CONCLUSIONS: These data demonstrate the utility of real-time PCR to quantitate platelets and other cell components in vascular thrombosis, which may facilitate the characterization and thus therapeutic intervention of a particular thrombotic event in both preclinical animal models and clinical conditions.

Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI(-/-)) on FeCl(3)-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI(-/-) mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI(+/-)) mice in 3.5% FeCl(3)-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI(-/-) and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl(3)-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.