The potential role of alpha 5 beta 1 (VLA-5) in leukocyte trafficking in zymosan-induced acute peritonitis was determined. In naive mice, similar to 98% of Gr1(high) cells (PMN) in bone marrow and circulation were alpha 5 beta 1-negative; these profiles were modestly affected by peritoneal injection of zymosan. In contrast, similar to 30% of Gr1(high) cells recruited by zymosan (24 h) to the peritoneal cavity expressed alpha 5 beta 1. With respect to F4/80(+) cells, similar to 60% of bone marrow and peripheral blood populations expressed alpha 5 beta 1, with similar to 90% positivity in resident cells of noninflamed peritoneum. Analysis of alpha 5 beta 1 expression revealed inflammation-dependent increased expression on Gr1(high) and F4/80(+) cells in bone marrow, blood, and peritoneal cavity. Blockade of alpha 5 beta 1, by an anti-alpha 5 mAb, attenuated zymosan-induced 24 h recruitment of Gr1(high) and F4/80(+) cells. At least one underlying mechanism of this action was reduction of cell adhesion and transmigration across microvascular vessels, as revealed by intravital microscopy. Confocal analyses indicated that deposition of fibronectin, the principal ligand for alpha 5 beta 1, was up-regulated significantly on and around the inflamed mesenteric microvasculature. These data suggest that the effects of alpha 5-blockade may be a result of inhibition of alpha 5 beta 1-dependent leukocyte adhesion to and migration along the fibronectin matrix. This is the first report that identifies a functional role for alpha 5 beta 1 in leukocyte trafficking during acute inflammation. J. Leukoc. Biol. 87: 877-884; 2010.

PURPOSE:To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model.METHODS:Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections.RESULTS:Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment.CONCLUSIONS:JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.

The rearrangement of asparagine to isoaspartate (isoD) is responsible for the deactivation of many functional proteins. However, the isoDGR motif, which is optimally presented as a conformationally controlled cyclic pentapeptide, binds selectively to α5β1 integrin (see the docking model) with an affinity comparable to that of the peptidic antitumor agent Cilengitide. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Recently, a new class of selective integrin alpha5beta1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.

OBJECTIVE:To evaluate the pharmacologic activity and tolerability of JSM6427, a potent and first selective small-molecule inhibitor of integrin alpha5beta1, in monkey and rabbit models of choroidal neovascularization (CNV).METHODS:JSM6427 selectivity for alpha5beta1 was evaluated by in vitro binding assays while the ability of JSM6427 to inhibit CNV was investigated in a laser-induced monkey model and a growth factor-induced rabbit model. Intravitreal injections of JSM6427 (100, 300, or 1000 microg) or vehicle were administered immediately after the CNV induction procedure and at weekly intervals for 4 weeks. Fluorescein angiography was performed weekly. Ocular tolerability was evaluated ophthalmoscopically and histologically in both models; additional assessments in monkeys included electroretinography, biomicroscopy, pathological examination, and analysis of JSM6427 pharmacokinetics.RESULTS:JSM6427 was highly selective for the alpha5beta1-fibronectin interaction. Weekly intravitreal injections of JSM6427 resulted in a statistically significant dose-dependent inhibition of CNV in laser-induced and growth factor-induced models without any ocular JSM6427-related adverse effects. JSM6427 was cleared through the systemic circulation with no evidence of systemic accumulation.CONCLUSIONS:Intravitreal JSM6427 provided dose-dependent inhibition of CNV in monkey and rabbit experimental models.CLINICAL RELEVANCE:JSM6427 may provide a new approach for the treatment of ocular neovascular diseases such as age-related macular degeneration in humans.

The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.

PURPOSE:The aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution. METHODS:Expression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin. RESULTS:Integrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. CONCLUSIONS:The apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin alpha 5 beta 1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin alpha 5 beta 1 inhibitor scaffolds for potential systemic treatment is described. (C) 2009 Elsevier Ltd. All rights reserved.

Integrins are heterodimeric transmembrane proteins, which mediate cell-cell and cell-extracellular matrix (ECM) interaction. We show, that an inhibitor of alpha5 beta1 integrin (alpha5beta1), JSM6427, attenuated glioma growth and decreased the density of microglia at the tumor border. 21 days after glioma cell injection into an experimental mouse model, the tumor volume was significantly smaller after treating animals for 14 days with JSM6427 as compared to controls. We could demonstrate the expression of integrin alpha5beta1 on both microglia and glioma cells using flow cytometry. In a slice culture we could compare glioma growth in the presence and absence of microglia. Slices injected with glioma cells were treated with the integrin inhibitor JSM6427 and showed a significant reduction in tumor size as compared to control. Depleting microglial cells from the slice culture by treatment with clodronate liposomes abrogated the effect of JSM6427 on glioma invasion indicating that the presence of microglia is required. We show further, that microglial migration, and proliferation was attenuated dose-dependently by JSM6427.

Starting from the structure of integrin alpha v beta 3 in a complex with a peptidic ligand plus SAR data on nonpeptidic ligands, we derived a new class of integrin alpha 5 beta 1 antagonists (1). Several synthesis strategies were applied to evaluate the chemical space around the essential pharmacophore groups R-1 to R-3 to obtain highly active and selective pyrrolidine derivatives as integrin alpha 5 beta 1 antagonists. Integrin selectivity was controlled by switching from a sulfonamide moiety to a mesitylene amide moiety for R3. This finding represents a general feature for modulating selectivity toward other related integrin receptors. On the basis of the encouraging results from various in vitro studies, the most active compounds were selected for further in vivo studies in animal models and preclinical development.

Integrins are transmembrane receptor proteins critical for growth and stabilization of vessels, but the mechanisms by which integrin activities are involved in neoangiogenesis of the eye remain unclear. Specific inhibitors to fibronectin receptor integrin α5β1 impeded pathological neovascularization in vivo. Our objective was to determine whether α5β1 plays a role in ocular angiogenesis, and whether a novel α5β1-inhibiting small molecule is able to reduce angiogenesis in a model of inflammatory corneal neovascularization. Corneal neovascularization was induced in C57Bl/6 mice by NaOH-application and debridement of the limbal epithelium. Mice were randomized into six groups receiving either no treatment, or intraperitoneal osmotic pumps delivering three different doses of integrin antagonist or control substance on day 10 after scraping. In order to quantify the neovascular response, flatmounts were stained with FITC-CD31. Integrin α5 expression was determined by immunohistochemistry and quantified by semiquantitative western blot analysis. Influence of integrin antagonist treatment on the mRNA expression of VEGF, bFGF and integrin α5 was quantified by real-time RT–PCR. Vascularized corneas demonstrated a strong up-regulation of integrin α5 within affected areas. Animals treated systemically with α5β1-inhibiting small molecule showed a significant inhibition and regression of corneal neovascularization. PCR analysis evinced a significant up-regulation of VEGF and integrin α5 mRNA levels in injured animals compared to controls, and a significant reduction of integrin α5 mRNA in substance-treated animals compared to control substance, but no significant differences of bFGF levels in all groups. Western blot analysis of integrin α5β1 protein expression showed a trend towards up-regulation in injured animals, both control substance-treated and those treated with the α5β1-inhibiting small molecule. Systemic delivery of an α5β1-inhibiting small molecule inhibits and regresses corneal neovascularization induced by mechanical-alkali burn corneal injury. These results suggest an essential role for the integrin α5β1 in pathological neovascular processes of the cornea. Integrin α5β1 inhibitors could become a new approach for treatment of neovascularization in the eye.

PURPOSE:Integrin alpha5beta1, a fibronectin receptor, is involved in endothelial cell migration and proliferation. Here we investigate the effect of JSM6427, an integrin alpha5beta1 inhibiting molecule, on the development of retinal vascular system using the mouse model of oxygen-induced retinopathy (OIR).METHODS:Endothelial cell migration and sprouting was analyzed in vitro using a 2D migration assay and a 3D sprouting/angiogenesis assay in fibrin gel. C57BL/C6 mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12 and returned to room air thereafter. Intravitreal injection of 40 microg JSM6427 was performed in each one eye on P14. On P17, vascular area, avascularized area, and neovascular blood vessel tufts were quantified after perfusion with fluorescein-coupled concanavalin A. The number of retinal neovascular cell nuclei was determined in hematoxylin-stained cross sections of the eyes. Integrin alpha 5 expression was determined by immunohistochemistry.RESULTS:In vitro, JSM6427 inhibits the migration of HUVEC and the tube formation induced by both bFGF and VEGF. In vivo, integrin alpha 5 expression was detectable in neovascular retinal blood vessels. Oxygen treatment (positive control) in comparison with no oxygen treatment (negative control) reduced significantly the vascularized area and increased the avascularized area. A single intravitreal injection of 40 microg JSM6427 resulted in a significant reduction of the vascularized area and the number of preretinal nuclei in comparison with the intravitreal injection of the vehicle while the avascularized area increased significantly.CONCLUSIONS:These results imply an essential role of integrin alpha5beta1 in the refining of the retinal vasculature in OIR and suggest JSM6427 may have a possible therapeutic function for neovascular disease.

B266 The fibronectin receptor alpha5beta1 integrin has been suggested to be a promising oncology target. However, proof-of-concept studies have so far been limited to function-blocking alpha5beta1 antibodies and non-selective peptide antagonists. Jerini AG was first to develop highly potent, orally available small molecule compounds that specifically inhibit alpha5beta1 integrin at sub-nanomolar IC50 values in an ELISA assay. These compounds were shown to affect a variety of oncogenic and metastatic processes in vitro. In particular, they inhibit adhesion of alpha5beta1-positive tumor cells (e.g. K562) to fibronectin at low nanomolar concentrations. Furthermore, the alpha5beta1 integrin antagonists diminish endothelial cell proliferation, migration, sprouting and alpha5beta1 integrin downstream signaling events like Erk phosphorylation. Different alpha5beta1 antagonists were used as a tool to demonstrate in vivo proof-of-concept in a variety of rodent tumor models. As a result, the compounds have significant invivo efficacy in multiple mouse tumor models. For example, they inhibit growth of HT29, A549, MDA-MB-231 and BxPC3 tumors in a dose-dependent manner by 50% to 85%. Besides, the compounds prolong animal survival in the clinically relevant, orthotopic rat tumor model 13762NF MTLn3 by significantly reducing lung metastasis. The alpha5beta1 integrin antagonists exert their effects by targeting of multiple oncogenic processes in vivo including tumor angiogenesis, tumor cell proliferation and metastasis. Treatment with Jerini’s development candidate is well tolerated. No effect on body weight gain and no adverse events were observed at tested doses of up to 200 mg/kg/day. Taken together, we are presenting sound evidence for the potential of selective alpha5beta1 antagonists as efficacious and safe treatment for multiple tumor indications.

Integrin alpha(5)beta(1) plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of alpha(5)beta(1) in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 microg/h ( approximately 1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective alpha(5)beta(1) antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 microg/h of JSM6427 was implanted 7 days after rupture of Bruch's membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in alpha(5)beta(1)-expressing cells that is blocked by JSM6427. These data suggest that alpha(5)beta(1) plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.