Discovery of an Orally Bioavailable Pan αv Integrin Inhibitor for Idiopathic Pulmonary Fibrosis.

The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvß3 and αvß5 significantly change the biological activities against αvß6 and αvß8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).

Targeting αvß3 and αvß5 integrins inhibits pulmonary metastasis in an intratibial xenograft osteosarcoma mouse model.

Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvß3 and αvß5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvß3 and αvß5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvß5 integrin is mainly found on tumor cells, whereas αvß3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvß3 and αvß5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease.

Nonpeptide alphavbeta3 antagonists. Part 11: discovery and preclinical evaluation of potent alphavbeta3 antagonists for the prevention and treatment of osteoporosis.

3-(S)-Pyrimidin-5-yl-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5e) and 3-(S)-(methylpyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5f) were identified as potent and selective antagonists of the alpha(v)beta(3) receptor. These compounds have excellent in vitro profiles (IC(50) = 0.07 and 0.08 nM, respectively), significant unbound fractions in human plasma (6 and 4%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in an in vivo model of bone turnover following once-daily oral administration, these two compounds were selected for clinical development for the treatment of osteoporosis.

Non-peptide alpha(v)beta(3) antagonists. Part 5: identification of potent RGD mimetics incorporating 2-aryl beta-amino acids as aspartic acid replacements.

A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements.

The role of alpha(v)beta3 integrins in vascular healing.

Alpha(v)beta3 integrins play an important role in vascular healing. Vascular injury is a stimulus for expression of alpha(v)beta3 by vascular cells and, among other effects, alpha(v)beta3 integrins function in the adhesion of activated platelets to endothelium. white cell/endothelium interactions, platelet-mediated thrombin generation, fibrin clot retraction by nucleated cells, smooth muscle cell (SMC) migration and proliferation, vascular cell apoptosis, and vascular remodeling. There are ten different animal models in which treatment with alpha(v)beta3 antagonists reduced the vascular response, including (neo)intima formation, after mechanical injury. These studies, along with mechanistic data derived from cell culture studies, provide compelling evidence that alpha(v)beta3 integrins are involved in vascular repair processes. The challenge is to develop a therapeutic agent that will prove effective in reducing restenosis in humans following percutaneous coronary intervention (PCI).

Synthesis and SAR of N-substituted dibenzazepinone derivatives as novel potent and selective alpha(V)beta(3) antagonists.

Synthesis and SARs of new integrin alpha(V)beta(3) antagonists based on an N-substituted dibenzazepinone scaffold are described. Variation of spacer and guanidine mimetic led to potent compounds exhibiting an IC(50) towards alpha(V)beta(3) in the nanomolar range, high selectivity versus integrin alpha(IIb)beta(3) and efficacy in functional cellular assays.

Nonpeptide alpha(v)beta(3) antagonists. Part 2: constrained glycyl amides derived from the RGD tripeptide.

Mimetics of the RGD tripeptide are described that are potent, selective antagonists of the integrin receptor, alpha(v)beta(3). The use of the 5,6,7,8-tetrahydro[1,8]naphthyridine group as a potency-enhancing N-terminus is demonstrated. Two 3-substituted-3-amino-propionic acids previously contained in alpha(IIb)beta(3) antagonists were utilized to enhance binding affinity and functional activity for the targeted receptor. Further affinity increases were then achieved through the use of cyclic glycyl amide bond constraints.

Non-peptide alpha(v)beta(3) antagonists. Part 3: identification of potent RGD mimetics incorporating novel beta-amino acids as aspartic acid replacements.

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.

Novel malonamide derivatives as alpha v beta 3 antagonists. Syntheses and evaluation of 3-(3-indolin-1-yl-3-oxopropanoyl)aminopropanoic acids on vitronectin interaction with alpha v beta 3.

In attempt to find novel integrin alphavbeta3 antagonists, we selected SC65811 and its guanidine analogue (1) as lead compounds. Modification of the glycine part of SC65811 led to a new series of malonamide derivatives that exhibited alphavbeta3 inhibitory activity. Among them, (R,S)-3-[3-[6-(3-benzylureido)indolin-1-yl]-3-oxopropanoylamino]-3- (pyridin-3-yl)propanoic acid (43a) showed not only potent activity with an IC50 value of 3.0 nM but also good selectivity for alphavbeta3 relative to alphaIIbbeta3, alpha5beta1, and alphavbeta5 with IC50 values of 19,000, 11,000, and 14 nM, respectively. Furthermore, optimization of 43a led to the most potent alphavbeta3 antagonist, (R,S)-3-(3-[6-[(4,5-dihydro-1H-imidazol-2-yl)amino]indolin-1-yl]-3-oxopropanoylamino)-3-(quinolin-3-yl)propanoic acid (431) with an IC50 value of 0.42 nM. The synthesis and the structure-activity relationships of these malonamide derivatives are presented.

Topical application of integrin antagonists inhibits proliferative retinopathy.

The expression of alphav-integrins is highly selective for angiogenic endothelial cells; ligation inhibition by cyclic RGD peptides prevents pathological neovascularization in tumor or retinopathy models to a large extent. We have previously demonstrated that proliferative retinopathy in a mouse model of retinopathy of prematurity (ROP model) can be reduced by more than 70%. To minimize systemic side effects and unwanted interference with responsive angiogenesis, we investigated topical application of cyclic RGD-peptides. In preliminary experiments, we could exclude any inhibiting effects of the carrier solution containing EDTA, Na2S, mannitol, hydroxyethyl starch, and benzalconium chloride on the inhibitory effect of cyclic RGD peptides. Retinal presence of small molecular-mass integrin antagonists after topical application was confirmed using fluorescein-labeled cyclic RGD peptide. Topical application of the peptide to the eye inhibited proliferative retinopathy in a dose-dependent fashion with a maximum of almost 50%. These results suggest that small molecular-mass peptide antagonists of alphav-type integrins are efficient in inhibiting proliferative retinopathy by topical application.

Disease-modifying activity of SB 273005, an orally active, nonpeptide alphavbeta3 (vitronectin receptor) antagonist, in rat adjuvant-induced arthritis.

OBJECTIVE: To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin avbeta3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA were orally dosed either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. RESULTS: SB 273005 is a potent antagonist of the closely related integrins, avbeta3 (Ki = 1.2 nM) and alphavbeta5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. CONCLUSION: Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the alphavbeta3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.

Reduction in atherosclerotic lesion size in pigs by alphaVbeta3 inhibitors is associated with inhibition of insulin-like growth factor-I-mediated signaling.

Insulin-like growth factor-I (IGF-I) is a potent stimulant of smooth muscle cell (SMC) migration and proliferation and has been implicated in the development of experimental atherosclerotic lesions. Because optimal stimulation of SMC in vitro by IGF-I requires ligand occupancy of alphaVbeta3, these studies were conducted to determine whether alphaVbeta3 antagonists would result in a change in lesion size and whether they could alter IGF-I-mediated actions. Clamps were placed on the carotid and femoral arteries of normal pigs that had been fed a high-cholesterol diet for 4 weeks. alphaVbeta3 inhibitors (SC-69000, SC-65811) (10(-6) mol/L) or saline were infused for 2 weeks into the peristenotic area. Lesion area, the number of SMC layers, and proliferating cell nuclear antigen positive cells were determined in a 1.2-mm segment of each artery. Lesion areas were 304 788+/-113 453 micron(2) (saline), compared with 149 799+/-35 456 micron(2) (SC-69000) (P<0.01). Lesion areas in arteries treated with SC-64258, a compound that does not bind to alphaVbeta3, were 310 284+/-160 467 micron(2), P=not significant. In a second experiment, lesion areas were 110 391+/-17 347 micron(2) (saline) and 59 533+/-17 568 micron(2) (SC-65811, P<0.001). Neointimal SMC layers were reduced by SC-65811 from 7.4+/-4.5 to 3.0+/-0.4 (P<0.001). To determine whether IGF-I action was altered, IGF binding protein-5, which is synthesized in response to IGF-I, was analyzed. IGF-I binding protein-5 mRNA abundance was reduced by 67+/-8% in the 6 lesions treated with SRL-69000 compared with saline controls (P<0.001). We conclude that alphaVbeta3 antagonists block the development of lesions in pigs that have been induced by a high-cholesterol diet and stenosis, and the effect of these compounds is associated with their ability to inhibit IGF-I-mediated signaling.

Dietary coenzyme Q10 supplementation alters platelet size and inhibits human vitronectin (CD51/CD61) receptor expression.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10, but a comprehensive explanation of its cardioprotective properties is still lacking. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet size and surface antigens was examined in human volunteers. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days. Receptor expression was measured by flow cytometry with monoclonal murine anti-human antibodies CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin). An increase of total serum CoQ10 level (from 0.6 +/- 0.1 to 1.8 +/- 0.3 micrograms/ml; p < 0.001) was found at protocol termination. Fluorescence intensity was higher for the large platelets when compared with the whole platelet population. Significant inhibition of vitronectin-receptor expression was observed consistently throughout ubiquinone treatment. Reduction of platelet size was observed at the end of CoQ10 supplementation. Inhibition of the platelet vitronectin receptor and a reduction of the platelet size are direct evidence of a link between dietary CoQ10 intake and platelets. These findings may not be fully explained by the known antioxidant and bioenergetic properties of CoQ10. Diminished vitronectin-receptor expression and reduced platelet size resulting from CoQ10 therapy may contribute to the observed clinical benefits in patients with cardiovascular diseases.

Neointima formation in injured hamster carotid artery is effectively prevented by the combination G4120 and quinapril.

The prevention of neointima formation by the tissue selective angiotensin converting enzyme (ACE) inhibitor quinapril and by the combination quinapril/G4120 (a platelet alpha Ub beta 3 and smooth muscle cell alpha v beta 3 antagonist) was investigated in a hamster carotid artery injury model. Quinapril at 10 mg/kg/day reduced neointima formation by about 45%, 1 and 2 weeks after injury to the artery, i.e. significantly better than the non-tissue selective ACE inhibitor captopril at 100 mg/kg/day. Quinapril did not decrease the early smooth muscle cell (SMC) proliferation in the media, but in agreement with its inhibition of the carotid artery ACE activity by 62%, SMC proliferation was reduced by 70% in the newly forming intima. To improve the inhibition of early medial SMC proliferation, quinapril (10 mg/kg/day) was complemented with G4120 (100 micrograms/kg/h). This combined treatment reduced the proliferation of medial SMCs to about 50% throughout the first week following injury, whereas intima SMC proliferation was reduced by 70% throughout treatment. Accordingly, the drug combination reduced neointima formation more potently than each drug separately by 70%. The disruption of medial elastic laminae, observed in the control and G4120 treated group, was consistently reduced when G4120 was complemented with quinapril. Thus, the present study shows in a hamster model of carotid artery injury, that combining drugs that prevent SMC migration and proliferation via different modes of action can lead to the effective prevention of neointima formation.