Design and Discovery of a Potent and Selective Inhibitor of Integrin αvß1.

Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvß1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvß1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvß1 inhibition.

Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

ATN-161 Ameliorates Ischemia/Reperfusion-induced Oxidative Stress, Fibro-inflammation, Mitochondrial damage, and Apoptosis-mediated Tight Junction Disruption in bEnd.3 Cells.

We have previously demonstrated the significance of endothelial cell-expressed α5ß1 integrin in ischemic stroke, having shown that α5ß1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood-brain barrier (BBB). In addition, inhibition of α5ß1 by the small peptide noncompetitive integrin α5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of α5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 µM) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin α5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting α5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.

Dual integrin αvß3 and αvß5 blockade attenuates cardiac dysfunction by reducing fibrosis in a rat model of doxorubicin-induced cardiomyopathy.

OBJECTIVE: The present study aimed to evaluate the protective role of cilengitide (CGT), an integrin αvß3 and αvß5 inhibitor, on doxorubicin (DOX)-induced myocardial fibrosis and cardiac dysfunction in a rat model. Methods. Forty male rats were randomly divided into four groups: DOX (n = 12), intraperitoneal (i.p.) injection of DOX 0.8 ∼ 1.0 mg/kg three times a week for up to 6 weeks, then saline i.p. three times a week for another 3 weeks; CGT (n = 8), CGT 10 mg/kg, i.p. three times a week for 9 weeks; DOX + CGT (n = 12), DOX and CGT co-administration as above for 6 weeks, then CGT alone for another 3 weeks; Control (n = 8), saline i.p. three times a week for 9 weeks. Echocardiography, serum procollagen I C-terminal propeptide (PICP) procollagen III N-terminal propeptide (PIIINP) and C telopeptide type I (CTX-I) were evaluated at baseline and 3, 6 and 9 weeks after initial DOX administration for all surviving rats. The heart tissues were then harvested for myocardial hydroxyproline (HYP) evaluation, qRT-PCR, and western blotting. Results. CGT attenuated DOX-induced eccentric remodeling by improving relative wall thickness at the 9th week. CGT also improved systolic function at the 9th week and diastolic function at the 6th and 9th week. CGT reduced myocardial HYP and serum PICP, PIIINP, CTX-I, and the PICP/PIIINP ratio. RT-PCR and western blot showed that CGT blocked the TGF-ß1/SMAD3 pathway and mitigating extracellular matrix turnover. Conclusions. CGT exerted a cardioprotective effect against doxorubicin-induced fibrosis and improved cardiac function.

Convallatoxin Promotes M2 Macrophage Polarization to Attenuate Atherosclerosis Through PPARγ-Integrin αvß5 Signaling Pathway.

INTRODUCTION: As the primary immune cells, macrophages play a key role in atherosclerotic progression. M2 macrophage polarization has been reported to promote tissue repair and attenuate plaque formation upon the expression of anti-inflammatory factors. Convallatoxin (CNT) is a natural cardiac glycoside with anti-inflammatory pharmacological properties. However, whether CNT protects against atherosclerosis (AS) and underlying mechanisms is unknown. This work was designed to explore the potential effects of CNT on atherosclerosis. METHODS: In this study, Apolipoprotein E deficiency (ApoE-/-) mice fed with high-fat diet were established, and CNT (50 or 100 µg/kg) were intragastrically administrated for 12 weeks every day. In vitro, RAW264.7 macrophages stimulated with ox-LDL were treated with CNT (50 or 100 nM) for 24 h. The specific PPARγ antagonist, GW9662, was used to block the PPARγ signaling pathway in vitro. Then, the atherosclerotic lesions, macrophage polarization markers, inflammatory cytokines and PPARγ signaling pathway were examined in further examinations. RESULTS: Our results showed that the atherosclerotic lesions were reduced by CNT, as demonstrated by the downregulation of serum lipid level and aortic plaque area in AS mice. Furthermore, we found that CNT treatment promoted the expression of M2 macrophage markers (Arg1, Mrc1, Retnla and Chi3l3), and decreased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), accompanied by the increase of anti-inflammatory factor (IL-10) in aortic vessels of AS mice. In ox-LDL-induced RAW264.7 cells, CNT administration also facilitated macrophages polarizing towards M2 subtype and inhibited inflammatory responses. Furthermore, both the in vivo and in vitro experiments showed CNT could increase the expression of PPARγ, Integrin αv and Integrin ß5, and GW9662 could block CNT-induced M2 macrophage polarization. CONCLUSION: Taken together, these data suggest that CNT may promote M2 macrophage polarization to exert an anti-atherosclerotic effect, partially through activating PPARγ-Integrin αvß5 signaling pathway.

Vitronectin-activated αvß3 and αvß5 integrin signalling specifies haematopoietic fate in human pluripotent stem cells.

OBJECTIVES: Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder-free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs. MATERIALS AND METHODS: A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN-mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells. RESULTS: We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial-haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvß3 and αvß5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking αvß3 and αvß5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both αvß3 and αvß5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, ß3 or ß5. CONCLUSION: The established VTN-based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.

Dual antagonists of α5ß1/αvß1 integrin for airway hyperresponsiveness.

Inhibition of integrin α5ß1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin α5ß1 by backbone replacement of known αvß1 integrin inhibitors. These integrin α5ß1 inhibitors also retain the nanomolar potency against αvß1 integrin, which shows promise for developing dual integrin α5ß1/αvß1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin αvß3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin α5ß1 inhibitors as potential novel asthma therapeutics.

Discovery of the first potent and selective αvß5 integrin inhibitor based on an amide-containing core.

Integrins αvß5 and αvß3 are closely related, proangiogenic members of the wider RGD-binding integrin family. Due to their high sequence homology, the development of αvß5-selective compounds has remained elusive to synthetic and medicinal chemists. Herein, we describe a survey of SAR around a series of amide-containing 3-aryl-succinamic acid-based RGD mimetics. This resulted in the discovery of α,α,α-trifluorotolyl 12 which exhibits 800 × selectivity for αvß5versus αvß3 with a pyrrolidine amide linker that confers selectivity for αvß5 by positioning a key aryl ring in the SDL of αvß5 with good complementarity; binding in this mode is disfavoured in αvß3 due to clashes with key residues in the ß3-subunit. Compound 12 exhibits selective inhibition by a cell adhesion assay, high passive permeability and solubility which enables potential use of this inhibitor as an αvß5-selective in vitro tool compound.

Selective Targeting of αvß5 Integrin in HepG2 Cell Line by RGDechi15D Peptide.

Recently, the research community has become increasingly concerned with the receptor αvß5, a member of the well-known integrin family. Different ongoing studies have evidenced that αvß5 integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. Remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. In this scenario, the availability of a selective αvß5 antagonist without 'off-target' protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. We recently reported a cyclic peptide, RGDechi15D, obtained by structure-activity studies. To our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvß5 integrin and not cross react with αvß3. Here we demonstrated the ability of the peptide to diminish both adhesion and invasion of HepG2 cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the PI3K pathway. The peptide, also decreases the formation of new vessels in endothelial cells. Taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma.

Redox-Responsive Polymeric RNAi Based on Multivalent Conjugation of siRNA for Improved Intracellular Delivery.

Learning from the design concept of antibody-drug conjugates (ADCs), we attempted to construct siRNA conjugated polymer brush by attaching a multiple of siRNA to the units of poly(amino acids) [poly(lysine) derivatives] through an intracellular cleavable disulfide bond. Note that the disulfide linkage is stable at extracellular milieu yet subjected to cleavage into free thiol residues at the intracellular reducing compartments. Consequently, ready release of arrays of active siRNA was achieved selectively in the intracellular compartments. Furthermore, tumor-targeted cyclic Asp-Gly-Arg (RGD) was conjugated to the aforementioned polymer brush in view that the RGD receptors (αVß3 and αVß5 integrins) were overexpressed over a wide spectrum of cancerous cells. Our subsequent results have achieved potent gene silencing in cultured cancerous cells from our proposed siRNA delivery construct. To our best knowledge, our proposed conjugate should be the first example of using an ADC platform in successful intracellular transportation of larger macromolecular biological payloads rather than small molecular chemotherapeutic drugs. Hence, the proposed strategy may serve as a promising avenue for targeted delivery of macromolecular pharmaceutical payloads.

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%).

Different epidermal growth factor receptor signaling pathways in neurons and astrocytes activated by extracellular matrix after spinal cord injury.

Spinal cord injury (SCI) is a serious central nervous system (CNS) trauma that results in permanent and severe disability. The extracellular matrix (ECM) can affect the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) by interacting with the ERK integrin subunits. In this study, we built a model of SCI with glial fibrillary acidic protein-green fluorescent protein (GFAP-GFP) and thymus cell antigen 1-yellow fluorescent protein-H (Thy1-YFPH) in mice that express specific transgenes in their astrocytes or neurons. Then, we collected spinal cord neurons or astrocytes by fluorescence-activated cell sorting (FACS). In this way, we investigated the SCI-induced phosphorylation of ERK1/2 and epidermal growth factor receptor (EGFR) in neurons and astrocytes, and we discovered that the SCI-induced EGFR signaling pathways differed between neurons and astrocytes. In the present study, we found that the Src-dependent phosphorylation of EGFR induced by SCI occurred only in neurons, not in astrocytes. This phenomenon may be due to the involvement of Thy-1, which promoted the binding between Src and EGFR in neurons after SCI. In addition, the expression of the integrin subunits after SCI differed between neurons and astrocytes. Our present study shows that the EGFR signaling pathway triggered by SCI in neurons differed from the EGFR signaling pathway triggered in astrocytes, a finding that may help to pave the way for clinical trials of therapies that inhibit EGFR signaling pathways after SCI.

The Design of Potent, Selective and Drug-Like RGD αvß1 Small-Molecule Inhibitors Derived from non-RGD α4ß1 Antagonists.

Up to 45 % of deaths in developed nations can be attributed to chronic fibroproliferative diseases, highlighting the need for effective therapies. The RGD (Arg-Gly-Asp) integrin αvß1 was recently investigated for its role in fibrotic disease, and thus warrants therapeutic targeting. Herein we describe the identification of non-RGD hit small-molecule αvß1 inhibitors. We show that αvß1 activity is embedded in a range of published α4ß1 (VLA-4) ligands; we also demonstrate how a non-RGD integrin inhibitor (of α4ß1 in this case) was converted into a potent non-zwitterionic RGD integrin inhibitor (of αvß1 in this case). We designed urea ligands with excellent selectivity over α4ß1 and the other αv integrins (αvß3, αvß5, αvß6, αvß8). In silico docking models and density functional theory (DFT) calculations aided the discovery of the lead urea series.

Integrin ανß5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats.

BACKGROUND: To date the TGF-ß1 activation mediated by integrin ανß5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανß5 inhibition in MyoFB differentiation. METHODS: Staining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation. RESULTS: SHR heart tissues revealed a higher TGF-ß1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-ß1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανß5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανß5 expression in SHR-CF at basal condition and after TGF-ß1 treatment, in comparison with WKY-CF. Inhibition of integrin ανß5 by cilengitide treatment led a decreased expression of ανß5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-ß1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανß5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανß5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF. CONCLUSION: Inhibition of integrin ανß5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανß5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

An RGD small-molecule integrin antagonist induces detachment-mediated anoikis in glioma cancer stem cells.

The malignancy of glioblastoma (GB) is primarily due to the ability of glioma cancer stem cells (GSC) to disseminate into surrounding brain tissues, despite surgery and chemotherapy, and to form new tumoral masses. Members of the RGD-binding integrin family, which recognize the arginine-glycine-aspartic acid (RGD) sequence present in components of the extracellular matrix, and which serve a crucial function in the dissemination of GCS, are overexpressed in GB. Small-molecule integrin antagonists (SMIAs) designed to recognize RGD-integrins may therefore be an effective tool for decreasing GB infiltration and recurrence. In the present study, in vitro pro-apoptotic and infiltrative effects elicited by the SMIA 1a­RGD in human GSC were investigated. Reverse transcription-quantitative polymerase chain reaction analysis revealed that, compared with normal human astrocytes, GSC grown on laminin-coated dishes overexpressed stemness markers as well as αvß3 and αvß5 integrins. In addition, dissociated GSC were identified to exhibit tumorigenic capacity when injected into immunodeficient mice. Using annexin/fluorescence-activated cell sorting analysis and ELISA nucleosome assays, it was identified that treatment of GSC with 25 µM 1a­RGD for 48 h elicited detachment­dependent anoikis not accompanied by necrosis-dependent cell death. A colorimetric proliferation assay indicated that 1a­RGD did not affect cell viability, but that, instead, it markedly inhibited GSC migration as assessed using a Transwell assay. Western blot experiments revealed a decrease in focal adhesion kinase and protein kinase B phosphorylation with a concomitant increase in caspase-9 and -3/7 activity following 1a­RGD treatment, suggesting that the pro-anoikis effects of 1a­RGD may be mediated by these molecular mechanisms. Western blot analysis revealed no changes in specific markers of autophagy, suggesting further that 1a­RGD-induced cell death is primarily sustained by anoikis-associated mechanisms. In conclusion, the results of the present study indicate that SMIA have potential as a therapeutic tool for decreasing GSC dissemination.

Non-Covalently Pre-Assembled High-Performance Near-Infrared Fluorescent Molecular Probes for Cancer Imaging.

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvß3 or αvß5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.

Continuous Infusion of Cilengitide Plus Chemoradiotherapy for Patients With Stage III Non-Small-cell Lung Cancer: A Phase I Study.

INTRODUCTION: Because of our previous preclinical results, we conducted a phase I study associating the specific αvß3/αvß5 integrin inhibitor cilengitide, given as a continuous infusion, with exclusive chemoradiotherapy for patients with stage III non-small-cell lung cancer. PATIENTS AND METHODS: A standard 3+3 dose escalation design was used. Cilengitide was given as a continuous infusion (dose levels of 12, 18, 27, and 40 mg/h), starting 2 weeks before and continuing for the whole course of chemoradiotherapy (66 Gy combined with platinum/vinorelbine), and then at a dose of 2000 mg twice weekly in association with chemotherapy. 2-Deoxy-2-[fluorine-18]fluoro-d-glucose positron emission tomography (PET) and computed tomography scans were performed before and after the first 2 weeks of cilengitide administration and then every 3 months. RESULTS: Of the 14 patients included, 11 were evaluable for evaluation of the dose-limiting toxicities (DLTs). One DLT, a tracheobronchial fistula, was reported with the 40 mg/h dose. No relevant adverse events related to cilengitide were observed overall. At the PET evaluation 2 months after chemoradiotherapy, 4 of 9 patients had a complete response and 4 had a partial response. The median progression-free and overall survival was 14.4 months (95% confidence interval [CI], 8.4 to not reached) and 29.4 months (95% CI, 11.73 to not reached), respectively. CONCLUSION: Cilengitide, given continuously with chemoradiotherapy, showed acceptable toxicity and gave encouraging clinical results.

Dual αvß3 and αvß5 blockade attenuates fibrotic and vascular alterations in a murine model of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibroblast activation and fibrosis of the skin and internal organs. Alterations in cell-integrin interaction are sufficient to initiate profibrotic processes. SSc fibroblasts express both αvß3 and αvß5 integrins and their activation induces myofibroblasts differentiation. The aim of the present study was to evaluate the effect of the anb3 and anb5 inhibitor, cilengitide, on the development of vascular and fibrotic changes in the chronic oxidant stress murine model of systemic sclerosis. SSc was induced in BALB/c mice by daily s.c. injections of HOCl for 6 weeks. Mice were randomized in three arms: HOCl alone (n=8), HOCl + Cilengitide (n=8), or Vehicle alone (n=8). Treatment with cilengitide 20 (mg/kg/i.p./day) was started 4 weeks after the first administration of HOCl and maintained throughout the remaining experimental period (2 weeks). Lung, skin, and heart fibrosis were evaluated by histology while kidney morphology by PAS staining. Collagen type I, focal adhesion kinase (FAK), and a-SMA were evaluated by immunostaining and p-FAK and TGF-ß1 by Western blot and gene expression. Both cutaneous and pulmonary fibrosis induced by HOCl were attenuated by cilengitide treatment. Cilengitide administration reduced a-SMA, TGF-ß1, and p-FAK expression and the increased deposition of fibrillar collagen in the heart and prevented glomeruli collapse in the kidneys. The inhibition of aνß3 and aνß5 integrin signaling prevented systemic fibrosis and renal vascular abnormalities in the reactive oxygen species model of SSc. Integrins aνß3 and aνß5 could prove useful as a therapeutic target in SSc.

High-Affinity RGD-Knottin Peptide as a New Tool for Rapid Evaluation of the Binding Strength of Unlabeled RGD-Peptides to αvß3, αvß5, and α5ß1 Integrin Receptors.

We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αvß3 but also detectable for both αvß5 and α5ß1, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αvß3. For assay validation, we finally determined IC50 values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αvß3, αvß5, α5ß1). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.

A phase I study of cilengitide and paclitaxel in patients with advanced solid tumors.

PURPOSE: Cilengitide is a potent and selective inhibitor of the integrins αvß3 and αvß5. The primary objective of this phase I clinical trial was to establish the maximum tolerated dose and determine safety/tolerability of cilengitide in combination with paclitaxel in patients with advanced solid tumors. Secondary objectives included the evaluation of the preliminary clinical outcomes. PATIENTS AND METHODS: Patients with advanced solid tumors experiencing disease progression on standard treatment were assigned to two different dose levels of cilengitide (2000 mg intravenously once or twice weekly) in combination with fixed-dose, weekly paclitaxel (90 mg/m2 intravenously). RESULTS: Twelve evaluable patients were treated per protocol. A single dose limiting toxicity (DLT) of grade 4 neutropenia was observed at the starting dose level of once weekly cilengitide. There were no grade ≥3 adverse events that occurred with >10% frequency. One patient achieved a partial response to therapy. Five patients experienced stable disease as best response, 3 of which discontinued study participation due to progressive, peripheral neuropathy. CONCLUSIONS: Cilengitide in combination with paclitaxel was well tolerated. Antitumor activity was observed. The recommended phase II dose is twice weekly cilengitide (2000 mg) with weekly paclitaxel (90 mg/m2). Further studies evaluating drugs that target this pathway are warranted.