αvβ3 (IC50 = 4 nM, αvβ3-Vitronectin interaction); αvβ5 (IC50 = 79 nM, αvβ5-Vitronectin interaction); αvβ3 (IC50 = 0.61 nM); αvβ5 (IC50 = 8.4 nM); α5β1 (IC50 = 14.9 nM); STAT3

An antagonist of the αvβ3 and αvβ5 integrin receptor is cilengitide (EMD 121974). In investigations assessing cell adhesion in human lung cancer cell lines UCLA-P3 or melanoma M21, respectively, clengitide reduced integrin-mediated binding to vitronectin with an IC50 of 0.4 and 0.4 μM [1]. In vitro, cilengitide demonstrates concentration- and time-dependent cytotoxic effects at concentrations higher than 1 μM [2].

Cilenegitide (10, 50, and 250 μg) was injected intraperitoneally three times a week into nude mice with M21-L melanoma tumors; the doses were demonstrated to suppress tumor growth and concurrently reduce tumor volume (55%, 75%, and 55%, respectively). Tumor weight (23%, 38%, and 61%, respectively) and 89% [2]. ILP administered with cilengitide alone, ILP administered with cilengitide plus melphalan, TNF, or both did not influence the systemic pharmacokinetics of cilengitide administered intraperitoneally in the rat model under study. After 10 minutes of intraperitoneal treatment, systemic levels of cilengitide reach about 20 μg/mL (about 35 μM) and keep rising to about 40 μg/mL (about 70 μM) within the first hour. After then, there is a 2.1-hour elimination half-life for serum levels of celibitide [3].

Integrin Binding Assay[Sci Rep. 2017 Jan 11;7:39805.]
The activity and selectivity of integrin ligands were determined by a solid-phase binding assay according to the previously reported protocol using coated extracellular matrix proteins and soluble integrins. The following compounds were used as internal standards: Cilengitide, c(RGDf(NMe)V) (αvβ3–0.54 nM, αvβ5–8 nM, α5β1–15.4 nM), linear peptide RTDLDSLRT4 (αvβ6–33 nM; αvβ8–100 nM) and tirofiban5 (αIIbβ3–1.2 nM).
Flat-bottom 96-well ELISA plates were coated overnight at 4 °C with the ECM-protein (1) (100 μL per well) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4; 3 × 200 μL) and blocked for 1 h at room temperature with TS-B-buffer (Tris-saline/BSA buffer; 150 μL/well; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA). In the meantime, a dilution series of the compound and internal standard is prepared in an extra plate, starting from 20 μM to 6.4 nM in 1:5 dilution steps. After washing the assay plate three times with PBS-T (200 μL), 50 ul of the dilution series were transfered to each well from B–G. Well A was filled with 100 ul TSB-solution (blank) and well H was filled with 50 ul TS-B-buffer. 50 ul of a solution of human integrin (2) in TS-B-buffer was transfered to wells H–B and incubated for 1 h at rt. The plate was washed three times with PBS-T buffer, and then primary antibody (3) (100 μL per well) was added to the plate. After incubation for 1 h at rt, the plate was washed three times with PBS-T. Then, secondary peroxidase-labeled antibody (4) (100 μL/well) was added to the plate and incubated for 1 h at rt. After washing the plate three times with PBS-T, the plate was developed by quick addition of SeramunBlau (50 μL per well, Seramun Diagnostic GmbH, Heidesee, Germany) and incubated for 5 min at rt in the dark. The reaction was stopped with 3 M H2SO4 (50 μL/well), and the absorbance was measured at 450 nm with a plate reader. The IC50 of each compound was tested in duplicate, and the resulting inhibition curves were analyzed using OriginPro 7.5G software. The inflection point describes the IC50 value. All determined IC50 were referenced to the activity of the internal standard.

Western Blot Analysis[Bioengineered. 2022 Feb;13(2):4557-4572.]
Cell Types: B16 and A375 cells
Tested Concentrations: 0, 5, 10, and 20 μg/mL
Incubation Duration: 12 hrs (hours)
Experimental Results: Suppressed PD-L1 expression and STAT3 phosphorylation at concentrations greater than 5 μg/mL.

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Apoptosis Analysis[3]
Cell Types: B16 and A375 cells
Tested Concentrations: 5 μg/mL
Incubation Duration: 12 hrs (hours)
Experimental Results: Resulted apoptosis rates in B16 and A375 cells of 15.27% and 14.89%, respectively.

Animal/Disease Models: Nude mice bearing M21-L melanoma tumors[1]
Doses: 10, 50, and 250 μg
Route of Administration: Dosed ip three times per week
Experimental Results: Demonstrated inhibition of tumor growth with a reduction in both tumor volume (55%, 75%, and 89%, respectively) and tumor weight (23%, 38%, and 61%, respectively), when compared to controls.

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Animal/Disease Models: Female C57BL/6 mice (6-8 weeks old) with B16 cells sc[Bioengineered. 2022 Feb;13(2):4557-4572.]
Doses: 50 mg/kg; with or without 10 mg/kg Anti-PD1 monoclonal antibody or isotype control ip every 3 days;
Route of Administration: intraperitoneal (ip)injection; daily
Experimental Results: Downregulated the expression of PD-L1 via STAT3 pathway and diminished the expression of PD-L1.

Dissolved in PBS; 100μg; i.p. injection

Human glioblastoma xenografts U87 MG

[1]. Assessment of the biological and pharmacological effects of the alpha nu beta3 and alpha nu beta5 integrinreceptor antagonist, Cilengitide (EMD 121974), in patients with advanced solid tumors. Ann Oncol. 2007 Aug;18(8):1400-7.

[2]. Combination therapy of cilengitide with belotecan against experimental glioblastoma. Int J Cancer. 2013 Aug 1;133(3):749-56.

[3]. The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model. Int J Cancer. 2012 Nov 13.

Background: Cilengitide, an antiangiogenic agent that inhibits the binding of integrins alpha(nu)beta(3) and alpha(nu)beta(5) to the extracellular matrix, was studied at two dose levels in cancer patients to determine the optimal biological dose. Patients and methods: The doses of cilengitide were 600 or 1200 mg/m(2) as a 1-h infusion twice weekly every 28 days. A novel dose escalation scheme was utilized that relied upon the biological activity rate. Results: Twenty patients received 50 courses of cilengitide with no dose-limiting toxic effects. The pharmacokinetic (PK) profile revealed a short elimination half-life of 4 h, supporting twice weekly dosing. Of the six soluble angiogenic molecules assessed, only E-selectin increased significantly from baseline. Analysis of tumor microvessel density and gene expression was not informative due to intrapatient tumor heterogeneity. Although several patients with evaluable tumor biopsy pairs did reveal posttreatment increases in tumor and endothelial cell apoptosis, these results did not reach statistical significance due to the aforementioned heterogeneity. Conclusions: Cilengitide is a well-tolerated antiangiogenic agent. The biomarkers chosen in this study underscore the difficulty in assessing the biological activity of antiangiogenic agents in the absence of validated biological assays.[1]

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The prognosis of patients diagnosed with glioblastoma remains dismal in spite of the current concomitant chemoradiotherapy with temozolomide. In particular, the resistance to temozolomide appears to be the greatest obstacle to the treatment of glioblastoma. In the present study, we evaluated in vitro and in vivo the antitumor effects of combination therapy of cilengitide with belotecan, a camptothecin derivate, to treat experimental glioblastoma. The therapeutic effects of the drugs on the U87MG and U251MG human glioblastoma cell lines were assessed using in vitro cell viability and apoptosis assays. The combination treatment group with cilengitide and belotecan enhanced the cytotoxic effects to the glioblastoma cell lines and increased the apoptosis of the tumor cells compared to monotherapy with either drug alone in vitro. Nude mice with established U87MG glioblastoma were assigned to the following four groups: control, cilengitide, belotecan and combination treatment. The volume of tumors and length of survival were also measured. Animals in the combination therapy group demonstrated a significant reduction of tumor volume and an increase in survival (p < 0.05). Immunohistochemistry revealed a decrease in angiogenesis by cilengitide and an increase in apoptosis by cilengitide and belotecan in vivo. The combination therapy of cilengitide with belotecan presented more cytotoxic effects compared to the monotherapy of either drug in vitro and in vivo. This combination protocol may serve as an alternative treatment option for glioblastoma.[2]

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Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.[3]

C29H41F3N8O9

702.68

702.295

C, 55.09; H, 6.85; N, 19.04; O, 19.03

199807-35-7

Cilengitide;188968-51-6; 199807-35-7 (TFA); 188969-00-8 (HCl)

129626550

cyclo[L-arginyl-glycyl-L-alpha-aspartyl-D-phenylalanyl-N-methyl-L-valyl] trifluoroacetic acid

cyclo[Arg-Gly-Asp-D-Phe-N(Me)Val].TFA

White to off-white solid powder

1.111

8

13

9

49

1110

4

CC(C)[C@H]1C(=O)N[C@H](C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](C(=O)N1C)CC2=CC=CC=C2)CC(=O)O)CCCN=C(N)N.C(=O)(C(F)(F)F)O

WHJCSACXAPYNTG-LOPTWHKWSA-N

InChI=1S/C27H40N8O7.C2HF3O2/c1-15(2)22-25(41)33-17(10-7-11-30-27(28)29)23(39)31-14-20(36)32-18(13-21(37)38)24(40)34-19(26(42)35(22)3)12-16-8-5-4-6-9-16;3-2(4,5)1(6)7/h4-6,8-9,15,17-19,22H,7,10-14H2,1-3H3,(H,31,39)(H,32,36)(H,33,41)(H,34,40)(H,37,38)(H4,28,29,30);(H,6,7)/t17-,18-,19+,22-;/m0./s1

2-[(2S,5R,8S,11S)-5-benzyl-11-[3-(diaminomethylideneamino)propyl]-7-methyl-3,6,9,12,15-pentaoxo-8-propan-2-yl-1,4,7,10,13-pentazacyclopentadec-2-yl]acetic acid;2,2,2-trifluoroacetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 16.67 mg/mL (23.72 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)