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

Cilengitide is a pentapeptide with cyclized RGD (Arg-Gly-Asp) motif. Cilengitide inhibits the attachment and migration of endothelial cells mediated by integrins αvβ3 and ανβ5[2]. Cilengitide, in cell adhesion studies evaluating the human melanoma M21 or UCLA-P3 human lung carcinoma cell lines, inhibits integrin-mediated binding to Vitronectin with IC50s of 0.4 and 0.4 μM[2]. With an IC50 of 2 μM, celenegitide prevents human umbilical vein endothelial cells from adhering to vitronectin[2]. Cilengitide (5 μg/mL; 12 h) induces apoptosis in B16 and A375 cells and inhibits the viability of melanoma cells in vitro (0–1 mg/mL; 24-72 h)[3]. B16 and A375 cells' ability to form colonies is inhibited by celenigitide (5 μg/mL, 10 μg/mL; 2 weeks)[3]. PD-L1 expression is reduced by inhibiting STAT3 phosphorylation with celenigitide (0–20 μg/mL; 12 h)[3].

In nude mice, cilengitide (ip at 10, 50, and 250 μg three times per week) inhibits the growth of M21-L melanoma tumors[2]. In the B16 murine melanoma model, cleengitide (50 mg/kg; intraperitoneal; daily) improves CD8+ T cell function and supports anti-PD1 efficacy with anti-PD1 monoclonal antibody[3].

Integrin Binding Assay[1]
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).[1]
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[3]
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[2]
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[3]
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.

[1]. A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins. Sci Rep. 2017 Jan 11;7:39805.

[2]. 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.

[3]. Cilengitide, an αvβ3-integrin inhibitor, enhances the efficacy of anti-programmed cell death-1 therapy in a murine melanoma model. Bioengineered. 2022 Feb;13(2):4557-4572.

Cilengitide is an oligopeptide.
Cilengitide has been used in trials studying the treatment of Sarcoma, Gliomas, Lymphoma, Leukemia, and Lung Cancer, among others.
Cilengitide is a cyclic Arg-Gly-Asp peptide with potential antineoplastic activity. Cilengitide binds to and inhibits the activities of the alpha(v)beta(3) and alpha(v)beta(5) integrins, thereby inhibiting endothelial cell-cell interactions, endothelial cell-matrix interactions, and angiogenesis. (NCI04)

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Drug Indication
Treatment of high-grade glioma

C₂₇H₄₀N₈O₇

588.66

588.302

C, 55.09; H, 6.85; N, 19.04; O, 19.02

188968-51-6

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

176873

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

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

White to light yellow solid powder

1.4±0.1 g/cm3

1.643

-2.46

7

8

9

42

1020

4

O=C1[C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N(C([H])([H])[H])C([C@@]([H])(C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])N([H])C([C@]([H])(C([H])([H])C(=O)O[H])N([H])C(C([H])([H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])/N=C(\N([H])[H])/N([H])[H])N1[H])=O)=O)=O)=O

AMLYAMJWYAIXIA-VWNVYAMZSA-N

InChI=1S/C27H40N8O7/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/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)/t17-,18-,19+,22-/m0/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: 100 mg/mL (169.88 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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