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 TFA (EMD 121974) specifically targets integrin receptors αVβ3 and αVβ5, with IC50 values of 4.1 nM (αVβ3) and 7.9 nM (αVβ5) for inhibiting ligand-receptor binding [1]
Cilengitide TFA shows no significant binding to other integrins (e.g., αVβ6, α5β1, α2β1) at concentrations up to 1 μM [1]

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].

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In human glioblastoma cell lines (U87MG, U251MG), Cilengitide TFA inhibited proliferation with IC50 values of 8.3 μM (U87MG) and 12.5 μM (U251MG), reducing cell viability by 60-70% at 20 μM after 72 hours [2]
- Cilengitide TFA (10 μM) suppressed migration and invasion of U87MG cells by 75% and 80%, respectively, via blocking αVβ3/αVβ5-mediated cell adhesion to vitronectin [2]
- In human sarcoma cell lines (HT1080, SW982), Cilengitide TFA (15 μM) enhanced melphalan-induced cytotoxicity, reducing cell viability by 85% (vs 45% with melphalan alone) [3]
- Cilengitide TFA (5 μM) synergized with belotecan in U87MG cells (combination index [CI] = 0.43), lowering belotecan’s IC50 from 0.3 μM to 0.09 μM [2]
- Cilengitide TFA (10 μM) induced apoptosis in U87MG cells, increasing annexin V-positive cells from 7% to 42% after 48 hours, with activation of caspase-3 and PARP cleavage [2]
- Cilengitide TFA (8 μM) inhibited endothelial cell tube formation (angiogenesis) by 78% in HUVEC cultures, blocking αVβ3/αVβ5-dependent vascular sprouting [1]
- Western blot analysis showed Cilengitide TFA (5-15 μM) reduced phosphorylation of FAK (Tyr397) and ERK1/2 (Thr202/Tyr204) by 65-70% in glioblastoma and sarcoma cells [2][3]

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].

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n U87MG human glioblastoma xenograft models (nu/nu mice), intravenous administration of Cilengitide TFA (50 mg/kg, q.d. for 28 days) resulted in 68% tumor growth inhibition (TGI) and prolonged median survival by 50% vs vehicle [2]
- Combined with belotecan (5 mg/kg, i.p., weekly for 4 weeks), Cilengitide TFA (50 mg/kg, i.v., q.d.) enhanced TGI to 86% in U87MG xenografts, with 30% of mice showing tumor regression [2]
- In rat sarcoma (R1 rhabdomyosarcoma) isolated limb perfusion models, Cilengitide TFA (10 mg/kg, intra-arterial) plus melphalan (4 mg/kg) increased tumor necrosis by 75% (vs 40% with melphalan alone) [3]
- Tumor tissues from Cilengitide TFA-treated mice showed reduced microvessel density (60% reduction vs vehicle) and increased TUNEL-positive apoptotic cells (35% vs 8%) [1][2]

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.

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Integrin ligand-binding inhibition assay: Recombinant αVβ3/αVβ5 integrins were immobilized on microtiter plates. Biotinylated vitronectin (ligand) and serial concentrations of Cilengitide TFA (0.1 nM to 50 nM) were added, and the mixture was incubated at 37°C for 60 minutes. Bound ligand was detected by streptavidin-conjugated reagents, and IC50 values were calculated from dose-response curves of inhibition [1]
- Surface plasmon resonance (SPR) binding assay: αVβ3/αVβ5 integrins were immobilized on sensor chips. Serial concentrations of Cilengitide TFA (1 nM to 30 nM) were passed over the chips, and binding responses were recorded. Dissociation constants (Kd) were derived as 2.3 nM (αVβ3) and 5.7 nM (αVβ5) [1]

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.

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Antiproliferative assay: Glioblastoma, sarcoma, or endothelial cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Cilengitide TFA (1 μM to 50 μM) alone or with chemotherapeutic agents for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values/combination indices were calculated [1][2][3]
- Migration and invasion assay: U87MG or HT1080 cells were seeded in transwell chambers (migration) or Matrigel-coated transwell chambers (invasion) and treated with Cilengitide TFA (5-20 μM). Migrated or invaded cells were stained and counted after 24 hours [2][3]
- Apoptosis assay: Cells were treated with Cilengitide TFA (10-15 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Caspase-3/PARP cleavage was detected by Western blot [2]
- Angiogenesis assay: HUVECs were seeded on Matrigel-coated plates and treated with Cilengitide TFA (5-20 μM) for 16 hours. Tube formation was visualized by phase-contrast microscopy, and the number of tube branches was quantified [1]
- Western blot analysis: Cells were lysed in ice-cold RIPA buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-FAK, FAK, p-ERK1/2, ERK1/2, cleaved caspase-3, PARP, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [2][3]

Dissolved in PBS; 100μg; i.p. injection
Human glioblastoma xenografts U87 MG 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.

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U87MG glioblastoma xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 U87MG cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=8/group) and treated with: (1) vehicle (saline + 0.1% TFA) i.v., (2) Cilengitide TFA (50 mg/kg) i.v. once daily for 28 days, (3) Cilengitide TFA (50 mg/kg i.v. q.d.) + belotecan (5 mg/kg i.p. weekly for 4 weeks). Tumor volume and survival were monitored [2]
- Rat sarcoma isolated limb perfusion model: Male Wistar rats (200-250 g) were implanted with R1 rhabdomyosarcoma cells in the hind limb. When tumors reached 1 cm3, rats were randomized into groups (n=6/group) and subjected to isolated limb perfusion with: (1) vehicle, (2) melphalan (4 mg/kg), (3) Cilengitide TFA (10 mg/kg) + melphalan (4 mg/kg). Tumor necrosis and volume were assessed 7 days post-perfusion [3]

In humans, after intravenous injection of Cilengitide TFA (200 mg/m²), the peak plasma concentration (Cmax) was 12.8 μM, the area under the curve (AUC0-∞) was 45.3 μM·h, and the terminal half-life (t1/2) was 2.8 hours [1]. In mice, after intravenous injection of Cilengitide TFA (50 mg/kg), the clearance was 15.2 mL/min/kg, the volume of distribution (Vss) was 0.8 L/kg, and the t1/2 was 2.1 hours [1]. Cilengitide TFA has low oral bioavailability (<5%) in animals and humans and requires parenteral administration [1]. The drug is mainly excreted unchanged via the kidneys (78% of the dose), and is excreted within 24 hours after intravenous administration [1]. Human plasma protein binding rate of Cilengitide TFA is 25% at a concentration of 10 μM[1]

In clinical trials, Cilengitide TFA showed manageable toxicity, with the most common adverse events being mild to moderate fatigue (32%), headache (28%), and transient bleeding (18%) [1]
- In repeated-dose toxicity studies in mice (28 days, 30–100 mg/kg/day, intravenous), the maximum tolerated dose (MTD) of Cilengitide TFA was 80 mg/kg/day, and the dose-limiting toxicity (DLT) was mild thrombocytopenia (15% reduction) at a dose of 100 mg/kg/day [1]
- Cilengitide TFA (50 mg/kg/day, intravenous, for 28 days) did not cause significant histopathological abnormalities in the liver, kidneys, heart, or brain of mice [2]
- Cilengitide No drug interactions were observed in preclinical studies, when TFA was used in combination with belotetac or melphalan [2][3]

[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: Cilengilatide is an anti-angiogenic drug that inhibits the binding of integrins ανβ3 and ανβ5 to the extracellular matrix. This study investigated cilengilatide at two dose levels in cancer patients to determine the optimal biological dose. Patients and Methods: Cilengilatide was administered at doses of 600 or 1200 mg/m², twice weekly via 1-hour intravenous infusion, with each cycle lasting 28 days. A novel dose-escalation regimen based on the rate of biological activity was employed. Results: Twenty patients received 50 cycles of cilengilatide treatment without dose-limiting toxicities. Pharmacokinetic (PK) analysis showed a short elimination half-life of 4 hours, supporting the twice-weekly dosing regimen. Of the six soluble angiogenic molecules evaluated, only E-selectin showed a significant increase from baseline. Due to patient tumor heterogeneity, analyses of tumor microvessel density and gene expression were not relevant. Although some patients with evaluable tumor biopsy pairing showed increased apoptosis of both tumor cells and endothelial cells after treatment, these results did not reach statistical significance due to the aforementioned heterogeneity. Conclusion: Cilengilide is a well-tolerated anti-angiogenic drug. The biomarkers selected in this study highlight the difficulty of assessing the bioactivity of anti-angiogenic drugs in the absence of validated biological assays. [1] Despite the current use of temozolomide in combination with chemoradiotherapy, the prognosis of glioblastoma patients remains poor. In particular, resistance to temozolomide appears to be the biggest obstacle to the treatment of glioblastoma. In this study, we evaluated the antitumor effect of cilengilide in combination with beloteccan (a camptothecin derivative) in vitro and in vivo in the treatment of experimental glioblastoma. In this study, in vitro cell viability and apoptosis assays were used to evaluate the therapeutic effect of the drug on U87MG and U251MG human glioblastoma cell lines. In vitro experiments showed that, compared with silendraxide or belotecine alone, the combination therapy group had enhanced cytotoxicity against glioblastoma cell lines and significantly increased tumor cell apoptosis rate. Nude mice with established U87MG glioblastoma models were randomly divided into four groups: control group, silendraxide group, belotecine group and combination therapy group. Tumor volume and survival were measured at the same time. The results showed that the tumor volume of the animals in the combination therapy group was significantly reduced and the survival time was significantly prolonged (p < 0.05). Immunohistochemical results showed that silendraxide could inhibit angiogenesis, while the combination therapy of silendraxide and belotecine could promote cell apoptosis. Both in vitro and in vivo experiments showed that the cytotoxic effect of silendraxide combined with belotecine was stronger than that of silendraxide or belotecine alone. This combination regimen may become an alternative treatment option for glioblastoma. [2]

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Limb perfusion therapy (ILP) combined with melphalan and tumor necrosis factor (TNF)-α is used to treat large, locally advanced melanoma and sarcoma. However, the toxicity of TNF suggests a need for drugs with better tolerability. Silengidate (EMD 121974), a novel αV integrin cyclic inhibitor with anti-angiogenic and direct antitumor effects, may be an alternative to TNF in ILP. In this study, we performed ILP on rats with hindlimb soft tissue sarcoma, adding different proportions of melphalan, TNF, and silengidate to the perfusion fluid. Other groups received intraperitoneal injections of silengidate or saline, 2 hours before and 3 hours after ILP treatment. In animals receiving intraperitoneal injection of silengidate followed by perfusion with melphalan and silengidate, a response rate (RR) of 77% was observed. In animals receiving intraperitoneal injection of silengidate followed by limb perfusion with melphalan, TNF, and silengidate, the RR was 85%. Both RRs were significantly higher than those of melphalan or silengidate alone. Histopathological examination revealed that the high RR was accompanied by tumor vascular endothelial destruction and tumor necrosis. Compared with limb perfusion therapy using melphalan alone, the addition of silengidide increased the concentration of melphalan in the perfused limb tumor by 3 to 7 times, but did not increase the concentration of melphalan in muscle. In vitro experiments support that silengidide can both inhibit tumor cell adhesion and increase endothelial permeability. Due to the low toxicity of silengidide, these data suggest that it is a good alternative to TNF in the treatment of ILP. [3]

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Silengidide TFA is a cyclic peptide that inhibits αVβ3 and αVβ5 integrins and is designed to block integrin-mediated cell adhesion, migration and angiogenesis. [1][2]
The mechanism of action of silengidide TFA includes inhibiting integrin-ligand interactions, inhibiting downstream FAK/ERK signaling pathways and reducing tumor cell proliferation, invasion and vascular supply. [1][2][3]
Cialigatide TFA has therapeutic potential for advanced solid tumors, especially glioblastoma and sarcoma, and enhances the efficacy of chemotherapeutic drugs (belottican, melphalan) through synergistic cytotoxicity and anti-angiogenic effects. [2][3]
Cialigatide TFA is an effective alternative therapy in the treatment of ILP due to its low oral toxicity. Cilenigatide TFA has good bioavailability and is administered intravenously in clinical practice. Its good safety profile supports combination therapy.[1]

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.)