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 (EMD 121974) targets integrin αvβ3 with a Ki value of 4.1 nM (human recombinant αvβ3) [1]
Cilengitide (EMD 121974) targets integrin αvβ5 with a Ki value of 79 nM (human recombinant αvβ5) [1]
Cilengitide (EMD 121974) shows low affinity for integrins αIIbβ3, α5β1, and αvβ6 (Ki > 1000 nM) [1]

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].

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In human recombinant integrin binding assays, Cilengitide (EMD 121974) (0.1-1000 nM) competitively inhibited [125I]-vitronectin binding to αvβ3 and αvβ5, with Ki values of 4.1 nM and 79 nM, respectively [1]
- In human glioblastoma U87MG cells, Cilengitide (EMD 121974) (1-100 μM) dose-dependently inhibited cell adhesion to vitronectin (αvβ3 ligand) and fibronectin (αvβ5 ligand); 100 μM reduced adhesion by 82% and 65%, respectively (p < 0.001) [1]
- Cilengitide (EMD 121974) (10-100 μM) suppressed migration and invasion of U87MG cells in Transwell assays; 50 μM reduced migration by 58% and invasion by 63% compared to vehicle (p < 0.01) [1]
- In murine melanoma B16F10 cells, Cilengitide (EMD 121974) (10 μM) enhanced the cytotoxicity of anti-PD-1 antibody, increasing T cell-mediated tumor cell lysis by 42% (p < 0.05) [3]
- Cilengitide (EMD 121974) (1-10 μM) upregulated MHC class I expression on B16F10 cells by 35% at 10 μM, promoting antigen presentation (flow cytometry analysis) [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].

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In a phase I/II clinical trial of patients with advanced solid tumors (n=84), intravenous administration of Cilengitide (EMD 121974) (0.25-20 mg/kg weekly) achieved disease stabilization in 32% of patients, with a median progression-free survival (PFS) of 3.8 months [2]
- In C57BL/6 mice bearing B16F10 melanoma xenografts, intraperitoneal injection of Cilengitide (EMD 121974) (5 mg/kg every other day) combined with anti-PD-1 antibody (10 mg/kg weekly) reduced tumor volume by 68% compared to anti-PD-1 monotherapy (p < 0.01) [3]
- Cilengitide (EMD 121974) + anti-PD-1 combination therapy prolonged median survival of tumor-bearing mice by 52% (42 days vs. 27.6 days in monotherapy group, p < 0.01) [3]
- In mice, Cilengitide (EMD 121974) (5 mg/kg, i.p.) increased intratumoral CD8+ T cell infiltration by 76% and reduced regulatory T cell (Treg) proportion by 38% (flow cytometry) [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.

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Integrin αvβ3/αvβ5 binding assay: Membrane preparations from cells expressing human recombinant αvβ3 or αvβ5 were incubated with [125I]-vitronectin and serial concentrations of Cilengitide (EMD 121974) (0.01 nM to 1 μM) at 25°C for 60 minutes; unbound radioligand was removed by filtration through glass fiber filters; bound radioactivity was measured by gamma counting; Ki values were calculated using the Cheng-Prusoff equation [1]
- Ligand competition assay: Microplates coated with vitronectin or fibronectin were incubated with Cilengitide (EMD 121974) (0.1-1000 nM) and fluorescently labeled αvβ3/αvβ5 integrin fragments; after 90 minutes of incubation at 37°C, unbound integrin was washed away; fluorescence intensity was measured, and inhibition curves were generated to confirm binding specificity [1]

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.

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Tumor cell adhesion assay: U87MG cells were seeded in 96-well plates coated with vitronectin or fibronectin; Cilengitide (EMD 121974) (1-100 μM) was added, and cells were incubated at 37°C for 2 hours; non-adherent cells were removed by washing; adherent cells were fixed, stained with crystal violet, and absorbance was measured at 570 nm to quantify adhesion rate [1]
- Migration and invasion assay: Transwell inserts (8 μm pores) were coated with fibronectin (migration) or Matrigel (invasion); U87MG cells (1×10⁵ cells/well) in serum-free medium containing Cilengitide (EMD 121974) (10-100 μM) were added to the upper chamber; medium with 10% fetal bovine serum was added to the lower chamber; after 24 hours (migration) or 48 hours (invasion), migrated/invaded cells were stained and counted under a microscope [1]
- T cell-mediated cytotoxicity assay: B16F10 cells were co-cultured with murine splenic T cells (effector:target ratio = 10:1) in the presence of Cilengitide (EMD 121974) (1-10 μM) and anti-PD-1 antibody (10 μg/mL); after 48 hours, cytotoxicity was assessed by lactate dehydrogenase (LDH) release assay, and lysis rate was calculated [3]
- MHC class I expression assay: B16F10 cells were treated with Cilengitide (EMD 121974) (1-10 μM) for 24 hours; cells were stained with anti-MHC class I antibody and analyzed by flow cytometry; mean fluorescence intensity (MFI) was normalized to vehicle control [3]

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.

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Murine melanoma xenograft model: 6-week-old female C57BL/6 mice were subcutaneously injected with 2×10⁶ B16F10 cells into the right flank; when tumors reached 100 mm³, mice were randomly divided into 4 groups (n=10 per group): vehicle control, Cilengitide (EMD 121974) monotherapy, anti-PD-1 monotherapy, combination therapy [3]
- Cilengitide (EMD 121974) was formulated in sterile physiological saline; administered via intraperitoneal injection at 5 mg/kg every other day for 3 weeks [3]
- Anti-PD-1 antibody was administered via intraperitoneal injection at 10 mg/kg once weekly for 3 weeks; tumor volume was measured twice weekly with calipers; mice were euthanized when tumors exceeded 2000 mm³, and survival time was recorded [3]
- Intratumoral immune cell analysis: At study end, tumors were harvested, dissociated into single-cell suspensions, stained with antibodies against CD8, CD4, and Foxp3, and analyzed by flow cytometry [3]
- Clinical trial protocol: Patients with advanced solid tumors (glioblastoma, melanoma, non-small cell lung cancer) were enrolled in a dose-escalation study; Cilengitide (EMD 121974) was administered as a 1-hour intravenous infusion at doses ranging from 0.25 to 20 mg/kg once weekly for 6 weeks per cycle; tumor response was assessed by RECIST criteria every 2 cycles [2]

In humans, Cilengitide (EMD 121974) exhibited linear pharmacokinetics at doses of 0.25–20 mg/kg; peak plasma concentration (Cmax) increased proportionally with dose, reaching 12.8 μg/mL at 20 mg/kg [2]. The terminal elimination half-life (t1/2) of Cilengitide (EMD 121974) in humans was 2.8 ± 0.6 hours [2]. The plasma clearance of Cilengitide (EMD 121974) was 15.2 ± 3.1 mL/min/kg, and the volume of distribution (Vd) was 0.38 ± 0.09 L/kg [2]. The plasma protein binding rate of Cilengitide (EMD 121974) in human plasma was 25 ± 4% (equilibrium dialysis method) [2]. The oral bioavailability of silengilide (EMD 121974) was <5% in preclinical studies (not detectable in human plasma after oral administration) [2]

In the Phase I/II clinical trials, the most common adverse events (AEs) of Cilengitide (EMD 121974) were fatigue (36%), nausea (28%), hypertension (22%), and headache (18%); 9% of patients experienced grade 3/4 adverse events (hypertension, thrombosis), which could be controlled with standard treatment [2]. - No significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) were observed in patients treated with Cilengitide (EMD 121974) [2]. - No significant changes in body weight, food intake, or histopathological results of major organs (liver, kidney, heart) were observed in mice treated with Cilengitide (EMD 121974) (5 mg/kg, intraperitoneal injection, for 3 weeks) [3]. No blood toxicity (anemia, leukopenia) was induced in humans or animals [2][3]

[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.

Silengilide is an oligopeptide. It has been used in therapeutic trials for various cancers, including sarcoma, glioma, lymphoma, leukemia, and lung cancer. Silengilide is a cyclic arginine-glycine-aspartic peptide with potential antitumor activity. It can bind to and inhibit the activity of αvβ3 and αvβ5 integrins, thereby suppressing endothelial cell interactions, endothelial cell-extracellular matrix interactions, and angiogenesis. (NCI04)

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Drug Indications
Treatment of high-grade gliomas Cialiglitatide (EMD 121974) is a cyclic RGD peptide that inhibits integrin αvβ3 and αvβ5 and is used to treat advanced solid tumors[1][2][3]
- Its mechanism of action includes blocking αvβ3/αvβ5-mediated cell adhesion, migration and angiogenesis, thereby inhibiting tumor progression; it can also enhance the efficacy of anti-PD-1 therapy by modulating the tumor immune microenvironment (increasing CD8+ T cell infiltration and reducing Treg cells)[3]
- Cialiglitatide (EMD 121974) showed clinical activity in patients with recurrent glioblastoma, with a disease control rate of 41% in subgroup analysis[2]
- Due to poor oral bioavailability, the drug needs to be administered intravenously, and its good toxicity profile supports its use in combination with immunotherapy[2][3]

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.)