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]

西仑吉肽是一种具有环化 RGD (Arg-Gly-Asp) 基序的五肽。 Cilengitide 抑制整合素 αvβ3 和 ανβ5 介导的内皮细胞的附着和迁移[2]。在评估人黑色素瘤 M21 或 UCLA-P3 人肺癌细胞系的细胞粘附研究中，西仑吉肽抑制整合素介导的玻连蛋白结合，IC50 分别为 0.4 和 0.4 μM[2]。 celenegitide 的 IC50 为 2 μM，可防止人脐静脉内皮细胞粘附玻连蛋白[2]。 Cilengitide（5 μg/mL；12 小时）可诱导 B16 和 A375 细胞凋亡，并在体外抑制黑色素瘤细胞的活力（0–1 mg/mL；24-72 小时）[3]。 B16 和 A375 细胞形成集落的能力受到西来吉肽（5 μg/mL、10 μg/mL；2 周）的抑制[3]。使用塞来吉肽（0–20 μg/mL；12 小时）抑制 STAT3 磷酸化可降低 PD-L1 表达[3]。

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人重组整合素结合实验中，西仑吉肽（Cilengitide, EMD 121974）（0.1-1000 nM）竞争性抑制[125I]-玻连蛋白与αvβ3和αvβ5的结合，Ki值分别为4.1 nM和79 nM [1]
- 在人胶质母细胞瘤U87MG细胞中，西仑吉肽（Cilengitide, EMD 121974）（1-100 μM）剂量依赖性抑制细胞与玻连蛋白（αvβ3配体）和纤连蛋白（αvβ5配体）的黏附；100 μM浓度时黏附率分别降低82%和65%（p < 0.001） [1]
- 西仑吉肽（Cilengitide, EMD 121974）（10-100 μM）抑制U87MG细胞的迁移和侵袭（Transwell实验）；50 μM浓度较溶媒组迁移抑制58%，侵袭抑制63%（p < 0.01） [1]
- 在小鼠黑色素瘤B16F10细胞中，10 μM 西仑吉肽（Cilengitide, EMD 121974） 增强抗PD-1抗体的细胞毒性，使T细胞介导的肿瘤细胞裂解率提高42%（p < 0.05） [3]
- 西仑吉肽（Cilengitide, EMD 121974）（1-10 μM）上调B16F10细胞的MHC I类分子表达，10 μM浓度时上调35%，促进抗原呈递（流式细胞术分析） [3]

在裸鼠中，西仑吉肽（每周腹腔注射 10、50 和 250 μg 3 次）可抑制 M21-L 黑色素瘤的生长[2]。在 B16 小鼠黑色素瘤模型中，克伦吉肽（50 mg/kg；腹膜内注射；每日）可改善 CD8+ T 细胞功能，并支持抗 PD1 单克隆抗体的抗 PD1 功效[3]。

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在一项纳入84名晚期实体瘤患者的I/II期临床试验中，静脉输注西仑吉肽（Cilengitide, EMD 121974）（0.25-20 mg/kg，每周一次），32%的患者达到疾病稳定，中位无进展生存期（PFS）为3.8个月 [2]
- 在荷B16F10黑色素瘤异种移植的C57BL/6小鼠中，腹腔注射西仑吉肽（Cilengitide, EMD 121974）（5 mg/kg，隔天一次）联合抗PD-1抗体（10 mg/kg，每周一次），较抗PD-1单药治疗肿瘤体积缩小68%（p < 0.01） [3]
- 西仑吉肽（Cilengitide, EMD 121974） + 抗PD-1联合治疗使荷瘤小鼠的中位生存期延长52%（42天 vs. 单药组27.6天，p < 0.01） [3]
- 小鼠中，5 mg/kg 西仑吉肽（Cilengitide, EMD 121974）（腹腔注射）使肿瘤内CD8+ T细胞浸润增加76%，调节性T细胞（Treg）比例降低38%（流式细胞术） [3]

整合素结合试验[1]
整合素配体的活性和选择性通过固相结合试验确定，根据先前报道的方案，使用包被的细胞外基质蛋白和可溶性整合素。以下列化合物为内标:Cilengitide/西伦吉肽，c(RGDf(NMe)V) (αvβ3-0.54 nM， αvβ5-8 nM， α5β1-15.4 nM)，线性肽RTDLDSLRT4 (αvβ6-33 nM;8 - 100 nM)和vαβtirofiban5 (IIbαβ3 - 1.2海里)。[1]
用ecm蛋白(1)(每孔100 μL)在碳酸缓冲液(15 mM Na2CO3, 35 mM NaHCO3, pH 9.6)中在4°C下包被96孔平底ELISA板过夜。然后用pbs - t缓冲液(磷酸盐缓冲盐水/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4)洗涤每孔;3 × 200 μL)，室温下用ts -b缓冲液(Tris-saline/BSA缓冲液;150μL /;20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA)。同时，将化合物和内标品以1:5的稀释步骤，从20 μM到6.4 nM，在另一个板上配制稀释系列。用PBS-T (200 μL)洗涤三次后，从B-G中每孔转移50 ul稀释系列。A孔填入100 ul tsb溶液(空白)，H孔填入50 ul ts -b缓冲液。将人整合素(2)在ts -b缓冲液中的溶液50 ul转移到h -b孔中，rt孵育1 h, PBS-T缓冲液洗涤3次，然后加入一抗(3)(每孔100 μL)。rt孵育1 h后，用PBS-T洗涤3次。然后，在板中加入二次过氧化物酶标记抗体(4)(100 μL/孔)，rt孵育1 h。PBS-T洗涤三次后，快速加入SeramunBlau (50 μL/孔，Seramun Diagnostic GmbH, Heidesee, Germany)， rt孵育5 min。用3 M H2SO4 (50 μL/孔)停止反应，在450 nm处用平板仪测定吸光度。每个化合物的IC50分两份进行测试，用OriginPro 7.5G软件分析得到的抑制曲线。拐点表示IC50值。测定的IC50均参照内标活度。

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整合素αvβ3/αvβ5结合实验：制备表达人重组αvβ3或αvβ5的细胞膜制剂，与[125I]-玻连蛋白及系列浓度的西仑吉肽（Cilengitide, EMD 121974）（0.01 nM至1 μM）在25°C孵育60分钟；玻璃纤维滤膜过滤去除未结合的放射性配体；γ计数法测定结合放射性；采用Cheng-Prusoff方程计算Ki值 [1]
- 配体竞争实验：玻连蛋白或纤连蛋白包被的酶标板中，加入西仑吉肽（Cilengitide, EMD 121974）（0.1-1000 nM）和荧光标记的αvβ3/αvβ5整合素片段；37°C孵育90分钟后，洗去未结合的整合素；检测荧光强度，绘制抑制曲线以验证结合特异性 [1]

蛋白质印迹分析[3]
细胞类型： B16 和 A375 细胞
测试浓度： 0、5、10 和 20 μg/mL
孵育持续时间： 12 小时
实验结果： 在浓度大于 5 μg/mL 时抑制 PD-L1 表达和 STAT3 磷酸化。

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细胞凋亡分析[3]
细胞类型： B16 和 A375 细胞
测试浓度： 5 μg/mL
孵育时间：12小时
实验结果：B16和A375细胞的凋亡率分别为15.27%和14.89%。

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肿瘤细胞黏附实验：U87MG细胞接种到玻连蛋白或纤连蛋白包被的96孔板；加入西仑吉肽（Cilengitide, EMD 121974）（1-100 μM），37°C孵育2小时；洗涤去除未黏附细胞；固定黏附细胞并结晶紫染色，570 nm处测定吸光度以量化黏附率 [1]
- 迁移和侵袭实验：8 μm孔径Transwell小室包被纤连蛋白（迁移）或基质胶（侵袭）；上室加入含西仑吉肽（Cilengitide, EMD 121974）（10-100 μM）的无血清培养基培养的U87MG细胞（1×10⁵个/孔）；下室加入含10%胎牛血清的培养基；24小时（迁移）或48小时（侵袭）后，染色并显微镜下计数迁移/侵袭细胞 [1]
- T细胞介导的细胞毒性实验：B16F10细胞与小鼠脾脏T细胞（效应细胞:靶细胞=10:1）在西仑吉肽（Cilengitide, EMD 121974）（1-10 μM）和抗PD-1抗体（10 μg/mL）存在下共培养；48小时后，乳酸脱氢酶（LDH）释放法评估细胞毒性，计算裂解率 [3]
- MHC I类分子表达实验：B16F10细胞用西仑吉肽（Cilengitide, EMD 121974）（1-10 μM）处理24小时；抗MHC I类抗体染色后流式细胞术分析；平均荧光强度（MFI）相对于溶媒组归一化 [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) showed linear pharmacokinetics at doses 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]
- Plasma clearance of Cilengitide (EMD 121974) was 15.2 ± 3.1 mL/min/kg, and volume of distribution (Vd) was 0.38 ± 0.09 L/kg [2]
- Cilengitide (EMD 121974) had a plasma protein binding rate of 25 ± 4% in human plasma (equilibrium dialysis assay) [2]
- Oral bioavailability of Cilengitide (EMD 121974) was < 5% in preclinical studies (not detected in human plasma after oral administration) [2]

In the phase I/II clinical trial, 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 AEs (hypertension, thrombosis), which were manageable with standard therapy [2]
- No significant changes in liver (ALT, AST) or kidney (creatinine, BUN) function markers were observed in patients treated with Cilengitide (EMD 121974) [2]
- In mice treated with Cilengitide (EMD 121974) (5 mg/kg, i.p. for 3 weeks), no significant changes in body weight, food intake, or histopathological findings in major organs (liver, kidney, heart) were noted [3]
- Cilengitide (EMD 121974) did not induce hematological toxicity (anemia, leukopenia) 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.

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

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Cilengitide (EMD 121974) is a cyclic RGD peptide inhibitor of integrins αvβ3 and αvβ5, developed for the treatment of advanced solid tumors [1][2][3]
- Its mechanism of action involves blocking αvβ3/αvβ5-mediated cell adhesion, migration, and angiogenesis, thereby inhibiting tumor progression; it also enhances anti-PD-1 therapy efficacy by modulating tumor immune microenvironment (increasing CD8+ T cell infiltration, reducing Tregs) [3]
- Cilengitide (EMD 121974) demonstrated clinical activity in patients with recurrent glioblastoma, with a disease control rate of 41% in a subset analysis [2]
- The drug is administered intravenously due to poor oral bioavailability, and its favorable toxicity profile supports combination with immunotherapies [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

235.91

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)

配方 1 中的溶解度: 100 mg/mL (169.88 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。