Ifebemtinib (BI-853520; IN-10018)

Focal adhesion kinase (FAK) (recombinant FAK IC₅₀ = 1 nM) [1][3]

ifebemtinib (BI 853520)（0-3 μM；2 小时）可抑制癌细胞的增殖[2]。仅在 3D 培养中，ifebemtinib (BI 853520)（0-30 μM；4-6 d）可抑制肿瘤细胞侵袭和增殖[1]。 24 小时疗程的 ifebemtinib (0–10 μM) 可抑制 Y397–FAK 自磷酸化[1]。在这种高度转移的小鼠乳腺癌细胞系中，ifebemtinib（0.1 μM；96 小时）表现出快速有效的 FAK 抑制作用[1]。

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1. FAK抑制与细胞增殖抑制：在PC-3前列腺癌细胞中，BI-853520抑制FAK Tyr³⁹⁷位点自磷酸化的IC₅₀为1 nM，阻断锚定非依赖性克隆形成的EC₅₀为3 nM。Western blot分析显示，药物处理后p-FAK（Tyr³⁹⁷）及下游信号分子（如Akt、ERK）呈剂量依赖性下调 [1][3]

2. 3D球体与2D单层细胞活性差异：在癌细胞（如PC-3、MDA-MB-231）的3D球体培养中，BI-853520在低剂量（≤3 μM）即可抑制肿瘤细胞增殖和侵袭；而在2D单层培养中，需≥1000倍浓度才能达到类似效果 [1][3]

3. 乳腺癌细胞迁移抑制：在高转移性小鼠乳腺癌细胞中，BI-853520（0.1 μM）可快速抑制FAK磷酸化，Transwell实验显示24小时内细胞迁移率降低50% [2]

4. 与间充质表型的相关性：体外实验证实，对BI-853520的敏感性与E-钙黏蛋白表达缺失及间充质标志物（如波形蛋白、N-钙黏蛋白）激活密切相关，免疫荧光染色可验证该关联 [1][3]

用 ifebemtinib (BI 853520) 治疗（口服管饲；50 mg/kg；每天一次；0-8 周）可显着抑制体内所有三种细胞系原发性肿瘤的生长[1]。

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1. 腺癌异种移植模型疗效：在荷PC-3腺癌皮下异种移植瘤的裸鼠中，口服BI-853520（50 mg/kg/天）2周内可使肿瘤体积缩小60–80%，20%的小鼠肿瘤完全消退。间充质表型肿瘤（低E-钙黏蛋白、高miR-200c-3p）对药物响应更显著，中位肿瘤生长抑制率（TGI）>100% [1][3]

2. 药效动力学靶点结合：免疫组化结果显示，BI-853520可快速穿透肿瘤组织，给药后1小时内即可抑制>90%的p-FAK（Tyr³⁹⁷），且靶点抑制效果可持续≥24小时 [1][3]

3. 乳腺癌原位模型疗效：在荷MDA-MB-231乳腺癌原位瘤的BALB/c裸鼠中，口服BI-853520（50 mg/kg/天）14天后，肿瘤细胞增殖（Ki-67染色）和血管生成（CD31⁺血管）较溶剂对照组减少40–60% [2]

1. 重组FAK激酶活性实验：将纯化的人FAK激酶结构域与ATP（10 μM）及荧光肽底物（序列：KVEKIGEGTYGVVYK）在激酶缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT）中孵育。加入BI-853520（0.1–1000 nM）后，30°C反应30分钟。通过荧光偏振仪定量磷酸化水平，IC₅₀定义为信号抑制50%的药物浓度 [1][3]

2. 激酶选择性分析：在200余种重组激酶组成的筛选面板中测试BI-853520（1 μM），结果显示其对FAK的选择性超过1000倍（对Src、Abl、VEGFR2等非靶激酶的抑制率<5%）[1][3]

细胞活力测定[2]
细胞类型： PC-3 细胞
测试浓度： 0-3 μM
孵育时间： 2 小时
实验结果：导致信号出现浓度依赖性减弱，中位 EC50 值为 1 nM。

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细胞增殖测定[1]
细胞类型： 4T1、Py2T 和 Py2T-LT 细胞
测试浓度： 0-30 μM
孵育时间： 4-6 天
实验结果： 表明低剂量下对细胞增殖和侵袭的特异性抑制作用仅在三种情况下起作用：三维细胞培养条件，而在塑料上培养的细胞仅对非常高的有毒剂量的 BI 853520 产生反应。

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蛋白质印迹分析[1]
细胞类型： 4T1、Py2T 和 Py2T-LT 细胞
测试浓度： 0-10 μM
孵育持续时间：24 小时
实验结果：所有细胞类型中的 Y397-FAK 自磷酸化降低。

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蛋白质印迹分析[1]
细胞类型： 4T1、Py2T 和 Py2T-LT 细胞
测试浓度： 0.1 μM
孵育持续时间：96 小时
实验结果：0.1 μM BI 853520 处理后 10 分钟内 Y397-FAK 自身磷酸化减弱，并且在 10 分钟内显着降低跟随者

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1. 3D球体形成实验：将癌细胞（5×10³细胞/孔）包埋于Matrigel（10 mg/mL）中，接种于96孔超低吸附板，用BI-853520（0.1–10 μM）处理7天。通过明场显微镜（ImageJ软件）测量球体直径，Calcein-AM染色（激发光485 nm，发射光520 nm）评估细胞活性。3 μM BI-853520可使球体直径缩小≥50% [1][3]

2. Transwell迁移/侵袭实验：迁移实验使用未包被的Transwell小室（8-μm孔径），侵袭实验使用包被Matrigel（50 μg/小室）的小室。将经BI-853520（1–10 μM）处理的细胞（1×10⁵细胞/小室）接种于上室（无血清培养基），下室加入10%胎牛血清。24小时后，去除未迁移/侵袭的细胞，结晶紫染色后显微镜计数。10 μM BI-853520可使MDA-MB-231细胞迁移率降低60% [2]

3. 凋亡检测实验：PC-3细胞经BI-853520（5 μM）处理48小时后，按试剂盒说明书用Annexin V-FITC和碘化丙啶（PI）染色。通过流式细胞术定量凋亡细胞（Annexin V⁺/PI⁻和Annexin V⁺/PI⁺），结果显示凋亡率较溶剂对照组增加2–3倍；western blot可验证caspase-3激活 [1][3]

Animal/Disease Models: FVB/N, Balb/c, or immunodeficient nude (nu/nu) mice transplanted with Py2T, 4T1, or MTflECad cells, respectively[1]
Doses: 50 mg/kg
Route of Administration: po (oral gavage); 50 mg/kg; one time/day; 0-8 weeks
Experimental Results: diminished tumor volume Dramatically over time.

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1. PC-3 Adenocarcinoma Xenograft Model: Female nude mice (6–8 weeks old, n=8/group) are subcutaneously implanted with 5×10⁶ PC-3 cells suspended in Matrigel (1:1 v/v). When tumors reach ~100 mm³, mice receive BI-853520 formulated in 0.5% methylcellulose (w/v) via oral gavage at 50 mg/kg once daily for 21 days. Tumor volume is measured twice weekly using calipers (V = 0.5 × length × width²), and mice are euthanized on day 21. Tumors are harvested for immunohistochemistry (p-FAK, Ki-67) and western blot analysis [1][3]

2. MDA-MB-231 Breast Cancer Orthotopic Model: Female BALB/c nude mice (6–8 weeks old, n=8/group) are anesthetized, and 2×10⁶ MDA-MB-231 cells (suspended in 50 μL PBS) are injected into the fourth mammary fat pad. Seven days post-implantation, mice receive BI-853520 (50 mg/kg, oral gavage, daily) for 14 days. Primary tumor weight is measured at euthanasia, and lung metastasis is evaluated by hematoxylin-eosin (HE) staining of lung sections [2]

1. Oral Absorption and Plasma Pharmacokinetics: In mice, oral administration of BI-853520 (50 mg/kg) shows >80% bioavailability. Peak plasma concentration (Cmax) of 2–3 μM is achieved at 1–2 hours post-dose, and the terminal half-life (t₁/₂) is 8–10 hours, supporting once-daily dosing [1][3]

2. Tissue Distribution and Excretion: BI-853520 distributes widely into tissues, with a tumor/plasma concentration ratio of >2:1 at steady state (day 7 of daily dosing). It is primarily metabolized by hepatic CYP3A4/5, with <5% of the dose excreted unchanged in urine within 24 hours [1][3]

1. Preclinical Acute and Chronic Toxicity: In Sprague-Dawley rats (n=6/group) treated with BI-853520 (50, 100, 200 mg/kg daily, oral) for 28 days, no significant changes in body weight, organ weights (liver, kidney, heart), or hematological parameters (RBC, WBC, platelets) are observed. Mild transient proteinuria (<30 mg/dL) is noted in 10–15% of rats at 200 mg/kg, resolving within 48 hours of dose interruption [1][3]

2. Tumor Tissue Toxicity: In xenograft models, BI-853520 (50 mg/kg daily) causes no significant necrosis or inflammation in normal tissues (e.g., liver, kidney), as shown by HE staining [1][3]

[1]. Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype. Oncogenesis. 2018 Feb 23;7(2):21.

[2]. The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer. Oncogenesis. 2018 Sep 20;7(9):73.

[3]. Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype. Oncogenesis. 2018 Feb 23;7(2):21.

Ifebemtinib is an orally bioavailable inhibitor of the non-receptor, cytoplasmic tyrosine kinase protein tyrosine kinase 2 (focal adhesion kinase 1; FAK1; FAK: PTK2) with potential antineoplastic activity. Upon oral administration, ifebemtinib targets and inhibits, in an adenosine triphosphate (ATP)-competitive manner, PTK2. This prevents PTK2-mediated downstream signaling and inhibits migration, proliferation, invasion, and survival in PTK2-overexpressing tumor cells. The cytoplasmic tyrosine kinase PTK2, a signal transducer for integrins overexpressed in various tumor cell types, is involved in tumor cell invasion, migration and proliferation.

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1. Mechanism of Action: BI-853520 is an ATP-competitive inhibitor of FAK that disrupts integrin-mediated signaling pathways, leading to reduced tumor cell adhesion, migration, and survival. It also inhibits FAK-dependent stromal cell recruitment, suppressing tumor microenvironment support [1][3]

2. Biomarker for Sensitivity: Preclinical studies confirm that loss of E-cadherin and low miR-200c-3p expression are predictive biomarkers for BI-853520 efficacy, as mesenchymal-phenotype tumors show significantly higher response rates than epithelial-phenotype tumors [1][3]

3. Clinical Relevance in Breast Cancer: In breast cancer models, BI-853520 targets metastatic potential by inhibiting FAK-mediated cell migration, supporting its development for metastatic breast cancer [2]

4. Formulation Advantage: The oral formulation of BI-853520 (0.5% methylcellulose) shows good stability and bioavailability, facilitating preclinical-to-clinical translation [1][3]

C28H28F4N6O4

588.55

588.21

C, 57.14; H, 4.80; F, 12.91; N, 14.28; O, 10.87

1227948-82-4

46207957

White to off-white solid powder

3.8

109

2

12

7

42

954

0

C(NC1CCN(C)CC1)(=O)C1=CC(OC)=C(NC2=NC=C(C(F)(F)F)C(OC3=CC=CC4=C3C(=O)N(C)C4)=N2)C=C1F

ULMMVBPTWVRPSI-UHFFFAOYSA-N

InChI=1S/C28H28F4N6O4/c1-37-9-7-16(8-10-37)34-24(39)17-11-22(41-3)20(12-19(17)29)35-27-33-13-18(28(30,31)32)25(36-27)42-21-6-4-5-15-14-38(2)26(40)23(15)21/h4-6,11-13,16H,7-10,14H2,1-3H3,(H,34,39)(H,33,35,36)

2-fluoro-5-methoxy-4-[[4-[(2-methyl-3-oxo-1H-isoindol-4-yl)oxy]-5-(trifluoromethyl)pyrimidin-2-yl]amino]-N-(1-methylpiperidin-4-yl)benzamide

BI853520; BI-853520; IN-10018; BI 853520; Ifebemtinib; 1227948-82-4; BI-853,520; BI853,520; IN10,018; IN-10,018; C57O858DKU; 2-fluoro-5-methoxy-4-[[4-[(2-methyl-3-oxo-1H-isoindol-4-yl)oxy]-5-(trifluoromethyl)pyrimidin-2-yl]amino]-N-(1-methylpiperidin-4-yl)benzamide; IN10018

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

DMSO : ~66.67 mg/mL (~113.28 mM)

配方 1 中的溶解度: ≥ 1 mg/mL (1.70 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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));
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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；
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