LY2874455

FGFR2 (IC50 = 2.6 nM); FGFR1 (IC50 = 2.8 nM); FGFR4 (IC50 = 6 nM); FGFR3 (IC50 = 6.4 nM); VEGFR2 (IC50 = 7 nM)

体外活性：在 RT-112 细胞、HUVEC、KATO-III 细胞和 SNU-16 细胞中，LY2874455 抑制 FGF/FGFR 介导的信号传导活性。 LY2874455 在 KMS-11、OPM-2、SNU-16 和 KATO-III 细胞中显示出 FGFR 依赖性抗增殖作用。激酶测定：用于检测 FGFR 磷酸化活性的生化过滤结合测定；反应混合物含有 8 mM Tris-HCl (pH 7.5)、10 mmol/L HEPES、5 mM 二硫苏糖醇、10 μM ATP、0.5 μCi 33P-ATP、10 mM MnCl2、150 mM NaCl、0.01% Triton X-100、4%二甲基亚砜、0.05 mg/mL 聚（Glu:Tyr）（4:1，平均分子量 20-50 kDa）以及 7.5、7.5 和 16 ng FGFR1、FGFR3 和 FGFR4，分别在室温放置 30 分钟，然后用 10% H3PO4 终止。将反应混合物转移至 96 孔 MAFB 过滤板，用 0.5% H3PO4 洗涤 3 次。风干后，用 Trilux 读数器读取板。细胞测定：细胞（每孔2,000个）首先在RPMI中生长6小时，并用LY2874455在37℃下处理3天。细胞在37℃染色4小时，然后在37℃溶解1小时。最后，使用读板器 (Spectra Max Gemini XS) 在 570 nm 处读取板的读数。

LY2874455 可有效抑制小鼠心脏组织中 FGF 诱导的 Erk 磷酸化，TED50 和 TED90 值分别为 1.3 和 3.2 mg/kg。在携带 RT-112、OPM-2 (DSMZ)、SNU-16 或 NCI-H460 异种移植物的小鼠中，LY2874455（3 mg/kg po）导致肿瘤生长的剂量依赖性抑制。

反应混合物包括以下成分：8 mM Tris-HCl (pH 7.5)、10 mM HEPES、5 mM 二硫苏糖醇、10 μM ATP、0.5 μCi ³³P-ATP、10 mM MnCl₂ 、150 mM NaCl、0.01% Triton X-100、4% DMSO、0.05 mg/mL 聚（Glu:Tyr）（4:1，平均分子量 20–50 kDa）和 7.5、7.5、分别为 16 ng 和 16 ng FGFR1、FGFR3 和 FGFR4。然后将反应混合物在室温下孵育30分钟，然后用10% H₃PO₄终止。将反应混合物转移至 96 孔 MAFB 过滤板后，进行 3 次 0.5% H₃PO₄ 洗涤。板风干后，使用 Trilux 读数器读取板的读数[1]。

采用多种多发性骨髓瘤细胞系——KMS-11 和 OPM-2 细胞、L-363 和 U266 细胞。每孔 2000 个细胞首先在 RPMI 中生长六小时，然后在 37°C 下应用 LY2874455 三天。 37°C 染色四小时后，细胞在相同温度下溶解一小时。最后，酶标仪在 570 nm 处读取酶标板[1]。

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基于细胞的Acumen和AlphaScreen SureFire测定分别检测膀胱癌症和人脐静脉内皮细胞中FGF9-和FGF2诱导的细胞外信号调节激酶磷酸化[1]
本文中使用的所有细胞系均未经过鉴定。过夜生长后，洗涤RT-112细胞（DSMZ），在37°C下在含有20 mg/mL牛血清白蛋白（BSA）的RPMI 16409中孵育3小时，并在37°℃下用LY2874455处理1小时，然后加入FGF9（500 ng/mL）20分钟。用甲醛（3.7%）固定板，然后用PBS洗涤3次，在-20°C下用冷甲醇孵育30分钟。用PBS洗涤板3次，并在4°C下振荡培养过夜。在加入磷酸化细胞外信号调节激酶（p-Erk）抗体后，将板在室温下孵育1小时，洗涤，然后用Alexa Fluor 488（Invitegen）孵育。用Acumen Explorer在加入碘化丙啶后读取平板。 在内皮基础培养基中生长过夜后，洗涤人脐静脉内皮细胞（HUVEC），并在37°C/5%CO2的相同培养基（1.5%血清和20mg/mL BSA）中孵育3小时。在加入LY2874455和FGF2（50 ng/mL，Sigma）15分钟后，将平板孵育1小时。移除培养基并加入裂解缓冲液后，在室温下摇动培养板10分钟。将裂解物转移到装有10μL反应混合物LY2874455的384孔板（Nunc）中。将平板密封，在室温下孵育2小时，并用EnVision06阅读器读取。

Mice: Mice (female, CD-1 nu/nu for RT-112, OPM-2, and NCI-H460 cells, and female, severe combined immunodeficient for SNU-16 cells) have their rear flanks subcutaneously implanted with a mixture of RT-112, OPM-2 (DSMZ), SNU-16, and NCI-H460 cells (RT-112: 2×10⁶ per animal; OPM-2: 10⁷ per animal; SNU-16: 10⁶ per animal; and NCI-H460: 3×10⁶ per animal) and Matrigel (1:1) mixed together. Solid tumors are grown by the implanted tumor cells. After tumors grow to a size of about 150 mm³, the animals are given oral doses of LY2874455 once (every day) or twice a day (TED90) at a dose of approximately 1 mg/kg (TED50) or 3 mg/kg (TED₉₀) in 10% Acacia to test the drug's effectiveness in these models. Twice a week, the body weight and tumor volume are measured.
Rats: In each group of four male rats, LY2874455 (1, 3, and 10 mg/kg) is dosed on day 0 and the vehicle (1% hydroxyethylcellulose, 0.25% polysorbate 80, and 0.05% Dow Corning antifoam 1510-US in purified water) is administered on day 1. After vehicle administration on day 1, at least 120 minutes of control data are gathered. After the last animal is dosed on day 0, data are gathered for about 20 hours.

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MSD-based in vivo target inhibition assays for measuring FGF2-induced Erk and VEGF-induced VEGFR2 phosphorylation in mouse heart tissues and also p-FRS2 in tumors[1]
Female nude mice (CD-1 nu/nu) were acclimated for 1 week before treatment. Animals were administered with LY2874455 formulated in 10% Acacia by oral gavage. Two hours after dosing, the animals were intravenously injected with mouse FGF2 (6 μg per animal) and sacrificed 10 minutes after injection. Animal heart was homogenized in cold lysis buffer containing phosphatase inhibitors. After centrifugation, the supernatants were collected and analyzed by MSD phospho-Erk ELISA (MSD) to determine tissue p-Erk level. The inhibitory activity of LY2874455 against VEGFR2 was assessed as described earlier except VEGF (6 μg per animal) and MSD phospho-Kdr ELISA (MSD) were used. The ELISA procedures were the same per manufacturer's recommendation (MSD) except that 0.2% SDS is added to the lysis buffer. TED50 (or TEC50) and TED90 (or TEC90) were defined as the dose or the concentration necessary to achieve 50% and 90% inhibition at this time point, respectively. SNU-16 and OPM-2 tumor xenograft tissues were homogenized in a Tris lysis buffer (MSD) containing beads. The lysate preparation and p-FRS2 determination were carried out as described earlier. To determine compound exposure, plasma samples were prepared and analyzed with a liquid chromatography/mass spectrometer–mass spectrometer system. Pharmacokinetic parameters were calculated with Watson LIMS information management system.

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Tumor xenograft models for assessing efficacy of LY2874455[1]
RT-112, OPM-2 (DSMZ), SNU-16, and NCI-H460 cells were grown as described earlier. The cells (RT-112: 2 × 106 per animal; OPM-2: 107 per animal; SNU-16: 106 per animal; and NCI-H460: 3 × 106 per animal) were mixed with Matrigel (1:1) and implanted subcutaneously into the rear flank of the mice (female, CD-1 nu/nu from Charles River Laboratories for RT-112, OPM-2, and NCI-H460 cells and female, severe combined immunodeficient from Charles River for SNU-16 cells). The implanted tumor cells grew as solid tumors. To test the efficacy of LY2874455in these models, the animals were orally dosed with approximately 1 mg/kg (TED50) or 3 mg/kg (TED90) of LY2874455 in 10% Acacia once (every day) or twice a day after tumors reached approximately 150 mm3. The tumor volume and body weight were measured twice a week.

[1]. Mol Cancer Ther . 2011 Nov;10(11):2200-10.

LY2874455 has been used in trials studying the treatment of Advanced Cancer.
Pan-FGFR Inhibitor LY2874455 is an orally bioavailable pan-inhibitor of fibroblast growth factor receptor (FGFR) family proteins, with potential antineoplastic activity. Upon oral administration, FGFR inhibitor LY2874455 binds to and inhibits FGFR subtypes 1 (FGFR1), 2 (FGFR2), 3 (FGFR3) and 4 (FGFR4), which results in the inhibition of FGFR-mediated signal transduction pathways. This inhibits both tumor angiogenesis and proliferation of FGFR-overexpressing tumor cells. FGFR, a family of receptor tyrosine kinases upregulated in many tumor cell types, plays a key role in cellular proliferation, cell survival and angiogenesis.

C21H19CL2N5O2

444.31

443.091

C, 56.77; H, 4.31; Cl, 15.96; N, 15.76; O, 7.20

1254473-64-7

46944259

Yellow solid powder

1.4±0.1 g/cm3

672.6±55.0 °C at 760 mmHg

360.5±31.5 °C

0.0±2.2 mmHg at 25°C

1.683

3.88

88.85

2

5

7

30

576

1

ClC1=CN=CC(Cl)=C1[C@@H](C)OC2=CC=C(NN=C3/C=C/C4=CN(CCO)N=C4)C3=C2

GKJCVYLDJWTWQU-CXLRFSCWSA-N

InChI=1S/C21H19Cl2N5O2/c1-13(21-17(22)10-24-11-18(21)23)30-15-3-5-20-16(8-15)19(26-27-20)4-2-14-9-25-28(12-14)6-7-29/h2-5,8-13,29H,6-7H2,1H3,(H,26,27)/b4-2+/t13-/m1/s1

2-[4-[(E)-2-[5-[(1R)-1-(3,5-dichloropyridin-4-yl)ethoxy]-1H-indazol-3-yl]ethenyl]pyrazol-1-yl]ethanol

LY 2874455; LY-2874455; LY2874455; 1254473-64-7; LY-2874455; UNII-E9M363811V; LY 2874455; 2-[4-[(E)-2-[5-[(1R)-1-(3,5-dichloropyridin-4-yl)ethoxy]-1H-indazol-3-yl]ethenyl]pyrazol-1-yl]ethanol; 1H-Pyrazole-1-ethanol, 4-((1E)-2-(5-((1R)-1-(3,5-dichloro-4-pyridinyl)ethoxy)-1H-indazol-3-yl)ethenyl)-; CHEMBL3828009; LY2874455

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: ≥ 2.5 mg/mL (5.63 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (5.63 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.63 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), 悬浮液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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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配方 4 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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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。