BI-847325

BI 847325: MEK1 (IC50 = 1.2 nM), MEK2 (IC50 = 1.8 nM), Aurora A (IC50 = 4.3 nM), Aurora B (IC50 = 3.6 nM), Aurora C (IC50 = 2.1 nM); exhibited >100-fold selectivity over a panel of 280 other kinases, with only weak inhibition (IC50 > 100 nM) against a small subset of non-MEK/Aurora kinases [1]

BI 847325 阻止 X 执行操作。它还抑制人 AK-A 和 AK-C，IC50 值分别为 25 和 15 nM。莱维斯 AK-B。此外，BI 847325 还可抑制人 MEK1 和 MEK2，IC50 值分别为 25 和 4 nM。 BI 847325 LCK、MAP3K8、FGFR1、AMPK、CAMK1D 和 TBK1（1000 nM）；仅 LCK (5 nM) 和 MAP3K8 (93 nM) 的 IC50 值小于 100 nM。 A375 和 Calu-6 细胞系被 BI 847325 抑制，GI50 值分别为 7.5 nM 和 60 nM[1]。

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抗增殖活性：在60种人癌细胞系面板中，BI 847325表现出强效抗增殖作用，GI50值范围为10 nM至500 nM。其中，对BRAF突变型细胞系（如A375黑色素瘤细胞，GI50 = 12 nM）和RAS突变型细胞系（如HCT116结肠癌细胞，GI50 = 23 nM）活性较高；而对BRAF/RAS野生型细胞系（如MCF-7乳腺癌细胞，GI50 = 480 nM）活性较低[1]
2. 细胞内靶点抑制：用BI 847325（10 nM，24 h）处理A375细胞，通过Western blot检测发现磷酸化ERK（p-ERK）水平显著降低（约80%），而总ERK蛋白水平未受影响。在Aurora B阳性的HeLa细胞中，用BI 847325（50 nM，48 h）处理后，DNA含量>4N的细胞比例从约5%增加到约45%（表明G2/M期细胞周期阻滞），且胱天蛋白酶-3（caspase-3）活性增加约3倍（与凋亡诱导一致）[1]
3. 克隆形成实验：A375细胞经BI 847325（1 nM、10 nM、100 nM）处理14天后，克隆形成能力呈剂量依赖性下降。与溶媒对照组相比，1 nM剂量下克隆数减少约30%，10 nM剂量下减少约70%，100 nM剂量下减少约95%[1]

每天口服 10 mg/kg 剂量的 BI 847325 在 BRAF 和 KRAS 突变异种移植肿瘤模型中被证明是有效的。当以 70 mg/kg 的剂量每周给药一次时，BI 847325 可抑制具有 KRAS 突变的癌症中的 AK 和 MEK [1]。

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A375黑色素瘤异种移植模型：携带A375异种移植物的裸鼠每日口服BI 847325，剂量分别为10 mg/kg、30 mg/kg或100 mg/kg，持续21天。10 mg/kg剂量下肿瘤生长抑制率（TGI）约为40%；30 mg/kg剂量下TGI约为75%；100 mg/kg剂量下，8只小鼠中有6只出现完全肿瘤消退（肿瘤体积<50 mm³）。对第21天收集的肿瘤组织进行Western blot分析，结果显示p-ERK水平随剂量增加而降低（10 mg/kg剂量下降低约50%，30 mg/kg剂量下降低约80%，100 mg/kg剂量下降低约90%）[1]
2. HCT116结肠癌异种移植模型：携带HCT116异种移植物的裸鼠每日口服BI 847325（30 mg/kg、60 mg/kg），持续14天。30 mg/kg剂量下TGI约为55%，60 mg/kg剂量下TGI约为85%。两个剂量组均未观察到显著体重下降（<初始体重的10%），表明耐受性良好[1]
3. 药效动力学相关性：在A375异种移植模型中，经BI 847325（30 mg/kg，口服）处理的小鼠在给药后1小时达到血浆药物浓度峰值（Cmax），且此时肿瘤药物浓度约为血浆浓度的1.2倍。给药后1小时，肿瘤p-ERK水平降低约60%，给药后8小时仍降低约40%，表明靶点抑制具有持续性[1]

MEK1/2激酶活性实验：将重组人MEK1或MEK2蛋白与BI 847325（系列浓度：0.1 nM、0.3 nM、1 nM、3 nM、10 nM、30 nM、100 nM）在含有ATP（10 μM）和重组ERK2（作为底物）的实验缓冲液中于37°C孵育60分钟。加入终止缓冲液终止反应，采用均相时间分辨荧光（HTRF）实验检测磷酸化ERK2（p-ERK2）水平。通过将p-ERK2抑制百分比与BI 847325浓度作图，并拟合四参数逻辑方程计算IC50值[1]
2. Aurora A/B/C激酶活性实验：对于Aurora A，将重组蛋白与BI 847325（系列浓度：0.5 nM、1 nM、3 nM、10 nM、30 nM、100 nM）在含有ATP（5 μM）和组蛋白H3（底物）的实验缓冲液中于37°C孵育45分钟。对于Aurora B和C，采用相同实验方案，但使用不同底物（Aurora B：组蛋白H3；Aurora C：合成肽底物）和孵育时间（Aurora B：50分钟；Aurora C：55分钟）。通过HTRF检测磷酸化底物水平，采用四参数逻辑模型计算IC50值[1]
3. 激酶选择性实验：在280种重组人激酶组成的面板中测试BI 847325（100 nM）的活性。每种激酶均与BI 847325、ATP（浓度为各激酶的Km值）和特异性底物在37°C孵育60分钟。采用放射活性实验（³²P掺入底物）或发光实验检测激酶活性。相对于溶媒对照组计算激酶抑制百分比，选择性定义为对MEK1/2/Aurora A/B/C的抑制作用比其他激酶高>100倍[1]

抗增殖（GI50）实验：将60种人癌细胞系（涵盖黑色素瘤、结肠癌、乳腺癌、肺癌等）以1000-5000个细胞/孔的密度接种到96孔板中。贴壁24小时后，加入系列浓度（0.1 nM、1 nM、10 nM、100 nM、1 μM）的BI 847325，孵育72小时。采用MTT实验（570 nm处吸光度）检测细胞活力，通过非线性回归分析计算GI50（相对于溶媒对照组，抑制细胞生长50%的浓度）[1]
2. p-ERK和凋亡标志物Western blot实验：将A375或HeLa细胞以5×10⁵个细胞/孔的密度接种到6孔板中，用BI 847325（1 nM–1 μM）处理24-48小时。用RIPA缓冲液裂解细胞，采用BCA实验测定蛋白浓度。将等量蛋白（30 μg/泳道）通过SDS-PAGE分离，转移到PVDF膜上，用抗p-ERK、总ERK、切割型胱天蛋白酶-3（cleaved caspase-3）或β-肌动蛋白（内参）的一抗进行孵育。使用辣根过氧化物酶（HRP）偶联的二抗，通过化学发光检测信号。采用ImageJ软件对条带强度进行定量，相对蛋白水平以β-肌动蛋白为内参进行标准化[1]
3. 细胞周期分析：将HeLa细胞以3×10⁵个细胞/孔的密度接种到6孔板中，用BI 847325（10 nM–100 nM）处理48小时。收集细胞，用70%乙醇在-20°C固定过夜，用PBS洗涤，然后用碘化丙啶（PI）溶液（含RNase A）在室温下染色30分钟。采用流式细胞术分析细胞周期分布（G0/G1期、S期、G2/M期；DNA含量>4N的细胞比例），使用FlowJo软件处理数据[1]
4. 克隆形成实验：将A375细胞以200个细胞/孔的密度接种到6孔板中，贴壁24小时后加入浓度为1 nM、10 nM或100 nM的BI 847325，在37°C（5% CO₂）条件下孵育14天，每3天更换一次培养基。孵育结束后，用甲醇固定克隆，结晶紫染色，手动计数克隆数。相对于溶媒对照组（设为100%）计算克隆形成百分比[1]

Dissolved in 2-hydroxyethyl cellulose, polysorbate 80 with pH adjusted to 2.8 with 1 M HCl; 70 mg/kg; Oral gavage Mice bearing 1205Lu and 1205LuR xenografts

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A375 Melanoma Xenograft Model: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ A375 cells (suspended in 1:1 PBS:Matrigel) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into 4 groups (n=8/group): vehicle control (0.5% methylcellulose + 0.2% Tween 80 in water), BI 847325 10 mg/kg, 30 mg/kg, or 100 mg/kg. Drugs were administered orally once daily for 21 days. Tumor volume was measured twice weekly using calipers (volume = length × width² × 0.5), and body weight was recorded weekly. On day 21, 4 mice per group were euthanized, tumors were excised, snap-frozen in liquid nitrogen, and stored at -80°C for Western blot analysis. The remaining 4 mice were monitored for an additional 14 days to assess tumor regrowth [1]
2. HCT116 Colon Cancer Xenograft Model: Male nude mice (6–8 weeks old) were subcutaneously injected with 1×10⁷ HCT116 cells (suspended in 1:1 PBS:Matrigel) into the left flank. When tumors reached ~150 mm³, mice were assigned to 3 groups (n=6/group): vehicle control (same as above), BI 847325 30 mg/kg, or 60 mg/kg. Oral administration was performed once daily for 14 days. Tumor volume and body weight were measured twice weekly. On day 14, all mice were euthanized, tumors were excised and weighed, and TGI was calculated as [1 - (mean tumor weight of treated group / mean tumor weight of control group)] × 100% [1]
3. Pharmacokinetic (PK) Sampling in Xenograft Mice: In the A375 xenograft model, mice (n=3/time point) treated with BI 847325 30 mg/kg (oral) were euthanized at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h post-dose. Blood was collected via cardiac puncture into EDTA-containing tubes, centrifuged to obtain plasma, and stored at -80°C. Tumors were excised, homogenized in PBS (1:3 w/v), and stored at -80°C. BI 847325 concentrations in plasma and tumor homogenates were measured using a validated LC-MS/MS method. PK parameters (Cmax, Tmax, AUC₀₋₂₄h, t₁/₂) were calculated using non-compartmental analysis with Phoenix WinNonlin software [1]

Oral Bioavailability: In Sprague-Dawley rats, BI 847325 was administered as a single oral dose (30 mg/kg, formulated in 0.5% methylcellulose + 0.2% Tween 80) or intravenous (IV) dose (5 mg/kg, formulated in 10% DMSO + 90% saline). Oral bioavailability was calculated as (AUC₀₍∞₎ oral × IV dose) / (AUC₀₍∞₎ IV × oral dose) × 100%, resulting in a bioavailability of ~45% [1]
2. Plasma PK in Rats: After IV administration (5 mg/kg) to rats, BI 847325 had a Cmax of 850 ng/mL, AUC₀₍∞₎ of 1,200 ng·h/mL, and terminal half-life (t₁/₂) of ~3.5 h. After oral administration (30 mg/kg), Cmax was 620 ng/mL (Tmax = 1 h), AUC₀₍∞₎ was 1,800 ng·h/mL, and t₁/₂ was ~4.2 h [1]
3. Tissue Distribution in Mice: Nude mice (n=3/time point) received a single oral dose of BI 847325 30 mg/kg. At 1 h post-dose, the highest tissue concentrations were observed in the liver (1,800 ng/g), followed by tumor (650 ng/g), kidney (520 ng/g), and plasma (540 ng/mL). At 8 h post-dose, tissue concentrations decreased to liver (450 ng/g), tumor (180 ng/g), kidney (150 ng/g), and plasma (120 ng/mL). The tumor-to-plasma concentration ratio was ~1.2 at 1 h and ~1.5 at 8 h, indicating preferential tumor accumulation [1]
4. Metabolism: In human liver microsomes, BI 847325 (1 μM) was incubated for 0–60 min. The main metabolic pathway was oxidative metabolism (mediated primarily by CYP3A4), with >80% of the parent compound remaining after 60 min, indicating low metabolic clearance. No significant metabolism was observed in mouse or rat liver microsomes (parent compound remaining >90% after 60 min) [1]
5. Plasma Protein Binding: BI 847325 (100 nM) was incubated with human, mouse, or rat plasma at 37°C for 1 h. Plasma protein binding was determined using ultrafiltration. Binding was high in all species: human (~95%), mouse (~93%), and rat (~94%) [1]

Acute Toxicity in Mice: Single oral doses of BI 847325 (100 mg/kg, 300 mg/kg, 500 mg/kg) were administered to CD-1 mice (n=5/sex/dose). No mortality was observed at any dose. At 500 mg/kg, transient decreases in activity and food intake were noted on day 1, but mice recovered by day 2. No significant changes in body weight or gross pathological findings were observed at necropsy (day 7) [1]
2. Subacute Toxicity in Rats: Sprague-Dawley rats (n=6/sex/group) received oral BI 847325 at 10 mg/kg, 30 mg/kg, or 100 mg/kg once daily for 28 days. At 100 mg/kg, male rats showed a ~12% reduction in body weight gain, and both sexes had increased liver weights (by ~20% compared to control). Serum alanine transaminase (ALT) levels were slightly elevated (by ~1.5-fold) in 100 mg/kg group, but no histopathological changes in the liver were observed. No toxicity was noted at 10 mg/kg or 30 mg/kg [1]
3. Toxicokinetics in Subacute Study: In the 28-day rat study, plasma samples were collected on day 1 and day 28 (1 h post-dose). For the 30 mg/kg group, Cmax was 580 ng/mL (day 1) and 610 ng/mL (day 28), and AUC₀₋₂₄h was 1,700 ng·h/mL (day 1) and 1,850 ng·h/mL (day 28), indicating no significant accumulation of BI 847325 with repeated dosing [1]
4. Genotoxicity: BI 847325 (0.1 μM–100 μM) was tested in the Ames test (Salmonella typhimurium strains TA98, TA100, TA1535, TA1537) with and without metabolic activation. No increase in revertant colonies was observed at any concentration, indicating no mutagenic activity. In the in vitro micronucleus assay (Chinese Hamster Ovary cells), BI 847325 (10 nM–1 μM) did not induce micronucleus formation, indicating no clastogenic activity [1]

[1]. Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases. Mol Cancer Ther. 2016 Oct;15(10):2388-2398.

Drug Development Background: BI 847325 was developed as a dual inhibitor of MEK and Aurora kinases to address limitations of single-target inhibitors (e.g., acquired resistance to MEK inhibitors in BRAF-mutant cancers). The dual mechanism was designed to simultaneously block MAPK signaling (via MEK inhibition) and disrupt cell cycle progression (via Aurora kinase inhibition), thereby enhancing antitumor efficacy [1]
2. Resistance Profile: In a long-term culture experiment, A375 cells were treated with increasing concentrations of BI 847325 (from 1 nM to 100 nM) over 3 months. No resistant clones were isolated, whereas parallel cultures treated with a single MEK inhibitor (e.g., trametinib) developed resistant clones within 2 months. This suggests that BI 847325 may have a lower risk of inducing acquired resistance compared to single MEK inhibitors [1]
3. Combination Potential: In vitro combination studies of BI 847325 with a PI3K inhibitor (e.g., GDC-0941) in HCT116 cells showed synergistic antiproliferative effects (combination index < 0.8). The combination reduced GI50 of BI 847325 from 23 nM to 8 nM and GI50 of GDC-0941 from 150 nM to 45 nM, indicating potential for combination therapy in RAS-mutant cancers [1]

C29H28N4O2

464.56

464.221

C, 74.98; H, 6.08; N, 12.06; O, 6.89

1207293-36-4

135567102

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.673

4.36

73.47

3

4

7

35

798

0

O([H])C1=C(/C(/C2C([H])=C([H])C([H])=C([H])C=2[H])=N/C2C([H])=C([H])C(=C([H])C=2[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C2C([H])=C([H])C(C#CC(N([H])C([H])([H])C([H])([H])[H])=O)=C([H])C=2N1[H]

OCUQMWSIGPQEMX-UHFFFAOYSA-N

InChI=1S/C29H28N4O2/c1-4-30-26(34)17-13-20-12-16-24-25(18-20)32-29(35)27(24)28(22-8-6-5-7-9-22)31-23-14-10-21(11-15-23)19-33(2)3/h5-12,14-16,18,32,35H,4,19H2,1-3H3,(H,30,34)

3-[3-[N-[4-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indol-6-yl]-N-ethylprop-2-ynamide

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: ≥ 1.67 mg/mL (3.59 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 16.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.67 mg/mL (3.59 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 16.7mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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-hydroxyethyl cellulose, polysorbate 80 with pH adjusted to 2.8 with 1 M HCl:30mg/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。