Entrectinib (NMS-E 628; RXDX101; ROZLYTREK)   中文名称: 恩曲替尼

TrkA (IC50 = 1 nM); TrkB (IC50 = 3 nM); TrkC (IC50 = 5 nM); ROS1 (IC50 = 12 nM); ALK (IC50 = 7 nM)

体外活性：Entrectinib 选择性阻断 ALK 依赖性细胞系的增殖，并有效抑制 ALK 依赖性信号传导。 Entrectinib 还高度抑制带有 EML4-ALK 重排的 NSCLC 细胞系 NCI-H2228 的细胞生长。激酶测定：Entrectinib 是一种有效的口服 Trk、ROS1 和 ALK 抑制剂；抑制 TrkA、TrkB、TrkC、ROS1 和 ALK，IC50 值分别为 1、3、5、12 和 7 nM。细胞测定：将 NLF、NLF-TrkB、SY5Y 或 SY5Y-TrkB 细胞接种于 96 孔板中，并暴露于不同浓度的药物（1、5、10、20、30、50 和 100 nM 的 entrectinib，1.5 μM Irino 和 50 μM TMZ，分别）一小时，然后添加 100 ng/mL BDNF。添加药物后 24、48 和 72 小时收获平板。使用标准 SRB 测定方案对板进行处理并分析细胞活力。

在携带 Karpas-299 和 SR-786 异种移植物的小鼠中，Entrectinib (po) 诱导肿瘤完全消退。在 NPM-ALK 转基因小鼠中，Entrectinib 诱导胸腺和淋巴结中观察到的肿瘤块完全消退。在NB异种移植模型中，Entrectinib联合治疗增强了常规化疗的疗效。

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在体内，连续十天口服携带Karpas - 299或SR - 786异种移植物的SCID小鼠，NMS - E628诱导肿瘤完全消退，离体分析表明，单次治疗后，剂量依赖性靶标调节可维持长达18小时。NMS‐E628在靶向T细胞表达人类NPM‐ALK的转基因小鼠白血病模型中也非常有效。后一种模型真实再现了人类ALCL的病理特征，用NMS - E628连续治疗NPM - ALK转基因小鼠仅3天，就能诱导胸腺和淋巴结中肿瘤块的完全消退。[3]

Entrectinib 抑制 TrkA、TrkB、TrkC、ROS1 和 ALK，IC50 值分别为 1、3、5、12 和 7 nM。它是一种强效且易于使用的 Trk、ROS1 和 ALK 口服抑制剂。

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涉及ALK酪氨酸激酶基因的染色体易位t(2;5)(p23;q35)导致NPM‐ALK融合蛋白的表达，该融合蛋白代表了间变性大细胞淋巴瘤亚群存活和增殖的驱动力。最近，在非小细胞肺癌患者中，ALK基因的染色体重排导致了一种新的融合变异EML4 - ALK，已被确定为一种低频事件，与EGFR和K - ras突变相互排斥。正如之前在NPM‐ALK中发现的那样，这种新的融合变体具有组成活性的ALK激酶，并被证明具有很强的致癌潜力。综上所述，这些发现支持了这样的假设，即ALK代表了肿瘤中含有易位ALK的ALCL和NSCLC患者癌症治疗的创新和有价值的靶点。[3]
在这里，我们进一步描述NMS - E628的临床前特征，NMS - E628是一种口服的ALK激酶活性小分子抑制剂。在广泛的人类肿瘤细胞系上的增殖分析表明，该化合物选择性地阻断ALK依赖性细胞系的增殖，并有效抑制ALK依赖性信号传导。[3]

将 NLF、NLF-TrkB、SY5Y 或 SY5Y-TrkB 细胞接种于 96 孔板中，并置于不同浓度的 entrectinib（1、5、10、20、30、50 和 100 nM、1.5 μM Irino 和 50 nM）中。 μM TMZ，分别）持续一小时。随后，添加 100 ng/mL 的 BDNF。添加药物后，在 24、48 和 72 小时后收获平板。准备好板，并使用 SRB 测定方案来分析细胞活力。

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体外实验及Western Blot分析[6]
为了确定肠替尼对TrkB磷酸化的抑制作用，在标准培养条件下，将细胞在10 cm3培养皿中培养到70-80%的合流度。细胞在2% FBS培养基中血清饥饿2小时，然后暴露于不同浓度的肠替尼(10 - 200 nM) 1小时。用100 ng/mL的TrkB配体，BDNF刺激细胞15分钟，然后收集总蛋白用于Western blots分析。用抗phospho Trk抗体(p-Trk, Tyr-490)或抗pan -Trk抗体确认Trk表达。使用抗phospho-Akt、抗phospho- erk1 /2抗体分析下游信号传导抑制作用，以总Akt、抗erk1 /2和肌动蛋白为负载对照。

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硫代胺B (SRB)测定[6]
采用Sulforhodamine B (SRB)法测定enterrectinib单用和与Irino-TMZ合用对表达trkb的NB细胞存活和生长的影响。将NLF、NLF- trkb、SY5Y或SY5Y- trkb细胞(5×103/孔)分别置于96孔板中，分别以不同浓度(1、5、10、20、30、50和100 nM的肠替尼、1.5 μM Irino和50 μM TMZ)暴露1小时，然后加入100 ng/mL的BDNF。在添加药物后24、48和72小时收获板。用标准SRB测定方案处理后分析细胞活力。所有体外实验一式三次，至少重复3次。

Male C57BL/6 mice (6-8 weeks old, 20-25 g; Bleomycin-induced pulmonary fibrosis model)[1].
20, 40, 60 mg/kg
Intragastric Administration; single daily for 7 days.

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Entrectinib (RXDX-101) is an orally available small molecule inhibitor of pan-Trk, Alk and Ros1 tyrosine kinases. It was dissolved in DMSO to obtain stocks for in vitro studies. For in vivo experiments, it was reconstituted in 0.5% methylcellulose (viscosity 400cP, 2% in H2O) containing 1% Tween 80 at a final dosing volume of 10 ml/kg (e.g., 0.2 ml for a 20 gm mouse). Entrectinib solution was stirred at RT for 30 min, and then sonicated in a water bath sonicator for 20 min. This formulation was made fresh every week. Animals were dosed BID, 7 days/week at 60 mg/kg.[6]

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In Vivo Experiments[6]
For the xenograft studies, animals were injected subcutaneously in the flank with 1 × 107 SY5Y-TrkB cells in 0.1 ml of Matrigel (BD Bioscience, Palo Alto, CA). Tumors were measured 2 times per week in 3 dimensions, and the volume calculated as follows: [(0.523xLxWxW)/1000]. Body weights were measured at least twice a week, and the dose of compound was adjusted accordingly. Treatment with entrectinib, Irino and TMZ started about 15–17 days after tumor inoculation when the average tumor size was 0.2 cm3. Mice were sacrificed when tumor volume reached 3 cm3. Tumors were harvested and flash frozen on dry ice for analysis of protein expression using Western blot. Tumor lysates were obtained using Fast Prep 24 System in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktail. The following antibodies were used for the Western blot: anti-TrkB (Abcam), anti-phospho- TrkB (Tyr816); anti-Trk (pan-Trk); anti-phospho-Akt (Ser473); anti-Akt; anti-phosphop44/42 Erk (Thr202/Tyr204); anti-p44/42 Erk; anti-Phospho-PLCγ1 (Tyr783) and anti- PLCγ1. Plasma was obtained at different times points after dosing for PK/PD studies.[6]

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Pharmacokinetic studies[6]
Entrectinib was dosed at 60 mg/kg BID, for the entire duration of the study. After the final dose was given, the blood samples were drawn from 4 mice per time point via retro-orbital bleeding and collected in heparinized tubes on wet ice. The plasma was then separated by centrifugation at 1200 g for 10 minutes at 4°C. The concentration of entrectinib (free base) was measured by LC-MS-MS. The pharmacokinetic analysis was performed using the Watson system, and plotted using GraphPad Prism (mean ±SD).

Absorption, Distribution and Excretion
Entrectinib has a Tmax of 4-5 h after administration of a single 600 mg dose. Food does not produce a significant effect on the extent of absorption.
After a single radio-labeled dose of entrectinib, 83% of radioactivity was present in the feces and 3% in the urine. Of the dose in the feces, 36% was present as entrectinib and 22% as M5.
Entrectinib has an apparent volume of distribution of 551 L. The active metabolite, M5, has an apparent volume of distribution of 81.1 L. Entrectinib is known to cross the blood-brain barrier.
The apparent clearance of entrectinib is 19.6 L/h while the apparent clearance of the active metabolite M5 is 52.4 L/h.

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Metabolism / Metabolites
CYP3A4 is responsible for 76% of entrectinib metabolism in humans including metabolism to the active metabolite, M5. M5 has similar pharmacological activity to entrectinib and exists at approximately 40% of the steady state concentration of the parent drug. In rats, six in vivo metabolites have been identified including N-dealkylated, N-oxide, hydroxylated, and glucuronide conjugated metabolites.

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Biological Half-Life
Entrectinib has a half-life of elimination of 20 h. The active metabolite, M5, has a half-life of 40 h.

Hepatotoxicity
In the prelicensure clinical trials of entrectinib in patients with NTRK fusion gene positive solid tumors and ROS1 fusion gene positive non-small cell lung cancer, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in 38% of entrectinib treated patients, but were above 5 times the upper limit of normal (ULN) in only 2% to 3% (although the incidence may have been underestimated as 4.5% of patients had no post-treatment liver function tests). In these trials that enrolled approximately 355 patients, entrectinib was discontinued early due to increased AST or ALT in 0.8% of patients. Thus, in preregistration trials of entrectinib there were no instances of clinically apparent liver injury with jaundice, but therapy was associated with a high rate of serum ALT elevations and the total clinical experience with its use has been limited. The product label for entrectinib recommends monitoring for routine liver tests before, at 2 week intervals during the first month of therapy, and monthly thereafter as clinically indicated.
Likelihood score: E* (unproven but suspect rare cause of clinically apparent liver injury).

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Protein Binding
Entrectinib is over 99% bound to plasma proteins.

[1]. Expert Opin Investig Drugs. 2015;24(11):1493-500.

[2]. Cancer Res (2015) 75 (15_Supplement): 5390.

[2]. Mol Cancer Ther (2009) 8 (12_Supplement): A244.

[4]. Thorac Cancer. 2022 Nov;13(21):3032-3041.

[5]. Clin Cancer Res. 2021 Feb 15;27(4):1184-1194.

[6]. Cancer Lett. 2016 Mar 28;372(2):179-86.

Pharmacodynamics
Entrectinib and its active metabolite suppress several pathways which contribute to cell survival and proliferation. This suppression shifts the balance in favor of apoptosis thereby preventing cancer cell growth and shrinking tumors.

C31H34F2N6O2

560.64

560.271

C, 66.41; H, 6.11; F, 6.78; N, 14.99; O, 5.71

1108743-60-7

25141092

Off-white to light yellow solid powder

1.3±0.1 g/cm3

717.5±60.0 °C at 760 mmHg

387.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.672

5.66

85.52

3

8

7

41

847

0

FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C1C([H])=C([H])C2=C(C=1[H])C(=NN2[H])N([H])C(C1C([H])=C([H])C(=C([H])C=1N([H])C1([H])C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)F

HAYYBYPASCDWEQ-UHFFFAOYSA-N

InChI=1S/C31H34F2N6O2/c1-38-8-10-39(11-9-38)25-3-4-26(29(19-25)34-24-6-12-41-13-7-24)31(40)35-30-27-17-20(2-5-28(27)36-37-30)14-21-15-22(32)18-23(33)16-21/h2-5,15-19,24,34H,6-14H2,1H3,(H2,35,36,37,40)

N-[5-[(3,5-difluorophenyl)methyl]-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide

Entrectinib, RXDX-101, NMS-E628; RXDX101; RXDX 101; Rozlytrek; RXDX-101; NMS-E628; Entrectinib (RXDX-101); entrectinibum; Entrectinib(rxdx-101); RXDX-101; NMS E628; NMS-E-628; trade name: ROZLYTREK

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 (4.46 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 (4.46 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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: ≥ 2.08 mg/mL (3.71 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 20.8 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 4 中的溶解度: 2.08 mg/mL (3.71 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100μL 20.8mg/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 5 中的溶解度: ≥ 2.08 mg/mL (3.71 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 20.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 6 中的溶解度: 5 mg/mL (8.92 mM) in 0.5% MC 0.5% Tween-80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。