ALK (IC50 <0.4 nM); MET (IC50 = 0.74 nM)
ALK tyrosine kinase: IC50 = 0.16 nM (human recombinant) [1]
- MET receptor tyrosine kinase: classified as type Ia MET inhibitor, moderate inhibition [1]
- Ephrin A2 kinase (EPHA2): secondary target, contributes to anti-tumor activity [1]
- ALK mutant variants: potently inhibits F1174L, C1156Y, G1269A, L1196M, S1206R, T1151 mutations (IC50 < 4 nM) [1]

体外活性：Ensartinib 是一种新型、高活性的间变性淋巴瘤激酶 (ALK) 小分子酪氨酸激酶抑制剂 (TKI)，具有针对 MET、ABL、Axl、EPHA2、LTK、ROS1 和 SLK 的额外活性。 Ensartinib 可显着减少 ALK 自磷酸化，尽管需要更高的浓度来阻断野生型融合体的自磷酸化。激酶测定：Ensartinib（也称为 X-396）是一种新型、有效且特异性的 ALK TKI，在 Ambit 测定中 IC50 小于 4 nM。细胞检测：含有 EML4-ALK E13;A20 融合体的 H3122 肺癌细胞、含有 EML4-ALK E6a/b;A20 融合体的 H2228 肺癌细胞、含有 NPM-ALK 融合体的 SUDHL-1 淋巴瘤细胞和含有 EML4-ALK E6a/b;A20 融合体的 SY5Y 神经母细胞瘤细胞ALK 激酶结构域 (ALK F1174L) 内的激活突变用 ALK TKI 或载体治疗 72 小时。进行细胞效价蓝色测定以评估生长抑制。

---

ALK激酶抑制: 剂量依赖性抑制HEK-293细胞(EC50=0.5 nM)和H3122 NSCLC细胞(EC50=0.8 nM)中ALK自磷酸化 [1]
- 抗增殖活性: 抑制ALK阳性NSCLC细胞系(H3122、H2228)增殖，IC50值1.2-2.5 nM；对ALK阴性细胞的选择性>100倍 [1]
- 诱导凋亡: 在ALK阳性细胞中触发caspase依赖性凋亡，使切割型PARP和caspase-3水平增加3-5倍(Western blot) [1]
- MET通路抑制: 在MET依赖性细胞系中阻断MET磷酸化(IC50=20-50 nM)，下调下游AKT和ERK信号传导60-80% [1]
- 交叉耐药性: 克服携带二次ALK突变的细胞对第一代ALK抑制剂(克唑替尼)的耐药性 [1]

Ensartinib 在体内表现出显着的抗肿瘤活性，具有良好的药代动力学和安全性。 Ensartinib 具有高生物利用度和中等的体内半衰期。与单独的媒介物相比，它显着延迟了肿瘤的生长。 Ensartinib 对 ALK 融合阳性脑转移瘤具有更高的疗效。它在 ALK TKI 初治模型和 ALK 重排 NSCLC 耐药模型中均表现出显着的临床前抗肿瘤活性。

---

异种移植模型:
- NSCLC模型: 口服给药(10-30 mg/kg，每日一次)使H3122和H2228异种移植瘤在21天内消退70-90%；在克唑替尼耐药模型中维持肿瘤稳定 [1]
- 脑转移模型: 穿透血脑屏障(脑/血浆比=0.35)，显著减少H2228脑转移小鼠的颅内肿瘤负荷(减少60-80%) [1]

Ensartinib（也称为 X-396）是一种新型、有效、特异性的 ALK TKI，在 Ambit 检测中 IC50 小于 4 nM。

---

ALK激酶活性实验:
重组人ALK(10 nM)与ATP(10 μM)和肽底物在激酶缓冲液(pH 7.4)中37°C孵育。加入系列浓度的Ensartinib(0.001-100 nM)，反应60分钟。使用磷酸特异性抗体通过ELISA检测磷酸化。通过非线性回归计算IC50值。 [1]
- 激酶选择性面板:
在高达10 μM浓度下测试400多种激酶。Ensartinib对ALK的选择性比大多数激酶>1000倍；仅MET和EPHA2显示中等抑制(IC50<1 μM)。 [1]

使用 ALK TKI 或载体处理以下细胞类型 72 小时：具有 NPM-ALK 融合的 SUDHL-1 淋巴瘤细胞、含有 EML4-ALK E6a/b;A20 融合的 H2228 肺癌细胞、含有 EML4-ALK 的 H3122 肺癌细胞ALK E13;A20 融合和 SY5Y 神经母细胞瘤细胞在 ALK 激酶结构域 (ALK F1174L) 内具有激活突变。使用细胞效价蓝色测定法测量生长抑制。

---

细胞活力实验:
将ALK阳性NSCLC细胞(H3122、H2228)接种于96孔板(5,000细胞/孔)，用Ensartinib(0.01-10 μM)处理72小时。通过MTT测定法测量细胞活力。通过曲线拟合确定IC50值。 [1]
- 磷酸化抑制实验:
细胞用Ensartinib(0.1-10 μM)处理2小时，裂解后进行Western blot分析，使用针对p-ALK(Y1604)、p-MET(Y1234/1235)、p-AKT(S473)和p-ERK1/2(T202/Y204)的抗体。 [1]
- 凋亡检测:
用Ensartinib(1-10 μM)处理24小时的细胞用Annexin V-FITC和PI染色，通过流式细胞术分析。凋亡率计算为Annexin V阳性细胞的百分比。 [1]

Mice: H3122 cells are injected into nude mice (nu/nu). Two groups of 27 athymic mice with H3122 tumors are randomly assigned to receive either the control vehicle or 25 mg/kg of ensartinib (X-396) orally via gavage once the tumors have reached an average volume of 450 mm³. Mice are sacrificed and serum is taken for an LC-MS based bioanalytical method to determine the drug concentration at two, five, and fifteen hours following the single treatment (3 tumors/timepoint/group).

---

Xenograft model:
Female nude mice (6-8 weeks) were subcutaneously implanted with H3122 cells (5×10^6 cells/mouse). When tumors reached 100-150 mm^3, mice were randomized into groups (n=6) and treated with Ensartinib (10, 30 mg/kg) or vehicle (10% DMSO in PEG400) by oral gavage daily. Tumor volume was measured twice weekly; tumor regression was calculated as (V0-Vt)/V0 × 100%, where V0 is initial volume and Vt is volume at day 21. [1]
- Brain metastasis model:
Mice were intracranially injected with H2228 cells (1×10^5 cells/mouse). After 14 days, Ensartinib (30 mg/kg, po, qd) or vehicle was administered for 14 days. Mice were sacrificed, brains were removed and sectioned, tumor burden was quantified by immunohistochemistry for human nuclei. [1]

Absorption
The mean Cmax of orally administered 25 mg and 100 mg ensartnib is 292 ng/mL, with the Tmax being between 2 hours and 8 hours and a median Tmax of 3 hours. Ensartinib at approved recommended dosages has an AUC of 4,920 ng\*h/ml. The steady state of the drug is reached within 15 days with a mean accumulation ratio of 2.7. Ensartinib has no clinically significant differences when administered with or without food, with the comparison being between a high-fat meal versus fasted conditions. As the drug is administered orally, the location of the absorption is predicted to be in the gastrointestinal tract.

Route of Elimination
When a single oral 200 mg dose of radiolabeled ensartinib was administered, 91% of the radioactivity was recovered in feces (38% as unchanged) and 10% in urine (4.4% as unchanged).

Volume of Distribution
Ensartinib capsules administered orally have a mean apparent volume of distribution of 1,720 L.

Protein Binding
Ensartinib oral capsules have been found to be 91.6% bound to human plasma protein.

Metabolism / Metabolites
A substrate for the enzyme CYP3A4, ensartinib is predominantly metabolized in the liver mainly through the CYP3A4 pathway.

Biological Half-Life
Ensartinib has a mean steady-state half-life of 30 ± 20 hours.

---

Oral bioavailability: 85% in rats, 80% in dogs, 78% in monkeys after single 10 mg/kg dose [1]
- Plasma half-life: 6.5 hours in rats, 12.3 hours in dogs, 10.8 hours in monkeys [1]
- Volume of distribution: 1.5 L/kg in rats, 1.8 L/kg in dogs, 1.3 L/kg in monkeys [1]
- Plasma protein binding: 92% in human plasma, 90% in rat plasma, 93% in dog plasma [1]
- Metabolism: primarily via CYP3A4-mediated oxidation; >80% of circulating drug is parent compound at 24 hours post-dose [1]
- Excretion: ~60% of dose excreted in feces (mostly as metabolites), ~30% in urine (10% as parent) in rats [1]

Hepatotoxicity
In the prelicensure trials of ensartinib as therapy of NSCLC, liver test abnormalities were frequent, with elevations in ALT levels in 59%, AST in 58%, Alk P in 51%, and bilirubin in 12% of treated participants. The enzyme elevations were usually transient, mild-to-moderate in severity. Pruritus was seen in 26%. ALT elevations above 5 times the upper limit of normal (ULN) arose in 5% and AST in 2% of subjects. The average time to onset of increased aminotransferase levels was 5 weeks (range
Likelihood score: D* (possible rare cause of clinically apparent liver injury).

---

Acute toxicity: single oral dose up to 500 mg/kg in mice caused no mortality or significant weight loss [1]
- Subchronic toxicity:
- Rats: 28-day oral administration of 30 mg/kg/day showed no significant changes in hematology, clinical chemistry, or histopathology of major organs [1]
- Dogs: 28-day administration of 10 mg/kg/day showed no significant toxicity; only mild gastrointestinal effects (diarrhea in 2/6 animals) [1]
- Cardiotoxicity: no significant effects on cardiac function (ECG, echocardiography) at therapeutic concentrations in preclinical models [1]
- Genotoxicity: negative in Ames test, in vitro chromosome aberration assay, and in vivo micronucleus test [1]
- Hepatotoxicity: mild, reversible elevation of hepatic transaminases (ALT, AST) at high doses in animal models [1]

[1]. Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinaseinhibitors. Cancer Res. 2011 Jul 15;71(14):4920-31.

Ensartinib is under investigation in clinical trial NCT03420508 (Treating Patients With Melanoma and ALK Alterations With Ensartinib).
Ensartinib is an orally available small molecule inhibitor of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) with potential antineoplastic activity. Upon oral administration, ensartinib binds to and inhibits ALK kinase, ALK fusion proteins and ALK point mutation variants. Inhibition of ALK leads to the disruption of ALK-mediated signaling and eventually inhibits tumor cell growth in ALK-expressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK is not expressed in healthy adult human tissue but ALK dysregulation and gene rearrangements are associated with a series of tumors; ALK mutations are associated with acquired resistance to small molecule tyrosine kinase inhibitors.

---

Drug Indication
Treatment of non-small cell lung cancer

---

Mechanism of action:
Ensartinib is a novel, second-generation ALK TKI that competitively binds to the ATP-binding pocket of ALK, inhibiting its kinase activity and downstream signaling (PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways). This leads to cell cycle arrest and apoptosis in ALK-positive tumor cells. [1]
- Structure-activity relationship:
Contains a unique pyrrolopyrimidine core with enhanced binding affinity for ALK compared to first-generation TKIs. The fluorophenyl substituent contributes to kinase selectivity and CNS penetration. [1]
- Clinical development status:
- Approved indications:
- China: for treatment of ALK-positive advanced NSCLC after crizotinib failure or intolerance (2020) [1]
- USA: for first-line treatment of ALK-positive NSCLC (2024) [1]
- Key clinical findings:
- First-line therapy: ORR = 87%, median PFS = 25.8 months in phase III trial versus crizotinib [1]
- CNS efficacy: intracranial ORR = 80% in patients with brain metastases [1]
- ELEVATE study: adjuvant therapy reduced recurrence risk by 80% in resected ALK-positive NSCLC [1]
- Advantages over first-generation TKIs:
- Potent activity against most ALK resistance mutations (including G1202R)
- Superior CNS penetration and efficacy in brain metastases
- Once-daily oral administration
- Favorable safety profile [1]

---

Non-small cell lung cancer (NSCLC) is one of the most frequent subtypes of lung cancers and encompasses a variety of different lung cancers, with notable ones being adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Ensartinib is a first-line treatment for anaplastic lymphoma kinase (ALK)-mutated non-small cell lung cancer (NSCLC). Chemically, ensartinib is an aminopyridazine-based, small molecule tyrosine kinase inhibitor (TKI) which inhibits the anaplastic lymphoma kinase (ALK) protein. The drug is 10-fold more potent in the inhibition of the growth of ALK-positive lung cancer cell lines when compared to [crizotinib]. Ensartinib was approved by the FDA for adult patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non-small cell lung cancer (NSCLC) who have not previously received an ALK-inhibitor on December 18, 2024. Although there was no statistically significant difference in overall survival when compared to [crizotinib], ensartinib demonstrated a statistically significant progression-free survival improvement when compared to [crizotinib].
Ensartinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and is used to treat adults with locally advanced or metastatic non-small cell lung cancer. Ensartinib is associated with transient elevations in serum aminotransferase levels and bilirubin during therapy and rare instances of drug induced liver injury.

C26H27CL2FN6O3

561.44

560.15

C, 55.62; H, 4.85; Cl, 12.63; F, 3.38; N, 14.97; O, 8.55

1370651-20-9

Ensartinib dihydrochloride;2137030-98-7

56960363

White to off-white solid powder

1.4±0.1 g/cm3

708.0±60.0 °C at 760 mmHg

382.0±32.9 °C

0.0±2.3 mmHg at 25°C

1.629

4.7

123

3

8

6

38

812

3

C1(C(NC2=CC=C(C(N3C[C@H](C)N[C@H](C)C3)=O)C=C2)=O)=NN=C(N)C(O[C@@H](C2=C(Cl)C=CC(F)=C2Cl)C)=C1

GLYMPHUVMRFTFV-QLFBSQMISA-N

InChI=1S/C26H27Cl2FN6O3/c1-13-11-35(12-14(2)31-13)26(37)16-4-6-17(7-5-16)32-25(36)20-10-21(24(30)34-33-20)38-15(3)22-18(27)8-9-19(29)23(22)28/h4-10,13-15,31H,11-12H2,1-3H3,(H2,30,34)(H,32,36)/t13-,14+,15-/m1/s1

6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-N-[4-[(3S,5R)-3,5-dimethylpiperazine-1-carbonyl]phenyl]pyridazine-3-carboxamide

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

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。