ALK (IC50 <0.4 nM); MET (IC50 = 0.74 nM)

Ensartinib (X-396) dihydrochloride has dual ALK/MET inhibitory activity and a potent IC50 of less than 0.4 nM and 0.74 nM, respectively. In H3122 lung cancer cells expressing EML4-ALK E13;A20, ensetinib dihydrochloride exhibits potency (IC50: 15 nM). In H2228 lung cancer cells expressing EML4-ALK E6a/b; A20, esartinib dihydrochloride is likewise effective (IC50: 45 nM). Moreover, X-376 has a high potency (IC50: 9 nM) in SUDHL-1 lymphoma cells that harbor NPM-ALK.

Ensartinib exhibits significant bioavailability and moderate half-lives in vivo, according to a pharmacokinetic analysis. Examined are Ensartinib (X-396)'sinvivo effects on H3122 xenografts. Ensartinib, 25 mg/kg bid, is administered to nude mice containing H3122 xenografts. When compared to the vehicle alone, ensartinib considerably slows the growth of tumors. Ensartinib appears to be well-tolerated in vivo in the xenograft experiments. Treatment with ensartinib has no effect on mouse weight. Mice given drugs seem healthy and do not show any symptoms of toxicity from the compounds. Additional systemic toxicity and toxico-kinetic studies are carried out in Sprague Dawley (SD) rats to further evaluate potential side effects of ensartinib. After ten days of repeated oral administration of Ensartinib at doses of 20, 40, and 80 mg/kg in SD rats, every animal survives to the end of the study. Ensartinib has been found to have a no significant toxicity (NST) level of 80 mg/kg. Ensartinib has an AUC of 66 μMΗhr and a C_max of 7.19 μM at NST levels[1].

In 96-well plates, cells are seeded at 25%–33% confluency and exposed to medications for viability experiments. Ensartinib (10, 30, 100, 300, and 1000 nM) is used to treat the human lung adenocarcinoma cell lines H3122 and H2228. Ensartinib (5, 10, 30, 100, and 300 nM) is used to treat SUDHL-1 lymphoma cells. Ensartinib (30, 100, 300, and 1000 nM) is used to treat SY5Y neuroblastoma cells. After adding Ensartinib for 72 hours, Cell Titer Blue Reagent is added, and the Spectramax spectrophotometer is used to measure the fluorescence. Every experimental point is set up in duplicate three times, and it is run at least twice independently. GraphPad Prism version 5 for Windows is used to calculate IC50s. With a log (inhibitor) vs. response formula, a nonlinear regression model is used to fit the curves[1].

Mice: H3122 cells are injected into naked mice (nu/nu). Following the tumors' average volume of 450 mm³, 27 athymic mice with H3122 tumors are randomly assigned to receive either the control vehicle or 25 mg/kg of ensartinib orally via gavage. Mice are killed two, five, and fifteen hours after the single treatment (3 tumors/timepoint/group), and serum is taken for an LC-MS based bioanalytical method to determine the drug concentration[1].

Absorption
The mean Cmax of 25 mg and 100 mg ensartinib orally was 292 ng/mL, Tmax ranged from 2 to 8 hours, and the median Tmax was 3 hours. The AUC of ensartinib at the approved recommended dose was 4,920 ngh/mL. The drug reached steady state within 15 days, with a mean cumulative rate of 2.7. There was no clinically significant difference between taking ensartinib with or without food; the comparison was made between a high-fat meal and a fasting state. As this drug is administered orally, its absorption site is expected to be in the gastrointestinal tract.
Elimination Route
After a single oral dose of 200 mg of radiolabeled ensartinib, 91% of the radioactive material was excreted in feces (38% unchanged) and 10% in urine (4.4% unchanged).
Volume of Distribution
The mean apparent volume of distribution of orally administered ensartinib capsules was 1720 L.
Protein Binding
Ensartinib oral capsules bind 91.6% to human plasma proteins.
Metabolites/Metabolites
Ensartinib is a substrate of the CYP3A4 enzyme and is primarily metabolized in the liver via the CYP3A4 pathway.
Biological Half-Life
The mean steady-state half-life of ensartinib is 30 ± 20 hours.

Hepatotoxicity
In the premarketing trial of ensartinib for the treatment of non-small cell lung cancer, abnormal liver function was relatively common. Elevated ALT was observed in 59% of subjects, elevated AST in 58%, elevated alkaline phosphatase (ALP) in 51%, and elevated bilirubin in 12%. These enzyme elevations were usually transient and mild to moderate. Pruritus was observed in 26% of subjects. ALT elevations exceeding 5 times the upper limit of normal (ULN) were observed in 5% of subjects, and AST elevations were observed in 2%. The average time to onset of elevated transaminase levels was 5 weeks (range).
Probability score: D (likely a rare cause of clinically significant liver injury).

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

Non-small cell lung cancer (NSCLC) is one of the most common subtypes of lung cancer, encompassing various types, among which adenocarcinoma, squamous cell carcinoma, and large cell carcinoma are relatively common. Ensartinib is a first-line treatment for anaplastic lymphoma kinase (ALK)-mutant NSCLC. Chemically, ensartinib is a small-molecule tyrosine kinase inhibitor (TKI) based on aminopyridazine that inhibits the anaplastic lymphoma kinase (ALK) protein. Compared to crizotinib, ensartinib is 10 times more potent in inhibiting the growth of ALK-positive lung cancer cell lines. On December 18, 2024, ensartinib was approved by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with ALK-positive locally advanced or metastatic NSCLC who have not previously received ALK inhibitor therapy. Although ensartinib did not show a statistically significant difference in overall survival compared to crizotinib, it demonstrated a statistically significant improvement in progression-free survival. Ensartinib is a small molecule ALK inhibitor used to treat adult patients with locally advanced or metastatic non-small cell lung cancer. Transient elevations in serum transaminase and bilirubin levels may occur during ensartinib treatment, and drug-induced liver injury may occur in rare cases. Ensartinib is an oral small molecule receptor tyrosine kinase anaplastic lymphoma kinase (ALK) inhibitor with potential antitumor activity. After oral administration, ensartinib binds to and inhibits the activity of ALK kinase, ALK fusion proteins, and ALK point mutants. Inhibition of ALK leads to the disruption of ALK-mediated signaling pathways, ultimately inhibiting the growth of ALK-expressing tumor cells. In addition, ensartinib also inhibits several other kinases, including receptor tyrosine kinase c-Met (hepatocyte growth factor receptor; HGFR; MET) and receptor tyrosine kinase C-ros oncogene 1 (ROS1). ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK is not expressed in healthy adult tissues, but ALK dysregulation and gene rearrangement are associated with a range of tumors; ALK mutations are associated with acquired resistance to small molecule tyrosine kinase inhibitors. Ensartinib is a small molecule drug that has reached Phase IV clinical trials (covering all indications), was first approved in 2024, and has three investigational indications. It is an anaplastic lymphoma kinase (ALK) inhibitor used to treat ALK-positive non-small cell lung cancer.

C26H29CL4FN6O3

634.357265233994

634.1

C, 49.23; H, 4.61; Cl, 22.35; F, 2.99; N, 13.25; O, 7.57

2137030-98-7

Ensartinib;1370651-20-9

138320013

White to light yellow solid powder

5

8

6

40

812

3

C[C@@H]1CN(C[C@@H](N1)C)C(=O)C2=CC=C(C=C2)NC(=O)C3=NN=C(C(=C3)O[C@H](C)C4=C(C=CC(=C4Cl)F)Cl)N.Cl.Cl

IERUINQRGJAECT-ISUJJMBGSA-N

InChI=1S/C26H27Cl2FN6O3.2ClH/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);2*1H/t13-,14+,15-;;/m1../s1

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

X-396 hydrochloride; X396 hydrochloride; X 396 hydrochloride; Ensartinib dihydrochloride; Ensartinib HCl; Ensartinib hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~35.71 mg/mL (~56.29 mM)
H2O : ~33.33 mg/mL (~52.54 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 14.29 mg/mL (22.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)