Ensartinib (X-396) is a potent and dual ALK/MET inhibitor with IC50s of <0.4 nM and 0.74 nM, respectively.
The ability of Ensartinib (X-396) to inhibit the growth of different cancer cell lines harboring ALK fusions or point mutations is tested. Ensartinib is potent in H3122 lung cancer cells harboring EML4-ALK E13;A20 (IC50: 15nM). Ensartinib is also potent in H2228 lung cancer cells harboring EML4-ALK E6a/b; A20 (IC50: 45 nM). Furthermore, X-376 is potent in SUDHL-1 lymphoma cells harboring NPM-ALK (IC50: 9 nM). X-376 also inhibits SY5Y neuroblastoma cells harboring ALK F1174L, MKN-45 gastric carcinoma cells harboring MET dependent, HepG2 cells and PC-9 lung cancer cell lines harboring EGFR exon 19 del with IC50s of 68 nM, 156 nM, 9.644 μM and 2.989 μM, respectively[1].
The effects of Ensartinib (X-396) in vivo against H3122 xenografts are examined. A pharmacokinetic study reveals that Ensartinib shows substantial bioavailability and moderate half-lives in vivo. Nude mice harboring H3122 xenografts are treated with Ensartinib at 25mg/kg bid. Ensartinib significantly delays the growth of tumors compared to vehicle alone. In the xenograft experiments, Ensartinib appears well-tolerated in vivo. Mouse weight is unaffected by Ensartinib treatment. Drug-treated mice appear healthy and do not display any signs of compound related toxicity. To further assess potential side effects of Ensartinib, additional systemic toxicity and toxico-kinetic studies are performed in Sprague Dawley (SD) rats. Following 10 days of repeated oral administration of Ensartinib at 20, 40, 80 mg/kg in SD rats, all animals survive to study termination. The no significant toxicity (NST) levels are determined to be 80mg/kg for Ensartinib. At NST levels, Ensartinib achieves an AUC of 66 μM×hr and a Cmax of 7.19 μM[1].
[1]. Lovly CM, et al. Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinaseinhibitors. Cancer Res. 2011 Jul 15;71(14):4920-31.

Ensartinib (X-396) has been evaluated for its efficacy in inhibiting the proliferation of various cancer cell lines characterized by ALK fusions or point mutations. It exhibits significant potency in H3122 lung cancer cells with EML4-ALK E13;A20 mutations (IC50: 15nM) and in H2228 lung cancer cells with EML4-ALK E6a/b; A20 mutations (IC50: 45 nM). Additionally, its efficacy extends to SUDHL-1 lymphoma cells containing NPM-ALK (IC50: 9 nM) and demonstrates inhibition in SY5Y neuroblastoma cells with ALK F1174L mutation, MKN-45 gastric carcinoma cells dependent on MET, HepG2 cells, and PC-9 lung cancer cell lines possessing EGFR exon 19 deletion, with IC50 values of 68 nM, 156 nM, 9.644 μM, and 2.989 μM, respectively.

The in vivo effects of Ensartinib (X-396) against H3122 xenografts were studied, showing that Ensartinib exhibits considerable bioavailability and moderate in vivo half-lives. Treatment of nude mice bearing H3122 xenografts with 25mg/kg bid of Ensartinib markedly inhibited tumor growth compared to the control. Ensartinib was well-tolerated, with no significant impact on mouse weight or indications of toxicity. Additional studies in Sprague Dawley (SD) rats, involving repeated oral doses of Ensartinib (20, 40, 80 mg/kg) over 10 days, confirmed survival of all subjects till study end without significant toxicity; the no significant toxicity (NST) threshold was established at 80mg/kg. At this NST level, Ensartinib showed an AUC of 66 μM×hr and a peak concentration (Cmax) of 7.19 μM[1].

For viability experiments, cells are seeded in 96-well plates at 25%-33% confluency and exposed to drugs.The human lung adenocarcinoma cell lines H3122 and H2228 are treated with Ensartinib (10, 30, 100, 300 and 1000 nM). SUDHL-1 lymphoma cells are treated with Ensartinib (5, 10, 30, 100 and 300 nM). SY5Y neuroblastoma cells are treated with Ensartinib (30, 100, 300 and 1000 nM). At 72 hours post Ensartinib addition, Cell Titer Blue Reagent is added and fluorescence is measured on a Spectramax spectrophotometer. All experimental points are set up in hextuplicate replicates and are performed at least two independent times. IC50s are calculated using GraphPad Prism version 5 for Windows. The curves are fit using a nonlinear regression model with a log (inhibitor) vs. response formula[1].