P-glycoprotein Mediates Resistance to the Anaplastic Lymphoma Kinase Inhiitor Ensartinib in Cancer Cells.

Ensartinib (X-396) is a promising second-generation small-molecule inhibitor of anaplastic lymphoma kinase (ALK) that was developed for the treatment of ALK-positive non-small-cell lung cancer. Preclinical and clinical trial results for ensartinib showed superior efficacy and a favorable safety profile compared to the first-generation ALK inhibitors that have been approved by the U.S. Food and Drug Administration. Although the potential mechanisms of acquired resistance to ensartinib have not been reported, the inevitable emergence of resistance to ensartinib may limit its therapeutic application in cancer. In this work, we investigated the interaction of ensartinib with P-glycoprotein (P-gp) and ABCG2, two ATP-binding cassette (ABC) multidrug efflux transporters that are commonly associated with the development of multidrug resistance in cancer cells. Our results revealed that P-gp overexpression, but not expression of ABCG2, was associated with reduced cancer cell susceptibility to ensartinib. P-gp directly decreased the intracellular accumulation of ensartinib, and consequently reduced apoptosis and cytotoxicity induced by this drug. The cytotoxicity of ensartinib could be significantly reversed by treatment with the P-gp inhibitor tariquidar. In conclusion, we report that ensartinib is a substrate of P-gp, and provide evidence that this transporter plays a role in the development of ensartinib resistance. Further investigation is needed.

Mass balance, metabolic disposition, and pharmacokinetics of [14C]ensartinib, a novel potent anaplastic lymphoma kinase (ALK) inhibitor, in healthy subjects following oral administration.

PURPOSE: Ensartinib is a novel, potent and highly selective inhibitor of anaplastic lymphoma kinase (ALK) that has promising clinical activity and low toxicity in patients with ALK-positive non-small cell lung cancer. This study was conducted to investigate the pharmacokinetics, metabolism and excretion of ensartinib following a single 200 mg/100 µCi oral dose of radiolabeled ensartinib to healthy subjects. METHODS: Six healthy male subjects were enrolled and administrated an oral suspension in a fasted state. Blood, urine and feces were collected. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of ensartinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and characterization. RESULTS: The mean total recovery was 101.21% of the radiolabeled dose with 91.00% and 10.21% excreted in feces and urine, respectively. Unchanged ensartinib was the predominant drug-related component in urine and feces, representing 4.39% and 38.12% of the administered dose, respectively. Unchanged ensartinib and its metabolite M465 were the major circulating components, accounting for the same 27.45% of the plasma total radioactivity (AUC0-24h pool), while other circulating metabolites were minor, accounting for less than 10%. Mean Cmax, AUC0-∞, T1/2 and Tmax values for ensartinib in plasma were 185 ng/mL, 3827 h ng/mL, 18.3 h and 3.25 h, respectively. The total radioactivity in plasma was cleared with terminal half-life of 27.2 h. Treatment with ensartinib was well tolerated, and no serious adverse events were reported. CONCLUSION: It was well tolerated in the six healthy male subjects following a single oral administration of 200 mg/100 µCi dose of ensartinib. Besides unchanged ensartinib, metabolite of M465 was the predominant circulating drug-related component. The drug was primarily eliminated in feces. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03804541.

New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK.

Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.