Ceritinib

NCI-H3122 (H3122) and HCC78 cells were obtained as described previously [7 (link)]. NCI-H1993 (H1993) and NCI-H596 (H596) were purchased from ATCC (Manassas, VA). All cells were maintained in RPMI 1640 medium (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS). All cells were grown at 37°C in a humidified atmosphere with 5% CO₂. Cells were plated in 96-well plates, allowed to attach overnight and then treated with or without kinase inhibitors for 72 hours. Cell viability was determined by CellTiter 96 Aqueous One solution proliferation kit (Promega, Madison, WI) according to the manufacture’s protocol. Inhibitory proliferation curves and the 50% inhibitory concentration (IC₅₀) were generated using the GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). Crizotinib and ceritinib were purchased from LC Laboratories (Woburn, MA) and Active Biochem (Maplewood, NJ), respectively. All reagents were dissolved in dimethyl sulfoxide (DMSO) and stored at −80°C.

Eight week old female nude mice were injected with 5 million cells subcutaneously (100 uL) in the hind flanks in 1:1 media plus matrigel solution (Corning). After tumors reached approximately 150 mm³, mice were randomized into three groups (7 mice and at least 11 tumors per group) and treated with vehicle (0.5% Tween 80 + 0.5% hydroxypropyl methylcellulose), crizotinib (LC Laboratories, 100 mg/kg once daily, 100 uL, p.o.), or ceritinib (LC laboratories, 50 mg/kg once daily, 100 uL, p.o.) for 25 days. Tumor volume (equation or volume = (length × width²) × 0.52) was evaluated twice per week with digital calipers using the Study Director software package (Studylog Systems). Body weights were measured twice weekly. An additional two MB 2141 xenograft mice were generated and once tumors reached 500 mm³ they were treated with crizotinib or ceritinib for 7 days for further genetic and pharmacodynamics analysis.