Entrectinib (Synonyms: NMS-E628, RXDX-101)

Cell lines

PC12, HT22 and SK-N-SH cells

Preparation Method

The rat adrenal pheochromocytoma cells PC12(FH0415) cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. mouse hippocampal neuron cells HT22 (FH1027) and human neuroblastoma cells SK-N-SH (FH0164) were cultured in DMEM mediumwith 10% FBS and 1% penicillin and streptomycin. All cells were incubated in a 37°C with 5% CO₂ atmosphere. To explore whether Entrectinib influences the cellular proliferation ability and apoptosis, cells were treated with 2.3, 4.2 and 4.3μM Entrectinib for 48h, respectively. After incubation, 10μL CCK-8 solution was added into each well and incubated at 37°C for 2h in the dark. Then, a microplate reader was used to measure the absorbance at a wavelength of 450nm. EdU cell proliferation detection kit was used for cell proliferation detection. Annexin V-FITC/PI kit was used for cell apoptosis analysis.

Reaction Conditions

2.3-4.3μM; 48h

Applications

Entrectinib dose-dependently reduced viability, suppressed EdU incorporation, and elevated apoptosis in PC12, HT22 and SK-N-SH nerve cells.

Animal models

athymic nu/nu mice

Preparation Method

Six-week-old athymic nu/nu mice were obtained from Jackson Laboratories. Mice were maintained at five per cage under humidity- and temperature-controlled conditions in a light/ dark cycle that was set at 12h intervals. For the xenograft studies, animals were injected subcutaneously in the flank with 1×10⁷ SY5Y-TrkB cells in 0.1ml of Matrigel. Tumors were measured 2 times per week in 3 dimensions, and the volume calculated as follows: [(0.523xLxWxW)/1000]. Body weights were measured at least twice a week, and the dose of compound was adjusted accordingly. Treatment with Entrectinib, Irino and TMZ started about 15–17 days after tumor inoculation when the average tumor size was 0.2cm³. Entrectinib was dosed at 60mg/kg BID by gavage for the entire duration of the study(7 weeks). After the final dose was given, the blood samples were drawn from 4 mice per time point via retro-orbital bleeding and collected in heparinized tubes on wet ice. The plasma was then separated by centrifugation at 1200g for 10 minutes at 4°C. The pharmacokinetic analysis was performed using the Watson system. Mice were sacrificed when tumor volume reached 3cm³ . Tumors were harvested and flash frozen on dry ice for analysis of protein expression using Western blot.

Dosage form

60mg/kg; BID for 7 weeks; p.o.

Applications

Entrectinib markedly suppressed SY5Y-TrkB xenograft growth, prolonged mouse event-free survival, and blocked TrkB, Akt and Erk phosphorylation in athymic nu/nu mice.

References:
[1] Tang Q, Dong J, Zhang F, et al. Entrectinib can induce nerve cell damage by inhibiting PI3K-AKT and TGF-? signaling pathways. Front Pharmacol. 2025;16:1489210.
[2] Iyer R, Wehrmann L, Golden RL, et al. Entrectinib is a potent inhibitor of Trk-driven neuroblastomas in a xenograft mouse model. Cancer Lett. 2016;372(2):179-186.