Entrectinib, a TRK/ROS1 inhibitor with anti-CNS tumor activity: differentiation from other inhibitors in its class due to weak interaction with P-glycoprotein

BACKGROUND: Studies evaluating the CNS penetration of a novel tyrosine kinase inhibitor, entrectinib, proved challenging, particularly due to discrepancies across earlier experiments regarding P-glycoprotein (P-gp) interaction and brain distribution. To address this question, we used a novel “apical efflux ratio” (AP-ER) model to assess P-gp interaction with entrectinib, crizotinib, and larotrectinib, and compared their brain-penetration properties. METHODS: AP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp–overexpressing cells. Brain penetration was studied in rat plasma, brain, and cerebrospinal fluid (CSF) samples after intravenous drug infusion. Unbound brain concentrations were estimated through kinetic lipid membrane binding assays and ex vivo experiments, while the antitumor activity of entrectinib was evaluated in a clinically relevant setting using an intracranial tumor mouse model. RESULTS: Entrectinib showed lower AP-ER (1.1–1.15) than crizotinib and larotrectinib (≥2.8). Despite not reaching steady-state brain exposures in rats after 6 hours, entrectinib presented a more favorable CSF-to-unbound concentration in plasma (CSF/C(u,p)) ratio (>0.2) than crizotinib and larotrectinib at steady state (both: CSF/C(u,p) ~0.03). In vivo experiments validated the AP-ER approach. Entrectinib treatment resulted in strong tumor inhibition and full survival benefit in the intracranial tumor model at clinically relevant systemic exposures. CONCLUSIONS: Entrectinib, unlike crizotinib and larotrectinib, is a weak P-gp substrate that can sustain CNS exposure based on our novel in vitro and in vivo experiments. This is consistent with the observed preclinical and clinical efficacy of entrectinib in neurotrophic tropomyosin receptor kinase (NTRK) and ROS1 fusion-positive CNS tumors and secondary CNS metastases.