Brain Distribution Study Following Intravenous Infusion in Rats

Male Sprague Dawley rats (n = 4 per time point) received a single i.v. bolus dose followed by i.v. infusion of the drug dissolved in vehicle (10 mM lactic acid and 5% glucose at pH 5.0). Animals were sacrificed 4–6 hours after the start of the infusion, and CSF, plasma, and brain samples were collected. The maximum feasible infusion time was 6 hours because entrectinib is a poorly soluble compound at the desired dose and needs to be dissolved in a vehicle with limited long-term tolerability. A detailed description of experimental design and collection schedule for plasma, CSF, and brain samples can be found in the Supplementary Materials and Supplementary Table 1.

The drugs were extracted from 10 µL (entrectinib, crizotinib, and their internal standards) or 5 µL (larotrectinib and its internal standard) of either rat CSF/plasma, plasma, or brain homogenate samples by a protein precipitation procedure. Brain samples were homogenized with a 3-fold volume of blank rat plasma and quantified against a rat plasma calibration curve. For entrectinib and crizotinib, CSF samples were diluted with an equal volume of blank rat plasma immediately after collection and were quantified against a calibration curve in CSF/plasma (1:1). For larotrectinib, the CSF samples were diluted with an equal volume of bovine serum albumin immediately after collection. The samples were further diluted with blank rat plasma and quantified against a calibration curve in rat plasma.

All extracts were analyzed using reverse-phase liquid chromatography with gradient elusion. The compounds were detected and quantified by tandem mass spectrometry. Calibration curves were obtained by performing a linear regression (weighted 1/x2) on the calibration standards with lower limits of quantification (LLOQ) of 0.05 ng/mL for entrectinib and crizotinib CSF/plasma samples, 1.00 ng/mL for entrectinib and crizotinib plasma samples, and 0.25 ng/mL for larotrectinib CSF and plasma samples.