Fig 5. In vivo analyses using mouse xenograft models.
(A) Anti-tumor effect of tirabrutinib in a mouse xenograft model. (B) Effect of tirabrutinib on BTK phosphorylation in the mouse xenograft model. A TMD8 cell suspension (0.1 mL, 1 × 108 cells/mL) was subcutaneously implanted in SCID mice under pentobarbital anesthesia. Animals were randomized into groups based on tumor volume calculated from the measurement of tumor diameter on the day after implantation. Tirabrutinib or 0.5% methyl cellulose was orally administered twice daily for 21 days, starting on day 0 (the group assignment day). Tumor diameter was measured, and tumor volume was calculated every 3 or 4 days after group assignment. (A) Tumor volume in the figure is expressed as the mean ± standard error in the 10 animals in each group on each measurement day. Dunnett test was used to compare tumor volume in the vehicle- and tirabrutinib-treated groups. A P-value of less than 5% was considered statistically significant. **: P < 0.01, ***: P < 0.001. Results of linear regression analysis in the tirabrutinib-treated group on day 21: The P-value of the slope was 0.0001. (B) Tumors were collected 1 h after the final administration. The mean fluorescence intensity (MFI) of BTK phosphorylation in each tumor cell with or without stimulation with H2O2 was determined. The difference in MFI between the H2O2-stimulated and unstimulated samples was expressed as the mean ± standard error in 4–8 animals in each group. The BTK phosphorylation inhibition rate (%) in the tirabrutinib-treated groups versus the vehicle group was calculated. BID, twice daily; BTK, Bruton’s tyrosine kinase; MFI, mean fluorescent intensity.