ONO-4641 and/or cyclosporine treatment alleviates the induction of BM failure in mice. CBF1 mice were subjected to 4 Gy TBI, and 5 × 10⁶ LN cells from B6 mice were administered via caudal vein injection to induce the aplastic anemia model. On day 14 after disease induction, peripheral blood cell count was determined by the hematology analyzer. (A) Schema of the ONO-4641 and/or cyclosporine treatment experiments. (B) CBF1 mice were treated daily with ONO-4641 (0.03, 0.1, or 0.3 mg/kg) or 0.5%MC (vehicle) by oral gavage from day 1 after disease induction. On day 14 after disease induction, peripheral blood cells were determined by a hematology analyzer. Indicated values are presented as means and individuals; n = 8 per group except for vehicle (n = 7). (C) CBF1 mice were treated daily with ONO-4641 0.3 mg/kg or 0.5%MC (vehicle) by oral gavage from day 4 after disease induction. Indicated values are presented as means and individuals; n = 8 per group except for vehicle (n = 10). (D) In the two-drug combination experiment, CBF1 mice were treated with either ONO-4641 0.1 mg/kg, cyclosporine 15 mg/kg (Cys), 0.5%MC alone, or ONO-4641 0.1 mg/kg + cyclosporine 15 mg/kg from day 1 after disease induction. Indicated values are presented as means and individuals; n = 8 per group except for vehicle (n = 7). Steel’s multiple comparison test was performed for comparison between the vehicle and the ONO-4641 0.03, 0.1, and 0.3 mg/kg groups, with a P value of less than 5%. ^#P < 0.05; ^##P < 0.01. Wilcoxon signed-rank sum test was performed for comparison between the vehicle group and the groups receiving TBI only; ONO-4641 0.1, 0.3 mg/kg; cyclosporine 15 mg/kg; or ONO-4641 0.1 mg/kg + cyclosporine 15 mg/kg, with a P value of less than 5%. *P < 0.05; **P < 0.01; ***P < 0.001. Wilcoxon signed-rank sum test was performed for comparison between the ONO-4641 0.1 mg/kg + cyclosporine 15 mg/kg and the ONO-4641 0.1 mg/kg or the cyclosporine 15 mg/kg groups, with a P value of less than 5%. +P < 0.05.

ONO-4641 treatment reduces the destruction of BM cells by inhibiting the infiltration of donor-derived T lymphocytes to BM. (A) The counts of total cells in the BM of mice that received TBI only, received TBI + LN cell infusion, or were untreated (normal mice) at the indicated days after disease induction. Indicated values are presented as means and individuals; n = 5 or 6 per group. (B) Representative hematoxylin and eosin–stained femur section (original magnification, 10×) in the treatment groups of TBI only with 0.5%MC (vehicle), TBI + LN cell infusion with vehicle, and TBI + LN cell infusion with ONO-4641 0.3 mg/kg. (C) Total cell counts in the BM of mice on day 14. Mice were treated daily with ONO-4641 at 0.03, 0.1, 0.3 mg/kg or with vehicle by oral gavage starting from day 1 after disease induction. Indicated values are presented as means and individuals; n = 8 per group except for vehicle (n = 7). (D) Representative flow cytometric analysis of IFN-γ or IL-17A expressions in donor-derived CD4⁺ and CD8⁺ T lymphocytes in the groups treated with vehicle or ONO-4641 0.3 mg/kg on day 14. (E) Absolute numbers of donor-derived CD4⁺ and CD8⁺ T-lymphocyte subsets in the groups treated with vehicle or ONO-4641 0.3 mg/kg on day 14. Indicated values are presented as means and individuals; n = 6 for the vehicle-treated group, and n = 5 for the group treated with ONO-4641 0.3 mg/kg. Steel’s multiple comparison test was performed for comparison between the vehicle and the ONO-4641 groups, with a P value of less than 5%. ^##P < 0.01. Wilcoxon signed-rank sum test was performed for comparison between the TBI-only and the TBI + LN cell infusion with vehicle or the ONO-4641 0.3 mg/kg groups, with a P value of less than 5%. *P < 0.05; **P < 0.01.

ONO-4641 treatment increases the number of HSPCs in the BM of model mice. Mice received TBI only with vehicle, TBI + LN cell infusion with 0.5%MC (vehicle), or TBI + LN cell infusion with ONO-4641 0.3 mg/kg treatment. Mice were treated daily with ONO-4641 0.3 mg/kg or vehicle for 13 days starting from day 1 after disease induction. On day 14, mouse femurs were collected for HSPC analysis. (A) Representative FACS dot plots of HSPCs. Data represent the expressions of Sca-1 and c-Kit on Lin⁻ BM cells (top panels) and the expressions of CD34 on Lin⁻ Sca1⁺ c-Kit⁺ BM cells (bottom panels) of TBI only with vehicle, TBI + LN cell infusion with vehicle, and TBI + LN cell infusion with ONO-4641 0.3 mg/kg treatment groups in the femur. (B) The numbers of HSPCs of TBI only with vehicle, TBI + LN cell infusion with vehicle, and TBI + LN cell infusion with ONO-4641 0.3 mg/kg treatment groups in the femur. (C) The number of CFU-GM progenitors was enumerated in an in vitro colony forming assay. Indicated values are presented as means and individuals; n = 8 per group except for vehicle (n = 10). Wilcoxon signed-rank sum test was performed for comparison between the TBI + LN cell infusion with vehicle and the TBI only with vehicle or the TBI + LN cell infusion with ONO-4641 0.3 mg/kg groups, with a P value of less than 5%. **P < 0.01; ***P < 0.001. Wilcoxon signed-rank sum test was performed for comparison between the TBI-only and the TBI + LN cell infusion with ONO-4641 0.3 mg/kg groups, with a P value of less than 5%. ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001.

ONO-4641 regulates plerixafor-induced mobilization of HSPCs in mice. (A) Mice were administered orally with 0.5%MC (vehicle) or ONO-4641 0.3 mg/kg or injected subcutaneously with PBS, plerixafor 3 mg/kg, or ONO-4641 + plerixafor (oral administration of ONO-4641 30 minutes before subcutaneous injection of plerixafor) for 8 days. At 3 hours after the last dosing, femurs were collected for BM and HSPC cell analysis. Blood was also collected for HSPC cell analysis. (A) The numbers of Lin⁻ Sca1⁺ c-Kit⁺ HSPC and BM cells in the femur. (B) The number of Lin⁻ Sca1⁺ c-Kit⁺ HSPCs in the blood. Indicated values are presented as means and individuals; n = 5 per group. Wilcoxon signed-rank sum test was performed for comparison between the vehicle and the ONO-4641 0.3 mg/kg, the plerixafor 3 mg/kg, or the ONO-4641 + plerixafor groups, with a P value of less than 5%. *P < 0.05. Wilcoxon signed-rank sum test was performed for comparison between the ONO-4641 + plerixafor and the ONO-4641 0.3 mg/kg or the plerixafor 3 mg/kg groups, with a P value of less than 5%. ⁺P < 0.05.