Ruxolitinib, a JAK1/JAK2 Selective Inhibitor Is Highly Efficacious in Corticosteroid Untreated and Refractory MHC-Mismatched Mouse Model of Acute GvHD

Introduction:

Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (REACH1: NCT02953678; REACH2: NCT02913261).

Methods:

Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2K^d) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells received from allogeneic cell transfer from donor C57BL/6 (H-2K^b) mice. Animals were dosed orally with vehicle or the potent and selective JAK1/JAK2 inhibitor, ruxolitinib (60 mg/kg) twice daily. Engraftment was analyzed for the proportion of donor and host leukocytes (CD45, H-2K^b, and H-2K^d). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analyses. Colon samples were sectioned at approximately 5 microns, and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3/STAT5, CD3+phosphoSTAT3/STAT5 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of ruxolitinib in corticosteroid refractory mice were evaluated by treating disease bearing BALB/c mice twice daily with an optimal oral dose of prednisolone (1 mg/kg) until the GvHD scores were comparable to vehicle treated mice. Steroid resistant animals were switched to ruxolitinib to determine the potential efficacy benefit in comparison to mice maintained on ruxolitinib treatment alone for the duration of the study.

Results:

Oral ruxolitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss, improving GvHD scores and had no detrimental effects on donor engraftment. Comparable efficacy was observed between prophylactic and therapeutic treatment with 60 mg/kg twice daily dosing regimens. Oral ruxolitinib administration in BALB/c mice achieved JAK1/JAK2 IC₅₀ coverage for 8 h with 60 mg/kg twice daily. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Ruxolitinib (60 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4⁺ T cells and CD8⁺ T cells in blood and spleen in groups treated with ruxolitinib, but significant reductions were detected in CD4⁺ T cells and CD8⁺ T cells in the inflamed colon tissue along with JAK/STAT pathway inhibition as measured by significant reductions in normalized phosphoSTAT3/STAT5 in T cells and colonic epithelial cells. Prednisolone was transiently effective until day 31 in reducing GvHD scores. Switching treatment regimens from prednisolone to ruxolitinib twice daily rapidly and significantly enhanced GvHD efficacy to a level comparable to ruxolitinib monotherapy. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy when treatment was resumed on day 33 after being halted on day 24. In addition, ruxolitinib significantly enhanced the survival of the recipient BALB/c mice in comparison to vehicle treated animals.

Conclusions:

Ruxolitinib, a JAK1/JAK2 selective inhibitor, significantly ameliorated GvHD severity with no detrimental effects on engraftment in both a steroid untreated and a corticosteroid refractory MHC-mismatched model of acute GvHD. Efficacy was associated with JAK/STAT pathway inhibition in colon and target tissue infiltrating T-cells. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy once treatment was resumed. In addition, ruxolitinib reduced disease burden and enhanced survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD.