Abstract
NF-κB is a transcription factor that controls expression of a number of genes that are important for mediating immune and inflammatory responses, such as IL-1 and TNF-α , which are both overproduced during graft versus host disease (GVHD). To determine whether the NF-κB signaling pathway plays a role in the pathophysiology of GVHD, we examined the affect of bortezomib (PS-341, Velcade®), a proteasome inhibitor whose mechansism of action includes but is not limited to inhibition of NF-κB, and PS-1145, a IKB kinase (IKK) inhibitor that selectively inhibits NF-κB, on the induction of GVHD. Lethally irradiated C57BL/6 (H-2b) mice that were transplanted with MHC-incompatible B10.BR (H-2k) bone marrow (BM) and spleen cells and treated with a single dose of bortezomib (1 mg/kg) on the day of BMT had significantly prolonged survival compared to untreated GVHD controls (50% versus 0% survival at 60 days). In contrast, extended or delayed administration of this agent on a twice weekly schedule that recapitulated current clinical practice guidelines resulted in a significant increase in mortality as all animals died within ten days of transplantation. Histological analysis of bortezomib-treated mice demonstrated that mortality was attributable to severe pathological damage in the colon, indicating that there was a narrow therapeutic window within which bortezomib could be administered. Toxicity was GVHD-dependent as syngeneic marrow transplant recipients treated with a similar schedule for four weeks (8 doses) all survived for 60 days and had no evidence of pathological damage when compared to vehicle-treated controls. Given the narrow therapeutic window observed with bortezomib, we examined whether more selective inhibition of NF-κB with PS-1145 could prevent GVHD without inducing toxicity and early mortality. In vitro studies demonstrated that PS-1145 induced apoptosis in alloactivated T cells, similar to bortezomib, although a higher concentration was required for equivalent results (8 μM versus 0.32 μM). To achieve a serum concentration of PS-1145 necessary for the induction of T cell apoptosis in vivo, PS-1145 was administered at a dose of 50 mg/kg on days 0–2 post transplantation to recipients of MHC-incompatible marrow grafts. Treatment with PS-1145 resulted in similar GVHD protection when compared to animals administered a single dose of bortezomib. Prevention of GVHD by bortezomib and PS-1145 was associated with equivalent reductions of serum cytokine levels of IL-2, IL-10 and TNF-α when compared to GVHD controls. To determine whether the selective inhibition of NF-κB with PS-1145 caused less toxicity in BMT recipients, mice were administered this agent for 10 consecutive days beginning on the day of transplant. In contrast to results observed with bortezomib, PS-1145-treated mice had no early mortality and had superior survival when compared to GVHD controls (100% versus 0% survival at 60 days). GVHD protection in these mice was associated with significantly reduced expansion of alloreactive donor T cells in the spleen without compromise of donor engraftment. These results confirm a critical role for NF-κB in the pathophysiology of GVHD and indicate that selective inhibition of NF-κB may have a superior therapeutic index and constitute a viable approach to reduce GVHD.
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