Successful Treatment of Animal Models of Acute Graft-Versus-Host Disease with Small-Molecule TNF Inhibitor

Tumor necrosis factors(TNF, including TNFα and TNFβ) have now been shown to be two of the major mediators of inflammation and can induce other inflammatory cytokines and proteases that orchestrate inflammatory responses. Recently, many reports have indicated that overexpression of TNF involved in the development of acute graft-versus-host disease(aGVHD), and TNF inhibitors have tremendous potential for the treatment of this disease. To date, 5 of the current biomacromolecular TNF inhibitors, including infliximab (IFX), etanercept(ETN), adalimumab, golimumab, and certolizumab pegol, are available for the part of the routine treatment of patients with rheumatoid arthritis, Crohn's disease, aGVHD and other inflammatory diseases. However, over the past years, several severe limitations of those biomacromolecular TNF inhibitors have also emerged, such as poor stability, exclusion from blood/brain barrier and inducing the development of antidrug antibodies (ADA). For this reason, small-molecule chemical compounds have been designed and developed as appropriate alternatives for overcoming those limitations associated with biomacromolecular inhibitors.

We previously reported a small molecular TNFα inhibitor C87, a lead chemical compound composed of (E)-4-(2-(4-chloro-3-nitrophenyl) which could directly binds to TNF-α, was designed by computer-aided drug design(CADD) and identified by virtual screening and cell-based assays in vitro, and it could attenuated mouse acute hepatitis mediated by TNFα and improve the mouse survival in vivo. As the docking template for virtual screening of above was based on the crystal structure of TNFβ·TNFR1 complex, C87 was supposed to has the potential to be a small molecule inhibitor of TNFβ which few reports to date have indicated . Therefore, in this study, the inhibition of C87 on TNFβ-induced cytotoxicity will be elucidated in detail in vitro, and more importantly, whether C87 could effectively blocked the development of aGVHD in animal model will also be further investigated.

To address those issues, we measured the inhibitory activity of C87 on TNF-mediated cytotoxicity in vitro or in vivo, the results indicated that C87, dissolved in a optimal clinical solvent, could potently and specifically inhibit the TNFβ-induced cytotoxicity in L929 cells in a dose-dependent fashion(IC50=1.59μM). C87 could effectively block TNFβ-induced the activation of Caspase3 and Caspase8 in L929 cells( cultured for 24 hours) and maintain the rate of cell survival up to 80%. In addition, C87 could also suppress the up-regulation of IL-1β and TNFβ itself induced by TNFβ, which was comparable with the TNFβ-neutralizing antibody treatment. Strikingly, C87 could directly bind to TNFβ with high affinity indicated by SPR assay(KD=750 nM). Most importantly, by using a acute GVHD mouse model, low dose C87 (2mg/kg), as single drug treatment for GVHD, could prevent the development of aGVHD in experimental mice efficiently by intraperitoneal administered. The survival rate of low dose C87 groups were 100%(control, 20%), and Low dose C87 also significantly attenuated the aGVHD severity in the damaged organs (such as skin and liver) by using histopathological analysis. And the results of further identification of inflammatory factors in the serum of the treated mice, comparing to the control mice, indicated that more than five kind of cytokines, including IL-2, IL-6, IL-9, IL-18 and GRO-α, were markedly decreased, similar potency to the CSA.

Taken together, we demonstrated that small molecular inhibitor C87 could inhibit both TNFα and TNFβ-induced cytotoxicity and effectively block TNF-triggered signaling activation. Importantly, in addition to its efficacy in vitro, C87 showed the exciting treatment effect in aGVHD mouse model at low dose. Thus, our results extended previous work and suggested that C87 is one of the first effective small-molecule inhibitors of TNF identified to date in the treatment for aGVHD in animal model and has the potential to be a novel small molecular agent in treatment for TNF- mediated inflammatory disease in the future.