Objective  To conduct preliminary studies on the biological activity in vitro and in vivo and pharmacokinetics of novel receptor interacting protein 1 (RIP1) kinases inhibitors, and to provide a reference basis for the subsequent development of new drugs.

Methods  Using GSK2982772 as a positive reference, inhibitory activity of novel RIP1 kinases inhibitors against recombinant human and murine RIP1 kinases was assessed by ADP-Glo methodology. Tumour necrosis factor-α (TNF-α) and apoptosis inhibitor (quinoline-val-asp-difluorophenoxymethylketone, Q-VD-OPh) were used to establish a cell necrosis model to assess the level of inhibition of cell necrosis by RIP1 kinases inhibitor in human tissue lymphoma cells (U-937) and mouse fibroblasts (L-929). The pharmacokinetics of RIP1 kinases inhibitors after intravenous and oral administration to mice was examined. Intravenous injection of mTNF-α in mice induces systemic inflammatory response syndrome (SIRS), and oral administration of a RIP1 kinases inhibitor evaluates the potential for treating inflammation by preventing body temperature loss.

Results  The novel RIP1 kinases inhibitors RIPK1-093, 095, and 106 had comparable inhibitory activities against recombinant human-derived RIP1 kinases (4.55 nmol/L, 7.38 nmol/L, and 10.93 nmol/L) to the positive reference compound (18.93 nmol/ L), and inhibitory activity against recombinant murine RIP1 kinases (5.70 nmol/L, 8.44 nmol/L, 70.58 nmol/L) was superior to the positive reference compound (2 035.00 nmol/L). Inhibition of necrotic activity against U-937 cells (4.92 nmol/L, 2.95 nmol/L, and 6.84 nmol/L) was comparable to that of the positive reference compound (5.37 nmol/L), and inhibition of necrosis activity on L-929 cells (8.59 nmol/L, 6.54 nmol/L, 41.34 nmol/L) was superior to the positive reference compound (489.40 nmol/L); in mouse pharmacokinetics, it had low clearance (P<0.05) and significantly higher oral exposure (P<0.05), noteworthy the oral exposure of RIPK1-106 was elevated about 4-fold. In the SIRS model, oral treatment of the 10 mg/kg dose group showed a comparable loss of body temperature as the positive reference compound at 6h and significantly reduced at 24h (P<0.05), and the 3 mg/kg dose group showed a significant reduction in body temperature loss at 6h (P<0.05) and a significant improvement of animal mortality at 24 h.

Conclusion  The in vitro and in vivo activity and pharmacokinetic profile of the novel RIP1 kinases inhibitors can be further developed and investigated as new drugs for the treatment of inflammatory and autoimmune diseases.

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