Establishment And Development Of Screening Platform For Janus Kinase Inhibitor

Currently,there are four JAK inhibitor drugs on the market,including Tofacitinib,Baricitinib,Ruxolitinib and Upadacitinib.they all have tumor risks,and anemia side effects caused by non-selective inhibition of JAK2 target.Therefore,the development of a JAK1 inhibitor with better safety and higher selectivity is the current market demand.Meanwhile,the highly selective JAK3 inhibitor NIBR3049 was 100 times less active in STAT5 phosphorylation of CTLL-2 cells than JAK1/3 inhibitor,suggesting that JAK3 is not critical in the ?c cytokines signaling pathways.Thus,in terms of marketing strategy,develop a JAK1/3 inhibitor is simpler due to the high homology of JAK1 and JAK3 in the kinase domain.In order to develop JAK1/3 inhibitors,this paper describes the establishment of screening platforms for JAK1/3 and JAK2 targets respectively at the cellular level.What's more,a rat CIA model was established to investigate the efficacy of compounds KYDR001 and Tofacitinib and their effects on blood biochemistry,in order to verify the screening results of these two established cell screening platforms.Objective: To establish JAK1/3 and JAK2 target screening platforms at cellular level respectively,and evaluate the feasibility of the experimental system for the determination results.The efficacy and biochemical effects of the compounds were evaluated by the rat CIA model to verify the screening results of those two established cell screening platforms.Methods: Establishing JAK1/3 and JAK2 target cell screening platform: firstly,the optimal stimulant concentration was found in literature,then the optimal cell density and incubation time under the maximum response window was found through different cell density gradient and different incubation time fluorescent response.The final experimental system was determined by eliminating the influence of FBS and DMSO.What's more,Multiple evaluations of positive drugs tofacitinib and Baricitinib was established,and evaluate whether the 95% confidence intervals were acceptable.Establishment of CIA rat platform: Through the establishment of CIA rat model,the efficacy of the compound in CIA rats was evaluated by the improvement of paw symptom score,hind paws increment,disease progression,white blood cell and lymphocyte count,which confirmed the successful establishment of JAK1/3 inhibitory active cell screening model.Furthermore,the potential inhibitory activity of the compound on the JAK2 target was evaluated by the blood biochemical indexes of CIA rats,including RBC count,hemoglobin,hematocrit and reticulocyte,and compared with the blood biochemical indexes of Tofacitinib to confirm the successful establishment of the JAK2 target inhibitory active cell screening platform.Results: successfully established the CTLL–2 cell proliferation experiment for JAK1/3targets inhibition evaluation and established TF-1 cell proliferation experiment for JAK2 target inhibition.Compound KYDR001 is 8.3 times inhibitory activity on JAK1 over JAK2 on enzymology.At cellular level,KYDR001 respectively to JAK1/3,JAK2 IC50 half inhibitory concentration is 255.5 n M,496.6 n M(with batch tofacitinib for 213.6 n M and765.2 n M),show less selective than tofacitinib at a cellular level.The efficacy of tofacitinib and KYDR001 was successfully evaluated in the rat CIA model,and the efficacy of KYDR001 at the dose of 30mg/kg/qd was considered to be equal to or slightly worse than Tofacitinib 10mg/kg/qd.Base on the results of PK-PD and the compounds plasma protein binding rate of rats,speculate that KYDR001 was well evaluated in the CTLL-2 cell proliferation experiment.With the efficacy result in CIA model that KYDR001 30mg/kg/qd is equal to or slightly worse than Tofacitinib 10mg/kg/qd,it was found that KYDR001 had slightly stronger inhibition of erythrocyte count,hemoglobin,hematocrit and reticulocyte than Tofacitinib.Which was consistent with the results of KYDR001's inhibition of TF-1 cell proliferation.Moreover,the blood biochemistry inhibition results of tofacitinib 10mg/kg/qd group is corresponding to the FDA reported data in erythrocyte count,hemoglobin,and hematocrit,suggesting that the blood biochemical indexes of the rat CIA model can evaluate the inhibitory effect of testing compounds on JAK2 to a certain extent,which has certain guiding significance for future hematological toxicity studies.Conclusion: CTLL-2 and TF-1 cell proliferation inhibition evaluation models targeting at JAK1/3 and JAK2 were successfully established respectively.And mutual verification results were observed in the establishment of rat CIA model.At the same time,it was found that the three biochemical indexes of red blood cell count,hemoglobin and hematocrit in the rat CIA model could evaluate the inhibitory effect of the compound on JAK2 to some extent.