Studies On The Antithrombotic Effect And Mechanism Of Direct Factor Xa Inhibitor TY1703

Objectives:The anti-thrombotic activity and mechanism of direct factor Xa inhibitor TY1703 in vitro and in vivo were studied by enzyme activity assay and various animal thrombosis models,and the PK-PD status was preliminarily discussed to provide preclinical pharmacological basis for its development as a new drug.Methods:1.In vitro anticoagulant activity andmechanism of action The in vitro enzyme activity assay method was established by the chromogenic substrate method and the continuous rate method to study the in vitro anticoagulant activity of TY1703 and the specificity for inhibition of FXa.2.In vivo anticoagulant activity and antithrombotic effect The anti-venous thrombosis effect of TY1703 was examined in a blood block or electrical injury with stenosis induced rat venous thrombosis model by evaluating the effect of ig TY1703single dose of different doses,different dosing time and multiple doses on thrombus wet and dry weight;The anti-arteriovenous thrombosis effect of TY1703 was examined in a rat arteriovenous bypass thrombosis model by evaluating the effect of TY1703 on thrombosis;The anti-artery thrombosis effect of TY1703 was examined in an electrically induced rat carotid thrombosis model by registering the thrombosis formation time;The bleeding risk of TY1703 compared with apixaban at the equivalent dose was examined in rat tail-bleeding model by measuring the bleeding time and the amount of bleeding;the anti-thrombotic mechanism of TY1703 was evaluated the effect on coagulation system and fibrinolysis system by detecting laboratory markers including anticoagulation factor?FIIa,FIXa,FXa,FXIa?activity,PT,TT and APTT.3.Dog PK-PD study The pharmacokinetics and pharmacodynamics of TY1703in dog plasma were evaluated by establishing the LC-MS/MS method determining the concentration and the chromogenic substrate method determining FXa activity and its correlation.Results:1.The mechanism of TY1703 In the in vitro enzyme activity assay,TY1703 and apixaban selectively inhibited FXa activity with Ki values of 1.5 nM and 1.2 nM,respectively,and had no significant inhibition of other coagulation factors such as FIIa,FIXa,and FXIa.In the rat thrombosis model experiment,TY1703 and apixaban inhibited the activity of FXa in a concentration manner(1¹0 mg·kg^-1),and had no significant effect on other coagulation factors and fibrinolytic systems.2.Antithrombotic effect TY1703 dose-dependently(0.3³0 mg·kg^-1)decreased thrombus wet weight and dry weight,and the wet weight and dry weight of thrombus were the lowest,the drug effect was the best at the time about 6h after administration,the antithrombotic effect of TY1703 was maintained for more than 10 hours.TY1703dose-dependently(1¹0 mg·kg^-1)extended the time of carotid thrombosis,the anti-venous thrombosis,arteriovenous thrombosis and arterial thrombosis were similar to apixaban at the same dose.3.Effects on bleeding TY1703 can significantly prolong the bleeding time,increase the amount of bleeding in a dose-dependent manner(1¹0 mg·kg^-1),and the bleeding side effects is the strongest at the time about 6h after administration.The results of the risk assessment suggest that TY1703 has better safety.4.Effects on coagulation TY1703 significantly prolonged PT in rat plasma,and had no significant effect on APTT and TT.The results were similar to the equal dose apixaban.5.Dog PK-PD study The plasma concentration increased rapidly after administration of TY1703 in dogs,and reached a maximum of 2170 ng·ml^-11 at 1 hour,then the plasma concentration gradually decreased with time and was similar to the trend of anti-FXa activity,the FXa inhibition rate reached a maximum of 81.7%at 2 h and remained above 50%within 6 h after administration,then the FXa inhibition rate gradually decreased with time.The plasma concentration and the FXa inhibitiong rate were only 42ng·ml^-1 and 4.8%.There was a high correlation between the plasma concentration and the FXa inhibition rate?r=0.9333,P<0.001?.Conclusions:TY1703 had sinificantly anti-venous thrombosis effect,anti-artery thrombosis effect and anti-arteriovenous thrombosis effect in a dose-dependent manner,the mechanism of its anti-thrombosis effect was to inhibit FXa activity with highly specific.According to the evaluation of benefit risk,TY1703 may be safer.According to the PK-PD study,there is a high correlation between the plasma concentration and the FXa inhibition rate of TY1703?r=0.9333,P<0.001?.