Dual inhibition of HY023016 based on binding properties of platelet membrane receptor subunit glycoprotein Ibα and thrombin exosites.

Thrombin has long been suggested as a desirable antithrombotic target, but anti-thrombin therapy without anti-platelet thereby has never achieved the ideal effect. HY023016 is a novel compound, in our previous study, it exerted better anti-thrombotic than dabigatran etexilate. The present study aims to illustrate the excess anti-thrombotic molecular mechanisms of HY023016 through thrombin anion exosites and the platelet membrane receptor subunit glycoprotein Ibα (GPIbα). HY023016 strongly inhibited the conversion of fibrinogen to fibrous may via blocking thrombin exosite I. We also discovered that HY023016 remarkably inhibited exosite II by a loss of affinity for the γ'-peptide of fibrinogen and for heparin. Furthermore, a solid phase binding assay revealed that HY023016 inhibited ristocetin-induced washed platelets bind to von Willebrand factor (vWF). In GST pull-down assay, HY023016 decreased the binding of recombinant vWF-A1 to GPIbα N-terminal. Thus, HY023016 provides an innovative idea for designing multi-targeted anti-thrombotic drugs and laying a scientific foundation for reducing "total thrombosis risk" in a clinical drug treatment.

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model.

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system.

Antidepressant-like effect of evodiamine on chronic unpredictable mild stress rats.

Evodiamine is a major alkaloid compound extracted from the dry unripened fruit Evodia fructus (Evodia rutaecarpa Benth., Rutaceae), which has a variety of pharmacological activities. The present study aims to determine the antidepressant-like effect of evodiamine in a rat model of chronic unpredictable mild stress (CUMS). We identified that evodiamine could reverse the following CUMS-induced behavioural deficits and biochemical changes in rats: the decreases of sucrose preference, number of crossings, 5-HT and NA levels, as well as the increase of immobility time. Evodiamine treatments also ameliorated the corticosterone hypersecretion induced by CUMS. Furthermore, we found that evodiamine was able to up-regulate the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin-related kinase B (pTrkB) without altering TrkB. This study suggests potential antidepressant-like effect of evodiamine on CUMS rats, and its underlying mechanisms can be potentially linked to their modulating effects on the monoamine transmitters and BDNF-TrkB signaling in the hippocampus.

Antithrombotic activity of HY023016, a novel Dabigatran prodrug evaluated in animal thrombosis models.

INTRODUCTION: Thrombin is a multifunctional trypsin-like serine protease that plays key roles in coagulation and thrombogenesis. HY023016, a novel Dabigatran prodrug, is an oral direct thrombin inhibitor. The purpose of this study was to compare the anti-thrombotic activities and haemorrhagic effects of HY023016 with Dabigatran etexilate and tetramethylpyrazine in several animal thrombosis models. METHODS: To investigate drug exposure, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the pharmacokinetic profile of HY023016. After single intragastric administrations of HY023016, Dabigatran etexilate or tetramethylpyrazine, the anti-thrombotic activities were evaluated through rabbit jugular vein thrombosis model, rat inferior vena cava thrombosis model, ex vivo rabbit platelet aggregation assay, in vivo rabbit coagulation assay, and direct thrombin binding assay. Meanwhile, we evaluated the effect of HY023016 on expression of tissue factor (TF) by RT-PCR. Rabbit cuticle bleeding assay and mouse tail bleeding assay were applied to evaluate the effects of HY023016 on haemorrhage. RESULTS: Pharmacokinetic parameters indicated that HY023016 can convert to Dabigatran and tetramethylpyrazine. Our studies showed that HY023016 was able to significantly inhibit thrombus formation in a dose-dependent manner in rabbit and rat models (P<0.05). Similarly, it was able to dose-dependently inhibit thrombin- or ADP-induced platelet aggregation, prolonging the activated partial thromboplastin time (APTT) and prothrombin time (PT), inhibiting the activity of thrombin and inhibiting thrombin- or ADP-induced expression of TF (P<0.05 or 0.01). Dabigatran etexilate was also able to dose-dependently and significantly inhibit thrombus formation (P<0.01) but was unable to affect ADP-induced platelet aggregation and expression of TF. In contrast, tetramethylpyrazine could only exhibit mild antithrombotic activity compared with HY023016 and Dabigatran etexilate (P<0.05). HY023016 could prolong bleeding time (P<0.001), but the prolongations were significantly less than Dabigatran etexilate (P<0.05). CONCLUSION: HY023016 showed thrombosis-inhibition activities comparable to those of Dabigatran etexilate, but better than those of tetramethylpyrazine. The attendant bleeding risk of HY023016 was lower than Dabigatran etexilate in rabbits and mice.