Application of DOCK2 inhibitor CPYPP in preparation of antithrombotic drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a DOCK2 inhibitor CPYPP in preparation of antithrombotic drugs. The invention provides an application of a DOCK2 inhibitor CPYPP in preparation of an antithrombotic drug. The DOCK2 inhibitor CPYPP can inhibit the expansion of platelets on the surface of fibrinogen and the retraction of thrombus, and inhibit the adhesion of the platelets on the surface of collagen; a flow cytometer detects that the CPYPP can reduce the exposure of P-selectin on the surface of platelets, integrate the activation level of alpha IIb beta 3 and inhibit the activation of the platelets; the CPYPP can be used for inhibiting the formation of laser-induced microthrombus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of DOCK2 inhibitor CPYPP in the preparation of anti-thrombotic drugs. Background Art

[0002] Thrombotic diseases seriously threaten human life and health. Vascular embolic diseases such as myocardial infarction and stroke have become the number one cause of death among Chinese residents. Currently, the commonly used antithrombotic drugs in clinical practice mainly include anticoagulants and antiplatelet drugs. During the treatment process, various adverse reactions such as liver and kidney damage and bleeding may occur. Therefore, the development of safe and efficient antithrombotic drugs based on new targets is of great clinical significance.

[0003] 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedion e (CPYPP) is a small molecule inhibitor of DOCK2 that binds to the DOCK2 DHR-2 domain and inhibits DOCK2 in a dose-dependent manner (IC 50 CPYPP has an activity on the guanine nucleotide exchange factor of Rac1, significantly reduces lymphocyte chemotaxis and T lymphocyte activation, and can also inhibit the activation of macrophages and neutrophils, inhibit the formation of neutrophil extracellular traps, and regulate immune responses. However, there are no relevant studies on the effect of CPYPP on platelet function and thrombosis. Summary of the invention

[0004] The purpose of the present invention is to provide the use of DOCK2 inhibitor CPYPP in the preparation of anti-thrombotic drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of DOCK2 inhibitor CPYPP in preparing anti-thrombotic drugs.

[0007] Preferably, the final concentration of the DOCK2 inhibitor CPYPP in the anti-thrombotic drug is 23-27 μmol / L.

[0008] The present invention also provides the use of DOCK2 inhibitor CPYPP in preparing a drug for inhibiting the formation of thrombus.

[0009] Preferably, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting thrombus formation is 23-27 μmol / L.

[0010] The present invention also provides the use of DOCK2 inhibitor CPYPP in the preparation of a drug for inhibiting platelet function.

[0011] Preferably, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet function is 23-27 μmol / L.

[0012] Preferably, the platelet functions include expansion function, adhesion function and thrombus retraction.

[0013] The present invention also provides the use of DOCK2 inhibitor CPYPP in preparing a drug for inhibiting platelet activation.

[0014] Preferably, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet activation is 23-27 μmol / L.

[0015] Preferably, the platelet activation is platelet activation induced by CRP.

[0016] Beneficial effects of the present invention:

[0017] DOCK2 inhibitor CPYPP can inhibit the expansion of platelets on the surface of fibrinogen and the retraction of thrombi, and inhibit the adhesion of platelets on the surface of collagen; flow cytometry detection found that CPYPP can reduce the exposure of P-selectin on the surface of platelets and the activation level of integrated αIIbβ3, inhibiting platelet activation; in vivo experiments found that CPYPP can inhibit laser-induced microthrombosis. Therefore, CPYPP significantly inhibits platelet function and activation, significantly inhibits thrombosis, and is a new type of antithrombotic compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the test on the effect of DOCK2 inhibitor CPYPP on platelet expansion function, where A is the expansion result diagram of washed platelets at different time points, B is the statistical diagram of the expansion area of ​​washed platelets, and C is the expansion phase of washing;

[0019] Figure 2 This is the test result of the effect of DOCK2 inhibitor CPYPP on platelet adhesion function, where A is the result of platelet adhesion and aggregation at different time points, and B is the statistical diagram of the area ratio of adhered platelets;

[0020] Figure 3 The results of the test on the effect of DOCK2 inhibitor CPYPP on platelet plug retraction are shown in Figure 1, where A is the result of plug retraction at different time points, and B is the statistical diagram of plug retraction area;

[0021] Figure 4This is the test result of the effect of DOCK2 inhibitor CPYPP on platelet activation, where A is the expression of P-selectin on the platelet surface, and B is the activation level of integrin αIIbβ3;

[0022] Figure 5 These are the test results of the effect of DOCK2 inhibitor CPYPP on thrombosis, where A is the platelet aggregation result at different time points in the site of arteriolar injury (scale: 20 μm), B is the curve of platelet fluorescence intensity changing with time, and C is the area under the curve of captured platelets during thrombosis formation. DETAILED DESCRIPTION

[0023] The present invention provides application of DOCK2 inhibitor CPYPP in preparing anti-thrombotic drugs.

[0024] In the present invention, the final concentration of the DOCK2 inhibitor CPYPP in the antithrombotic drug is 23-27 μmol / L, preferably 24-26 μmol / L, and more preferably 25 μmol / L.

[0025] The present invention also provides the use of DOCK2 inhibitor CPYPP in preparing a drug for inhibiting the formation of thrombus.

[0026] In the present invention, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting thrombus formation is 23 to 27 μmol / L, preferably 24 to 26 μmol / L, and more preferably 25 μmol / L.

[0027] The present invention also provides the use of DOCK2 inhibitor CPYPP in the preparation of a drug for inhibiting platelet function.

[0028] In the present invention, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet function is 23-27 μmol / L, preferably 24-26 μmol / L, and more preferably 25 μmol / L.

[0029] In the present invention, the platelet functions include expansion function, adhesion function and thrombus retraction.

[0030] The present invention also provides the use of DOCK2 inhibitor CPYPP in preparing a drug for inhibiting platelet activation.

[0031] In the present invention, the final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet activation is 23 to 27 μmol / L, preferably 24 to 26 μmol / L, and more preferably 25 μmol / L.

[0032] In the present invention, the platelet activation is platelet activation induced by CRP.

[0033] In the present invention, the inhibition of platelet activation is to inhibit the expression of platelet surface activation factor P-selectin and the activation level of integrated αIIbβ3.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1 Preparation of washed platelets

[0036] 1 mL of venous blood was collected from C57BL / 6 mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), centrifuged at 90×g for 12 min at 25°C, and the supernatant was collected into a 5 mL centrifuge tube and allowed to stand at 37°C for 20 min. Then, 1 / 5 volume of ACD solution [85 mmol / L sodium citrate, 21 mmol / L citric acid, 83 mmol / L D-glucose, pH 6.5] of the platelet suspension was added, and then an equal amount of PGE1 (final concentration of 0.2 μM) was added. After gentle inversion and mixing, the blood was centrifuged at 620×g for 10 min at 25°C. After discarding the supernatant, HTG solution (130 mmol / L NaCl, 5 mmol / L KCl, 12 mmol / L NaHCO3, 0.4 mmol / L Na2HPO4, 1 mmol / L MgCl2·6H2O, 10 mmol / L HEPES buffer, 5 mmol / L D-glucose, pH 7.4) to obtain washed platelets.

[0037] Example 2 Effect of DOCK2 inhibitor CPYPP on platelet expansion function

[0038] Fibrinogen (100 μg / mL) was coated in the plate overnight at 4°C, then washed with PBS buffer, and blocked with 1% bovine serum albumin (BSA) at 25°C for 1 h to obtain a fibrinogen-coated plate. The washed platelets in Example 1 were diluted to 1×10 7 / L; 300 μL washed platelets were pre-incubated with 1‰ dimethyl sulfoxide (DMSO) (control group, Veh) and DOCK2 inhibitor CPYPP (final concentration 25 μmol / L, CPYPP group) for 10 min, and then incubated in a fibrinogen-coated plate at 37°C for 5 min, 15 min and 30 min, respectively, to wash away the unattached washed platelets, fix with paraformaldehyde at 25°C for 20 min, break the membrane and block for 5 min; add rhodamine-labeled phalloidin (1 μg / mL) and incubate in the dark for 30 min, then seal the platelets, observe the platelet expansion under a confocal fluorescence microscope, and classify and count the platelet expansion morphology. The results are shown in the figure. Figure 1 shown.

[0039] from Figure 1 It can be seen that the expansion area of ​​washed platelets in the CPYPP group on the fibrinogen surface at different time points (5min, 15min and 30min) was smaller than that in the control group; and the expansion phase of washed platelets in the CPYPP group at 15min and 30min was significantly slower than that in the control group. It can be seen that the DOCK2 inhibitor CPYPP can significantly inhibit the expansion of platelets.

[0040] Example 3 Effect of DOCK2 inhibitor CPYPP on platelet adhesion function

[0041] The flow chamber channels were coated with collagen (100 μg / mL), washed with PBS after overnight at 4°C, and blocked with 1% BSA at room temperature for 1 h to obtain a well plate; 200 μL of whole blood (mouse venous blood in Example 1) was pre-incubated with 1‰ dimethyl sulfoxide (DMSO) (control group, Veh) and DOCK2 inhibitor CPYPP (final concentration 25 μmol / L, CPYPP group) for 10 min, and then platelets were labeled with DyLight-488 fluorescently labeled CD42c antibody (0.5 μg / mL) at 25°C for 20 min to obtain labeled whole blood; the labeled whole blood was added to the well plate and incubated at 40 dyne / cm 2 The flow rate was tested, and images were collected under a fluorescence microscope at 1min, 3min, 5min and 10min. The area ratio of adhered platelets was calculated using ImageJ software. The results of platelet adhesion and area ratio are shown in Figure 2 shown.

[0042] from Figure 2 It can be seen that under the shear force of arterial blood flow, the adhesion of washed platelets on the collagen surface in the CPYPP group was significantly reduced compared with the control group. It can be seen that the DOCK2 inhibitor CPYPP can significantly inhibit the adhesion function of platelets.

[0043] Example 4 Effect of DOCK2 inhibitor CPYPP on platelet plug retraction

[0044] The washed platelets in Example 1 were diluted to 3×10 8 The washed platelets were pre-incubated with 1‰ dimethyl sulfoxide (DMSO) (control group, Veh) and DOCK2 inhibitor CPYPP (final concentration 25 μmol / L, CPYPP group) for 30 min to obtain the pre-incubated washed platelets; 100 μL of the pre-incubated washed platelets were added to 200 μL of human platelet-poor plasma, inverted and mixed, and thrombin (Thrombin, 1 U / mL) was added to start the retraction reaction of the washed platelet plug. The retraction of the plug was observed at 30 min, 60 min, 90 min and 120 min, and the plug area was calculated using ImageJ software. The statistical results of the contraction and plug area are shown in the figure. Figure 3 shown.

[0045] from Figure 3 It can be seen that the volume of the washed platelet plug in the CPYPP group was significantly reduced at 30min, 60min and 90min compared with the washed platelets in the control group. It can be seen that CPYPP can significantly inhibit the retraction of platelet plugs.

[0046] Example 5 Effect of DOCK2 inhibitor CPYPP on platelet activation

[0047] The washed platelets in Example 1 were diluted to 5×10 7The washed platelets were pre-incubated with 1‰ dimethyl sulfoxide (DMSO) (control group, Veh) and DOCK2 inhibitor CPYPP (final concentration 25 μmol / L, CPYPP group) for 30 min to obtain pre-incubated washed platelets; adenosine diphosphate (ADP, 30 μmol / L), thrombin (Thrombin, 0.1 U / mL), 0.3 μg / mL collagen-related peptide CRP [CRP(0.3)] and 0.5 μg / mL collagen-related peptide CRP [CRP(0.5)] were added to 20 μL of pre-incubated washed platelets and incubated for 10 min to activate the washed platelets to obtain activated washed platelets; no agonists (ADP, Thrombin, CRP) were added to resting platelets (Rest group); 5 μL of resting and activated washed platelets were added FITC-labeled P-selectin antibody was incubated at 25°C in the dark for 20 min, and then flow cytometry was used to detect the expression of P-selectin on the platelet surface. 5 μL of PE-labeled JON / A antibody was added to the resting and activated washed platelets, and then incubated at 25°C in the dark for 20 min. Flow cytometry was used to detect the activation level of integrin αIIbβ3 on the platelet surface. The results of the platelet surface P-selectin expression and the activation level of integrin αIIbβ3 are shown in Figure 2. Figure 4 shown.

[0048] from Figure 4 It can be seen that after pre-incubation of platelets with the DOCK2 inhibitor CPYPP, the exposure of CRP-induced P-selectin and the activation level of αIIbβ3 were significantly reduced compared with the control group. It can be seen that CPYPP can significantly inhibit CRP-induced platelet activation.

[0049] Example 6 Effect of DOCK2 inhibitor CPYPP on thrombosis

[0050] C57BL / 6 mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were randomly divided into two groups, Veh group and CPYPP group;

[0051] Injection solution for the CPYPP group: DOCK2 inhibitor CPYPP was first dissolved in polyethylene glycol 300, and then diluted with an equal volume of normal saline. The final concentration of CPYPP was 25 mg / mL. Injection solution for the control group: polyethylene glycol 300 solution, prepared with polyethylene glycol 300 and normal saline in a volume ratio of 1:1.

[0052] The mice in the CPYPP group and the Veh group were intraperitoneally injected with the corresponding injection solution, with an injection volume of 0.2 mL / mouse;

[0053] The mice in the CPYPP group and the Veh group were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg / kg body weight) according to their body weight. The external jugular vein was isolated and cannulated, and then the cremaster muscle of the mice was isolated, and the blood flow was observed under a fluorescent intravital microscope. DyLight-488 fluorescently labeled CD42c antibody (0.1 mg / kg body weight) was injected into the jugular vein, and cremaster arterioles with a diameter of 30-60 μm were selected as laser injury targets to produce vascular injury. The platelet aggregation at the injury site was recorded within 3 min, and the fluorescence signal of the platelets was analyzed. The results Figure 5 shown.

[0054] from Figure 5 It can be seen that the platelet aggregation, platelet fluorescence intensity and corresponding area under the curve in the vascular injury site of CPYPP pretreated mice were significantly reduced compared with those in the control group. It can be seen that CPYPP can significantly inhibit thrombosis.

[0055] As can be seen from the above examples, the present invention provides the use of DOCK2 inhibitor CPYPP in the preparation of anti-thrombotic drugs. DOCK2 inhibitor CPYPP can inhibit the expansion of platelets on the surface of fibrinogen and the retraction of thrombi, and inhibit the adhesion of platelets on the surface of collagen; flow cytometry detection found that CPYPP can reduce the exposure of P-selectin on the surface of platelets and the activation level of integrated αIIbβ3, inhibiting platelet activation; CPYPP can inhibit laser-induced microthrombosis.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of DOCK2 inhibitor CPYPP in the preparation of anti-thrombotic drugs.

2. The use according to claim 1, characterized in that: The final concentration of the DOCK2 inhibitor CPYPP in the anti-thrombotic drug is 23-27 μmol / L.

3. Application of DOCK2 inhibitor CPYPP in the preparation of drugs for inhibiting thrombus formation.

4. The use according to claim 3, characterized in that: The final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting thrombus formation is 23-27 μmol / L.

5. Application of DOCK2 inhibitor CPYPP in the preparation of drugs for inhibiting platelet function.

6. The use according to claim 5, characterized in that: The final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet function is 23-27 μmol / L.

7. The use according to claim 6, characterized in that: The platelet functions include expansion function, adhesion function and thrombus retraction.

8. Application of DOCK2 inhibitor CPYPP in the preparation of drugs for inhibiting platelet activation.

9. The use according to claim 8, characterized in that: The final concentration of the DOCK2 inhibitor CPYPP in the drug for inhibiting platelet activation is 23-27 μmol / L.

10. The use according to claim 9, characterized in that: The platelet activation is platelet activation induced by CRP.

Images