Application of mGluR5 agonist CHPG in preparation of antithrombotic drugs

Abstract

The invention belongs to the technical field of medicine, particularly relates to application of mGluR5 agonist CHPG in preparation of antithrombotic drugs, and proves that the mGluR5 agonist CHPG has an antithrombotic effect for the first time through platelet function detection, activation index analysis, a FeCl3 induced mouse artery injury model and protein phosphorylation detection. Researches show that the mGluR5 agonist CHPG significantly reduces the risk of thrombosis by inhibiting platelet activation, and has an antithrombotic application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of mGluR5 agonist CHPG in preparation of an antithrombotic drug. BACKGROUND

[0002] Platelets participate in the core link of the coagulation mechanism, and abnormal activation thereof can induce pathological thrombus formation, and further cause acute vascular occlusion events, ischemic events and multiple organ dysfunction. Current mainstream anti-platelet drugs in clinical practice include cyclooxygenase (COX) inhibitors (such as aspirin), P2Y12 receptor inhibitors (such as clopidogrel) and platelet glycoprotein GPIIb / IIIa receptor antagonists (such as tirofiban). The above drugs generally have the defect of single target, resulting in clinical challenges such as drug resistance and increased risk of bleeding during treatment. Developing new antithrombotic agents with multiple target mechanisms and controllable bleeding risk has become an urgent scientific problem to be solved.

[0003] CHPG is a selective mGluR5 agonist (English name: (RS)-2-Chloro-5-hydroxyphenylglycine (abbreviation: CHPG), Chinese name: (RS)-2-chloro-5-hydroxyphenyl glycine), which has been confirmed to have multiple pharmacological activities such as neuroprotection, anti-inflammatory, antioxidant and metabolic regulation. Its main function is to selectively activate mGluR5 and regulate neuroprotection and inflammatory response. Although the patent with the publication number CN115379847A discloses the potential of the compound in the treatment of psychomotor disorders, the research on its antithrombotic effect has not been reported so far, and its antithrombotic mechanism and clinical application value still need to be further explored. SUMMARY

[0004] The application provides application of mGluR5 agonist CHPG in preparation of an antithrombotic drug.

[0005] The specific implementation is achieved by the following technical solutions. The application provides application of mGluR5 agonist CHPG in preparation of an antithrombotic drug, wherein the Chinese name of the CHPG is (RS)-2-chloro-5-hydroxyphenyl glycine, and the structural formula of the (RS)-2-chloro-5-hydroxyphenyl glycine is as follows: .

[0006] Preferably, the antithrombotic drug is a drug for inhibiting platelet-mediated thrombus formation.

[0007] Preferably, the antithrombotic drug is a drug for inhibiting activation of platelets or aggregation of platelets.

[0008] Further, the platelet aggregation refers to the platelet aggregation induced by collagen, ADP or thrombin.

[0009] Preferably, the anti-thrombotic drug is a drug for inhibiting the ATP release of platelets.

[0010] Preferably, the anti-thrombotic drug is a drug for inhibiting the P-selectin expression.

[0011] Preferably, the anti-thrombotic drug is a drug for inhibiting the P-selectin binding with fibrin.

[0012] Preferably, the anti-thrombotic drug is a drug for inhibiting the platelet-mediated inflammatory response.

[0013] Preferably, the anti-thrombotic drug is a drug for inhibiting the platelet plaque retraction function.

[0014] Beneficial effects: The innovation of the present application is to first confirm the anti-thrombotic activity of CHPG as a selective mGluR5 agonist. Through a series of experimental verification, including: platelet aggregation experiment, plaque retraction experiment, platelet ATP release function detection, platelet activation marker detection, FeCl3-induced mouse mesenteric artery thrombosis model evaluation, and related protein phosphorylation level detection, it is confirmed that CHPG plays an anti-thrombotic effect through PI3K-Akt, MAPK and PKC signaling pathways. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The dose-effect curve of CHPG on collagen-induced platelet aggregation is shown; Figure 2 The quantitative analysis of the inhibitory effect of CHPG on platelet aggregation rate under collagen induction is shown; Figure 3 The dose-effect curve of CHPG on thrombin-induced platelet aggregation is shown; Figure 4 The inhibition statistical graph of CHPG on platelet aggregation rate under thrombin induction is provided; Figure 5 The result graph of the effect of CHPG on P-selectin expression level detected by flow cytometry is shown; Figure 6 The result graph of the effect of CHPG on P-selectin expression rate detected by flow cytometry is shown; Figure 7 The inhibition efficiency of CHPG on collagen-induced platelet ATP release is quantified; Figure 8 The result graph of the fibrinogen binding rate under the intervention of CHPG determined by flow cytometry is shown; Figure 9Statistical chart of the effect of CHPG intervention on fibrinogen binding rate measured by flow cytometry; Figure 10 System comparison of the spatiotemporal regulation effect of different concentrations of CHPG treatment on platelet patch retraction function; Figure 11 Statistical chart of the size of platelet patch retraction in the platelet patch retraction experiment with different concentrations of CHPG and different times; Figure 12 Results chart showing the time-dependent inhibition of platelet accumulation in the injury model by different doses of CHPG treatment. DETAILED DESCRIPTION

[0016] The specific embodiments of the present application are further described in detail below, but the present application is not limited to these embodiments, and any improvement or replacement made in the basic spirit of the present embodiments is within the scope of protection of the claims of the present application.

[0017] Example 1 This example verifies the application efficiency of CHPG (selective mGluR5 agonist) in the preparation of antithrombotic drugs through in vitro experiments: The Chinese name of the CHPG is (RS)-2-chloro-5-hydroxyphenyl glycine, and the structural formula of the (RS)-2-chloro-5-hydroxyphenyl glycine is as follows: .

[0018] (1) Preparation of platelet suspension: Preparation of human platelet suspension: Venous blood samples were collected from healthy volunteers approved by the Ethics Committee of Guizhou Medical University (no drug treatment for 10 days before blood collection), and anticoagulated with 3.8% sodium citrate. The whole blood was centrifuged at 250×g for 15 min (25℃) to obtain platelet-rich plasma (PRP). After mixing PRP with Tyrode's buffer at a ratio of 1:1, prostaglandin 1 (PGE1) (50 ng·mL -1 ) and apyrase (0.01 U·mL -1 ) were added in turn, and the precipitate was collected by centrifugation at 700×g for 10 min. The precipitate was resuspended in Tyrode's buffer and repeated the centrifugation step, finally obtaining a platelet suspension of 2.5×10 11 / mL (37℃ equilibration for 15 min for standby).

[0019] Mouse platelet suspension preparation: The experimental mice were anesthetized by intraperitoneal injection of sodium pentobarbital (2%, 10 mg / kg) and then venous blood was collected. The citrate dextrose (ACD) anticoagulant was mixed at a ratio of 1:9. The blood sample was mixed with Tyrode's buffer at a ratio of 1:1, and then centrifuged at 180 x g for 12 min to obtain platelet-rich plasma. After the addition of prostaglandin 1 (PGE1) (50 ng·mL -1 , 1:1000), the precipitate was collected by centrifugation at 800 x g for 10 min. The precipitate was resuspended in Tyrode's buffer and supplemented with 1 M EDTA. The centrifugation step was repeated to complete the platelet washing, and the washed platelets were obtained.

[0020] (2) Platelet aggregation experiment: The platelet aggregation experiment is a classic method for observing the reactivity of platelets to different agonists.

[0021] The Chrono-Log 700 platelet aggregometer (Wisdom Medical Devices Co., Ltd., USA) was used to quantitatively analyze the platelet aggregation effect induced by different agonists. The experimental system included three independent repeated detection groups, which evaluated the platelet aggregation level and ATP release induced by Collagen and Thrombin after treatment with 0.1% dimethyl sulfoxide (DMSO) and gradient concentrations of compounds at 37°C for a specific time.

[0022] The Chrono-Log aggregation analyzer used for detecting platelet aggregation needs to be turned on in advance to preheat it to 37°C: The operation process is as follows: (1) Pre-equilibrium: platelet suspension (2.5 x 10 11 cells / ml) was incubated at 37°C for 3 min after standing at room temperature for 10 min; (2) Activation treatment: 1 μg·mL -1 Collagen and 0.025 U·mL -1 Thrombin were added, and the platelet aggregation was detected on the platelet aggregometer; (3) Intervention experiment: blank control group (0.1% DMSO) and experimental group (CHPG final concentration 25 μM, 50 μM, pretreatment for 3 min) were set; the platelet aggregation rate was detected (as shown in Figure 1 、 2 , 3, 4); (4) Synchronous detection: 2 μL luciferase antibody was added under light-proof conditions, and the ATP release amount induced by collagen was detected in real time. The ATP release amount (%) was recorded and analyzed (the detection results are shown in Figure 7 ).

[0023] The experimental data showed that CHPG had a concentration-dependent inhibitory effect on collagen and thrombin-induced platelet aggregation (seeFigures 1-4 ), and significantly reduced the release level of ATP in the collagen-activated platelet process.

[0024] (3) Flow cytometry detection: The number of washed platelets was adjusted to 5 x 10 10 / mL, the concentration of the stimulant snake venom (Convulxin) was adjusted in the aggregometer, and 100 μL of platelet suspension was taken into a light-proof EP tube after 3 min of pretreatment with different concentrations (25 μM, 50 μM) of CHPG in the experimental group. CaCl2 (final concentration 1 mM) and 2.5 μL of flow detection antibody (isotype control antibody was used for negative control) were added in turn. After 5 min of incubation, the snake venom was added for 5 min of induction, and finally 200 μL of 4% paraformaldehyde solution was added to terminate the reaction. The expression level of P-selectin (as shown in Figure 5 、 6 ) and the binding rate of fibrinogen (as shown in Figure 8 、 9 ) were detected by BD Accuri C6 plus flow cytometer (San Jose, CA, USA). The data showed that CHPG significantly inhibited the α-granule release induced by snake venom, reducing the expression of P-selectin and the binding rate of fibrinogen.

[0025] Mechanism of platelet function regulation and granule secretion inhibition strategy: There is a close correlation between the biological effects of platelets and the release of active substances in the cells. Activated platelets can release more than 300 kinds of bioactive molecules, which not only have self-activation enhancement effect, but also mediate platelet-cell interaction network. The intracellular storage granules of platelets are mainly divided into three types: dense granules, α-granules, and lysosomes, among which the exocytosis process of α-granules and dense granules plays a decisive role in the physiological function of platelets. Inhibiting platelet granule secretion has become an effective strategy for the prevention and treatment of antithrombosis and atherosclerosis. P-selectin, which is specifically expressed on the membrane surface of α-granules, is a sensitive marker of platelet activation, and its expression level is positively correlated with the degree of platelet activation. Experimental data showed that the expression of P-selectin was significantly down-regulated in the CHPG treatment group ( Figure 5 、 6 ), and the release of ATP, a characteristic secretion indicator of dense granules, was also reduced (p<0.05) ( Figure 7 ). Moreover, the inhibition effect showed a clear concentration gradient characteristic, and there was a significant statistical difference between each experimental group.

[0026] Integrin αIIbβ3 activation inhibition verification: Integrin αIIbβ3 mediates fibrinogen binding through conformational rearrangement, which constitutes the key molecular mechanism of thrombus signal amplification. Fibrinogen binding rate directly reflects the level of integrin αIIbβ3 activation, which is the core index to evaluate the ability of platelet aggregation. To verify the regulatory effect of CHPG on this pathway, the fibrinogen-integrin αIIbβ3 binding rate was quantitatively detected by flow cytometry. The experimental results show that CHPG significantly reduces the fibrinogen-integrin αIIbβ3 binding rate Figure 8 、 9 ), confirming its inhibitory effect on the integrin activation process.

[0027] (4) Platelet patch retraction: After pre-incubating human washed platelet suspension with 0.1% DMSO or gradient concentration of drug CHPG (25 μM, 50 μM), it is mixed with CaCl2, fibrinogen and thrombin (final concentration 0.2 U / mL) in gradient, and reacted at 37°C. The suspension contraction state is observed at 0, 15, 25, 35, 45, 55 min, and the patch retraction dynamic is recorded synchronously (as shown in Figure 10 、 11 ), and the experimental data confirm that CHPG significantly inhibits the patch retraction process by interfering with integrin αIIbβ3 transmembrane signal transduction.

[0028] Clot retraction experiment in vitro is an experiment to evaluate platelet function. Clot retraction is mediated by the "outside-in" signal pathway of platelets (via platelet integrin αIIbβ3), which can make the fibrin clot shrink and thus cause the blood clot to become smaller and more solid, which is essential for the formation of irreversible platelet thrombus. This experiment simulates the process of platelet thrombus formation in vitro, and can also evaluate the function of platelets and the role they play in thrombus formation. This process has been shown to be mediated by a receptor (integrin αIIbβ3 receptor) present on the surface of platelets, which mediates platelet aggregation and participates in thrombus formation. CHPG can inhibit platelet patch retraction, thus indicating that CHPG can weaken the "outside-in" signal transduction mediated by integrin αIIbβ3.

[0029] (5) Ferric chloride-induced mesenteric artery thrombosis model in mice: Healthy C57 mice (18-22 g) were randomly divided into solvent control group (normal saline) and CHPG administration group. CHPG was administered intraperitoneally for seven days, once a day, with dosages of 5 mg / kg and 10 mg / kg, respectively. After administration, the mice were subjected to ferric chloride-induced injury of the mesentery.

[0030] 1) Extract mouse washed platelets according to the method described herein, adjust the platelet concentration to 1 x 10 12 / mL, incubate the platelets with calcein (final concentration 10 μg / ml) in a 37°C incubator for 30 min in the dark to label the platelets with fluorescence; -1 2) Take another male mouse as a recipient mouse, and extract the platelets of the above concentration labeled with fluorescence into an insulin needle, fix the mouse on a tail vein injection operating table, slowly inject the platelets (0.1 ml / 10 g) into the recipient mouse through the tail vein, release the mouse to promote the circulation of the injected platelets in its body, and about 5-10 min later, anesthetize the mouse by intraperitoneal injection according to the body weight (the anesthetic dose is slightly lower); 3) Open the abdominal cavity of the mouse, place the mesentery of the mouse on a glass dish, keep it wet with normal saline, and place it under a fluorescence microscope for bright field observation and select an artery with a diameter of 60-80 μm to move to the center of the field; 4) Turn off the bright field, turn on the mercury lamp, and observe whether there are fluorescently labeled platelets flowing rapidly in the blood vessels to confirm that the blood flow of the mouse is normal; 5) Use tweezers to clamp a 2 x 2 mm filter paper that has been cut in advance, soak it in a 10% FeCl3 solution, then place the filter paper horizontally on the artery in the field of view, cover the injured artery for 2.5 min, then remove it, gently rinse it with filtered normal saline or PBS, and absorb the excess water with a filter paper piece; 6) Turn on the fluorescence, and under a 4x objective, observe the gradual accumulation of platelets and thrombus formation at the injured site, record the thrombus formation time, and take videos and photos at specific time points for recording, as shown in FIG. 1, CHPG can significantly delay the thrombus formation time; Figure 12 7) After observing the thrombus formation of the artery, the blood vessel occlusion (no fluorescent platelets pass downstream) time is more than 60 s, and the time point is recorded as the occlusion time of the artery; if the artery downstream still has fluorescent platelets passing rapidly after 60 min, the blood vessel is not completely occluded, and the observation is stopped. The results show that the solvent control group is completely occluded at 7 min, while the fluorescent platelets pass rapidly downstream of the artery after 60 min in the administration group.

[0031] Statistical processing ​​The above experiments were repeated at least three times, and the experimental data were processed by Graphpad Prism 9.5 statistical software, and the results were expressed as mean ± standard error. The comparison among multiple groups was first subjected to homogeneity of variance test, and the single factor analysis of variance (one-way ANOVA) was used for the groups with equal variance, and the least significant difference (LSD) test was used for pairwise comparison. If the variance is not equal, Tamhance's T2 test is performed, and the t test is used for pairwise comparison. P<0.05 is considered statistically significant.

[0032] The above examples and verification examples only express the specific embodiments of the present application, which are described in detail, but cannot be understood as limiting the protection scope of the present application. It should be pointed out that for ordinary skilled persons in the art, without departing from the technical scheme concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application, and the dosage form of CHPG drug does not belong to the protection scope of the present application.

Claims

1. The application of the mGluR5 agonist CHPG in the preparation of antithrombotic drugs, characterized in that, The Chinese name of the CHPG is (RS)-2-chloro-5-hydroxyphenylglycine, and the structural formula of (RS)-2-chloro-5-hydroxyphenylglycine is as follows: 。 2. The application as described in claim 1, characterized in that, The antithrombotic drug is a drug used to inhibit platelet-mediated thrombus formation.

3. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is a drug used to inhibit platelet activation or platelet aggregation.

4. The application as described in claim 3, characterized in that, Platelet aggregation refers to platelet aggregation induced by collagen, ADP, or thrombin.

5. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is a drug used to inhibit the release of ATP from platelets.

6. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is used to inhibit the expression of P-selectin.

7. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is used to inhibit the activation of integrin αIIbβ3.

8. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is a drug used to inhibit platelet-mediated inflammatory responses.

9. The application as described in claim 1 or 2, characterized in that, The antithrombotic drug is used to inhibit the retraction function of platelet plaques.

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