Pharmaceutical composition for treating thrombosis through synergistic interaction and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a combined medicine for treating thrombosis through synergistic interaction and application of the combined medicine. The invention relates to a pharmaceutical composition for preventing and treating diabetes, which is characterized in that the pharmaceutical composition comprises 1, 2, 3, 4, 6-pentagalloyl-beta-D-glucose and geniposide, and the weight ratio of the 1, 2, 3, 4, 6-pentagalloyl-beta-D-glucose to the geniposide is 1: (0.4-8). Researches prove that when geniposide and 1, 2, 3, 4, 6-pentagalloyl-beta-D-glucose are combined for use, nerve cell injury caused by glutamic acid and thrombin can be synergistically protected, thrombosis formation can be resisted, activation of MKK3, MKK6 and p38 in a p38MAPK signal channel can be inhibited, and a p38-MKK3 axis can be blocked. The invention can be used for targeted drugs for treating thrombosis. And the compound can be conveniently prepared into solid preparation medicines such as tablets, capsules, solid dispersions, suspensions or granules together with a carrier.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a synergistic and potentiating combination drug for treating thrombosis and its application. Background Art

[0002] Thrombosis is the basic pathological process of many diseases such as myocardial infarction, ischemic stroke, and stroke. It has a high incidence rate and has become the most serious disease threatening the health of the middle-aged and elderly people in the world. According to a survey report by the World Health Organization, deaths caused by thrombotic diseases due to atherosclerosis have ranked first. Currently, commonly used anti-thrombotic drugs in clinical practice include aspirin, clopidogrel, etc., but long-term use is prone to the risk of gastrointestinal bleeding. Therefore, safe and effective natural drugs are expected.

[0003] Targeted therapy strategies have become a new model for the treatment of thrombosis. Activation of the p38MAPK signaling pathway plays a key role in platelet activation and thrombosis, and MAPK inhibitors can reduce thrombotic events. Therefore, the treatment of thrombosis with p38MAPK signaling pathway inhibitors has great clinical application prospects.

[0004] 1,2,3,4,6-pentagalloyl-β-D-glucose is a natural polyphenol compound derived from natural medicines such as red peony root, and geniposide is an iridoid glycoside compound derived from natural medicines such as rehmannia root. 1,2,3,4,6-pentagalloyl-β-D-glucose and geniposide have antithrombotic effects and can inhibit the p38MAPK signaling pathway. At present, there is no pharmaceutical composition that combines 1,2,3,4,6-pentagalloyl-β-D-glucose and geniposide to enhance antithrombotic effects and inhibit the p38MAPK signaling pathway.

[0005] According to the needs of the world's high-thrombosis population, the application prospects of targeted drugs for treating thrombosis and the advantages of combined use of natural medicines, the present invention combines 1,2,3,4,6-pentagalloyl-β-D-glucose with geniposide to form a targeted drug composition for treating thrombosis and inhibiting the p38MAPK signaling pathway. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to overcome the defects of the prior art and provide the use of 1,2,3,4,6-pentagalloyl-β-D-glucose and geniposide in the preparation of a combined anti-thrombotic targeted drug. Another purpose of the present invention is to provide a new pharmaceutical composition.

[0007] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] A synergistic and effective combination drug for treating thrombosis, the combination drug is 1,2,3,4,6-pentagalloyl-β-D-glucose and geniposide.

[0009] Furthermore, the combination of 1,2,3,4,6-pentagalloyl-β-D-glucose and geniposide can synergistically inhibit the activation of the p38MAPK signaling pathway and enhance the anti-thrombotic effect.

[0010] Furthermore, in the combination drug, the weight ratio of 1,2,3,4,6-pentagalloyl-β-D-glucose to geniposide is 1:1.5.

[0011] Furthermore, the above-mentioned pharmaceutical composition for treating thrombosis includes preparations such as granules, tablets, suspensions, solid dispersions, capsules or suspensions.

[0012] The invention also provides the use of the combined medicine, which is used for preparing antithrombotic medicine.

[0013] The invention relates to the use of the pharmaceutical composition for treating thrombosis in the preparation of a drug for treating thrombosis.

[0014] Beneficial effects: Compared with the prior art, the pharmaceutical composition provided by the present invention can synergistically enhance the protection against nerve cell damage caused by glutamate and thrombin, inhibit the activation of the p38MAPK signaling pathway, and resist thrombosis. The above pharmaceutical composition can effectively prevent and treat the occurrence of various cardiovascular and cerebrovascular thrombotic events, and can be used as a targeted drug for anti-thrombotic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the results of neuronal apoptosis in each treatment in Experimental Example 2 of the present invention.

[0016] Figure 2 These are the staining intensity results of cardiac erythrocytes in each treatment in Experimental Example 3 of the present invention.

[0017] Figure 3 These are the results of protein expression levels of the p38MAPK pathway in each treatment in Experimental Example 4 of the present invention.

[0018] Figure 4 This is the result of the effects of various treatments on the p38-MKK3 axis in Experimental Example 4 of the present invention.

[0019] Figure 5 It is the chemical structural formula of two monomer drugs in the present invention. DETAILED DESCRIPTION

[0020] In order to make the content of the present invention more easily understood, the present invention is further explained below according to the specific embodiments of the present invention and in conjunction with the accompanying drawings, and it is understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0021] Example 1 Synergistic effect experiment of 1,2,3,4,6-pentagalloyl-β-D-glucose (PGG) combined with geniposide (GE)

[0022] To evaluate whether the combination of PGG and GE has a synergistic effect, the synergistic protective effect of the drugs was evaluated using the PC12 cell injury model induced by glutamate (20uM) and the BV2 cell injury model induced by thrombin (100uM). First, the OD values ​​of PGG and GE on PC12 and BV2 cells were detected by CCK8 method, and the inhibition rate was calculated as follows:

[0023] Inhibition rate = (OD value of drug group - OD value of model group) / (OD value of blank group - OD value of model group)

[0024] Then, the IC value of the drug was obtained by fitting the inhibition rate using GraphPad Prism 6. 50 The results are shown in Table 1. PGG and GE have strong inhibitory effects on PC12 cell and BV2 cell damage, among which the IC 50 The smallest, with the strongest ability to inhibit nerve cell damage.

[0025] Table 1 IC of single drug against PC12 and BV2 50 value

[0026]

[0027] Note: PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide

[0028] To further evaluate the synergistic effect of the drug combination (PGG+GE), the drug combination dose was set according to a constant ratio two drug combination (Constant Ratio Two Drug Combination), and the two-drug combination setting for each group was 0.125IC 50 , 0.25 IC 50 , 0.5 IC 50 、1 IC 504 doses. The OD values ​​of the drugs alone and in combination were detected using glutamate-induced PC12 cells and thrombin-induced BV2 cell models, and the inhibition rate was calculated (the formula is the same as above). The "Chou-Talalay" model was then used to calculate the combination index (CI) of the two-drug combination, and the calculation method is as follows:

[0029] CI=D1 / Dx1+D2 / Dx

[0030] Where D1 and D2 are the doses of the two drugs required to produce the X effect when used together; Dx1 and Dx2 are the doses of the two drugs when used alone to produce the X effect. If CI < 1, it indicates that the drug combination has a synergistic effect, where CI is in the range of 0.5-0.7, it indicates that the drug combination has a strong synergistic effect, and CI is in the range of 0.3-0.5, it indicates that the drug combination has a strong synergistic effect.

[0031] The results are shown in Table 2. The inhibition rate of the drug monomer increases with the increase of dose. The inhibition rate of PC12 and BV2 cell damage is less than 55%, indicating that they have a certain protective effect on nerve cell damage. After the drug combination, the inhibition rate of each group increased significantly, and each group increased by about 20%. The CI of the 8 dose combinations is in the range of 0.35-0.7, among which the CI of 3 groups is in the range of 0.3-0.5, and the CI of 5 groups of dose combinations is in the range of 0.5-0.7, indicating that the combination of drugs has a strong synergistic effect. This research experiment shows that the combination of PGG and GE has a strong synergistic protective effect on nerve cell damage caused by glutamate and thrombin within a certain dose setting range.

[0032] Example 2 Experiment on the inhibition of neuronal apoptosis by the combination of PGG and GE

[0033] To further investigate the synergistic protective effect of PGG and GE combined on neuronal cell injury, flow cytometry was used to evaluate the apoptosis rate of the drugs alone and in combination on glutamate-induced PC12 injury and thrombin-induced BV2 injury. The results showed (Table 3, Figure 1 A, 1B), the apoptosis rate of PC12 and BV2 models was significantly higher than that of the blank group (P < 0.001). Compared with the model group, the apoptosis rate of PC12 and BV2 was significantly reduced after single and combined use of drugs (P < 0.001), while the apoptosis rate of the combination of PGG and GE was significantly lower than that of the single drug (P < 0.001, P < 0.01, P < 0.05). This result shows that the combination of PGG and GE has a stronger synergistic inhibitory effect on the apoptosis of damaged nerve cells and has therapeutic uses for nerve damage caused by glutamate and thrombin.

[0034] Table 2 Inhibition rate and CI of cell damage by single drug use and combination drug use

[0035]

[0036] Note: CI: combination drug index; PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide

[0037] Table 3 Effects of single and combined use of drugs on the apoptosis rate of PC12 and BV2 (X±S)

[0038]

[0039] Note: Compared with the blank group, ▲▲P<0.001; compared with the model group, **P<0.001; compared with the combination of drugs, ###P<0.001, ##P<0.01, #P<0.05; PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide

[0040] Example 3 Anti-thrombotic experiment of PGG combined with GE

[0041] To evaluate the antithrombotic synergistic effect of PGG combined with GE, phenylhydrazine (PHZ) was used to induce a zebrafish thrombosis model. The zebrafish cardiac erythrocyte staining intensity was used as an indicator, and aspirin was used as a positive drug to evaluate the antithrombotic effect of the drugs alone and in combination. The staining of zebrafish cardiac erythrocytes in each group was observed under a microscope, and images were collected. The image processing software imagepro plus 6.0 was used to quantitatively analyze the cardiac erythrocyte staining intensity (SI). The antithrombotic efficacy was further analyzed using the following formula:

[0042] Antithrombotic efficiency (%) = (SI 药物 -SI 模型 ) / (SI 空白 -SI 模型 )×100%.

[0043] From Table 4, Figure 2 The results showed that compared with the blank group (a), the staining intensity of cardiac erythrocytes in the zebrafish model group (b) was significantly reduced (P < 0.01), indicating that the thrombosis model was successful; compared with the model group, the staining intensity of cardiac erythrocytes in the three dose groups of PGG (gi) and GE (df) alone and in combination (j-1) at low, medium and high doses, and the aspirin group (c) was significantly increased (P < 0.05, P < 0.01, P < 0.001), and the relationship was dose-response, indicating that the drugs alone and in combination had significant anti-thrombotic effects. The anti-thrombotic effect of the high and medium dose groups of the drug combination was significantly stronger than that of the same dose of the drug alone group (P < 0.05, P < 0.001). In addition, there was no significant difference between the medium dose group of the drug combination and the aspirin group. The antithrombotic efficiency results (Table 5) showed that compared with the use of single drugs, the antithrombotic efficiency of the drug combination in each dose group was improved. Among them, the antithrombotic efficiency of the medium-dose combination was close to that of the aspirin group, and the antithrombotic efficiency of the high-dose combination was greatly improved.

[0044] Table 4 Effects of drug combination on zebrafish cardiac erythrocyte staining intensity (X±S)

[0045]

[0046] Note: Compared with the blank group, ▲▲P<0.01; compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the drug combination and equal dose groups, #P<0.05, ##P<0.001; PHZ: phenylhydrazine; As: aspirin;

[0047] PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide; SI: cardiac erythrocyte staining intensity

[0048] Table 5 Antithrombotic efficacy of single and combined drugs in zebrafish thrombosis model

[0049]

[0050] Note: PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide; As: aspirin

[0051] This result shows that the anti-thrombotic effect of PGG combined with GE is significantly enhanced, and it has therapeutic use in anti-thrombotic treatment.

[0052] Example 4 Inhibitory effect of combined use of PGG and GE on p38MAPK signaling pathway

[0053] The activation of p38MAPK signaling pathway plays a key role in platelet activation and thrombosis. To investigate the targeted therapeutic effect of PGG combined with GE on thrombosis caused by p38MAPK signaling pathway, Western blot was used to detect the effects of single and combined use of drugs and SB203580 (p38MAPK inhibitor) on the phosphorylation levels of MKK3, MKK6 and p38 proteins in PC12 cells induced by glutamate. The results are shown in Table 6. Figure 3 Compared with the blank group, PC12 cells induced by glutamate could significantly increase the phosphorylation levels of MKK3, MKK6 and p38 proteins (P<0.01); compared with the model group, SB203580, drugs alone and combined use could significantly reduce the phosphorylation levels of MKK3, MKK6 and p38 proteins (P<0.01); compared with drugs alone, drug combination could significantly inhibit the phosphorylation levels of MKK3, MKK6 and p38 proteins (P<0.01, P<0.05). In addition, there was no significant difference in the inhibitory level of MKK6 protein phosphorylation between SB203580 and PGG+GE.

[0054] Table 6 Effects of single and combined use of drugs on the expression levels of MKK3, MKK6 and p38 phosphorylated proteins

[0055]

[0056] Note: Compared with the blank group, ▲▲P<0.01; compared with the model group, **P<0.01; compared with the combination drug, #P<0.05, ##P<0.01;

[0057] PGG: 1,2,3,4,6-pentagalloyl-β-D-glucose; GE: geniposide

[0058] The effects of PGG and GE combined on the p38-MKK3 axis were further investigated. Thrombin-induced BV2 cell injury model and glutamate-induced PC12 cell injury model were used to evaluate the effects of single and combined use of drugs on the p38-MKK3 axis by immunoprecipitation. Figure 4 Compared with the blank group, the binding of p38 / p-p38 and MKK3 in PC12 and BV2 cells of the model group increased; compared with the model group, the binding of p38 / p-p38 and MKK3 decreased to varying degrees after drug monomer and combined intervention. Among them, compared with single administration, combined drug treatment significantly reduced the binding of p38 / p-p38 and MKK3, blocked the p38-MKK3 axis, and inhibited the activation of p38 by activated MKK3.

[0059] The above results show that although PGG and GE alone can inhibit the phosphorylation expression levels of MKK3, MKK6 and p38 proteins and weaken the p38-MKK3 axis, the combination of the two drugs can enhance the inhibition of the phosphorylation expression levels of MKK3, MKK6 and p38 proteins, block the p38-MKK3 axis, inhibit the activation of p38 by activated MKK3, and have a strong inhibitory effect on the activation of the p38MAPK signaling pathway, and have a targeted therapeutic use against thrombosis.

[0060] In summary, the present invention combines PGG and GE (structural formula see Figure 5 ) combination has a synergistic effect on the protection of nerve cell damage, anti-thrombotic effect and inhibition of p38MAPK signaling pathway activation.

[0061] The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or improvements based on the technical solution and inventive concept of the present invention without departing from the principle and technical scope of the present invention. Such substitutions or improvements should all be included in the protection scope of the present invention.

Claims

1. A synergistic combination drug for treating thrombosis, characterized in that: The combined drug is 1,2,3,4,6-pentagalloyl-β-D-glucose (CAS No.: 14937-32-7) and geniposide (CAS No.: 24512-63-8).

2. The synergistic combination drug for treating thrombosis according to claim 1, characterized in that: The combined drug is used in a combined administration manner.

3. The combination drug according to claim 1, characterized in that The weight ratio of 1,2,3,4,6-pentagalloyl-β-D-glucose to geniposide is 1:1.

5.

4. The use of the combined drug according to claim 1, characterized in that: It is used to prepare drugs for the treatment of thrombosis.

5. The use of the combined drug according to claim 1, characterized in that: The preparations include solid preparations such as tablets, capsules, solid dispersions, suspensions, etc.

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