Metrnl protein and clopidogrel combined medicine composition for treating thrombus and application of Metrnl protein and clopidogrel combined medicine composition

Abstract

The invention relates to the field of molecular biology and biological medicine, and particularly discloses a synergistic effect of combined application of Metrnl protein and a clinical first-line therapeutic drug clopidogrel for treating thrombus, and the Metrnl protein has important significance in the field of thrombotic disease treatment. According to the invention, mice are divided into four groups: a solvent control group, a Metrnl proteome, a clopidogrel group and a drug combination group. A FeCl3 induced thrombus experiment shows that thrombus formation can be inhibited by independently using Metrnl protein and clopidogrel, the thrombus formation inhibition effect is stronger by combined medication, and a synergistic effect is achieved. In addition, in the aspect of untoward effects of bleeding, compared with a clopidogrel single drug group, the risk of bleeding of the drug combination group is not obviously increased. The metrnl protein and clopidogrel are combined to enhance the antithrombotic curative effect without increasing the adverse reaction of bleeding, so that the metrnl protein and clopidogrel have good treatment potential and safety.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and biomedicine, and more specifically, to a pharmaceutical composition for the combined treatment of thrombosis with Metrnl protein and clopidogrel, and its application. Background Technology

[0002] The Metrnl protein consists of 311 amino acids, is encoded by the Metrnl gene, and is approximately 35 kDa in size. The amino acid sequence of the human Metrnl protein can be found in NP_001004431.1. Chinese patent application CN200980137344.4, entitled "Therapeutic Use of Growth Factor METRNL", publication number CN102164611A, discloses that METRNL is a neurosurvival and growth factor with neuroprotective and / or neurogenic effects, which can be used to treat diseases, disorders, or damage of the nervous system; Chinese patent CN201310525184.9 discloses the application of Metrnl protein in the preparation of hypoglycemic drugs; Chinese patent CN201310525181.5 discloses the application of Metrnl protein in the preparation of lipid-lowering drugs or health foods; Chinese patent CN201810743032.9 discloses the anti-atherosclerotic use of Metrnl; and Chinese patent CN201710778140.5 discloses the antithrombotic use of Metrnl.

[0003] Thrombotic diseases are a group of cardiovascular and cerebrovascular diseases that seriously threaten human life and health, with typical examples including ischemic stroke and myocardial infarction. Clinically, commonly used antithrombotic treatments mainly rely on antiplatelet drugs, such as the P2Y12 receptor antagonist clopidogrel, as first-line drugs. However, the therapeutic efficacy of these drugs in clinical use is often limited by the contradiction between "antithrombotic benefits and bleeding risks": on the one hand, sufficiently potent antithrombotic effects are needed to prevent thrombus formation; on the other hand, drug-induced bleeding adverse reactions need to be controlled. Therefore, how to enhance antithrombotic efficacy while avoiding bleeding risks has always been a core technical challenge in this field. To address this problem, several technologies have attempted to improve treatment safety through combination drug therapy. For example, patent application CN200810048929.6 discloses an antithrombotic drug composition comprising an effective dose of an ADP receptor antagonist and indobufen. The two drugs have a synergistic effect, achieving an antithrombotic effect while reducing the dosage of a single drug, thereby reducing adverse reactions. Patent application CN201010291389.1 discloses a drug combination for inhibiting thrombus formation, with trifluralin and clopidogrel as active ingredients. Patent application CN201410238841.6 discloses a drug composition composed of clopidogrel and resveratrol for antithrombotic treatment. All of the above-mentioned prior art utilizes a combination drug strategy, achieving an antithrombotic effect while reducing drug dosage through synergistic drug action, thereby reducing the risk of bleeding.

[0004] However, the core idea of ​​existing technical solutions still focuses on reducing dosage while maintaining efficacy, that is, reducing the drug dosage to reduce side effects while ensuring antithrombotic efficacy. This approach fails to substantially solve the problem of enhancing antithrombotic efficacy without increasing the risk of bleeding. Therefore, there is an urgent need to develop novel treatment regimens to enhance antithrombotic efficacy without increasing the risk of bleeding. Currently, there are no reports on the application of Metrnl protein combined with clopidogrel for synergistic treatment of thrombosis without increasing the risk of bleeding. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a pharmaceutical composition that has a synergistic effect in inhibiting thrombus formation without increasing the risk of adverse bleeding associated with single-drug therapy.

[0006] A second objective of this invention is to address the shortcomings of the prior art by providing an application of a pharmaceutical composition in the preparation of a drug for inhibiting thrombosis.

[0007] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0008] A pharmaceutical composition that synergistically inhibits thrombus formation, the composition comprising the Metrnl protein and a P2Y12 receptor antagonist.

[0009] In the above-mentioned pharmaceutical composition with synergistic effect in inhibiting thrombus formation, preferably, the P2Y12 receptor antagonist is clopidogrel.

[0010] In the above-mentioned drug composition with synergistic inhibitory effect on thrombus formation, preferably, the weight ratio of the Metrnl protein to the P2Y12 receptor antagonist is (0.01-1.0):(1-10).

[0011] In the above-mentioned pharmaceutical composition with synergistic inhibitory effect on thrombus formation, preferably, the weight ratio of the Metrnl protein to the P2Y12 receptor antagonist is 0.12:4.

[0012] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0013] Application of Metrnl protein in combination with P2Y12 receptor antagonist drugs in the preparation of drugs that inhibit thrombosis.

[0014] In the above applications, preferably, the P2Y12 receptor antagonist is clopidogrel.

[0015] In the above applications, preferably, the weight ratio of the Metrnl protein to the P2Y12 receptor antagonist drug is (0.01-1.0):(1-10).

[0016] In the above applications, preferably, the weight ratio of the Metrnl protein to the P2Y12 receptor antagonist drug is 0.12:4.

[0017] In the above applications, preferably, the drug further includes one or more pharmaceutically acceptable auxiliary ingredients.

[0018] In the above applications, preferably, the pharmaceutically acceptable auxiliary ingredient is an excipient.

[0019] In the above applications, preferably, the pharmaceutically acceptable auxiliary ingredient is a filler.

[0020] In the above applications, preferably, the pharmaceutically acceptable auxiliary ingredient is a diluent.

[0021] More preferably, in the above applications, the drug comprises the active ingredient Metrnl protein and a P2Y12 receptor antagonist, as well as an appropriate amount of adjuvant ingredients.

[0022] In the above applications, preferably, the drug is prepared into a clinically acceptable pharmaceutical formulation according to conventional pharmaceutical preparation methods. The active pharmaceutical ingredient can be the same or different conventional dosage forms in the art, and the dosage form can be in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably a powder, granules, tablet, capsule, pill, solution, suspension, or injection. The preferred route of administration is injection or oral administration. Preferred routes of injection include intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0023] The advantages of this invention are as follows: Mice were divided into a solvent control group, a Metalnl protein group, a clopidogrel group, and a combination therapy group. Through FeCl3-induced thrombosis experiments, it was found that both Metalnl protein and clopidogrel alone could inhibit thrombus formation. The antithrombotic effect of the combination therapy group was significantly better than either single-drug group, indicating a synergistic effect between Metalnl protein and clopidogrel in inhibiting thrombus formation. Simultaneously, by assessing adverse bleeding reactions using indicators such as tail bleeding time, it was found that the combination therapy group did not show a significant increase in bleeding risk compared to the clopidogrel monotherapy group. These results demonstrate that the combination therapy of Metalnl protein and clopidogrel enhances antithrombotic efficacy without increasing adverse bleeding reactions, exhibiting good therapeutic potential and safety. Attached Figure Description

[0024] Appendix Figure 1 Synergistic effect of Metrnl protein combined with clopidogrel in the treatment of thrombosis. A. The antithrombotic effect of clopidogrel increases with increasing dose. n=3 per group. B. The risk of bleeding increases with increasing dose of clopidogrel. n=4 per group. C. Metrnl protein combined with clopidogrel has a synergistic effect in the treatment of thrombosis. n=8-10. *P<0.05, ***P<0.001, NS, no significance.

[0025] Appendix Figure 2 The combined use of Metrnl protein and clopidogrel did not increase the risk of bleeding. A. Metrnl protein did not increase the bleeding time associated with clopidogrel. (n=8 per group) B. Metrnl protein did not increase the amount of bleeding associated with clopidogrel. (n=8 per group) *P<0.05, **P<0.01, NS, no significance. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0027] Metrnl protein

[0028] In this invention, the Metrnl protein used can be naturally occurring, for example, it can be isolated and purified from mammals.

[0029] Furthermore, the Metrnl protein can also be artificially prepared, for example, using conventional genetic engineering techniques. Any suitable Metrnl protein is applicable to this invention. The Metrnl protein includes the full-length Metrnl protein or its bioactive fragment. As one specific embodiment, the Metrnl protein can be a human Metrnl protein with a size of 35 kDa, and its amino acid sequence is shown in NP_001004431.1. As another specific embodiment, the Metrnl protein can be a rat or mouse Metrnl protein, and the amino acid sequences of rat and mouse Metrnl proteins are shown in patent ZL2016100890727. The amino acid sequences of Metrnl proteins formed by substitution, deletion, or addition of one or more amino acid residues are also included in this invention. The Metrnl protein or its bioactive fragment includes a portion of conserved amino acid substitution sequences, the substitution sequences of which do not affect its activity or retain part of its activity. Appropriate amino acid substitution is a well-known technique in the art, which can be easily implemented and ensures that the biological activity of known molecules is not altered. These techniques have led those skilled in the art to recognize that, generally speaking, altering a single amino acid in a non-essential amino acid region of a polypeptide does not change its biological activity.

[0030] All bioactive fragments of the Metrnl protein can be used in this invention. In this invention, a bioactive fragment of the Metrnl protein refers to a polypeptide that retains all or part of the function of the full-length Metrnl protein. Typically, the bioactive fragment retains at least 50%, 60%, and up to 99% or 100% of the activity of the full-length Metrnl protein.

[0031] This invention can also employ modified or altered Metrnl proteins, including all or some of their amino acids. For example, Metrnl proteins can be modified or altered to improve half-life, efficacy, metabolism, and / or protein potency. The modified or altered Metrnl protein can be a conjugate of a Metrnl protein, or a substitute or artificial amino acid thereof. The modified or altered Metrnl protein or gene may differ from the natural Metrnl protein or gene, but still possess the functions described in this invention without causing other adverse reactions or toxicity. In other words, any variation that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in this invention.

[0032] Example 1: Synergistic effect of Metrnl protein combined with clopidogrel in the treatment of thrombosis

[0033] I. Experimental Methods

[0034] Drug administration: Eight-week-old C57BL / 6J mice were randomly assigned to groups. Clopidogrel 1 mg / kg, 4 mg / kg, or 10 mg / kg was administered by gavage 2 hours before modeling; Metrnl protein 0.12 mg / kg was administered via tail vein injection immediately after modeling; combined administration was performed by administering clopidogrel 4 mg / kg and Metrnl protein 0.12 mg / kg in the same manner; the solvent control group was administered physiological saline by gavage and PBS by tail vein injection.

[0035] Thrombosis modeling and measurement: Before modeling, Rhodamine 6G was injected into the tail vein to observe the thrombus. After the injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg / kg). An incision was made along the midline of the abdomen from below the manubrium of the sternum towards the abdomen (avoiding damage to blood vessels). The mice were placed on their left side in a culture dish (approximately 10 cm in diameter). An inverted glass dish (approximately 3 cm in diameter) was placed next to the mouse, level with the opening in the abdomen. The small intestine of the mouse was carefully pulled out with forceps (avoiding pinching blood vessels and causing bleeding) and spread flat on the dish. Warm physiological saline was added to maintain the temperature and humidity of the mouse's intestine. Under a microscope, a suitable field of view of the mesenteric artery was selected. A piece of filter paper (1.5 mm × 3 mm) soaked in 10% FeCl3 solution was carefully placed on the mesenteric artery. After 1 minute, the filter paper was removed, and the thrombus formation was observed under a microscope. The entire process was recorded using a computer-controlled microscope for photography and measurement.

[0036] Bleeding model and measurement: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg / kg). After complete anesthesia, the mice were placed on a 37°C constant temperature pad. A mark was made 3 mm from the tip of the mouse's tail with a ruler. The tail tip was cut off at the mark and immediately immersed in 10 mL of 37°C physiological saline. After the blood flow stopped, the bleeding time was recorded.

[0037] II. Results

[0038] In clopidogrel dosage studies, it was found that as the dose increased, the antithrombotic effect of clopidogrel significantly increased, but the risk of bleeding also significantly increased. Figure 1 (A and 1B). Therefore, 4 mg / kg was selected as the experimental dose for subsequent experiments. It was found that both Metrnl protein (0.12 mg / kg) and clopidogrel (4 mg / kg) alone could inhibit thrombus formation, and the combination therapy group (Metrnl protein 0.12 mg / kg + clopidogrel 4 mg / kg) showed significantly better anti-thrombus formation effects than either Metrnl protein or clopidogrel alone. Figure 1 C). The boundary is defined as 1161 seconds (s) from the highest point of the solvent control group. Figure 1 (C, red line) The efficacy rate of Metrnl protein treatment was determined to be 62.5% (5 / 8), the efficacy rate of clopidogrel treatment was 40% (4 / 10), and the efficacy rate of combined Metrnl protein and clopidogrel treatment was 100% (10 / 10). Using the q-value method, the efficacy of the two drugs combined was calculated as q = 100% / (62.5% + 40% - 62.5% × 40%) = 1.290. Since the q-value is > 1.15, the combination of Metrnl protein and clopidogrel has a synergistic effect in inhibiting thrombus formation.

[0039] Example 2: Metrnl protein combined with clopidogrel treatment did not increase bleeding adverse reactions.

[0040] I. Experimental Methods

[0041] Drug administration: Eight-week-old C57BL / 6J mice were randomly divided into groups. Clopidogrel 4 mg / kg was administered by gavage 2 hours before modeling; Metrnl protein 0.12 mg / kg was administered by tail vein injection immediately after modeling; combined drug administration was performed by administering clopidogrel 4 mg / kg and Metrnl protein 0.12 mg / kg in the same manner; the solvent control group was administered physiological saline by gavage and PBS by tail vein injection.

[0042] Bleeding Model and Measurement: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg / kg). After complete anesthesia, the experiment began. Mice were placed on a 37°C constant-temperature mat, and a mark was made 3 mm from the tip of the tail. The tail tip was cut off at the mark and immediately immersed in 10 mL of 37°C physiological saline. After blood flow stopped, the bleeding time was recorded, and the blood was gently mixed and frozen at -80°C. Blood samples from another mouse were collected, and 320, 160, 80, 40, 20, 10, and 0 μL were added to 10 mL of physiological saline, gently mixed, and frozen at -80°C. The next day, all blood samples were thawed, and 200 μL was added to a 96-well plate and measured using a microplate reader at a wavelength of 405 nm. The amount of bleeding in the mice was then calculated based on a known blood volume standard curve.

[0043] II. Experimental Results

[0044] from Figure 2 As can be seen from AB, compared with the solvent control group, treatment with clopidogrel alone increased bleeding time. Figure 2 A) and blood loss ( Figure 2 B). Compared with the clopidogrel monotherapy group, there was no significant difference in bleeding time and bleeding volume in the combination therapy group (Metrnl protein 0.12 mg / kg + clopidogrel 4 mg / kg).

[0045] comprehensive Figure 1 C and Figure 2 The results of the AB study show that the combination of Metrnl protein and clopidogrel as active ingredients has a significant synergistic effect on thrombus formation, and the combined use is significantly better than the single-drug group in inhibiting thrombus formation; moreover, the treatment of Metrnl protein combined with clopidogrel does not increase the bleeding time and bleeding volume of clopidogrel.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition having a synergistic effect in inhibiting thrombus formation, characterized in that, The drug consists of the Metrnl protein and a P2Y12 receptor antagonist.

2. The pharmaceutical composition with synergistic inhibitory effect on thrombus formation according to claim 1, characterized in that, The P2Y12 receptor antagonist is clopidogrel.

3. The pharmaceutical composition with synergistic inhibitory effect on thrombus formation according to claim 2, characterized in that, The weight ratio of the Metrnl protein to the P2Y12 receptor antagonist drug is (0.01-1.0):(1-10).

4. The pharmaceutical composition with synergistic inhibitory effect on thrombus formation according to claim 3, characterized in that, The weight ratio of the Metrnl protein to the P2Y12 receptor antagonist drug is 0.12:

4.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for inhibiting thrombosis.

6. The application according to claim 5, characterized in that, The drug also includes one or more pharmaceutically acceptable adjuvants.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable auxiliary ingredient is an excipient.

8. The application according to claim 6, characterized in that, The pharmaceutically acceptable auxiliary ingredient is a filler.

9. The application according to claim 6, characterized in that, The pharmaceutically acceptable auxiliary ingredient is a diluent.

10. The application according to claim 5, characterized in that, The pharmaceutical preparation is in the form of granules, capsules, tablets, suspensions, emulsions, solutions, or injections. The drug can be administered to individuals in need of treatment via oral, intravenous, intravenous drip, intramuscular, or subcutaneous injection routes, depending on the dosage form.

Images