Medicament for simultaneously reducing risks of bleeding and microvascular occlusion

Abstract

The invention provides a medicine for simultaneously reducing the risks of bleeding and microvascular occlusion. Specifically, the invention provides a composition, the composition comprises (a) a first active component with a therapeutically effective amount, and the first active component is clopidogrel or a pharmaceutically acceptable salt thereof; (b) a therapeutically effective amount of a second active ingredient, wherein the second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof; and (c) a therapeutically effective amount of a third active ingredient, wherein the third active ingredient is ginsenoside Rg1 or a pharmaceutically acceptable salt thereof. The first active component, the second active component and the third active component can generate a bidirectional synergistic protection effect, can effectively reduce microvascular occlusion, can effectively prevent bleeding, reduces the dosage of a single drug, and reduces the toxicity of the drug.

Description

[0001] This application is a divisional application of the application with the application number "201810634906.7", the application date "June 15, 2018", and the patent name "Drug for Simultaneously Reducing the Risks of Bleeding and Microvascular Occlusion". Technical Field

[0002] The present invention relates to the field of drugs. Specifically, the present invention relates to a drug for simultaneously reducing the risks of bleeding and microvascular occlusion. Background Art

[0003] Ischemia reperfusion injury refers to the phenomenon that when blood flow is restored to tissues or organs after a period of ischemia, the injury of the tissues or organs does not decrease but instead worsens, which is the continuation of ischemic injury and the manifestation of aggravated injury. Ischemia reperfusion injury of important organs or tissues such as the heart, brain, kidney, liver, and skeletal muscle seriously endangers human life and health. During surgical treatments such as thrombolytic surgery or vascular stent surgery, risks such as bleeding and microvascular occlusion often occur.

[0004] How to effectively prevent and treat ischemia reperfusion injury of various tissues and organs in the human body, reduce it, and improve bleeding and microvascular occlusion are the difficulties and hotspots in current basic medical and clinical medical research.

[0005] Therefore, there is an urgent need in the art to develop a drug with high efficacy and low side effects for ischemia reperfusion injury, bleeding, and microvascular occlusion. Summary of the Invention

[0006] The purpose of the present invention is to provide a drug with high efficacy and low side effects for ischemia reperfusion injury, bleeding, and microvascular occlusion.

[0007] In the first aspect of the present invention, there is provided a composition, which comprises:

[0008] (a) A therapeutically effective amount of a first active ingredient, and the first active ingredient is clopidogrel or a pharmaceutically acceptable salt thereof;

[0009] (b) A therapeutically effective amount of a second active ingredient, and the second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof; and

[0010] (c) A therapeutically effective amount of a third active ingredient, and the third active ingredient is ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

[0011] In another preferred example, the pharmaceutically acceptable salt is a sulfate, a bisulfate, or a hydrochloride.

[0012] In another preferred embodiment, the active ingredients of the composition consist of (a) a therapeutically effective amount of a first active ingredient, which is clopidogrel or a pharmaceutically acceptable salt thereof; and (b) a therapeutically effective amount of a second active ingredient, which is salvianolic acid B or a pharmaceutically acceptable salt thereof; and (c) a therapeutically effective amount of a third active ingredient, which is ginsenoside Rg1.

[0013] In another preferred embodiment, the content range of the first active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; preferably 10% to 99%; more preferably 20% to 99%.

[0014] In another preferred embodiment, the content range of the second active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; more preferably 1% to 90%; more preferably 1% to 80%.

[0015] In another preferred embodiment, the content range of the third active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; preferably 10% to 99%; more preferably 20% to 99%.

[0016] In another preferred embodiment, the weight ratio of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5 - 8:0.5 - 6:0.6 - 12, preferably 1 - 6:0.5 - 4:1 - 10, more preferably 2 - 4:1 - 3:3 - 7.

[0017] In another preferred embodiment, the weight ratio of the second active ingredient to the third active ingredient is 0.5 - 6:0.6 - 12, preferably 0.5 - 4:1 - 10, more preferably 1 - 3:3 - 7.

[0018] In another preferred embodiment, the total weight ratio of the first active ingredient to the second active ingredient is 0.5 - 8:0.5 - 9, preferably 0.5 - 8:0.5 - 6, preferably 1 - 6:0.5 - 4, more preferably 2 - 4:1 - 3.

[0019] In another preferred embodiment, the total weight ratio of the first active ingredient to the third active ingredient is 0.5 - 8:0.6 - 12, preferably 1 - 6:1 - 10, more preferably 2 - 4:3 - 7.

[0020] In another preferred embodiment, the purity of clopidogrel or a pharmaceutically acceptable salt thereof is 80 - 100%, preferably 90% - 100%.

[0021] In another preferred embodiment, the purity of salvianolic acid B or its pharmaceutically acceptable salt is 80-100%, preferably 90%-100%.

[0022] In another preferred embodiment, the purity of ginsenoside Rg1 or its pharmaceutically acceptable salt is 80-100%, preferably 90%-100%.

[0023] In another preferred embodiment, the composition is a pharmaceutical composition.

[0024] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0025] In a second aspect of the present invention, there is provided a medicine box, which comprises:

[0026] (A) A first preparation containing clopidogrel or its pharmaceutically acceptable salt;

[0027] (B) A second preparation containing salvianolic acid B or its pharmaceutically acceptable salt;

[0028] (C) A third preparation containing ginsenoside Rg1 or its pharmaceutically acceptable salt; and

[0029] (D) Instructions for use.

[0030] In another preferred embodiment, the first preparation, the second preparation and the third preparation are independent preparations.

[0031] In another preferred embodiment, at least one of the first preparation, the second preparation and the third preparation is an independent preparation.

[0032] In another preferred embodiment, the first preparation, the second preparation and the third preparation are combined preparations.

[0033] In another preferred embodiment, the first preparation is a solid preparation, preferably a tablet.

[0034] In another preferred embodiment, the second preparation is a liquid preparation, preferably an injection, an infusion or a freeze-dried preparation.

[0035] In another preferred embodiment, the third preparation is a liquid preparation, preferably an injection, an infusion or a freeze-dried preparation.

[0036] In another preferred embodiment, the instructions for use state that the first preparation, the second preparation and the third preparation are used in combination to prevent and / or treat ischemia-reperfusion injury, reduce bleeding and / or microvascular embolism.

[0037] In another preferred embodiment, the first preparation, the second preparation and the third preparation are administered simultaneously, separately or sequentially.

[0038] In another preferred embodiment, the first preparation, the second preparation and the third preparation are administered before, during or after an ischemia-reperfusion operation, a vascular stent operation or a thrombolytic operation.

[0039] In another preferred embodiment, the weight ratio of the first active ingredient, the second active ingredient and the third active ingredient is 0.5-8:0.5-6:0.6-12, preferably 1-6:0.5-4:1-10, more preferably 2-4:1-3:3-7.

[0040] In the third aspect of the present invention, there is provided a combination of active ingredients, which combination of active ingredients comprises the following components:

[0041] (1) A therapeutically effective amount of a first active ingredient, which first active ingredient is clopidogrel or a pharmaceutically acceptable salt thereof;

[0042] (2) A therapeutically effective amount of a second active ingredient, which second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof; and

[0043] (3) A therapeutically effective amount of a third active ingredient, which third active ingredient is ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

[0044] In another preferred embodiment, in the combination of active ingredients, at least one active ingredient is independent.

[0045] In another preferred embodiment, the weight ratio of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5-8:0.5-6:0.6-12, preferably 1-6:0.5-4:1-10, more preferably 2-4:1-3:3-7.

[0046] In the fourth aspect of the present invention, there is provided a use of the composition as described in the first aspect of the present invention, or the kit as described in the second aspect of the present invention, or the combination of active ingredients as described in the third aspect of the present invention, for including: (i) preparing a drug for improving bleeding during a vascular stent operation and reducing microvascular obstruction; (ii) preparing a drug for improving bleeding during a thrombolytic operation and reducing microvascular obstruction; and / or (iii) preparing a drug for preventing and / or treating ischemia-reperfusion injury.

[0047] In another preferred embodiment, the ischemia-reperfusion injury includes bleeding injury and microvascular obstruction injury.

[0048] In another preferred embodiment, the vascular stent operation is a vascular stent operation combined with an anticoagulant drug.

[0049] In another preferred embodiment, the thrombolytic surgery is a thrombolytic surgery combined with an anticoagulant drug.

[0050] In another preferred embodiment, the ischemia-reperfusion injury is selected from the group consisting of: cardiovascular ischemia-reperfusion injury, cerebrovascular ischemia-reperfusion injury, extracirculatory ischemia-reperfusion injury, acute arterial embolism reperfusion injury, traumatic shock reperfusion injury, surgical reperfusion injury, organ transplantation reperfusion injury, burns, or reperfusion injury caused by frostbite or blood circulation disorders caused by thrombosis.

[0051] In another preferred embodiment, the cardiovascular ischemia-reperfusion injury includes myocardial infarction, myocardial fibrosis, myocardial ischemia, myocardial necrosis, myocardial hypertrophy, heart failure or myocardial microvascular obstruction.

[0052] In a fifth aspect of the present invention, there is provided a use of a compound combination, the compound combination comprising (a) salvianolic acid B or a pharmaceutically acceptable salt thereof and (b) ginsenoside Rg1 or a pharmaceutically acceptable salt thereof, the compound combination for preparing a pharmaceutical composition or preparation, the pharmaceutical composition or preparation for including: (i) improving bleeding in vascular stent surgery and reducing microvascular obstruction; (ii) improving bleeding in thrombolytic surgery and reducing microvascular obstruction; and / or (iii) reducing the side effects of anticoagulant drugs.

[0053] In another preferred embodiment, the vascular stent surgery is a vascular stent surgery combined with an anticoagulant drug.

[0054] In another preferred embodiment, the thrombolytic surgery is a thrombolytic surgery combined with an anticoagulant drug.

[0055] In another preferred embodiment, the side effects of the anticoagulant drug are side effects produced by the anticoagulant drug when used in vascular stent surgery or thrombolytic surgery.

[0056] In another preferred embodiment, the side effects of the anticoagulant drug include bleeding and / or microcirculation disorder.

[0057] In another preferred embodiment, the anticoagulant drug is selected from the group consisting of: vitamin K antagonists, antiplatelet aggregation drugs, thrombin IIa inhibitors, factor Xa inhibitors, or combinations thereof.

[0058] In another preferred embodiment, the vitamin K antagonist is selected from the group consisting of: warfarin.

[0059] In another preferred embodiment, the antiplatelet aggregation drug is selected from the group consisting of: aspirin, ticlopidine, clopidogrel, abciximab, eptifibatide or tirofiban, or combinations thereof.

[0060] In another preferred embodiment, the thrombin IIa inhibitor is selected from the group consisting of: bivalirudin, argatroban, dabigatran etexilate, hirudin.

[0061] In another preferred embodiment, the factor Xa inhibitor is selected from the group consisting of: apixaban, rivaroxaban, edoxaban, or a combination thereof.

[0062] In another preferred embodiment, the weight ratio of salvianolic acid B or a pharmaceutically acceptable salt thereof to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5 - 6:0.6 - 12, preferably 0.5 - 4:1 - 10, more preferably 1 - 3:3 - 7.

[0063] In the sixth aspect of the present invention, there is provided a method for simultaneously reducing the risks of bleeding and microvascular obstruction, said method comprising the step of: administering to a subject in need thereof the composition as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention.

[0064] In another preferred embodiment, the subject is a patient undergoing a surgery selected from the group consisting of: ischemia-reperfusion surgery, vascular stent surgery, or thrombolysis surgery.

[0065] In another preferred embodiment, the subject is a human and non-human mammal.

[0066] In another preferred embodiment, the subject is a human.

[0067] In another preferred embodiment, the non-human mammals include (but are not limited to): pets (such as dogs, cats), livestock (such as cows, sheep, horses, pigs), various zoo animals (such as pandas, elephants), etc.

[0068] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is the hemodynamic detection in Example 1 of the present invention.

[0070] Figure 2 It is the Prussian blue staining diagram in Example 2 of the present invention.

[0071] Figure 3 It is the quantitative bar chart of the cardiac bleeding area of each group of rats in Example 2 of the present invention.

[0072] Figure 4 It is the Sirius red staining result in Example 3 of the present invention.

[0073] Figure 5 It is the hematoxylin and eosin staining result in Example 4 of the present invention.

[0074] Figure 6 This is the Castrales staining diagram in Example 5 of the present invention.

[0075] Figure 7 This is the bar chart of the quantitative results of cardiac microvascular obstruction in each group of rats in Example 5 of the present invention.

[0076] Figure 8 This is the Mallory phosphotungstic acid hematoxylin (PTAH) staining in the examples of the present invention Detailed implementation manners

[0077] Through extensive and in-depth research, the inventor of the present invention unexpectedly found that the combination of anticoagulant drugs (such as clopidogrel), salvianolic acid B and ginsenoside Rg1 can effectively reduce microvascular obstruction and effectively prevent bleeding in ischemia-reperfusion injury, as well as in vascular stent surgery and thrombolytic surgery. The synergistic therapeutic effect of the combination is significantly better than the individual use of the three, and at the same time can reduce the dosage of individual drugs and reduce the toxicity of the drugs. On this basis, the present invention was completed.

[0078] Anticoagulant drugs are often used in ischemia-reperfusion, vascular stent surgery and thrombolytic surgery. However, the conditions of different patients (such as age, weight, disease condition, etc.) often vary, which leads to the difficulty in using anticoagulant drugs. When the dosage of anticoagulant drugs is too low, it often leads to insufficient anticoagulant effect, resulting in phenomena such as microvascular obstruction (or embolism); while when the dosage of anticoagulant drugs is too high, it often leads to an increased risk of bleeding.

[0079] Unexpectedly, the present invention provides a dual or two-way synergistic protection effect, that is, it can effectively reduce microvascular obstruction and effectively prevent bleeding, thereby greatly improving the efficacy of anticoagulant drugs and reducing their side effects, and at the same time can significantly reduce the surgical risks of ischemia-reperfusion, vascular stent surgery and thrombolytic surgery (including the risk of microvascular obstruction and bleeding (especially the risk of hemorrhagic necrosis).

[0080] Specifically, the animal experiments of the present invention show that although "clopidogrel + salvianolic acid B" will lead to an increased risk of microvascular obstruction, and "clopidogrel + ginsenoside Rg1" will also lead to an increased risk of microvascular obstruction, unexpectedly, "clopidogrel + salvianolic acid B / ginsenoside Rg1" significantly reduces the risk of microvascular obstruction (and is better than "salvianolic acid B + ginsenoside Rg1").

[0081] Similarly, animal experiments have shown that while "clopidogrel + salvianolic acid B" increases the bleeding risk and "clopidogrel + ginsenoside Rg1" also increases the bleeding risk, unexpectedly, "clopidogrel + salvianolic acid B / ginsenoside Rg1" significantly reduces the bleeding risk (and is superior to "salvianolic acid B + ginsenoside Rg1").

[0082] Term

[0083] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0084] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0085] As used herein, the components of the term "pharmaceutically acceptable carrier" refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0086] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs.

[0087] As used in this invention, "prevention" and "treatment" include delaying and terminating the progression of a disease, or eliminating the disease, and do not require 100% inhibition, eradication, and reversal. In some embodiments, compared to the levels observed in the absence of the composition or pharmaceutical composition of this invention, the composition or pharmaceutical composition of this invention prevents, alleviates, inhibits, and / or reverses ischemic reperfusion injury by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0088] Active ingredient

[0089] In this invention, the first active ingredient is clopidogrel or a pharmaceutically acceptable salt thereof, the second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof, and the third active ingredient is ginsenoside Rg1.

[0090] Clopidogrel is an anticoagulant and antiplatelet drug used for the prevention and treatment of cardiovascular, cerebrovascular, and other arterial circulation disorders caused by high platelet aggregation, such as recent onset stroke, myocardial infarction, and diagnosed peripheral arterial disease.

[0091] Salvianolic acid B has a strong antioxidant effect and is one of the natural products with the strongest known antioxidant effects; it significantly improves the neurological deficits in rats with cerebral ischemia-reperfusion injury, manifested as improving behavioral disorders and significantly reducing the area of cerebral infarction; it has a delayed protective effect on cardiac microvascular endothelial cells; it has a preventive and therapeutic effect on atherosclerosis; and it has a protective effect on preconditioned cardiac cells.

[0092] Ginsenoside Rg1 has the effects of slowing heart rate and two-way blood pressure regulation; dilating blood vessels, improving neural plasticity, enhancing learning and memory, anti-aging, anti-fatigue, improving immunity, assisting in anti-tumor, and repairing sexual function.

[0093] As used herein, "clopidogrel" refers to clopidogrel or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the above components.

[0094] As used herein, "salvianolic acid B" refers to salvianolic acid B or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the above components.

[0095] As used herein, "ginsenoside Rg1" refers to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the above components.

[0096] In the present invention, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention with an acid or a base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salts formed by the compounds of the present invention with acids. Acids suitable for forming salts include, but are not limited to: inorganic acids such as toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid. A preferred class of salts is the salts formed by the compounds of the present invention with bases. Bases suitable for forming salts include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, etc., organic bases such as ammonia, triethylamine, diethylamine, etc. A preferred class of salts is the salts formed by the compounds with metal ions. Metal ions suitable for forming salts include, but are not limited to: sodium ions, magnesium ions, potassium ions, calcium ions, etc.

[0097] A typical pharmaceutically acceptable salt of clopidogrel is clopidogrel sulfate, clopidogrel bisulfate, or clopidogrel hydrochloride.

[0098] A typical pharmaceutically acceptable salt of salvianolic acid B is magnesium salvianolate or calcium salvianolate.

[0099] Representatively, the chemical structural formulas of clopidogrel sulfate, salvianolic acid B, and ginsenoside Rg1 are as follows:

[0100]

[0101] Composition, kit, combination of active ingredients, and pharmaceutical composition

[0102] The present invention provides a composition, which comprises:

[0103] (a) A therapeutically effective amount of a first active ingredient, which is clopidogrel or a pharmaceutically acceptable salt thereof;

[0104] (b) A therapeutically effective amount of a second active ingredient, which is salvianolic acid B or a pharmaceutically acceptable salt thereof; and

[0105] (c) A therapeutically effective amount of a third active ingredient, which is ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

[0106] In another preferred embodiment, the content range of the first active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; preferably 10% to 99%; more preferably 20% to 99%.

[0107] In another preferred embodiment, the content range of the second active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; more preferably 1% to 90%; more preferably 1% to 80%.

[0108] In another preferred embodiment, the content range of the third active ingredient is 0.01% to 99.99% based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; preferably 10% to 99%; more preferably 20% to 99%.

[0109] In another preferred embodiment, the weight ratio of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 is 0.5 - 8:0.5 - 6:0.6 - 12, preferably 1 - 6:0.5 - 4:1 - 10, more preferably 2 - 4:1 - 3:3 - 7.

[0110] In another preferred embodiment, the purity of the clopidogrel or a pharmaceutically acceptable salt thereof is 80 - 100%, preferably 90% - 100%.

[0111] In another preferred embodiment, the purity of the salvianolic acid B or a pharmaceutically acceptable salt thereof is 80 - 100%, preferably 90% - 100%.

[0112] In another preferred example, the purity of the ginsenoside Rg1 is 80 - 100%, preferably 90% - 100%.

[0113] When necessary, the composition may further include a pharmaceutically acceptable carrier to form a pharmaceutical composition (drug).

[0114] Typically, the composition further includes a pharmaceutically acceptable carrier to form a pharmaceutical composition, and the pharmaceutical composition includes:

[0115] (a) A therapeutically effective amount of a first active ingredient, which is clopidogrel or a pharmaceutically acceptable salt thereof;

[0116] (b) A therapeutically effective amount of a second active ingredient, which is salvianolic acid B or a pharmaceutically acceptable salt thereof;

[0117] (c) A therapeutically effective amount of a third active ingredient, which is ginsenoside Rg1 or a pharmaceutically acceptable salt thereof; and

[0118] (d) A pharmaceutically acceptable pharmaceutical carrier.

[0119] The pharmaceutical composition containing the first active ingredient, the second active ingredient and the third active ingredient according to the present invention, in addition to various dosage forms suitable for oral administration, can also be various external administration preparations or other parenteral administration preparations. For example, the external administration preparation of the present invention can be further prepared into (including but not limited to): liniments, tinctures, oils, ointments, plasters, pastes, ironing agents, patches, patches, film-forming agents, membranes, gels, cataplasms, acupoint application agents, sprays, aerosols, implants, emulsions, etc. by adding excipients such as surfactants, transdermal absorption promoters, preservatives, solvents, antioxidants, humectants, pH regulators, colorants, fragrances, etc. For ischemic reperfusion injury, the preferred dosage forms include: various dosage forms for oral administration, implants, injections.

[0120] It should be understood that in the present invention, the carrier is not particularly limited and is a commonly used material in the art, and its type, usage method and source are well known to those skilled in the art.

[0121] Examples of pharmaceutically acceptable carrier parts include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffering agents, chelating agents, thickening agents, pH regulators, transdermal penetration enhancers, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0122] In another preferred example, in the said pharmaceutical composition, the content ranges of the first active ingredient and the second active ingredient, as well as the mass ratio of the first active ingredient and the second active ingredient, are as described in the above-mentioned composition.

[0123] The present invention also provides an active ingredient composition, and the active ingredient combination includes the following components: (1) a therapeutically effective amount of a first active ingredient, and the first active ingredient is clopidogrel or its pharmaceutically acceptable salt;

[0124] (2) a therapeutically effective amount of a second active ingredient, and the second active ingredient is salvianolic acid B or its pharmaceutically acceptable salt; and

[0125] (3) a therapeutically effective amount of a third active ingredient, and the third active ingredient is ginsenoside Rg1 or its pharmaceutically acceptable salt.

[0126] In another preferred example, the weight ratio of clopidogrel or its pharmaceutically acceptable salt, salvianolic acid B or its pharmaceutically acceptable salt, and ginsenoside Rg1 is 0.5 - 8:0.5 - 6:0.6 - 12, preferably 1 - 6:0.5 - 4:1 - 10, more preferably 2 - 4:1 - 3:3 - 7.

[0127] In the said active ingredient combination, the first active ingredient, the second active ingredient, and the third active ingredient can be independent of each other, or can be combined together and exist in the form of an active ingredient composition. Preferably, in the said active ingredient combination, at least one active ingredient is independent.

[0128] The present invention also provides a medicine box, and the medicine box includes:

[0129] (A) a first preparation containing clopidogrel or its pharmaceutically acceptable salt;

[0130] (B) a second preparation containing salvianolic acid B or its pharmaceutically acceptable salt;

[0131] (C) a third preparation containing ginsenoside Rg1 or its pharmaceutically acceptable salt; and

[0132] (D) Instructions for Use.

[0133] In another preferred example, the Instructions for Use state that the first preparation, the second preparation and the third preparation are used in combination to prevent and / or treat ischemia-reperfusion, ischemia and / or microvascular occlusion injury.

[0134] In another preferred example, the first preparation, the second preparation and the third preparation are administered simultaneously, separately or sequentially in the prevention and / or treatment of ischemia-reperfusion injury, ischemia and / or microvascular occlusion.

[0135] In the medicine box of the present invention, it should be understood that the first preparation, the second preparation and the third preparation in the medicine box can be independent of each other, or any two or three of the preparations can be combined.

[0136] For example, the second preparation and the third preparation are combined preparations, while the first preparation is independent. Typically, the medicine box of the present invention includes: a tablet (the first preparation) and an injection (the combined preparation of the second preparation and the third preparation), wherein the tablet contains clopidogrel or a pharmaceutically acceptable salt thereof, and the injection contains salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

[0137] In another preferred example, the first preparation, the second preparation and the third preparation are independent preparations.

[0138] In another preferred example, at least one of the first preparation, the second preparation and the third preparation is an independent preparation.

[0139] In another preferred example, the first preparation, the second preparation and the third preparation are combined preparations.

[0140] In another preferred example, in the medicine box, the weight ratio of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5 - 8:0.5 - 6:0.6 - 12, preferably 1 - 6:0.5 - 4:1 - 10, more preferably 2 - 4:1 - 3:3 - 7.

[0141] The composition, active ingredient combination, pharmaceutical composition and medicine box of the present invention can all be prepared by conventional methods and equipment.

[0142] Use and Administration Route

[0143] The present invention provides a composition, combination of active ingredients, pharmaceutical composition or kit as described herein for use in the preparation of a medicament for: (i) preparing a medicament for improving bleeding during vascular stent surgery and reducing microvascular obstruction; (ii) preparing a medicament for improving bleeding during thrombolysis surgery and reducing microvascular obstruction; and / or (iii) preparing a medicament for preventing and / or treating ischemia-reperfusion injury.

[0144] Preferably, the ischemia-reperfusion injury includes (but is not limited to): cardiovascular and cerebrovascular ischemia-reperfusion injury, extracirculatory ischemia-reperfusion injury, acute arterial embolism reperfusion injury, traumatic shock reperfusion injury, surgical reperfusion injury, organ transplantation reperfusion injury, reperfusion injury caused by burns, frostbite or blood circulation disorders caused by thrombosis.

[0145] In another preferred embodiment, the ischemia-reperfusion injury includes bleeding injury and microvascular obstruction injury.

[0146] In another preferred embodiment, the cardiovascular ischemia-reperfusion injury includes myocardial infarction, myocardial fibrosis, myocardial ischemia, myocardial necrosis, myocardial hypertrophy, heart failure or myocardial microvascular obstruction

[0147] The first active ingredient, second active ingredient and third active ingredient in the composition, combination of active ingredients, pharmaceutical composition and kit according to the present invention can have a synergistic effect on the prevention and treatment of ischemia-reperfusion injury, bleeding and microvascular obstruction, enhance the effect of treating ischemia-reperfusion injury, reduce the dosage of individual drugs, and reduce the toxicity of the drugs.

[0148] Before, simultaneously or after using the composition, combination of active ingredients, pharmaceutical composition and kit of the present invention, other active substances for treating ischemia-reperfusion injury or surgical operations for treating ischemia-reperfusion injury can be used in combination.

[0149] During the combined use of drugs, the interaction of drugs is classified into additive effect, synergistic effect and antagonistic effect according to the effect when the drugs are used together. The synergistic effect means that the effect when the combined drugs are used together is significantly enhanced compared with the individual use, the additive effect means that the effect when the combined drugs are used together is equivalent to the individual use, and the antagonistic effect means that the effect when the combined drugs are used together is smaller than the individual use. In the present invention, it is first discovered that the combined use of the first active ingredient, second active ingredient and third active ingredient has a synergistic effect.

[0150] When preventing and / or treating ischemia-reperfusion injury, the administration methods of the present invention include sequentially administering the first active ingredient, second active ingredient and third active ingredient, or simultaneously administering the first active ingredient, second active ingredient and third active ingredient.

[0151] The pharmaceutical preparation should match the administration route. When using a pharmaceutical composition or preparation, a safe and effective amount of the drug is administered to a desired subject (such as a human or non-human mammal). Among them, the safe and effective daily dose of the first active ingredient is usually at least about 0.1 mg, and in most cases does not exceed about 2500 mg. Preferably, the dose is 1 mg - 500 mg; the safe and effective amount of the second active ingredient is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg. Preferably, the dose range is 0.1 mg to 2500 mg. The safe and effective amount of the third active ingredient is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg. Preferably, the dose range is 0.1 mg to 2500 mg. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician. When the first active ingredient, the second active ingredient, and the third active ingredient are administered sequentially, there is no special requirement for the administration interval. The first active ingredient, the second active ingredient, and the third active ingredient in the composition, the combination of active ingredients, the pharmaceutical composition, and the kit of the present invention are administered simultaneously or successively by the same or different routes, including but not limited to: oral administration, injection administration, intratumoral administration, implantation administration, intracavitary administration, anal administration, transdermal administration, internal and external application;

[0152] Preferred injection administrations include: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary injection.

[0153] The present invention also provides the use of a compound combination, the compound combination includes salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof, and the compound combination is used to prepare a pharmaceutical composition or preparation, and the pharmaceutical composition or preparation is used for including: (i) improving bleeding during vascular stent surgery and reducing microvascular obstruction; (ii) improving bleeding during thrombolysis surgery and reducing microvascular obstruction; and / or (iii) reducing the side effects of anticoagulant drugs.

[0154] In the present invention, the combination of salvianolic acid B and ginsenoside Rg1 can reduce the side effects of anticoagulant drugs. The anticoagulant drugs are not particularly limited. Typically, the anticoagulant drugs include (but are not limited to): vitamin K antagonists, antiplatelet aggregation drugs, thrombin IIa inhibitors, factor Xa inhibitors, or combinations thereof.

[0155] Typically, the vitamin K antagonists include (but are not limited to): warfarin.

[0156] Typically, the antiplatelet aggregation drugs include (but are not limited to): aspirin, ticlopidine, clopidogrel, abciximab, eptifibatide or tirofiban, or combinations thereof.

[0157] Typically, the thrombin IIa inhibitors include (but are not limited to): bivalirudin, argatroban, dabigatran etexilate, hirudin.

[0158] Typically, the factor Xa inhibitors include (but are not limited to): apixaban, rivaroxaban, edoxaban, or a combination thereof

[0159] Preferably, the weight ratio of salvianolic acid B or a pharmaceutically acceptable salt thereof to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5 - 6:0.6 - 12, more preferably 0.5 - 4:1 - 10, still more preferably 1 - 3:3 - 7.

[0160] The present invention also provides a method for simultaneously reducing the risks of bleeding and microvascular obstruction, said method comprising the step of administering to a subject in need thereof a composition, combination of active ingredients, pharmaceutical composition or kit as described in the present invention.

[0161] In another preferred embodiment, the subject is a human and non - human mammal. Representative non - human mammals include (but are not limited to): pets (such as dogs, cats), livestock (such as cows, sheep, horses, pigs), various zoo animals (such as pandas, elephants, tigers), etc.

[0162] The main advantages of the present invention include:

[0163] 1. In the present invention, the combined use of clopidogrel, salvianolic acid B and ginsenoside Rg1 significantly improves the clinical efficacy of ischemia - reperfusion, ischemia and / or microvascular obstruction, significantly improves the adverse effects of ischemia - reperfusion injury on the structure and function of organs, and significantly improves the risks of myocardial hemorrhage caused by thrombolytic drugs or anticoagulant drugs during PCI (coronary artery stenting) or thrombolytic therapy, as well as heart failure and cardiovascular obstruction induced thereby.

[0164] 2. In the present invention, the combined use of salvianolic acid B and ginsenoside Rg1 can significantly improve the side effects such as bleeding or microcirculation disorders of anticoagulant drugs (such as clopidogrel).

[0165] The following specific examples are used to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0166] Example 1

[0167] Improvement of cardiac function in rats with ischemia - reperfusion by the combined use of clopidogrel and salvianolic acid B / ginsenoside Rg1

[0168] 1.1 Experimental materials

[0169] 1.1.1 Experimental animals

[0170] Wistar rats: 90 males, provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, and raised in the SPF-class animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The temperature was maintained at 22 ± 2°C, with a 12-hour light cycle, standard diet, and free access to water.

[0171] 1.1.2 Drugs

[0172] Salvianolic acid B and ginsenoside Rg1 were purchased from Shanghai Yousi Biotechnology Co., Ltd. (purity ≥ 99%). Clopidogrel was purchased from Sanofi (Hangzhou) Pharmaceutical Co., Ltd.

[0173] Methods for preparing the drugs: ① Weigh 1.5 mg of salvianolic acid B and dissolve it in 1 ml of normal saline, filter through a microporous membrane, and vortex for 3 minutes until the drug is completely dissolved. ② Weigh 1.5 mg of ginsenoside Rg1 and dissolve it in 1 ml of normal saline, filter through a microporous membrane, and vortex for 3 minutes until the drug is completely dissolved. ③ Weigh 0.43 mg of salvianolic acid B and 1.07 mg of ginsenoside Rg1 respectively, mix them evenly (total amount is 1.5 mg), dissolve in 1 ml of normal saline, filter through a microporous membrane, and vortex for 3 minutes until the mixture is completely dissolved to obtain a 2:5 drug combination. ④ Weigh 0.675 mg of clopidogrel and dissolve it in 1 ml of normal saline, filter through a microporous membrane, and vortex for 3 minutes until the drug is completely dissolved.

[0174] 1.2 Experimental methods

[0175] 1.2.1 Preparation of myocardial ischemia-reperfusion injury model

[0176] After weighing the rats, the clopidogrel group and the groups combined with clopidogrel were given 6.75 mg / kg of clopidogrel by gavage before surgery, and anesthetized with a mixture of 60 mg / kg of ketamine and 6 mg / kg of xylazine hydrochloride by intraperitoneal injection. The rats were fixed on the operating board in the dorsal position, the chest hair was removed, and the skin was disinfected with iodine tincture. An intravenous indwelling needle sleeve was inserted into the trachea and connected to a ventilator, with the respiratory rate set at 80 breaths per minute, the respiratory ratio of 1:1, and the tidal volume of 18 ml. The skin was longitudinally incised about 1 cm to the left of the sternal border, the muscles were bluntly separated, and the third and fourth rib spaces were opened and fixed with a retractor. The thymus was clamped with a hemostat and gently pulled upward to expose the upper part of the heart. The left anterior descending coronary artery was found between the pulmonary artery conus and the left atrial appendage, and a non-invasive suture needle was used to pass a 5-0 silk thread through 2 mm below the origin of the left anterior descending coronary artery and tied. After successful ligation of the left anterior descending branch, the myocardial tissue became pale. After 40 minutes, the non-invasive suture was cut to restore perfusion of the ischemic myocardium. After reperfusion, the muscles and skin were sutured, and at the same time, tail vein administration was carried out. The rats were placed in a 37-degree constant temperature system for warming and then returned to the cage after waking up. The sham operation was the same as above except that the left anterior descending coronary artery was not ligated. Hemodynamic evaluation was performed 7 days after reperfusion.

[0177] 1.2.2 Animal grouping and administration

[0178] Rats weighing about 220 g were randomly divided into the following 9 groups: sham operation group, ischemia-reperfusion model group, salvianolic acid B group (dosage: 15 mg / kg), ginsenoside Rg1 group (dosage: 15 mg / kg), salvianolic acid B / ginsenoside Rg1 combination group (where the dosage of salvianolic acid B was 4.29 mg / kg and the dosage of ginsenoside Rg1 was 10.71 mg / kg), clopidogrel group (dosage: 6.75 mg / kg), clopidogrel + salvianolic acid B combination group (where the dosage of clopidogrel was 6.75 mg / kg and the dosage of salvianolic acid B was 15 mg / kg), clopidogrel + ginsenoside Rg1 combination group (where the dosage of clopidogrel was 6.75 mg / kg and the dosage of ginsenoside Rg1 was 15 mg / kg), clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group (where the dosage of clopidogrel was 6.75 mg / kg, the dosage of salvianolic acid B was 4.29 mg / kg, and the dosage of ginsenoside Rg1 was 10.71 mg / kg). There were 10 rats in each group.

[0179] 1.2.3 Hemodynamic detection

[0180] After rats were anesthetized by intraperitoneal injection of a mixture of 60 mg / kg ketamine and 6 mg / kg xylazine hydrochloride, the right common carotid artery was isolated and a Millar catheter was inserted. Hemodynamic indexes such as carotid artery pressure, left ventricular pressure, maximum left ventricular systolic velocity, maximum left ventricular diastolic velocity, left ventricular end-systolic pressure, and left ventricular systolic pressure were recorded using a Powerlab8 / 30 physiological recorder (ML870, ADINSTRUMENTS).

[0181] 1.2.4 Statistical methods

[0182] All experimental data were expressed as mean ± standard deviation (Mean ± SD). SPSS19.0 software was used for statistical analysis of the data. One-way analysis of variance (one-way ANVOVA) with a completely randomized design was used for comparison among multiple groups, and the Bonferroni method was used for pairwise comparison among the means of multiple samples; P < 0.05 was considered statistically significant.

[0183] 1.3 Experimental results

[0184] The hemodynamic detection results of different experimental groups are shown in Table 1 and Figure 1 as follows,

[0185] Table 1 Hemodynamic detection results

[0186]

[0187] Note: *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham operation group, #p < 0.05, ##p < 0.01, p < 0.001 vs. model group, △p < 0.05, △△p < 0.01, △△△p < 0.001 vs. clopidogrel group, n ≥ 8.

[0188] As can be seen from Table 1 and Figure 1 it can be seen that compared with the sham operation control group, the maximum systolic rate, maximum diastolic rate and systolic blood pressure of the left ventricle in the ischemia-reperfusion model group were significantly decreased, and the end-diastolic pressure was significantly increased. Compared with the ischemia-reperfusion model group, the maximum systolic rate in the salvianolic acid B / ginsenoside Rg1 group was significantly increased; compared with the ischemia-reperfusion model, the maximum systolic rate of the left ventricle in the combination of clopidogrel + ginsenoside Rg1 was significantly increased, and compared with clopidogrel alone, the maximum systolic rate of the left ventricle was significantly increased. Compared with the ischemia-reperfusion model, the maximum systolic rate, maximum diastolic rate and systolic blood pressure of the left ventricle in the combination of clopidogrel + salvianolic acid B / ginsenoside Rg1 were significantly increased, and compared with clopidogrel alone, the maximum systolic rate and systolic blood pressure of the left ventricle in the combination of clopidogrel + salvianolic acid B / ginsenoside Rg1 were significantly increased.

[0189] Example 2

[0190] Improvement of myocardial hemorrhage in rats with ischemia-reperfusion by combination of clopidogrel and salvianolic acid B / ginsenoside Rg1

[0191] 2.1 Experimental materials

[0192] 2.1.1 Experimental animals

[0193] The experimental animals were the same as those in 1.1.1 of Example 1.

[0194] 2.2 Experimental methods

[0195] 2.2.1 Preparation of myocardial ischemia-reperfusion model

[0196] The method was the same as that in 1.2.1 of Example 1 for the preparation of myocardial ischemia-reperfusion model.

[0197] 2.2.2 Animal grouping and administration

[0198] The method was the same as that in 1.2.2 of Example 1 for animal grouping and administration.

[0199] 2.2.3 Prussian blue staining

[0200] The thickness of the paraffin section was 4 μm. First, the paraffin tissue was baked (65 °C, 45 - 50 minutes), and then dewaxed to the aqueous phase (xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, rinsed with distilled water for 1 minute); Prussian blue staining solution was dropped onto the section and incubated for 45 minutes, then rinsed with distilled water for 2 minutes; the section was placed in eosin staining solution and incubated for 10 minutes, and rinsed with tap water for 3 seconds; dehydrated and then cleared with xylene (75% ethanol for 2 minutes, 95% ethanol for 2 minutes, absolute ethanol for 3 minutes, xylene for 15 minutes), and sealed with neutral balsam.

[0201] 2.2.4 Prussian blue staining quantification

[0202] After Prussian blue staining, the blue dots represent bleeding. Each sample of Prussian blue staining was photographed at the same magnification, and the bleeding area and the left ventricular area were quantified, and the percentage of the bleeding area in the left ventricle was calculated.

[0203] 2.3 Experimental results

[0204] The results of Prussian blue staining are as Figure 2 shown. It can be seen that compared with the sham operation control group, the left ventricular bleeding in the ischemia-reperfusion model group was obvious; compared with the ischemia-reperfusion injury model group, the bleeding phenomenon in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was significantly improved, and the bleeding phenomenon in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was more significantly improved than that in the salvianolic acid B / ginsenoside Rg1 group; while there was no obvious improvement in the bleeding conditions in the single salvianolic acid B group, single ginsenoside Rg1 group, single clopidogrel group, clopidogrel + salvianolic acid B group, and clopidogrel + ginsenoside Rg1 group compared with the ischemia-reperfusion model group.

[0205] The quantitative results of the bleeding area of the hearts of rats in each group shown by Prussian blue staining are as Table 2 and Figure 3 shown:

[0206] Table 2 Quantitative results of the bleeding area of the hearts of rats in each group

[0207]

[0208] Note: *p < 0.05, **p < 0.01, ***p < 0.001 vs sham operation group, #p < 0.05, ##p < 0.01, p < 0.001 vs model group, n ≥ 6.

[0209] As can be seen from Table 2, compared with the sham operation control group, the left ventricular hemorrhage in the ischemia-reperfusion injury model group increased significantly. The group treated with clopidogrel alone had a certain bleeding risk but was improved compared with the model group. The combination of clopidogrel and salvianolic acid B and the combination of clopidogrel and ginsenoside Rg1 both increased the bleeding area compared with the use of clopidogrel alone, aggravating the bleeding risk and showing no improvement effect. However, unexpectedly, it was found that the combination of clopidogrel and salvianolic acid B / ginsenoside Rg1 could significantly reduce the bleeding area and significantly reduce the bleeding risk compared with clopidogrel alone. Therefore, it can be concluded that salvianolic acid B / ginsenoside Rg1 can significantly reduce the bleeding risk.

[0210] Example 3

[0211] Combination of clopidogrel and salvianolic acid B / ginsenoside Rg1 improves myocardial fibrosis in rats with ischemia-reperfusion

[0212] 3.1 Experimental materials

[0213] 3.1.1 Experimental animals

[0214] The experimental animals were the same as those in 1.1.1 of Example 1.

[0215] 3.2 Experimental methods

[0216] 3.2.1 Preparation of myocardial ischemia-reperfusion model

[0217] The method was the same as that in 1.2.1 of Example 1 for the preparation of myocardial ischemia-reperfusion model.

[0218] 3.2.2 Animal grouping and administration

[0219] The method was the same as that in 1.2.2 of Example 1 for animal grouping and administration.

[0220] 3.2.3 Sirius red staining

[0221] Bake the paraffin sections at 65 °C for 45 - 50 minutes, then place them in xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, and rinse with running water for 1 minute for dewaxing; drop the Sirius red staining solution on the sections and incubate for 1 hour, then rinse with running water for 2 minutes; dehydrate and then permeabilize with xylene (absolute ethanol for 1 minute, xylene for 10 minutes), and seal with neutral gum.

[0222] 3.3 Experimental results

[0223] The results of Sirius red staining were as Figure 4As shown, compared with the sham operation control group, there was a large amount of collagen deposition in the left ventricle of the ischemia-reperfusion model group, resulting in myocardial fibrosis; compared with the ischemia-reperfusion model group, the combined group of clopidogrel + salvianolic acid B / ginsenoside Rg1 and the group of salvianolic acid B / ginsenoside Rg1 had significantly reduced collagen deposition and significantly improved myocardial fibrosis, and the combined group of clopidogrel + salvianolic acid B / ginsenoside Rg1 reduced collagen deposition and improved fibrosis more significantly than the group of salvianolic acid B / ginsenoside Rg1; while in the groups of salvianolic acid B alone, ginsenoside Rg1 alone, clopidogrel alone, the group of clopidogrel + salvianolic acid B, and the group of clopidogrel + ginsenoside Rg1, there was no significant reduction in collagen and no significant improvement in fibrosis compared with the ischemia-reperfusion model group.

[0224] Example 4

[0225] Combined use of clopidogrel and salvianolic acid B / ginsenoside Rg1 improves myocardial structure in rats with ischemia-reperfusion

[0226] 4.1 Experimental materials

[0227] 4.1.1 Experimental animals

[0228] The experimental animals were the same as those in 1.1.1 of Example 1.

[0229] 4.2 Experimental methods

[0230] 4.2.1 Preparation of myocardial ischemia-reperfusion model

[0231] The method was the same as that in 1.2.1 of Example 1 for the preparation of myocardial ischemia-reperfusion model.

[0232] 4.2.2 Animal grouping and drug administration

[0233] The method was the same as that in 1.2.2 of Example 1 for animal grouping and drug administration.

[0234] 4.2.3 Hematoxylin-eosin staining

[0235] The paraffin tissues were baked (65 °C, 45 - 50 minutes), and then dewaxed to the aqueous phase (xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, running water rinse for 1 minute); placed in hematoxylin staining solution for 15 minutes of staining, and then rinsed with running water for 4 minutes; differentiated with 1% hydrochloric acid ethanol for 10 seconds (the differentiation time was adjusted according to the standing time of the differentiation solution), placed in water and rinsed with running water for 5 minutes; stained with eosin staining solution for 1 minute and then placed in water for 1 minute; dehydrated and then cleared with xylene (75% ethanol for 2 minutes, 95% ethanol for 4 minutes, absolute ethanol for 4 minutes, xylene for 15 minutes), and sealed with neutral gum.

[0236] 4.3 Experimental results

[0237] The results of hematoxylin-eosin staining were as follows Figure 5 shown. Compared with the sham operation control group, the cell arrangement in the ischemia-reperfusion model group was irregular, necrosis occurred, and a large number of inflammatory cells infiltrated; compared with the ischemia-reperfusion model group, the cell arrangement in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group and the salvianolic acid B / ginsenoside Rg1 group was relatively regular, the number of necrotic cells decreased, and the inflammatory infiltration was significantly improved. Moreover, compared with the salvianolic acid B / ginsenoside Rg1 group, the cell arrangement in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was more regular, necrosis was further reduced, and inflammatory infiltration was further improved; compared with the ischemia-reperfusion model group, the cell arrangement, necrosis, and inflammatory infiltration in the single salvianolic acid B group, single ginsenoside Rg1 group, single clopidogrel group, clopidogrel + salvianolic acid B group, and clopidogrel + ginsenoside Rg1 group showed a certain degree of improvement.

[0238] Example 5

[0239] Improvement of myocardial microvascular disorders in rats with ischemia-reperfusion by the combination of clopidogrel and salvianolic acid B / ginsenoside Rg1

[0240] 5.1 Experimental materials

[0241] 5.1.1 Experimental animals

[0242] The experimental animals were the same as those in 1.1.1 of Example 1.

[0243] 5.2 Experimental methods

[0244] 5.2.1 Preparation of myocardial ischemia-reperfusion model

[0245] The method was the same as that in 1.2.1 of Example 1 for the preparation of myocardial ischemia-reperfusion model.

[0246] 5.2.2 Animal grouping and drug administration

[0247] The method was the same as that in 1.2.2 of Example 1 for animal grouping and drug administration.

[0248] 5.2.3 Castrales staining

[0249] Bake the paraffin tissue sections (65 °C, 45 - 50 minutes), then dewax to water phase (xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, running water rinse for 1 minute); add GENMED Fixative Solution onto the sections and incubate at room temperature for 72 hours; remove the Fixative Solution and add Clearing Solution to incubate for 2 minutes; remove the Clearing Solution and add GENMED Mordant Solution to incubate at room temperature for 8 minutes; remove the GENMED Mordant Solution and add Clearing Solution to incubate for 2 minutes; remove the Clearing Solution and add GENMED Mayer's Solution to incubate at room temperature for 5 minutes; remove the GENMED Mayer's Solution and add Clearing Solution to incubate for 5 minutes; remove the Clearing Solution and add GENMED Orange Staining Solution to incubate for 45 minutes; remove the GENMED Orange Staining Solution and add Clearing Solution to incubate for 2 minutes; remove the Clearing Solution and add GENMED Fuchsin Solution to incubate for 2 minutes; remove the GENMED Fuchsin Solution and add Clearing Solution to incubate for 2 minutes; remove the Clearing Solution and add GENMED Acidic Solution to incubate for 2 minutes; remove the GENMED Acidic Solution and add Clearing Solution to incubate for 2 minutes; remove the Clearing Solution and add GENMED Staining Solution to incubate for 15 minutes; remove the GENMED Staining Solution and add Clearing Solution to incubate for 2 minutes; dehydrate and then permeabilize with xylene (absolute ethanol for 1 minute, xylene for 15 minutes), and mount with neutral balsam.

[0250] 5.2.3 Quantitative Method for Castellanos Staining

[0251] Using a BX51 microscope, take pictures of the infarct lesion area of each sample one by one, trying to achieve full coverage of the infarct area. Since the infarct areas of animals in each group are different, the number of pictures taken for each heart sample ranges from 10 to 30 to ensure full coverage of the infarct lesion area of each sample. Collect and count the number of microvascular occlusions in each picture. Define a microvascular occlusion when the number of aggregated red blood cells, platelets, or white blood cells is greater than or equal to 4. Based on the number of infarcted blood vessels in the fields of view taken for each animal, perform statistics on each group of animals to obtain the average number of infarcted blood vessels and the standard deviation for each group of animals.

[0252] 5.3 Experimental Results

[0253] As shown in the Castellanos staining photos Figure 6As shown, compared with the sham operation control group, there were a large number of microvascular obstructions in the ischemia-reperfusion injury model group, with red blood cells, platelets, and white blood cells clustering and accumulating in the microvessels. Compared with the ischemia-reperfusion model group, the number of obstructed microvessels in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was significantly reduced, and the microvascular disorder was significantly improved. Compared with the salvianolic acid B / ginsenoside Rg1 group, the number of microvascular infarcts in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was reduced more significantly, and the effect of improving microvascular disorder was more obvious. However, compared with the ischemia-reperfusion model group, there was no obvious improvement in microvascular obstruction in the single salvianolic acid B group, single ginsenoside Rg1 group, single clopidogrel group, clopidogrel + salvianolic acid B group, and clopidogrel + ginsenoside Rg1 group.

[0254] The quantitative results of the number of microvascular obstructions in the hearts of rats in each group stained with Castales are shown in Table 3 and Figure 7 as follows:

[0255] Table 3 Quantitative microvascular obstruction in the hearts of rats in each group

[0256]

[0257]

[0258] Note: *p < 0.05, **p < 0.01, ***p < 0.001 vs sham operation group, #p < 0.05, ##p < 0.01, p < 0.001 vs model group, n ≥ 6.

[0259] Table 3 can more objectively and comprehensively evaluate the improvement effect of drugs on microcirculation disorders. The quantitative results in Table 3 show that the number of obstructed microvessels in the ischemia-reperfusion injury model group was significantly increased compared with the sham operation control group (P < 0.01). The combination of clopidogrel and salvianolic acid B / ginsenoside Rg1 significantly reduced the number of obstructed microvessels compared with the ischemia-reperfusion model group, and the microvascular disorder was very significantly improved (p < 0.001). Whether clopidogrel was combined with salvianolic acid B or ginsenoside Rg1, the number of microvascular obstructions increased compared with clopidogrel alone, increasing the risk of embolism. However, unexpectedly, the combination of clopidogrel and salvianolic acid B / ginsenoside Rg1 significantly reduced the number of microvascular obstructions compared with clopidogrel alone, significantly reduced the risk of embolism, and significantly improved myocardial microvascular disorders. Therefore, it can be concluded that salvianolic acid B / ginsenoside Rg1 can significantly reduce the risk of microvascular obstruction.

[0260] Example 6

[0261] Improvement of rhabdomyolysis in ischemia-reperfusion rats by the combination of clopidogrel and salvianolic acid B / ginsenoside Rg1

[0262] 6.1 Experimental materials

[0263] 6.1.1 Experimental animals

[0264] The experimental animals are the same as those in 1.1.1 of Example 1.

[0265] 6.2 Experimental methods

[0266] 6.2.1 Preparation of myocardial ischemia-reperfusion model

[0267] The method is the same as that for preparing the myocardial ischemia-reperfusion model in 1.2.1 of Example 1.

[0268] 6.2.2 Animal grouping and administration

[0269] The method is the same as that for animal grouping and administration in 1.2.2 of Example 1.

[0270] 6.2.3 Mallory phosphotungstic acid hematoxylin (PTAH) staining

[0271] Bake the paraffin tissue (65 °C, 45 - 50 minutes), then dewax to the aqueous phase (xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, rinse with distilled water for 2 minutes); put it into potassium permanganate solution for oxidation for 10 minutes; wash with tap water for 1 minute, distilled water for 1 minute, put it into oxalic acid solution for bleaching for 2 minutes; wash with tap water for 1 minute, distilled water for 1 minute, put it into Mallory phosphotungstic acid-hematoxylin solution for staining for 48 hours; directly differentiate in 95% alcohol for 30 seconds, dehydrate rapidly with absolute ethanol and then clarify with xylene (absolute ethanol for 30 seconds, xylene for 15 minutes), and seal with neutral gum.

[0272] 6.3 Experimental results

[0273] The results of Mallory phosphotungstic acid hematoxylin (PTAH) staining of striated muscle tissue are as Figure 8 shown. Compared with the sham operation control group, a large amount of striated muscle tissue was dissolved and broken in the ischemia-reperfusion model group; compared with the ischemia-reperfusion model group, the dissolution of striated muscle P was significantly reduced in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group and the salvianolic acid B / ginsenoside Rg1 group, and compared with the salvianolic acid B / ginsenoside Rg1 group, the dissolution of striated muscle was further reduced in the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group; while compared with the ischemia-reperfusion model group, there was no obvious improvement in the dissolution of striated muscle in the single salvianolic acid B group, single ginsenoside Rg1 group, single clopidogrel group, clopidogrel + salvianolic acid B group and clopidogrel + ginsenoside Rg1 group.

[0274] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A pharmaceutical composition having a dual or bidirectional synergistic protective effect, which can effectively reduce microvascular obstruction and effectively prevent bleeding, improve the efficacy of anticoagulant drugs and reduce their side effects, and is composed of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof, and the weight ratio is 2-4:1-3:3-7.

2. The pharmaceutical composition according to claim 1, wherein, the pharmaceutical composition can simultaneously significantly reduce the surgical risks of ischemia-reperfusion injury, vascular stent surgery, and thrombolytic surgery.

3. The pharmaceutical composition according to claim 2, wherein, the ischemia-reperfusion injury includes bleeding injury and microvascular obstruction injury; the vascular stent surgery is a vascular stent surgery combined with an anticoagulant drug; the thrombolytic surgery is a thrombolytic surgery combined with an anticoagulant drug.

4. The pharmaceutical composition according to claim 3, wherein, the ischemia-reperfusion injury is selected from the following group: cardiovascular ischemia-reperfusion injury, cerebrovascular ischemia-reperfusion injury, extracirculatory ischemia-reperfusion injury, acute arterial embolism reperfusion injury, traumatic shock reperfusion injury, surgical reperfusion injury, organ transplantation reperfusion injury, burns, or reperfusion injury caused by frostbite or blood circulation disorders caused by thrombosis.

5. The pharmaceutical composition according to claim 4, wherein, the cardiovascular ischemia-reperfusion injury includes myocardial infarction, myocardial fibrosis, myocardial ischemia, myocardial necrosis, myocardial hypertrophy, heart failure, or myocardial microvascular obstruction.

6. The pharmaceutical composition according to claim 1, wherein, the side effects of the anticoagulant drug are the side effects produced by the anticoagulant drug when used in vascular stent surgery or thrombolytic surgery.

7. The pharmaceutical composition according to claim 1, wherein, the side effects of the anticoagulant drug include bleeding and / or microcirculation disorders.

8. The pharmaceutical composition according to claim 1, wherein, the anticoagulant drug is selected from the following group: vitamin K antagonists, antiplatelet aggregation drugs, thrombin IIa inhibitors, factor Xa inhibitors, or combinations thereof; or the antiplatelet aggregation drugs are selected from the following group: aspirin, ticlopidine, clopidogrel, abciximab, eptifibatide, or tirofiban, or combinations thereof.

9. The pharmaceutical composition according to claim 8, wherein, the vitamin K antagonist is selected from the following group: warfarin.

10. The pharmaceutical composition according to claim 8, wherein, the thrombin IIa inhibitor is selected from the following group: bivalirudin, argatroban, dabigatran etexilate, hirudin.

11. The pharmaceutical composition according to claim 8, wherein, the factor Xa inhibitor is selected from the following group: apixaban, rivaroxaban, edoxaban, or combinations thereof.

12. The pharmaceutical composition according to claim 1, wherein, Pharmaceutically acceptable salts Preferred salts are salts formed by the compounds of the present invention and acids; acids suitable for forming salts include, but are not limited to: inorganic acids such as toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid, glutamic acid; a class of preferred salts are salts formed by the compounds of the present invention and bases, bases suitable for forming salts include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, etc., ammonia water, triethylamine, diethylamine and other organic bases; a class of preferred salts are salts formed by the compounds and metal ions, metal ions suitable for forming salts include, but are not limited to: sodium ions, magnesium ions, potassium ions, calcium ions, etc.

13. The pharmaceutical composition according to claim 12, characterized in that, the pharmaceutically acceptable salt of clopidogrel is clopidogrel sulfate, clopidogrel bisulfate, or clopidogrel hydrochloride; the pharmaceutically acceptable salt of salvianolic acid B is magnesium salvianolate B or calcium salvianolate B.

14. The pharmaceutical composition according to claim 3, characterized in that, the surgical risks of the vascular stent surgery and thrombolysis surgery include the risks of microvascular obstruction and bleeding.

15. The pharmaceutical composition according to claim 14, characterized in that, the bleeding risk is the risk of hemorrhagic necrosis.

16. The pharmaceutical composition according to any one of the above claims, characterized in that, the safe and effective daily dosage of clopidogrel or its pharmaceutically acceptable salt is usually at least about 0.1 mg, and in most cases does not exceed about 2500 mg; preferably, the dosage is 1 mg - 500 mg; the safe and effective amount of salvianolic acid B or its pharmaceutically acceptable salt is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg; preferably, the dosage range is 0.1 mg to 2500 mg; the safe and effective amount of ginsenoside Rg1 is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg, preferably, the dosage range is 0.1 mg to 2500 mg.

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