WO2023232090A1 - 治疗出血性疾病的化合物及其组合物 - Google Patents

治疗出血性疾病的化合物及其组合物 Download PDF

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Publication number
WO2023232090A1
WO2023232090A1 PCT/CN2023/097545 CN2023097545W WO2023232090A1 WO 2023232090 A1 WO2023232090 A1 WO 2023232090A1 CN 2023097545 W CN2023097545 W CN 2023097545W WO 2023232090 A1 WO2023232090 A1 WO 2023232090A1
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Prior art keywords
ginsenoside
salvianolic acid
active ingredient
acid
saponin
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PCT/CN2023/097545
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English (en)
French (fr)
Inventor
姜宝红
王琳琳
王艺宇
张雨菡
张凌霄
陈斯琪
申海尚
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中国科学院上海药物研究所
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Publication of WO2023232090A1 publication Critical patent/WO2023232090A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of medicine, and in particular to a group of compounds and compositions thereof and their use in preventing and/or treating bleeding disorders.
  • the object of the present invention is to provide a pharmaceutical composition for preventing and/or treating tissue and organ hemorrhagic diseases, especially cerebral hemorrhage and hemorrhagic stroke.
  • the present invention provides the use of the salvianolic acid B/ginsenoside Rg1 pharmaceutical composition for preparing drugs for treating, alleviating and/or preventing bleeding disorders.
  • a combination of active ingredients for use in preparing a medicament for treating, alleviating and/or preventing bleeding disorders.
  • the combination of active ingredients includes:
  • a first active ingredient selected from the group consisting of: salvianolic acid, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts or esters, and salvianolic acid-containing extracts , or their combination;
  • a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or their combination.
  • the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.
  • the salvianolic acid is selected from the following group: salvianolic acid B (Salvianolic acid B, Sal B), salvianolic acid A (Salvianolic acid A, Sal A), salvianolic acid C (Salvianolic acid C, Sal C), isosal C (Isosalvianolic acid C), salvianolic acid D (Salvianolic acid D, Sal D), salvianolic acid E (Salvianolic acid E, Sal E), salvianolic acid F (Salvianolic acid F, Sal F), salvianolic acid G (Salvianolic acid G, Sal G), salvianolic acid H (Salvianolic acid H, Sal H), salvianolic acid I (Salvianolic acid I, Sal I), salvianolic acid J (Salvianolic acid J, Sal J), salvianolic acid K (Salvianolic acid K, SalK), salvianolic acid L ( Salvianolic acid L, Sal L), salvianolic acid M (
  • the ginsenoside is selected from the following group: ginsenoside Rg1 (Ginsenoside Rg1), ginsenoside Re (Ginsenoside Re), ginsenoside Rg2 (Ginsenoside Rg2), ginsenoside Rh1 (Ginsenoside Rh1), ginsenoside F1 (Ginsenoside F1), Notoginsenoside R1 (Notoginsenoside R1), Notoginsenoside R2 (Notoginsenoside R2), Notoginsenoside R3 (Notoginsenoside R3), Notoginsenoside R6 (Notoginsenoside R6), Notoginsenoside M (Notoginsenoside M) , Notoginsenoside N, Notoginsenoside Rt, Notoginsenoside T3, Notoginsenoside FP1, Notoginsenoside RW1, Notoginsenoside Fh7 (Notoginsenoside F
  • the salvianolic acid is salvianolic acid B
  • the ginsenoside is ginsenoside Rg1.
  • the first active ingredient includes a purified product of salvianolic acid or a pharmaceutically acceptable salt thereof.
  • the purified product has a purity of ⁇ 90% based on salvianolic acid, preferably ⁇ 95%, more preferably ⁇ 98% or 99%, based on the total weight of the purified product. count.
  • the first active ingredient includes a salvianolic acid extract with a salvianolic acid content C1 ⁇ 30 wt%, wherein the content C1 is based on the weight of salvianolic acid.
  • the content of salvianolic acid C1 in the extract is ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the second active ingredient includes a total saponin extract with a ginsenoside content C2 ⁇ 30 wt%, wherein the content C2 is based on the weight of total saponins.
  • the content of ginsenosides C2 in the extract is ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the weight ratio of the first active ingredient and the second active ingredient is 1-100:100-1, 1-50:50-1, 1-20:20-1, 1-10: 10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1-5:5-1, 1-4:4- 1. 1-3:3-1, or 1-2:2-1.
  • the weight ratio of the first active ingredient and the second active ingredient is 5:2-1:10, such as 5:2, 5:5, 2:5, 1:5, or 1: 10.
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.0), or 5:(1.2-3.8), or 5:(1.5-3.5).
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).
  • the weight ratio of the first active ingredient and the second active ingredient is 2:(2-20.0), or 2:(5-20), or 2:(10-20).
  • the weight ratio of the first active ingredient and the second active ingredient is 2:(5-20), or 2:(19.9-20.1), or 1:10.
  • the first active ingredient is salvianolic acid B
  • the second active ingredient is ginsenoside Rg1.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as lyophilized preparations), gas dosage forms, semi-solid dosage forms.
  • the bleeding disease is selected from: bleeding diseases of various tissues and organs.
  • the tissue or organ is selected from the following group: brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or a combination thereof.
  • the hemorrhagic disease is a cerebral hemorrhagic disease.
  • the brain hemorrhagic disease is selected from the following group: hemorrhagic stroke, brain trauma, intracranial aneurysm rupture, cerebral vascular malformation hemorrhage, intracranial malignant tumor-induced hemorrhage, post-infarction hemorrhage, Bleeding after anticoagulant or thrombolytic therapy, bleeding from cerebral vascular rupture, traumatic cerebral hemorrhage, hyperglycemia-induced cerebral hemorrhage, or combinations thereof.
  • the hemorrhagic stroke is caused by hemorrhagic transformation.
  • the cerebral hemorrhage disease includes cerebral hemorrhage caused by drugs, preferably cerebral hemorrhage caused by thrombolytic drugs, and more preferably cerebral hemorrhage caused by alteplase.
  • the drug is used for a purpose selected from the following group:
  • the dosage form is selected from the following group: injections (such as injections or powder injections), oral preparations (such as capsules, tablets, pills, powders, granules, syrups, oral liquids or tinctures), Sublingual preparations, respiratory tract administration preparations, skin administration preparations, mucosal administration preparations, more preferably, the dosage form is an injection.
  • a pharmaceutical composition for preparing a medicament for treating, alleviating and/or preventing bleeding disorders comprising:
  • a first active ingredient selected from the group consisting of: salvianolic acid, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts or esters, and salvianolic acid-containing extracts , or their combination;
  • a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or their combination;
  • the weight ratio of the first active ingredient and the second active ingredient is 1-100:100-1, 1-50:50-1, 1-20:20-1, 1-10: 10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1-5:5-1, 1-4:4- 1. 1-3:3-1, or 1-2:2-1.
  • the weight ratio of the first active ingredient and the second active ingredient is 5:2-1:10, such as 5:2, 5:5, 2:5, 1:5, or 1: 10.
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.0), or 5:(1.2-3.8), or 5:(1.5-3.5).
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).
  • the salvianolic acid is salvianolic acid B
  • the ginsenoside is ginsenoside Rg1.
  • the first active ingredient is salvianolic acid B
  • the second active ingredient is ginsenoside Rg1.
  • an active ingredient for preparing a medicament for treating, alleviating and/or preventing bleeding disorders the active ingredient being selected from the following group:
  • Salvianolic acid its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, or derivatives thereof, and extracts containing salvianolic acid;
  • Ginsenosides their stereoisomers, their crystal forms, their pharmaceutically acceptable salts or esters, or their derivatives, and extracts containing ginsenosides.
  • a method of treating, alleviating and/or preventing a bleeding disorder in a subject in need thereof comprising administering a medicament to the subject in need thereof, the medicament comprising The following active ingredients:
  • a first active ingredient selected from the group consisting of: salvianolic acid, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts or esters, and salvianolic acid-containing extracts , or their combination;
  • a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or their combination.
  • the bleeding disease is selected from: bleeding diseases of various tissues and organs.
  • the tissue or organ is selected from the following group: brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or a combination thereof.
  • Figure 1 shows representative images of cerebral hemorrhage (A) and quantitative results of SalB/Rg1 reducing hemorrhage compared with the hemorrhage model group (B).
  • Figure 2 shows that SalB/Rg1 has a synergistic effect in preventing and treating collagenase-induced cerebral hemorrhage.
  • A Representative image of bleeding;
  • B Quantitative results of SalB/Rg1 reducing bleeding compared with the bleeding model group;
  • C The percentage of each group inhibiting bleeding compared with the model group;
  • D Bliss method to evaluate the synergistic effect result.
  • Figure 3 shows the experimental scheme of SalB/Rg1 inhibitingreteplase (tPA)-induced cerebral hemorrhage and extending the tPA thrombolysis time window.
  • A Hemorrhage model system induced by reperfusion andreteplase (tPA) 4.5 hours after infarction.
  • B Hemorrhage model system induced by reperfusion withreteplase (tPA) 6 hours after infarction.
  • C Hemorrhage model system induced by reperfusion with alteplase (tPA) 9 hours after infarction.
  • Figure 4 shows that SalB/Rg1 reduces the bleeding score of tPA-induced intracerebral hemorrhage.
  • A Representative diagram of bleeding picture.
  • Figure 5 shows the behavioral assessment using Longa score, Bederson score, mNSS score and suspension test. Compared with the sham operation group, ***P ⁇ 0.001; compared with the bleeding model group, #P ⁇ 0.05, ##P ⁇ 0.01.
  • Figure 7 shows that SalB/Rg1 improves the survival rate of tPA-induced cerebral hemorrhage animals. Compared with the sham operation group, ***P ⁇ 0.01, **P ⁇ 0.001; compared with the bleeding model group, ###P ⁇ 0.001.
  • Figure 9 shows that SalB/Rg1 has a synergistic effect in reducing tPA-induced bleeding.
  • A Representative image of bleeding.
  • B Bleeding score.
  • Figure 10 shows representative images (top) and quantitative data (bottom) of bleeding scores to evaluate the therapeutic effect of SalB/Rg1 in an animal model of hemorrhagic stroke. Compared with the sham operation group, ***P ⁇ 0.001; compared with the bleeding model group, &P ⁇ 0.05.
  • Figure 11 shows that SalB/Rg1 improves neurological behavior in animals with hyperglycemia-induced hemorrhage. Compared with the sham operation group, ***P ⁇ 0.001; compared with the bleeding model group, &P ⁇ 0.05.
  • the inventor After extensive and in-depth research and extensive screening and testing, the inventor has provided a pharmaceutical composition containing salvianolic acid B and ginsenoside Rg1 as active ingredients for use in the treatment of bleeding disorders. It has been verified by animal experiments that the composition of the present invention can effectively reduce the amount of intracerebral bleeding in mice with hemorrhagic stroke, and can improve the neurological behavior of animals with cerebral hemorrhage.
  • the pharmaceutical composition of the present invention shows a better therapeutic effect on hemorrhagic diseases (especially hemorrhagic stroke) and can be used for the treatment of hemorrhagic diseases in various tissues and organs. On this basis, the present invention was completed.
  • the terms “comprises,” “includes,” 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.”
  • stereoisomer is intended to include all isomeric forms (e.g., enantiomeric Isomers, diastereomers and geometric isomers (or conformational isomers): such as R and S configurations containing asymmetric centers, (Z) and (E) isomers with double bonds, etc. Therefore, individual stereochemical isomers of the active ingredients of the invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) fall within the scope of the invention.
  • the active ingredients of the present invention may be amorphous, crystalline or mixtures thereof.
  • pharmaceutically acceptable salts refer to salts of compounds of the active ingredients of the present invention with acids or bases suitable for use as pharmaceuticals.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • One preferred class of salts is the salts of the active ingredient compounds of the invention with acids.
  • Acids suitable for forming salts include, but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, 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, benzenesulfonic acid and other organic acids; as well as aspartic acid, glutamic acid and other acidic amino acids.
  • One preferred class of salts is the salts of the active ingredient compounds of the invention with bases.
  • Bases suitable for forming salts include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, magnesium carbonate, calcium carbonate, sodium carbonate, sodium bicarbonate, and sodium phosphate, and organic bases such as ammonia, triethylamine, and diethylamine.
  • inorganic bases such as sodium hydroxide, potassium hydroxide, magnesium carbonate, calcium carbonate, sodium carbonate, sodium bicarbonate, and sodium phosphate
  • organic bases such as ammonia, triethylamine, and diethylamine.
  • Another type of preferred salt is the salt formed by the active ingredient of the present invention and a metal ion, including but not limited to magnesium salt, sodium salt, calcium salt, potassium salt, etc.
  • esters refers to an ester of a compound of an active ingredient of the present invention with an acid or alcohol suitable for use as a pharmaceutical.
  • a preferred class of esters is one or more hydroxyl groups of the active ingredient of the invention with an acid. The ester formed.
  • Suitable ester-forming acids include, but are not limited to: phosphoric acid, 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, benzenesulfonic acid, etc.; another preferred ester is the ester formed by the carboxyl group of the active ingredient of the present invention and alcohol.
  • Alcohols suitable for forming esters include but are not limited to: C1 -C6 alkyl-OH, such as methanol, ethanol, n-propanol, isopropanol, etc.
  • Prevention and “treatment” in the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination and reversal.
  • a composition or pharmaceutical composition described herein prevents, reduces, inhibits and/or reverses cerebral hemorrhage injury compared to levels observed in the absence of a composition or pharmaceutical composition described herein. For example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
  • the term "salvianolic acid” refers to a compound selected from the following group: salvianolic acid B, salvianolic acid A, salvianolic acid C, isoSalC, salvianolic acid D, salvianolic acid E, Salvianolic acid F, salvianolic acid G, salvianolic acid H, salvianolic acid I, salvianolic acid J, salvianolic acid K, salvianolic acid L, salvianolic acid M, salvianolic acid N, salvianolic acid T, Salvianolic acid U, salvianolic acid Y, caffeic acid, salvianoside, protocatechuic acid, protocatechuic acid, ferulic acid, isoferulic acid, Salvinal, Ailanthoidol, rosmarinic acid, lithospermic acid, salvianoside, Catechol, dimethyllithosperm B, tanshinone I, dihydrotanshinone I, tanshindiol-A
  • ginsenoside refers to a compound selected from the following group: ginsenoside Rg1, ginsenoside Re, ginsenoside Rg2, ginsenoside Rh1, ginsenoside F1, notoginsenoside R1, notoginsenoside R2 , notoginseng saponin R3, notoginseng saponin R6, notoginseng saponin M, notoginseng saponin N, notoginseng saponin Rt, notoginseng saponin T3, notoginseng saponin FP1, notoginseng saponin RW1, notoginseng saponin Fh7, wild camellia Glycoside D, camelin U, 20-glucose ginsenoside Rf, malonate ginsenoside Rg1, bamboo saponin L5, safflower ginsenoside R1, pseudoginsenoside RT3, saponin 10, saponin 1, saponin 2, saponin 3, Ginsenoside Ra
  • the present invention provides a pharmaceutical composition that can be used to prepare a treatment, alleviation and/or prevention of bleeding disorders, which includes a first active ingredient and a second active ingredient, wherein the first active ingredient is selected from the group consisting of: salvianolic acid, its stereo isomers, crystalline forms thereof, pharmaceutically acceptable salts or esters thereof, salvianolic acid-containing extracts, or combinations thereof.
  • the first active ingredient is selected from the group consisting of: salvianolic acid B, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts or esters, extracts containing salvianolic acid B objects, or their combinations.
  • the salvianolic acid B is represented by the following formula (I):
  • the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.
  • the purified product has a purity of ⁇ 90% based on salvianolic acid B, preferably ⁇ 95%, more preferably ⁇ 98% or 99%, based on the total amount of the purified product. Weight scale.
  • the first active ingredient includes a salvianolic acid extract with a salvianolic acid B content C1 ⁇ 30 wt%, wherein the content C1 is based on the weight of salvianolic acid.
  • the content C1 of salvianolic acid B in the extract is ⁇ 70%, preferably ⁇ 80%, More preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the second active ingredient is selected from the following group: ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof.
  • the second active ingredient is selected from the following group: ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salts or esters, and extraction containing ginsenoside Rg1 objects, or their combinations.
  • the ginsenoside Rg1 is represented by the following formula (II):
  • the second active ingredient includes a total saponin extract with a content C2 of ginsenoside Rg1 ⁇ 30 wt%, wherein the content C2 is based on the weight of total saponins.
  • the content C2 of ginsenoside Rg1 in the extract is ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the present invention provides the use of a pharmaceutical composition, which includes:
  • a first active ingredient selected from the group consisting of: salvianolic acid, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts, salvianolic acid-containing extracts, or their combination;
  • a second active ingredient selected from the group consisting of: ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts thereof, extracts containing ginsenosides, or their combination; and (c) a pharmaceutically acceptable carrier.
  • the present invention also provides the use of a combination of active ingredients, which includes:
  • a first active ingredient selected from the group consisting of: salvianolic acid, its stereoisomers, its crystalline forms, its pharmaceutically acceptable salts, salvianolic acid-containing extracts, or their combination;
  • a second active ingredient selected from the group consisting of: ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts thereof, extracts containing ginsenosides, or their combination.
  • the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.
  • the weight ratio of the first active ingredient and the second active ingredient is 1-100:100-1, 1-50:50 -1, 1-20:20-1, 1-10:10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1- 5:5-1, 1-4:4-1, 1-3:3-1, 1-2:2-1.
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-5.0), or 5:(1.2-3.8), or 5:(1.5-3.5).
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).
  • the weight ratio of the first active ingredient and the second active ingredient is 2:(2-20.0), or 2:(5-20), or 2:(10-20).
  • the weight ratio of the first active ingredient and the second active ingredient is 2:(5-20), or 2:(19.9-20.1), or 1:10.
  • the salvianolic acid is salvianolic acid B
  • the ginsenoside is ginsenoside Rg1.
  • the first active ingredient is salvianolic acid B
  • the second active ingredient is ginsenoside Rg1.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as lyophilized preparations).
  • the dosage form is selected from the following group: injections (such as injections or powder injections), oral preparations (such as capsules, tablets, pills, powders, granules, syrups, oral liquids or tinctures), More preferably, preferably, the dosage form is an injection.
  • the first active ingredient and the second active ingredient can be formulated separately or mixed together to form a formulation.
  • the pharmaceutical composition of the present invention contains a first active ingredient and/or a second active ingredient within a safe and effective amount.
  • the “safe and effective amount” refers to the amount of active ingredients that is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the active ingredient/dose of the present invention, and more preferably, contains 10-500 mg of the active ingredient/dose of the present invention.
  • the "one dose” is a capsule, tablet, injection, etc.
  • pharmaceutically acceptable carrier refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and low enough toxicity.
  • Cosmetic here refers to the ability of each component of the composition to be blended with the first active ingredient and/or the second active ingredient without significantly reducing the efficacy of the first active ingredient and/or the second active ingredient. effect.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • solid lubricants such as
  • the first active ingredient and the second active ingredient may be independent of each other, or may be combined together to form an active ingredient composition.
  • compositions and active ingredient combination of the present invention can be prepared using conventional methods and equipment.
  • the present invention provides the use of a pharmaceutical composition, active ingredient, combination of active ingredients or kit described herein for preparing a medicine or kit for preventing and/or treating hemorrhagic diseases. Disease, preferably prevention and/or treatment of hemorrhagic stroke.
  • the hemorrhagic disease is selected from hemorrhagic diseases of various tissues and organs.
  • the tissues and organs mentioned therein include, but are not limited to: brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or combinations thereof.
  • the hemorrhagic stroke refers to intracerebral hemorrhage caused by cerebral blood vessel wall lesions, necrosis, and rupture.
  • the main clinical manifestations are headache, vomiting, disturbance of consciousness, hemiplegia, sensory impairment, or a combination thereof.
  • the hemorrhagic stroke may be a hemorrhagic stroke caused by hemorrhagic transformation.
  • the application for preventing and/or treating hemorrhagic diseases, preventing and/or treating hemorrhagic stroke, etc. includes preventive applications and also includes post-improvement applications.
  • it includes administering the pharmaceutical composition or the composition of active ingredients of the present invention before, during, and/or after the hemorrhagic stroke to protect the tissues and organs after the hemorrhagic stroke. , fixes, or feature improvements or enhancements.
  • the pharmaceutical composition or combination of active ingredients used in the present invention only contains the first active ingredient salvianolic acid B and the second active ingredient ginsenoside Rg1.
  • the first active ingredient and the second active ingredient can also be administered in combination with other pharmaceutically acceptable compounds, including (but not limited to): antihypertensive drugs, hypolipidemic drugs drugs, hypoglycemic drugs, antioxidant drugs, tissue dehydration drugs, neuroprotective agents, etc.
  • the first active ingredient and the second active ingredient can be administered simultaneously, separately or sequentially during administration.
  • the administration mode of the first active ingredient and the second active ingredient is not particularly limited.
  • Representative administration modes include (but are not limited to): oral, rectal, gastrointestinal External (intravenous, intramuscular or subcutaneous), and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) Humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarder, such as paraffin; (f) Absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostea
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active ingredient can also be combined with the above-mentioned excipients One or more form microencapsulates.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils,
  • compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions may contain, in addition to the active ingredient, suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.
  • compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration of the active ingredients of the present invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.
  • the general range of the therapeutically effective dose of the active ingredients will be: about 1-2000 mg/day, about 10 - about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 80 mg/day.
  • the therapeutically effective dose will be administered in one or more doses.
  • the specific dose of the active ingredient of the present invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health, diet, individual response of the patient to be treated, the time of administration, the Severity of disease being treated, dosage form, mode of application, and concomitant medications.
  • the therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of the clinician or physician.
  • the active ingredient will be administered in multiple doses based on the patient's individual condition and in a manner that allows for the delivery of a therapeutically effective amount.
  • the present invention discovered the use of the salvianolic acid B/ginsenoside Rg1 pharmaceutical composition in the treatment of bleeding diseases.
  • the present invention found that the salvianolic acid B/ginsenoside Rg1 pharmaceutical composition has a synergistic effect on the treatment of hemorrhagic stroke, and can significantly reduce the amount of intracerebral bleeding.
  • Example 1 The effect of different ratio combinations of salvianolic acid B and ginsenoside Rg1 (SalB/Rg1) on inhibiting cerebral hemorrhage
  • Rats Male SD rats were purchased from Beijing Huafukang Biotechnology Co., Ltd., weighing 260-300 g. Rats were kept in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ⁇ 2°C), a 12-hour light and dark cycle, and free access to food and water.
  • Salvianolic acid B is abbreviated as SalB
  • ginsenoside Rg1 is abbreviated as Rg1.
  • Bleeding model control group (10:1)SalB/Rg1 group, (5:1)SalB/Rg1 group, (5:2)SalB/Rg1 group, (5:5)SalB/Rg1 group, (2:5) SalB/Rg1 group, (1:5)SalB/Rg1 group, (1:10)SalB/Rg1 group mice
  • the dosage volume is 5ml/kg
  • the solvent is 0.9% sodium chloride injection. This experiment adopted a double-blind method.
  • the EP tubes and pipette tips used in the pharmaceutical preparation preparation process were sterilized by a vertical pressure steam sterilizer.
  • a dental drill with a diameter of 0.5mm to drill a small hole with a diameter of about 1.0mm 0.5mm in front of the bregma and 3.5mm to the right of the midline.
  • 1 ⁇ l collagenase VII with a concentration of 0.5U/ ⁇ l into the striatum through a microsyringe.
  • the drilled area was sealed with bone wax, the skin was sutured, and adequate food and water were provided.
  • 5 mg/kg SalB/Rg1 was injected into the tail vein.
  • V is the hematoma volume of each brain slice (mm 3 ); A1 and A2 are the red hemorrhage areas on the rostral and caudal sides of the brain slice respectively (mm 2 ); D is the slice thickness (mm)]; the whole brain hematoma volume is 7 The sum of hematoma volumes in brain slices.
  • Hematoma volume (%) (hematoma volume/brain volume of the contralateral hemisphere) ⁇ 100%.
  • the animal model of collagenase VII-induced hemorrhage is a classic model for studying cerebral hemorrhage. Since collagenase is injected into the striatum at a targeted location, Figure 1A shows the occurrence of obvious hemorrhage in the striatum.
  • the hematoma volume inhibition rate was calculated using the following formula: (hematoma volume in bleeding model group - hematoma in salvianolic acid B/Rg1 administration group) Volume)/hematoma volume in the bleeding model group.
  • the hematoma inhibition rate of (10:1)SalB/Rg1 is 0.30 ⁇ 0.20%; the hematoma inhibition rate of (5:1)SalB/Rg1 is -10.05 ⁇ 0.34%; the hematoma inhibition rate of (5:2)SalB/Rg1 13.31 ⁇ 0.13%; the hematoma inhibition rate of (5:5)SalB/Rg1 is 11.92 ⁇ 0.09%; the hematoma inhibition rate of (2:5)SalB/Rg1 is 21.33 ⁇ 0.27%; (1:5)SalB/Rg1 The hematoma inhibition rate was 13.14 ⁇ 0.28%; the hematoma inhibition rate of (1:10) SalB/Rg1 was 23.95 ⁇ 0.24%.
  • Example 2 Salvianolic acid B and Rg1 have a synergistic effect in reducing collagenase-induced cerebral hemorrhage.
  • Rats Male SD rats were purchased from (Beijing Huafukang Biotechnology Co., Ltd.), weighing 260-300g. Rats were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ⁇ 2°C), a 12-hour light and dark cycle, and free access to food and water.
  • the animals in the bleeding model group were given normal saline; the animals in the SalB group were given 0.45 mg/kg salvianolic acid B; the animals in the Rg1 group were given 4.55 mg/kg ginsenoside Rg1; the animals in the SalB/Rg1 group were given 5 mg/kg salvianolic acid B.
  • /Rg1 (where salvianolic acid B and Rg1 are formulated at a mass ratio of 1:10).
  • physiological saline, salvianolic acid B, Rg1 or SalB/Rg1 combination was injected into the tail vein immediately after collagenase treatment.
  • S model , S SalB , S Rg1 and S SalB/Rg1 respectively represent the hematoma volume of the bleeding model, SalB, Rg1 and SalB/Rg1 groups.
  • I SalB , I Rg1 and I SalB/Rg1 represent SalB, I SalB/Rg1 respectively.
  • the ratio of the hematoma volume in the Rg1 and SalB/Rg1 groups to the hemorrhage model group, and the I value are calculated as shown in formulas 1, 2, and 3.
  • I SalB (S SalB /S model ) ⁇ 100% (Formula 1)
  • I Rg1 (S Rg1 /S model ) ⁇ 100% (Formula 2)
  • I SalB/Rg1 (S SalB/Rg1 /S model ) ⁇ 100% (Formula 3)
  • Figure 2A is a representative picture of each group of collagenase-induced cerebral hemorrhage. The hematoma area appears dark red.
  • Figure 2B is the quantitative result of hematoma volume. The hematoma volume ratio of the hemorrhage model group is (10.97 ⁇ 3.97%). Neither SalB alone (8.54 ⁇ 3.63%) nor Rg1 alone (11.03 ⁇ 2.38%) can significantly reduce the hematoma volume; SalB/Rg1 at 5mg/kg can significantly reduce hematoma volume (6.92 ⁇ 2.36%, #p ⁇ 0.05), and its effectiveness is statistically significant compared with Rg1 (&p ⁇ 0.05).
  • Figure 2C is the quantitative result of the hematoma volume inhibition rate. The inhibition rate of 5mg/kg SalB/Rg1 is significantly higher than that of Rg1 (36.95 ⁇ 0.22%), and has a more excellent efficacy (&p ⁇ 0.01).
  • Example 3 Salvianolic acid B combined with ginsenoside Rg1 inhibits cerebral hemorrhage caused by alteplase (tPA)
  • mice Male C57BL/6 mice (26 ⁇ 3 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ⁇ 2°C), a 12-hour light and dark cycle, and free access to food and water.
  • mice were anesthetized by intraperitoneal injection of Serta and xylazine, and the neck skin was prepared and disinfected with iodophor.
  • the skin was cut along the middle of the mouse's neck to expose the deep cervical myometrium and pretracheal muscles.
  • the common carotid artery was isolated and threaded to block the blood flow with a slipknot. Separate upward along the common carotid artery, tie a dead knot at the proximal end of the common carotid artery, prepare a suture at the distal end, and temporarily Do not tie it tightly.
  • microscissors to cut a small opening in the proximal end of the common carotid artery, and gently insert the silicone suture plug through the small opening to block the beginning of the main trunk of the middle cerebral artery. Tie the slip knot prepared on the distal end of the common carotid artery to the fixed suture bolt, and suture the skin. After the middle cerebral artery is temporarily occluded for a specified period of time, the suture site is opened, the suture bolt is removed, and the distal end of the common carotid artery is tied tightly for reperfusion. According to the different time points of tPA application, experimental studies were carried out in three batches.
  • the suture plug was removed at 4.5h, 6h, and 9h after middle cerebral artery occlusion in mice to achieve vascular reperfusion, and tPA was immediately administered to induce a hemorrhage model.
  • Each batch of experiments was set up with a sham operation group, a model group and a salvianolic acid B/Rg1 group (5mg/kg salvianolic acid B/Rg1).
  • the formula of salvianolic acid B/Rg1 is such that the ratio of salvianolic acid B and Rg1 is 5:2.
  • the hemorrhage model group and salvianolic acid B/Rg1 group of this study were both given 10 mg/kg tPA, while the Sham group was given normal saline at corresponding time points.
  • the preparation of SalB/Rg1 is such that the mass ratio of salvianolic acid B and Rg1 is 5:2.
  • the time points of SalB/Rg1 treatment are shown in Figure 3, with a total of 3 administrations. Reperfusion for 48 hours was used as the end point of the experiment.
  • the experimental plan design of each time window is shown in Figure 3. In order to ensure the objectivity of the research results, the experiment was conducted using a double-blind method until all experiments were completed and conclusions were drawn before the blinding was lifted.
  • the Longa score is evaluated on a scale from 0 to 4:
  • the Bederson score is rated on a 5-point scale:
  • the scores for each slice were added up and the total score was used as the evaluation metric.
  • the brain tissue was fixed in 4% neutral formaldehyde fixative for 48 hours, it was taken out and placed in an embedding frame. After running water for 3-5 hours, it was placed in an automatic dehydrator and dehydrated according to the set program. After dehydration was completed, it was embedded in paraffin. The embedded tissue was cut into paraffin sections with a thickness of 4 ⁇ m. Place the tissue slices on a baking machine and heat them at 68°C for 60 minutes. Take it off and dewax it sequentially through xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 5 minutes, and running water for 5 minutes. Stain with Nissl staining solution for 10 min and wash twice with distilled water.
  • FIG 4A Representative images of brain tissue hemorrhage are shown in Figure 4A.
  • Figure 4B shows the bleeding score. It can be seen that whether tPA is given at 4.5h, 6h or 9h, SalB/Rg1 intervention will significantly reduce the bleeding score (P ⁇ 0.01).
  • tPA was administered 4.5 hours after cerebral ischemia, the bleeding score of the hemorrhage model group was 3.75 ⁇ 0.71. After administration of SalB/Rg1, the bleeding score dropped to 2.20 ⁇ 1.93, which was significantly different from the hemorrhage model group (P ⁇ 0.01).
  • the bleeding score of the blood model group increased to 4.75 ⁇ 1.98 after administration of tPA 6 hours after cerebral ischemia, and dropped to 1.70 ⁇ 2.34 after administration of SalB/Rg1, which was significantly different from the bleeding model group (P ⁇ 0.01). Even in the experiment of administering tPA 9 hours after cerebral ischemia, there was still a highly significant difference between the SalB/Rg1 group and the hemorrhage model group (5.4 ⁇ 1.58 versus 3.6 ⁇ 1.43, P ⁇ 0.01).
  • SalB/Rg1 protects neurons in cortex and striatum
  • Nissl staining was performed on brain tissue sections, and the number of Nissl bodies was counted to determine whether SalB/Rg1 can protect neurons.
  • the Nissl staining results are shown in Figure 6.
  • the same positions in the cerebral cortex and striatum were selected for quantification in each picture.
  • the number of Nissl stain-positive cells in the cortex and striatum after modeling decreased significantly compared with the sham operation group (P ⁇ 0.001).
  • the number of Nissl stain-positive cells in the SalB/Rg1 group was significantly increased compared with the hemorrhage model group (P ⁇ 0.05). This result corresponds to the above-mentioned behavioral studies, suggesting that SalB/Rg1 may improve the neurobehavior of mice by protecting neurons in the cerebral cortex and striatum.
  • mice in each group are shown in Figure 7. Whether tPA was given at 4.5 hours, 6 hours or 9 hours after cerebral ischemia, the survival rate of mice after modeling was significantly reduced (P ⁇ 0.01). The survival rate of mice will be improved after giving SalB/Rg1. In the experiment of giving tPA 4.5 hours after cerebral ischemia, there was a significant difference in the improvement of survival rate after SalB/Rg1 intervention compared with the hemorrhage model group (80% vs 20%, P ⁇ 0.001).
  • the survival rate of the SalB/Rg1 group after 12 hours of reperfusion was 71.43%, while the survival rate of the hemorrhage model group was 44%; at 48 hours of reperfusion, the survival rate of the SalB/Rg1 group was 50%, and the survival rate of the hemorrhage model group was 50%. The group survival rate was 36%. Similar conclusions were drawn in the 9-hour experiment. After 12 hours of reperfusion, the survival rate of the SalB/Rg1 group was 90%, while the survival rate of the hemorrhage model group was 60%.
  • the survival rate of the SalB/Rg1 group was 70%, and the survival rate of the hemorrhage model group was 70%.
  • the survival rate of the model group was 40%. This result indicates that SalB/Rg1 can prolong the survival time of mice.
  • Example 4 MRI imaging evaluation of the effect of SalB/Rg1 on inhibiting cerebral hemorrhage
  • MRI data acquisition was performed on an 11.7T BioSpec 117/16USR MRI system.
  • a 2 ⁇ 2 phased array coil and a 72mm volume resonator are used for transmission and image acquisition respectively. All test animals were anesthetized using 1% to 1.5% isoflurane, and their body temperature was maintained at 37°C to ensure proper immobilization during MRI scanning. Monitor breathing and maintain a rate of 95-110 breaths/minute.
  • T2-weighted image Whole-brain T2WI uses the rapid acquisition and relaxation enhancement (RARE) sequence to obtain high-resolution images, flip angle 90°, 2500ms repetition time (TR), 6.5ms echo Wave time (echo time, TE) and 0.063 ⁇ 0.063 ⁇ 0.5mm 3 spatial resolution.
  • RARE rapid acquisition and relaxation enhancement
  • echo time TE
  • 0.063 ⁇ 0.063 ⁇ 0.5mm 3 spatial resolution 0.063 ⁇ 0.063 ⁇ 0.5mm 3 spatial resolution.
  • a multi-slice multi-echo (MSME) sequence was used with a flip angle of 90°, 2200 ms TR and 30 effective TE from 7.5 ms to 225 ms.
  • FLASH Fast Low-Angle Shot
  • Image analysis and data processing Use SPMMouse based on SPM8 (http://www.fil.ion.ucl.ac.uk/spm/software/spm8/) for motion correction and adjustment, using MATLAB 2021b (MathWorks, Natick, USA ) for data analysis. Other MRI modalities were rigidly co-registered with T2WI.
  • CBF Cerebral blood flow
  • SWI is a high-resolution 3D gradient echo MRI sequence that highlights differences between paramagnetic materials (such as deoxyhemoglobin, hemosiderin, and ferritin) and brain parenchyma, making the sequence also sensitive for the detection of microbleeds , paramagnetic substances (such as hemosiderin, deoxyhemoglobin and ferritin) show low signal in SWI images.
  • SWI scanned multiple layers of images from the olfactory bulb to the cerebellum, and scored the degree of bleeding in each layer. The sum of each layer was used as the bleeding score of the mouse and statistics were made. The results are shown in Figure 8. Consistent with the previous article, there was no bleeding in the sham operation group. There were 6 mice in the bleeding model group, 4 of which had parenchymal bleeding and the bleeding was serious, while only 6 mice in the SalB/Rg1 group had bleeding. Bleeding was detected in SWI in 2 animals (7.00 ⁇ 7.83 versus 0.50 ⁇ 0.76).
  • Example 5 Salvianolic acid B and ginsenoside Rg1 have a synergistic effect in inhibiting cerebral hemorrhage caused by alteplase
  • mice Male C57BL/6 mice (26 ⁇ 3 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ⁇ 2°C), a 12-hour light and dark cycle, and free access to food and water.
  • Example 3 The model of intracerebral hemorrhage in mice induced by alteplase (tPA) is shown in Example 3, which is the same as Scheme B in Figure 3 above.
  • mice Male C57BL/6 mice were randomly divided into four groups, bleeding model group, SalB group, Rg1 group, SalB/Rg1 Combined groups, 10 animals in each group.
  • the animals in the bleeding model group were given normal saline; the animals in the SalB group were given 3.57 mg/kg salvianolic acid B; the animals in the Rg1 group were given 1.43 mg/kg ginsenoside Rg1; the animals in the SalB/Rg1 group were given 5 mg/kg salvianolic acid B.
  • /Rg1 (where salvianolic acid B and Rg1 are formulated at a mass ratio of 5:2).
  • the animals were divided into tail veins and injected with normal saline, salvianolic acid B, ginsenoside Rg1 or SalB/Rg1 combination.
  • SalB/Rg1 has a synergistic effect in reducing alteplase (tPA)-induced bleeding
  • Figure 9A is a representative picture of each group of tPA-induced cerebral hemorrhage, and the bleeding site appears red.
  • Figure 9B is the quantitative result of the degree of bleeding.
  • the bleeding score of the bleeding model group is (4.75 ⁇ 1.98).
  • SalB alone (3.88 ⁇ 1.76) or Rgl alone (4.5 ⁇ 1.86) cannot significantly reduce the bleeding score; 5mg/kg SalB/Rgl can significantly reduce the bleeding score (1.7 ⁇ 2.34verus 4.75 ⁇ 1.98, p ⁇ 0.01), and is statistically different from Rg1 in effectiveness (1.7 ⁇ 2.34verus 4.5 ⁇ 1.86, p ⁇ 0.01).
  • mice Male healthy C57BL/6 mice, weighing 25g-28g, were purchased from Shanghai Slack Experimental Animal Company. After purchase, the animals were raised in the SPF-grade animal room of the Shanghai Institute of Materia Medica, with free access to food and water, 12 hours of light and 12 hours of darkness, and the temperature was controlled at 22 ⁇ 2°C. The experiment was carried out after three days of adapting to the environment. Before the experiment officially begins, the mice need to undergo behavioral training, including neurological damage severity scoring and suspension time training.
  • a mouse intracerebral hemorrhage animal model was prepared using high glucose and reperfusion stimulation. Inject 7 mg/kg glucose intraperitoneally 15 minutes before ischemia, and remove the suture plug 3 hours after ischemia to restore blood flow for 21 hours. Sham surgery was performed using the same procedure without suture occlusion.
  • mice were intravenously injected with 2% Evans blue 22 hours after ischemia to assess hemorrhagic transformation. After 2 h of circulation, the mice were transcardially perfused with saline and the brains were quickly removed. Then, bleeding assessment was performed by Heidelberg bleeding classification (Yaghi, S & Willey, J.Z 2017). Briefly, hemorrhagic transformation events were scored as follows: 0, indicating no blood; 1, indicating hemorrhagic infarction (HI) type I, with small petechiae at the edge of the infarct; and 2, HI type II, with more blood within the infarct area.
  • HI hemorrhagic infarction
  • SalB/Rg1 alleviates cerebral hemorrhage induced by hyperglycemia

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Abstract

一种治疗出血性疾病的药物组合物包括：丹酚酸作为第一活性成分；和人参皂苷作为第二活性成分。药物组合物能够用于预防和/或治疗组织、器官出血性疾病，尤其是出血性脑卒中，并能够改善出血性脑卒中相关神经症状。

Description

治疗出血性疾病的化合物及其组合物 技术领域
本发明涉及医药领域，具体涉及一组化合物及其组合物及其在预防和/或治疗出血性疾病的应用。
背景技术
出血性疾病对人类生命安全带来巨大威胁。在不同的病理基础上，脑部，脾脏等器官都会发生出血。创伤、血管破裂等导致的出血是常见的颅内出血性脑血管疾病。出血性脑卒中约占脑卒中总数的30％，系因脑血管壁病变、坏死、破裂导致的出血。出血性脑卒中与缺血性脑卒中相比，具有更高的死亡率和长期残疾风险。目前，临床出血性脑卒中治疗策略包括及时诊断、积极控制血压、纠正潜在的凝血缺陷，以及可能的手术干预。在出血性脑卒中的治疗药物方面，患者的选择余地非常有限，需要开发更多的治疗药物，满足患者减少神经损伤和优化神经功能的治疗需求。现有技术对单体化合物及其组合物治疗出血性脑卒中的应用进行了系统研究。以往，在出血性脑卒中领域上述化合物以及组合物的作用效果未知。
因此，本领域有必要开发能够预防和/或治疗组织、器官出血性疾病，尤其是出血性脑卒中的药物组合物。
发明内容
本发明的目的是提供一种用于预防和/或治疗组织、器官出血性疾病，尤其是脑出血、以及出血性脑卒中的药物组合物。具体地，本发明提供了丹酚酸B/人参皂苷Rg1药物组合物用于制备治疗、缓解和/或预防出血性疾病的药物的用途。
在本发明的第一方面，提供了一种活性成分的组合用于制备治疗、缓解和/或预防出血性疾病的药物的用途，所述的活性成分的组合包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合；和
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合。
在另一优选例中，所述的活性成分的组合由(a)第一活性成分和(b)第二活性成分构成。
在另一优选例中，所述丹酚酸选自下组：丹酚酸B(Salvianolic acid B，Sal B)、丹酚酸A(Salvianolic acid A，Sal A)、丹酚酸C(Salvianolic acid C，Sal C)、异Sal C(Isosalvianolic acid C)、丹酚酸D(Salvianolic acid D，Sal D)、丹酚酸E(Salvianolic acid E，Sal E)、丹酚酸F(Salvianolic acid F，Sal F)、丹酚酸G(Salvianolic acid G，Sal G)、丹酚酸H(Salvianolic  acid H，Sal H)、丹酚酸I(Salvianolic acid I，Sal I)、丹酚酸J(Salvianolic acid J，Sal J)、丹酚酸K(Salvianolic acid K，SalK)、丹酚酸L(Salvianolic acid L，Sal L)、丹酚酸M(Salvianolic acid M，Sal M)、丹酚酸N(Salvianolic acid N，Sal N)、丹酚酸T(Salvianolic acid T，Sal T)、丹酚酸U(Salvianolic acid U，Sal U)、丹酚酸Y(Salvianolic acid Y，Sal Y)、咖啡酸(Caffeic Acid)、丹参素(Danshensu)、原儿茶酸(Protocatechuic acid)、原儿茶醛(Protocatechuic aldehyde)、阿魏酸(Ferulic acid)、异阿魏酸(Isoferulic Acid)、Salvinal、Ailanthoidol、迷迭香酸(Rosmarinic acid)、紫草酸(Lithospermic acid)、丹参苷(Salviaflaside)、儿茶酚(Catechin)、二甲基紫草酸B(dimethyl lithospermate B)、丹参酮I(tanshinone I)、二氢丹参酮I、丹参二醇-A(tanshindiol-A)、丹参酮IIA(tanshinone IIA)、隐丹参酮(cryptotanshinone)、丹参新酮(miltirone)、去氢丹参新酮(dehydro miltirone)、异丹参酮I(isotanshinone I)、丹参新醌甲(Danshenxinkun A)，或其组合。
在另一优选例中，所述人参皂苷选自下组：人参皂苷Rg1(Ginsenoside Rg1)、人参皂苷Re(Ginsenoside Re)、人参皂苷Rg2(Ginsenoside Rg2)、人参皂苷Rh1(Ginsenoside Rh1)、人参皂苷F1(Ginsenoside F1)、三七皂苷R1(Notoginsenoside R1)、三七皂苷R2(Notoginsenoside R2)、三七皂苷R3(Notoginsenoside R3)、三七皂苷R6(Notoginsenoside R6)、三七皂苷M(Notoginsenoside M)、三七皂苷N(Notoginsenoside N)、三七皂苷Rt(Notoginsenoside Rt)、三七皂苷T3(Notoginsenoside T3)、三七皂苷FP1(Notoginsenoside FP1)、三七皂苷RW1(Notoginsenoside RW1)、三七皂苷Fh7(Notoginsenoside Fh7)、野山茶苷D(Yesanchinoside D)、野山茶苷U(Yesanchinoside U)、20-葡萄糖人参皂苷Rf(20-O-glucoginsenoside R)、丙二酸人参皂苷Rg1(Malonyl-ginsenoside Rg1)、竹节皂苷L5(Chikusetsu saponin L5)、红花人参皂苷R1(koryoginsenoside R1)、拟人参皂苷RT3(Pseudoginsenoside RT3)、皂苷10(saponin 10)、皂苷1(saponin 1)、皂苷2(saponin 2)、皂苷3(saponin 3)、人参皂苷Ra3(Ginsenoside Ra3)、人参皂苷Rb1(Ginsenoside Rb1)、人参皂苷Rb2(Ginsenoside Rb2)、人参皂苷Rb3(Ginsenoside Rb3)、人参皂苷Rc(Ginsenoside Rc)、人参皂苷Rd(Ginsenoside Rd)、人参皂苷Rg3(Ginsenoside Rg3)、丙二酸人参皂苷Rb1(Malonyl-ginsenoside Rb1)、丙二酰人参皂苷Rd(Malonyl-ginsenoside Rd)、6'-O-乙酰人参皂苷Rg3(6”-O-acetylginsenoside Rg3)、人参皂苷F2(Ginsenoside F2)、人参皂苷CK(Ginsenoside CK)、人参皂苷Mc(Ginsenoside Mc)、20(S)-人参皂苷Rg3(20(S)-Ginsenoside Rg3)、20(R)-人参皂苷Rg3(20(R)-Ginsenoside Rg3)、20(R)-人参皂苷Rh2(20(R)-Ginsenoside Rh2)、20(S)-人参皂苷Rh2(20(S)-Ginsenoside Rh2)、三七皂苷R4(Notoginsenoside R4)、三七皂苷K(Notoginsenoside K)、绞股蓝皂苷XVII(Gypenoside XVII)、绞股蓝皂苷IX(Gypenoside IX)、绞股蓝皂苷XV(Gypenoside XV)、绞股蓝皂 苷VIII(Gypenoside VIII)、三七皂苷Fa(Notoginsenoside Fa)、三七皂苷Fc(Notoginsenoside Fc)、三七皂苷Fe(Notoginsenoside Fe)、三七皂苷D(Notoginsenoside D)、三七皂苷L(Notoginsenoside L)、三七皂苷L*(Notoginsenoside L*)、三七皂苷O(Notoginsenoside O)、三七皂苷P(Notoginsenoside P)、三七皂苷Q(Notoginsenoside Q)、三七皂苷S(Notoginsenoside S)、三七皂苷T(Notoginsenoside T)、三七皂苷FP2(Notoginsenoside FP2)、三七皂苷Ftl(Notoginsenoside Ftl)、三七皂苷ST-4(Notoginsenoside ST-4)、三七皂苷FZ(Notoginsenoside FZ)、三七皂苷Fh1(Notoginsenoside Fh1)，或其组合。
在另一优选例中，所述的丹酚酸为丹酚酸B，并且所述的人参皂苷为人参皂苷Rg1。
在另一优选例中，所述的第一活性成分包括丹酚酸或其药学上可接受的盐的纯化产物。
在另一优选例中，所述的纯化产物中，按丹酚酸计，纯度≥90％，较佳地≥95％，更佳地≥98％或99％，按所述纯化产物的总重量计。
在另一优选例中，所述的第一活性成分包括丹酚酸含量C1≥30wt％的丹酚酸提取物，其中所述含量C1按丹酚酸的重量计。
在另一优选例中，所述提取物中，丹酚酸的含量C1≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
在另一优选例中，所述的第二活性成分包括人参皂苷的含量C2≥30wt％的总皂苷提取物，其中所述含量C2按总皂苷的重量计。
在另一优选例中，所述提取物中，人参皂苷的含量C2≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为1-100:100-1、1-50:50-1、1-20:20-1、1-10:10-1、1-9:9-1、1-8:8-1、1-7:7-1、1-6:6-1、1-5:5-1、1-4:4-1、1-3:3-1、或1-2:2-1。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:2-1:10，例如5:2、5:5、2:5、1:5、或1:10。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1-4.0)、或5:(1.2-3.8)、或5:(1.5-3.5)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)、或5:(1.9-3.1)、或5:(2-3)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为2:(2-20.0)、或2:(5-20)、或2:(10-20)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为2:(5-20)、或2:(19.9-20.1)、或1:10。
在另一优选例中，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
在另一优选例中，所述药物组合物的剂型选自下组：液体制剂(如溶液、 乳液、悬浮液)、固体制剂(如冻干制剂)、气体剂型、半固体剂型。
在另一优选例中，所述的出血性疾病选自：各组织器官的出血性疾病。
在另一优选例中，所述的组织器官选自下组：脑、心、肝、脾、肺、肾、胃、肠、动脉、静脉、肌肉、神经，或其组合。
在另一优选例中，所述的出血性疾病为脑部出血性疾病。
在另一优选例中，所述的脑部出血性疾病选自下组：出血性脑卒中、脑外伤、颅内动脉瘤破裂、脑血管畸形出血、颅内恶性肿瘤诱发出血、梗死后出血、抗凝或溶栓治疗后出血、脑血管破裂出血、损伤性脑出血、高血糖诱发的脑出血，或其组合。
在另一优选例中，所述的出血性脑卒中为出血转化导致。
在另一优选例中，所述的脑部出血性疾病包括药物引起的脑出血，较佳地为溶栓药物引起的脑出血，更佳地为阿替普酶引起的脑出血。
在另一优选例中，所述药物用于选自下组的用途：
a)减轻脑血肿；
b)改善脑出血动物的神经行为；
c)保护脑出血动物的大脑皮层和纹状体神经元；
d)提高脑出血动物的生存率。
在另一优选例中，所述剂型选自下组：注射剂(如注射液或粉针剂)、口服制剂(如胶囊剂、片剂、丸剂、散剂、颗粒剂、糖浆、口服液或酊剂)、舌下含服制剂、呼吸道给药制剂、皮肤给药制剂、粘膜给药制剂，更佳地，所述剂型为注射剂。
在本发明的第二方面，提供了一种药物组合物用于制备治疗、缓解和/或预防出血性疾病的药物的用途，所述药物组合物包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合；和
(c)药学上可接受的载体。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为1-100:100-1、1-50:50-1、1-20:20-1、1-10:10-1、1-9:9-1、1-8:8-1、1-7:7-1、1-6:6-1、1-5:5-1、1-4:4-1、1-3:3-1、或1-2:2-1。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:2-1:10，例如5:2、5:5、2:5、1:5、或1:10。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1-4.0)、或5:(1.2-3.8)、或5:(1.5-3.5)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)、或5:(1.9-3.1)、或5:(2-3)。
在另一优选例中，所述的丹酚酸为丹酚酸B，并且所述的人参皂苷为人参皂苷Rg1。
在另一优选例中，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
在本发明的第三方面，提供了一种活性成分用于制备治疗、缓解和/或预防出血性疾病的药物的用途，所述活性成分选自下组：
(1)丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、或其衍生物、和含丹酚酸的提取物；以及
(2)人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、或其衍生物、和含人参皂苷的提取物。
在本发明的第四方面，提供了一种治疗、缓解和/或预防有需要的受试者的出血性疾病的方法，所述方法包括给有需要的受试者施用药物，所述药物包括下述活性成分：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合；和
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合。
在另一优选例中，所述的出血性疾病选自：各组织器官的出血性疾病。
在另一优选例中，所述的组织器官选自下组：脑、心、肝、脾、肺、肾、胃、肠、动脉、静脉、肌肉、神经，或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了脑出血代表图像(A)和相较于出血模型组，SalB/Rg1降低出血的定量结果(B)。
图2显示了SalB/Rg1在防治胶原酶诱导的脑出血方面具有协同作用。(A)出血代表图像；(B)相较于出血模型组，SalB/Rg1降低出血的定量结果；(C)各组相较于模型组抑制出血的百分比；(D)Bliss法评价协同作用的结果。相较于模型组，#p<0.05；相较于Rg1组，&p<0.05，&&p<0.01。
图3显示了SalB/Rg1抑制阿替普酶(tPA)诱发的脑出血并延长tPA溶栓时间窗的实验方案。(A)梗死4.5小时复灌给与阿替普酶(tPA)诱发的出血模型体系。(B)梗死6小时复灌给与阿替普酶(tPA)诱发的出血模型体系。(C)梗死9小时复灌给与阿替普酶(tPA)诱发的出血模型体系。
图4显示了SalB/Rg1降低tPA诱发脑出血的出血评分。(A)出血代表图 像。(B)出血评分。与假手术组相比，***P<0.001；与出血模型组相比，#P<0.05，##P<0.01；n＝10。
图5显示了采用Longa评分、Bederson评分、mNSS评分和悬挂实验进行行为学评估。与假手术组相比，***P<0.001；与出血模型组相比，#P<0.05，##P<0.01。
图6显示了：(A)SalB/Rg1保护给予tPA后神经元的尼氏染色代表图；(B)皮层尼氏小体定量结果；(C)纹状体尼氏小体定量结果。与假手术组相比,***P<0.001；与出血模型组相比,#P<0.05，##P<0.01。比例尺＝250μm，n＝6。
图7显示了SalB/Rg1提高tPA诱发脑出血动物的生存率。与假手术组相比，***P<0.01，**P<0.001；与出血模型组相比，###P<0.001。
图8显示了SalB/Rg1降低小鼠出血评分的脑组织SWI代表图和出血评分的定量结果。n＝6-7。
图9显示了SalB/Rg1在降低tPA诱发的出血方面具有协同作用。(A)出血代表图像。(B)出血评分。(C)Bliss模型评价协同效果。与假手术组相比，***P<0.001；与出血模型组相比，#P<0.05，##P<0.01；与Rg1组相比，&&P<0.01；n＝10。
图10显示了在出血性脑卒中的动物模型中评估SalB/Rg1治疗效果的出血评分代表性图像(上)和量化数据(下)。与假手术组相比，***P<0.001；与出血模型组相比，&P<0.05。
图11显示了SalB/Rg1改善高血糖诱发出血动物的神经行为。与假手术组相比，***P<0.001；与出血模型组相比，&P<0.05。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种包含丹酚酸B与人参皂苷Rg1作为活性成分的药物组合物在治疗出血性疾病中的用途。经动物实验验证，本发明的组合物能够有效减少出血性脑卒中小鼠的脑内出血量，并能改善脑出血动物的神经行为。本发明的药物组合物对出血性疾病(尤其是出血性脑卒中)表现出更优异的改善治疗效果，可用于多种组织器官的出血性疾病治疗。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“立体异构体”意在包括所有的同分异构形式(如对映 异构，非对映异构和几何异构体(或构象异构体))：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明活性成分的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
本发明活性成分可以是无定形的、晶型或其混合物。
如本文所用，“药学上可接受的盐”指本发明活性成分的化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明活性成分的化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明活性成分的化合物与碱形成的盐。适合形成盐的碱包括但并不限于：氢氧化钠、氢氧化钾、碳酸镁、碳酸钙、碳酸钠、碳酸氢钠、磷酸钠等无机碱，氨水、三乙胺、二乙胺等有机碱。另一类优选的盐是本发明活性成分与金属离子形成的盐，包括但不限于镁盐、钠盐、钙盐、钾盐等等。
如本文所用，“药学上可接受的酯”指本发明活性成分的化合物与酸或醇形成的适合用作药物的酯一类优选的酯为本发明的活性成分的一个或多个羟基与酸形成的酯，适合形成酯的酸包括但并不限于：磷酸、甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等；另一类优选的酯为本发明的活性成分的羧基与醇形成的酯，适合形成酯的醇包括但并不限于：C1-C6烷基-OH，如甲醇、乙醇、正丙醇、异丙醇等。
本发明所述的“预防”和“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述组合物或药物组合物时观察到的水平相比，本发明所述组合物或药物组合物将脑出血损伤预防、减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％。
在本发明中，所用的术语“丹酚酸”是指选自下组的化合物：丹酚酸B、丹酚酸A、丹酚酸C、异SalC、丹酚酸D、丹酚酸E、丹酚酸F、丹酚酸G、丹酚酸H、丹酚酸I、丹酚酸J、丹酚酸K、丹酚酸L、丹酚酸M、丹酚酸N、丹酚酸T、丹酚酸U、丹酚酸Y、咖啡酸、丹参素、原儿茶酸、原儿茶醛、阿魏酸、异阿魏酸、Salvinal、Ailanthoidol、迷迭香酸、紫草酸、丹参苷、儿茶酚、二甲基紫草酸B、丹参酮I、二氢丹参酮I、丹参二醇-A、丹参酮IIA、隐丹参酮、丹参新酮、去氢丹参新酮、异丹参酮I、丹参新醌甲，或其组合。
在本发明中，所用的术语“人参皂苷”是指选自下组的化合物：人参皂苷Rg1、人参皂苷Re、人参皂苷Rg2、人参皂苷Rh1、人参皂苷F1、三七皂苷R1、三七皂苷R2、三七皂苷R3、三七皂苷R6、三七皂苷M、三七皂苷N、三七皂苷Rt、三七皂苷T3、三七皂苷FP1、三七皂苷RW1、三七皂苷Fh7、野山茶 苷D、野山茶苷U、20-葡萄糖人参皂苷Rf、丙二酸人参皂苷Rg1、竹节皂苷L5、红花人参皂苷R1、拟人参皂苷RT3、皂苷10、皂苷1、皂苷2、皂苷3、人参皂苷Ra3、人参皂苷Rb1、人参皂苷Rb2、人参皂苷Rb3、人参皂苷Rc、人参皂苷Rd、人参皂苷Rg3、丙二酸人参皂苷Rb1、丙二酰人参皂苷Rd、6'-O-乙酰人参皂苷Rg3、人参皂苷F2、人参皂苷CK、人参皂苷Mc、20(S)-人参皂苷Rg3、20(R)-人参皂苷Rg3、20(R)-人参皂苷Rh2、20(S)-人参皂苷Rh2、三七皂苷R4、三七皂苷K、绞股蓝皂苷XVII、绞股蓝皂苷IX、绞股蓝皂苷XV、绞股蓝皂苷VIII、三七皂苷Fa、三七皂苷Fc、三七皂苷Fe、三七皂苷D、三七皂苷L、三七皂苷L*、三七皂苷O、三七皂苷P、三七皂苷Q、三七皂苷S、三七皂苷T、三七皂苷FP2、三七皂苷Ftl、三七皂苷ST-4、三七皂苷FZ、三七皂苷Fh1，或其组合。
如本发明所用，术语“SalB”与“丹酚酸B”可以互换使用；术语“Rg1”与“人参皂苷Rg1”可以互换使用。
第一活性成分
本发明提供了可用于制备治疗、缓解和/或预防出血性疾病的药物组合物，其包括第一活性成分和第二活性成分，其中第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合。
在本发明优选的实施方式中，第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合。所述丹酚酸B如下式(I)所示：
在另一优选例中，所述的第一活性成分包括丹酚酸B或其药学上可接受的盐的纯化产物。
在另一优选例中，所述的纯化产物中，按丹酚酸B计，纯度≥90％，较佳地≥95％，更佳地≥98％或99％，按所述纯化产物的总重量计。
在另一优选例中，所述的第一活性成分包括丹酚酸B含量C1≥30wt％的丹酚酸提取物，其中所述含量C1按丹酚酸的重量计。
在另一优选例中，所述提取物中，丹酚酸B的含量C1≥70％，较佳地≥80％， 更佳地≥90％或≥95％，按提取物的干重计。
第二活性成分
本发明中，第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合。
在本发明的优选实施方式中，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合。所述人参皂苷Rg1如下式(II)所示：
在另一优选例中，所述的第二活性成分包括人参皂苷Rg1的含量C2≥30wt％的总皂苷提取物，其中所述含量C2按总皂苷的重量计。
在另一优选例中，所述提取物中，人参皂苷Rg1的含量C2≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
药物组合物、活性成分组合、药盒
本发明提供了一种药物组合物的用途，所述药物组合物包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐、含丹酚酸的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐、含人参皂苷的提取物、或它们的组合；和(c)药学上可接受的载体。
本发明还提供了一种活性成分组合的用途，所述活性成分组合包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐、含丹酚酸的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐、含人参皂苷的提取物、或它们的组合。
在另一优选例中，所述的活性成分的组合由(a)第一活性成分和(b)第二活性成分构成。
在本发明的优选实施方式中，以上所述的药物组合物和活性成分组合中，所述第一活性成分和第二活性成分的重量比为1-100:100-1、1-50:50-1、 1-20:20-1、1-10:10-1、1-9:9-1、1-8:8-1、1-7:7-1、1-6:6-1、1-5:5-1、1-4:4-1、1-3:3-1、1-2:2-1。
优选地，所述第一活性成分和第二活性成分的重量比为5:(1-5.0)、或5:(1.2-3.8)、或5:(1.5-3.5)。
更优选地，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)、或5:(1.9-3.1)、或5:(2-3)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为2:(2-20.0)、或2:(5-20)、或2:(10-20)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为2:(5-20)、或2:(19.9-20.1)、或1:10。
在本发明的优选实施方式中，所述丹酚酸为丹酚酸B，并且所述人参皂苷为人参皂苷Rg1。
在本发明的进一步优选实施方式中，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
所述药物组合物的剂型选自下组：液体制剂(如溶液、乳液、悬浮液)、固体制剂(如冻干制剂)。
在另一优选例中，所述剂型选自下组：注射剂(如注射液或粉针剂)、口服制剂(如胶囊剂、片剂、丸剂、散剂、颗粒剂、糖浆、口服液或酊剂)，更佳地，优选地，所述剂型为注射剂。
本发明的药物组合物中，第一活性成分和第二活性成分可以分别制成制剂或混合在一起制成制剂。
本发明的药物组合物包含安全有效量范围内第一活性成分和/或第二活性成分。其中“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明活性成分/剂，更佳地，含有10-500mg本发明活性成分/剂。较佳地，所述的“一剂”为一个胶囊、药片、针剂等。
本发明中，“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和第一活性成分和/或第二活性成分相互掺和，而不明显降低第一活性成分和/或第二活性成分的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
在所述的活性成分组合中，第一活性成分和第二活性成分可以是相互独立的，也可以使组合在一起，以活性成分组合物的形成存在。
本发明的药物组合物、活性成分组合均可采用常规方法和设备进行制备。
用途和施用方法
本发明提供了一种本文所述药物组合物、活性成分、活性成分的组合或药盒的用途，用于制备一药物或药盒，所述药物或药盒用于预防和/或治疗出血性疾病，优选为预防和/或治疗出血性脑卒中。
本发明中，所述的出血性疾病选自各组织器官的出血性疾病。其中所述的组织器官包括但不限于：脑、心、肝、脾、肺、肾、胃、肠、动脉、静脉、肌肉、神经，或其组合。
本发明中，所述的出血性脑卒中是因指脑血管壁病变、坏死、破裂导致的脑内出血。临床主要表现为头痛、呕吐、意识障碍、偏身瘫痪、感觉障碍，或其组合。所述出血性脑卒中可以是出血转化导致的出血性脑卒中。
本发明中，所述预防和/或治疗出血性疾病、预防和/或治疗出血性脑卒中等应用，包括在预防性的应用，也包括事后改善性的应用。例如，对于出血性脑卒中，包括在出血性脑卒中之前、之中、和/或之后施用本发明的药物组合物或活性成分的组合物，以对发生出血性脑卒中后的组织器官进行保护、修复或功能的改善或增强。
优选地，用于本发明用途的药物组合物或活性成分的组合中，仅包含所述第一活性成分丹酚酸B与第二活性成分人参皂苷Rg1。
本发明的药物组合物或活性成分的组合中，第一活性成分与第二活性成分还可以与其他药学上可接受的化合物联合给药，包括(但并不限于)：降血压药、降血脂药、降糖药、抗氧化药、组织脱水药、神经保护剂等。
本发明的药物组合物、活性成分的组合或药盒中，第一活性成分和第二活性成分可以在施用时同时给药、分别给药或顺序给药。
本发明的药物组合物、活性成分的组合或药盒中，第一活性成分和第二活性成分的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的 一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明活性成分的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明的药物组合物、活性成分的组合或药盒中，按丹酚酸B和人参皂苷Rg1总量计，活性成分的治疗有效剂量的一般范围将是：约1-2000mg/天、约10-约1000mg/天、约10-约500mg/天、约10-约250mg/天、约10-约100mg/天，或约10-约80mg/天。治疗有效剂量将以一个或多个剂量给予。然而，应理解，对于任何特定患者的本发明活性成分的特定剂量将取决于多种因素，例如，待治疗的患者的年龄、性别、体重、一般健康状况、饮食、个体响应，给予时间、待治疗的疾病的严重性、剂型、应用模式和伴用药物。给定情况的治疗有效量能用常规实验测定，并在临床医生或医师能力和判断范围内。在任何情况中，所述活性成分将基于患者的个体情况以多个剂量给予并以允许递送治疗有效量的方式给予。
本发明的主要优点包括：
1.与现有技术相比，本发明发现了丹酚酸B/人参皂苷Rg1药物组合物在治疗出血性疾病中的用途。
2.本发明发现了丹酚酸B/人参皂苷Rg1药物组合物对出血性脑卒中的治疗具有协同作用，其能够显著减少脑内出血量。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1丹酚酸B和人参皂苷Rg1(SalB/Rg1)不同比例组合抑制脑出血的效果
1.实验材料
1.1实验动物
雄性SD大鼠，购自北京华阜康生物科技股份有限公司，体重260-300g。大鼠饲养在中国科学院上海药物研究所SPF级动物房，房间内恒温(22±2℃)，光照和黑暗周期为12小时，自由摄食饮水。
1.2主要试剂和药品
表1材料与试剂
1.3主要仪器
表2实验仪器

2.实验方法
2.1实验分组
48只雄性SD大鼠依据体重随机分成8组，每组6只，造模时使用胶原酶浓度为0.5U/μl。
丹酚酸B缩写为SalB，人参皂苷Rg1缩写为Rg1。出血模型对照组、(10：1)SalB/Rg1组、(5：1)SalB/Rg1组、(5：2)SalB/Rg1组、(5：5)SalB/Rg1组、(2：5)SalB/Rg1组、(1：5)SalB/Rg1组、(1：10)SalB/Rg1组小鼠 给药体积5ml/kg，溶媒为0.9％氯化钠注射液。本实验采取双盲法，药物制剂配置过程中所用到的EP管和移液枪枪头均经过立式压力蒸汽灭菌锅灭菌。
2.2胶原酶Ⅶ-S诱导的脑出血动物建造
术前将手术用品在高压蒸汽灭菌锅内120℃灭菌30min备用。将舒泰麻醉的大鼠俯卧位固定于手术台上，用酒精和碘伏对头皮进行消毒。于双耳连线中点做长约2cm的纵行切口，用镊子顿性剥离皮下组织，暴露出颅骨，使用3％H2O2破坏颅骨的骨膜，暴露出前后囟，调节立体定位仪使前后囟位于同一平面。使用直径0.5mm的牙科钻，于前囟前0.5mm，中线向右3.5mm处钻取直径约1.0mm的小孔，通过微量注射器将浓度0.5U/μl，1μl胶原酶Ⅶ缓慢注入纹状体部位，钻孔处用骨蜡封住，缝合皮肤，给予充足的食物和水。注射胶原酶后立即尾静脉注射5mg/kg SalB/Rg1。
2.3血肿体积定量
大鼠实施安乐死后，整个脑组织被切成2毫米厚的连续七张脑片。使用Image Pro Plus(6.0版本)软件定量每一张脑片红色区域的血肿面积，依据血肿面积计算血肿体积。血肿体积计算公式V＝(∑(A1+A2)/2)*D(mm3)。V为每张脑片的血肿体积(mm3)；A1、A2分别为脑片嘴侧面和尾侧面红色出血区域面积(mm2)；D为切片厚度(mm)]；全脑血肿体积是7张脑片血肿体积的总和。
考虑到血肿带来左右半球体积不规则，血肿体积比用出血对侧半球脑体积进行校对。血肿体积(％)＝(血肿体积/出血对侧半球脑体积)×100％。
2.4数据统计
数据用表示，并采用Graph Pad Prism 9.0进行处理。对于符合正态分布的数据，采用单因素方差分析法(One-way ANOVA)进行统计分析，并用Tukey进行组间差异检验。对于不符合正态分布的数据，采用Kruskal-Wallis检验。当p<0.05时，认为组间存在显著性差异。
3.结果
胶原酶Ⅶ诱发的出血动物模型是研究脑出血的经典模型，由于胶原酶被定点注射到纹状体的区域，图1A显示了纹状体区域出现明显的出血灶。通过对出血灶进行定量，以及比较不同比例丹酚酸B/Rg1组合抑制脑出血的效果，血肿体积抑制率采用如下公式计算：(出血模型组血肿体积-丹酚酸B/Rg1给药组血肿体积)/出血模型组血肿体积。(10:1)SalB/Rg1的血肿抑制率为0.30±0.20％；(5:1)SalB/Rg1的血肿抑制率为-10.05±0.34％；(5:2)SalB/Rg1的血肿抑制率为13.31±0.13％；(5:5)SalB/Rg1的血肿抑制率为11.92±0.09％；(2:5)SalB/Rg1的血肿抑制率为21.33±0.27％；(1:5)SalB/Rg1的血肿抑制率为13.14±0.28％；(1:10)SalB/Rg1的血肿抑制率为23.95±0.24％。
实施例2丹酚酸B与Rg1在降低胶原酶诱导的脑出血方面具有协同作用
1.实验材料
1.1实验动物
雄性SD大鼠，购自(北京华阜康生物科技股份有限公司)，体重260-300g。大鼠饲养在中国科学院上海药物研究所SPF级动物房，房间内恒温(22±2℃)，光照和黑暗周期为12小时，并自由获得食物和水。
1.2主要试剂和药品、主要仪器同前文
2.实验方法
2.1胶原酶Ⅶ-S诱导的脑出血动物模型制备方法同前文
2.2实验分组
依据前文，丹酚酸B和Rg1不同比例对脑血肿的抑制效果不同，其中丹酚酸B和Rg1在1：10的比例下，抑制脑血肿的效果更为突出。本实施例对丹酚酸B/Rg1组合的协同效果进行考察，采用Bliss独立统计学模型评价协同效果。
雄性SD大鼠随机分为四组，出血模型组、SalB组、Rg1组、SalB/Rg1组合组，每组10只动物。出血模型组动物给与生理盐水；SalB组动物给与0.45mg/kg丹酚酸B；Rg1组动物给与4.55mg/kg人参皂苷Rg1；SalB/Rg1组动物给与5mg/kg丹酚酸B/Rg1(其中丹酚酸B和Rg1按照质量比为1:10配制)。依据动物分组，在胶原酶处理后立即尾静脉注射生理盐水、丹酚酸B、Rg1或者SalB/Rg1组合。
2.3血肿体积定量同前文
2.4Bliss独立统计学模型
为了探究SalB和Rg1在治疗脑出血方面是否具有协同作用，采用Bliss独立统计学模型，在抑制血肿体积上进行了验证。首先进行了以下计算，S模型、SSalB、SRg1和SSalB/Rg1分别代表出血模型、SalB、Rg1和SalB/Rg1组的血肿体积，ISalB、IRg1和ISalB/Rg1分别代表SalB、Rg1和SalB/Rg1组的血肿体积与出血模型组的比值，I值计算如公式1、2和3所示。
ISalB＝(SSalB/S模型)×100％(公式1)
IRg1＝(SRg1/S模型)×100％(公式2)
ISalB/Rg1＝(SSalB/Rg1/S模型)×100％(公式3)
通过Bliss独立统计学模型评估SalB和Rg1治疗脑出血的协同作用。假设Independence为SalB和Rg1计算得出的协同作用理论值。根据Bliss独立统计学模型，Independence计算如公式4所示。
Independence＝ISalB×IRg1(公式4)
为了简化协同效应的统计检验，经Kolmogorov-Smirnov检验数据是否符合正态分布后，进行了对数尺度上的转换得到方程如公式5所示。
InSSalB+InSRg1-InS模型-InSSalB/Rg1>0(公式5)
依据统计学方法，使用单因素方差分析和Tukey的多重比较测试分析来比较二者之间的差异性。若P<0.05，则认为SalB和Rg1具有协同作用。
2.5数据统计
统计学分析研究中量化的数据用表示，并采用Graph Pad Prism 9.0进行处理。对于符合正态分布的数据，采用单因素方差分析法(One-way ANOVA)进行统计分析，并用Tukey进行组间差异检验。对于不符合正态分布的数据，采用Kruskal-Wallis检验。当p<0.05时，认为组间存在显著性差异。
3.实验结果
图2A是胶原酶诱导脑出血的各组代表图，血肿部位呈现深红色。图2B是血肿体积的定量结果，出血模型组的血肿体积比为(10.97±3.97％)，单独的SalB(8.54±3.63％)或者单独的Rg1(11.03±2.38％)都不能显著下调血肿体积；5mg/kg的SalB/Rg1能够显著下调血肿体积(6.92±2.36％，#p<0.05)，并且相较于Rg1的有效性具有统计学意义(&p<0.05)。图2C是血肿体积抑制率的定量结果，5mg/kg的SalB/Rg1的抑制率相较于Rg1显著提高(36.95±0.22％)，具有更加优异的疗效(&p<0.01)。
协同效果采用Bliss独立统计学模型进行评价。依据前文给出的公式，结果显示Sal/Rg1组与独立组具有显著的统计学差异(p<0.05),说明SalB和Rg1组合在降低脑血肿方面具有非常显著的协同效果。
实施例3丹酚酸B联合人参皂苷Rg1抑制阿替普酶(tPA)引起的脑出血
1实验材料
雄性C57BL/6小鼠(26±3g)购自北京华阜康生物科技有限公司。小鼠饲养在中国科学院上海药物研究所SPF级动物房，房间内恒温(22±2℃)，光照和黑暗周期为12小时，并自由获得食物和水。
2实验的药品、试剂与仪器见前文。
本实施例所用的特殊试剂如下：
表3实验药品与试剂信息表
3.实验方法
3.1阿替普酶(tPA)诱导小鼠脑出血的模型
实验前对所有小鼠进行行为学评价，确保所有小鼠神经行为功能正常。腹腔注射舒泰复合赛拉嗪麻醉小鼠，颈部备皮，碘伏消毒。沿小鼠颈部正中剪开皮肤，暴露颈深肌膜和气管前肌，分离出颈总动脉并穿线以活结阻断血流。沿颈总动脉向上分离，在颈总动脉的近心端系一死结，在远心端备一缝合线，暂 不系紧。用显微剪在颈总动脉的近心端剪一小口，将硅胶线栓从小口处轻轻插入，阻塞了大脑中动脉主干的起始部。将颈总动脉远心端上所备活结系紧固定线栓，缝合皮肤。大脑中动脉短暂闭塞到规定时间后拆开缝合部位，取出线栓，将颈总动脉远心端结系紧，进行再灌注。依据tPA应用的时间点不同，分三批开展实验研究，分别为小鼠大脑中动脉闭塞4.5h、6h、9h撤除线栓实现血管再灌注，并立即给予tPA诱导出血模型。每批实验都设置假手术组、模型组和丹酚酸B/Rg1组(5mg/kg丹酚酸B/Rg1)。其中丹酚酸B/Rg1的配制是丹酚酸B和Rg1的比例为5：2。
该研究的出血模型组和丹酚酸B/Rg1组均在给予10mg/kg tPA的背景下，Sham组则在相应时间点给予生理盐水。SalB/Rg1的配制为丹酚酸B和Rg1的质量比为5：2。SalB/Rg1治疗时间点见图3，一共给药3次。再灌注48h作为实验终点，各个时间窗的实验方案设计如图3。为保证研究结果的客观性，实验采用双盲法进行，直至实验全部完成并得出结论再揭盲。
3.2行为学评价
3.2.1.Longa评分
Longa评分的评估范围为0到4：
0分，正常，无神经功能缺损；
1分，右肢体(瘫痪测)不能完全伸展，轻度神经功能缺损；
2分，行走时向瘫痪测(右侧)转圈，中度神经功能缺损；
3分，行走时向瘫痪测(右侧)倾倒，重度神经功能缺损；
4分，不能自发行走，有意识丧失。
3.2.2 Bederson评分
Bederson评分采用5分制评分：
0分，无缺陷；
1分，失去前肢屈曲；
2分，失去前肢屈曲，侧推阻力较低；
3分，单向盘旋；
4分，纵向旋转或癫痫活动；
5分，没有运动。
3.2.3悬挂实验
将实验鼠置于传统用的鼠笼盖上，轻轻震动鼠笼盖促使实验鼠紧握鼠笼盖，随后迅速翻转鼠笼盖，记录后肢离开笼盖的最长潜伏期。以90s为分界值，超过90s按90s记，不足90s按实际时间记录。每次实验重复3次并取其最好成绩记录。
3.2.4 mNSS(Modified Neurological Severity Score)评分
表4 mNSS评分表

3.3出血评分
用注射用生理盐水配置成2％的伊文思蓝染液，按4mg/kg剂量进行尾静脉注射。2h后用10ml 0.9％生理盐水进行心脏灌流。取出脑组织用OLYMPUS-BX51显微镜拍照。出血程度通过HT分级进行评分:0＝无血；1＝HI I，梗死边缘有小瘀点；2＝HI II，梗死区内有较多汇合的瘀点，但无占位作用；3＝PH I，血凝块不超过梗死面积的30％，有轻度占位效应；4＝PH II，超过梗死体积30％的致密血凝块，具有明显的占位效应。将每个切片的评分相加，使用总分作为评价指标。
3.4尼氏染色
脑组织在4％中性甲醛固定液中固定48h后，取出放入包埋框中，流水冲3-5h后放入自动脱水机，按所设定程序脱水，脱水完成后石蜡包埋。包埋后的组织切成厚度为4μm的石蜡切片。将组织切片置于烤片机上68℃加热60min后 取下，依次经过二甲苯15min；无水乙醇5min；95％乙醇5min；75％乙醇5min；流水冲洗5min进行脱蜡。用尼氏染色液染色10min后用蒸馏水洗涤两次。
3.5数据统计方法
数据以表示。对于正态分布变量，采用方差分析(ANOVA)和Bonferroni事后检验来评估组间的差异。如果只有两组进行比较，则应用未配对的双尾Student's t test。采用卡方检验(Chi-Squared Test)探讨两组比值之间的差异。P<0.05为差异有统计学意义。
4结果
4.1 SalB/Rg1降低阿替普酶(tPA)诱发的出血
脑组织出血代表图如图4A所示。随着tPA使用时间的推后，出血模型组的出血评分也随之升高，表明出血随着延迟给予tPA而愈发严重。图4B显示了出血评分，可以看出无论是4.5h、6h还是9h给予tPA，SalB/Rg1干预后均会显著降低出血评分(P<0.01)。脑缺血4.5h后给予tPA，出血模型组的出血评分3.75±0.71，给予SalB/Rg1后出血评分降至2.20±1.93，相较于出血模型组具有显著性差异(P<0.01)。脑缺血6h给予tPA后血模型组的出血评分升至4.75±1.98，给予SalB/Rg1后出血评分降至1.70±2.34，相较于出血模型组具有显著性差异(P<0.01)。即使在脑缺血9h给予tPA实验中，SalB/Rg1组与出血模型组相比，仍有极显著性差异(5.4±1.58versus 3.6±1.43,P<0.01)。
4.2 SalB/Rg1改善神经行为
在图5中，造模后的小鼠神经行为评分几乎都与假手术组呈现极显著性差异。在脑缺血4.5h给予tPA实验中，除悬挂实验外其余三个评分在SalB/Rg1组都有显著降低。在脑缺血4.5h给予tPA实验中，SalB/Rg1组显著降低了mNSS评分和延长了悬挂时间(P<0.05)。而Longa评分和Bederson评分虽然没有显著降低，但有降低评分的趋势。神经行为学结果表明，SalB/Rg1可以改善脑出血动物的神经行为。
4.3 SalB/Rg1保护皮层和纹状体的神经元
皮层和纹状体区域的神经元受损均会对小鼠的行为学产生严重影响。因此对脑组织切片进行尼氏染色，通过对尼氏小体数量的统计明确SalB/Rg1能否保护神经元。尼氏染色结果如图6所示，在经尼氏染色后的脑组织图片中，在每张图片的大脑皮层和纹状体中选择相同位置进行定量。在4.5h和6h的实验中，造模后皮层和纹状体的尼氏染色阳性细胞数量与假手术组相比均有显著下降(P<0.001)。而SalB/Rg1组的尼氏染色阳性细胞数量均较出血模型组有显著增多(P<0.05)。这一结果与上述行为学相对应，提示SalB/Rg1可能通过保护大脑皮层和纹状体中的神经元进而改善小鼠的神经行为。
虽然在脑缺血9h给予tPA实验中，给予SalB/Rg1干预后未改善神经行为评分，但在尼氏染色中SalB/Rg1组皮层区域(2513.67±429.51 versus 1694.50±272.71,P<0.001)和纹状体区域(2963.33±508.18 versus 2026.17± 481.92,P<0.001)的阳性细胞数量与出血模型组相比依旧有显著性差异。
4.4 SalB/Rg1提高延迟给予阿替普酶后的生存率
各组小鼠生存曲线如图7所示。无论是脑缺血4.5h给予tPA、6h给予tPA还是9h给予tPA，造模后小鼠生存率显著降低(P<0.01)。而给予SalB/Rg1后均会提高小鼠生存率。在脑缺血4.5h给予tPA实验中，SalB/Rg1干预后与出血模型组相比生存率的提高有显著性差异(80％versus 20％,P<0.001)。在脑缺血6h给予tPA实验中，再灌注12h后SalB/Rg1组生存率为71.43％，而出血模型组生存率为44％；再灌注48h时SalB/Rg1组生存率为50％，出血模型组生存率为36％。在9h的实验中也得出相似结论，再灌注12h后SalB/Rg1组生存率为90％，而出血模型组生存率为60％；再灌注48h时SalB/Rg1组生存率为70％，出血模型组生存率为40％。这一结果表明SalB/Rg1可以延长小鼠的生存时间。
实施例4.MRI影像学评价SalB/Rg1抑制脑出血的效果
1实验材料
1.1实验动物的购买和饲养条件同前
1.2实验药品与试剂同前
1.3实验仪器与材料
表5实验仪器与材料信息表
2实验方法
2.1模型制备和处理方案同实施例3，图3中的方案B。
2.2小动物核磁共振成像
MRI数据采集在11.7T BioSpec 117/16USR的MRI系统上进行。分别采用2×2相控阵线圈和72mm体积谐振器进行传输和图像采集。所有受试动物均使用1％至1.5％的异氟醚麻醉，其体温保持在37℃，以确保在核磁扫描过程中进行适当的固定。监测呼吸情况，并保持95-110次/分钟的速率。
T2加权成像(T2-weighted image,T2WI):全脑T2WI用快速采集与松弛增强(RARE)序列获得高分辨率的图像，翻转角度90°，2500ms重复时间(repetition time,TR)，6.5ms回波时间(echo time,TE)和0.063×0.063×0.5mm3空间分辨率。对于T2AMP，使用多层多回波(MSME)序列，翻转角度为90°，2200ms TR和30有效TE，从7.5ms到225ms。
磁敏感加权成像(Susceptibility Weighted Imaging,SWI):对于SWI，使用血流补偿快速低角度拍摄(Fast Low-Angle Shot,FLASH)序列获得血管异常，TR＝350ms，TE＝12ms，矩阵大小＝200×200，空间resolution＝0.08×0.08mm2，切片厚度＝0.8mm。
动脉自旋标记(Arterial spin labeling,ASL):ASL图像使用漂移校正的 稳定脉冲成像和标记(drift-corrected steady pulsed imaging and labeling,dSPIL)序列获得的，TE＝3.0ms，TR＝5000ms，时间重复次数为32次。图像分析及数据处理：使用基于SPM8(http://www.fil.ion.ucl.ac.uk/spm/software/spm8/)的SPMMouse进行运动校正和调整，使用MATLAB 2021b(MathWorks,Natick,USA)进行数据分析。其他MRI形态与T2WI进行刚性共配准。
利用最小二乘算法将信号强度数据拟合到单指数衰减曲线中，基于T2MAP计算全脑的T2-弛豫时间。脑血流(CBF)图像使用MATLAB编写的自定义程序进行处理。使用ITK-SNAP软件手动创建不同MRI模式下的ROI。脑血流量评价方法:将脑组织划分为梗死区、梗死边缘区和对侧局域，并测量区域内的CBF值。
2.3数据统计方法
数据以表示。对于正态分布变量，采用单向方差分析(ANOVA)和Bonferroni事后检验来评估组间的差异。如果只有两组进行比较，则应用未配对的双尾Student’s t tes，P<0.05为差异有统计学意义。
3结果
3.1磁共振成像显示SalB/Rg1降低出血评分
通过MRI中SWI对脑实质中的出血进行检测。SWI是一种高分辨率3D梯度回波MRI序列，可突出顺磁性物质(例如脱氧血红蛋白、含铁血黄素和铁蛋白)与脑实质之间的差异，从而使该序列对检测到微出血也很敏感，顺磁性物质(如含铁血黄素、脱氧血红蛋白和铁蛋白)在SWI图像中显示低信号。
SWI从嗅球到小脑共扫描了多层图像，对每层进行出血程度评分，每层进行加和作为该小鼠的出血分数并进行统计。结果如图8所示，与前文一致，假手术组未出现出血，出血模型组共6只小鼠，其中有4只出现实质出血且出血较为严重，而SalB/Rg1组6只小鼠中仅有2只在SWI中检测到出血(7.00±7.83versus 0.50±0.76)。
实施例5丹酚酸B和人参皂苷Rg1在抑制阿替普酶引起的脑出血方面具有协同作用
1.实验材料
雄性C57BL/6小鼠(26±3g)购自北京华阜康生物科技有限公司。小鼠饲养在中国科学院上海药物研究所SPF级动物房，房间内恒温(22±2℃)，光照和黑暗周期为12小时，并自由获得食物和水。
2.实验的药品、试剂与仪器见前文。
3.实验方法
3.1阿替普酶(tPA)诱导小鼠脑出血的模型见实施例3，同前文图3中的方案B
3.2出血评分同实施例3中的出血评分方法
3.3实验动物分组和给药
雄性C57BL/6小鼠随机分为四组，出血模型组、SalB组、Rg1组、SalB/Rg1 组合组，每组10只动物。出血模型组动物给与生理盐水；SalB组动物给与3.57mg/kg丹酚酸B；Rg1组动物给与1.43mg/kg人参皂苷Rg1；SalB/Rg1组动物给与5mg/kg丹酚酸B/Rg1(其中丹酚酸B和Rg1按照质量比为5：2配制)。依据动物分组尾静脉注射生理盐水、丹酚酸B、人参皂苷Rg1或者SalB/Rg1组合。
3.4 Bliss独立统计学模型如实施例2所述
3.5数据统计方法
数据以表示。对于正态分布变量，采用方差分析(ANOVA)和Bonferroni事后检验来评估组间的差异。如果只有两组进行比较，则应用未配对的双尾Student’s t test。采用卡方检验(Chi-Squared Test)探讨两组比值之间的差异。P<0.05为差异有统计学意义。
4.结果
4.1 SalB/Rg1在降低阿替普酶(tPA)诱发的出血方面具有协同作用
图9A是tPA诱发脑出血的各组代表图，出血部位呈现红色。图9B是出血程度的定量结果，出血模型组的出血评分为(4.75±1.98)，单独的SalB(3.88±1.76)或者单独的Rgl(4.5±1.86)都不能显著下调出血评分；5mg/kg的SalB/Rgl能够显著下调出血评分(1.7±2.34verus 4.75±1.98，p<0.01)，并且相较于Rg1的有效性具有统计学差异(1.7±2.34verus 4.5±1.86，p<0.01).
协同效果采用Bliss独立统计学模型进行评价(图9C)。依据前文给出的公式，结果显示Sal/Rg1组与独立组具有显著的统计学差异(p<0.01)，说明SalB和Rg1组合在降低溶栓药物诱发的脑出血方面具有非常显著的协同效果。
实施例6.SalB/Rg1抑制高血糖加剧的脑出血
1.实验材料
雄性健康C57BL/6小鼠，体重为25g～28g，购自上海斯莱克实验动物公司。动物购回后饲养于上海药物研究所SPF级动物房，自由摄食饮水，12小时光照12小时黑暗，温度控制在22士2℃，三天适应环境后开展实验。实验正式开始前，需对小鼠进行行为学训练，包括神经损害严重程度评分和悬挂时间训练。
2.实验方法
2.1出血性小鼠模型的制备
用高葡萄糖和再灌注刺激制备小鼠脑出血动物模型。在缺血前15分钟腹膜内注射7mg/kg葡萄糖，并在缺血3小时后取出线栓以恢复血流21小时。使用相同的程序进行假手术，没有线栓阻塞。
2.2实验设计与分组
为了评估SalB/Rg1对高血糖诱发的脑出血的影响，将所有小鼠随机分为三组(n＝8-10)。假手术小鼠给予生理盐水作为正常对照，其余两组通过高糖加再灌注诱导出血，出血模型组小鼠给予生理盐水作为模型对照，SalB/Rg1给药组小鼠给予6.25mg/kg SalB/Rg1考察治疗效果。24h后进行神经行为测试。
2.3配药与给药方案
为保证研究结果的客观性，实验采用双盲法进行，直至研究结束再进行揭盲。SaB/Rg1的配制:SalB和Rgl粉末按5：2比例称量充分混合，经旋涡振荡器完全溶解后，用微孔滤膜过滤至无菌的EP管内分装，于-20℃冻存，待用。
2.4行为学评估，见实施例3的方法介绍
2.5出血评估
缺血后22h后小鼠静脉注射2％伊文思蓝评估出血转化。循环2h后，用盐水经心灌注小鼠并迅速取出大脑。然后，通过Heidelberg出血分类(Yaghi,S&Willey,J.Z 2017)进行出血评估。简而言之，出血性转化事件被评分为：0分表示没有血液；1分表示I型出血性梗死(HI)，梗死边缘有小瘀点；2分表示HI型II，梗塞区域内有更多融合的瘀点，但无占位作用；3分表示实质出血(PH)I型，血凝块不超过梗塞面积的30％，具有轻微的占位作用；4分表示PH II型，致密血凝块超过梗塞面积的30％，具有显着的占位效应。
2.6统计学方法
使用GraphPad Prism 6.0进行数据分析，所有计量数据均用表示，方差齐性检验采用单因素方差分析(One-way ANOVA)，t检验法比较各组间的差异，p<0.05时有统计学意义。
3实验结果
3.1 SalB/Rg1减轻高血糖诱发的脑出血
静脉注射伊文斯蓝便于观察出血点，出血点始终位于伊文斯蓝染色边缘(图10A)。根据出血点的数量和范围确定出血评分。出血模型组的出血点相当明显。与出血模型组相比，6.25mg/kg SalB/Rg1显着下调出血评分(1.5±0.20vs 2.22±0.22，p<0.05)。通过修正的Bederson、Longa评分、悬挂时间和mNSS评分检测神经功能预后，神经行为结果显示SalB/Rg1处理呈现改善趋势(图11)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种活性成分的组合用于制备治疗、缓解和/或预防出血性疾病的药物的用途，其特征在于，所述的活性成分的组合包括：
    (a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合；和
    (b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合。
  2. 如权利要求1所述的用途，其特征在于，所述丹酚酸选自下组：丹酚酸B、丹酚酸A、丹酚酸C、异SalC、丹酚酸D、丹酚酸E、丹酚酸F、丹酚酸G、丹酚酸H、丹酚酸I、丹酚酸J、丹酚酸K、丹酚酸L、丹酚酸M、丹酚酸N、丹酚酸T、丹酚酸U、丹酚酸Y、咖啡酸、丹参素、原儿茶酸、原儿茶醛、阿魏酸、异阿魏酸、Salvinal、Ailanthoidol、迷迭香酸、紫草酸、丹参苷、儿茶酚、二甲基紫草酸B、丹参酮I、二氢丹参酮I、丹参二醇-A、丹参酮IIA、隐丹参酮、丹参新酮、去氢丹参新酮、异丹参酮I、丹参新醌甲，或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述人参皂苷选自下组：人参皂苷Rg1、人参皂苷Re、人参皂苷Rg2、人参皂苷Rh1、人参皂苷F1、三七皂苷R1、三七皂苷R2、三七皂苷R3、三七皂苷R6、三七皂苷M、三七皂苷N、三七皂苷Rt、三七皂苷T3、三七皂苷FP1、三七皂苷RW1、三七皂苷Fh7、野山茶苷D、野山茶苷U、20-葡萄糖人参皂苷Rf、丙二酸人参皂苷Rg1、竹节皂苷L5、红花人参皂苷R1、拟人参皂苷RT3、皂苷10、皂苷1、皂苷2、皂苷3、人参皂苷Ra3、人参皂苷Rb1、人参皂苷Rb2、人参皂苷Rb3、人参皂苷Rc、人参皂苷Rd、人参皂苷Rg3、丙二酸人参皂苷Rb1、丙二酰人参皂苷Rd、6'-O-乙酰人参皂苷Rg3、人参皂苷F2、人参皂苷CK、人参皂苷Mc、20(S)-人参皂苷Rg3、20(R)-人参皂苷Rg3、20(R)-人参皂苷Rh2、20(S)-人参皂苷Rh2、三七皂苷R4、三七皂苷K、绞股蓝皂苷XVII、绞股蓝皂苷IX、绞股蓝皂苷XV、绞股蓝皂苷VIII、三七皂苷Fa、三七皂苷Fc、三七皂苷Fe、三七皂苷D、三七皂苷L、三七皂苷L*、三七皂苷O、三七皂苷P、三七皂苷Q、三七皂苷S、三七皂苷T、三七皂苷FP2、三七皂苷Ftl、三七皂苷ST-4、三七皂苷FZ、三七皂苷Fh1，或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述的丹酚酸为丹酚酸B。
  5. 如权利要求1所述的用途，其特征在于，所述的人参皂苷为人参皂苷Rg1。
  6. 如权利要求1所述的用途，其特征在于，所述的活性成分的组合由(a)第一活性成分和(b)第二活性成分构成。
  7. 如权利要求1所述的用途，其特征在于，所述第一活性成分和第二活性成分的重量比为5:2-1:10。
  8. 如权利要求1所述的用途，其特征在于，所述的出血性疾病为脑部出血性疾病。
  9. 如权利要求8所述的用途，其特征在于，所述的脑部出血性疾病包括药物引起的脑出血，较佳地为溶栓药物引起的脑出血，更佳地为阿替普酶引起的脑出血。
  10. 如权利要求1所述的用途，其特征在于，所述药物用于选自下组的用途：
    a)减轻脑血肿；
    b)改善脑出血动物的神经行为；
    c)保护脑出血动物的大脑皮层和纹状体神经元；
    d)提高脑出血动物的生存率。
  11. 一种药物组合物用于制备治疗、缓解和/或预防出血性疾病的药物的用途，其特征在于，所述药物组合物包括：
    (a)第一活性成分，所述第一活性成分选自下组：丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸的提取物、或它们的组合；
    (b)第二活性成分，所述第二活性成分选自下组：人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷的提取物、或它们的组合；和
    (c)药学上可接受的载体。
  12. 如权利要求11所述的用途，其特征在于，所述的丹酚酸为丹酚酸B。
  13. 如权利要求11所述的用途，其特征在于，所述的人参皂苷为人参皂苷Rg1。
  14. 如权利要求11所述的用途，其特征在于，所述第一活性成分和第二活性成分的重量比为5:2-1:10。
  15. 一种活性成分用于制备治疗、缓解和/或预防出血性疾病的药物的用途，其特征在于，所述活性成分选自下组：
    (1)丹酚酸、其立体异构体、其晶型、其药学上可接受的盐或酯、或其衍生物、和含丹酚酸的提取物；以及
    (2)人参皂苷、其立体异构体、其晶型、其药学上可接受的盐或酯、或其衍生物、和含人参皂苷的提取物。
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