Application of sulfated carbohydrates in treatment of cerebrovascular diseases

Abstract

The invention provides an application of a sulfated saccharide and / or a material containing the sulfated saccharide in treatment of cerebrovascular diseases. Specifically, the invention provides an application of a sulfated saccharide and / or a material containing the sulfated saccharide, the sulfated saccharide and / or the material containing the sulfated saccharide are / is used for preparing a pharmaceutical composition, and the pharmaceutical composition is used for preventing and / or treating cerebrovascular diseases. The sulfated saccharide and / or the material containing the sulfated saccharide can reach the focus position of the cerebrovascular disease to play a role through different nanocrystallization modes and delivery modes, promote apoptosis of neutrophils, further influence recruitment of macrophages, promote neuronal recovery, improve cognition, recover neural functions and treat the cerebrovascular disease.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to the use of sulfonated sugars in treating cerebrovascular diseases. Background Art

[0002] Cardiovascular and cerebrovascular diseases are one of the leading causes of death and disability worldwide. Ischemic cardiovascular and cerebrovascular diseases cause local blood supply insufficiency due to vascular stenosis, atherosclerosis, infarction, etc., which eventually lead to tissue lesions or necrosis. What is more serious is that the average life expectancy in the world is increasing, and the prevalence of cardiovascular and cerebrovascular diseases is also increasing, which brings greater challenges to the prevention and treatment of cardiovascular and cerebrovascular diseases. Ischemic stroke is one of the more common diseases among cardiovascular and cerebrovascular diseases. Clinically, it can mainly achieve a certain therapeutic effect through mechanical thrombectomy and drug thrombolysis. This treatment method uses recanalizing agents to dissolve thrombi or inhibit thrombus formation and restore blood perfusion in the ischemic area. However, thrombolytic therapy has strict time limits. It can only effectively alleviate damage if it is implemented within the treatment window period. Therefore, it is also greatly limited in clinical treatment.

[0003] In order to cope with the limitations of the window period for treating ischemic stroke, some neuroprotectants have been developed for stroke treatment. However, most neuroprotectants have difficulty in successfully reaching the lesion site through the blood-brain barrier or have poor targeting, making it difficult to reach drug therapeutic concentrations, which seriously affects the effectiveness of the drug. In addition, intravenous injection can also cause systemic toxicity. Due to the limitations of the above-mentioned shortcomings, neuroprotectants have not been widely used in clinical treatment.

[0004] Therefore, there is a need in the art for a new material that can replace traditional neuroprotective agents for the treatment of cerebrovascular diseases. Summary of the invention

[0005] The purpose of the present invention is to provide an application of sulfonated sugars and / or materials containing sulfonated sugars in treating cerebrovascular diseases.

[0006] In a first aspect of the present invention, there is provided a use of sulfonated sugars and / or materials containing sulfonated sugars for preparing a pharmaceutical composition for preventing and / or treating cerebrovascular diseases.

[0007] In another preferred embodiment, the cerebrovascular disease is ischemic cerebrovascular disease.

[0008] In another preferred embodiment, the cerebrovascular disease is selected from the group consisting of transient ischemic attack, stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral arteriosclerosis, cerebral arterial steal syndrome, or a combination thereof.

[0009] In another preferred embodiment, the cerebrovascular disease is selected from the group consisting of transient ischemic attack (TIA), cerebral thrombosis, cerebral embolism, cerebral stroke, cerebral infarction, or a combination thereof.

[0010] In another preferred embodiment, the stroke is selected from the group consisting of ischemic stroke and hemorrhagic stroke.

[0011] In another preferred embodiment, the ischemic stroke is selected from the following group: reversible ischemic neurological deficit (RIND), progressive stroke, and complete stroke.

[0012] In another preferred embodiment, the ischemic stroke is selected from the following group: early stroke and late stroke.

[0013] In another preferred embodiment, the prevention and / or treatment of cerebrovascular disease has one or more characteristics selected from the following group:

[0014] 1. Suppress infarct volume;

[0015] 2. Reduce the release of neutrophil extracellular traps;

[0016] 3. Promote the recruitment of M2 macrophages;

[0017] 4. Improve the patient's movement;

[0018] 5. Increase the number of M2 microglia;

[0019] 6. Inhibit apoptosis of cells in lesion areas;

[0020] 7. Increase the number of neurons and / or protect neurons from damage.

[0021] In another preferred embodiment, the prevention and / or treatment of cerebrovascular diseases also has the characteristic of promoting angiogenesis.

[0022] In another preferred embodiment, the improving the patient's movement includes one or more of increasing the patient's cognition of exploring new things and reducing stray steps.

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

[0024] In another preferred embodiment, the cerebrovascular disease refers to a cerebrovascular disease with nerve damage.

[0025] In another preferred embodiment, the cerebrovascular disease refers to a cerebrovascular disease in which brain neurons are damaged.

[0026] In another preferred embodiment, the cerebrovascular disease refers to a cerebrovascular disease in which motor neurons are damaged.

[0027] In another preferred embodiment, the cerebrovascular disease refers to a cerebrovascular disease with impaired brain function.

[0028] In another preferred embodiment, the sulfonated sugars include sulfonated oligosaccharides and sulfonated polysaccharides.

[0029] In another preferred embodiment, the sulfonated sugar is selected from the group consisting of sulfonated chitosan, sulfonated chito-oligosaccharide, sulfonated dextran, heparin sulfate, cellulose sulfate, chondroitin sulfate, or a combination thereof.

[0030] In another preferred embodiment, the sulfonated sugar is sulfonated chitosan.

[0031] In another preferred embodiment, the weight average molecular weight of the sulfonated sugar is 5,000 to 100,000, preferably 20,000 to 80,000.

[0032] In another preferred embodiment, the weight average molecular weight of the sulfonated polysaccharide is 10,000 to 100,000, preferably 20,000 to 80,000.

[0033] In another preferred embodiment, the weight average molecular weight of the sulfonated oligosaccharide is 5000-10,000, preferably 7000-8000.

[0034] In another preferred embodiment, the sulfur content of the sulfonated sugar is 5%-20%, preferably 10%-20%.

[0035] In another preferred embodiment, the method for preparing the sulfonated sugar comprises the following steps:

[0036] Under an inert gas atmosphere and in the presence of an organic solvent, a sulfonating reagent is prepared by using chlorosulfonic acid and DMF, and then mixed with a sugar solution to react and obtain sulfonated sugars.

[0037] In another preferred embodiment, the method for preparing the sulfonated sugar comprises the following steps:

[0038] (a1) mixing chlorosulfonic acid and DMF to prepare a sulfonating reagent solution;

[0039] (a2) dissolving sugar in a mixed solution of formamide and formic acid to prepare a sugar solution;

[0040] (a3) Under an inert gas atmosphere, a sulfonation reagent solution and a sugar solution are mixed to react to obtain sulfonated sugars.

[0041] In another preferred embodiment, the method for preparing the sulfonated sugar comprises the following steps:

[0042] (a1) 5 mL of chlorosulfonic acid was added dropwise to 50 mL of DMF and reacted for 30 min to prepare a sulfonation reagent solution;

[0043] (a2) dissolving 2.5 g of sugar in a mixed solution of 50 mL of formamide and 2 mL of formic acid, stirring and mixing to prepare a sugar solution;

[0044] (a3) under an inert gas atmosphere, adding the sulfonation reagent solution prepared in step (a1) to the sugar solution, reacting at 45-55° C. for 2 h to obtain a reaction mixture;

[0045] (a4) adding 500-1000 mL of anhydrous ethanol to the reaction mixture in step (a3) ​​to precipitate the product, repeatedly washing with anhydrous ethanol at least 3 times, and finally dialysis post-treatment to obtain the final product.

[0046] In another preferred embodiment, the material containing sulfonated sugars is selected from the following group: self-assembled nanoparticles containing sulfonated sugars, liposome microspheres containing sulfonated sugars, hydrogels containing sulfonated sugars, or a combination thereof.

[0047] In another preferred embodiment, the content of sulfonated sugar in the sulfonated sugar-containing material exceeds 1wt%, preferably exceeds 2wt%, more preferably exceeds 10wt%, for example 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%.

[0048] In another preferred embodiment, the size of the self-assembled nano-microspheres containing sulfonated sugars is less than 500 nm, preferably less than 300 nm, such as 80-120 nm.

[0049] In another preferred embodiment, in the self-assembled nano-microspheres containing sulfonated sugars, the mass fraction of the sulfonated sugars is greater than 8 wt %, preferably greater than 10 wt %, for example 13.6%.

[0050] In another preferred embodiment, the self-assembled nano-microspheres containing sulfonated sugars are obtained by coupling sulfonated sugars with hydrophobic molecules using a coupling agent to obtain amphiphilic molecules, which are then self-assembled.

[0051] In another preferred embodiment, the hydrophobic molecule is selected from the following group: oleic acid, L-lactic acid, or a combination thereof.

[0052] In another preferred embodiment, the coupling agent is a coupling agent for carboxyl groups and hydroxyl groups and / or amino groups.

[0053] In another preferred embodiment, the coupling agent is selected from the following group: EDC, DCC, or a combination thereof.

[0054] In another preferred embodiment, the self-assembled nanoparticles containing sulfonated sugars are prepared by the following method, which comprises the following steps:

[0055] (b1) providing sulfonated sugars and hydrophobic molecules;

[0056] (b2) adding the sulfonated sugar solution dropwise into the hydrophobic molecule solution, and then adding a coupling agent to perform a coupling self-assembly reaction;

[0057] (b3) dialyzing the reaction mixture obtained in step (b2), and post-treating to obtain the self-assembled nanospheres.

[0058] In another preferred embodiment, the sulfonated sugar is dissolved in water to prepare a sulfonated sugar solution.

[0059] In another preferred embodiment, the water is ultrapure water.

[0060] In another preferred embodiment, in the sulfonated sugar solution, the concentration of the sulfonated sugar is 100-500 mg / mL, for example, 250 mg / mL.

[0061] In another preferred embodiment, the hydrophobic molecule is dissolved in DMF to prepare the hydrophobic molecule solution.

[0062] In another preferred embodiment, in the hydrophobic molecule solution, the volume concentration of the hydrophobic molecules is 0.5-5%, for example, 1%.

[0063] In another preferred embodiment, the volume ratio of the sulfonated sugar solution to the hydrophobic molecule solution is 1:1-5, for example, 1:1.5.

[0064] In another preferred embodiment, the self-assembled nanospheres containing sulfonated sugars are prepared by the following method, which includes the following steps: adding the sulfonated sugar solution dropwise into the oleic acid solution, adding 3.4 mL of 95% EDC solution after sufficient stirring, reacting for 24 hours, and finally dialyzing the supernatant with a 3000Da dialysis bag for 7 days, and obtaining the final product after freeze-drying.

[0065] In another preferred embodiment, the size of the liposome microspheres containing sulfonated sugars is less than 500 nm, preferably less than 300 nm, such as 80-120 nm.

[0066] In another preferred embodiment, in the liposome microspheres containing sulfonated sugars, the mass fraction of the sulfonated sugars is greater than 20 wt %, preferably greater than 30 wt %, for example 32.6%.

[0067] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are modified liposomes for delivering sulfonated sugars, and the modified substances used are selected from the following group: one or more of neutrophil membranes, erythrocyte membranes, and homing peptides.

[0068] In another preferred embodiment, the lipid components of the liposome microspheres containing sulfonated sugars are selected from the following group: cholesterol, phosphatidylcholine, phosphatidylserine, phosphatidylcholine, or a combination thereof.

[0069] In another preferred embodiment, the lipid components of the liposome microspheres containing sulfonated sugars are a combination of cholesterol, phosphatidylcholine and phosphatidylserine.

[0070] In another preferred embodiment, the molar ratio of cholesterol, lecithin and phosphatidylserine is 0.8-1.2:0.8-1.2:1-1.5, for example, 1:1:1.33.

[0071] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are assembled by liposome components at the interface of an organic solvent, and then the organic solvent is volatilized to obtain a liposome film, and then a sulfonated sugar aqueous solution is added to obtain the liposome microspheres containing sulfonated sugars.

[0072] In another preferred embodiment, the concentration of the sulfonated sugar in the sulfonated sugar aqueous solution is 10 mg / mL.

[0073] In another preferred embodiment, the organic solvent is a low boiling point organic solvent selected from the following group: tetrahydrofuran, dichloromethane, or a combination thereof.

[0074] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are prepared by the following method, which comprises the following steps:

[0075] (c1) dissolving the liposome components in an organic solvent and volatilizing the organic solvent to obtain a liposome film;

[0076] (c2) mixing the liposome film obtained in step (c1) with an aqueous solution of sulfonated sugar, and hydrating the mixture to obtain the liposome microspheres containing the sulfonated sugar.

[0077] In another preferred embodiment, the method further comprises the steps of:

[0078] (c3) mixing the liposome microspheres containing the sulfonated sugar with the neutrophil membrane and sonicating them to obtain the sulfonated chitosan liposome microspheres embedded with the neutrophil membrane.

[0079] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are prepared by the following method, which comprises the following steps:

[0080] dissolving cholesterol, lecithin and phosphatidylserine in dichloromethane at a molar ratio of 1:1:1.33, volatilizing the dichloromethane by rotary evaporation to obtain a thin film, and adding a 10 mg / mL sulfonated sugar aqueous solution to obtain liposome microspheres containing the sulfonated sugar; and

[0081] Optional step: HL-60 cells were centrifuged and resuspended in cell buffer (cell density 5×10 6 / mL), the neutrophil membrane was obtained by cell disruptor ultrasound, and after freeze-drying, it was mixed with 1.5mL of liposome microspheres containing sulfonated sugars and ultrasounded to finally obtain sulfonated chitosan liposome microspheres embedded with neutrophil membranes.

[0082] In another preferred embodiment, in the hydrogel containing sulfonated sugars, the mass fraction of the sulfonated sugars is greater than 0.5 wt %, preferably greater than 1 wt %, for example 2%.

[0083] In another preferred embodiment, the hydrogel component in the hydrogel containing sulfonated sugars is an ingredient or a polymerization product thereof selected from the following group: collagen, gelatin, chitosan, alginate, hyaluronic acid, or a combination thereof.

[0084] In another preferred embodiment, the hydrogel containing sulfonated sugars is methacryloyl gelatin.

[0085] In another preferred embodiment, the hydrogel containing sulfonated sugars is a hydrogel using gelatin as a skeleton and modified with methacrylic anhydride.

[0086] In another preferred embodiment, the hydrogel containing sulfonated sugars is prepared by the following method, which includes the following steps: preparing a hydrogel component solution, then adding a modifier dropwise to react, obtaining a hydrogel after dialysis and post-treatment, then adding the freeze-dried hydrogel and the sulfonated sugars into a buffer solution, and obtaining the hydrogel containing sulfonated sugars after UV crosslinking.

[0087] In another preferred embodiment, the concentration of the hydrogel component solution is 5-20%, such as 10%.

[0088] In another preferred embodiment, the modifier is methacrylic anhydride.

[0089] In another preferred embodiment, the buffer is PBS buffer.

[0090] In another preferred embodiment, the hydrogel containing sulfonated sugars is prepared by the following method, which includes the following steps: firstly reacting gelatin with methacrylic anhydride and then freeze-drying to obtain GelMA, then preparing a 10% GelMA hydrogel and adding SCS, and obtaining the hydrogel containing sulfonated sugars after UV crosslinking.

[0091] In another preferred embodiment, the hydrogel containing sulfonated sugars is prepared by the following method, which comprises the following steps: preparing 10% gelatin solution with 5 g of gelatin, adding 5 mL of methacrylic anhydride dropwise, reacting at 60° C. for 2 h, adding 200 mL of PBS to terminate the reaction, dialyzing and freeze-drying to obtain the final hydrogel, dissolving the freeze-dried GelMA and SCS in PBS at concentrations of 10% and 0.2%, respectively, and obtaining a methacryloyl gelatin hydrogel containing sulfonated sugars through UV crosslinking.

[0092] In another preferred embodiment, the dosage form of the pharmaceutical composition is a solution, a suspension, a granule, an injection, a microsphere, a gel, a tablet, or a capsule.

[0093] In another preferred embodiment, the pharmaceutical composition is administered by injection.

[0094] In another preferred embodiment, the pharmaceutical composition is administered by in situ injection at the lesion site, intravenous injection, orbital injection, or intramuscular injection.

[0095] In another preferred embodiment, the dosage of the sulfonated sugar is 1 μg to 1000 μg.

[0096] In another preferred embodiment, in the pharmaceutical composition, the sulfonated sugar accounts for 1 wt% to 99 wt% of the total weight of the pharmaceutical composition.

[0097] In another preferred embodiment, the drug further comprises a pharmaceutically acceptable excipient, and the excipient is selected from: one or more of a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent, an antioxidant, a carbohydrate, a fat, a vitamin, an amino acid, a trace element or a protein.

[0098] In a second aspect of the present invention, a method for preventing and / or treating cerebrovascular diseases is provided, the method comprising: administering sulfonated sugars and / or materials containing sulfonated sugars to a subject in need thereof.

[0099] In another preferred embodiment, the subject is a human or non-human mammal.

[0100] In a third aspect of the present invention, a self-assembled nanoparticle containing sulfonated sugar is provided. The self-assembled nanoparticle containing sulfonated sugar is obtained by coupling sulfonated sugar with hydrophobic molecules through a coupling agent to obtain amphiphilic molecules, and then self-assembling.

[0101] Wherein, the hydrophobic molecule is selected from the following group: oleic acid, L-lactic acid, or a combination thereof.

[0102] In another preferred embodiment, the self-assembled nanoparticles containing sulfonated sugars are as described in the first aspect of the present invention.

[0103] In another preferred embodiment, the sulfonated sugar is as described in the first aspect of the present invention.

[0104] In the fourth aspect of the present invention, a liposome microsphere containing sulfonated sugars is provided. The liposome microsphere containing sulfonated sugars is a modified liposome for delivering sulfonated sugars, and the modified substance used is selected from the following group: one or more of neutrophil membrane, red blood cell membrane, and homing peptide.

[0105] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are as described in the first aspect of the present invention.

[0106] In another preferred embodiment, the sulfonated sugar is as described in the first aspect of the present invention.

[0107] In the fifth aspect of the present invention, a liposome microsphere containing sulfonated sugar is provided. The liposome microsphere containing sulfonated sugar is assembled by liposome components at the interface of an organic solvent, and then the organic solvent is volatilized to obtain a liposome film, which is then added with a sulfonated sugar aqueous solution.

[0108] In another preferred embodiment, the liposome microspheres containing sulfonated sugars are as described in the first aspect of the present invention.

[0109] In another preferred embodiment, the sulfonated sugar is as described in the first aspect of the present invention.

[0110] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 The infrared signature of sulfonated chitosan is shown.

[0112] Figure 2 Shown are the immunofluorescence images of neutrophil extracellular traps after the action of different polysaccharide materials.

[0113] Figure 3 The infrared characteristics of glycerol-sulfonated chitosan microspheres are shown.

[0114] Figure 4 The particle size of glycerol-sulfonated chitosan microspheres measured by dynamic light scattering is shown.

[0115] Figure 5Transmission electron micrographs of neutrophil membrane-encrusted sulfonated chitosan liposome microspheres are shown.

[0116] Figure 6 It shows that FITC-grafted sulfonated chitosan is encapsulated in the microspheres embedded with cell membrane liposomes.

[0117] Figure 7 The infrared characteristic images and nuclear magnetic resonance hydrogen spectrum of GelMA hydrogel are shown.

[0118] Figure 8 Shown are typical images of TTC staining of brain tissue 1, 3, and 7 days after direct injection of sulfonated chitosan.

[0119] Fig. 9 Shown are H&E-stained sections of brain tissue 1, 3, and 7 days after direct injection of sulfonated chitosan, with yellow arrows representing blood vessels.

[0120] Fig.10 Shown are immunofluorescence images of brain tissues 1, 3, and 7 days after direct injection of sulfonated chitosan, with green representing neutrophils and red representing neutrophil extracellular traps.

[0121] Fig.11 Shown are immunofluorescence images of brain tissues 1, 3, and 7 days after direct injection of sulfonated chitosan, with green representing macrophages.

[0122] Fig.12 Shown are typical flow cytometry images and quantitative images of macrophage polarization in brain tissue 1, 3, and 7 days after direct injection of sulfonated chitosan.

[0123] Fig.13 Shown are typical images of the open field test in mice 1, 3, and 7 days after direct injection of sulfonated chitosan, as well as quantitative graphs of movement distance and movement time in the middle area.

[0124] Fig.14 Shown are typical images of TTC staining of brain tissue 1, 3, and 7 days after injection of sulfonated chitosan microspheres.

[0125] Fig.15 Shown are representative flow cytometric images of microglial polarization in infarcted brain 1, 3, and 7 days after direct injection of sulfonated chitosan microspheres.

[0126] Fig.16 Shown are typical images of TTC staining 1, 3, and 7 days after injection of neutrophil-encrusted sulfonated chitosan liposome microspheres.

[0127] Fig.17 Shown are immunofluorescence images of brain tissues 1, 3, and 7 days after injection of neutrophil-encrusted sulfonated chitosan liposome microspheres, with green representing neuronal cells.

[0128] Fig.18 Shown are immunofluorescence images of brain tissues 1, 3, and 7 days after injection of neutrophil-encrusted sulfonated chitosan liposome microspheres, with red representing apoptotic cells.

[0129] Fig.19 Quantitative graphs of staggered analysis 3 and 10 days after intracranial injection of sulfonated chitosan-containing hydrogels are shown.

[0130] Fig. 20 Shown are the quantitative graphs of the balance beam analysis 3 and 10 days after intracranial injection of sulfonated chitosan containing hydrogels. DETAILED DESCRIPTION

[0131] After extensive and in-depth research, the inventors discovered for the first time that sulfonated sugars can promote / repair nerves, thereby treating cerebrovascular diseases caused by nerve damage.

[0132] The experimental study of the present invention shows that in the transient middle cerebral artery occlusion reperfusion model, sulfonated sugars and materials containing sulfonated sugars are injected into the lesion site through the orbit and can slow down the expansion of the infarct volume; histological evaluation found that the material can regulate immune response and promote neural recovery; behavioral evaluation found that the material can improve neural damage and cognitive function, and has a good therapeutic effect.

[0133] This new material is expected to be used clinically to treat cerebrovascular diseases, which are becoming more and more common in an aging society. Based on this, the present invention was completed.

[0134] the term

[0135] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0136] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0137] Sulfonated sugars

[0138] Sulfonated sugars are products obtained by sulfonation of sugars.

[0139] Sulfonation is a reaction process that introduces sulfonic acid groups (-SO3H) or sulfonyl chloride groups (-SO3Cl) into organic molecules. The sulfonating agents used are usually sulfur trioxide, concentrated sulfuric acid, fuming sulfuric acid, etc. Chlorosulfonic acid, sulfur dioxide plus chlorine, sulfur dioxide plus oxygen, and sodium sulfite are sometimes used as sulfonating agents. Sulfonation methods include liquid phase sulfonation and gas phase sulfonation. The sulfonation process in which the sulfonic acid group replaces the hydrogen on the carbon atom is called direct sulfonation; the sulfonic acid group replaces the halogen or nitro group on the carbon atom is called indirect sulfonation.

[0140] The sulfonated carbohydrate in the present invention is selected from one or more of sulfonated chitosan, sulfonated chito-oligosaccharide, sulfonated dextran, heparin, heparin sulfate, and cellulose sulfate. It is prepared by sulfonating chitosan, dextran, etc. as raw materials using methods known in the art. Preferably, the sulfonated carbohydrate in the present invention is sulfonated chitosan.

[0141] The molecular weight of the sulfonated sugar of the present invention is 5000-350000Da, and the sulfur content is 5%-30%. In another preferred embodiment, the molecular weight of the sulfonated sugar is 5000-300000Da, and the sulfur content is 1%-20%.

[0142] use

[0143] The present invention provides a use of sulfonated sugars and / or materials containing sulfonated sugars for preparing a pharmaceutical composition for preventing and / or treating cerebrovascular diseases.

[0144] In some embodiments, the pharmaceutical composition is used to treat cerebrovascular diseases such as lower limb ischemia and ischemic stroke. In some embodiments, the pharmaceutical composition is used to treat diseases related to nerve damage caused by ischemia and improve cognitive function. In some embodiments, the pharmaceutical composition has the effects of regulating immunity, reducing inflammation and promoting angiogenesis in cerebral blood vessels. In some embodiments, the drug combination is used to regulate the inflammatory response caused by various cerebrovascular diseases and neurodegenerative diseases. In some embodiments, the drug combination is used to regulate the behavioral changes of neutrophils, microglia, and astrocytes.

[0145] The sulfonated sugars and / or materials containing sulfonated sugars of the present invention can reach the lesions of cerebrovascular diseases through different nano-formulation methods and delivery methods to exert their effects. The sulfonated sugars of the present invention have the functions of promoting apoptosis of neutrophils and thus affecting the recruitment and polarization of macrophages, promoting angiogenesis, promoting neuronal recovery and improving cognition in ischemic microenvironment cerebrovascular diseases, which are beneficial to alleviating the deterioration of neurodegenerative diseases and have the effect of treating neuroinflammation.

[0146] The research results of the present invention show that sulfonated sugars as active molecules can reduce the infarct volume after stroke reperfusion and improve nerve damage, and are expected to be applied to the clinical treatment of cerebrovascular diseases such as cerebral infarction and stroke.

[0147] Compositions and methods of administration

[0148] The pharmaceutical composition of the present invention comprises a safe and effective amount of sulfonated sugars and / or materials containing sulfonated sugars, and a pharmaceutically acceptable excipient or carrier. Generally, the composition of the present invention contains 1-2000 μg / dose of sulfonated sugars, and more preferably, contains 10-500 μg / dose of sulfonated sugars. Preferably, the "one dose" is one dose of injection.

[0149] "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 sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

[0150] The administration method of the active ingredient or composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0151] Compositions for parenteral injection may include 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.

[0152] The active ingredients of the present invention can be administered alone or in combination with other therapeutic agents. In another preferred embodiment, the composition further comprises one or more other therapeutic agents. In certain embodiments, the active ingredients of the present invention are used simultaneously with other agents as part of a combined treatment regimen in the same or separate formulations, or are used sequentially with the other agents.

[0153] The general range of the therapeutically effective dose of the composition of the active ingredient of the present invention will be: about 1-200 μg / day, about 1-about 100 μg / day, about 1-about 50 μg / day, about 1-about 25 μg / day, about 1-about 10 μg / day, or about 1-about 5 μg / day. The therapeutically effective dose will be given in one or more doses. However, it should be understood that the specific dose of the compound of the present invention for any particular patient will depend on a variety of factors, for example, the age, sex, body weight, general health, diet, individual response of the patient to be treated, the severity of the disease to be treated, the activity of the specific compound administered, dosage form, application mode and concomitant medication. The therapeutically effective amount of a given situation can be determined by routine experiments and is within the scope of the clinician or physician's ability and judgment. In any case, the active ingredient or composition will be given in multiple doses based on the individual situation of the subject of administration and in a manner that allows the delivery of a therapeutically effective amount.

[0154] Examples of subjects to which the active ingredient or composition of the present invention is administered include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).

[0155] The main advantages of the present invention include:

[0156] The sulfonated sugars and / or materials containing sulfonated sugars of the present invention can achieve immune regulation of cerebrovascular diseases, protection of neurons, and improvement of cognition and movement through their own biological effects without adding factors or cells. High doses of sulfonated sugars can be delivered to the lesion site through different delivery methods to achieve the best therapeutic effect. The therapeutic effect is mainly reflected in the inhibition of neutrophil infiltration / promotion of neutrophil apoptosis while recruiting more M2 macrophages; changes in the microenvironment can protect neuronal cells from damage, thereby achieving improvement in cognition and recovery of motor function.

[0157] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0158] Example 1

[0159] Preparation of sulfonated sugar (SCS):

[0160] In an anhydrous and oxygen-free environment and an ice bath, 5 mL of HClSO3 was added dropwise to 50 mL of N, N-dimethylformamide (DMF), and the reaction was continued for 30 min to obtain a sulfonated reagent for standby use; 2.5 g of chitosan (Mw≈300,000) was dissolved in a mixed solution of 50 mL of formamide and 2 mL of formic acid to form a uniform and stable system under strong mechanical stirring, and then the prepared sulfonated reagent was added under the protection of argon, and the water bath heating temperature was maintained at 45-55°C and the reaction was continued for 2 h; after the reaction was completed, 500-1000 mL of anhydrous ethanol was added to precipitate the product, which was repeatedly washed with anhydrous ethanol for at least 3 times, and finally dialyzed and freeze-dried to obtain the final sulfonated chitosan as the sulfonated sugar in Example 1.

[0161] Figure 1 The infrared characteristic graph of the SCS prepared in Example 1 is shown, and Table 1 shows the physical properties of the SCS.

[0162] Table 1 Physical properties of SCS

[0163] sample Number average molecular weight (Da) Weight average molecular weight (Da) Sulfur content (%) SCS 42000 47600 12.99

[0164] Example 2

[0165] Regulatory effects of sugars modified with different groups on neutrophils:

[0166] After the neutrophils were plated, they were stimulated with 1 μg / mL LPS for 12 hours to simulate the inflammatory microenvironment, and 4 μg / mL of sulfonated sugar (SCS), heparin (HEP), chitosan quaternary ammonium salt (HACC) and carboxylated chitosan (CCS) were added respectively and cultured for a further 24 hours.

[0167] Figure 2 The amount of neutrophil extracellular traps released after neutrophils were treated with different sugars is shown.

[0168] It can be seen that the release of neutrophil extracellular traps from quaternary ammonium salts of chitosan and carboxylated chitosan is high, indicating that quaternary ammonium salts of sugars and carboxylated sugars are not suitable as therapeutic drugs, while sulfonated sugars have excellent effects.

[0169] Example 3

[0170] Synthesis of glycerol-sulfonated sugar microspheres (SCSNPs):

[0171] The SCS solution was added dropwise to the oleic acid solution, and after sufficient stirring, 3.4 mL of 95% EDC solution was added dropwise and reacted for 24 h. Finally, the supernatant was dialyzed with a 3000 Da dialysis bag for 7 days and the final product was obtained after freeze-drying.

[0172] Figure 3 The infrared characteristic image of glycerol-sulfonated sugar microspheres is shown. Figure 4The particle size of glycerol-sulfonated sugar microspheres is shown.

[0173] It can be seen that glycerol-sulfonated sugar microspheres were successfully prepared, and the size of the microspheres was about 80-120 nm.

[0174] Example 4

[0175] Neutrophil membrane-embedded sulfonated glycoliposome microspheres (MLS)

[0176] Cholesterol, phosphatidylcholine and phosphatidylserine were dissolved in dichloromethane at a molar ratio of 1:1:1.33, and the dichloromethane was evaporated by rotary evaporation to obtain a thin film, and 10 mg / mL SCS aqueous solution was added to hydrate to obtain liposome microspheres containing SCS. HL-60 cells were centrifuged and resuspended in cell buffer (cell density 5×10 6 / mL), the neutrophil membrane was obtained by ultrasonication of a cell disruptor, and after freeze-drying, it was mixed with 1.5mL of liposome microspheres containing SCS and ultrasonicated to finally obtain sulfonated sugar liposome microspheres embedded with neutrophil membranes.

[0177] Figure 5 It is a transmission electron micrograph of sulfonated glycoliposome microspheres embedded in the membrane of neutrophils, indicating that sulfonated glycoliposome microspheres embedded in the membrane of neutrophils were successfully synthesized. Figure 6 Flow cytometry data showed that most of the SCS were encapsulated in the microspheres.

[0178] Example 5

[0179] Synthesis of methacryloyl gelatin (GelMA) containing sulfonated sugars:

[0180] 5 g of gelatin was prepared into a 10% gelatin solution, 5 mL of methacrylic anhydride was added dropwise, reacted at 60°C for 2 h, 200 mL of PBS was added to terminate the reaction, and the final product was obtained by dialysis and freeze-drying. The freeze-dried GelMA and SCS were dissolved in PBS at concentrations of 10% and 0.2%, respectively, and methacryloyl gelatin hydrogel containing sulfonated sugar was obtained by UV cross-linking.

[0181] Figure 7 The infrared characteristic images and nuclear magnetic resonance hydrogen spectrum of GelMA hydrogel are shown.

[0182] Example 6

[0183] A transient middle cerebral artery occlusion-reperfusion model was constructed. Immediately after reperfusion, 150 μL of SCS was injected into the orbits of the rats. After 1 day, 3 days, or 7 days, part of the brain tissue was removed for histological analysis or flow cytometry, and part for behavioral evaluation.

[0184] Figure 8Shown is 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain sections after SCS injection. Figure 8 It can be seen that SCS can significantly inhibit infarct volume and help curb the progression of stroke; Fig. 9 The H&E-stained sections shown indicate that nuclear deformities and nuclear chromatin edge aggregation were suppressed after SCS injection; Figure 10-11 Immunofluorescence showed that SCS injection reduced neutrophil infiltration and the release of neutrophil extracellular traps (NETs), but promoted macrophage recruitment. Fig.12 Flow cytometry data showed that most of the recruited macrophages were M2 macrophages, which are beneficial to tissue repair. Fig.13 This is the movement trajectory diagram of the open field experiment. It can be seen from the figure that SCS can improve movement and increase its cognition of exploring new things. The recovery of cognitive function is closely related to the connection between neurons, so SCS can protect / repair neurons.

[0185] This example shows that the injection of SCS in the early stage of ischemia-reperfusion is beneficial to inhibit the infiltration of neutrophils, but can recruit more M2 macrophages to promote tissue repair. At the same time, the change of microenvironment is beneficial to protect / repair neurons, thereby improving the cognitive ability of mice.

[0186] Example 7

[0187] A transient middle cerebral artery occlusion-reperfusion model was established. Immediately after reperfusion, 100 μL of glycerol-sulfonated sugar microspheres (SCSNP) were injected intraorbitally. After 1, 3 or 7 days of feeding, brain tissues were removed for histological analysis or flow cytometry.

[0188] Fig.14 The figure shows 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain slices after SCSNP injection. As can be seen from the figure, compared with the PBS group, the cerebral infarction volume at different time points in the SCSNP group was significantly reduced, indicating that SCSNP is beneficial to curb the deterioration of stroke; Fig.15 Flow cytometry data showed that under the action of SCSNP, the number of M2 phenotype microglia in the infarcted hemisphere increased significantly at 1 and 3 days, while more microglia tended to be steady-state at 7 days, indicating that SCSNP can regulate the inflammatory response process of microglia and thus improve the course of stroke.

[0189] This example shows that the injection of SCSNP in the early stage of ischemia-reperfusion is beneficial to regulate the phenotype of microglia, promote their polarization to the M2 phenotype, and accelerate the return of microglia to homeostasis after stroke, thereby promoting tissue repair.

[0190] Example 8

[0191] A transient middle cerebral artery occlusion-reperfusion model was established. The mice were fed for three days after reperfusion and then injected with 150 μL of neutrophil membrane-embedded sulfonated glycoliposome microspheres (MLS) via the orbital cavity. The mice were fed for 3, 7 or 14 days, and the brain tissues were removed for histological analysis.

[0192] Fig.16 Shown is TTC staining of brain sections after SCS injection. Fig.16 It can be seen that sulfonated glycoliposome microspheres embedded with neutrophil membrane can significantly reduce the infarct volume and are beneficial to the treatment of stroke; Figure 17-18 Immunofluorescence staining showed that MLS administration was beneficial to the increase of neuron number and inhibited the apoptosis of cells in the lesion site.

[0193] This example shows that the injection of MLS in the late stage of ischemia-reperfusion is beneficial to the recovery of stroke. The reduction of infarct volume can be clearly seen through TTC staining. Immunofluorescence staining proves that MLS is beneficial to the recovery of brain function, protects neurons from damage, and can also inhibit cell apoptosis, which is helpful for the recovery of stroke.

[0194] Example 9

[0195] A transient middle cerebral artery occlusion-reperfusion model was constructed. The mice were fed for five days after reperfusion, the skin was cut open, and 6 μL of 10% GelMA hydrogel (containing 0.1% LAP initiator) containing 1.2 μg SCS was directly injected through the skull. After UV cross-linking, the skin was sutured and the mice were fed for 3 and 10 days for behavioral evaluation.

[0196] Fig.19 and Fig. 20 The rows are foot-fault test and bean walking analysis. The data show that the addition of SCS is beneficial to reducing the number of times the mouse's feet fall off the grid and reducing the time to pass the balance beam, indicating that the mouse's motor ability has improved.

[0197] This example shows that the injection of a hydrogel containing SCS in the late stage of ischemia-reperfusion is also beneficial for the treatment of ischemic stroke, especially in the improvement of motor function in mice, which further indicates that the neurological function of mice has recovered.

[0198] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of sulfonated sugars and / or materials containing sulfonated sugars, characterized in that: Used for preparing a pharmaceutical composition, which is used for preventing and / or treating cerebrovascular diseases.

2. The use according to claim 1, characterized in that The cerebrovascular disease is selected from the group consisting of transient ischemic attack, cerebral stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, cerebral arteriosclerosis, cerebral arterial steal syndrome, or a combination thereof.

3. The use according to claim 1, characterized in that The prevention and / or treatment of cerebrovascular disease has one or more characteristics selected from the following group: (1) Suppression of infarct volume; (2) reduce the release of neutrophil extracellular traps; (3) Promote the recruitment of M2 macrophages; (4) Improve the patient's movement; (5) Increase the number of M2 microglia; (6) Inhibit apoptosis of cells in the lesion site; (7) Increase the number of neurons and / or protect neurons from damage.

4. The use according to claim 1, characterized in that The cerebrovascular disease refers to a cerebrovascular disease in which nerves are damaged.

5. The use according to claim 1, characterized in that The sulfonated sugar is selected from the group consisting of sulfonated chitosan, sulfonated chito-oligosaccharide, sulfonated dextran, heparin sulfate, cellulose sulfate, chondroitin sulfate, or a combination thereof.

6. The use according to claim 1, characterized in that The sulfonated sugars have one or more characteristics selected from the group consisting of: (1) The weight average molecular weight of the sulfonated sugar is 5,000 to 100,000, preferably 20,000 to 80,000; (2) The sulfur content of the sulfonated sugar is 5%-20%, preferably 10%-20%.

7. The use according to claim 1, characterized in that The material containing sulfonated sugar is selected from the group consisting of self-assembled nanoparticles containing sulfonated sugar, liposome microparticles containing sulfonated sugar, hydrogels containing sulfonated sugar, or a combination thereof.

8. The use according to claim 1, characterized in that The dosage form of the pharmaceutical composition is solution, suspension, granule, injection, microsphere, gel, tablet, capsule.

9. A self-assembled nanoparticle containing sulfonated sugars, characterized in that: The self-assembled nano-microspheres containing sulfonated sugars are obtained by coupling sulfonated sugars with hydrophobic molecules using a coupling agent to obtain amphiphilic molecules, which are then self-assembled. Wherein, the hydrophobic molecule is selected from the following group: oleic acid, L-lactic acid, or a combination thereof.

10. A liposome microsphere containing sulfonated sugars, characterized in that: The liposome microspheres containing sulfonated sugars are modified liposomes for delivering sulfonated sugars, and the modified substances used are selected from the following group: one or more of neutrophil membranes, erythrocyte membranes, and homing peptides.

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