Exosome composition containing baicalein or derivative thereof

Abstract

The invention relates to an application of an exosome composition containing baicalein or a derivative thereof in treatment of nasal-nasosinusitis, the baicalein and the derivative thereof can achieve a synergistic treatment effect, part or all of the baicalein or the derivative thereof in the exosome composition is loaded in exosome, and the nasal-nasosinusitis especially eosinophilic chronic nasosinusitis is treated. The exosome is used for loading the baicalein or the derivative thereof, so that the solubility of the baicalein or the derivative thereof can be improved, the in-vivo circulation time of the baicalein or the derivative thereof can be prolonged, the therapeutic activity of the medicine can be retained, and the infiltration capacity of the baicalein or the derivative thereof in the nasal mucosa can be enhanced, which is a particularly preferable embodiment of the invention.

Description

[0001] This application is a divisional application of Chinese patent application 202311227597.9, filed on September 22, 2023, with the invention name “An exosome composition comprising baicalein or its derivatives and its use in the treatment of rhinitis”. Technical Field

[0002] The present invention relates to the field of medical technology, and in particular to an exosome composition comprising baicalein or a derivative thereof and an application of the exosome composition, especially an application in treating rhinitis. Background Art

[0003] The anatomical structures of the nasal cavity and paranasal sinuses are interrelated. Therefore, in clinical practice, although rhinitis and sinusitis are distinguished, they are often interrelated and usually occur at the same time. After the release of the evidence-based diagnosis and treatment guidelines for sinusitis and nasal polyps developed by the European Society of Allergy and Clinical Immunology in 2007 based on the method of evidence-based medicine, rhinitis and sinusitis are collectively referred to as rhinosinusitis (including nasal polyps) in clinical practice at home and abroad, which refers to inflammation of the nasal cavity and paranasal sinuses, manifested as two or more clinical features of nasal and paranasal sinus inflammation, including at least one of nasal obstruction / congestion and / or rhinorrhea (front and back rhinorrhea), with or without facial pressure pain / swelling, with or without hyposmia / loss of smell; nasal endoscopy reveals nasal polyps and / or mucopurulent secretions from the middle nasal meatus and / or swelling or obstruction of the middle nasal meatus mucosa; nasal CT scan reveals changes in the ostiomeatal complex or mucosa in the sinuses. It is divided into acute and chronic according to the duration of symptoms. Acute rhinosinusitis refers to symptoms that last for less than 12 weeks and can be completely relieved, while chronic rhinosinusitis refers to symptoms that last for more than 12 weeks and are not completely relieved or may worsen. The 2020 European Sinusitis and Nasal Polyps Opinion divides chronic sinusitis into type 1 (Th1 type), type 2 (Th2 type) and type 3 (Th17 type) according to different immune inflammation types. Type 2 is also commonly referred to as eosinophilic chronic sinusitis in clinical practice, and types 1 and 3 are also called non-type 2, which is non-eosinophilic chronic sinusitis.

[0004] Although the current routine clinical treatment for chronic sinusitis is still the topical use of corticosteroids as the first-line treatment, and there is no more precise symptomatic treatment for different subtypes, since chronic sinusitis subtypes have different immune responses and inflammatory characteristics, such as type 2 inflammation and non-type 2 inflammation, the efficacy of these treatments varies greatly, causing many patients who are insensitive to the response to treatments that are actually ineffective.

[0005] Baicalein is one of the flavonoids with the highest content in Scutellaria baicalensis, and baicalin is a glycoside compound formed by the combination of baicalein and a molecule of glucuronic acid. Both exist simultaneously in Scutellaria baicalensis. Baicalein and baicalin have similar pharmacological activities, including anti-inflammatory, antioxidant, and free radical scavenging, and both have extensive biotransformation in the body.

[0006] Similar to mesenchymal stem cells, induced pluripotent stem cells have strong differentiation and regeneration capabilities, but induced pluripotent stem cells have the characteristics of unlimited proliferation and can differentiate into various cell types required by various organs and tissues of the human body. Induced pluripotent stem cells are not directly derived from human organs and tissues, but are a type of stem cells that are edited through genes, so there are no social ethical issues in terms of their source, and induced pluripotent stem cells are easier to obtain than mesenchymal stem cells. Therefore, induced pluripotent stem cell exosomes are easier to mass produce and prepare than mesenchymal stem cell exosomes, and are easier to source.

[0007] Exosomes are extracellular vesicles secreted by cells, which have unique properties such as low immunogenicity, innate stability, high delivery efficiency and the ability to cross the blood-brain barrier, as well as many unique advantages such as tissue repair and anti-inflammation. Exosomes have also received extensive attention and research as nanocarriers for drugs. Summary of the invention

[0008] The present invention relates to an exosome composition containing baicalein or its derivatives and its use in treating rhinosinusitis, wherein rhinosinusitis is especially eosinophilic chronic sinusitis. The use of exosomes to encapsulate baicalein or its derivatives can improve the solubility of baicalein or its derivatives, prolong its in vivo circulation time, retain the drug therapeutic activity, and enhance the permeability of baicalein or its derivatives in the nasal mucosa. The exosome composition containing baicalein or its derivatives of the present invention has a better effect on treating rhinosinusitis than the treatment effect of using exosomes alone, using baicalein or its derivatives alone, and sequentially or simultaneously giving independent exosomes and independent baicalein or its derivatives.

[0009] The first aspect of the present invention is to provide an exosome composition comprising baicalein or a derivative thereof, wherein the exosome composition comprises a combination of baicalein or a derivative thereof and exosomes as an active ingredient. In one embodiment of the present invention, only a portion of the baicalein or a derivative thereof in the exosome composition is loaded in the exosome. In one embodiment of the present invention, all of the baicalein or a derivative thereof in the exosome composition is loaded in the exosome. In the present invention, "loaded in exosomes" includes at least two forms of existence, baicalein or a derivative thereof permeates into the exosome, and baicalein or a derivative thereof is associated with the exosome.

[0010] In some embodiments of the present invention, the loading concentration of baicalein or its derivatives is 2-500 μg / mL, calculated as baicalein, for example, 2.1 μg / mL, 2.5 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 20 0μg / mL, 210μg / mL, 220μg / mL, 230μg / mL, 240μg / mL, 250μg / mL, 260μg / mL, 270μg / mL , 280μg / mL, 290μg / mL, 300μg / mL, 310μg / mL, 320μg / mL, 330μg / mL, 340μg / mL, 350μg / mL, 360 μg / mL, 370 μg / mL, 380 μg / mL, 390 μg / mL, 400 μg / mL, 410 μg / mL, 420 μg / mL, 430 μg / mL, 440 μg / mL, 450 μg / mL, 460 μg / mL, 470 μg / mL, 480 μg / mL, 490 μg / mL, 495 μg / mL, etc. In the present invention, "loading concentration" refers to the concentration of baicalein or its derivatives loaded in exosomes in the exosome composition.

[0011] In some embodiments of the present invention, in the exosome composition, the amount of baicalein or its derivatives loaded in the exosomes accounts for 5%-100% of the total amount of baicalein or its derivatives in the exosome composition, calculated as baicalein, for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc.

[0012] The second aspect of the present invention is to provide the use of the exosome composition containing baicalein or its derivatives in the preparation of a medicament for treating rhinosinusitis;

[0013] Also provided is the use of exosomes and baicalein or its derivatives in preparing a medicine for treating rhinosinusitis.

[0014] The third aspect of the present invention is to provide a method for preparing the exosome composition comprising baicalein or its derivatives.

[0015] In some embodiments of the present invention, the exosome composition comprising baicalein or its derivatives is prepared by incubation, ultrasound, freeze-thaw, extrusion or saponin treatment.

[0016] In one embodiment of the present invention, the incubation method comprises: preparing baicalein or its derivatives into a solution of appropriate concentration, and incubating the solution with the exosomes. In one embodiment of the present invention, an appropriate concentration of baicalein is co-incubated with exosomes; preferably, in the incubation system, the final concentration of baicalein is 5-500 μg / mL (e.g., 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL). mL, 220μg / mL, 230μg / mL, 240μg / mL, 250μg / mL, 260μg / mL, 270μg / mL, 280μg / mL, 290μ g / mL, 300μg / mL, 310μg / mL, 320μg / mL, 330μg / mL, 340μg / mL, 350μg / mL, 360μg / mL, 370 μg / mL, 380μg / mL, 390μg / mL, 400μg / mL, 410μg / mL, 420μg / mL, 430μg / mL, 440μg / mL, 45 0μg / mL, 460μg / mL, 470μg / mL, 480μg / mL, 490μg / mL, 495μg / mL, etc.), for example, it can be 50-300μg / mL,The final concentration of exosomes is 2-3000 μg / mL (e.g., 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL mL, 110μg / mL, 120μg / mL, 130μg / mL, 140μg / mL, 150μg / mL, 160μg / mL, 170μg / mL, 180μg / mL, 190μg / m L, 200μg / mL, 210μg / mL, 220μg / mL, 230μg / mL, 240μg / mL, 250μg / mL, 260μg / mL, 270μg / mL, 280μg / mL, 290μg / mL, 300μg / mL, 350μg / mL, 400μg / mL, 450μg / mL, 500μg / mL, 550μg / mL, 600μg / mL, 650μg / mL, 7 00μg / mL, 750μg / mL, 800μg / mL, 850μg / mL, 900μg / mL, 950μg / mL, 1000μg / mL, 1500μg / mL, 2000μg / mL, 2500μg / mL, 2600μg / mL, 2700μg / mL, 2800μg / mL, 2900μg / mL, 2950μg / mL, 2960μg / mL, 2970μg / mL, 2980μg / mL, 2990μg / mL, 2995μg / mL, etc.), for example, it can be 50-200μg / mL; more preferably, the incubation conditions are 0℃-5℃, 1-25h, or, 20℃-35℃, 1-4h. ,

[0017] In one embodiment of the present invention, the ultrasonic method comprises: mixing an exosome suspension with a solution of baicalein or a derivative thereof, and ultrasonicating the mixed solution using an ultrasonic instrument to obtain the exosome composition containing baicalein or a derivative thereof.

[0018] In one embodiment of the present invention, the freeze-thaw method comprises: mixing the exosome suspension and the baicalein or its derivative solution, freezing them in a refrigerator, then thawing and refreezing them, and repeating the cycle several times to obtain the exosome composition containing baicalein or its derivative.

[0019] The fourth aspect of the present invention is to provide a pharmaceutical composition, which comprises the exosome composition described in the first aspect of the present invention, or further contains a pharmaceutically acceptable excipient. In one embodiment of the present invention, only a portion of baicalin or its derivatives in the pharmaceutical composition are loaded in exosomes. In one embodiment of the present invention, all of the baicalin or its derivatives in the pharmaceutical composition are loaded in exosomes. According to the present invention, the pharmaceutical composition can be in various formulations known in the art, such as: liquid preparations (including but not limited to suspensions), solid preparations (including but not limited to tablets, powder injections, etc.), semi-solid preparations (including but not limited to ointments).

[0020] In one embodiment of the present invention, the pharmaceutical composition is a suspension, which, in addition to containing an exosome composition comprising baicalein or a derivative thereof, may further contain a buffer, and the buffer may be a buffer commonly used in the art, including but not limited to PBS buffer, DPBS buffer, Tris buffer, Hepes buffer, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and the like.

[0021] In one embodiment of the present invention, the pharmaceutical composition is a lyophilized preparation, which may further contain a cryoprotectant in addition to the exosome composition containing baicalein or its derivatives. The cryoprotectant may be a cryoprotectant commonly used in the art, including but not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc. For the lyophilized preparation, water for injection or buffer may be added to reconstitute it into a suspension before use.

[0022] According to the present invention, in the pharmaceutical composition, the content of exosomes and the content of baicalein or its derivatives (calculated as baicalein) can be independently 0.1-500 μg, for example, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, respectively. μg, 150μg, 160μg, 170μg, 180μg, 190μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg , 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg, 495μg, etc.When the pharmaceutical composition is a suspension, the concentration of the exosomes and the concentration of baicalein or its derivatives calculated as baicalein can be independently 2-500 μg / mL, for example, 2.1 μg / mL, 2.5 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL. g / mL, 200μg / mL, 210μg / mL, 220μg / mL, 230μg / mL, 240μg / mL, 250μg / mL, 260μg / mL, 270 μg / mL, 280μg / mL, 290μg / mL, 300μg / mL, 310μg / mL, 320μg / mL, 330μg / mL, 340μg / mL, 35 0μg / mL, 360μg / mL, 370μg / mL, 380μg / mL, 390μg / mL, 400μg / mL, 410μg / mL, 420μg / mL, 4 30μg / mL, 440μg / mL, 450μg / mL, 460μg / mL, 470μg / mL, 480μg / mL, 490μg / mL, 495μg / mL, etc.

[0023] The fifth aspect of the present invention is to provide the use of the pharmaceutical composition in the preparation of a drug for treating rhinosinusitis.

[0024] According to the present invention, the exosome composition containing baicalein or its derivatives can be administered by various administration methods known in the art, such as oral administration, nasal administration, subcutaneous injection, external application or intravenous injection, etc. In a preferred embodiment, nasal administration is adopted. The dosage of exosomes and the dosage of baicalein or its derivatives calculated as baicalein are independently 0.1-500 μg, for example, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg 60μg, 170μg, 180μg, 190μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg, 330μ g, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg, 495μg, etc.

[0025] The sixth aspect of the present invention is to provide a method for treating a disease, by administering an effective amount of the exosome composition comprising baicalein or its derivatives described in the present invention, or the pharmaceutical composition described in the present invention, to a patient in need of treatment.

[0026] According to the present invention, the baicalein or its derivatives are selected from one or more of the group consisting of baicalein, a pharmaceutically acceptable salt of baicalein, baicalin and a pharmaceutically acceptable salt of baicalin.

[0027] According to the present invention, the pharmaceutically acceptable salt of baicalein and the pharmaceutically acceptable salt of baicalin can be selected from the salt of organic acid and the salt of inorganic acid, respectively. The inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid. The organic acid can be acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid.

[0028] In some embodiments of the present invention, the rhinosinusitis is chronic rhinosinusitis, preferably eosinophilic chronic sinusitis, with or without nasal polyps.

[0029] According to the present invention, the treatment includes reducing or alleviating or inhibiting inflammation of the mucous membrane of the nasal cavity and / or paranasal sinuses, repairing the mucous membrane of the nasal cavity and / or paranasal sinuses, and inhibiting the thickness of the mucous membrane of the nasal cavity and / or paranasal sinuses.

[0030] According to the present invention, the exosomes may be exosomes from human or mammalian cells.

[0031] The mammals include, but are not limited to, ungulates such as cattle, sheep, horses, pigs, rodents such as rabbits, rats, mice, guinea pigs, primates such as monkeys and gorillas, cats, dogs, etc. In one embodiment of the present invention, the exosomes are exosomes of human cells.

[0032] The cells may be normal somatic cells and / or stem cells, and the cells include but are not limited to vascular endothelial cells, dendritic cells, T cells (e.g., Foxp3 + Treg cells), neurons, oligodendrocytes, astrocytes, microglia, embryonic stem cells, adult stem cells (e.g., hematopoietic stem cells, neural stem cells, mesenchymal stem cells derived from bone marrow, umbilical cord, fat, umbilical cord blood, amnion, (placental) chorion, dental pulp, thymus, synovium, etc.), etc.

[0033] Induced pluripotent stem cells (iPSCs) technology can also be used to reprogram terminally differentiated somatic cells into pluripotent stem cells by introducing specific genes (such as Oct4, Sox2, Klf4, c-Myc, etc.), and iPSCs can be used to obtain exosomes derived from iPSCs. In the present invention, the exosomes of iPSCs are abbreviated as "iPS-EV". iPSCS technology can be used to prepare proprietary induced pluripotent stem cells using the patient's own somatic cells, and then exosomes are obtained from the patient's proprietary induced pluripotent stem cells. Used in the patient's own body, it can greatly reduce the possibility of immune rejection.

[0034] In some embodiments of the present invention, the exosomes are exosomes derived from pluripotent stem cells. In some embodiments of the present invention, the pluripotent stem cell exosomes are exosomes derived from induced pluripotent stem cells, exosomes derived from mesenchymal stem cells, or exosomes derived from embryonic pluripotent stem cells. In some embodiments of the present invention, the pluripotent stem cell exosomes are human pluripotent stem cell exosomes, such as exosomes derived from human induced pluripotent stem cells, exosomes derived from human mesenchymal stem cells, or exosomes derived from human embryonic pluripotent stem cells.

[0035] Exosomes can be prepared by culturing the corresponding cells in vitro and isolating them from the cell culture fluid by cell autocrine. The culture of each type of cell can be carried out using methods and culture media known in the art. For example, the culture medium for human skin fibroblasts can be DMEM+15% FBS+NEAA, the culture medium for umbilical cord mesenchymal stem cells can be DMEM+10% FBS, and the culture medium for neuronal cells can be B27+Neurobasal+GlutaMAX.

[0036] In order to increase the production of exosomes in cell culture, the content of chemical substances in the culture medium can be adjusted according to the characteristics of the cultured cells, for example: retaining the chemical substances necessary for cell growth in the culture medium, removing or reducing the chemical substances or their content that are not necessary for cell growth in the culture, so that the cell growth is in a "starved" state, so as to achieve the effect of increasing the production of exosomes in the culture. For example, for culturing stem cells to obtain stem cell exosomes, L-ascorbic acid or its salt, selenium or its salt, insulin, etc. can be added to the basic culture medium of stem cells, such as DMEM, F12 or DMEM / F12, to induce stem cells to secrete a large amount of exosomes. For example, the exosome secretion inducer described in the Chinese patent application CN112920991A is incorporated into this application in its entirety.

[0037] In the present invention, various exosome separation methods in the art can be used to separate and obtain exosomes from cell culture fluid.

[0038] Exosomes can be separated and concentrated by differential ultracentrifugation, which can precipitate different impurities in the sample under different centrifugal forces to obtain purer exosomes. This is currently the most commonly used method for separating and concentrating exosomes. This method can improve the purity of the product and reduce the aggregation of exosomes by combining ultrafiltration with a 0.22μm or 0.45μm pore size filter membrane.

[0039] Exosomes are a subpopulation of extracellular vesicles with a diameter ranging from 30 to 150 nm. Exosomes can also be separated based on particle size using methods such as ultrafiltration, size exclusion chromatography, hydrostatic filtration dialysis, and asymmetric field flow separation.

[0040] Commercial exosome extraction kits such as Exo-spin can also be used TM 、ExoQuick TM 、Invitrogen TM And other products to isolate exosomes.

[0041] The extracted exosomes can be suspended in a buffer, such as a phosphate buffer, and stored frozen at -80°C to 4°C. They can also be stored in a lyophilized form by lyophilization with the addition of a cryoprotectant, such as trehalose.

[0042] Definition of terms:

[0043] “And / or” will be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0044] "Including" and "comprising" have the same meaning, are intended to be open ended and allow but not require the inclusion of additional elements or steps. When the terms "including" or "comprising" are used herein, the terms "consisting of" and / or "consisting essentially of" are also included and disclosed.

[0045] "Pharmaceutically acceptable excipient" refers to any ingredient other than the exosomes described herein and having substantially non-toxic and non-inflammatory properties in a patient, including but not limited to any and all solvents, dispersion media or other liquid carriers, dispersion or suspension aids, diluents, isotonic agents, preservatives, colorants, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure concentration adjusters, pH adjusters, buffers, chelating agents, cryoprotectants and / or fillers, as appropriate for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art. Exemplary antimicrobial or antifungal agents include but are not limited to benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, and the like, and combinations thereof. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and the like, and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Standard curve of baicalein concentration and absorbance.

[0047] Figure 2 : H&E stained sections of mouse sinus tissue.

[0048] Figure 3 :Statistical graph of olfactory epithelium and nasal mucosal thickness in mouse sinus tissue. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the examples. It should be noted that the examples cannot be used as a limitation on the protection scope of the present invention, and those skilled in the art understand that any improvements and changes made on the basis of the present invention are within the protection scope of the present invention.

[0050] Conventional reagents used in the following examples are all commercially available. The biological experiments performed are conventional biological experiments in the art and can be performed according to the instructions in the corresponding experimental manual or kit instructions.

[0051] Example 1 Preparation of exosomes from human induced pluripotent stem cells

[0052] (1) Cell culture: iPSC cells (product of Guodian (Beijing) Pharmaceutical Technology Co., Ltd.) were expanded and cultured (using the Essential 8TM Medium kit (purchased from Thermo Fisher, catalog number: A1517001) for routine expansion culture) until the logarithmic growth phase and in good condition, and digested into small cell clusters (3-10 cells) according to the technical manual of the Versene Solution kit (purchased from Thermo Fisher, catalog number: 15040066), and then plated on a 10 cm cell culture dish coated with vitronectin (Recombinant Human Vitronectin purchased from Peprotech, catalog number 140-09). After adhesion, the coverage rate reached 60% to 70%, and cultured overnight at 37°C in an atmosphere of 95% air and 5% CO2. When iPSCs were in good condition of adhesion, the medium was replaced with GDEV medium (product of Guodian (Beijing) Pharmaceutical Technology Co., Ltd.). After 24 hours, the medium was replaced and the cell culture supernatant was collected. The previous step was repeated, and a batch of cells could be collected 3 times.

[0053] (2) Isolation of exosomes: The cell culture supernatant obtained by inducing and culturing cells in step (1) was centrifuged at 3000g for 15 min at room temperature to remove dead cells and cell debris. The supernatant was collected and filtered through a 0.22 μm filter; the filtrate was transferred to a Centricon Plus-70 (100 kDa) ultrafiltration tube for concentration and centrifuged at 3000g for 30 min at room temperature. The concentrate was diluted with DPBS (Dulbecco's phosphate buffered saline) at a ratio of about 1:100, and then concentrated again using the same device to obtain an exosome solution.

[0054] Example 2 Preparation of exosomes from human umbilical cord mesenchymal stem cells

[0055] (1) Sample processing: 10 cm of fresh umbilical cord tissue was collected and washed with physiological saline containing penicillin-streptomycin solution (double antibody) (100×) (purchased from Gibco) and cut into 1 mm pieces with ophthalmic scissors. 2 The small pieces were transferred to a cell culture bottle, and DMEM / F12 culture medium containing 10% serum replacement and penicillin-streptomycin (both purchased from Gibco) was added, and the cells were cultured in a 5% CO2, 37°C incubator.

[0056] (2) Cell culture: During the primary culture of step (1), the medium was changed for the first time after 1 week, and then once every 3 to 4 days. When the cells grew to 80% to 90% confluence, they were digested and passaged with trypsin / EDTA digestion solution (purchased from Gibco) and transferred to new culture dishes for culture.

[0057] (3) Isolation of exosomes: When the cell concentration grows to 80-90%, human mesenchymal stem cell serum-free culture medium (MSC XF Medium, purchased from Biological Industries), continue to culture, replace the medium after 24 hours, collect the cell culture supernatant, repeat the previous step, and a batch of cells can be collected 3 times; the cell culture supernatant collected 3 times was centrifuged for 20 minutes (3000g / min) to remove cells and cell debris, and centrifuged at 3000g / min at room temperature for 30 minutes using Centricon Plus-70 (100kDa) ultrafiltration tubes (purchased from Sigma) to obtain exosomes.

[0058] Example 3 Preparation of exosome composition containing baicalein

[0059] 1.1 Weigh 5 mg of baicalein powder and add 1.15 mL of DMSO to obtain a 16 mM DMSO stock solution. Divide into 10 tubes and store in a -80°C refrigerator away from light.

[0060] 1.2 Take out the sample with a density of 10 from the -80℃ refrigerator 11 / mL (density is 10 11 Thaw 100 μL of iPSC exosomes (about 108 μg / mL exosome concentration is about 1 mg / mL) at room temperature, add 290 μL of PBS solution, and then add 10 μL of 16 mM baicalein DMSO solution in step 1.1. At this time, the concentration of baicalein in the system is 0.4 mM (about 108 μg / mL), and the system contains 2.5% DMSO. Vortex to mix, and place at room temperature away from light for 2 h, or place in a 4°C refrigerator overnight.

[0061] 1.3 Transfer the solution to a 100K ultrafiltration tube, set the centrifugal force to 14000×g, and centrifuge for 5 minutes.

[0062] 1.4 Add 400 μL of PBS solution containing 2.5% DMSO to the ultrafiltration tube, set the centrifugal force to 14000×g, and centrifuge for 5 minutes. Repeat this washing step four times and collect the last washing solution.

[0063] 1.5 Add 400 μL of PBS solution without DMSO, set the centrifugal force to 14000×g, and centrifuge for 5 minutes.

[0064] 1.6 Use a 20μL pipette to carefully aspirate the drug-loaded exosomes in the ultrafiltration tube and transfer them to a 0.2mL centrifuge tube. The tube can be stored in a -80℃ refrigerator.

[0065] Example 4 Detection of the drug loading amount of baicalin in the exosome composition containing baicalin

[0066] 1.1. Prepare the standard solution of baicalin.

[0067] 1) Take 10 μL of 16 mM baicalein DMSO solution and add 10 μL of DMSO to obtain a baicalein DMSO solution with a concentration of 8 mM.

[0068] 2) Take 10 μL of the above 8 mM baicalein solution and add it to 390 μL of PBS solution to obtain a standard solution with a concentration of 200 μM.

[0069] 3) Take 200 μL of the above 200 μM standard solution, add 200 μL of PBS solution containing 2.5% DMSO to obtain a standard solution with a concentration of 100 μM.

[0070] 4) According to the above dilution method, dilute in sequence to obtain standard solutions with concentrations of 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, and 0 μM.

[0071] 1.2 UV-visible spectrophotometry with an enzyme-labeled instrument

[0072] 1) Prepare the standard curve solution.

[0073] Take 4.5 μL of each standard solution prepared in 1.1 and place it in a 0.2 mL centrifuge tube. 11 3 μL of iPSC exosomes per mL. The gradient concentration of baicalein in the system was 120 μM, 60 μM, 30 μM, 15 μM, 6 μM, 3 μM, 0.6 μM, and 0 μM (blank control). All contained 1.5% DMSO.

[0074] 2) Dilute the sample solution

[0075] The exosome composition containing baicalein prepared in Example 3 was taken as a sample, 3 μL was taken, and 4.5 μL of PBS solution containing 2.5% DMSO was added. At this time, the content of DMSO was 1.5%.

[0076] 3) Add 6 μL of the standard curve solution and diluted sample solution (3 parallel wells) to the microplate. Then set 338 nm as the absorption wavelength in the M5 microplate reader and measure the OD value. The absorbance values ​​of the 3 parallel wells of the sample solution are shown in Table 1.

[0077] 4) In Origin8.0 software, the concentration of the standard curve solution was used as the horizontal axis, and the OD value after deducting the blank absorption was used as the vertical axis. Linear fitting was performed and the linear equation and R were obtained. 2 Values, such as Figure 1 shown.

[0078] 5) Calculate the concentration of baicalin in the diluted sample according to the standard curve equation. Multiply the concentration by 2.5 to obtain the loading concentration of baicalin in the exosome composition containing baicalin prepared in Example 3.

[0079] Table 1 Absorbance values ​​of three parallel wells of diluted sample solution

[0080] parallel holes Parallel hole 1 Parallel hole 2 Parallel hole 3 Absorbance value (au) 0.0320 0.0306 0.0317

[0081] Substituting the absorbance values ​​of the three parallel wells of the diluted sample solution into the standard curve y=0.0016x, the concentrations of baicalin in the diluted sample were obtained to be 20 μM, 19.13 μM, and 19.81 μM, with an average concentration of 19.65 μM. The loading concentration of baicalin in the exosome composition containing baicalin prepared in Example 3 was 19.65 μM×2.5=49.12 μM.

[0082] The drug loading rate of baicalein % = 49.12 μM / 400 μM×100% = 12.28%.

[0083] The applicant adopted the above method, changed the incubation conditions, different exosome final concentrations in the incubation system and different baicalin concentrations, and measured the loading concentration of baicalin in the exosome composition containing baicalin. The results are as follows:

[0084] (1) PBS system, containing 2.5×10 8 exosomes / mL, 200μM baicalin

[0085]

[0086]

[0087] (2) PBS system, containing 3.0×10 11 exosomes / mL, 1mM baicalein

[0088] temperature Incubation time Baicalin concentration in exosomes Drug loading rate RT 3h 420μM 42.0％

[0089] Example 5: Treatment of eosinophilic sinusitis with an exosome composition containing baicalein

[0090] Eosinophilic sinusitis modeling scheme for mice: Weigh an appropriate amount of papain and dissolve it in PBS solution to prepare a papain solution with a final concentration of 1 mg / ml. Alternately drip 5 μL / mouse in the nose in the morning and evening for modeling, 20 μL / mouse / day.

[0091] Grouping scheme: Mice were induced with eosinophilic rhinitis model using papain intranasally (days 0-2 and 7-10), and then divided into PBS group, PC (positive control) group, EXO-1 (exosome) group, baicalein (hereinafter abbreviated as BC) group, and BC+EXO-1 (exosome composition containing baicalein) group and administered drugs respectively.

[0092] The other normal mice were induced with saline (days 0-2 and 7-10) as the Sham normal control group.

[0093] On days 7-13 after modeling, the Sham group, PBS group, PC group, EXO-1 group, BC group, and BC+EXO-1 group were intervened by intranasal drops of PBS, PBS, Nasonex, iPSC exosomes, baicalein, and an exosome composition containing baicalein in the morning and evening, respectively. On day 14, the mouse sinus tissues were collected, embedded in paraffin, and stained with hematoxylin & eosin to observe the nasal mucosa.

[0094] Group setting and drug preparation instructions

[0095] 1) Sham group: normal control group, modeling was performed with normal saline and PBS was administered;

[0096] 2) PBS group: placebo group, using papain-induced rhinitis model, PBS administration;

[0097] 3) PC group: positive control group, using papain-induced rhinitis model, Nasonex (Nasonex: mometasone furoate nasal spray), 10 μL / time, once in the morning and evening;

[0098] 4) EXO-1 group: exosome treatment group, using papain-induced rhinitis model, iPSC exosomes were administered, the final concentration was 100 μg / ml, 10 μL / time (exosome content was 1 μg), once in the morning and once in the evening;

[0099] 5) BC group: baicalein treatment group, using papain-induced rhinitis model, baicalein at a concentration of 100 μg / mL (with 2% DMSO as solvent), 1 μg / time, about 10 μL / time, once in the morning and evening;

[0100] 6) BC+EXO-1 group: The group treated with the exosome composition containing baicalein used the papain-induced rhinitis model, and baicalein with a final concentration of 100 μg / mL (in 2% DMSO as a solvent) was co-incubated with 100 μg / mL iPSC exosomes (4°C for 20 h, or room temperature for 3 h), and 10 μL / time (exosome content of 1 μg, baicalein content of 1 μg) was dripped into the nose once in the morning and evening.

[0101] Experimental procedures

[0102] 1) Thirty male C57BL / 6J mice weighing 18-22 g were acclimated for one week and then randomly divided into 6 groups according to the animal numbers in Table 1, with 5 mice in each group.

[0103] 2) Grouping was performed according to the table below. On days 0-2 and 7-10, groups 2-6 (PBS group and multiple treatment groups) received papain drops alternately into the left and right nostrils, 20 μL / mouse. Group 1 (sham group) received an equal volume of normal saline drops into the nostrils;

[0104] 3) On days 7-13, groups 3-6 received alternating intranasal drops of PC, EXO-1, BC, and BC+EXO-1, respectively, while groups 1 (sham group) and 2 (PBS group) received intranasal drops of an equal volume of PBS solution;

[0105] 4) On the 14th day, the mouse sinus tissues were obtained, fixed, and embedded in paraffin. HE staining was performed to observe the morphology. Three locations were selected for each section to calculate the thickness of the nasal mucosa. The average value of the nasal mucosa thickness at three locations of each section was taken for data statistics.

[0106] Animal experiment groups:

[0107] Experimental Results

[0108]

[0109] Epithelial cell damage was assessed by H&E staining. Figure 2It can be seen that the olfactory epithelial mucosa of the nasal mucosa in the Sham group was intact and the cilia were intact. The nasal mucosa of the PBS group was severely damaged, and the nasal mucosa showed atrophy of the olfactory epithelial mucosa, loose and damaged cells, some cell nuclei were condensed and darkly stained, vacuolar changes, and cilia fell off. The olfactory epithelial mucosa of the nasal mucosa in the PC group was atrophy, the cells were loose and damaged, the cell nuclei were condensed and darkly stained, and vacuolar changes, but some cilia fell down. The olfactory epithelial mucosa of the nasal mucosa in the BC group was intact, but the cilia fell off. The olfactory epithelial mucosa of the nasal mucosa in the EXO-1 group had some cilia falling off. The olfactory epithelial mucosa of the nasal mucosa in the BC+EXO-1 group was intact and the cilia were intact. It can be seen from the H&E staining sections that the nasal mucosa of the BC+EXO-1 group was intact and the cilia did not fall off, and the degree of damage to the nasal mucosa was reduced compared with the PBS group, the PC group, and the groups using BC or EXO-1 alone.

[0110] H&E stained sections were used to evaluate the thickening of the nasal mucosa. Thickening of the nasal mucosa usually indicates chronic inflammation of the nasal cavity, repeated episodes of rhinitis, or long-term chronic local irritation. Figure 3 It can be seen that in the saline modeling Sham group, the thickness of the nasal mucosa was about 30μm, and in the PBS group, the nasal mucosa thickened to about 40μm due to the continuous stimulation of inflammation and edema. Compared with the PBS group, the Nesonade PC group and the BC group using baicalein alone did not show obvious inhibition of nasal mucosal thickening. The overall average thickness of the nasal mucosa in the exosome EXO-1 group was lower than that in the PBS group, but there was no significant difference. The exosome composition containing baicalein, the BC+EXO-1 group, had the best therapeutic effect. After treatment, the nasal mucosal thickness of mice in the BC+EXO-1 group reached the level of the Sham group, indicating that exosomes containing baicalein can effectively inhibit the inflammatory response of the nasal mucosa and have a good therapeutic effect on chronic sinusitis.

[0111] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An exosome composition comprising baicalein or a derivative thereof, characterized in that: Comprising a combination of baicalein or its derivatives and exosomes as active ingredients; The baicalein or its derivative is selected from one or more of the group consisting of baicalein, a pharmaceutically acceptable salt of baicalein, baicalin and a pharmaceutically acceptable salt of baicalin; The exosomes are exosomes derived from human induced pluripotent stem cells; Only a portion of the baicalein or its derivatives in the exosome composition is loaded into the exosomes, or all of the baicalein or its derivatives in the exosome composition are loaded into the exosomes; The loading concentration of baicalein or its derivatives is 2-500 μg / mL in terms of baicalein; Calculated as baicalein, in the exosome composition, the amount of baicalein or its derivatives loaded in the exosomes accounts for 5%-100% of the total amount of baicalein or its derivatives in the exosome composition.

2. The exosome composition according to claim 1, characterized in that The exosome composition is obtained by mixing the exosomes with the baicalein or a derivative thereof and incubating the mixture.

3. The exosome composition according to claim 2, characterized in that The incubation comprises: co-incubating an appropriate concentration of baicalein or its derivatives with exosomes; in the incubation system, the final concentration of baicalein or its derivatives is 5-500 μg / mL in terms of baicalein, and the final concentration of exosomes is 2-3000 μg / mL; the incubation conditions are 0°C-5°C, 1-25h, or 20°C-35°C, 1-4h.

4. The exosome composition according to claim 3, characterized in that In the incubation system, the final concentration of baicalein or its derivatives is 5-100 μg / mL in terms of baicalein, and the final concentration of exosomes is 2-100 μg / mL.

5. A pharmaceutical composition, characterized in that A composition comprising the exosomes according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is a suspension, which contains a buffer in addition to an exosome composition containing baicalein or a derivative thereof, and the concentration of the exosomes and the concentration of baicalein or a derivative thereof calculated as baicalein are independently 2-500 μg / mL, respectively.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is a lyophilized preparation, which contains a cryoprotectant in addition to an exosome composition containing baicalein or a derivative thereof, and the content of the exosomes and the content of baicalein or a derivative thereof calculated as baicalein are independently 0.1-500 μg, respectively.

8. The exosome composition according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 7, characterized in that: The pharmaceutically acceptable salts of baicalein and baicalin are respectively selected from salts of organic acids and salts of inorganic acids.

9. The exosome composition or pharmaceutical composition according to claim 8, characterized in that The inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid.

10. The exosome composition or pharmaceutical composition according to claim 8, characterized in that The organic acid is acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid.

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