New application of rapamycin or derivative thereof

Abstract

The invention provides a new application of rapamycin or a derivative thereof. The invention provides application of rapamycin or pharmaceutically acceptable salt, ester, prodrug or metabolite thereof in preparation of a medicine. The medicine is used for preventing or treating allergic rhinitis. Experiments show that rapamycin can significantly improve various symptoms of allergic rhinitis model mice. The traditional Chinese medicine composition can effectively relieve nasal cavity expansion and hyperemia of mice and reduce the frequency of sneezing, rhinorrhea and nose scratching behaviors, so that the allergic rhinitis inflammation is relieved.

Description

Technical Field

[0001] The present invention relates to the field of medicine. Specifically, the present invention relates to new uses of rapamycin or its derivatives. More specifically, the present invention relates to new uses of rapamycin or its pharmaceutically acceptable salts, esters, prodrugs or metabolites. Background Art

[0002] Allergic rhinitis (AR) is an abnormal nasal reaction caused by increased sensitivity to certain allergens. It is a common nasal disease characterized by sudden and recurrent episodes of nasal itching, sneezing, runny nose, and nasal congestion. Sneezing is most severe in the early morning and during sleep, and some patients experience symptoms of allergic conjunctivitis, such as redness, itching, and tearing of the eyes. AR affects approximately 10 to 40 years of age. With the advancement of industrialization, modern lifestyles, and environmental pollution, the incidence of AR has been increasing globally, with an average global prevalence of 10% to 25%. Although not life-threatening, the nasal and ocular symptoms (conjunctivitis) and related organ symptoms (such as bronchial asthma and otitis media) caused by AR can lead to sleep disturbances, loss of appetite, general weakness, fatigue, mood disorders, decreased attention span, and learning difficulties, significantly impacting people's physical health, work, and study, and reducing their quality of life.

[0003] Currently, the use of corticosteroid nasal sprays for the treatment of allergic rhinitis is internationally recognized as an effective treatment and has been recommended by the American Association for the Advancement of Science (ARIA) as a first-line treatment. Although corticosteroids are currently considered relatively safe, allergic rhinitis cannot be cured and requires long-term use, which carries certain risks. Loratadine is often used as a first-line treatment for mild allergic rhinitis, but it has side effects such as dry mouth, which some patients may not tolerate well. More effective treatments for allergic rhinitis are needed clinically. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] The present invention is accomplished based on the following findings of the inventors:

[0006] Allergic rhinitis (AR) is an immune-mediated allergic inflammatory reaction caused by inhaled allergens, primarily through the action of immunoglobulin E (IgE). Its primary manifestations include sneezing, nasal congestion, nasal itching, and runny nose. The pathogenesis of AR is complex. Allergens stimulate B cells in the nasal epithelium to produce IgE. Subsequently, IgE activates mast cells, promoting the release of inflammatory mediators such as histamine, IL-4, and IL-6, triggering an inflammatory response including increased plasma exudation and mucus secretion in the nasal mucosa. Therefore, alleviating the inflammatory response may be an important strategy for treating AR.

[0007] Rapamycin (RAPA), whose chemical name is: (3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexahydro-9,27-dihydroxy- 3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxazacyclohexacyclopenta ...

[0008]

[0009] Rapamycin (also known as "sirolimus") is a new macrolide immunosuppressant. Developed as early as the 1970s, it was initially used as a low-toxic antifungal drug. In 1977, it was discovered to have immunosuppressive effects. In 1989, RAPA began to be tested as a new drug for the treatment of organ transplant rejection. From the results of animal experiments and clinical applications, it is a new immunosuppressant with good efficacy, low toxicity, and no nephrotoxicity. It is now often used as a drug to maintain the immune capacity of transplanted organs (especially kidney transplants) to slow down the immune rejection reaction after organ transplant surgery. During the experiment, the inventor unexpectedly discovered that rapamycin, as an active ingredient, can relieve allergic rhinitis inflammation after nasal administration, effectively relieve nasal dilation and congestion, and improve behaviors such as scratching the nose, sneezing, and runny nose.

[0010] Therefore, in the first aspect of the present invention, the present invention proposes the use of rapamycin or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof in the preparation of a medicament for preventing or treating allergic rhinitis. The results of animal experiments show that rapamycin can significantly improve various symptoms of allergic rhinitis model mice. It can effectively relieve nasal dilation and congestion in mice, reduce the frequency of sneezing, runny nose and nose scratching, and thus reduce the inflammation of allergic rhinitis. Based on this, rapamycin is expected to become an effective drug for preventing or treating allergic rhinitis, and further clinical studies can be carried out to verify its application value.

[0011] According to an embodiment of the present invention, the drug is administered through the nasal cavity, thereby effectively alleviating the symptoms of allergic rhinitis.

[0012] According to an embodiment of the present invention, the dosage form of the drug is selected from the group including but not limited to liquid dosage form and gel dosage form.

[0013] It should be noted that the "liquid dosage form" described in the present invention refers to a liquid dispersion system for oral or external use in which the active ingredient rapamycin is dispersed in a liquid medium in a certain form. The liquid dispersion system may be homogeneous or heterogeneous, and the present invention does not specifically limit the liquid medium. The "gel dosage form" described in the present invention is a semi-fluid semi-solid dosage form, which is usually made by mixing the active ingredient rapamycin with suitable excipients (such as polymer materials, moisturizers, preservatives, etc.). The gel has a certain viscosity and plasticity and can form a stable gel state at a specific location. The present invention does not specifically limit the excipient, as long as it can form a gel state with rapamycin and ensures that it does not have any negative impact on the pharmacological activity of rapamycin.

[0014] In the second aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: rapamycin or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof as an active ingredient, and optionally a pharmaceutically acceptable excipient or carrier. The results of animal experiments show that rapamycin can significantly improve various symptoms of allergic rhinitis model mice. It can effectively relieve nasal dilation and congestion in mice, reduce the frequency of sneezing, runny nose and scratching of the nose, thereby reducing the inflammation of allergic rhinitis. Based on this, the pharmaceutical composition containing rapamycin can effectively prevent or treat allergic rhinitis.

[0015] As used herein, the term "active ingredient" refers to a substance with biological activity that can affect an organism, including treating or preventing a disease, or improving certain functions of an organism.

[0016] As used herein, the term "pharmaceutically acceptable excipients or carriers" refers to all pharmaceutical materials other than the principal drug that are added to a formulation to improve its formability, efficacy, stability, and safety. These materials can generally be categorized as solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

[0017] According to an embodiment of the present invention, the carrier includes but is not limited to exosomes and hydrogels. Since rapamycin is insoluble in water, the inventors use exosomes and / or hydrogels as carriers. Among them, when exosomes are used to load rapamycin, since the exosomes themselves are hydrophobic and have low water solubility, they can not only accurately deliver the active ingredient to the target area, but also reduce adverse reactions to the body by virtue of their good biocompatibility. At the same time, the hydrogel can effectively encapsulate rapamycin and accurately control its release rate by virtue of its unique three-dimensional network structure. In addition, the hydrogel can form a gel state in the nasal cavity, firmly adhere to the mucosal surface, effectively reduce the rapid loss of the drug, significantly increase the contact time of the drug with the nasal mucosa, and then prolong the action time of the drug, and ultimately achieve a significant improvement in the therapeutic effect.

[0018] According to an embodiment of the present invention, the exosomes are derived from plant cells or animal cells. The present invention does not specifically limit the source cell type of the exosomes, as long as these cells can secrete exosomes and can extract exosomes from them. Specifically, the plant cells may include but are not limited to carrot cells, ginseng cells, Arabidopsis cells, etc.; the animal cells may include but are not limited to stem cells (such as bone marrow mesenchymal stem cells, adipose stem cells, etc.), immune system cells (such as B lymphocytes, dendritic cells, T lymphocytes, etc.) and epithelial cells (such as alveolar epithelial cells, renal tubular epithelial cells, etc.).

[0019] According to an embodiment of the present invention, the animal cells include stem cells.

[0020] According to an embodiment of the present invention, the stem cells are derived from, but not limited to, umbilical cord, bone marrow, embryo, placenta, fat, bone, cartilage, periodontium, synovium, muscle, lung, liver, and pancreas.

[0021] According to an embodiment of the present invention, the stem cells include but are not limited to: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, synovial mesenchymal stem cells, and thymus mesenchymal stem cells.

[0022] It should be noted that, in the present invention, the above-mentioned mesenchymal stem cells can generally be obtained commercially or by methods known in the art. The purchase or acquisition of the above-mentioned mesenchymal stem cells, especially human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells (hBMSCs), placental mesenchymal stem cells, and amniotic membrane mesenchymal stem cells, should be carried out in compliance with local laws and regulations, including regulations on cell therapy, import and export of biological materials, ethics and privacy protection. When the exosomes described in the present invention are obtained from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, and amniotic membrane mesenchymal stem cells, these stem cells should also be within 14 days of fertilization without undergoing in vivo development.

[0023] According to an embodiment of the present invention, the stem cells are derived from animals including but not limited to mice, rats, rabbits, dogs, pigs, and primates.

[0024] According to an embodiment of the present invention, the stem cells are human umbilical cord mesenchymal stem cells.

[0025] According to an embodiment of the present invention, the hydrogel is selected from the group consisting of but not limited to gelatin, sodium alginate hydrogel, carboxymethyl chitosan hydrogel, polyvinyl alcohol hydrogel, povidone hydrogel, and carbomer hydrogel.

[0026] According to an embodiment of the present invention, the carrier is the exosome, and the ratio of rapamycin or its pharmaceutically acceptable salt, ester, prodrug or metabolite to the exosome is (170-230) μg:2.0×10 10 particles. For example, 170 μg: 2.0×10 10 particles, 180 μg: 2.0 × 10 10 particles, 190 μg: 2.0 × 10 10 particles, 200 μg: 2.0 × 10 10 particles, 210 μg: 2.0 × 10 10 particles, 220 μg: 2.0 × 10 10 particles, 230 μg: 2.0 × 10 10Particles, etc., or can be within the range of any of the above values. Thus, it can be ensured that the exosomes can efficiently encapsulate sufficient rapamycin or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof, thereby enhancing the therapeutic effect.

[0027] According to an embodiment of the present invention, the carriers are the exosomes and the hydrogel. The exosomes and the rapamycin or its pharmaceutically acceptable salt, ester, prodrug, or metabolite are configured in the above-mentioned ratio. The present invention does not specifically limit the amount of the hydrogel. As long as the hydrogel can effectively encapsulate the exosomes and rapamycin or its pharmaceutically acceptable salt, ester, prodrug, or metabolite without affecting the normal efficacy of the drug, the requirements of the present invention are met.

[0028] According to an embodiment of the present invention, the pharmaceutical composition is administered through the nasal cavity, thereby effectively treating allergic rhinitis.

[0029] According to an embodiment of the present invention, the dosage form of the pharmaceutical composition includes but is not limited to a liquid dosage form and a gel dosage form.

[0030] In a third aspect, the present invention provides the use of the pharmaceutical composition described in the second aspect in the preparation of a medicament for preventing or treating allergic rhinitis. Animal experimental results have shown that rapamycin can significantly improve multiple symptoms in mice with allergic rhinitis. It can effectively alleviate nasal dilation and congestion in mice, reduce the frequency of sneezing, runny nose, and nasal scratching, and thus reduce the inflammation of allergic rhinitis. Based on this, the pharmaceutical composition containing rapamycin can effectively prevent or treat allergic rhinitis.

[0031] In a fourth aspect, the present invention provides a method for treating allergic rhinitis. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable dose of rapamycin or a pharmaceutically acceptable salt, ester, prodrug, or metabolite thereof to a subject.

[0032] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0034] Figure 11 is a graph showing the morphology and particle concentration of exosomes (hUC-MSC-EXO, referred to as "EXO") derived from human umbilical cord mesenchymal stem cells (HUC-MSCs) according to Example 1 of the present invention. In which:

[0035] A is the transmission electron microscopy scanning result of EXO;

[0036] B is the experimental results of EXO's nanoparticle tracking analysis technology;

[0037] Figure 2 This is a transmission electron microscopy scanning morphology characterization result of the system after human umbilical cord mesenchymal stem cell exosomes loaded with rapamycin (RAPA) (abbreviated as "EXO-RAPA") according to Example 2 of the invention;

[0038] Figure 3 The results of the construction and identification of the allergic rhinitis (AR) model mice in Example 3 of the present invention are as follows:

[0039] A is a comparison table of behavioral scores for mice in constructing AR animal models, with scores >5 indicating successful model construction;

[0040] B is a statistical graph of behavioral scores of wild type mice (WT) and AR mice after the stimulation phase;

[0041] C is a statistical graph showing the score changes of AR model mice at different stimulation days during the stimulation process;

[0042] D is the comparison image of the nose of WT mice and AR mice after the stimulation phase;

[0043] Figure 4 This is a flow chart of nasal administration of human umbilical cord mesenchymal stem cell exosomes loaded with rapamycin (EXO-RAPA) according to Example 4 of the present invention, a comparison image of the nose after treatment, and a statistical graph of changes in behavioral scores during treatment, wherein:

[0044] A is a diagram of the nasal administration of EXO-RAPA;

[0045] B is the comparative image of the nose of mice in each group after nasal administration of EXO-RAPA;

[0046] C is a statistical graph showing the score changes of mice in each group at different treatment days after nasal administration of EXO-RAPA;

[0047] Figure 5After nasal administration of human umbilical cord mesenchymal stem cell exosomes loaded with rapamycin (EXO-RAPA) in Example 5 of the present invention, the mRNA levels of inflammatory factors in the nasal mucosa of mice were detected by QPCR experiments, wherein:

[0048] A is the statistical graph of relative expression of IL-1β mRNA in mouse nasal mucosa;

[0049] B. Statistical graph of relative expression of IL-6 mRNA in mouse nasal mucosa;

[0050] Figure 6 This is a statistical graph showing changes in behavioral scores during nasal administration of the hydrogel loaded with EXO-RAPA (Gel-EXO-RAPA) in Example 6 of the present invention, and the mRNA levels of inflammatory factors in the nasal mucosa of mice detected by QPCR experiments after treatment, wherein:

[0051] A is a statistical graph showing the score changes of mice in each group at different treatment days after nasal administration of Gel-EXO-RAPA;

[0052] B is the statistical graph of relative expression of IL-1β mRNA in mouse nasal mucosa;

[0053] C is the statistical graph of the relative expression of IL-6 mRNA in mouse nasal mucosa. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are described in detail below, which are intended to explain the present invention but are not to be construed as limiting the present invention.

[0055] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0056] For the sake of clarity, only some numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" and any variations thereof in the specification and claims of the present invention and the above-mentioned drawings are intended to cover non-exclusive inclusions.

[0058] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0059] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0060] The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0061] The term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in SM Berge et al., J. Pharmaceutical Sciences, 66, 1-19, 1977. Pharmaceutically acceptable salts formed with non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates, and organic acid salts, such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates, or other methods described in the literature, such as ion exchange methods, to obtain these salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium / quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0062] The term "ester" is represented by the formula -OC(O)R or -C(O)OR, where R can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl.

[0063] The term "prodrug" refers to a compound that is converted into rapamycin in vivo. Such conversion is affected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissues. The prodrug compound of the present invention can be an ester. In the prior invention, esters that can be used as prodrugs include phenyl esters, aliphatic (C1-C 24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For a complete discussion of prodrugs, please refer to the following literature: T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270, and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0064] The term "metabolite" refers to a product resulting from the in vivo metabolism of a specific compound or salt thereof. Metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized using assays such as those described herein. Such products can be obtained by subjecting the compound to oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention encompasses metabolites of the compound, including metabolites produced by contacting a compound of the invention with a mammal for a sufficient period of time.

[0065] The term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or veterinary use.

[0066] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients enhance the handling properties of pharmaceutical formulations, i.e., by increasing flowability and / or cohesiveness, making the formulation more suitable for direct compression.

[0067] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering the drug herein to an individual in need.

[0068] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0069] The term "subject" may also be referred to as a "subject." In many embodiments of the methods of the present disclosure, the subject treated by the methods of the present disclosure is ideally a human subject, although it should be understood that the methods described herein are effective for all vertebrate species, which are intended to be included in the term "subject." Thus, a "subject" can include a human subject for various medical purposes, such as a prophylactic treatment for treating an existing condition or disease or for preventing the onset of a condition or disease, or an animal (non-human) subject for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates, such as humans, monkeys, and apes; bovines, such as cattle and oxen; ovines, such as sheep; caprines, such as goats; porcines, such as pigs and hogs; equines, such as horses, donkeys, and zebras; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits and hares; and rodents, including mice and rats. An animal can be a transgenic animal. In some embodiments, the subject is a human, including but not limited to fetal, neonatal, infant, adolescent, and adult subjects. Furthermore, a "subject" can include a patient suffering from or suspected of suffering from a condition or disease. Therefore, the terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human.

[0070] With respect to a drug or pharmacologically active agent, the terms "effective dose," "effective amount," or "therapeutically effective amount" refer to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For oral dosage forms of the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0071] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0072] Example 1: Preparation of human umbilical cord mesenchymal stem cell-derived exosomes loaded with rapamycin (EXO-RAPA)

[0073] In this embodiment, since RAPA is insoluble in water, a common exosome carrier is used to load RAPA to explore the efficacy of RAPA. Primary mesenchymal stem cells extracted from human umbilical cord were cultured, and when the cells grew to 70-80% confluence, 0.25% trypsin was used for digestion and passage, and the passage ratio was 1:2. Stable 2-3 generation human umbilical cord mesenchymal stem cells were selected. When the cell confluence reached 60-70%, the complete culture medium was replaced with a complete culture medium containing 10% human umbilical cord mesenchymal stem cell serum without exosomes (purchased from Punosai Company) and continued to be cultured for 48 hours. The human umbilical cord mesenchymal stem cell culture supernatant was collected and centrifuged at 300g for 10 minutes, 3000g for 25 minutes, and 10000g for 1 hour. After gradient centrifugation, the supernatant was ultrafiltrated and concentrated (molecular weight cutoff of 100kDa) to obtain an exosome solution.

[0074] The exosome solution was characterized by transmission electron microscopy and nanoparticle tracking analysis. Figure 1 As shown, the particles in the exosome solution of human umbilical cord mesenchymal stem cells are spherical vesicle-like structures, which are consistent with the extracellular vesicle structure of exosomes ( Figure 1 A); Under the condition of 500-fold dilution, the particle concentration is 4.0×10 7 Particles / mL, therefore, the original exosome particle concentration was 2.0×10 10 Particles / mL, with a particle size of 40-200 nm, which is consistent with the particle size of exosomes ( Figure 1B).

[0075] The above results indicate that the particle solution obtained from the human umbilical cord mesenchymal stem cell culture in this example is an exosome solution (hereinafter referred to as "EXO solution"). The obtained particles have a vesicle-like structure with a particle size of 40 to 200 nm, and are exosomes derived from human umbilical cord mesenchymal stem cells (hereinafter referred to as "EXO").

[0076] Example 2: Preparation of EXO-RAPA drug system

[0077] 1 mg of RAPA was first dissolved in 20 μL of dimethyl sulfoxide (DMSO). Then, the RAPA dissolved in DMSO was slowly added dropwise to 1 mL of EXO solution (particle concentration of 2.0×10 10 After the addition was completed, the solution was ultrasonically concentrated by ultrafiltration using an ultrafiltration centrifuge tube (molecular weight cut-off of 100 kDa) to remove free RAPA and prepare an EXO-RAPA drug system.

[0078] To characterize the loading efficiency of RAPA, the EXO-RAPA drug system was added with lysis buffer (1% protease inhibitor cocktail and 1% PMSF were added to the Biyuntian western blot lysis buffer and placed at 4°C for 0.5 h. The loading amount of RAPA was then detected using high performance liquid chromatography. The morphology and size of EXO-RAPA were characterized by transmission electron microscopy.

[0079] The results are as follows Figure 2 As shown in the results, under ultrasonic conditions, the loading ratio of EXO to RAPA is: 2.0×10 10 The particles can load 198.0±27.0μg RAPA; transmission electron microscopy scanning results show that the constructed EXO-RAPA drug system is still nanoscale in size.

[0080] The above results show that RAPA can be successfully loaded using the ultrasonic method to prepare the EXO-RAPA drug system.

[0081] Example 3: Construction of an allergic rhinitis (AR) mouse model

[0082] Thirty male BALB / C mice aged 6-7 weeks and weighing 20-25g were randomly divided into 5 groups, with 6 mice in each group. The first group was the WT group, and the other 4 groups were used to construct AR model mice. (1) Sensitization stage: a physiological saline solution containing 10% ovalbumin (OVA) and 10% aluminum hydroxide was prepared, and each mouse was given an intraperitoneal injection of 200μL of OVA solution on the 1st, 7th and 14th days respectively. (2) Provocation stage: each mouse was given nasal drops of OVA solution daily from the 15th to the 21st day, with 20μL dripped into each nostril for 7 consecutive days. The control group mice were given an equal amount of physiological saline. After the provocation, the amount of nasal discharge, the number of sneezes and nose scratching of the mice within 30 minutes were observed and recorded every two days and scored. Figure 3 As shown in A, the total score of each group of mice was calculated using the superposition method. The total score for successful model construction must be greater than 5 points. The mouse noses were photographed for comparison.

[0083] The results are as follows Figure 3 As shown, the results showed that compared with WT mice, the scores of the constructed AR model mice were greater than 5 points ( Figure 3 B), and the scores of the mice showed an upward trend during the stimulation phase, exceeding 5 points on the 18th day, and then the scores of each AR mouse remained above 5 points ( Figure 3 C). From the photos of the mice's noses, compared with WT mice, the AR model mice had obvious runny nose, and the nasal cavity was dilated and congested ( Figure 3 D).

[0084] The above results show that AR model mice with a score greater than 5 points were constructed, and compared with WT mice, AR model mice showed obvious sneezing, runny nose and nose scratching behaviors in the nose, and the nasal cavity was dilated and accompanied by congestion, which is more consistent with AR symptoms, that is, AR model mice were successfully constructed.

[0085] Example 4: Nasal administration of RAPA to treat AR mice

[0086] Based on Example 3 above, an experiment on the therapeutic effect of rapamycin on AR was conducted. The 24 mice in the AR group were randomly divided into 4 groups of 6 mice each, and 6 mice in the WT group were treated with nasal administration of EXO-RAPA. Pure EXO was used as the solvent control group, and budesonide, a common drug for treating AR on the market, was used as the positive control group. The treatment regimen is shown in Table 1:

[0087] Table 1

[0088]

[0089] Each group of mice received nasal administration, 2 times a day, 10 μL each time, 20 μL per day, for a total of 14 days, during which OVA nasal challenge was continued every day ( Figure 4 A) Every two days after dosing and challenge, the mice were observed and scored according to a scoring table: the amount of nasal discharge, the number of sneezes, and the number of nasal scratchings were observed and recorded over a 30-minute period, and the total score was calculated using the cumulative method. The mouse noses were photographed for comparison.

[0090] The results are as follows Figure 4 As shown in the results, the AR group still showed obvious runny nose behavior, nasal dilation and congestion in the nose. The EXO group only had slight improvement after treatment, while the positive control group budesonide showed significant improvement. EXO-RAPA also had significant improvement but did not exceed the effect of budesonide ( Figure 4 B) In terms of the scores of mice during treatment, compared with the AR group, the scores of EXO-RAPA gradually decreased from greater than 5 to less than 5 during treatment, which was similar to the improvement trend of budesonide in the positive control group. Both significantly alleviated the sneezing, runny nose, and nose scratching behaviors of AR mice, while EXO did not reduce the scores to below 5. Figure 4 C), indicating that RAPA played a major therapeutic role.

[0091] The above results show that after nasal administration of EXO-RAPA, the runny nose behavior and nasal dilation accompanied by congestion of AR can be significantly improved, and the superimposed score also shows that the sneezing, runny nose and nose scratching behavior of AR mice are significantly relieved. The therapeutic effect is slightly weaker than that of the positive control group budesonide, and the relief effect of EXO is not obvious. Therefore, it can be inferred that RAPA treatment has significantly alleviated the symptoms of AR mice.

[0092] Example 5: RAPA affects the level of nasal inflammation in AR mice

[0093] In this example, mice in each group that completed the above experiment were euthanized, and mouse serum was first obtained and tested by ELISA to detect the levels of pro-inflammatory cytokines IL-1β and IL-6 in the body.

[0094] The results show that Figure 5 It can be seen that compared with the AR group of mice, the levels of proinflammatory cytokines IL-1β and IL-6 in the serum were significantly reduced through nasal administration of EXO-RAPA, indicating that EXO-RAPA can alleviate the level of nasal inflammation, while there was no significant change after nasal administration of EXO compared with AR, but there was a slight improvement. The positive control group budesonide had a more obvious improvement on the inflammation of AR mice.

[0095] The above results show that nasal administration of EXO-RAPA can significantly improve the inflammation level of AR, and the improvement effect is slightly weaker than that of the positive control group budesonide, while the improvement effect of EXO is not significant. Therefore, it can be inferred that RAPA treatment reduces the inflammation level of AR mice.

[0096] Example 6: Hydrogel can enhance the therapeutic effect of EXO-RAPA on AR mice

[0097] In order to improve the retention time of the drug in the nasal cavity, the EXO-RAPA for nasal administration was further optimized, and a hydrogel-loaded EXO-RAPA drug system was prepared. The preparation method is as follows: first, 200 μL of the EXO-RAPA solution prepared above was concentrated by ultrafiltration tube (molecular weight cut-off was 100kD) to a volume of 100 μL, and then 10 mg of gelatin was dissolved in 100 μL of heated PBS solution. After complete dissolution, 100 μL of the above-mentioned concentrated EXO-RAPA solution was added, stirred, and cooled to 4 ° C to obtain the hydrogel system (abbreviated as Gel-EXO-RAPA) of EXO-RAPA. 12 AR model mice were reconstructed and randomly divided into 2 groups for nasal administration of EXO-RAPA and Gel-EXO-RAPA. The treatment plan is the same as in Example 4 above, wherein the nasal administration of Gel-EXO-RAPA is to slowly drip 20 μL of gel system into the mouse nasal cavity, which cannot completely fill the nasal cavity and can be administered multiple times until absorbed. Every two days after dosing and challenge, the mice were observed and scored using a scoring table: the amount of nasal discharge, the number of sneezes, and the number of nasal scratchings within 30 minutes were observed and recorded, and the total score was calculated using the cumulative method. Mice in each group that completed the experiment were euthanized, and their serum was obtained and tested by ELISA to measure the levels of the pro-inflammatory cytokines IL-1β and IL-6.

[0098] The results showed that, compared with the EXO-RAPA group, Gel-EXO-RAPA significantly alleviated the sneezing, runny nose and nose scratching behaviors of AR mice. After the treatment, the score dropped to 4 ( Figure 6 A). By Figure 6 As shown in Figures B and 6C, compared with the mice in the EXO-RAPA group, the levels of proinflammatory cytokines IL-1β and IL-6 in the serum were reduced by 15% and 11.5%, respectively, through nasal administration of Gel-EXO-RAPA, which was better than the simple treatment effect of EXO-RAPA.

[0099] The above results show that after nasal administration, Gel-EXO-RAPA can more significantly improve the sneezing, runny nose and nose scratching behaviors of AR mice than EXO-RAPA, and also more significantly improve the inflammation level of AR. The therapeutic effect is stronger than EXO-RAPA and close to the positive control group budesonide. Therefore, the hydrogel enhances the retention time of RAPA in the nasal cavity and can enhance the symptom relief of AR mice by RAPA, indicating the potential enhanced medicinal properties of the hydrogel.

[0100] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0101] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of rapamycin or a pharmaceutically acceptable salt, ester, prodrug or metabolite thereof in the preparation of a medicament for preventing or treating allergic rhinitis.

2. The use according to claim 1, characterized in that The drug is administered through the nasal cavity.

3. The use according to claim 1, characterized in that The dosage form of the drug is selected from the group including but not limited to liquid dosage form and gel dosage form.

4. A pharmaceutical composition, characterized in that include: Rapamycin or its pharmaceutically acceptable salt, ester, prodrug or metabolite is used as an active ingredient, and optionally a pharmaceutically acceptable excipient or carrier.

5. The pharmaceutical composition according to claim 4, characterized in that The carrier includes but is not limited to exosomes and hydrogels.

6. The pharmaceutical composition according to claim 5, characterized in that The exosomes are derived from plant cells or animal cells; Optionally, the animal cells include stem cells; Optionally, the stem cells are derived from, including but not limited to, umbilical cord, bone marrow, embryo, placenta, fat, bone, cartilage, periodontium, synovium, muscle, lung, liver, pancreas; Optionally, the stem cells are mesenchymal stem cells; Optionally, the stem cells include but are not limited to: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, synovial membrane mesenchymal stem cells, and thymus mesenchymal stem cells; Optionally, the stem cells are derived from animals including but not limited to mice, rats, rabbits, dogs, pigs, and primates.

7. The pharmaceutical composition according to claim 6, characterized in that The hydrogel is selected from the group consisting of, but not limited to, gelatin, sodium alginate hydrogel, carboxymethyl chitosan hydrogel, polyvinyl alcohol hydrogel, povidone hydrogel, and carbomer hydrogel.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is administered through the nasal cavity.

9. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition includes but is not limited to liquid dosage form and gel dosage form.

10. Use of the pharmaceutical composition according to any one of claims 4 to 9 in the preparation of a medicament for preventing or treating allergic rhinitis.

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