Pharmaceutical compositions for treating mast cell-mediated inflammatory diseases

Abstract

The present invention relates to the use of atovaquone for the treatment and prevention of acute and chronic inflammatory diseases in mammals, including mast cell-mediated inflammatory diseases.

Description

[Technical Field]

[0001] The present invention relates to the technical field of medicine, specifically to the field of pharmacotherapy of inflammatory diseases. [Background technology]

[0002] The inflammatory response is a tissue cellular response that occurs in vivo in response to pathogenic microbial infection, trauma, anaphylaxis, and other conditions. It is physiologically and pathophysiologically beneficial and is necessary for wound healing, relief from viral or bacterial infection, and recovery. However, excessive inflammatory responses can be harmful, resulting in allergic and allergy-like reactions, hypersensitivity, and idiopathic inflammation. Mast cells are central players in the initiation and maintenance of inflammation.

[0003] Mast cells are one of the key effector cells in the inflammatory response process, a key link between the nervous and immune systems, and are involved in various pathological processes such as postoperative pain and skin inflammation. Compared to other innate immune cells, mast cells have a significant spatial advantage, being located close to peripheral nerve endings and responding quickly to the activation of sensory nerve fibers. Activated by neuropeptides, mast cells release various "inflammatory cytokines" and "chemokines," which are involved in the recruitment of various naturally occurring immune cells, further contributing to the inflammatory cascade and sensitization of peripheral sensory afferents.

[0004] The peripheral nervous system receives stimuli from changes in the external environment and transmits that information to the central nervous system, creating various sensations. Various receptors distributed in the peripheral nervous system play important roles, and G protein-coupled receptors are one of the many receptors that play a key role in olfaction, taste, pain, itch, and other sensations. The recently discovered G protein-coupled receptor family is primarily distributed in the peripheral nervous system, and the function of MrGPRS receptors has attracted increasing attention from researchers in related fields. MrGPRS is a type of G protein-coupled receptor related to the Mas gene. Young et al. analyzed the hydrophobicity map of the Mas amino acid sequence and, with the aid of an algorithm that can accurately predict the transmembrane segments of many proteins, found that Mas contains seven hydrophobic transmembrane domains, suggesting that this protein belongs to the G protein-coupled receptor (GPCR) family.

[0005] MrgprX2 was initially found to be highly expressed in human DRG small-diameter injury sensory neurons and was hypothesized to play an important role in injury perception. It was also found to be highly expressed in mast cells, and recent studies have shown that it is also expressed in human eosinophils and basophils and mediates their degranulation. Mast cells, as one of the key effector cells in the immune response, play a crucial role connecting the nervous and immune systems. Due to their proximity to nerve terminals, mast cells have a distinct spatial advantage over other innate immune cells and are the first to respond to sensory nerve activation. Upon mast cell activation, they release various inflammatory cytokines and chemokines, which act on specific receptors on sensory nerve terminals, triggering the release of neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP) from the nerve terminals. This then activates receptors on mast cells, inducing further mast cell degranulation and the release of numerous inflammatory factors and chemokines, leading to neurogenic inflammation. In addition to these local interactions between mast cells and sensory nerves, nociceptive signals also induce Ca ions in nerve terminals. 2+Neurogenic peptides such as SP and VIP can activate mast cells through MrgprX2 receptors, which are amplified by interneuronal axon potentials, triggering excitation of terminal fibers and generating nerve signals that subsequently travel along axons to the central nervous system, suggesting that MrgprX2 contributes to the development of neurogenic inflammation and pain.

[0006] Recent studies have demonstrated that MrgprB2- / - mice exhibit significantly reduced inflammatory and nociceptive hypersensitivity responses compared to control mice in both postoperative and CFA-induced inflammatory pain models. Furthermore, MrgprB2- / - mice exhibit significantly reduced recruitment of innate immune cells at the injury site, leading to the hypothesis that mast cell-neuron interactions may be involved: SP released after tissue injury activates mast cells via MrgprB2, leading to the release of cytokines and chemokines and the recruitment of immune cells, contributing to inflammation and pain. As research into MrgprB2 / MrgprX2 continues to deepen, the function of MrgprB2 / MrgprX2 is becoming increasingly important. This receptor is not only widely involved in non-IgE-mediated hypersensitivity, neurogenic inflammation, pain, and convulsive responses, but also promotes innate immune responses in the skin and intestine against noxious stimuli and pathogen invasion. Therefore, this receptor is considered a potential target for the development of anti-inflammatory and analgesic drugs. Summary of the Invention

[0007] The present invention provides a drug for treating and preventing acute and chronic inflammatory diseases in mammals, including mast cell-mediated inflammatory diseases, the drug being atovaquone. [ka]

[0008] Atovaquone, a hydroxy 1,4-naphthoquinoline, is a naphthoquinone analog of coenzyme Q and is active against several protozoan parasites. Its site of action against protozoan parasites is the cytochrome B C1 binding site (binding site III). The drug reversibly binds to an 11,500 Da molecular motif on a peptide. Dihydroorotate dehydrogenase, a key enzyme in pyridine biosynthesis, connects mitochondria via electron transfer via coenzyme Q, inhibiting pyridine synthesis by blocking electron transfer. Mitochondrial electron transfer via coenzyme Q involves many metabolic enzymes, and atovaquone inhibits electron transfer, thereby inhibiting the activity of these enzymes, resulting in broad-spectrum antiparasitic activity.

[0009] While the broad antiparasitic activity of atovaquone has been widely studied, its other pharmacological activities have not been extensively explored. The present invention provides a novel use of atovaquone for the treatment and prevention of various disorders associated with excessive inflammatory responses by inhibiting mast cell degranulation. These uses relate to the effective treatment and prevention of various inflammatory diseases involving mast cells, including, but not limited to, allergies and atopic diseases (allergic asthma, eczema, rhinitis), mast cell activation syndrome (MCAS), physical and chemical urticaria, spontaneous urticaria, Crohn's disease, inflammatory bowel disease, dermatitis and contact dermatitis, arthritis and rheumatoid arthritis, skin, tissue, or systemic reactions to stings or other allergic or allergy-like stimuli, canine mastocytosis, and bovine and porcine allergies and inflammation. Furthermore, it relates to allergic dermatitis or other allergic diseases such as skin eczema, dermatitis, atopic dermatitis, contact dermatitis, neurodermatitis and urticaria in adults or in children or infants.

[0010] In contrast to the existing use of corticosteroid therapy for the treatment and / or prevention of reactions involving excessive inflammation, which have significant side effects and use-dependence, results from numerous user-reported populations of atovaquone indicate a low side effect profile and no significant drug dependence with long-term use.

[0011] Therefore, the present invention has important implications for the treatment and prevention of various inflammatory diseases.

[0012] One aspect of the present invention provides a pharmaceutical composition comprising the active ingredient atovaquone.

[0013] The pharmaceutical composition inhibits mast cell degranulation to treat and prevent various diseases associated with excessive inflammatory responses, including, but not limited to, allergies and atopic diseases (allergic asthma, eczema, rhinitis), mast cell activation syndrome (MCAS), physical and chemical urticaria, spontaneous urticaria, Crohn's disease, inflammatory bowel disease, dermatitis and contact dermatitis, arthritis and rheumatoid arthritis, skin, tissue, or systemic reactions to stings or other allergic or allergy-like stimuli, canine mastocytosis, bovine and porcine allergies and inflammation, etc. Furthermore, the pharmaceutical composition relates to allergic dermatitis or other allergic diseases, such as skin eczema, dermatitis, atopic dermatitis, contact dermatitis, neurodermatitis, urticaria, etc., in adults, children, or infants.

[0014] Some embodiments of the present invention relate to pharmaceutical compositions comprising the active drug ingredient atovaquone and a pharmaceutically acceptable carrier or diluent, and in some embodiments, the pharmaceutical composition is an ointment, emulsion, emulsified paste, solution, patch, gel, aerosol, tablet, granule, injectable solution, etc.

[0015] In some embodiments, the pharmaceutical composition is adapted for administration by inhalation, vaporizer, nebulizer, or aerosol. In some embodiments, the pharmaceutical composition is formulated for oral administration, oral administration, or sublingual administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration, intramuscular administration, or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intrathecal administration or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical administration.

[0016] In some embodiments, the active pharmaceutical ingredient is present in the pharmaceutical composition at a concentration of 0.0001 to 100 mg / ml, preferably 0.0001 to 50 mg / ml.

[0017] In another aspect, the present invention provides a method for treating an immune system disease, the method comprising administering an effective amount of a pharmaceutical composition of the present invention.

[0018] In some embodiments, the immune system disease is allergy or atopy. In some embodiments, the immune system disease is mast cell activation syndrome ("MCAS"), physical and chemical urticaria, spontaneous urticaria, Crohn's disease, inflammatory bowel disease, dermatitis and contact dermatitis, arthritis and rheumatoid arthritis, skin, tissue, or systemic reactions to stings or other allergic or allergy-like stimuli, canine mastocytosis, bovine or porcine allergies and inflammation, etc. Furthermore, the present invention relates to allergic dermatitis or other allergic diseases, such as skin eczema, dermatitis, atopic dermatitis, contact dermatitis, neurodermatitis, urticaria, etc., in adults, children, or infants. Allergic and inflammatory diseases of the respiratory and digestive systems include, but are not limited to, asthma, rhinitis, allergic pneumonia, allergic enteritis, etc.

[0019] In some embodiments, the immune disease is a disease associated with CB1 or CB2 receptor dysregulation or mast cell cAMP dysregulation, hi some embodiments, the immune disease is a disease associated with overactivation of CB1 or CB2 receptors or mast cell cAMP inhibition.

[0020] In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered by oral administration. In some embodiments, the pharmaceutical composition is delivered sublingually. In some embodiments, the pharmaceutical composition is administered by injection. In some embodiments, the pharmaceutical composition is administered topically, preferably by topical transdermal administration.

[0021] In some embodiments, atovaquone is administered in an amount of less than 1.5 mg per dose. In some embodiments, atovaquone is administered in an amount of 1000 ng per dose. In some embodiments, atovaquone is administered in an amount of less than 500 ng per dose. In some embodiments, atovaquone is administered in an amount of 100 ng per dose. In some embodiments, atovaquone is administered in an amount of 10 ng per dose.

[0022] In some embodiments, the pharmaceutical composition is administered as needed. In some embodiments, the pharmaceutical composition is administered once daily. In some embodiments, the pharmaceutical composition is administered two to four times daily. In some embodiments, the pharmaceutical composition is administered two to four times weekly. In some embodiments, the pharmaceutical composition is administered once weekly. In some embodiments, the pharmaceutical composition is administered once every two weeks.

[0023] These and other aspects of the invention are described in more detail below. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the inhibition rate of mast cell degranulation by drugs. [Figure 2] This is a dynamic observation diagram of calcium ion changes when substance P acts on different receptors. [Figure 3] Calcium ion flow curves at different drug concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0025] Active drug ingredients In a first aspect, the present invention provides a pharmaceutical composition having atovaquone as a primary active ingredient.

[0026] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition comprising the active ingredient atovaquone and a pharmaceutically acceptable carrier or diluent. The active ingredient may further comprise a second active substance different from atovaquone, which may be selected from the group consisting of antihistamines, corticosteroid hormones, mast cell stabilizers (e.g., sodium cromoglycate), leukotriene receptor antagonists (e.g., montelukast), antimicrobial agents (for fungal, bacterial, viral, chlamydial, and mycoplasmal infections, e.g., itraconazole), drugs containing the active ingredient of licorice extract (e.g., glycyrrhizin, diammonium glycyrrhizinate), calmodulin phosphatase inhibitors (e.g., tacrolimus, pimecrolimus), immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate), monoclonal antibodies (e.g., dupilumab), and rheumatoid and immunological agents expected to treat allergic inflammation.

[0027] Active pharmaceutical ingredient content In exemplary embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 0.001 μg / ml, at least 0.01 μg / ml, at least 0.5 μg / ml, or at least 1 μg / ml. In some embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, or 25 μg / ml. In some embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, or 50 μg / ml.

[0028] Common drug formulations The pharmaceutical compositions may be in any form suitable for human or veterinary use, including ointments, emulsions, emulsified pastes, solutions, patches, gels, aerosols, tablets, granules, injectables, and the like.

[0029] Pharmaceutical compositions may be formulated for administration by any route of administration suitable for human or veterinary use, including enteral and parenteral routes of administration.

[0030] In various embodiments, the formulated pharmaceutical composition is for administration by inhalation, the formulated pharmaceutical composition is for oral, buccal, or sublingual administration, the formulated pharmaceutical composition is for intravenous, intramuscular, or subcutaneous administration, and the formulated pharmaceutical composition is for topical administration, e.g., topical dermal administration, topical mucosal administration.

[0031] Mucosal administration formulations are administered as dry powder formulations, typically lyophilized, consisting of a dry form of a bioactive agent having an appropriate particle size or range of particle sizes, typically for nasal administration. The minimum particle size suitable for deposition in the nasal or pulmonary passages is typically about 0.5 microns median mass equivalent aerodynamic diameter (MMEAD), typically about 1 micron MMEAD, and more typically about 2 microns MMEAD. The maximum particle size suitable for deposition in the nasal cavity is typically about 10 microns MMEAD, typically about 8 microns MMEAD, and more typically about 4 microns MMEAD. Nasally inhalable powders within these size ranges can be produced by a variety of conventional techniques, including jet milling, spray drying, solvent precipitation, and supercritical fluid condensation. These suitable MMEAD dry powders can be administered to patients via conventional dry powder inhalers (DPIs), which rely on the patient's breathing to disperse the powder into the nebulizer volume after pulmonary or nasal inhalation. Alternatively, the dry powder can be administered via an air-assisted device that uses an external power source, such as a piston pump, to disperse the powder into a nebulized volume.

[0032] Pharmaceutical compositions suitable for oral / buccal / sublingual administration Formulations for oral, buccal, or sublingual administration contain a predetermined amount of the subject polypeptide therapeutic as an active ingredient in the form of capsules, flat capsules, pills, tablets, dragees (using a flavored matrix, typically sucrose and gum arabic or yarrow), powders, granules, or a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion, or a melt or syrup, or lozenges (using an inert matrix, e.g., gelatin and glycerol, or sucrose and gum arabic), and / or mouthwash, etc. In addition to the active compound, suspensions also contain co-suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite clay, agar, and yarrow gum, and mixtures thereof.

[0033] In solid dosage forms (such as capsules, tablets, pills, dragees, powders, granules, etc.) for oral, buccal, or sublingual administration, the therapeutic agent(s) can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dibasic calcium phosphate, and / or (1) fillers or extenders such as starch, lactose, sucrose, dextrose, mannitol, and / or silicic acid, (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic, and (3) humectants such as glycerol. (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginates, certain silicates, and sodium carbonate; (5) solution retardants such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetearyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, and mixtures thereof; and (10) coloring agents. For capsules, tablets, and pills, pharmaceutical compositions can also contain buffering agents. Solid compositions of a similar type can also be used as fillers for soft-filled and hard-filled gelatin capsules, particularly using excipients such as lactose or lactose and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and fluxes. In addition to the active ingredient, the liquid dosage forms may further contain water or other solvents, solubilizing agents, and inert diluents commonly used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of dehydrated sorbitol, and mixtures thereof.Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0034] Pharmaceutical compositions suitable for injection For intravenous, intramuscular, subcutaneous, or localized injection, the active ingredient is in the form of a non-pyrogenic, parenterally acceptable aqueous solution having appropriate pH, isotonicity, and stability. Those skilled in the art can prepare appropriate solutions using, for example, isotonic media (e.g., sodium chloride injection, Ringer's injection, lactated Ringer's injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0035] In various embodiments, the unit-dosage form is a vial, an ampoule, a bottle, or a pre-filled syringe. In some embodiments, the unit-dosage form contains 0.01 μg, 0.1 μg, 0.5 μg, 1 μg, 2.5 μg, 5 μg, 10 μg, 12.5 μg, 25 μg, 50 μg, 75 μg, or 100 μg of the atovaquone composition. In some embodiments, the unit-dosage form contains 125 μg, 150 μg, 175 μg, or 200 μg of the atovaquone composition. In some embodiments, the unit-dosage form contains 250 μg of the atovaquone composition.

[0036] In some embodiments, the pharmaceutical composition in unit dosage form is in a solid form suitable for dissolution, such as a lyophilized material.

[0037] Pharmaceutical compositions and formulations suitable for topical administration may include transdermal patches, ointments, detergents, emulsified pastes, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily matrices, thickeners, and the like may be necessary or desirable.

[0038] General Dosage Range In vivo and / or in vitro assays may optionally be employed to help determine optimal dosage ranges for use. The precise dosage to be employed in a formulation will also depend on the route of administration and the severity of the symptoms, and should be based on the judgment of the skilled artisan and each individual's circumstances. Effective amounts may be based on dose-response curves derived from in vitro or animal model test systems.

[0039] Method of Use: Method of Treating Mast Cell-Mediated Inflammatory Diseases In another aspect, a method for treating an individual suffering from an immune system disorder is provided. In a typical embodiment, the immune system disorder is a mast cell-associated inflammatory disorder, and the immune system disorders to be treated include, but are not limited to, mast cell activation syndrome ("MCAS"), physical and chemical urticaria, spontaneous urticaria, Crohn's disease, inflammatory bowel disease, dermatitis and contact dermatitis, arthritis and rheumatoid arthritis, skin, tissue, or systemic reactions to stings or other allergic or allergy-like stimuli, canine mastocytosis, bovine and porcine allergies and inflammation, etc. Furthermore, the present invention relates to allergic dermatitis or other allergic disorders, such as skin eczema, dermatitis, atopic dermatitis, contact dermatitis, neurodermatitis, urticaria, etc., in adults, children, or infants. Allergic inflammatory disorders of the respiratory and digestive systems include, but are not limited to, asthma, rhinitis, allergic pneumonia, allergic enteritis, etc.

[0040] As used herein, the term "treatment" and the like generally refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative, in terms of complete or partial prevention of a disease, condition, or its symptoms, and / or therapeutic, in terms of partial or complete cure of a disease or condition and / or adverse reactions (e.g., symptoms) resulting from the disease or condition. As used herein, "treatment" includes any treatment of a disease or condition in a mammal, particularly a human, including (a) preventing the onset of the disease or condition in an individual susceptible to the disease or condition but not yet diagnosed with the disease or condition, (b) inhibiting the disease or condition (e.g., halting its progression), or (c) alleviating the disease or condition (e.g., causing remission of the disease or condition, providing improvement in one or more symptoms). Improvement of any condition can be readily assessed according to standard methods and techniques known in the art. Individuals treated by the present methods include those suffering from an undesirable condition or disease, as well as those at risk of developing the condition or disease.

[0041] The term "therapeutically effective dose" or "effective amount" refers to the dose or amount administered to produce the desired effect. The exact dose or amount will depend on the purpose of the treatment and can be determined by one of ordinary skill in the art using known techniques.

[0042] In some embodiments, the pharmaceutical composition is administered once daily, 2-4 times daily, 2-4 times weekly, once weekly, or once every two weeks.

[0043] Depending on the condition being treated, the compositions may be administered alone or sequentially, either simultaneously or in combination with other treatments.

[0044] The present invention provides pharmaceutical compositions comprising atovaquone, which have been shown to have significant anti-inflammatory activity, and such compositions can be used to treat immunological conditions, such as those associated with excessive inflammatory responses, such as abnormally increased release of histamine from mast cells.

[0045] Example The following examples are offered by way of illustration only and not by way of limitation.

[0046] Example 1: Inhibition study of mast cell degranulation by atovaquone The experimental method for the β-hexosaminidase assay includes the following steps: 1. Prepare a PNAG / HEPES buffer / 0.1% Triton-100 / glycine solution at room temperature. 2. Remove the LAD2 cells from the 37℃ incubator, count the cells, and extract an appropriate amount of cell suspension (estimated at 5,000-10,000 cells / well) according to your experimental cell needs. 3. Centrifuge the LAD2 cell suspension at 4°C for 5 min, and remove the supernatant after 3000 rps. 4. After adding HEPES buffer, mix the cells evenly into the solution. 5. Transfer the cell suspension to a 96-well plate, 80 μl per well. 6. According to the experimental design, the test drug atovaquone was added to the corresponding position, and the positive control SP (neuropeptide P substance), negative control QWF (MRGPRX2 receptor antagonist) and blank control positions were reserved. Each drug was used three times. 7. Incubate the cells and drug for 30 minutes. 8. Add SP to all plate wells except for the blank control and incubate for 30 min. After centrifugation at 3000 rps for 9.5 min at low temperature, 50 μl of the supernatant was taken and added to a supernatant plate containing 50 μl / well of PNAG, and incubated for 90 min. 10. After removing the supernatant, add 150 μL / well of 0.1% Triton-100 to the remaining solution, and then add 50 μL / well of the solution to a cell lysis plate to which 50 μL / well of PNAG had been added in advance. Incubate for 90 minutes. 11. After removing the two plates, add 50ul / well of glycine solution to each well. m. Absorbance value detection, 12, percent mast cell degranulation.

[0047] Calculate according to the formula: Percent degranulation = 2 x supernatant plate absorbance value / (supernatant plate absorbance value + 4 x cell lysis plate absorbance value) x 100%.

[0048] Test Results: As can be seen from the LAD2 mast cell degranulation percentage graph in Figure 1, the addition of atovaquone and QWF significantly improved LAD2 mast cell degranulation in the SP stimulation experiment of LAD2 cells, with atovaquone being far superior to QWF. That is, atovaquone can significantly inhibit mast cell degranulation.

[0049] Example 2: Intracellular calcium ion mobility experiments The incubation buffer consisted of 0.5 μI Fluo-3, 2 μI Pluronic F-127, and 997.5 μI CIB (calcium imaging buffer: 125 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 10 mM HEPES, 20 mM glucose, 1.2 mM NaHCO3, and 20 mM sucrose, pH 7.4). Different concentrations of atovaquone (0, 1, 10, 100 nM, and 1 μM) were added to the incubation buffer. An incubation buffer without atovaquone was used as a negative control. LAD2 cells were washed twice with CIB and then added to the required incubation buffer. After a 30-min incubation, the cells were washed twice with CIB and then seeded into a 96-well plate for calcium imaging. For calcium imaging, cells were magnified 200x and photographed once per second under blue light. After substance injection, [Ca 2+ Cells were confirmed as responding if the ] increased by at least 50%.

[0050] The experimental results are shown in Figures 2 and 3: Figure 2 shows the dynamic changes in calcium ion mobilization when substance P acts on different receptors: MrgA1, MrgX2, and NK1R (5 nM acts on NK1R, 50 nM acts on MrgX2, and 10 μM acts on MrgA1). This set of images was taken from left to right at 5-second intervals for 120 seconds. The top row of images shows cells with atovaquone treatment, and the bottom row shows cells without atovaquone treatment. The effects of atovaquone treatment on calcium ion mobility via MrgA1 and NK1R were not as strong as those on MrgX2 treatment.

[0051] FIG. 3 shows that different concentrations of atovaquone resulted in a decrease in calcium ion mobility, and at atovaquone concentrations above 10 nM / L, calcium ions began to show no significant mobility.

[0052] Thus, atovaquone significantly reduced the inflammatory activity of mast cells.

Claims

1. A pharmaceutical composition for treating or preventing a mast cell-mediated inflammatory disease, said pharmaceutical composition comprising the active ingredient atovaquone.

2. 2. The pharmaceutical composition according to claim 1, wherein the concentration of the active ingredient atovaquone is 0.0001 to 100 mg / ml.

3. The pharmaceutical composition of claim 1 or 2, further comprising a second active ingredient.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is in the form of an ointment, emulsion, emulsified paste, solution, patch, gel, aerosol, tablet, granule, or injection.

6. Use of atovaquone in a pharmaceutical composition for treating or preventing a mast cell-mediated inflammatory disease.

7. 7. The use according to claim 6, wherein the inflammatory diseases are allergies and atopy, mast cell activation syndrome (MCAS), physical and chemical urticaria, spontaneous urticaria, Crohn's disease, inflammatory bowel disease, dermatitis and contact dermatitis, arthritis and rheumatoid arthritis, skin, tissue or systemic reactions to stings or other allergic or allergy-like stimuli, canine mastocytosis, bovine, porcine allergies and inflammations.

8. 8. The use according to claim 7, wherein the inflammatory diseases further include skin eczema, dermatitis, atopic dermatitis, contact dermatitis, neurodermatitis, urticaria in adults or children or infants.

9. The use according to any one of claims 6 to 8, wherein the pharmaceutical composition is administered by systemic or local administration.

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