WO2020244508A1 - Nadh和/或nadph抗过敏药物和/或抗过敏保健食品中的应用 - Google Patents

Nadh和/或nadph抗过敏药物和/或抗过敏保健食品中的应用 Download PDF

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Publication number
WO2020244508A1
WO2020244508A1 PCT/CN2020/093955 CN2020093955W WO2020244508A1 WO 2020244508 A1 WO2020244508 A1 WO 2020244508A1 CN 2020093955 W CN2020093955 W CN 2020093955W WO 2020244508 A1 WO2020244508 A1 WO 2020244508A1
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Prior art keywords
allergic
injection
nicotinamide adenine
adenine dinucleotide
sodium salt
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PCT/CN2020/093955
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English (en)
French (fr)
Inventor
陈建生
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泓博元生命科技（深圳）有限公司
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Publication of WO2020244508A1 publication Critical patent/WO2020244508A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • This application belongs to the field of drug and health food research and development, and particularly relates to nicotinamide adenine dinucleotide and its sodium salt and/or nicotinamide adenine dinucleotide phosphate and its sodium salt in anti-allergic drugs and/or anti-allergic health care Application in food.
  • Allergies are a series of hypersensitivity phenomena caused by human body contacting some of the allergic factors in the environment that have little effect on ordinary people, that is, the phenomenon of excessive immunity of the human body to certain foreign substances. Allergies include: immediate allergic reactions (also known as IgE-mediated allergic reactions), antibody-dependent and cytotoxic allergic reactions, immune complex-mediated allergic reactions, and delayed allergic reactions.
  • common anti-allergic drugs include: antihistamines, glucocorticoids, anti-leukotrienes, intranasal decongestants, etc., all of which have certain adverse reactions and require repeated medication to control symptoms. Allergies are easy to recur.
  • nicotinamide adenine dinucleotide treats allergic diseases, such as contact dermatitis, but it is only limited to external application, and the treatment effect is poor, and it is easy to relapse.
  • nicotinamide adenine dinucleotide and its sodium salt NADH
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • this application provides the application of nicotinamide adenine dinucleotide and its sodium salt and/or nicotinamide adenine dinucleotide phosphate and its sodium salt in anti-allergic drugs and/or anti-allergic health foods It is used to solve the technical defects of anti-allergic drugs in the prior art, such as adverse reactions, repeated medications and easy recurrence of allergies.
  • This application provides a nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) in anti-allergic drugs and/or anti-allergic health care Application in food.
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • a composition of nicotinamide adenine dinucleotide and its sodium salt (NADH) and nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) is used in anti-allergic drugs and/or anti-allergic health foods.
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • the allergic reaction of the anti-allergic drug and/or anti-allergic health food is selected from any one of allergic asthma, allergic rhinitis, allergic conjunctivitis, drug allergy, food allergy, pollen allergy and oral allergy Or multiple.
  • the mass ratio of nicotinamide adenine dinucleotide and its sodium salt (NADH) to nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) is (1-10) :(1 ⁇ 10).
  • the nicotinamide adenine dinucleotide is reduced nicotinamide adenine dinucleotide.
  • the allergic reaction of the anti-allergic drug and/or anti-allergic health food is selected from the group consisting of: IL10, TGF- ⁇ , and IFN- ⁇ immunosuppressive cytokine regulated immune response.
  • the dosage form of the anti-allergic drug and/or anti-allergic health food is oral or injection.
  • the oral agent is selected from any one or more of tablets, powders, capsules, granules, pills, suspensions, syrups, mixtures, powders, oral liquids and dripping pills.
  • the injection is selected from: solution injection, suspension injection, emulsion injection and sterile powder for injection; injection method is selected from intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, spinal injection and joint injection Internal injection.
  • the anti-allergic medicine and/or the auxiliary material of the anti-allergic health food is selected from: mannitol, microcrystalline cellulose, magnesium stearate, carboxymethyl cellulose, calcium hydrogen phosphate, starch, sucrose, dextrin, Lactose, calcium sulfate, mannitol, microcrystalline cellulose, talc, micronized silica gel, magnesium stearate, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, sodium alginate, polyethylene glycol , Magnesium aluminum silicate, polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl fiber, carboxymethyl cellulose, sodium bicarbonate, citric acid, tartaric acid, stearic acid, calcium stearate, hydrogenated vegetable oil , Purified water, calcium hydrogen phosphate and water for injection, oil for injection (sesame oil, tea oil), ethanol, glycerin
  • the medicine and/or health food does not contain lactose.
  • the oral single dose of nicotinamide adenine dinucleotide and its sodium salt or nicotinamide adenine dinucleotide phosphate and its sodium salt is 0.05-10 mg/kg, and the number of oral administrations per day is 1-3 Times;
  • the single injection dose of nicotinamide adenine dinucleotide and its sodium salt or nicotinamide adenine dinucleotide phosphate and its sodium salt is 0.05-2 mg/kg.
  • the anti-allergic drug and/or anti-allergic health food does not contain other than nicotinamide adenine dinucleotide and its sodium salt and/or nicotinamide adenine dinucleotide phosphate and its sodium salt Anti-allergic active ingredients.
  • the dosage of NADH and/or NADPH can be flexibly adjusted according to the severity of the allergic reaction, the method of administration and the specific physiological factors of the allergic patient;
  • the safe dosage of NADH and/or NADPH for a single oral administration is 0.05 ⁇ 10mg/kg, preferably 0.1 ⁇ 4mg/kg
  • the safe dose of NADH and/or NADPH for oral administration is 0.05 ⁇ 30mg/kg, preferably 0.1 ⁇ 12mg/kg, more preferably 0.4 ⁇ 2mg/kg
  • the single safe dose for injection of NADH and/or NADPH is 0.05-2 mg/kg, preferably 0.1-1 mg/kg.
  • NADH and/or NADPH are used as active ingredients in the preparation of anti-allergic drugs or health foods to provide a long-acting natural anti-allergic drug or health food with immunomodulatory effects; further, this application provides In the technical solution, the oral preparation used is to treat delayed-onset allergies, and NADH and/or NADPH can regulate immunity, so as to achieve long-term effects and improve allergic constitution to treat allergies.
  • this application provides a nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) in anti-allergic drugs and / Or application in anti-allergic health food.
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • taking NADH/NADPH can induce Treg cells to secrete immunosuppressive cytokines IL10, TGF- ⁇ , etc., thereby inhibiting the immune response of TH2, blood basophils and tissue mast cells, reducing sIgE synthesis while increasing
  • the synthesis of sIgG and sIgA can reduce allergic symptoms; at the same time, it can stimulate the TH1 response of allergens, produce cytokines IFN- ⁇ and TGF- ⁇ , inhibit TH2, blood basophilic and tissue mast cell immune response, thereby reducing
  • the synthesis of sIgE also increases the synthesis of sIgG and sIgA, thereby reducing allergic symptoms.
  • NADH and/or NADPH anti-allergic drugs and/or anti-allergic health foods solves the technical defects in the prior art that anti-allergic drugs have some adverse reactions, need to be used repeatedly, and allergies are prone to recurrence.
  • the embodiments of this application provide the application of NADH and/or NADPH in anti-allergic drugs and/or anti-allergic health foods, which are used to solve the problems of anti-allergic drugs that have some adverse reactions, need to be used repeatedly, and allergies are prone to recurrence in the prior art.
  • Technical defects providing a long-acting natural anti-allergic drug or health food with immunomodulatory effect.
  • This application provides a nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) in anti-allergic drugs and/or anti-allergic health care Application in food.
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • taking NADH/NADPH can induce Treg cells to secrete immunosuppressive cytokines IL10, TGF- ⁇ , etc., thereby inhibiting the immune response of TH2, blood basophils and tissue mast cells, reducing sIgE synthesis while increasing The synthesis of sIgG and sIgA can ultimately relieve allergic symptoms.
  • taking NADH/NADPH can also stimulate the TH1 response of allergens, produce cytokines IFN- ⁇ and TGF- ⁇ , and inhibit TH2, blood basophilic and tissue mast cell immunity Response, thereby reducing the synthesis of sIgE and increasing the synthesis of sIgG and sIgA, ultimately reducing allergic symptoms.
  • the allergic reactions of anti-allergic drugs and/or anti-allergic health foods prepared from NADH and/or NADPH are selected from: allergic asthma, allergic rhinitis, allergic conjunctiva Any one or more of inflammation, drug allergy, food allergy, pollen allergy, and oral allergy.
  • the allergic reaction of the anti-allergic drug and/or anti-allergic health food is selected from: IL10, TGF- ⁇ and IFN- ⁇ immunosuppressive Immune response regulated by cytokines.
  • the dosage forms of anti-allergic drugs and/or anti-allergic health foods prepared from NADH and/or NADPH are selected from: tablets, injections, powders, capsules, granules, pills, suspensions, Any one or more of syrup, mixture, powder, oral liquid and dripping pills.
  • the coating of the coated tablet is acid Stable coating.
  • the anti-allergic drugs and/or anti-allergic health food supplements are selected from :Mannitol, microcrystalline cellulose, magnesium stearate, carboxymethyl cellulose, dibasic calcium phosphate, starch, sucrose, dextrin, lactose, calcium sulfate, talc, micronized silica gel, methyl cellulose, hydroxypropyl Methyl cellulose, ethyl cellulose, sodium alginate, polyethylene glycol, magnesium aluminum silicate, polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl fiber, sodium bicarbonate, citric acid, tartaric acid , Stearic acid, calcium stearate, hydrogenated vegetable oil, purified water, and water for injection, oil for injection (sesame oil
  • the prepared drugs and/or health foods do not contain lactose as an auxiliary material to prevent lactose from reacting with NADH and/or NADPH to reduce the efficacy of the drug.
  • nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide A single oral dose of adenine dinucleotide phosphate and its sodium salt (NADPH) is 0.05-10mg/kg; nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide adenine dinucleotide
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH uronic acid phosphate and its sodium salt
  • NADPH uronic acid phosphate and its sodium salt
  • C3H/HeJ peanut allergic mice were prepared by the following method: 6-week-old female C3H/HeJ mice were purchased from Shanghai Experimental Animal Center. The test animals were reared in a room with an average temperature of 21°C to 23°C and a relative humidity of 40% to 70% under specific non-pathogenic conditions, and a 12-hour light/dark cycle.
  • mice in the sensitized group were given intraperitoneal injection (ip) of 30 mg CPE (peanut extract) per day by intraperitoneal injection (ip) of peanut homogenate (80 mg/each) every day. Rats, pups that have never been exposed to peanuts were used as negative controls. Then, through skin tests and allergic reactions (by measuring vascular leakage, monitoring clinical symptoms, rectal temperature, respiratory rate and measuring serum mast cell protease-1 (mmcp-1)-a specific marker of mast cell degranulation) The test confirms the successful establishment of the sensitized mouse model.
  • CPE peanut extract
  • peanut homogenate 80 mg/each
  • C3H/HeJ 6-week-old C3H/HeJ female mice (strain: C3H/HeJ, purchased by Shanghai Experimental Animal Center), IL10, TGF- ⁇ , IFN- ⁇ and IgE antibodies (purchased by Sigma-aldrich), NADH (provided by yourself).
  • mice Randomly take 6 wild-type mice and 24 peanut-sensitized mice and randomly divide the peanut-sensitized mice into 4 groups, each with 6 mice.
  • the grouping is as follows:
  • Treatment group 1 C3H/HeJ peanut allergic mice (injected with NADH 1mg/kg body weight);
  • Treatment group 2 C3H/HeJ peanut allergic mice (injection of NADH 5mg/kg body weight);
  • Treatment group 3 C3H/HeJ peanut allergic mice (injected with NADH 10mg/kg body weight);
  • Control group 4 C3H/HeJ peanut allergic mice (injection of physiological saline 10mg/kg body weight)
  • C3H/HeJ wild-type mice and C3H/HeJ peanut allergic mice were injected intraperitoneally with 30mg CPE (peanut extract)/mouse after sensitization.
  • the blank control group was injected with normal saline 10 mg/kg body weight
  • the treatment groups 1 to 3 were injected with NADH 1 mg/kg body weight, 5 mg/kg body weight, and 10 mg/kg body weight respectively
  • the treatment group 4 was injected with normal saline 10 mg/kg body weight.
  • the intracanthal venous blood of each group of mice was taken to detect the contents of the cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ and IgE in the peripheral blood serum of the mice.
  • C3H/HeJ peanut allergic mice treated with different concentrations of NADH can increase the levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ in peripheral blood and reduce the content of allergenic protein IgE to varying degrees.
  • Table 1 is a numerical table of NADH injection to increase the levels of cellular immunosuppressive factors IL10, TGF- ⁇ and IFN- ⁇ and decrease the level of IgE antibodies in the allergic mouse model.
  • the C3H/HeJ peanut allergic mice in the test examples of this application were prepared by the following method: 6-week-old female C3H/HeJ mice were purchased from Shanghai Experimental Animal Center. The test animals were reared in a room with an average temperature of 21°C-23°C and a relative humidity of 40%-70% under specific non-pathogenic conditions, and a 12-hour light/dark cycle.
  • mice in the sensitized group were given intraperitoneal injection (ip) of 30 mg CPE (peanut extract) per day by intraperitoneal injection (ip) of peanut homogenate (80 mg/each) every day. Rats, pups that have never been exposed to peanuts were used as negative controls. Then through skin tests and allergic reactions (by measuring vascular leakage, monitoring clinical symptoms, rectal temperature, respiratory rate and measuring serum mast cell protease-1 (mmcp-1)-a specific marker for mast cell degranulation) Confirm the successful establishment of the sensitized mouse model.
  • CPE peanut extract
  • peanut homogenate 80 mg/each
  • C3H/HeJ 6-week-old C3H/HeJ female mice (strain: C3H/HeJ, purchased by Shanghai Experimental Animal Center), IL10, TGF- ⁇ , IFN- ⁇ and IgE antibodies (purchased by Sigma-aldrich), NADH (provided by yourself).
  • mice Six wild-type mice and 24 peanut-sensitized mice were randomly selected, and the peanut-sensitized mice were randomly divided into 4 groups with 6 mice in each group.
  • the grouping is as follows:
  • Treatment group 1 C3H/HeJ peanut allergic mice (during the sensitization process, they were given a general diet and orally supplemented with NADH 1 mg/kg body weight (including excipients);
  • Treatment group 2 C3H/HeJ peanut allergic mice (in the sensitization process, they are given a general diet and orally supplemented with NADH 10mg/kg body weight (including excipients);
  • Treatment group 3 C3H/HeJ peanut allergic mice (in the course of sensitization, they were given a general diet and supplemented with NADH 20mg/kg body weight (including excipients);
  • Control group C3H/HeJ peanut allergic mice (during the sensitization process, given the general diet and the same amount of excipients as the treatment group 3)
  • C3H/HeJ wild-type mice given a general diet
  • C3H/HeJ mice were supplemented with different concentrations of NADH during the peanut sensitization process, and the mice in each group were raised 21 days later.
  • Mice in each group were sensitized by intraperitoneal injection of 30mg CPE (peanut extract)/mouse.
  • mice After sensitization by injection of CPE, blood from the intracanthal veins of each group of mice was taken to detect the levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ and IgE in the peripheral blood serum of mice.
  • C3H/HeJ peanut allergic mice treated with different concentrations of NADH can increase the levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ in peripheral blood and reduce the content of sensitizing protein IgE to varying degrees.
  • agarose-protein G Thermo Fisher Scientific, Rockford, IL
  • Table 2 is a numerical table showing that oral NADH increases the levels of cellular immunosuppressive factors IL10, TGF- ⁇ and IFN- ⁇ and decreases the level of IgE antibodies in the allergic mouse model.
  • IFN- ⁇ TGF- ⁇ IL-10 IgE Blank group 420 ⁇ 4.1pg/ml 42.23 ⁇ 3.56pg/ml 9.27 ⁇ 0.89pg/ml 2.12 ⁇ 0.36KU/L Control group 226 ⁇ 8.33pg/ml 26.85 ⁇ 6.24pg/ml 6.11 ⁇ 1.41pg/ml 4.67 ⁇ 0.55KU/L Treatment group 1 298 ⁇ 7.82pg/ml 29.03 ⁇ 4.11pg/ml 7.12 ⁇ 0.98pg/ml 4.01 ⁇ 0.29KU/L Treatment group 2 404 ⁇ 3.35pg/ml 40.09 ⁇ 5.17pg/ml 9.11 ⁇ 1.60pg/ml 2.67 ⁇ 0.21KU/L Treatment group 3 383 ⁇ 6.06pg/ml 36.53 ⁇ 6.27pg/ml 8.18 ⁇ 0.95pg/ml 3.21 ⁇ 0.61KU/L
  • PBMC peripheral blood of healthy children of the same age and peripheral blood of children with food allergy (PBMC), from Shenzhen Children's Hospital.
  • IL10, TGF- ⁇ , IFN- ⁇ and IgE antibodies purchased by Sigma-aldrich
  • NADH provided by yourself.
  • Healthy group PBMC (10 ⁇ 7/ml) of healthy children were suspended and cultured in the culture medium (normal saline 0.2mg/ml was added to the culture medium;
  • Blank group PBMC (10 ⁇ 7/ml) of children with food allergies were suspended and cultured in the medium (normal saline 0.2mg/ml was added to the medium);
  • Treatment group 1 PBMC (10 ⁇ 7/ml) of children with food allergies were suspended and cultured in the medium (NADH 0.02mg/ml was added to the medium);
  • Treatment group 2 PBMC (10 ⁇ 7/ml) of children with food allergies were suspended and cultured in the culture medium (NADH 0.1mg/ml was added to the culture medium);
  • Treatment group 3 PBMC (10 ⁇ 7/ml) of children with food allergies are suspended and cultured in the medium (NADH 0.2mg/ml is added to the medium)
  • PBMC treated with different concentrations of NADH can increase the content of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ in peripheral blood and reduce the content of sensitizing protein IgE to varying degrees;
  • Table 3 is a numerical table for adding NADH to the culture medium to cultivate the peripheral blood (PBMC) of children with food allergies, increasing the levels of immunosuppressive factors IL10, TGF- ⁇ and IFN- ⁇ of PBMC cells of children with food allergies and reducing the level of IgE antibodies.
  • PBMC peripheral blood
  • the C3H/HeJ peanut allergic mice in the test examples of this application were prepared by the following method: 6-week-old female C3H/HeJ mice were purchased from Shanghai Experimental Animal Center. The test animals were reared in a room with an average temperature of 21°C-23°C and a relative humidity of 40%-70% under specific non-pathogenic conditions, and a 12-hour light/dark cycle.
  • mice in the sensitized group were given intraperitoneal injection (ip) of 30 mg CPE (peanut extract) per day by intraperitoneal injection (ip) of peanut homogenate (80 mg/each) every day. Rats, pups that have never been exposed to peanuts were used as negative controls. Then through skin tests and allergic reactions (by measuring vascular leakage, monitoring clinical symptoms, rectal temperature, respiratory rate and measuring serum mast cell protease-1 (mmcp-1)-a specific marker for mast cell degranulation) Confirm the successful establishment of the sensitized mouse model.
  • CPE peanut extract
  • peanut homogenate 80 mg/each
  • C3H/HeJ 6-week-old C3H/HeJ female mice (strain: C3H/HeJ, purchased by Shanghai Experimental Animal Center), IL10, TGF- ⁇ , IFN- ⁇ and IgE antibodies (purchased by Sigma-aldrich), NADPH (provided by yourself).
  • mice Six wild-type mice and 24 peanut-sensitized mice were randomly selected, and the peanut-sensitized mice were randomly divided into 4 groups with 6 mice in each group.
  • the grouping is as follows:
  • Treatment group 1 C3H/HeJ peanut allergic mice (injected with NADPH 1mg/kg body weight);
  • Treatment group 2 C3H/HeJ peanut allergic mice (injected with NADPH 5mg/kg body weight);
  • Treatment group 3 C3H/HeJ peanut allergic mice (injected with NADPH 10mg/kg body weight);
  • Control group 4 C3H/HeJ peanut allergic mice (injection of physiological saline 10mg/kg body weight)
  • C3H/HeJ wild-type mice and C3H/HeJ peanut allergic mice were injected intraperitoneally with 30mg CPE (peanut extract)/mouse after sensitization.
  • the blank control group was injected with normal saline 10 mg/kg body weight
  • the treatment groups 1-3 were injected with NADPH 1 mg/kg body weight, 5 mg/kg body weight, and 10 mg/kg body weight respectively
  • the treatment group 4 was injected with normal saline 10 mg/kg body weight.
  • the intracanthal venous blood of each group of mice was taken to detect the contents of the cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ and IgE in the peripheral blood serum of the mice.
  • C3H/HeJ peanut allergic mice treated with different concentrations of NADPH can increase the levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ in peripheral blood and reduce the content of allergenic protein IgE to varying degrees.
  • Table 4 is a numerical table showing that injection of NADPH increases the levels of cellular immunosuppressive factors IL10, TGF- ⁇ and IFN- ⁇ and decreases the level of IgE antibodies in the allergic mouse model.
  • Treatment group 3 426 ⁇ 5.22pg/ml 48.4 ⁇ 2.46pg/ml 9.81 ⁇ 0.47pg/ml 3.29 ⁇ 0.34KU/L
  • the C3H/HeJ peanut allergic mice in the test examples of this application were prepared by the following method: 6-week-old female C3H/HeJ mice were purchased from Shanghai Experimental Animal Center. The test animals were reared in a room with an average temperature of 21°C to 23°C and a relative humidity of 40% to 70% under specific non-pathogenic conditions, and a 12-hour light/dark cycle. The mice in the sensitized group were given peanut homogenate every day (80 mg/each).
  • mice were given intraperitoneal injection (i.p) injection of 30 mg CPE (peanut extract)/mouse, and pups that had never been exposed to peanuts were used as negative controls. Then through skin tests and allergic reactions (by measuring vascular leakage, monitoring clinical symptoms, rectal temperature, respiratory rate and measuring serum mast cell protease-1 (mmcp-1)-a specific marker for mast cell degranulation) Confirm the successful establishment of the sensitized mouse model.
  • CPE peanut extract
  • mmcp-1 serum mast cell protease-1
  • C3H/HeJ 6-week-old C3H/HeJ female mice (strain: C3H/HeJ, purchased by Shanghai Experimental Animal Center), IL10, TGF- ⁇ , IFN- ⁇ and IgE antibodies (purchased by Sigma-aldrich), NADPH (provided by yourself).
  • mice Six wild-type mice and 24 peanut-sensitized mice were randomly selected, and the peanut-sensitized mice were randomly divided into 4 groups with 6 mice in each group.
  • the grouping is as follows:
  • Treatment group 1 C3H/HeJ peanut allergic mice (in the course of sensitization, they were given a general diet and supplemented with NADPH 1 mg/kg body weight (including excipients);
  • Treatment group 2 C3H/HeJ peanut allergic mice (in the course of sensitization, they are given a general diet and orally supplemented with NADPH 10mg/kg body weight (including excipients);
  • Treatment group 3 C3H/HeJ peanut allergic mice (in the course of sensitization, they are given a general diet and orally supplemented with NADPH 20mg/kg body weight (including excipients);
  • Control group C3H/HeJ peanut allergic mice (during the sensitization process, given the general diet and the same amount of excipients as the treatment group 3)
  • C3H/HeJ wild-type mice given a general diet
  • C3H/HeJ mice were fed with different concentrations of NADPH during the peanut sensitization process for 21 days.
  • Mice in each group were sensitized by intraperitoneal injection of 30mg CPE (peanut extract)/mouse.
  • mice After injection of CPE sensitization, the intracanthal venous blood of each group of mice was taken, and the serum levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ and IgE in the peripheral blood of mice were detected.
  • C3H/HeJ peanut allergic mice treated with different concentrations of NADPH can increase the levels of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ in peripheral blood and reduce the content of allergenic protein IgE to varying degrees.
  • Table 5 is a numerical table showing that oral administration of NADPH increases the levels of cellular immunosuppressive factors IL10, TGF- ⁇ and IFN- ⁇ and decreases the level of IgE antibodies in the allergic mouse model.
  • PBMC peripheral blood of children with food allergy
  • Healthy group PBMC (10 ⁇ 7/ml) of healthy children were suspended and cultured in the medium (with 0.2mg/ml normal saline added to the medium);
  • Blank group PBMC (10 ⁇ 7/ml) of children with food allergies were suspended and cultured in the medium (normal saline 0.2mg/ml was added to the medium);
  • Treatment group 1 PBMC (10 ⁇ 7/ml) of children with food allergies are suspended and cultured in the medium (add NADPH 0.02mg/ml in the medium);
  • Treatment group 2 PBMC (10 ⁇ 7/ml) of children with food allergies were suspended and cultured in the medium (add NADPH 0.1mg/ml in the medium);
  • Treatment group 3 PBMC (10 ⁇ 7/ml) of children with food allergies are suspended and cultured in the medium (add NADPH 0.2mg/ml in the medium)
  • PBMC treated with different concentrations of NADPH can increase the content of cellular immunosuppressive factors IL10, TGF- ⁇ , IFN- ⁇ and reduce the content of sensitizing protein IgE in different degrees.
  • the control group was given loratadine tablets (10 mg/tablet), which was used according to the drug instructions for one month.
  • Experimental group 1 was given NADH 10 mg, once a day on an empty stomach in the morning, for 1 month.
  • Experimental group 2 was given NADPH 10mg, once a day on an empty stomach in the morning, for 1 month.
  • the experimental group 3 was given NADH:NADPH 5mg:5mg, once a day on an empty stomach in the morning, for 1 month.
  • this application provides a nicotinamide adenine dinucleotide and its sodium salt (NADH) and/or nicotinamide adenine dinucleotide phosphate and its sodium salt (NADPH) in anti-allergic drugs and / Or application in anti-allergic health food.
  • NADH nicotinamide adenine dinucleotide and its sodium salt
  • NADPH nicotinamide adenine dinucleotide phosphate and its sodium salt
  • taking NADH/NADPH can induce Treg cells to secrete immunosuppressive cytokines IL10, TGF- ⁇ , etc., thereby inhibiting the immune response of TH2, blood basophils and tissue mast cells, reducing sIgE synthesis while increasing
  • the synthesis of sIgG and sIgA can reduce allergic symptoms; at the same time, it can stimulate the TH1 response of allergens, produce cytokines IFN- ⁇ and TGF- ⁇ , inhibit TH2, blood basophilic and tissue mast cell immune response, thereby reducing
  • the synthesis of sIgE also increases the synthesis of sIgG and sIgA, thereby reducing allergic symptoms.
  • NADH and/or NADPH anti-allergic drugs and/or anti-allergic health foods solves the technical defects in the prior art that anti-allergic drugs have some adverse reactions, need to be used repeatedly, and allergies are prone to recurrence.

Abstract

属于药品、保健食品研发领域，尤其涉及NADH和／或NADPH在抗过敏药物和／或抗过敏保健食品中的应用。提供了一种NADH和／或NADPH在抗过敏药物和／或抗过敏保健食品中的应用。服用NADH／NADPH，可诱导Treg细胞分泌免疫抑制细胞因子IL10、TGF-β等，从而抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状；同时，还可刺激过敏原特异的TH1反应，产生细胞因子IFN一γ和TGF-β，抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，从而减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状；解决了现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷，提供一种长效、具有免疫调节作用的天然抗过敏药物或保健食品。

Description

NADH和/或NADPH抗过敏药物和/或抗过敏保健食品中的应用 技术领域
本申请属于药品、保健食品研发领域，尤其涉及烟酰胺腺嘌呤二核苷酸及其钠盐和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐在抗过敏药物和/或抗过敏保健食品中的应用。
背景技术
过敏为人体接触环境中部分对一般人影响不大的过敏原因子后，所引发的一系列超敏反应现象，也即人体对于某些外源性物质过度免疫的现象。过敏包括：即发性过敏反应(又称IgE介导型过敏反应)、抗体依赖型和细胞毒杀过敏反应、免疫复合体媒介过敏反应以及迟发性过敏反应等。
现有技术中，常见的抗过敏的药物包括：抗组胺药、糖皮质激素、抗白三烯药、鼻内减充血剂等，这些药物均存在一定不良反应，且需反复用药控制症状，过敏容易复发。为克服上述不良反应，有若干最新报道烟酰胺腺嘌呤二核苷酸治疗过敏疾病，比如接触性皮炎，但只限于外涂，且治疗效果差，易复发。
因此，研发出烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)抗过敏药物和/或抗过敏保健食品中的应用，用于解决现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷，成为了本领域技术人员亟待解决的问题。
申请内容
有鉴于此，本申请提供了烟酰胺腺嘌呤二核苷酸及其钠盐和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐在抗过敏药物和/或抗过敏保健食品中的应用，用于解决现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷。
本申请提供了一种烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺 腺嘌呤二核苷酸磷酸及其钠盐(NADPH)在抗过敏药物和/或抗过敏保健食品中的应用。
优选地，一种烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)组合物在抗过敏药物和/或抗过敏保健食品中的应用。
优选地，所述抗过敏药物和/或抗过敏保健食品的过敏反应选自：过敏性哮喘、过敏性鼻炎、过敏性结膜炎、药物过敏、食物过敏、花粉过敏及口腔过敏中的任意一种或多种。
优选地，所述组合物中，烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)的质量比为(1～10):(1～10)。
优选地，所述烟酰胺腺嘌呤二核苷酸为还原型烟酰胺腺嘌呤二核苷酸。
优选地，所述抗过敏药物和/或抗过敏保健食品的过敏反应选自：IL10、TGF-β以及IFN-γ免疫抑制细胞因子所调控的免疫反应。
优选地，所述抗过敏药物和/或抗过敏保健食品的剂型为口服剂或注射剂。
优选地，所述口服剂选自：片剂、粉剂、胶囊剂、颗粒剂、丸剂、混悬剂、糖浆剂、合剂、散剂、口服液以及滴丸中的任意一种或多种。
优选地，所述注射剂选自：溶液型注射剂、混悬性注射剂、乳剂型注射剂和注射用灭菌粉末；注射方式选自皮内注射、皮下注射、肌内注射、静脉注射、脊椎注射和关节内注射。
优选地，所述抗过敏药物和/或抗过敏保健食品的辅料选自：甘露醇、微晶纤维素、硬脂酸镁、羧甲基纤维素、磷酸氢钙、淀粉、蔗糖、糊精、乳糖、硫酸钙、甘露醇、微晶纤维素、滑石粉、微粉硅胶、硬脂酸镁、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、海藻酸钠、聚乙二醇、硅酸镁铝、聚乙烯吡咯烷酮、羧甲基淀粉钠、低取代羟丙基纤维、羧甲基纤维素、碳酸氢钠、枸橼酸、酒石酸、硬脂酸、硬脂酸钙、氢化植物油、纯化水、磷酸氢钙以及注射用水、注射用油(麻油、茶油)、乙醇、甘油、丙二醇、聚乙二醇、苯甲酸苄酯、二甲基乙酰胺、油酸乙酯、乳酸乙酯中的任意一种或多种；
所述药物和/或保健食品中不含有乳糖。
优选地，所述烟酰胺腺嘌呤二核苷酸及其钠盐或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的口服单次剂量是0.05～10mg/kg，日口服次数为1～3次；
所述烟酰胺腺嘌呤二核苷酸及其钠盐或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的注射单次剂量为0.05～2mg/kg。
优选地，所述抗过敏药物和/或抗过敏保健食品中，除烟酰胺腺嘌呤二核苷酸及其钠盐和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐外不含有其它抗过敏活性成分。
在实际应用时，可根据过敏反应的严重程度、给药方式及过敏患者具体的生理相关因素，灵活调整NADH和/或NADPH的用量；其中，单次口服NADH和/或NADPH的安全剂量为0.05～10mg/kg，优选是0.1～4mg/kg，日口服合适的NADH和/或NADPH的安全剂量为0.05～30mg/kg，优选是0.1～12mg/kg，更优选0.4～2mg/kg；当用于注射时，可以制备成常规的注射剂，注射NADH和/或NADPH的单次安全剂量是0.05～2mg/kg，优选是0.1～1mg/kg。
本申请中，NADH和/或NADPH是作为活性成分应用在制备治疗抗过敏药物或保健食品中，提供一种长效、具有免疫调节作用的天然抗过敏药物或保健食品；进一步地，本申请提供的技术方案中，所采用的口服制剂，治疗的是迟发性过敏，利用的是NADH和/或NADPH能够调节免疫力，从而达到长效、改善过敏体质的作用来治疗过敏。
综上所述，本申请提供了一种烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)在抗过敏药物和/或抗过敏保健食品中的应用。本申请提供的技术方案中，服用NADH/NADPH，可诱导Treg细胞分泌免疫抑制细胞因子IL10、TGF-β等，从而抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状；同时，还可刺激过敏原特意的TH1反应，产生细胞因子IFN-γ和TGF-β，抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，从而减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状。本申请提供的NADH和/或NADPH抗过敏药物和/或抗过敏保健食品中的应用，解决了现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷，提供一种 长效、具有免疫调节作用的天然抗过敏药物或保健食品。
具体实施方式
本申请实施例提供了NADH和/或NADPH在抗过敏药物和/或抗过敏保健食品中的应用，用于解决现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷，提供一种长效、具有免疫调节作用的天然抗过敏药物或保健食品。
下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
为了更详细说明本申请，下面结合实施例对本申请提供的NADH和/或NADPH抗过敏药物和/或抗过敏保健食品中的应用，进行具体地描述。
本申请提供了一种烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)在抗过敏药物和/或抗过敏保健食品中的应用。
本申请提供的技术方案中，服用NADH/NADPH，可诱导Treg细胞分泌免疫抑制细胞因子IL10、TGF-β等，从而抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，减少sIgE合成同时增加sIgG和sIgA合成，最终减轻过敏症状或者，服用NADH/NADPH，还可刺激过敏原特意的TH1反应，产生细胞因子IFN-γ和TGF-β，抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，从而减少sIgE合成同时增加sIgG和sIgA合成，最终减轻过敏症状。
同时，进一步结合实施例1～6可以得出，NADH和/或NADPH对于过敏反应具有良好的抑制和治疗作用。
根据实验统计可得，本申请提供的技术方案中，由NADH和/或NADPH制得的抗过敏药物和/或抗过敏保健食品的过敏反应选自：过敏性哮喘、过敏性鼻炎、过敏性结膜炎、药物过敏、食物过敏、花粉过敏及口腔过敏中的任意一种或多种。
进一步地，根据NADH和/或NADPH作用靶点，本申请实施例提供的技术方案中，抗过敏药物和/或抗过敏保健食品的过敏反应选自：IL10、TGF-β以及IFN-γ免疫抑制细胞因子所调控的免疫反应。
在实际应用的过程中，由NADH和/或NADPH制得的抗过敏药物和/或抗过敏保健食品的剂型选自：片剂、注射剂、粉剂、胶囊剂、颗粒剂、丸剂、混悬剂、糖浆剂、合剂、散剂、口服液以及滴丸中的任意一种或多种。
为有效防止的NADH和/或NADPH在胃酸不稳定、易分解，分解后其生物利用度降低，为有效提高生物利用度本申请实施例提供的技术方案中，包衣片剂的包衣为酸性稳定包衣。
为使得NADH和/或NADPH产品可以维持较好的形态、便于服用以及提高产品保存期限的设计需求，本申请实施例提供的技术方案中，抗过敏药物和/或抗过敏保健食品的辅料选自：甘露醇、微晶纤维素、硬脂酸镁、羧甲基纤维素、磷酸氢钙、淀粉、蔗糖、糊精、乳糖、硫酸钙、滑石粉、微粉硅胶、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、海藻酸钠、聚乙二醇、硅酸镁铝、聚乙烯吡咯烷酮、羧甲基淀粉钠、低取代羟丙基纤维、碳酸氢钠、枸橼酸、酒石酸、硬脂酸、硬脂酸钙、氢化植物油、纯化水、及注射用水、注射用油(麻油、茶油)、乙醇、甘油、丙二醇、聚乙二醇、苯甲酸苄酯、二甲基乙酰胺、油酸乙酯、乳酸乙酯中的任意一种或多种。
本申请提供的技术方案中，所制得的药品和/或保健食品中，不含有乳糖作为辅料，以防止乳糖与NADH和/或NADPH发生反应而降低药效。
根据临床实验测定结果，兼顾以良好的抗过敏效果及较低的不良反应，本申请实施例提供的技术方案中，烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)的口服单次剂量是0.05～10mg/kg；烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)的合适单次注射剂量为0.05～2mg/kg。
实施例1
本实施例中，C3H/HeJ花生过敏小鼠用下述方法制备得到：6周龄雌性C3H/HeJ小鼠，购自上海实验动物中心。受试动物在特定的无致病性条件下， 在平均温度为21℃～23℃，相对湿度40％～70％的房间里，12小时的光照/黑暗循环的饲养。
致敏组小鼠每天灌胃花生匀浆(单次80mg/每只)，在致敏措施实施后的第21天，给予小鼠腹腔内注射(i.p)注射30mg CPE(花生提取物)/小鼠，从未接触过花生的幼鼠作为阴性对照。然后，通过皮肤实验和过敏反应(通过测定血管渗漏、监测临床症状、直肠温度、呼吸频率和测量血清中肥大细胞蛋白酶-1(mmcp-1)-肥大细胞脱颗粒的特异性标记物)的检测确定致敏小鼠模型的成功建立。
1.1动物及实验试剂：
6周龄C3H/HeJ雌性小鼠(品系：C3H/HeJ，上海实验动物中心购买)，IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)，NADH(自备)。
1.2实验方法
随机取6只野生型小鼠和24只花生致敏小鼠并将花生致敏小鼠随机分为4组，每组6只小鼠。分组如下：
①、空白组：C3H/HeJ野生型小鼠(注射生理盐水10mg/kg体重)；
②、处理组1：C3H/HeJ花生过敏小鼠(注射NADH 1mg/kg体重)；
③、处理组2：C3H/HeJ花生过敏小鼠(注射NADH 5mg/kg体重)；
④、处理组3：C3H/HeJ花生过敏小鼠(注射NADH 10mg/kg体重)；
⑤、对照组4：C3H/HeJ花生过敏小鼠(注射生理盐水10mg/kg体重)
将C3H/HeJ野生型小鼠和C3H/HeJ花生过敏小鼠均腹腔注射30mg CPE(花生提取物)/小鼠致敏后。空白对照组注射生理盐水10mg/kg体重，处理组1～3分别注射NADH 1mg/kg体重、5mg/kg体重、10mg/kg体重，处理组4注射生理盐水10mg/kg体重。
在用药后的16小时，取各组小鼠目内眦静脉血,检测小鼠外周血血清中细胞免疫抑制因子IL10、TGF-β、IFN-γ和IgE的含量。
1.3结果
不同浓度的NADH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含 量。
涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将血清样品在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。用相应的抗体检测样品。
用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量结果见表1。
表1为注射NADH增加过敏性小鼠模型细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表1
分组 IFN-γ TGF-β IL-10 IgE
空白组 426±3.54pg/ml 41.26±4.08pg/ml 9.63±0.77pg/ml 2.32±0.29KU/L
对照组 219±6.63pg/ml 29.85±5.23pg/ml 5.79±2.06pg/ml 4.27±0.31KU/L
处理组1 388±5.02pg/ml 36.63±3.95pg/ml 8.02±1.18pg/ml 3.38±0.24KU/L
处理组2 437±2.05pg/ml 43.09±4.28pg/ml 9.07±2.25pg/ml 2.22±0.19KU/L
处理组3 418±5.53pg/ml 39.46±5.66pg/ml 9.81±1.39pg/ml 2.52±0.48KU/L
实施例2
本申请试验例中C3H/HeJ花生过敏小鼠用下述方法制备得到：6周龄雌性C3H/HeJ小鼠，购自上海实验动物中心。受试动物在特定的无致病性条件下，在平均温度为21℃-23℃，相对湿度40％-70％的房间里，12小时的光照/黑暗循环的饲养。
致敏组小鼠每天灌胃花生匀浆(单次80mg/每只)，在致敏措施实施后的第21天，给予小鼠腹腔内注射(i.p)注射30mg CPE(花生提取物)/小鼠，从未接触过花生的幼鼠作为阴性对照。然后通过皮肤实验和过敏反应(通过测定血管渗 漏、监测临床症状、直肠温度、呼吸频率和测量血清中肥大细胞蛋白酶-1(mmcp-1)-肥大细胞脱颗粒的特异性标记物)的检测确定致敏小鼠模型的成功建立。
2.1动物及实验试剂：
6周龄C3H/HeJ雌性小鼠(品系：C3H/HeJ，上海实验动物中心购买)，IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)，NADH(自备)。
2.2实验方法
随机取6只野生型小鼠和24只花生致敏小鼠，并将花生致敏小鼠随机分为4组，每组6只小鼠。分组如下：
①、空白组：C3H/HeJ野生型小鼠(给予一般饮食)；
②、处理组1：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADH 1mg/kg体重(含辅料)；
③、处理组2：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADH 10mg/kg体重(含辅料)；
④、处理组3：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADH 20mg/kg体重(含辅料)；
⑤、对照组：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食和与处理组3等量的辅料)
将C3H/HeJ野生型小鼠(给予一般饮食)和C3H/HeJ小鼠在花生致敏过程中补充不同浓度NADH，各组小鼠饲养21天后。各组小鼠均腹腔注射30mg CPE(花生提取物)/小鼠致敏。
注射CPE致敏后，取各组小鼠目内眦静脉血，检测小鼠外周血血清中细胞免疫抑制因子IL10、TGF-β、IFN-γ和IgE的含量。
3.3结果
不同浓度的NADH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量。涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将血清样品在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为 了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。
用相应的抗体检测样品。用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量结果见表2。
表2为口服NADH增加过敏性小鼠模型细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表2
分组 IFN-γ TGF-β IL-10 IgE
空白组 420±4.1pg/ml 42.23±3.56pg/ml 9.27±0.89pg/ml 2.12±0.36KU/L
对照组 226±8.33pg/ml 26.85±6.24pg/ml 6.11±1.41pg/ml 4.67±0.55KU/L
处理组1 298±7.82pg/ml 29.03±4.11pg/ml 7.12±0.98pg/ml 4.01±0.29KU/L
处理组2 404±3.35pg/ml 40.09±5.17pg/ml 9.11±1.60pg/ml 2.67±0.21KU/L
处理组3 383±6.06pg/ml 36.53±6.27pg/ml 8.18±0.95pg/ml 3.21±0.61KU/L
实施例3
3.1实验材料及实验试剂：
同龄的健康儿童外周血和食物过敏儿童外周血(PBMC),来自深圳市儿童医院。IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)，NADH(自备)。
3.2实验方法
随机取健康儿童的外周血和食物过敏儿童外周血(PBMC),将PBMC悬浮于有10％自体血浆的培养基(RPMI-1640；Mediatech)中，并在37℃，5％CO2细胞培养箱中培养分组如下：
①、健康组：健康儿PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入生理盐水0.2mg/ml；
②、空白组：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入生理盐水0.2mg/ml)；
③、处理组1：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加NADH 0.02mg/ml)；
④、处理组2：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入NADH 0.1mg/ml)；
⑤、处理组3：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入NADH 0.2mg/ml)
⑥、在各组PBMC的培养基中加入不同浓度NADH体外培养72小时后通过ELISA检测5组外周血中血清中IgE，IL-10，IFN-γ和TGF-β的含量。
3.3结果
不同浓度的NADH处理的PBMC能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量；
涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将培养基上清液在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。用相应的抗体检测样品。用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADH处理的食物过敏儿童的PBMC，能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量结果见表3。
表3为在培养基中添加NADH培养食物过敏儿童外周血(PBMC)，增加食物过敏儿童PBMC细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表3
Figure PCTCN2020093955-appb-000001
Figure PCTCN2020093955-appb-000002
实施例4
本申请试验例中C3H/HeJ花生过敏小鼠用下述方法制备得到：6周龄雌性C3H/HeJ小鼠，购自上海实验动物中心。受试动物在特定的无致病性条件下，在平均温度为21℃-23℃，相对湿度40％-70％的房间里，12小时的光照/黑暗循环的饲养。
致敏组小鼠每天灌胃花生匀浆(单次80mg/每只)，在致敏措施实施后的第21天，给予小鼠腹腔内注射(i.p)注射30mg CPE(花生提取物)/小鼠，从未接触过花生的幼鼠作为阴性对照。然后通过皮肤实验和过敏反应(通过测定血管渗漏、监测临床症状、直肠温度、呼吸频率和测量血清中肥大细胞蛋白酶-1(mmcp-1)-肥大细胞脱颗粒的特异性标记物)的检测确定致敏小鼠模型的成功建立。
4.1动物及实验试剂：
6周龄C3H/HeJ雌性小鼠(品系：C3H/HeJ，上海实验动物中心购买)，IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)，NADPH(自备)。
4.2实验方法
随机取6只野生型小鼠和24只花生致敏小鼠，并将花生致敏小鼠随机分为4组，每组6只小鼠。分组如下：
①、空白组：C3H/HeJ野生型小鼠(注射生理盐水10mg/kg体重)；
②、处理组1：C3H/HeJ花生过敏小鼠(注射NADPH 1mg/kg体重)；
③、处理组2：C3H/HeJ花生过敏小鼠(注射NADPH 5mg/kg体重)；
④、处理组3：C3H/HeJ花生过敏小鼠(注射NADPH 10mg/kg体重)；
⑤、对照组4：C3H/HeJ花生过敏小鼠(注射生理盐水10mg/kg体重)
将C3H/HeJ野生型小鼠和C3H/HeJ花生过敏小鼠均腹腔注射30mg CPE(花生提取物)/小鼠致敏后。空白对照组注射生理盐水10mg/kg体重，处理组1-3分别注射注射NADPH 1mg/kg体重、5mg/kg体重、10mg/kg体重，处理组4注射生理盐水10mg/kg体重。
在用药后的16小时，取各组小鼠目内眦静脉血,检测小鼠外周血血清中细胞免疫抑制因子IL10、TGF-β、IFN-γ和IgE的含量。
4.3结果
不同浓度的NADPH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量。
涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将血清样品在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。
用相应的抗体检测样品。用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADPH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量结果见表4。
表4为注射NADPH增加过敏性小鼠模型细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表4
分组 IFN-γ TGF-β IL-10 IgE
空白组 418±2.33pg/ml 45.36±3.65pg/ml 10.25±0.69pg/ml 2.57±0.31KU/L
对照组 262±4.58pg/ml 30.48±6.01pg/ml 6.01±1.62pg/ml 6.26±0.54KU/L
处理组1 329±3.83pg/ml 40.05±4.02pg/ml 6.88±0.51pg/ml 4.22±0.37KU/L
处理组2 397±2.16pg/ml 38.63±2.21pg/ml 12.54±0.29pg/ml 4.09±0.41KU/L
处理组3 426±5.22pg/ml 48.4±2.46pg/ml 9.81±0.47pg/ml 3.29±0.34KU/L
实施例5
本申请试验例中C3H/HeJ花生过敏小鼠用下述方法制备得到：6周龄雌性C3H/HeJ小鼠，购自上海实验动物中心。受试动物在特定的无致病性条件下，在平均温度为21℃～23℃，相对湿度40％～70％的房间里，12小时的光照/黑暗循环的饲养。致敏组小鼠每天灌胃花生匀浆(单次80mg/每只)。
在致敏措施实施后的第21天，给予小鼠腹腔内注射(i.p)注射30mg CPE(花生提取物)/小鼠，从未接触过花生的幼鼠作为阴性对照。然后通过皮肤实验和过敏反应(通过测定血管渗漏、监测临床症状、直肠温度、呼吸频率和测量血清中肥大细胞蛋白酶-1(mmcp-1)-肥大细胞脱颗粒的特异性标记物)的检测确定致敏小鼠模型的成功建立。
5.1动物及实验试剂
6周龄C3H/HeJ雌性小鼠(品系：C3H/HeJ，上海实验动物中心购买)，IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)，NADPH(自备)。
5.2实验方法
随机取6只野生型小鼠和24只花生致敏小鼠，并将花生致敏小鼠随机分为4组，每组6只小鼠。分组如下：
①、空白组：C3H/HeJ野生型小鼠(给予一般饮食)；
②、处理组1：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADPH1mg/kg体重(含辅料)；
③、处理组2：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADPH10mg/kg体重(含辅料)；
④、处理组3：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食并口服补充NADPH 20mg/kg体重(含辅料)；
⑤、对照组：C3H/HeJ花生过敏小鼠(在致敏过程中给予一般饮食和与处理组3等量的辅料)
将C3H/HeJ野生型小鼠(给予一般饮食)和C3H/HeJ小鼠在花生致敏过程中补充不同浓度NADPH的各组小鼠饲养21天后。各组小鼠均腹腔注射30mg  CPE(花生提取物)/小鼠致敏。
注射CPE致敏后，取各组小鼠目内眦静脉血,检测小鼠外周血血清中细胞免疫抑制因子IL10、TGF-β、IFN-γ和IgE的含量。
5.3结果
不同浓度的NADPH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量。
涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将血清样品在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。
用相应的抗体检测样品。用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADPH处理的C3H/HeJ花生过敏小鼠能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量结果见表5。
表5为口服NADPH增加过敏性小鼠模型细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表5
分组 IFN-γ TGF-β IL-10 IgE
空白组 433±3.2pg/ml 40.9±2.8pg/ml 10.01±0.52pg/ml 2.68±0.68KU/L
对照组 254±5.11pg/ml 20.12±5.35pg/ml 7.29±0.93pg/ml 6.92±0.44KU/L
处理组1 246±7.64pg/ml 25.36±3.15pg/ml 7.96±1.01pg/ml 5.06±0.49KU/L
处理组2 389±2.65pg/ml 43.21±4.12pg/ml 12.50±0.87pg/ml 2.94±0.81KU/L
处理组3 426±3.43pg/ml 40.67±8.01pg/ml 11.00±1.26pg/ml 3.21±0.24KU/L
实施例6
6.1实验材料及实验试剂：
同龄的健康儿童外周血和食物过敏儿童外周血(PBMC)，来自深圳市儿童 医院。IL10、TGF-β、IFN-γ和IgE抗体(Sigma-aldrich购买)。
6.2实验方法
随机取健康儿童的外周血和食物过敏儿童外周血(PBMC),将PBMC悬浮于有10％自体血浆的培养基(RPMI-1640；Mediatech)中，并在37℃，5％CO2细胞培养箱中培养分组如下：
①、健康组：健康儿PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入生理盐水0.2mg/ml)；
②、空白组：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入生理盐水0.2mg/ml)；
③、处理组1：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加NADPH 0.02mg/ml)；
④、处理组2：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入NADPH 0.1mg/ml)；
⑤、处理组3：食物性过敏儿童PBMC(10^7/ml)悬浮于培养基中培养(培养基中加入NADPH 0.2mg/ml)
在各组PBMC的培养基中加入不同浓度NADPH体外培养72小时后通过ELISA检测4组外周血中血清中IgE，IL-10，IFN-γ和TGF-β的含量。
6.3结果
不同浓度的NADPH处理的PBMC能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、IFN-γ的含量并减少致敏蛋白IgE的含量。
涂有5μg/mL重组蛋白的ELISA板过夜，然后用2％BSA(98％的PBS)封闭。将培养基上清液在1％BSA中以1：500倍稀释，然后进行1：3连续稀释。为了检测IgE，用琼脂糖-蛋白G(Thermo Fisher Scientific，Rockford，IL)处理血清50分钟，然后将1:20稀释的样品上样到ELISA板上。用相应的抗体检测样品。用SureBlue TMB底物(KPL，Gaithersburg，MD)反应，并用TMB终止溶液(KPL，Gaithersburg，MD)终止反应。用Epoch ELISA读数器(BioTek，Winooski，VT)读取结果。与对照组相比不同浓度的NADPH处理的食物过敏儿童的PBMC，能够不同程度的增加外周血中细胞免疫抑制因子IL10、TGF-β、 IFN-γ的含量并减少致敏蛋白IgE的含量结果见表6。
表6为在培养基中添加NADPH培养食物过敏儿童外周血(PBMC)，增加食物过敏儿童PBMC＝细胞免疫抑制因子IL10、TGF-β和IFN-γ水平及减少IgE抗体水平数值表。
表6
分组 IFN-γ TGF-β IL-10 IgE
健康组 115.24±9.51pg/L 84.26±6.52pg/ml 50.21±5.0pg/ml 342±5.9U/ml
空白组 70.29±6.66pg/L 45.37±4.12pg/ml 29.87±4.69pg/ml 602±9.91U/ml
处理组1 86.32±7.44pg/L 62.13±7.21pg/ml 33.62±3.23pg/ml 452±12.5U/ml
处理组2 109.68±5.27pg/L 88.35±5.56pg/ml 49.37±5.71pg/ml 334±6.45U/ml
处理组3 99.88±5.61pg/L 90.14±3.9pg/ml 46.96±3.59pg/ml 403±7.43U/ml
实施例7
60例过敏性哮喘和60例过敏性鼻炎患者，其中，男40例，女40例，12岁以下儿童40例。发病原因诊断为化学接触、呼吸道吸入等所引起的迟发性过敏，已排除器质性病变、外伤等非过敏性病例。随机平均分为4组，分别为对照组、实验1组(NADH 10mg)，实验2组(NADPH 10mg)，实验3组(NADH:NADPH 5:5mg)，各组性别、年龄、病程等经统计学处理，差异无显著性意义，具有可比性。
对照组给予氯雷他定片(10mg/片)，按药品说明书使用，连续服用一个月。
实验1组给予NADH 10mg，每日早晨空腹一次，连续服用1个月。
实验2组给予NADPH 10mg，每日早晨空腹一次，连续服用1个月。
实验3组给予NADH:NADPH 5mg:5mg，每日早晨空腹一次，连续服用1个月。
诊断标准
无效：患者症状完全没有改善，且有加重迹象；
显效：患者症状有显著改善，但是仍未完全消失；
有效：患者症状完全消失。
副反应：嗜睡、头晕、注意力不集中等。
治疗结果：
考察各组治疗的总有效率(％)，以及停药后1个月、3个月、6个月的复发率(％)，结果见表7。
表7
Figure PCTCN2020093955-appb-000003
从上表7结果可知，与对照组相比较，本申请实验1组、2组和3组，对于过敏性鼻炎、过敏性哮喘的治疗效果与氯雷他定的治疗效果相当，证明NADH、NADPH及其组合能够有效治疗迟发性过敏性疾病。在复发率方面，6个月后的复发率均低于23％，远远低于对照组的87.5％，证明NADH和/或NADPH能够显著降低过敏性疾病的复发率。
在治疗过程中，对照组发生副反应18例，而各实验组均无副反应发生。
综上所述，本申请提供了一种烟酰胺腺嘌呤二核苷酸及其钠盐(NADH)和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐(NADPH)在抗过敏药物和/或抗过敏保健食品中的应用。本申请提供的技术方案中，服用NADH/NADPH，可诱导Treg细胞分泌免疫抑制细胞因子IL10、TGF-β等，从而抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状；同时，还可刺激过敏原特意的TH1反应，产生细胞因子IFN-γ和TGF-β，抑制TH2、血液嗜碱性和组织肥大细胞的免疫反应，从而减少sIgE合成同时增加sIgG和sIgA合成，从而减轻过敏症状。本申请提供的NADH和/或NADPH抗过敏药物和/或抗过敏保健食品中的应用，解决了现有技术中，抗过敏药物存在一些不良反应、需反复用药及过敏易复发的技术缺陷，提供一种长效、具有免疫调节作用的天然抗过敏药物或保健食品。
以上所述仅是本申请的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。

Claims (10)

  1. 一种烟酰胺腺嘌呤二核苷酸及其钠盐和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐在抗过敏药物和/或抗过敏保健食品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，一种烟酰胺腺嘌呤二核苷酸及其钠盐和烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的组合物在抗过敏药物和/或抗过敏保健食品中的应用。
  3. 根据权利要求1所述的应用，其特征在于，所述抗过敏药物和/或抗过敏保健食品的过敏反应选自：过敏性哮喘、过敏性鼻炎、过敏性结膜炎、药物过敏、食物过敏、花粉过敏及口腔过敏中的任意一种或多种。
  4. 根据权利要求2所述的应用，其特征在于，所述组合物中，烟酰胺腺嘌呤二核苷酸及其钠盐与烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的质量比为(1～10):(1～10)。
  5. 根据权利要求1或2所述的应用，其特征在于，所述烟酰胺腺嘌呤二核苷酸为还原型烟酰胺腺嘌呤二核苷酸。
  6. 根据权利要求1或2所述的应用，其特征在于，所述抗过敏药物和/或抗过敏保健食品的过敏反应选自：IL10、TGF-β以及IFN-γ免疫抑制细胞因子所调控的免疫反应。
  7. 根据权利要求1或2任意一项所述的应用，其特征在于，所述抗过敏药物和/或抗过敏保健食品的剂型为口服剂或注射剂。
  8. 根据权利要求7所述的应用，其特征在于，所述口服剂选自：片剂、粉剂、胶囊剂、颗粒剂、丸剂、混悬剂、糖浆剂、合剂、散剂、口服液以及滴丸中的任意一种或多种，所述注射剂选自：溶液型注射剂、混悬性注射剂、乳剂型注射剂和注射用灭菌粉末；注射方式选自皮内注射、皮下注射、肌内注射、静脉注射、脊椎注射和关节内注射。
  9. 根据权利要求1所述的应用，其特征在于，所述烟酰胺腺嘌呤二核苷酸及其钠盐或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的口服单次剂量是0.05～10mg/kg，日口服次数为1～3次；
    所述烟酰胺腺嘌呤二核苷酸及其钠盐或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐的注射单次剂量为0.05～2mg/kg。
  10. 根据权利要求1所述的应用，其特征在于，所述抗过敏药物和/或抗过敏保健食品中，除烟酰胺腺嘌呤二核苷酸及其钠盐和/或烟酰胺腺嘌呤二核苷酸磷酸及其钠盐外不含有其它抗过敏活性成分。
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