WO2022166641A1 - 核苷酸的新用途 - Google Patents

核苷酸的新用途 Download PDF

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WO2022166641A1
WO2022166641A1 PCT/CN2022/073440 CN2022073440W WO2022166641A1 WO 2022166641 A1 WO2022166641 A1 WO 2022166641A1 CN 2022073440 W CN2022073440 W CN 2022073440W WO 2022166641 A1 WO2022166641 A1 WO 2022166641A1
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nucleotide
ump
imp
gmp
allergic rhinitis
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PCT/CN2022/073440
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English (en)
French (fr)
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李勇
刘欣然
徐美虹
陈玉松
曾峥
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陈玉松
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Priority to EP22748920.0A priority Critical patent/EP4108245A4/en
Priority to US17/759,297 priority patent/US20230158056A1/en
Priority to JP2022549816A priority patent/JP7427801B2/ja
Publication of WO2022166641A1 publication Critical patent/WO2022166641A1/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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids

Definitions

  • the present invention relates to the technical field of drug development, the technical field of functional food, and in particular to the new use of nucleotides.
  • Allergic diseases refer to a class of diseases in which the body produces a large number of antibodies after exposure to one or more sensitizing substances (allergens), thereby causing tissue or organ dysfunction or damage. It mainly includes allergic rhinitis, asthma, atopic Dermatitis, allergic gastrointestinal disorders and food allergies. Allergic diseases have become a common public health problem, and the incidence is increasing year by year, causing a huge medical burden to patients. At present, the main treatment method for allergic diseases is chemical drug therapy. Although the disease can be relieved to a certain extent, drug treatment cannot change the course of allergic diseases. Relapse, and the long-term use of chemotherapy may have a negative impact on the body, so it cannot fundamentally achieve a cure. Therefore, it is obviously necessary to explore an effective method that can effectively relieve or treat allergic diseases.
  • ingesting nucleotides can reduce the symptoms of allergic rhinitis in mice, relieve splenomegaly caused by allergy, significantly reduce the level of histamine in serum and nasal lavage fluid, and effectively regulate inflammatory factors, thereby preventing, The role of the treatment of allergic rhinitis. If this technology is applied to the health industry, the health care and medical needs of patients with allergic diseases can be fully met.
  • the purpose of the present invention is to provide the use of a nucleotide preparation with relatively low molecular weight and fast absorption in the preparation of drugs for preventing and treating diseases such as allergic rhinitis or functional foods for relieving symptoms of allergic rhinitis. It provides a new way to prevent and treat allergic rhinitis and other diseases. At the same time, establish a stable, effective and reproducible experimental animal model method.
  • One aspect of the present invention provides an application of a nucleotide in the preparation of a drug for preventing and treating allergic rhinitis.
  • Another aspect of the present invention provides the use of a nucleotide in a functional food for relieving allergic rhinitis symptoms.
  • the nucleotides are four or five 5' single nucleotides or a nucleotide mixture in the form of a sodium salt thereof, and various nucleotides are converted into CMP, AMP, UMP, GMP.
  • the total amount of IMP molecules is 100%, and the effective mass percentage of each nucleotide in terms of CMP, AMP, UMP, GMP and IMP molecules are: CMP 23-78%, AMP 6-44%, UMP 7- 40%, GMP 7-51%, IMP 0-2.5%.
  • the effective percentages of various nucleotides in terms of CMP, AMP, UMP, GMP, IMP molecules are respectively: CMP 42-43%, AMP 16-17%, UMP 21-22%, GMP 18%, IMP 0-2.5%.
  • the effective percentages of various nucleotides are calculated as CMP, AMP, UMP, GMP, IMP molecules: CMP 43%, AMP 17%, UMP 21%, GMP 18%, IMP 1 %.
  • the medicine is powder, tablet, soft and hard capsule or oral liquid.
  • the functional food is powder, liquid beverage, preferably milk powder, dairy product or bakery product.
  • nucleotides significantly reduce histamine (Histamine, HIS) content in serum and nasal lavage fluid with allergic rhinitis symptoms, and significantly reduce serum IgG, IgE, IL-4, IL-12 , IL-10 was significantly increased;
  • the experimental animal model used was a model of allergic rhinitis in BALB/c mice induced by a specific amount of ovalbumin (OVA) combined with a specific amount of aluminum hydroxide gel immune adjuvant.
  • OVA ovalbumin
  • the present invention discovers that the existing substance - nucleotides, has the function of preventing and treating allergic rhinitis and other diseases, and discovers that it is used in the preparation of medicines for preventing and treating allergic rhinitis and other diseases or functional foods for relieving allergic rhinitis symptoms use in.
  • Animal experiments have confirmed that ingestion of nucleotides can effectively prevent and treat allergic diseases by relieving splenomegaly caused by allergy, significantly reducing histamine levels in serum and nasal lavage fluid, and effectively regulating inflammatory factors. It has significant preventive and therapeutic effects on allergic rhinitis and other diseases.
  • Fig. 1 The effect of nucleotides on HIS content in mouse serum and nasal lavage fluid
  • Fig. 2 The effect of nucleotides on the serum IgE content of mice
  • Figure 3 The effect of nucleotides on serum IgG levels in mice.
  • Nucleotides in this example are composed of five 5' mononucleotides or their sodium salts in the form of percentages by mass: CMP 43%, AMP 17%, UMP 21%, GMP 18%, IMP 1% mix.
  • Nucleotides in this example are mixed in the form of five 5' mononucleotides or their sodium salts according to the mass percentage: CMP 78%, AMP 6%, UMP 8%, GMP 7%, IMP 1%.
  • the preparation method is the same as in Example 1.
  • the nucleotides in this example are mixed in the form of five 5' mononucleotides or their sodium salts according to the mass percentage: CMP 23%, AMP 44%, UMP 25%, GMP 7%, IMP 1%.
  • the preparation method is the same as in Example 1.
  • the nucleotides described in the present invention are mixed in the form of five 5' mononucleotides or their sodium salts according to the mass percentage: CMP 23%, AMP 17%, UMP 40%, GMP 19%, IMP 1% .
  • the preparation method is the same as in Example 1.
  • the nucleotides described in the present invention are mixed in the form of five 5' mononucleotides or their sodium salts according to the mass percentage: CMP 24%, AMP 17%, UMP 7%, GMP 51%, IMP 1% .
  • the preparation method is the same as in Example 1.
  • mice 6-8 week old BALB/c healthy female mice (SPF grade, experimental animal use license number: SYXK (Beijing) 2011-0039; experimental animal production license) provided by the Laboratory Animal Center of Peking University Medical Department No.: SCXK (Beijing) 2011-0012), weighing 18-22 g, a total of 50 animals were kept in separate cages in the Laboratory Animal Department of Peking University School of Medicine, 5 animals per cage, with free food and water.
  • the temperature range of the animal room is 25°C ⁇ 1°C, the relative humidity is 50% to 60%, and the indoor lighting is controlled at a 12h/12h light-dark cycle rhythm.
  • mice were randomly divided into 5 groups according to body weight, 10 mice in each group, namely blank control group, model control group, low-dose nucleotide group, medium-dose nucleotide group, and nucleotide group. high-dose group.
  • the mice in the blank control group did not undergo the modeling process, while the mice in the other groups were given the corresponding test substances by gavage at a fixed time every day after the model was established.
  • Nucleotide low dose group was given 0.3g/kg.bw nucleotide
  • nucleotide medium dose group was given 0.6g/kg.bw nucleotide
  • nucleotide high dose group was given 1.2g/kg.bw nucleotide .
  • the model control group and blank control group were given the same amount of sterile distilled water. The intervention period was 14 days, during which food and water were ad libitum.
  • organ coefficient (organ weight/body weight)*100.
  • mice After 14 days of gavage, the mice were anesthetized and blood was collected into a 1.5 mL centrifuge tube, left to centrifuge and the serum was separated. The content of HIS in serum was detected by Elisa kit. After the mice were sacrificed, their heads were cut off, and the pre-cooled saline was drawn with a 1 mL syringe and carefully inserted into the nasopharynx from the mouse trachea, and the saline was slowly pushed out. And separate the supernatant from the lavage fluid. The content of HIS in nasal lavage fluid was detected by Elisa kit.
  • mice After 14 days of gavage, the mice were anesthetized and blood was collected into a centrifuge tube, left to stand and centrifuged to separate serum. The content of IgG and IgE in serum was detected with Elisa kit.
  • mice 14 days after gavage, the mice were anesthetized and blood was taken into a centrifuge tube, and the serum was separated by centrifugation. The levels of IL-4, IL-10 and IL-12 in serum were detected by Elisa kit.
  • the spleen coefficient of allergy model mice was significantly higher than that of the blank control group, but the spleen coefficient of the mice in the low-dose nucleotide group after intervention was significantly lower than that of the model control group, indicating that nucleotide intervention can reduce splenomegaly caused by allergy. , has a certain significance for the treatment of allergies.
  • a has a statistical difference compared with the blank control group
  • b has a statistical difference compared with the model control group.
  • the high-dose nucleotide group can significantly reduce the serum IgE content, which has a significant effect on the treatment of allergies.
  • the high-dose nucleotide group can significantly reduce the serum IgG content, which has a significant effect on the treatment of allergies.
  • the serum IL-10 in the model control group was significantly lower, while the serum IL-10 level in the mice in the high-dose nucleotide group after intervention was significantly higher than that in the model control group, and its content was close to that in the blank control group. mouse. It shows that nucleotide intervention has a significant effect on correcting the disorder of serum inflammatory factors in allergic mice.
  • a has a statistical difference compared with the blank control group
  • b has a statistical difference compared with the model control group.
  • the serum IL-10 of the mice in the model control group was significantly reduced, while the serum IL-10 level of the mice in the high-dose nucleotide group of Example 2-5 after intervention was significantly higher than that of the model control group. Close to the blank control group of healthy mice. It shows that nucleotide intervention has a significant effect on correcting the disorder of serum inflammatory factors in allergic mice.
  • nucleotides can play a role in the treatment of allergic diseases by alleviating splenomegaly caused by allergy, significantly reducing the level of histamine in serum and nasal lavage fluid, and effectively regulating inflammatory factors. It shows that it has significant efficacy in treating allergic rhinitis and other diseases, and has the potential as a new drug for treating allergic diseases, and its suitable concentration range is 0.6-1.2g/kg.bw daily intake.

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Abstract

核苷酸的一种新用途，具体地涉及核苷酸在制备预防、治疗过敏性鼻炎等疾病的药物或缓解过敏性鼻炎症状的功能性食品中的用途。所述核苷酸是由CMP、AMP、UMP、GMP、IMP五种核苷酸的混合物。核苷酸分子量小，摄入人体吸收较快且生物利用率高。摄入核苷酸可减轻过敏性鼻炎症状，缓解由过敏引起的脾肿大，显著降低血清和鼻腔灌洗液中组胺水平，有效调节炎症因子，从而起到治疗过敏性疾病的作用。可用于制备预防、治疗过敏性鼻炎等疾病的药物或缓解过敏性鼻炎症状的功能性食品，并为膳食干预方式调节免疫功能提供了一种途径。

Description

核苷酸的新用途 技术领域
本发明涉及药物开发技术领域，功能性食品技术领域，具体是核苷酸的新用途。
背景技术
过敏性疾病是指机体接触某种或多种致敏物质(过敏原)后产生大量抗体，从而引起组织或器官功能障碍或损伤的一类疾病，其主要包括过敏性鼻炎、哮喘、特应性皮炎、过敏性胃肠道疾病和食物过敏。过敏性疾病已成为一种普遍的公共健康问题，发病率正逐年增加，对患者造成了巨大的医疗负担。目前，过敏性疾病最主要的治疗方式是化学药物治疗，虽然在一定程度上可缓解病情，但药物治疗并不能改变过敏性疾病的进程，在停药之后也无长期持续药效，病情很容易复发，且长期使用化学药物治疗可能对机体产生负面影响，因而也就不能从根本上达到治愈的效果。因此，探索一种能够有效缓解或治疗过敏性疾病的有效方法显然十分必要。
通过研究发现，摄入核苷酸可减轻小鼠过敏性鼻炎症状，缓解由过敏引起的脾肿大，显著降低血清和鼻腔灌洗液中组胺水平，有效调节炎症因子，从而起到预防、治疗过敏性鼻炎的作用。若将此项技术应用于健康产业中可以充分满足过敏疾病患者的保健、医疗需求。
发明内容
本发明的目的在于提供一种分子量较低、吸收快的核苷酸制剂在制备预防、治疗过敏性鼻炎等疾病的药物或缓解过敏性鼻炎症状的功能性食品中的用途，为通过膳食补充剂来预防、治疗过敏性鼻炎等疾病提供一种新途径。与此同时，建立一种稳定有效、可重复的实验动物模型方法。
为实现上述发明目的，本发明采用以下技术方案：
本发明一方面提供了一种核苷酸在制备预防、治疗过敏性鼻炎药物中的应用。
本发明另一方面提供了一种核苷酸在缓解过敏性鼻炎症状的功能性食品中的应用。
上述技术方案中，进一步地，所述核苷酸是四种或五种5’单核苷酸或者其钠盐形式的核苷酸混合物，各种核苷酸折合成CMP、AMP、UMP、GMP、IMP分子计总量为100％，其中各核苷酸有效质量百分含量以CMP、AMP、UMP、GMP、IMP分子计分别为：CMP 23-78％、AMP 6-44％、UMP 7-40％、GMP 7-51％、IMP 0-2.5％。
上述技术方案中，进一步地，各种核苷酸有效百分含量以CMP、AMP、UMP、GMP、IMP分子计分别为：CMP 42-43％、AMP 16-17％、UMP 21-22％、GMP 18％、IMP 0-2.5％。
上述技术方案中，进一步地，各种核苷酸有效百分含量以CMP、AMP、UMP、GMP、 IMP分子计分别为：CMP 43％、AMP 17％、UMP 21％、GMP 18％、IMP 1％。
上述技术方案中，进一步地，所述药物为粉剂、片剂、软硬胶囊或口服液。
上述技术方案中，进一步地，所述功能性食品为粉剂、液体饮料，优选奶粉、奶制品或烘焙制品。
上述技术方案中，进一步地，核苷酸显著降低有过敏性鼻炎症状的血清、鼻腔灌洗液中组胺(Histamine，HIS)含量，使血清IgG、IgE、IL-4、IL-12显著降低，IL-10显著升高；所用实验动物模型为以特定量卵白蛋白(OVA)联合特定量氢氧化铝凝胶免疫佐剂致BALB/c小鼠过敏性鼻炎的模型。
本发明发现了现有物质——核苷酸，具有预防、治疗过敏性鼻炎等疾病的功能，发现了其在制备预防、治疗过敏性鼻炎等疾病的药物或缓解过敏性鼻炎症状的功能性食品中的用途。动物实验证实摄入核苷酸能够通过缓解由过敏引起的脾肿大，显著降低血清和鼻腔灌洗液中组胺水平，有效调节炎症因子，从而起到预防、治疗过敏性疾病的作用，表明其具有显著的预防、治疗过敏性鼻炎等疾病的功效。
附图说明
图1核苷酸对小鼠血清及鼻腔灌洗液中HIS含量的影响；
图2核苷酸对小鼠血清IgE含量的影响；
图3核苷酸对小鼠血清IgG含量的影响。
具体实施方式
以下结合具体实施例对本发明作进一步说明，但不以任何方式限制本发明。
实施例1
1.本实施例核苷酸是由五种5’单核苷酸或者其钠盐的形式按照质量百分含量：CMP 43％、AMP 17％、UMP 21％、GMP 18％、IMP 1％比例混合。
2.具体制备方式如下：
(1)将五种5’单核苷酸或者其钠盐分别进行检测，合格后备用。
(2)将质检合格的五种5’单核苷酸或者其钠盐过60目筛备用。
(3)按比例计算称取所需的各单核苷酸样品量，全部加入后进行总混，混合时间不低于40分钟。所得样品常温保存。
实施例2
本实施例核苷酸是由五种5’单核苷酸或者其钠盐的形式按照质量百分含量：CMP 78％、AMP 6％、UMP 8％、GMP 7％、IMP 1％比例混合。制备方法同实施例1。
实施例3
本实施例核苷酸是由五种5’单核苷酸或者其钠盐的形式按照质量百分含量：CMP 23％、AMP 44％、UMP 25％、GMP 7％、IMP 1％比例混合。制备方法同实施例1。
实施例4
本发明所述核苷酸是由五种5’单核苷酸或者其钠盐的形式按照质量百分含量：CMP 23％、AMP 17％、UMP 40％、GMP 19％、IMP 1％比例混合。制备方法同实施例1。
实施例5
本发明所述核苷酸是由五种5’单核苷酸或者其钠盐的形式按照质量百分含量：CMP 24％、AMP 17％、UMP 7％、GMP 51％、IMP 1％比例混合。制备方法同实施例1。
实验验证
一、材料与方法
1.样品：上述实施例1-5中所得的核苷酸样品。
2.实验动物：由北京大学医学部实验动物中心提供的6-8周龄BALB/c健康雌性小鼠(SPF级，实验动物使用许可证号：SYXK(京)2011-0039；实验动物生产许可证号：SCXK(京)2011-0012)，重18～22g，共50只，分笼饲养于北京大学医学部实验动物部，每笼5只，自由进食和饮水。动物室温度范围25℃±1℃，相对湿度50％～60％，室内照明控制在12h/12h光暗周期节律。
3.过敏性鼻炎动物模型建立：模型为卵白蛋白联合4％氢氧化铝凝胶免疫佐剂致小鼠过敏性鼻炎模型。具体流程分为两部分：(1)初始致敏：在第0天、第7天、第14天和第21天，向小鼠腹腔内注射含25μg卵白蛋白及2mg氢氧化铝凝胶的200μL生理盐水中致敏。(2)再次激发；在初始致敏后的第23、25和27天，向小鼠双侧鼻腔滴注含500μg卵白蛋白的生理盐水20μL，两侧鼻腔轮流滴注，每侧10μL。对照组用生理盐水代替。每次鼻腔激发时的20分钟内，观察记录小鼠打喷嚏及鼻瘙痒动作次数，模型组打喷嚏和鼻瘙痒次数显著高于对照组即视为模型建立成功。
4.分组与剂量：按体重将小鼠随机分为5个组，每组10只，分别为空白对照组、模型对照组、核苷酸低剂量组、核苷酸中剂量组、核苷酸高剂量组。空白对照组小鼠不经历造模过程，而其他组小鼠在模型建成后每日以固定时间灌胃给予各组相应受试物。核苷酸低剂量组给予0.3g/kg.bw核苷酸，核苷酸中剂量组给予0.6g/kg.bw核苷酸，核苷酸高剂量组给予1.2g/kg.bw核苷酸。模型对照组和空白对照组均给予等量无菌蒸馏水。干预周期为14天，期间自由进食和饮水。
5.主要仪器与试剂：Bio-Rad酶标仪(美国)；卵白蛋白(美国)，氢氧化铝凝胶佐剂(中国)，Elisa试剂盒(中国)。
6.实验方法：
6.1体重及脏器系数测定：灌胃14天后，称小鼠体重，麻醉取血后立即颈椎脱臼处死，取肝脏、肾脏、脾脏和胸腺，去尽筋膜，用滤纸吸干脏器表面血污并称重，计算各脏器系数。公式为脏器系数＝(脏器重量/体重)*100。
6.2血清及鼻腔灌洗液中组胺含量测定：灌胃14天后，小鼠麻醉后取血至1.5mL离心管中，静置离心并分离血清。用Elisa试剂盒检测血清中HIS含量。小鼠处死后断其头部，用1mL注射器吸取预冷的生理盐水从小鼠气管处小心插入至鼻咽部，将生理盐水缓慢推出，用离心管在小鼠鼻孔处承接鼻腔灌洗液，离心并分离灌洗液上清。用Elisa试剂盒检测鼻腔灌洗液中HIS含量。
6.3血清中免疫蛋白测定：灌胃14天后，小鼠麻醉后取血至离心管中，静置离心并分离血清。用Elisa试剂盒检测血清中IgG和IgE的含量。
6.4血清中炎症因子测定：灌胃14天后，小鼠麻醉后取血至离心管中，静置离心并分离血清。用Elisa试剂盒检测血清中IL-4、IL-10和IL-12的含量。
7.统计方法：所有结果均以均数±标准差表示，统计检验采用SPSS 23.0软件进行单因素方差分析，以p<0.05作为差异具有统计学意义。
二、实施例1实验结果
1.核苷酸对小鼠体重及脏器系数的影响
由表1结果可见，各组小鼠的体重、肾脏系数、胸腺系数差异均无统计学意义(p>0.05)。过敏模型对照组肝脏系数较空白对照组有显著升高，而核苷酸干预后的三个组小鼠肝脏系数均较模型对照组显著降低，接近空白对照组水平。说明核苷酸干预可改善过敏小鼠可能发生的一些肝损伤症状。
过敏模型小鼠脾脏系数均显著高于空白对照组，但核苷酸低剂量组干预后小鼠脾脏系数较模型对照组有显著降低，说明核苷酸干预可减轻由过敏引起的脾肿大情况，对于过敏的治疗有一定的意义。
表1 实施例1核苷酸对小鼠体重及重要脏器系数的影响(
Figure PCTCN2022073440-appb-000001
n＝10)
Figure PCTCN2022073440-appb-000002
Figure PCTCN2022073440-appb-000003
注：a与空白对照组有相比有统计学差异，b与模型对照组相比有统计学差异。
由图1可见，模型对照组血清及鼻腔灌洗液中的HIS含量均显著高于空白对照组，说明模型建立非常成功，小鼠局部和全身的过敏反应明显。而核苷酸干预的高剂量组小鼠表现为血清HIS含量的显著降低；另一方面，在鼻腔灌洗液HIS含量中，核苷酸中剂量组和核苷酸高剂量组的HIS含量也显著降低。表明核苷酸的摄入具有一定治疗过敏的作用。
由图2可见，与模型对照组比较，核苷酸高剂量组可显著降低血清IgE含量，对于治疗过敏有显著的效果。
由图3可见，与模型对照组比较，核苷酸高剂量组可显著降低血清IgG含量，对于治疗过敏有显著的效果。
由表2可见，核苷酸高剂量组可显著降低过敏模型小鼠血清IL-4和IL-12的水平。
与空白对照组相比，模型对照组小鼠血清IL-10显著降低，而核苷酸高剂量组干预后小鼠血清IL-10水平显著高于模型对照组，其含量接近空白对照组健康小鼠。表明核苷酸干预对于纠正过敏小鼠血清炎症因子紊乱有很显著的作用。
表2 实施例1核苷酸对小鼠血清炎症因子的影响(x±s，n＝10)
Figure PCTCN2022073440-appb-000004
注：a与空白对照组有相比有统计学差异，b与模型对照组相比有统计学差异。
三、实施例2～5实验结果
由表3可见，实施例2～5的核苷酸高剂量组可显著降低过敏模型小鼠血清IL-4和IL-12的水平。
与空白对照组相比，模型对照组小鼠血清IL-10显著降低，而实施例2—5的核苷酸高剂量组干预后小鼠血清IL-10水平显著高于模型对照组，其含量接近空白对照组健康小鼠。表明核苷酸干预对于纠正过敏小鼠血清炎症因子紊乱有很显著的作用。
表3 实施例2～5核苷酸对小鼠血清炎症因子的影响(x±s，n＝10)
Figure PCTCN2022073440-appb-000005
由表4可见，实施例2～5的核苷酸高剂量组可显著降低过敏模型小鼠血清HIS和鼻腔HIS的含量。
表4 实施例2～5核苷酸对小鼠血清HIS和鼻腔HIS的含量的影响(x±s，n＝10)
Figure PCTCN2022073440-appb-000006
四、实验结论
本研究通过设立空白对照组、模型对照组作为对照组，探讨核苷酸的预防、治疗过敏性鼻炎等疾病的作用。动物实验结果表明，核苷酸可以通过缓解由过敏引起的脾肿大、显著降低血清和鼻腔灌洗液中组胺水平、有效调节炎症因子，从而起到治疗过敏性疾病的作用。表明其具有显著的治疗过敏性鼻炎等疾病的功效，具备作为一种新型治疗过敏性疾病药物的潜力，其适宜浓度范围为每天摄入0.6～1.2g/kg.bw。
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。

Claims (8)

  1. 核苷酸在制备预防、治疗过敏性鼻炎药物中的应用。
  2. 核苷酸在缓解过敏性鼻炎症状的功能性食品中的应用。
  3. 根据权利要求1或2所述的应用，其特征在于，所述核苷酸是四种或五种5’单核苷酸或者其钠盐形式的核苷酸混合物，各种核苷酸折合成CMP、AMP、UMP、GMP、IMP分子计总量为100％，其中各核苷酸有效百分含量以CMP、AMP、UMP、GMP、IMP分子计分别为：CMP 23-78％、AMP 6-44％、UMP 7-40％、GMP 7-51％、IMP 0-2.5％。
  4. 根据权利要求3所述的应用，其特征在于，各种核苷酸有效百分含量以CMP、AMP、UMP、GMP、IMP分子计分别为：CMP 42-43％、AMP 16-17％、UMP 21-22％、GMP 18％、IMP 0-2.5％。
  5. 根据权利要求3所述的应用，其特征在于，各种核苷酸有效百分含量以CMP、AMP、UMP、GMP、IMP分子计分别为：CMP 43％、AMP 17％、UMP 21％、GMP 18％、IMP 1％。
  6. 根据权利要求1所述的应用，其特征在于，所述药物为粉剂、片剂、软硬胶囊或口服液。
  7. 根据权利要求2所述的应用，其特征在于，所述功能性食品为粉剂、液体饮料，优选奶粉、奶制品或烘焙制品。
  8. 根据权利要求1或2所述的应用，其特征在于，核苷酸显著降低有过敏性鼻炎症状的血清、鼻腔灌洗液中组胺(Histamine，HIS)含量，使血清IgG、IgE、IL-4、IL-12显著降低，IL-10显著升高。
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