WO2004037862A1 - Ig/gamma speciale resistante aux allergenes et methode de preparation - Google Patents

Ig/gamma speciale resistante aux allergenes et methode de preparation Download PDF

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Publication number
WO2004037862A1
WO2004037862A1 PCT/CN2003/000333 CN0300333W WO2004037862A1 WO 2004037862 A1 WO2004037862 A1 WO 2004037862A1 CN 0300333 W CN0300333 W CN 0300333W WO 2004037862 A1 WO2004037862 A1 WO 2004037862A1
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WO
WIPO (PCT)
Prior art keywords
igy
specific
allergy
weight
preparation
Prior art date
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Ceased
Application number
PCT/CN2003/000333
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English (en)
Chinese (zh)
Inventor
Shing Paau
Rongjian Yang
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JASON MEDICAL GROUP (FAR EAST) Ltd
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JASON MEDICAL GROUP (FAR EAST) Ltd
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Priority to AU2003242074A priority Critical patent/AU2003242074A1/en
Publication of WO2004037862A1 publication Critical patent/WO2004037862A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from eggs
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • the invention relates to an antibody and a preparation method thereof, in particular to a composite IgY against various allergic diseases and a preparation method thereof.
  • Allergic diseases are abnormal immune reactions that occur in the body by external antigen substances.
  • Clinical manifestations such as bronchial asthma and bronchitis, allergic rhinitis, allergic laryngitis, allergic dermatitis, etc., are stubborn symptoms and it is quite difficult to treat. It is usually treated with chemicals such as hormones and epinephrine. It is regrettable that the currently widely used treatment schemes and their drugs are only local anti-inflammatory or short-term relief of symptoms and imperfect symptoms, not "cause treatment”. What's more, the side effects are quite strong.
  • corticosteroids can cause adverse reactions in the central nervous system, musculoskeletal, and endocrine systems, induce fungal infections in the oropharynx and esophagus, and even be susceptible to fatal herpes virus infections. It is more harmful to children and can inhibit children's growth. Affect the development of children's bones and gonads.
  • Adrenergic drugs and 2 -receptor agonists can also cause cardiovascular diseases such as hypertension and arrhythmia, skeletal muscle tremor (such as hand tremor), and even cause patients with adrenal insufficiency or failure; in severe cases, Causes cardiac arrest and dies. Based on long-term observations and statistical analysis, the international medical community has revealed that excessive use of salbutamol and ⁇ 2 -receptor agonists is the bane of increasing asthma mortality in previous years.
  • the medical community has developed a specific immune method to address the mechanism caused by "specific reactions” caused by certain "allergens” in the human body ( (“Desensitization therapy”), which stimulates the body to produce “blocking antibodies” by repeated injections of specific allergens, and is combined with the "allergens” that reenter the body to form a complex and is netted Clearance of the endothelial system.
  • An object of the present invention is to provide an antibody preparation that is safe, specific, and easy to use for allergic diseases such as asthma and a method for preparing the same.
  • A hay fever allergens: Artemisia pollen, ragweed pollen, ragtail, Timothy grass, rye, tree pollen such as Platanus, Maple, Poplar, Elm, Cypress, Gooseberry, Mulberry, Willow, Pine, Plane Wood, Birch, Ginkgo Wait.
  • allergen protein or antigen components identified by the most common representative allergenic pollen in various places, such as ragweed, wormwood as Hrt VI (scientific name), Dactylis as Arab al (scientific name), weeping birch It is Dac gl (scientific name), Hinoki cypress is Bet VI (scientific name), Cunninghamia lanceolata is Cry IC (scientific name), etc.
  • the representative and most common allergen protein or antigen component is used as the representative allergen of this allergenic pollen. According to the type of allergenic pollen screened out, several commercially available allergenic pollen extracts or extractants can be selected as representative pollen allergens.
  • cockroach antigen ingredients or cockroach extracts such as "black-breasted big cockroach extract”
  • cockroach homemade extracts or cut off the wings and feet of the cockroaches and put them into a high-speed homogenizer to stir them into pieces.
  • Allergens can be made using pet sebaceous gland secretions, saliva or urine egg extracts.
  • these pets' representative allergen proteins can be directly used as the raw material of the antigen.
  • the main allergen protein for cats is Fel dl
  • the allergen protein for dog saliva is Ca dl.
  • the single or mixed allergen extracts or preparations of each group are added with Freund's adjuvant at a ratio of 1-10: 1-10, and then placed in a high-speed homogenizer and stirred at high speed to form a milky slurry.
  • IgE can be extracted from the serum of humans, especially patients with allergies, and off-the-shelf IgE immunoglobulins can also be purchased as antigen raw materials.
  • IgE immunoglobulin is formulated with Freund's adjuvant at a ratio of 1-10: 1-10, placed in a high-speed homogenizer, and pulverized and homogenized at a high speed at 10,000 to 30,000 rpm to prepare an IgE antigen;
  • the same immunization technology can also be applied to immunize laying ducks with various single or composite allergen antigens and IgE antigens;
  • the filtered and sterilized product can also be freeze-dried with a freeze dryer to obtain a series of anti-allergen-specific IgY crude extracts or anti-IgE-specific IgY crude extracts.
  • the anti-allergen-specific IgY and anti-IgE-specific IgY crude extracts prepared by the above process were passed through an ion exchange column and a gel exchange column, respectively, to obtain pure IgY;
  • IgY prepared from different antigens are also strictly labeled according to their respective different coding marks to distinguish them.
  • One anti-allergy-specific IgY or multiple anti-allergy-specific IgY mixtures and anti-IgE-specific IgY are 1 to 10: Mix in a proportion of 0-10, then add a certain proportion of distilled water or physiological saline to prepare a solvent;
  • Anti-IgE specific IgY dry powder and specific anti-allergen-specific IgY or multiple anti-allergic specific IgY mixture dry powder can also be mixed directly in the ratio of 1-10: 0-10;
  • the above-mentioned multiple anti-allergy specific IgY mixtures are selected from a variety of specific anti-allergen-specific IgYs, and they are firstly pressed according to 1-10: 1-10 (2 types) or 1-10: 1-10: 1- 10 (3 types) or 1 1: 10: 1—10: 1—10: 1—10 (4 types) or the like and made by uniform mixing;
  • a series of anti-allergen-specific composite IgY solvents or powders prepared by the present invention are directly added to various nebulizers, and atomized by the nebulizer to form mist-like particles for patients to inhale the trachea, lungs or nasopharynx Can produce effective therapeutic and preventive effects.
  • This atomizer can be a jet pneumatic atomizer (including electric pump type, compression air pump type, foot air pump or compressed oxygen) or an ultrasonic wave atomizer, or any other type of atomizer.
  • the series of anti-allergen-specific composite IgY dry powder prepared by the present invention can also be directly filled into a variety of Dry Powder inhalers for use by patients.
  • dry powder inhalers There are two types of dry powder inhalers: single-dose dry powder inhalers are divided into two types: rotary inhalers (Spinhaler) and rotary inhalers (Rotahaler). (DisKhaler) and vortex inhaler (Turbuhaler).
  • the patient can easily inhale the anti-allergen complex IgY combination into the trachea, lungs or nasopharynx by operating the dry powder inhaler.
  • a series of anti-allergen compound IgY solvents or powders prepared can be diluted with water (distilled water or physiological saline, etc.) and then added to various spray tanks to prepare various sprays.
  • a booster must be added during filling. Frequently used boosters are Chlorofluorocarbon (CFC) or nitrogen (N 2 ).
  • CFC Chlorofluorocarbon
  • N 2 nitrogen
  • the anti-allergen compound IgY prepared by the present invention is stored in a spray tank at a certain pressure to make a portable pressure-type quantitative inhalation product. When hand pressure drives the quantitative valve, a fully atomized IgY solution can be sprayed. Patients can inhale this anti-allergen compound IgY into the trachea or lungs by simply holding the outlet of the spray can, inhaling with the pressure spray nozzle, or inhaling through the "mist tank".
  • the spray can be added with "booster” or without “booster”, and the prepared anti-allergen compound IgY can be diluted with water at normal pressure. It can be poured into a tank and put into a regular spray nozzle to make a spray. When using, just point the outlet of this constant-pressure fixed-quantity spray tank towards the nose and spray the misty liquid into the nose.
  • the invention Based on the in-depth study of the immunological mechanism of allergic diseases such as asthma, the invention accurately grasps the two most critical influencing factors of external allergens and internal specific IgE, and creatively applies passive immune technology to prepare a series of Anti-allergen-specific IgY and an anti-IgE-specific IgY. On the one hand, it clears and inhibits allergens entering the airway, on the other hand, it directly inhibits and reduces specific IgE levels, which effectively prevents the occurrence of allergic symptoms. Meanwhile, the anti-allergen prepared by the present invention Specific composite IgY and anti-IgE specific IgY is a unique pure natural biological preparation. This natural ingredient is directly sprayed into the respiratory tract, instead of being injected or taken into the human circulatory system.
  • Example 1 Preparation of dust mite specific IgY and detection titer
  • Lipoprotein adding the centrifuged slurry to an ultrafiltration machine, passing through an ultra-micro membrane, and filtering and sterilizing; lyophilizing the filtered and sterilized product using a freeze dryer to obtain a dust mite-specific IgY crude Extraction of dry powder finished product; purification of crude extract by ion transfer column and gel transfer column, to obtain dust-mite-resistant pure IgY;
  • Example 2 Immunization of laying ducks and preparation of duck dust mite-specific IgY
  • Immunized elite breeding hens with the prepared dust mite antigen by subcutaneous injection and intravenous injection in wings The combined method of booster immunization with each shot reaches 1 to 2 ml of antigen per injection, which is equivalent to about 5000 to 10,000 dust mites.
  • the booster injection is performed every two weeks, and a total of 3 immunizations are made within one month. 20 days after the second immunization, the immune eggs produced by the chickens were retrieved;
  • the slurry after centrifugation is added to an ultrafiltration machine, passed through an ultra-micro membrane, and filtered and sterilized; the filtered and sterilized product is freeze-dried with a freeze dryer to obtain a dust mite-specific crude IgY extract Finished dry powder product; the crude extract was purified by ion transfer column and gel transfer column to obtain dust-mite-resistant pure IgY;
  • Example 3 Preparation of specific anti-Artemisia pollen, ragweed pollen, quinoa pollen and algal grass pollen-specific IgY.
  • Antigen was prepared from wormwood pollen, ragweed pollen, quinoa pollen and algae pollen, respectively. First, the four collected pollen were collected. Use high-speed pulper to pulverize at a high speed of 1000 rpm, then add Freund's adjuvant at a ratio of 5: 4, and then homogenize at high speed in a high-speed pulper at 20,000 rpm to obtain four milky pollen protein antigens;
  • the diluted diluent with adjusted pH value is further stirred and homogenized, then it is cooled to 4 ° C and left for 12 hours; the diluent is added to a high-speed centrifugal separator and centrifuged at 8000-12000rpm for 20 minutes at high speed; The separated supernatant was put into an ultrafilter for ultrafiltration and concentrated 16 times.
  • Alginic acid was added to the concentrated slurry at a ratio of 0.3%, and the mixture was thoroughly stirred; then added to a high-speed centrifuge to centrifuge, the supernatant was removed to remove lipoproteins; the centrifuged slurry was added to an ultrafiltration machine, and ultramicronized Membrane, filtering and sterilizing; freeze-drying the filtered and sterilized product with a freeze dryer to obtain a dried IgY crude extract of anti-Artemisia pollen, ragweed pollen, quinoa pollen and algae pollen specific dry powder; The crude extract was purified by an ion transfer column and a gel transfer column to obtain pure IgY specific to Artemisia selengensis pollen, ragweed, pollen, and algae pollen.
  • the prepared breeding hens were immunized with the prepared Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, Aspergillus niger, and Aspergillus flavus antigens, respectively, and the same method was used to strengthen the immune system by combining subcutaneous injection and wing vein injection.
  • Each injection of chicken reaches 1.5 ml of antigen, which is equivalent to about 50-100 antigen protein.
  • the adjuvant is pulverized and homogenized at a high speed of 20,000 rpm in a high-speed homogenizer to obtain a milky cockroach protein antigen;
  • Example 2 Using the obtained antigen, immunizing breeder chickens in the same manner as in Example 1 and preparing an anti-cockroach allergen-specific IgY in the same manner.
  • the immunoglobulin IgE purchased from the market is mixed with Freund's adjuvant at a ratio of 3: 5, and then added to a high-speed homogenizer and homogenized at a high speed of 1,000 rpm to obtain an IgE antigen;
  • Example 8 Preparation of an anti-asthma combination nebulizer Dust mite anti-specific IgY powder and 100g humilis pollen-specific anti-anti-workers IgYlOOg g E and specific IgY was added 100g dry thoroughly mixed in a small mixer, to obtain an anti-asthmatic specific complex IgY300g;
  • Anti-dust mite-specific IgY dry powder 100g and anti-cockroach allergen-specific IgY dry powder 100g and anti-IgE-specific IgY dry powder 100g are added to a small mixer and fully mixed to make an anti-allergy-specific composite IgY dry powder 300 g;
  • Antifungal allergen-specific pure dry powder IgYlOOg, anti-pet allergy-specific pure IgY dry powder lOOg, anti-dust mite-specific IgY dry powder lOOg is added to a small mixer and fully mixed to make an anti-allergy specific composite IgY dry powder 300g;
  • the anti-dust mite-specific IgY prepared by the method of the present invention has a high antibody binding titer against dust mite allergens.
  • the anti-dust mite IgY prepared by the antigen preparation method and the enhanced immunity method of the present invention has a certain broad spectrum.
  • it also has "antigen crossover” on the same genus. "Other dust mites (such as” house dust mites ”) can also be suppressed.
  • Experimental example 2 Dust mite and house dust mite were added and mixed with Freund's adjuvant to prepare dust mite antigen and house dust mite antigen. The binding titer of the duck anti-dust mite IgY to these two antigens was detected by ELISA and method.
  • the test results show that using the method of the present invention to immunize laying ducks, the duck-specific dust mite-specific IgY prepared can completely achieve the same effect as the immunized chickens.
  • the obtained duck specific dust mite-specific IgY still has a high binding titer and has a certain broad spectrum.
  • the specific anti-artemisia pollen-specific gY prepared by immunizing hens by the method of the present invention has a high antibody binding titer against the allergen Artemisia selengensis pollen and has a certain broad spectrum It also has a certain inhibitory effect on pollen belonging to the same genus Yarrow that has "antigen cross". This reminds us that the pollen-resistant specific wormwood prepared by the method of the present invention can be used for all kinds of Patients with grass pollen allergy are used for prevention and treatment.
  • each kind of anti-pollen-specific IgY can be universal to allergies caused by pollen of the same genus. Pollens of grasses also need to use representative dogtail, Timothy grass, rye, etc. as antigens to prepare specific IgY against pollen and other pollen, and they are allergic to the pollen of grasses that have "antigen crossover" in the same genus. Are applicable.
  • the anti-fungal specific IgY antibody prepared by using the antigen preparation method and the immunization method of the present invention has a relatively high binding titer and high binding titer to other molds of the same genus; thus, further verification There is a high degree of antigenic cross between these five species.
  • the broad-spectrum specificity of the anti-mold IgY prepared by the present invention is also very strong. As long as the specific IgY prepared against a certain mold is prepared, it will also have a good effect on allergies caused by other molds with "antigen cross".
  • Experimental example 5 The ELISA method was used to detect the anti-cat allergy-specific IgY antibody-binding titer of the cat with the prepared detection antigen.
  • the titer of the test result was 1: 128. It was proved that the anti-cat allergy-specific IgY prepared according to the method of the present invention has quite satisfactory cat allergen suppression effect.
  • Experimental Example 6 The ELISA method using the cockroach protein antigen prepared in the method listed in item (1) of this example as the detection antigen, and the prepared anti-cockroach allergy-specific IgY antibody binding titer against the cockroach allergen was detected.
  • the potency is 1: 256.
  • Validated anti-pet allergic characteristics prepared by the method of the present invention The effect of the opposite sex IgY is very ideal.
  • the 50 patients were divided into two groups at any time.
  • the treatment group used an anti-allergic "normal-pressure quantitative spray can” and the control group used a "normal-pressure quantitative spray can” equipped with a placebo.
  • both groups of patients had severe allergic rhinitis, persistent nasal congestion, nasal itching, paroxysmal sneezing, a large amount of serous snot, and a loss of smell.
  • 3 people had hyposmia
  • 2 people had hyposmia.

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  • Health & Medical Sciences (AREA)
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  • Organic Chemistry (AREA)
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  • Pulmonology (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Molecular Biology (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract

La présente invention concerne un composite de l'Ig/gamma pouvant résister à des types de maladie se rapportant à l'irritabilité et sa méthode de préparation. La méthode de préparation de l'Ig/gamma spéciale résistante aux allergènes consiste à: cribler et isoler les allergènes responsables de l'irritabilité; sur la base du criblage et du classement combinés, préparer un antigène anti-allergène simple et un antigène anti-allergène composite, respectivement; préparer un anti-IgE; préparer un oeuf immune; préparer un produit brut de l'Ig/gamma spéciale résistant aux différents types d'allergènes et purification du produit brut de l'Ig/gamma anti-IgE; préparer un composite spécial de l'Ig/gamma résistant à un ou plusieurs allegènes spéciaux. Le produit de l'invention résiste à la pénétration d'un allergène dans la trachée, diminue le taux de l'IgE spéciale, et empêche l'apparition effective de symptômes allergiques. En outre, la méthode de préparation mise en oeuvre est purement biologique, sûre et agit rapidement. Elle ne comporte aucune toxine ni ne présente d'effets secondaires ou de réactions négatives.
PCT/CN2003/000333 2002-10-25 2003-05-08 Ig/gamma speciale resistante aux allergenes et methode de preparation Ceased WO2004037862A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003242074A AU2003242074A1 (en) 2002-10-25 2003-05-08 Special igy of resist allergic and its preparation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNB021460264A CN1286527C (zh) 2002-10-25 2002-10-25 抗过敏特异性IgY及其制剂
CN02146026.4 2002-10-25

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WO2004037862A1 true WO2004037862A1 (fr) 2004-05-06

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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103913578B (zh) * 2014-03-11 2016-01-20 安徽润敏江生物科技有限公司 一种蟑螂过敏原制剂的质量控制方法
CN105541999A (zh) * 2016-01-29 2016-05-04 西北农林科技大学 一种制备IgY过程中的去脂液的配方及其配制方法
CN107893090B (zh) * 2017-10-20 2020-05-15 国家海洋局第三海洋研究所 土曲霉h768的发酵化合物在制备抗过敏药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1250056A (zh) * 1999-09-08 2000-04-12 中德联合研究院 抗幽门螺杆菌鸡卵黄免疫球蛋白产品及其应用
CN1354187A (zh) * 2001-12-02 2002-06-19 重庆和润实业(集团)有限公司 抗幽门螺杆菌细胞毒素鸡卵黄抗体其制备及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1250056A (zh) * 1999-09-08 2000-04-12 中德联合研究院 抗幽门螺杆菌鸡卵黄免疫球蛋白产品及其应用
CN1354187A (zh) * 2001-12-02 2002-06-19 重庆和润实业(集团)有限公司 抗幽门螺杆菌细胞毒素鸡卵黄抗体其制备及其应用

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CN1513558A (zh) 2004-07-21
AU2003242074A1 (en) 2004-05-13

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