WO2006019183A1 - Immunité des muqueuses et vaccin par voie muqueuse et système d'inoculation protectrice utilisant le sensibilisateur - Google Patents

Immunité des muqueuses et vaccin par voie muqueuse et système d'inoculation protectrice utilisant le sensibilisateur Download PDF

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WO2006019183A1
WO2006019183A1 PCT/JP2005/015379 JP2005015379W WO2006019183A1 WO 2006019183 A1 WO2006019183 A1 WO 2006019183A1 JP 2005015379 W JP2005015379 W JP 2005015379W WO 2006019183 A1 WO2006019183 A1 WO 2006019183A1
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vaccine
mucosal
mucosal immunity
nasal
iga
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PCT/JP2005/015379
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Japanese (ja)
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Hiroshi Kido
Dai Mizuno
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The University Of Tokushima
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to mucosal immunity, a mucosal vaccine using the sensitizer and a vaccination system, and more particularly, preferential and effective antigen-specific secretory immunoglobulin A (igA), particularly
  • the present invention relates to mucosal immunity technology that enables production by selective induction.
  • Non-patent Documents 1 and 2 since the defense against infection depends on a small amount of IgG leached into the mucosa, its effectiveness is limited (Non-patent Documents 1 and 2). Furthermore, since IgG antibodies have high specificity for antigens, they are ineffective or less effective for mutant antigens and mutant viruses. In contrast, IgA antibodies are effective because they have a broad spectrum of cross-reactivity with antigens, and exhibit neutral activity against mutant viruses. In this way, infection prevention in the natural infection route is blood Despite the importance of mucosal secretory IgA antibody as a responsible factor for mucosal immunity compared to IgG antibodies in Infection protection by induction or mucosal immunity was hopeless.
  • Nasal vaccine In order to protect against infection with IgA, which is considered to be the most effective, an attempt was made to directly inoculate the nasal liquid split antigen, but it was pointed out that IgA production was low. ing. In order to increase IgA antibody production, attempts have been made to enhance the induction of IgA antibody by enhancing the induction of mucosal immune response by using E. coli heat-labile toxin or cholera toxin as an adjuvant to the split antigen. Therefore, it is difficult to put it to practical use because the safety of the adjuvant is not guaranteed.
  • live virus is an active ingredient, it is highly adsorbed and invaded by cells, and is excellent in initializing and sustaining immunity, but since mild influenza symptoms appear, severe infection with influenza Disadvantages such as high-risk patients and elderly people who are prone to change are not known.
  • Ambroxol (generic name ambroxol; CAS registry number 18638-91-5) and its hydrochloride, amproxol hydrochloride (generic name ambroxol hydrochlrode; CAS registry number 23828-92-4) are safe and have few side effects. It is widely used as an expectorant, from infants to the elderly, and is used for the treatment of diseases such as acute bronchitis and bronchiectasis (Non-patent Document 6).
  • Non-patent Document 7 Bromohexine (generic name bromhexine; CAS registry number 3572-43-8) and its hydrochloride, bromhexine hydrochrolide hydrochloride (generic name bromhexine; CAS registry number 6 1 1-75-6) are also expectorants. In addition, it is used as a therapeutic agent for chronic bronchitis.
  • Bromhexine is related to the metabolic precursor of ambroxol (amp ampoxol is a metabolite of promhexine), and ambroxol is superior in water solubility and toxicity compared to bromhexine. Is low. Furthermore, regarding the use of the above-mentioned compounds, the invention of use utilizing the action of amproxol, bromhexine and their pharmacologically acceptable salts to inhibit the growth of influenza virus, ie, an anti-influenza drug (this case (According to Kido, the largest inventor) is known (Patent Document 1).
  • influenza virus that supports this invention of invention is due to the following actions by the above compounds: (a) Inhibition of airway proteaase that induces infectivity of influenza virus induction of substance preventing adsorption penetration of virus particles into cells (infection) in this stage; (b) that facilitates secretion of mucosal immune substances IgA and I g G after the virus infection; and (c ) Inhibiting the release of inflammatory cytokines.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2 0 0 3-1 5 5 2 3 0
  • Non-Patent Document 1 Japanese Clinical Practice, Vol. 61 (No. 11), 1993-2000, 2003
  • Non-Patent Document 2 Pediatric Internal Medicine, Vol. 35 (No. 10), 1739-1741, 2003
  • Patent Document 3 Pediatric Internal Medicine, Vol. 35 (No. 10), 1714-1717, 2003
  • Non-Patent Document 4 New England Journal of Medicine, Vol. 350 (No. 9), 893-903, 2004
  • Non-Patent Document 5 Lancet, Vol. 363 (No. 9427), 2122-2127. 2004
  • Non-Patent Document 6 Lxmg, Vol. 163, pp. 337-344, 1985
  • Non-Patent Document 7 Research in Experimental Medicine, 196, 389-398, 1997
  • Non-Patent Document 8 European Journal of Medical Research ⁇ 2nd pp. 129-132, 1997
  • Non-Patent Document 9 Inflammation Research, Vol. 48, 86-93, 1999
  • sensitization of mucosal immunity means “a state in which mucosal immune cells in the living body are selectively activated or activated”. Such a state (sensitization of mucosal immunity) is caused by a secretory IgA antibody and an IgA production-promoting cycle by transmucosal administration of an antigen. Both repulsive in, for example, TGF- ⁇ ⁇ production, can be detected or confirmed by a very marked increase.
  • antigens and adjuvants were co-inoculated at the same time in the local area, such as subcutaneously or intramuscularly, thereby inducing local inflammation there and enhancing the ability to present antigens.
  • ambroxol hydrochloride and its analogs are administered orally, subcutaneously, intramuscularly, etc. in a period of about 3 days before or 3 days after the antigen or vaccination, separately or independently of the antigen. .
  • intraperitoneal administration is possible.
  • This administration temporarily activates the cells responsible for mucosal immunity in the body before antigen inoculation (sensitization of mucosal immunity).
  • the desired antigen or vaccine is inoculated into the nasal cavity, oral cavity, respiratory tract, etc., in the sensitized organism, the secretory IgA antibody against the pathogen in the mucosa such as the nasal cavity, intestinal tract, lungs, etc.
  • Production is extremely effective and preferential, particularly selectively induced, and mucosal immunity is established (emergence of mucosal immunity).
  • the present invention is based on the above findings (1) and (2), and according to the present invention, the following means for solving the above-described problems are provided.
  • the mucosa immunosensitizing agent according to 2 above which contains as an active ingredient at least one compound selected from the group consisting of ampoampoxol and its analogs in an amount capable of sensitizing mucosal immunity. .
  • a mucosal vaccine kit comprising the sensitizer for mucosal immunity of 2 or 3 above and the antigen, vaccine or mucosal vaccine of 4 or 5 above. In order to facilitate and smooth use in vaccination, this kit can be commercialized by appropriately combining a sensitizer for mucosal immunity with a desired antigen and the like in advance.
  • 7 An allergy treatment system characterized in that (A) sensitization of mucosal immunity and (B) emergence of mucosal immunity are carried out in the order of (A) and (B).
  • FIG. 9 An allergy treatment kit comprising the sensitizer according to 8 above and an allergen administered transmucosally or orally.
  • Figure 1 shows the effect of ambroxol hydrochloride on influenza virus growth in the nasal cavity (a) and alveoli (b) after nasal inactivation of influenza vaccine and in the nasal cavity (c) and alveoli (d) after subcutaneous inoculation. The effect of this is shown.
  • Figure 2 shows the effect of ambroxol hydrochloride on the production of anti-influenza specific antibodies (IgA, IgG) in the nasal mucosa by nasal inoculation (a) and subcutaneous inoculation (b).
  • IgA, IgG anti-influenza specific antibodies
  • Figure 3 shows the effect of ampoule hydrochloride on the production of anti-influenza antibodies (IgA> IgG) in the alveolar mucosa by nasal inoculation (a) and subcutaneous inoculation (b).
  • Figure 4 shows the effect of ambroxol hydrochloride on the production of anti-influenza antibodies (IgA. IgG) in blood by nasal vaccination (a) and subcutaneous vaccination (b).
  • Figure 5 shows the effect of ambroxol hydrochloride on the amount of TGF- ⁇ produced in the nasal cavity (a) and alveoli (b) mucosa by nasal inoculation of the inactivated influenza vaccine.
  • Figure 6 shows the dose of ambroxol hydrochloride on the anti-influenza antibody production in alveolar lavage fluid (a) and (b) and blood (c) and (d) by nasal inoculation of inactivated influenza vaccine. The influence of the frequency of administration is shown.
  • Mucosal immunity is the strongest infection protection mechanism possessed by animals, including humans, against infectious pathogens such as the respiratory tract and intestinal tract.
  • This invention uses a conventional inactivated vaccine that is commercially available. By using it, it was possible to obtain such mucosal immunity in animals including all humans, and freed these animals from suffering from the above infections. This is the gospel of all centuries.
  • the vaccination according to the present invention is oral or nasal, so it is much simpler than conventional subcutaneous injection and is not painful at all. Can be vaccinated easily and quickly without causing fear.
  • the allergy treatment system is extremely effective in improving and treating IgE-dependent type I allergies such as atopic type 1 bronchial asthma, allergic rhinitis, hay fever and food allergies.
  • allergen administration routes are oral or nasal, there is no pain compared to injection.
  • the mucosal immunity sensitizer according to the present invention has withstood many years of clinical use and evaluation as an expectorant, and its safety has already been confirmed. Furthermore, this vaccination is done by mouth or transmucosal and does not use a syringe or needle, so it is not painful or painful and does not cause fear in infants. Therefore, the vaccination system according to the present invention guarantees much higher safety and effectiveness than conventional live vaccines and adjuvant vaccines, and enables non-invasive vaccination in a safe state. At the same time, this invention strongly enhances the credibility of the people for health and medical administration as a result of the above.
  • the present invention prevents both a decrease in gross domestic product and an increase in medical expenses due to the spread or epidemic of infectious diseases, and contributes to so-called rich country strong production (the country is rich and the industry is strengthened).
  • mucosal immunity is acquired by first performing “sensitization of mucosal immunity” and then “emergence of mucosal immunity” in order. Sensitization of mucosal immunity is carried out by administering a mucosal immunity sensitizer orally, subcutaneously or intramuscularly. The emergence of mucosal immunity is usually by inoculating commercially available inactivated vaccines or antigens, for example, transmucosal, oral, etc., specifically via the nasal, nasal, respiratory tract, eye drops, medications, etc. Do.
  • This system is applicable not only to chick but also mucosal immunity of laboratory animals, livestock, poultry, pets, fish, etc. Allergy treatment system:
  • allergens are used instead of the inactivated vaccines and antigens in the vaccination system described above. Allergens are administered by, for example, transmucosal, oral, etc., specifically, nasal, respiratory tract, taking, etc.
  • Mucosal immunity sensitizer :
  • This sensitizer contains as an active ingredient ampoules and its analogs (analogous compounds) in amounts sufficient for mucosal immunization.
  • ampoules and its analogs analogous compounds
  • ambroxol and its analogs as used in the present invention means “amp mouth quinol, bromhexine, and pharmacologically acceptable salts thereof”.
  • the active ingredient include amproxol, amproxol hydrochloride, bromhexine, bromhexine hydrochloride and the like. These compounds can be used alone or in combination of two or more thereof.
  • affinity similarity relationship between Ambrochitool and its analogs, CAS registration number for chemical properties, drug a action, use, etc. are as shown in the “Background Technology” section above.
  • the LD50 of ambroxol hydrochloride is intraperitoneal 268 mg and oral 2,720 mg in mice, and intraperitoneal 380 mg and oral 13,400 mg in rats.
  • the LD 50 (weight / kg body weight) of bromhexine is greater than 10 g for rabbits.
  • the reproductive toxicity of ambroxol at the oral dose (weight Z body weight kg), litter 500 mg significantly reduced the birth weight of the offspring (F1), and both rats 3,000 mg and 500 mg It has been reported that fetal body weight decreased, and the maximum no-effect level was 50 mg / kg body weight in rats and 40 mg / kg body weight in rabbits. In the above dose, teratogenicity was not detected in both rats and rabbits.
  • ampoules hydrochloride is an expectorant of capsules (45 mgZ dose / capsule), and proxin hydrochloride is an injection solution (4 mgZ dose / ampoule).
  • proxin hydrochloride is an injection solution (4 mgZ dose / ampoule).
  • As an airway mucus solubilizer it has been manufactured by Boehringer ⁇ ⁇ Ngelheim (Germany) for more than 10 years, and is commercially available. It has been investigated. As a result, there are no known reports of serious side effects, and the safety of these compounds has been widely established around the world.
  • Administration and timing of mucosal immunity sensitizer is an expectorant of capsules (45 mgZ dose / capsule), and proxin hydrochloride is an injection solution (4 mgZ dose / ampoule).
  • known routes such as oral, subcutaneous and intramuscular can be used.
  • oral administration is preferred because it is simple and can be easily carried out by infants without pain.
  • Mucosal immunity sensitizer form :
  • Mucosal immunity sensitizer It is necessary to vary the dose based on the age, body weight, administration route, etc. of the subject to be administered, such as human animals, but for example, oral dose of ambroxol hydrochloride Body weight kg / day is about lOOmg to about lmg, desirably in the range of about 20 mg to about 5 mg.
  • Antigens and vaccines are examples of:
  • Antigens used for the emergence of mucosal immunity include desired antigens derived from eukaryotic cells, prokaryotic cells, pathogens, viruses, chlamydia, etc., and as vaccines, commercially available or conventional inactivated vaccines such as A desired vaccine such as a factor vaccine containing a bacterium, virus, toxoid, or pathogen component as an active ingredient, or a split vaccine can be used.
  • viruses such as droplet infection or respiratory tract infection viruses such as adeno, corona, influenza, measles, rubella, mumbus, and new pneumonia SARS, enteroviruses such as entero, polio, rota, etc.
  • inactivated vaccines In the use of inactivated vaccines, bacterial vaccines, such as commercially available DP (diphtheria / pertussis) mixed inactivated vaccines, and other HIV inactivated vaccines, a remarkable infection protection effect by mucosal immunity can be obtained. Since the live vaccine itself induces cellular immunity and mucosal immunity, the vaccination system according to the present invention is unnecessary and is not applicable. In addition, live vaccines cannot be used for the emergence of mucosal immunity according to the present invention.
  • DP diphtheria / pertussis
  • the active ingredient of the live vaccine is a proliferative attenuated bacterium or attenuated virus, etc.
  • the establishment of cellular immunity is based on the premise that the amount of antigen is amplified by its own proliferation in the body of a live vaccine recipient. Therefore, the amount of antigen in live vaccine is about 1 / 10,000 or less than that of inactivated vaccine.
  • the growth and amplification of proliferative attenuated pathogens which are effective antigens in live vaccines, are rather inhibited or prevented, so these antigens are quantitatively recognized immunologically. Cannot be done. As a result, cellular immunity is not established, mucosal immunity is not created, and mucosal immunity is not established. Allergen:
  • Allergens used for emergence of mucosal immunity include mites, pet animals, insects, grass Known or commercially available purified allergens derived from this pollen, woody pollen, fungus, food, etc. can be used: eg: Der p I, Der f I, Der ml, Eur m I from cats, Fed from cats dl, Amb a I from Bubususa, Cry jl, Cry j II from cedar, Asp fl from Aspergillus, Gal dl from chicken eggs, Gal d II, etc.
  • Antigen, vaccine or allergen inoculation or administration route eg: Der p I, Der f I, Der ml, Eur m I from cats, Fed from cats dl, Amb a I from Bubususa, Cry jl, Cry j II from cedar, Asp fl from Aspergillus, Gal dl from chicken eggs, Gal d II, etc.
  • the mucosa is the most desirable route for inoculating the mucosal immune response.
  • a histologically known mucosa in the nostril, trachea, airway, eyelid, oral cavity, intestinal tract, etc. can be used as the inoculation route. Inoculation or administration of antigen, vaccine or allergen to mucosa:
  • Inoculation of the antigen, vaccine or allergen into the mucous membrane can be performed, for example, by nasal, oral, ophthalmic, respiratory tract and the like.
  • Specific examples include parenteral 'mucosal, for example, liquid formulation antigens, vaccines or allergens, nasal drops, eye drops, sprayed airway arches and dripping onto mucous membranes.
  • parenteral 'mucosal for example, liquid formulation antigens, vaccines or allergens, nasal drops, eye drops, sprayed airway arches and dripping onto mucous membranes.
  • it can be performed by taking an antigen such as a syrup, a roach, a powder, a tablet, a vaccine, swallowing, or the like.
  • inactivated split influenza vaccine Floating egg-derived floating fluid inoculated with influenza virus A Aichi / 68/2 / H3N2 (lx lO 8 Plaque forming unit (PFU)) (Kawasaki Medical University ⁇ Department of Microbiology, Masanobu Ouchi
  • the split-type influenza vaccine was created using the following procedure. ⁇ Propiolactone (Wako Pure Chemicals Co., Ltd. Japan 'Osaka) was added to a suspension of virus that was dialyzed overnight with 0.004M PBS (Yukara Bio Inc. Japan ⁇ Tokyo ⁇ Shiga) to a final concentration of 8 nM. And incubated for 18 hours in an ice bath.
  • ⁇ -propiolactone was hydrolyzed by incubation at 37 ° C for 1.5 hours.
  • Tween 20 (Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1%, and then Jetyl ether (Wako Pure Chemical Industries, Ltd.) in an amount equal to Tween was added, and the mixture was mixed by inverting at 4 for 2 hours.
  • the aqueous layer was recovered by centrifuging this solution at 2000 rpm for 5 minutes.
  • the Jetyl ether was removed from the water layer using the Automatic Environmental Speed Vac System (SAVANT INSTRUMENTS, INC., USA). This was filtered with a Millex 0.45 ⁇ filter (MILLIPORE USA 'Massachusetts) and used as an inactivated split influenza vaccine (Chemical and Serum Therapy Laboratory ⁇ Kikuchi Laboratory ⁇ Kumamoto).
  • the split-type influenza vaccine prepared by the above method is diluted with PBS so that it becomes a 0 ⁇ 2 ⁇ ⁇ / 2 ⁇ 1 solution, and this is ⁇ ⁇ each in both nasal cavities of mice. A total of 2 ⁇ 1 nasally was administered.
  • the same split influenza vaccine was diluted with 0.2p g / 50 pl so that solution PBS, which was administered to the mice neck subcutaneously. ⁇ for the control group PBS in the same amount as the cutin solution was administered.
  • secondary immunization was performed in the same manner as the first immunization.
  • Ambroxol hydrochloride (Nippon Belinger Ingelheim Japan ⁇ Kobe) was intraperitoneally administered daily as a 200 pl PBS solution at a dose of 10 mg / kg body weight from 2 days before and 2 days after vaccine administration.
  • mice Two weeks after secondary immunization, mice underwent laparotomy and incision of the trachea under Pentobarbi anaesthesia, and 3Fr (Atom Medical Co., Ltd. Japan) Tokyo) was inserted into the lung, and 1 ml of physiological saline was injected to collect the solution. This was repeated three times, and a total of 3 ml was used as the lung lavage fluid.
  • an Atom vein catheter was inserted from the incised trachea into the nasal cavity, 1 ml of physiological saline was injected, and the fluid that came out of the nose was collected. This solution was used as a nasal wash.
  • blood was collected from the heart, and serum was prepared by centrifugation at 5000 rpm for 10 minutes.
  • Virus infection experiment and evaluation of infectivity titer The egg-derived suspension of the same influenza virus A Aichi / 68/2 / H3N2 strain used for the preparation of the inactivated split-type influenza vaccine in Experimental Example 1 A total of 6.6 ⁇ 10 4 PFU / 3pl was instilled into both nasal cavities to infect the nasal mice of the week. Three days after the infection, nasal cavity and lung lavage fluid were prepared in the same manner as in Example 3 above, and used for evaluation of virus infectivity. The evaluation of virus infectivity was performed using A549 cells (provided by Professor Masanobu Ouchi, Department of Microbiology, Kawasaki Medical School).
  • A549 cells were cultured using 5% fetal calf serum (FCS) / DMEM (Gibco America New York). A549 cells were subcultured to 6-well culture plates (Grainer-Germany Stuttgart) at 100% confluence and replaced with serum-free medium after 24 hours. Nasal influenza infected mice in each Ueru, lung wash was added dropwise in 500Myu1, and culture was performed at 16 hours 37 12 hours C0 2 incubator scratch. To this was added 1% PBS of red blood cells collected from guinea pigs, and the mixture was allowed to stand at room temperature for 5 minutes.
  • FCS fetal calf serum
  • DMEM Gibco America New York
  • the cells were washed with lmM Ca 2+ / Mg 2+ PBS, and the cells that aggregated erythrocytes were counted as virus-infected cells, and the virus infectivity was evaluated (Infection and Immunity, 39, 879-888). Page, 1983).
  • IgG fraction was purified from lung lavage fluid from influenza-treated and virus-infected mice by affinity chromatography using a Rec-Protein G Sepharose 4B column (ZYMED LABORTORIES INC, San Francisco, USA).
  • Anti-mouse IgA 'IgG (SIGMA) was bound to a BrCN-activated Sepharose 4B column (Amersham bioscience USA' New Jersey) column and passed through Protein G From this fraction, the IgA fraction was purified by chromatography using this fraction.
  • Anti-fluenza-specific IgA and IgG were purified from the IgA and IgG fractions by binding the inactivated split influenza vaccine used for immunization to the BrCN-activated Sepharose column and using the antigen affinity mouthmatography using this. .
  • Coupling of an inactivated split influenza vaccine as a ligand to the column was performed by a binding reaction using O. IM NaHCO 3 /0.5M NaCl buffer (pH 8.5), and free ligand was added to 0.1M acetic acid / 0.5 After removal with M NaCl buffer ( ⁇ 8 ⁇ 5), neutralization was performed with PBS ( ⁇ 7.5).
  • Each affinity matrix was subjected to affinity binding reaction and free antibody removal with PBS ( ⁇ 7.5) and elution with Glycine-HCl buffer (pH 2.8). The eluted fraction was immediately neutralized with 0.5M Tris-HCl buffer ( ⁇ 9.0), lysed with MilliQ water, lyophilized, and dissolved in PBS before use.
  • the contents of anti-influenza IgA and IgG in nasal cavity, lung lavage fluid and serum were quantified by ELISA assay.
  • the ELISA assay was performed by partially modifying the method of the Mouse ELISA quantitation kit of BETHYL LABORATORIES (American Power, Texas).
  • BETHYL LABORATORIES American Power, Texas.
  • Vaccine lpg, urushi serum albumin (BSA, SIGMA, Missouri) lpg / ml PBS solution ⁇ was added to each well and allowed to stand overnight at 43 ⁇ 4: A reaction was performed.
  • wash solution 50 mM Tris, 0.14 M NaCl, 0.05% Tween20, pH 8.0.
  • wash solution 50 mM Tris, 0.14 M NaCl, 0.05% Tween20, pH 8.0.
  • sample / conjugate diluent 50mM Tris, 0.15M NaCl, 1% BSA, 0.05% Tween20, pH8.0
  • Goat anti-mouse IgA or IgG-HRP (BETHYL LABORATORIES INC.) was used as a secondary antibody, and a color reaction was performed using TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Inc. United States, Maryland). The reaction was stopped by adding ⁇ 2 ⁇ H 2 S0 4 (Wako Pure Chemical Industries, Ltd.) to each well, and the absorbance at 450 nm was measured with SPECTRA max PLUS 384. As a standard for quantification, 10 ng of anti-influenza IgA and IgG purified from the above-mentioned lung lavage fluid, and the absorbance obtained in the same manner as described above were used.
  • TGF- ⁇ The amount of TGF- ⁇ secretion in the nasal cavity and lung lavage fluid was quantified by ELISA assay.
  • the ELISA assay was performed using a TGF- ⁇ ELISA kit (BIOSOURCE INTERNATIONAL USA, California) according to the usage attached to the kit.
  • inactivated split influenza octynein O. lpg prepared in Experimental Example 1 was administered as a PBS solution to both nasal cavities of BALB / c mice in ⁇ aliquots.
  • the same and the same amount as the vaccine used for nasal inoculation was injected subcutaneously into the neck of BALB / c mice as a 50 ⁇ l PBS solution.
  • the second immunization was performed in the same manner as the first immunization.
  • the ampoule hydrochloride was administered intraperitoneally every day as a 200 ⁇ l PBS solution so that lOmg / kg body weight of the mouse was 2 to 2 days before vaccine administration.
  • Figure 1 shows the virus infectivity in nasal lavage fluid (a) and lung lavage fluid (b) after nasal inoculation, and the virus infection titer in nasal lavage fluid (c) and lung lavage fluid (d) in subcutaneous inoculation (n -15 ⁇ 20, average person SD; +, significance level by t-test is p ⁇ 0.01 for the vaccine group.
  • Fig. 1 (a) and (b) in the case of nasal inoculation, the virus growth is hardly suppressed by administration of ⁇ cutin alone. However, the use of amproxol hydrochloride significantly suppressed the growth of the virus.
  • Fig. 1 (c) and (d) the effect of using amproxol hydrochloride was not observed in the subcutaneous inoculation.
  • the ampoline hydrochloride specifically activates only the cells responsible for mucosal immunity (sensitization of mucosal immunity by ambroxol hydrochloride) and passed through the inactivated vaccine under such sensitization. It shows that mucosal inoculation establishes infection protection and mucosal immunity against pathogens in the nasal cavity and lung mucosa (emergence of mucosal immunity by vaccine).
  • the inactivated split influenza vaccine O.lg prepared in Experimental Example 1 was administered as a PBS solution to both nasal cavities of BALB / c mice in ⁇ .
  • the same and the same amount as the vaccine used for nasal inoculation was injected subcutaneously into the neck of BALB / c mice as a 50 ⁇ l PBS solution. 4 weeks later, in the same way as the first immunization Secondary immunization was performed.
  • Ambroxol hydrochloride was intraperitoneally administered daily as a 200 ⁇ l PBS solution so that lOmg / kg body weight of mice was obtained 2 to 2 days after vaccine administration. Each control group received the same volume of PBS.
  • Figure 2 shows the amount of anti-influenza antibody production in the nasal lavage fluid after nasal inoculation (a) and subcutaneous ingestion (b), respectively.
  • inactivated split influenza vaccine O In nasal inoculation, inactivated split influenza vaccine O. lpg prepared in Experimental Example 1 was administered as a PBS solution to both nasal cavities of BALB / c mice at ⁇ .
  • the same and the same amount as the vaccine used for nasal inoculation was injected subcutaneously into the neck of BALB / c mice as a 50 ⁇ l PBS solution.
  • the second immunization was performed in the same manner as the first immunization.
  • Ambroxol hydrochloride was intraperitoneally administered daily as a 200 ⁇ l PBS solution so that the concentration was 10 mg / kg body weight of mice 2 to 2 days before vaccine administration. Each control group received the same volume of PBS. Two weeks after the second immunization, the mice were sacrificed, lung lavage fluid was prepared in the same manner as in Experimental Example 3 above, and the production of anti-influenza antibodies (IgA and IgG) was quantified using these. Figure 3 shows the results. result
  • Figure 3 shows the amount of anti-influenza antibody produced in the lung lavage fluid after nasal inoculation (a) and subcutaneous inoculation (b).
  • the white bar in the figure indicates IgA, and the black bar indicates IgG.
  • N 15-20, mean soil SD; +, p ⁇ 0.01 for the vaccinated group).
  • Fig. 3 (a) in the case of nasal inoculation, administration of amproxol hydrochloride significantly increased the amount of IgA in the lung lavage fluid produced by vaccine administration alone. However, this effect was very significant for IgA, a factor responsible for mucosal immunity, and IgG showed almost no increase in production.
  • Fig. 3 (b) IgA production was observed in the lung lavage fluid even in the case of subcutaneous inoculation, but no increase in IgA production by ampoline hydrochloride was observed.
  • the activated split influenza vaccine O. lpg prepared in Experimental Example 1 was administered as a PBS solution and administered to both nasal cavities of BALB / c mice at ⁇ .
  • the same and the same amount as the vaccine used for nasal inoculation was injected subcutaneously into the neck of BALB / c mice as a 50 ⁇ l PBS solution.
  • the second immunization was performed in the same manner as the first immunization.
  • Ambroxol hydrochloride was intraperitoneally administered daily as a 200 ⁇ l PBS solution so that the concentration was 10 mg / kg body weight of mice 2 to 2 days after vaccine administration.
  • Each control group received the same volume of PBS.
  • Mice were sacrificed 2 weeks after the second immunization, serum prepared in the same manner as in Experimental Example 3 was quantified anti-influenza antibodies (IgA and I g G) production amount using these. The results are shown in Fig. 4. result
  • Figure 4 shows the amount of anti-influenza antibody produced in the blood by nasal inoculation (a) and subcutaneous inoculation (b), respectively.
  • the white bar in the figure indicates IgA, and the black bar indicates IgG.
  • N 15-20, average person SD; +, p ⁇ 0.01 for the vaccinated group).
  • the cytokine TGF- ⁇ - acts at the stage where T lymphocytes receiving signals from antigen-presenting cells stimulate B cells to produce antibodies. Therefore, the effect of amproxol hydrochloride on TGF- ⁇ secretion in both nasal cavity and alveolar mucosa by nasal vaccination with the vaccine was investigated.
  • the activated split-type influenza vaccine O. lpg prepared in Experimental Example 1 was administered as a PBS solution to both nasal cavities of BALB / c mice by ⁇ . Four weeks later, the second immunization was performed in the same manner as the first immunization.
  • Ambroxol hydrochloride was intraperitoneally administered daily as a 200 ⁇ l PBS solution so that the body weight was 10 mg / kg body weight from 2 to 2 days before vaccine administration. Each control group received the same volume of PBS. Two weeks after the second immunization, the mice were sacrificed, nasal washes were prepared in the same manner as in Experimental Example 3 above, and the amount of TGF- ⁇ sputum secretion was quantified. The results are shown in Fig. 5. Result
  • FIG. 5 shows the TGF- ⁇ ⁇ concentration in each mucous membrane of the nasal cavity (a) and alveoli (b) after nasal vaccination.
  • the ampoule hydrochloride is once a day of vaccination or peritoneally as a 200 ⁇ 1 PBS solution to reach 1, 3, and lOmg / kg body weight of mice for 5 days from 2 to 2 days before vaccination.
  • Administered In nasal inoculation, influenza inactivated vaccine O. lpg was administered as a ⁇ PBS solution to both nasal cavities of BALB / c mice.
  • secondary immunization was performed in the same manner as the first immunization.
  • Ambroxol hydrochloride was administered intraperitoneally as a 200 ⁇ l PBS solution once daily on the day of vaccine administration or for 5 days from 2 to 2 days prior to administration to give 1, 3, and lOmg / kg body weight of mice.
  • mice were sacrificed, and nasal lavage fluid, alveolar lavage fluid, and serum were collected, and the amounts of these anti-influenza IgA and IgG antibodies were respectively determined. The result is shown in Fig. 6. Result
  • FIG. 6 shows the amount of each antibody produced by IgA (a) in alveolar mucosa, IgG (b) in alveolar mucosa, IgA (c) in blood, and IgG (d) in blood.
  • IgA antibody in the alveolar lavage fluid increased depending on the dose even after a single dose of ampoline hydrochloride, and it exceeded 3 mg / kg body weight. There was a significant difference (p ⁇ 0.01) in the concentration of. Among the concentrations tested, the strongest effect was obtained at 10 mg / kg body weight. Increase the frequency of administration for 5 days after 2 to 2 days before administration. In the case of continuous administration of acid ambroxol, a significant increase (p ⁇ 0.05) was observed only at 10 mg / kg compared to a single dose. By administering 10 mg / kg five times to the vaccine-only treatment, an approximately 3-fold increase in IgA antibody was observed.
  • ampoline hydrochloride increased the production of antigen-specific IgA preferentially in a concentration-dependent manner.
  • the minimum amount required for intraperitoneal administration of ambrochitool hydrochloride as a sensitizer for mucosal immunity was 10 mg / kg.
  • Reference Example 1 Sugi pollen allergens Cry jl and Cry J2 [manufactured by Seikagaku Corporation (Japan)] in place of influenza inactivated vaccine, using 0.1 ⁇ g / nasal dose, as in Examples 2 and 3 The production of specific IgA antibody against the allergen can be confirmed. However, anti-Cry Jl and 2 monoclonal antibodies [manufactured by Seikagaku Corporation (Japan)] are used for antibody measurement by ELISA.
  • This invention Used in the manufacturing and sales of vaccines for livestock, livestock, poultry, fish, pets, etc.
  • This invention is a technology that enables the practical use of mucosal immunity, and since this mucosal immunity is the most important and powerful means of preventing infection, it relates to the pharmaceutical industry, veterinary medicine industry, vaccination.

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Abstract

L'invention concerne un système d'inoculation protectrice comprenant d'abord de 'sensibiliser une compétence immunologique des muqueuses' et ensuite de 'développer une immunité des muqueuses' et un système servant à traiter une allergie. Ce système d'inoculation protectrice grâce à l'utilisation de l'immunité des muqueuses ou d'un anticorps IgA spécifique est un moyen tout à fait sans danger, effectif et efficace pour éviter des infections telles que des infections respiratoires et des infections de l'appareil digestif. Le système servant à traiter une allergie, lequel est établi en administrant de façon orale ou transmuqueuse un allergène, est un moyen qui fait date contribuant largement au traitement et à la prévention d'une allergie de type I IgE-dépendante.
PCT/JP2005/015379 2004-08-18 2005-08-18 Immunité des muqueuses et vaccin par voie muqueuse et système d'inoculation protectrice utilisant le sensibilisateur WO2006019183A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02235823A (ja) * 1989-03-09 1990-09-18 Watanabe Mitsuko 抗アレルギー剤
JPH0761934A (ja) * 1993-08-25 1995-03-07 Yakult Honsha Co Ltd ワクチンの効果増強剤及び効果増強食品
JPH11515023A (ja) * 1995-11-01 1999-12-21 メデヴァ ホールディングス ビー．ヴィ． インフルエンザワクチン組成物
JP2002003395A (ja) * 2000-04-19 2002-01-09 Eisai Co Ltd 粘膜免疫賦活剤
JP2002326953A (ja) * 2001-03-19 2002-11-15 Grisotech Sa 経粘膜法により吸収されるワクチン
JP2003155230A (ja) * 2001-09-04 2003-05-27 Techno Network Shikoku Co Ltd 抗インフルエンザ薬

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02235823A (ja) * 1989-03-09 1990-09-18 Watanabe Mitsuko 抗アレルギー剤
JPH0761934A (ja) * 1993-08-25 1995-03-07 Yakult Honsha Co Ltd ワクチンの効果増強剤及び効果増強食品
JPH11515023A (ja) * 1995-11-01 1999-12-21 メデヴァ ホールディングス ビー．ヴィ． インフルエンザワクチン組成物
JP2002003395A (ja) * 2000-04-19 2002-01-09 Eisai Co Ltd 粘膜免疫賦活剤
JP2002326953A (ja) * 2001-03-19 2002-11-15 Grisotech Sa 経粘膜法により吸収されるワクチン
JP2003155230A (ja) * 2001-09-04 2003-05-27 Techno Network Shikoku Co Ltd 抗インフルエンザ薬

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Title
GIBBS B.F.: "Ambroxol inhibits IgE-dependent mediator secretion from human skin mast cells", INFLAMMATION RESEARCH, vol. 49, SUPPLEMENT 1, 2000, pages S17 - S18, XP002996873 *
KIDO H.: "Ko-Influenza Sayo o Shimesu Kyotanzai no Fushigi, Aratana Yakuri Sayo to Kijo", ALLERGOLOGY, vol. 16, no. 3, 2003, pages 232 - 239, XP002996872 *
NISHIKAWA M.: "Seitai Bogyo Inshigun no Bunpitsu o Sokushin suru Ensan Ambroxol no Ko-Influenza Koka", SHIKOKU ACTA MEDICA, vol. 58, no. 3, 15 June 2002 (2002-06-15), pages 162 - 167, XP002996871 *

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