WO2006104336A1 - Préparation pour la prévention, le soulagement et le traitement de la dermatite atopique - Google Patents

Préparation pour la prévention, le soulagement et le traitement de la dermatite atopique Download PDF

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Publication number
WO2006104336A1
WO2006104336A1 PCT/KR2006/001128 KR2006001128W WO2006104336A1 WO 2006104336 A1 WO2006104336 A1 WO 2006104336A1 KR 2006001128 W KR2006001128 W KR 2006001128W WO 2006104336 A1 WO2006104336 A1 WO 2006104336A1
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WO
WIPO (PCT)
Prior art keywords
staphylococcus aureus
antibody
set forth
composition
lysate
Prior art date
Application number
PCT/KR2006/001128
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English (en)
Inventor
Jong Bae Park
Jung Woo Kim
Si Yong Yang
Mi Ok Jang
Soon Oh Shin
Original Assignee
Dan Biotech Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dan Biotech Inc. filed Critical Dan Biotech Inc.
Publication of WO2006104336A1 publication Critical patent/WO2006104336A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60J—WINDOWS, WINDSCREENS, NON-FIXED ROOFS, DOORS, OR SIMILAR DEVICES FOR VEHICLES; REMOVABLE EXTERNAL PROTECTIVE COVERINGS SPECIALLY ADAPTED FOR VEHICLES
    • B60J1/00—Windows; Windscreens; Accessories therefor
    • B60J1/20—Accessories, e.g. wind deflectors, blinds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B9/00—Screening or protective devices for wall or similar openings, with or without operating or securing mechanisms; Closures of similar construction
    • E06B9/52—Devices affording protection against insects, e.g. fly screens; Mesh windows for other purposes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60J—WINDOWS, WINDSCREENS, NON-FIXED ROOFS, DOORS, OR SIMILAR DEVICES FOR VEHICLES; REMOVABLE EXTERNAL PROTECTIVE COVERINGS SPECIALLY ADAPTED FOR VEHICLES
    • B60J3/00—Antiglare equipment associated with windows or windscreens; Sun visors for vehicles
    • B60J3/002—External sun shield, e.g. awning or visor
    • B60J3/005—External sun shield, e.g. awning or visor for side windows
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • 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
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B9/00—Screening or protective devices for wall or similar openings, with or without operating or securing mechanisms; Closures of similar construction
    • E06B9/52—Devices affording protection against insects, e.g. fly screens; Mesh windows for other purposes
    • E06B2009/524—Mesh details
    • E—FIXED CONSTRUCTIONS
    • E06—DOORS, WINDOWS, SHUTTERS, OR ROLLER BLINDS IN GENERAL; LADDERS
    • E06B—FIXED OR MOVABLE CLOSURES FOR OPENINGS IN BUILDINGS, VEHICLES, FENCES OR LIKE ENCLOSURES IN GENERAL, e.g. DOORS, WINDOWS, BLINDS, GATES
    • E06B9/00—Screening or protective devices for wall or similar openings, with or without operating or securing mechanisms; Closures of similar construction
    • E06B9/52—Devices affording protection against insects, e.g. fly screens; Mesh windows for other purposes
    • E06B2009/527—Mounting of screens to window or door

Definitions

  • the present invention relates to a composition for the prevention, alleviation, and treatment of atopic dermatitis. More particularly, the present invention relates to a composition, based on antibodies to Staphylococcus aureus enterotoxins and Staphylococcus aureus lysates and optionally to Streptococcus pyogenes lysates, for the prevention, alleviation and treatment of atopic dermatitis.
  • Atopy is the clustering of allergic syndromes on the skin, respiratory mucosa, the conjunctiva, intestinal mucosa, and other sites in certain individuals who exhibit atopic factors.
  • atopic factor-carrying individuals suffers from itching, eruptions, hives, angioedema, sneezing, sniveling, conjunctivitis, tearing, etc.
  • allergic disorders caused by atopic factors include atopic dermatitis, allergic rhinitis, asthma, allergic conjunctivitis, allergic enteritis, and atopic rashing, which are caused alone or in combination depending on various factors of patients, such as genetic factors, environment, age, etc.
  • Atopic dermatitis is representative of the allergic diseases from which individuals having atopic allergies suffer.
  • Atopic dermatitis is prevalent all over the world, and 0.7% of the adult population and 3-5% of children younger than 5 years old suffer from this disease. Furthermore, the number of patients with atopic dermatitis rapidly increases every year, mainly due to environmental factors.
  • the mechanism of atopic dermatitis is as follows: If an allergen is introduced through the homy layer into the epidermal cells, macrophages engulf and decompose the allergen. When the allergen is too abundant to be decomposed completely, macrophages generate information about the existence of the untreated allergen, which is transferred through T cells to eosinophils dispersed in the blood, leading to the collection of eosinophils at the site of invasion of the allergen. The eosinophils accumulated at the affected site secrete excess chemokines to cause inflammation. This excessive response is the cause of the allergic reactioa
  • Itching is typical of the syndromes of atopic dermatitis. If a patient with atopic dermatitis repeatedly scratches an itchy spot on the skin, secondary abrasion may be developed. Also, erythema appears on various skin spots, such as the palm, lower eyelid, etc., of patients suffering from atopic dermatitis. There may be increased risk of dermal infection with Staphylococcus aureus. In addition, abnormal dermal syndromes including skin dryness, change in skin color, and keratinization develop, and the level of IgE in the blood increases.
  • Atopic diseases are worsened by IgE against Staphylococcus aureus or by Staphylococcus aureus enterotoxin B (SEB), which stimulates T-cell responses.
  • SEB acts as the main cause of atopic diseases, making it more difficult to cure atopic diseases.
  • steroid-based agents exhibit an excellent antiphlogistic and immunosuppressive effect, but cause various adverse effects, including excessive hair growth, pigment reduction, susceptibility to infections, skin shrinkage, acne, thin skin, capillary fragility, etc., upon long- term use.
  • systemic symptoms may be induced by hormones, or steroid resistance, which causes the aggravation of systemic symptoms when the intake of the steroid-based agents is ceased.
  • Korean Patent Application No. 2000-0021196 discloses a selective antibacterial composition comprising farnesol and/or xylitol. It explains that the selective antibacterial composition has inhibitory activity against Staphylococcus aureus relevant to atopy, but does not influence the growth of Staphylococcus epidermidis on healthy skin, thereby curing atopic dermatitis.
  • Korean Patent Application No. 2003-0053370 discloses a cosmetic composition for atopic skin (skin affected by atopic dermatitis), comprising a mixture of extracts from Sophora flavescens Ait, Bletilla striata, Anemarrhena asphodeloides, and Ophiopogon japonicus, as an active ingredient
  • This cosmetic composition is described to have antibacterial, antifungal, and anti-inflammatory activities and to show moisturizing and free radical-scavenging effects.
  • the cosmetic composition can cure skin dryness and eczema, both characteristic of atopic skin, and alleviate itching in addition to preventing and treating secondary microbacterial infection.
  • 2003-0053370 is likely to have inhibitory activity not only against harmful bacteria, but also against skin flora. Although it is described as being selective for Staphylococcus aureus, the composition of Korean Patent Application No. 2000-0021196 is found to significantly inhibit the growth of skin flora as well. Particularly, it is impossible for this composition to remove SEB, which is a persistent cause of atopy.
  • a pharmaceutical composition for local use in the prevention, alleviation and treatment of atopic dermatitis, which comprises respective antibodies to enterotoxins and lysates of Staphylococcus aureus, and optionally an antibody to a Staphylococcus aureus lysate in combination with a pharmaceutically acceptable vehicle.
  • a cosmetic composition for the prevention and alleviation of atopic dermatitis, which comprises respective antibodies to an enterotoxin and a lysate of Staphylococcus aureus, and optionally an antibody to a Staphylococcus aureus lysate in combination with a pharmaceutically acceptable vehicle.
  • FIG. 1 is an electrophoretogram showing a gene coding for Staphylococcus aureus enterotoxin B in accordance with the present invention.
  • FIG. 2 is a schematic diagram showing the structure of the recombinant expression plasmid pDQSEB carrying a DNA sequence (SEB) coding for Staphylococcus aureus enterotoxin B.
  • FIG. 3 is an electrophoretogram of the recombinant expression vector pDQSEB according to the present invention.
  • FIG. 4 shows electrophoretograms for SDS-PAGE (A) and Western blotting (B) with the recombinant Staphylococcus aureus enterotoxin B antigen obtained in accordance with the present inventioa
  • FIG. 5 is a graph showing results of ELISA with sera from laying hens immunized with the recombinant Staphylococcus aureus enterotoxin B antigen according to the present invention.
  • FIG. 6 is a graph showing results of ELISA with egg yolks from laying hens immunized with the recombinant Staphylococcus aureus enterotoxin B antigen according to the present invention.
  • FIG. 7 provides electrophoretograms for SDS-PAGE (A) and Western blotting (B) with an antibody to the recombinant Staphylococcus aureus enterotoxin B antigen in accordance with the present invention.
  • FIG. 8 is a graph showing blood IgE levels which decrease as the yolk antibodies prepared in accordance with the present invention are administered.
  • respective antibodies against SEB and Staphylococcus aureus lysates, together with an antibody against Streptococcus pyogenes lysates show an excellent and synergistic effect in preventing, alleviating and treating atopic dermatitis, so that the antibodies can be utilized in a pharmaceutical composition for local application as well as in a cosmetic composition.
  • a pharmaceutical composition for local application as well as in a cosmetic composition.
  • the present invention provides a locally applicable pharmaceutical composition for the prevention, alleviation and treatment of atopic dermatitis, which comprises respective antibodies against SEB and Staphylococcus aureus lysates, in combination with a pharmaceutically acceptable vehicle.
  • the pharmaceutical composition for local use comprises an antibody against the enterotoxin of Staphylococcus aureus in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 5 wt%, and an antibody against a lysate of Staphylococcus aureus in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 2 wt%, on the basis of the total weight of the composition.
  • the pharmaceutical composition for local use may further comprise an antigen against Streptococcus pyogenes lysates, showing improved effects of preventing, alleviating and treating atopic dermatitis.
  • the antibody to Streptococcus pyogenes lysates may be contained in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 2 wt%, based on the total weight of the composition.
  • respective antibodies against SEB, Staphylococcus aureus lysates, and Streptococcus pyogenes lysates can be prepared as follows.
  • antibodies against a Staphylococcus aureus enterotoxin, a Staphylococcus aureus lysate, and a Streptococcus pyogenes lysate can be prepared by immunizing a mammal with an antigen (e.g., selected from a Staphylococcus aureus enterotoxin, a Staphylococcus aureus lysate, a Streptococcus pyogenes lysate, and combinations thereof), and isolating antibodies from the serum of the immunized mammal. Details of these processes are well known in the art.
  • the antigen is injected into laying hens which then lay eggs. From the yolk of the laid eggs, respective yolk antibodies to a Staphylococcus aureus enterotoxin, a Staphylococcus aureus lysate, and a Streptococcus pyogenes lysate are isolated.
  • the antibodies used in the present invention are the above- mentioned "yolk antibodies” because they can be readily prepared in a high concentration. Details of these processes are given below. It is preferred that a Staphylococcus aureus enterotoxin, useful as an antigen in the present invention, be prepared through genetic recombination.
  • a genetic recombination method for preparing a pathogenic factor comprises (1) constructing a DNA sequence coding for the pathogenic factor; (2) operatively linking the DNA sequence to an expression control sequence of a vector to give a recombinant expression vector; (3) transforming a host cell with the recombinant expression vector; (4) growing the transformed host cell to express the pathogenic factor; and (5) separating and purifying the pathogenic factor from the culture.
  • the pathogenic factor is a Staphylococcus aureus enterotoxin, and preferably the Staphylococcus aureus enterotoxin B (SEB) that induces and aggravates atopic dermatitis.
  • SEB Staphylococcus aureus enterotoxin B
  • a DNA sequence encoding a Staphylococcus aureus enterotoxin can be chemically synthesized or prepared through PCR, with the genomic DNA of Staphylococcus aureus serving as a template. Primers useful for this PCR may be synthesized with reference to the base sequences available from GenBank.
  • the isolation of the genomic DNA from Staphylococcus aureus can be achieved by disrupting a cell mass collected from a culture of Staphylococcus aureus in a lysis buffer, centrifuging the cell lysate, and removing proteins and RNA from the supernatant to extract pure DNA.
  • Culture media for Staphylococcus aureus are generally nutrient broth, but are not limited thereto.
  • Preferable growth conditions for Staphylococcus aureus include a temperature from 30 to 40 ° C and a time period of 10 to 20 hours.
  • the DNA sequence thus obtained may be cloned in a suitable vector.
  • a recombinant expression vector identified as pDQSEB, which contains and can express a DNA sequence (SEB) encoding a Staphylococcus aureus enterotoxin.
  • vector means a DNA molecule used as a vehicle to deliver exogeneous genetic materials into host cells.
  • recombinant expression vector means a circular DNA molecule in which an exogeneous genetic material is operatively linked so that it can be expressed in host cells.
  • the vector may be a plasmid, a phage genome, or a potential genome insert.
  • examples of the vector used in the present invention include pUC8, pBR322, pQE80L, pET/Rb, pGEX, and pET28a, with preference for pQE80L.
  • the recombinant expression vector pDQSEB of the present invention is constructed from the vector pQE80L.
  • a host cell transformed or transfected with the above-described recombinant expression vector constitutes another aspect of the present invention.
  • transformation means the genetic alteration of a host cell resulting from the introduction, uptake, either into the host genome or as an extra-genomic (cytoplasmic) factor, and expression of a foreign DNA molecule.
  • transfection as used herein, means the introduction of a recombinant expression vector into a host cell irrespective of whether any coding sequence is expressed or not
  • Host cells used in the present invention may be prokaryotic or eukaryotic. Preferable is a host which is superior in terms of DNA introduction and expression efficiency. Eukaryotes or prokaryotes, such as Escherichia spp., Pseudomona spp., Bacillus spp., Streptomyces spp., fungi, yeasts, etc., may be used, with Escherichia spp. being preferred.
  • a typical technique such as DEAE-dextran, calcium phosphate, electroporation, etc., may be utilized.
  • the transformed host cell can be grown in a nutrient broth suitable for the production of a Staphylococcus aureus enterotoxin using a well-known method.
  • the transformed host cells may be cultured on a small or large scale using a lab or industrial reactor containing a proper culture medium. Cell culturing is performed with a suitable nutrient broth containing carbon and nitrogen sources and minerals. Proper media may be commercially available or may be prepared with reference to literature (e.g., catalog from the American Type Culture Collection).
  • the Staphylococcus aureus enterotoxin expressed in the transformed host cells may be purified using a protein separation method well known in the art.
  • the separation of Staphylococcus aureus enterotoxins from the nutrient broth may be achieved by illustrative, non- limitative, conventional techniques including centrifugation, filtration, extraction, spray drying, evaporation, and/or precipitation.
  • Staphylococcus aureus enterotoxins can be purified through various techniques well known in the art, including chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), electrophoresis, fractional solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.
  • chromatography e.g., ion exchange, affinity, hydrophobicity, and size exclusion
  • electrophoresis e.g., electrophoresis
  • fractional solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE e.g., SDS-PAGE, or extraction.
  • a lysate of Staphylococcus aureus which is utilized as an antigen in the present invention, may be all or part of the Staphylococcus aureus constituents obtainable by cell disruption using a sonicator or a homogenizer.
  • a lysate of Streptococcus pyogenes also serving as an antigen, may be all or part of the Streptococcus pyogenes constituents obtainable by cell disruption with the aid of a sonicator or a homogenizer.
  • cells are cultured in a conventional manner and the culture is centrifuged at 0 to 10 0 C at 3,000 to 9,000 rpm for 5 to 15 min to yield a cell pellet which is then allowed to stand at -10 to -30°C for 10 to 20 hours and slowly thawed.
  • the cells were resuspended in an appropriate buffer (e.g., PBS), followed by centrifiigation at 0 to 10 0 C at 3,000 to 9,000 rpm for 5 to 15 min.
  • the cell pellet thus obtained was resuspended in an appropriate buffer (e.g., PBS) and disrupted using a sonicator to give a lysate of Staphylococcus aureus or Streptococcus pyogenes. In this process, as many cell proteins can be obtained as possible without being destroyed.
  • an appropriate buffer e.g., PBS
  • the immunization of laying hens with the antigen prepared according to the present invention can be achieved using a technique well known in the art.
  • the laying hens available in the present invention are preferably chickens which show a high laying efficiency, such as white leghorn, rhode island, hyline brown, etc., but not are limited thereto.
  • Antigens may be injected into chickens via various routes, such as intramembranous, intraocular, intramuscular, and subcutaneous routes.
  • Adjuvants such as Freund's complete adjuvants or incomplete adjuvants, may be utilized to stimulate the immune response to the antigen of the present invention. Additional antigen injection (boosting) can be performed until sufficient antibodies are obtained. Preferably, immunization is conducted about three times at intervals of about 2 weeks, and optionally at intervals of 6 to 10 weeks. While being collected from the immunized laying hens, eggs are measured for the level of the induced antibody. If there is no increasing change in antibody level, the eggs are finally recovered.
  • Additional antigen injection boosting
  • immunization is conducted about three times at intervals of about 2 weeks, and optionally at intervals of 6 to 10 weeks. While being collected from the immunized laying hens, eggs are measured for the level of the induced antibody. If there is no increasing change in antibody level, the eggs are finally recovered.
  • the quantitative determination of the induced antibody can be achieved by a conventional method.
  • an enzyme linked immunosorbent assay (ELISA) and a microliter method may be used, with preference for ELISA.
  • ELISA enzyme linked immunosorbent assay
  • the antibody separated from the yolk of the egg immunized according to the present invention is reacted with an antibody attached to the surface of a microplate and then combined with a secondary antibody, followed by adding a substrate to the antibody complex to develop a color through an enzymatic reaction.
  • Absorbance at a specific wavelength is measured to determine the amount of the antibody.
  • the yolk antibody according to the present invention can be obtained by a series of processes such as diluting the yolk of the recovered egg in distilled water, adjusting the pH of the dilution to 4.0 to 6.0, freezing the dilution, thawing the frozen yolk, centrifuging the yolk solution, and filtering the supernatant.
  • the yolk separated from the recovered egg is added to distilled water to create an approximately 5- to 15-fold dilution, and the pH of the dilution is adjusted to about 5.0.
  • the dilution is centrifuged at 5,000 to 15,000x g, at 1 to 10°C for 20 to 40 min. The supernatant thus obtained is forced through a filter.
  • the pharmaceutical composition of the present invention may be formulated into a dosage form for local use, such as ointments, gels, creams, liniments, lotions, etc.
  • a dosage form for local use such as ointments, gels, creams, liniments, lotions, etc.
  • suitable ones such as those who are skilled in the art can readily determine suitable ones.
  • an ointment which is formulated with the active ingredient, an ointment base, and, optionally, additives in a proper ratio, is provided.
  • Ointment bases available to the present invention are well known in the art. Examples of ointment bases useful in the art include higher fatty acids or esters thereof (e.
  • adipic acid myristic acid, palmitic acid, stearic acid, oleic acid, adipic acid ester, myristic acidester, palmitic acid ester, sebacic acid dimethyl, lauric acid hexyl, isooctanic acid cetyl, etc), waxes (e.g, spermaceti, beeswax, cerecin, etc.), surfactants (e.g, polyoxyethylenealkylether phosphoric acid ester, etc.), higher alcohols (e.g, cetanol, stearylalcohol, cetostearylalcohol, etc.), silicon oil (e.g., dimethylpolysiloxane, methylphenylpolysiloxane, glycolmethylpolysiloxane, silicon glycolpolymer etc.), hydrocarbons (e.
  • hydropbilic Vaseline g, hydropbilic Vaseline, white Vaseline, purified lanolin, fluid paraffin, etc.
  • water e. g., water, moisturizers (e. g., glycerin, propyleneglycol, butyleneglycol, sorbitol, etc.), and/or infection preventives.
  • moisturizers e. g., glycerin, propyleneglycol, butyleneglycol, sorbitol, etc.
  • infection preventives e.g., glycerin, propyleneglycol, butyleneglycol, sorbitol, etc.
  • a gel agent which can be formulated with the active ingredient in combination with a gel base and, optionally, additives, is provided.
  • Gel bases available to the present invention are well known in the art Examples of the gel bases useful in the present invention include alcohols (e. g, ethanol, isopropylalcohol, etc.), water, gelling agents (e. g, carboxyvinylpolymer, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, alginic acid propyleneglycol ester etc.), neutralizing agents (e. g., triethanolamine, diisopropanolamine, sodium hydroxide, etc.), surfactants (e.
  • sesquioleic acid sorbitan 1,3-bis(trimethoxy)-2-butanedioleic acid sorbitan
  • trioleic acid sorbitan 1,3-bis(trimethoxy)-2-butanedioleic acid sorbitan
  • monooleic acid sorbitan monostearic acid sorbitan
  • monolauric acid sorbitan monostearic acid polyethyleneglycol, polyoxyethylenenonylether, polyoxyethylenelaurylether, etc.
  • infection preventives g., sesquioleic acid sorbitan, trioleic acid sorbitan, monooleic acid sorbitan, monostearic acid sorbitan, monolauric acid sorbitan, monostearic acid polyethyleneglycol, polyoxyethylenenonylether, polyoxyethylenelaurylether, etc.
  • cream bases available in the present invention are well known in the art.
  • the cream bases useful in the present invention include higher fatty acid esters (e. g., myristic acid ester, palmitic acid ester, sebacic acid diethyl, lauric acid hexyl, isooctanoic acid cetyl, etc.), lower alcohols (e. g., ethanol, isopropanol, etc.), carbohydrates (e. g., fluid paraffin, squalane, etc.), polyhydric alcohols (e.
  • propyleneglycol 1,3-butyleneglycol etc.
  • higher alcohols e. g., 2-hexyldecanol, cetanol, 2- octyldodecanol etc.
  • emulsifying agents e. g., polyoxyethylenealkyl ethers, fatty acid esters, polyethyleneglycol fatty acid ester, etc.
  • preservatives e. g., paraoxybenzoic acid ester, etc.
  • a liniment which can be formulated with the active ingredient in combination with a liniment base, examples of which include alcohols (e. g., rnonohydric alcohols, such as ethanol, propanol, isopropanol, etc., polyhydric alcohols such as polyethylene glycol, polypropylene glycol, butylene glycol, etc.), water, fatty acid esters (e. g., esters of adipic acid, sebacic acid, myristic acid, etc.), and surfactants (e.
  • alcohols e. g., rnonohydric alcohols, such as ethanol, propanol, isopropanol, etc.
  • polyhydric alcohols such as polyethylene glycol, polypropylene glycol, butylene glycol, etc.
  • water e. g., esters of adipic acid, sebacic acid, myristic acid, etc.
  • surfactants e.
  • An additional embodiment of the present invention is an aerosol which may be formulated with a liquid or suspension of the active ingredient and a propellant
  • a liquid or suspension of the active ingredient a general diluent may be used, and a diluent for injections may also be used.
  • the propellant it may be a typical one. For instance, liquefied gas propellants, such as chlorofluorocarbons, e.
  • the aerosol may include a co-solvent, a buffer, and, optionally, a coloring agent, a preservative, perfume, etc.
  • the pharmaceutical composition of the present invention may be applied in an effective amount of approximately 0.1 ⁇ g to 500 mg per kg of body weight, preferably approximately 1 ⁇ g to 250 mg per kg of body weight, and most preferably approximately 2 ⁇ g to 100 mg per kg of body weight.
  • the application amount of the pharmaceutical composition may vary depending on various factors including the severity of the patient's condition and drug resistance.
  • a cosmetic composition for the prevention and alleviation of atopic dermatitis which comprises an antibody to a Staphylococcus aureus enterotoxin and an antibody to a Staphylococcus aureus lysate in combination with a cosmetically acceptable vehicle.
  • the cosmetic composition comprises the anti-Staphylococcus aureus enterotoxin antibody in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 5 wt%, based on the total weight of the composition, and the anti-Staphylococcus aureus lysate antibody in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 2 wf%, based on the total weight of the composition.
  • the cosmetic composition may further comprise an antibody to a Streptococcus pyogenes lysate, showing an improvement in view of the prevention and alleviation of atopic dermatitis.
  • the anti-Streptococcus pyogenes lysate antibody may be contained in an amount from 0.001 to 10 wt%, and preferably in an amount from 0.01 to 2 wt%, based on the total weight of the composition.
  • antibodies to the Staphylococcus aureus enterotoxin, Staphylococcus aureus lysates and Streptococcus pyogenes lysates may be prepared, as in the above-mentioned pharmaceutical composition.
  • the cosmetic composition of the present invention may be formulated into typical cosmetic forms, such as face lotions, lotions, creams, gels, etc. Although the kind and concentration of available cosmetically acceptable vehicles vary greatly according to the form of cosmetic, those who are skilled in the art can determine suitable ones.
  • cosmetically acceptable vehicles include purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, low alcohols, etc., but are not limited thereto. If necessary, whitening agents, moisturizers, anti-inflammatory agents, anti-bacterial agents, anti-fungal agents, vitamins, UV blocking agents, anti-acne agents, perfumes, and/or dyes may be used in the cosmetic composition.
  • Useful oils are represented by hydrogenated vegetable oils, castor oils, cotton seed oils, olive oils, palm-kernel oils, jojoba oils, or avocado oils.
  • waxes useful in the cosmetic composition beeswax, spermaceti, carnauba, candelilla, montan, cerecin, liquid paraffin, or lanolin may be used.
  • examples are stearic acid, linoleic acid, linolenic acid, and oleic acid for useful fatty acids, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol for useful fatty acid alcohols, and isopropyl myristate, isopropyl palmitate, and butyl stearate for useful fatty acid esters, but are not limited thereto.
  • surfactants useful in the present invention include anionic surfactants, such as sodium stearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; cationic surfactants, such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; amphoteric surfactants, such as alkylaminoethylglycine hydrochloride and lecithin; non-ionic surfactants, such as glycerin monostearate, sorbitan monostearate, sucrose fatty acid ester, propylene glycol monostearate, polyoxyethylene oleylether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanolarnide, polyoxypropylene glycol, polyoxyethylene castor oil, and polyoxyethylene lanolin.
  • anionic surfactants such as sodium stearate, sodium cetylsul
  • a humectant is glycerin, 1,3-butyulene glycol, or propylene glycol. Ethanol or isopropanol is exemplified for the lower alcohol.
  • the thickening agent include sodium alginate, sodium caseinate, gelatin agar, xanthan rubber, starch, cellulose ether (e. g., hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxyl propylmethyl cellulose), polyvinyllpyrrolidone, polyvinylalcohol, polyethylene glycol, and sodium carboxymethyl cellulose, but are not limited thereto.
  • Examples are butylated hydroxytoluene, butylated hydroxyanisole, propyl galate, citric acid, and ethoxyquin for antioxidants; disodium edentate and ethanehydroxy diphosphate for chelating agents; citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate for buffers; and methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dehydroacetic acid, salicylic acid and benzoic acid for preservatives, but are not limited thereto.
  • a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
  • a total genomic DNA was prepared from Staphylococcus aureus (Christopher LJ et al. 1986, J. Bacteriol, vol.166, pp.29-33).
  • Staphylococcus aureus was inoculated in a sterile nutrient broth and incubated overnight at 37 0 C with shaking at 200 rpm, followed by isolating the total genomic DNA from the cells.
  • the preparation of total genomic DNA was conducted according to the instructions in the manufacturer's manual (DNeasy Tissue Kit, QIAGEN). PCR was performed with the prepared total genomic DNA serving as a template.
  • primers suitable for the amplification of the SEB gene were synthesized on the basis of the GenBank database (NCBI GenBank Ml 1118). Using a set of the primers (forward primer: 5'-gga tec gag agt caa cca gat c-3 1 , SEQ. ID. NO.: 1 , and backward primer: 5'-gtc gac ggt act eta taa gtg cc-3', SEQ. ID.
  • PCR for the SEB started with 95°C pre- denaturation for 5 min and was carried out with 30 cycles of denaturing temperature at 95°C for 1 min, annealing temperature at 54°C for 1 min and extending temperature at 72°C for 2 min, finally followed by 72°C extension for an additional 10 min [MiniCyclerTM (MJ Research. Inc. USA)].
  • the PCR product thus obtained was run on 1% agarose gel (SeaKem LE agarose, BMA. USA) for 30 min in the presence of an electric field of 100V, using an electrophoresis kit (RunOne Electrophoresis Cell, Embi Tec. USA), and visualized with ethidium bromide staining.
  • FIG. 1 shows the result of electrophoresis wherein the PCR product amplified under the above conditions was run (lane 2), along with a 100 bp ladder DNA marker (lane 1). As shown in FIG. 1, a gene about 780bp in size was obtained by PCR under the above conditions.
  • the base sequence of the PCR product was found to share 100% homology with the gene coding to Staphylococcus aureus enterotoxin B as analyzed by the BLAST program from the NCBI (National Center for Biotechnology Information).
  • the base sequence of the gene of Staphylococcus aureus enterotoxin B, amplified by PCR, is listed in SEQ. ID. NO. 3.
  • a clone harboring the gene was digested with BamH I and Sal I .
  • the DNA fragment excised was inserted into the expression vector PQE 8OL (QIAGEN.
  • FIG. 2 A competent cell Ml 5 was transformed with the recombinant expression vector and positive clones were selected in the presence of appropriate antibiotics (ampicillin and kanamycin), followed by agarose gel electrophoresis to confirm the transformation (FIG. 3).
  • EXAMPLE 2 Expression of Recombinant SEB Gene in E. coli
  • the positive clone selected in Example 1 was inoculated in 5 ml of an LB broth (DUCHEFA Co., Netherland) and cultured at 37°C with shaking. Subsequently, the overnight culture was added to 100 ml of the same fresh medium and grown until the cells reached an absorbance A600 of 0.5-0.6. Then, the cells were further cultured for an additional 3.5 to 4 hours in the presence of 0.6 mM of IPTG (isopropyl- ⁇ -D-thio-galactopyranoside, DUCHEFA Co. Netherlands).
  • IPTG isopropyl- ⁇ -D-thio-galactopyranoside
  • the cell pellet thus obtained was put in a freezer.
  • the frozen cell mass was thawed on ice for 10 min and then resuspended in a lysis buffer (10OmM sodium phosphate (NaH 2 PO4), 30OmM sodium chloride (NaCl), 1OmM imidazole, pH 8.0).
  • Lysozyme, RNase and DNase was added in a concentration of 1 mg/ml to the suspension, which was then allowed to stand for 15 min on ice.
  • the cells lysed through this procedure were disrupted with a sonicator and centrifuged at 10,000xg for 20 min, followed by separately collecting the supernatant and the pellet to obtain the recombinant protein.
  • Example 2 Western blotting was performed to examine whether the recombinant protein obtained in Example 2 was Staphylococcus aureus enterotoxin B or not First, a commercially available wild-type SEB protein was used as a positive control while a protein of a different kind expressed with the same vector as used for the recombinant SEB protein was used as a negative control. Two sheets of polyacrylamide gel were prepared simultaneously. Also, a protein treated in one tube was divided into two halves, which were then loaded on the two 12% SDS-PAGE gel sheets, respectively, followed by electrophoresis.
  • the membrane After being washed three times with TBST, the membrane was incubated at room temperature for 2 hours with a 5,000-fold dilution of the secondary antibody mouse IgG (Sigma Co. USA) in 20 ml of TBST. The membrane was washed three times for 5 min each time and once with TNM (10OmM Tris-Cl pH9.5, 10OmM NaCl, 5mM MgC12), Mowed by color development with BCIP (5-Bromo4-Chloro-3-Indolyl Phosphate, Amersham Pharmacia Bioscience, UK) and NBT (Nitro blue tetrazolium, Amersham Pharmacia Bioscience, UK).
  • TNM Tris-Cl pH9.5
  • 10OmM NaCl 5mM MgC12
  • FIG. 4 shows blots visualized after SDS-PAGE (A) and Western blotting (B), in which a commercially available wild-type SEB (lane 1), the SEB expressed in the transformed E. coli (lane 2), and a different protein expressed in the same vector (lane 3) were run, along with a marker (BIO-RAD, USA). As shown in FIG. 4, both the wild-type SEB and the recombinant SEB were detected at about 28kDa. On the other hand, no bands were detected in the lane for the different protein expressed in the same vector.
  • the recombinant SEB protein identified in Example 3 was purified using the QIAExpressionist Kit (QIAGEN Co. USA). First, cell mass was obtained by cell growth in 1 liter of an LB broth and centrifugation as in Example 1. A supernatant obtained through disruption with a sonicator and centrifugation as in Example 2 was mixed and reacted for 16 hours with the Ni-NTA agarose provided in the kit under reftigeration. Subsequently, the protein was loaded on a polypropylene tube equipped with frits.
  • lysates of the bacteria were prepared through the following procedure.
  • Cells were inoculated in a nutrient LB broth in a 1:1000 dilution ratio and cultured overnight at 37°C with shaking.
  • the overnight culture was added to a large volume of the same fresh medium in a 1 : 100 dilution ratio and grown at 37°C for 8 hours with shaking.
  • centrifugation at 6,000 rpm at 4 0 C for 10 min, the cell pellet thus obtained was kept overnight at -2O 0 C.
  • the frozen cell mass was slowly thawed in ice water and then resuspended in 5 ml/g FW (fresh weight) of a buffer. Again, centrifugation at 4 0 C at 6,000 rpm for 10 min was followed by resuspension in 5 ml/g FW of PBS.
  • the cells were disrupted with a sonicator (BRANSON SONIFIER 450, Branson Ultrasonic. USA) to give a Staphylococcus aureus lysate and a Streptococcus pyogenes lysate, independently.
  • Example 4 In order to produce yolk antibodies to the recombinant SEB protein purified in Example 4, the lysate of Staphylococcus aureus, and the lysate of Streptococcus pyogenes, these antigens were injected into ISA-BROWN lineage laying hens.
  • An antigen mixture of 6:2:2 SEB protein: Staphylococcus aureus lysate: Streptococcus pyogenes lysate was emulsified with an equal volume of a Freund's complete adjuvant (Sigma Co. USA). 1 ml of the emulsion was primarily injected into each of four chest sites of laying hens.
  • Example 6 The anti sera and the eggs obtained in Example 6 were analyzed for antibody titer using
  • ELISA Enzyme-Linked Immunosorbent Assay
  • 100 ⁇ l of a carbonate-bicarbonate buffer dilution (pH 9.6) containing the recombinant SEB protein purified in Example 4 at a concentration of 5 ⁇ g/ml was added to each well of a microplate [Microtest IH flexible Assay plate (Falcon 3911), BD Biosciences, USA], followed by incubation overnight at 4°C to attach the protein to the well.
  • the plate thus coated with the protein was washed three times with a washing buffer (0.02M phosphate buffer saline, 0.13M NaCl, 0.05% Tween20, pH 7.2), and 175 ⁇ l of a 3% bovine serum albumin (BSA) buffer (pH 7.3, Sigma Co. USA) was added to each well. Subsequently, the plate was allowed to stand for 2 hours at room temperature.
  • a washing buffer 0.02M phosphate buffer saline, 0.13M NaCl, 0.05% Tween20, pH 7.2
  • BSA bovine serum albumin
  • the anti-sera and the eggs, obtained in Example 6 were diluted by a factor of 5,000 each, followed by serial three-fold dilutions with the same dilution buffer.
  • the resulting dilutions were added to and reacted at 37°C for 2 hours with the SEB attached to the wells.
  • a 5,000-fold diluted alkaline phosphatase-conjugated rabbit anti-chicken IgG (Jackson ImmunoResearch Laboratories Inc. USA) was reacted at 37°C for 2 hours with the primary antibodies.
  • the antibody titer continuously increased with time after the immunization of laying hens with the antigens of the present invention, peaking four weeks after immunization, and then decreased. The same pattern of change was found in the antibody titers of both the sera and the eggs.
  • Example 6 Western blotting analysis was conducted with the recombinant and the wild- type SEB protein. The same procedure as in Example 3 was conducted for this Western blotting analysis. Proteins were used in equal amounts.
  • As a primary antibody the yolk antibody obtained by immunizing laying hens with the recombinant SEB protein was diluted by a factor of 10,000.
  • AP conjugated anti-chicken IgY Jackson ImmunoResearch Laboratories Inc. USA
  • FIG. 7 shows blots visualized after SDS-PAGE (A) and Western blotting (B).
  • the yolk separated from the eggs laid by the immunized hens of Example 6 was diluted 10- fold with distilled water. After its pH was adjusted to 5.0, the yolk dilution was frozen overnight at - 20°C. The frozen yolk dilution was thawed at room temperature, followed by centrifijgation at 100Ox g at 4 0 C for 30 min. The supernatant thus obtained was forced through a filter to separate the water soluble fraction, which was then lyophilized for storage.
  • DEAE ion-exchange chromatography was utilized for the purification of the yolk antibodies. A column filled with DEAE was equilibrated with a 0.025M phosphate buffer. After sample loading, the column was washed and eluted with 0.25M phosphate buffer (pH 8.0) in fractions of 3 ml of the protein. Purity was measured using a spectrometer (280nm).
  • Example 9 To examine whether or not the yolk antibody separated in Example 9 could defend against wild-type SEB in animals, a transdermal test was carried out on mice. Per animal, wild-type SEB was used in an amount of 5 ⁇ g while the yolk antibody was used in amounts of 10 and 50 mg (LeClaire RD et al.2002, Infec. frnmun. vol.70, pp.2278-2281). BALB/c mice (Samtako BioKorea Korea) 8 weeks old were divided into 6 groups, and the backs thereof were shaved. 200 ⁇ l of the wild-type SEB, the yolk antibody, or PBS was uniformly spread over a piece of gauze 1x1 cm in size.
  • Example 10 Using ELISA, the sera stored in Example 10 were analyzed for blood IgE level according to the administration of the yolk antibody.
  • a Mouse IgE ELISA set commercially available from BD Biosciences, was utilized for the analysis.
  • purified capture anti-mouse IgE was diluted to 2 ⁇ g/ml in PBS, and 100 ⁇ l of the dilution was aliquoted into each well of a microplate (Maxisorp plate, NUNC. Denmark) and incubated overnight at 4°C.
  • the plate coated with the IgE was washed three times with a washing buffer (0.02M phosphate buffer, 0.13M NaCl, 0.05% Tween20, pH 7.2), followed by adding an aliquot of 200 ⁇ l of a 3% BSA buffer (pH 7.3, Sigma Co. USA) to each well. The plate was allowed to stand at room temperature for 30 min and washed three times with the same buffer. Serial dilutions of the sera of Example 10 were prepared as samples, along with a blank and a standard. The standard was prepared by diluting purified mouse IgE in series from 0.5 ⁇ g/ml to a predetermined concentration.
  • the standard, the blank and the samples were loaded in triplicate in an amount of 100 ⁇ l per well onto the microplate, and then incubated for one hour at room temperature or overnight at 4 0 C.
  • Into the microplate was aliquoted 100 ⁇ l of a 3% BSA containing biotinylated anti- mouse IgE (BD Biosciences) at a concentration of 2 ⁇ g/ml, and incubation was conducted for one hour at room temperature. Washing six times with PBST was followed by aliquoting 100 ⁇ l of a dilution of 1:1,000 avidin-HRP conjugate and 3% BSA and then by incubating at room temperature for 30 rnin.
  • a phosphate-citrate buffer Sigma Co.
  • ABTS 3-ethylbenzthiazoline-6- sulfonic acid, Sigma Co. USA
  • EL-800 BIO-TEK Instrument Inc. USA
  • the blood IgE level As compared with that of the SEB-administered group, the blood IgE level, as shown in FIG.
  • EXAMPLE 12 Decrease in the Population of Staphylococcus aureus and Streptococcus pyogenes According to the
  • Example 9 Into sterile test tubes was aliquoted 10 ml of a Staphylococcus aureus or Streptococcus pyogenes culture with 1x109 CFU/ml, and then 1, 3, 5, 10 and 20 mg of the yolk antibody obtained in Example 9 was added thereto. During shaking incubation at 37 0 C, the culture was sampled at an amount of 1 ml at intervals of 2, 4 and 6 hours. Thermal treatment at 60°C for 20 min was conducted before cold storage. The cells were analyzed for change in population using competitive sandwich ELISA.
  • the yolk antibody of the present invention can suppress the effects of Staphylococcus aureus and Streptococcus pyogenes on atopic dermatitis as well as preventing the activity of Staphylococcus aureus enterotoxins.
  • EXAMPLE 13 Preparation of Cosmetic Composition
  • 0.25 g of a yolk antibody to Staphylococcus aureus enterotoxin, 0.25 g of a yolk antibody to a Staphylococcus aureus lysate, 83.5 g of distilled water, 4.8 g of the moisturizer glycerin, 0.05 g of the chelating agent EDTA, and 0.15 g of the thickening agent KOH were mixed at 70°C to give an aqueous phase.
  • 4 g of olive oil, 3 g of stearic acid, and 3 g of a surfactant were mixed well at 7O 0 C to give an oily phase.
  • the aqueous phase and the oily phase were mixed and dispersed with stirring at 7000 to 8000 rpm to produce a cosmetic composition 1.
  • a cosmetic composition 2 was prepared in the same manner as in the cosmetic composition 1, except that 0.5 g of a yolk antibody to Staphylococcus aureus enterotoxin was employed instead of 0.25 g of the antibody to Staphylococcus aureus enterotoxin and 0.25 g of the antibody to Staphylococcus aureus lysate.
  • antibodies to Staphylococcus aureus enterotoxins and Staphylococcus aureus lysates are useful for the prevention, alleviation, and treatment of atopy.

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Abstract

La présente invention concerne une préparation pour la prévention, le soulagement et le traitement de la dermatite atopique, qui comprend respectivement des anticorps anti-entérotoxines de Staphylococcus aureus et anti-lysats de Staphylococcus aureus, et, éventuellement, anti-lysats de Streptococcus pyogenes.
PCT/KR2006/001128 2005-03-28 2006-03-28 Préparation pour la prévention, le soulagement et le traitement de la dermatite atopique WO2006104336A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2011059110A1 (fr) * 2009-11-13 2011-05-19 株式会社さいわいメディックス Agent thérapeutique pour le psoriasis ou la dermatite atopique

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KR20160024516A (ko) 2014-08-26 2016-03-07 김판식 피부질환에 유용한 도꼬마리 추출액의 제조방법 및 도꼬마리 추출액

Non-Patent Citations (3)

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Title
IDE F. ET AL.: "Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis", PEDIATR. INT., vol. 46, no. 3, 2004, pages 337 - 341 *
SOHN M.H. ET AL.: "Effect of staphylococcal enterotoxin B on specific antibody production in children with atopic dermatitis", ALLERGY ASTHMA PROC., vol. 24, no. 1, 2003, pages 67 - 71 *
YISHIOKA T. ET AL.: "DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C", IMMUNOLOGY, vol. 108, no. 4, 2003, pages 562 - 569 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011059110A1 (fr) * 2009-11-13 2011-05-19 株式会社さいわいメディックス Agent thérapeutique pour le psoriasis ou la dermatite atopique
JP2011105614A (ja) * 2009-11-13 2011-06-02 Saiwai Medix:Kk 乾癬又はアトピー性皮膚炎治療剤
EP2500034A1 (fr) * 2009-11-13 2012-09-19 Kabushiki Kaisha Saiwai Medix Agent thérapeutique pour le psoriasis ou la dermatite atopique
CN102724999A (zh) * 2009-11-13 2012-10-10 株式会社幸福医科 干癣或特应性皮炎的治疗药物
EP2500034A4 (fr) * 2009-11-13 2013-07-24 Saiwai Medical Kk Agent thérapeutique pour le psoriasis ou la dermatite atopique

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