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WO1999064066A1 - Agents induisant une tolerance immunitaire et contenant de la keratine dure ou une substance ressemblant a la keratine - Google Patents

Agents induisant une tolerance immunitaire et contenant de la keratine dure ou une substance ressemblant a la keratine Download PDF

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Publication number
WO1999064066A1
WO1999064066A1 PCT/JP1999/002821 JP9902821W WO9964066A1 WO 1999064066 A1 WO1999064066 A1 WO 1999064066A1 JP 9902821 W JP9902821 W JP 9902821W WO 9964066 A1 WO9964066 A1 WO 9964066A1
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WO
WIPO (PCT)
Prior art keywords
hard keratin
keratin
substance
cells
immune tolerance
Prior art date
Application number
PCT/JP1999/002821
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English (en)
Japanese (ja)
Inventor
Kazutoshi Horie
Kazuyoshi Masuda
Ryuji Suzuki
Original Assignee
Shionogi & Co., Ltd.
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 Shionogi & Co., Ltd. filed Critical Shionogi & Co., Ltd.
Priority to AU39552/99A priority Critical patent/AU3955299A/en
Publication of WO1999064066A1 publication Critical patent/WO1999064066A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • 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
    • A61P37/02—Immunomodulators

Definitions

  • the present invention provides immunity useful as an agent for preventing or treating various diseases caused by unnecessary immune responses in vivo, such as skin diseases accompanied by inflammation mediated by T cells or dependent on T cells. It relates to a tolerance inducer.
  • Inflammatory skin diseases involving cellular immunity by T cells include psoriasis, vitiligo, pemphigus vulgaris, dermatomyositis, alopecia areata, systemic alopecia, etc.
  • Atopic dermatitis, contact dermatitis, granuloma and the like are known to involve foreign antigens. Because the causes of these diseases are not fully understood and it is difficult to isolate foreign antigens from the living environment, all of them are often intractable, and pharmacotherapy is the main treatment method. . At that time, steroids and antihistamines are often used as first-line drugs, and if no effect is seen, immunosuppressants are used. However, the use of these drugs may cause serious side effects, and there are concerns about complications associated with prolonged treatment. In addition, since external preparations are used in many cases, they greatly affect the patient's compliance in terms of appearance and the time required to apply the drug.
  • TGF- / 3 by-stander antigen-specific inhibitory cytotoxicity
  • TGF- / 3 by-stander antigen-specific inhibitory cytotoxicity
  • Methods for suppressing or treating related allergic diseases and the like are known (W093 / 16724, Tokuhyo 8-504745).
  • specific examples of antigens to be administered in this document are only collagen, inhibitory epitope, glucagon, insulin, S-antigen, inuichi photoreceptin 'retinoid binding protein (IRBP), and the like. None is described.
  • Keratin is generally classified into hard keratin, which is a structural protein derived from animal hair, feathers, nails, horns, hooves, and scales, and soft keratin, which is derived from the stratum corneum of the skin. , The properties are quite different.
  • hard keratin contains about 5 to 45% of high sulfur protein, and the content of cysteine residues is remarkably higher than that of soft keratin (RDBFraser et al., Academic Press, New Yourk, P470 (1973))
  • the soft keratins are the labile protein compared to hard keratin, even if preparing a pharmaceutical formulation containing the same, stable therapeutic effect rather becomes hard to reproduce, also stored It is expected that there will be problems in terms of stability, etc., and there is a concern that compliance with patients who require long-term drug administration may be impaired.
  • soft keratin must be purified from animal skin tissue or produced using advanced genetic engineering techniques. Formulation techniques are also practically based on aqueous solutions or aqueous suspensions. It is generally considered disadvantageous for industrial production as compared to hard keratin, because it must be limited to a conventional formulation.
  • Egg shell membrane protein is considered to be a type of hard keratin in that it has more than 10% cystine, and there have been examples of it that have been used for the treatment of trauma such as burns. K. Maeda, Y. Sasaki (1981) Burns, 8, 313-316; An experience of hen-egg membrane as a biological dressing) .
  • these treatments are based on collagen It promotes production and the like, and is completely different from a therapeutic method that induces immune tolerance by oral or transmucosal administration.
  • the present inventors have conducted intensive studies in view of the above, and as a result, administration of hard keratin or a hard keratin-like substance has revealed that T cell proliferation and anti-cytokeratin that are reactive to cytokeratin, a typical skin antigen protein, can be obtained.
  • the present inventors have found that the IgG antibody titer can be suppressed efficiently and that it is effective against dermatitis and the like, and the present invention described below has been completed.
  • An immune tolerance inducer characterized by containing hard keratin or a hard keratin-like substance hereinafter also referred to as the present preparation.
  • the immune tolerance inducing agent according to the above (1) which is an agent for preventing or treating a skin disease accompanied by inflammation mediated by T cells or dependent on T cells.
  • Hard keratin is a structural protein or its partial peptide derived from animal hair, feathers, nails, horns, hooves or scales
  • hard keratin-like substance is a structural protein or its partial peptide derived from eggshell membrane
  • a method for inducing immune tolerance comprising administering hard keratin or a hard keratin-like substance.
  • a method for preventing or treating a skin disease accompanied by inflammation mediated by T cells or dependent on T cells which comprises administering hard keratin or a hard keratin-like substance.
  • Hard keratin which is the active ingredient of this formulation, is generally known as the hair, feathers, claws, horns, and hooves of mammals such as humans, pigs, and horses, birds such as chickens, and fish and other animals. Also, it broadly refers to structural proteins derived from epithelium such as scales and the like.For example, in the aforementioned literature (RDBFraser et al., Academic Press, New Yourk, P470 (1973)), the protein composition is low in weight ratio. 40-85% of sulfur protein, 5-45% of high-sulfur protein, 1-30% of high glycine protein, and the single-chain molecular weight of each protein is about 50,000 and 10,000-30,000, respectively. , And 5,000 to 10,000, and the percentage (%) of cysteine residues is described as 4%, 10 to 30%, and 5 to 11%, respectively.
  • the hard keratin-like substance broadly means a substance which is physically and chemically similar to the above-mentioned hard keratin and its origin is not particularly limited, but a structural protein derived from eggshell membranes of birds or the like is preferably used. .
  • the hard keratin or hard keratin-like substance can be soluble or insoluble in water, and can be contained in the present preparation in various forms depending on the dosage form, preparation method, and the like of the present preparation.
  • the solid is used in the form of finely pulverized, usually having a particle size of about 1 ⁇ m to about 100 ° m, preferably about 1 ⁇ m to about 100 ⁇ m, and more preferably about 1 ⁇ m to about 100 ⁇ m. 1 m to about 50 m. However, if it can be prepared, it may be 1 m or less.
  • hard keratin or hard keratin-like substance such as feather fine powder (also known as feather powder (FP), Ishihara Yakuhin Co., Ltd.), eggshell membrane powder (ESM-3) P20 series, the company), EM protein (type P or L, etc., Cupid Co., Ltd.) may be used.
  • the content of hard keratin or hard keratin-like substance in the present preparation varies depending on the form of the preparation and the like, but is usually about 0.001 to 90% by weight, preferably about 0.01 to 50% by weight, based on the whole preparation.
  • the administration route of this preparation is preferably oral or transmucosal (nasal, pulmonary, etc.).
  • the product may be a solid preparation (eg, powder, granules, tablets, capsules, aerosol, etc.) or a liquid preparation (aqueous solution, aqueous suspension, W / 0 emulsion, etc.). It can be prepared by appropriately applying a well-known method.
  • the present formulation may optionally contain various pharmaceutical additives (eg, excipients, binders, disintegrants, dissolving agents, surfactants, suspending agents, emulsifiers, Agent, pH regulator, coating agent Etc.), and the amount of addition may be appropriately set.
  • an appropriate pharmaceutically active ingredient eg, an anti-inflammatory drug, an anti-allergic drug, a steroid, an immunosuppressant, an antibacterial, an antibiotic, an antifungal, an immunomodulator
  • an appropriate pharmaceutically active ingredient eg, an anti-inflammatory drug, an anti-allergic drug, a steroid, an immunosuppressant, an antibacterial, an antibiotic, an antifungal, an immunomodulator
  • Sitekines, chemokines, cholera toxins, lipopolysaccharides, etc. may be contained in the present preparation.
  • the dosage of this drug varies depending on the type of disease, the age of the patient, etc., but is usually about 0.1 to 50 m.g / kg per adult in terms of the weight of hard keratin or hard keratin-like substance. / Day. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 A graph examining the inhibitory effect on ⁇ cell proliferation in mice immunized subcutaneously with cytokeratin (see Example 1).
  • FIG. 2 A graph examining the production of IgG antibody in mice immunized subcutaneously with cytokeratin for suppression of IgG antibody production (see Example 2).
  • FIG. 3 A graph examining the relationship between the inhibitory effect on T cell proliferation and induced regulatory cells in mice immunized subcutaneously with cytokeratin (see Example 3).
  • FIG. 4 is a graph showing the transition of the dermatitis score in the case of continuous oral administration of a finely divided product of feather-derived hard keratin (FP) to mice without dermatitis (Example 4). See).
  • FP feather-derived hard keratin
  • FIG. 5 is a graph showing the change in pruritus when a finely pulverized feather-derived hard keratin (FP) was continuously orally administered to mice without dermatitis three times in total (see FIG. See Example 4.)
  • FP finely pulverized feather-derived hard keratin
  • FIG. 6 A graph showing the transition of the dermatitis score when a finely pulverized feather-derived hard keratin (FP) was orally administered once to mice that developed dermatitis (Example). 5).
  • FIG. 7 A graph showing the change in pruritus after a single continuous oral administration of finely ground feather-derived hard keratin (FP) to mice with dermatitis (see Example 5). .
  • a finely pulverized feather-derived hard keratin Ishihara Pharmaceutical Co., Ltd., particle size 6.28 ⁇ m; hereinafter abbreviated as FP
  • Cytokeratin purified from pig skin by anion exchange chromatography hereinafter referred to as CK was used. At least No. 4, 5, 7, 8, 17, It was confirmed that 18 cytokeratins were contained.
  • CK was immunized subcutaneously (25 ⁇ g / mouse, day 8) with hind leg foot together with Freund's complete adjuvant (FCA (H37Ra)).
  • FCA Freund's complete adjuvant
  • the popliteal lymph node lymphocytes were collected by passing the stainless steel of the 200 mesh, suspension 1% penicillin and scan streptomycin, 2X10- S M 2- mercaptoethanol, in RPMI 1640 medium containing 10% ⁇ shea calf serum It became cloudy and had a concentration of 5 ⁇ 10 f cells / ml.
  • the lymphocyte proliferation test was performed using a 96-well microplate. After plating 5 ⁇ 10 5 cells / 100/1 lymph node cells per well, add 0-200 ug / ml CK ( ⁇ ) (final). The cells were cultured at a concentration of 0-100 ⁇ g / ml) at 37 ° C and 5% CO for 3 days.
  • CK cytokeratin
  • FCA Freund's complete adjuvant
  • the basic procedure for ELISA was based on the previous report (Microbiol. Immunol. 36, 873-884 (1992)). A mouse IgG2a heron antibody was used. The determination of the serum antibody titer was performed by a significant difference test method, and was expressed as the maximum dilution at which the absorbance at 450 nm in the ELISA showed a value significantly higher than the negative control (P ⁇ 0.01).
  • the serum CK antibody titer was strongly suppressed in the FP oral administration group as compared with the control group administered with 2 mM Tris-HCl buffer (pH 8.0). That is, it was revealed that oral administration of FP suppressed humoral immunity to CK.
  • Example 3 (Function of regulatory cells)
  • CK was immunized subcutaneously (25 ⁇ g / mouse, day 8) in the hind paw together with Freund's complete adjuvant (FCA (H37Ra)), and lymph nodes below the knee were collected 10 days after the immunization (dayl8).
  • FCA Freund's complete adjuvant
  • the below-knee lymph node lymphocytes were collected by passing the stainless steel of the 200 mesh, 1% penicillin and scan streptomycin, 2X10- S M 2- mercaptoethanol, RPMI1640 medium containing 10% ⁇ shea fetal serum At a concentration of 1 ⁇ 10 7 cells / ml.
  • the lymphocyte proliferation test was performed using a 96-well microplate and plated with 5 ⁇ 10 S cells / 50 ⁇ l of responder cells per well (2 Tris-HCl buffer ( ⁇ 8.0) administered group). 0.5, 1 or 2 ⁇ 10 5 overnight cells were added, and CK was added to a final concentration of 0 or 50 ug / ml. After culturing for 3 days at 37 ° C and 5% CO ; pulse labeling was performed for 4 hours with 3 H-thymidine (1 ⁇ Ci / 201 1 / ⁇ ). Proliferating cells were monitored by radioactivity of 3 il thymidine as measured by Topcount (Packard).
  • a suspension for oral use is prepared by suspending 10 g of fine crushed feather-derived hard keratin (FP; Ishihara Yakuhin) in 100 ml of 2 mM Tris-HCl buffer (pH 8.0).
  • FP fine crushed feather-derived hard keratin
  • An oral suspension is prepared by suspending 10 g of eggshell membrane powder (ESM-3P20, Ishihara Yakuhin Co., Ltd.) as a hard keratin-like substance in 100 ml of 2 ⁇ Tris-HCl buffer (pH 8.0).
  • Hard keratin eg, FP
  • hard keratin-like substance eg, ESM—3P20
  • 43.2 g of corn starch and 43.2 g of lactose are added and mixed, and then 100 ml of a 3% aqueous hydroxypropyl cellulose solution is added, and the mixture is kneaded and granulated.
  • Magnesium stearate (lg) is added to the granules obtained by drying the mixture, mixed, and tableted to produce tablets.
  • This formulation is useful for autoimmune diseases such as allergy in humans and other mammals and rheumatoid arthritis, as well as various diseases caused by unnecessary immune reactions in the body, such as rejection during organ transplantation. On the other hand, it is effective.
  • This product can efficiently suppress the proliferation of cytokeratin-reactive T cells, which are a typical antigen protein of the skin, and the titer of anti-cytokeratin IgG antibody.
  • various skin diseases with inflammation dependent on T cells such as psoriasis, vitiligo, pemphigus vulgaris, dermatomyositis, alopecia areata, systemic alopecia, atopic dermatitis, contact dermatitis, granuloma, etc. Suitable for prevention or treatment.
  • this preparation uses a stable and relatively easily available hard keratin or hard keratin-like substance as an active ingredient, it is easy to industrially produce and is advantageous in terms of patient compliance.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention se rapporte à des agents induisant une tolérance immunitaire, qui se caractérisent en ce qu'ils contiennent de la kératine dure ou une substance ressemblant à la kératine. Ces agents s'avèrent utiles pour prévenir ou traiter diverses affections cutanées où l'immunité cellulaire par des lymphocytes T joue un rôle, par exemple le psoriasis, le vitiligo, le pemphigus vulgaire, la dermatomyosite, l'alopécie zonale, la calvitie généralisée, la dermatite atopique, la dermatite de contact et la granulomatose.
PCT/JP1999/002821 1998-06-09 1999-05-28 Agents induisant une tolerance immunitaire et contenant de la keratine dure ou une substance ressemblant a la keratine WO1999064066A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39552/99A AU3955299A (en) 1998-06-09 1999-05-28 Immune tolerance-inducing agents containing hard keratin or keratin-like substance

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP16027298 1998-06-09
JP10/160272 1998-06-09

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WO1999064066A1 true WO1999064066A1 (fr) 1999-12-16

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003146895A (ja) * 2001-08-31 2003-05-21 Almado Co Ltd 卵殻膜含有錠剤
JP2003245055A (ja) * 2002-02-25 2003-09-02 Q P Corp 肌改善用食品組成物及び肌改善方法
JP2003246741A (ja) * 2002-02-25 2003-09-02 Q P Corp 経口用肌改善剤及び肌改善用食品組成物、並びに肌改善方法
JP2004321095A (ja) * 2003-04-25 2004-11-18 Q P Corp 飲食品組成物
JP2012097049A (ja) * 2010-11-04 2012-05-24 Hydrox Kk 皮膚外用用組成物
JP2020505442A (ja) * 2017-01-31 2020-02-20 ケラメディックス インクＫｅｒａｍｅｄｉｘ Ｉｎｃ． 脱毛防止又は発毛促進用注射用組成物
JP2022554305A (ja) * 2019-10-28 2022-12-28 中国医学科学院薬物研究所 ケラチンｂｄ－４、その調製および医薬組成物および使用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040232A1 (fr) * 1995-06-07 1996-12-19 Autoimmune, Inc. Tolerance induite par voie orale pour le traitement de maladies de la peau impliquant une inflammation induite par les lymphocytes t

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040232A1 (fr) * 1995-06-07 1996-12-19 Autoimmune, Inc. Tolerance induite par voie orale pour le traitement de maladies de la peau impliquant une inflammation induite par les lymphocytes t

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003146895A (ja) * 2001-08-31 2003-05-21 Almado Co Ltd 卵殻膜含有錠剤
JP2003245055A (ja) * 2002-02-25 2003-09-02 Q P Corp 肌改善用食品組成物及び肌改善方法
JP2003246741A (ja) * 2002-02-25 2003-09-02 Q P Corp 経口用肌改善剤及び肌改善用食品組成物、並びに肌改善方法
JP2004321095A (ja) * 2003-04-25 2004-11-18 Q P Corp 飲食品組成物
JP2012097049A (ja) * 2010-11-04 2012-05-24 Hydrox Kk 皮膚外用用組成物
JP2020505442A (ja) * 2017-01-31 2020-02-20 ケラメディックス インクＫｅｒａｍｅｄｉｘ Ｉｎｃ． 脱毛防止又は発毛促進用注射用組成物
JP2022554305A (ja) * 2019-10-28 2022-12-28 中国医学科学院薬物研究所 ケラチンｂｄ－４、その調製および医薬組成物および使用
US12098176B2 (en) 2019-10-28 2024-09-24 Institute Of Materia Medica, Chinese Academy Of Medical Sciences Keratin BD-4, preparation method, and pharmaceutical composition and use thereof

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Publication number Publication date
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