WO1999064066A1

WO1999064066A1 - Immune tolerance-inducing agents containing hard keratin or keratin-like substance

Info

Publication number: WO1999064066A1
Application number: PCT/JP1999/002821
Authority: WO
Inventors: Kazutoshi Horie; Kazuyoshi Masuda; Ryuji Suzuki
Original assignee: Shionogi & Co., Ltd.
Priority date: 1998-06-09
Filing date: 1999-05-28
Publication date: 1999-12-16
Also published as: AU3955299A

Abstract

Immune tolerance-inducing agents characterized by containing hard keratin or a keratin-like substance. These agents are useful in preventing or treating various skin diseases in which cellular immunity by T cell participates, for example, psoriasis, vitiligo, vulgar pemphigus, dermatomyositis, area alopecia, general hairlessness, atopic dermatitis, contact dermatitis and granuloma.

Description

Specification

Immune tolerance inducer containing hard keratin or hard keratin-like substance

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. Background art

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.

In recent years, in diseases involving the immune system, attempts have been made to suppress the abnormally enhanced immune response by regulating the immune system specifically for the causative antigen, instead of suppressing the entire immune system. One of them is a method of inducing immunological tolerance specifically using the pathogenic antigen itself or a part thereof. An example of this is desensitization therapy, but its mechanism of action is still unclear and is not an established treatment method. On the other hand, by administering an antigen (bystander antigen) specific to the diseased site transmucosally or orally, not limited to the etiological antigen, by-stander antigen-specific inhibitory cytotoxicity (TGF- / 3) induce regulatory cells to produce autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, organ transplant rejection, or T cells. Methods for suppressing or treating related allergic diseases and the like are known (W093 / 16724, Tokuhyo 8-504745). However, 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. Nothing is described.

More recently, the researchers have suggested that skin I, IV, collagen, keratins 1, 5, 10, 14 and 14 and transgluminases 1, 5 which are commonly present in the skin, can be treated as a treatment for skin diseases. A method of oral administration as an antigen was reported (W096 / 40232). However, all of the keratins described in the literature are so-called soft keratins derived from the stratum corneum of the skin, and hard keratins are not described at all.

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. Normally, 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)) _o the other hand, 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. In addition, 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) . However, 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.

In other words, it effectively gives a high therapeutic effect to inflammatory skin diseases and the like in which cellular immunity caused by τ cells is involved, and provides industrially simple production and stable supply by efficiently inducing immune tolerance. There is also a need for the development of new tolerogenic drugs that are advantageous in terms of patient compliance. Disclosure of the invention

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.

(1) An immune tolerance inducer characterized by containing hard keratin or a hard keratin-like substance (hereinafter also referred to as the present preparation).

(2) The immunological tolerance inducer according to the above (1), which induces immune tolerance by transmucosal administration or oral administration.

(3) 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.

(4) Hard keratin is a structural protein or its partial peptide derived from animal hair, feathers, nails, horns, hooves or scales, and hard keratin-like substance is a structural protein or its partial peptide derived from eggshell membrane The immunological tolerance inducing agent according to the above (1), wherein

(5) A method for inducing immune tolerance, comprising administering hard keratin or a hard keratin-like substance.

(6) 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.

(7) A method of using hard keratin or a hard keratin-like substance to produce an immune tolerance inducing agent.

(8) Use of hard keratin or a hard keratin-like substance for the manufacture of a prophylactic or therapeutic agent for skin diseases associated with inflammation mediated by T cells or dependent on T cells. Law.

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. Preferably, the solid is used in the form of finely pulverized, usually having a particle size of about 1 μm to about 100 ° m, preferably about 1 μm to about 100 μm, and more preferably about 1 μm to about 100 μm. 1 m to about 50 m. However, if it can be prepared, it may be 1 m or less. In addition, commercially available products containing a high proportion of 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. In addition to the above-mentioned active ingredients, 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. In addition, if necessary, 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) may be contained in the present preparation. Sitekines, chemokines, cholera toxins, lipopolysaccharides, etc.).

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 τ 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).

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.)

(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). . BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES Examples of the present invention will be shown below, but these do not limit the present invention in any way. In each test, a finely pulverized feather-derived hard keratin (Ishihara Pharmaceutical Co., Ltd., particle size 6.28 μm; hereinafter abbreviated as FP) was used as hard keratin. 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.

Example 1 (suppression of cell-mediated immunity)

Oral administration of FP suspended in 2πιΜ Tris-HCl buffer (ρΗ80) to a final concentration of 0 to 4 _mg / ml to BALB / c mice (6 W, male) for 5 days daily (0 to lmg FP) After equivalent / 250 j 1 / mouse / day, day 0 to 4), CK was immunized subcutaneously (25 μg / mouse, day 8) with hind leg foot together with Freund's complete adjuvant (FCA (H37Ra)). Ten days after immunization (dayl8), lymph nodes below the knee were collected. 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 (ΙΟΟμΙΟΟ) (final). The cells were cultured at a concentration of 0-100 μg / ml) at 37 ° C and 5% CO for 3 days. After 3 days of culture, pulse labeling was performed for 4 hours together with ³ H-thymidine (1 Ci / 20 JUL 1 / well). Proliferating cells ³ H was measured by shoulder stop counting (Packard) - thymidine Ri monitor one was by the radioactivity. As a control group (vehicle), 2 mM Tris-HCl buffer (pH 8.0) was used. Further, as a comparative test, the same experiment was conducted by administering the above-mentioned CK, which is a soft keratin, instead of FP. (result)

As shown in FIG. 1, proliferation of CK added to the medium was suppressed in the lymph node cells in the FP oral administration group as compared with the control group. Since T cells mainly grow in this experimental system, it was clarified that oral administration of FP suppressed the growth of CK-specific T cells.

In addition, as shown in Table 1, the growth of CK-specific T cells was strongly suppressed in the FP oral administration group as compared with the CK administration group. (table 1 )

Oral administration agent dosage% inhibition ¹⁾

lliy / illUUiafcJ / Llay

Purified cytokeratin 0.01 17.3

1 40.8

Feather powder 0.01 64.3

1 91.4

1) (vehicle-administered group CK50 _t ig / ml upon addition cpm) (cpm upon addition sample administration group CK50 _f ig / ml)

X 100 (cpm with CK50 g / ml added to vehicle administration group) — (cpm without GK added to vehicle administration group) Example 2 (suppression of humoral immunity)

FP suspended in 2 mM Tris-HCl buffer (pH 8.0) to a final concentration of 0 to 4 mg / ml was orally administered to BALB / c mice (6 W, male) daily for 5 days (0 to lmgFP equivalent / After 250 JUL 1 / mouse / day, day 0 to 4), cytokeratin (CK) was immunized subcutaneously (25 μg / mouse, day 8) with hind leg foot together with Freund's complete adjuvant (FCA (H37Ra)). Serum antibody titers to CK (total IgG) on day 21 after immunization were examined by enzyme-linked immunosorbent assay (ELISA). 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).

(Result)

As shown in FIG. 2, 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)

For the preparation of responder cells, 2 mM Tris-HCl buffer (pH 8.0) alone or For preparation of Yure overnight cells, FP suspended in 2 mM Tris-HCl buffer (PH8.0) to a final concentration of 0 or 4 mg / ml was applied to BALB / c mice (6 W, male) for 5 days each day. Oral administration (0.01, lmg FP equivalent / 250 1 / mouse / day, day 0 to 4) was performed. Next, CK was immunized subcutaneously (25 μg / 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). 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 μl of responder cells per well (2 Tris-HCl buffer (ρΗ8.0) 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 / ゥ). Proliferating cells were monitored by radioactivity of ³ il thymidine as measured by Topcount (Packard).

(Result)

As shown in FIG. 3, the growth of the responder cells in the 2 mM Tris-HCl buffer (pH 8.0) administration group was suppressed by coexisting the module cells in the FP administration group. As a result, it became clear that regulatory cells induced by oral administration of FP suppressed the proliferation of T cells in the vicinity, either directly or through humoral factors. . Example 4 (Protective effect of FP on DS / Nh mice)

FP DS / Nh mice suspended in 2 mM Tris-HCl buffer (pH 8.0) (Exp. AnH

225-229 (1997)) to prevent spontaneous dermatitis. In the experiment, four-week-old DS / Nh mice () were transferred to a conventional environment, and preliminarily reared for one week, and then administered with 2 mM Tris-HCl buffer (pH 8), Tris-HCl buffer containing FP. A total of four groups (n = 7) were set up (0.2, 1, 5 mg / mouse / day), and continuous oral administration for 5 days was performed 3 times at 3-week intervals. Dermatitis score up to 11 weeks after arrival (skin of each individual The scoring was evaluated according to the severity of the inflammation (0 to 5) and pruritus (the cumulative number of seconds of reptile behavior during 5 minutes of observation).

($ α)

The results are shown in FIGS. In the group treated with Tris-HCl buffer containing FP, the suppression of the dermatitis score and the increase in pruritus associated with exacerbation of dermatitis were suppressed as compared with the group treated with the Tris-HCl buffer, confirming a preventive effect. Example 5 (Therapeutic effect of FP on DS / Nh mice)

The therapeutic effect of FP suspended in 2 mM Tris-HCl buffer (PH8.0) on spontaneous dermatitis of DS / Nh mice Exp. Anim. 46, 225-229 (1997)) was examined. In the experiment, 4-week-old DS / Nh mice () were bred in a conventional environment for 12 weeks, divided into groups so that the dermatitis score was not biased, and 2 mM Tris-HCl buffer (pH 8) administration group, FP A total of two groups (n = 6), a group containing the Tris-HCl buffer (1 mg / mouse / day), were administered and oral administration was performed for 5 days. The dermatitis score (scored from 0 to 5 according to the severity of dermatitis of each individual) and pruritus (the number of seconds of reptile behavior in 5 minutes of observation) were evaluated up to 30 days after the start of administration. .

(Result)

The results are shown in FIGS. In the FP-containing Tris-HCl buffer administration group, the suppression of the dermatitis score and the increase in pruritus associated with the exacerbation of dermatitis were suppressed, as compared with the Tris-HCl buffer administration group, confirming the therapeutic effect. Formulation Example 1

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).

Formulation Example 2

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 2πιΜ Tris-HCl buffer (pH 8.0). Formulation Example 3

Hard keratin (eg, FP) or hard keratin-like substance (eg, ESM—3P20) 9.6 To 4 g, 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. Industrial applicability

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. Or 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. In addition, since 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.

Claims

The scope of the claims

1. An immune tolerance inducing agent characterized by containing hard keratin or a hard keratin-like substance.

2. The immunological tolerance inducer according to claim 1, which induces immune tolerance by transmucosal or oral administration.

3. The agent for inducing tolerance according to claim 1, which is an agent for preventing or treating a skin disease associated with inflammation mediated by T cells or dependent on T cells.

4. Hard keratin is a structural protein or a partial peptide derived from animal hair, feathers, nails, horns, hooves or scales, and hard keratin-like substance is a structural protein derived from eggshell membrane or a part thereof. 2. The immune tolerance inducing agent according to claim 1, which is a peptide.

5. A method for inducing immune tolerance, comprising administering hard keratin or a hard keratin-like substance.

6. A method for preventing or treating a skin disease associated with inflammation mediated by T cells or dependent on T cells, which comprises administering hard keratin or a hard keratin-like substance.

7. A method of using hard keratin or a hard keratin-like substance to produce an immune tolerance inducing agent.

8. A method of using hard keratin or a hard keratin-like substance for the manufacture of a prophylactic or therapeutic agent for a skin disease associated with inflammation mediated by or dependent on T cells.