JP4673068B2 - Th1 type allergic disease treatment composition - Google Patents

Description

  The present invention relates to a pharmaceutical composition used for the prevention and / or treatment of a Th1-type allergic disease, and particularly to a pharmaceutical composition used for the prevention and / or treatment of Th1-type atopic dermatitis or Th1-type bronchial asthma. Is.

  Atopic dermatitis (hereinafter referred to as AD) is a typical chronic skin disorder, and its main feature is that it has pruritus, chronic repetitiveness and genetic background (Non-patent literature). 1-5).

  Traditionally, AD has been thought to be due to elevated serum IgE levels and immunopathology resulting from a Th2 response. In fact, as many researchers have reported, AD patients with high serum IgE levels (exogenous AD) account for about 80% of the total and are preferential in peripheral blood T cells and lesion sites It shows a bias toward Th2 cells and expression of Th2 chemokines such as CCL17 (TARC) and CCL22 (MDC) (Non-Patent Documents 1 to 5).

  However, AD patients with normal range of IgE levels, called endogenous AD, have slightly different immunological characteristics, namely Th1 cytokines IFN-γ, CXCL9 (Mig), CXCL10 (IP-10), It has the property of increased expression of IFN-related chemokines such as CXCL11 (I-TAC) (Non-patent Documents 2 to 8). Furthermore, recent studies have revealed that immunological features in skin lesions are completely different depending on the stage of AD (Non-patent Documents 2 and 5). Lymphocytes expressing Th2 cytokines are dominant in skin lesions of acute AD in early childhood, while lymphocytes expressing IFN-γ (Th1 cytokines) in skin lesions of chronic AD patients, Furthermore, monocytic cells expressing IL-12 (a cytokine that promotes differentiation into Th1 cells) are observed (Non-patent Documents 2 and 5).

  The present inventors recently revealed that it is possible to induce AD in mice independently of IgE but completely dependent on increased IL-18 secretion from keratinocytes (Non-Patent Document 9). 10). It is known that a caspase-1-transgenic mouse (KCASP1Tg) secreting a large amount of IL-18 from keratinocytes spontaneously develops AD-like dermatitis in an SPF (specific pathogen-free) environment (Patent Document 1). . The present inventors have found that by deleting the IL-18 gene in the KCASP1Tg, AD does not develop even if it has the Stat6 gene, which is an IL-4 signaling factor essential for IgE induction. On the contrary, it was found that an IgE-independent Th1-type AD model mouse can be produced by deleting the Stat6 gene in the above KCASP1Tg (Non-Patent Documents 9 and 10), and patent applications have been filed for these model mice (Patent Document 2).

  On the other hand, bronchial asthma has also been considered as a Th2-type disease induced by the cooperative action of allergens, IgE, and cytokines produced by helper T2 cells (Th2 cytokines). In particular, it is well known that bronchial asthma is caused by Th2 cytokines (eg IL-4, IL-5, IL-9, IL-13, etc.) and GM-CSF (granulocyte macrophage colony stimulating factor). ing.

  However, there are reports that some patients with bronchial asthma have a Th1 cell-dominated immune response and cases that have both Th1 and Th2 cell immune responses. Can be classified into two types: Th1 cell induced bronchial asthma (Th1 type bronchial asthma), Th2 cell induced bronchial asthma (Th2 type bronchial asthma) and Th1 cell / Th2 cell mixed induced bronchial asthma (mixed bronchial asthma) . In Th1-type bronchial asthma, IgE production is not observed, or even if it is observed, it is almost normal. In addition, it is a characteristic of Th1-type bronchial asthma that is exacerbated by infection.

  When the present inventors stimulate antigen-specific Th1 cells with antigen and IL-18, they produce not only Th1 cytokines but also IL-13 and various chemokines, which are effector molecules responsible for allergic responses, in large quantities. Based on this finding, it is possible to induce bronchial asthma (Th1-type bronchial asthma) that cannot be induced by antigen nasal nasal drop by nasalizing antigen and IL-18 in mice encapsulating antigen-specific Th1 cells. (Non-Patent Document 11).

  Moreover, the possibility that IL-18 induces interstitial pneumonia has been reported (Non-patent Document 12), and the possibility of inducing COPD (chronic respiratory failure) has also been suggested. Therefore, interstitial pneumonia and COPD are probably considered as advanced forms of Th1 bronchial asthma.

By the way, as a treatment for atopic dermatitis, external use of steroids, external use of immunosuppressive agents, internal use of antiallergic agents and the like are currently performed. As treatment of bronchial asthma, steroid inhalation, inhalation of a sympathetic nerve agent having a bronchodilator action, inhalation of an antiallergic agent, or internal use of these agents are performed. Further, for AD and bronchial asthma that develop due to IgE, IgE neutralization therapy with an anti-IgE antibody is also performed. However, Th1-type AD and Th1-type bronchial asthma are resistant to the above-mentioned treatment methods, and an appropriate treatment method has not yet been established. In addition, Th1 type bronchial asthma is incorporated in intractable asthma.
International Publication WO01 / 95710A1 Pamphlet (International Publication Date: December 20, 2001) JP 2004-41123 A (publication date: February 12, 2004) Grewe, M., et al. A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis.Immunol. Today 19, 359-361 (1998). Leung, DYM & Bieber, T. Atopic dermatitis. Lancet 361, 151-160 (2003). Novak, N., Bieber, T. & Leung, DYM Immune mechanisms leading to atopic dermatitis. J. Allergy Clin. Immunol. 112, S128-S139 (2003). Spergel, JM & Paller, ASP Atopic dermatitis and the atopic march. J. Allergy Clin. Immunol. 2003, S118-S127 (2003). Leung, DYM, Boguniewicz, M., Howell, MD, Nomura, I. & Hamid, QA New insights into atopic dermatitis. J. Clin. Invest. 113, 651-657 (2004). Simon, D., Borelli, S., Braathen, L. & Simon, H. Peripheral blood mononuclear cells from IgE- and non-IgE-associated allergic atopic eczema / dermatitis syndrome (AEDS) demonstate increased capacity of generating interleukin-13 but diffr in their potential of synthesizing interferon-γ.Allergy 57, 431-435 (2002). Jeong, C.-W., et al. Differntial in vivo cytokine mRNA expression in lesional skin of intrinsic vs. extrinsic atopic dermatitis patients using semiuantitative RT-PCR. Clin. Epx. Allergy 33, 1717-1724 (2003). Klunker, S., et al. A second step of chemotaxis after transendothelial migration: Keratinocytes undergoing apoptosis release IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-γ-inducible α-chemoattractant for T cell chemotaxic toward epidermis in atopic dermatitis. J. Immunol. 171, 1078-1084 (2003). Konishi, H., et al. IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE / stat6 under specific pathogen-free conditions.Proc. Natl. Acad. Sci. USA 99, 11340-11345 (2002). Tsutsui, H., Yoshimoto, T., Hayashi, N., Mizutani, H. & Nakanishi, K. Induction of allergic inflammation by interleukin-18 in experimental animal models. Immunol. Rev. 202, 115-138 (2004). Sugimoto, T., et al. IL-18 acts on memory Th1 cells to induce airway inflammation and hyperresponsiveness in a naive host mouse.J. Exp. Med. 199, 535-545 (2004). Hoshino, T., et al. Interleukin-18 (IL-18) in synergy with IL-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia. , 1289-1298 (2002).

  As described above, the present inventors have made extensive studies on Th1-type AD and Th1-type bronchial asthma. As a result, IL-18 induces dermatitis and asthma in Th1-type AD and bronchial asthma. It has already been announced that it is the main factor (for example, patent document 2 etc.). It is also known that IL-18 induces the production of IL-4, IL-13, GM-CSF, IL-8, IFN-γ, and IFN-γ related chemokines.

However, even if it has been experimentally strongly suggested that it is a disease inducer, it is not possible to prevent or treat the disease simply by inhibiting the function of the inducer. It is a well-known fact. For example, IL-5 and IL-13 are cytokines whose expression level is increased in asthma, but when anti-IL-5 antibody or anti-IL-13 antibody is administered, the increase in eosinophils is suppressed. It has been reported that airway hypersensitivity is not suppressed (Ichinose, M., et al. Cytokine-directed therapy in asthma. Curr. Drug Targets Inflamm. Allergy. Sep; 3 (3): 263-269 (2004). Nagai, H., et al. The role of interleukin-5 (IL-5) in allergic airway hyperresponsiveness in mice. Ann. NY Acad. Sci. Oct31; 796: 91-96 (1996).)
Therefore, in Th1-type AD and Th1-type bronchial asthma, it is considered to be very meaningful to study how the pathology changes when IL-18 or IFN-γ is inhibited. Such research results have not yet been reported.

  In addition, as described above, the current treatment of AD and bronchial asthma is intended for Th2 type pathologies caused by an increase in IgE due to allergens, and is less effective for Th1 type patients. Development of therapeutic agents and treatment methods suitable for AD patients and bronchial asthma patients is awaited.

  The present invention has been made in view of the above-mentioned problems, and its purpose is to prevent Th1-type allergic diseases such as Th1-type atopic dermatitis and Th1-type bronchial asthma without effective treatment methods and drugs. And providing an effective pharmaceutical composition.

The present inventors diligently studied to solve the above-mentioned problems, and have developed anti-IL-18 neutralizing antibodies or Th1 type AD model mice or Th1 type bronchial asthma model mice independently developed by the present inventors. It has been found that administration of an anti-IFN-γ neutralizing antibody can suppress the onset of AD or asthma, and the present invention has been completed.

  That is, the pharmaceutical composition according to the present invention is used for preventing and / or treating Th1-type atopic dermatitis or Th1-type bronchial asthma, and includes an IL-18 inhibitor and / or an IFN-γ inhibitor. It is characterized by that. The IL-18 inhibitor is preferably an anti-IL-18 neutralizing antibody, and the IFN-γ inhibitor is preferably an anti-IFN-γ neutralizing antibody.

  The kit according to the present invention is a kit used for preventing and / or treating Th1 type atopic dermatitis or Th1 type bronchial asthma, and comprises at least a reagent for measuring IgE level in a sample, IL- It comprises a pharmaceutical composition comprising a reagent for measuring 18 levels and an IL-18 inhibitor and / or an IFN-γ inhibitor. The IL-18 inhibitor is preferably an anti-IL-18 neutralizing antibody, and the IFN-γ inhibitor is preferably an anti-IFN-γ neutralizing antibody.

  The method for preventing and / or treating Th1-type atopic dermatitis or Th1-type bronchial asthma according to the present invention comprises the steps of measuring IgE level and IL-18 level in a sample derived from a subject, the IgE level and IL-18 level. A IL-18 inhibitor and / or an IFN-γ inhibitor for a subject whose IgE level is within the normal range and whose IL-18 level exceeds the normal range And a step of administering. The IL-18 inhibitor is preferably an anti-IL-18 neutralizing antibody, and the IFN-γ inhibitor is preferably an anti-IFN-γ neutralizing antibody.

  The composition, kit, and method having the above-described configuration enable effective prevention and / or treatment of Th1-type AD or Th1-type bronchial asthma.

  The pharmaceutical composition, kit and method of the present invention have the effect of providing a means for effectively preventing and / or treating Th1-type AD or Th1-type bronchial asthma that could not be effectively treated conventionally. In addition, it has the effect of preventing the onset of interstitial pneumonia and chronic respiratory failure, which is considered to be a progressive form of Th1-type bronchial asthma.

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

[Pharmaceutical composition]
The pharmaceutical composition according to the present invention only needs to contain an IL-18 inhibitor and / or an IFN-γ inhibitor, and is used for preventing and / or treating a Th1-type allergic disease.

  In the present specification, Th1-type allergic disease means a disease that develops due to an antigen and infection and does not accompany IgE production or has an IgE level within a normal range.

  Examples of the Th1-type allergic disease include Th1-type atopic dermatitis (AD), Th1-type bronchial asthma and the like.

  As described in detail in the Examples, the present inventors have experimentally demonstrated that administration of anti-IL-18 neutralizing antibody or anti-IFN-γ neutralizing antibody to Th1-type AD model mice does not cause AD. Confirmed. Further, administration of anti-IL-18 neutralizing antibody or anti-IFN-γ neutralizing antibody to a Th1-type bronchial asthma model mouse suppresses asthma symptoms, and IL-18 gene knockout (KO) mouse or IFN-γ It was experimentally confirmed that the Th1-type bronchial asthma model mouse prepared using the gene KO mouse suppresses asthma symptoms in the same manner as the mouse administered with the neutralizing antibody.

  Based on such experimental data by the present inventors, it is clear that IL-18 inhibitors and IFN-γ inhibitors can effectively suppress Th1-type allergic diseases, particularly Th1-type AD and Th1-type bronchial asthma. is there.

  Here, the “inhibiting substance” is not particularly limited as long as it acts to impair the normal function of IL-18 or IFN-γ. Therefore, the “inhibitor” includes a substance that inhibits transcription from IL-18 gene or IFN-γ gene, a substance that inhibits translation from IL-18 mRNA or IFN-γ mRNA, IL-18 protein, or IFN-γ protein. And a substance that inhibits the functional expression of mature IL-18 protein or IFN-γ protein.

  IL-18 (IL-18 as a mature protein) inhibitor can be obtained by the following method. For example, stimulation of skin keratinocytes with protein A derived from Staphylococcus aureus, or stimulation of airway epithelial cells with LPS derived from Escherichia coli induces the production of IL-18. Thus, an IL-18 inhibitor can be obtained by measuring whether IL-18 production is suppressed. However, the screening method for IL-18 inhibitor is not limited to this.

  Moreover, an IFN-γ (IFN-γ as a mature protein) inhibitor can be obtained by the following method. For example, when Th1 cells are antigen-stimulated, or NK cells are stimulated with IL-12 and IL-18, IFN-γ is produced, but addition of an inhibitor candidate substance to this system suppresses IFN-γ production. IFN-γ inhibitory substance can be obtained by measuring whether or not. However, the screening method for the IFN-γ inhibitor is not limited to this.

  More specific IL-18 inhibitor may include an anti-IL-18 neutralizing antibody, and more specific IFN-γ inhibitor may include an anti-IFN-γ neutralizing antibody. Here, “antibody” means an immunoglobulin (IgA, IgD, IgE, IgG, IgM and fragments thereof), and examples thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, and the like. .

  Antibodies can be obtained by various known methods (for example, HarLow et al., “Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, New York (1988)”, Iwasaki et al., “Monoclonal antibody hybridoma and ELISA, Kodansha (1991)”). It can be manufactured if it follows. Whether or not the prepared antibody has neutralizing activity can be confirmed, for example, according to the screening method for the IL-18 inhibitor or the screening method for the IFN-γ inhibitor. In addition, a known anti-IL-18 neutralizing antibody or anti-IFN-γ neutralizing antibody may be used.

  Further, the antibody is preferably an antibody derived from an animal to be administered. This is because when an antibody derived from a different animal is administered, it is recognized as a foreign protein and inactivated / eliminated by the immune system.

  The application target of the pharmaceutical composition of the present invention is not limited to humans. For example, the pharmaceutical composition of the present invention containing anti-IL-18 neutralizing antibody or anti-IFN-γ neutralizing antibody for humans. When applying the composition, the antibody is preferably a human antibody or a humanized antibody. Human antibodies and humanized antibodies can be obtained by known methods (eg, Riechman, Nature 332, 323-327 (1988), Queen, Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989), International Patent Application Publication Number. WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, WO96-34101, etc.).

  The pharmaceutical composition of the present invention may contain only one of an IL-18 inhibitor or an IFN-γ inhibitor, or may contain both.

  The pharmaceutical composition of the present invention can be administered to a mammal by any oral, parenteral or topical route. The content of the IL-18 inhibitor or IFN-γ inhibitor as an active ingredient can be appropriately selected according to the weight and condition of the subject to be treated, the condition of the disease to be treated, and the specific administration route selected. Good.

  The IL-18 inhibitor or IFN-γ inhibitor can be administered alone or in combination with a pharmaceutically acceptable carrier or diluent by any of the above routes of administration, Administration can be performed as single or multiple doses. More specifically, the pharmaceutical compositions of the present invention can be administered in a wide variety of dosage forms. Tablets, capsules, lozenges, troches, hard candy, powders, sprays, creams, paints, suppositories, jelly, gels, pastes, lotions, ointments, aqueous suspensions Can be combined with various pharmaceutically acceptable inert carriers in the form of injection solutions, elixirs, syrups and the like. Carriers include solid diluents or fillers, sterile aqueous media, various non-toxic organic solvents and the like. Furthermore, an appropriate sweetness and / or aroma can be imparted to the oral pharmaceutical composition. Usually, the active ingredient is present in the dosage form at a concentration level ranging from 5% to 70% by weight (preferably 10% to 50% by weight).

  For oral administration, tablets containing various excipients (eg, microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine) can be combined with various disintegrants (eg, starch (preferably corn , Potato, or tapioca starch), alginic acid, and silicate complexes) or with granulating binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and gum arabic). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very useful for tablet formation. The same type of solid composition can also be used as a filler in gelatin capsules. Suitable materials in this regard include lactose and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration, the active ingredients can be combined with various sweetening amounts or flavors, colorings, or dyes, optionally further emulsifying and / or Suspending agents can be combined with the above diluents (eg, water, ethanol, propylene glycol, glycerin, and various combinations thereof).

  For parenteral administration, either sesame or peanut oil or aqueous propylene glycol can be used as a solvent. If desired, the aqueous solution should be appropriately buffered (preferably pH> 8) and the liquid diluent should first be made isotonic. These aqueous solutions are suitable for intravenous injection purposes. Oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmacological techniques well known to those skilled in the art. In addition, the compounds of the invention can also be administered topically when treating inflammatory skin conditions. In this case, it can be preferably carried out with creams, jellies, gels, pastes, and ointments according to standard pharmaceutical methods.

〔kit〕
The kit according to the present invention comprises at least a reagent for measuring IgE levels in a sample, a reagent for measuring IL-18 levels in a sample, and a pharmaceutical composition comprising an IL-18 inhibitor and / or an IFN-γ inhibitor. It only has to be provided. The kit of the present invention is used for preventing and / or treating Th1-type allergic diseases, specifically, for example, Th1-type atopic dermatitis or Th1-type bronchial asthma.

  Since the pharmaceutical composition containing an IL-18 inhibitor and / or an IFN-γ inhibitor provided in the kit has already been described in the above section [Pharmaceutical composition], description thereof is omitted here.

  By using the kit of the present invention, the IgE level and the IL-18 level are measured in advance using a reagent for measuring the IgE level in the sample and a reagent for measuring the IL-18 level in the sample. If it is confirmed whether or not there is a Th1 type using a pharmaceutical composition containing an IL-18 inhibitor and / or an IFN-γ inhibitor (ie, the pharmaceutical composition according to the present invention) more efficiently It becomes possible to prevent and / or treat allergic diseases.

  The sample may be a biological sample derived from the target animal to which the pharmaceutical composition of the present invention contained in the kit is applied, and examples thereof include blood, body fluid, sputum, washing fluid, excretory fluid, biopsy tissue, and the like. be able to.

  The IgE level in the sample can be determined, for example, by measuring the IgE concentration in the sample. Therefore, the reagent for measuring the IgE level in the sample is not particularly limited as long as it is a reagent used in a known IgE concentration measurement method. In general, as an IgE concentration measurement method, an immunological measurement method using an anti-IgE antibody such as an ELISA method is widely used. Therefore, as an example of a reagent for measuring the IgE level provided in this kit, an anti-IgE antibody that can bind to IgE of the target animal species, a labeled secondary antibody, a coloring reagent, a washing buffer, an ELISA plate, etc. Can be mentioned. However, it is not limited to these.

  The IL-18 level in the sample can be determined, for example, by measuring the IL-18 concentration in the sample or by measuring the IL-18 mRNA level in the sample.

  When measuring the IL-18 concentration, an immunological measurement method using an anti-IL-18 antibody such as an ELISA method can be used in the same manner as the measurement of the IgE concentration. Therefore, as an example of a reagent for measuring the IL-18 level provided in this kit, an anti-IL-18 antibody that can bind to IL-18 of the target animal species, a labeled secondary antibody, a coloring reagent, a washing buffer And ELISA plates. However, it is not limited to these.

  Moreover, when measuring an IL-18 mRNA level, the method of measuring a well-known mRNA can be used, and the reagent which measures the IL-18 level in the sample with which this kit is equipped is used for these well-known methods. If it is a reagent, it will not specifically limit. Specific examples include RT-PCR method, real-time RT-PCR method, Northern hybridization method, DNA array method and the like.

  For example, when measuring the IL-18 level in a sample by RT-PCR method or real-time RT-PCR method, as an example of a reagent for measuring IL-18 level provided in this kit, IL-18 mRNA of the animal species of interest Primer sets that can amplify (cDNA), reagents for preparing RNA from tissues or cells, reverse transcriptase, buffers used for reverse transcription, heat-resistant DNA polymerase, PCR reagents, real-time PCR reagents, PCR Tubes, PCR plates and the like can be mentioned. However, it is not limited to these.

  The reagent for measuring IgE level in the sample, the reagent for measuring IL-18 level in the sample, and reagents and instruments other than the pharmaceutical composition according to the present invention, which are provided in the kit of the present invention, are particularly limited. Not.

[Prevention and treatment methods]
The method for preventing and / or treating a Th1-type allergic disease according to the present invention comprises the steps of measuring IgE level and IL-18 level in a sample derived from a subject, and comparing the IgE level and IL-18 level with a normal level. And administering an IL-18 inhibitor and / or an IFN-γ inhibitor to a subject whose IgE level is within the normal range and whose IL-18 level exceeds the normal range. Anything to do.

  Hereinafter, the above steps will be described in order.

  In the step of measuring IgE level and IL-18 level in a sample derived from a subject (hereinafter referred to as “measurement step” for convenience), the subject is not particularly limited and widely includes animals in general. Preferably there is. As animals other than humans, for example, pet animals such as dogs and cats are preferable. The sample is as described in the above [Kit] section.

  According to the studies by the present inventors, it has been clarified that, in the Th1-type allergic disease, the IgE level is within the normal range, but the IL-18 level is increased. Therefore, it is significant from the viewpoint of medical economy as well as improving the treatment efficiency to measure the IgE level and IL-18 level of the subject in advance and diagnose the subject as being a Th1-type allergic disease.

  The method for measuring the IgE level and the method for measuring the IL-18 level are not particularly limited, and can be appropriately selected from known methods as described in the above [Kit] section.

  In addition, as an index of the pathological condition of the Th1-type allergic disease, there may be mentioned infections other than the above IgE and IL-18, the influence of environmental factors (dust, gas, etc.) and the like. Therefore, it is preferable to comprehensively judge the index and determine whether or not it is a Th1-type allergic disease.

  In the next step, the IgE level and IL-18 level measured in the measurement step are compared with normal levels (hereinafter referred to as “comparison step” for convenience). Here, the normal level means the level of IgE and IL-18 in a normal healthy individual. The normal level is preferably measured using the same cell, tissue, or body fluid in the same animal species as the non-analyte-derived sample to be compared as the sample. The normal level is more preferably the average level of a normal healthy individual population.

  It is known that the IgE level and the IL-18 level differ depending on the measurement method and the facility, and it is difficult to define the normal level range with specific numerical values. Therefore, it is preferable to determine whether or not it is within the normal range by accumulating the measured values of normal healthy individuals measured by the same method for each facility, and comparing the measured values with the measured level of the subject using the background data. .

  In the next step, an IL-18 inhibitor and / or IFN-γ inhibition is performed on a subject in which the IgE level is within the normal range and the IL-18 level exceeds the normal range in the comparison step. A substance is administered (hereinafter referred to as “administration step” for convenience).

  The IL-18 inhibitor and the IFN-γ inhibitor are as described in the above section [Pharmaceutical composition], and examples thereof include an anti-IL-18 neutralizing antibody and an anti-IFN-γ neutralizing antibody. it can. Each of the IL-18 inhibitor and / or IFN-γ inhibitor may be administered alone, but is preferably administered in combination with a pharmaceutically acceptable carrier or diluent. The carrier or diluent is also as described in the above section [Pharmaceutical composition].

  There are no particular limitations on the route of administration, the number of administrations, and the like. What is necessary is just to select the administration route suitable for a subject, the frequency | count of administration, etc. suitably.

  In addition, in the prevention and / or treatment method of this invention, it cannot be overemphasized that processes other than the said measurement process, a comparison process, and an administration process may be included, and the content of the process which can be included is not limited.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments can be obtained by appropriately combining technical means disclosed in different embodiments. The form is also included in the technical scope of the present invention.

[ Reference Example 1: Inhibitory action by IL-18 inhibition and IFN-γ inhibition in Th1-type atopic dermatitis model mice]
(1) Animal used NC / Nga mice (female, 6-8 weeks old, Japanese Charles River) were purchased and bred in an SPF (specific pathogen-free) environment. The number of animals in one group was 4-6.

(2) Reagents, etc.
SpA derived from S. aureus Cowan1 strain was purchased from Calbio Chem (La Jolla, CA). PE-conjugated anti-IA ^d , FITC-conjugated anti-CD3, PE-conjugated anti-CD8, FITC-conjugated anti-CD4 and PE-conjugated anti-Gr-1 were purchased from PharMingen (San Diego, Calif.). A hybridoma producing anti-IL-4 mAb (11B11) and a hybridoma producing anti-IFN-γ mAb (6A2) were obtained from ATCC (American Type Culture Collection). Anti-IL-18 rabbit polyclonal antibodies are available in the literature (Sugimoto, T., et al. IL-18 acts on memory Th1 cells to induce airway inflammation and hyperresponsiveness in a naive host mouse. J. Exp. Med. 199, 535-545. (2004).) Was used. Recombinant mouse IL-12 was purchased from R & D (Minneapolis, Minn.). Recombinant mouse IL-18 was purchased from MBL (Nagoya, Japan). Recombinant human IL-2 was provided by Shionogi. As the cell culture solution, RPMI 1640 containing 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mM of 2-ME and 2 mM L-glutamine was used.

(3) Production of Th1-type atopic dermatitis (AD) model mouse SDS (Sodium dodecyl salfate) was dissolved in sterilized distilled water to prepare a 4% solution. In addition, SpA was dissolved in sterilized distilled water and adjusted to an appropriate concentration so that the coating amount per animal was 10 μg, 100 μg, and 200 μg.

  A 4% SDS solution was applied to the shaved mouse, and SpA was applied 2 minutes later. Application was performed every day for 4 weeks. For scoring skin symptoms, clinical severity score (Matsuda, H., et al. Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC / Nga mice. Int. Immunol. 9, 461-466 (1997)) Each symptom was scored according to the score shown in Table 1, and the severity of the skin symptom was evaluated based on the total score.

  The results are shown in FIGS. 1 (A) and (B). As is clear from FIG. 1 (A), the application of 4% SDS and SpA causes dermatitis, whose main symptoms are dryness and lichenification of the skin, and curettage in spite of the SPF environment. It was done. Further, as is clear from FIG. 1 (B), the severity of dermatitis depends on the dose of SpA, and by applying 200 μg of SpA in combination with 4% SDS, more than 7 moderate or more Dermatitis was caused in all cases. In addition, the onset of significant dermatitis was not recognized by the application of SDS alone or SpA alone.

(4) Analysis using Th1-type AD model mouse 4-1. Serum IL-18 and serum IgE
Blood was collected over time from the tail vein, and serum IL-18 and IgE concentrations were measured. The measurement followed the method described in Non-Patent Document 9.

  The results are shown in FIG. As is apparent from the graph on the left side of FIG. 2, when 200 μg of SpA was applied in combination with 4% SDS, the IL-18 concentration in the serum was significantly increased (p <0.01) compared to before application. . Although an increasing tendency was observed in the application of SpA alone, it was not significant as compared to before application. Application of SDS alone did not increase the serum IL-18 concentration. On the other hand, as is apparent from the graph on the right side of FIG. 2, the IgE concentration in serum did not increase in any group. Therefore, it was shown that IL-18 is involved in the onset of dermatitis in this Th1-type AD model mouse and there is an IgE-independent mechanism.

4-2. Histological observation The skin lesions were sampled, fixed in formalin and prepared in paraffin-embedded sections according to a conventional method. Hematoxylin and eosin (HE) staining or toluidine blue staining was applied and observed with an optical microscope. In addition, the number of mast cells in 5 fields randomly selected at low magnification (40 times) was counted, and the relationship with the plasma histamine concentration was analyzed.

  FIG. 3 shows a microscopic image of the skin lesion. As is apparent from FIG. 3, thickening of the epidermis and dermis depending on time was observed. In the dermis, inflammatory changes were observed in which various leukocytes such as neutrophils, eosinophils, mast cells, and lymphocytes were infiltrated with high density, and were particularly prominent in lesions 4 weeks after the start of application. A severely inflamed site consisting of neutrophils and eosinophils was observed subcutaneously in the HE-stained image at 4 weeks, which was strongly enlarged. Although not shown in FIG. 3, an almost normal tissue image was observed over 4 weeks when SDS alone was applied.

  FIG. 4 shows changes in the number of mast cells (left graph) and plasma histamine concentration (right graph) observed with a microscope. As is clear from FIG. 4, the number of mast cells and plasma histamine concentration increased in parallel with the severity of dermatitis by application of SDS and SpA. On the other hand, no change was observed in the single application of SDS.

  Next, in order to identify the type of infiltrating cells in the lesion, observation was performed using a confocal laser microscope. That is, the collected skin was immediately embedded in an OTC compound and stored at −80 ° C. A frozen section having a thickness of 4 μm was prepared, fixed with PBS containing 3% paraformaldehyde for 30 minutes, and blocked with 1% bovine serum albumin. After washing with PBS, the mixture was reacted with each FITC-labeled or PE-labeled monoclonal antibody described in (2) for 1 hour. Each labeled antibody was used at a 50-fold dilution. After washing with PBS, immunostained images of each section were evaluated using a confocal laser microscope (Olympus IX81).

  FIG. 5 shows a confocal laser microscope observation image. An increase in CD3-positive T cells was observed in the epidermis and dermis of the lesioned part of the mouse applied with 4% SDS and 200 μg SpA for 4 weeks. CD4 positive T cells and CD8 positive T cells were almost the same level. In addition, many local accumulations of Gr-1 positive neutrophils were observed in the dermis. More interestingly, the number of MHC class II positive cells was extremely increased. On the other hand, when only SDS was applied, a slight accumulation of neutrophils (Gr-1 positive) was observed, but other findings were not observed.

  From the above observation results, it was clarified that T cells and neutrophils infiltrated at a high density in the AD model mice as much as mast cells and eosinophils.

4-3. Cytokine and chemokine expression in skin lesions Total RNA was extracted from the collected skin lesions using an RNeasy kit (Quiagen, Basel, Switzerland) according to the instruction manual. RT-PCR using this total RNA was described in the literature (Ogushi, I., et al. Nuclear factor kB decoy oligodeoxynucleotides prevent endotoxin-induced fatal liver failure in a murine model. Hepatology 38, 335-344 (2003)). Went in the way.

  The results are shown in FIG. As is clear from FIG. 6, IL-12 (p40) was expressed from day 1 to day 14. On the other hand, IL-12 (p35) was constantly weakly expressed. IFN-γ expression was similar to IL-12 (p40). TFN-α began to express on day 1 or 2, and then its expression increased gradually and continuously until day 28. It is interesting that IL-13 becomes weakly expressed after day 14, and is relatively behind the expression of IFN-γ. On the other hand, IL-4 was not expressed.

  These cytokine expression kinetics indicate that IFN-γ and its upstream cytokine IL-12 are induced immediately after the expression of TNF-α and IL-13 is induced, This suggests that a Th1-like reaction is predominantly activated in the skin lesion.

  As is clear from FIG. 6, the expression of chemokines involved in the accumulation of T cells and neutrophils was detected. That is, on the first day, MIF, IP-10 and I-TAC, which are IFN-induced chemokines involved in the accumulation of macrophages and T cells, are expressed, and the expression induction of IL-12 and IFN-γ is clearly I was in sync. At the same time, MIP-1α and MIP-1β began to be expressed. These are chemokines involved in the accumulation of various inflammatory cells such as macrophages, Th1 cells, mast cells, eosinophils. Thereafter, MIP-2, a chemokine that accumulates neutrophils, was expressed in mice. On the other hand, the expression levels of eotaxin and RANTES did not change after application of SpA / SDS (data not shown).

  From the above results, it was revealed that cytokines and chemokines preferentially involved in the Th1-type inflammatory reaction were sequentially induced in the skin lesions after the SpA / SDS challenge.

4-4. Effects of anti-IL-18 neutralizing antibody and anti-IFN-γ neutralizing antibody Since cytokines and chemokines related to Th1 were selectively induced in skin lesions of the AD model mice, endogenous IL-18 and In order to confirm whether or not IFN-γ is a cause of progression of skin symptoms, the following analysis was performed.

  On the day before application of 4% SDS and SpA (200 μg) (day 0), 500 μg / mouse or anti-IFN-γ neutralization of the anti-IL-18 neutralizing antibody (anti-IL18 rabbit polyclonal antibody) described in (2) above An antibody (anti-IFN-γ mAb (6A2)) 1000 μg / mouse was administered intraperitoneally. Anti-IL-18 neutralizing antibody prepared at 2.5 mg / mL using PBS, anti-IFN-γ neutralizing antibody prepared at 5 mg / mL using PBS, both injection pumps 200 μL was administered. The application of SpA / SDS was started from the day after the first antibody administration, and the same dose as the first administration was intraperitoneally administered every 7 days for anti-IL-18 neutralizing antibody and every 5 days for anti-IFN-γ neutralizing antibody. . The severity of skin symptoms was evaluated using the scores shown in Table 1 at 1 week and 2 weeks after the start of application. Also, skin was collected at 2 weeks and subjected to histological observation.

  FIG. 7 shows the skin severity of mice administered with and without antibody. Ctrl in the figure indicates a group (control group) to which SpA / SDS was applied daily and no antibody was administered, α-IL-18 was applied to SpA / SDS daily, and anti-IL-18 neutralization The group to which the antibody was administered (anti-IL-18 group) is shown, and α-IFN-γ represents the group to which SpA / SDS was applied every day and the anti-IFN-γ neutralizing antibody was administered (anti-IFN group). As is clear from FIG. 7, the AD score of any antibody-administered group was kept low, and no significant dermatitis development was observed. On the other hand, in the control group, the score increased to about 5 points, and significant dermatitis developed. In addition, the mouse | mouth which administered the control antibody showed a score comparable as the control group (data not shown). As a control antibody for the anti-IL-18 antibody, a γ-globulin fraction of normal rabbit serum before immunization is used, and as a control antibody for the anti-IFN-γ antibody, it matches the class of the anti-IFN-γ antibody. In addition, an antibody that does not react with the mouse was used.

  FIG. 8 shows a histological observation image. The first row is an optical microscope image of a HE-stained paraffin section, the second row is an optical microscope image of a toluidine blue-stained paraffin section, the third and fourth stages are immunostained on a frozen section, and a confocal laser microscope It is the image observed by.

  In the anti-IL-18 group, inflammatory changes were observed in the dermis, but there was little change in the epidermis. There was no change in the number of T cells and mast cells, and no neutrophil infiltration was observed. MHC class II positive cells were at a lower level compared to the control group, but remained in an increased state.

  In the anti-IFN-γ group, no obvious inflammatory changes were observed in the epidermis and dermis. There was no clear infiltration of T cells, mast cells, and neutrophils even when observed with a confocal laser microscope. Furthermore, MHC class II expression was reduced to the level of normal mice.

  On the other hand, in the control group, similarly to the images shown in FIGS. 5 and 6, an inflammation image in which leukocytes such as T cells, neutrophils and mast cells were infiltrated with high density was observed. In addition, when the control antibody was administered, an inflammation image similar to that in the control group was observed (data not shown).

  From the above results, endogenous IL-18 and endogenous IFN-γ are essential for the onset of AD in this AD model mouse, and it is possible to suppress the onset of AD by inhibiting these functions. It became clear.

4-5. Th1 / Th2 differentiation in regional lymph nodes First, it was confirmed whether or not T cells can respond to SpA after applying SpA / SDS to the skin. That is, cervical lymph nodes were collected 4 weeks after the start of application from mice applied with 4% SDS and SpA (200 μg) every day. The collected lymph node cells were cultured in a medium containing each concentration of SpA for 3 days to confirm whether the cells proliferated in response to SpA. For cell growth, ³ H-TdR was added 16 hours before the end of the culture, and the amount of ³ H-TdR incorporated into the cells was measured after the end of the culture.

  The results are shown in FIG. As is apparent from FIG. 9, the cells after applying SpA / SDS proliferated depending on the SpA concentration. On the other hand, the cells before applying SpA / SDS did not proliferate. Therefore, it was revealed that SpA-specific T cells were accumulated in the regional lymph nodes by application of SpA / SDS.

  Next, it was examined whether lymph node cells differentiated into Th1 or Th2. That is, two groups of a 4% SDS and SpA (200 μg) application group and a 4% SDS single application group were provided, and cervical lymph nodes were collected over time. CD4 positive lymph node cells were selected using MACS. The CD4 positive for 3 to 5 mice in each group was pooled and cultured for 48 hours on a plate on which an anti-CD3 antibody was immobilized, and then the concentrations of IFN-γ, IL-13 and IL-4 in the supernatant were measured. An ELISA kit (Genzyme Techne, Boston, MA) was used for these measurements.

  The results are shown in FIG. As is apparent from FIG. 10, cells derived from mice applied with SpA / SDS once or several times produced a large amount of IFN-γ as compared with the case where SDS was applied alone (upper left graph). However, IL-4 was not detected in any group (lower right graph). In addition, IL-13 was produced in a large amount in cells derived from mice coated with SpA / SDS for 7 days or more (lower left graph).

  From the above results, it was shown that local CD4-positive T cells differentiate into Th1 cells immediately after the start of application of SpA / SDS, and Th1 cells receive IFN-γ and IL upon receiving TCR stimulation and IL-18 stimulation. -13 was produced.

  Whether IL-18 induces differentiation of naive CD4 positive T cells into Th cells that produce both IFN-γ and IL-13 (hereinafter referred to as “inflammatory-induced Th cells”) in cooperation with IL-12 In order to confirm whether or not, the following experiment was conducted. That is, naive CD4-positive cells of NC / Nga-derived lymph nodes are cultured in a Th1 environment supplemented with IL-18, and solid-phased IFN-γ produced by stimulation with anti-CD3 antibody and / or IL-18. And IL-13 were measured.

  The results are shown in FIG. As is clear from FIG. 11, CD4-positive T cells stimulated with IL-18 in a Th1 environment produced IFN-γ and IL-13. On the other hand, Th1 cells produced the same level of IFN-γ but not IL-13. This result was consistent with our previous knowledge that Th1 cells produce IL-13 in response to both TCR-mediated stimulation and IL-18. Proinflammatory Th cells produced much larger amounts of IL-13 than Th1 cells in response to the same stimulus. Conversely, the amount of IFN-γ produced in response to stimulation with anti-CD3 antibody and IL-18 in both pro-inflammatory Th cells and Th1 cells is equal to the amount of IFN-γ produced from the response to anti-CD3 antibody alone. It was the same level. Therefore, antigen stimulation and IL-18 had a synergistic effect on IL-13 production by these uniquely differentiated inflammation-induced Th cells.

4-6. Relationship between inflammation-induced Th cell generation and IL-18 In order to confirm whether IL-18 is essential for the generation of inflammation-induced Th cells by application of SpA / SDS, the following experiment was conducted. That is, from the mouse not coated with SpA / SDS (normal mouse), the mouse coated with SpA / SDS, and the mouse treated with anti-IL-18 neutralizing antibody and coated with SpA / SDS, 2 weeks from the start of coating The cervical lymph node was collected in the eye, and CD4 positive cells were isolated and cultured on a plate on which an anti-CD3 antibody was immobilized. IFN-γ and IL-13 in the culture supernatant were measured, and the ratio of normal mouse-derived cells to the concentration in the culture supernatant was calculated. The administration conditions for the anti-IL-18 neutralizing antibody were the same as the administration conditions described in 4-4 above.

  The results are shown in FIG. CD4 positive cells derived from SpA / SDS-coated mice produced significantly larger amounts of IFN-γ and IL-13 than normal mice (Ctrl in the figure). On the other hand, CD4 positive cells derived from SpA / SDS-coated mice administered with anti-IL-18 neutralizing antibody produced normal mouse levels of IFN-γ and IL-13 (α-IL-18 in the figure).

  From this result, it was revealed that IL-18 is essential for the generation of inflammation-induced Th cells.

[Example 2: Inhibitory action by IFN-γ inhibition and IL-18 inhibition in Th1-type bronchial asthma model mice]
(1) Animal used
Specific pathogen free (SPF) female BALB / c mice were purchased from Jackson Laboratory. An αβTCR transgenic (Tg) mouse (DO11.10) that recognizes OVA _323-339 (polypeptide consisting of amino acids 323 to 339 of OVA) from Dr. D. Loh (Washington University, St. Louis, MO). Genetic background BALB / c) was obtained. For the transgenic mice, all experiments were heterozygous for the transgene. IL-4 receptor α chain gene KO mice (IL-4RαKO) were purchased from Jackson Laboratory (USA). The IFN-γ gene KO mouse (IFN-γKO) was a gift from Prof. Iwakura, University of Tokyo. IL-18 gene KO mice (IL-18KO) were distributed by Shizuo Akira et al. At Hyogo College of Medicine. Any of the above KO mice are normal in appearance and have an abnormality only in the immune response using the knocked out cytokine. All mice were bred under an SPF condition at an animal facility at Hyogo College of Medicine (Nishinomiya, Japan) and used at 6-10 weeks of age.

(2) Reagents etc. Recombinant mouse IL-12 was purchased from R & D (Minneapolis, Minn.). Recombinant mouse IL-18 was purchased from MBL (Nagoya, Japan). Anti-mouse IL-18 antibody (anti-IL18 rabbit polyclonal antibody) has been published in the literature (Sugimoto, T., et al. IL-18 acts on memory Th1 cells to induce airway inflammation and hyperresponsiveness in a naive host mouse. J. Exp. Med. 199, 535-545 (2004).) Was used after purification. Anti-mouse IL-4 (11B11) and anti-mouse IFN-γ (XMG1.2) were purified and used. Recombinant mouse IL-13Rα2 / Fc Chimera was purchased from R & D (Minneapolis, Minn.). PE (Phycoerythrin) labeled anti-Gr-1 antibody was purchased from PharMingen (San Diego, Calif.).

(3) Production of Th1-type bronchial asthma model mouse 3-1. In vitro induction of OVA-specific Th1 cells RPMI 1640 loaded with 10% FBS (fetal calf serum), 50 μM 2-ME (ME: mercaptoethanol), 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin In addition to 3 ml 6-well plates, naive spleen CD4 ⁺ CD62L ⁺ T cells (CD4 and CD62 expressing T cells; 1 × 10 ⁵ / ml) from DO11.10 Tg mice were added to antigen-presenting cells (APC; radiation) for 7 days. Stimulated with IL-2 (100 pM) and OVA323-339 (1 μM) in the presence of BALB / c splenocytes from which T cells were removed by irradiation) (1 × 10 ⁶ / ml). In order to induce the T cells into Th1 cells, IL-12 (10 ng / ml) and anti-IL-4 antibody (11B11; 10 μg / ml) were added to the medium. Such antigen stimulation was performed twice.

3-2. Production of model mice OVA-specific Th1 cells (5-10 × 10 ⁶ cells / mouse) induced in vitro were intravenously administered to BALB / c mice to produce passive Th1-type bronchial asthma model mice. Further, active Th1-type bronchial asthma model mice were prepared by subcutaneously administering OVA and CFA (Complete Freund Adjuvant) to BALB / c mice and various knockout mice. Specifically, OVA (SIGMA, USA) was dissolved in PBS to a concentration of 200 μg / mL, and 500 μL of each mouse mixed with the same amount of CFA was subcutaneously administered (final concentration: OVA: 50 μg / mL). Mouse, CFA: 250 μL / mouse). These mice were used for the following experiments 2 to 4 weeks after administration.

(4) Experimental method PBS (control), OVA, or OVA + IL-18 was intranasally administered to Th1 type bronchial asthma model mice prepared as described above for 3 consecutive days.

  AHR (respiratory tract resistance) was measured using mouse and software of Pulmos-I (MIPS, Osaka, Japan) for inhalation of β-methacholine (Mch) in mice. Mice were placed in the chamber, first exposed to saline aerosol (baseline), and then exposed to increasing concentrations of methacholine (5, 10, 15 and 20 mg / ml). Each was measured for 3 minutes after 2 minutes of exposure, reflecting an enhanced pressure wave shape and a change in breathing time amplitude.

  For BALF (alveolar lavage fluid), 0.5 mL of PBS was divided into 3 times per mouse, and alveolar lavage was performed, and the total number of cells in the collected BALF was counted. In addition, BALF was centrifuged and then stained with Dif-Quik (Baxter Healthcare Corp, Miami, FL), and distinguished based on morphology and staining characteristics.

  Lung immunohistology was performed as follows. That is, the collected lung tissue was immediately embedded in an OTC compound and stored at −80 ° C. A frozen section having a thickness of 7 μm was prepared, fixed with PBS containing 3% paraformaldehyde for 30 minutes, and blocked with 1% bovine serum albumin. After washing with PBS, it was reacted with a PE-labeled anti-Gr-1 antibody (diluted 50 times) for 1 hour. After washing with PBS, evaluation was performed using a confocal laser microscope (Olympus IX81).

(5) Experimental results 5-1. In vivo-induced Th1-type bronchial asthma model mouse A normal mouse is immunized subcutaneously with OVA + CFA to induce a Th1 environment in normal Balb / c mice. When OVA (100 μg / day) and IL-18 (500 ng / day) were administered intranasally for 3 days to this mouse, the AHR elevation, airway inflammation, neutrophils in BALF, Infiltration of eosinophil was observed, and a pathological condition of Th1-type bronchial asthma was exhibited.

  The results of AHR are shown in FIG. The graph on the left (DO11.10Th1) shows the results for the Th1 cell-transferred mice, and the graph on the right (OVA + CFA) shows the results for the mice immunized subcutaneously with OVA and CFA. Control in the figure represents that PBS was administered intranasally, and OVA + IL-18 represents that OVA and IL-18 were administered intranasally. As is clear from FIG. 13, AHR increased in all mice when OVA and IL-18 were administered intranasally.

5-2. Effect of IL-13 and IL-4Rα on Th1-type bronchial asthma FIG. 14 shows PBS (Control), OVA + IL-18 or OVA + IL-18 + IL-13Rα2 / Fc Chimera against wild type mice immunized subcutaneously with OVA and CFA. The results of AHR measurement were shown when nasal administration was administered intranasally and PBS (Control) or OVA + IL-18 was intranasally administered to IL-4RαKO mice immunized subcutaneously with OVA and CFA. As is clear from FIG. 14, when IL-13Rα2 / Fc Chimera was simultaneously administered to neutralize IL-13, the increase in AHR was not suppressed (left side of FIG. 14). Further, IL-4RαKO mice subjected to OVA + CFA immunization showed an increase in AHR by intranasal administration of OVA and IL-18 as in the case of wild type mice (right side of FIG. 14).

FIG. 15 shows that when wild type mice immunized subcutaneously with OVA and CFA were not administered intranasally (unchallenge), when OVA + IL-18 was administered intranasally, and IL immunized subcutaneously with OVA and CFA The types and number of cells in BALF when OVA + IL-18 was administered intranasally to -4RαKO mice were shown. In the figure, Mo. Are monocytes, Eo. Is eosinophil, Lym is lymphocyte, Neu. Represents neutrophils. As is clear from FIG. 15, when OVA + IL-18 was administered intranasally to wild-type mice immunized subcutaneously with OVA and CFA, eosinophils in BALF increased markedly. On the other hand, the number of eosinophils in BALF of IL-4RαKO mice was significantly smaller than that of wild-type mice.
5-3. Effect of IFN-γ on Th1 type bronchial asthma FIG. 16 shows PBS (Control), OVA + IL-18 or OVA + IL-18 + in Th1 cell-transfected mice (DO11.10Th1) or mice immunized subcutaneously with OVA and CFA. The results of AHR when anti-IFN-γ antibody (anti IFN-γ) was administered intranasally are shown. As is clear from FIG. 16, the increase in AHR was suppressed by administration of anti-IFN-γ antibody in any mouse.

  FIG. 17 shows cells in BALF when mice immunized subcutaneously with OVA and CFA were administered intranasally with PBS (Control), OVA + IL-18, or OVA + IL-18 + anti-IFN-γ antibody (anti IFN-γ). The kind and number of are shown. In the figure, Mo. Are monocytes, Eo. Is eosinophil, Lym is lymphocyte, Neu. Represents neutrophils. As is clear from FIG. 17, neutrophil infiltration in BALF was suppressed by administration of anti-IFN-γ antibody, but eosinophils were not affected.

FIG. 18 shows the results of AHR when IFN-γKO mice immunized subcutaneously with OVA and CFA were administered intranasally with PBS (Control) or OVA + IL-18, and IFN-γKO mice immunized subcutaneously with OVA and CFA. On the other hand, the results are shown in which the type and number of cells in BALF when OVA + IL-18 was administered intranasally were compared with the type and number of cells in BALF of wild-type mice treated with the same treatment. As is clear from FIG. 18, no increase in AHR was observed in the model produced with IFN-γKO mice (left in FIG. 18). Moreover, in the model produced by the IFN-γKO mouse, neutrophil infiltration in BALF was suppressed, but eosinophil infiltration was enhanced as compared with the wild type.
5-4. Induction of endogenous IL-18 by LPS (Lipopolysaccharide) and Th1 type bronchial asthma by OVA + LPS FIG. 19 shows PBS (Cont), LPS, OVA + LPS, OVA + LPS + anti-IFN in mice immunized subcutaneously with OVA and CFA. The results of AHR when γ antibody or OVA + LPS + anti-IL-18 antibody was administered intranasally are shown. As is clear from FIG. 19, AHR was not increased only by administration of LPS, and AHR was increased by administration of OVA + LPS. On the other hand, when an anti-IFN-γ antibody or an anti-IL-18 antibody was administered at the same time, any increase in AHR was suppressed.

  FIG. 20 shows the types and number of cells in BALF of mice subjected to the above treatments. In the figure, Mo. Are monocytes, Eo. Is eosinophil, Lym is lymphocyte, Neu. Represents neutrophils. As is clear from FIG. 17, neutrophil infiltration in BALF was suppressed when anti-IFN-γ antibody or anti-IL-18 antibody was administered simultaneously.

  FIG. 21 shows a case in which mice immunized subcutaneously with OVA and CFA were not administered intranasally (unchallenge), OVA + LPS was administered intranasally, and OVA + LPS + anti-IL-18 antibody was administered intranasally. The results of immunohistological observation using a PE-labeled anti-Gr-1 antibody for the lungs collected from each mouse are shown. As is clear from FIG. 21, neutrophil infiltration was enhanced when OVA + LPS was administered intranasally, but neutrophil infiltration was suppressed by simultaneous administration of anti-IL-18 antibody.

  FIG. 22 shows AHR when intranasally administered PBS (Control) or OVA + LPS to IFN-γKO mice immunized subcutaneously with OVA and CFA, or IL-18KO mice immunized subcutaneously with OVA and CFA. The results are shown in comparison with AHR when OVA + LPS was administered intranasally to wild type mice immunized subcutaneously with OVA and CFA. As is clear from FIG. 22, neither IFN-γKO mice nor IL-18KO mice showed an increase in AHR seen in wild-type mice.

  The present invention provides a pharmaceutical composition used for the prevention and / or treatment of Th1-type atopic dermatitis or Th1-type bronchial asthma, and can be used in the pharmaceutical industry.

(A) is a photograph showing the skin condition of a Th1-type AD model mouse, and (B) is a graph showing the AD severity score. It is a graph which shows the result of having measured the IL-18 density | concentration and IgE density | concentration in serum of Th1 type | mold AD model mouse. It is an optical microscope observation image of the skin lesion part of Th1 type | mold AD model mouse. It is a graph which shows the change of the number of mast cells in the skin lesion part of a Th1 type AD model mouse, and plasma histamine density | concentration. It is a confocal laser microscope observation image of the skin lesion part of Th1 type AD model mouse. It is an image which shows the RT-PCR result of various cytokine and chemokine in the skin lesion part of Th1 type | mold AD model mouse. It is a graph which shows the score of the severity of AD in the Th1-type AD model mouse which administered the anti- IL-18 neutralizing antibody or the anti- IFN-γ neutralizing antibody. It is the optical microscope observation image and the confocal laser microscope observation image of the skin lesion part of Th1 type AD model mouse which administered the anti- IL-18 neutralizing antibody or the anti- IFN-gamma neutralizing antibody. It is a graph which shows the result of a responsiveness test to SpA of the cervical lymph node cell of Th1-type AD model mouse. It is data which shows the result of having measured the IFN-gamma, IL-13, and IL-4 density | concentration of a culture supernatant in the test which confirms Th1 / Th2 differentiation of the cervical lymph node cell of a Th1-type AD model mouse. It is a graph which shows the result of having measured IFN-gamma, IL-13, IL-4 density | concentration, and IL-10 of a culture supernatant in the differentiation induction experiment of naive CD4 positive T cell. IFN-γ of culture supernatant of SpA / SDS-coated mouse-derived cells and SpA / SDS-coated mouse-derived cells administered with anti-IL-18 neutralizing antibody in a test to confirm the relationship between inflammation-induced Th cell generation and IL-18 It is a graph which shows the result of having measured IL-13 and measuring the ratio with respect to the density | concentration in the culture supernatant of a normal mouse origin cell. It is a graph which shows the result of AHR at the time of administering OVA + IL-18 intranasally with respect to the mouse | mouth which immunized subcutaneously with the OVA specific Th1 cell transfer mouse | mouth and OVA and CFA. OVA + IL-18 or OVA + IL-18 + IL-13Rα2 / Fc Chimera was administered intranasally to wild type mice immunized subcutaneously with OVA and CFA, and OVA + IL against IL-4RαKO mice immunized subcutaneously with OVA and CFA It is a graph which shows the result of AHR at the time of administering -18 intranasally. When OVA + IL-18 was administered intranasally to wild type mice immunized subcutaneously with OVA and CFA, and when OVA + IL-18 was administered intranasally to IL-4RαKO mice immunized subcutaneously with OVA and CFA It is the graph which showed the kind and number of the cell in BALF. It is the graph which showed the result of AHR when OVA + IL-18 or OVA + IL-18 + anti-IFN-γ antibody was intranasally administered to a mouse transfected with OVA-specific Th1 cells or a mouse immunized subcutaneously with OVA and CFA. It is the graph which showed the kind and number of the cell in BALF when the OVA + IL-18 or OVA + IL-18 + anti-IFN-γ antibody was intranasally administered to the mouse immunized subcutaneously with OVA and CFA. Results of AHR when intranasally administered OVA + IL-18 to IFN-γKO mice immunized subcutaneously with OVA and CFA, and intranasally administered OVA + IL-18 to IFN-γKO mice immunized subcutaneously with OVA and CFA It is the graph which showed the kind and number of the cell in BALF at the time of doing. It is the graph which showed the result of AHR when LPS, OVA + LPS, OVA + LPS + anti-IFN-γ antibody or OVA + LPS + anti-IL-18 antibody was intranasally administered to mice immunized subcutaneously with OVA and CFA. It is the graph which showed the kind and number of cells in BALF when LPS, OVA + LPS, OVA + LPS + anti-IFN-γ antibody or OVA + LPS + anti-IL-18 antibody was intranasally administered to mice immunized subcutaneously with OVA and CFA . From mice immunized subcutaneously with OVA and CFA, mice immunized subcutaneously with OVA and CFA and further administered intranasally with OVA + LPS, and mice immunized subcutaneously with OVA and CFA and administered intranasally with OVA + LPS + anti-IL-18 antibody It is the confocal laser microscope observation image of the extract | collected lung. It is the graph which showed the result of AHR when OVA + LPS was intranasally administered to the IFN-γKO mouse immunized subcutaneously with OVA and CFA, or the IL-18KO mouse immunized subcutaneously with OVA and CFA.

Claims (9)

A pharmaceutical composition used for preventing and / or treating Th1-type bronchial asthma and comprising an anti-IFN-γ neutralizing antibody . The pharmaceutical composition according to claim 1, further comprising an IL-18 inhibitor. The pharmaceutical composition according to claim 2, wherein the IL-18 inhibitor is an anti-IL-18 neutralizing antibody. A kit used for preventing and / or treating Th1-type bronchial asthma ,
A kit comprising a pharmaceutical composition comprising at least a reagent for measuring IgE level in a sample, a reagent for measuring IL-18 level in a sample, and an anti-IFN-γ neutralizing antibody . The kit according to claim 4, further comprising an IL-18 inhibitor. The kit according to claim 5, wherein the IL-18 inhibitor is an anti-IL-18 neutralizing antibody. Measuring IgE and IL-18 levels in a sample from a subject;
A step of comparing the IgE level and IL-18 level with a normal level; and for a subject whose IgE level is within a normal range and whose IL-18 level exceeds the normal range, in anti-IFN-γ Administering a sum antibody ,
A method for preventing and / or treating Th1-type bronchial asthma , wherein the subject is a non-human animal . The method for preventing and / or treating Th1-type bronchial asthma according to claim 7, further comprising the step of administering an IL-18 inhibitor to the subject. The method for preventing and / or treating Th1-type bronchial asthma according to claim 8, wherein the IL-18 inhibitor is an anti-IL-18 neutralizing antibody.

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