JP6653120B2 - Anti-inflammatory drugs based on IKAROS inhibition - Google Patents

Description

The present invention relates to anti-inflammatory drugs.

  Inflammatory diseases such as allergic diseases (for example, hay fever, allergic conjunctivitis, asthma, atopic dermatitis, Crohn's disease, ulcerative colitis) have been increasing in incidence. On the other hand, current inflammation control technologies mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), steroid drugs or immunosuppressive drugs targeting immune cells such as lymphocytes, and their side effects, for example, induction of infectious diseases. Is a problem.

  As described in Patent Documents 1 to 3, research on IKAROS has been performed. In addition, there are non-patent documents 1 to 18 as studies on TLR3 and inflammation.

Japanese Patent No. 3556228 Japanese Patent Publication No. 10-507070 Japanese Patent Publication No. 11-512284

Nakamura N, Tamagawa-Mineoka R, Ueta M, Kinoshita S, Katoh N. Toll-Like Receptor 3 Increases Allergic and Irritant Contact Dermatitis. J Invest Dermatol. 2014 Sep 17.doi: 10.1038 / jid.2014.402. Ueta M, Sotozono C, Koga A, Yokoi N, Kinoshita S. Usefulness of a New Therapy Using Rebamipide Eyedrops in Patients with VKC / AKC Refractory to Conventional Anti-Allergic Treatments.Allergol Int. 2014 63: 75-81 Ueta M, Mizushima K, Naito Y, Narumiya S, Shinomiya K, Kinoshita S. Suppression of polyI: C-inducible gene expression by EP3 in murine conjunctival epithelium. Immunol Lett. 2013 Sep 12. pii: S0165-2478 (13) 00121 -1. Ueta M, Sotozono C, Yokoi N, Kinoshita S: Rebamipide Suppresses PolyI: C-Stimulated Cytokine Production in Human Conjunctival Epithelial Cells.J Ocul Pharmacol Ther. 2013 Sep; 29 (7): 688-93. Ueta M, Kinoshita S: Ocular surface inflammation is regulated by innate immunity.Prog Retin Eye Res. 2012; 31 (6): 551-575. Ueta M, Matsuoka T, Sotozono C, Kinoshita S: Prostaglandin E2 Suppresses Poly I: C-Stimulated Cytokine Production Via EP2 and EP3 in Immortalized Human Corneal Epithelial Cells.Cornea.2012; 31 (11): 1294-1298. Ueta M, Tamiya G, Tokunaga K, Sotozono C, Ueki M, Sawai H, Inatomi T, Matsuoka T, Akira S, Narumiya S, Tashiro K, Kinoshita S: Epistatic interaction between Toll-like receptor 3 (TLR3) and prostaglandin E receptor 3 (PTGER3) genes. J Allergy Clin Immunol. 2012; 129 (5): 1413-1416.e11. Ueta M, Matsuoka T, Yokoi N, Kinoshita S: Prostaglandin E2 suppresses polyinosine-polycytidylic acid (polyI: C) -stimulated cytokine production via prostaglandin (EP) 2 and 3 in human conjunctival epithelial cells.Br J Ophthalmol. 2011; 95 ( 6): 859-863. Ueta M, Matsuoka T, Yokoi N, Kinoshita S: Prostaglandin E receptor subtype EP3 downregulates TSLP expression in human conjunctival epithelium. Br J Ophthalmol. 2011; 95 (5): 742-743. Ueta M, Mizushima K, Yokoi N, Naito Y, Kinoshita S: Gene-expression analysis of polyI: C-stimulated primary human conjunctival epithelial cells. Br J Ophthalmol. 2010; 94 (11): 1528-1532. Ueta M, Kinoshita S: Innate immunity of the ocular surface.Brain Res Bull. 2010; 81 (2-3): 219-228. Ueta M, Uematsu S, Akira S, Kinoshita S: Toll-like receptor 3 enhances late-phase reaction of experimental allergic conjunctivitis. J Allergy Clin Immunol. 2009; 123 (5): 1187-1189. Ueta M, Matsuoka T, Narumiya S, Kinoshita S: Prostaglandin E receptor subtype EP3 in conjunctival epithelium regulates late-phase reaction of experimental allergic conjunctivitis.J Allergy Clin Immunol. 2009; 123 (2): 466-471. Kaniwa N, Saito Y, Aihara M, Matsunaga K, Tohkin M, Kurose K, Sawada J, Furuya H, Takahashi Y, Muramatsu M, Kinoshita S, Abe M, Ikeda H, Kashiwagi M, Song Y, Ueta M, Sotozono C , Ikezawa Z, Hasegawa R: JSAR research group.HLA-B locus in Japanese patients with anti-epileptics and allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis. Pharmacogenomics. 2008; 9 (11): 1617-1622. Ueta M: Innate immunity of the ocular surface and ocular surface inflammatory disorders.Cornea. 2008; 27 (Suppl 1): S31-40. Ueta M, Sotozono C, Inatomi T, Kojima K, Hamuro J, Kinoshita S: Association of Fas Ligand gene polymorphism with Stevens-Johnson syndrome. Br J Ophthalmol. 2008; 92 (7): 989-991. Ueta M, Sotozono C, Inatomi T, Kojima K, Tashiro K, Hamuro J, Kinoshita S: Toll like receptor 3 gene polymorphisms in Japanese patients with Stevens-Johnson syndrome. Br J Ophthalmol. 2007; 91 (7): 962-965 . Ueta M, Hamuro J, Kiyono H, Kinoshita S: Triggering of TLR3 by polyI: C in human corneal epithelial cells to induce inflammatory cytokines. Biochem Biophys Res Commun. 2005; 331 (1): 285-294.

  The present inventors have found expression of IKAROS in epithelial cells which has not been reported so far, have found a method of inducing IKAROS in cultured human mucosal epithelium and cultured human epidermis, and have completed the present invention. In the present invention, noting that not only immune cells but also epithelial cells are involved in the inflammatory state, a novel inflammatory control method targeting the inflammatory state has been developed. Thereby, local administration is facilitated by targeting epithelial cells, and drastic reduction of side effects is expected. Accordingly, the present invention provides the following.

Although prostaglandins (PG) _{E 2} may be also referred to as inflammatory substances, the result of the present invention, since the suppressing IKAROS found to be an inflammatory factor in epithelial, certain inflammatory, IKAROS, EP3 (PGE ₂ receptor 3), TLR3 (Toll-like receptor 3); TLR3 is a type I that recognizes double-stranded RNA (dsRNA) derived from a virus and exhibits strong antiviral action. It is one of the innate immune system molecules that promotes the production of interferon and works in defense against the virus.) Related inflammations (eg, Stevens-Johnson syndrome, asthma, contact dermatitis, acute dermatitis, atopic dermatitis, Allergic conjunctivitis and atopic keratoconjunctivitis), which function as inhibitors and as anti-inflammatory drugs Proven to be usable. The knowledge of the existing literature that discusses the relationship between prostaglandin (PG) E2 and polyI: C and allergy cannot be used to predict the therapeutic agent for IKAROS-upregulated disease of the present invention. Therefore, the present invention can be useful as a highly specific anti-inflammatory drug not found in the prior art. Accordingly, the present invention provides the following.
(1) An anti-inflammatory drug comprising an IKAROS (IKZF1) inhibitor.
(2) The anti-inflammatory agent according to item 1, wherein the IKAROS inhibitor is selected from the group consisting of PGE ₂ , anti-IKAROS siRNA, and anti-IKAROS antibody.
(3) The anti-inflammatory drug according to item 1 or 2, wherein the anti-inflammatory drug targets epithelial tissue.
(4) The anti-inflammatory drug according to any one of items 1 to 3, wherein the inflammation is an inflammation caused by IKAROS.
(5) The anti-inflammatory drug according to any one of items 1 to 4, wherein the inflammation is an inflammation caused by an increased expression of IKAROS.
(6) The anti-inflammatory drug according to any one of items 1 to 5, wherein the inflammation is an inflammation involving an epithelium caused by IKAROS.
(7) The anti-inflammatory drug according to any one of items 1 to 6, wherein the inflammation is an inflammation associated with IKAROS and TLR3 in epithelium.
(8) The anti-inflammatory drug according to any one of items 1 to 7, wherein the inflammation is caused by an allergic disease.
(9) The inflammation is any one of items 1 to 8, which is selected from the group consisting of Stevens-Johnson syndrome, asthma, contact dermatitis, acute dermatitis, atopic dermatitis, allergic conjunctivitis and atopic keratoconjunctivitis. Or the anti-inflammatory agent according to item 1.
(10) The anti-inflammatory agent according to any one of items 1 to 9, which is a topical administration agent.
(11) The anti-inflammatory agent according to item 10, wherein the topical administration agent is an external preparation, an eye drop, a nasal drop, or an inhalant.
(12) The anti-inflammatory drug according to any one of items 1 to 11, wherein the anti-inflammatory drug is for treating or preventing inflammation in skin or mucosal tissues.
(13) The anti-inflammatory agent according to item 12, wherein the mucosal tissue is an eye, an oral cavity, a nasal cavity, a pharynx, an airway, a vagina, a urethra, and / or a digestive tract.
(14) The anti-inflammatory drug according to any one of items 1 to 13, wherein the anti-inflammatory drug is for treating or preventing inflammation in the eye.
(15) The following steps (1) to (3):
(1) contacting a cell containing a nucleic acid encoding a reporter protein under the control of the IKAROS gene or the transcriptional regulatory region of the gene with a test substance;
(2) a step of measuring the expression level of an IKAROS gene or an IKAROS protein or a reporter protein in the cells; and (3) an IKAROS gene or an IKAROS protein or a reporter protein as compared to the case where the expression level is measured in the absence of a test substance. A screening method for an anti-inflammatory substance, comprising a step of selecting a test substance having a reduced expression level as a candidate for an anti-inflammatory substance.
(16) A method for screening an anti-inflammatory substance, comprising contacting IKAROS with a test substance and selecting a test substance having an ability to bind to IKAROS as a candidate for the anti-inflammatory substance.
(17) The following steps (1) to (3):
(1) contacting IKAROS with a cell membrane fraction of a target cell of an inflammatory disease in the presence of a test substance;
(2) a step of measuring the amount of IKAROS bound to the cell membrane fraction; and (3) a test wherein the amount of IKAROS bound to the cell membrane fraction is reduced as compared to the case where the measurement is performed in the absence of the test substance. 17. The screening method according to item 15 or 16, comprising a step of selecting a substance as a candidate for an anti-inflammatory substance.
(18) any one of items 15 to 17, further comprising applying a test substance selected as a candidate for an anti-inflammatory substance to an inflammation model and testing whether or not to suppress an inflammatory response in the model; The method described in the section.
(19) The following steps (1) to (3):
(1) contacting a target cell of an inflammatory disease with a test substance in the presence and absence of IKAROS;
(2) measuring the degree of the inflammatory response in the cells under each condition; and (3) suppressing the inflammatory response in the presence of IKAROS, as compared to the case where the measurement is performed in the absence of the test substance. A method for screening an anti-inflammatory substance, comprising the step of selecting a test substance that did not suppress the inflammatory response in the absence of IKAROS as a candidate for a substance exhibiting an anti-inflammatory effect by inhibiting the function of IKAROS.
(20) The method according to any one of items 15 to 19, further comprising a step of inducing an inflammatory response in the target cell.
(21) any one of items 15 to 20, characterized in that an inflammatory response is induced by polyI: C, interleukin-Iα (IL-1α), tumor necrosis factor (TNF) α or H ₂ O ₂ stimulation. The method described in the section.
(22) The method according to any one of items 15 to 21, wherein the cell contains an epithelial cell or is related to an epithelium.
(23) The epithelial cells are skin epidermal cells, esophageal epithelial cells, gastric epithelial cells, vaginal epithelial cells, uterine epithelial cells, thymic epithelial cells, bladder epithelial cells, prostate epithelial cells, mammary epithelial cells, conjunctival epithelial cells or corneal epithelium 23. The method according to item 22, wherein the cell is a cell.
(24) The method according to item 22 or 23, wherein the epithelial cell is a skin epidermal cell, a conjunctival epithelial cell, or a corneal epithelial cell.
(25) The method according to any one of items 22 to 24, wherein the epithelial cells are mucosal epithelial cells that express keratin 5.
(26) The method according to any one of items 15 to 25, wherein the inflammation is an inflammation caused by an increased expression of IKAROS.
(27) The method according to any one of items 15 to 26, wherein the inflammation is an inflammation involving an epithelium caused by IKAROS.
(28) The method according to any one of items 15 to 27, wherein the inflammation is an inflammation involving IKAROS and TLR3 in epithelium.
(29) A composition for increasing or inducing the expression of IKAROS gene, comprising polyI: C.
(30) The composition according to item 29, wherein the increase or induction of the expression of the IKAROS gene is in epithelial cells.
(31) An isolated cell in which the expression of IKAROS gene is increased or induced.
(31A) The isolated cell according to item 31, wherein the cell is keratin-positive.
(32) The cell according to item 31 or 31A, wherein the increase or induction of the expression of the IKAROS gene is caused by polyI: C.
(33) The cell according to item 31, 31A or 32, wherein the cell is an epithelial cell.
(34) An inflammation model animal in which the expression of IKAROS gene is increased or induced.
(35) The animal according to item 34, wherein the increase or induction of the expression of the IKAROS gene is caused by polyI: C.
(36) The animal according to item 34 or 35, wherein the inflammation model includes inflammation on an ocular surface.
(37) The animal according to any one of items 34 to 36, wherein the increase or induction of the expression of the IKAROS gene is due to a genetic modification.
(38) The animal according to any one of items 34 to 37, wherein the genetic modification includes modification to express the IKAROS gene in an epithelial-specific manner.
(39) The animal according to any one of items 34 to 38, wherein the animal causes cutaneous mucosal inflammation.
(40) The animal according to any one of items 34 to 39, wherein the animal comprises a vector operably linked to a keratin 5 promoter and comprising a nucleic acid sequence encoding the IKAROS gene.
(41) The animal according to any one of items 34 to 40, wherein the animal is a rodent.
(41A) The animal according to any one of items 34 to 41, wherein the inflammation model animal has cell infiltration in at least one selected from the group consisting of a skin tissue and a mucosal tissue.

  In the present invention, it is contemplated that one or more of the features described above may be provided in further combination in addition to the explicit combinations. Still further embodiments and advantages of the invention will be apparent to those skilled in the art upon reading and understanding the following detailed description, as appropriate.

  The present invention provides a novel anti-inflammatory treatment method targeting IKAROS (IKZF1) of epithelial cells.

  The prevalence of inflammatory diseases such as allergic diseases is steadily increasing, and the current method of controlling inflammation is mainly steroid drugs or immunosuppressive drugs that target immune cells such as lymphocytes, and their side effects are problematic. However, according to the present invention, not only immune cells but also epithelial cells ("epidermis" covering the body surface and "epithelium (narrow definition)" constituting mucous membranes of luminal organs) are greatly involved in inflammatory pathology. Focusing on this, a novel method of controlling inflammation targeting epithelial cells can be developed, and by using epithelial cells as the target, local administration is facilitated and drastic reduction of side effects is expected.

  According to the present invention, a new method for controlling inflammation targeting epithelial cells can be developed, focusing on the fact that epithelial cells are involved in the onset of inflammatory pathology as factors located upstream of immune cells. By controlling the kinetics and activation of immune cells using epithelial cells as targets, local administration is facilitated, and there is a substantial merit that drastic reduction of side effects is expected. An inflammation control model from a point of interest that has not been performed so far is also provided. Pioneering new inflammation control methods targeting epithelial cells that are completely different from existing inflammation control methods will contribute to the realization of a safe and secure society by providing appropriate treatments and extending healthy life expectancy. Furthermore, the development of a novel inflammatory control method that controls the dynamics and activation of immune cells by targeting epithelial cells will lead to a major turning point in inflammatory control methods. The novel method of controlling inflammation targeting epithelial cells is expected to drastically reduce side effects because local administration with a control compound is easy, and will be a major player in the treatment of inflammation in the future.

  In addition to exploring new compounds related to IKAROS present in epithelial cells, the mechanism of action can be elucidated at the molecular level, which can lead to the exploration of further molecular targets and drug seeds. In addition, a novel evaluation system for tissue permeability is constructed to facilitate the search for compounds that selectively act on epithelial cells. Elucidation of the mechanism of action at the molecular level reveals the mechanism of inflammation control through epithelial cells, leading to prevention of allergic diseases, treatment with medicines and medical supplies, and safe and secure quality (chemical materials, cosmetics, foods, health foods). Connect with. As a result, we can directly contribute to the improvement of human life in the future in terms of a safe and secure society by providing appropriate treatment methods and extending healthy life expectancy, as well as in a wide range of industries (medical, pharmaceutical, chemical, food, etc.) ) Can be expected.

FIG. 1 shows a technique for inducing IKAROS in human epidermal and epithelial cells. FIG. 1a shows the time course of IKAROS mRNA expression in primary cultures of human conjunctival epithelial cells exposed to 10 μg / ml polyI: C (poly I: C). 0 hours, 1 hour, 3 hours, 6 hours and 10 hours are shown from the left. Quantitative data was normalized to the expression of the housekeeping gene, GAPDH. The vertical axis shows the rise of specific mRNA for the 0 hr sample. FIG. 1b shows IKAROS mRNA expression in primary human conjunctival epithelial cells exposed to 10 μg / ml polyI: C (poly I: C) for 10 hours. The left shows the medium, and the right shows polyI: C. Quantitative data was normalized to the expression of the housekeeping gene, GAPDH. The vertical axis indicates the elevation of specific mRNA relative to the unstimulated sample. Data are representative of triplicate experiments and represent the mean ± SEM from experiments performed with 3 wells per group. FIG. 1c shows the expression of IKAROS mRNA in primary cultures of keratinocyte cells, which are the majority of cells constituting the epidermis, exposed to 10 μg / ml polyI: C (poly I: C) for 10 hours. . Quantitative data was normalized to the expression of the housekeeping gene, GAPDH. The vertical axis indicates the elevation of specific mRNA relative to the unstimulated sample. Data are representative of triplicate experiments and represent the mean ± SEM from experiments performed with 3 wells per group. FIG. 2 shows the state of an animal in overexpression of IKAROS and the like. The upper photograph is a comparison of the abdomen of a transgenic mouse (left) and a wild mouse (right) of IKAROS (IK1). The lower left panel is a comparison of the face of the IKAROS (IK1) transgenic mouse (top) and the wild mouse (bottom). The lower right panel is a comparison of the back of IKAROS (IK1) transgenic mice (right) and wild mice (left). FIG. 3 shows the expression of IKAROS mRNA and its inhibitory effect with an inhibitor. Figure 3a, polyl in primary cultured human conjunctival epithelial cells: upregulation of IKAROS mRNA of C-induced (10 hours stimulation), and show that was significantly inhibited by the addition of 100 [mu] g / ml PGE ₂ in its raised (left control, central polyl: C stimulation alone, right, polyl: a C stimulation + 100μg / ml PGE ₂ added).. Figure 3b, polyl in primary human keratinocytes (keratinocyte) cells: upregulation of IKAROS mRNA of C-induced (10 hours stimulation), and it was significantly inhibited by the addition of 100 [mu] g / ml PGE ₂ in its raised It is shown (left control center polyl: C stimulation alone, right, polyl: a C stimulation + 100μg / ml PGE ₂ addition.). FIG. 4 shows the structure and construction procedure of the expression vector (pK5-Ikzfl-2A-EGFP) prepared in the present invention. The upper part shows the structure of each element. Keratin 5 promoter is depicted on the left and the structure of Ikzfl-P2A-EGFP is shown on the right. The expression vector (pK5-lkzfl-2A-EGFP) prepared by inserting it into the Spe1 and NotI sites located downstream of the promoter of pBSK5 (human keratin 5 promoter in pBluescript II KS +) is shown below. FIG. 5 shows a restriction map of the produced expression vector (pK5-Ikzfl-P2A-EGFP) (SEQ ID NO: 5). The left side shows the map of the vector, and the right side is a blot showing the DNA cleavage pattern. The restriction enzymes used are as shown in the figure. From the left side, molecular weight markers, and lanes 1 to 7 show SalI, SalI + NotI, SpeI + NotI, Acc65I, EcoRI, and BamHI. The right side is also a molecular weight marker. There is a SalI recognition sequence (GTCGAC) site at positions 2223 to 2228 of SEQ ID NO: 5. The sequence of human Keratin 5 promoter region exists from position 2229 to position 16654 of SEQ ID NO: 5. Position 2763 of SEQ ID NO: 5 is T, but it is C in the NCBI database. A SpeI recognition site (ACTAGT) exists at positions 16655 to 16660 of SEQ ID NO: 5. The sequence of Ikzf1 isoform 1 (DNA (1545 bp; NM_001025597) exists at positions 16667 to 18211 of SEQ ID NO: 5. The P2A sequence exists at positions 18212 to 18277 of SEQ ID NO: 5. From position 18284 of SEQ ID NO: 5 EGFP (720 bp) is present at position 19003. bGH polyA is present at positions 19020 to 19309 of SEQ ID NO: 5. A NotI recognition sequence (GCGGCCGC) is present at positions 19310 to 19317 of SEQ ID NO: 5. FIG. 6 shows the results of examination of pCR analysis conditions for selecting pK5-Ikzfl-2A-EGFP founder mice (condition 1). As condition 1, a K5p-F1 binding to the Keratin5 promoter and a BGH-R3 primer binding downstream of bGH polyA were designed. In this figure, each primer sequence and PCR analysis conditions are shown. FIG. 7 shows the results of examination of pCR analysis conditions for selecting pK5-Ikzfl-2A-EGFP founder mice (condition 2). As condition 2, a K5p-F6 and a K5p-R2 primer which bind to the Keratin5 promoter upstream site were designed. In this figure, each primer sequence and PCR analysis conditions are shown. FIG. 8 shows the preparation of the pK5-Ikzfl-2A-EGFP transgene. The upper part shows the configuration of the transgene, and the lower part shows the cleavage pattern by the restriction enzyme. Specifically, the expression vector was digested with restriction enzymes SalI and Natl. As a result, the plasmid vector sequence was separated, and the expression cassette portion (about 17.1 kbp) was purified. The lower part confirms that the cutting is correct. FIG. 9 shows the results of confirming the expression of IKAROS and GAPDH by PCR. Regarding each lane, the leftmost is an empty lane, the second is a molecular weight ladder marker (each indicated in units of 100 bp), and an arrow is attached at a position of 500 bp. The third and fourth from the left indicate mononuclear cells, with and without IKAROS RT, respectively. Put another, the sixth from the left is the molecular weight ladder marker again, and the seventh and eighth from the left indicate the conjunctival epithelium, with RT with IKAROS and without RT, respectively. Put another, the 10th from the left is the molecular weight ladder marker again, and the 11th and 12th from the left indicate mononuclear cells, each of which has RT of GAPDH (28 cycles) and no RT. Put another, the 14th from the left is the molecular weight ladder marker again, the 15th and 16th from the left indicate the conjunctival epithelium, and indicate the presence and absence of RT of GAPDH (28 cycles), respectively. FIG. 10 shows a HE-stained photograph of skin tissue (auricle) in an IKZF1Tg mouse (Example 5). The left column shows wild type, and the right column shows IKZF1 transgenic. Bars indicate 100 μm (upper) or 50 μm (lower). FIG. 11 shows a HE-stained photograph of skin tissue (back epidermis) in IKZF1Tg mouse (Example 5). The left column shows wild type, and the right column shows IKZF1 transgenic. The upper part shows 50 times and the lower part shows 100 times. Bars indicate 100 μm (upper) or 50 μm (lower). FIG. 12 shows an HE-stained photograph of a mucosal tissue (eyelid) in an IKZF1Tg mouse (Example 5). The left column shows wild type, and the right column shows IKZF1 transgenic. The upper part shows 50 times and the lower part shows 100 times. Bars indicate 100 μm (upper) or 50 μm (lower). FIG. 13 shows an HE-stained photograph of mucosal tissue (tongue) in an IKZF1Tg mouse (Example 5). The left column shows wild type, and the right column shows IKZF1 transgenic. The upper part shows 50 times and the lower part shows 100 times. Bars indicate 100 μm (upper) or 50 μm (lower). FIG. 14 is a photograph showing an expression analysis of IKZF1 protein in human conjunctival tissue (Steven-Jones syndrome (SJS) corneal scarred conjunctival tissue) (Example 6). From the top, HE staining (bars indicate 100 μm), photographs stained with goat anti-IKZF antibody (bars indicate 100 μm), and photographs stained with goat IgG (bars indicate 100 μm) are shown. (Stained with goat anti-IKZF antibody, but not with control goat IgG. FIG. 15 shows a photograph showing the expression analysis of IKZF1 protein in human conjunctival tissue (Steven-Jones syndrome (SJS) corneal scarred conjunctival tissue) (Example 6). From the top, HE staining (bars indicate 100 μm), photographs stained with goat anti-IKZF antibody (bars indicate 100 μm), and photographs stained with goat IgG (bars indicate 100 μm) are shown. It is stained with goat anti-IKZF antibody but not with control goat IgG. FIG. 16 shows a photograph showing the expression analysis of IKZF1 protein in human control conjunctival tissue (conjunctival flaccidity) (Example 6). From the top, HE staining (bars indicate 100 μm), photographs stained with goat anti-IKZF antibody (bars indicate 100 μm), and photographs stained with goat IgG (bars indicate 100 μm) are shown. It is stained with goat anti-IKZF antibody but not with control goat IgG. FIG. 17 shows a photograph showing the expression analysis of IKZF1 protein in human control conjunctival tissue (conjunctival flaccidity) (Example 6). From the top, HE staining (bars indicate 100 μm), photographs stained with goat anti-IKZF antibody (bars indicate 100 μm), and photographs stained with goat IgG (bars indicate 100 μm) are shown. It is stained with goat anti-IKZF antibody but not with control goat IgG.

  Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Thus, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept unless specifically stated otherwise. It should also be understood that the terms used in this specification are used in a meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The present invention is based, at least in part, on the discovery that IKAROS is associated with the epithelium and contributes to exacerbation of inflammatory diseases including conjunctivitis, dermatitis and the like. These findings indicate that IKAROS can be used not only as an inflammatory marker but also as a drug target for inflammatory diseases. That is, known inhibitors of IKAROS are useful for the prevention and / or treatment of inflammatory diseases, and novel IKAROS inhibitors, and thus prevention of inflammatory diseases, using IKAROS proteins and cells / animals expressing the same.・ It is also possible to search for a substance to be a therapeutic drug.

(IKAROS protein and nucleic acid encoding the same)
In the present specification, “IKAROS” (Icarus) is a known protein also called “IKZF1”, and Genbank Accession No. : NP_001207694 (human) <its nucleic acid sequence NM_0012207655.2> or Genbank Accession No. : NP — 001020768 (mouse) <amino acid sequence of human or mouse IKAROS represented by SEQ ID NO: 2 or 4 (the nucleic acid sequence is SEQ ID NO: 1 or 3, respectively), known as its nucleic acid sequence NM — 0010255597.2>, or It is a protein containing an amino acid sequence substantially identical to this. In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation.

As used herein, IKAROS refers to cells of humans and other warm-blooded animals (eg, mice, rats, cows, monkeys, dogs, pigs, sheep, rabbits, guinea pigs, hamsters, chickens, etc.) (eg, neutrophils, etc.). Alternatively, the protein may be isolated and purified from a tissue [for example, blood or the like] by a known protein separation and purification technique. IKAROS is a "zinc finger" DNA binding protein of the Kruppel family, one of such regulatory networks essential for normal lymphocyte production. IKAROS acts as an evolutionarily conserved "master switch" that causes hematopoiesis and governs transcriptional regulation at the earliest stages of ontogenesis and differentiation of lymphocytes (Georgopolous et al., 1994, Cell. Georgopoulous et al., 1992, Science, 258: 808-812; Hahm et al., 1994, Mol. Cell Biol., 14: 7111-12323; Molnar and Georgegopolous, 1994, MoI. Biol., 14: 8292-8303; Wang et al., 1996, Immunity, 5: 537-549; Winandy et al., 1995, Cell, 83. 289-299; Molnar et
al. , 1996, J.A. of Immunol. Sun et al., 156: 585-592; , 1996, EMBO J .; , 15: 5358-5369; Hansen et al. , 1997, Eur. J. Immunol, 27: 3049-3058; Georgepopoulous et al. , 1997, Ann. Rev .. Immunol. , 15: 155-176; Brown et al. , 1997, Cell, 91: 845-854; and Klug et al. , 1998, Proc. Natl. Acad. Sci. ScL USA, 95: 657-662). The programmed expression and function of the IKAROS gene is tightly controlled by alternative splicing of IKAROS pre-mRNA, which produces eight different Ikaros isoforms. The eight Ikaros isoforms (Ik-1, Ik-2, Ik-3, Ik-4, Ik-5, Ik-6, Ik-7, and Ik-8) all share a common carboxy (C) terminus. This C-terminal domain has a transcriptional activation motif and two IKAROS isoforms necessary for homodimer or heterodimer formation and interaction with other proteins. And a zinc finger motif.

"IKAROS" used in the present invention is represented by the amino acid sequence represented by SEQ ID NO: 2 or 4 or an amino acid sequence substantially identical thereto, and includes functional equivalents thereof. (A) to (e) of:
(A) the amino acid sequence represented by SEQ ID NO: 2 or 4;
(B) an amino acid sequence represented by SEQ ID NO: 2 or 4, wherein one or more amino acids are deleted, added, inserted or substituted, and imparts an activity to promote an inflammatory response;
(C) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and imparting an activity to promote an inflammatory response;
(D) an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 1 or 3;
(E) an amino acid sequence which is encoded by a DNA which hybridizes with a DNA having a complementary sequence of the nucleotide sequence of SEQ ID NO: 1 or 3 under stringent conditions, and which imparts an activity to promote an inflammatory reaction.

  Specifically, the human IKAROS protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 is an ortholog amino acid sequence of another mammal, or the human IKAROS protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4; Examples include the amino acid sequence of a splice variant, allelic variant or polymorphic variant of the ortholog.

  Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code. As used herein, “homology” refers to an optimal alignment (preferably, the algorithm employs sequence alignment for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. (Which can take into account the introduction of gaps in one or both)), the percentage (%) of identical and similar amino acid residues to all overlapping amino acid residues. “Similar amino acids” means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), and polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr), small side chain amino acids (Gly, Ala, Ser, Thr, Met), etc. Amino acids classified into groups are included. It is expected that substitutions with such similar amino acids will not change the phenotype of the protein (ie, are conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various documents (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

  In the present specification, the comparison of similarity, identity and homology between an amino acid sequence and a base sequence is calculated using BLAST, a tool for sequence analysis, using default parameters. The search for the identity can be performed using, for example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) 2.2.26 (issued on 2011.10.30). In the present specification, the value of identity usually refers to a value when the above-mentioned BLAST is used and alignment is performed under default conditions. However, if a higher value appears due to a change in the parameter, the highest value is used as the value of the identity. When the identity is evaluated in a plurality of areas, the highest value among them is set as the identity value. Similarity is a numerical value calculated for similar amino acids in addition to identity. When NCBI BLAST is used, for example, the calculation can be performed under the following conditions (expected value = 10; gap is allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993), which is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)). )], Needleman et al. Mol. Biol. , 48: 444-453 (1970) [the algorithm is incorporated in the GAP program in the GCG software package], and the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [ The algorithm is incorporated into the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated in the FASTA program in the GCG software package], and the like can be preferably used as well.

  The stringent condition in the above (e) is, for example, a condition described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC ( Sodium chloride / sodium citrate) / hybridization at 45 ° C., followed by 0.2 × SSC / 0.1% SDS / one or more washes at 50 to 65 ° C. Hybridization conditions that provide the same stringency can be appropriately selected.

  More preferably, the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or 4” is at least about 90%, preferably about 95% Above, more preferably about 96% or more, even more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more identity.

  "A protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or 4" is a protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or 4, It is a protein having substantially the same function as the protein consisting of the amino acid sequence represented by No. 2 or 4.

  The modified amino acid sequence such as IKAROS used in the present invention is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, and particularly preferably 1 to 2. The amino acid may have been inserted, substituted or deleted, or added to one or both termini. The modified amino acid sequence is preferably an amino acid sequence having one or more (preferably one or several or 1, 2, 3, or 4) conservative substitutions in an amino acid sequence such as IKAROS. There may be. Here, “conservative substitution” means that one or more amino acid residues are substituted with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, a case where a certain hydrophobic residue is substituted with another hydrophobic residue, a case where a certain polar residue is substituted with another polar residue having the same charge, and the like can be mentioned. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. As specific examples, non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  Here, “substantially the same function” means that the properties are qualitatively the same, for example, physiologically or pharmacologically, and the degree of the function (eg, about 0.1 to Quantitative factors such as about 10-fold, preferably about 0.5 to about 2-fold) and the molecular weight of the protein may be different. Although the detailed role of IKAROS in vivo is unknown at this time, a protein having an activity to promote an inflammatory response can be regarded as a "protein having substantially the same function".

  Therefore, in the present specification, a “functional equivalent” means any functional substance having the same target function as the target entity, that is, having substantially the same function but having a different structure. A thing. Therefore, when referring to “IKAROS or a functional equivalent thereof” or “a group consisting of IKAROS and a functional equivalent thereof”, a fragment, mutant or variant of IKAROS (for example, a modified amino acid sequence, etc.) besides IKAROS itself. ) Having one or more of the biological functions of IKAROS, as well as those that can be converted to IKAROS or a fragment, mutant or variant of IKAROS at the time of action (eg, IKAROS itself or (Including nucleic acids encoding fragments, variants or variants of IKAROS, and vectors, cells, etc. containing the nucleic acids). “IKAROS or a functional equivalent thereof” or “a group consisting of IKAROS and a functional equivalent thereof” is a representative example of at least one factor selected from the group consisting of IKAROS and a fragment thereof. In the present invention, it is understood that functional equivalents of IKAROS can be used in the same manner as IKAROS, even if not specifically mentioned.

  Here, the activity of promoting the inflammatory response can be measured using, for example, an in vitro or in vivo inflammatory model (for example, the cell or animal model of the present invention) described below.

  As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed depending on the purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically recited herein (eg, 11 and the like) are also suitable as lower limits. obtain. In the case of a polynucleotide, nucleotides of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides can be mentioned. A length represented by a non-integer integer (eg, 11, etc.) may also be appropriate as a lower limit. It is understood herein that if such a laminin chain functions as a factor in its activity, eg, growth promotion or maintenance, the fragment itself is also within the scope of the invention. In accordance with the present invention, the term "activity" refers herein to the function of a molecule in the broadest sense. Activity generally includes, but is not limited to, the biological, biochemical, physical or chemical function of the molecule. The activity may be, for example, to activate, promote, stabilize, inhibit, suppress, or destabilize enzyme activity, the ability to interact with other molecules, and the function of other molecules. , Stability, and the ability to localize to a particular intracellular location. Where applicable, the term also relates to the function of the protein complex in the broadest sense. As used herein, the term "biological function", when referring to a gene or a nucleic acid molecule or polypeptide related thereto, refers to a specific function that the gene, nucleic acid molecule or polypeptide can have in a living body. Examples thereof include, but are not limited to, generation of a specific antibody, enzymatic activity, provision of resistance, and the like. As used herein, a biological function may be exerted by a “biological activity”. As used herein, “biological activity” refers to an activity that a certain factor (eg, a polynucleotide, a protein, etc.) may have in a living body, and exerts various functions (eg, a transcription promoting activity). For example, an activity in which interaction with one molecule activates or inactivates another molecule is also included. When two factors interact, their biological activity is determined by the binding between the two molecules and the resulting biological changes, such as when one molecule is precipitated with an antibody to another. When the molecules also co-precipitate, the two molecules are considered bound. Therefore, looking at such co-precipitation is one of the judgment methods. For example, if a factor is an enzyme, its biological activity includes that enzyme activity. In another example, where an agent is a ligand, the ligand involves binding to the corresponding receptor. Such a biological activity can be measured by techniques well known in the art. Thus, “activity” indicates or reveals binding (either directly or indirectly); affects response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indices, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of upstream or downstream protein or some other amount following, for example, some stimulation or event. A similar measure of function may be mentioned.

  As the IKAROS protein in the present invention, for example, (i) 1 to 30, preferably 1 to 10, more preferably 1 to (5, 4, 3 or 5) in the amino acid sequence represented by SEQ ID NO: 2 or 4 2) an amino acid sequence in which amino acids have been deleted; (ii) 1 to 30, preferably 1 to 10, more preferably 1 to a number (5, 4, (Iii) the amino acid sequence represented by SEQ ID NO: 2 or 4, 1 to 30, preferably 1 to 10, more preferably 1 to a number (5, 4); (Iv) 1 to 30, preferably 1 to 10, more preferably 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 2 or 4; 5, 4, 3 Properly 2) number of amino acid sequence are substituted with other amino acids, or (v) be such as protein comprising the amino acid sequence comprising a combination thereof are included.

  When the amino acid sequence is inserted, deleted, added or substituted as described above, the position of the insertion, deletion, addition or substitution is not particularly limited as long as the protein can promote an inflammatory reaction.

  Here, as a technique for artificially deleting, adding, inserting or substituting amino acids, for example, conventional site-specific mutagenesis is introduced into a DNA encoding the amino acid sequence represented by SEQ ID NO: 2 or 4. And then expressing this DNA by a conventional method. Examples of the site-directed mutagenesis method include a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), and PCR using a mutagenesis primer. Method and the like.

  Preferred examples of IKAROS include, for example, a human protein (Genbank Accession No. NP_443204) consisting of the amino acid sequence represented by SEQ ID NO: 2, or an ortholog thereof in another mammal (eg, a mouse IKAROS protein (SEQ ID NO: 4, Genbank Accession No. NP_084072), allelic variants, polymorphic variants [eg, single nucleotide polymorphisms (SNPs)], and the like.

The “nucleic acid encoding IKAROS” used in the present invention encodes the amino acid sequence represented by SEQ ID NO: 2 or 4 shown in the above (a) to (e) or an amino acid sequence substantially identical thereto. A nucleic acid containing a base sequence Specifically, the following (f) to (j):
(F) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or 4,
(G) in the amino acid sequence represented by SEQ ID NO: 2 or 4, one or more amino acids are deleted, added, inserted or substituted, and a base sequence encoding an amino acid sequence that imparts an activity to promote an inflammatory response;
(H) a nucleotide sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2 or 4, and encoding an amino acid sequence that imparts an activity to promote an inflammatory response;
(I) the base sequence represented by SEQ ID NO: 1 or 3,
(J) a nucleotide sequence that hybridizes under stringent conditions to DNA having a complementary strand sequence to the nucleotide sequence represented by SEQ ID NO: 1 or 3, and encodes an amino acid sequence that imparts an activity to promote an inflammatory response Base sequence,
And nucleic acids having the formula:

  Here, the gene may be any of DNA such as cDNA or genomic DNA, or RNA such as mRNA, and is a concept including both a single-stranded nucleic acid sequence and a double-stranded nucleic acid sequence. Also, in the present specification, the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 are DNA sequences for convenience, but when an RNA sequence such as mRNA is shown, thymine (T) is replaced with uracil (U) To be interpreted as

  Preferred examples of the nucleic acid encoding IKAROS include, for example, human IKAROS cDNA (Genbank Accession No. NM_052972) consisting of the nucleotide sequence represented by SEQ ID NO: 1, or its ortholog in other mammals (eg, mouse IKAROS cDNA ( SEQ ID NO: 3, Genbank Accession No. NM — 029796), allelic variants, polymorphic variants [eg, single nucleotide polymorphisms (SNPs)], and the like.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are interchangeable and used interchangeably herein, and refer to a polymer of amino acids of any length. This polymer may be linear, branched or cyclic. Amino acids may be natural or non-natural, and may be modified amino acids. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is done. The protein of the present invention (for example, IKAROS) can be prepared by incorporating a DNA encoding each chain gene of interest into an appropriate vector and expressing this in either eukaryotic or prokaryotic cells in each host. A desired protein can be obtained by introducing using an expression vector and expressing each chain. Host cells that can be used to express IKAROS are not particularly limited, and prokaryotic host cells such as Escherichia coli and Bacillus subtilis, and true host cells such as yeast, fungi, insect cells, plants and plant cells, and mammalian cells. Nuclear hosts are included. The vector constructed to express the target IKAROS or the like is transformed, transfected, conjugated, protoplast fusion, electroporation, particle gun technique, calcium phosphate precipitation, Agrobacterium method, direct microinjection, etc. Can be introduced into a host cell. The IKAROS or the like can be obtained by growing the cells containing the vector in an appropriate medium to produce the IKAROS or the like used in the present invention and purifying from the cells or the medium. Purification can be performed using size exclusion chromatography, HPLC, ion exchange chromatography, immunoaffinity chromatography, or the like.

  As used herein, "amino acid" may be natural or non-natural as long as it satisfies the purpose of the present invention.

  As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are interchangeable and are used interchangeably herein, and refer to a polymer of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". The term “oligonucleotide derivative” or “polynucleotide derivative” includes oligonucleotides or polynucleotides containing nucleotide derivatives or unusual bonds between nucleotides, and is used interchangeably. Specific examples of such an oligonucleotide include 2′-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is converted to an N3'-P5 'phosphoramidate bond, an oligonucleotide derivative in which ribose and a phosphodiester bond in the oligonucleotide are converted to a peptide nucleic acid bond, and uracil in the oligonucleotide is C- Oligonucleotide derivative substituted with 5-propynyluracil, oligonucleotide derivative substituted with uracil in the oligonucleotide with C-5 thiazoleuracil, cytosine in the oligonucleotide with C-5 propynylcytosine Substituted oligonucleotide derivative, oligonucleotide derivative in which cytosine in oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivative in which ribose in DNA is substituted by 2′-O-propyl ribose And oligonucleotide derivatives in which ribose in the oligonucleotide is substituted with 2'-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof, as well as explicitly stated sequences. Is contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, “nucleotide” may be natural or non-natural.

  As used herein, the term “gene” refers to a factor that defines a genetic trait. Usually they are arranged in a certain order on the chromosome. Genes that define the primary structure of proteins are called structural genes, and genes that control their expression are called regulatory genes. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid”.

(Description of a preferred embodiment)
A description of the preferred embodiment is set forth below, but it should be understood that this embodiment is exemplary of the invention and that the scope of the invention is not limited to such preferred embodiments. It should be understood that those skilled in the art can also easily make modifications, changes and the like within the scope of the present invention by referring to the following preferred embodiments. It should also be understood that any of the embodiments may be combined.

(Anti-inflammatory drug through inhibition of IKAROS (IKZF1))
In one aspect, the present invention provides anti-inflammatory agents, including IKAROS (IKZF1) inhibitors. Before the present invention was disclosed, it was not known that IKAROS was involved in inflammation, and the present inventors found the involvement for the first time, and found that IKAROS inhibitors suppressed or eliminated inflammation. It was confirmed by this.

In one embodiment, the IKAROS inhibitor can include PGE ₂ , anti-IKAROS siRNA, anti-IKAROS antibody, and the like. In certain embodiments, compounds that inhibit the action of polyI: C can be used as IKAROS inhibitors.

In one embodiment, as the compound that inhibits the action of polyI: C, prostaglandin E ₂ (PGE ₂ ), rebamipide <the 62nd Autumn Meeting of the Japanese Society of Allergology (http: //jja.jsaweb) .jp / am / view.php? pubdate = 20121025 & dir = 2012a & number = 12a_on640O64-4); UetaM, Sotozono C, Yokoi N, Kinoshita S: Rebamipide Suppresses PolyI: C-StimulatedCytokine Production in Human Conjunctival Epithelial Cells. J Ocul PharmacolTher. 2013 Sep; 29 (7): 688-93. And WO 2013108644). )>, As well as EP3 agonists and EP2 agonists (for reference, Ueta M, Matsuoka T, Yokoi N, Kinoshita S: Prostaglandin E2 suppresses polyinosine-polycytidylic acid (polyI: C) -stimulated cytokine production via prostaglandin (EP) 2 and 3 in human conjunctival epithelial cells.Br J Ophthalmol. 2011; 95 (6): 859-863 .; and Ueta M, Sotozono C, Yokoi N, Kinoshita S: Rebamipide Suppresses PolyI: C-Stimulated Cytokine Production in Human Conjunctival Epithelial Cells. J Ocul Pharmacol Ther. 2013 Sep; 29 (7): 688-93.). Although not wishing to be bound by theory, it is thought that polyI: C is mediated through TLR3, and may also be mediated through MDA5 or RIG-1, which uses IPS1 as an adapter factor. IPS1 is an adapter factor for MDA5 and RIG-1, and it has been reported that polyI: C-induced cytokines in the conjunctival epithelium are suppressed even in IPS-1KO mice (Ueta M, Kawai T, Yokoi N, Akira S , Kinoshita S: Contribution of IPS-1 to polyI: C-induced cytokine production in conjunctival epithelial cells. Biochem Biophys Res Commun. 2011; 404 (1): 419-423).

  In one embodiment, the anti-inflammatory drug targets epithelial tissue or cells. Previously, epithelial cells were thought to be unrelated to inflammation, but the present inventors have found that this is involved. By controlling the kinetics and activation of immune cells using epithelial cells as targets, local administration is facilitated, and there is a substantial merit that drastic reduction of side effects is expected. Compounds having poor tissue permeability and easily acting on epithelial cells are preferred. The epithelial cells referred to in the present invention refer to “epidermal” covering the body surface and “epithelium (strictly defined)” constituting the mucous membrane of a luminal organ. Although it has only been thought to constitute a structural and functional barrier to foreign antigens, it has become clear that epithelial cells are significantly involved in controlling inflammation. The development of a new inflammation control method targeting epithelial cells that are completely different from existing inflammation control methods can be said to contribute to the realization of a safe and secure society by providing appropriate treatment methods and extending healthy life expectancy.

In one embodiment, the inflammation targeted by the present invention is IKAROS-induced inflammation. Although inflammation is to follow a complex path, the present invention, where IKAROS has been found to be involved in inflammation, TLR3, EP3 routes (prostaglandin E ₂ type 3 receptor) A link with inflammation via the present invention has also been found in the present invention, and inflammation relating to these pathways is specific inflammation and can be preferably used as a subject to be treated by the present invention.

  In one embodiment, the inflammation targeted by the present invention is inflammation due to up-regulation of IKAROS. As used herein, “enhanced expression” refers to expression at a significantly higher level than normal expression.

In another embodiment, the inflammation is an epithelium-related inflammation due to IKAROS. In a more particular embodiment, the inflammation targeted by the present invention is an inflammation involving IKAROS and TLR3 and / or EP3 in the epithelium. Without wishing to be bound by theory, since polyI: C is a ligand for TLR3, the target of the present invention may be, based on the information revealed in the present invention, an inflammation involving IKAROS and TLR3. It can be said that it was not known that such inflammation involving both IKAROS and TLR3 could be targeted. Therefore, PGE ₂ and polyl: only findings existing literature discussing the relationship between the C and the allergy is unpredictable therapeutic agent for enhanced expression diseases of this IKAROS. Further, as far as the inventors know, there is no document reporting the relationship between TLR3 and IKAROS. Therefore, it can be said that the treatment of "IKAROS and TLR3-related inflammation" with the present IKAROS inhibitor cannot be predicted only from the knowledge of the literature relating to "TLR3-related inflammation".

  In certain embodiments, the inflammation targeted by the present invention is due to an allergic disease, typically, Stevens-Johnson syndrome, asthma, contact dermatitis, acute dermatitis, atopic dermatitis, allergic Examples include, but are not limited to, conjunctivitis and atopic keratoconjunctivitis. Epithelial cells constitute a structural and functional barrier to foreign antigens. However, in recent years, it has been reported using mouse models that these epithelial cells are significantly involved in the pathology of inflammatory diseases. Mice in which the NEMO molecule required for NFkB activation is specifically deficient in intestinal epithelium develop inflammatory bowel disease (Nenci A, et al. Nature 2007, 446 (7135): 557-61). In addition, TSLP (Thymic structural lymphopoietin) identified as a lymphocyte growth factor causes atopic dermatitis by being expressed in the epidermis (Yoo J, et al. J Exp Med 2005, 202: 541-9), It causes bronchial asthma by being expressed in the tracheal epithelium (Zhou B, et al. Nat Immunol 2005, 6, 1047-1053). From these, it is understood that, as for the inflammation and inflammatory diseases targeted by the present invention, these epithelium-related diseases can be mentioned as typical examples.

  In another embodiment, the anti-inflammatory agent of the present invention can be administered as a topical agent (eg, topical, ophthalmic, nasal or inhalant). Although not wishing to be bound by theory, it is possible to control the kinetics and activation of immune cells using the target as epithelial cells, thereby facilitating local administration. And, since local administration becomes possible, drastic reduction of side effects is expected. In addition, since local administration targeting epithelial cells requires only a small amount of a necessary dose of a regulatory molecule for expressing the effect, a small amount is expected to be effective. There is also an advantage that it is not necessary to consider absorption distribution metabolic excretion (ADME). This is because it is not necessary to reach the deep tissue. In addition, the design of a control molecule that is stored for a long time on the epithelial cell surface makes it possible to further reduce the dose required for the manifestation of the effect, and further reduce the side effects. According to the present invention, since the epithelial cells are targeted for control, local administration (application, transdermal administration, ophthalmic administration, and nasal administration) of the regulatory compound is possible, and the side effect that is the greatest barrier to drug development is achieved. Achieve objective reduction. Conventional methods for controlling inflammation, which directly suppress and control immune cells, are apt to fall into an immunodeficiency state and often induce infections. However, the method of controlling inflammation through epithelial cells developed in this study suppresses and controls only local immune cells through epithelial cells. Reduction can be expected. The inflammation control technology of the present invention through an inflammation control mechanism by epithelial cells may be a major player in future inflammation treatment, replacing conventional inflammation control technology. Although not wishing to be bound by theory, the present invention aims to create a regulatory molecule suitable for local administration for the purpose of acting on epithelial cells from the beginning, and to produce an activity that produces a local effect by producing a small amount in tissue. Taking into account that it is a tissue hormone receptor with, it is in harmony with the target's natural function in the body, and is a drug for conventional systemic administration (eg, various antibiotics, atropine, steroids, NSAIDS, β1 agonist, β2 agonist, PGE1, salazosulfapyridine, FK506, cyclosporine, VEGF antibody, heparin, adrenaline). On the other hand, even at present, some are developed from the beginning on the premise of local administration because they are difficult to use or cannot be used for the whole body (side effects or effects cannot be expected). Representatives include Botox (blepharospasm, hemifacial spasm, spastic torticollis, strabismus, facial tic, hyperhidrosis, migraine), xalatan, hyaluronic acid preparation (joint injection), blast spray (wound healing), aptamer ( Macugen: age-related maculopathy). Since the present invention is an inflammation control technology targeting epithelial cells, it can be locally administered for the purpose of acting on epithelial cells from the beginning, so that side effects associated with pharmacokinetics and systemic administration do not need to be considered so much. , A wide selection is possible.

  Until now, the target of inflammation control was immune cells, so that the inevitable side effects such as induction of infection by systemically administered drugs, diabetes, gastric ulcer, osteoporosis, cataract and glaucoma have been unavoidable. According to the present invention, since the epithelial cells are targeted for control, local administration (application, transdermal administration, ophthalmic administration, and nasal administration) of the regulatory compound is possible, and the side effect that is the greatest barrier to drug development is achieved. It is thought that it will achieve the target reduction. One of the challenges in the drug discovery process is the difficulty in designing compounds that act as specifically as possible on organs and tissues that are disease sites.However, inflammation control methods targeting epithelial cells are directly applied to the affected area by local administration. It is possible to act on only certain epithelial cells, and has a great advantage that a control molecule can be designed without considering ADME (A: absorption, D: distribution, M: metabolism, E: excretion).

  In one preferred embodiment, the anti-inflammatory agent of the present invention is for treating or preventing inflammation in mucosal tissue.

  In another embodiment, the anti-inflammatory agent of the present invention is for treating or preventing inflammation in the eyes or skin.

  In a further embodiment, an anti-inflammatory agent of the invention is for treating or preventing inflammation in the eye.

  In a further embodiment, the anti-inflammatory agent of the present invention is an external preparation, eye drops, nasal drops or inhalants.

  The present invention provides a prophylactic and / or therapeutic agent for an inflammatory disease, comprising an IKAROS inhibitor, for example, a substance that inhibits the expression or function of IKAROS expression.

  As used herein, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and are defined as long as they can achieve the intended purpose. It can also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) (eg, an “inhibitor” is an agent that “inhibits” an object of interest. ). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (eg, hormones, ligands, signal transducers, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.)) , These complex molecules, but are not limited thereto. As a factor specific to a polynucleotide, typically, a polynucleotide having a certain degree of sequence homology to the sequence of the polynucleotide (for example, a sequence having a complementarity of 70% or more), Examples include, but are not limited to, polypeptides such as transcription factors that bind to the promoter region. As a factor specific to a polypeptide, typically, an antibody specifically directed to the polypeptide, a derivative thereof or an analog thereof (eg, a single-chain antibody), and the polypeptide is a receptor Or, when the ligand is a specific ligand or receptor, or when the polypeptide is an enzyme, its substrate includes, but is not limited to.

(Substance that inhibits IKAROS expression)
In the present invention, “a substance that inhibits the expression of IKAROS” refers to any level of transcription level of nucleic acid encoding IKAROS (IKAROS gene), level of post-transcriptional regulation, level of translation into IKAROS protein, level of post-translational modification, etc. May be used. Therefore, examples of the substance that inhibits the expression of IKAROS include a substance that inhibits the transcription of the IKAROS gene (eg, Antigene), a substance that inhibits the processing of the initial transcript to mRNA, and a substance that inhibits the transport of mRNA to the cytoplasm. Substances that inhibit IKAROS translation from mRNA (eg, antisense nucleic acid, miRNA) or degrade mRNA (eg, siRNA, ribozyme), substances that inhibit post-translational modification of early translation products, etc. . Any substance acting at any stage can be preferably used, and more preferably, a substance selected from the group consisting of the following (1) to (3) is exemplified.
(1) an antisense nucleic acid against a transcript of the IKAROS gene,
(2) a ribozyme nucleic acid for a transcript of the IKAROS gene,
(3) A nucleic acid having RNAi activity against a transcript of the IKAROS gene or a precursor thereof.

  Here, a preferable example of the transcript includes mRNA.

  As a substance that specifically inhibits the translation of mRNA of IKAROS gene into IKAROS (or degrades mRNA), preferably, a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or a part thereof And nucleic acids containing

  The nucleotide sequence substantially complementary to the nucleotide sequence of the mRNA of the IKAROS gene refers to the binding of the mRNA to the target sequence under physiological conditions of an IKAROS-producing cell (eg, neutrophil) in the mammal to be administered. Means a base sequence having a degree of complementarity that can inhibit its translation (or cleave the target sequence). Specifically, for example, a base sequence completely complementary to the base sequence of the mRNA (ie, , A base sequence of the complementary strand of mRNA), and a base sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more homology with respect to the overlapping region.

  "Homology of base sequences" in the present invention is determined by using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap is allowed; filtering = ON; Match score = 1; mismatch score = −3).

More specifically, the base sequence complementary or substantially complementary to the base sequence of the mRNA of the IKAROS gene includes the following (k) or (l):
(K) a base sequence complementary or substantially complementary to the base sequence represented by SEQ ID NO: 1;
(L) a base sequence that hybridizes under stringent conditions with a complementary strand sequence of the base sequence represented by SEQ ID NO: 1 and a sequence that encodes a protein having an activity to promote an inflammatory reaction; Or a substantially complementary base sequence;
Is mentioned. Stringent conditions are described above.

  Preferred examples of the mRNA of the IKAROS gene include human IKAROS mRNA containing the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_052972), or their orthologs in other mammals (eg, mouse IKAROS (SEQ ID NO: 3, Genbank Accession No. NM — 029796)), splice variants, allelic variants, polymorph variants, and the like.

  The nucleotide sequence of the mRNA of the IKAROS gene and the "part of the complementary or substantially complementary nucleotide sequence" are those that can specifically bind to the mRNA of the IKAROS gene and that translate the protein from the mRNA. There are no particular restrictions on the length or position of the target sequence as long as it can inhibit (or degrade the mRNA), but from the viewpoint of sequence specificity, a portion complementary or substantially complementary to the target sequence should have at least 10 bases. As mentioned above, preferably, it contains about 15 bases or more.

Specifically, as a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the IKAROS gene or a part thereof, one of the following (1) to (3) is preferable. Will be:
(1) an antisense nucleic acid against IKAROS gene mRNA;
(2) a ribozyme nucleic acid for the mRNA of the IKAROS gene;
(3) A nucleic acid having RNAi activity against mRNA of IKAROS gene or a precursor thereof.

(1) Antisense nucleic acid against IKAROS gene mRNA The “antisense nucleic acid against IKAROS gene mRNA” in the present invention includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. A nucleic acid having a function of suppressing protein synthesis by forming a specific and stable duplex with and binding to a target mRNA.

  Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is a base such as found in DNA or RNA And nucleotides having a configuration permitting the attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, as well as unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, e.g., those with labels known in the art, capped, methylated, substituted for one or more natural nucleotides with analogs, modified with intramolecular nucleotides Having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nal peptide, poly-L-lysine, etc.), sugars (eg, monosaccharides, etc.) having side-chain groups, interactive compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, , Metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) Is also good. As used herein, “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or the like, or functional groups such as ethers, amines, and the like. It may be converted.

  As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, an RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNaseH and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNaseH, the target sequence may be not only the sequence in the mRNA but also the sequence of the intron region in the early translation product of the IKAROS gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the IKAROS gene using a homology search program such as BLAST or FASTA.

  The target region of the antisense nucleic acid of the present invention is not particularly limited in its length, as long as the hybridization of the antisense nucleic acid results in the inhibition of translation into protein: IKAROS. The entire sequence or partial sequence of the mRNA may be as short as about 10 bases in a short one, or the entire sequence of mRNA or early transcript in a long one. In consideration of the easiness of synthesis, antigenicity, intracellular transportability, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, is preferred, but not limited thereto. Specifically, the 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region of the IKAROS gene A 3'-end palindrome region or a 3'-end hairpin loop may be selected as a preferred target region of an antisense nucleic acid, but is not limited thereto.

  Furthermore, the antisense nucleic acid of the present invention not only hybridizes to the mRNA and early transcript of the IKAROS gene and inhibits translation into proteins, but also binds to these genes, which are double-stranded DNA, to form a triplex ( (Antigene) that can form a triplex) and inhibit transcription into RNA.

The nucleotide molecule constituting the antisense nucleic acid may be a naturally occurring DNA or RNA. However, various nucleotides may be used to improve stability (chemical and / or counterpart enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as a nuclease, a phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate or the like. It can be replaced with a phosphate residue. Further, the hydroxyl group at the 2′-position of the sugar (ribose) of each nucleotide is represented by —OR (R ＝ CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

  The conformation of the sugar moiety of RNA is predominantly C2'-endo (S-type) and C3'-endo (N-type), and single-stranded RNA exists as an equilibrium of both, but double-stranded. Is fixed to N-type. Therefore, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to the N-type by crosslinking 2 ′ oxygen and 4 ′ carbon to impart strong binding ability to the target RNA. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS. Et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides). Nucleotides Nucleic Acids, 22, 1619-1621, 2003) can also be preferably used.

  The antisense oligonucleotide of the present invention determines the target sequence of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the IKAROS gene, and uses a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman). Etc.) to synthesize a sequence complementary thereto. In addition, antisense nucleic acids containing the various modifications described above can be chemically synthesized by a method known per se.

(2) Ribozyme nucleic acid for IKAROS gene mRNA Another preferable example of a nucleic acid containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of IKAROS gene mRNA or a part thereof is, Ribozyme nucleic acids that can be specifically cleaved internally are included. “Ribozyme” in a narrow sense refers to RNA having an enzymatic activity for cleaving nucleic acid, but in the present specification, it is used as a concept including DNA as long as it has a sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and viruses, and hammerhead and hairpin types are known. The hammerhead type exerts enzymatic activity at about 40 bases, and converts several bases at both ends (about 10 bases in total) adjacent to the hammerhead structure into a sequence complementary to a desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the further advantage that it does not attack genomic DNA since it uses only RNA as substrate. When the mRNA of the IKAROS gene takes a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Further, when the ribozyme is used in the form of an expression vector containing a DNA encoding the ribozyme, a hybrid ribozyme in which a sequence modified from tRNA is further linked in order to promote the transfer of a transcript to the cytoplasm. [Nucleic Acids Res. , 29 (13): 2780-2788 (2001)].

(3) siRNA against mRNA of IKAROS gene
In the present specification, a double-stranded RNA consisting of an oligo RNA complementary to the mRNA of the IKAROS gene and a complementary strand thereof, so-called siRNA, is also complementary or substantially complementary to the base sequence of the mRNA of the IKAROS gene. It is defined as being included in a nucleic acid containing a base sequence or a part thereof. When a short double-stranded RNA is introduced into a cell, mRNA complementary to the RNA is degraded, a phenomenon called so-called RNA interference (RNAi), which has long been known in nematodes, insects, plants, and the like. Since this phenomenon was confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative technique to the above antisense nucleic acids and ribozymes.

  siRNA can be prepared based on the cDNA sequence information of the target gene, for example, based on Elbashir et al. (Genes Dev., 15, 188-200 (2001)), Teramoto et al. (FEBS Lett. 579 (13): p2878-82 (2005)). Can be designed according to the rules proposed by The siRNA target sequence has in principle a length of 15 to 50 bases, preferably 19 to 49 bases, more preferably 19 to 27 bases, for example, AA + (N) 19 (AA followed by 19 Base sequence), AA + (N) 21 (base sequence of 21 bases following AA) or A + (N) 21 (base sequence of 21 bases following A).

  The nucleic acid of the present invention may have an additional base at the 5 'or 3' end. The length of the additional base is usually about 2 to 4 bases, and the total length of the siRNA is 19 bases or more. The additional base may be DNA or RNA, but use of DNA may improve the stability of the nucleic acid in some cases. Such additional base sequences include, for example, ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', tttt-3. ', Uuuuu-3' and the like, but are not limited thereto.

  In addition, the siRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents a deoxythymidine residue of deoxyribonucleic acid) is added. . In addition, blunt ends (blunt ends) without terminal addition may be used.

  In the siRNA, the sense strand and the antisense strand may have different base numbers. For example, “aiRNA” in which the antisense strand has a protruding portion sequence (overhang) at the 3 ′ end and the 5 ′ end. Can be mentioned. A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand (Sun, X., et al., Nature Biotechnology Vol 26). No. 12 p1379, International Publication No. WO2009 / 029688 pamphlet).

  Although the position of the target sequence is not particularly limited, it is desirable to select the target sequence from the 5'-UTR and about 50 bases from the initiation codon, and from a region other than the 3'-UTR. With respect to the target sequence candidate group selected based on the above rules and the like, whether or not there is homology in the 16-17 base continuous sequence in the mRNA other than the target is determined by BLAST (http: //www.ncbi.nlm). Nih.gov/BLAST/) or other homology search software to confirm the specificity of the selected target sequence. For the target sequence for which specificity has been confirmed, a sense strand having a TT or UU 3 ′ terminal overhang at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 base and TT or A double-stranded RNA consisting of an antisense strand having a 3 'overhang of UU may be designed as siRNA. In addition, for a short hairpin RNA (shRNA) which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5 to 25 bases) capable of forming a loop structure is appropriately selected, and the above sense strand and antisense strand are combined with each other. It can be designed by linking via a linker sequence.

  The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Such sites include, for example, siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer (registered trademark) Expression Vector design tool insert tool (insertion tool) http://www.ambion.com/jp/techlib/misc/silencer_converter.html), GeneSeer provided by RNAi Codex (http://codex.chl.edu/scripts/newsearch. Not done.

  The ribonucleoside molecule constituting the siRNA may also be modified in the same manner as in the case of the antisense nucleic acid in order to improve stability, specific activity and the like. However, in the case of siRNA, if all the ribonucleoside molecules in the natural RNA are replaced with the modified form, the RNAi activity may be lost, so it is necessary to introduce the minimum modified nucleoside capable of functioning the RISC complex. .

Specifically, in order to improve the stability (chemical and / or counter-enzymatic) or specific activity (affinity with RNA) of a part of the nucleotide molecule constituting the siRNA as the modification, Can be replaced with RNAs subjected to various chemical modifications (Usman and Cedergren, 1992, TIBS 17, 34; see Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting the siRNA is replaced with a chemically modified phosphate such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. Can be substituted for a residue. Further, the hydroxyl group at the 2′-position of the sugar (ribose) of each nucleotide is represented by —OR (R ＝ CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH _{2 CH 2 NHC (NH) NH 2} , CH 2 CONHCH 3, CH 2 CH 2 CN , etc.), may be replaced by fluorine atom (-F). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned. In addition, the method for modifying an antisense nucleic acid described in the above (1) can be used. Alternatively, chemical modification (2′-deoxylation, 2′-H) for substituting a part of RNA in siRNA for DNA may be performed. Alternatively, an artificial nucleic acid (LNA: Locked Nucleic Acid) in which the 2′- and 4′-positions of sugar (ribose) are cross-linked with —O—CH ₂ — and the conformation is fixed to N-type may be used.

  In addition, the sense strand and the antisense strand that constitute the siRNA are connected to a ligand, a peptide, a sugar chain, an antibody, a lipid, a positive charge or a molecular structure that specifically recognizes a receptor present on the cell surface via a linker. It may be chemically bonded to oligoarginine, a Tat peptide, a Rev peptide, an Ant peptide, or the like that adsorbs and penetrates the surface.

  The siRNA is obtained by synthesizing the sense strand and the antisense strand of the target sequence on the mRNA using a DNA / RNA automatic synthesizer and denaturing the same in a suitable annealing buffer at about 90 to about 95 ° C. for about 1 minute. It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing a short hairpin RNA (shRNA) that is a precursor of siRNA and cleaving it using a dicer.

  In the present specification, a nucleic acid designed to be capable of producing an siRNA for the mRNA of the IKAROS gene in a living body is also referred to as a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the mRNA of the IKAROS gene or one of them. Is defined as being included in the nucleic acid containing the moiety. Examples of such a nucleic acid include an expression vector constructed to express the above-described shRNA and siRNA. An shRNA is an oligo containing a base sequence in which the sense strand and the antisense strand of the target sequence on the mRNA are linked by inserting a spacer sequence having a length (for example, about 5 to 25 bases) capable of forming an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with a DNA / RNA automatic synthesizer. Vectors for expressing shRNA include a tandem type and a stem loop (hairpin) type. The former is a tandem linkage of the expression cassette of the sense strand of the siRNA and the expression cassette of the antisense strand, and forms a double-stranded siRNA (dsRNA) by expressing and annealing each strand in the cell. It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in a cell and processed by a dicer to form dsRNA. As a promoter, a polII promoter (for example, a CMV immediate-early promoter) can be used, but a polIII promoter is generally used in order to accurately perform transcription of short RNA. Pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase PRNA promoter, human valine-tRNA promoter and the like. In addition, a sequence in which four or more Ts are continuous is used as a transcription termination signal.

  The thus constructed siRNA or shRNA expression cassette is then inserted into a plasmid vector or a viral vector. As such a vector, a viral vector such as a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes virus, a Sendai virus, and an animal cell expression plasmid are used.

  The siRNA can be obtained, for example, from 394 Applied Biosystems, Inc. based on nucleotide sequence information. It can be chemically synthesized according to a conventional method using a DNA / RNA automatic synthesizer such as a synthesizer. See, for example, Caruthers et al. , 1992, Methods in Enzymology 211, 3-19, Thompson et al. , WO 99/54459, Wincott et al. , 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al. , 1997, Methods Mol. Bio. , 74, 59, Brennan et al. , 1998, Biotechnol Bioeng. , 61, 33-45, Usman et al. , 1987 J .; Am. Chem. Soc. , 109, 7845, Scaninge et al. , 1990 Nucleic Acids Res. , 18, 5433, and U.S. Patent No. 6,001,311. Specifically, it can be synthesized using a nucleic acid protecting group (for example, a dimethoxytrityl group at the 5 'end) and a coupling group (for example, a phosphoramidite at the 3' end) known to those skilled in the art. That is, the protecting group at the 5 'end is deprotected with an acid such as TCA (trichloroacetic acid), and the coupling reaction is performed. Next, after capping with an acetyl group, the following condensation reaction of nucleic acids is performed. In the case of siRNA containing modified RNA or DNA, modified RNA (for example, 2′-O-methyl nucleotide, 2′-deoxy-2′-fluoro nucleotide) may be used as a raw material, and a coupling reaction may be performed. Can be adjusted as appropriate. In the case of introducing a phosphorothioate bond in which the phosphoric acid binding portion is modified, a voage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide) can be used.

  Alternatively, the oligonucleotides may be synthesized separately and linked together after synthesis, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al. International Publication WO 93/23569; Shabarova et al. 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al. And / or by hybridization after deprotection. It may be connected to a cord. siRNA molecules can also be synthesized by tandem synthesis. That is, both siRNA strands are synthesized as a single continuous oligonucleotide separated by a cleavable linker, which is then cleaved to produce separate siRNA fragments, which are hybridized and purified. . The linker may be a polynucleotide linker or a non-nucleotide linker.

  The synthesized siRNA molecules can be purified using methods known to those skilled in the art. For example, a method of purifying by gel electrophoresis or a method of purifying using high performance liquid chromatography (HPLC) may be mentioned.

  Another preferred example of a nucleic acid containing a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the IKAROS gene or a part thereof includes a microRNA (miRNA) targeting the mRNA. miRNA can also be prepared according to the method described above for siRNA.

  A nucleic acid containing a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the IKAROS gene or a part thereof is provided in a special form such as a liposome or a microsphere, or other molecules are added thereto. It can be provided in a form. Examples of additional forms that can be used include polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton, and lipids that enhance the interaction with cell membranes or increase the uptake of nucleic acids. (Eg, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside linkage. Other groups include capping groups specifically located at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  The IKAROS expression inhibitory activity of these nucleic acids can be examined using a transformant into which a nucleic acid encoding IKAROS has been introduced, an IKAROS gene expression system in vivo or in vitro, or a translation system of IKAROS protein in vivo or in vitro. Can be.

  The substance that inhibits the expression of IKAROS in the present invention is not limited to a nucleic acid containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the mRNA of the IKAROS gene as described above, or a nucleic acid containing a part thereof. Other substances such as low molecular weight compounds may be used as long as they directly or indirectly inhibit. Such a substance can be obtained, for example, by the screening method of the present invention described below.

(Substance that inhibits the function of IKAROS)
In the present invention, "a substance that inhibits the function of IKAROS" may be any substance as long as it inhibits the function of IKAROS once produced functionally.

Specifically, examples of the substance that suppresses the function of IKAROS include an antibody against IKAROS. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a method for producing an antibody or antiserum known per se. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (Ab ') Fragments such as ₂ , scFv, scFv-Fc, minibodies, diabodies and other genetically engineered conjugate molecules, or molecules having a protein stabilizing action such as polyethylene glycol (PEG) Modified derivatives thereof may be used.

  In a preferred embodiment, an antibody against IKAROS is used as a pharmaceutical for human administration, and therefore, the antibody (preferably a monoclonal antibody) is an antibody having a reduced risk of showing antigenicity when administered to humans. Specific examples include fully human antibodies, humanized antibodies, mouse-human chimeric antibodies and the like, and particularly preferred are fully human antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. Although fully human antibodies can be produced from human-human (or mouse) hybridomas, human antibody-producing mice and phage display methods must be used to stably provide a large amount of antibodies at low cost. It is desirable to manufacture using.

  Another preferred substance that inhibits the function of IKAROS is a low molecular weight compound suitable for Lipinski's Rule. Such a compound can be obtained, for example, using the screening method of the present invention described below.

  Since a substance that inhibits the expression or function of IKAROS exhibits an activity of suppressing an inflammatory response promoted by IKAROS, it is useful as an anti-inflammatory agent for the prevention and / or treatment of inflammatory diseases such as inflammatory bowel disease. is there.

  Therefore, a medicament containing a substance that inhibits the expression or function of IKAROS can be used as an agent for preventing and / or treating an inflammatory disease (anti-inflammatory agent).

(Drug containing antisense nucleic acid, ribozyme nucleic acid, siRNA and its precursor)
The antisense nucleic acid of the present invention, which can complementarily bind to a transcript of the IKAROS gene and inhibit protein translation from the transcript, and a homologous (or complementary) to a transcript (mRNA) of the IKAROS gene ) An siRNA (or ribozyme) having a base sequence and capable of cleaving the transcript by targeting the transcript, and a shRNA that is a precursor of the siRNA (hereinafter, collectively referred to as “nucleic acid of the present invention” ) Suppresses the expression of IKAROS in vivo and suppresses the inflammatory response, and thus can be used as an anti-inflammatory agent.

  Pharmaceuticals containing the nucleic acid of the present invention have low toxicity, and can be used directly as a liquid or as a pharmaceutical composition of an appropriate dosage form in a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, Can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.) to dogs, monkeys, etc.).

  When the nucleic acid of the present invention is used as the above-mentioned anti-inflammatory agent, it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention is inserted alone or in an operable manner into an appropriate mammalian cell expression vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. can do. The nucleic acid can be administered as it is or together with an auxiliary for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and administered locally into the trachea as an inhalant.

  Further, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated alone or together with a carrier such as a liposome (injection) and administered intravenously, subcutaneously, or the like. .

  The nucleic acids of the invention may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

  As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. May be included. Such an injection can be prepared according to a known method. The injection can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository for rectal administration may be prepared by mixing the above nucleic acid with a usual suppository base.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained, for example, usually in an amount of about 5 to about 500 mg per dosage unit dosage form, particularly about 5 to about 100 mg for injection, and about 10 to about 250 mg for other dosage forms.

  The dose of the above-mentioned medicine containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptoms, administration route and the like. For example, for example, inflammatory bowel disease such as ulcerative colitis (UC) and Crohn's disease (CD) When used for the treatment / prevention of disease (IBD), the nucleic acid of the present invention is usually administered in a single dose of about 0.01 to about 20 mg / kg body weight, preferably about 0.1 to about 10 mg / kg. It is convenient to administer about kg body weight, more preferably about 0.1 to about 5 mg / kg body weight, by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

  Each of the above-described compositions may appropriately contain other active ingredients as long as the composition does not cause an undesirable interaction with the nucleic acid of the present invention.

(Antibodies against IKAROS, pharmaceuticals containing low molecular weight compounds that inhibit the expression or function of IKAROS)
An antibody against IKAROS or a low-molecular compound that inhibits the expression or function of IKAROS can inhibit the production or function of IKAROS. Therefore, since these substances inhibit the expression or function of IKAROS in a living body, they can be used as prophylactic and / or therapeutic agents for inflammatory diseases.

  Pharmaceuticals containing the above-mentioned antibodies and low molecular weight compounds have low toxicity, and can be used as they are as liquids or as pharmaceutical compositions in appropriate dosage forms in humans or mammals (eg, rats, rabbits, sheep, pigs, cattle, cats). , Dogs, monkeys, etc.) can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.).

  The above-mentioned antibody or low molecular compound may be administered as it is, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the above-mentioned antibody or low-molecular compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

  As compositions for parenteral administration, for example, injections, suppositories and the like are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. May be included. Such an injection can be prepared according to a known method. The injection can be prepared, for example, by dissolving, suspending or emulsifying the antibody or low-molecular compound or its salt of the present invention in a sterile aqueous liquid or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) add of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a conventional suppository base.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The antibody or low-molecular compound is usually contained in an amount of about 5 to about 500 mg, preferably about 5 to about 100 mg for an injection, and about 10 to about 250 mg for other dosage forms.

  The dose of the above-mentioned medicine containing the above-mentioned antibody or low-molecular compound or a salt thereof varies depending on the administration subject, target disease, symptoms, administration route, etc., for example, when used for the treatment or prevention of IBD. Is usually about 0.01 to about 20 mg / kg of body weight, preferably about 0.1 to about 10 mg / kg of body weight, more preferably about 0.1 to about 5 mg of antibody or low molecular weight compound as a single dose. It is convenient to administer intravenously about 1 to 5 times a day, preferably about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

  Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody or low-molecular compound.

  Pharmaceutical compositions containing the above-mentioned antisense nucleic acid, ribozyme nucleic acid, siRNA and its precursor against IKAROS, and pharmaceutical compositions containing an antibody against IKAROS, a low-molecular compound that suppresses the expression or function of IKAROS, and the like are used as anti-inflammatory agents. For the treatment, prevention, or prevention of progress of inflammatory diseases. Specific examples of inflammatory diseases include inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD), and inflammatory disease such as rheumatoid arthritis (RA), Behcet's disease, and Castleman's disease. Any inflammation in which IKAROS is involved in exacerbating its pathology, including but not limited to autoimmune diseases, is included in the inflammatory diseases targeted by the present invention.

  Treatment of an inflammatory disease with a pharmaceutical composition containing the above-mentioned antisense nucleic acid, ribozyme nucleic acid, siRNA and its precursor against IKAROS, and a pharmaceutical composition containing an antibody against IKAROS, a low molecular compound that suppresses the expression or function of IKAROS, etc. When used for prophylaxis, it may be used alone or in combination with one or more drugs having an anti-inflammatory effect.

  Examples of the concomitant drug include, but are not particularly limited to, mesalazine, corticosteroids (eg, betamethasone, prednisolone, hydrocortisone, dexamethasone, etc.), nonsteroidal anti-inflammatory drugs (NSAIDs; eg, salicylic acid, anthranilic acid, etc.) System, aryl acid system, propionic acid system, oxicam system, pilin system), anti-TNFα antibody (influximab, adalimumab), antirheumatic drug (eg, immunomodulator such as actarit, immunosuppressant such as methotrexate, anti-TNFα antibody) And biological preparations such as etanercept).

(Screening of drug candidate compounds or anti-inflammatory agents for diseases such as inflammation)
As described above, inhibiting the expression and / or function of IKAROS suppresses the inflammatory response. Therefore, compounds that inhibit the expression and / or function of IKAROS can be used as prophylactic and / or therapeutic agents (anti-inflammatory agents) for inflammatory diseases.

  Therefore, cells that produce or have increased expression of IKAROS can be used as a tool for screening for anti-inflammatory substances by using the expression level and / or function of IKAROS (or IKAROS gene) as an index. Can be.

  When screening for a compound that inhibits the expression or function of IKAROS, the screening method comprises culturing cells capable of producing IKAROS in the presence and absence of a test substance, and expressing the IKAROS under both conditions. Including comparing the amount or degree of function. Compounds that inhibit the function of IKAROS can also be screened by testing the ability to bind to the purified IKAROS protein and the activity of inhibiting the binding between the IKAROS protein and its binding protein (eg, an antibody to IKAROS). it can.

  Therefore, in one aspect, the present invention provides the following steps (1) to (3): (1) a cell capable of producing IKAROS, for example, under the control of an IKAROS gene or a transcription regulatory region of the gene. Contacting a cell containing a nucleic acid encoding a reporter protein with a test substance; (2) measuring the expression level of an IKAROS gene or an IKAROS protein or a reporter protein in the cell; Screening for an anti-inflammatory substance comprising a step of selecting a test substance having a reduced expression level of an IKAROS gene or an IKAROS protein or a reporter protein as a candidate for an anti-inflammatory substance, as compared to the case where the measurement is carried out in the presence of Provide a way.

  The cells having the ability to produce IKAROS used in the above-mentioned screening method may be human or other mammalian cells that naturally express them or a biological sample containing the same (eg, blood, tissue, organ, etc.). There are no particular restrictions. In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance may be administered to the living body itself, and after a certain period of time, those biological samples may be isolated. You may.

  Examples of the cells having the ability to produce IKAROS include various transformants prepared by a known and commonly used genetic engineering technique. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells, and CHO cells are preferably used.

  Specifically, it hybridizes under stringent conditions to DNA encoding IKAROS (that is, the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence having complementarity to the nucleotide sequence; DNA containing a nucleotide sequence encoding a polypeptide having the same function as the protein consisting of the amino acid sequence represented) is prepared by ligating downstream of a promoter in an appropriate expression vector and introducing into a host animal cell. be able to.

  The method for preparing the gene encoding IKAROS will be described below.

  The gene encoding IKAROS can be obtained by a conventional genetic engineering method (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory (Cold) Spring Harbor Laboratory Press) or the like. That is, the DNA encoding IKAROS is, for example, a cDNA or cDNA derived from a cell or tissue that produces the aforementioned IKAROS by synthesizing an appropriate oligonucleotide as a probe or primer based on the nucleotide sequence represented by SEQ ID NO: 1. Cloning can be performed from the library using a hybridization method or a PCR method. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd edition (above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

The DNA base sequence can be determined using known kits, for example, Mutan ^™ -Super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped Duplex method, The conversion can be carried out according to a method known per se such as the Kunkel method or a method analogous thereto.

  The cloned DNA can be used as it is depending on the purpose, or after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can be added using a suitable synthetic DNA adapter.

  Next, using the obtained IKAROS gene, an IKAROS protein can be produced and obtained according to a conventional genetic engineering method.

  For example, a culture can be obtained by preparing a plasmid capable of expressing the IKAROS gene in a host cell, introducing the plasmid into the host cell, transforming the plasmid, and then culturing the transformed host cell (transformant). What is necessary is just to acquire IKAROS from a thing. Examples of the plasmid include a plasmid that contains genetic information that can be replicated in a host cell and can replicate autonomously, is easily isolated and purified from the host cell, and can function in a host cell. And a gene in which a gene encoding IKAROS is introduced into an expression vector having a detectable marker. Various types of expression vectors are commercially available.

  For example, an expression vector used for expression in Escherichia coli is an expression vector containing a promoter such as lac, trp, or tac, and these are commercially available from Pharmacia, Takara Bio, or the like. Restriction enzymes used to introduce a gene encoding IKAROS into the expression vector are also commercially available from Takara Bio Inc. If it is necessary to direct even higher expression, a ribosome binding region may be ligated upstream of the DNA encoding IKAROS. As the ribosome binding region to be used, Guarante L. et al. (Cell 20, p543) and those described in reports by Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).

  Also, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. It can also be used. Any promoter may be used as long as it is appropriate for the host used for gene expression. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, β-actin A gene promoter, aP2 gene promoter and the like are used. Among them, EF-α promoter, CAG promoter, CMV promoter, SRα promoter and the like are preferable.

  As the expression vector, in addition to the above, those containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori) and the like, if desired, may be used. it can.

As the selection marker include dihydrofolate reductase gene (hereinafter sometimes abbreviated as dhfr, methotrexate (MTX) resistance), the ampicillin resistance gene (hereinafter sometimes abbreviated as # 038 ^r), neomycin resistance gene ( Hereinafter, G418 resistance, which may be abbreviated as neo ^r , may be used. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a thymidine-free medium.

  By transforming a host with an expression vector containing the DNA encoding IKAROS described above, an IKAROS-expressing cell can be produced.

Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, mammalian cells, and the like. As mammalian cells, for example, HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells ( Hereinafter, CHO (dhfr ⁻ ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells, and the like can be used. For example, E. coli and the like can be preferably mentioned from the viewpoint of facilitating large-scale preparation of IKAROS.

  The plasmid obtained as described above can be introduced into the host cell by a conventional genetic engineering method. The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, the cultivation is performed in a medium containing an appropriate carbon source, a nitrogen source, and trace nutrients such as vitamins as appropriate. The culture method may be any of solid culture and liquid culture, and preferably includes liquid culture such as aeration and stirring culture.

  Transformation can be performed by a calcium phosphate coprecipitation method, a PEG method, an electroporation method, a microinjection method, a lipofection method, or the like. For example, the method described in Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973) can be used.

  The transformed cells obtained as described above, the mammalian cells having the ability to naturally produce IKAROS, or the tissues and organs containing the cells are, for example, a minimum essential medium (MEM containing about 5 to 20% fetal calf serum). ) [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 1967, 199 (519)]. )], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH of the medium is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C., and aeration and / or agitation are added as necessary.

  The IKAROS protein may be obtained by combining methods generally used for isolation and purification of general proteins. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and IKAROS may be purified from the culture supernatant in the same manner as described above. When the IKAROS protein accumulates in the cells of the transformant obtained by the above culture, for example, the transformant is collected by centrifugation or the like, and then the cells are disrupted or lysed. For example, the protein may be solubilized and purified by using various chromatography steps such as ion exchange, hydrophobicity, and gel filtration alone or in combination. An operation of restoring the higher-order structure of the purified protein may be further performed.

  In carrying out the screening of the present invention, examples of the test substance include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. May be a novel substance or a known substance.

  When selecting a substance that decreases the expression level of IKAROS or the IKAROS gene, or a substance that reduces the function of IKAROS, control cells that are not contacted with a test substance can also be used as a control. Here, “does not come into contact with the test substance” means that the same amount of solvent (blank) as the test substance is added instead of the test substance, or that the expression level of IKAROS or IKAROS gene or the function of IKAROS is affected. The case where a negative control substance not given is added is also included.

  The contact of the test substance with the cells can be performed, for example, by the above-described medium or various buffers (eg, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetate buffer). Buffer, etc.) and incubating the cells for a certain period of time. The concentration of the test substance to be added varies depending on the type (solubility, toxicity, etc.) of the compound, but is appropriately selected, for example, from about 0.1 nM to about 100 μM. The incubation time includes, for example, about 10 minutes to about 24 hours.

  When the cells producing IKAROS are provided in the form of a non-human mammal individual, the condition of the animal individual is not particularly limited. For example, an animal model of an inflammatory disease in which inflammation is induced by a drug or genetic modification (for example, IBD such as mice in which colitis is induced by drugs such as dextran sodium sulfate (DSS) and 2.4.6-trinitrobenzenesulfonic acid (TNBS), and genetically modified mice such as IL-10 knockout mice and IL-2 knockout mice Model animals, collagen-induced arthritis (CIA) mice, SKG mice, PD-1 knockout mice, K / BxN mice, RA model animals such as Synoviolin Tg mice, etc.). The breeding conditions of the animals used are not particularly limited, but it is preferable that the animals be bred in an environment of SPF grade or higher. The contact of the test substance with the cells is performed by administering the test substance to the animal individual. The administration route is not particularly limited, and examples include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. Although the dose is not particularly limited, for example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days. .

  Alternatively, the above screening method can also be performed by contacting a test substance with an extract of the cells or IKAROS isolated and purified from the cells, instead of the cells having the ability to produce IKAROS.

  Therefore, in another aspect, the present invention provides an anti-inflammatory substance comprising contacting IKAROS with a test substance, and selecting a test substance having an ability to bind to IKAROS as a candidate for the anti-inflammatory substance. A screening method is provided. IKAROS that can be used can be isolated or purified from an extract of a cell capable of producing IKAROS, or from the cell.

  In one embodiment, in the screening method of the present invention, the following steps (1) to (3) are performed: (1) IKAROS is brought into contact with a cell membrane fraction of target cells of an inflammatory disease in the presence of a test substance. (2) measuring the amount of IKAROS bound to the cell membrane fraction; and (3) reducing the amount of IKAROS bound to the cell membrane fraction as compared to the case where the measurement is performed in the absence of the test substance. Selecting a test substance as a candidate for an anti-inflammatory substance.

  In certain embodiments, the present invention utilizes a unique evaluation system for membrane permeability and tissue permeability using stratified conjunctival epithelial cells. In this evaluation system, compounds that selectively act on epithelial cells can be selected.

  In a further embodiment, the present invention can use ocular surface inflammation as a system for assessing inflammation. The ocular surface is a mucosal tissue that can be easily observed, and has the advantage that inflammation evaluation is relatively easy in macroscopic and histological analysis. Further, ocular surface inflammation is generally treated by topical administration by eye drops, and the ocular surface inflammation model is superior in evaluating the inflammation-suppressing action of the novel compound. In addition, in the present invention, a mouse model of allergic conjunctivitis that is excellent in evaluating the effect of suppressing inflammation can be used. As such an inflammation model, those exemplified in the examples can be used, but not limited thereto.

  In one embodiment, the method of the present invention further comprises applying the test substance selected as a candidate for the anti-inflammatory substance to an inflammatory model, and assaying whether or not to suppress an inflammatory response in the model. Including.

(Measurement of IKAROS gene or IKAROS expression level)
The present invention provides a method for screening an anti-inflammatory substance, which comprises comparing the expression of the protein (gene) in a cell capable of producing IKAROS in the presence and absence of a test substance. I do. The cells used in this method, the type of the test substance, the mode of contact between the test substance and the cells, and the like are the same as described above.

  The expression level of IKAROS is determined to be stringent with the nucleic acid capable of hybridizing with the above-described DNA encoding IKAROS under stringent conditions, that is, the nucleotide sequence represented by SEQ ID NO: 1 or 3 or a nucleotide sequence complementary thereto. It can be measured at the RNA level by detecting the mRNA of the IKAROS gene using a nucleic acid (DNA) that can hybridize under the conditions (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”). Alternatively, the expression level can also be measured at the protein level by detecting these proteins using an antibody against IKAROS described above (hereinafter sometimes referred to as “the detection antibody of the present invention”).

Therefore, more specifically, the present invention provides:
(A) Cells having the ability to produce IKAROS are cultured in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions is measured using the nucleic acid for detection of the present invention. And (b) culturing cells having the ability to produce IKAROS in the presence and absence of a test substance, and comparing the protein under both conditions. And a method for screening an anti-inflammatory substance, characterized by measuring and comparing the amount of the anti-inflammatory substance using the detection antibody of the present invention.

That is, screening of a substance that changes the expression level of IKAROS can be performed as follows.
(I) Test on normal or disease models (eg, DSS or TNBS-induced colitis model) and non-human mammals (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) After a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) after administration of the substance, blood or a specific organ (eg, brain) Etc.) or a tissue or cell isolated from an organ.

IKAROS mRNA can be quantified by extracting mRNA from cells or the like by a usual method, or can be quantified by Northern blot analysis known per se. On the other hand, the amount of IKAROS protein can be quantified by Western blot analysis or various immunoassay methods described in detail below.
(Ii) A cell expressing the IKAROS gene (for example, a transformant into which IKAROS has been introduced) is prepared according to the above-described method. After the incubation (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 days to 3 days), IKAROS contained in the cell culture or mRNA encoding the same was purified as described in (i) above. And can be quantified and analyzed.

  Detection and quantification of the expression level of the IKAROS gene (mRNA) can be carried out using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom by a known method such as Northern blotting or RT-PCR. Specifically, by using a polynucleotide having at least 15 contiguous bases in the base sequence of the IKAROS gene and / or a polynucleotide complementary thereto as a primer or a probe, the presence or absence of the expression of the IKAROS gene in RNA and the expression thereof Level can be detected and measured. Such a probe or primer can be designed based on the base sequence of the IKAROS gene using, for example, primer 3 (http://primer3.sourceforge.net/) or vector NTI (manufactured by Infomax). .

When the Northern blot method is used, the above-mentioned primer or probe is labeled with a radioisotope (such as ³² P, ³³ P: RI) or a fluorescent substance, and the resulting RNA is transferred to a nylon membrane or the like according to a conventional method. After the hybridization with the primer or probe (DNA or RNA) and the formed double strand of RNA as a signal derived from a label (RI or fluorescent substance) of the primer or probe, a radiation detector ( BAS-1800II, manufactured by Fuji Film Co., Ltd.) or a method of detecting and measuring with a fluorescence detector. Further, using AlkPhos Direct Labeling and Detection System (Amersham PharmaciaBiotech), the probe is labeled according to the protocol, and the probe is hybridized with RNA derived from cells. A method of detecting and measuring with Major STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.

  When the RT-PCR method is used, a pair of cDNAs prepared based on the sequence of the IKAROS gene are prepared by preparing a cDNA from the cell-derived RNA according to a conventional method and using the cDNA as a template to amplify the target IKAROS gene region. A method of hybridizing a primer (a positive strand binding to the above-mentioned cDNA (-strand) and a reverse strand binding to the + strand) and performing a PCR method according to a conventional method to detect the amplified double-stranded DNA obtained Can be exemplified. The amplified double-stranded DNA was detected by a method for detecting labeled double-stranded DNA produced by performing the PCR using primers previously labeled with RI or a fluorescent substance. A method in which double-stranded DNA is transferred to a nylon membrane or the like in accordance with a conventional method, and the above-mentioned labeled primer is used as a probe, hybridized with the probe, and detected can be used. The generated labeled double-stranded DNA product can be measured with an Agilent 2100 bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and ABI PRIME 7900 Sequence Detection System (Applied Biosystem) is used to detect the reaction. You can also.

  The expression of the IKAROS gene in cells to which the test substance has been added is 2/3 times or less, preferably 1/2 times or less, and more preferably 1/3 times, as compared with the expression level in control cells to which no test substance is added. In the following cases, the test substance can be selected as an IKAROS gene expression inhibitor.

  Screening for a substance that changes the expression level of IKAROS can also be performed by a reporter gene assay using a transcriptional regulatory region of the IKAROS gene. Here, the “transcription regulatory region” generally refers to a range of several kb to several tens of kb upstream of the chromosomal gene, and includes, for example, (i) 5′-race method (5′-RACE method) (for example, 5′-RACE method). A step of determining the 5 'end by a conventional method such as' -full Race Core Kit (manufactured by Takara Bio Inc.), oligocap method, S1 primer mapping, etc .; (ii) Genome Walker Kit ( A 5 ″ -upstream region is obtained using Clonetech Co., Ltd.), and the obtained upstream region can be identified by a method including a step of measuring promoter activity.

  A reporter protein expression vector is constructed by ligating a reporter protein-encoding nucleic acid (hereinafter, referred to as “reporter gene”) in a form operable downstream of the transcriptional regulatory region of the IKAROS gene. The vector may be prepared by a method known to those skilled in the art. That is, "Molecular Cloning: A Laboratory Manual 2nd edition" (1989), Cold Spring Harbor Laboratory Press, "Current Protocols In Molecular Biology, (1987), J. and J. Wo., 1987." And the like, and the transcriptional regulatory region of the IKAROS gene cut out according to the usual genetic engineering techniques can be incorporated into a plasmid containing a reporter gene.

  As reporter proteins, β-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), β-galactosidase (GAS), green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein ( CFP), red fluorescent protein (RFP) and the like.

  A reporter gene prepared by operatively linking the transcriptional regulatory region of the prepared IKAROS gene is inserted into a vector that can be used in a cell into which the reporter gene is to be introduced, using a conventional genetic engineering technique, and the plasmid is inserted into the plasmid. It can be prepared and introduced into a suitable host cell. Stable transformed cells can be obtained by culturing in a medium under selection conditions according to the selectable marker gene carried on the vector. Alternatively, a reporter gene in which a transcription regulatory region of the IKAROS gene is operably linked may be transiently expressed in a host cell.

  In addition, as a method for measuring the expression level of a reporter gene, a method corresponding to each reporter gene may be used. For example, when using a luciferase gene as a reporter gene, the transformed cells are cultured for several days, and then an extract of the cells is obtained. Then, the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity is obtained. By measuring, the promoter activity can be detected. At this time, a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.

As a method for measuring the amount of IKAROS protein, specifically, for example,
(I) Quantifying IKAROS in a sample solution by reacting the detection antibody of the present invention with a sample solution and labeled IKAROS competitively, and detecting a labeled protein bound to the antibody. How and
(Ii) After reacting the sample solution, the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously, the labeling agent on the insolubilized carrier A method of measuring IKAROS in a sample solution by measuring the amount (activity) of the sample is exemplified.

Detection and quantification of the IKAROS protein expression level can be determined according to a known method such as Western blotting using an antibody that recognizes IKAROS. In the Western blot method, an antibody recognizing IKAROS is used as a primary antibody, and then a secondary antibody is bound to a primary antibody labeled with a radioisotope such as ¹²⁵ I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), or the like. And labeling with a labeled antibody, and measuring the signal derived from these labeled substances with a radiation meter (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like. After using an antibody recognizing IKAROS as a primary antibody, detection was performed using ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech) according to the protocol, and multi-biomagnifier STORM860 (manufactured by Amersham Pharmacia Biotech) was used. It can also be measured.

  The form of the above antibody is not particularly limited, and may be a polyclonal antibody using IKAROS as an immunogen, or a monoclonal antibody. Furthermore, at least a continuous amino acid sequence constituting IKAROS may be used. An antibody having an antigen-binding property to a polypeptide consisting of 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids can also be used.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Public. John Wiley and Sonny. Section 11.12-11.13).

  In the quantification method (ii), it is preferable that the two kinds of antibodies recognize different portions of IKAROS. For example, if one antibody is an antibody that recognizes the N-terminal of IKAROS, one that reacts with the C-terminal of the protein can be used as the other antibody.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can be used for binding the antibody or antigen to the labeling agent.

  The method for quantifying IKAROS using the detection antibody of the present invention is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex, which corresponds to the amount of the antigen in the sample solution, is determined chemically or physically. Any measuring method may be used as long as it is detected by a means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are suitably used. In terms of sensitivity and specificity, it is preferable to use, for example, a sandwich method described later.

  In insolubilizing the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing / immobilizing a protein or enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

  In the sandwich method, the sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and further reacted with another labeled detection antibody of the present invention (secondary reaction). By measuring the amount or activity of the labeling agent, IKAROS in the sample solution can be quantified. The primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed with a time delay. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the immobilized antibody or the labeled antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

  The antibody for detection of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.

  In the competition method, after the IKAROS in the sample solution and the labeled IKAROS are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), IKAROS in the sample solution is quantified by measuring the labeling amount of either B or F. In this reaction method, a liquid phase method in which a soluble antibody is used as an antibody and B / F separation is performed using polyethylene glycol or a secondary antibody to the antibody (primary antibody), or a solid phase is used as the primary antibody An antibody is used (direct method), or a solid antibody is used as a primary antibody, and an immobilization method using an immobilized antibody as a secondary antibody (indirect method) is used.

  In the immunometric method, the IKAROS in the sample solution and the immobilized IKAROS are subjected to a competitive reaction against a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated, or the IKAROS in the sample solution is reacted with the IKAROS in the sample solution. An excess amount of the labeled antibody is allowed to react, and then immobilized IKAROS is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any one of the phases is measured to quantify the amount of antigen in the sample solution.

  In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of IKAROS in the sample solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used.

  In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measurement system of IKAROS by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.

  For example, "Radio immunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radio immunoassay continued" edited by Hiroshi Irie (published in 1974), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Technologies (Part A)), ibid., Vol. 73 (Immunochemical Techniques (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) can be referred to.

  As described above, the amount of IKAROS in cells can be quantified with high sensitivity by using the detection antibody of the present invention.

  For example, in the above-mentioned screening method, the expression amount (mRNA amount or protein amount) of IKAROS in the presence of the test substance is about 20% or more, preferably about 30%, as compared with the case in the absence of the test substance. As described above, more preferably, when the inhibition is about 50% or more, the test substance can be selected as a candidate for an IKAROS expression inhibitor, and thus as an anti-inflammatory substance.

  Alternatively, in the above screening method, a cell containing a reporter gene under the control of a transcriptional regulatory region intrinsic to the IKAROS gene can be used instead of a cell expressing the IKAROS gene. Such a cell may be a cell, tissue, organ or individual of a transgenic animal into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of the IKAROS gene has been introduced. When such cells are used, the expression level of IKAROS can be evaluated by measuring the expression level of a reporter gene using a conventional method.

(Measurement of IKAROS function)
The screening method of the present invention can also be performed using as an index whether or not a test substance inhibits the function of IKAROS.

  Since IKAROS is a protein mainly secreted into the blood from neutrophils, substances capable of binding to IKAROS protein include IKAROS protein and its binding partner protein (eg, IKAROS receptor on target cell surface) It is thought that by blocking the interaction with, the function of IKAROS can be inhibited. Therefore, a candidate for an IKAROS function inhibitor can be screened using the binding ability to IKAROS as an index.

  For example, a test substance is adsorbed to each well of a well plate, an IKAROS solution labeled with an appropriate labeling agent is added to each well, incubated, the liquid phase is removed, and after washing, the amount of label bound to the solid phase is measured. By doing so, a test substance having the ability to bind to IKAROS can be detected. Instead of directly labeling IKAROS, IKAROS bound to the solid phase can be detected using a labeled anti-IKAROS antibody. Alternatively, a solution of a test substance is passed through a carrier (eg, an affinity column) on which IKAROS is immobilized, and the test substance retained on the carrier is converted into a substance capable of binding to IKAROS, that is, a candidate for an anti-inflammatory substance. Can also be selected.

Whether or not the candidate substance thus obtained actually has an anti-inflammatory action is confirmed by applying the candidate substance to an inflammation model and testing whether or not to suppress the inflammatory response in the model. be able to. In vivo and in vitro models can be used as such an inflammation model. Examples of the in vivo model include a DSS-induced colitis model (a non-human animal drinking water containing 3 to 5% (w / v) of DSS having a molecular weight of 5,000 to 10,000 for 5 to 10 days). IBD models such as TNBS-induced colitis model (which can be prepared by dissolving TNBS in 50% ethanol and rectally administering to non-human animals in an amount of, for example, 50 μg / g body weight). , CIA model (can be prepared by immunizing non-human animals with type II collagen emulsified with Complete Freund's adjuvant), CAIA model (monoclonal antibody cocktail recognizing epitopes in CB11 of type II collagen, Can be prepared by injecting into an animal). DOO but can, without limitation. On the other hand, as an in vitro model, a culture system of target cells (eg, intestinal epithelial cells in IBD, synovial cells in RA, etc.) in inflammatory diseases (eg, Caco-2 cell culture system, synovial tissue derived from RA patients) Culture system of synovial fibroblasts, etc.), and the like, but are not limited thereto. These in vitro models may be stimulated with inflammatory cytokines such as TNFα, active oxygen such as H ₂ O ₂ , LPS or the like, or with inflammatory cytokine-producing cells such as monocytes, macrophages, and neutrophils, as necessary. Complex culture (for example, using a Transwell ^TM culture system or the like, target cells (eg, Caco-2 cells) in the upper compartment, and inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW 264.7 cells) in the lower compartment , Respectively, can induce an inflammatory reaction.

  Whether or not a candidate substance has an anti-inflammatory action can be determined based on whether or not the inflammatory reaction in the above-described inflammation model has been suppressed by the addition of the candidate substance. For example, in the above-mentioned drug-induced colitis model animal, changes in body weight (suppression of body weight loss due to onset of colitis or degree of recovery from body weight loss, etc.), shortening of colon length, damage of epithelium at the site of colon inflammation The presence or absence of the anti-inflammatory effect and / or the degree thereof can be determined by using the degree of inflammatory cell infiltration and the like as an index. On the other hand, when a monolayer culture system of intestinal epithelial cells, which is an in vitro inflammatory bowel disease model, is used, an index of inflammatory response is determined by using a decrease in transepithelial electrical resistance (TER), LDH production, and an increase in IL-8 expression. The degree can be evaluated.

  In another preferred embodiment of the present invention, a substance inhibiting the function of IKAROS can be screened by testing the activity of inhibiting the binding between the IKAROS protein and its binding protein (for example, the IKAROS receptor). Although the IKAROS receptor and biological material that binds to IKAROS have not been identified yet, since IKAROS is a protein secreted mainly from neutrophils into the blood, it is expressed on the surface of target cells in inflammatory diseases. It is strongly suggested that among these proteins, a physiological binding partner of IKAROS exists. Therefore, the membrane fraction of the target cell (for example, in the case of intestinal epithelial cells, the membrane fraction on the lamina propria side) is isolated by a conventional method, and the solid phase is immobilized on a suitable carrier, and the target substance is subjected to solid phase separation. And contacting the solid phase with the labeled IKAROS protein in the absence of the test substance and comparing the amount of IKAROS bound to the cell membrane fraction in the absence of the test substance with the cell membrane fraction in the presence of the test substance When the amount of bound IKAROS is significantly low, the test substance can be selected as a candidate for an anti-inflammatory substance.It is determined whether or not the candidate substance thus selected has an anti-inflammatory action as described above. Can be confirmed by the following method.

  In still another embodiment of the present invention, a substance that inhibits the function of IKAROS and exhibits an anti-inflammatory effect can be screened in one step using the above-described in vitro inflammation model. The method comprises the following steps (1) to (3): (1) a step of bringing target cells of an inflammatory disease into contact with a test substance in the presence and absence of IKAROS; (2) under each condition Measuring the degree of the inflammatory response in said cells; and (3) inhibiting the inflammatory response in the presence of IKAROS, Provided is a method for screening an anti-inflammatory substance, which comprises a step of selecting, as a candidate for a substance exhibiting an anti-inflammatory action by inhibiting the function of IKAROS, a test substance that has not suppressed the inflammatory response.

The method may further include, if necessary, a step of inducing inflammation simultaneously with or before and after the step (1). As a method of inducing inflammation, for example, in addition to polyI: C, stimulation with inflammatory cytokines such as interleukin-Iα (IL-1α) and TNFα, active oxygen such as H ₂ O ₂ , LPS, or monocytes And complex culture with inflammatory cytokine-producing cells such as macrophages and neutrophils. In a preferred embodiment, for example, using a Transwell ^TM culture system or the like, target cells (eg, Caco-2 cells) are used in the upper compartment, and inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW264) are used in the lower compartment. .7 cells) in a monolayer culture. In this case, IKAROS is added to the medium in the lower compartment. LPS or the like may be further added to the medium to cause inflammation. The test substance is usually added to the medium in the lower compartment. For example, to screen for a component contained in food that can be absorbed into the intestinal tract and inhibit the function of IKAROS, or an IKAROS function inhibitor that can be administered orally. For example, the test substance can be added to the medium in the upper compartment.

  In certain embodiments, for the methods of the present invention, it is preferred that the target cells comprise epithelial cells. More specifically, the epithelial cells are skin epidermal cells, esophageal epithelial cells, gastric epithelial cells, vaginal epithelial cells, uterine epithelial cells, thymic epithelial cells, bladder epithelial cells, prostate epithelial cells, mammary epithelial cells, conjunctival epithelial cells or corneal epithelial cells Epithelial cells, preferably, but not limited to, skin epidermal cells, conjunctival epithelial cells or corneal epithelial cells. Alternatively, the epithelial cells are mucosal epithelial cells that express keratin 5. For cells expressing keratin 5, see Okuma et al. , Immunity. (2013) 38: 450-60 and Liang et al. , J Biomed Sci. (2009) 16: 2.

  In a further embodiment, the inflammation targeted by the screening of the present invention is inflammation due to increased expression of IKAROS. In certain embodiments, the inflammation targeted by the screens of the present invention is inflammation involving the epithelium due to IKAROS. In certain embodiments, the inflammation targeted by the screens of the present invention is inflammation involving IKAROS and TLR3 in the epithelium. In certain embodiments, the inflammation targeted by the present invention is an allergic disease, typically, Stevens-Johnson syndrome, asthma, contact dermatitis, acute dermatitis, atopic dermatitis, allergic conjunctivitis and Atopic keratoconjunctivitis and the like can be mentioned, but are not limited thereto.

  The substance that inhibits the expression or function of IKAROS, obtained by using any of the above screening methods of the present invention, is useful as a medicament for preventing and / or treating inflammatory diseases.

  When the compound obtained by using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned low molecular weight compound that inhibits the expression or function of IKAROS, It can be administered orally or parenterally to a human or mammal (eg, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) at a dosage. it can.

(IKAROS gene expression increase / enhancement / inducer)
In one aspect, the present invention provides a composition for increasing or inducing the expression of an IKAROS gene, comprising polyI: C. polyI: C (also referred to as "polyI: C", "polyI: C polynucleotide") is a polyI: C polynucleotide containing inosinic acid residues (I) and cytidylic acid residues (C). A double-stranded polynucleotide molecule (either RNA or a combination of DNA or DNA and RNA) that induces the production of inflammatory cytokines, such as interferons, and is a synthetic double-stranded RNA analog. Generally, it consists of one strand consisting entirely of cytosine-containing nucleotides and one strand consisting entirely of inosine-containing nucleotides, but other configurations are possible. By way of example, each strand may contain both cytosine-containing nucleotides and inosine-containing nucleotides. In some cases, either or both strands may further include one or more non-cytosine or non-inosine nucleotides. See, for example, Non-Patent Document 9 for polyI: C.

  Thus, as used herein, "polyI: C", "polyI: C" or "polyI: C polynucleotide" is a double-stranded polynucleotide molecule (RNA or DNA or a combination of DNA and RNA), Each strand comprises at least six adjacent inosinic acid or cytidylic acid residues, or six adjacent residues selected from inosinic acid and cytidylic acid in any order (e.g., IICIIC or ICICIC); Are capable of inducing or promoting the production of at least one inflammatory cytokine, such as interferon. The polyI: C polynucleotide is generally about 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75. , 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000 or more residues in length. We do not consider the upper limit to be important. A preferred polyI: C polynucleotide may have a minimum length of about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides, with a maximum length of about 1000. , 500, 300, 200, 100, 90, 80, 70, 60, 50, 45 or 40 nucleotides.

  Each strand of the polyI: C polynucleotide may be a homopolymer of inosinic acid or cytidylic acid residues, or each strand may be a heteropolymer containing inosinic acid and cytidylic acid residues. Is also good. In each case, the polymer, as described above, will have one or more non-native regions if there is at least one contiguous region of six I, six C or six I / C residues. It may be interrupted by inosinic acid or non-cytidylic acid residues such as uridine. Generally, each strand of a polyI: C polynucleotide will have no more than one non-I / C residue per six I / C residues, more preferably 8, 10, 12, 14, 16, 18, 20, Contains no more than 1 non-I / C residue for every 22, 24, 26, 28 or 30 I / C residues.

  Inosinic acid or cytidylic acid (or other) residues in a polyI: C polynucleotide are known in the art, provided that the ability of the polyI: C polynucleotide to promote the production of inflammatory cytokines such as interferons is retained. It may be derivatized or modified as known. Non-limiting examples of derivatives or modifications include, for example, azide modification, fluoro modification, or the use of a thioester (or similar) bond instead of the natural phosphodiester bond to improve stability in vivo. PolyI: C polynucleotides can also be complexed with molecules, eg, with positively charged poly-lysine and carboxymethyl cellulose, or with positively charged synthetic peptides, eg, to increase their resistance to degradation in vivo. It may be modified by formation. The polyI: C polynucleotide is generally included in the compositions of the present invention in an amount of about 0.001 mg to 1 mg per unit dose of the composition.

  In one embodiment, the up-regulation or induction of the IKAROS gene is in epithelial cells. Although not wishing to be bound by theory, it is possible to develop a novel method of controlling inflammation targeting epithelial cells, and by using epithelial cells as the target, local administration becomes easy and drastic reduction of side effects is expected. . In addition, by providing a cell model targeting epithelial cells, a novel method of controlling inflammation targeting epithelial cells can be developed. The cells of the present invention are useful for screening for drugs that can be easily administered locally by controlling the kinetics and activation of immune cells using the epithelial cell as the target, and have the substantial merit that the side effects are expected to be drastically reduced. is there. An inflammation control model from a point of interest that has not been performed so far is also provided. Pioneering new inflammation control methods targeting epithelial cells that are completely different from existing inflammation control methods will contribute to the realization of a safe and secure society by providing appropriate treatment methods and extending healthy life expectancy. Cells have significance as a development tool. Furthermore, the development of a novel inflammatory control method that controls the dynamics and activation of immune cells by targeting epithelial cells will lead to a major turning point in inflammatory control methods. The novel method of controlling inflammation targeting epithelial cells is expected to drastically reduce side effects because local administration with a control compound is easy, and will be a major player in the treatment of inflammation in the future.

  The cells of the present invention also facilitate, inter alia, the search for compounds that act selectively on epithelial cells. Elucidation of the mechanism of action at the molecular level reveals the mechanism of inflammation control through epithelial cells, leading to prevention of allergic diseases, treatment with medicines and medical supplies, and safe and secure quality (chemical materials, cosmetics, foods, health foods). Connect with. As a result, we can directly contribute to the improvement of human life in the future in terms of a safe and secure society by providing appropriate treatment methods and extending healthy life expectancy, as well as in a wide range of industries (medical, pharmaceutical, chemical, food, etc.) ) Can be expected.

(Cells in which the expression of IKAROS gene is increased or induced)
In one aspect, there is provided a cell in which the expression of an IKAROS gene is increased or induced. Here, “IKAROS gene expression has been increased or induced” means that the level of IKAROS gene expression has been increased or induced in cells existing in a normal state. Although it is thought that such a level can vary depending on the cell type, the term “IKAROS gene expression is increased or induced” as used in the present invention means that the expression level of the IKAROS gene is higher than that of the normal state depending on each cell type. Note that expression is elevated or induced.

  The cells of the present invention are preferably isolated cells. It is the first time that a cell in which the expression of the IKAROS gene has been increased or induced can be provided under an artificial environment other than the state in which it occurs in nature. As used herein, “isolated” means that naturally associated substances are at least reduced, and preferably substantially free, in normal circumstances. Thus, an isolated cell, tissue, or the like refers to a cell, tissue, or the like, which is substantially free from other accompanying substances (eg, other cells, proteins, nucleic acids, and the like) in its natural environment.

  In one embodiment, the cell in which the expression of the IKAROS gene is increased or induced by the present invention is one in which the expression of the IKAROS gene is increased or induced by polyI: C. Any form described in (IKAROS gene expression increase / enhancement / induction agent) can be used as polyI: C.

  In yet another embodiment, the cells of the invention are epithelial cells. In one specific embodiment, the cells of the invention may be keratin 5 positive cells.

(Animal in which expression of IKAROS gene is increased or induced)
In one aspect, there is provided an animal having increased or induced expression of an IKAROS gene. Here, the expression “IKAROS gene expression is increased or induced” means that the expression level of the IKAROS gene is increased or induced in an animal existing in a normal state. Although it is thought that such a level may vary depending on the type of animal, the expression “IKAROS gene expression is increased or induced” as used in the present invention means that the expression level of IKAROS gene is higher than that of the normal state depending on the type of animal. Note that expression is elevated or induced.

  In one embodiment, the animal of the present invention is an inflammation model animal. This is the first animal in which an inflammatory model has been provided as an animal in which the expression of the IKAROS gene has been increased or induced, and the inflammatory model has been provided by increasing or inducing the expression of such an IKAROS gene in a state other than the natural state. It is the first time.

  In one embodiment, the animal in which the expression of the IKAROS gene is increased or induced by the present invention is one in which the expression of the IKAROS gene is increased or induced by polyI: C. Any form described in (IKAROS gene expression increase / enhancement / induction agent) can be used as polyI: C.

  In one embodiment, the inflammation model comprises inflammation on the ocular surface. Although not wishing to be bound by theory, as shown in the present invention, it is thought that instillation of polyI: C increases the expression of the IKAROS gene on the ocular surface and causes inflammation. It has also been shown that epithelial-specific expression causes cutaneous mucosal inflammation. Thus, without wishing to be bound by theory, it is understood that application of polyIC to other mucous membranes or skin will cause inflammation as well. The inflammation caused by instillation of polyIC may be milder than that of Tg mice, which is thought to be due to the difference in the expression level of IKAROS.

  In another embodiment, the animal in which the expression of the IKAROS gene of the present invention is increased or induced is due to a genetic modification. Preferably, the genetically modified animal of the present invention comprises a vector comprising a nucleic acid sequence encoding the IKAROS gene operably linked to the keratin 5 promoter. As a specific embodiment, such a genetically modified animal (transgenic animal (also referred to as Tg animal)) is an IKAROS transgenic animal (also referred to as IKAROS Tg), and a vector in which the IKAROS gene is inserted into the keratin 5 promoter. (E.g., mouse) ES cells can be produced by introducing the keratin 5-expressing cell-specific IKAROS overexpressing animal (e.g., mouse). This animal (eg, mouse) has been found to cause inflammation of the skin and mucous membranes, and such findings are provided by the present invention for the first time.

  In one preferred embodiment, the inflammation model animal has at least one cell infiltration selected from the group consisting of skin tissue and mucosal tissue.

  The animals of the present invention include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), and are preferably model animals, eg, rodents (eg, mouse , Rats, etc.), and primates.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. et al. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Experimental Medicine “Gene Transfer & Expression Analysis Experiments”, Yodosha, 1997, etc., and these are relevant parts (may be all) in this specification. Is incorporated by reference.

  For DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M .; J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.C. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.A. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z .; et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T. (I996). Bioconjugate Technologies, Academic Press, and the like, and the relevant portions are incorporated herein by reference.

  For example, the oligonucleotides of the invention can be synthesized herein by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). is there. For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), or a controlled pore glass polymer support (Sarin et al.). Natl. Acad. Sci. USA 85: 7448-7451) can also be used to prepare methylphosphonate oligonucleotides.

  In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". In this specification, the phrase "within a range of two values" includes the two values per se.

  The references cited herein, such as scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically described.

  The present invention has been described above by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

  Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto. When applicable, the handling of biological samples, etc. was performed in compliance with the standards prescribed by the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology.

(Example 1: Method for inducing IKAROS expression in human epithelial cells and epidermal cells)
In this example, induction of IKAROS expression in human epithelial cells and epidermal cells was demonstrated. The experimental methods and results are shown below.

(Materials and methods)
-Cultured human conjunctival epithelial cells Acquisition method: The conjunctival epithelial layer was separated by Dsipase from unnecessary conjunctival tissue removed during surgery and obtained.

Culture method: The separated conjunctival epithelial layer was separated with 0.05% Trypsin-EDTA and then cultured with CnT-20 (CELLnTEC).
-Cultured human epidermal cells Acquisition method: used was purchased from Thermo (Product No. C0055C; Human Epidermal Keratinocytes, adult (HEKa))
Culture method: Culture was performed in EpiLife (GIBCO) with HKGS (GIBCO # S0015) added.
• polyI: C (Poly I: C) (obtained from Invivogen (Poly (I: C) High Molecular Weight Synthetic analog of dsRNA-TLR3 ligand, Catalog # tlrl-pic)). Poly I: C stimulation was performed by adding a stock solution to the cell culture so that the final concentration was the indicated concentration.
-The measurement of mRNA was performed as follows. Briefly, RNA was extracted from cells using the RNeasy Mini Kit obtained from Qiagen, and cDNA was transcribed from RNA using ReverTra Ace, random primer, dNTP, and RNase inhibitor obtained from TOYOBO. Quantitative PCR was performed using this cDNA sample. Quantitative PCR was performed using StepOne plus of Applied Biosystem, using TaqMan probe (Hs00958474) of IKAROS and TaqMan (registered trademark) Fast Advanced Master Mix.

(result)
The results are shown in FIG. As shown in FIG. 1 a, stimulation of cultured human conjunctival epithelial cells with 10 μg / ml polyI: C (poly I: C) resulted in an increase in IKAROS mRNA over time (the values in the graph are as follows: 0). After 1 hour, after 1 hour 0.9, after 3 hours 2.5, after 6 hours 10.6, after 10 hours 121.2). As shown in FIG. 1b, in cultured human conjunctival epithelial cells, IKAROS mRNA expression increased several times or more 10 hours after stimulation with 10 μg / ml polyI: C (poly I: C) (the values in the graph are as follows) : Medium 1, polyIC 8.8). As shown in FIG. 1c, in cultured human epidermal cells, 10 hours after stimulation with 10 μg / ml polyI: C (poly I: C), IKAROS mRNA expression was more than 10 times (the numbers in the graph are as follows: Medium 1, polyIC 11.4).

  Thus, it has been demonstrated that polyI: C (poly I: C) stimulation can increase the expression of IKAROS. Thus, in this Example, a method for inducing IKAROS in cultured human mucosal epithelium and cultured human epidermis was found. Until now, there has been no report on the expression of IKAROS in epithelial cells, and thus, in this example, a breakthrough discovery was made.

(Example 2: Skin and mucosal inflammation due to strong expression of IKAROS)
In the present example, it was confirmed that when IKAROS is strongly expressed in mucosal epithelium and epidermal cells, cutaneous mucosal inflammation occurs. In addition, the present invention has demonstrated that an IKAROS transgenic mouse can be prepared and used as a skin mucosa inflammation model. The experimental methods and results are shown below.

(Materials and methods)
(Transgenic animal production)
-Production of transgenic mice: A gene obtained by adding a keratin-5 promoter specifically expressed in epidermal cells or mucosal epithelium to the Ikaro isoform of IKAROS (an isoform containing all Exon1-7) was added to a mouse embryo. Then, transgenic mice in which the IkAR isoform of IKAROS is strongly expressed in the skin and mucosa were produced. Details will be described below.

  First, a K5-Ikzfl-2A-EGFP transgenic mouse (transgene: K5-lkzfl-2A-EGFP) was prepared. First, construction of an Ikzf1 gene expression vector, which is a vector used, and examination of PCR conditions for selection of founder mice will be described.

(Construction of expression vector)
FIG. 4 shows the structure and the construction procedure of the produced expression vector (pK5-Ikzfl-2A-EGFP). The protein coding region of the Ikzfl gene (isoform 1; 1545 bp), pocine teschovirus-12A (P2A; 57 bp), the protein coding region of the EGFP gene (720 bp), and the bGHpolyA signal (bovine growthhormone porphyry. 2663 bp) was produced by gene synthesis (SEQ ID NO: 5, FIG. 5 shows a schematic diagram of the vector). This was inserted into the Spe1 and NotI sites located downstream of the promoter of pBSK5 (human keratin 5 promoter in pBluescript II KS +) produced by the inventor, thereby producing an expression vector (pK5-lkzfl-2A-EGFP). (FIG. 4).

  In order to confirm the structure of the produced expression vector, it was digested with a restriction enzyme and the electrophoresis pattern was confirmed. The observed DNA cleavage pattern was consistent with that expected (FIG. 5). Further, the nucleotide sequence of the joint site was determined (SEQ ID NO: 5). When the nucleotide sequence of pBSK5 (human keratin 5 promoter in pBluescriptII KS +) used in the present invention was confirmed, there was one difference within the confirmed range from the NCBI database (position 2763 of SEQ ID NO. But in the NCBI database it was C). Since there is a report using this plasmid (Tarutani et al. 1997, Proc. Natl. Acad. Sci. USA 94 7400-7405), the produced expression vector was used as it is in the next step.

(Examination of PCR conditions for selection of founder mice)
PCR primers for confirming the introduction of the transgene (K5-Ikzfl-2A-EGFP) in the production of transgenic mice were designed, and the PCR conditions were examined.

  As condition 1, a K5p-F1 primer that binds to the Keratin5 promoter and a BGH-R3 primer that binds downstream of bGH polyA were designed (SEQ ID NOs: 6 and 7). FIG. 6 shows each primer sequence and PCR analysis conditions. As condition 2, a K5p-F6 primer and a K5p-R2 primer that bind to the Keratin5 promoter upstream site were designed (SEQ ID NOS: 8 and 9). FIG. 7 shows each primer sequence and PCR analysis conditions. As a result of setting conditions using a sample in which a pK5-Jkzfl-2A-EGFP expression vector and genomic DNA purified from a wild-type mouse were mixed, the analysis conditions were set so that the sensitivity could be detected even when one copy of the transgene was inserted. It could be set (FIGS. 6-7).

  Thus, a vector (Ikzll gene expression vector) was constructed, and the examination of PCR conditions for selection of founder mice was confirmed.

(Purification of expression vector)
In order to prepare an expression cassette (K5-Ikzfl-2A-EGFP) to be used for fertilized egg injection, an expression vector (pK5-lkzfl-2A-EGFP) was digested with restriction enzyme and a DNA fragment was purified.

(Preparation of transgene)
The expression vector was digested with the restriction enzymes SalI and Natl. Thus, the plasmid vector sequence was separated, and the expression cassette portion (about 17.1 kbp) was purified (FIG. 8). The linear DNA fragment obtained by these operations was used as a transgene, and the amount required for fertilized egg injection was prepared.

  From the above, the purification of the expression vector as a transgene was completed.

  Next, vector introduction (DNA injection) was performed. The prepared transgene (transgene: K5-lkzfl-2A-EGFP) was injected into pronuclear stage fertilized eggs and transplanted into recipient female mice.

(DNA injection)
1 to 2 pl of the prepared transgene (4.0 to 6.0 ng / μl) was injected into 302 pronuclear stage fertilized eggs (BALB / c strain). Of the transgene-injected pregnancies, 217 in good condition were transplanted into 9 recipient female mice (Table 1).

  From the above, it is confirmed that the vector was successfully introduced by DNA injection.

(Observation)
-The prepared transgenic mouse was observed with the naked eye to observe the inflammatory state, and a photograph was appropriately taken.

(Observation results)
The results are shown in FIG. Observation of the resulting transgenic mouse revealed that the mouse naturally developed inflammation in the skin and mucous membranes. Specifically, it was shown that in the transgenic mouse in which the IKAROS IK1 isoform is strongly expressed in the skin mucosa, inflammation occurs in the skin and eyes. Thus, in this example, it was demonstrated that when IKAROS is strongly expressed in mucosal epithelium and epidermal cells, cutaneous mucosal inflammation occurs.

(Example 3. polyI: a drug that suppresses IKAROS mRNA induced by C stimulation)
In this example, drugs that suppress IKAROS mRNA induced by polyI: C stimulation in cultured human conjunctival epithelial cells and cultured human epidermal cells were searched.

(Materials and methods)
-Cultured human conjunctival epithelial cells: obtained in the same manner as in Example 1.
-Cultured human epidermal cells: Obtained in the same manner as in Example 1.
• polyI: C: obtained as in Example 1.
- prostaglandin (PG) _{E 2} were used _{(PGE 2 Cayman Chemical # 14010)} . )
Using the same method as in Example 1, polyI: C-induced (10-hour stimulation) IKAROS mRNA expression increase was induced in cultured human conjunctival epithelial cells. Then, polyl: a C alone was compared the expression of IKAROSmRNA between group with addition of 100μg / ml PGE _2. Also in cultured human epidermal cells, polyl: a C alone was compared the expression of IKAROSmRNA between group with addition of 100μg / ml PGE _2.

(result)
The results are shown in FIG. As shown in FIG. 3, 100 μg / ml PGE ₂ suppressed the increase in IKAROS mRNA expression in cultured human conjunctival epithelial cells (FIG. 3a) (the numerical values in the graph are as follows: medium 1, polyI: C) 8.8, polyI: C + PGE2 1.9, polyI: C + rebamide 2.7). Similarly, 100 μg / ml PGE ₂ suppressed the increase in IKAROS mRNA expression in cultured human conjunctival epidermal cells (FIG. 3b) (the values in the graph are as follows: medium 1, polyI: C 11.4, (polyI: C + PGE2 2.7, polyI: C + rebamipide 2.8). Thus, in this example, it was demonstrated that there is a drug that suppresses IKAROS mRNA induced by polyI: C stimulation in cultured human conjunctival epithelial cells and cultured human epidermal cells.

(Example 4: Confirmation of IKAROS expression using PCR)
In this example, an experiment was performed to confirm whether IKAROS can be expressed in conjunctival tissue in a living body.

(Materials and methods)
Separate the conjunctival epithelium from the conjunctival tissue that is no longer needed at the time of surgery using Dispase.
RNA was extracted using the RNeasy mini kit obtained from the company. CDNA was transcribed from RNA using ReverTra Ace, random primer, dNTP, and RNase inhibitor obtained from TOYOBO. The cDNA was subjected to PCR according to the following method and conditions.
Reagent: TaKaRa Taq CAT # R001B
Equipment: ABI GeneAmp PCR system 9700
・ PCR reaction solution composition
TaKaRaTaq (5units / μl) 0.25μl
10 × PCR Buffer 5μl
dNTP Mixture (2.5mM each) 4μl
20μM F-Primer 1.5μl (Final 0.6μM)
20μM R-Primer 1.5μl (Final 0.6μM)
Template 2μl
Distilled water 35.75μl
Total 50μl
・ Primers used
GAPDH F 784435 K6148E12 (5'-CCATCACCATCTTCCAGGAG-3 '; SEQ ID NO: 10)
R 784435 K6148F01 (5'-CCTGCTTCACCACCTTCTTG-3 '; SEQ ID NO: 11)
(The above information is created from NM_002046 Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mRNA), F is 320-339, and R is 895-876. The size of the purified product is 575 bp. )
Ikaros F Primer A 768773 K5949A11 (GCTGATGAGGGTCAAGACAT; SEQ ID NO: 12)
R Primer C 768773 K5949B01 (AGCTTCGGCCACAATATCCA; SEQ ID NO: 13).
(In the information of IKAROS, F is information of Exon2 and R is information of Exon6. When these are used, Ik1 is 622 bp and Ik2 is 361 bp.)
・ PCR conditions
95 ℃ 5min → 95 ℃ 30sec → 72 ℃ 7min → 10 ℃ ∞
57 ℃ 30sec
72 ℃ 30sec × 28 (GAPDH) or 40 (Ikaros) cycle
・ Electrophoresis
1.2% agarose gel
Sample 5μ
Marker 100 base ladder.

(result)
The results of RT-PCR of IKAROS using monocytes and conjunctival epithelium are shown in FIG. As shown, expression of IKAROS mRNA (the bands in this example indicate IK1 and IK2) was observed in both monocytes and conjunctival epithelium. This result indicates that IKAROS can be expressed in human conjunctival epithelium.

(Example 5: Tissue analysis of IKZF1 transgenic mouse)
In this example, further analysis was performed on the transgenic (Tg) mouse of IKZF1 prepared in Example 2, and the tissue analysis was performed. The procedure and results are shown below.

(Method)
Various organs (skin tissues (including auricles, back epidermis, etc.) from one 5-week-old wild-type mouse (mouse born from the same parent and not overexpressing the IKZF1 gene) and one IKZF1Tg mouse prepared in Example 2 ), Mucosal tissues (including the eyelids and tongue)) were excised, paraffin-embedded sections were prepared according to a conventional method, and hematoxylin and eosin (HE) tissues were applied. Changes were observed. The above experiments were commissioned to the Non-Clinical Research Department, Research Division, Shinyaku Research Center Co., Ltd. (Eniwa, Hokkaido).

(result)
Histopathological analysis of IKZF1Tg mice revealed the following findings (results of HE tissue staining analysis of one 5-week-old wild-type mouse and one IKZF1Tg mouse).
(1) Skin tissue 1 (auricle)
In the IKZF1Tg mouse, slight localized intradermal dermal cell infiltration was observed (FIG. 10).
(2) Skin tissue 2 (back epidermis)
In IKZF1Tg mice, slight localized epidermal hyperplasia and slight localized intradermal dermal cell infiltration were observed (FIG. 11).
(3) Mucosal tissue 1 (eyelid)
Slight localized blepharitis submucosal cell infiltration was observed in IKZF1Tg mice (FIG. 12).
(4) Mucosal tissue 2 (tongue)
In the IKZF1Tg mouse, slight localized filamentous papillary spine cell layer thickening and slight localized subpapillary cell infiltration were observed (FIG. 13).

(Conclusion)
From the above, since the mouse used in this example had the IKZF1 gene bound to the keratin 5 promoter, the mouse overexpressing IKZF1 specifically in keratin 5 positive cells showed cutaneous mucosal inflammation. It can be said that it was a tendency to be.

(Example 6: Expression analysis of IKZF1 protein in human conjunctival tissue)
In this example, expression analysis of IKZF1 protein in human conjunctival tissue was performed. The description is given below.

(Method)
The ocular surface conjunctival tissue resected and discarded during surgery from a human patient who agreed to be used for the experiment was cryo-embedded and sectioned. Thereafter, the section was subjected to HE staining and fluorescent immunostaining. In fluorescent immunostaining, Gocam polyclonal ab106787 of abcam was diluted 100-fold as an anti-IKZF1 antibody, and Goat IgG of DAKO was used as an isotype control at the same concentration. Alexa488-labeled anti-Goat IgG antibody was used as the secondary antibody.

(result)
In both the conjunctival tissue of SJS and control conjunctival tissue (conjunctival flaccidity), the nuclei of the conjunctival epithelium were stained with anti-IKZF1 antibody in addition to the infiltrating cells.
SJS supracorneal scarred conjunctival tissue 1 (FIG. 14)
SJS conjunctival tissue 2 (Fig. 15)
Control conjunctival tissue 1 (FIG. 16)
Control conjunctival tissue 2 (FIG. 17)
(Conclusion)
In human conjunctival tissue in vivo, the expression of IKZF1 protein in human conjunctival epithelial cells was confirmed by immunostaining.

(Example 7: Screening method for novel anti-inflammatory treatment targeting IKAROS of epithelial cells)
The present inventors have reported that TLR3 promotes skin inflammation and ocular surface mucosal inflammation (Nakamura N, Tamagawa-Mineoka R, Ueta M, Kinoshita S, Katoh N. Toll-Like Receptor 3 Increases Allergic. and Irritant Contact Dermatitis.J Invest Dermatol.2014 Sep 17.doi: 10.1038 / jid.2014.402 .; and Ueta M, Uematsu S, Akira S, Kinoshita S: Toll-like receptor 3 enhances late-phase reaction of experimental allergic conjunctivitis. J Allergy Clin Immunol. 2009; 123 (5): 1187-1189.). It has also been reported that polyI: C and TLR3 increase the expression of inflammatory-related substances such as IFN-beta, TSLP, RANTES, MCP1, IP-1, IL-6, and IL-8 in epithelial cells (Ueta M, Hamuro J, Kiyono H, Kinoshita S: Triggering of TLR3 by polyI: C in human corneal epithelial cells to induce inflammatory cytokines. Biochem Biophys Res Commun. 2005; 331 (1): 285-294; Ueta M, Kinoshita S: Innate immunity of the ocular surface.Brain Res Bull. 2010; 81 (2-3): 219-228; and Ueta M, Mizushima K, Yokoi N, Naito Y, Kinoshita S: Gene-expression analysis of polyI: C-stimulated primary human conjunctival epithelial cells. Br J Ophthalmol. 2010; 94 (11): 1528-1532.

  In the present invention, TLR3 stimulation induces the expression of IKAROS in skin mucosal epithelial cells, and the strong expression of IKAROS in epithelial cells causes skin mucosal inflammation. , Suggest an important role in TLR3-induced mucocutaneous inflammation. As described above, in the present example, an experiment is conducted for the purpose of demonstrating a screening method for a drug that can be used for a novel anti-inflammatory treatment targeting IKAROS in epithelial cells.

(Method 1)
Using a cultured epithelial cell, a drug showing an inhibitory action on polyI: C-induced IKAROS expression is selected. Specifically, an agent that suppresses polyI: C-induced IKAROS expression is selected using human epidermal cells in which IKAROS expression is induced by polyI: C. The inhibitory effect of IKAROS expression is verified by quantitative PCR.

(Method 2)
Using an epithelium-specific IKAROS transgenic mouse, a drug that suppresses skin mucosal inflammation of the mouse is selected. Specifically, a candidate drug is applied to the auricle of an epithelium-specific IKAROS transgenic mouse for a certain period of time, the thickness of the ear is reduced, and the number of inflammatory cells is analyzed to examine the presence or absence of an inhibitory effect on inflammation. Then, a drug having an anti-inflammatory effect is selected. In addition, IKAROS expression in the pinna after the administration of the drug is analyzed, and a drug that suppresses IKAROS expression is selected.

(result)
It is understood that any of Methods 1-2 can screen for agents and / or anti-inflammatory agents that modulate IKAROS expression.

(Example 8: Experiment on tetOnsystem IKZF1 shRNA-expressing HaCaT cells)
In this example, tetOnsystem IKZF1 shRNA-expressing HaCaT cells were prepared, and in a state in which IKAROS expression was induced with polyI: C, IKAROS expression was knocked down by tetramycin administration, and changes in inflammatory-related substances were analyzed. The experimental conditions are shown below.

(Materials and methods)
・ Poly I: C (polyriboinosinic acid-polyribocytidic acid)
・ Knockout Single Vector Inducible RNAi System (Clontech 630933)
• tetOnsystem IKZF1 shRNA-expressing HaCaT cells are prepared by transfection as follows.

That is, MISSION esiRNA (SIGMA) is used for the IKZF1 gene. The siRNA is transfected into HacaT cells using Lipofectamine RNAiMAX (Life Technologies).
For confirmation of expression, transfected cells were treated with PolyI: C to induce IKZ1 expression, total RNA was extracted from PolyI: C-treated cells, and the expression of IKZF1 and control gene was quantified by qPCR. .
-Construction of an inducible expression vector using the tetON system and confirmation of its activity are performed as follows.
-The expression vector is prepared as follows.

Four types of shRNAs for the IKZF1 gene are designed and produced by incorporating the shRNA into a pSingle-rTS-shRNA vector.
For confirmation of expression, transfected cells were treated with PolyI: C to induce IKZ1 expression, total RNA was extracted from PolyI: C-treated cells, and knockdown of IKZF1 gene expression was temporarily performed by qPCR. It is examined by sex development.
-Establishment of IKZF1 shRNA-expressing HaCaT cells is performed as follows.
-The inducible shRNA expression vector obtained in the previous step is prepared for transfection, transfected into HaCaT cells, and clones are isolated by limiting dilution and drug selective culture.
-With regard to the expression confirmation, the expression of the IKF1 gene in the cells treated with PolyI: C was examined using qPCR for the obtained 20 clones, and the cell clones showing knockdown were subjected to expansion culture and cryopreservation.

(result)
From the above, the above-described experimental system of this example reveals an inflammation suppression mechanism by IKAROS-specific knockdown.

(Example 9. Novel anti-inflammatory treatment targeting IKAROS in epithelial cells)
In this example, a novel anti-inflammatory therapy targeting IKAROS in epithelial cells using an IKAROS inhibitor as identified in the present invention such as Example 4 can be demonstrated. The method for confirming the inflammatory effect is described below.

(Method 1)
Using a cultured epithelial cell, a drug showing an inhibitory action on polyI: C-induced IKAROS expression is selected. Specifically, an agent that suppresses polyI: C-induced IKAROS expression is selected using human epidermal cells in which IKAROS expression is induced by polyI: C. The inhibitory effect of IKAROS expression is verified by quantitative PCR.

(Method 2)
Using an epithelium-specific IKAROS transgenic mouse, a drug that suppresses skin mucosal inflammation of the mouse is selected. Specifically, a candidate drug is applied to the auricle of an epithelium-specific IKAROS transgenic mouse for a certain period of time, the thickness of the ear is reduced, and the number of inflammatory cells is analyzed to examine the presence or absence of an inhibitory effect on inflammation.

  (Method 3) Immunostaining is performed using skin tissue collected at the biopsy of acute Stevens-Johnson syndrome or at the biopsy of atopic dermatitis to confirm the presence or absence of increased expression of IKAROS. .

(result)
In any of the methods 1 to 3, the inflammatory effect of the anti-inflammatory agent of the present invention can be confirmed.

  As described above, the present invention has been exemplified by the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the appended claims. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Understood.

  According to the present invention, there is provided an agent for preventing and / or treating a disease associated with cell migration, targeting IKAROS, which is a different factor from the targets of therapeutic agents up to now. By providing therapeutic agents with a new mechanism of action, existing therapies will not be effective in reversing the disease, or will not be sufficiently effective in reversing the disease. Continued use of a therapeutic agent can provide better treatment even for inflammatory disease patients who have acquired resistance to the therapeutic agent. Furthermore, the present invention can also be used for the purpose of preventing inflammatory diseases before they occur, and the purpose of preventing the recurrence of illness in patients whose inflammatory diseases have remitted temporarily. Further, according to the present invention, it is possible to screen for a candidate substance for a novel prophylactic / therapeutic agent for inflammatory diseases, which exerts a therapeutic or preventive action by inhibiting the expression or function of IKAROS.

SEQ ID NO: 1: IKAROS human nucleic acid sequence (NM_0012207655.2)
SEQ ID NO: 2: IKAROS human amino acid sequence (NP — 00127694)
SEQ ID NO: 3: IKAROS mouse nucleic acid sequence (NM — 0010255597.2)
SEQ ID NO: 4: IKAROS mouse amino acid sequence (NP — 001020768)
SEQ ID NO: 5: Expression vector (pK5-Ikzfl-2A-EGFP) prepared in Example (FIG. 5)
SEQ ID NO: 6: K5p-Fl primer SEQ ID NO: 7: BGH-R3 primer SEQ ID NO: 8: K5p-F6 primer SEQ ID NO: 9: K5p-R2 primer SEQ ID NO: 10: GAPDH F784435K6148E12 primer SEQ ID NO: 11: GAPDH R784435K6148F01 primer SEQ ID NO: 12: Ikaros F PrimerA 768773 K5949A11 primer SEQ ID NO: 13: Ikaros R Primer C 768773 K5949B01 primer

Claims (11)

An anti-inflammatory agent for treating or preventing inflammation in a patient with Stevens-Johnson syndrome, comprising an IKAROS (IKZF1) inhibitor selected from the group consisting of PGE2, anti-IKAROS siRNA, and anti-IKAROS antibody . A topical agent, an anti-inflammatory agent according to claim 1. The following steps (1) to (3):
(1) the IKAROS gene or cell comprising a nucleic acid encoding a reporter protein under the control of the transcriptional regulatory region of the gene, contacting the test substance;
(2) a step of measuring the expression level of an IKAROS gene or an IKAROS protein or a reporter protein in the cells; and (3) an IKAROS gene or an IKAROS protein or a reporter protein as compared to the case where the expression level is measured in the absence of a test substance. A method for screening an anti-inflammatory substance for treating or preventing inflammation in a patient with Stevens-Johnson syndrome, comprising a step of selecting, as a candidate for an anti-inflammatory substance, a test substance having a reduced expression level of.   A method for treating or preventing inflammation in a patient with Stevens-Johnson syndrome, comprising: bringing IKAROS into contact with a test substance; and selecting a test substance capable of binding to IKAROS as a candidate for an anti-inflammatory substance. A screening method for anti-inflammatory substances. The following steps (1) to (3):
(1) contacting a target cell of an inflammatory disease with a test substance in the presence and absence of IKAROS;
(2) measuring the degree of the inflammatory response in the cells under each condition; and (3) suppressing the inflammatory response in the presence of IKAROS, as compared to the case where the measurement is performed in the absence of the test substance. Selecting a test substance that did not suppress the inflammatory response in the absence of IKAROS as a candidate for a substance exhibiting an anti-inflammatory effect by inhibiting the function of IKAROS, comprising: A method for screening an anti-inflammatory substance for treatment or prevention. The method according to claim 5 , wherein an inflammatory response is induced by polyI: C, interleukin-Iα (IL-1α), tumor necrosis factor (TNF) α or H2O2 stimulation. 7. The method of any one of claims 3, 5 or 6 , wherein the cells comprise epithelial cells. The composition for use in a method for screening an anti-inflammatory substance for treating or preventing inflammation in a patient with Stevens Johnson syndrome according to claim 5, comprising polyI: C as a substance that induces an inflammatory response . The isolated cells in which the expression of IKAROS gene is increased or induced, the use of the screening method of the anti-inflammatory agent for treating or preventing inflammation in a patient Stevens-Johnson syndrome. Use of a non-human animal with increased or induced expression of an IKAROS gene as an inflammation model animal for use in a method for screening an anti-inflammatory substance for treating or preventing inflammation in a patient with Stevens-Johnson syndrome. The use as the inflammation model animal according to claim 10 , wherein the inflammation model animal has cell infiltration in at least one selected from the group consisting of skin tissue and mucosal tissue.