JP2012197227A - Therapeutic agent for ischemic cerebrovascular disorder - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent for ischemic cerebrovascular disorders.SOLUTION: The therapeutic agent for ischemic cerebrovascular disorders comprises a substance capable of inhibiting the function of interleukin 17, interleukin 23 or γδT cells or the invasion of γδT cells into the brain.

Description

  The present invention relates to a therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of interleukin 17, interleukin 23 or γδ T cells, or a substance that inhibits infiltration of γδ T cells into the brain.

Stroke is a leading cause of death and disability worldwide, but no effective treatment has yet been established that can be initiated in the subacute or late phase of cerebral infarction ^(1,2) . 1, 2). Recently, lymphocyte recruitment and activation has been implicated in the progression of cerebral ischemic reperfusion (I / R) injury, but the role of specific lymphocyte subpopulations and cytokines in stroke It is still not clearly understood.

On the other hand, there is a lot of data suggesting that the innate immune response plays an important role in ischemic reperfusion (I / R) injury. Furthermore, it has recently been suggested that T lymphocytes play a role as mediators of I / R disorders in the subacute inflammatory phase. T cells have been identified by immunohistochemistry 24 hours after reperfusion in the postischemic brain and appear to be located in the border region of the infarct (typically ^4,5 near the blood vessels) Yes (Non-Patent Documents 4 and 5). In addition, studies so far suggest that CD4-positive T lymphocytes and IFNγ play an indispensable role in cerebral ischemic injury ³ (Non-patent Document 3). However, I / since R is the role of IFN gamma at fault is debatable ⁶ (Non-Patent Document 6), T lymphocyte subsets and inflammation mediators, whether really involved in the development of cerebral infarction It is unknown.

Sacco RL, Chong JY, Prabhakaran S, Elkind MS. Experimental treatments for acute ischaemic stroke. Lancet. 369, 331-341 (2007) Ooboshi et al. Postischemic gene transfer of interleukin-10 protects against both focal and global brain ischemia. Circulation. 111, 913-919 (2006) Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation. 113, 2105-2112 (2006) Schroeter M, Jander S, Witte OW, Stoll G. Local immune responses in the rat middle cerebral artery occlusion. J Neuroimmunol. 55, 195-203 (1994) Jander S, Karemer M, Schroeter M, Witte OW, Stoll G. Lymphocytic infiltration and expression of intercellular adhesion molecule-1 in photochemically induced ischemia of the rat cortex.J Cereb Blood Flow Metab. 15, 42-51 (1995) Lambertsen KL et al. A role for interferon-gamma in focal cerebral ischemia in mice.J Neuropathol Exp Neurol. 63, 942-955 (2004)

  It is an object of the present invention to provide a therapeutic agent for ischemic cerebrovascular disorders, particularly ischemic cerebrovascular disorders in the acute phase or subacute phase.

As a result of intensive studies to solve the above problems, the present inventor has found that the function of interleukin 17 (IL-17), interleukin 23 (IL-23) or γδT cells or the infiltration of γδT cells into the brain. It has been found that the above-mentioned ischemic cerebrovascular disorder can be improved by suppressing it, and the present invention has been completed.
That is, the present invention is as follows.
(1) An ischemic cerebrovascular disorder therapeutic agent comprising a substance that inhibits the function of interleukin-17.
(2) A therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of interleukin 23.
(3) A therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of γδT cells or infiltration of the γδT cells into the brain.
(4) An interleukin 17 production inhibitor comprising a substance that inhibits the function of interleukin 23.
(5) An interleukin 17 production inhibitor comprising a function that inhibits the function of γδT cells or infiltration of the γδT cells into the brain.

  In the present invention, examples of the substance that inhibits the function of interleukin 17 include an antibody against interleukin 17, or an inhibitory nucleic acid of the interleukin 17 gene. Examples of the substance that inhibits the function of interleukin 23 include, for example, interleukin 23. Examples include an antibody against leukin 23, or an inhibitory nucleic acid of interleukin 23 gene. Examples of substances that inhibit the function of γδ T cells or infiltration of the γδ T cells into the brain include, for example, antibodies against γδ T cells or γδ T cell reception Inhibitory nucleic acids of body genes.

  In the present invention, the “inhibitory nucleic acid” includes an antisense nucleic acid, a decoy nucleic acid, microRNA, shRNA, or siRNA for each of the genes.

  In the present invention, the cerebrovascular disorder to which the substance that inhibits the function of interleukin 17, or the function of γδT cells or the substance that inhibits infiltration of the γδT cells into the brain is applied is 3 days after ischemia. Or later. Moreover, it is preferable that the cerebrovascular disorder to which the substance that inhibits the function of interleukin 23 is applied is within 24 hours after ischemia.

  The present invention provides a new treatment for ischemic cerebrovascular disorders. In the present invention, the substance that suppresses the function of IL-17, IL-23, or γδT cells, or the infiltration of γδT cells into the brain, ischemic cerebrovascular disorder by suppressing the production of IL-17 or IL-23 Can be improved.

It is a figure which shows the relationship between the defect | deletion of IL-17 or IL-23, and improvement of a cerebral ischemic injury. It is a figure which shows the relationship between IL-17 and IL-23, and the inflammatory reaction produced in the case of a cerebral ischemic injury. It is a figure which shows the expression of IL-23 and IL-17 in an ischemic brain. It is a figure which shows the effect of (gamma) delta T lymphocyte depletion in an ischemic brain tissue. It is a schematic diagram which shows the production mechanism of IL-23 and IL-17 after cerebral ischemia induction. It is a figure which shows the mRNA expression level of chemokine in an ischemic brain tissue. It is a figure which shows the result of having performed intracellular FACS dyeing | staining about various surface markers.

The present invention will be described in detail.
1. Overview The present invention has been completed based on the finding that IL-23 and IL-17 play a major role in the development of cerebral infarction and the accompanying neurological deficits in a cerebral ischemia model.
Specifically, invasive inflammatory cells produced IL-23 within 24 hours after the introduction of cerebral ischemia (FIG. 5). Produced IL-23 induced IL-17 production of invading γδ T lymphocytes. On days 3 and 4, the subacute phase of cerebral ischemia, IL-17 stimulates ischemic brain macrophages, microglia cells, and various other cells to produce inflammatory cytokines and other factors did. Thus, IL-23 induces IL-17-producing γδ T lymphocytes, which maintain inflammatory responses to ischemic brain injury, and apoptotic neuronal death during the subacute phase of cerebral ischemia (FIG. 5).

  Analysis using gene-deficient mice shows that IL-23 functions immediately after ischemia-reperfusion injury (I / R injury), whereas IL-17 is in the subacute phase (3 days after I / R injury) ) Became necessary. This indicates that IL-23 expression increases 1 day after I / R, and IL-17 producing cells enter the brain after 3 days.

  IL-17-producing cells do not appear in IL-23 gene-deficient mice (IL-23-KO mice) having an I / R disorder, and from this, IL-17-producing cells induced by IL-23 It is shown to be a critical effector for the progression of brain damage. Moreover, intracellular cytokine staining showed that γδT lymphocytes, not CD4 positive helper T cells, were the major source of IL-17. Depletion of γδT lymphocytes by gene disruption or anti-TCRγδ antibody treatment improved brain I / R damage to the level observed in IL-17 deficient mice. Furthermore, it was observed that the administration of anti-TCR γδ antibody provides a neuroprotective effect even when administered 24 hours after the onset of cerebral ischemia (acute phase). These findings indicate that γδT lymphocytes that have entered the brain activated by IL-23 mainly produce IL-17 and cause an inflammatory response to ischemic brain injury. Thus, γδT lymphocytes may be a therapeutic target for late inflammatory events that exacerbate early injury in cerebral ischemia.

2. Substances that inhibit the function of IL-17, IL-23 or γδT cells or the infiltration of γδT cells into the brain "inhibit function" means that each substance acts on IL-17, IL-23 or γδT cells Inhibiting the activation of cytokines or cells, suppressing the expression of the respective cytokines or cells, suppressing the production of the respective cytokines, suppressing the migration of γδT cells to the lesion, or IL-17 And inhibit IL-23 signaling.
In addition, “inhibiting infiltration of γδT cells into the brain” means suppressing the migration of γδT cells to the lesion in the brain.

  Therefore, there is no limitation on the “substance” as long as it has the function of the IL-17, IL-23 or γδT cell or the activity of inhibiting the infiltration of γδT cell into the brain. For example, as a substance that inhibits the function of IL-17, IL-23 or γδT cells, or a substance that inhibits infiltration of γδT cells into the brain, antibodies against these cytokines or cells, surface proteins of these cytokines or cells Inhibitory nucleic acids for genes encoding (surface antigens or receptors) such as antisense nucleic acids, decoy nucleic acids, microRNAs, shRNAs or siRNAs.

(1) Anti-IL-17 antibody, anti-IL-23 antibody or anti-γδ T cell antibody Among antibodies that can be contained in the therapeutic agent or inhibitor of the present invention, anti-IL-17 antibody, anti-IL-23 antibody and anti-γδ T cell An antibody can be prepared as follows.

(A) Preparation of polyclonal antibody
(i) Preparation of antigen and solution thereof In producing the above antibody, it is necessary to first prepare and obtain a protein to be used as an immunogen (antigen).
As the antigen protein, purified IL-17, purified IL-23 and γδ T cell surface antigens or receptors can be used, but are not limited thereto. For example, one or several amino acid sequences of these proteins are used. It is also possible to use a protein having an amino acid sequence in which these amino acids are deleted, substituted or added, and having IL-17, IL-23 or anti-γδ T cell activity.
The amino acid sequence information of human IL-17 and IL-23 can be obtained from NCBI database GeneID: 51561 (IL-23) 3605 (IL-17). In addition, commercially available antibodies against purified IL-17, purified IL-23 and γδT cells can be used, for example, human IL-17 (R & D), human IL-23 (R & D), γδT. (Becton Dickison).

  Next, the obtained purified protein is dissolved in a buffer solution to prepare an antigen solution, and a known adjuvant can be added as necessary. Immunization is performed by administering the purified IL-17 or IL-23, or a solution containing the surface antigen or receptor of γδ T cells to a mammal (eg, mouse, rat, rabbit, etc.). Administration is mainly performed by injecting intravenously, subcutaneously or intraperitoneally.

The collection of serum (antiserum) obtained by the immunization is not limited, but is performed, for example, 1 to 28 days after the last administration. The antiserum can be collected from the blood of the immunized animal according to a conventional method.
The desired antiserum is screened from the antisera collected from each of the immunized animals. As the screening method, a known immunoassay can be performed according to the conventional method. The antibody contained in the target antiserum by the above screening is a polyclonal antibody. When it is necessary to purify the antibody, for example, a known purification method such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel chromatography or the like can be used alone or in combination of two or more. May be adopted and implemented as appropriate.

(B) Preparation of monoclonal antibody Antigen and its solution can be prepared in the same manner as in the preparation of the polyclonal antibody.
For the immunization method, the dose using the antigen solution, the administration interval and the number of administrations, the same methods and conditions as in the production of the polyclonal antibody can be employed.
The collection of antibody-producing cells (antibody-producing cells) obtained by the above immunization is not limited, but is preferably performed, for example, 1 to 14 days after the last administration, more preferably 2 to 4 days .

As antibody-producing cells, for example, spleen cells, lymph node cells, peripheral blood cells and the like are preferable, and spleen cells are more preferable.
A fused cell (hybridoma) can be obtained by performing cell fusion between the collected antibody-producing cells and myeloma cells.

  As myeloma cells, for example, cell lines that are generally available in mammals such as mice can be used. Specifically, cell lines that have drug selectivity and cannot grow on HAT selection media (hypoxanthine, aminopterin and thymine-containing media) in an unfused state, but can grow in a state fused with antibody-producing cells. preferable. Specifically, as mouse myeloma cells, for example, PAI, P3X63-Ag.8.U1 (P3U1), NS-I and the like can be used. The cell fusion is performed, for example, by mixing antibody-producing cells and myeloma cells in a culture medium for animal cell culture such as RPMI-1640 medium without serum. The mixing ratio of antibody-producing cells and myeloma cells is arbitrary, for example 5: 1.

  In general, the fusion reaction is preferably performed in the presence of a cell fusion promoter, and as the promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons or the like can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

  The cells after the fusion reaction are cultured using, for example, a HAT selection medium. After the above culture, cells in which growth on the HAT selection medium is observed become fused cells (hybridomas).

The target hybridoma is screened from the hybridoma obtained by the above culture. The screening method is not particularly limited, and a known immunoassay method (for example, ELISA or the like) can be performed according to the usual method.
Cloning of the target hybridoma by the above screening, that is, establishment of a monoclonal antibody-producing cell line can be generally performed by selecting colonies derived from one cell by culturing using a limiting dilution method or the like.

  As a method for collecting a monoclonal antibody from the hybridoma obtained by the above cloning, a cell culture method or ascites formation method can be generally employed. These methods are well known in the art.

  When it is necessary to purify the antibody at or after the collection of the monoclonal antibody, one or more known purification methods such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, and gel chromatography can be used. A combination can be adopted and implemented as appropriate.

In the present invention, humanized antibodies, human antibodies, and fragments of the antibodies can also be used.
A human-type antibody (CDR-grafted antibody) is a monoclonal antibody produced by genetic engineering, and specifically, part or all of the complementarity determining region of the hypervariable region is derived from a mouse monoclonal antibody. The antibody is a complementarity determining region of a hypervariable region, a framework region of the variable region is a framework region of a variable region derived from human immunoglobulin, and a constant region thereof is a constant region derived from human immunoglobulin. Human-type antibodies can be prepared by methods well known in the art.

  The human antibody is an antibody in which all regions including the variable region of the heavy chain and the constant region of the heavy chain and the variable region of the light chain and the constant region of the light chain are derived from a gene encoding human immunoglobulin. Say. The human antibody is prepared in the same manner as the above-described polyclonal antibody or monoclonal antibody by preparing a transgenic animal that produces a human antibody by a method well known in the art and immunizing the transgenic animal with an antigen. Can be manufactured.

The antibody fragment means a portion containing the antigen-binding region of the antibody or a portion derived from the region, and specifically F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), scFv (single chain Fv), dsFv (disulphide stabilized) and the like.

(2) Gene inhibitory nucleic acid
Inhibitory nucleic acid of IL-17 gene, IL-23 gene or γδ T cell receptor gene means a nucleic acid that suppresses its gene function or gene expression, for example, antisense nucleic acid, decoy nucleic acid, microRNA, shRNA or siRNA Etc. These inhibitory nucleic acids can suppress the expression of the gene. The base sequence of the gene to be suppressed is known, and sequence information can be obtained for each. The GenBank accession numbers for each gene are shown below.
IL-17 gene: NM010552.3 (mouse), NM 002190.2 (human)
IL-23 gene: NM031252 (mouse), NM 016584.2 (human)
γδ T cell receptor γ chain gene: NM 000073.2 (human)
γδ T cell receptor δ chain gene: NM000732 or NM001040651 (human)
Genomic sequence including γδ T cell receptor δ chain gene: NG001332 (human)

The base sequence of each gene is represented by the following SEQ ID NO.
Mouse IL-17 gene: SEQ ID NO: 1
Human IL-17 gene: SEQ ID NO: 2
Mouse IL-23 gene: SEQ ID NO: 3
Human IL-23 gene: SEQ ID NO: 4
Human γδ T cell receptor γ chain gene: SEQ ID NO: 5
Human γδ T cell receptor δ chain gene: SEQ ID NOs: 6, 7
Genomic sequence comprising the human γδ T cell receptor δ chain gene: SEQ ID NO: 8

  Here, since IL-23 is a heterodimer of IL-23p19 and IL-12p40, it targets either or both of IL-23p19 and IL-12p40 in order to suppress the expression of the IL-23 gene. It can be.

(2-1) Antisense nucleic acid Antisense nucleic acid is a single strand that can bind to mRNA (sense) or DNA (antisense) sequence of IL-17 gene, IL-23 gene or γδ T cell receptor gene (target gene) Nucleic acid sequence (either RNA or DNA). The length of the antisense nucleic acid sequence is at least about 14 nucleotides, preferably about 14-100 nucleotides. Antisense nucleic acid binds to the gene sequence to form a double strand and suppresses transcription or translation.

  Antisense nucleic acids can be produced using chemical synthesis methods or biochemical synthesis methods known in the art. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a gene recombination technique can be used.

  Antisense nucleic acids are introduced into cells by, for example, various gene transfection methods such as DNA transfection or electroporation, or by using viral vectors.

(2-2) Decoy nucleic acid In the present invention, a decoy nucleic acid can bind to a transcription factor of IL-17 gene, IL-23 gene or γδ T cell receptor gene and suppress promoter activity. The decoy nucleic acid of the present invention means a short decoy nucleic acid containing a binding site for a transcription factor. The nucleic acid is introduced into a cell, and the transcription factor binds to the nucleic acid, thereby returning to the original genome binding site of the transcription factor. Is competitively inhibited, and as a result, the expression of the transcription factor is suppressed. Specifically, a decoy nucleic acid is a nucleic acid that can bind to a target binding sequence or an analog thereof.

  Examples of preferred decoy nucleic acids of the present invention include, for example, nucleic acids that can bind to a transcription factor that binds to the promoter of the above gene. The decoy nucleic acid can be designed as a single strand or a double strand including the complementary strand based on the promoter sequence of the gene. The length is not particularly limited, and is 15 to 60 bases, preferably 20 to 30 bases.

  The nucleic acid may be DNA or RNA, or may include modified nucleic acids and / or pseudo-nucleic acids within the nucleic acid. Further, these nucleic acids, variants thereof, or nucleic acids containing these in the molecule may be single-stranded or double-stranded, and may be circular or linear. A variant is a nucleotide sequence in which one or several bases (for example, 1 to 10, 1 to 5, or 1 to 2) of the decoy nucleic acid sequence are deleted, substituted, or added. And a nucleic acid having a function of inhibiting the promoter activity of the gene, that is, a function of binding to a transcription factor.

The decoy nucleic acid used in the present invention can be produced using a chemical synthesis method or a biochemical synthesis method known in the art. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a gene recombination technique can be used. In addition, after isolating or synthesizing a base sequence serving as a template, a PCR method or a gene amplification method using a cloning vector can also be used. Furthermore, the desired nucleic acid may be produced by cleaving the nucleic acid obtained by these methods using a restriction enzyme or the like and binding using a DNA ligase. Furthermore, in order to obtain a more stable decoy nucleic acid in a cell, chemical modification such as alkylation or acylation can be added to a base or the like. A method for producing a variant of a decoy nucleic acid can be synthesized by a method known in the art, for example, by site-directed mutagenesis. Site-directed mutagenesis is well known in the art, and commercially available kits such as GeneTailor ^™ Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.) (Manufactured by Takara Bio Inc.)).

  For analysis of the transcriptional activity of the promoter when decoy nucleic acid is used, a commonly performed luciferase assay, gel shift assay, Western blotting method, FACS analysis method, RT-PCR, etc. can be employed. Kits for performing these assays are also commercially available (eg, promega dual luciferase assay kit).

(2-3) RNA interference In the present invention, synthetic small nucleic acid molecules capable of regulating gene expression by RNA interference (RNAi) in cells, such as siRNA, microRNA (miRNA), and shRNA molecules can be used.
When siRNA (small interfering RNA) molecules are used, various RNAs corresponding to the target gene can be targeted. Examples of such RNA include mRNA, variants obtained by alternative splicing of the target gene, and post-transcriptionally modified RNA of the target gene. If alternative splicing results in a family of transcripts that are distinguished by the use of appropriate exons, siRNA molecules can be used to inhibit expression of exon portions or conserved sequences.

In general, the target sequence on the mRNA can be selected from a cDNA sequence corresponding to the mRNA, preferably in the region 50-100 nucleotides downstream from the start codon. However, the target sequence may be located in the 5 ′ or 3 ′ untranslated region or the region near the start codon.
siRNA molecules can be designed based on criteria well known in the art. For example, as the target segment of the target mRNA, a continuous 15-30 base, preferably 19-25 base segment, preferably starting with AA (most preferred), TA, GA or CA can be selected. The GC ratio of siRNA molecules is 30-70%, preferably 35-55%.

siRNAs can be generated as single stranded hairpin RNA molecules that fold on their nucleic acid to generate a double stranded portion. siRNA molecules can be obtained by conventional chemical synthesis. Alternatively, siRNA molecules can be generated biologically using expression vectors containing sense and antisense siRNA sequences.
In order to introduce siRNA into cells, a method of linking siRNA synthesized in vitro to plasmid DNA and introducing it into cells, a method of annealing double-stranded RNA, and the like can be employed.

  The present invention can also use shRNA to produce RNAi effects. shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a partial region of a single strand forms a complementary strand with another region.

shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is set as sequence A and the complementary strand to sequence A is set as sequence B, these sequences are linked in the order of sequence A, spacer, and sequence B so that they are present in one RNA strand. Design to be 45-60 bases in length. The sequence A is a sequence of a partial region of the target gene, and the target region is not particularly limited, and any region can be a candidate. The length of the sequence A is 19 to 25 bases, preferably 19 to 21 bases.
Furthermore, this invention can suppress the expression of the said gene using microRNA. MicroRNA (miRNA) is a single-stranded RNA with a length of about 20-25 bases that exists in cells, and is considered to have a function to regulate the expression of other genes (non-coding RNA) It is a kind of. miRNAs are produced as a nucleic acid that forms a hairpin structure that is produced by being processed when transcribed into RNA and suppresses the expression of a target sequence.
Since miRNA is also an inhibitory nucleic acid based on RNAi, it can be designed and synthesized according to shRNA or siRNA.

3. Ischemic cerebrovascular disorder therapeutic agent and IL-17 production inhibitor As shown in FIG. 5, IL-23 is released from inflammatory cells upon ischemic brain injury, and γδT cells are stimulated by the stimulation. IL-17 is produced by γδ T cells. Therefore, in the present invention, a substance that inhibits the function of IL-17, IL-23, or γδT cells, or a substance that inhibits the infiltration of γδT cells into the brain can suppress the production of IL-17. Can improve ischemic cerebrovascular disorder. Therefore, the above substance can be used as an ischemic cerebrovascular disorder therapeutic agent and an IL-17 production inhibitor.

  The ischemic cerebrovascular disorder to be applied in the present invention includes cerebral infarction and a transient ischemic attack (TIA) which is considered as a precursor. Cerebral infarction is classified into four types: atherothrombotic cerebral infarction, cardiogenic cerebral embolism, lacunar infarction and other cerebral infarctions (NINDS: National Institute of Neurological Disorders and Stroke). The therapeutic agent of the present invention is applied to any of the above cerebral infarction and TIA. Moreover, spinal cord infarction can be illustrated as a disease used as the object of application in this invention.

  Here, the period of ischemic cerebrovascular disorder to be applied in the present invention is the acute phase and the subacute phase. The “acute phase” is a period within 24 hours after ischemia, and the “subacute phase” is basically within 3 days after ischemia, but ischemia gradually worsens due to the nature of vascular lesions. If you do, it can be after 3 days.

  In the present invention, the substance that inhibits the function of IL-17 or the function of γδT cells or the infiltration of γδT cells into the brain can be applied to cerebrovascular disorders 3 days after ischemia or later. A substance that inhibits the function of -23 can be applied to cerebrovascular disorders within 24 hours after ischemia.

Administration of the therapeutic agent or inhibitor of the present invention into the body can be carried out by a known method such as parenteral or oral, and is not limited, but is preferably parenteral administration.
Formulations (parenteral agents, oral agents, etc.) used in these various usages are excipients, fillers, extenders, binders, wetting agents, disintegrating agents, lubricants, surfactants, dispersions commonly used in drug production. An agent, a buffer, a preservative, a solubilizer, a preservative, a flavoring agent, a soothing agent, a stabilizer, an isotonic agent and the like can be appropriately selected and used, and can be prepared by a conventional method.

  The dosage of the therapeutic agent or inhibitor of the present invention is generally determined by the active ingredient in the preparation (anti-IL-17 antibody, anti-IL-23 antibody, anti-γδT antibody, or IL17 gene, IL-23 gene or γδT cell receptor). In consideration of the mixing ratio of the inhibitory nucleic acid of the body gene), the age, weight, type and progress of the disease, route of administration, number of administrations, administration period, etc. should be set appropriately. be able to.

The case where the therapeutic agent or inhibitor of the present invention is used as a parenteral or oral preparation will be specifically described below.
When used as a parenteral preparation, its form is not generally limited. For example, any of intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. There may be.

  In the case of various injections, for example, it can be provided in the state of a unit dose ampoule or a multi-dose container, or in the form of a lyophilized powder that is re-dissolved in a solution at the time of use. In addition to the above-mentioned active ingredient, the parenteral preparation can contain various known shaping materials and additives according to various forms as long as the effects of the active ingredient are not impaired. For example, in the case of various injections, water, glycerol, propylene glycol, aliphatic polyalcohols such as polyethylene glycol, and the like can be mentioned.

The dose of parenteral agent (per day) is not limited. For example, in the case of various injections, generally, the above-mentioned active ingredient (in the case of antibody) is 300 μg per 1 kg of the subject (patient) body weight. It is preferably -30 mg / day, more preferably 3-8 mg / day.
When used as an oral preparation, its form is generally not limited, and for example, any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product which is redissolved when used. In addition to the above-mentioned active ingredient, the oral preparation can contain various types of excipients and additives according to various forms as long as the effect of the active ingredient is not impaired. For example, binder (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), filler (lactose, sugar, corn starch, potato starch, calcium phosphate, sorbitol, glycine, etc.), lubricant (magnesium stearate, talc, Polyethylene glycol, silica, etc.), disintegrating agents (various starches, etc.), and wetting agents (sodium lauryl sulfate, etc.).

The dose (per day) of the oral preparation is generally 1 μg to 100 mg / day, preferably 1-10 mg / day, per 1 kg body weight of the application subject (patient) of the above-mentioned active ingredient.
When antisense nucleic acid is administered, it is 1 μg to 100 mg / day, preferably 1-10 mg / day, per 1 kg body weight of the subject (patient), and is administered continuously or once to several times a day. Is done. The administration route is preferably parenteral administration (for example, intravenous injection).

When siRNA, shRNA or miRNA is administered, the effective amount is not particularly limited as long as it is sufficient to cause RNAi-mediated degradation of the target mRNA. One skilled in the art can readily determine the effective amount to be administered to a subject taking into account factors such as the subject's height and weight, age, sex, route of administration, or local or systemic administration. Generally, the siRNA, shRNA or miRNA is at an intracellular concentration of about 1 nM to about 100 nM, preferably 2 nM to 50 nM, for parenteral administration (eg, intravenous injection).
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Materials and methods mouse
IL-17 ^-/-(29) , IL-23p19 ^-/-(11) , IFN γ ^-/- , IL-6 ^-/- and γδ TCR ^-/- knockout (KO) mice derived from C57BL / 6 As well as wild type C57BL / 6 mice.

Preparation of inflammatory cells of cerebral infarction Cerebral ischemia was induced in mice. At each time point, mice were cardiac perfused with cold PBS to remove blood cells. The forebrain was excised from the cerebellum and fully suspended in RPMI-1640. Suspended on a shaker for 45 minutes at 37 ° C with type IV collagenase (1 mg / ml, Sigma-Aldrich, St. Louis, MO, USA) and DNaseI (50 μg / ml, Roche, Basel, Switzerland) The solution was enzyme treated. Inflammatory cells were isolated by 37% / 70% Percoll (GE Healthcare, Chalfont St. Giles, Bucks., UK) density gradient centrifugation according to known method ¹⁹ . During this procedure, inflammatory cells were removed and washed with RPMI-1640 for further analysis.

Depletion of γδ T lymphocytes Anti-TCR γδ antibody (UC7-13D5) was used to deplete γδ T lymphocytes. Mice were administered PBS or 250 μg of the antibody 7 days before (ip) or 24 hours after induction of cerebral ischemia. Whether to administer PBS or antibody was randomly selected. Seven days after induction of cerebral ischemia, mice were sacrificed and infarct size was measured by anti-MAP2 immunostaining. There was no significant difference in body weight or CBF reduction between PBS-treated or antibody-treated mice and TCR γδ KO mice.

Statistical analysis Data are expressed as mean + S.D. A one-way analysis of variance (ANOVA) followed by a post hoc multiple comparison test (Dunnett's correction) was performed to analyze differences between groups of 3 or more mice. Between the two groups of mice, Unpaired Student's t test was performed to determine the significance of the difference. P <0.05 was considered significant.

Mouse cerebral ischemia model To conduct local cerebral ischemia experiments, 9-17 week old male mice (body weight 20-30 g) were used. There was no significant difference in body weight between wild type mice and all KO groups.

Cerebral ischemia was induced by known method ¹⁵ . In summary, mice were anesthetized with halothane in a 70% nitrous oxide / 30% oxygen mixture. The head temperature was maintained at 36 ° C with a heating lamp. Cerebral blood flow (CBF) in the parietal cortex was measured through the skull before and during ischemia by laser Doppler velocimetry in the ipsilateral parietal bone. The quiescent CBF value for each mouse was considered the baseline for that mouse and the change in CBF value after inducing cerebral ischemia was expressed as a percentage of the quiescent value. After ligating the right common carotid artery (CCA), the right middle cerebral artery (MCA) was occluded with 7-0 nylon monofilament (ETHILONE) using a tip with a round tip. A filament (11 mm long) was inserted from the right CCA and advanced to the internal carotid artery (ICA). The distance from the suture tip to the right CCA branch was approximately 9 mm. While the right MCA was occluded, laser Doppler flow velocity measurement confirmed that CBF decreased by more than 60%. Sixty minutes after cerebral ischemia, the filament was withdrawn and the right MCA area was reperfused. No cerebral vascular abnormalities (evaluated by Evans blue staining) were observed in IL-17, IL-23p19, IL-6 or IFNγKO mice.

On days 1 and 4 after reperfusion, 2% 2,3,5-triphenyltetrazolium chloride (TTC) was obtained from continuous coronal slices (thickness 1 mm) from the brain to measure infarct volume. ). Samples were obtained on day 7 and mice were fixed by cardiac perfusion with cold PBS followed by 4% paraformaldehyde / PBS. Serial coronal slices (thickness 1 mm) collected from the brain were embedded in paraffin sections and immunostained with anti-microtubule associated protein 2 (MAP2). Infarct areas identified by TTC and anti-MAP2 staining were measured in each section using Photoshop Creative Suite 3 (Adobe, San Jose, CA, USA) and the volume of the infarct area was calculated according to known methods ¹⁶ .

Neurological evaluation
Neurological function was blindly assessed using a 4-point scale neurological score. The evaluation contents of each point are as follows.
0: No observable defects
1: Forelimb flexion
2: Resistance to the pressing force from the side decreases (no turning)
3: Same behavior as 2 (with turning)

TUNEL staining On the 4th day after reperfusion, brain DNA sections at caudoputamen level were evaluated for genomic DNA cleavage using the TUNEL kit (In Situ Cell Death Detection Kit POD, Roche) . In the periphery, positively stained cells on the cortex were counted. Positive cells were considered to have spread 1 mm behind the bregma and 2.5 mm outside the midline ³⁰ . The number of positive cells (per 0.1 square mm) in three different regions was expressed as an average value.

Quantitative real-time PCR
Invasive inflammatory cells prepared by ischemic brain tissue or Percoll gradient centrifugation were lysed in RNAiso (Takara, Shiga, Japan) for RNA preparation. Real-time PCR was performed on cDNA samples using Power SYBR Green (Applied Biosystems, Foster City, CA, USA). Relative quantitative values were expressed as 2 ^-ΔCt . In the formula, ΔCt is the difference between the mean Ct values of duplicate measurements of the sample and the control (endogenous hypoxatin phosphoribosyltransferase 1 (HPRT1)).

Intracellular cytokine staining For invasive inflammatory cells collected by Percoll for surface and intracellular cytokine staining, 50 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 1 μg / ml ionomycin (Sigma -Aldrich) and 1 μM brefeldin A (eBioscience, San Diego, Calif., USA) for 4.5 hours. Surface staining was performed for 15 minutes with the corresponding mixture of fluorescently labeled antibodies. These invading inflammatory cells derived from ischemic brain tissue were classified by different surface markers. The relationship between each inflammatory cell and the surface marker is as follows.
Macrophages: CD45 high CD11b high;
Microglia cells: CD45 high in CD11b;
Neutrophils: CD45 high CD11b high Gr-1 + cells;
T lymphocytes: CD45 high CD3 + cells;
γδ T lymphocytes: CD45 high CD3 + TCR γδ + cells

  After surface staining, cells are suspended in Fixation buffer (eBioscience) and intracellular cytokines using anti-IL-17-APC, anti-IFNγ-APC and anti-TNF α-PE (eBioscience) as per manufacturer's protocol Staining was performed.

FACS analysis
FACS analysis was performed on a FACSAria instrument (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

2. Results Among the several types of T cells and cytokines involved in brain I / R injury, we focused on Th17, a newly identified T lymphocyte subset. The reason is that these cells have been shown to play an essential role in experimental autoimmune encephalomyelitis (EAE). EAE is a well-established model of T cell-mediated brain and spinal inflammation. In this model, IL-17 and other inflammatory mediators derived from Th17 cells are involved in the development and progression of EAE ^7-9 . Th17 has been shown to differentiate from naive CD4 positive T cells by in vitro stimulation with IL-6 and TGFβ. Furthermore, IL-23, a heterodimer of IL-23p19 and IL-12p40, has been shown to be essential for inducing Th17 in vivo ¹⁰ . IL-23 is mainly produced from antigen-presenting cells such as dendritic cells. IL-23 has been demonstrated to be an important cytokine for the development of experimental autoimmune encephalomyelitis (EAE) ^11,12 . In addition, both IL-23 and IL-17 have been reported to modify many inflammatory responses in the central nervous system ^13,14 .

In order to determine the functional importance of cytokines for cerebral infarction (especially IL-17, IL-23p19, IL-6 and IFNγ), we have An experimental cerebral ischemia model was applied ¹⁵ .
The results are shown in FIG. FIG. 1 shows that IL-17 or IL-23 deficiency attenuated cerebral ischemic injury. Each panel of FIG. 1 is as follows.
(A) The result of measuring the time course of the volume of the infarct portion by TTC staining on the first day and the fourth day.
(B) Anti-MAP2 immunostaining of day 7 brain section.
(C) Neurological scores of WT and KO mice on day 1 and day 4.
(D) Results of TUNEL staining on day 4 brain section (scale bar: 100 μm).
*: P <0.05, **: P <0.01, ***: P <0.001 vs wild type (universal ANOVA using Dunnett's correction, error bar, sd).
The number of mice is indicated on the bar.

As shown in FIG. 1a, the volume of the infarct area was evaluated on days 1 and 4 by 2,3,5-triphenyltetrazolium chloride (TTC) staining ¹⁶ . Surprisingly, there was no significant difference in infarct volume between wild type (WT) and IFNγ knockout (KO) mice (FIG. 1a). However, in IL-23p19 KO mice, it was observed that the volume of the infarct portion was significantly reduced on day 1 and day 4 as compared to wild-type mice (FIG. 1a). IL-17 KO mice also showed a marked decrease in infarct volume on the 4th day, but no extreme decrease was observed on the 1st day, indicating the very early (acute phase) of the disorder. : IL-24p19 functions within 24 hours after ischemia), while IL-17 plays a role in the subacute phase of the disorder.

  In addition, the anti-microtubule associated protein 2 (MAP2) immunostaining on the 7th day confirmed that the volume of the infarct was significantly reduced in association with the IL-17 and IL-23p19 deficiency. (Fig. 1b). Interestingly, IL-6 deficiency did not affect the increase in infarct volume caused by I / R (FIG. 1b).

On day 4, it was confirmed that neuronal deficits were reduced in both IL-17 KO mice and IL-23p19 KO mice, and IL-17 and IL-23p19 showed neuropathy caused by I / R damage. It is suggested that it promotes (Fig. 1c). The progression of ischemic brain injury is associated with apoptotic neuronal death that occurs in the periphery (adjacent region around the ischemic center) hours or days after cerebral ischemia ^17,18 . As expected, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling) staining in the peripheral area of the ischemic brain revealed that IL-17 KO mice and IL-23p19 KO mice than wild-type and IFNγ KO mice. It became clear that there were few apoptotic neurons in mice (Fig. 1d).

Next, mononuclear cells were isolated from the ischemic brain by Percoll gradient centrifugation ¹⁹ and separated into fractions using flow cytometry to clarify which type of cells produced inflammatory cytokines.
The results are shown in FIG. FIG. 2 shows that IL-17 and IL-23 enhanced the inflammatory response that occurs later in the cerebral ischemic injury. Each panel in FIG. 2 is as follows.
(A) Time course of the absolute number of various inflammatory cells that have entered the ischemic brain.
(B) Comparison of day 3 invasive inflammatory cell populations between WT and KO mice.
(C) Mean fluorescence intensity (MFI) of MHC class II expression in macrophages of day 3 invading inflammatory cells between WT and KO mice.
Intact (no treatment) mouse, n = 2;
WT mice and KO mice, n = 4 each.
(D) Relative changes in mRNA expression levels of inflammatory cytokines and other inflammatory factors (compared to intact mice by quantitative real-time PCR using ischemic brain tissue).
Intact mice, n = 2, 1st day: WT, n = 4; IL-17KO, n = 3; IL-23p19KO, n = 3; and 4th day: WT, n = 5; IL-17KO, n = 4; IL-23p19KO, n = 3.
(E) Comparison of TNFα production of macrophages and microglial cells assessed by intracellular FACS.
The MFI was as follows:
WT macrophages, 701; IL-17KO macrophages, 421; and WT microglia cells, 28.4; IL-17KO microglia cells, 28.7.
Data are representative of two independent experiments.
(F) Comparison of mRNA expression levels of inflammatory cytokines in day 3 invasive inflammatory cells.
Relative values compared to intact mice are shown.
WT and IL-17KO, n = 3 each.
*: P <0.05, **: P <0.01 vs wild type (one-way ANOVA with Dunnett's correction (d) or bilateral Student's t-test (f)).
Error bar, sd.

  As shown in FIG. 2a, the absolute number of CD45 positive inflammatory cells, most of which are macrophages and microglial cells, increased to day 3 and began to decrease on day 6. The number of invading macrophages increased rapidly on the third day, but decreased extremely on the sixth day. In contrast, there was no noticeable change in the number of microglia cells. The number of invading T lymphocytes and γδ T lymphocytes continued to increase until day 3, and the number of CD4 helper T cells gradually increased until day 6. There were no significant differences in cell population and macrophage MHC class II expression between wild type and KO mice (FIGS. 2b, c). However, more T lymphocytes and γδT lymphocytes invaded the ischemic brain of IL-17 KO mice and IL-23p19 KO mice compared to wild type mice.

To elucidate the molecular mechanism for the association of IL-17 and IL-23 in cerebral ischemia, the expression level of inflammatory mediators was assessed by quantitative real-time PCR (FIG. 2d).
IL-1 β and TNF α are neurotoxic cytokines, and matrix metalloproteinases (MMPs) (eg, MMP-3 and MMP-9) are responsible for blood brain barrier (BBB) breakdown and neuroinflammatory responses. It has been reported that it is related ^20-22 . Further, both the IL-1 beta and MMP-9 is ²³ to promote apoptotic neuronal death have been ^{suggested, 24.}

Expression of all these factors in the whole brain was low in both IL-23p19 KO and IL-17 KO mice after 4 days of I / R (FIG. 2d). However, chemokine mRNA levels did not change between wild-type and KO mice (FIG. 6).
FIG. 6 shows chemokine mRNA expression levels in ischemic brain tissue.
Intact mice (n = 2), WT (n = 5), IL-17KO mice (n = 4) and IL-23p19KO mice (n = 3). All obtained differences were calculated by one-way ANOVA (error bar, sd) using Dunnett's correction.
Therefore, the decrease in infarct volume in IL-17 KO and IL-23p19 KO mice is probably due to a decrease in these neurotoxic inflammatory factors. Some of these (IL-1β, TNFα and MMP-3) had already been reduced in the brains of IL-23p19 KO mice from day 1. This result indicates that IL-23 acts upstream of IL-17, and that IL-17 plays an important role in the subacute phase of cerebral ischemia (FIG. 5).

  Intracellular cytokine staining revealed that TNFα was produced in large amounts from invading macrophages but not from microglial cells (FIG. 2e). TNFα production from macrophages was reduced in IL-17 KO mice (FIG. 2e). Consistent with this, TNFα and IL-1β mRNA levels in day 3 invasive inflammatory cells were significantly reduced in IL-17 KO mice (FIG. 2f).

Next, the expression of IL-23 and IL-12 in the ischemic brain was examined.
The results are shown in FIG. FIG. 3 shows the expression of IL-23 and IL-17 in the ischemic brain. In FIG. 3, each panel is as follows.
(A) Time course of IL-23 and IL-12 mRNA expression in invasive inflammatory cells of wild type mice (compared to intact mice).
Intact, n = 2;
On day 1 and day 3, n = 3 respectively.
(B) Comparison between IFNγ-producing cells and IL-17-producing cells in invading T lymphocytes between mice.
Data are typical of two independent experiments.
(C) Time course of the number of invading IL-17 positive cells or IFN γ positive cells in the ischemic brain.
Error bar, sd (n = 3 for each time point).
(D) Sources of IL-17 and IFNγ in the ischemic brain. IL-17 positive or IFNg positive cell populations were further analyzed for TCRgd or CD4 expression levels. Data are representative of 3 independent experiments.

  We found that IL-23p19, IL-12p35 and IL-12p40 mRNA levels were high at day 1 in invasive inflammatory cells and then decreased (FIG. 3a). Next, intracellular FACS analysis was performed using invasive inflammatory cells to identify IL-17 producing cells in ischemic brain tissue. Specific staining of IL-17 and IFNγ was confirmed using KO cells (FIG. 3b). Importantly, IFN γ producing cells are present in IL-17 KO and IL-23p19 KO mice at a level similar to that found in wild type mice, and IL-17 producing cells are also IFN γ Present in KO mice. However, IL-17-producing cells were not detected in invading mononuclear cells derived from IL-23 KO mice (FIG. 3b). These data indicate that the IL-23 / IL-17 axis and the IL-17 / IFN γ axis are independent of each other in cerebral ischemia.

  The number of IL-17 positive cells in ischemic brain tissue was first measurable on day 3 and decreased on day 6 (FIG. 3c). Similarly, the number of IFNγ positive cells increased up to day 3 and decreased on day 6 (FIG. 3c).

  To determine the source of IL-17 and IFNγ, intracellular FACS staining was performed on various surface markers (FIG. 7). Cerebral ischemia was induced in several mice, and invading inflammatory cells were collected from the ischemic brain on day 3 and pooled. Using these cells, intracellular FACS was performed using various surface markers to (a) detect the source of IL-17, and (b) detect the source of IFNγ. Data are representative of 3 or more independent experiments.

  All IL-17 positive cells and IFN γ positive cells were also CD45 positive. Approximately 90% of IL-17 positive cells and 85% of IFN γ positive cells were CD3 positive T lymphocytes. None of the cells were double positive for IL-17 and IFNγ. Surprisingly, almost all IL-17 producing T lymphocytes were CD4 negative but γδ TCR positive (FIG. 3d). On the other hand, approximately 60% of IFNγ producing T lymphocytes were CD4 positive and γδ TCR negative. These data indicate that the source of IL-17 is γδ T cells rather than Th17 cells, and the source of IFN γ is Th1 cells.

To test the effect of γδ T lymphocyte depletion on cerebral ischemia, we used γδ TCR-deficient mice or mice treated with anti-TCR γδ antibody (UC7-13D5) ²⁵ .
The results are shown in FIG. FIG. 4 shows the effect of γδ T lymphocyte depletion in ischemic brain tissue. In FIG. 4, each panel is as follows.
(A) Comparison of infarct volume assessed by anti-MAP2 immunostaining.
(B) The effect of depletion of IL-17-producing γδ T lymphocytes was evaluated by intracellular FACS using invasive inflammatory cells.
(C) mRNA expression of inflammatory cytokines in day 3 invasive inflammatory cells. Relative expression compared to intact mice is shown.
WT mice and antibody treated mice, n = 3 each.
(D) Infarct volume of mice treated with PBS or antibody for 24 hours after induction of cerebral ischemia.
****: P <0.024. *: P <0.05, ***: P <0.001 vs wild type (one-way ANOVA with Dunnett's correction (a) or two-sided Student's t-test (c, d), error bar) , Sd).
The number of mice is indicated on the bar.

  It was observed that the volume of the infarct portion was significantly reduced in the antibody-treated mouse and the γδ TCR KO mouse (FIG. 4a). The present inventors confirmed that administration of this antibody almost completely depleted brain γδ T lymphocytes and IL-17 positive T lymphocytes of ischemic brain tissue (FIG. 4b). The administration of the antibody also significantly reduced the mRNA expression level of inflammatory cytokines in the invading inflammatory cells on day 3 (Fig. 4c), and the same effect as the knockout of IL-17 in mice was obtained (Fig. 2c). ). Furthermore, it was observed that a neuroprotective effect was obtained when anti-TCR γδ antibody was administered even 24 hours after the occurrence of cerebral ischemia (FIG. 4d).

Recent studies, the ?? T lymphocytes are the primary source of IL-17, to play an essential role in the liver and intestine to prevent bacterial infection it has been revealed ²⁶ . IL-23 but not IL-6 directly activates IL-17 secretion from NKT lymphocytes ²⁸ . Shibata et al. Say that these γδ T lymphocytes that produce IL-17 (but not IFN-γ or IL-4) develop in the thymus and are called naturally occurring IL-17 producing cells ²⁵ reported. Since γδ T lymphocytes in the ischemic brain produce IL-17 and not IFNγ, and because IL-23 is essential for the development of IL-17 producing cells, we It was speculated that invading γδ T lymphocytes were derived from such naturally occurring IL-17-producing γδ T lymphocytes. The inventors have shown that some combinations of IL-17 and IFNγ, or combinations of IL-17 and some other mediators derived from γδ T lymphocytes, are responsible for the progression of brain I / R damage. Could not rule out the possibility of being important. However, since similar infarct volume reduction is observed in IL-17 KO and TCR γδ KO mice, our study shows that IL-17 is the most potent effector of γδ T lymphocytes Proved that. It is interesting to determine whether IL-17 producing γδ T lymphocytes are important in I / R injury to other tissues.

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According to the present invention, IL-23-induced IL-17 production from γδ T lymphocytes in the subacute phase of cerebral ischemia promotes the inflammatory response of invading macrophages and promotes apoptotic neuron death. Indicated. Based on these findings, substances that inhibit the function of γδ T lymphocytes that produce IL-23 and IL-17 have become a novel therapeutic target for ischemic cerebrovascular disorders, and the treatment time for neuroprotection against cerebral ischemia has been increased. Can be secured.
In the present invention, a substance that inhibits the function of interleukin 17, interleukin 23 or γδ T cells or infiltration of γδ T cells into the brain is useful as a therapeutic agent for ischemic cerebrovascular disorder.

Claims (17)

  A therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of interleukin-17.   A therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of interleukin 23.   A therapeutic agent for ischemic cerebrovascular disorder comprising a substance that inhibits the function of γδT cells or infiltration of the γδT cells into the brain.   The therapeutic agent according to claim 1, wherein the substance that inhibits the function of interleukin 17 is an antibody against interleukin 17 or an inhibitory nucleic acid of interleukin 17 gene.   The therapeutic agent according to claim 4, wherein the inhibitory nucleic acid of the interleukin 17 gene is an antisense nucleic acid, decoy nucleic acid, microRNA, shRNA or siRNA against the interleukin 17 gene.   The therapeutic agent according to claim 2, wherein the substance that inhibits the function of interleukin 23 is an antibody against interleukin 23 or an inhibitory nucleic acid of interleukin 23 gene.   The therapeutic agent according to claim 6, wherein the inhibitory nucleic acid of the interleukin 23 gene is an antisense nucleic acid, decoy nucleic acid, microRNA, shRNA or siRNA against the interleukin 23 gene.   The therapeutic agent according to claim 3, wherein the substance that inhibits the function of γδ T cells or the infiltration of the γδ T cells into the brain is an antibody against γδ T cells or an inhibitory nucleic acid of a γδ T cell receptor gene.   The therapeutic agent according to claim 8, wherein the inhibitory nucleic acid of the γδ T cell receptor gene is an antisense nucleic acid, decoy nucleic acid, microRNA, shRNA or siRNA against the γδ T cell receptor gene.   The therapeutic agent according to claim 1 or 3, wherein the cerebrovascular disorder is 3 days after ischemia or later.   The therapeutic agent according to claim 2, wherein the cerebrovascular disorder is within 24 hours after ischemia.   An interleukin 17 production inhibitor comprising a substance that inhibits the function of interleukin 23.   An interleukin-17 production inhibitor comprising a substance that inhibits the function of γδT cells or infiltration of the γδT cells into the brain.   The inhibitor according to claim 12, wherein the substance that inhibits the function of interleukin 23 is an antibody against interleukin 23 or an inhibitory nucleic acid of interleukin 23 gene.   The inhibitor according to claim 13, wherein the substance that inhibits the function of γδ T cells or the infiltration of the γδ T cells into the brain is an antibody against γδ T cells or an inhibitory nucleic acid of a γδ T cell receptor gene.   The inhibitor according to claim 14, wherein the inhibitory nucleic acid of the interleukin 23 gene is an antisense nucleic acid, decoy nucleic acid, microRNA, shRNA, or siRNA against the interleukin 23 gene.   The therapeutic agent according to claim 15, wherein the inhibitory nucleic acid of the γδ T cell receptor gene is an antisense nucleic acid, decoy nucleic acid, microRNA, shRNA or siRNA against the γδ T cell receptor gene.