JP2017057173A - Composition for treating brain damage disease targeting TIM-3 and screening method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for preventing or treating brain damage diseases, the composition comprising TIM-3 (T-cell immunoglobulin and mucin domain protein 3) inhibitor as an active ingredient, and also provide a screening method for a therapeutic agent for brain damage disease, the method using TIM-3.SOLUTION: The present invention provides a pharmaceutical composition for preventing or treating brain damage diseases, based on the following findings: TIM-3 protein plays a role as a modulator in brain injury induced during the state of hypoxia caused by ischemia; and expression of TIM-3 is regulated by HIF-1, which regulates the expression of genes occurring in hypoxia. Therefore, the composition according to the present invention can be usefully used for the treatment and prevention of cerebral nervous system diseases associated with hypoxia.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a pharmaceutical composition for the prevention or treatment of brain injury diseases comprising a TIM-3 (T-cell immunoglobulin and mucin domain protein 3) inhibitor as an active ingredient, and a screening for therapeutic agents for brain injury diseases using TIM-3. Regarding the method.

  Cerebral ischemia causes complex pathophysiological changes, ultimately causing brain damage, particularly in the penumbrial area surrounding the ischemic core of ischemic tissue. Such changes include activation of resident cells, generation of inflammatory mediators and infiltration of inflammatory cells. According to the results of clinical experiments, the inflammatory response due to cerebral ischemia seems to be related to the onset of brain injury, but much is not yet known about the inflammatory response associated with this.

  TIM-3, a member of the T-cell immunoglobulin and mucin domain protein family, is a first-type helper T that negatively regulates TH1-dependent immune responses. Although identified for the first time as a cell (TH1) -specific surface molecule, in subsequent studies, TIM-3 was found to be TH17 cells, Tregs, NK cells, protein leukocytes (monocytes), resinous cells, mast cells. And is expressed from various types of epithelial cells, including microglia, and regulates not only adaptive immunity but also innate immunity It has been revealed. According to recent research results, TIM-3 plays an important role in regulating the activation of congenital epithelial cells and acts as an activation marker or activation limiting factor depending on the environment. In animal models and the human body, TIM-3 has been shown to be closely related to a variety of immune related diseases including infections, autoimmune diseases and cancer. Interestingly, TIM-3 is said to exhibit various functions depending on the cell type and environment (Non-Patent Document 1). For example, suppression of TIM-3 in epigenetic virus infection and tumors increases the effector function of depleted T cells, while increased TIM-3 signaling is Th-1-mediated EAE ( It has been shown to improve experimental autoimmune encephalomyelitis). In addition, decreased TIM-3 levels on CD4 + CD25-T cells in autocidal hepatitis contributed to impaired immune regulation, whereas TIM-3 on CD4 + and CD8 + T cells was overexpressed in late hepatitis C .

  The physiological response to hypoxia is a heterodimer consisting of an oxygen-regulated alpha-subunit and a constitutive beta-subunit (constitutive β-subunit). It is known to be mainly mediated by HIF (hypoxia-inducible factor) -1, which is a heterodimeric transcription factor. HIF-1 complexes bind to hypoxic-response elements (HREs) of various genes associated with adaptation to hypoxia. Interestingly, HIF-1 regulates cellular responses not only in a hypoxic environment but also in an inflammatory environment and is thought to play an important role in the pathogenesis of many inflammation-related diseases. In in vivo and in vitro experiments, HIF-1 has been shown to be essential for bone marrow cell-mediated inflammatory responses such as bone marrow cell migration. HIF-1 activity was also associated with pathogenic inflammatory responses following ischemic lung and intestinal damage. Therefore, HIF-1 is regarded as a core regulator that regulates inflammation-related signal transmission.

  The central nervous system (CNS), on the other hand, was known to be an immune-privileged region, but recent studies have accelerated innate and acquired adaptive immune responses. It has been reported that it has an elaborate monitoring system that can be triggered. In the immune response of the CNS, glial cells that function as the main epithelial cells recognize minute changes in the brain and respond quickly to pathophysiological stimuli.

  As a result of studies based on the conventional reports as described above, the present inventors have increased TIM-3 expression in microglia and astrocytes in a hypoxic environment, It was newly discovered that such increased expression of TIM-3 affects the infiltration of neutrophils into hypoxic penumbra. Such infiltration is known as a major cause of ischemic brain damage. The present inventors have also clarified the fact that HIF-1 regulates the oxygen-dependent expression of TIM-3 in glial cells, and completed the present invention from the results of such experiments.

US2014 / 0099254A1

Han, G .; , Et al. (2013) Cramer, T.A. et al. HIF-1 alpha is essential for myeloid cell-mediated inflation. Cell 112, 645-57 (2003) Zhang, L.M. et al. Estrogens stimulates microglia and brain recovery from hypoxia-ischemia in normotic butt diabetic female. J. et al. Clin. Invest. 113, 85-5 (2004) Swanson, R.M. A. et al. A semi-automated method for measuring brain inform volume. J. et al. Cereb. Blood Flow Metab. 10, 290-93 (1990) Frank, M.M. G. Wieseler-Frank, J .; L. , Watkins, L .; R. & Maier, S .; F. isolation of high enriched and quiet microglia from adult hippocampus: immunophenotypic and functional charactaristics. J. et al. Neurosci. Methods 151, 121-30 (2006) Weinstein, D.C. Isolation and purification of primary rodent astrocycles. Curr. Protoc. Neurosci. Chapter 3, Unit 35 (2001) Chang, C.I. Y. et al. Dual function of myeloperoxidase in rotenone-exposed brain-resident immuno cells. Am. J. et al. Pathol. 179, 964-79 (2011) Huang, Z .; et al. Effects of cerebral ischemia in mificant in neuron nitrite synthase. Science 265, 1883-885 (1994) Bloom, B.W. et al. The hypoxic response of tumors is dependent on the microenvironment. Cancer Cell 4, 133-46 (2003) Kutner, R.A. H. , Zhang, X. Y. & Reiser, J .; Production, contention and titration of pseudotyped HIV-1-based lenticular vectors. Nat. Protoc. 4,495-05 (2009) Zhang, L.M. et al. Estrogens stimulates microglia and brain recovery from hypoxia-ischemia in normotic butt diabetic female. J. et al. Clin. Invest. 113, 85-95 (2004) Wang, G .; L. & Semenza, G .; L. Characteristic of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. et al. Biol. Chem. 268, 21513-21518 (1993) Bergeron, M.M. Yu, A .; Y. , Solway, K .; E. , Semenza, G .; L. & Sharp, F.A. R. Induction of hypoxia-inducible factor-1 (HIF-1) and it's target genes following focal ischaemia in rat brain. Eur. J. et al. Neurosci. 11, 4159-4170 (1999) Williams, R.A. The Mouse Brain Library http: // www. mbl. org / atlas165 / atlas165_start (1999) Franklin, K.M. B. J. et al. & Paxinos, G.M. The Mouse Brain in Stereocoordinates 3rd edn (Elsevier / Academic Press, 2008) Gerriets, T .; et al. Noninvasive quantification of brain edema and the spacecapping effect in rat stroke models using magnetic resonance imaging. Stroke 35, 566-571 (2004) Le, D.C. A. et al. Caspase activation and neuroprotection in caspase-3-definitive microphone after in vivo cerebral ischemia and in vitro oxygen glucose deprivation. Proc. Natl Acad. Sci. USA99, 15188-15193 (2002) Broughton, B.M. R. Reutens, D .; C. & Sovey, C.I. G. Apoptotic machinery after cerebral ischemia. Stroke40, e331-e339 (2009) Chaitanya, G .; V. Steven, A .; J. et al. & Babu, P.A. P. PARP-1 cleavage fragments: signatures of cell-death protocols in neurogeneration. Cell Commun. Signal. 8, 31 (2010) McColl, B.M. W. Rothwell, N .; J. et al. & Allan, S .; M.M. Systematic stimulus potentials the exact phase and CXC chemokin responses to experiential brains and excurbate brain vein dimensions. J. et al. Neurosci. 27, 4403-. 4412 (2007) Chen, H.C. et al. Anti-CD11b monoclonal antibodies redismatic cell damage after transient focal cerebral ischemia in rat. Ann. Neurol. 35,458-. 463 (1994) Kobayashi, S .; D. , Voyich, J. et al. M.M. Burlake, C .; & DeLeo, F.A. R. Neutrophils in the innate immune response. Arch. Immunol. Ther. Exp. (Warsz). 53,505-517 (2005) Murkinati, S.M. et al. Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutropil recruitment. FASEB J.H. 24, 788-798 (2010) Muir, K.M. W. Tyrrell, P .; Sattar, N .; & Warburton, E .; Inflammation and ischemic stroke. Curr. Opin. Neurol. 20, 334-. 342 (2007) Saijo, K .; & Glass, C.I. K. Microcellular cell origin phenotypes in health and disease. Nat. Rev. Immunol. 11,775-. 787 (2011) Ren, X. et al. Regulatory B cells limit CNS inflation and neurological defects in murine experomental stroke. J. et al. Neurosci. 31, 8556-. 8563 (2011) Kleinschnitz, C.I. et al. Post-stroke inhibition of induced NADPH oxidase type 4 presents oxidative stress and neurodegeneration. PLoS Biol. 8, pii: e1000479 (2010)

  An object of the present invention is to provide a pharmaceutical composition for preventing or treating a brain injury disease that suppresses this expression or activity targeting TIM-3 (T-cell immunoglobulin and mucin domain protein 3).

  Another object of the present invention is to provide a method for screening for a therapeutic agent for brain injury using TIM-3.

  In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a brain injury disease comprising an inhibitor of TIM-3 (T-cell immunoglobulin and mucin domain protein 3) as an active ingredient.

  In one embodiment of the invention, the TIM-3 inhibitor binds to, reacts with, or modulates the expression of TIM-3, such as directly or indirectly binding to TIM-3. A substance that can specifically suppress or reduce expression or activity, and includes organic or inorganic compounds, proteins, antibodies, peptides, nucleic acid molecules, and the like. In one embodiment of the present invention, the TIM-3 inhibitor is an antagonistic antibody or a fragment thereof that specifically binds to or reacts with TIM-3 and specifically suppresses or decreases the activity of TIM-3. However, it is not limited to this. In one embodiment of the present invention, the TIM-3 inhibitor is a nucleic acid molecule that directly or indirectly suppresses the expression of the TIM-3 gene. Examples of such a nucleic acid molecule include a TIM-3 gene or Examples include, but are not limited to, antisense nucleotides, siRNA, shRNA, or miRNA for the fragment.

  In one embodiment of the present invention, the TIM-3 protein consists of an amino acid sequence of sequence number 1, and the TIM-3 gene consists of a sequence of sequence number 2.

  In one embodiment of the present invention, the TIM-3 inhibitor suppresses the expression or activity of an upstream gene of the TIM-3 gene or an expression regulatory site of the TIM-3 gene, thereby expressing TIM-3. The action which suppresses.

  In one embodiment of the present invention, the TIM-3 inhibitor suppresses the expression or activity of HIF-1 (hypoxia-inducible factor-1).

  In one embodiment of the present invention, the TIM-3 inhibitor decreases the expression or activity of a neutrophil chemotactic factor to inhibit neutrophil migration and invasion, thereby It represents the preventive or therapeutic effect of the injured disease.

  The present invention also includes (a) a step of treating a candidate substance in a cell or animal model in which TIM-3 is expressed, and (b) a step of measuring the expression or activity level of TIM-3 after the candidate substance treatment And (c) selecting a candidate substance having a decreased expression or activity level of TIM-3 as compared to a control group not treating the candidate substance, and a screening method for a therapeutic agent for a brain injury disease.

  In one embodiment of the present invention, the screening method further includes the step of additionally analyzing whether the candidate substance selected in the step (c) suppresses the expression or activity of HIF-1 compared to the control group. Including.

  In one embodiment of the present invention, the method of measuring the step (b) and / or analyzing the expression or activity of the HIF-1 is performed by immunohistochemical staining, PCR, RT-PCR, Western blot, ELISA or protein chip. However, the method is not limited to this.

  In one embodiment of the present invention, the cell expressing TIM-3 is a glial cell, but is not limited thereto. In one embodiment of the present invention, the animal model is a hypoxia-ischemia brain injury disease model, but is not limited thereto.

  Examples of brain injury diseases to which the present invention can be applied include, but are not limited to, cerebral infarction, stroke, hypoxic brain injury, ischemic brain disease, and medium wind. According to one embodiment of the present invention, the brain injury disease is an inflammation-related brain injury that occurs in a hypoxia environment.

  The present inventors have the role of TIM-3 protein as a modulator in brain injury induced by hypoxia caused by ischemia, and the expression of TIM-3 regulates gene expression generated in hypoxia To be regulated by HIF-1. Therefore, the present invention may be usefully used for the treatment and prevention of cranial nervous system diseases associated with hypoxia, for example, cerebral infarction, stroke, hypoxic brain injury, ischemic brain disease and medium wind disease. it can.

FIG. 5 shows that TIM-3 is expressed in hypoxia-induced brain regions of a mouse model of hypoxic ischemic stroke. (A) TIM-3 transcription level from 24 hours after induction of hypoxic-ischemic stroke, from contralateral cortex (C, boxed region) from mouse model and from ipsilateral cortex (I, boxed region) from which stroke was induced Brain tissue was removed and measured. The results obtained by the reverse transcriptase gene amplification method were measured using the image J program and quantified to reflect the expression of actin. HIF-1α transcription levels were expressed as a hypoxic positive control. The right panel shows TTC staining of brain cross-sectional tissue of a mouse model of hypoxic ischemic stroke induction. (B) Western blot analysis shows expression of TIM-3 and HIF-1 proteins (n = 3). The relative degree of TIM-3 expression was expressed as a significant value measured from three independent experiments. (C) TIM-3 expression is confirmed through immunohistochemistry using Tim-3 antibody in the ipsilateral cortex and contralateral cortex of the hypoxic-ischemic stroke mouse brain cross section and expresses TIM-3 The number of cells was measured by number per mm ² . (D) The immunohistochemical method was measured using a TIM-3 and a hypoprobe-1 (red, to detect hypoxic regions) antibody after removing a brain section from a mouse in which hypoxic ischemic stroke was induced. . Scale bars, 50 μm (× 20); 50 μm (× 40). (E, f) Brain cells were separated from ipsilateral and contralateral sites from 3 animals per group, and GFAP (f) capable of representing astrocytes and Iba-1 (e ), And TIM-3 antibody was added all at once and analyzed using a FACS (fluorescence-based cell sorter). This result represented relative TIM-3 levels in gated microglia and was confirmed through three independent experiments. FIG. 5 shows that HIF-1α binds to the TIM-3 promoter and regulates its expression in primary cultured glial cells. (A) BV2 cells are maintained in a 20% O ₂ or 1% O ₂ environment for 24 hours, and then the cell surface expression of TIM-3 is stained with PE-conjugated TIM-3 antibody to obtain fluorescence-using cells. Analysis was performed using a sorter. The results obtained through three independent experiments were represented as bar graphs, and the change in mean value (± sd) compared to normal environment samples. (B) Glial cells cultured from mice were cultured for 24 hours in a normal environment and a hypoxic environment, and the cells were confirmed for TIM-3 expression through a plague cytochemistry method using Tim-3 antibody. . (C, d) Glial cells and neurons primarily cultured from mice were cultured in normal and hypoxic environments for 24 hours, and the expression levels of TIM-3 and actin were measured by the reverse transcriptase gene amplification method. The mean value change (± sd) is shown graphically through experiments in which expression changes were repeated three times independently (NS, statistics not valid, Student-Newman-Keuls test). (E) Glial cells derived from mice were cultured in normal and hypoxic environments for 24 hours, and immunoprecipitation purification was carried out with HIF-1 antibody and control group IgG. The results are shown graphically through three independent experiments. (F) Glial cells derived from HIF-1α ^{+ f / + f} mice were infected with Ad-GFP or Ad-Cre / GFP virus, and TIM-3-luciferase reporter (Tim-3 promoter gene was transferred to the infected cells). The containing vector) constructs were transfected and cultured in normal and hypoxic environments for 24 hours. Promoter gene expression activity was expressed as ratio of luciferase activity / β-galactosidase activity. (G, h) The reverse transcriptase gene amplification method (g) and the Western blot analysis method (h) were performed in a normal environment and a hypoxic environment for 24 hours using a primer and an antibody. This data was represented through three independent experiments independently. The graph shows TIM-3 gene transcription and protein expression levels compared to cells infected with Ad-GFP in a hypoxic environment. IP, immunoprecipitation TIM-3 blockade indicates a significant reduction in brain damage induced after hypoxic-ischemic stroke. (A) Representative view showing an image of a TTC-stained brain section from a hypoxic-ischemic stroke model mouse treated with IgG (n = 12) and 100 μg of TIM-3 blocking antibody (n = 12). Infarct volume was analyzed through the image J program and the damaged ipsilateral site expressed as a percentage. (B) Representative of MRI (magnetic resonance imaging) obtained from mice treated with TIM-3 antibody (n = 4) and IgG (n = 4) 24 hours after induction of hypoxic-ischemic stroke Photo. (C) T2-MRI (magnetic resonance imaging) obtained from mice treated with TIM-3 antibody (n = 4) and IgG (n = 4) 24 hours after induction of hypoxic ischemic stroke Representative photo. (D) The degree of edema formation was obtained through T2-weighted MRI images and ADC map. (E) 24 hours after induction of hypoxic-ischemic stroke, NeuN (neuronal cell) cleaved caspase-3 (detection of cell death was detected in brain sections obtained from mice treated with TIM-3 antibody and mice treated with IgG. Representative photograph measured by a confocal microscope after immunohistochemical method. Scale bar, 50 μm. The graph shows the average number of cells per mm ² stained with NeuN and the cleaved caspase-3 antibody. (F) 24 hours after induction of hypoxic-ischemic stroke, the full-length PARP protein (in the contralateral and ipsilateral cortex obtained from mice treated with TIM-3 antibody and mice treated with IgG ( Western blot photograph showing the expression of a protein representing cell death). The graph shows the level of full-length PARP in comparison. All data were expressed as significant values from three independent experiments. FIG. 5 shows that TIM-3 blocking antibodies reduce neutrophil migration. Reverse transcriptase gene amplification method (a) and Western blot analysis method (b) were used to measure MPO expression in hypoxic ischemic stroke model mice treated with IgG and TIM-3 blocking antibodies. The graph shows the MPO level compared. (C) 24 hours after induction of hypoxic ischemic stroke, immunohistochemistry was performed with MPO and Gr-1 antibodies from brain sections obtained from mice treated with TIM-3 antibody and mice treated with IgG, Representative photograph measured with a confocal microscope. Scale bar, 50 μm. The graph shows the average number of cells stained with MPO and Gr-1 antibody per mm ² (± sd). (D) Cerebral cortex derived from hypoxic-ischemic stroke model mice and (e) Brain sections obtained at the basal site were subjected to immunohistochemistry using MPO and Gr-1 antibodies, and MPO and Gr-1 The number of cells stained per mm ² was counted. Blocking TIM-3 both in vivo and in vitro shows that the expression of two representative neutrophil chemotaxis factors is reduced. (A) Glial cells (2 × 10 ⁵ ) primarily cultured from mice were laid on a transchamber low chamber, and TIM-3 and control group IgG antibody were first treated as shown, followed by spleen cells 5 × 10 ⁵ is placed on the upper chamber. After culturing for 24 hours in a hypoxic condition, the degree of migration to spleen cell low chamber was analyzed using a fluorescence-based cell sorter. The percentage of Gr-1 ^high CD11b ^high cells that migrated to the low chamber through three independent experiments was the mean ± s. Expressed with d. (B) Gr-1 ^high CD11b ^high neutrophils are isolated from bone marrow of C57BL / 6 mice and cultured in a hypoxic environment together with gliocytes treated with IgG and TIM-3 antibody. The results obtained from three independent experiments represent the degree of reduction when IgG treated cells are viewed as 1. (C) Reverse transcriptase gene amplification was performed on tissues obtained from a hypoxic-ischemic stroke model treated with IgG and TIM-3. (D) The graph shows the results corrected with actin (n = 3). (E) Glial cells derived from mice are treated with IgG and TIM-3 antibody and cultured for 24 hours in normal and hypoxic environments. CXCL1 and IL-1beta transcription levels were determined by reverse transcriptase gene amplification. The graph is derived from the results of three independent experiments. NS, not effective. Hypoxia-induced neutrophil migration is shown to decrease in an environment devoid of HIF-1. (A) Glial cells (2 × 10 ⁵ ) primarily cultured from HIF-1α ^{+ f / + f} mice were infected with Ad-GFP or Ad-Cre / GFP virus, and placed on the low well of the transwell, 5 × 10 ⁵ Is placed on the upper chamber. After culturing for 24 hours under hypoxia, the extent to which spleen cells migrated to low chamber was analyzed using a fluorescence-based cell sorter. (B) Gr-1 ^high CD11b ^high neutrophils were isolated from bone marrow of C57BL / 6 mice, and glial cells primarily cultured from HIF-1α ^{+ f / + f} mice were subjected to Ad-GFP or Ad-Cre / GFP virus. And incubate in a hypoxic environment like neutrophil cells. The results obtained through three independent experiments showed altered migration of neutrophil cells compared to glial cells from HIF-1α ^{+ f / + f} mice infected with Ad-GFP. (C) CXCL1 and IL-1beta transcript levels were confirmed by culturing glial cells infected with Ad-GFP or Ad-Cre / GFP virus in normal and hypoxic environments for 24 hours. (D) The graph is the result obtained from real-time quantitative PCR. It is an experimental result which shows that the brain damage induced | guided | derived by the hypoxic-ischemic-stroke is reduced in a LysM-Hif-1 (alpha) <^-/-> transformed mouse. (A) Reverse transcriptase gene amplification was performed using primers displayed on glial cells cultured from HIF-1α ^{+ f / + f} or LysM-Hif-1α ^{− / −} mice. (B) TIM-3 gene transcription levels from HIF-1α ^{+ f / + f} or LysM-Hif-1 ^{α − / −} mice (n = 3) contralateral cortex and ipsilateral cortex in which ischemic stroke was induced Confirmed from the derived brain tissue. (C) TTC-stained brain section from HIF-1α ^{+ f / + f} (n = 12) or LysM-Hif-1α ^{− / −} mice (n = 12) in which hypoxic ischemic stroke was induced for 24 hours A representative diagram showing an image. Infarct volume was analyzed through the image J program and the damaged ipsilateral site expressed as a percentage. (D) 24 hours after hypoxic stroke, NeuN (an antibody that detects nerve cells) cleaved caspase-3 (cell death) in brain slices obtained from HIF-1α ^{+ f / + for} LysM-Hif-1α ^{− / −} mice A representative photograph measured by a confocal microscope after immunohistochemistry with a detection antibody). Scale bar, 50 μm. The graph shows the average number of cells per mm ² stained with NeuN and the cleaved caspase-3 antibody. (± sd 3 independent experiments) Experimental results show that LV-TIM3-GFP inoculation of LysM-Hif-1a-/-mice increases cerebral infarction area and neurological sequelae. (A) Using an IVI spectrum system (Xenogen IVIS-200), mice inoculated with lentivirus overexpressing PBS, GFP, and mice inoculated with lentivirus overexpressing TIM-3 and GFP The representative figure which measured the fluorescence image (excitation filter, from 445 to 490 nm, and emission filter, from 515 to 575 nm). (B) Representative view showing an image of a brain section stained with TTC from a mouse inoculated with LV-TIM3-GFP or LV-GFP. (C, d) Infarct size (c, n = 6 for LV-GFP or n = 5 for LV-TIM3-GFP) and neurological sequelae (d, n = 6 for reach group) are hypoxic-ischemic Examined 24 hours after induction of stroke. It is a schematic diagram of a TIM-3 related event that can occur in a hypoxic brain environment. Hypoxia-dependent HIF-1a activity increases TIM-3 expression in microglia and astrocytes. Activation of the HIF-1 / TIM-3 axis induces neutrophil attractant production and neutrophil infiltration in hypoxic areas. Unusual neutrophil infiltration phenomena induce an excessive inflammatory response, which in turn is responsible for the pathophysiological environment of the brain. TIM-3 shRNA was infected with primary culture cells or BV2 microglia cells under 1% oxygen condition and 20% oxygen condition, respectively. 10A shows the result of primary cultured glial cells, 10B shows the result of BV2 microglia cells, 10A shows the results of PCR analysis, and b shows the results of immunocytochemistry. Represent.

  TECHNICAL FIELD The present invention relates to a composition for treating brain injury diseases caused by hypoxia such as ischemic stroke and a screening method for a therapeutic agent for brain injury diseases, and specifically, TIM (T-cell immunoglobulin and mucin domain protein) -3 suppression. A composition for treating a brain injury disease comprising an agent as an active ingredient, and (a) a step of treating a candidate substance in a cell or animal in which TIM-3 is expressed, and (b) a level of expression or activity of TIM-3. The present invention relates to a screening method for a therapeutic agent for a brain injury disease, comprising a step of measuring, and (c) selecting a candidate substance having a decreased expression or activity level of TIM-3 compared to a control group not treated with the candidate substance.

  Cerebral ischemia causes a series of pathophysiological changes and induces brain damage. Inflammatory mediator production and penetration is an important step in causing brain damage, and clinical and research results suggest that the degree of brain damage due to cerebral ischemia is very closely related to the inflammatory condition. It has increased. Therefore, there is a growing interest in strategies for developing therapeutic agents for cranial nervous system diseases that target inflammation control. To date, however, there has been a limit that very little information is known about the inflammatory response associated with ischemic brain disease.

  The present invention relates to brain injury caused by a hypoxia (hypoxia) state occurring after ischemia, which is associated with inflammatory cell penetration and inflammatory response, where TIM-3 is abnormal. It is characterized by demonstrating that it affects the death of brain cells and the reduction of cerebral infarction sites. The present invention relates to brain damage induced by hypoxia caused by ischemia, in which TIM-3 protein plays a role as a modulator, and TIM-3 expression is expressed in hypoxia. Based on the research results of being regulated by HIF-1 that regulates According to one embodiment of the present invention, expression of TIM-3 is increased in hypoxia-induced brain cells of a hypoxic-ischemic mouse model mouse model (Hypoxia-ischemia mouse model). (FIG. 1), TIM-3 expression was regulated by HIF-1 (FIG. 2). Also, TIM-3 blockade reduces cerebral infarction and brain cell death following hypoxic ischemia (FIG. 3), reduces neutrophil migration to the brain and migration-related cytokines. Confirmed (FIG. 4). Hypoxia-induced neutrophil migration and brain damage are also reduced in the hypoxic-ischemic stroke model of HIF-1-deficient mice (FIG. 6), which can increase TIM-3 expression in the mice. Brain damage increased again. Such a result represents an association between HIF-1 / TIM-3 axis and brain injury in a hypoxic environment.

  Therefore, the present invention relates to a pharmaceutical composition for preventing or treating brain injury diseases, for example, cerebral infarction, stroke, hypoxic brain injury, ischemic brain disease and medium wind disease, comprising a TIM-3 inhibitor as an active ingredient. Can be provided. As used herein, the term “prophylaxis” refers to the administration of a therapeutic agent (eg, a prophylactic or therapeutic agent) or a combination of therapeutic agents that manifests, recurs, or develops in a subject with signs of brain injury. Means to prevent. As used herein, the term “treatment” means to improve or regulate symptoms or any one or more physical parameters of a patient with a brain injury disorder or delay its development or progression, It doesn't matter whether it is recognized or not. The pharmaceutical composition of the invention comprises one or more pharmaceutically acceptable carriers, excipients or diluents. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifier, an antiseptic and the like. Carriers suitable for use include, but are not limited to, aqueous media including saline, phosphate buffered saline, minimal essential medium (MEM) or MEM in HEPES buffer.

  In addition, the pharmaceutical compositions of the present invention are formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. be able to. Dosage forms may be in the form of powders, granules, refined, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. The pharmaceutical compositions of the present invention may be administered by intramuscular, subcutaneous, transdermal, intravenous, intranasal, intraperitoneal or oral route, preferably by intramuscular or subcutaneous route. The dosage of the composition is appropriately selected according to various factors such as the administration route, the age, sex, weight and severity of the animal.

  The pharmaceutical composition of the present invention is formulated in the following various oral or parenteral dosage forms, but is not limited thereto. First, solid preparations for oral administration include tablets, pills, powders, granules, hard or soft capsules, and such solid preparations contain at least one or more active ingredients of the present invention. Formulated with excipients. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions or syrups, but include various excipients in addition to commonly used simple diluents such as water and liquid paraffin. May be. The pharmaceutical composition of the present invention can also be administered parenterally, and parenteral administration is based on a method of injecting a subcutaneous injection, an intravenous injection, an intramuscular injection, or an intrathoracic injection. In this case, in order to formulate into a dosage form for parenteral administration, the active ingredient of the present invention is mixed with water together with a stabilizer or a buffer to produce a solution or suspension, which is then administered in unit of ampoules or vials. Can be manufactured in molds. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations or suppositories. As the non-aqueous solvent or suspension, propylene glycol (polypropylene glycol), polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate may be used. In addition, the pharmaceutical composition of the present invention may be administered to mammals such as mice, rats, domestic animals and humans by various routes, and examples thereof include oral, rectal, intravenous, muscle, subcutaneous, intrauterine. For example, dura mater or intracerebral intravascular injection. The pharmaceutical composition of the present invention is administered by selecting an appropriate method according to the patient's year, sex and weight.

  Hereinafter, the present invention will be described in more detail with reference to examples. The following examples are merely for explaining the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples. is there.

Experimental Materials and Methods <1-1> Experimental Animals Mice with HIF-1α ^{+ f / + f} (HIF-1α-floxed alleles) produced by Dr. Randall Johnson were used. Bone marrow lineage cells lacking HIF-1α were produced from crossbreeding of HIF-1α ^{+ f / + f} mice and LysM-Cre transformed mice (Non-patent Document 2). Eight week old male C57BL / 6 mice (Orient Bio) were used for in vivo and in vitro experiments.

<1-2> Measurement of volume of hypoxic cerebral ischemia model and infarction (infarct) H / I was measured for C57BL / 6 male mice (8 weeks, Orient Bio) by the method as described in Non-Patent Document 3. Induced. Briefly, mice are anesthetized with Zoletil (Virbac) and Rompun (Bayer) (4: 1) to expose the right common carotid artery of each mouse and 4-0 surgical silk. (silk). The incision site was sutured and the mice were allowed to recover for 2 hours with excess food and water. Systemic hypoxia was induced by exposure to 8% oxygen / balance N2 in a temperature controlled hypoxia chamber (BioSpherix, C-474). Such a transient unilateral cerebral ischemia model produces reproducible brain damage in the ipsilateral hemisphere, but not in the contralateral hemisphere. For TIM-3-blocking experiments, mice were treated with 100 μg rat IgG2a, kisotype (eBioscience, 16-4321) or anti-TIM-3 monoclonal antibody (eBioscience, RMT-) 30 min after H / I. 3-23) was injected intravenously. After 24 hours of H / I, the mice were killed, the brains were removed, immediately cut into 2 mm thick sections, and incubated with TTC at 37 ° C. for 30 minutes. The image of the section was observed with a stereo microscope (Zeiss, Stereo Discovery. V20) equipped with a camera. Infarct volume was measured in an indirect manner to compensate for edema of the infarcted tissue, calculated as a percentage of the ratio of damaged area to hemispheric area, and corrected for hemispheric swelling due to edema. The formula for calculating the infarct volume is as follows (Non-patent Document 4):
Infarct volume (%) = [(contralateral hemisphere-healthy area of ipsilateral hemisphere) / contralateral hemisphere] × 100

<1-3> Measurement of magnetic resonance imaging (Magnetic resonance imaging)
Mice are fixed to the animal bed and placed under the MRI measurement equipment (Bruker7T BioSpec) and then anesthetized during the video measurement. A T2-weighted image was obtained using Rapid Acquisition with a Relaxation Enhancement sequence. 18 adjacent axial slices of 0.7 mm thickness were obtained [matrix 256 × 256; field of view = 20 × 20 mm; TR (Repetition Time) = 2,500 ms; TE (Echo Time) = 35 ms; acquisition time = 4 Min; no gap]. The ADC (approximate diffusion coefficient) map was obtained by a diffusion-weighted image using a spin-echo sequence. For this reason, eight adjacent axis images were obtained [thickness 0.7 mm, matrix 256 × 128, field of view = 20 × 20 mm, TR = 2,000 ms, TE = 26.936 ms, acquisition time = 16 minutes, 1 average, b values = 45,350, 1,000 and 2,000 s per mm ² , no gap]. The ADC map was obtained with a scanner. Edema volume was obtained from T2-weighted images and ADC maps were obtained from Image J analyzer. Oedema volume (%) = [(Ipsilateral volume-contralateral volume) / contralateral volume] x 100.

<1-4> Separation of microglia and astrocytes from mouse brain tissue Microglia were separated from brain tissue by a known method (Non-patent Document 5). Briefly, brains were removed from perfused mice and divided into ipsilateral and contralateral hemispheres, then sharpened to 250 μgml ⁻¹ collagenase IV / DNase I. After the treatment, it was decomposed by incubation at 37 ° C. for 45 minutes. The cell lysate was fractionated at 1,000 g for 25 minutes with a 50/70% Percoll gradient. Microglia were collected at the interface between the 50 and 70% bands and washed with HBSS (hanks' balanced salt solutions) (Welgene). The purity of the separated microglia was measured by FACS analysis. Astrocytes were separated by a known method (Non-patent Document 6). Briefly, cell suspensions from brain tissue were fractionated at 1,000 g for 25 minutes with a 30/60% Percoll gradient. Astrocytes were collected at the PBS / 30% interface. The purity of the separated astrocytes was measured by FACS analysis using an anti-GFAP antibody (Cell Signaling Technology, # 3670, 1: 500).

<1-5> Glial cells and neuron-enhanced midbrain culture Primary mixed glial cells were cultured from mouse cerebral cortex after 1 to 3 days (Non-patent Document 7). ). The percentage of microglia in mouse mixed glial cultures was determined to be 30.50% by FACS analysis using anti-CD11b antibody (eBioscience, 11-0112, 5 μgml ⁻¹ ). Neuron-enriched midbrain cells were cultured from mice on the 14th embryonic day (Non-patent Document 7). Briefly, ventral mesencephalic tissues are dissected, incubated with CMF-HBSS (Ca2 +, Mg2 + -free HBSS) for 10 minutes, and 0.01% trypsin in CMF-HBSS. ) For 9 minutes at 37 ° C. Cultures were supplemented with 10% fetal bovine serum, 6 mg ml ⁻¹ glucose, 204 mg ml ⁻¹ L-glutamine and 100 Uml ⁻¹ penicillin / streptomycin (P / S) for inhibition of trypsin (Dulbecco's modified Eagle's medium) and then crushed to separate into single cells. Cells were dispensed into poly-D-lysine (5 mg ml ⁻¹ ) and laminin (0.2 mg ml ⁻¹ ) coated plates (2 × 10 ⁶ cells per well).

<1-6> Adenovirus transduction (Adenoviral transduction)
Non-proliferating adenovirus (AD-GFP / Cre) in which the Cre recombinase gene is expressed under the control of the cytomegalovirus promoter was purchased from Vector Biolabs. Reporter Ad-GFP was used as a control group (Vector Biolabs). For adenovirus transduction, primary mixed glial cells were cultured from HIF-1α ^{+ f / + f} mice and infected with Ad-GFP or Ad-GFP / Cre for 24 hours [MOI (multiplicity of infection). ) = 100]. The infection efficiency measured by flow cytometry was about 50%.

<1-7> ChIP Assay A ChIP assay was performed using a ChIP assay kit (Upstate Biotechnology). Mouse primary mixed glial cells were cultured for 24 hours in a hypoxic environment, immediately fixed with 1% formaldehyde / phosphate-buffered saline, and sonicated for 500-1,000- A bp DNA fragment was obtained. Chromatin was immunoprecipitated with 5 μg anti-HIF-1α (Novus, NB100-134) or rabbit IgG. The immunoprecipitated DNA was amplified with a promoter pair specific for the TIM-3-promoter [F, 5′-CCTGCTGCCTTGGAATTTTGC-3 ′ (order number 3); and R, 5′-GAGTACTTGGCAGGGGAAATC-3 ′ (order number 4)].

<1-8> Neutrophil migration assay (Netrophil migration assay)
Based on the binding of FITC-conjugated anti-CD11b (eBioscience, 11-0112, 5 μgml ⁻¹ ) and PE-conjugated anti-Gr-1 (Ly6G) (eBioscience, 12-5931, 2 ugml ⁻¹ ), FACS Aria Neutrophils were isolated using a system (BD Bioscience). Sorted neutrophils were added to 24-well plates containing primary mouse mixed glia in the upper chamber of the Transwell. The cells were cultured for 24 hours at 1% or 20% oxygen conditions. The migration was measured using a haematocytometer and flow cytometry.

<1-9> Measurement of neurological sequelae The neurological sequelae was evaluated using a neurological scoring system (Non-patent Document 8). The neurological scores of mice are as follows: 0, normal motor function; 1, contralateral torso and forelimb reflexes by lifting the tail; and forelimb lifting by; 2, turning to the contralateral side when the mouse was held by the tail; 3, and the biasing to the contralateral side; Loss of spontaneous motor activity.

<1-10> Immunohistochemistry
Brains were removed for immunohistochemistry, fixed and embedded in paraffin. A microtome was used to cut coronal sections (10-mm thickness) through the infarct site and mounted on the slide. Paraffin was removed and sections were washed with PBS-T and blocked with 10% bovine serum albumin for 2 hours. The following primary antibodies were then applied: goat anti-TIM-3 (Santa Cruz Biotechnology, sc-30326, 2 μgml ⁻¹ ), rat anti-Gr-1 (Ly6G) (eBioscience, MPO (Dako, A0398, 10 μgml). ^-1 ), rabbit anti-Iba-1 (Wako, # 019-19741, 2 μgml ⁻¹ ), rabbit anti-cleaved case-3 (Cell Signaling Technology, # 9662S, 1: 300), mouse antiMileNeup # MAB377,10μgml ^-1). pimonidazole (pimonidazole) (Hypoxyprobe-1, Natural Pharma The hypoxic region was detected using ia International (Non-Patent Document 9), and images were obtained using a confocal microscope (Carl Zeiss LSM510), measuring TIM-3 expression in primary glial cells. For this purpose, mouse primary mixed glial cells were fixed with methanol, washed with PBS-T, and cultured at 4 ° C. with anti-TIM-3 antibody (R & D Systems, AF1529, ¹ μgml ⁻¹ ).

<1-11> TIM-3 Promoter Assay A 1,517-bp fragment of the mouse TIM-3 promoter (−1,517 to +1 with respect to the start codon) was PCR-amplified from genomic DNA, and PGL3 basic vector (Promega) Cloned into. Site-directed mutagenesis of each HRE was performed using mutant primers and Phusion High-Fidelity DNA polymerase (NEB). All constructs were confirmed by DNA sequencing. Lipofectamine 2000 (Invitrogen) was used to transfect mouse primary mixed glial cells (primary mixed glial cells). After transfection, the cells were cultured for 24 hours under 1% or 20% oxygen conditions, and the reporter gene activity was measured with a luciferase assay system (Promega). Beta-galactosidase activity was measured for normalization of transfection efficiency.

<1-12> Analysis of Western Blot The right and left hemispheres of H / I mice were dissected, and protease inhibitors [2 mM phenylmethylsulfonfluoride, 100 μgml ⁻¹ leueptin, 10 μgml ⁻¹ peptin, ¹ μmml2 peptin, ¹ μgml- ¹ Homogenized by pellet pellet (Fisher) with ice-cooled RIPA buffer containing [EDTA]. The homogenized product was centrifuged at 12,000 rpm for 30 minutes at 4 ° C., and the upper layer liquid was collected. Samples were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and cultured with the following primary antibodies: goat anti-TIM-3 (R & D Systems, AF1529, 0.1 μgml ⁻¹ ), Mouse anti-PARP (Zymed, 33-3100, 2 μgml ⁻¹ ), rabbit anti-MPO (Dako, A0398, 2 μgml ⁻¹ ), goat anti-Iba-1 (Abcam, ab5076, 0.5 μgml ⁻¹ ), mouse anti-GFAP (Cell Signaling Technology, # 3670, 1: 1,000), mouse anti-NeuN (Millipore, # MAB377, 1 μgm) l ⁻¹ ), mouse anti-α-tubulin (Sigma, T5168, 1: 5,000), microtubule-associated protein 2 (Millipore, # MAB3418, ¹ μgml ⁻¹ ), glutamate decalboxycylamine 110 μg, ¹ , Peroxidase-conjugate goat anti-rabbit (Bio-Rad, # 170-6515, 1: 5,000), peroxidase-conjugated rabbit anti-goat (Zymed, R-21259, 1: 5,000), peroxydase crate anti-mouse (Bio-Rad, # 170-6516, 1: 5,000). Results were visualized using an enhanced chemiluminescence system and quantified with a densitometric analysis (Image J software, NIH). All experiments were performed independently and repeated at least three times.

<1-13> RT-PCR analysis Total RNA was isolated using Easy-Blue (iNtRON), and cDNA was synthesized using avian myeloblastosis reverse transcriptase (TaKaRa) according to the manufacturer's instructions. PCR was performed in a continuous reaction of 25-30 cycles. All experiments were performed independently and repeated at least three times, and PCR products were quantified using NIH Image J and normalized to actin. Real-time PCR was performed using the QuantFast SYBR Green PCR kit (Qiagen). Real-time PCR was performed using the Roche LightCycler 480 Real-Time PCR System (Roche Applied Science) and the LightCycler 480 Quantification Software Version 1.5.

The primers used for quantitative PCR are as follows:
For IL-1β (forward) 5′-GGATGGAGACATGAGCACCT-3 ′ (order number 5) and (reverse) 5′-TCCATTGAGGTGGAGAGCTTT-3 ′ (order number 6);
(Forward) 5'-TGCACCCAAACCGAAGTCCAT-3 '(order number 7) and (reverse) 5'-TTGTCAGAAGCCAGGTTCAC-3' (order number 8) for CXCL1;
(Forward) 5′-CTCATCAGTTGCCACTTCC-3 ′ (order number 9) and (reverse) 5′-TCATCTTCACTGTTCTAGACCAC-3 ′ (order number 10) for HIF-1α;
(Forward) 5′-TGTCGTGGAGTCTACTGGTGTCTCTC-3 ′ (order number 11) and (reverse) 5′-CGTGGTTCACACCCATCACAA-3 ′ (order number 12) for GAPDH.

Other PCR primer sequences used are as follows:
(Forward) 5'-CCCTGCAGGTTACACTCTACC-3 '(order number 13) and (reverse) 5'-GTATCCTGCAGCAGTAGGGTC-3' (order number 14);
(Forward) 5′-AGCCCTTAACCCTTCTGCCACTT-3 ′ (order number 15) and (reverse) 5′-GAAATCATTATACATTGCATATATAGAGACAT-3 ′ (order number 16) for HIF1α;
(Forward) 5′-AGGATAGAGACTGGATTTGCCTG-3 ′ (order number 17) and (reverse) 5′-GTGGTGATGCCAGTGTGTCA-3 ′ (order number 18);
For IL-1β (forward) 5′-TACAGGCTCCCGAGATGAACAACAA-3 ′ (order number 19) and (reverse) 5′-TGGGGAAGCATCATAAAACAGTCC-3 ′ (order number 20);
(Forward) 5'-CGCTCGCTTCTCTGTGCAGC-3 '(order number 21) and (reverse) 5'-GTGGCTATGAACTTCGGTTTGG-3' (order number 22) for CXCL1;
(Forward) 5'-CATGTTTGAGCACTTCAACACCCC-3 '(order number 23) and (reverse) 5'-GCCATCTCCTCTCGAGATCTAG-3' (order number 24).

<1-14> Flow cytometry
All staining steps were performed in the dark and blocked with BD Fc Block. Freshly obtained microglia and astrocytes were stained with the following antibodies: rabbit anti-Iba-1 (Wako, # 019-19714, ¹ μgml ⁻¹ ) followed by Alexa 488-conjugated chicken anti-rabit (Invitrogen, A21441, 2 μgml ⁻¹ ), and PE-conjugated anti-mouse TIM-3 (eBioscience, RMT-3-23, 2 μgml ⁻¹ ) or isotype control Ab (eBioscience, 2 μgml ⁻¹ ) for 30 minutes at 4 ° C. For intracellular staining, cells were fixed and permeabilized for 20 minutes using IC fixation / permeabilization buffer (eBioscience) and permeabilized (pe rmeabilization) buffer twice, incubated with anti-GFAP (Cell Signaling Technology, # 3672, 1: 500) in permeabilization buffer for 30 minutes, Alexa 488-conjugated chicken anti-mouse (Invitrogen, A21200), 2 μgml- ¹ Stained with Data were analyzed with Cell-Quest software (BD Bioscience) and Flow Jo software (Treestar) packages.

<1-15> Lentivirus production and stereotaxic injection
The coding sequence of TIM-3 (GE Dharmacon) is PLL3.7. PLL3.7. Conjugated to EF1α plasmid (Addgene, Inc.). EF1α-TIM3 was produced. Recombinant lentivirus LV-TIM3-GFP was constructed using the plasmid. As a control group, a lentiviral vector (LV-GFP) expressing only GFP was prepared. Lentivirus was titrated using flow cytometry (Non-Patent Document 10). LV-TIM3-GFP or LV-GFP was injected using a stereotaxic instrument. Each mouse received four intracranial injections of lentivirus (20 μl containing 5 × 10 ⁶ TUml ⁻¹ in the right hemisphere). Collected cells were analyzed by Western blotting using FACS and anti-GFP antibody (Santacruz, sc-9996, 1: 1,000) for in vitro fluorescence imaging. Whole body in vivo imaging was performed using Caliper Life Science's Xenogen IVIS Spectrum [excitation filters at 445 to 490 nm, emission filters at 515 to 575 nm].

<1-16> Analysis of data All data are expressed as mean ± s. Displayed with d. Post-hoc comparisons (Student-Newman-Keuls test) were performed using SigmaPlot 10.0. Neurological scores were evaluated by nonparametric statistical processing. Two groups (IgG vs anti-TIM-3, HIF-1α ^{+ f / + f} mice vs LysM-Hif-1α ^{− / −} mice, LV-GFP injection LysM-Hif-1α ^{− / −} mice vs LV-TIM3-GFP injection Comparison between LysM-Hif-1α ^{− / −} ) was analyzed by Mann-hitney U-tests.

Increased TIM-3 expression in the hypoxic penumbra To investigate the molecular mechanism underlying the interdependent relationship between ischemic brain injury and inflammation, we Candidate molecules that could play a major role in the pathophysiological inflammatory response due to cerebral hypoxia-ischaemia (H / I) were investigated. For this reason, after unilateral ligation of the right carotid artery, a transient unilateral ischemia mouse model in which systemic hypoxia was induced (Non-patent Document 11) ). After obtaining tissue from the contralateral and penumbral cortex areas 24 hours after H / I, the expression levels of various inflammation-related molecules were investigated at the RNA and protein levels. As a result, it was found that the transcriptional level of TIM-3 (T-cell immunoglobulin and mucin domain-3) increased much higher in the contralateral regions in the ipsilateral penumbra. In addition, it was confirmed that TIM-3 protein was also increased from the contralateral region in the ipsilateral penumbra (FIGS. 1a and b). The ipsilateral semi-shaded area was reported to have a high transcript and protein level of HIF-1, which is a positive control group under hypoxia (Non-patent Document 12; and Non-patent Document 13).

  In order to confirm the above results, immunohistochemistry was performed on coronal sections of H / I mice using an antibody against TIM-3 (Non-Patent Document 14; and Non-Patent Document 15). As a result, it was confirmed that the number of TIM-3-positive cells was greatly increased in the ipsilateral semi-shade so as to agree with the above results (FIG. 1c). Furthermore, it was confirmed that TIM-3 was highly expressed in the hypoxia half-shade of H / I mice stained with hypoxyprobe-1 using pimonidazole (hypoxyprobe-1), which is a hypoxia marker ( FIG. 1d).

  These results indicate that TIM-3 expression is up-regulated in hypoxic half-shadows, suggesting that TIM-3 can play a role in pathophysiological changes due to cerebral ischemia.

Upregulation of TIM-3 expression in hypoxic glial cells The inventors investigated that after H / I any cell exhibits TIM-3 upregulation. As a result of Western blot analysis, activated microglia marker Iba-1 (ionized calcium binding adapter molecule-1) and activated stars in the ipsilateral cortex of H / I mice 24 hours after H / I. The protein expression level of GFAP (glial fibrous acid protein), which is a glial cell marker, was even higher than that of the contralateral cortex. On the other hand, the expression levels of neuronal cell markers such as NeuN (neuronal nuclei), microtubule-associated protein 2 and glutamate decarboxylase are determined by the semi-shadow cortex tissue. Decreased.

  Therefore, the present inventors investigated the expression level of TIM-3 in microglia and astrocytes 24 hours after H / I. As a result of immunohistochemistry, many regions of TIM-3-expressing cells in the ipsilateral cortex of H / I mice appeared Iba-1 positive. Strong expression of TIM-3 was also observed in GFAP-immunoreactive astrocytes in the ipsilateral cortex. Furthermore, as a result of FACS (Fluorescence-activated cell sorting) analysis of brain cells isolated from H / I mice, hypoxia-ischaemia leads to microglial and astrocyte activity. Represents increased TIM-3 expression. Microglia that express high levels of Iba-1 and astrocytes that express high levels of GFAP greatly increase in ipsilateral penumbra after 24 hours of H / I, It means that glial cells and astrocytes were activated in a hypoxic environment. In addition, TIM-3 expression is significantly higher in Iba-1-positive microglia and GFAP-positive astrocytes isolated from the ipsilateral cortex than in the contralateral region. Appeared (FIGS. 1e, f).

  These results support the fact that TIM-3 expression is greatly increased in microglia and astrocytes activated under hypoxia.

HIF-1-dependent increase of TIM-3 in hypoxic environment Based on the results of the experiment, the present inventors changed the expression of TIM-3 in glia cells by oxygen tension. Whether it could be done was studied using BV2 microglia and primary cultured glial cells. BV2 cells were cultured for 24 hours in normoxic (20% O ₂ ) or hypoxic (1% O ₂ ) conditions, and TIM-3 cell surface levels were determined by FACS analysis. It was measured. Interestingly, TIM-3 expression was greatly increased in hypoxic conditions (Figure 2a). Immunocytochemistry analysis results also show that TIM-3 expression in mouse primary mixed glial cells is greatly increased in a hypoxic environment compared to a normoxic environment (FIG. 2b). In addition, the present inventors have confirmed that the transcription level of TIM-3 was increased in primary mixed glial cells in a hypoxic environment, but not increased in primary neuronal cells (Fig. 2c, d). Such results indicate that hypoxia induces TIM-3 expression in glial cells.

HIF-1 is a major transcriptional regulator of many genes in a hypoxic environment. To investigate whether the up-regulation of TIM-3 stimulated by hypoxia in glial cells is mediated by HIF-1, we have identified anti-HIF-1α antibodies and potential HRE consensus sequences (HIF- A ChIP assay (chromatin immunoprecipitation assay) was performed using TIM-3 promoter regions (responsible elements (HRE) consensus sequences). As shown in FIG. 2e, HIF-1α was able to bind to the HRE-containing TIM-3 promoter region in primary mixed glial cells in a hypoxic environment. Furthermore, to confirm the above results, we investigated the activity of the TIM-3 promoter in HIF-1α-deficient glial cells. After culturing primary mixed glial cells from HIF-1α ^{flox / flox} (HIF-1α ^{+ f / + f} ) mice, adenovirus-Cre / GFP (Ad-Cre / GFP) or control group GFP (green fluorescent protein (GFP) ) Was encoded with the adenovirus encoding (Ad-GFP)). The efficiency of virus infection was confirmed using FACS, and cells were transfected with TIM-3 luciferase reporter (-1,517 / + 1), and then TIM-3 promoter activity was measured. As expected, TIM-3 promoter activity in hypoxic environments was greatly increased in the control group Ad-GFP-infected glial cells (HIF1α ^{+ f / + f} ), but Ad-Cre / GFP-infected, HIF -1α-deficient glial cells (HIF1α ^{Δ / Δ} ) were greatly reduced (FIG. 2f). Site-directed mutation of potential HREs of the TIM-3 promoter greatly reduced the hypoxia-dependent increase in luciferase activity compared to the wild-type reporter. In addition, the increase in TIM-3 transcript and protein by hypoxic stimulation was significantly suppressed in Ad-Cre / GFP-infected HIF-1α-deficient glial cells (FIGS. 2g and h).

  These results indicate that TIM-3 expression is regulated in a HIF-1-dependent manner in a hypoxic environment.

Reduction of brain damage due to TIM-3 inhibition in mouse H / I model Since TIM-3 was up-regulated in glial cells of the H / I mouse model, the inventors post-cerebral H / I brain The role of TIM-3 induced by hypoxia was investigated. Therefore, the effect of TIM-3-inhibitory antibody on brain damage 24 hours after H / I was investigated using TTC (2,3,5-triphenyltetrazolium) staining. As shown in FIG. 3a, it was confirmed that the TTC-negative region was greatly reduced in the mouse intravenously injected with 100 μg of TIM-3-suppressing antibody compared to the control group IgG-injected mouse. Such results indicate that TIM-3-suppressing antibodies can reduce brain damage in a hypoxic environment.

  Edema, which is a life threatening result of cerebral infarction, appears with inflammation and ischemic brain injury (Non-patent Document 16). Therefore, the inventors investigated the effect of TIM-3-suppression on H / I-induced edema formation. To observe infarct area and edema formation, T2-weighted magnetic resonance images were acquired from day 1 to day 7 of H / I. Similar to the results obtained from TTC staining, infarct and edema formation in the ipsilateral hemisphere of TIM-3-antibody-injected mice on day 1 of H / I compared to IgG-injected mice The edema formation and infarct reduction persisted on days 3, 5, and 7 (FIGS. 3c, d).

  To further investigate the relationship between post-H / I brain injury and TIM-3, the effect of TIM-3-inhibitory antibodies on neuronal cell death is considered to be an important role in cerebral ischemia. It investigated by measuring the expression of caspase-3 which is an effector protease (cell non-patent literature 17; and non-patent literature 18). As a result of immunohistochemistry, the expression of caspase-3 was greatly increased in neuronal cells in the ipsilateral cortical region of IgG-treated H / I mice, whereas such increase was observed in mice treated with TIM-3 inhibitory antibody. It was greatly reduced (Fig. 3e). Next, ischemic cell killing with a marker of caspase-3 activity cleaved by caspase-3 in the ipsilateral and contralateral cortex of H / I mice treated with control group IgG or TIM-3-suppressor antibody The level of PARP (poly (ADP-ribose) polymerase) related to the above was measured (Non-patent Document 19). As shown in FIG. 3f, expression of full length PARP was greatly reduced in the ipsilateral cortical tissue of control IgG-injected H / I mice, but not in TIM-3-suppressing antibody-injected H / I mice. It was.

  Such results indicate that suppression of TIM-3 can greatly reduce infarct sites and neuronal cell death after cerebral ischemia in mice.

Reduction of Neutrophil Infiltration by TIM-3 Inhibition According to various studies, neutrophils are rapidly infiltrated in the ischemic brain within hours and are involved in the development of inflammatory responses and brain damage ( Non-patent document 20; and Non-patent document 21). Since glial cells are one of the cells that respond primarily to brain injury that exhibits associated activity within minutes after the onset of ischemia, we have identified TIM- HIF-1-dependent increase of 3 affects neutrophil infiltration into the ischemic penumbra, and downregulation of TIM-3's ability to collect neutrophils We hypothesized that brain damage after blood could be reduced. Thus, first, the expression of two typical neutrophil markers, MPO (myeloperoxidase) and Gr-1 (granulocyte receptor-1), was measured, and at 24 hours after H / I, It was confirmed that cells positive for the marker (MPO ⁺ Gr-1 ⁺ ) greatly increased in the penumbral cortex and striatum compared to the region. Next, the present inventors investigated whether glial cells (gial cells) can collect Gr-1 ^high CD11b ^high neutrophils in a hypoxic environment. Does it contain control group cells of mouse embryonic fibroblasts known to isolate spleen cells from C57BL / 6 mice and collect primary mixed glial cells or epithelial cells at the site of injury? In a Transwell system not containing the cells, the cells were cultured for 24 hours under oxygen conditions of 1 or 20% (Non-patent Document 22). In the presence of glial cells or mouse fetal fibroblasts, Gr-1 ^high CD11b ^high cells migrated very much into the lower chamber in a hypoxic environment, but only a few cells in a normic environment moved. However, such hypoxia-dependent increase in Gr-1 ^high CD11b ^high cell migration was greatly reduced in the absence of glial cells. Such results suggest that glial cells may be involved in collecting Gr-1 ^high CD11b ^high cells in a hypoxic environment.

Next, we tested the effect of TIM-3-suppression on the infiltration of neutrophils into ipsilateral hemispheres at 24 hours after H / I. The results of reverse transcription-PCR (RT-PCR) and Western blot analysis on cortical tissue of H / I mice showed that the level of MPO expression was much higher in TIM-3-suppressing antibody-treated mice compared to control group IgG-treated mice (FIGS. 4a and 4b). Experimental results of immunohistochemistry on the coronal surface of the ipsilateral cortex also show that MPO ⁺ Gr-1 ⁺ cells are greatly reduced by TIM-3-suppressing antibody treatment (FIG. 4c). Such a result was also confirmed by an immunohistochemical experiment using an anti-neutrophil and an anti-MPO antibody. Also, the effect of TIM-3 inhibition on neutrophil infiltration was measured at various time points using the coronal plane of many ipsilateral regions of the H / I brain (bregma-2 to +2). As shown in FIGS. 4d, e, even fewer numbers of MPO ⁺ Gr-1 ^{+ in} the semi-shadow cortex and striatum of mice that suppressed TIM-3 at all observation time points (1-7 days). Cells were observed.

  The above results strongly suggest that TIM-3 is associated with infiltration of neutrophils into the damaged brain in a hypoxic environment.

Reduction of neutrophil recruitment due to TIM-3 blockade To further specifically measure the effect of TIM-3 on neutrophil migration, glial cells in the hypoxic environment It was investigated whether the ability to replenish was affected by TIM-3 blockade. Using a Transwell system, primary glial cells are plated in the lower chamber, pretreated with TIM-3-suppressing antibody or control IgG, and then spleen cells in the upper chamber (Splenocytes) was loaded. Cells were cultured for 24 hours under 1% oxygen conditions, and the percentage of Gr-1 ^high CD11b ^high cells in the lower chamber was determined by FACS analysis. As a result, it was confirmed that Gr-1 ^high CD11b ^high cells in the lower chamber in a hypoxic environment were greatly reduced by 10 mg of TIM-3-suppressor antibody compared to control group IgG (FIG. 5a).
In order to further verify the above results, migration of bone marrow (BM) -derived Gr-1 ^high CD11b ^high cells in a hypoxic environment was investigated. Gr-1 ^high CD11b ^high
Cells were separated from BM cells and plated in the upper chamber, and the lower chamber contained TIM-3-suppressor antibody or control group IgG-treated primary mixed glia in 1% oxygen conditions. glial cells) was loaded. Consistent with the above results, migration of BM-derived Gr-1 ^high CD11b ^high cells to the lower chamber was greatly reduced by TIM-3-suppressor antibody treatment compared to control IgG treatment (FIG. 5b). ). These results clearly show the role of glial TIM-3 in neutrophil recruitment to the hypoxic region after cerebral ischemia.

Decrease of neutrophil chemotactics by TIM-3 suppression Neutrophil infiltration or infiltration of the site of injury is regulated by chemoattractants, which are neutrophils in the postischemic brain Prior to infiltration, it is adjusted upward (Non-patent Document 23). Therefore, the present inventors investigated the influence of TIM-3 inhibition on the levels of IL-1β and CXCL1 that act as neutrophil chemotactic factors in ischemic brain (Non-patent Document 24). At 30 minutes after H / I, mice were injected intravenously with 100 mg TIM-3-suppressor antibody or control group IgG. After 24 hours, IL-1β and CXCL1 transcription levels were examined in ipsilateral and contralateral cortical tissues. As shown in FIGS. 5c, d, all transcript levels of IL-1β and CXCL1 were greatly increased in the ipsilateral cortical regions of H / I mice injected with control IgG, but this effect is -3- Much decreased in mice injected with inhibitory antibodies.

  To further investigate the role of glial TIM-3, the effect of TIM-3 blockade on IL-1β and CXCL1 expression levels was investigated. The cells were treated with TIM-3-suppressor antibody or control group IgG and cultured for 24 hours under 1% oxygen or 20% oxygen conditions. Consistent with the above results, the levels of IL-1β and CXCL1 transcripts increased in IgG-treated control cells cultured in 1% oxygen compared to 20% oxygen, but this increase Was greatly reduced in cells treated with TIM-3-suppressor antibody (FIGS. 5e, f).

  These results indicate that cell TIM-3 is a factor that plays an important role in the pathogenesis of cerebral ischemia through the regulation of neutrophil infiltration.

HIF-1 deficiency reduces neutrophil migration and infarcts Based on the discovery that HIF-1α regulates TIM-3 expression in hypoxic glial cells, we have developed HIF-1 It was investigated whether -1α affects neutrophil recruitment ability of glial cells in a hypoxic environment. Primary mixed glial cells cultured from HIF-1α ^{+ f / + f} mice were infected with Ad-GFP or Ad-GFP / Cre and 1% or 20% oxygen with splenocytes in a transwell system The culture was performed for 24 hours under the above conditions. The percentage of Gr-1 ^high CD11b ^high cells in the lower chamber in a hypoxic environment was determined by the control group Ad-GFP-infection when spleen cells were cultured with Ad-GFP / Cre-infected HIF-1α-deficient glial cells. It was greatly reduced compared to cells. On the other hand, the number of Gr-1 ^high CD11b ^high cells that migrated under 20% oxygen conditions was not significantly different between HIF-1α-deficient and normal cells (FIG. 6a). Next, we found that the number of migrated BM-derived Gr-1 ^high CD11b ^high cells was greatly reduced by culturing with HIF-1α-deficient glial cells in 1% oxygen condition. Discovered (Figure 6b). Also, IL-1β and CXCL1 in Ad-GFP / Cre-infected HIF-1α-deficient glial cells that do not show hypoxia-dependent increase in TIM-3 compared to control group Ad-GFP-infected cells. The hypoxia-dependent increase in was greatly reduced (FIGS. 6c, d).

It is known that microglia become resident myeloid cells in the brain (Non-patent Document 25). In order to confirm the role of glial cell HIF-1α, the inventors of the present invention used LysMCre-HIF-1α ^{+ f / + f} (LysM-Hif-1α ^{− / −} ) mice specifically lacking HIF-1α in bone marrow cells. The degree of brain damage after H / I was investigated. First, the present inventors measured the level of HIF-1α in primary microglia of LysM-Hif-1α ^{− / −} mice. As shown in FIG. 7a, the levels of HIF-1α transcripts were very low in LysM-Hif-1α ^{− / −} mouse microglia compared to HIF-1α ^{+ f / + f} . At 24 hours after H / I, the level of TIM-3 transcripts was also lower in the eastern cortical region of LysM-Hif-1α ^{− / −} mice (FIG. 7b). We found that the TTC staining-negative region was greatly reduced in LysM-Hif-1α ^{− / −} mice compared to HIF-1α ^{+ f / + f} mice, which was 24 hours of H / I. Later, it represents the role of microglia HIF-1α in brain injury (FIG. 7c). The expression of caspase-3 was also greatly reduced in LysM-Hif-1α ^{− / −} mouse neuron cells compared to HIF-1α ^{+ f / + f} mice (FIG. 7d). Furthermore, no significant increase in IL-1β and CXCL1 expression was detected in the ipsilateral cortex of LysM-Hif-1α ^{− / −} mice 24 hours after H / I.

  These results indicate that in hypoxia, HIF-1α is closely associated with TIM-3-related neutrophil infiltration and linked brain damage.

Effects of TIM-3 blockade and HIF-1α deficiency on NDS In order to investigate whether reduced infarct volume and neuronal cell death are associated with improved neurological function, known methods are used. The NDS (neurologic defect score) was measured using the H / I model (Non-Patent Document 26; and Non-Patent Document 27). Neurological sequelae are contralateral torso and forelimb flexion, circling to the contralateral side, leaning to the contralateral side. It was measured by the lateral side at rest, and spontaneous motor activity (spontaneous motor activity). Neurological sequelae due to H / I were reduced in mice treated with TIM-3-suppressing antibodies compared to IgG-treated mice. NDS for IgG treated mice was 2.8 ± 0.8 (± sd) 20 hours after H / I, whereas NDS for TIM-3-suppressed antibody treated mice was 0.8 ± 0.8. 0.8 (Table 1; P = 0.012; Mann-Whitney U-test).

Such results indicate that TIM-3 is associated with neural function in a hypoxic environment. Next, the present inventors, for HIF-1α ^{+ f / + f} mice (n = 10) and LysM-Hif-1α ^{− / −} mice (n = 11), when 24 hours after H / I NDS was measured. HIF-1α ^{+ f / + f} mice were observed to have leaning behavior and absence of spontaneous motor function, but not to LysM-Hif-1α ^{− / −} mice. Mean NDS in LysM-Hif-1α ^{− / −} mice was much lower than in HIF-1α ^{+ f / + f} mice (Table 2; 1.2 ± 0.6 vs. 2.6 ± 1.1, P = 0) .0008)

  These results indicate that the HIF-1α / TIM-3 axis (axis) is closely related not only to cerebral infarct volume and pathophysiological inflammatory response, but also to neurological function.

Increased nerve damage by TIM-3 in HIF-1α-deficient mice We investigated whether TIM-3 could affect the traits of HIF-1α-deficient mice after H / I. Therefore, a lentiviral vector (LV-TIM3-GFP) expressing TIM-3 and GFP was produced. First, after investigating whether lentivirus can infect glial cells, we observed that TIM-3 expression was greatly increased in GFP-positive-CD11b ^high CD45 ^low glial cells of lentivirus-injected mice did. The virus was injected into the right hemisphere of LysM-Hif-1α ^{− / −} mice using a stereotaxic instrument. Control group mice were injected with LV-GFP expressing only GFP. Four intracranial injections were made in the right hemisphere of each mouse (Figure 8a). H / I was injected into LysM-Hif-1α ^{− / −} mice with LV-TIM3-GFP or LV-GFP and induced 5 days later, the infarct size and neurological results were 24 hours I investigated later. As shown in FIGS. 8b and c, the TTC-stained-negative region was greatly increased in the LV-TIM3-GFP-injected mice (n = 5) compared to the control group LV-GFP-injected mice (n = 6). . In addition, the mean NDS for LysM-Hif-1α ^{− / −} mice injected with LV-TIM3-GFP was higher than that of LV-GFP-injected control group mice (FIG. 8d) (1.1 ± 0.7 vs. 2). .3 ± 0.8, P = 0.046). Such a result is a result showing again the relationship between the HIF-1 / TIM-3 axis and brain damage in a hypoxic environment.

Analysis of TIM-3 inhibitory activity using shRNA against TIM-3 The experiments in the above examples were carried out using an antibody against TIM-3, and the inventors further suppressed TIM-3. As another method that could be used, the availability of shRNA against TIM-3 was confirmed. For this reason, a lentivirus expressing a shRNA against TIM-3 or a control range virus was first produced in a primary cultured glial cell (FIG. 10A) or V2 microglia (FIG. 10B) (Santacruz # sc-72015-V). Cells were infected according to the instructions provided by Thereafter, the infected cells are cultured for 24 hours under 1% or 20% oxygen condition, and TIM-3 is expressed using reverse transcriptase chain reaction analysis, immunocytochemistry and flow cytometry. These experiments were obtained as results from three independent repeated experiments and expressed as meanSD.

  As a result of the analysis, as shown in FIG. 10, the shRNA against TIM-3 used in the experiment of the present invention was effectively required to express Tim-3 when compared with the control group, In the group treated with shRNA against TIM-3 under hypoxic conditions, it was shown that the increase in TIM-3 expression was inhibited compared to the group treated with the control group.

  Therefore, in view of these results, a TIM-3 inhibitor comprising an antibody or shRNA against TIM-3 that can inhibit TIM-3 expression or activity effectively inhibits TIM-3 expression or activity. Thus, it has been found that such inhibitors can be used as formulations for the prevention or treatment of brain injury diseases.

  So far, the present invention has been studied focusing on its preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Accordingly, the disclosed embodiments are to be considered in an illustrative rather than a restrictive perspective. The scope of the present invention is shown not in the above description but in the claims, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (13)

  A pharmaceutical composition for preventing or treating a brain injury disease comprising a TIM-3 (T-cell immunoglobulin and mucin domain protein 3) inhibitor as an active ingredient.   The composition according to claim 1, wherein the TIM-3 inhibitor is a compound, protein, antibody, peptide, or nucleic acid capable of specifically suppressing or decreasing TIM-3 expression or activity. object.   The antibody is an antagonistic antibody or a fragment thereof that binds to or reacts with TIM-3 represented by the amino acid sequence of SEQ ID NO: 1 and specifically suppresses or decreases the activity of TIM-3. A composition according to claim 2 characterized.   The composition according to claim 2, wherein the nucleic acid is an antisense nucleotide, siRNA, shRNA, or miRNA against the TIM-3 gene represented by the base sequence of sequence number 2 or a fragment thereof.   The composition according to claim 1, wherein the TIM-3 inhibitor suppresses the expression or activity of HIF-1 (hypoxia-inducible factor-1).   2. The composition of claim 1, wherein the TIM-3 inhibitor decreases the expression or activity of a neutrophil chemotactic factor.   The composition according to any one of claims 1 to 6, wherein the brain injury disease is selected from the group consisting of cerebral infarction, stroke, hypoxic brain injury, ischemic brain disease and medium wind. . (A) treating a candidate substance in a cell or animal model in which TIM-3 is expressed;
(B) measuring the expression or activity level of TIM-3 after the candidate substance treatment;
(C) screening a candidate substance for treating a brain injury disease, comprising selecting a candidate substance having a decreased expression or activity level of TIM-3 as compared to a control group not treating the candidate substance.   The measurement in the step (b) is performed by a method selected from the group consisting of immunohistochemical staining, PCR, RT-PCR, Western blot, ELISA, or protein chip. the method of.   9. The method according to claim 8, further comprising the step of additionally analyzing whether the selected candidate substance suppresses the expression or activity of HIF-1 compared to the control group.   9. The method of claim 8, wherein the cell is a glial cell.   9. The method of claim 8, wherein the animal model is a hypoxia-ischemia brain injury disease model.   13. The method according to any one of claims 8 to 12, wherein the brain injury disease is selected from the group consisting of cerebral infarction, stroke, hypoxic brain injury, ischemic brain disease and medium wind.

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