WO2010021516A9 - Nouvelle utilisation de lipocaline 2 pour traiter des lesions cérébrales - Google Patents

Nouvelle utilisation de lipocaline 2 pour traiter des lesions cérébrales Download PDF

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WO2010021516A9
WO2010021516A9 PCT/KR2009/004676 KR2009004676W WO2010021516A9 WO 2010021516 A9 WO2010021516 A9 WO 2010021516A9 KR 2009004676 W KR2009004676 W KR 2009004676W WO 2010021516 A9 WO2010021516 A9 WO 2010021516A9
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lcn2
astrocytes
cells
protein
cell
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WO2010021516A2 (fr
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석경호
이신려
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경북대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • the present invention relates to a novel use of lipocalin 2 for the treatment of brain injury, and more particularly, a composition for treating brain injury containing a lipocalin 2 (Lipocalin 2, LCN2) inhibitor and a method for treating brain injury by administering the same. It is about.
  • a lipocalin 2 Lipocalin 2, LCN2
  • Astrocytes are the most abundant glia cell type in the brain (Barres, BA et al. 2000, Curr Opin Neurobiol 10: 642-648 Aschner, M. 1998, Neurotoxicology 19: 269-281). Astrocytes provide neuronal metabolism and nutritional support and modulate synaptic activity. They are involved in the formation and maintenance of blood-brain barriers for neuroprotection, antioxidant defense, ion and pH homeostasis, and neuronal-glial networks. In addition to the physiological role of astrocytes in this intact central nervous system (CNS), astrocytes are key responders to damage to the CNS under various physiological conditions such as injury, ischemia, infection and neurodegeneration. (Farina, C. et al.
  • Astrocytes become reactive in response to all forms of CNS damage and undergo a process called reactive astrocytosis (Correa-Cerro, LS et al. 2007, J Neuropathol Exp Neurol 66: 169-176; Sofroniew , MV 2005, Neuroscientist 11: 400-407; Pekny, M. et al. 2005, Glia 50: 427-434).
  • astrocytes proliferate to fill gaps, increase the amount of cytoplasm, protuberance and branching, glial fibrillary acidic protein (GFAP), nestin and nonmethine ( conventional morphological changes such as increased expression of intermediate fibers such as vimetin).
  • GFAP glial fibrillary acidic protein
  • reactive astrocytosis is beneficial or detrimental.
  • Reactive astrocytosis is associated with the protection of nerve damage and the promotion of vascular brain barrier repair.
  • reactive astrocytosis is also associated with potentially harmful effects such as inhibiting the regeneration of axons that act as barriers and increasing the secretion of pro-inflammatory and neurotoxic mediators (Silver, J. et. al. 2004, Nat Rev Neurosci 5: 146-156).
  • lcn2 lipocalin 2
  • MET mesenchymal-epithelial transition
  • Lcn2 is an inducer of endogenous epithelial cells (Yang, J. et al, 2002, Mol Cell 10: 1045-1056) and stimulates transformed cells to regulate invasion and metastasis (Hanai, J. et al. 2005 J Biol Chem 280: 13641-13647; Lee, HJ et al.
  • lcn2 promotes tubulogenesis by regulating morphogenesis of epithelial cells (Lee, HJ et al. 2006, Int J Cancer 118: 2490-2497).
  • Lcn2 is a member of the lipocalin family and binds to or carries lipids and other hydrophobic molecules (Flower, DR et al. 2000, Biochim Biophys Acta 1482: 9-24; Kjeldsen, L. et al. 2000, Biochim Biophys Acta) 1482: 272-283. Lcn2 also contains 24p3 (Flower, DR, et al. 1991. Biochem Biophys Res Commun 180: 69-74), SIP24 (24 kDa superinducible protein) (Hamilton, RT et al.
  • Lcn2 neurotrophil gelatinase-associated lipocalin, a human homologue of lcn2
  • NGAL neurotrophil gelatinase-associated lipocalin, a human homologue of lcn2
  • lcn2 has been suggested as an indicator of active kidney injury (Mori, K. et al. 2007, Kidney Int 71: 967-970). In various forms of gastrointestinal damage, lcn2 facilitates cell regeneration by promoting cell migration (Playford, RJ et al. 2006, Gastroenterology 131: 809-817). However, in vivo studies in mice lacking lcn2 refute the role of lcn2 in protecting kidneys (Berger, T. et al. 2006, Proc Natl Acad Sci USA 103: 1834-1839). In addition, mice lacking lcn2 did not have iron sequestration, which increased the susceptibility to bacterial infection.
  • lcn2 prevents degradation of NACP (Yan, L. et al. 2001, J Biol Chem 276: 37258-37265), and acute phase protein (Liu, Q. et al. 1995, J Biol Chem 270: 22565 -22570) and has been suggested to act as adipokine associated with insulin resistance (Yan, QW et al. 2007, Diabetes 56: 2533-2540; Zhang, J. et al. 2008, Mol Endocrino l 22: 1416-1426). Recently two cellular receptors for lcn2 have been identified.
  • LCN2 is proactively involved in astrocytosis, which inhibits the regeneration of neurons, and thus, by inhibiting LCN2, it is possible to promote the regeneration of neurons after brain injury and to complete the present invention. Reached.
  • the present invention provides a composition for treating brain injury containing an LCN2 inhibitor.
  • the present invention relates to a method of treating brain injury by administering a composition for treating brain injury containing an LCN2 inhibitor.
  • Astrocytes one of the brain's most abundant glial cells, provide neuronal metabolic and nutritional support and modulate synaptic activity.
  • astrocytes amplify, increase the expression of intermediate filament proteins, and become hypertrophic. This process is called (reactive) astrocytosis.
  • Lipocalin 2 is a member of the lipocalin family that binds to small hydrophobic molecules and is involved in various physiological processes and can be highly induced in inflammatory and neoplastic diseases.
  • LCN2 is considered to be an autocrine mediator of reactive astrocytosis. This conclusion is based on the results confirming that LCN2 is involved in the regulation of apoptosis, morphology and migration of astrocytes as follows.
  • LCN2 increases the apoptosis sensitivity of astrocytes to cytotoxic substances. That is, it accelerates the cell death of astrocytes by cytotoxic substances. LCN2 accelerates astrocytic cell death induced by NO, as well as necrosis cell death induced by H 2 O 2 or paraquat.
  • lcn2 is strongly induced by lipopolysaccharide (LPS) and TNF- ⁇ and weakly by a mixture of serum withdrawal, PMA, INF- ⁇ and gangliosides. This means that the expression and secretion of lcn2 in astrocytes increases under inflammatory conditions in the CNS.
  • LPS lipopolysaccharide
  • LCN2 glial fibrillary acidic protein
  • NO amplifies the expression of GFAP in astrocytes (Brahmachari, S. et al. 2006. I J Neurosci 26: 4930-4939). Since the present invention confirmed that LCN2 can also induce the expression of GFAP, it was investigated whether NO is involved in LCN2 action in astrocytes. LCN2 induces an increase in NO production in astrocytes, similar to that of LPS. Expression of GFAP induced by LCN2 by the NOS inhibitor NMMA is blocked, and the sensitivity to cell death induced by LCN2 is attenuated. This means that NO plays an important role in cell death, GFAP expression and morphological changes of astrocytes mediated by LCN2.
  • Rho subfamily of small G proteins is involved in the regulation of astrocyte morphology (Boran, MS et al. 2007. J Neurochem 102: 216-230; Chen, CJ et al. 2006. stellation. Eur J Neurosci 23: 1977-1987; Suidan, HS et al. 1997. Glia 21: 244-252; John, GR et al. 2004. J Neurosci 24: 2837-2845; Ramakers, GJ et al. 1998. Exp Cell Res 245: 252- 262; Holtje, M. et al. 2005. J Neurochem 95: 1237-1248; Hall, A. 2005.
  • Rho protein's relevance in morphological changes of astrocytes induced by LCN2 was investigated.
  • LCN2 induces activation of Rho, while ROCK inhibitors block morphological changes induced by LCN2 and inhibit the expression of GFAP. This means that the Rho / ROCK pathway is involved in morphological regulation of astrocytes.
  • LCN2 a secretory protein of astrocytes, performs two functions that determine the pathway for the shape and function of activated astrocytes (FIG. 36).
  • LCN2 is a protein that leads to the process of astrocytes in astrocytes, so it can be controlled by controlling this protein.
  • Astrocytes are a major cause of inhibition of regeneration of CNS neurons. Therefore, it is possible to suppress astrocytosis by inhibiting the expression of the lcn2 gene or inhibiting the activity of the LCN2 protein, and promote regeneration of CNS neurons as astrocytosis is suppressed.
  • neural cell regeneration ie brain injury treatment is required for all types of brain injury, it is not particularly limited, but degenerative nerves, including, for example, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease and multiple sclerosis Diseases, stroke, trauma, brain infections, Creutzfeldt-Jakob disease, brain inflammatory diseases, and the like.
  • degenerative nerves including, for example, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease and multiple sclerosis Diseases, stroke, trauma, brain infections, Creutzfeldt-Jakob disease, brain inflammatory diseases, and the like.
  • the present invention provides a composition for treating brain injury containing an inhibitor of LCN2.
  • the inhibitor of LCN2 may be an activity inhibitor of LCN2 or an expression inhibitor of LCN2.
  • the activity inhibitor of LCN2 may be, for example, but not limited to, peptides, polypeptides, proteins, peptide replicas, compounds, and biologics.
  • it may be an anti-LCN2 antibody capable of neutralizing the activity of LCN2.
  • the anti-LCN2 antibody may be a polyclonal antibody or a monoclonal antibody.
  • Antibodies of the invention can be prepared by conventional methods well known in the art of immunology using LCN2 protein as an antigen.
  • Polyclonal antibodies can be used in one of ordinary skill in the art from several warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats. That is, the antigen is immunized to the animal via intraperitoneal, intramuscular, intraocular or subcutaneous injection. Immunity to the antigen can be increased using an adjuvant, for example Freund's complete adjuvant or incomplete adjuvant. Following booster immunization, a small sample of serum is collected and tested for reactivity to the desired antigen. Once the titer of the animal reaches a steady state in terms of its reactivity to the antigen, large amounts of polyclonal immune serum can be obtained by bleeding weekly or by bleeding the animal.
  • an adjuvant for example Freund's complete adjuvant or incomplete adjuvant.
  • Monoclonal antibodies can also be generated using known techniques (Kennettm McKearn and Bechtol (eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses , Plenum Press, 1980). Monoclonal antibodies immunize animals with LCN2 protein as an immunogen, fusion of splenocytes of the immunized animal with myeloma cells to produce hybridomas, select hybridomas that selectively recognize LCN2 protein, and select high It can be prepared by culturing the bridoma and separating the antibody from the culture medium of the hybridoma.
  • the monoclonal antibody of the present invention can be prepared by injecting the above-mentioned hybridomas producing an anti-LCN2 antibody selectively recognizing LCN2 protein into an animal and separating it from the ascites of the recovered animal after a certain period of time after the injection. Can be.
  • inhibitortion of gene expression includes inhibition of gene transcription and translation into protein. In addition, not only the gene expression is completely stopped, but also the expression is reduced.
  • Antisense molecules are most commonly used as a method of inhibiting gene expression. Antisense molecules inhibit the expression of target genes by inhibiting transcriptional initiation by triple chain formation, transcriptional inhibition by hybridization at sites where local open loop structure is formed by RNA polymerase, and in RNA where synthesis is in progress. Inhibition of transcription by hybrid formation, inhibition of splicing by hybridization at the junction of introns and exons, inhibition of splicing by hybridization at the site of splicosomal formation, transition from nucleus to cytoplasm by hybridization with mRNA Inhibition of translation initiation by hybridization at the site of translation initiation factor binding. They inhibit the expression of target genes by inhibiting transcription, splicing or translation processes.
  • the antisense molecule used in the present invention may inhibit the expression of the target gene by any of the above actions.
  • Representative antisense molecules include triplets, ribozymes, RNAi, or antisense nucleic acids.
  • the triple agent allows the initiation of transcription to be suppressed by winding around double helix DNA to form a three-stranded helix (Maher et al., Antisense Res. And Dev ., 1 (3): 227, 1991; Helene, C., Anticancer Drug Design , 6 (6): 569, 1991).
  • Ribozymes are RNA enzymes that possess the ability to specifically cleave single-stranded RNA. Ribozymes inhibit protein expression of target genes by recognizing and site-specific cleavage of specific nucleotide sequences in target RNA molecules (Cech, J. Amer. Med. Assn ., 260: 3030, 1998; Sarver et al., Science 247: 1222-1225, 1990).
  • RNAi RNA interference
  • RNA interference is a method of inhibiting gene expression at the transcriptional level or at the post-transcriptional level by using RNA of hairpin type small molecule that acts in sequence (Mette et al., EMBO J. , 19: 5194-). 5201, 2000).
  • the small molecule RNA used in the RNAi method is a double-stranded RNA molecule having homology with the target gene.
  • RNA molecules As a method of preparing the RNA molecule in the above, known chemical synthesis methods and enzymatic methods can be used.
  • the chemical synthesis of RNA molecules can use the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999), and the enzymatic synthesis of RNA molecules is T7, T3.
  • phage RNA polymerases such as SP6 RNA polymerase are disclosed in the literature (Milligan and Uhlenbeck, Methods Enzymol . 180: 51-62, 1989).
  • Antisense nucleic acids refer to DNA or RNA molecules that are at least partially complementary to a target mRNA molecule (Weintraub, Scientific American , 262: 40, 1990). In cells, antisense nucleic acids hybridize with their corresponding mRNAs to form double-stranded molecules that inhibit protein translation by inhibiting mRNA translation of target genes (Marcus-Sakura, Anal. Biochem ., 172: 289, 1988). The antisense nucleic acid may be prepared by any suitable method known in the art, preferably in the form of oligonucleotides.
  • the antisense oligonucleotides may be used in chemical synthesis, for example, such as phosphoramidite chemistry, which is sulfided with tetraethylthiuram disulfide in acetonitrile as described in Tetrahedron Lett ., 1991, 32, 30005-30008. It can manufacture very easily.
  • the LCN2 inhibitor may be administered by any route as long as the LCN2 inhibitor can be directed to the lesion, i.
  • the compositions of the present invention may be used in various forms including topical (including buccal, sublingual, skin and intraocular), parenteral (including subcutaneous, intradermal, intravascular and intraarticular) or transdermal administration. Routes may also be administered, preferably parenterally, most preferably directly at the site where the beta amyloid has been deposited.
  • the LCN2 inhibitor can be administered to a subject by suspending in a suitable diluent, which diluent is used for the purpose of protecting and maintaining the cells and facilitating use when infused into the desired brain tissue.
  • a suitable diluent which diluent is used for the purpose of protecting and maintaining the cells and facilitating use when infused into the desired brain tissue.
  • the diluent may include physiological saline, PBS, HBSS buffer solution, plasma, cerebrospinal fluid or blood components.
  • the LCN2 inhibitor may be used in admixture with a pharmaceutically acceptable carrier according to conventional methods.
  • suitable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the composition may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.
  • compositions of the invention can be formulated using methods well known in the art to provide rapid or delayed release of the active ingredient after administration to a subject.
  • the formulations may be in the form of tablets, powders, pills, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.
  • the formulation will be convenient in a single dosage form.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease, and an effective dose level may include the severity of the disease; The age, body weight, health and sex of the patient; Sensitivity to the drug of the patient; Time of administration, route of administration, and rate of excretion; Duration of treatment; It may be determined according to factors including drugs used in combination or coincidental with the composition of the present invention and other factors known in the medical field.
  • the pharmaceutical composition of the present invention may vary the effective amount depending on the extent of the disease, preferably 1 to 10,000 g / weight kg / day, more preferably 10 to 1000 mg / kg / day The effective amount may be repeated several times a day.
  • LCN2 inhibitors can inhibit astrocytosis, which inhibits the regeneration of neurons, they can promote neuronal regeneration.
  • LCN2 inhibitors can be used to treat a variety of diseases that require neuronal regeneration, ie, any disease associated with brain damage.
  • LCN2 is involved in the upper stages in the process of astrocytosis, so LCN2 inhibitors can effectively inhibit astrocytosis and promote neuronal regeneration.
  • Figure 1 shows the results of analysis of protein expression and changes in lcn2 mRNA in C6 cells transfected with sense or antisense lcn2 cDNA (S3: sense lcn2 transfectants; AS7: antisense lcn2 transfectants).
  • Figure 2 shows the change in cell viability by SNP, H 2 O 2 and paraquat in C6 cells transfected with sense or antisense lcn2 cDNA (S3: sense lcn2 transfectant; AS7: antisense lcn2 transfectant).
  • Figure 3 shows the results of the analysis of lcn2 expression in C6 cells transfected with adenovirus vectors containing lcn2 DNA fused with GFP.
  • Figure 4 shows the change in cell viability by NO donors SNP, H 2 O 2 and paraquat in C6 cells transfected with adenovirus vectors comprising lcn2 cDNA fused with GFP.
  • 5 shows recombinant mouse LCN2 protein.
  • FIG. 6 shows changes in cell viability when stellate cells were treated with LCN2 alone and when treated with SNP, H 2 O 2 or paraquat.
  • Figure 7 shows the changes in necrosis and apoptosis cell death rate of astrocytes when LCN2 was treated with astrocytes alone and when treated with SNP, H 2 O 2 or paraquat.
  • Figure 9 shows the results confirming whether the LCN2 protein affects the cell cycle distribution of C6 cells and astrocytes.
  • FIG. 10 shows changes in cell viability when astrocytes and C6 cells were treated with iron chelator (DFO) alone and when treated with NO donor SNAP.
  • DFO iron chelator
  • FIG. 11 shows changes in cell viability when treated with iron donors (FC) alone and when treated with SNAP in astrocytes and C6 cells.
  • FIG. 12 shows the change in cell viability when C6 cells were treated with LCN2 and SNAP and co-treated with the cyder pores and iron complexes.
  • FIG. 13 shows changes in Bim RNA and protein amounts in culture of C6 cells treated with LCN2.
  • Figure 14 shows the changes in the amount of Bim RNA and protein in the culture of astrocytes treated with LCN2.
  • Figure 15 shows the results of analyzing the change in the amount of LCN2 expression by the mixture of SNP, LPS, serum clearance (SW), PMA, IFN- ⁇ , TNF- ⁇ and gangliosides.
  • Figure 16 shows the results of analyzing the change in the secretion amount of LCN2 by LPS.
  • Figure 17 shows the results of analyzing the change in the expression amount of LCN2 receptor lcn2R / 24p3R in C6 cells and astrocytes.
  • Figure 18 shows the results of analyzing the changes in morphological changes, protuberance length and cell viability when astrocytes were exposed to LCN2, Forskolin or dbcGMP.
  • Figure 19 shows the morphological changes of astrocytes with the dose of LCN2 and the time of exposure to LCN2.
  • FIG. 20 shows the results of analyzing changes in GFAP mRNA and protein expression levels of astrocytes with the dose of LCN2 and time exposed to LCN2.
  • Figure 21 shows the change in cell viability when co-treatment with SNP and LCN2, Forskolin or dbcGMP in astrocytes.
  • Figure 22 shows the results of analyzing the changes in the LCN2 protein or GFAP expression when treated with Forskolin or dbcGMP in astrocytes.
  • Figure 23 shows the results of analyzing the changes in GFAP mRNA and protein levels by SNP.
  • Figure 24 shows the effect of LCN2 on the NO production of astrocytes.
  • Figure 25 shows the effect of polymyxin B (PB) on the NO production of astrocytes induced by LCN2.
  • PB polymyxin B
  • Figure 26 shows the effect of the NOS inhibitor NMMA on GFAP expression in astrocytes induced by LCN2.
  • 29 shows the effect of Y27632 on the expression of GFAP induced by LCN2.
  • Figure 31 shows the results confirmed the effect of LCN2, Forskolin or dbcGMP on the migration of astrocytes using an in vitro wound treatment assay.
  • Fig. 34 shows the result of confirming the effect on the generation of radial glial cells by injecting LCN2 mRNA into the zebrafish embryo.
  • Figure 35 shows the change in the projection thickness, length and number of radial glial cells by injecting the mRNA of LCN2 into the embryo of zebrafish.
  • FIG. 36 is a schematic showing that LCN2 is involved at higher stages in sensitization and morphological changes of astrocytes to cell death.
  • lipocalin 2 antibody inhibits astrocytosis of astrocytes.
  • Lipopolysaccharide (LPS) obtained from E. coli 0111: B4 was prepared by phenol extraction and gel filtration chromatography.
  • Rho Kinase (ROCK) inhibitor Y27632 was purchased from Calbiochem (La Jolla, Calif.). Recombinant human TNF-a and mouse IFN-g proteins were purchased from R & D Systems (Minneapolis, MN). Iron-saturated enterochelin (0.7 kDa) was purchased from EMC Microcollections GmbH (Tuebingen, Germany). All other compounds were purchased from Sigma Chemical Co. unless otherwise noted.
  • C6 rat glioma cells were maintained in DMEM (Dulbecco's modified Eagle medium) supplemented with 5% heat inactivated fetal bovine serum (FBS) (GibcoBRL Gaithersburg, MD), gentamicin (50 mg / ml).
  • Astrocyte cultures were prepared from the brains of three-day-old ICR mice (Samtako Co .; Osan, Korea) by the method of McCarthy and de Vellis. Whole brains were disassociated in DMEM supplemented with 10% FBS, 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco-BRL).
  • Cells were seeded in 75 cm 2 tissue culture flasks coated with poly D-lysine (Falcon, Becton Dickinson and Company Franklin Lakes, NJ). Cells were incubated at 37 ° C. in 5% CO 2 humid air. The culture medium was changed after every 3 days thereafter. Secondary pure cultures of astrocytes were obtained by shaking the culture of mixed glial cells at 250 rpm overnight and the culture medium was removed. Astrocytes were harvested using trypsin-EDTA and then centrifuged at 1,000 rpm for 10 minutes.
  • astrocytes were inoculated at 1 x 10 5 cells per sphere on sterile cover slips on a 24 sphere plate, then fixed for 4 minutes with 4% formaldehyde and washed twice with PBS. Samples were blocked with 1% BSA dissolved in PBS-Tween 20 and placed in PBS containing 3% BSA and mouse anti-GFAP antibody (1:30 dilution) (Biogenex; San Ramon, CA).
  • the spheres were washed twice with PBS-Tween 20 followed by 2.5 mg / ml of Hoechst 33342 fluorochrome (Molecular Probes Eugene, OR) and anti-mouse IgG-fluorescein isothiocyanate (FITC) -conjugated secondary antibody (BD Biosciences; San Jose , CA). Samples were observed using a fluorescence microscope (Olympus BX50 Tokyo, Japan). Microscopic images were processed with MetaMorph Imaging System (Molecular Devices; Sunnyvale, Calif.). Treatment of astrocytes was performed with a slight modification of known methods (Wilhelmsson, U. et al. 2004. J Neurosci 24: 5016-5021; Boran, MS et al. 2007. J Neurochem 102: 216-230). The average projection length is based on the longest projection for each cell from five microscopic regions containing at least 100 cells optionally selected.
  • Example 5 Flow cytometry of apoptosis and cell cycle
  • Astrocytes were detached using trypsin-EDTA and washed twice with cold PBS. The cells were then resuspended in 250 ml of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 pH 7.4) and then placed in 3 ⁇ l of FITC-conjugated annexin V (Molecular Probes) according to the manufacturer's instructions. It was. The cells were then vortexed lightly and left under dark conditions for 15 minutes at room temperature. After adding Propidium iodide (20 mg / ml), flow cytometry was performed for 1 hour with FACSAria (BD Biosciences).
  • binding buffer 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 pH 7.4
  • FITC-conjugated annexin V Molecular Probes
  • Cells were suspended by PBS-5 mM EDTA and analyzed by adding 100% ethanol drops for analysis of cell cycle distribution.
  • RNase A 40 mg / ml
  • Propidium iodide 100 mg / ml was added and left to stand for 30 minutes.
  • the percentage of cells at each stage of the cell cycle was determined by flow cytometry using FACSCalibur (BD Biosciences).
  • Cells in 6-spheres were lysed in three lysis solutions (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride). Protein concentration in cell lysate was determined using Bio-Rad Protein Assay Kit (Bio-Rad; Hercules, CA). For each sample, the same amount of protein was separated by 12% SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences Piscataway, NJ).
  • the cell lysate containing a similar amount of Rho was left standing with GST-Rhotekin immobilized on agarose, and the coprecipitate was subjected to anti-Rho Western blot assay to evaluate the amount of Rho protein bound to GTP. It is known that the anti-Rho antibodies used herein can recognize RhoA, RhoB and RhoC.
  • RT-PCR was performed using total RNA isolated from C6 rat glioma cells.
  • Lcn2 cDNA sequences of rats with sense or antisense orientation were PCR amplified from pooled cDNA using target sequence specific primers by Gateway cloning (Invitrogen).
  • the sequence of the PCR primer for gateway cloning is as follows: sense lcn2 forward, 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGCT CCA CC ATG GGC CTG GGT GTC CTG TGT -3 '(SEQ ID NO: 1) sense lcn2 reverse , 5'-GGGG ACC ACT TTG TAC AAG AAA GCT GGG TTG TT GTC AAT GCA TTG GTC GGT -3 '(SEQ ID NO: 2) antisense lcn2 forward, 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGCT CCA CC ATG TCA GTT GTC AAT GCA TTG GTC -3 '(SEQ ID NO: 3) Antisense lcn2 Reverse, 5'-GGGG ACC ACT TTG TAC AAG AAA GCT GGG TTG TT ATG GGC CTG GGT GTC CTG TG T-3' (SEQ ID NO: 4) .
  • the forward primer was used by introducing the attB1 sequence (underlined) followed by the Kozak sequence (CCACC) and the gene specific sequence (bold).
  • reverse primers were used by introducing an attB2 sequence (underlined) followed by a gene specific sequence (bold).
  • PCR products were cloned into the pDONR207 dornor vector (Invitrogen), and the sequence was reconfirmed with (Macrogen Inc. Seoul, Korea) and then converted to the pDS-GFP-XB destination vector (Invitrogen).
  • C6 cells were transformed with lipofectAMINE reagent (Invitrogen) with sense or antisense rat lcn2 cDNA with 4 ⁇ g of GFP (Green fluorescent protein) tag.
  • An empty pEGFP vector was used as a control for stable expression of lcn2.
  • Stable transformants were selected in the presence of G418 (800 ⁇ g / ml) 2 days after transformation. Up or down regulation of lcn2 mRNA or protein in stable transformants was confirmed by RT-PCR or Western blot.
  • Recombinant adenovirus (Ad-lcn2-GFP) expressing rat lcn2-GFP was developed by Newgex Inc. Produced by (Seoul, Korea). That is, cDNA encoding rat lcn2-GFP was inserted into pShuttle-cytomegalovirus vector (Clontech Palo Alto, Calif.). The pShuttle-cytomegalovirus vector containing rat lcn2-GFP was linearized with Pme I and cotransformed with BJ5183 E. coli with a vector of pAdEasy-1 (Clontech) adenovirus.
  • Transformed cells were placed on agarose plates containing kanamycin and each colony was checked for the presence of a suitable transformant. After sequence identification, stocks of recombinant adenovirus were expanded by infection and extraction with HEK-293A cells. Adenovirus was semi-purified from high titer supernatants of infected HEK-293A cells. Supernatants were clarified by centrifugation to remove cell debris and stored at -80 ° C. C6 cells were infected with adenovirus expressing GFP or rat lcn2-GFP for 2 days and observed by fluorescence microscopy. More than 100 cells were identified from several randomly selected regions. Adenoviruses expressing GFP were used as controls.
  • Recombinant mouse LCN2 protein was prepared as known (Yang, J. et al. 2002, Mol cell 10: 1045-1056). In other words, the recombinant LCN2 protein does not synthesize siderophores.
  • E. coli From the BL21 strain It was expressed as glutathione S-transferase (GST) fusion protein. Proteins were purified by using glutathione-Sepharose 4B beads (Amersham Biosciences) and then eluting with thrombin or glutathione.
  • enterokeline EMC Microcollections GmbH
  • EMC Microcollections GmbH saturated with 5-fold molar excess of ions was mixed with recombinant LCN2 protein.
  • zymosan A S. cerevisiae BioParticles, Alexa Fluor 594 conjugate (Molecular Probes)
  • zymosan A S. cerevisiae BioParticles, Alexa Fluor 594 conjugate (Molecular Probes)
  • Molecular Probes Alexa Fluor 594 conjugate
  • Cell migration was measured using a 48-ball Boyden chamber (Neuro Probe, Inc. Gaithersburg, MD). Various concentrations of LCN2 protein or compound in DMEM were placed on a lower wall separated from the upper wall by a polycarbonate filter (8-mm pore size, 25 x 80 mm Neuro Probe, Inc.) without polyvinylpyrrolidone. Located. Cells were recovered by treatment with trypsin, resuspended in DMEM and added to the upper chamber at 1 ⁇ 10 4 cells / sphere. Cells were left at 37 ° C. under 5% CO 2 . Cells were then fixed with methanol for 10 minutes and stained with modified Giemsa stain (Sigma) for 1 hour.
  • scratch wounds were made using a 10 ⁇ l pipette tip in a confluent cell monolayer on a 24-neck culture plate and 10% FBS containing 100 U / ml penicillin and 100 ⁇ g / ml streptomycin. Refilled with DMEM including. Cells were left at 37 ° C. under 5% CO 2 while the monolayer moved to the wound area. The wound area was observed under a microscope (Olympus CK2) (magnification, x 100). Relative cell migration distance was determined by measuring the width of the wound and subtracting it from the initial value (Bassi, R. et al. 2008, J Neurooncol 87: 23-33). A total of three regions were randomly selected and calculated for each sphere. T results are expressed as multiples of the increase in travel distance.
  • Embryos were recovered from crosses and raised in egg water at 28.5 ° C. and developed according to days after fertilization (Park, HC et al. J Neurosci 25: 6836-6844; Kucenas, S. et al. 2008. Nat Neurosci 11: 143 -151).
  • AB and Tg (GFAP: egfp) fish Bosset, RL et al. 2006, Gene Expr Patterns 6: 1007-1013
  • Rat lcn2 cDNA including full-length open reading frame, was subcloned into the pCSDest vector for mRNA injection (Villefranc, JA et al. 2007, Dev Dyn 236: 3077-3087.) mRNA was expressed in the Message Machine Kit (Ambion Austin, TX). Produced as. The production of lcn2 mRNA was confirmed by gel electrophoresis of transcriptional reactants (data not shown). 100 pg of lcn2 mRNA was injected into egg yolk at one to two cell stages. Expression of LCN2 protein in embryos injected with mRNA was confirmed by immunocytochemistry (data not shown).
  • the embryos were fixed at 4 ° C. overnight with AB Fix (4% paraformaldehyde, 8% sucrose, 1 ⁇ PBS), embedded in 1.5% agarose / 30% sucrose and immersed in liquid nitrogen and frozen in frozen 2-methyl butane.
  • a 10 ⁇ m transreverse section was recovered using a cryostat microtome.
  • the following primary antibodies were used for immunocytochemistry: mouse antibody against Zrf-1 (1: 400 dilution, University of Oregon Monoclonal Antibody Facility), rabbit polyclonal anti-LCN2 / NGAL antibody (1: 100 dilution, Santa Cruz Biotech).
  • Example 15 Increases Sensitivity of Astrocytes to Cytotoxic Stimulators
  • lcn2 is involved in the survival and apoptosis of various types of cells
  • the following three methods were used to investigate how lcn2 is involved in the apoptosis of activated astrocytes: 1) Transfection of C6 glial cells with either sense or antisense lcn2 cDNA Stable overexpression or knockdown of lcn2 by; 2) transient expression mediated by adenovirus of lcn2 in C6 cells; And 3) treatment of primary astrocytic cultures or C6 with recombinant LCN2 protein.
  • Transient overexpression of lcn2 was achieved by using an adenovirus vector containing lcn2 cDNA fused with GFP (FIG. 3). Expression of lcn2 increased the sensitivity of C6 glial cells to NO donors SNP, H 2 O 2 and paraquat (FIG. 4).
  • LCN2 protein was prepared and tested for potential cytotoxic effects (FIG. 5). LCN2 protein sensitized primary astrocytic cultures to cell death, whereas LCN2 protein alone did not affect astrocytic survival (FIG. 6). Similar results were obtained from C6 glial cells (data not shown). In order to confirm the properties of cell death, the effect of increasing the cell death of LCN2 protein was evaluated by staining with propidium iodide (PI) and annexin V and flow cytometry (FIG. 7).
  • PI propidium iodide
  • LCN2 Treatment of LCN2 protein increased the sensitivity of apoptosis (PI ⁇ / annexin V + or PI + / annexin V + ) as well as necrosis (PI + / annexin V ⁇ ) of astrocytes.
  • the effect of increasing cell death of LCN2 protein was observed from a dose dependent and statistically significant effect of 0.1 ng / ml of LCN2 (FIG. 8). However, there was no significant effect on cell cycle distribution of C6 glial or primary astrocytic cells (FIG. 9).
  • the iron supply to the cells reduces the amount of transferrin receptor (TfR1) expression and increases the amount of ferritin.
  • TfR1 transferrin receptor
  • the influx of iron into the cell reduces the amount of expression of Bim protein, a proapoptosis protein.
  • Bim protein transferrin receptor 1
  • intracellular mammalian ciderpore iron complex binds to lcn2 and is released out of the cell by exocytosis. Depletion of iron in cells leads to upregulation of Bim, a proapoptotic molecule.
  • Example 17 Expression and Control of lcn2 and lcn2 Receptors (lcn2R / 24p3R) in Astrocytes
  • lcn2 has been proposed as an acute phase protein (Liu, Q. et al. 1995. J Biol Chem 270: 22565-22570) and expression of lcn2 was regulated by immune stimulators in macrophages (Liu, Q. et al. 1995 J Biol Chem 270: 22565-22570; Meheus, LA et al. 1993. J Immunol 151: 1535-1547; Cowland, JB et al. 2003. J Immunol 171: 6630-6639. Therefore, it was confirmed whether the expression of lcn2 is regulated by immunity or other stimulator in the phase.
  • LCN2 induced morphological changes in astrocytes in addition to apoptosis sensitization effects.
  • astrocytes were exposed to LCN2 protein, the number of cell processes increased without affecting cell viability (FIG. 18).
  • cyclic AMP (cAMP) and cyclic GMP (cGMP) are known to induce similar morphological changes (Boran, MS et al. 2007. J Neurochem 102: 216-230; Hu, W. et al. 2008.
  • Forskolin and dibutyryl cyclic GMP (dbcGMP) were used for comparison ( Cell Mol Neurobiol 28: 519-528) (FIG. 18).
  • Morphological changes in astrocytes induced by LCN2 were dose and time dependent (FIG. 19). Significant changes occurred at 12 ng / ml of LCN2 and 12 hours after exposure thereto. They have been shown to increase the expression of lcn2 in astrocytes (FIGS. 15 and 16) and to induce activation of glial cells (Jou, I. et al. 2006. Am J Pathol 168: 1619-1630; Yoon, HJ et al. 2008. Mol Cells 25: 99-104).
  • LCN2 Changes in the cell processes induced by LCN2 are made by upregulation of the expression of GFAP mRNA and protein, which is dependent on the dose and time of LCN2 (FIG. 20). Morphological changes in this type of astrocytes are similar to those occurring in in vivo reactive astrocytes (Sofroniew, MV 2005. Neuroscientist 11: 400-407).
  • Hypertrophy and increased GFAP expression are two features of reactive astrocytic cells that appear after all forms of neuronal damage in vivo (Wilhelmsson, U. et al. 2004. J Neurosci 24: 5016-5021). Changes in astrocyte morphology induced by LCN2 were confirmed to be associated with phenotypes sensitive to cell death. Forskolin and dbcGMP, which induce changes in astrocytic morphology similar to those induced by LCN2, also led to phenotypes sensitive to cell death (FIG. 21). dbcGMP increased LCN2 expression while forskolin did not (FIG. 22).
  • the amount of NO produced induced by LCN2 was similar to that of LPS and was not offset by polymyxin B treatment to rule out the possibility of LPS contamination in recombinant LCN2 preparations (FIG. 25).
  • Recombinant GST protein prepared in the same manner as LCN2 was also used as a control to exclude the possibility of contamination of LPS (FIG. 25).
  • NO production induced by LPS / IFN- ⁇ was completely offset by polymyxin B treatment.
  • Expression of GFAP induced by LCN2 was blocked by the NOS inhibitor NMMA (FIG. 26).
  • the increase in cell death sensitivity induced by LCN2 was attenuated by NMMA (FIG. 27). The above results indicate that NO plays an important role in astrocytic cell death, GFAP expression and morphological changes mediated by LCN2.
  • Rho subfamily of small G proteins is known to be involved in the regulation of astrocyte morphology (Boran, MS et al. 2007. J Neurochem 102: 216-230; Chen, CJ et al. 2006. stellation. Eur J Neurosci 23 : 1977-1987; Suidan, HS et al. 1997. Glia 21: 244-252; John, GR et al. 2004. J Neurosci 24: 2837-2845; Ramakers, GJ et al. 1998. Exp Cell Res 245: 252 Hol262, M. et al. 2005. J Neurochem 95: 1237-1248; Hall, A. 2005. Biochem Soc Trans 33: 891-895).
  • Rho protein in the morphological changes of astrocytes induced by LCN2 was investigated.
  • the Rho / ROCK pathway appears to play an important role in the action of LCN2 on astrocyte morphology according to the following results: 1) ROCK inhibitor Y27632 partially blocked morphological changes induced by LCN2 (FIG. 28); 2) Y27632 also inhibited the expression of GFAP induced by LCN2 (FIG. 29) and 3) LCN2 induced the activation of Rho (FIG. 30).
  • Y27632 had no effect on cell viability at the concentrations used in this example (data not shown).
  • Activation of Rho induced by LCN2 was initiated 1 hour after LCN2 stimulation and continued up to 24 hours (FIG. 30).
  • a zebrafish model was used to confirm the expression of lcn2 and its functional role in in vivo reactive astrocytes.
  • the expression of lcn2 in zebrafish CNS was examined. Labeling of zebrafish embryos with anti-LCN2 polyclonal antibodies showed no signal on day 2 after fertilization (data not shown), but LCN2 + cells were identified in the whole brain and spine on day 3 (A, B in FIG. 33). ). LCN2 antibodies in whole brain sections labeled microglia-like cells in the form of microglia (arrows in A of FIG. 33).
  • Tg (gfap-egfp) embryos which were anti-Zrf-1 markers of radial glial protuberances. It was labeled with an antibody.
  • Tg (gfap-egfp) embryos express EGFP in radial glial cells under the control of the gfap promoter (Bernardos, RL et al. 2006. Gene Expr Patterns 6: 1007-1013). By labeling the processes (Trevarrow, B. et al. 1990.
  • compositions of the present invention can be used to treat brain damage.

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Abstract

La présente invention concerne une composition contenant un inhibiteur de lipocaline 2 (LCN2) permettant de traiter des lésions cérébrales ainsi qu'une méthode permettant de traiter des lésions cérébrales par administraiton de la composition. Lorsque le système nerveux central est lésé, des astrocytes sont activés induisant une astrocytose. L'astrocytose est un facteur principal provoquant des lésions de cellules nerveuses et supprimant la régénération des cellules nerveuses lésées. Le LCN2 induisant principalement l'astrocytose, des lésions supplémentaires des cellules nerveuses suite à des lésions cérébrales peuvent être évitées grâce à l'utilisation du l'inhibiteur LCN2. En outre, la régénération des cellules nerveuses lésées peut être favorisée.
PCT/KR2009/004676 2008-08-21 2009-08-21 Nouvelle utilisation de lipocaline 2 pour traiter des lesions cérébrales WO2010021516A2 (fr)

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