KR101575093B1 - Composition comprising lipocalin 2 inhibitor for preventing and treating inflammatory pain - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory pain comprising a lipocalin 2 inhibitor as an active ingredient. The expression level of the LCN2 protein according to the present invention is markedly increased in the inflammatory pain state, and the response to thermal or mechanical pain stimulation is reduced in the CFN-treated LCN2 gene-deficient mouse as compared with the normal mouse, Infiltration is reduced, expression of TNF-a, IL-l [beta] and IL-8 is reduced, and spinal cord microcellular activity is significantly reduced. In addition, treatment of recombinant LCN2 protein in normal mice increases sensitivity to thermal or mechanical pain stimuli, increases neutrophil infiltration in tissues, and increases expression of TNF-a, IL-l [beta], and IL-8 , Which significantly increases the activity of spinal cord microglia. In addition, when LCN2 inhibitors are treated in an animal model of inflammatory pain, there is a mitigating effect on inflammatory pain. Therefore, the LCN2 inhibitor according to the present invention can be usefully used for the prevention or treatment of inflammatory pain.

Description

[0001] The present invention relates to a composition for preventing or treating inflammatory pain comprising lipocalin 2 inhibitor as an active ingredient,

The present invention relates to a composition for preventing or treating inflammatory pain, which comprises a lipocalin 2 inhibitor as an active ingredient.

Inflammation processes are a series of complex biochemical and cellular events that are activated in response to the presence of foreign substances or tissue damage, causing swelling and pain. The inflammation process is a defense mechanism against foreign pathogenic factors, but it can cause pain even when there is no stimulation of toxic substances. The local inflammatory response releases proinflammatory cytokinins and upregulates cyclooxygenase-2 to synthesize prostaglandins, sensitizing primary afferent neurons and causing peripheral nerve cells To activate neuroimmunity. Thus, ultimately, thermal or mechanical hypersensitivity occurs. When an inflammatory reaction occurs, proteins such as pentraxins, C-reactive protein, serum amyloid A, serum amyloid P and Rabs are expressed, so that the protein can be used as a marker of chronic inflammatory disease. However, the role of the peripheral and central proinflammatory cascades in the inflammatory response of the protein and in the development of chronic inflammatory pain is unclear.

On the other hand, LCN2 (Lipocalin 2) binds or carries lipids and other hydrophobic molecules as a member of the lipocalin family (Flower, DR et al. 2000, Biochim Biophys Acta 1482: 9-24; Kjeldsen, L. et al 2000, Biochim Biophys Acta 1482: 272-283). LCN2 is also known as 24p3 (Flower, DR, et al. 1991. Biochem Biophys Res Commun 180: 69-74), SIP24 (24 kDa superinducible protein) (Hamilton, RT et al 1985, J Cell Physiol 123: 201-208 ) And NGAL (a human homologue of lcn2) (Borregaard, N. et al. 2006, Biometals 19: 211-215; Kjeldsen, L. et al. 1993, J Biol Chem 268: 10425-10432 ). In addition, LCN2 has been shown to be important in both cell apoptosis and survival (Nelson, AM et al. 2008, J Clin Invest 118: 1468-1478; Yousefi, S. et al. 2002, Cell Death Differ 9: 595- Tong, Z. et al. 2003, Biochem J 372: 203-210; Tong, LR et al., 2001, Science 293: 829-834; , 2002, Mol Cell 10: 1045 (1986)), play a central role in inducing cell differentiation in the kidney during embryogenesis (Yang et al., 2005, Biochem J 391: -1056), it is known that kidneys are protected from ischemic injury (Mishra, J. et al., 2004, J Am Soc Nephrol 15: 3073-3082; Mori, K. et al 2005, J Clin Invest 115: 610- 621). LCN2 has also been suggested as an indicator of kidney damage (Mori, K. et al. 2007, Kidney Int 71: 967-970). In various forms of gastrointestinal injury, LCN2 facilitates mucosal regeneration by promoting cell migration (Playford, R. J. et al. 2006, Gastroenterology 131: 809-817).

As described above, various roles of LCN2 have been clarified, but the role of LCN2 in inflammatory pain has not yet been elucidated.

Accordingly, the present inventors have found that LCN2 can induce chronic inflammatory pain, and by inhibiting LCN2, chronic inflammatory pain can be prevented or treated, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing or treating chronic inflammatory pain comprising lipocalin 2 inhibitor as an active ingredient.

The present invention also provides a method for preventing or treating chronic inflammatory pain comprising administering to a subject a lipocalin 2 inhibitor.

The present invention provides a composition for preventing or treating inflammatory pain comprising lipocalin 2 (hereinafter also referred to as "LCN2") inhibitor as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in detail.

In the present invention, the CFN-treated LCN2-deficient mice have decreased responses to thermal or mechanical pain stimuli as compared with normal mice, and the infiltration of neutrophils into the tissues is reduced, and TNF-α, IL-1β and IL-8 And the activity of spinal cord macrophages is remarkably reduced. In addition, treatment of recombinant LCN2 in normal mice increases sensitivity to thermal or mechanical pain stimuli, increases penetration of neutrophils in tissues, increases expression of TNF-a, IL-l [beta], and IL-8 , Which significantly increases the activity of spinal cord microglia. In addition, when the LCN2 neutralizing antibody of the present invention is treated, there is a mitigating effect on inflammatory pain. Therefore, the LCN2 inhibitor according to the present invention can be usefully used for the prevention or treatment of inflammatory pain.

Inflammatory pain of the present invention is pain caused by inflammation, including chronic inflammatory pain or acute inflammatory pain.

In the present invention, "prevention or treatment" means to include elimination, relief and inhibition of pain and the like.

The LCN2 inhibitors of the present invention include their expression inhibitors and activity inhibitors.

The expression inhibitor may be at least one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the LCN2 gene, RNAi, a small hairpin RNA, a small interfering RNA, and ribozyme Lt; / RTI > The activity inhibitor of the LCN2 protein is selected from the group consisting of a compound specifically binding to a lipocalin 2 receptor (24p3R) protein, a compound specifically binding to an LCN2 protein, a peptide, a peptide mimetic, a substrate analog, But it is not limited thereto.

In the present invention, "expression inhibition" means to inhibit gene transcription and to inhibit translation into protein. Also included are those in which the expression of the gene is not only completely stopped but also the expression is reduced. As a method for suppressing the expression of a gene, it is most common to use an antisense molecule. The antisense molecule inhibits the expression of the target gene by inhibiting transcription initiation by triple chain formation, inhibiting transcription by hybrid formation at a site where a local open loop structure is formed by RNA polymerase, Inhibition of splicing by hybridization at the junction of introns and exons, inhibition of splicing by formation of hybrids at spliceosome-forming sites, and formation of hybrids with mRNAs Inhibition of translation into the cytoplasm, inhibition of translation initiation by hybridization at the translation initiation factor binding site, and the like. They inhibit the transcription, splicing or translation process and thus inhibit the expression of the target gene.

Examples of the antisense molecule include triple, ribozyme, RNAi, or antisense nucleic acid. Triple helix is wrapped around double helix DNA to form a triple helix so that transcription initiation is inhibited (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 have the ability to specifically cleave single-stranded RNA. Ribozymes inhibit protein expression of a target gene by recognizing and specifically cleaving a specific nucleotide sequence in a target RNA molecule (Cech, J. Amer. Med. Assn., 260: 3030, 1998; Sarver et al. Science 247: 1222-1225, 1990). RNAi (RNA interference) is a method of inhibiting gene expression at the transcription level or post transcription level using small molecule RNA of the hairpin type, which acts as a nucleotide sequence-specific (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. As a method for producing the RNA molecule, known chemical synthesis methods and enzymatic methods can be used. For example, the chemical synthesis of RNA molecules can be performed using the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999) And SP6 RNA polymerase have been disclosed (Milligan and Uhlenbeck, Methods Enzymol. 180: 51-62, 1989). Antisense nucleic acid refers to a DNA or RNA molecule that is at least partially complementary to a target mRNA molecule (Weintraub, Scientific American, 262: 40, 1990). In the cell, the antisense nucleic acid hybridizes with the corresponding mRNA to form a double-stranded molecule, thereby inhibiting mRNA detoxification of the target gene and suppressing protein expression (Marcus-Sakura, Anal. Biochem., 172: 289, 1988). The antisense nucleic acid can be prepared by any suitable method known in the art, preferably in the form of oligonucleotides. The antisense oligonucleotides can be synthesized by methods such as chemical synthesis, for example phosphoamidite chemistry, which is sulfated with tetraethylthiuram disulfide in acetonitrile as described in the literature (Tetrahedron Lett., 1991, 32, 30005-30008) Can be produced very easily.

The siRNA for LCN2 of the present invention is composed of a sense sequence of 15 to 30 mers selected in the nucleotide sequence of the mRNA of the gene encoding the LCN2 protein and an antisense sequence complementarily binding to the sense sequence, , And the sense sequence is preferably composed of 25 bases though not particularly limited thereto.

The antisense nucleotide binds (hybridizes) to a complementary base sequence of DNA, immature-mRNA or mature mRNA, as defined in the Watson-Crick base pair, to interfere with the flow of genetic information as a protein in DNA will be. The nature of antisense nucleotides that are specific for the target sequence makes them exceptionally multifunctional. Because antisense nucleotides are long chains of monomeric units they can be readily synthesized against the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means to study target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel type of inhibitor, as there have been many advances in the field of oligonucleotide chemistry and in the field of nucleotide synthesis showing improved cell adsorption, affinity of target binding and nuclease resistance.

The peptide mimetics inhibit the activity of the LCN2 protein by inhibiting the binding domain of the LCN2 protein. Peptide mimetics may be peptides or non-peptides and may include amino acids linked by non-peptide bonds, such as psi bonds (Benkirane, N., et al. J. Biol. Chem., 271: 33218-33224, Lt; / RTI > Also included within the scope of the present invention are "conformationally constrained" peptides, cyclic mimetics, at least one exocyclic domain, a binding moiety (binding amino acid) Can be. Peptide mimetics are structurally similar to the secondary structural features of the LCN2 protein and are expressed as antibodies (Park, BW et al. Nat Biotechnol 18, 194-198, 2000) or water soluble receptors (Takasaki, W. et al. Nat Biotechnol 15, 1266- (Wrighton, NC et al., Nat Biotechnol 15, 1261-1265, 1997), which can mimic the inhibitory properties of gigantic molecules such as, for example, .

The aptamer is a single-chain DNA or RNA molecule, which has high affinity for a specific chemical molecule or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) (J. Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990; M. Famulok, et al. DS Wilson and Szostak, Annu Rev. Biochem., 68, 611-647, 1999). Aptamers can specifically bind to a target and modulate the activity of the target, for example, by blocking the function of the target through binding.

The antibody can specifically and directly bind to LCN2 and effectively inhibit the activity of LCN2. As the antibody specifically binding to the LCN2, it is preferable to use a polyclonal antibody or a monoclonal antibody. The antibody specifically binding to LCN2 may be prepared by a known method known to those skilled in the art, and commercially available LCN2 antibody can be purchased and used. The antibody may be prepared by injecting an immunogen-causing LCN2 protein into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen may be injected intramuscularly, intraperitoneally or by subcutaneous injection, and may generally be administered with an adjuvant to increase the antigenicity. Blood can be collected periodically from the external host and the antibody formed can be collected by collecting sera showing specificity to the antigen and the titer formed.

The present invention contemplates the use of LCN2 inhibitors in a variety of routes including topical (including buccal, sublingual, subcutaneous and intradermal administration), parenteral (including subcutaneous, intradermal, intravascular and intraarticular administration) And preferably parenterally, but is not limited thereto. The LCN2 inhibitor can be administered to a subject suspended in a suitable diluent which is used to protect and maintain the cells and to facilitate their use in injecting into the desired brain tissue. The diluent may be physiological saline, a buffer solution such as PBS or HBSS, plasma, cerebrospinal fluid or blood components.

The LCN2 inhibitor of the present invention can be used in admixture with a pharmaceutically acceptable carrier according to a conventional method. Examples of suitable carriers are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, the composition may further contain a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The compositions of the present invention may be formulated using methods well known in the art so as to provide for rapid or delayed release of the active ingredient after administration to the individual. Formulations may be in the form of tablets, powders, pills, emulsions, solutions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders and the like. The formulation will conveniently be in a disposable dosage form.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease, and the effective dose level is the severity of the disease; Age, weight, health, sex of the patient; The sensitivity of the patient to the drug; Administration time, route of administration and rate of release; Treatment period; Factors including, but not limited to, the drugs used in combination with or contemporaneously with the composition of the present invention used, and other factors known in the medical field. Preferably, the effective amount of the pharmaceutical composition of the present invention may vary depending on the severity of the disease, but is preferably 1 to 10000 μg / kg body weight / day, more preferably 10 to 1000 μg / kg body weight / day May be repeatedly administered several times a day in an effective amount.

The LCN2 inhibitor according to the present invention can be added to foods or beverages for the purpose of preventing or improving inflammatory pain. Examples of the foods include various foods, beverages, gums, tea, vitamin complexes, functional foods, and the like. Specific nutritional foods (eg, crude oil, spirits, baby food, etc.) (Eg soy sauce, soybean paste, hot pepper paste, mixed sauce, etc.), sauces, confections (eg, snacks), candies, chocolates, gums, ice cream, dairy products , Fruit drinks, vegetable beverages, fermented beverages, etc.) and natural seasonings. The food, beverage or food additive may be prepared by a conventional production method.

The term "functional food" as used herein refers to a food group that is imparted with added value to function or express the function of the food by physical, biochemical, biotechnological techniques, etc., or to control the biological defense rhythm of the food composition, Means a food which has been designed and processed so as to sufficiently express the body's control function with respect to the living body. Specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

The amount of the LCN2 inhibitor in the food or beverage may comprise from 0.001% to 90% by weight, preferably from 0.1% to 50% by weight of the total food, and in the case of beverage, 0.001% g to 2 g, preferably 0.01 g to 0.1 g. However, in the case of long-term intake intended for health and hygiene purposes or for health control purposes, it may be lower than the above range. It is not limited to the above range since it can be used in an amount of more than the above range because there is no problem.

The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient like a normal food composition. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, , Protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

The present invention also provides a method of preventing or treating inflammatory pain comprising the step of administering to a subject a lipocalin 2 inhibitor.

The inflammatory pain includes chronic inflammatory pain or acute inflammatory pain.

The route of administration of the lipocalin 2 inhibitor may be administered topically (including buccal, sublingual, subcutaneous, and intrathecal), parenteral (including subcutaneous, intradermal, intravascular and intraarticular) And the like.

The object is selected from the group consisting of a canine, a feline, a wild animal, a bovine animal, a deer and an animal, a giraffe and an animal, a curry animal and a camel, a hippopotamus, a horse and an animal, a macroscopic animal, a rhinoceros animal, a weasel, But are not limited to, primates.

The present invention also provides a composition for diagnosing inflammatory pain comprising an agent for measuring the level of mRNA or protein of lipocalin 2 gene.

In the present invention, "diagnosis" means confirming a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of inflammatory pain.

In the present invention, " agent for measuring mRNA level " includes a sense and an antisense primer complementary to mRNA of lipocalin 2 gene, or a probe.

As used herein, the term "primer" refers to a primer that is hybridized under appropriate conditions in a suitable buffer solution (for example, four different nucleoside triphosphates and a polymer such as DNA, RNA polymerase or reverse transcriptase) Refers to single stranded oligonucleotides that can serve as a starting point for DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primers of the present invention can be chemically synthesized using methods known in the art such as, for example, the phosphoramidite solid support method. Further, it can be modified by methylation, capping or the like by a known method.

The term " probe " in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to mRNA and is labeled to confirm the presence or absence of a specific mRNA . The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like

In the present invention, " agent for measuring protein level " includes an antibody specific to a protein encoded by the gene.

The present invention also provides an inflammatory pain kit comprising the composition.

The diagnostic kit of the present invention comprises one or more other component compositions, solutions or devices suitable for the assay method. For example, the kit of the present invention can be used to carry out PCR using genomic DNA derived from a sample to be analyzed, a primer set specific for the lipocalin 2 gene of the present invention, a suitable amount of DNA polymerase (for example, Taq polymerase, dNTP mixture, PCR buffer solution and water. The PCR buffer may contain KCl, Tris-HCl and MgCl _2. In addition, components necessary for conducting electrophoresis to confirm whether the PCR product is amplified can be further included in the kit of the present invention.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing RT-PCR. The RT-PCR kit contains test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase and the like, in addition to the respective primer pairs specific for the lipocalin 2 gene Enzymes such as reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific to the gene used as a quantitative control.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof.

In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

In addition, the kit of the present invention may be a kit comprising essential elements necessary for performing ELISA. An ELISA kit comprises an antibody specific for a marker protein and comprises an agent that measures said protein level. The ELISA kit may comprise a reagent capable of detecting an antibody that has formed the antigen-antibody complex, for example, a labeled secondary antibody, chromopores, an enzyme (such as an antibody) and its substrate. In addition, antibodies specific for the quantitative control protein may be included. The antigen-antibody complex means a conjugate of a protein encoded by lipocalin 2 gene and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through a signal label of a detection label have. Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto.

The present invention also provides a method for providing information for predicting the risk of inflammatory pain comprising measuring the level of mRNA of the lipocalin 2 gene or the protein encoded by the gene from a biological sample.

Biological samples in the present invention include, but are not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine.

In the present invention, the level of mRNA expression is determined by PCR, RT-PCR, Competitive RT-PCR, Realtime RT-PCR, RNase protection But are not limited to, methods such as RPA (RNase protection assay), Northern blotting and DNA chip.

In the present invention, the level of protein expression can be measured by an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (ELISA), or an immunoassay using an antigen-antibody binding reaction using an antibody that specifically binds to a lipocalin 2 gene- Immunoassay, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays ), A fluorescence activated cell sorter (FACS), and a protein chip, but the present invention is not limited thereto.

Through these methods, the level of mRNA of the lipocalin 2 gene or the level of the protein encoded by the gene in the biological sample is compared with the mRNA or protein expression level in the normal group (control group), and the lipocalin 2 gene Can be diagnosed as inflammatory pain and the risk of inflammatory pain can be predicted.

In addition, the present invention provides a method for screening a therapeutic agent for inflammatory pain comprising the step of treating a biological sample with a candidate compound for the prophylaxis or treatment of inflammatory pain, and measuring the level of mRNA or protein expression of the lipocalin 2 gene. When the candidate substance is treated in a biological sample, when the expression of lipocalin 2 mRNA or protein is decreased, it can be judged to be a therapeutic agent for inflammatory pain.

The lipocalin 2 inhibitor of the present invention can alleviate the response to pain caused by inflammation and thus can be usefully used for the prevention or treatment of inflammatory pain.

Figure 1 shows the expression pattern of LCN2 in CFA-treated mice (A: expression confirmation at the protein level; B: expression confirmation at the mRNA level; contra: opposite side; ipsi: one side)
Fig. 2 is a view of the femoral tissue of a mouse treated with CFA using H & E staining.
FIGS. 3 and 4 show the infiltration of neutrophils and macrophages in CFA-treated mice using immunofluorescence.
Figure 5 shows the relationship between the sensitivity to pain and LCN2 in mice treated with CFA (A: confirmation of response to thermal stimulation, B: confirmation of response to mechanical stimulation, WT (contra): opposite side of normal mouse; KO (contra): opposite side of LCN2 deficient mice; KO (ipsi): one side of LCN2 deficient mice).
6 shows the expression pattern of 24p3R in CFA-treated mice (A: confirmation of the relationship between neutral leukocyte infiltration and 24p3R; B: confirmation of the relationship between macrophage infiltration and 24p3R).
Figure 7 shows the results of the observation of edema, tissue damage and inflammatory cell infiltration in CFA-treated mice (A and B: indicating the size of edema and edema, C: observing the follicular tissue of the mice, D and E: ≪ / RTI >
Figure 8 shows the expression of MPO activity and the expression of an immunological mediator in CFA-treated mice (A: MPO activity, B: expression of TNF-α, C: expression of IL-1β; D: Expression of IL-8)
FIG. 9 is a graph showing the expression of TNF-a and IL-1 beta when the recombinant LCN2 was treated with a mouse (A: TNF-α; B: IL-1β)
FIG. 10 is a graph showing a response to a thermal pain stimulus, an analysis of histopathology, and an infiltration of inflammatory cells when the recombinant LCN2 was treated in mice (A: response to thermal pain stimulation; B: histopathological analysis; C: Confirmation of infiltration of inflammatory cells by immunofluorescence)
Fig. 11 is a graph showing the relationship between the activity of LCN2 and microsomes of the spinal cord (A: confirmation of microglia activity when LCN2 is deficient; B: confirmation of microglia activity when recombinant LCN2 was treated with mouse)
Fig. 12 shows the result of confirming the activity of microglia according to the elapsed time of treatment with CFA.
Fig. 13 shows the results of confirming the activity of astrocytes according to the elapsed time of treatment with CFA.
14 to 16 are diagrams for confirming the mitigation effect on inflammatory pain of the LCN2 neutralizing antibody.

Hereinafter, preferred embodiments, experimental examples, and production examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and production examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, experimental examples and production examples.

[ Example  1] mice exposed to chronic inflammatory pain inducers Tribe  Organization (hindpaw tissue )in LCN2  ( lipocalin2 ) And confirmation of inflammatory cell infiltration

One. CFA  ( complete Freund`s adjuvant ) To mice

To identify the role of LCN in chronic inflammatory pain, anesthetize with wild type C57BL / 6 mice (Samtaco Inc., Osan, South Korea) with 3% oxygen and 2% isoflurane, (Left hindpaws) of the mice were injected intraperitoneally into the left lower limb of the mouse with a complete inflammatory pain inducing factor CFA (complete Freund's adjuvant; 0.5 mg / mg; Sigma-Aldrich, St. Louis, MO) Lt; / RTI >

2. Immunofluorescent staining  Used LCN2  Identification of Protein Expression

To confirm the expression of LCN2 protein over time after CFA injection, mice injected with CFA in Example 1-1 were euthanized, fixed with 0.1 M 4% paraformaldehyde, and immersed in PBS . Lumbar spinal cord segments and hindpaw tissue in the spinal cord were cut out and fixed with the same fixative solution for one day. Thereafter, the rats and the trunk were cryopreserved overnight at 4 ° C in 0.1M PBS containing 30% sucrose. Using a freeze-holding device, a 10-um fusiform tissue was prepared and placed on a slide covered with gelatin. Spinal cord of 30 μm size was stored in 0.1% PBS. Background staining was enhanced with 4% normal serum in 0.3% Triton X-100 for 90 min at room temperature. (Rabbit, 1: 200; Sigma-Aldrich, St. Louis, Mo.), CD4 (mouse, 1: 200; Abcam, Cambridge, MA ) Or Ly6G (rat, 1: 200; eBioscience, San Diego, Calif.) And Iba-1 (mouse, 1: 1000; Wako) were used as the primary antibodies and FITC or Cy3 Immunostaining was performed using a secondary antibody (1: 200; Jackson ImmunoResearch, West Grove, Pa.) And visualized with a fluorescence microscope. The expression of LCN2 in the follicular tissue at various times after CFA injection is shown in Fig.

As shown in Fig. 1 (A), LCN2 expression was induced in the fascial tissue of CFA-injected side before 6 hours of CFA injection, and LCN2 expression was found to be highest after 12 hours of CFA injection. In addition, LCN2 expression began to decrease after 3 days of CFA injection, and it was confirmed that almost no LCN2 protein was found after 7 days of injection.

 3. RT - PCR On by LCN2  Identification of expression pattern.

To identify a role in chronic inflammatory pain of LCN2, the mouse of Example 1-1 was euthanized and PBS was perfused through the artery to remove blood. Subsequently, the lumbar spinal cord, dorsal root ganglion (DRGs) and fascia were rapidly cleaved in the mouse. The spinal cord area corresponding to L4-L6 was divided into ipsilateral side and contralateral side. The site was immediately frozen with liquid nitrogen and homogenized with Trizol reagent (Life Technologies, Carlsbad, Calif.) To extract mRNA. 2 ug of total RNA from the four sites was reverse transcribed with cDNA using first strand cDNA synthesis kit (MBI Fermentas, Hanover, Germany). Then, using the one-step SYVBR®Primescript ™ RT-PCR kit (Perfect Real-Time; Takara Bio Inc., Tokyo) and the ABI Prism® 7000 sequence detection system (Applied Biosystems, Foster City, CA) Real-time PCR was performed. To calculate the relative changes in gene expression, the 2 ^{-ΔΔ CT} method was used. The primers of Table 1 below were used for the PCR. To analyze the PCR products, 10 μl of each PCR product was electrophoresed on 1% agarose gel and the expression of LCN2 was confirmed by UV. GAPDH was used as an internal control. Figure 1B shows the mRNA expression pattern of LCN2.

Base sequence SEQ ID NO: LCN2 forward primer 5`CCCCATCTCTGCTCACTGTC-3` One LCN2 reverse primer 5`-TTTTTCTGGACCGCATTG-3` 2 GAPDH forward primer 5`-TGGGCTACACTGAGCACCAG-3` 3 GAPDH reverse primer 5`-GGGTGTCGCTGTTGAAGTCA-3` 4

As shown in Fig. 1B, it was confirmed that the expression of LCN2 mRNA was significantly increased in the unilateral fascial tissue injected with CFA into mouse, and the expression was the highest at 12 days after injection and gradually decreased . On the other hand, it was confirmed that no change in the expression pattern of LCN2 mRNA was observed in the contralateral hippocampus (the side without CFA injection).

4. LCN2  The relationship between expression and neutrophil and macrophage infiltration

After the mouse was injected with CFA, H & E (hematoxylin and eosin) staining was performed on the follicular tissue (using the follicular tissue of mice not treated with CFA as a control). (10 μm) was fixed with the same fixative as the fixative of Example 1-2, and H & E staining was performed and observed with an optical microscope (Olympus B-50, Tokyo). The results are shown in Fig.

In addition, immunofluorescence was performed with antibodies against Iba-1, which is an antibody against Ly6G, which is a marker of neutrophils in Example 1-2, and macrophages, and the relationship between macrophage and neutrophil infiltration and LCN2 expression Respectively. The results are shown in Figures 3 and 4.

As shown in FIGS. 2 to 4, neutrophils infiltrated into the follicular tissues of mice injected with CFA. In particular, neutrophils and macrophages were observed after 12 hours of CFA injection, and LCN2 MRNA and protein expression was highest. In particular, immunostaining for LCN2 protein and Ly6G together confirmed that they were mostly associated with LCN2 and neutrophilic leukocyte infiltration. In addition, it was confirmed that the LCN2 protein and Iba-1 partially coexist. In addition, at 3 days or 7 days after the CFA injection, it was confirmed that Ly6G was not detected in the posterior tissues and neutral neutrophils disappeared, but Iba-1 was detected.

In contrast, LCN2 expression did not occur in the control group, and neutrophil and macrophage infiltration did not occur.

Taken together, these results indicate that expression of LCN2 by neutrophils and macrophages is associated with the development of chronic inflammatory pain.

[ Example  2] LCN2  Identify hypersensitivity to pain in deficient mice

CFA was expressed in LCN2-deficient mice (Dr.Shizo Akira, Osaka University, Japan; mice lacking the LCN2 gene; hereinafter referred to as LCN2-deficient mice) and LCN2 The behavior of normal mice and LCN-deficient mice for thermal or mechanical stimulation was compared for 6 hours to 14 days after treatment or non-treatment with mice lacking LPS (hereinafter referred to as normal mice).

One. LCN2  Reduced thermal hyperalgesia in deficient mice

First, in order to confirm the relationship between LCN2 and thermal hyperalgesia (related to inflammation), the degree of avoidance response to the electrically generated thermal stimulation of LCN2-deficient mice and normal mice was compared.

Specifically, Hargreaves, et. The mice were acclimated on a glass surface maintained at 30 DEG C according to the method described in al., and the plantar surface of the tails was subjected to thermal stimulation generated by a current of 0.5A. Thereafter, the time required for the mice to avoid the latter from stimulation was automatically measured. The intensity of radiant heat was adjusted so that the reference PWL (paw withdrawal latency) was 9 to 12 seconds. To obtain the average PWL value, the experiment was performed 3 times and the interval between the tests was 5 minutes. The results are shown in Fig. 5A.

As shown in Fig. 5A, after treatment with CFA, LCN2-deficient mice showed decreased thermal hyperalgesia compared to normal mice.

2. LCN2  Mechanically in deficient mice Allodynia ( mechanical allodynia ) Reduction effect confirmation

In order to confirm the association with LCN2 and inflammatory pain, the response of LCN2-deficient mice or normal mice injected with CFA to sensitivity to mechanical stimuli in vetral hind paws was measured.

More specifically, in order to quantify the mechanical sensitivity of the legs, mice were placed on a metal mesh floor in a clear plastic box, and left and right legs were weighed using 2.0 grams and 4.0 grams of von Frey hair To determine the threshold value of the stimulation intensity required to avoid the foot, and the 50% PWT (paw withdrawal threshold) of the mice was measured by Dixon's up-down method. The result is shown in Fig. 5B.

As shown in Fig. 5B, it was confirmed that the mechanical allodynia of one foot of LCN-deficient mice was decreased from 6 hours to 7 days after CFA injection.

[ Example  3] CFA  After the induction inflammatory reaction, LCN2  The receptor 24 p3R Confirmation of expression of

In order to confirm the relationship between chronic inflammatory pain and LCN2, CFA was injected into mice (using normal mice (WT) and LCN2 deficient mice), and 24p3R expression was confirmed.

More specifically, immunofluorescence was performed using antibodies against 24p3R (rabbit, 1: 200; Sigma-Aldrich, St. Louis, Mo.), Ly6G and Iba-1 as primary antibodies by the method of Example 1-2 . The results of confirming the expression pattern of 24p3R are shown in Fig. 6 (A: double immunofluorescence results for 24p3R and Ly6G; B: double immunofluorescence for 24p3R and Iba-1).

As shown in FIG. 6, it was confirmed that expression of 24p3R was not observed in the rear of normal mice not injected with CFA. In contrast, when CFA was injected into normal or LCN2-deficient mice, 24p3R expression was strongly induced.

Also, as a result of performing double immunofluorescence, it was confirmed that Ly6G (marker for neutrophils) and 24p3R were present in the late stage at 12 hours after injection of CFA injection into normal mice (see A in FIG. 6). In addition, it was confirmed that 24p3R and Iba-1 (markers for macrophages) were present simultaneously in the late mouse of the normal mouse at this time (see FIG. 6B). On the contrary, in the follicular tissue of LCN2-deficient mice, it was confirmed that Ly6G-positive cells, neutrophils and Iba-1-positive cells, macrophages were significantly decreased.

As a result, it was confirmed that the expression of LCN2 was related to the tissue infiltration of neutrophils and macrophages but not to the expression of 24p3R.

[ Example  4] LCN2 And local inflammatory response

One. CFA  After injection, confirmation of the production of edema in LCN2 deficient mice.

In order to confirm the relationship between LCN2 and chronic inflammatory pain, CFA was injected into normal and LCN2-deficient mice by the method of Example 1-1, and the degree of swelling of the paw (scale of degree of inflammation response) The thickness of the foot was used to quantify.

The dorsoventral thickness of the central region of the posterior fascia was measured using a caliper. Were measured at 6 hours and 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days and 14 days after CFA injection, and the units were expressed in millimeters. The results are shown in Figs. 7A and 7B.

As shown in FIGS. 7A and 7B, it was confirmed that the thickness of the group was increased from the 6 hours after the injection to the normal mice by 14 days. In contrast, LCN2-deficient mice showed significantly decreased group thickness increase compared to normal mice, while the opposite group of CFA injected mice showed a change in group thickness in both normal and LCN2-deficient mice (See Fig. 7B).

2. CFA  After injection, LCN  Identification of histopathological features of deficient mice

Through H & E staining, CFA was injected into normal and LCN2 deficient mice and histopathological analysis was performed. The results are shown in C to E of Fig.

As shown in Fig. 7C to E, it was confirmed that, in the case of LCN2-deficient mice, tissue damage and infiltration of inflammatory cells were markedly reduced as compared with normal mice.

3. CFA  After injection, LCN  Of deficient mice MPO  Active measurement

MPO activity measurement was performed by the method described in Liang, D. Y et al. Specifically, LCN2-deficient mice and normal mice were injected into CFA or PBS, and the follicles were separated, washed with PBS, and resuspended in 200 μl of lysis buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxychlolate, 0.1% sodium dodecyl sulfate, 50 mM Tris HCl (pH 7.5), and 2 mM EDTA). Tissue samples were homogenized, centrifuged at 13,400 g for 15 min at 4 ° C, and the MPO activity of the supernatant was measured using a peroxidase assay kit (Anaspec, San Jose, Calif.). The results are shown in Fig. 8A.

As shown in Fig. 8A, it was confirmed that neutrophil infiltration was significantly reduced in the follicle of LCN2-deficient mice.

4. CFA  After injection, LCN  Expression of inflammatory mediators in deficient mice

MRNA expression of TNF- [alpha], IL-1 [beta] and IL-8 was confirmed by the method of Example 1-3 using the primers shown in Table 2 below. The results are shown in Figs. 8B to 8D.

Base sequence SEQ ID NO: TNF-α forward primer 5`-ATGGCCTCCTCATCAGTTG-3` 5 TNF-α reverse primer 5`-TTGGTTTGCTACGACGTG-3` 6 IL-1? Forward primer 5`-AAGTTGACGGACCCCAAAAGAT-3` 7 IL-1? Reverse primer 5`-TGTTGATGTGCTGCTGCGA-3` 8 IL-8 forward primer 5`-GACATACTCCAAACCTTTCCACC-3` 9 IL-8 reverse primer 5`-AACTTCTCCACAACCCTCTGC-3` 10

As shown in FIGS. 8B to 8D, in the case of the rear flank of LCA2-deficient mice after the CFA treatment of normal mice and the CFA of the posterior flank of the mice after the injection of CFA, TNF-alpha , And mRNA expression of IL-1 [beta] and IL-8 was significantly reduced.

5. Recombination in macrophages LCN2  After treatment, TNF -α and IL -1β

Primary mouse peritoneal macrophages were obtained in the following manner.

Mice were intraperitoneally administered with 3 ml of 3% sterile thioglycollate (BD Bioscience). Four days later, peritoneal exudate cells were collected by washing with PBS. 1x10 < ⁶ > cells were inoculated into a 6-well polystyrene culture dish. Thereafter, the cells were incubated for 3 hours, and the cells were attached to the dish and washed. Thereafter, non-adherent cells were removed by washing three times with PBS. Cells were then cultured for 24 hours. Cells were cultured with LCN2 protein (10 μg / ml) for 8 hours, total RNA was collected, and RT-PCR was performed on TNF-α and IL-1β. The results are shown in Fig.

As shown in Fig. 9, when LCN2 was treated in macrophages, expression of TNF-α and IL-1β was significantly increased.

[ Example  5] Recombination LCN2  When treated, confirm the appearance of chronic inflammatory pain

1. Recombination LCN2  After treatment, confirm the effect of thermal hyperalgesia reduction

In order to reaffirm the relationship between LCN2 and thermal hyperalgesia (related to inflammation) in the same manner as in Example 2-1, 0.1 μg or 1.0 μg of LCN2 was injected into the soles of normal mice, The degree of avoidance response to pain for the generated thermal stimuli was compared. The results are shown in Fig. 10A.

As shown in Fig. 10A, it was confirmed that when the recombinant LCN2 was treated, thermal hyperalgesia was induced depending on the treatment capacity. After 6 hours of treatment with 0.1 μg of recombinant LCN2, thermal hyperalgesia was induced and continued until 2 days after treatment.

In addition, when 1.0 μg of LCN2 was treated, it was confirmed that thermal hyperalgesia was induced 3 hours after treatment and continued for 6 days.

2. Recombination LCN2  After treatment, Tribe  Histopathological analysis of tissue

After the recombinant LCN2 treatment, the follicular tissues were stained with H & E to confirm inflammatory infiltration and tissue damage. The results are shown in Figs. 10B and 10C.

As shown in Fig. 10B, it was confirmed that when the recombinant LCN2 was treated at the rear of the mice, inflammatory infiltration and tissue damage occurred.

3. Recombination LCN2  After treatment, Tribe  Identification of inflammatory infiltration of tissue

After the recombinant LCN2 treatment, immunostaining was carried out using anti-Ly6G antibody in mouse tail tissue. The results are shown in Fig. 10C.

As shown in Fig. 10C, infiltration of Ly6G-positive neutrophils was confirmed after 12 hours of the recombinant LCN2 treatment.

[ Example  6] LCN2 However, glial cell ) And astrocytes ( astroctyte ) To determine the role of regulation of activity.

One. LCN2 In the regulation of activity on microglia.

In order to confirm the effect of LCN2 on microglia, the activity of microglia in spinal cord at 12 hours, 3 days, and 7 days after treatment of CFA with normal and LCN2 deficient mice was confirmed. In addition, normal mice were treated with recombinant LCN2, and then the activity of microglia was confirmed.

More specifically, By the method of Example 1-2 Iba-1 positive cells were identified by confirming the expression pattern of Iba-1 using an antibody against Iba-1 (mouse, 1: 200; Wako) as a primary antibody. The results are shown in Fig. 11 (A: confirmation of microbial cell activity in LCN-deficient mice; B: confirmation of microbial cell activity in case of treatment with recombinant LCN2) and Fig.

As shown in FIG. 11 and FIG. 12, in normal mice, the number of Iba-1 positive macrophages was significantly increased in the dosal horn of the spinal cord. Strong Iba-1 immunoreactivity was confirmed after 12 hours of CFA treatment, and it was confirmed that it continued until 3 to 7 days after.

On the contrary, in the spinal cord of LCN2-deficient mice, the morphological characteristics of micrographic cells and immune activity of Iba-1 were remarkably decreased, but in the contralateral spinal cord of CFA-treated normal and LCN2-deficient mice, It was confirmed that the morphological characteristics of the cells and the immunological activity of Iba-1 did not appear.

In addition, when the recombinant LCN2 was injected into the normal mouse, the activity of the microglial cell was increased in the unilateral spinal cord, but the activity of the microglial cell was not significantly increased in the contralateral spinal cord. In the normal mouse without LCN2 injection, Was not observed.

2. LCN2 Identification of the Role for the Regulation of Activity on Stromal Cells

In order to confirm the influence of LCN2 on astrocytes, the activity of astrocytes in spinal cord at 12 hours, 3 days, and 7 days after treatment of CFA with normal and LCN2 deficient mice was confirmed. Specifically, the expression pattern for GFAP was confirmed by using the antibody against GFAP as a primary antibody in the same manner as in Example 1-2. The results are shown in Fig.

As shown in Fig. 13, in normal mice, the number of GFAP-positive astrocytes was significantly increased in the dosal horn of the spinal cord. After 12 hours of CFA treatment, strong Iba-1 immunoreactivity was confirmed and continued until 3 to 7 days.

On the other hand, it was confirmed that GFAP-positive astrocytes in one spinal cord of LCN2-deficient mice were reduced, but the morphological characteristics of micrographic cells and immune activity of Iba-1 appeared in the contralateral spinal cord of normal and LCN2-deficient mice not treated with CFA Respectively.

[ Example  7] LCN2  Identification of the anti-inflammatory effects of neutralizing antibodies

1 μg of LCN2 neutralizing antibody was administered to the rat white part of the mouse 30 minutes before CFA injection and then quantified for pain response and group thickness for 6 hours, 1 day, 2 days and 3 days (after CFA treatment, 6 hours, 1 day, 2 days and 3 days). The results are shown in Figs. 14 to 16.

As shown in Figs. 14 to 16, in the group to which the LCN2 neutralizing antibody was administered, it was confirmed that it was thinner than that in the untreated group, and it was confirmed that there was no change in the contralateral group. In addition, when the LCN2 neutralizing antibody was administered, it was confirmed that the PWL and the PWT were increased compared with the case without administration.

As a result, it was confirmed that LCN2 neutralizing antibody, which is an LCN2 inhibitor, has an inflammatory pain suppressing effect.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition comprising lipocalin 2 inhibitor for preventing and          가과 자 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for LCN2 <400> 1 ccccatctct gctcactgtc 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for LCN2 <400> 2 tttttctgga ccgcattg 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for GAPDH <400> 3 tgggctacac tgagcaccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for GAPDH <400> 4 gggtgtcgct gttgaagtca 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for TNF-a <400> 5 atggcctcct catcagttg 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for TNF-a <400> 6 ttggtttgct acgacgtg 18 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for IL-1B <400> 7 aagttgacgg accccaaaag at 22 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for IL-1B <400> 8 tgttgatgtg ctgctgcga 19 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for IL-8 <400> 9 gacatactcc aaacctttcc acc 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for IL-8 <400> 10 aacttctcca caaccctctg c 21

Claims (15)

A pharmaceutical composition for preventing or treating inflammatory pain comprising an antibody that specifically binds to lipocalin 2 protein as an active ingredient. The pharmaceutical composition for preventing or treating inflammatory pain according to claim 1, wherein the inflammatory pain is chronic inflammatory pain or acute inflammatory pain. The pharmaceutical composition for preventing or treating inflammatory pain according to claim 1, wherein the antibody is a neutralizing antibody. delete delete A food composition for preventing or ameliorating inflammatory pain comprising an antibody that specifically binds to lipocalin 2 protein as an active ingredient. 7. The food composition of claim 6, wherein the inflammatory pain is chronic inflammatory pain or acute inflammatory pain. A method for alleviating inflammatory pain by administering an antibody that specifically binds to lipocalin 2 protein to an individual other than a human. 9. The method of claim 8, wherein the object is selected from the group consisting of a canine, a feline, a wild boar, an animal, a bovine animal, a deer and an animal, a giraffe and an animal, A primate, a rabbit, a rodent, and a primate excluding a human. A composition for diagnosing inflammatory pain comprising a primer that specifically binds to the lipocalin 2 gene or an antibody that specifically binds to the lipocalin 2 protein as an agent for measuring mRNA or lipocalin 2 protein level of lipocalin 2 gene . delete delete And measuring the expression level of the lipocalin 2 gene mRNA or protein from the biological sample. 14. The method according to claim 13, wherein the biological sample is at least one selected from the group consisting of tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine. And measuring the level of mRNA or protein expression of the lipocalin 2 gene by treating the biological sample with an agent for the prophylaxis or treatment of inflammatory pain or a candidate for treatment.

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