KR20170096350A

KR20170096350A - Composition for alleviating neuropathic pain comprising jmjd3 inhibitor

Info

Publication number: KR20170096350A
Application number: KR1020160017625A
Authority: KR
Inventors: 윤태영; 이지윤; 최혜영; 장대식; 주봉건
Original assignee: 경희대학교 산학협력단
Priority date: 2016-02-16
Filing date: 2016-02-16
Publication date: 2017-08-24
Also published as: KR101811012B1

Abstract

The present invention provides a composition for alleviating neuropathic pain comprising a JMJD3 activity inhibitor.

Description

Technical Field [0001] The present invention relates to a composition for alleviating neuropathic pain including JMJD3 activity inhibitor,

The present invention relates to a composition for relieving neuropathic pain comprising a JMJD3 activity inhibitor and a health functional food.

Pain is a sensory, emotional, and unpleasant experience that is expressed in terms of actual or potential tissue damage, and is one of the physiological responses to life threatening or defense against strong external stimuli that protects the body from danger It plays an important role.

Pain is divided into nocieptive pain due to tissue damage and neuropathic pain, which does not involve tissue damage but is caused by a nervous system adverse reaction. Inflammatory pain is known to be closely related to the inflammatory response accompanied by tissue damage and is naturally extinguished by healing of the injured area.

Neuropathic pain is a chronic neurological disease that occurs when the nervous system is damaged by various causes such as trauma, inflammation, ischemic injury, or metabolism (Gilron et al., 2006; Barn, 2006). In general, neuropathic pain involves spontaneous pain without any stimulation and hyperalgesia or allodynia caused by external stimuli. Hyperalgesia is defined as an abnormal pain response to noxious stimuli, and allodynia is known to be a pain response caused by innocuous stimuli (Woolf and Mannion, 1999).

In particular, patients with spinal cord injury experience neuropathic pain within a few months of complete or partial spinal nerve damage (70%) (Berens, 1997; Christensen and Hulsebosch, 1997; Yezierski, 1996 Yu et al., 2003) , Neuropathic toxicosis may ultimately develop into chronic neuropathic pain (Richards et al., 1980).

The pain caused by spinal cord injury can be seen in three major areas, including the above-noted pain in the rostral area of the injury, at-level pain in the vicinity of the damaged area, or in the same dermatome, (Siddall et al., 1997; Siddall and cousins, 1997). The pain caused by spinal cord injury varies from mild to severe pain, usually leading to chronic pain, causing pain to patients. In addition, neuropathic pain not only threatens the quality of life of the patient because it accompanies mental stress such as sleeping disorder, anxiety and depression as well as pain itself, but also enormous treatment costs (25 million won per person per year for spinal cord injury patients) (Cairns et al., 1996; Segatore, 1994).

Neuropathic pain is known to be difficult to relieve pain with conventional analgesics. Neuropathic pain inhibitors include anticonvulsants such as Gabapentin, Pregabalin, acting on the calcium channel, and anticonvulsants such as Carbamazepine acting on the sodium channel; Serotonin Norepinephrine Reuptake Inhibitor (SNRI) antidepressants; And opioids, which are used only in emergencies, are clinically used.

Although the drugs used for neuropathic pain are mostly used in clinical practice, they all cause similar side effects, which may cause adverse effects in some cases, but have a poor therapeutic effect. Although neuropathic pain is still experiencing severe pain in spite of the development of various analgesic or analgesic methods, the mechanism of neuropathic pain has not been clearly established, It is evident that sexual pain can not be treated properly.

On the other hand, histone methylation means methylation in the lysine and arginine residues of the histone protein, and each residue has three different forms of methylation. Depending on the type of histone methylation, the relevant gene may be active or inactive. In general, methylation of lysines present in H3K9, H3K27 and H4K20 inactivates genes and methylation of lysine in H3K4, H3K36 and H3K79 promotes gene activity.

Inhibition of transcription by the H3K27-methyl mark regulates the expression of transcription of lineage-specific (LS) genes in lineage-committed progenitor cells (Mikkelsen et al., 2007). H3K27me3 plays an important role in pluripotentcy and plasticity during embryonic stem (ES) cells, polycomb-mediated gene silencing, and X chromosome inactivation (Schuettengruber et al 2006; Trojer et al., 2006).

H3K27 is methylated by polycomb compelx (PRC2) proteins, and H3K27me is able to inhibit the expression of genes at the developmental stage. When three methylations are made to the 27th lysine of the protein (H3K27me3), the expression of the gene is inhibited by aggregation of chromatin. H3K27me3 plays an important role in developmental fate and cell growth regulation (Sparmann and van Lohuizen, 2006). Recently, UTX and JMJD3, which are H3K27-specific demethylating enzymes that remove the methyl mark of H3K27me, have been reported (Agger et al., 2007; De Santa et al., 2007; Lan et al. 2007; Lee et al., 2007).

JMJD3 has been known to be involved in various biological pathways such as neuronal maturation and differentiation of immune cells. However, despite the close association with JMJD3 cell maintenance and differentiation, the relationship between neuropathic pain and neuropathic pain caused by central nerve injury has not been elucidated. No attempts were made.

The present inventors have clarified the interrelationship between neuropathic pain and JMJD3 gene, and based on the above understanding, provide a fundamental treatment method for neuropathic pain.

The present invention aims at clarifying the correlation between JMJD3 protein and neuropathic pain in order to solve the problems of the prior art described above, and to provide a method of preventing or treating neuropathic pain fundamentally.

According to one aspect of the present invention, there is provided a composition for relieving neuropathic pain comprising JMJD3 activity inhibitor as an active ingredient.

In one embodiment, the JMJD3 activity inhibitor may be selected from the group consisting of an antibody or an antigen-binding fragment thereof, an uterus, an siRNA, an shRNA, a microRNA, an inhibitory compound, and a pharmaceutically acceptable salt thereof.

In one embodiment, the inhibitory compound may be a compound of Formula 1:

[Chemical Formula 1]

In one embodiment, the inhibiting compound may be a compound of the following formula (2).

(2)

In one embodiment, the inhibiting compound may be a compound of the following formula (3).

(3)

In one embodiment, the inhibitory compound may be a compound of formula (4).

[Chemical Formula 4]

In one embodiment, the inhibiting compound may be a compound of formula (5).

[Chemical Formula 5]

In one embodiment, the neuropathic pain can be induced by damage of the central nervous system.

According to another aspect of the present invention, there is provided a health functional food for preventing or ameliorating neuropathic pain comprising JMJD3 activity inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment, the health functional food may be prepared in any one form selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings.

According to another aspect of the present invention, there is provided a method for the treatment of JMJD3 comprising: (a) contacting cells expressing JMJD3 and a test agent; (b) determining the modulatory effect of the test agent on JMJD3 expression; And (c) comparing the expression level of the JMJD3 in the cells contacted with the test agent and in a control group not in contact with the test agent, for screening a therapeutic agent for neuropathic pain.

In one embodiment, the test agent may be selected from the group consisting of a polypeptide, a small organic molecule, a polysaccharide, and a polynucleotide.

According to another aspect of the present invention there is provided a method of treating neuropathic pain comprising administering to a non-human subject a JMJD3 activity inhibitor.

The present invention provides a method for effectively alleviating neuropathic pain based on a clear understanding of the molecular mechanism of JMJD3, and relates to the treatment, prevention, diagnosis, or the study of neuropathic pain caused by damage to the central nervous system And the like.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

Figure 1 shows the correlation between inhibition of JMJD3 and central neuropathic pain using GSK-J4, which is known as the JMJD3 inhibitor.
FIG. 2 shows the JMJD3 inhibitory effect of a compound according to an embodiment of the present invention.
Figure 3 confirms the mitigating effect of Swertisin's central neuropathic pain.
Fig. 4 shows the mitigation effects of protocatechuic acid in the treatment of central neuropathic pain.
Figure 5 confirms the mitigating effect of Gallic acid on central neuropathic pain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. When an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. It is not intended that the invention be limited to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', amino acid sequences from left to right, amino to carboxyl.

Hereinafter, the present invention will be described in more detail.

The "neuropathic pain" may be defined as a pain initiated or induced by a primary impairment or dysfunction of the nervous system by the IASP (IASP, Classification of chronic pain, 2nd Edition, IASP Press (2002) .

The neuropathic pain may be induced by peripheral nerve injury or central nerve injury, including peripheral nervous system disorders or damage, multiple sclerosis including spinal cord injury, trigeminal neuralgia, diabetes neuropathy, Pain following pharmacotherapy of cancer, atypical facial pain, post herpetic neuralgia, and neurological disorders, including phantom limb pain, HIV infection, cancer, alcoholism, cancer therapy, Neuropathic pain caused by the central nervous system, but preferably it may be neuropathic pain induced by damage of the central nervous system.

The "neuropathic pain induced by central nerve injury" refers to neuropathic pain initiated or induced by primary damage, dysfunction, or temporary disruption of the central nervous system, It can be defined as the nervous system (Abbadie C., 2005). Symptoms and differences associated with neuropathic pain induced by peripheral and central nervous system damage have been reported in detail in previous literature (Jensen et al., 2003; Klein et al., 2005).

The " alleviation " may mean any action in which administration of the composition improves or alleviates neuropathic pain.

The above-mentioned " activity inhibitor " may mean a substance that blocks or delays the biological pathway in which the JMJD3 protein is involved, by inhibiting or decreasing the expression of the JMJD3.

The JMJD3 activity inhibitor may be any one selected from the group consisting of an antibody or an antigen-binding fragment thereof, an abstamator, an siRNA, a shRNA, a microRNA, an inhibitory compound and a pharmaceutically acceptable salt thereof. Or more, but is not limited thereto.

The " antibody " includes monoclonal antibodies and chimeric antibodies, humanized antibodies, and human antibodies thereto, and may include antibodies already known in the art in addition to the novel antibodies. The antibody may comprise a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, so long as the antibody has the property of binding specifically recognizing the JMJD3 protein. A functional fragment of a molecule of an antibody means a fragment having at least an antigen binding function and may be Fab, F (ab ') 2, F (ab') 2 or Fv.

Quot; siRNA " means a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a complementary sequence. Since siRNA can inhibit the expression of a target gene, it can be provided as an efficient gene knockdown method or gene therapy method. The siRNA is not limited to the complete pairing of the double-stranded RNA portions that are mated with each other, but the siRNA may be paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one of the chains) And a portion that is not achieved may be included. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by the RNAi effect. The adhesive end structure can be a structure having a 3 'end protruding structure and a 5' end protruding structure. The number of protruding bases is not limited. In addition, the siRNA may include a low molecular RNA (for example, a natural RNA molecule such as a tRNA, a rRNA, or a viral RNA, or an artificial RNA molecule) at a protruding end of one end to the extent that the effect of suppressing the expression of the target gene can be maintained. The siRNA end structure does not need to have a truncation structure on both sides, and may be a step-loop structure in which the terminal region of the double-stranded RNA is connected by linker RNA. The siRNA according to the present invention may be a complete form having polynucleotide pairing itself, that is, a form that is directly introduced into a cell through two transformation processes in which an siRNA is directly synthesized in vitro, Single-stranded oligonucleotide fragments and their reverse-phase trefoil can be derived from single-stranded polynucleotides separated by a spacer, for example, siRNA expression vectors or siRNA expression vectors or PCR-derived siRNAs The expression cassette may be introduced into the cell through a transformation or infection process.

The "shRNA" is to overcome the disadvantages of expensive biosynthesis cost of siRNA, short-time maintenance of RNA interference effect due to low cell transfection efficiency, and the like. It is known that adenovirus, lentivirus and plasmid expression vector system , And it is well known that such shRNA is converted into an siRNA having an accurate structure by the siRNA processing enzyme (Dicer or Rnase III) existing in the cell to induce silencing of the target gene .

In one embodiment, the inhibitory compound may be a compound of Formula 1:

[Chemical Formula 1]

The compound of Chemical Formula 1 may be used in combination with 5-hydroxy-2- (4-hydroxyphenyl) -7-methoxy-6 - [(2S, 3R, 4R, 5S, 6R) -3,4,5-trihydroxy- oxan-2-yl] chromen-4-one. The compound is commonly referred to as Swertisin, and is contained as an active ingredient in plants such as spruce.

(2)

The compound of formula (2) may be named 3,4-dihydroxybenzoic acid. The compound is generally called protocatechuic acid, and may be derived from plants such as green tea and bokbunja.

(3)

The compound of formula (3) may be named 3,4,5-trihydroxybenzoic acid. The compound is usually called gallic acid and may be derived from plants such as green tea, maple leaf, and grass.

In one embodiment, the inhibitory compound may be a compound of formula (4).

[Chemical Formula 4]

The compound of Chemical Formula 4 may be named as (2R) -3- (3,4-dihydroxyphenyl) -2 - [(E) -3- (3,4-dihydroxyphenyl) prop-2-enoyl] oxypropanoic acid. The compounds are commonly referred to as rosmarinic acid, and may be derived from plants such as perilla leaves, barley, and the like.

In one embodiment, the inhibiting compound may be a compound of formula (5).

[Chemical Formula 5]

The compound of formula (5) can be prepared by reacting [(2R, 3R, 4R, 5R, 6R) -6- [2- (3,4- dihydroxyphenyl) ethoxy] -4,5-dihydroxy- , 5R, 6S) -3,4,5-trihydroxy-6-methyloxan-2-yl] oxymethyloxan-3-yl] (E) -3- (3,4-dihydroxyphenyl) prop-2-enoate The compound is commonly referred to as Forsythiaside, and may be derived from the fruit of plants such as forages and forages.

The JMJD3 activity inhibitor may be administered in the form of oral delivery, parenteral delivery. The composition may be administered via any conventional route so long as it can reach the target tissue. For example, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrathecal administration, intraperitoneal administration, intraperitoneal administration, It is not.

The JMJD3 activity inhibitor may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form, and may further comprise carriers, excipients and diluents used in the preparation of the pharmaceutical composition.

The carrier, excipient and diluent may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral, but is not limited thereto.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by adding to the alpha-linolenic acid and fractions thereof from the perilla oil at least one excipient such as starch, calcium Carbonate, sucrose, lactose, or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may also be used.

The liquid preparation for oral administration may be a suspension, a solution, an emulsion, a syrup, etc. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solutions and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and ethyl oleate. The base of the suppository can be witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin.

A pharmaceutical composition comprising the JMJD3 activity inhibitor as an active ingredient can be administered to a subject in a pharmaceutically effective amount. The " pharmaceutically effective amount " means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dosage level will depend on the type of disease, severity, activity of the drug, Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

The health functional food may refer to a food prepared or processed by using raw materials or ingredients having useful functions in the human body according to the Health Functional Foods Act No. 6727. The functional properties include nutrients Or for the purpose of obtaining a beneficial effect for health use such as physiological action. However, the type of the food to be ingested for the purpose of improving health is not particularly limited.

The health functional food refers to a food prepared and processed by using a raw material or a component having a useful function in the human body according to the Health Functional Food Act No. 6727. The functional property refers to the nutrient Or for the purpose of obtaining a beneficial effect for health use such as physiological action.

The above-mentioned health functional foods may contain usual food additives, and, unless otherwise specified, the above-mentioned food additives are classified according to the standard and the standard of the relevant items according to the general rules of the Food Additives Ordinance approved by the Korean Food and Drug Administration It can be judged whether it is suitable or not. The food additives described in the above-mentioned Food Additives Code include chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as persimmon extract, licorice extract, crystalline cellulose, high- A preservative formulation, a tar coloring agent, and the like.

The health functional food may be variously used for foods and beverages for preventing or improving thrombosis-related diseases including arterial thrombosis and venous thrombosis. For example, various health foods such as various foods, beverages, gums, tea, vitamins, , Food additives and the like.

The health functional food may be variously used for foods and beverages for preventing or ameliorating neuropathic pain related diseases. For example, the health functional food may be used in a variety of foods, beverages, gums, tea, vitamin complex, health functional supplement, Can be used.

The health functional food may contain 1.0 to 10.0% by weight of JMJD3 activity inhibitor based on the total weight for the purpose of preventing or improving neuropathic pain. If the JMJD3 activity inhibitor is less than 1.0 wt%, the effect of improving neuropathic pain may not be sufficiently realized. If the JMJD3 activity inhibitor is more than 10.0 wt%, the product may not have its original quality or cost efficiency may be deteriorated and unexpected side effects may be caused .

In addition, the health functional food may be manufactured and processed into any one form selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings for the purpose of preventing or improving neuropathic pain.

Specifically, the health functional food in the form of tablets may be prepared by granulating a mixture of JMJD3 activity inhibitor, excipient, binder, disintegrant and other additives by a conventional method, compression molding with a lubricant or the like, Can be produced by molding. The health functional food of the tablet form may contain a mating agent and the like if necessary, and may be sieved to a suitable skin care agent if necessary.

The hard capsule of the capsule type health functional food may be prepared by filling a normal hard capsule with a mixture of JMJD3 activity inhibitor and excipient such as excipient or a granular product thereof or a gelatinized granule. The soft capsule may contain JMJD3 activity inhibitor And additives such as excipients and the like may be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The above-mentioned ring-type health functional food can be prepared by molding a mixture of JMJD3 activity inhibitor, excipient, binder, disintegrant and the like by an appropriate method. If necessary, the preparation may be mixed with white sugar or other suitable skin care agent or starch, talc, The substance may be irritated.

The granular health functional food may be prepared by granulating a mixture of JMJD3 activity inhibitor, excipient, binder, disintegrant and the like by a suitable method, and may contain a flavoring agent, a mating agent and the like if necessary.

Further, the definitions of the above excipients, binders, disintegrants, lubricants, mating agents, flavoring agents and the like are described in documents known in the art, and the functions and the like may be the same or similar.

The term " contact " is used in its ordinary meaning to mean to combine two or more agents (e.g., two polypeptides) or to bind a preparation and a cell (e.g., a protein and a cell). For example, a recombinant polynucleotide encoding two polypeptides can be used to bind two or more agents in a test tube or other container or to bind a test agent and a cell or cell lysate to a test agent, To coexpress the two polypeptides in the cell or cell lysate.

The term "agent" or "test agent" includes any substance, molecule, element, compound, entity, or combination thereof . For example, a protein, a polypeptide, a small organic molecule, a polysaccharide, a polynucleotide, etc., and may be a natural product, a synthetic compound or a chemical compound or a combination of two or more substances. have. Unless otherwise defined, the agents, materials and compounds may be used interchangeably.

The screening method can utilize various biochemical and molecular biological techniques known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third ; Protein binding assays (in vitro pooled assays), EMSA, < RTI ID = 0.0 > EMSA, < / RTI & (Such as phosphorylated assays), yeast-2 hybrid assays, non-immunoprecipitation assays, immunoprecipitation Western blot assays, immuno-co-localization assays, and the like. Can be carried out by various known methods.

The compound used for the screening may be a low molecular weight compound having a therapeutic effect. For example, the low molecular weight compound may have a weight of about 1000 Da, such as 400 Da, 600 Da, or 800 Da. Depending on the purpose, the compound may constitute a part of the compound library, and the number of the compounds constituting the library may vary from several tens to several millions. The compound library can be used in combination with peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on a template such as benzodiazepines, hydantoins, biaryls, carbocycles and polycycle compounds (such as naphthalene, But are not limited to, thiourea, azine, acridine, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles such as triazine, indole, thiazolidine and the like.

In addition, biologics may be used for the screening method. The biologics refers to a cell or a biomolecule, and refers to a substance produced using protein, nucleic acid, carbohydrate, lipid, or in vivo or in vitro cell system. The biomolecules may be used alone or in combination with other biomolecules or cells. The biomolecule may include, for example, a protein or biological organic material found in a polynucleotide, a peptide, an antibody, or other plasma.

The type and concentration of the cells used in the screening method and the amount and type of the test substance may be varied depending on the specific experimental method and the type of the test agent to be used, and those skilled in the art will be able to select an appropriate amount. As a result, a substance which increases or inhibits the activity of JMJD3 in the presence of the test substance can be selected as a candidate substance as compared with a control substance not in contact with the test substance. About 80% reduction, about 80% reduction, about 75% reduction, about 70% reduction, about 65% reduction, about 60% reduction, about 90% reduction, about 90% reduction, %, Less than about 55%, less than about 50%, less than 45%.

According to one embodiment, it is possible first to determine whether the test agent is capable of modulating the biological activity of JMJD3. Specifically, the primary assay can identify the modulating agent that modulates the biological activity of the polypeptide by analyzing the biological activity of the isolated JMJD3 in the presence of the test agent.

The primary assays can be assessed for modulation of various biological activities of JMJD3. For example, the test agent may be assayed for the level of expression of JMJD3, e.g., activity to modulate transcription or translation. The test agent may also be assayed for its activity to modulate the intracellular level or stability of JMJD3, such as post-translational modification or hydrolysis.

The primary assay can be used to identify modulatory agents associated with the biological activity of JMJD3 and then secondarily test whether the test agent can alleviate neuropathic pain. For example, the test agent may be a neuropathic pain Lt; RTI ID = 0.0 > and / or < / RTI >

The primary and secondary assays can use intact JMJD3 and its fragments, analogs, or functional equivalents. Fragments that can be used in these assays generally can possess one or more of the biological activities of JMJD3. Also, a fusion protein comprising said fragment or analog may be used for screening for the test agent. The functional equivalent of JMJD3 includes amino acid deletion, insertion, or substitution, but retains the same bioactivity as JMJD3, and thus can be used to carry out the screening method of the present invention.

A variety of assays routinely practiced in the art can be used to identify agents that modulate JMJD3. Preferably, the agent can be screened with a cell based assay system. For example, in a typical cell-based assay (i.e., a second assay) for screening, the reporter gene activity (e.g., enzyme activity) is measured in the presence of the test agent and compared to the activity of the reporter gene in the absence of the test agent .

The reporter gene may encode any detectable polypeptide (reaction or reporter polypeptide) known in the art, for example, a polypeptide detectable by fluorescence or phosphorescence or a polypeptide detectable by the enzyme activity it possesses have. Detectable reponse polypeptides include, for example, luciferase, alpha-glucuronidase, alpha-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, enhanced green fluorescent protein and human Secreted < / RTI > alkaline phosphatase.

In such cell-based assays, test agents (e. G., Peptides or polypeptides) may be expressed by other vectors present in the host cells. In some methods, a library of test agents can be encoded by a library of such vectors (e.g., a cDNA library). The library can be prepared using methods known in the art (Sambrook et al. And Ausubel et al., Supra) or from a variety of commercial sources.

In addition to the cell-based assays, they can also be screened by non-cell based methods. The method can be used for example in mobility shift DNA-binding assays, methylation and uracil interference assays, DNase and hydroxyl radical footprinting assays ), Fluorescence polarization and UV cross-linking or chemical cross-linkers. A general overview is given in Ausubel et al. (Ausubel et al., Supra, chapter 12, DNA-Protein Interaction). Techniques for separating co-associating proteins, including nucleic acids and DNA / RNA binding proteins, include cleavable cross-linkers dithiobis (succinimidylpropionate) and 3,3'-sulfosuccinimidyl-propionate (McLaughlin, Am. J. Hum. Genet., 1996; Tang, 1996; Lingner, 1996; Chodosh, 1986).

Hereinafter, the present invention will be described in more detail by way of examples. However, the embodiments of the present invention are intended to assist the understanding of the invention, and the scope of protection of the invention is not limited by the following examples.

Experimental Example 1: Preparation of an animal model of central neuropathic pain

Adult male rats (body weight 220-250 g) at 7 weeks of age were used as animal models. The rats were housed in a 12 hour light cycle room and allowed free access to water and food. Animal experiments were carried out in accordance with the guidelines for animal experiments at Kyung Hee University.

To induce CNS pain in animal models, spinal cord T8-T10 was exposed after intraperitoneal injection of chloral hydrate (500 mg / kg). An animal model with spinal cord injuries was used, with a weight of 10g removed from a certain height to give similar trauma to human spinal cord injury, and trauma intensity was controlled using a NYU impactor designed to be computerized.

After the rat was fixed in the clamp of the NYU impactor, a weight of 10 g at 25 mm height was dropped freely to damage the spinal cord T9. Data on the damage intensity calculated from the computer was used to confirm that the damage was uniformly applied within the error range, and the damaged area was sealed. The injured area was disinfected with povidone solution, and two cages were placed in a cage. The bladder was artificially massaged three times a day to promote urination.

Experimental Example 2: Measurement of mechanical allodynia

The spinal cord was subjected to mechanical stimulation on the damaged animal model and the pain change was measured to measure the reduction or mitigation effect of neuropathic pain.

Spinal cord injured animals were placed in a transparent plastic box (10 cm × 10 cm) on a wire net (3 mm × 3 mm). At least 20 minutes before the start of the sensory test, the adaptation was made.

The von-Frey filaments were applied to both soles. The tactile (mechanical) sensitivities were measured using the stiff nylon von Frey single filament series (3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18mN, Stoelting, WoodDale, IL, USA, equivalent in grams to 0.4, 0.6 , 1.0, 2.0, 4.0, 6.0, 8.0 and 15.0). The foot avoidance response to the increased mechanical stimuli was assessed by applying sufficient force. The present prefilament was applied to each foot for 3 to 4 seconds to a complete bend. The 50% threshold was confirmed using the up-down method (Chaplan et al., 1994).

Among the eight bouffray filaments of 0.4 to 15 g, a middle filament of 2.0 g (4.31 mN) was first used and it began to identify the most sensitive region at various sites. If there is no evasive response to the foot, repeat with the next thick filament. When the avoidance response appears, the weaker filament was applied to the sensitive area. Stimulation was performed at intervals of 2 seconds.

(G) of von Frey hair showing a 50% avoidance response by foot stimulation. The mean value was calculated by measuring both feet. It was judged that mechanical allodynia occurred when the avoidance response threshold was decreased statistically significantly compared with before spinal cord injury induction.

Experimental Example 3: Measurement of heat hyperalgesia

The spinal cord was subjected to thermal stimulation and the pain change was measured on the damaged animal model, and the reduction or mitigation effect of neuropathic pain was measured.

Measurement of hyperalgesia was performed by the Hargreaves method using a plantar test device (810 plantar test, IITC, USA) (Hargreaves et al., 1988).

Spinal cord injured animals were placed on top of the glass plate, sealed with a rectangular transparent acrylic box, and allowed to adapt for 20 minutes. At the bottom of the glass plate, a movable laser beam is located and the surface of the plantar surface of the animal can be continuously heated. The thermal intensity of the laser beam was controlled by obtaining an average paw withdrawal latency of about 15 seconds and the time limit was limited to 20 seconds to prevent tissue damage. Three thermal stimuli were applied to both feet at 5 to 10 minute intervals. The mean time of the foot evasion latency of each foot was measured, and the avoidance time of both feet was measured and the mean value was calculated.

Experimental Example 4: Correlation between JMJD3 and CNS pain

The spinal cord injury animal model of Experimental Example 1 was used to confirm the correlation between JMJD3 and central neuropathic pain. JMJD3 and central neuropathic pain, the analgesic effect of the JMJD3 inhibitor was observed.

After 30 days of spinal cord injury, intrathecal injection of GSK-J4, known as JMJD3 inhibitor, was applied to an animal model of CNS pain, and von Frey test and Hagreaves test were used to evaluate mechanical allodynia and pain Hyperalgesia were measured.

Mechanical allodynia and hyperalgesia were measured, and the analgesic effect was maximal after 30 minutes of injecting GSK-J4 into the spinal canal, and the analgesic effect continued for 90 to 120 minutes (Fig. 1).

These results suggest that the activity of JMJD3 and neuropathic pain are closely related to each other and that neuropathic pain can be alleviated or improved by down-regulating JMJD3 activity.

Experimental Example 5: Confirmation of inhibitory effect of a specific compound on JMJD3

Addition of JMJD3 to histone in vitro confirmed the inhibitory effect of JMJD3 indirectly through the level of H3K27me, since H3K27 was demethylated and the level of H3K27me decreased.

The levels of H3K27me were almost unchanged (Fig. 2) when JMJD3, swertissin, protocatechuic acid, gallic acid, rosemary acid, and formic acid were separately added to the histone and the level of H3K27me was measured. That is, the above results suggest that the compounds can effectively inhibit the activity of JMJD3.

Experimental Example 6: Confirmation of the effect of Swertisin on central neuropathic pain

After 30 days of spinal cord injury, mechanical allodynia and hyperalgesia were measured by von Frey test and Hagreaves test after spinal cord injection of swell gland into animal model of CNS pain.

Similar to GSK-J4, which is known as a JMJD3 inhibitor, the analgesic effect was maximal after 30 minutes of spinal tube injection and the analgesic effect continued for about 90 minutes (FIG. 3).

Experimental Example 7: Confirmatory effect of protocatechuic acid on central neuropathic pain

After 30 days of spinal cord injury, intraperitoneal injection of protocatechuic acid was performed in an animal model of CNS pain, and mechanical allodynia and hyperalgesia were assessed by von Frey test and Hagreaves test. Respectively.

Similar to GSK-J4, which is known as the JMJD3 inhibitor, the analgesic effect was maximal after 30 minutes of intraperitoneal injection and the analgesic effect continued for about 90 minutes (FIG. 4).

Experimental Example 8: Effect of gallic acid on pain relief of central neuropathic pain

After 30 days of spinal cord injury, mechanical allodynia and hyperalgesia were measured by von Frey test and Hagreaves test after intraperitoneal injection of gallic acid in an animal model of CNS pain.

Similar to GSK-J4, which is known as the JMJD3 inhibitor, the analgesic effect was maximal after 30 minutes of intraperitoneal injection and the analgesic effect continued for about 120 minutes (FIG. 5).

The experimental results suggest that swertissin, protocatechuic acid, gallic acid, rosemary acid, and formic acid can effectively inhibit JMJD3 and may be useful as a therapeutic agent for improving the central nervous pain.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

A composition for relieving neuropathic pain comprising JMJD3 activity inhibitor as an active ingredient.

The method according to claim 1,
Wherein said JMJD3 activity inhibitor is at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof, an alphamer, an siRNA, an shRNA, a microRNA, an inhibitory compound and a pharmaceutically acceptable salt thereof.

3. The method of claim 2,
Wherein the inhibiting compound is a compound of formula < RTI ID = 0.0 >
[Chemical Formula 1]

3. The method of claim 2,
Wherein the inhibiting compound is a compound of formula (2).
(2)

3. The method of claim 2,
Wherein the inhibiting compound is a compound of formula (3).
(3)

3. The method of claim 2,
Wherein the inhibiting compound is a compound of formula (4)
[Chemical Formula 4]

3. The method of claim 2,
Wherein the inhibiting compound is a compound of the following formula (5).
[Chemical Formula 5]

8. The method according to any one of claims 1 to 7,
Wherein said neuropathic pain is induced by damage of the central nervous system.

A health functional food for preventing or ameliorating neuropathic pain comprising JMJD3 activity inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

10. The method of claim 9,
Wherein the active ingredient is one selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings.

(a) contacting cells expressing JMJD3 and a test agent;
(b) determining the modulatory effect of the test agent on JMJD3 expression; And
(c) comparing the expression level of said JMJD3 in cells contacted with said test agent and in a control not contacted with said test agent.

12. The method of claim 11,
Wherein the test agent is at least one selected from the group consisting of a polypeptide, a small organic molecule, a polysaccharide, and a polynucleotide.

A method for treating neuropathic pain comprising administering to a non-human subject a JMJD3 activity inhibitor.