WO2015025995A1 - Pharmaceutical composition and method for preventing or treating diseases associated with epilepsy or convulsions by targeting microrna - Google Patents

Abstract

Disclosed is a pharmaceutical composition for preventing or treating diseases associated with epilepsy or convulsions, the composition containing a material targeting miR-203. Disclosed are a marker capable of being used for the diagnosis or prognosis measurements of diseases associated with epilepsy or convulsions, and a use of the marker. The composition of the present invention, unlike existing therapeutic agents depending on allopathy, can restore a glycine receptor, thereby fundamentally treating various diseases caused by the reduction in expression level of the glycine receptor, for example, diseases associated with epilepsy or convulsions.

Description

Pharmaceutical compositions and methods for preventing or treating epilepsy or seizure related diseases that target microRNAs

The present application relates to the treatment of epilepsy or seizure diseases that target specific miRNAs.

Epilepsy is a chronic neurological disorder in which the brain's irregular electrical excitation causes the brain to generate electricity and cause seizures and cramps. Epilepsy is caused by a number of causes, epidemiological studies have reported that more than one-third of patients have a history of pathological changes or brain damage in the brain, and the main causes are stroke, congenital malformations, and head. Trauma, encephalitis, brain tumors, degenerative encephalopathy, heredity, premature infants, damage before and after delivery.

Epilepsy is a disease that is not usually treated with drug therapy but relies on regulation. However, more than 30% of epilepsy patients do not control seizures even with drugs of various mechanisms of action, and thus, there is a need for the development of new mechanisms for the control and / or treatment.

Recent attempts have been made to develop therapeutics using micro RNA suppressors.

International Patent Publication No. WO 2013/045652 discloses the treatment of epilepsy using inhibitors of miR-134.

European Publication No. 2436784 also discloses the diagnosis and treatment of colorectal cancer with miR-203.

There is still a need to develop or control drugs for seizure diseases such as epilepsy based on new mechanisms.

The present application seeks to develop miRNA modulators targeting miRNAs to treat epilepsy or seizure related diseases.

In one aspect, the present disclosure provides a pharmaceutical composition for treating or preventing epilepsy or seizure related diseases, comprising a substance capable of inhibiting the activity of miR-203 and a pharmaceutically acceptable carrier.

In one embodiment according to the invention, the substance capable of inhibiting the activity of the miR-203 activity according to the present invention is in particular the interaction with the 3'-UTR of the GLRβ subunit (GLRB, glycine receptor beta subunit) of the miR-203 A substance capable of inhibiting action, and in another embodiment, a substance capable of inhibiting the activity of the activity of miR-203 herein is a substance capable of inhibiting its expression.

In one embodiment according to the present application, the substance capable of inhibiting the activity of miR-203 according to the present application is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the miR-203 Such nucleic acid molecules include, for example, RNA, DNA, antagonists, antisense molecules, siRNAs, shRNAs, 2′-O-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, phosphorothioate-backbone ribonucleotides, This includes, but is not limited to, decoy oligonucleotides, peptide nucleic acid (PNA) oligonucleotides or locked nucleic acid (LNA) oligonucleotides. In one embodiment according to the present disclosure, a nucleic acid molecule that may be used herein is an antisense comprising a sequence that is partially or wholly complementary to the nucleotide sequence of the first or second to seventh or eighth nucleotide sequences of SEQ ID NO: 1 Oligonucleotides. Antisense oligonucleotides according to the present disclosure may be used without modifications or may include one or more modifications in view of the effects according to the present disclosure, eg, one or more nucleotides making up thereof are LNA, or sugars of one or more nucleotides making up the same. One or more phosphothioates, or combinations thereof, may be included in this 2'-0-methylation, or backbone thereof, but is not limited thereto. For example, an antisense oligonucleotide according to one embodiment of the present application may be 5'-CAUUUCAC-3 ', 5'-AUUUCAC-3', 5'-CAUUUCA-3 ', 5'-AUUUCA-3' or 5'-CUAGUGGUCCUAAACAUUUCAC Contains -3 '.

Seizure related diseases, which cause seizures, in which the compositions, kits and methods according to the invention can be used, include stroke, hippocampus, cerebral palsy, congenital malformations, central nervous system infection, hypoxia, brain tumors, traumatic brain injury, neurodegenerative diseases, metabolic Seizures due to disease, autoimmune disease, or unknown cause, but not limited thereto.

The composition according to the invention is in particular delivered to the brain and may be formulated for, but not limited to, intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration, or oral administration.

In another aspect, the present disclosure also provides a method of contacting a miR-203 RNA with a test substance; And

Determining the activity of miR-203 RNA in contact with the test substance, wherein the activity of the contacted miR-203 is reduced compared to the activity of miR-203 RNA of a control group not in contact with the test substance. It provides a method for screening a substance for treating or preventing epilepsy or seizure-related diseases, which is selected as a candidate.

MiR-203 RNA used in the method according to the invention is provided in the form of cells expressing it, the activity of which is the 3'-UTR of the miR-203 RNA and its target GLRβ subunit (GLRB, glycine receptor beta subunit) It can be determined by analyzing the interaction with.

In other embodiments, the present disclosure also provides miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451 miRNA selected from the group consisting of, miR-455, miR-99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 The present invention relates to a kit for diagnosing a seizure disease comprising a substance for detecting the sequence, including a nucleotide sequence of the gene, a complementary sequence thereof, or a fragment thereof.

In another embodiment the disclosure is miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451 miRNA selected from the group consisting of, miR-455, miR-99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 Detecting the expression of one or more of the markers; And correlating the expression level of the detected marker with a diagnosis or prognosis of an epilepsy or seizure related disease of a test subject, to detect the marker to provide information necessary for diagnosing or prognosis of an epilepsy or seizure disease. Provide a way to.

The step of associating in the method herein further comprises using non-marker clinical information of the seizure disease test subject, for example, the age, sex, weight, diet, body mass of the subject. , But not limited to, underlying disease, EEG, type of seizure, brain MRI, brain CT, or cerebrospinal fluid.

In an embodiment according to the present invention, the step of associating the expression level of the determined markers is compared with a detection result for each of the markers determined in the normal control group. Among the markers, miR-21, miR-129-5p, and miR-132 , miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, miR-455, miR-199a-5p, miR-666-3p, miR- Expression of 708 and miR-805 was increased in comparison with the control group, and miR-99b *, miR-206, miR-468 and miR-574-3p in the markers were decreased in expression.

In another aspect, the present disclosure provides a method for treating or preventing epilepsy or seizure related diseases through inhibition of miR203 activity in cells or tissues of a subject, in particular brain cells or brain tissues or brain.

Furthermore, the present disclosure also provides for the treatment or prevention of epilepsy or seizure related diseases in a subject, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an miR203 activity inhibitor. Provide a method.

Furthermore, the present application also provides a substance capable of inhibiting the activity of miR-203 for use in the treatment or prevention of epilepsy or seizure related disease in a subject. In one embodiment according to the present application, a substance capable of inhibiting the activity of miR-203 is delivered to the brain.

The epilepsy treatment composition targeting miR203 according to the present invention has a new mechanism of action by restoring glycine receptors unlike conventional epilepsy therapeutics, and various diseases caused by lowering the expression level of glycine receptors, for example, epilepsy It is possible to fundamentally treat diseases related to seizures including hypertension, and it may be particularly useful for patients who have difficulty in controlling seizures with conventional epilepsy treatments.

1A and 1B show the results of microarray analysis of miRNA expression in hippocampus (a) and cortex (b) in chronic epilepsy mice with spontaneous recurrent seizure (SRS). More than two-fold expression changes and P -values versus controls indicated only up- and down-regulated miRNAs meeting the conditions of <0.05. The color represents the Z-score, with red () representing high expression (high Z-score) and green (right top and bottom left) low expression. miR-203 is shown to be elevated in both hippocampus and cortex. In FIG. 1A, a red color corresponds to a left upper portion and a lower right portion based on an intersection line, and a green color corresponds to a right upper side and a lower left side. In FIG. 1B, a red color corresponds to a right side and a green color corresponds to a left side.

1C shows the target mRNA of miR-203. Target prediction software (TargetScan, PicTar, and microT) was used to confirm that the 3'-untranslated site (UTR) of mouse Glycine receptor-β (GLRB) was the target of mmu-miR-203. The 5 'seed sequence of miR-203 is underlined.

1D shows that miR-203 is significantly upregulated in the hippocampus of chronic epilepsy mice by real-time PCR. Expression of miR-203 is shown relative to the expression level of mRNA encoding GAPDH (glyceraldehyde phosphate dehydrogenase). * P <0.05. ns = not significant.

Figure 2a confirms the effect of miR-203 on GLRB protein expression, GLRB concentration was reduced after transduction of miR-203 into neuro-2a cells measured by Western blot (left) and intensity (densitometer) Right) is the result.

Figure 2b confirms the effect of miR-203 on GLRB protein expression in other aspects, the luciferase reporter gene containing the 3'-UTR of GLRB and miR-203 when delivered to HeLa cells together luciferase The result is a reduced signal.

FIG. 2C shows that when miR-203 and AM203 are co-introduced into neuro-2a cells, the GLRB concentration may be offset by miR-203, and this may be achieved by Western blot (left) and densitometry (right). It is the result measured. All measurements in FIGS. 2A-2C were performed in triplicate. * P <0.05, and ** P <0.01.

3A-3D show the therapeutic effect of AM203 administered nasal. After chronic administration of AM203 to mice with chronic epilepsy, the concentration of GLRB in the hippocampus was increased compared to the control treatment (Elecrosis-control), while there was no effect on the expression in the cortex, Western blot (a) and hippocampal den It is the result measured by cytometry (b) and cortical densimetry (c). Electroencephalography showed a decrease in SRS in chronic epilepsy mice intranasally administered AM203 (d). SRS monitoring for 14 days prior to treatment and for another 14 days after treatment showed that intranasal AM203 administration reduced the number of SRS episodes by 71.5% (e). ** P <0.01, ns = not significant

The present application is based on the finding that reduced expression of glycine receptors by miR-203 is associated with seizure related diseases.

Thus, in one aspect, the present application relates to a pharmaceutical composition for treating or preventing epilepsy or seizure related diseases, comprising a substance capable of inhibiting the activity of miR-203 and a pharmaceutically acceptable carrier.

As used herein, the term “miR” or “microRNA” refers to 21-23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. As used herein, the mature sequence of miRNA can be obtained from the miRNA database (http://www.mirbase.org). As of August 2012, the miRNA database (19th edition, miRBase) registered 25,141 mature miRNAs from 193 species. Generally, microRNAs are transcribed into precursors of about 70-80 nt (nucleotide) in length with a hairpin structure called pre-miRNA, and are then cut and matured by Dicer, an RNAse III enzyme. MicroRNAs form ribonucleocomplexes called miRNPs that cleave target genes through complementary binding to target sites or inhibit translation. More than 30% of human miRNAs are present in clusters, transcribed into one precursor, and cleaved to form the final mature miRNA.

MiR-203 herein is not limited to this theory but is expressed in the brain, particularly the hippocampus and cortex, binds to the 3 'untranslated site of mRNA encoding the glycine receptor, inhibits its expression, and thereby glycine receptors in the brain. Decrease the concentration. The sequence of miR-203 herein is of mammalian origin, eg, from human, mouse, or rat. In one embodiment according to the present application is a human-derived, mature sequence comprising the [5'GUGAAAUGUUUAGGACCACUAG 3 '(SEQ ID NO: 1)], as well as bulbs sequence ([5'-GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAACAGUUCAACAGUUCUGUAGCGCAAUU GUGAAAUGUUUAGGACCACUAG ACCCGGCGGGCGCGGCGACAGCGA- 3 ' ( SEQ ID NO: 2) will be.

 Inhibiting the activity of miR-203 herein means inhibiting or interfering with the intracellular actions or functions of miR-203, which typically miR-203 directly binds to its target such as mRNA molecules encoding glycine receptors. Or inhibiting the function of miR-203 directly using a small molecule inhibitor, an antibody or a fragment of an antibody, or indirectly using an inhibitor or a small interfereing RNA molecule. Furthermore, interference or inhibition of the activity of miR-203 includes directly or indirectly inhibiting the activity of precursor sequences (SEQ ID NO: 2) and mature sequences (SEQ ID NO: 1). Inhibition of the activity of miR-203 also includes the inhibition of miR-203 transcription to lower its intracellular concentration.

As used herein, the term “substance capable of inhibiting the activity of miR-203” includes any substance capable of inhibiting its expression and / or activity. Such materials include, for example, small molecule compounds, antagonists, antisense molecules, small hairpin RNA molecules (shRNA), fire-fighting RNA molecules (siRNA), seed target LNA (Locked Nucleic Acid) oligonucleotides, decooligonucleotides, aptamers, Ribozymes, or antibodies that recognize DNA: RNA hybrids.

As used herein, the term “antisense oligonucleotide” encompasses nucleic acid-based molecules having a sequence complementary to all or a portion of the miRNA, in particular the seed sequence of the miRNA, to form a duplex with the miRNA. will be. Thus, the term "antisense oligonucleotide" as used herein may be referred to as a "complementary nucleic acid-based inhibitor."

The antisense oligonucleotides include various molecules, for example ribonucleic acid (RNA), deoxyribonucleic acid (DNA), antagonists, 2'-0-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, phosphorophores Thioate-backbone ribonucleotides, peptide nucleic acid (PNA) oligonucleotides or locked nucleic acid (LNA) oligonucleotides. Preferably ribonucleic acid. The ribonucleic acid includes small stranded RNA (shRNA), short hairpin RNA (shRNA), and ribozyme.

LNA is a modified ribonucleotide that has a locked form, including an additional bridge between the 2 'to 4' carbon of the ribose sugar moiety, so that the oligonucleotide with LNA has improved thermal stability.

Peptide nucleic acids (PNAs) include peptide-based backbones instead of sugar-phosphate backbones. The 2'-0-modified oligonucleotide is preferably a 2'-0-alkyl oligonucleotide, more preferably a 2'-OC _1-3 alkyl oligonucleotide, most preferably a 2'-0-methyl oligonucleotide to be.

The antisense oligonucleotides include narrow sense antisense oligonucleotides, antagonists and inhibitory RNA molecules.

As used herein, the term “antigomere” is a single-strand chemically modified oligonucleotide, used for the silencing of endogenous microRNAs. Antagonists include sequences that are not complementary at the Arganoute 2 (Ago 2) cleavage site, or the base is modified with, for example, 2 'methoxy groups, 3' cholesterol groups, phosphorothioates such that Ago2 cleavage is inhibited It has a complementary sequence to the target sequence. The antagonists according to the invention have a sequence that is at least partially or completely complementary to miR-203. In one embodiment, the antagonist comprises one or more modifications (eg, 2'-0-methyl-sugar modifications, or 3 'cholesterol modifications). In other embodiments, the antagonist comprises one or more phosphorothioate linkages and at least partially has a phosphorothioate backbone. Antagonists suitable for inhibiting miR-203 according to the present application have a length of 7-50 nucleotides, in particular 10-40 nucleotides, more particularly 15-30 nucleotides, even more particularly 15-25 nucleotides, especially 16-19 nt, but are limited thereto. It is not.

The term "complementary" as used herein means that the antisense oligonucleotides are sufficiently complementary to selectively hybridize to miR-203 targets under certain hybridization or annealing conditions, preferably physiological conditions, and are partially or partially substantially complementary. It has the meaning encompassing both substantially complementary and perfectly complementary, and preferably means completely complementary. Substantially complementary means, but not completely complementary, complementary enough to bind to the target sequence and have a sufficient effect to interfere with the activity of miR-203.

The term “nucleic acid” herein includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof, including, for example, peptide nucleic acids (PNAs) or mixtures thereof. Nucleic acids may also be single or double stranded, and may encode molecules including polypeptides, mRNA, microRNA or siRNA, and the like.

In one embodiment according to the invention, a substance capable of inhibiting the activity of miR-203 may bind complementarily to all or a portion of the precursor and / or mature sequence of miR-203, in particular the seed sequence, thereby inhibiting its activity. Antisense oligonucleotides. Inhibition of this activity is to inhibit transcription of miR-203 and / or binding of miR-203 with a target mRNA. Antisense oligonucleotides according to the present disclosure may or may not include the nucleotides constituting them or the backbones (skeletons) linking them to one or more of the following modifications. That is, the antisense oligonucleotide may include 2 or more phosphothioate in LNA, or 2'-O- methylation of one or more nucleotides constituting the same, or one or more nucleotides constituting the same.

In one embodiment according to the present disclosure, the antisense oligonucleotide or nucleic acid molecule of the present disclosure comprises a sequence complementary to all or part of the seed sequence of miR-203. Seed sequences are sequences conserved in various species that are very important for recognition of target molecules of miRNA (Krenz, M. et al., J. Am. Coll. Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et. al., Annu. Rev. Physiol. 62: 321 (2000). Since miRNA binds to the target through the seed sequence, when the seed sequence is inhibited from interacting with the target, translation of the target mRNA can be effectively suppressed. In one embodiment according to the present disclosure comprises a sequence that is partially or completely complementary to the nucleotide sequence of the first or second to seventh or eighth nucleotide sequence of SEQ ID NO: 1, eg, an antisense oligonucleotide herein 5'-CAUUUCAC-3 '5'-AUUUCAC-3', 5'-CAUUUCA-3 ', 5'-AUUUCA-3' or 5'-CUAGUGGUCCUAAACAUUUCAC-3 ', one or more constituting the oligonucleotide Each nucleotide may be 2'-0-methylated, or each one or more of the nucleotides is LNA, or one or more of the chemical bonds constituting the backbone may be phosphothioate, but may not include such modifications.

The present application is based on the discovery that miRNA-203 excessively inhibits the expression of glycine receptor GlyR and thus is involved in the development of epilepsy. Epilepsy is associated with deviant excitatory neurotransmission due to non-functional inhibitory ion channels and neurotransmitters. Glycine, along with γ-aminobutyric acid, is a major neurotransmitter in the central nervous system, but the role of glycine and glycine receptors (GlyR) in epilepsy is not clear. Glysine binding to post-synaptic GlyR opens anion channels, leading to Cl- ions, which hyperpolarizes post-synaptic membranes and reduces neuronal fire. Inhibiting or modifying these synapses causes neurons to become over-excited, resulting in pain, cramping and hyperekplexia.

Thus, through the inhibition of miR-203 activity herein, it can be used for the treatment of epilepsy or seizure related diseases.

In one embodiment according to the invention is used for the treatment of epilepsy. Epilepsy is a generic term that describes about 40 clinically different symptoms, which can lead to neurological dysfunction caused by lesions or functional disorders of the brain tissue, resulting in various neurological symptoms: sudden loss of consciousness, convulsions, and mental disorders. Or sensory disorders, characterized by an unstimulated seizure of persistent recurrence.

Epilepsy includes chronic epilepsy and status epilepticus (SE) that lasts for more than 30 minutes once occurring. Epilepsy includes partial epilepsy that indicates partial epilepsy seizures and general epilepsy that indicates global epilepsy seizures when classified by seizure. When categorizing by cause, there may be idiopathic disease that has no specific cause except genetic factors, symptom that causes lesions that cause epilepsy in brain images such as MRI or CT, and lesions that cause epilepsy. Includes hidden epilepsy, which is presumed to be abnormal but is not found in current brain imaging.

As used herein, the term “seizure related disease” includes brain related diseases involving seizures and causes seizure, such as stroke; encephalitis; Hippocampal hardening; Cerebral palsy and congenital malformations; Seizures related to CNS infection; Hypoxia; Brain tumors; Traumatic brain injury; Angioplasty; Neurodegenerative diseases; Metabolic diseases such as hypoglycemia, glycogen accumulation, and pyruvate dehydrogenase deficiency; Multiple sclerosis; Autoimmune diseases, such as systemic lupus erythematosus, can be cited and include diseases in which seizures occur without knowing the cause. The compositions herein are used to treat seizures derived from seizure related diseases.

As used herein, the term "treatment", "mitigation" or "improvement" means any action that improves or beneficially alters the symptoms of a related disease by administration of the composition. Those skilled in the art to which the present application belongs, will be able to know the exact criteria of the disease, to determine the degree of improvement, improvement and treatment with reference to the data presented by the Korean Medical Association.

As used herein, the term "prevention" means any action that inhibits or delays the development of a related disease. It will be apparent to those skilled in the art that the compositions herein can prevent the initial symptoms, or related diseases, if administered before they appear.

The composition of the present invention is a compound which maintains / increases the solubility and / or absorption of one or more active ingredients or the active ingredients having the same or similar function in relation to the treatment of a disease, in addition to a substance capable of inhibiting the activity of miR-203 of the present application. It may further contain. It may also optionally further comprise chemotherapeutic agents, anti-inflammatory agents, antiviral agents and / or immunomodulators and the like.

The composition may further comprise one or more pharmaceutically acceptable diluents, carriers and / or adjuvants in addition to the above-mentioned active ingredients. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have. It can be prepared in a variety of formulations and dosage forms such as, for example, solution, emulsion and liposome formulations. For example, the active ingredient may be mixed into a solid carrier or excipient of a pharmaceutically acceptable liquid and / or fine powder to prepare into tablets, capsules, gels, syrups or suppositories. The compositions according to the invention can also be prepared in suspension using aqueous, nonaqueous or mixed media. The aqueous suspension may further comprise substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol and / or dextran.

The administration method of the composition according to the present application is not particularly limited, and known administration methods of inhibitors may be applied, and parenteral administration (for example, intranasal, intravenous, subcutaneous, intraperitoneal or topical) may be applied according to a desired method. Administration), or by intranasal infusion is preferred to obtain a rapid therapeutic effect. The compositions herein can also be delivered by a variety of routes and can be administered, for example, via infusion or bolus injection, epidermis or mucosa (oral mucosa, anal mucosa, intestinal mucosa, etc.). The compositions herein can also be systemically or topically administered.

Furthermore, the composition of the present application is preferably introduced into the central or peripheral nerve through a suitable route. Suitable routes include intraventricular or intrathecal administration. Such administration can be accomplished using a catheter connected to the reservoir. Formulations with aerosols can also be used to administer through the lungs via inhalers or nebulizers. Formulations for intravenous administration, subcutaneous injection, intrathecal injection, inhalation, or oral administration are not excluded as long as the effect according to the present invention occurs.

In one embodiment according to the present application is administered nasal. During nasal administration, as delivered to the brain along the olfactory pathway, the effect of the present composition can be enhanced. Nasal refers to the space in the nasal cavity, which is divided left and right by the nasal septum, and intranasal administration refers to the delivery of a composition herein to any tissue of the nasal epithelium. Intranasal acceptable carriers may be included for intranasal administration of the compositions herein, wherein the carrier is one or more suitable solid or filler diluents suitable for administration to any part of the nasal epithelium of a mammal, preferably a human. Or encapsulated material. Typically, the carrier may be a liquid, solution, suspension, gel, ointment, lotion, or a combination thereof. Preferably, the carrier is a pharmaceutically acceptable aqueous carrier. The compositions of the present invention can be prepared in various unit dosage forms. Such forms include, but are not limited to, nasal drops, nasal sprays, nasal gels, nasal ointments, and nasal powders.

The carrier may also include a delivery enhancer, wherein the intranasal delivery enhancer includes agglutination inhibitors, dose modifiers, pH control agents, degrading enzyme inhibitors, mucolytic or mucus removers, cilia stabilize reagents, membrane permeation promoters (eg For example, surfactants, bile salts, phospholipids or fatty acid additives, mixed micelles, liposomes, or carriers, alcohols, enamines, nitric oxide donor mixtures, long-chain amphiphilic molecules, small Hydrophobic penetration enhancers, sodium or salicylic acid derivatives, glycerol esters of acetoacetic acid, cyclodextrins or beta-cyclodextrin derivatives, medium chain fatty acids, chelating reagents, amino acids or salts thereof, N-acetylamino acids or salts thereof, degradation to selected membrane components Enzymes, fatty acid synthesis inhibitors, cholesterol synthesis inhibitors or nitric oxide stimulants, chitosan, and chito To modulate epithelial conjugation physiology such as derivatives, vasodilators, selective delivery promoters and intranasal mucosal delivery, the compositions herein can be effectively combined, bound, stored, encapsulated or stabilized in active agents, Stable carriers, carriers, support materials, or complex-forming species.

The composition according to the present application is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically or therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, It can be determined according to the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the concurrent drug and other factors well known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Dosages vary widely depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, and severity of the disease. Appropriate dosage is, for example, a drug accumulated in the patient's body. Depending on the amount of and / or the specific potency of the polynucleotide used. It can generally be calculated on the basis of EC ₅₀ , which has been determined to be effective in in vivo animal models and in vitro, for example from 0.01 μg to 1 g per kg of body weight, in unit time periods of daily, weekly, monthly or yearly It may be administered once or several times per unit period, or may be continuously administered for a long time using an infusion pump. The number of repeated doses is determined in consideration of the time the drug stays in the body, the drug concentration in the body, and the like. Even after treatment according to the course of the disease treatment, the composition may be administered for relapse.

The active ingredients according to the invention, for example antisense oligonucleotides, can be used in the composition on their own or in the form of pharmaceutically acceptable salts. A pharmaceutically acceptable salt is one which minimizes the undesirable toxicity while maintaining the biological activity of the polynucleotide according to the present application. Such salts are, for example, base addition salts formed with metal cations such as zinc, calcium, bisbus, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium and the like and salts formed with organic amino acids, or ammonia, N It may include, but is not limited to, salts formed with cations derived from N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine. no.

In one embodiment according to the present application, the active ingredient of the present application, such as antisense oligonucleotide, is negatively charged due to the nature of the nucleotides that make up it. Because of the lipophilic nature of cell membranes, uptake of antisense oligonucleotides into cell membranes may be reduced. Absorption disturbances due to this polarity can be solved through the prodrug approach described below (Crooke, RM (1998) in Crooke, ST Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140).

In other embodiments, the present disclosure provides miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, of miRNA selected from the group consisting of miR-455, miR-99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 The present invention relates to a kit for diagnosing a seizure disease comprising a nucleotide sequence, a complementary sequence thereof, or a fragment thereof.

The nucleotide sequence of the miRNA molecules can be found in miRBase (http://www.mirbase.org).

The miRNA molecules are differentially expressed miRNAs that are up- or down-expressed in the hippocampus of chronic epilepsy mice, as shown in Table 1. Most preferably the miRNA molecule is miR-203.

Diagnostic kits of the invention include strokes that cause epilepsy or seizures; encephalitis; Hippocampal hardening; Cerebral palsy and congenital malformations; Seizures related to CNS infection; Hypoxia; Brain tumors; Traumatic brain injury; Angioplasty; Neurodegenerative diseases; Metabolic diseases including hypoglycemia, glycogen accumulation disease and pyruvate dehydrogenase deficiency; Multiple sclerosis; Autoimmune diseases including systemic lupus erythematosus; Or it may be used for the diagnosis of seizure-related diseases including unknown cause seizures, most preferably in the diagnosis of epilepsy diseases.

The kit of the present invention may further include other components in addition to the above components. For example, when the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus). (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

According to a preferred embodiment of the invention, the kit of the invention may be a microarray. According to a preferred embodiment of the present invention, the kit of the present invention is a gene amplification kit.

When the kit of the present invention is a microarray, the probe is immobilized on the solid surface of the microarray. When the kit of the present invention is a gene amplification kit, it includes a primer. Probes or primers are miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR- At least one of 451, miR-455, miR-99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 Recognized as having a sequence complementary to the sequence. As used herein, the term "complementary" means, as defined above, to have a complementarity sufficient to selectively hybridize to the nucleotide sequence under a given hybridization or annealing conditions, and the probe or primer of the present invention. May have one or more mismatch sequences as long as it can selectively hybridize to the nucleotide sequence in addition to being completely complementary.

The nucleotide sequence of the miRNA of the present invention, which should be referred to when preparing a primer or probe, may be identified in miRBase, and the primer or probe may be designed with reference to this sequence.

In another embodiment the present application provides miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR Among markers consisting of -451, miR-455, miR-99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 Detecting one or more expressions; And correlating the expression level of the detected marker with a diagnosis or prognosis of an epilepsy or seizure related disease of a test subject, detecting the marker to provide information necessary for diagnosis or prognosis of an epilepsy or seizure disease. It is about how to.

The associating step in the method according to the present invention is to compare the expression level of the determined markers with the detection results for the respective markers determined in the normal control group, among which miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, miR-455, miR-199a-5p, miR-666-3p, miR-708 And miR-805 is increased in expression compared to the control, miR-99b *, miR-206, miR-468 and miR-574-3p of the markers are reduced in expression.

Furthermore, the method according to the present application may further use non-marker clinical information of the seizure disease test subject when diagnosing or prognosticing epilepsy or seizure related disease of the test subject. Non-marker clinical information of such test subjects includes, but is not limited to, age, sex, weight, diet, body mass, underlying disease and EEG, seizure type, brain MRI, brain CT, or cerebrospinal fluid test of the subject.

The degree of expression of the miRNAs analyzed by the kits or methods of the invention, such as reverse transcriptase (RT) -PCR or real-time-PCR methods (Sambrook, J. et al., Molecular Cloning. When the degree of expression measured by Laboratory Manual, 3rded.Cold Spring Harbor Press (2001)) is 1.5 times or more, preferably 2 times or more, compared to the normal control, the subject from which the analytical sample is taken Epilepsy or seizure disease, particularly epilepsy disease, or having a high risk of development.

In another aspect, the present disclosure provides a method of treating a miR-203 RNA with a test substance; And determining the activity of miR-203 RNA in contact with the test substance, wherein the activity of the contacted miR-203 is reduced compared to the activity of miR-203 RNA of a control group not in contact with the test substance. The present invention relates to a method for screening a substance for treating or preventing epilepsy or seizure disease, in which case it is selected as a candidate.

In one embodiment according to the present invention, the miR-203 RNA is provided in the form of cells expressing it, and the activity is analyzed by expression of the miR-203 RNA. For example, after contacting a cell expressing miR-203 with a test substance, the change in u-expression of miR-203 is compared to control cells prior to or without contact, whereby a change, in particular a decrease in expression level, is observed. What is present is selected as a candidate. The expression level of miR-203 used in the method according to the present invention can be performed using a known method such as Northern blot, RT-PCR, hybridization method using a microarray.

In another embodiment the miR-203 RNA is provided in the form of cells expressing it, the activity of the miR-203 RNA and its target GLRβ subunit (GLRB, glycine receptor beta subunit) of the 3'-UTR and Is determined by the analysis of interactions. For example, after contacting a cell expressing miR-203 with a test substance, the degree of interaction of the 3'-UTR and miR-203 of the GLRβ subunit is compared to control cells before or without contact, Candidates are screened for variations, especially decreases, in interactions. Methods for detecting RNA-RNA interactions for use in the methods according to the invention are known in the art and see, for example, those described in RNA Walk (Lusting et al., Nucleic Acids Res. 2010; 38 (1): e5. ) Or Yeat two hybrid system (Piganeau et al., RNA 2006; 12: 177-184) and the like, see RNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011).

The type of cell used in the present method and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and a person skilled in the art will be able to select an appropriate cell type, amount and / or conditions. As a result of the experiment, as a candidate, a substance which results in a decrease in miR-203 RNA activity in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance. Less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60% A reduction, about 55% reduction, about 50% or less reduction, about 45% or less reduction, about 40% or less reduction, about 30% or less reduction, or about 20% or less reduction, but the range is not excluded.

By "test substance" herein is meant a substance which is expected to inhibit the activity of miR-203 RNA as described above, such as low molecular weight compounds, high molecular weight compounds, mixtures of compounds (e.g., natural extracts or cell or tissue cultures) Or biopharmaceuticals (eg, proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzyme and DNAzyme), or sugars and lipids, and the like. The test substance may be a polypeptide having two or more amino acid residues, such as 6, 10, 12, 20 or less or more than 20 such as 50 amino acid residues. The test substance can be obtained from a library of synthetic or natural compounds and methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA) and Available from MycoSearch (USA). Test materials can be obtained by a variety of combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the required synthetic library method, “1-bead 1-compound” library method, and synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like.

In one embodiment according to the present invention for the purpose of the screening of drugs for treating epilepsy or seizure related diseases it can be used that the compound has a low molecular weight therapeutic effect. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

Biologics can also be used for screening, for example. Biologics refers to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids or substances produced using cellular systems in vivo and ex vivo. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organics found in polynucleotides, peptides, antibodies, or other plasma.

The present application is based on the discovery that miRNA-203 is involved in the development of epilepsy by excessively inhibiting the expression of the glycine receptor GlyR. In another embodiment, the present application is directed to a cell or tissue of a subject, particularly in a brain cell or brain tissue or brain. It provides a method for treating or preventing epilepsy or seizure-related diseases through the inhibition of miR203 activity.

Furthermore, the disclosure also provides a method of treating an epilepsy or seizure related disease in a subject, comprising administering to the subject in need thereof a therapeutically or prophylactically effective amount of an miR-203 activity inhibitor. Or provide preventive measures.

Furthermore, the present application also provides a substance capable of inhibiting the activity of miR-203 for use in the treatment or prevention of epilepsy or seizure related disease in a subject. In one embodiment according to the invention, substances capable of inhibiting the activity of miR-203 are delivered to the brain in particular.

For the miR-203 activity inhibitor, the miR-203 activity regulation or inhibition, the administration method, the kind of the disease to be treated, etc., which are used in the method of the present application, reference may be made to the foregoing.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

The experimental method used in this example is as follows.

Chronic Epilepsy Mouse Model Preparation

This study was conducted with the approval of Korea Advanced Institute of Science and Technology (KAIST) in Korea and Institutional Animal Care and Use Commitee (IACUC) of Seoul National University Hospital. The committee was accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Male C57BL / 6J mice (55 weeks old, 22-25 g) were treated with methylscopolamine (1 mg / kg ip, Sigma-Aldrich, USA). After 30 minutes, pilocarpine (330 mg kg ⁻¹ , Sigma-Aldrich) was intraperitoneally injected into mice to induce SE (status epilepticus). 40 minutes after the beginning of SE, diazepam (5 mg / kg, Samjin Pharmaceutical, Seoul, Korea) was injected intraperitoneally into mice (Racine scale ≧ IV) to stop the ongoing seizures. After SE, 5% glucose solution was fed for 2 days until all animals started eating normal food pellets. Mice were reared at a 12-hour light cycle and unlimited access to water and food. Chronic epilepsy is defined as having a frequent attack of 60 days after SRS (4-5 on the Racine scale).

miRNA microarray

Mice were deeply anesthetized after 60 days of SE with pilocarpine and sacrificed with guillotine. The brain was immediately removed and the hippocampus and whole cortex were excised from the remaining brain structures. Total RNA was isolated from hippocampus and cortex using Trizol® (Invitrogen, USA) according to the manufacturer's method. The miRNA expression profile was investigated using the mouse miRNA microarray 8 × 15K kit (detecting 567 mouse miRNA) (Agilent Technilogies, Inc. USA) according to the manufacturer's method. GenSpring GX, version 7.3.1 (Agilent Technologies) was used as the manufacturer's method to normalize and analyze the data. The numerical value of 0.01 or less was set to 0.01. Seven samples (three normal and four interstitial) were tested on each microarray chip. Upregulation was defined as a two-fold increase in expression relative to the control and a P -value of 0.05 or less relative to the control. Down-regulation was defined as a two-fold decrease in expression for the control and a P-value of 0.05 or less for the control.

Real time PCR of miRNA

Mice were deeply anesthetized after 60 days of SE with pilocarpine and sacrificed with guillotine. The brain was immediately removed and the hippocampus and whole cortex were excised from the remaining brain structures. Total RNA was isolated from hippocampus and cortex using Trizol® (Invitrogen, USA) according to the manufacturer's method. Real-time PCR of miRNA was performed using TaqMan® MicroRNA Assay (Life Technologies, USA) according to the manufacturer's method. The conditions were repeatedly performed for 40 cycles of heat treatment consisting of 95 ° C. 15 seconds and 60 ° C. 60 seconds after heat treatment at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes. All real time reactions were performed in multiples on the ABI PRISM 7000 Sequence-Applies Biosystem. Relative expression was calculated using a comparative threshold cycle and each miRNA concentration was titrated according to the amount of snoRNA202 measured using an endogenous snoRNA detection kit (Ambion-Applied Biosystem).

Target gene prediction of miRNA

The miRNA mature sequences disclosed herein were obtained from miRBase (http://www.mirbase.org). Potential miRNA target positions in the mouse gene 3′-nontranslated site (UTR) were determined by TargetScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de/), and microT ( http://diana.cslab.ece.ntua.gr/microT/).

Pass in inbit

Lipofectamine® 2000 (Life Technologies, USA) was used according to the manufacturer's method for Neuro-2a cells (American Type Culture Collection, with 50 nM AM203 and 50 nM miR-203 duplexes (or scrambled miRNA duplexes; Bioneer, Korea). USA). For western blot, homogenates of cells were obtained 48 hours after transfection.

Luciferase Assay

A total 3'-UTR sequence of human GLRβ subunit (GLRB, glycine receptor beta subunit) was cloned into the luciferase reporter construct (SwitchDB, USA). HeLa cells were dispensed in 24-well plates at 5 × 10 ⁴ cell concentrations per well in RPMI medium containing 10% fetal bovine serum. After 24 hours, Lipofectamine 2000 was used to deliver 83 ng / ml luciferase expression vector and 50 nM miRNA-203 duplex (or scrambled miRNA duplex (Bioneer)) into cells. Luciferase activity was measured after 48 hours using the Luciferase Reporter 1000 Assay System (Promega, USA) according to the manufacturer's method.

Treatment of Antagonists in Chronic Epilepsy Mice

Intranasal administration of antagonists was performed as previously described (Lee, ST, et al . (2012) miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model. Ann Neurol , 72, 269-277). In summary, for intranasal administration of antagonists, the anesthetized mice were placed in supine position with their heads upright. AM203 (2'-O-methylated-5'-cuagugguccuaaacauuucac-3 '; 5 nmol in 24 μl of 0.1% v / v diethylpyrocarbonate-treated distilled water; Bioneer, Korea) was added every 2 minutes. Nostrils were changed, and 4 μl of each pipette was administered (6 fractions in total). Control mice received an equal volume of vehicle.

Western Blot for Invivo Model

Anesthetized mice were sacrificed with guillotine and brains were immediately removed. Homogenates of the brain regions (hippocampus, and cortex) were prepared and sequentially subjected to western blot using GLRB (Santa Cruz Biotechnology, USA) antibodies. Immune response proteins were visualized with enhanced chemiluminescent reagents (Pierce, USA) and scanned using a GS-700 scanner. The optical density of each band was measured using Image-J software (National Institute of Health, USA). , relative to the β-actin band.

Invivo Electrophysiology

In vivo EEG and surgical procedures were performed as previously described (Jeon, D. et al. (2011) A cell-free extract from human adipose stem cells protects mice against epilepsy. Epilepsia , 52, 1617-1626) . In summary, to monitor EEG, mice underwent electroencephalography two weeks before pilocarpine injection. Animals were anesthetized by intraperitoneal injection of 1% ketamine (30 mg / kg) and xylazine hydrochloride (4 mg / kg) for surgery, using stereotaxic apparaqtus (Kopf Instruments, USA). It was performed by. Electroencephalograms were obtained using a tungsten electrode (0.005 inch, 2 MΩ), from AP-1.8 mm, L 2.1 mm and DV 0.8-1.0 mm (cortex) from Bregma, including the bottom of the cerebellum. Was located within the right hemisphere. Using a digital electroencephalography system (Comet XL; Astro-Med, Inc. USA), the electrical activity was amplified (x 1200) and recorded and passed through a bandpass-filtered at 0.1 to 70 Hz, 400 Digitized at -Hz sampling rate (AS 40). Electrophysiological data were analyzed offline using PSG Twin 4.2 (Astro-Med, Inc). EEG signals in chronic epilepsy mice were recorded continuously for 14 days beginning 60 days after SE. After repeated intranasal treatment of AM203 or vehicle (48 hour intervals), signals were recorded continuously for another 14 days. Data for 14 days between the pre- and post-treatment portions of the experiments were averaged.

Statistical analysis

Heat maps for miRNA expression were generated using Z-scores, which were calculated as follows: Z-score = [(raw value for expression of each miRNA-for all miRNA expressions) Mean values / standard deviation of all miRNAS expressions.] Two groups were compared using Student's-t test, and three or more groups were compared using Krushall-Wallis analysis of variance P obtained from Krushall-Wallis test. When the value was <0.05, the Mann-Whitney U test was used to post-hoc the comparison between the groups.Two-tailed P- values below 0.05 were statistically significant. It was.

Example 1 Identification of Differentiated miRNAs in the Brain of Mice with Chronic Epilepsy

Microarrays were used to investigate miRNA expression in chronic epilepsy mice 60 days after pilocarpine-induced SE. Compared with normal mice, the expression of four miRNAs was reduced and the expression of eleven miRNAs was increased in the hippocampus of chronic epilepsy mice (FIG. 1A, Table 1). In the cortex, 10 miRNAs were up-regulated and none were down-regulated (FIG. 1B, Table 1). Five (miR-199b, miR-203, miR-223, miR-451, and miR-455) were upregulated in both hippocampus and cortex (Table 1).

Among the differentially expressed miRNAs, a target prediction program was selected that miR-203 was able to bind to the 3'-untranslated site of Glrb (the gene encoding GLRB) (FIG. 1C). Real-time PCR analysis confirmed that miR-203 was significantly upregulated in the hippocampus of chronic epilepsy mice (1.78 fold vs. control; FIG. 1D; P = 0.041). The difference did not appear to be significant in the cortex (FIG. 1D).

Table 1 Up- or Down-regulated mRNA in Chronic Epilepsy Mice Changes in manifestation hippocampus cortex Both hippocampus and cortex Adjusted up miR-21 miR-199a-5p miR-199b miR-129-5p miR-199b miR-203 miR-132 miR-199b miR-223 miR-142-3p miR-203 miR-451 miR-155 miR-223 miR-455 miR-199b * miR-451 miR-199b miR-455 miR-203 miR-666-3p miR-223 miR-708 miR-451 miR-805 miR-455 Down-regulated miR-99b * None None miR-206 miR-468 miR-574-3p

Example 2 Reduction of GLRB Protein Concentration by miR-203

At 48 hours after transfection with miR-203, the concentration of GLRB protein was reduced in Neuro-2a cells. Furthermore, in HeLa cells transfected with miR-203 and a luciferase vector containing 3'UTR of Glrb , miR-203 reduced the luciferase signal at 48 hours, which miR-203 translated into Glrb Directly inhibits (FIG. 2B; P <0.001). In addition, when the antagonist (AM203) against miR-203 was transfected, it was shown that the decrease in GLRB protein concentration was suppressed (FIG. 2C; P = 0.024 vs. miR-203 alone).

Example 3 Increasing GLRB Protein Concentration in Mice with Chronic Epilepsy by miR-203 Antagonmere AM203

Chronic epilepsy mice were treated intranasally with 5 nmol of AM203 or saline on days 0 and 2. Western blots of hippocampal tissues collected on day 10 showed lower GLRB protein concentrations in chronic epilepsy mice than in normal mice ( P = 0.002) and administration of AM203 increased GLRB concentrations ( P = 0.002). P = 0.031) (FIGS. 3A and B). In contrast, the amount of GLRB was low in the cortex and GLRB was lower in the cortex of SRS mice, but AM203 did not appear to affect the expression of GLRB in the cortex (FIGS. 3A and B).

Example 4 Reduction of SRS in Mice with Chronic Epilepsy by AM203

EEG in chronic epilepsy mice was monitored for 14 days, intranasally treated with AM203 twice at 48-hour intervals, and then monitored for an additional 14 days. The frequency of SRS episodes was similar before and after treatment in mice administered saline (average number of episodes / 14 days ± standard deviation = 37.8 ± 5.2 vs. 32.8 ± 5.4 after treatment; P = 0.69; FIG. 3E). Whereas after treatment with AM203 it was significantly lower (36.6 ± 5.9 episodes / 14 days before treatment vs. 10.4 ± 4.0 episodes / 14 days after treatment; P = 0.003; FIG. 3E).

Intranasal administration of miR-203's antagonist AM203 restored GLRB expression and reduced seizures in chronic epilepsy mice, suggesting that miR-203-GLRB interactions are involved in the development of epilepsy. Indicates.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Claims (22)

A pharmaceutical composition for treating or preventing epilepsy or seizure related diseases, comprising a substance capable of inhibiting the activity of miR-203 and a pharmaceutically acceptable carrier. According to claim 1, The substance capable of inhibiting the activity of miR-203 is a substance capable of inhibiting the interaction of the miR-203 GLRβ subunit (GLRB, glycine receptor beta subunit) with the 3'-UTR Pharmaceutical composition for treating or preventing phosphorus, epilepsy or seizure related diseases. The pharmaceutical composition of claim 1, wherein the substance capable of inhibiting the activity of miR-203 is a substance capable of inhibiting its expression. According to claim 1, wherein the substance capable of inhibiting the activity of miR-203 epilepsy or a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the miR-203, epilepsy or Pharmaceutical compositions for treating or preventing seizures-related diseases. The method of claim 4, wherein the nucleic acid molecule is RNA, DNA, antagonists, antisense molecules, siRNA, shRNA, 2'-0-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribo A pharmaceutical composition for treating or preventing epilepsy or seizure related diseases, which is a nucleotide, a decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide or a locked nucleic acid (LNA) oligonucleotide. 5. The epilepsy according to claim 4, wherein the nucleic acid molecule is an antisense oligonucleotide comprising a sequence partially or entirely complementary to the nucleotide sequence of the first or second to seventh or eighth nucleotide sequences of SEQ ID NO: 1. Or a pharmaceutical composition for treating or preventing seizures-related diseases. The antisense oligonucleotide of claim 6, wherein the antisense oligonucleotide comprises 2 or more phosphothioates in the LNA, or a sugar of one or more nucleotides constituting it, 2'-0- methylated, or a backbone thereof, or a combination thereof. It comprises a combination of, epilepsy or seizure-related disease treatment or prevention pharmaceutical composition. The method of claim 6 or 7, wherein the antisense oligonucleotide is 5'-CAUUUCAC-3 ', 5'-AUUUCAC-3', 5'-CAUUUCA-3 ', 5'-AUUUCA-3' or 5'- CUAGUGGUCCUAAACAUUUCAC-3 'A pharmaceutical composition for treating or preventing phosphorus, epilepsy or seizure related diseases. The method of claim 1, wherein the seizure-related disease is stroke, hippocampal hardening, cerebral palsy, congenital malformation, central nervous system infection, hypoxia, brain tumor, traumatic brain injury, neurodegenerative disease, metabolic disease, autoimmune disease, or unknown cause. Pharmaceutical composition for treating or preventing seizures, epilepsy or seizure-related diseases caused by seizures. 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the composition is delivered to the brain. The pharmaceutical composition of claim 10, wherein the composition is a formulation for intranasal administration, intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration, or oral administration. contacting the miR-203 RNA with a test substance; And Determining the activity of miR-203 RNA in contact with the test substance, Substance for treating or preventing epilepsy or seizure-related diseases is selected as a candidate if the activity of the contacted miR-203 is reduced compared to the activity of the miR-203 RNA of a control group not in contact with the test substance. Screening method. The method of claim 12, wherein the miR-203 RNA is provided in the form of cells expressing it and the activity is determined by expression analysis of the miR-203 RNA. 13. The miR-203 RNA of claim 12, wherein the miR-203 RNA is provided in the form of a cell expressing it, wherein the activity is 3'-UTR of the miR-203 RNA and its target GLRβ subunit (GLRB, glycine receptor beta subunit) Which is determined by analysis of the interaction with. The method of claim 12, wherein the seizure-related disease is due to stroke, hippocampus hardening, cerebral palsy, congenital malformation, central nervous system infection, hypoxia, brain tumor, traumatic brain injury, neurodegenerative disease, metabolic disease, autoimmune disease, or unknown cause A method of screening a substance for treating or preventing seizures, seizure diseases. miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, miR-455, miR Nucleotide sequence of miRNA selected from the group consisting of -99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805, its complement A kit for diagnosing a seizure disease comprising the sequence detection material, including an enemy sequence or a fragment of the sequence. miR-21, miR-129-5p, miR-132, miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, miR-455, miR Expression of one or more of miRNA markers selected from the group consisting of: -99b *, miR-206, miR-468, miR-574-3p, miR-199a-5p, miR-666-3p, miR-708 and miR-805 Detecting; And Detecting the marker to provide information necessary for the diagnosis or prognosis of epilepsy or seizure disease, the method comprising correlating the expression level of the detected marker with a diagnosis or prognosis of epilepsy or seizure related disease of a test subject. Way. 18. The method of claim 17, wherein said associating further uses non-marker clinical information of said seizure disease test subject. 19. The method of claim 18, wherein the non-marker clinical information of the test subject is one or more of age, sex, weight, diet, body mass, underlying disease, EEG, seizure type, brain MRI, brain CT, or cerebrospinal fluid test of the subject. Way. 18. The method of claim 17, wherein the associating comprises comparing the expression level of the determined markers with the detection results of the respective markers determined in the normal control group, wherein miR-21, miR-129-5p, and miR-132 are among the markers. , miR-142-3p, miR-155, miR-199b *, miR-199b, miR-203, miR-223, miR-451, miR-455, miR-199a-5p, miR-666-3p, miR- The expression of 708 and miR-805 is an increase in the amount of expression compared to the control, wherein the miR-99b *, miR-206, miR-468 and miR-574-3p of the marker is that the amount of expression is reduced. A method of treating or preventing epilepsy or seizure related disease in a subject, comprising administering a therapeutically or prophylactically effective amount of an miR-203 activity inhibitor to a subject in need thereof for treating or preventing an epilepsy or seizure related disease. A substance capable of inhibiting the activity of miR-203 for use in the treatment or prevention of epilepsy or seizure related disease in a subject.

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