KR101615424B1 - Composition and method for treating neurodegenerative diseases targeting miRNA - Google Patents

Abstract

The present invention discloses pharmaceutical compositions for the prevention or treatment of neurological diseases through microRNA regulation. The present disclosure also discloses markers that can be used for the diagnosis or prognosis of neurological diseases and their uses. The composition of the present invention exhibits a neuroprotective effect and can be useful for the prevention or treatment of acute, subacute or chronic neurodegenerative diseases.

Description

[0001] The present invention relates to a pharmaceutical composition and method for preventing or treating a neurodegenerative disease,

This disclosure relates to the treatment of neurodegenerative diseases that target specific miRNAs.

As the lifespan of humans increases, the incidence of neurodegenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, Alzheimer's disease, diabetic retinopathy, multiple infarct dementia and disc macular degeneration is also increasing. The number of neurodegenerative disease patients is estimated to be 24 million worldwide. In addition, diseases caused by other types of neurodegenerative diseases such as strokes or other trauma or injuries are increasing year by year. In Korea, as of the end of 2003, the number of elderly people aged 65 or older, who need to receive medical treatment or care for their neurological diseases such as dementia and stroke, should reach 640,000, , And the proportion of elderly population arithmetically will reach 870,000 in 2015 and 1.64 million in 2030. The management costs for these will exceed 4.8 trillion won in 2015 and reach 9 trillion by 2030 .

There is a need for the development of therapeutic agents for neuroprotection that effectively treat these acute and chronic neurodegenerative diseases and / or prevent damage to neuronal cell death or neuronal cells.

Recent attempts have been made to develop therapeutic agents using microRNA inhibitors.

U.S. Published Unexamined Patent Application, Publication No. 2010-0286249 relates to a method for diagnosing asthma by measuring the expression level of a plurality of miRNAs including miR-466.

Korean Laid-Open Publication No. 2012-0032003 relates to a method for screening a diet that produces milk having an immunomodulatory action. By identifying a diet or substance that increases or decreases the expression level of microRNA containing miR-466, A method of screening a diet or substance to produce breast milk having action.

The present invention provides miRNA-targeted miRNA modulators to provide a method for treating neurodegenerative disease-related diseases.

In one embodiment, the disclosure provides a pharmaceutical composition comprising a substance capable of inhibiting the activity of one or more molecules of microarray (miR) -466 or microarray (miR) -let7, and a pharmaceutically acceptable carrier. Or a pharmaceutically acceptable salt thereof.

In one embodiment according to the present invention, the substance capable of inhibiting the activity of the one or more molecules is a substance capable of inhibiting its expression, for example, a nucleic acid molecule capable of binding to all or a part of the nucleotide sequence of the molecule , Such as RNA, DNA, antigens, antisense molecules, siRNA, shRNA, 2'-O-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, phosphorothioate-backbone ribonucleotides, decoy oligonucleotides, PNA but are not limited to, peptide nucleic acid oligonucleotides or LNA (oligonucleotide) oligonucleotides.

The miR-466 that is the target of the present invention is derived from human in one embodiment and is represented by SEQ ID NO: 1 or 2. The nucleic acid molecule capable of inhibiting its activity is the first to eighth nucleotide sequences of the nucleotide sequence of SEQ ID NO: Or an antisense oligonucleotide comprising a sequence complementary to all or a part of the 11th to 18th nucleotide sequences of SEQ ID NO: 2.

The target miR-let7 is human-derived, and is represented by SEQ ID NO: 3 or SEQ ID NO: 4, and the nucleic acid molecule capable of inhibiting the activity of miR-let7 is the nucleotide sequence of SEQ ID NO: 3 2 to 8, or the 12th to 18th nucleotide sequence of SEQ ID NO: 4, or a complementary sequence to all or part of the nucleotide sequence.

In one embodiment, the antisense oligonucleotides used herein are modified, for example one or more of the nucleotides constituting the antisense oligonucleotide is a 2 ' -O-methylated LNA, or a sugar of one or more nucleotides constituting it, Or a combination thereof, but is not limited thereto.

In one embodiment, the antisense oligonucleotide capable of inhibiting the activity of miR-466 is SEQ ID NO: 5 (5'-CACACAUA-3 ') and the antisense oligonucleotide capable of inhibiting the activity of miR- (5'-CUACCUC-3 ').

The composition of the present invention may be useful for the treatment or prevention of neurodegenerative diseases including acute, subacute or chronic neurodegenerative diseases.

In another embodiment, the present invention also provides a method comprising: contacting at least one molecule selected from the group consisting of miR-466 and miR-let7 with a test substance; And determining the activity of the molecule contacted with the test substance, wherein if the contacted molecular activity is reduced compared to the activity of the control RNA not contacted with the test substance, the candidate substance is selected , A method for screening a substance for treating or preventing a neurodegenerative disease.

The present invention also provides a kit for the diagnosis of neurodegenerative diseases, comprising a substance for detecting a nucleotide sequence of a miRNA selected from the group consisting of miR-466 and miR-let7, a complementary sequence thereof, or a sequence comprising a fragment thereof.

The present invention also provides a method of detecting miR-467, comprising detecting the expression of one or more of miRNA markers selected from the group consisting of miR-466 and miR-let7 in Invitro; And a step of correlating the expression level of the detected marker with a diagnosis or prognosis of a neurodegenerative disease of a subject to be examined, to provide information necessary for diagnosis or prognosis of a neurodegenerative disease .

The composition capable of regulating microarray expression according to the present application exhibits a neuroprotective effect through inhibition of cellular apoptosis and neuroinflammation reaction, for example, prevention or treatment of a neurodegenerative disease, or development of a neuroprotective drug after a neurodegenerative disease .

FIG. 1 is a microarray analysis result using total miRNA extracted from each hemisphere of the brain 24 hours after induction of ICH in rats.
FIG. 2 shows the results of analysis of miRNA expression of cerebral cortex, basal ganglia, and hematoma itself according to the brain position after cerebral hemorrhage in rat by quantitative reverse transcription neutralizing enzyme chain reaction.
FIG. 3 shows the results of analysis of cytoprotective effect after treatment with ant thromb after thrombin treatment in the in vitro thrombin toxicity model.
FIG. 4A shows the result of comparing the neuronal recovery with the control group by following the intracranial administration of miR-let7 and tumor nerve after inducing the cerebral hemorrhage in the rat.
FIG. 4B shows the result of comparing the neuronal recovery with the control group by following the intracranial administration of miR466 and natomore after the induction of cerebral hemorrhage in the rat, followed by the examination of the neural function score.
FIGS. 5A and 5B show results of western blotting of changes in the expression levels of the target gene and the lower-stage protein, respectively, after treatment of (miR-let7) antagomir with ICH-induced mice.
FIGS. 5C and 5D show results of western blotting of changes in the expression levels of the target gene and the lower-stage protein, respectively, after treatment with (miR-466) antigomere for ICH-induced mice.

The present invention is based on the discovery that it is possible to prevent or treat neurodegenerative diseases through modulation of microarray expression.

Thus, in one aspect, the invention provides a pharmaceutical composition for treating or preventing neurodegenerative diseases, comprising a substance capable of inhibiting the activity of one or more molecules selected from the group consisting of miR-let7 and miR-466, and a pharmaceutically acceptable carrier ≪ / RTI >

As used herein, the term " miR "or" microRNA "or microRNA comprises 21 to 23 noncoding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation . The maturation sequences of the miRNAs used herein can be obtained from the miRNA database (http://www.mirbase.org). As of August 2012, 25,141 mature miRNAs from 193 species are registered according to the miRNA database (19th edition, miRBase). Generally, microRNAs are transcribed into precursors of about 70-80 nt (nucleotide) long with a hairpin structure called pre-miRNA, and then matured by cleavage by the RNAse III enzyme Dicer. MicroRNAs form a ribonucleotide complex called miRNP, which cleaves the target gene by complementary binding to the target site, or inhibits translation. Over 30% of human miRNAs are present in clusters, which are transcribed into a single precursor and then cleaved to form the final mature miRNA.

MiR-466 is expressed in the brain, particularly in the basal ganglia and cortex, and is not limited to this theory, and includes insulin-like growth factor receptor 1 and insulin-like growth factor receptor 2 lt; RTI ID = 0.0 > receptor < / RTI > receptor 2) and regulates its expression. The sequence of miR-466 herein is of human origin and the mature sequence includes the 5'-UAUGUGUGUGUGUAUGUCCAUG-3 '(SEQ ID NO: 1) as well as the precursor sequence 5'-CAUGUGUAUAUAUGUGUGUGUGUAUGUCCAUGUGUGUAUAUGAAUAUACAUACACACACACAUACACACACGUGCAAGCACACACA-3' (SEQ ID NO: 2).

Herein, miR-let7 is expressed in the brain, particularly the basal gang, though not limited to this theory, and binds to the 3 ' untranslated region of mRNA encoding insulin-like growth factor receptor 1 and regulates its expression. The sequence of miR-let7 herein is of human origin and the matured sequence includes the 5'-UGAGGUAGUAGGUUGUAUGGUU-3 '(SEQ ID NO: 3) as well as the precursor sequence 5'-GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUUAGAGUUACACCCUGGGAGUUAACUGUACAACCUUCUAGCUUUCCUUGGAGC-3' (SEQ ID NO: 4).

 Herein, inhibition of the activity of miR-let7 and miR-466 is meant to inhibit or interfere with their intracellular action or function. Typically, the microenalve has its targets such as insulin-like growth factor receptor 1 and insulin Directly inhibit the binding of mRNA molecules encoding pseudo-growth factor receptor 2, or directly inhibit the function of these microRNAs using fragments of small molecule inhibitors, antibodies or antibodies, or inhibitors or small interfering small interfering RNA molecules. Further, the inhibition or inhibition of miR-let7 and miR-466 activity includes inhibiting the activity of a precursor or mature sequence, directly or indirectly. Inhibition of the activity of miR-let7 and miR-466 also includes suppressing their transcription (expression) and lowering their intracellular concentration.

The term "substance capable of inhibiting the activity of microalpha" herein includes any substance capable of inhibiting its expression and / or activity. Such materials include, for example, antigens, antisense molecules, small hairpin RNA molecules (shRNA), small interfering RNA molecules (siRNA), seeded LNA (Locked Nucleic Acid) oligonucleotides, decoy oligonucleotides, aptamers, ribozymes, Or an antibody that recognizes a DNA: RNA hybrid.

As used herein, the term "antisense oligonucleotide" is intended to encompass nucleic acid-based molecules capable of forming duplexes with miRNAs having a sequence complementary to all or part of the target miRNA, will be. Thus, the term "antisense oligonucleotide" as used herein may be referred to as "complementary nucleic acid-based inhibitor ".

The antisense oligonucleotides include various molecules such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), antagomir, 2'-O-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, Thioate-backbone ribonucleotide, a PNA (peptide nucleic acid) oligonucleotide or a LNA (locked nucleic acid) oligonucleotide.

Preferably a ribonucleic acid. The ribonucleic acid includes double interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme.

LNA is a modified ribonucleotide with an additional bridge between the 2 'to 4' carbons of the ribose sugar site to have a locked form, whereby the oligonucleotide with the LNA has improved thermal stability.

PNA (peptide nucleic acids) include peptide-based backbones instead of sugar-phosphate backbones in polynucleotides. The 2'-O-modified oligonucleotide is preferably a 2'-O-alkyl oligonucleotide, more preferably a 2'-OC _{1 -3} alkyl oligonucleotide, and most preferably a 2'-O-methyl oligonucleotide to be.

The antisense oligonucleotides include antisense oligonucleotides, antigomes and inhibitory RNA molecules in the narrow sense.

As used herein, the term " antagomir "is a single-stranded chemically modified oligonucleotide that is used for the silencing of endogenous microRNAs. Antagomir contains sequences that are not complementary to the Arganoute 2 (Ago 2) cleavage site, or that the base is modified to, for example, a 2 'methoxy group, a 3' cholesterol group or a phosphorosioate to inhibit Ago2 cleavage And has a complementary sequence to the target sequence. An antagonist according to the present invention has a sequence which is at least partially or completely complementary to the microalgae according to the present invention. In one embodiment, the antigomere comprises one or more modifications (e. G., 2'-O-methyl-sugar modifications, or 3'cholesterol modifications). In other embodiments, the antigomere comprises at least one phosphorocytoate linkage and at least partially has a phosphorothioate backbone. Suitable antagomir lengths for inhibiting the activity of microalgae according to the present invention are 7-50 nucleotides, especially 10-40 nucleotides, more particularly 15-30 nucleotides, even more particularly 15-25 nucleotides, especially 16-19 nt , But is not limited thereto. For example, 5'-CACACAUA-3 'complementary to 5'-UAUGUGUG-3', which is a seed sequence of 5'-CUACCUC-3 'miR-466, which is a complementary sequence to 5'-GAGGUAG- 3 '. < / RTI >

As used herein, the term "complementary" means that the antisense oligonucleotide is sufficiently complementary to the target of microenergy according to the present invention under certain hybridization or annealing conditions, preferably physiological conditions, Is meant to encompass both partially partially substantially complementary and perfectly complementary, and preferably means completely complementary. Substantially complementary, although not entirely complementary, is intended to mean a complementarity sufficient to bind to the target sequence and to produce an effect according to the present invention, i. E. Sufficient to interfere with the activity of the microarray.

As used herein, the term "nucleic acid" includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof such as peptide nucleic acids (PNA) or mixtures thereof. The nucleic acid can also be single or double stranded and can encode molecules including polypeptides, mRNA, microRNA or siRNA, and the like.

In one embodiment according to the present disclosure, a substance capable of inhibiting the activity of a microarray can be complementarily bound to all or a part of the microarray pre- and / or mature sequence, in particular the seed sequence, Is an antisense oligonucleotide capable of inhibiting its activity. The inhibition of the activity is to inhibit the transcription of microalgae according to the present invention and / or its binding to the target mRNA. An antisense oligonucleotide according to the present invention may or may not include one or more of the following modifications of the backbone (skeleton) connecting the nucleotides or nucleotides constituting the antisense oligonucleotide. That is, the antisense oligonucleotide may comprise at least one phosphorothioate in its backbone, wherein at least one nucleotide constituting the antisense oligonucleotide is 2-methylated at the sugar of the LNA, or at least one nucleotide constituting it.

In one embodiment according to the present application, the antisense oligonucleotide or nucleic acid molecule of the present invention comprises a sequence complementary to all or part of the seed sequence of a microarray according to the present invention. Seed sequences are conserved sequences that are conserved in a variety of species that are critical for the recognition of target molecules of miRNAs (Krenz, M. et al., J. Am. Coll Cardiol. 44: 2390-2397 (2004); H. Kiriazis, et al., Rev. Physiol. 62: 321 (2000)). Since miRNA binds to the target through the seed sequence, it can effectively suppress the translation of the target mRNA when the interaction of the seed sequence with the target is inhibited. Herein, the seed sequence of miR-466 is represented by the first or eighth nucleotide sequence of SEQ ID NO: 1, or the 11th to 18th nucleotide sequence of SEQ ID NO: The seed sequence of miR-let7 of the present invention is represented by the second or eighth nucleotide sequence of SEQ ID NO: 3 or the 12th to 18th nucleotides of SEQ ID NO: 4.

Thus, in one embodiment according to the present invention, the nucleic acid molecule capable of inhibiting the activity of miR-466 is the first to eighth nucleotide sequences of SEQ ID NO: 1 or all of the 11th to 18th nucleotide sequences of SEQ ID NO: For example, an antisense oligonucleotide of the present invention may be 5'-CACACAUA-3 '(SEQ ID NO: 5).

In another embodiment, the nucleic acid molecule capable of inhibiting the activity of the miR-let7 of the present invention is a nucleic acid molecule of the second to eighth nucleotide sequences of SEQ ID NO: 3, or all or part of the nucleotide sequences of the 12th to 18th nucleotides of SEQ ID NO: For example, an antisense oligonucleotide of the present invention may be 5'-CUACCUC-3 '(SEQ ID NO: 6).

Wherein one or more nucleotides constituting the oligonucleotide are 2'-O-methylated, or each of the one or more nucleotides is LNA, or at least one of the chemical bonds constituting the backbone may be a phosphothioate, . ≪ / RTI >

Microenes according to the present invention exhibit neuroprotective effects.

The term "neuroprotection" as used herein means protecting nerve cells of the brain, central nervous system or peripheral nervous system (especially in the brain or spinal cord) from killing and / or damage. It also means to delay or prevent the treatment, the onset of a neurological disease affecting the nervous system due to the above protection.

In another aspect, neuroprotection herein includes neuroprotection following injury to the brain, central nervous system, or peripheral nervous system, wherein damage is caused by, or limited to, chemical, toxic, infectious, radiation and / or traumatic injury It does not.

The extract or composition according to the present invention can be used for the prevention or treatment of various diseases which are effective by direct neuroprotection or which slow down disease progression by controlling the pathological mechanism of the disease. Such diseases include, but are not limited to, acute, subacute, or chronic neurodegenerative diseases.

Thus, through inhibition of one or more of miR-466 or miR-let7 according to the present invention, it can be used for the treatment or prevention of neurodegenerative diseases.

As used herein, "acute neurodegenerative disease" includes, but is not limited to, various types of diseases associated with the death or impairment of nerve cells, including cerebral blood flow deficit, local brain trauma, brain injury and spinal cord injury, , Reperfusion following embolic occlusion and thrombotic occlusion, acute ischemia, perinatal hypoxia-ischemic injury, cardiac arrest and any type of intracranial hemorrhage (e. G., Epidural, subdural, subarachnoid and intracerebral) Includes cerebral ischemia or cerebral infarction due to my lesions (including bruises, penetrating, compression, and lacerations). In one embodiment, the acute neurodegenerative disease is the result of stroke, cerebral infarction, cerebral hemorrhage, head injury, or spinal cord injury.

"Subacute neurodegenerative disease" as used herein means a neurological dysfunction disorder occurring over a period of several days or weeks, including, but not limited to, dehydrative diseases such as multiple sclerosis, neurogenic tumor syndrome, subacute combined degeneration, subacute necrotizing encephalitis, and subacute sclerosing encephalitis.

The term "chronic neurodegenerative disease" as used herein includes, but is not limited to, senile dementia, vascular dementia, diffuse white matter disease (Vince Disease), dementia of endocrine or metabolic origin, dementia caused by head trauma and diffuse brain injury, Alzheimer's disease, peptic disease, diffuse rheumatoid disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration (Schie-Drager's syndrome), neurodegeneration Amyotrophic lateral sclerosis, incompatibility, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's Diseases, Parkinson's disease, synucleinopathies, primary progressive aphasia, ancestral malformation, macado-Joseph disease / spinal cord cerebellum (Including atopic dermatitis), motor neuron diseases including olivary schwannomas, giardi < RTI ID = 0.0 > Lutret < / RTI > disease, training and false training paralysis, spinal cord and spinal cord training muscle atrophy (Kennedy disease), multiple sclerosis, Welfare-Kugelberg-Welander disease, ankylosing spondylitis, progressive multifocal < RTI ID = 0.0 > Prion diseases including cerebral palsy, hereditary autonomic ataxia (Lilly-Day Syndrome), Creutzfeldt-Jakob, Gerstmann-Straussler-Shinkler disease, Kurus disease and fatal hereditary insomnia, or cerebral palsy. In one embodiment, the chronic neurodegenerative disease includes Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis or cerebral palsy. In one embodiment according to the present application, it is used for the prevention or treatment of cerebral hemorrhage or stroke.

In one embodiment according to the present application, it is used for the treatment or prevention of stroke. Stroke is a disease that causes death or disability of a patient due to neurological deficit due to occlusion or breakdown of cerebrovascular vessels. It promotes inflammation reaction, nerve and excitability and self-destruction even after the initial neuronal cell necrosis after stroke. Secondary < / RTI > nerve damage progresses, and the composition according to the present invention exhibits a neuroprotective effect against such primary and secondary nerve damage.

The composition according to the present invention provides a protective effect against nerve damage caused by the above-mentioned neurological diseases and is administered to a subject in need of prevention or treatment of a neurodegenerative disease in an effective amount in a preventive or therapeutic manner, Can be effectively used for prevention or treatment.

As used herein, the term "treatment ", easing, or amelioration refers to any act that improves or alleviates symptoms of a related disease upon administration of the composition. Those skilled in the art will be able to ascertain the precise criteria of the disease by referring to the data provided by the Korean Medical Association, and to judge the degree of improvement, improvement, and treatment of the disease.

As used herein, the term "prophylactic" means any act that inhibits or delays the onset of a related disorder. It will be apparent to those skilled in the art that the compositions herein may prevent early onset symptoms, or related disorders when administered prior to appearance.

The composition of the present invention may further comprise one or more active ingredients exhibiting the same or similar functions in association with the treatment of a disease other than the substance capable of inhibiting the activity of microalbumin according to the present invention or maintaining / increasing the solubility and / ≪ / RTI > Also optionally, it may further comprise a chemotherapeutic agent, an anti-inflammatory agent, an antiviral agent and / or an immunomodulator.

The composition of the present invention may further comprise one or more pharmaceutically acceptable diluents, carriers and / or adjuvants in addition to the above-mentioned active ingredients. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have. For example, as solutions, emulsions and liposomes, and the like. For example, the active ingredient may be formulated into tablets, capsules, gels, syrups or suppositories by mixing with pharmaceutically acceptable liquid and / or solid carriers or excipients of fine powder. The composition according to the invention may also be prepared as a suspension in an aqueous, non-aqueous or mixed medium. The aqueous suspension may further comprise a material that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol and / or dextran.

The method of administering the composition according to the present invention is not particularly limited, and a known method of administering the inhibitor can be applied. Depending on the intended method, parenteral administration (for example, intranasal, intravenous, subcutaneous, ) Or oral administration. In order to obtain a rapid therapeutic effect, administration by intra-nasal injection is preferable. The compositions herein may also be delivered in a variety of routes including, for example, infusion or bolus injection, epidermal or mucosal (oral mucosa, anal mucosa, intestinal mucosa, etc.). The compositions herein may also be administered systemically or topically.

Further, the composition of the present invention is preferably introduced into the central nervous or peripheral nerves through appropriate routes. Suitable routes include intraventricular or intrathecal administration. Such administration can be accomplished using a catheter connected to the reservoir. Administration via the lung via an inhaler or sprayer may also be used, formulated as an aerosol. It is not intended to exclude formulations for intravenous administration, subcutaneous injection, intracerebral intramuscular injection, inhalation administration, or oral administration, so long as the effects according to the present invention arise.

In one embodiment according to the present application. Upon nasal administration, the effect of the composition can be enhanced as it is delivered to the brain along the olfactory nerve pathway. The nasal cavity refers to the space in the nose separated by the septa, and the intranasal administration refers to the delivery of the composition of the present invention to any tissue of the nasal epithelium. Nasal administration for the intranasal administration of the composition of the present application, which carrier comprises one or more suitable solid or filler diluents suitable for administration to any part of the mammalian, preferably human, nasal epithelium, Or an encapsulating material. Typically, the carrier can 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 may be prepared in various unit dosage forms. Such forms include, but are not limited to, nasal drops, nasal sprays, nasal gels, nasal ointments, and powders for nasal passages.

The carrier may also include a delivery enhancer, which may include, but is not limited to, an aggregation inhibitor, a dosage modifier, a pH control agent, a degradative enzyme inhibitor, a mucolytic dissolution or dextrin, a ciliostatic reagent, a membrane permeation enhancer For example, one or more of a surfactant, a bile salt, a phospholipid or a fatty acid additive, a micelle, a liposome, or a carrier, an alcohol, an enamine, a nitric oxide donor mixture, a long-chain amphipathic molecule, Hydrophobic penetration enhancers, sodium or salicylic acid derivatives, glycerol esters of acetoacetic acid, cyclodextrin or beta-cyclodextrin derivatives, medium chain fatty acids, chelating reagents, amino acids or salts thereof, N-acetylamino acids or salts thereof, Enzymes, fatty acid synthesis inhibitors, cholesterol synthesis inhibitors or nitric oxide stimulating substances, chitosan, The present compositions can be effectively combined, combined, stored, encapsulated, or stabilized to stabilize active pharmaceutical agents, such as modulating agents, vasodilators, selective delivery enhancers and intranasal mucosal delivery of epithelial junctional physiology, Stable carriers, carriers, support materials or complex-forming species, and the like.

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 the effective dose level will depend on the type, severity, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage ranges vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease, and a proper dose may be, for example, And / or the degree of specific activity of the polynucleotide used. It may be calculated on the basis of the EC ₅₀ generally measured as effective in the in vivo animal model and in vitro, for example from 0.01 μg to 1 g per kg of body weight, and may be calculated on a daily, weekly, monthly or yearly basis , May be administered once or several times per unit period, or may be continuously administered for a long period using an infusion pump. The number of repeated administrations is determined in consideration of the duration of the drug in the body, the drug concentration in the body, and the like. The composition may be administered for recurrence, even after treatment according to the course of the disease treatment.

The active ingredients according to the present invention, for example antisense oligonucleotides, may be used in the composition as such or in the form of pharmaceutically acceptable salts. A pharmaceutically acceptable salt means that the undesirable toxicity is minimized while maintaining the biological activity of the polynucleotide according to the present invention. Such salts include salts formed with metal cation such as, for example, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, But are 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 ingredients of the present invention, such as antisense oligonucleotides, are negatively charged due to the nature of the nucleotides thereof. Due to the lipophilic nature of the cell membrane, the uptake of antisense oligonucleotides into the cell membrane can be reduced. Absorption inhibition due to this polarity can be overcome through the prodrug approach described below (Crooke, RM (1998) in Crooke, ST Antisense research and Application, Springer-Verlag, Berlin, Germany, vol. 103-140).

In another embodiment, the disclosure relates to a diagnostic kit for a seizure disorder comprising a nucleotide sequence of a miRNA selected from the group consisting of miR-466 and miR-let7, 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 in the brain of stroke mice, as shown in Fig.

The diagnostic kit of the present invention can be used for diagnosing neurodegenerative diseases, particularly stroke diseases, as described above.

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 applied to a PCR amplification process, the kit of the present invention may optionally comprise reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

According to a preferred embodiment of the present invention, the kit of the present 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-phase surface of the microarray. When the kit of the present invention is a gene amplification kit, it includes a primer. The probe or primer specifically recognizes one or more of miR-466 and miR-let7 according to the present invention and has a sequence complementary to the above sequence. The term "complementary " as used herein means having complementarity to selectively hybridize to the nucleotide sequence under certain hybridization or annealing conditions, as defined above, and the probe or primer of the present invention May have one or more mismatch nucleotide sequences so long as they are capable of selectively hybridizing to the nucleotide sequence in addition to being fully complementary.

The nucleotide sequence of the miRNA of the present invention to be referred to in the preparation of the primer or probe can be confirmed by miRBase, and the primer or the probe can be designed by referring to this sequence.

In another embodiment, the invention provides a method comprising: detecting expression of one or more of a marker consisting of miR-466 and miR-let7 from a sample; And a method of detecting the marker to provide information necessary for diagnosis or prognosis of a neurodegenerative disease, comprising the step of associating the expression level of the detected marker with the diagnosis or prognosis of a neurodegenerative disease of a test subject .

In the method according to the present invention, the associating step compares the expression amount of the determined marker with the detection result for each of the markers determined in the normal control group, wherein the marker has an increased expression amount as compared with the control.

Further, the method according to the present invention can further use the non-marker clinical information of the subject to be examined at the time of diagnosing or prognosing a neurodegenerative disease of the test subject. The non-marker clinical information of the subject includes, but is not limited to, the age, sex, weight, diet, body mass, baseline disease, blood test, brain MRI, brain CT, or cerebrospinal fluid examination.

The degree of expression of the miRNA analyzed by the kit or method of the present invention, such as RT (reverse transcriptase) -PCR or real-time-PCR (see Sambrook, J. et al., Molecular Cloning. When the degree of expression measured by the method described in the Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) is 1.5 times or more, preferably 2 times or more, as compared with the normal control, It is judged that the patient is suffering from a metamorphic disorder or is at an increased risk.

In another aspect, the present invention provides a method of detecting a nucleic acid comprising contacting a test substance with one or more RNAs of a microenvironment according to the present invention; And determining the activity of the microen- ene in contact with the test substance, wherein the activity of the contacted microen- ene is determined by comparing the activity of the microen- ene of the control not contacted with the test substance And selecting the candidate substance as a candidate substance when it is decreased.

In one embodiment according to the present application, the microenal according to the present invention is provided in the form of a cell expressing it, and the activity is analyzed by the expression of these microalenia. For example, a cell expressing microalgae according to the present invention may be contacted with a test substance, and then the change in the amount thereof may be compared with a control cell before or after the contact, Candidate materials are selected. The amount of expression of microalgae used in the method according to the present invention can be performed using known methods such as Northern blotting, RT-PCR, hybridization using a microarray, and the like.

In yet another embodiment, the microen- gen is provided in the form of a cell expressing it, and the activity is determined by analyzing the interaction of the micro-antigen and the target with the 3'-UTR of the target. For example, after contacting a cell expressing microalgae according to the present invention with a test substance, the degree of interaction of the target gene with the 3'-UTR is compared with that of the control cell before or after the contact, Variations, especially reductions, are selected as candidates. Methods for detecting RNA-RNA interactions used in the methods according to the present application are known in the art and are described for example in RNA Walk (Lusting et al., Nucleic Acids Res. 2010; 38 (1): e5 ) Or yeast two hybrid system (Piganeau et al., RNA 2006; 12: 177-184), and may be referred to RNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011).

The type of cells used and the amount and type of the test substance used in the present method will depend on the specific experimental method used and the kind of the test substance. Those skilled in the art will be able to select appropriate cell types, amounts and / or conditions. As a result of the experiment, a substance which causes a decrease in the activity of microalgae in the presence of the test substance as compared with the control group not in contact with the test substance is selected as a candidate substance. Less than about 99%, less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70% less, less than about 65% , About 55% reduction, about 50% reduction, about 45% reduction, about 40% reduction, about 30% reduction, about 20% reduction or less.

As used herein, the term "test substance" means a substance that is expected to inhibit the activity of microalbumin according to the present invention as described above, and may be a low molecular weight compound, a high molecular weight compound, a mixture of compounds But are not limited to, proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes and DNAzymes) or sugars and lipids. The test substance may be a polypeptide having two or more amino acid residues, such as six, ten, twelve, twenty or fewer or more than twenty such as 50 amino acid residues. The test materials can be obtained from libraries of synthetic or natural compounds, and methods for 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 Available from MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution The desired synthetic library method, the bead 1-compound library method, and the synthetic library method using affinity chromatography screening. Methods for synthesis of 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 screening a drug for treating neurodegenerative diseases, a compound having a therapeutic effect of a low molecular weight may be used. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

For example, biologics can also be used for screening. Biologics refers to a cell or a biomolecule, which refers to a substance produced using biomolecules, proteins, nucleic acids, carbohydrates, lipids, or in vivo and in vitro cell systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organic materials found in polynucleotides, peptides, antibodies, or other plasma.

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

Example

The experimental method used in this embodiment is as follows.

Mouse model manufacturing

This study was conducted under the approval of IACUC (Institutional Animal Care and Use Committee) of Seoul National University Hospital. The committee was authorized by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Male SD (Sprague-Dawley) (Korea Bio, Korea) rats weighing 200-220 g were used in the experiments. Intracerebral hemorrhage (ICH) was induced as follows. The rats were placed in a stereotaxic frame (David Kopf instruments, Tukunga, CA, USA) after intraperitoneal administration of 1% ketamine 30 mg / kg. Then, the needle was inserted into the striatum through a tunnel of Tiandu, and a 30 gauge hamiltonian needle was inserted. Bacterial collagenase type VII (Sigma) 0,23 UNIT in 1 μl of PBS was added to the left striatum (position: 3.0 mm left lateral to the midline, 0.2 mm posterior to bregma, and 6 mm below the skull) And were orally administered to the predetermined coordinates. The balloon was then closed with bone wax and the incision site was closed. The rats were allowed to recover from anesthesia, and vital signs were continuously measured.

miRNA microarray analysis and target gene discovery

After 24 hours of induction of ICH, 4 rats were anesthetized and the brains were harvested. Total miRNAs were extracted from each hemisphere of the brain using the miRNeasy Mini Kit (Qiagen, Valencia, USA) according to the manufacturer's instructions. The concentration of each RNA sample was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Respectively. The miRNA extracted from each hemisphere was hybridized using GenoExplorer microRNA array labeling kit (GenoSensor Corporation, USA) according to the manufacturer's instructions and labeled with GenoExplorer microRNA chips (GenoSensor Corporation) containing 1088 miRNAs according to the manufacturers' Respectively. The hybridized microRNAs were then scanned and analyzed using an Axon GenePix 4000B scanner and GenePix Pro software (Molecular Devices, USA). The heat map of the miRNA expression pattern was visualized according to the Z-score. The Z-score was calculated as follows: (Raw individual value of the miRNA expression-mean of the miRNA expressions) / Standard deviation of the miRNA expression.

For miRNAs with elevated expression, four major miRNA target prediction sites, TargetScan (www.targetcan.org), Pictar (http://pictar.mdc-berlin.de/), DIANA microRNA.org (www.microRNA .org), and miRecords (http://mirecords.biolead.org/) to find possible targets for elevated miRNAs. MiRNAs that are both elevated in the ICH model and the in vitro thrombin toxicity model and that are predicted to modulate genes that are likely to have a therapeutic effect at the target prediction site have been screened. Followed by constructing an antagonistic sequence of a single strand having a phosphorothioate backbone with a cholesterol residue at the 3 'end. The constructed sequence is as follows: miR-let7 < RTI ID = 0.0 > 5'-CUACCUC-3 '; miR-LeT7: 5'-UGAGGUAGUAGGUUGUAUGGUU-3 '; miR-466 < / RTI > Antigomere: 5'-CACACAUA-3 '; miR-466: 5'-UAUGUGUGUGUGUAUGUCCAUG-3 '. The synthesized microen enantiomer was used in a therapeutic effect experiment (Fig. 3) in Invitro and a therapeutic effect experiment (Fig. 3, 5) in Invivo.

Real-time PCR of miRNA

MiRNAs selected by microarray analysis were verified by qRT-PCR (reverse transcription-polymerase chain reaction). Expression of miRNA was measured using GenoExplorer miRNA qRT-PCR kit (GenoSensor Corporation) and miRNA specific primer (GenoSensor Corporation).

Each microarray probe is as follows; miR-466, 5'-UAUGUGUGUGUGUAUGUCCAUG-3 '; Let7, 5'-UGAGGUAGUAGGUUGUAUGGUU-3 '; miR-24-2, 5'-GUGCCUACUGAGCUGAAACAGU-3 '; miR-142-5p, 5'-CAUAAAGUAGAAAGCACUACU-3 '; miR-196, 5'-UAGGUAGUUUCAUGUUGUUGGG-3 '; miR-30d, 5'-UGUAAACAUCCCCGACUGGAAG-3 '. Each microarray primer used was a commercialized primer (GenoSensor Corporation, Tempe, AZ). Amplification specific conditions are as follows: PCR conditions: 40-60 cycles (90 ° C for 20s, 60 ° C for 15s, and 70 ° C for 30s).

The comparative threshold cycle (Ct) was calculated by 2-ΔCt expression and normalized to the RNU6B (GenoSensor Corporation) value of each sample as a control. Each qRT-PCR (quantitative reverse transcription PCR) was performed three times.

Differences in MicroRNA Expression by Region

Since collagenase-induced major lesions of ICH are present in the basal ganglia, cortical, basal and hematomic segments were isolated 24 h after induction of ICH using minute aggregates to assess miRNA expression by region. Cortical and basal ganglia miRNAs of the hemispherical hemisphere were compared with those of the opposite hemispheres respectively. The results are shown in FIG. As shown, miR-466 and miR-let7 all increased after hemorrhage. Especially, miR-let7 significantly increased the expression level in the basal ganglia after cerebral hemorrhage.

Invitro Thrombin  Toxic model

Thrombin is known to cause nerve cell death, which simulates nerve damage after ICH. Therefore, in vitro thrombin toxicity model was constructed by treating thrombin (Sigma, USA) with PC12 cell line (ATCC, USA). PC12 cells were grown in RPMI 1640 (Gibco BRL, USA) medium containing 100 units / ml penicillin and 100 ug / ml streptomycin (Sigma), 10% horse serum (Sigma) and 5% fresh bovine serum At 37 ° C, 5% CO ₂ and 100% humidity. Thrombin was treated with 200 U / ml and 500 U / ml over 6 to 24 hours. Cell viability analysis was performed using the WST-1 assay kit (Roche) according to the manufacturer's instructions. Absorbance was measured at 450 nm (630 nm in reference group) using a Multiskan JX microplate reader (Thermo Fisher Scientific, USA). Higher values indicate better survival. As a result of treatment with various concentrations of thrombin, 50% of the cells were killed by 200 U / ml of thrombin and 90% of cells were killed by 500 U / ml of thrombin. In this study, 500 U / RTI ID = 0.0 > miRNA. ≪ / RTI >

Measuring the effectiveness of treatment with Invit

In order to investigate the therapeutic potential of antigomir, we first used an in vitro damage model. To evaluate the neuroprotective effect in the in vitro thrombin model, each antagomere constructed as described above was mixed with 500 IU / ml of thrombin in the culture medium for 24 hours. Each of the antagonists was transferred to cells using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions. Cell viability was measured using the WST-1 assay kit (Roche) 24 hours after the application of thrombin and antagonism. Controls were treated with the same volume of PBS.

Measuring Inhibitory Therapeutic Effect

In order to determine the therapeutic effect of microRNA control, nasal administration of antagomir after ICH induction was performed as previously described (Lee, ST, et al. (2012) Ann Neurol, 72, 269-277) . In summary, 5 nmol of antagomiR-466 or antagomiR let7 in 24 μl of 0.1% diethyl pyrocarbonate-treated distilled water was administered intranasally to the nostrils immediately after induction of ICH, immediately before recovery in anesthesia . The control group received the same volume of medium.

Brain water content measurement

To measure cerebral edema, after general anesthesia, 72 hours after induction of ICH, the brain was harvested and separated into two hemispheres and cerebellum. The wet weight of each brain sample was then immediately measured and dried for 24 hours in a gravity oven and then the dry weight was measured. The brain water content is expressed by the following formula: [(wet weight-dry weight) / (wet weight) X 100 (%)].

Immunochemical analysis

After 72 hours of ICH induction, 4 rats in each group were anesthetized and maintained in the heart with 50 ml of cold saline in 0.1 mol / l phosphorylation buffered saline and 50 ml of 4% paraformaldehyde. Brains were harvested and fixed in 4% paraformaldehyde for 24 h, then treated with 30% sucrose for 24 h for cryoprotection. The sections were cut into 30-μm sections using a cryostat (Leica CM 1900, Germany). One reference section containing the Tiandubong insertion site and a section located 1 mm each before and after the reference section were selected. Three sections were immunostained with myeloperixodase (MPO, 1: 200, DAKO, Baltimore, USA) and Ox-42 (1: 500, Chemicon, Temecula, USA) Cell death was detected by performing TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, which detects DNA fragmentation during death. Total cell numbers per slice were converted to cell density.

Nerve recovery assessment

In order to evaluate the functional improvement after miRNA regulation, two limb placing tests (Kim JM et al, Brain Research 2007; 1183: 43-50; Jung KH et al., Stroke 2004; 35: 1744-1749). This test consisted of 4 domains with 8 points as the total score. The first and second domains measure the two leg position tests, which assess the sensory integration of the forelimbs and hind limbs, respectively, by monitoring the response to tactile and proprioceptive stimuli. The rats were hung from the edge of the table with the front legs, placed on the edge of the table, and moved freely. The third and fourth domains were lightly pulled on the forelimbs and hind limbs, respectively, and then restoration and repositioning were investigated. Each domain was scored as 0 for normal response, 1 for weak or delayed response, and 2 for no response.

Western blot  analysis

Anesthetized animals (normal, intracerebral hemorrhage control, and intracerebral hemorrhagic fever treated mice, 4 mice from each group) were perfused with 10 mL of cold saline followed by Western blotting after preparing homogenates of each brain . The antibodies used in the Western blot are as follows: Insulin like growth factor 1 receptor (IGF1R; Santa Cruz, USA), IGF2R (Santa Cruz), high mobility group AT-hook 2 (Cell Signaling Technology, USA) binding protein 1 (IGF2BP1; Abcam Incorporated, USA), and glyceraldehydes 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, USA). The expression patterns of the following proteins were also examined: serine threonine protein kinase (Akt; Santa Cruz, USA), phosphorylated Akt (p-Akt; Santa Cruz, CA, USA), B-cell lymphoma-extra large (BcL-xL; Santa Cruz, USA), and Bcl-2 associated protein (Bax; Santa Cruz, USA). Western blotting was visualized using enhanced chemiluminescent reagents (Pierce, USA) according to manufacturer's instructions and the results were scanned (GS-700; Bio-Rad, USA). Western blot results were optimized for GAPDH and calculated using the Image-J program (National Institutes of Health, USA).

Statistical analysis

Consecutive values were expressed as mean value standard deviation. The heat map for miRNA expression was generated using the Z-score, which was calculated as follows: Z-score = [(raw value for expression of each miRNA - Standard deviations of all miRNAS expressions]. Mann-Whitney U test was used to compare each group and a two-tailed p-value below 0.05 was considered statistically significant. The Bonferroni method was used for post-hoc intergroup comparison after repeated variance analysis for functional recovery assessment.

Example 1: Differentially expressed in the brain of a stroke model miRNA  Identification

Since human hemorrhage and experimental hemorrhage models are located in the basal ganglia, microinjection analysis was performed according to brain location. As described in the experimental method, the brain was extracted 24 hours after the intracerebral hemorrhage using the cerebral hemorrhage model prepared by administering collagenase to the predetermined coordinates in the rat, and RNA was isolated from the hemisphere in which hemorrhage occurred and the hemisphere opposite the hemisphere. In addition, microRNA expression patterns of both sides were analyzed by microarray method and confirmed by quantitative RT-PCR method.

The results are shown in FIG. A total of 1088 microarrays were analyzed by microarray method, and it was confirmed that 8 microarrays were significantly increased after cerebral hemorrhage. The most increased microalbumin was miR-466 and subsequently increased in the order of let7, miR-24-2, and miR-142-5p. (A) in paired t - test with a p - value of less than 0.05 and (b) microalbumin (B) with a p - value of less than 0.05 and a difference of more than two in the hemisphere hemisphere.

As shown in FIG. 2, both microRNAs of miR-466 and miR-let7 were increased after cerebral hemorrhage, and miR-let7 was significantly increased in the basal ganglia after cerebral hemorrhage.

As shown in FIG. 3, in the thrombin toxicity model in which thrombin was added to the PC12 cell line and imitated nerve damage after in vitro hemorrhage, miR-466, miR-let7, miR -24-2, < / RTI > Expression was confirmed. As a result, it was confirmed that miR-let7 increased expression level after 24 hours of thrombin treatment.

We also examined miR-466, miR-let7, miR-24-2, and miR-142-5p target genes in the microarray database to identify candidate miR-let7 and miR- 466 were selected.

Example 2 Micro-AL  Therapeutic Effect and Mechanism through Control

In order to inhibit the increase in the expression of the microallenes selected in Example 1, a complementary sequence was prepared and the neuroprotective effect was confirmed in an ex vivo thrombin toxicity model and a rat cerebral hemorrhage model as described in the experimental method.

The results are shown in FIG. As shown above, miR-466 and miR-let7 complementary sequences exhibited neuronal cell protection effects when these complementary sequences were suppressed by administration in an ex vivo thrombin toxicity model.

As shown in FIG. 4A, when the let7 complementary sequence was administered to the white cerebral hemorrhage model, the cerebral edema decreased on the third day, and after the fifth week, the neurological function was recovered from the fourth week, which is superior to the control. In addition, as shown in FIG. 4B, when the miR-466 complementary sequence was administered to the white cerebral hemorrhage model, the brain edema did not decrease but the neurological function recovery was excellent from the 2nd week.

The expression of target genes and lower stage proteins of the microRNA miR-let7 and miR-466 was analyzed by western blotting for the purpose of identifying the therapeutic mechanism. The results are shown in Figures 5A-5D. As shown in FIGS. 5A and 5B, the expression of insulin-like growth factor receptor 1 was increased and the phosphorylated Akt was also increased upon administration of miR-let7 and tadmoride. As shown in FIGS. 5c and 5d, the increase in the expression of insulin-like growth factor receptor 1 and 2 and phosphorylated Akt were also found to increase with administration of miR-466 antigomere.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> SNU R & DB Foundation <120> Composition and method for treating neurodegenerative diseases          targeting miRNA DP <130> DP201312002P <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <400> 1 uaugugugug uguaugucca ug 22 <210> 2 <211> 86 <212> RNA <213> Homo sapiens <400> 2 cauguguaua uaugugugug uguaugucca uguguguaua ugaauauaca uacacacaca 60 cauacacaca cgugcaagca cacaca 86 <210> 3 <211> 22 <212> RNA <213> Homo sapiens <400> 3 ugagguagua gguuguaugg uu 22 <210> 4 <211> 84 <212> RNA <213> Homo sapiens <400> 4 gcauccgggu ugagguagua gguuguaugg uuuagaguua cacccuggga guuaacugua 60 caaccuucua gcuuuccuug gagc 84 <210> 5 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> antagomir for miR-466 <400> 5 cacacaua 8 <210> 6 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> antagomir for miR-let7 <400> 6 cuaccuc 7

Claims (20)

A pharmaceutical composition for the treatment or prevention of cerebral hemorrhage comprising a substance capable of inhibiting the activity of microarray (miR) -466 and a pharmaceutically acceptable carrier,
The substance capable of inhibiting the activity of miR-466 may be a first to eighth nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence of 10 to 20 nucleotides in length including a sequence complementary to the 11th to 18th nucleotide sequence of SEQ ID NO: Antisense oligonucleotide, for the treatment or prevention of cerebral hemorrhage.
The method according to claim 1,
Wherein the composition further comprises a substance capable of inhibiting the activity of microRNA (miR) -let7, wherein the substance capable of inhibiting microRNA (miR) Or an antisense oligonucleotide of 10 to 20 nucleotides in length comprising a sequence complementary to a 12th to 18th nucleotide sequence of SEQ ID NO: 4.
delete delete delete 3. The method according to claim 1 or 2,
Wherein the antisense oligonucleotide comprises at least one nucleotide consisting of 2'-O-methylated LNA (Locked Nucleic Acid) or a sugar of one or more nucleotides constituting it, or at least one phosphothioate in the backbone of the oligonucleotide, &Lt; / RTI &gt; or a combination thereof.
3. The method of claim 2,
The antisense oligonucleotide capable of inhibiting the activity of miR-466 is represented by SEQ ID NO: 5,
Wherein the antisense oligonucleotide capable of inhibiting the activity of the miR-let7 is represented by SEQ ID NO: 6.
delete delete 3. The method according to claim 1 or 2,
Wherein said composition is delivered to the brain.
3. The method according to claim 1 or 2,
Wherein said composition is formulated for intranasal administration, intravenous administration, subcutaneous injection, intracerebroventricular injection, inhalation administration, or oral administration.
delete delete delete delete delete Detecting the expression of miR-466 marker in Invitro; And
And correlating the expression level of the detected marker with the diagnosis or prognosis of the subject's brain hemorrhage.
18. The method of claim 17,
Wherein the method further comprises detecting expression of a miR-let7 marker.
18. The method of claim 17,
The non-marker clinical information may further include at least one of age, gender, weight, dietary habit, body mass index, underlying disease, blood test, brain MRI, brain CT, Or brain spinal fluid examination.
19. The method of claim 18,
Wherein the associating step compares the expression amount of the determined marker with the detection result for each marker determined in the normal control group, wherein the expression of the marker is increased as compared with the control.

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