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WO2015025995A1 - Composition pharmaceutique et méthode de prévention ou de traitement de maladies associées à l'épilepsie ou aux convulsions par ciblage de micro-arn - Google Patents

Composition pharmaceutique et méthode de prévention ou de traitement de maladies associées à l'épilepsie ou aux convulsions par ciblage de micro-arn Download PDF

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WO2015025995A1
WO2015025995A1 PCT/KR2013/007701 KR2013007701W WO2015025995A1 WO 2015025995 A1 WO2015025995 A1 WO 2015025995A1 KR 2013007701 W KR2013007701 W KR 2013007701W WO 2015025995 A1 WO2015025995 A1 WO 2015025995A1
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mir
epilepsy
seizure
disease
activity
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Korean (ko)
Inventor
이상건
노재규
김만호
주건
정근화
이순태
전대종
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SNU R&DB Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications

Definitions

  • 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.
  • European Publication No. 2436784 also discloses the diagnosis and treatment of colorectal cancer with miR-203.
  • the present application seeks to develop miRNA modulators targeting miRNAs to treat epilepsy or seizure related diseases.
  • 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.
  • 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 GLR ⁇ subunit (GLRB, glycine receptor beta subunit) of the miR-203
  • GLRB GLR ⁇ subunit
  • a substance capable of inhibiting the activity of the activity of miR-203 herein is a substance capable of inhibiting its expression.
  • the substance capable of inhibiting the activity of miR-203 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
  • 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.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • 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.
  • an antisense oligonucleotide 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.
  • 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.
  • the present disclosure also provides a method of contacting a miR-203 RNA with a test substance
  • 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 GLR ⁇ subunit (GLRB, glycine receptor beta subunit) It can be determined by analyzing the interaction with.
  • GLRB glycine receptor beta subunit
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method 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.
  • 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.
  • 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.
  • FIG. 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.
  • a red color corresponds to a left upper portion and a lower right portion based on an intersection line
  • a green color corresponds to a right upper side and a lower left side.
  • FIG. 1B a red color corresponds to a right side and a green color corresponds to a left side.
  • Target prediction software (TargetScan, PicTar, and microT) was used to confirm that the 3'-untranslated site (UTR) of mouse Glycine receptor- ⁇ (GLRB) was the target of mmu-miR-203.
  • UTR 3'-untranslated site
  • GLRB mouse Glycine receptor- ⁇
  • 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.
  • 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).
  • the present application is based on the finding that reduced expression of glycine receptors by miR-203 is associated with seizure related diseases.
  • 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.
  • miRNA refers to 21-23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation.
  • 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.
  • miRNAs 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ribonucleic acid 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.
  • PNAs Peptide nucleic acids
  • 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.
  • 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.
  • the antagonist comprises one or more modifications (eg, 2'-0-methyl-sugar modifications, or 3 'cholesterol modifications).
  • 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.
  • complementary 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.
  • 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.
  • PNAs peptide nucleic acids
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • miR-203 activity can be used for the treatment of epilepsy or seizure related diseases.
  • 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.
  • SE status epilepticus
  • Epilepsy includes partial epilepsy that indicates partial epilepsy seizures and general epilepsy that indicates global epilepsy seizures when classified by seizure.
  • hidden epilepsy which is presumed to be abnormal but is not found in current brain imaging.
  • 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.
  • treatment 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.
  • 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.
  • 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.
  • 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.
  • other conventional additives such as buffers and bacteriostatic agents can be added.
  • 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.
  • 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.
  • 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.
  • 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.).
  • compositions herein can also be systemically or topically administered.
  • 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.
  • nasal refers to the space in the nasal cavity, which is divided left and right by the nasal septum
  • 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.
  • the carrier may be a liquid, solution, suspension, gel, ointment, lotion, or a combination thereof.
  • 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
  • composition according to the present application is administered in a pharmaceutically effective amount.
  • 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.
  • EC 50 which has been determined to be effective in in vivo animal models and in vitro, for example from 0.01 ⁇ g 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.
  • the active ingredient of the present application 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).
  • 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.
  • 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.
  • 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.
  • the probe is immobilized on the solid surface of the microarray.
  • the kit of the present invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RT reverse transcriptase
  • 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.
  • 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.
  • the miR-203 RNA is provided in the form of cells expressing it, the activity of the miR-203 RNA and its target GLR ⁇ 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 GLR ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the compound has a low molecular weight therapeutic effect.
  • compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • substances capable of inhibiting the activity of miR-203 are delivered to the brain in particular.
  • the miR-203 activity inhibitor 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.
  • the experimental method used in this example is as follows.
  • mice 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).
  • 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.
  • 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.
  • 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/).
  • 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.
  • GLRB human GLR ⁇ subunit
  • glycine receptor beta subunit human GLR ⁇ subunit
  • RPMI fetal bovine serum
  • 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.
  • 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).
  • the anesthetized mice were placed in supine position with their heads upright.
  • AM203 (2'-O-methylated-5'-cuagugguccuaaacauuucac-3 '; 5 nmol in 24 ⁇ l of 0.1% v / v diethylpyrocarbonate-treated distilled water; Bioneer, Korea) was added every 2 minutes. Nostrils were changed, and 4 ⁇ l of each pipette was administered (6 fractions in total). Control mice received an equal volume of vehicle.
  • 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 ⁇ -actin band.
  • mice 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) .
  • 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 M ⁇ ), 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.
  • 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.
  • 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).
  • 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).
  • Chronic epilepsy mice were treated intranasally with 5 nmol of AM203 or saline on days 0 and 2.
  • 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).
  • 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.
  • 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).

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Abstract

L'invention concerne une composition pharmaceutique utilisée pour prévenir ou traiter des maladies associées à l'épilepsie ou aux convulsions, la composition contenant un matériau ciblant miR-203. L'invention concerne un marqueur qui peut être utilisé pour les mesures de diagnostic ou de pronostic de maladies associées à l'épilepsie ou aux convulsions, et une utilisation du marqueur. La composition de la présente invention, contrairement aux agents thérapeutiques existants de type allopathique, peut restaurer un récepteur de glycine, ce qui permet essentiellement de traiter diverses maladies provoquées par la réduction du niveau d'expression du récepteur de glycine, par exemple, les maladies associées à l'épilepsie ou aux convulsions.
PCT/KR2013/007701 2013-08-21 2013-08-28 Composition pharmaceutique et méthode de prévention ou de traitement de maladies associées à l'épilepsie ou aux convulsions par ciblage de micro-arn Ceased WO2015025995A1 (fr)

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CN107982537A (zh) * 2017-11-17 2018-05-04 厦门大学 针对microRNA-155的治疗性药物及其应用
CN107961380A (zh) * 2017-11-23 2018-04-27 清华大学 试剂在制备药物中的用途、筛选药物的方法以及药物组合物
CN107961380B (zh) * 2017-11-23 2020-05-05 清华大学 试剂在制备药物中的用途、筛选药物的方法以及药物组合物
US10844380B1 (en) 2019-06-17 2020-11-24 Biorchestra Co., Ltd. Uses for prevention or treatment of brain diseases using microrna
US11198908B2 (en) 2019-06-17 2021-12-14 Biorchestra Co., Ltd. Method for diagnosis of Alzheimer's disease using microRNA
US11542503B2 (en) 2019-06-17 2023-01-03 Biorchestra Co., Ltd. Uses for prevention or treatment of brain diseases using microRNA
US12084718B2 (en) 2019-06-17 2024-09-10 Biorchestra Co., Ltd. Method for diagnosis of Alzheimer's disease using microrna
CN116004628A (zh) * 2023-01-03 2023-04-25 苏州大学 一种预防或治疗慢性疼痛的miRNA靶点及其应用

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