WO2013039271A1 - Modèle animal de dysfonctionnement du lobe frontal induit par une lésion cérébrale, et composition pour atténuer un dysfonctionnement du lobe frontal - Google Patents

Modèle animal de dysfonctionnement du lobe frontal induit par une lésion cérébrale, et composition pour atténuer un dysfonctionnement du lobe frontal Download PDF

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WO2013039271A1
WO2013039271A1 PCT/KR2011/006847 KR2011006847W WO2013039271A1 WO 2013039271 A1 WO2013039271 A1 WO 2013039271A1 KR 2011006847 W KR2011006847 W KR 2011006847W WO 2013039271 A1 WO2013039271 A1 WO 2013039271A1
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frontal lobe
type calcium
dysfunction
calcium channel
cobalt
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PCT/KR2011/006847
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English (en)
Korean (ko)
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김대수
김정진
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한국과학기술원
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/30—Animals modified by surgical methods
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the present invention relates to a model animal, and specifically relates to a model animal for irreversible frontal lobe dysfunction due to brain injury and a composition for reducing the irreversible frontal lobe dysfunction.
  • ADHD Attention Deficit and Hyperactivity Disorder
  • Parkinson's disease (Taylor et al., Brain , 109 (5): 845-883, 1985), Alzheimer's disease (Michon et al, J. Neurol., Neurosurg. Psych ., 57: 805-809, 1994) , Neurorodegenerative disease such as frontotemporal dementia (PR Talbot, J. Neural Transm. Suppl ., 47: 125-132, 1996), schizophrenia, PJ Reading, J. Royal Soc. Med.
  • the frontal lobe dysfunction is caused by hypoxia of the frontal lobe
  • the development of a therapeutic agent that can reversibly repair the frontal lobe damage has been a major concern, but this attempt has not been successful. Therefore, instead of the recovery of the frontal lobe damaged by suboptimal measures, it is necessary to develop a symptom improver that can improve the functional disorders caused by the frontal lobe damage, in particular, the behavioral disorder.
  • conventional prefrontal injury animal models include non-competitive non-human schizophrenia caused by prolonged administration of non-competitive N-methyl-D-aspartate (NMDA) / glutamate receptor antagonists such as phencyclidine (PCP). Primates have been reported (Jentsch et al ., Neuroscientist , 6 (4): 263-270, 2000).
  • NMDA N-methyl-D-aspartate
  • PCP glutamate receptor antagonists
  • Primates have been reported (Jentsch et al ., Neuroscientist , 6 (4): 263-270, 2000).
  • the animal model is mainly a model animal for severe frontal lobe dysfunction, such as schizophrenia, and is not suitable as a model animal for symptoms such as ADHD or prefrontal epilepsy, but has an advantage of being similar to human because of the use of primates.
  • the development of successful prefrontal dysfunction improvers using the same has not been reported.
  • Another object of the present invention is to provide a composition for improving frontal lobe dysfunction developed using the frontal lobe dysfunction model animal.
  • a frontal lobe dysfunction model animal except for a human implanted with a cobalt wire in the frontal lobe of the brain.
  • composition for treating or improving frontal lobe dysfunction comprising a T-type calcium channel inhibitor or an inhibitor of expression of the ⁇ 1G subunit of a T-type calcium channel as an active ingredient.
  • the screening step of determining whether any compound or natural product inhibits T-type calcium channel activity provides a screening method of a material for treating or improving frontal lobe dysfunction comprising a step of confirming whether or not the compound or natural product selected in the selection step improves frontal lobe dysfunction in the animal.
  • the cobalt line of the present invention In the model animal in which the cobalt line of the present invention is inserted into the frontal lobe, low frequency increased electric excitement, hyperactivity, and cognitive decline, which are characteristic of frontal lobe dysfunction, are observed, and thus can be usefully used as a prefrontal dysfunction model animal. In addition, it can be usefully used for the screening of the frontal lobe dysfunction improving material showing the symptoms in common. In addition, by administering a T-type calcium channel inhibitor to the prefrontal dysfunction model animals, the number of spikes observed in EEG decreases and the behavioral disorders of these animal models improve, so that the T-type calcium channel inhibitor is a frontal lobe dysfunction.
  • these T-type calcium channel inhibitors do not restore the damaged frontal lobe, but rather thetheta frequencies due to crosstalk between the prefrontal cortex (PFC) and the medial thalamus (MD) of the damaged frontal lobe. It can be a breakthrough frontal lobe improver in that it can reduce frontal lobe-specific epilepsy and hyperactivity disorder.
  • FIG. 1 is a brain and brain magnetic resonance image (a) of the cobalt line insertion mouse prepared according to an embodiment of the present invention, tissue staining pictures of the area induced damage by cobalt line insertion (b), cobalt line insertion site A graph showing the number of erythrocytes, ghost cells, and neurons in (c) and immunoblotting results showing the expression of VEGF expression at the cobalt line insertion site (d):
  • W tungsten
  • F L left prefrontal cortex
  • AU Arbitrary unit (number of neurons in the right frontal cortex / number of neurons in the left frontal cortex).
  • Figure 2 is a cobalt wire insertion mouse prepared according to an embodiment of the present invention (a) and when homocysteine thiolactone (homocysteine thiolactone) administered to the mouse (b) continuously recording the brain waves of the frontal lobe and other brain areas Electroencephalogram (EGG):
  • F L left prefrontal cortex
  • MD medial dorsal thalamus (mediodorsal thalamus),
  • T R right temporal cortex
  • T L left temporal cortex
  • W tungsten
  • Co Cobalt
  • Figure 3 is a graph measuring the distance of action over time in the open field of the cobalt-line-inserted mouse and the control group prepared according to an embodiment of the present invention (a), the copper wire pattern recorded the moving copper in the open field (b) a copper wire recording diagram (locomotor pattern); Graph (c) showing the total distance traveled for 60 minutes; A graph showing the central exploration distance after 5 minutes and 60 minutes after the start of the test (d) and the degree of stereotypic circling (e):
  • Co ⁇ 28d 28 days after cobalt line insertion.
  • Figure 4 is a graph showing the difference in learning ability in cobalt wire inserted mouse prepared according to an embodiment of the present invention and the control group:
  • Context The memory of the place where the horror memory was formed.
  • FIG. 5 shows the electroencephalogram of the cobalt-line-inserted mouse prepared according to an embodiment of the present invention when zonisamide, phenytoin, and T-type calcium channel inhibitor ethosuximide, which is an epilepsy treatment,
  • a graph (b) that records the change (a) and the number of interstitial spikes:
  • PHT phenytoin
  • FIG. 6 is a photograph showing changes in T-type currents in lentiviral vectors with CaV3.1 target shRNA, ⁇ 1G subunit of T-calcium channel, and cobalt-line-inserted mice infected with lentiviral vectors with scrambled shRNA as a control, respectively.
  • (a) graph recording the current density at ⁇ 60 mV
  • (b) 3 showing the results of a bicoherence analysis between the prefrontal cortex and MD thalamus in cobalt-line inserted mice infected with the lentiviral vector.
  • sh 3.1-DM or sh 3.1 Ca V 3.1 shRNA infection
  • MD medial dorsal thalamus
  • PFC prefrontal cortex
  • FIG. 7 is a graph showing changes in theta wave and normalized coherence in the frontal cortex (PFC) of Ca V 3.1 ⁇ / ⁇ cobalt gland and Ca V 3.1 + / + cobalt gland.
  • Electroencephalograph showing changes in EEG on the frontal lobe and MD thalamus 6 days after cobalt line transplantation, and graphs showing changes in spikes per hour after 6 days of cobalt line transplantation, and first 5 days after the start of the exercise test 6 days after cobalt line transplantation.
  • Copper chart showing movement patterns for minutes and last 5 minutes and graph (c) showing the distance of movement for the first 5 minutes and the last 5 minutes after the start of the exercise test 6 days after cobalt line implantation.
  • a frontal lobe dysfunction model animal except for a human implanted with a cobalt wire in the frontal lobe of the brain.
  • the model animal may be a mammal, and the mammal may belong to rodents, warrant trees, rabbits, carnivorous trees, carnivorous trees, base trees, or bovine trees except mice.
  • the cobalt gland can be implanted into the prefrontal cortex of the frontal lobe of the brain.
  • the model animals include traumatic brain damages, brain tumors, fetal hypoxia, neurodegenerative disease, schizophrenia, menchiitis, meningitis, vitamin B12 deficiency, hydrocephalus (hydrocephalus), multiple sclerosis, CNS lupus can be a model animal for frontal lobe damage caused by.
  • the neurodegenerative disease may be Alzheimer's disease or anterior temporal lobe dementia.
  • the frontal lobe injury is neurocongnitive deficit, delusion, speech or movement problem, mental handicap, personality change, broca aphasia. May cause aphathy, dysarthria, apraxia, agnosia, amensia, inattentiveness, impaired concentration, including Broca's aphasia .
  • the prefrontal dysfunction model animal production can be performed by inserting a cobalt line into the frontal lobe part of the animal's brain using a stereotaxic device.
  • the insertion position of the cobalt line And depth can be adjusted according to the type of animal and brain size.
  • the inventors of the present invention produced a model animal in which cobalt lines were inserted into the frontal lobe (see FIG. 1A).
  • Mice prepared by inserting the cobalt wire into the frontal lobe showed hemorrhagic teeth reflecting neovascularization at the cobalt wire insertion site 5 days after insertion, which was not found in the tungsten wire inserted as a control.
  • the results of T2-weighted magnetic resonance imaging (MRI) also revealed that the frontal cortex was more severely damaged in the cobalt-inserted mouse than in the tungsten-inserted mouse (see FIG. 1A).
  • Matoxylin / eosin staining of the damaged areas identified by the MR image showed an increase in ghost cells, a clear signal of hypoxic damage at the insertion site of the cobalt gland, and inflammatory neoplasms visualized by blood vessels filled with red blood cells. Angiogenesis was observed (see FIGS. 1B and 1C).
  • VEGF vascular endothelial growth factor
  • the present inventors observed the characteristic EEG observed in the frontal lobe dysfunction of the cobalt-line inserted mouse in order to confirm whether the damage of the frontal cortex actually causes prefrontal dysfunction.
  • EEG electroencephalogram
  • wires ie wires made of metals such as tungstec, copper and aluminum, did not induce interstitial spikes when inserted into the frontal cortex. 9-10 days after insertion, the prefrontal stromal spike spread to the left frontal lobe and MD thalamus (see FIG. 2B). Between 11 and 30 days post-insertion, there was a secondary generalization of single spikes or an ictal discharge with whole-body convulsion (see FIG. 2A).
  • Corticothalamic and interstitial cortices of interstitial spikes from the frontal cortex with intermittent secondary generalization by administering a subspasm level (550 mg / kg) of homocysteine thiolactone (HT) (corticocortial) propagation was possible in a similar fashion within an hour (see FIG. 2B).
  • HT homocysteine thiolactone
  • mice inserted with cobalt lines were inserted with cobalt lines.
  • the behavior was monitored by an open-field test at 6, 14, and 28 days, and the activity of the cobalt line compared to the control group was increased. A significant increase could be observed (see FIGS. 3A and 3C).
  • their movement was confined to the wall of the open field, the search toward the center was found to be limited compared to the control (see Figs. 3b and 3d). This tendency increased as the duration of the cobalt line insertion increased, and more specifically, the activity was analyzed by drawing traces of the frontal lobe dysfunction animal model. It was confirmed that homotypy (stereotypy), which is a turning behavior, was observed (see FIGS.
  • the present inventors performed a conditioned fear learning test to determine whether memory loss observed in frontal lobe dysfunction seen in humans is also observed in cobalt-inserted mice.
  • a frontal lobe prefrontal dysfunction animal model was able to observe a marked decrease in memory (see Figure 4). Therefore, low frequency EEG, homology, and cognitive decline, which are characteristic of frontal lobe dysfunction, are observed in mice inserted with the cobalt gland of the present invention, therefore, animals in which the cobalt gland is inserted into the frontal lobe of the brain are model animals. It was confirmed that it can be usefully used.
  • the present inventors measured the electroencephalogram after administering a drug known as an antiepileptic agent to the cobalt gland insert animal, in order to confirm whether the epileptic spikes appearing in the cobalt gland insert animal is reduced by administration of the epileptic treatment.
  • a drug known as an antiepileptic agent
  • cobalt-induced epilepsy was reduced (FIGS. 5A and 5B).
  • the present inventors were able to suppress cobalt pre-induced interstitial spikes when administration of ethosuximide, a treatment for absence seizure, which is known to inhibit thalamic triggers (FIGS. 5A and 5B).
  • Ca V 3.1 which is an ⁇ 1G subunit of T-type calcium channel, is a major T-type Ca 2+ channel subunit that produces a trigger (multiple ignition pattern) in the parathalamic relay neuron, Ca V 3.1.
  • Lentiviruses with -specific shRNAs were introduced into the MD thalamus and confirmed that the viral infection significantly reduced Ca V 3.1 protein and T-type calcium current in the MD market (FIGS. 6A and 6B).
  • knockdown of Ca V 3.1 in the MD thalamus significantly reduced neurological and behavioral abnormalities in these model animals (see FIGS. 6C and 6D).
  • the number of frontal lobe-specific spikes after 6 days of insertion in these model animals at Ca V 3.1 knockdown decreased (FIGS. 6E and 6F) and reduced overaction (FIGS. 6G and 6H).
  • a1G subunit of the T-type calcium channel of the abnormal symptom sagittal nucleus such as hyperactivity and reduced memory due to prefrontal dysfunction due to irreversible damage
  • inhibitors of T-type calcium channel inhibitors or expression of ⁇ 1G subunits of T-type calcium channels eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding ⁇ 1G subunits
  • ⁇ 1G subunits of T-type calcium channels eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding ⁇ 1G subunits
  • composition for treating or improving frontal lobe dysfunction comprising a T-type calcium channel inhibitor or an inhibitor of expression of the ⁇ 1G subunit of the T-type calcium channel as an active ingredient.
  • the T-type calcium channel inhibitor is ethosuximide, mibefradil, tetramethrin, SUN-N8075, eponidipine, trivalent metal ions, Ni 2+ , U-92032 (7-[[4- [bis (4-fluorophenyl) methyl] -1-piperazinyl] methyl] -2-[(2-hydroxyethyl) amino] 4- (1-methylethyl) -2 , 4,6-cycloheptatrien-1-one, penfluridol, fluspirilene, valproate, zoninsamide, TTA-A2 (Kraus et al ., J Pharmacol.
  • TTA-P2 (Dreyfus et al ., J. Neurosci ., 30 (1): 99-109, 2010) or ⁇ 1G subtypes of T-type calcium channels. It may be an antibody or aptamer that specifically binds to the unit, wherein the trivalent metal ion is Y 3+ , La 3+ , Ce 3+ , Nd 3+ , Gd 3+ , Ho 3+ , Er 3 + Or Yb 3+ .
  • the inhibitor of the expression of the ⁇ 1G subunit of the T-type calcium channel is antisense nucleotide, siRNA (small interfering RNA), shRNA (short hairpin RNA) or specific for the gene encoding the ⁇ 1G subunit Micro RNA (miRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA Micro RNA
  • the frontal lobe dysfunction is traumatic brain damages (brain tumor), brain tumor (fetal hypoxia), neurodegenerative disease (neurodegenerative disease), schizophrenia, meningitis (menigitis) , Vitamin B12 deficiency, hydrocephalus, multiple sclerosis, central nervous system lupus (CNS lupus) may be caused.
  • the T-type calcium channel inhibitor or the inhibitor of expression of the ⁇ 1G subunit of the T-type calcium channel is increased vascular cells, ghost cells or vascular endothelial growth factor due to hypoxia damage Symptoms of frontal lobe dysfunction due to increased expression of (VEGF) can be alleviated.
  • the symptoms of the frontal lobe dysfunction include neurocongnitive deficit, delusion, speech or movement problem, mental handicap, personality change, broca aphasia Acathy, dysarthria, apraxia, agnosia, amensia, inattentiveness, impaired concentration, including Broca's aphasia.
  • the T-type calcium channel inhibitor or the inhibitor of expression of the ⁇ 1G subunit of the T-type calcium channel prevents the formation of T-type calcium currents involved in propagation of abnormal brain waves into the thalamus nucleus caused by frontal lobe injury. You can block.
  • the composition can ameliorate frontal lobe dysfunction without reversible recovery of the damaged frontal lobe.
  • the therapeutically effective amount of the T-type calcium channel inhibitor or the inhibitor of expression of the ⁇ 1G subunit of the T-type calcium channel may vary depending on several factors, such as the method of administration, the site of interest, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such considerations when determining the effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E. W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
  • compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents.
  • Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of a T-type calcium channel inhibitor or an expression inhibitor of the ⁇ 1G subunit of a T-type calcium channel, for example, Merck Index, 13 th ed., Merck & Co. Inc.
  • Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives can be added.
  • diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.
  • the composition of the present invention comprises 0.0001 to 10% by weight of the compound, preferably 0.001 to 1% by weight, based on the total weight of the composition.
  • composition may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, but is preferably parenterally administered, but is not limited thereto.
  • Formulations for parenteral administration include powders, granules, tablets, capsules, sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. It may be used in the form of a formulation, and preferably, an external skin pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasma agent may be prepared and used. It is not limited to this. Compositions of topical administration may be anhydrous or aqueous, depending on the clinical prescription.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, pills and the like.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and aerosols.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be.
  • Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient.
  • the daily dosage of the composition according to the present invention is 0.0001 mg to 300 mg, preferably 0.001 mg to 200 mg, and more preferably administered once to several times a day.
  • the screening step of determining whether any compound or natural product inhibits the activity or expression of T-type calcium channel Selecting a compound or natural product identified as inhibiting the activity or expression of the T-type calcium channel in the screening step; And it provides a screening method of a material for treating or improving frontal lobe dysfunction comprising a step of confirming whether or not the compound or natural product selected in the selection step improves frontal lobe dysfunction in animals.
  • the compound may be a synthetic compound or a purely purified compound from a natural product
  • the natural product may be a mineral, an organic material or an extract extracted from the mineral or organic material
  • the organic material may be a microorganism, a plant or Animals or their tissues, organs, organs or secretions.
  • the screening step may be performed by measuring a change in T-type calcium current after treatment of the compound or natural product to cells expressing T-calcium calcium channel, the T-type calcium current
  • the measurement of may be performed by methods well known in the art, such as patch clamps.
  • Mishra and Hermsmeyer confirmed the T-type calcium currents distinguished from the L-type calcium currents through patch clamp experiments on vascular muscle cells of rats. to identify the current is completely gone, mibe plastic dill the T-type bar that identify a selective inhibitor of calcium channel, and (. Mishra and Hermsmeyer, Circ Res , 75:.
  • the frontal lobe dysfunction is characterized by traumatic brain damages, brain tumors, fetal hypoxia, neurorodegenerative disease, schizophrenia, meningitis ), Vitamin B12 deficiency, hydrocephalus, multiple sclerosis, and central nervous system lupus (CNS lupus).
  • the T-type calcium channel inhibitor or the inhibitor of expression of the ⁇ 1G subunit of the T-type calcium channel is increased vascular cells, ghost cells or vascular endothelial growth due to hypoxia damage Symptoms of frontal lobe dysfunction due to increased expression of factor (VEGF) can be alleviated.
  • VEGF factor
  • the symptoms of the frontal lobe dysfunction include neurocongnitive deficit, delusion, speech or movement problem, mental handicap, personality change, broca aphasia Acathy, dysarthria, apraxia, agnosia, amensia, inattentiveness, impaired concentration, including Broca's aphasia.
  • the animal is a non-human animal, and may be a frontal lobe dysfunction model animal, and the frontal lobe dysfunction model animal is a frontal lobe model in which a cobalt wire is implanted into the frontal lobe of the brain. It may be an animal, and the model animal may belong to a primate tree, a rodent tree except a mouse, a rabbit tree, a carnivorous tree, a carnivorous tree, a base tree, or a woo tree.
  • the identifying step is to administer the compound or natural product selected in the selection step to the experimental animal; Measuring EEG, behavioral or cognitive impairment in the animal to which the compound or natural product has been administered; And it can be carried out by selecting a compound or natural product is reduced the number of spikes (eskele) of the brain waves or reduced behavioral or cognitive impairment compared to the control group not administered the compound or natural product.
  • Example 1 Preparation of frontal lobe dysfunction animal model
  • mice C57BL / 6J mice (Bio Model System Park), 10 to 20 weeks old, were bred and treated according to the regulations of the Experimental Animal Steering Committee of the Korea Advanced Institute of Science and Technology (KAIST). Mice remained freely accessible to water and feed, with a light and dark cycle of 12 hours.
  • KAIST Experimental Animal Steering Committee of the Korea Advanced Institute of Science and Technology
  • a cobalt gland reported to cause hypoxia was inserted into the right frontal lobe to produce a prefrontal dysfunction animal model.
  • the head of the mouse (B6 mouse between 10 and 20 weeks) is fixed to a stereotaxic device, and the cobalt wire (Alfa Aesar) is a stereosteric part of the holder. Inserted using.
  • the cobalt line was 0.5 mm thick and inserted into the right frontal lobe 2.6 mm forward from bregma (reference point of coordinates), 1.8 mm laterally from the central axis, and 1.3 mm rearward.
  • the EEG electrode was put together with the cobalt wire, it was closed using dental cement.
  • the suture was closed using a surgical thread.
  • the inventors inserted a wire made of a metal such as tungsten, copper, aluminum as a control in the same manner as in Example 1, to prepare a metal wire insertion mouse.
  • mice produced by inserting cobalt line in frontal lobe The brain was extracted from the mouse prepared by inserting cobalt line in frontal lobe. After 5 days of cobalt line insertion, neovascularization due to hypoxia was generated at the cobalt line inserted site Reflective hemorrhagic teeth appeared, which was not seen in mice with tungsten wire used as a control (FIG. 1A). Subsequently, T2-weighted magnetic resonance imaging (MRI) of these mice also revealed that the frontal cortex was more severely damaged in the cobalt-inserted mouse than in the tungsten-inserted mouse ( 1a).
  • MRI magnetic resonance imaging
  • the white dotted line in the arrow and magnetic resonance image in the brain extraction picture of Figure 1a represents the position where the cobalt line (right) or tungsten line (left) is inserted, F L is the left frontal lobe, F R is the right frontal lobe.
  • the extracted brain was fixed in 4% formalin-containing PBS (phosphate buffered saline) for 24 hours, and then paraffin blocks were prepared using a microtome and 5 ⁇ m. Thicknesses of flakes were prepared. After removing paraffin, matoxylin / eosin staining was performed, which was observed by light microscopy.
  • FIGS. 1B and 1C the portion shown by the dotted box on the leftmost image represents the enlarged portion on the right side, the arrow in the middle photo of the right panel represents red blood cells, and the arrow in the far right photo of the right panel represents a ghost cell. Indicates.
  • the present inventors observed the characteristic electroencephalogram of the frontal lobe dysfunction in order to check whether the animal model produced by inserting the cobalt line of Example 1 into the right frontal lobe can be used as a prefrontal dysfunction animal model.
  • EEG electrodes were inserted into the left, right frontal lobe, and left and right temporal lobes to measure the electroencephalogram (EEG) of the animal model, and the brain waves were observed for 30 days.
  • the EEG electrode was inserted simultaneously with the cobalt wire.
  • the coordinates were calculated and positioned using a stereotaxic device such as a cobalt line, and the EEG electrode was inserted.
  • EEG recording was performed at the same time as video recording after the recovery period of 3-4 days.
  • Homocysteine thiolactone was administered at a dose of 550 mg / kg at which no seizure occurred, resulting in interstitial and interstitial transmission of interstitial spikes from PFCs with intermittent secondary generalization. (FIG. 2B). No interstitial spikes were observed in mice without the cobalt line inserted at this dose.
  • prefrontal epilepsy in the cobalt line model appears to be active and neural circuit-dependent, but not by time dependent diffusion of cobalt ions from the PFC into the MD thalamus or other brain regions.
  • the present inventors attempted to confirm the characteristic behavior of prefrontal dysfunction in order to check whether the animal model prepared by inserting the cobalt line of Example 1 into the right frontal lobe can be used as a prefrontal dysfunction animal model.
  • the mouse is carefully placed in an open field test kit (a box of acrylic square bottom, 40 ⁇ 40 ⁇ 50 cm), and the distance traveled in the kit for 1 hour and 1/2 cycle is determined by digital video recording. Monitoring at minute intervals The open field test was performed between 18 and 22 hours and EthoVision (Noduls, USA) was used to analyze the video images.
  • the present inventors observed that a decrease in cognitive function related to frontal lobe is observed in the animal model of the present invention.
  • a fear-learning memory test was performed to confirm.
  • the mouse In order to perform a fear conditioning test, the mouse repeatedly gave an electrical stimulus with a specific sound on the first day, causing the mouse to remember fear. On the second day, two tests were carried out. The first confirmed the context of the space where the horror memory was formed by the electrical stimulation on the first day, and the second the memory of the sound in which the horror memory was formed by the electrical stimulation. ) Was tested.
  • the freezing time of the mouse was measured to confirm memory. Since the mouse does not show movement when it is afraid, the freezing time will be long if the mouse is remembering, otherwise the freezing time will be reduced. This time was measured to examine the cognitive function of the cobalt wired mouse.
  • the present inventors measured the electroencephalogram after intraperitoneally administering a drug known as an antiepileptic agent to the cobalt gland inserted animal, in order to confirm whether the epileptic spikes appearing in the cobalt gland inserted animal is reduced by administration of the antiepileptic agent.
  • zonisamide zonisamide
  • phenytoin phenytoin
  • the electroencephalogram for 2 hours.
  • cobalt-induced epilepsy was reduced when the epilepsy treatment was administered systemically (FIGS. 5A and 5B).
  • the present inventors were able to suppress cobalt pre-induced interstitial spikes when dose of 150 mg / kg of ethosuximide, a treatment for absence seizure, known to inhibit thalamic triggers ( 5a and 5b).
  • the present inventors considered that the major T- type Ca 2+ channel subunits that Ca V 3.1 supports the firing pin (multiple ignition) in the thalamus cortical relay neurons, introducing a lentivirus having a specific shRNA Ca V 3.1- to MD thalamus It was.
  • a synthetic oligonucleotide SEQ ID NO: 1, 5'-CGGAATTCCGG GAAGATCGTAGATAGCAAA ttcaagaga TTTGCTATCTACGATCTTC TTTTTGATATCTAGACA-3 '
  • was inserted into the sh Lentisyn3.4G lentiviral vector (Macrogen LentiVector Institute, Korea).
  • the sh Lentisyn3.4G lentiviral vector is designed to express shRNA from the U6 promoter and to express the improved green fluorescent protein from the synapsin promoter.
  • the target sequence is a sequence that does not overlap any other mRNA on the database of the National Bioinformatics Center except Ca V 3.1, and a scrambled version of the Ca V 3.1 shRNA oligonucleotide (SEQ ID NO: 2: 5'-CGGAATTCCGG GTAAGTGAACTGACAAGAA ttcaagaga TTCTTGTCAGTTCACTTAC TTTTTGATATCTAGACA-3 ' ) was inserted into the shLentisyn3.4G vector and used as a control.
  • the recombinant lentiviral vector was produced and then concentrated commercially (Macrogen LintiVector Institute). Lentiviruses of 2 ⁇ 10 6 transduction units / ml were used and these viruses were injected onto the ipsilateral MD thalamus using Nanofil 33G smoothing needle, Nanofil syringe (World Precision Instrument) and micro syringe pump (Eicom), respectively. Seven days after virus infection, epidural electrodes were implanted for electroencephalography, and electroencephalography was recorded for 30 days with video monitoring. As a result, it was confirmed that the viral infection significantly reduced Ca V 3.1 protein and T-type calcium current in MD thalamus (FIGS. 6A and 6B).
  • FIGS. 6C and 6D show that knockdown of Ca V 3.1 in the MD thalamus significantly reduced neurological and behavioral abnormalities in these model animals (see FIGS. 6C and 6D).
  • FIG. 6C the gray dotted line indicates the range of theta frequency.
  • FIG. 6E the left panel is an EEG representing the interstitial spike after 6 days of knockdown before the knockdown
  • FIG. 6F is a graph showing the number of spikes measured.
  • FIG. 6G is a copper wire recording diagram showing the movement path of the first 5 minutes and the last 5 minutes during the 60-minute exercise test after Ca V 3.1 knockdown
  • FIG. 6H is a graph showing the total travel distance in this case.
  • Example 7 Based on the results of Experimental Example 7, the inventors inserted a cobalt line into the frontal lobe of a Ca V 3.1 -/- knockout mouse by the method of Example 1, thereby inserting a Ca V 3.1 -/- knockout cobalt line insertion mouse. Prepared.
  • T-type calcium channels of the symptoms sisanghaek such as hyperactivity and memory loss due to more than the frontal function by irreversible damage a1G (Ca V 3.1) subunit
  • the role is very important and it has been demonstrated that the abnormal symptoms can be treated or ameliorated by blocking their function or expression.
  • inhibitors of T-type calcium channel inhibitors or expression of ⁇ 1G subunits of T-type calcium channels eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding ⁇ 1G subunits
  • ⁇ 1G subunits of T-type calcium channels eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding ⁇ 1G subunits
  • compositions of the present invention are given below.
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
  • the frontal lobe dysfunction model animal produced using the cobalt line according to the present invention exhibits hyperactivity and prefrontal hyper-excitability of the frontal lobe.
  • These model animals can be used as model animals for the screening of therapeutic agents for prefrontal dysfunction, and such prefrontal dysfunction symptoms are reduced by the administration of T-type calcium channel inhibitors, which is reversible recovery of prefrontal injury. Rather, because it is achieved through the prevention of cross-frontal-thalamic crosstalk occurring after frontal lobe injury, T-type calcium channel inhibitors may be usefully used as a composition for alleviating frontal lobe dysfunction.
  • SEQ ID NO: 1 is the DNA sequence corresponding to a Ca V 3.1 specific shRNA used for Ca V 3.1 gene knockout.
  • SEQ ID NO: 2 is a scrambled DNA sequence that randomly changes a portion of the sequence of Ca V 3.1 specific shRNA used as a control.

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Abstract

L'invention concerne un modèle animal de dysfonctionnement du lobe frontal induit par une lésion cérébrale, et l'utilisation de ce modèle. Plus spécifiquement, l'invention concerne un modèle animal de dysfonctionnement du lobe frontal, dans lequel un fil de cobalt a été implanté dans le lobe frontal du cerveau; et une composition destinée à traiter ou à atténuer un dysfonctionnement du lobe frontal, ladite composition comprenant un principe actif sous la forme d'un suppresseur des canaux calcium de type T, impliqués dans la propagation d'ondes cérébrales anormales dans les noyaux thalamiques suite à une lésion du lobe frontal.
PCT/KR2011/006847 2011-09-16 2011-09-16 Modèle animal de dysfonctionnement du lobe frontal induit par une lésion cérébrale, et composition pour atténuer un dysfonctionnement du lobe frontal WO2013039271A1 (fr)

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WO2023022504A1 (fr) * 2021-08-17 2023-02-23 한국과학기술원 Oligonucléotide antisens ciblant le gène cav3.1 et ses utilisations

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KR20060026315A (ko) * 2004-09-20 2006-03-23 한국과학기술연구원 Ｔ-타입 칼슘채널 길항제로 유효한 신규 피페라지닐알킬이소옥사졸 유도체
WO2009093774A1 (fr) * 2008-01-23 2009-07-30 Korea Institute Of Science And Technology Procédé de traitement d'un trouble anxieux par régulation du canal calcique de type t
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KR20060026315A (ko) * 2004-09-20 2006-03-23 한국과학기술연구원 Ｔ-타입 칼슘채널 길항제로 유효한 신규 피페라지닐알킬이소옥사졸 유도체
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WO2023022504A1 (fr) * 2021-08-17 2023-02-23 한국과학기술원 Oligonucléotide antisens ciblant le gène cav3.1 et ses utilisations

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