WO2013039271A1 - Brain-damage-induced frontal lobe dysfunction model animal and composition for alleviating frontal lobe dysfunction - Google Patents

Abstract

The present invention relates to a brain-damage-induced frontal lobe dysfunction model animal and to a use for same. More specifically, the present invention relates to: a frontal lobe dysfunction model animal in which a cobalt wire has been implanted into the frontal lobe of the brain; and to a composition for treating or alleviating frontal lobe dysfunction, the composition comprising an active ingredient in the form of a suppressor of T-type calcium channels involved in the propagation of abnormal brain-waves into thalamic nuclei following frontal-lobe damage.

Description

Frontal Lobe Dysfunction Model due to Brain Injury

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.

The key neurological corals of the frontal lobe dysfunction in neurological and mental disorders are abnormal oscillations or excitations (McAllister and Price, Compr . Psychiatry , 28: 14-21, 1987; Barry et al ., Clin Neurophysiol, 114: 171-183, 2003 ; Wienbruch et al, Clin Neurophysiol, 114:.. 2052-2060, 2003; Tan and Appleton, Arch Dis Child, 90:...... 57-59 2005; Beleza et al., Epilepsia , 50: 550-555, 2009). For example, 1-7 Hz EEG appears in schizophrenia (Elbert et al., Biol. Psychiatry, 32: 595-606, 1992) and is enhanced in Attention Deficit and Hyperactivity Disorder (ADHD). Theta rhythm (Barry et al., Clin. Neurophysiol ., 114: 171-183, 2003), and frontal lobe epilepsy (FLE), a frontal lobe epilepsy (FLE), which appears in childhood Develop ADHD-like symptoms (Hernandez et al ., Adv. Behav. Biol ., 50: 103-111, 2001; Gonzalez-Heydrich et al ., Epilepsy Behav ., 10: 384-388, 2007) Resulting in schizophrenic-like symptoms (Adachi et al ., Seizure , 9: 328-335., 2000; Helmstaedter, Epilepsy Behav . 2: 384-395, 2001; Patrikelis et al ., Epilepsy Behav ., 14: 19 -26, 2009).

The most common causes of such frontal lobe dysfunction are hypoxic damage in the frontal cortex induced by various conditions including traumatic brain damages, brain tumors and fetal hypoxia. Representative causes have been suggested (Damasio and Anderson, Clin. Neuropsychol ., 339-375, 1985; Owen et al ., Neuropsychologia , 28: 1021-1034, 1990, Owen et al ., Brain , 116: 1159-1175, 1993; Shallice and Burgess, Brain , 114: 727-741, 1991; Stuss and Gow, Cogn.Behav . Neurol ., 5: 272-282, 1992; Pigula et al ., J. Pediatr. Surg ., 28: 310 -316, 1993; H.ckel and Vaupel, J. Natl. Cancer Inst . 93: 266-276, 2001). In addition, 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. , 84: 349-353, 1991), meningitis (Mendez and Zander, Psychosomatics , 33 (2): 216-217, 1992), vitamin B12 deficiency (Blundo et al ., Neurol. Sci ., 32 (1)). : 101-105, 2011), hydrocephalus, Saito et al., Dement.Geriatr. Cogn. Disord.Extra , 1: 202-211, 2011), multiple sclerosis, Foong et al., Brain , 120 : 15-26, 1997), central nervous system lupus (CNS lupus, Paran et al., Ann. Rheum. Dis ., 68: 812-816, 2009) have also been reported as causes of frontal lobe dysfunction.

Since 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.

Currently, as a treatment for hyperactivity disorder, a kind of frontal lobe dysfunction, central nervous stimulants such as ritalin and methylphen, which are methylphenidate, have been used. It is known to have a short-term effect on mitigation of hyperactivity and impulsivity, but the long-term effects have not been verified (Kim et al ., Korean Neuropsychiatr. Assoc ., 4: 683-699, 1998).

In addition, even if a diarrhea treatment is developed, it is difficult to verify the treatment effect due to the absence of an appropriate animal model system.

In this regard, 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).

However, 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. In addition to the disadvantages of excessive cost, the development of successful prefrontal dysfunction improvers using the same has not been reported.

Therefore, there is a demand for development of a prefrontal dysfunction model animal that can represent various levels of prefrontal dysfunction and can be manufactured at low cost.

It is an object of the present invention to provide a frontal lobe dysfunction model animal in order to solve various problems including the above problem.

Another object of the present invention is to provide a composition for improving frontal lobe dysfunction developed using the frontal lobe dysfunction model animal.

According to one aspect of the invention there is provided a frontal lobe dysfunction model animal, except for a human implanted with a cobalt wire in the frontal lobe of the brain.

According to another aspect of the present invention, there is provided a composition for treating or improving frontal lobe dysfunction comprising a T-type calcium channel inhibitor or an inhibitor of expression of the α1G subunit of a T-type calcium channel as an active ingredient.

In addition, according to another aspect of the invention, the screening step of determining whether any compound or natural product inhibits T-type calcium channel activity; Selecting a compound or natural product identified as inhibiting the activity 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 the animal.

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. It can be usefully used as a medicine for improvement of In particular, 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.

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,

Co: cobalt,

F ^L : left prefrontal cortex,

F ^R : right prefrontal cortex,

c: control, and

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 ^R : right prefrontal cortex,

F ^L : left prefrontal cortex,

MD: medial dorsal thalamus (mediodorsal thalamus),

T ^R : right temporal cortex,

T ^L : left temporal cortex,

W: tungsten, and

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):

Control: control,

Co: cobalt,

Co ≤ 6d: within 6 days of cobalt wire insertion,

Co ≤ 14d: within 14 days after cobalt line insertion, and

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.

Cue: Memory of the sound in which horror memory is 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, Here is a graph (b) that records the change (a) and the number of interstitial spikes:

Veh: only excipients,

ZNS: Johnny'samide,

PHT: phenytoin, and

Etho: Ethosuccimid.

FIG. 6 is a photograph showing changes in T-type currents in lentiviral vectors with CaV3.1 target shRNA, α1G 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. Dimensional graph (c), graph showing changes in theta frequecy in the frontal cortex (PFC), electroencephalogram (e) showing changes in brain waves in the frontal lobe and MD thalamus 6 days after cobalt line implantation, Graph showing spike number change after 6 days of cobalt line implantation (f), and copper transcript (g) showing movement pattern for the first 5 minutes and last 5 minutes after the start of the exercise test 6 days after cobalt line implantation And a graph (h) showing the distance to move for the first 5 minutes and the last 5 minutes after the start of the exercise test 6 days after the cobalt line implantation:

sh C-MD or sh C: control shRNA infection,

sh 3.1-DM or sh 3.1: Ca _V 3.1 shRNA infection,

θ: theta frequency

MD: medial dorsal thalamus,

PFC: prefrontal cortex, and

F: Frontal lobe.

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.

Hereinafter, the present invention will be described in detail.

According to one aspect of the invention there is provided 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.

In the model animal 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. At this time, the neurodegenerative disease may be Alzheimer's disease or anterior temporal lobe dementia.

In the model animal, 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. In the preparation of the prefrontal dysfunction model animal, 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).

To measure cobalt-induced damage at the molecular level, we identified the expression level of vascular endothelial growth factor (VEGF), which plays a key role in angiogenesis in response to hypoxia. As a result, a significant increase in VEGF protein was observed after 5 days of cobalt wire insertion compared to tungsten wire inserted mice (see FIG. 1D). These results suggest that the insertion of cobalt gland causes hypoxia-like damage in the frontal cortex.

Based on the above results, 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. Specifically, we analyzed the electroencephalogram (EEG) of mice in which cobalt lines were inserted into the right frontal lobe. As a result of observing EEG through the EEG electrodes inserted into the left, right frontal and temporal lobes of the animal model mouse, a single spike activity was observed in the right frontal lobe between 5 and 6 days after cobalt line insertion (see FIG. 2A). ). However, other 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). On the other hand, when the same dose of HT was administered to mice without cobalt grafts, no epileptic spikes were seen.

We observed behavioral and cognitive changes in mice inserted with cobalt lines. After the cobalt line was inserted, 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). In addition, 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. 3B and 3E). The homology has been reported to be representatively observed in prefrontal dysfunction animals (Jones, Compr. Psychiatry , 6 (5): 323-335, 1965; Davids et al ., Brain Res. Rev. 42: 1-21, 2003 ).

In addition, 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. As a result, when systemic administration of an antiepileptic agent including zonisamide and phenytoin was found, cobalt-induced epilepsy was reduced (FIGS. 5A and 5B). In addition, 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).

The propagation of interstitial spikes into the MD thalamus and the inhibition of epilepsy by ethosuccimides suggest that the thalamus may be involved in the development of frontal lobe epilepsy. To confirm this, we examined cobalt pre-induced frontal lobe epilepsy in mice that caused right injuries in MD thalamus, and the occurrence of anterior spikes in these mice was alleviated. However, the right MD market impairment had no effect on cortical epilepsy when administered bicuculline methobromide (BMB), which induces cortical epilepsy as a GABA _A antagonist with thalamic-independent mechanisms (see FIGS. 5A and 5B). These results suggest that cobalt pre-induced frontal lobe epilepsy requires the role of thalamic neurons.

Accordingly, the present inventors consider that Ca _V 3.1, which is an α1G 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). In addition, 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).

This therapeutic effect on prefrontal epileptic symptoms of knockdown of MD thalamic specific Ca _V 3.1 gene was also observed in Ca _V 3.1 ^{− / −} knockout mice known to eliminate thalamic trigger spikes (FIG. 7).

As described above, the inventors have a very important role for the a1G (Ca _V 3.1) 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, By blocking its function or expression it has been demonstrated that the abnormal symptoms can be treated or ameliorated. Thus, inhibitors of T-type calcium channel inhibitors or expression of α1G subunits of T-type calcium channels (eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding α1G subunits) are irreversible to brain damage. It can be usefully used to treat or ameliorate prefrontal dysfunction.

Therefore, according to another aspect of the present invention, there is provided a composition for treating or improving frontal lobe dysfunction comprising a T-type calcium channel inhibitor or an inhibitor of expression of the α1G subunit of the T-type calcium channel as an active ingredient.

In the composition, 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. , 335 (2): 409-417, 2010), TTA-P2 (Dreyfus et al ., J. Neurosci ., 30 (1): 99-109, 2010) or α1G 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⁺ .

In the composition, the inhibitor of the expression of the α1G 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 α1G subunit Micro RNA (miRNA).

In the composition, 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.

In the composition, the T-type calcium channel inhibitor or the inhibitor of expression of the α1G 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.

At this time, 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.

In the composition, the T-type calcium channel inhibitor or the inhibitor of expression of the α1G 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 α1G 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.

The 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 α1G 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. In addition, 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. Furthermore, 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).

The 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.

The 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. As the 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. As the 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.

In addition, according to another aspect of the invention, 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.

In the screening method, 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.

In the screening method, 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. In this regard, 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:. 144-148, 1994), Furukawa et T- Patch clamps on Xenopus eggs and BHK cells transduced to express type calcium channels have revealed that the R (-) isomer of eponidipine is a selective inhibitor of T-type calcium channels (Furukawa et. al ., Br. J. Pharmacol ., 143: 1050-1057, 2004).

In the screening method, 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).

In the screening method, the T-type calcium channel inhibitor or the inhibitor of expression of the α1G 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.

At this time, 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.

In the screening method, 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.

In the screening method, 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.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

Example 1: Preparation of frontal lobe dysfunction animal model

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.

A cobalt gland reported to cause hypoxia was inserted into the right frontal lobe to produce a prefrontal dysfunction animal model.

In frontal lobe dysfunction mouse production, 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. When the EEG electrode was put together with the cobalt wire, it was closed using dental cement. In the case of surgery for behavioral experiments without inserting the EEG electrode, the suture was closed using a surgical thread.

Comparative Example: Fabrication of Other Metal Wire Insert Animals

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.

Experimental Example 1: Analysis of frontal lobe damage in animal models of prefrontal dysfunction

Mouse 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). 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. In order to histologically analyze the cell death in the damaged area identified by the MR image, 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 μm. Thicknesses of flakes were prepared. After removing paraffin, matoxylin / eosin staining was performed, which was observed by light microscopy. As a result, an increase in ghost cells, an obvious signal of hypoxic damage, and inflammatory neovascularization visualized as red blood cells-filled blood vessels were observed at the cobalt gland insertion site (see FIGS. 1B and 1C). In FIG. 1B, 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.

Experimental Example 2: Molecular level analysis of frontal lobe damage in prefrontal dysfunction animal model

To measure cobalt-induced damage at the molecular level, we performed immunoblotting as follows. First, brains isolated on ice were sectioned to a thickness of 1 mm using a brain matrix (RBMA-200C, WPI), and then sonicated in a homogenization buffer containing a protease inhibitor. 30-50 μg of protein was loaded on 10% polyacrylamide Tris-HCl gel, followed by electrophoresis, transfer to nitrocellulose membrane (Protran, Whatman), and then anti-β-actin antibody (Santa Cruz Biotechnology) as a control. In addition, immunoblotting was performed using an anti-VEGFa antibody (Santa Cruz Biotechnology) as an experimental group. As a result, a significant increase in VEGF protein was observed after 5 days of cobalt insertion compared with tungsten insertion mice (see FIG. 1D). These results suggest that the insertion of cobalt gland causes hypoxia-like damage in the frontal cortex.

Experimental Example 3 EEG Observation of Prefrontal Dysfunction

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. After inserting the cobalton wire and the EEG electrode, EEG recording was performed at the same time as video recording after the recovery period of 3-4 days.

After inserting the cobalt wire and the EEG electrode, 10 C57BL / 6J mice that sent a four-day recovery period were allowed to roam freely in the EEG chamber (EEG chamber, box on the bottom of the square), and the EEG signal was sent to the Grass model 7H polygraph. After amplification using (Grass Technologies, USA), it was digitized at a sampling rate of 500 Hz through DIGIDATA 1320A (Molecular Devices, USA). The data was then obtained using pClamp9.2 software (Molecular Devices, USA).

As a result of observing EEG through the EEG electrodes inserted into the left, right frontal and temporal lobes of the animal model mouse, a single spike activity was observed in the right frontal lobe between 5 and 6 days after cobalt line insertion (see FIG. 2A). ). However, other wires made in the comparative examples, i.e. wires made of metals such as tungsten, 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).

Homocysteine thiolactone (HT) 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. Thus, the development of 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.

Experimental Example 4: Measurement of behavioral change and cognitive change in prefrontal dysfunction animal model

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.

Control mice without cobalt lines divided into 6 days (n = 5), 14 days (n = 5), and 28 days (n = 9), depending on the time when the cobalt line was inserted (n = 9) And behavior. The mouse is carefully placed in an open field test kit (a box of acrylic square bottom, 40 ㅧ 40 ㅧ 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.

We observed behavioral and cognitive changes in mice inserted with cobalt lines. After the cobalt line was inserted, 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. Significant increase was observed (FIGS. 3A and 3C). In addition, their movement was limited to the wall of the open field, the search toward the center was found to be limited compared to the control (Fig. 3b and 3d). This tendency increased as the duration of the cobalt line insertion increased, and more specifically, the activity was analyzed by drawing the traces of the frontal lobe dysfunction animal model. It could be confirmed that the behavior of turning (stereotypy) is observed (Fig. 3b and 3e). It has been reported that homology is typically observed in attention deficit hyperactivity disorder or schizophrenia (Jones, Compr. Psychiatry , 6 (5): 323-335, 1965; Davids et al ., Brain Res. Rev. 42: 1 -21, 2003).

Experimental Example 5 Measurement of Changes in Cognitive Function in Frontal Lobe Animal Model

In order to confirm whether the animal model produced by inserting the cobalt line of Example 1 into the right frontal lobe can be used as a frontal lobe damaged animal model, 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.

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.

As a result, as shown in Figure 4, it was observed that the cognitive function is reduced in the animal model in which the cobalt line is inserted. That is, in the case of the cobalt wired mouse, the freezing time is significantly lowered in both the context and the cue test, which indicates that the cobalt wired mouse has the pain of electric stimulation. Remind them that they are not remembering properly.

Experimental Example 6 Observation of EEG Changes in Prefrontal Dysfunction Animal Models by Various Compounds

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. Specifically, zonisamide (zonisamide) was 60 mg / kg, phenytoin (phenytoin) was administered at a dose of 100 mg / kg and measured the electroencephalogram for 2 hours. As a result, it was found that cobalt-induced epilepsy was reduced when the epilepsy treatment was administered systemically (FIGS. 5A and 5B). In addition, 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).

This suggests that the propagation of interstitial spikes into the MD thalamus and the inhibition of epilepsy by ethosuccimides may be involved in the development of frontal lobe epilepsy. To confirm this, we investigated cobalt pre-induced frontal lobe epilepsy in mice that caused right injuries in the MD market, and the occurrence of anterior spikes in these mice was alleviated. However, the right MD market impairment had no effect on cortical epilepsy when administered bicuculline methobromide (BMB), which induces cortical epilepsy as a GABA _A antagonist with thalamic-independent mechanisms (see FIGS. 5A and 5B). These results suggest that cobalt pre-induced frontal lobe epilepsy requires the role of thalamic neurons.

Experimental Example 7: CaV3.1 Knockout

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. Specifically, 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). In addition, 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). In FIG. 6C, the gray dotted line indicates the range of theta frequency. 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). In FIG. 6E, the left panel is an EEG representing the interstitial spike after 6 days of knockdown before the knockdown, and 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, and FIG. 6H is a graph showing the total travel distance in this case.

Experimental Example 8: Experiment with CaV3.1 Knockout Mouse

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.

As a result, MD thalamus-specific Ca _V therapeutic effect on the frontal lobe epilepsy symptoms knockdown of 3.1 genes known to remove the trigger spikes awards Ca _V 3.1 ^{- / -} knockout mice (Kim et al, Neuron, 31 . : 35-45, 2001) (FIG. 7).

As shown in Experimental Examples 7 and 8, the inventors have found that the ratio of 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. Thus, inhibitors of T-type calcium channel inhibitors or expression of α1G subunits of T-type calcium channels (eg, antisense nucleotides, siRNAs or shRNAs specific for genes encoding α1G subunits) are irreversible to brain damage. It can be usefully used to treat or ameliorate prefrontal dysfunction.

The preparation examples for the compositions of the present invention are given below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

1-1: Preparation of Powder

2 g of ethosuccimid

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

1-2: Preparation of Tablets

Etosuccimid 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3: Preparation of Capsule

Etosuccimid 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4: Preparation of Ring

1 g of ethosuccimid

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

1-5: Preparation of Granules

Etosuccimid 150 mg

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

As described above, 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.

Claims (21)

Prefrontal dysfunction model animals, except humans, who have a cobalt wire implanted in the frontal lobe of the brain. The method of claim 1, The model animal belongs to the tree, rodent, rabbit, carnivorous, carnivorous, base or lumberjack, Frontal lobe dysfunction model animals. The method of claim 1, In the model animal the cobalt gland is implanted in the prefrontal cortex of the frontal lobe of the brain, Frontal lobe dysfunction model animals. A composition for treating or improving frontal lobe dysfunction comprising a T-type calcium channel inhibitor or an inhibitor of expression of the α1G subunit of a T-type calcium channel as an active ingredient. The method of claim 4, wherein 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- α1G of cycloheptatrien-1-one, penfluridol, fluspirilene, valproate, valproate, zoninsamide, TTA-A2, TTA-P2 or T-type calcium channels The composition is an antibody or aptamer that specifically binds to a subunit. The method of claim 5, The trivalent metal ion is Y ³⁺ , La ³⁺ , Ce ³⁺ , Nd ³⁺ , Gd ³⁺ , Ho ³⁺ , Er ³⁺ or Yb ³⁺ . The method of claim 4, wherein The inhibitor of expression of the α1G subunit of the T-type calcium channel is an antisense nucleotide, siRNA (small interfering RNA), shRNA (short hairpin RNA) or micro RNA (miRNA) specific for the gene encoding the α1G subunit. Phosphorus composition. The method of claim 4, wherein The frontal lobe dysfunction includes traumatic brain damages, brain tumors, fetal hypoxia, neurodegenerative disease, schizophrenia, meningitis, meningitis, vitamin B12 deficiency, Hydrocephalus, multiple sclerosis, caused by the central nervous system (CNS lupus), the composition. The method of claim 4, wherein The T-type calcium channel inhibitor or the inhibitor of expression of the α1G subunit of the T-type calcium channel may be an increase in vascular cells, an increase in ghost cells, or an expression of vascular endothelial growth factor (VEGF) due to hypoxia injury. A composition for alleviating symptoms of prefrontal dysfunction caused by an increase. The method of claim 4, wherein Wherein said T-type calcium channel inhibitor or inhibitor of expression of the α1G subunit of said T-type calcium channel blocks the formation of T-type calcium currents involved in propagation of aberrant brain waves into the thalamus nucleus caused by frontal lobe injury. The method of claim 4, wherein Wherein said T-type calcium channel inhibitor or inhibitor of expression of the α1G subunit of said T-type calcium channel is capable of ameliorating prefrontal dysfunction without reversible recovery of an impaired frontal lobe. Screening to determine whether any compound or natural product inhibits the activity or expression of T-type calcium channels; Selecting a compound or natural product identified as inhibiting the activity or expression of the T-type calcium channel in the screening step; And The screening method of the material for improving frontal lobe dysfunction comprising the step of identifying whether the compound or natural product selected in the selection step improves frontal lobe dysfunction in the animal. The method of claim 12, The compound is a screening method is a compound or a purely purified compound from a natural compound. The method of claim 12, The natural product is a screening method of minerals, organics or extracts extracted from the minerals or organics. The method of claim 14, Wherein said organic material is a microorganism, plant or animal or a tissue, organ, organ or secretion thereof. The method of claim 12, The screening method is performed by measuring the change in T-type calcium current after treatment of the compound or natural product to cells expressing T-calcium calcium channel. The method of claim 16, The measurement of the T-type calcium current is carried out using a patch clamp. The method of claim 12, The frontal lobe dysfunction includes traumatic brain damages, brain tumors, fetal hypoxia, neurodegenerative disease, schizophrenia, meningitis, meningitis, vitamin B12 deficiency, Screening method, which is caused by hydrocephalus, multiple sclerosis, central nervous system lupus (CNS lupus). The method of claim 12, Wherein said animal is a non-human prefrontal dysfunction model animal. The method of claim 19, The non-human prefrontal dysfunction model animal is a model animal of claim 1, the screening method. The method of claim 12, The identifying step comprises administering the compound or natural product selected in the selecting 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 screening the compound or natural product with reduced spike number of brain waves or reduced behavioral or cognitive impairment as compared to the control group that is not administered the compound or natural product.

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