WO2009093774A1

WO2009093774A1 - Method for treatment of anxiety disorder by regulating t-type calcium channel

Info

Publication number: WO2009093774A1
Application number: PCT/KR2008/001983
Authority: WO
Inventors: Hee-Sup Shin; Sukchan Lee; Huisu Kim
Original assignee: Korea Institute Of Science And Technology
Priority date: 2008-01-23
Filing date: 2008-04-08
Publication date: 2009-07-30
Also published as: KR20090081307A; KR100958291B1

Abstract

The present invention relates to a composition for the prevention and treatment of anxiety disorder containing a T-type calcium channel blocker, and a method for the prevention and treatment of anxiety disorder by administering the said composition into mediodorsal thalamus (MD) of a subject with anxiety disorder. When the T-type calcium channel blocker mibefradil is administered into MD of the phospholipaseβ4 (PLCβ4, expressed mainly in MD) knock-out mouse, fear memory extinction which has been reduced is restored to normal level. When mibefradil and efonidipine are administered into MD of the wild type mouse, fear memory is lost faster than usual and fear memory recall is retarded. Therefore, a T-type calcium channel blocker can be effectively used for the prevention and treatment of anxiety disorder.

Description

[DESCRIPTION]

[invention Title]

METHOD FOR TREATMENT OF ANXIETY DISORDER BY REGULATING T-TYPE CALCIUM CHANNEL

[Technical Field]

The present invention relates to a composition for the prevention and treatment of anxiety disorder containing a T-type calcium channel blocker, and a method for the prevention and treatment of anxiety disorder by administering the said composition into mediodorsal thalamus (MD) .

[Background Art] Calcium channel plays an important role in excitation-contraction coupling mechanism shown in muscle cells of skeleton, blood vessel and heart and in regulating the secretion of neurotransmitters in the central or peripheral nervous system. The calcium channel is classified by physiological and molecular biological characteristics into high-voltage dependent L-type calcium channel and low-voltage dependent T-type calcium channel. L-type calcium channel plays an important role in delivering calcium ions through cell membranes of vascular muscle, skeletal muscle and smooth muscle, and further is involved in the regulation of muscle contraction and in the secretion of neurotransmitters in endocrine glands and nervous tissues. T-type calcium channel is dominant in central muscle cells, endocrine gland of adrenal and heart cells such as sinoauricular node, which plays an important role in heart beating along with L-type calcium channel and is also involved in calcium inflow into muscle cells based on the small potential difference. Thus, it is expected to regulate the fibroblast proliferation, blood pressure, and abnormal secretion of neurotransmitters by using an antagonist of the calcium channel. And in fact, a calcium channel regulator has been a major target of studies to develop a drug for the treatment of heart disease and various psychomotor disorders.

Anxiety disorder includes phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder. Phobic disorder is the state of feeling fear for a specific object or under specific circumstance and therefore avoiding constantly the object or the circumstance. Social phobia, acrophobia, claustrophobia and expectation anxiety are all included in phobic disorder. Generalized anxiety disorder is the state of constant anxiety in every day life widely and persistently. Obsessive compulsive disorder is a problem of repeating specific behavior or thought regardless of one's will. Compulsive idea or anankastic behavior might be normal to some degree, but when the compulsive idea or anankastic behavior disturbs every day life or annoys mind and body, it is diagnosed as obsessive compulsive disorder. In this case, anankastic personality such as accuracy, perfectionism, and principlism is shown as well. Post-traumatic stress disorder is a problem or illness that someone thinks or dreams or re-experiences a horrible accident that he or she had been through which is something that every common people does not experience generally such as war, airplane crash, rape, collapse (Sampoong department collapse) and thereby the person becomes insensible to the outside world and exhibits abnormal symptoms of autonomic nervous system. Somatoform disorder exhibits physical symptoms which might be the signs of a disease, but in reality, most of the physical symptoms at this time have nothing to do with a disease but caused by a mental conflict. Sometimes, a mental conflict or a psychological factor can cause real physical symptoms or diseases, which are called psycho-physiological disorders. In most cases, when a mental conflict is expressed via physical symptoms, it can be done unconsciously. Therefore, it is quite different from malingering, the physical symptoms pretended by a patient deliberately. It is rarely expressed as dissociative disorder, which is the state of losing a part of mental functions temporarily like in ^ΛDr. Jekyll and Mr. Hyde'.

Mediodorsal thalamus (MD) is a part of limbic system, which is linked to prefrontal cortex involved in emotion, amygdala and hippocampus one another. However, the mechanism of MD in relation to emotional act has not been disclosed, yet. Phospholipase β4 (PLCβ4) is largely expressed in MD, but hardly expressed in prefrontal cortex or amygdala playing an important role in fear extinction. The present inventors generated PLCβ4 knock-out mice and confirmed that the PLCβ4 knock-out mice exhibited serious defect of memory extinction, so that they could be used as an anxiety disorder animal model.

The present inventors completed this invention by confirming that when mibefradii, the most representative T- type calcium channel blocker, was administered to MD of PLCβ4 knock-out mouse, memory extinction which had been deficient in the mouse was recovered and when mibefradil and efonidipine were co-administered to MD of a wild-type mouse, fear memory was lost fast and the recall of the extinct memory was retarded. [Disclosure] [Technical Problem]

It is an object of the present invention to provide a composition for the prevention and treatment of anxiety disorder containing a T-type calcium channel blocker and a method for the prevention and treatment of anxiety disorder using the same.

[Technical Solution]

To achieve the above object, the present invention provides a composition for the prevention and treatment of anxiety disorder containing a T-type calcium channel blocker. The present invention also provides a method for the treatment of anxiety disorder containing the step of administering a pharmaceutically effective dose of a T-type calcium channel blocker to a subject with anxiety disorder.

The present invention further provides a method for the prevention of anxiety disorder containing the step of administering a pharmaceutically effective dose of a T-type calcium channel blocker to a subject with anxiety disorder.

In addition, the present invention provides a use of a T-type calcium channel blocker for the development of the drug for the prevention and treatment of anxiety disorder. Hereinafter, the present invention is described in detail .

The present invention provides a composition for the prevention and treatment of anxiety disorder containing T- type calcium channel blocker.

In this invention, "T-type calcium channel blocker" indicates a material that is capable of inhibiting selectively the functions of T-type calcium ion channel, which can be selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts, and sera. These compounds can be either novel compounds or well-informed compounds. The candidate compound can form a salt. For example, the salts of the candidate compounds are physiologically acceptable acids

(inorganic acids) or bases (organic acids) and a physiologically acceptable acid added salt is more preferred. The salts are exemplified by the salts of inorganic acids (ex. hydrochloric acid, phosphoric acid, hydrobromic acid and sulfuric acid) and organic acids (ex. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methyl sulfonic acid and benzene sulfonic acid) .

The T-type calcium channel blocker herein is preferably selected from the group consisting of mibefradil, tetramethrin (Sumitomo, Japan) , ethosuximide (Sigma, USA) , SUN-N8075 (Daiichi Suntory Biomedical Research Co Ltd, Japan), efonidipine (Masumiya et al. , Life Sci., 2000, 68 (3) : 345-51) , triply charged ions such as Ni²⁺, Y³⁺, La³⁺, Ce³⁺, Nd³⁺, Gd³⁺, Ho³⁺, Er³⁺ and Yb³⁺ (Mlinar et al., J. Physiol., 1993, 469:639-52), U-92032 (7- [ [4- [bis (4- fluorophenyl) methyl] -l-piperazinyl]methyl] -2- [ (2- hydroxyethyl) amino] 4- (1-methylethyl) -2,4,6- cycloheptatrien-1-one, Xu and Lee, J. Pharmacol. Exp. Ther., 1994, 268: 1135-1142), penfluridol, fluspirilene (Enyeart et al., MoI. Pharmacol. , 1992, 42 (2 ): 364-72 ) and valproate (Macdonald and Kelly, Epilepsia, 1994, 35 Suppl 4: S41-50), but not always limited thereto.

The T-type calcium channel blockers have cross- reactivity to the sub-types of the other calcium channel, but those who are capable of inhibiting selectively T-type calcium channel alone without cross-reactivity to other sub-types are preferred.

The T-type calcium channel blocker used in this invention can be screened by the method well known to those in the art, in addition to the above compounds. For example, the method of Song, et al (Br. J. Pharmacol. , 129 (5) : 893-900, 2000) or the method described in Korean Patent Publication No. 2006-94776 or No. 2006-128148 can be used, but not always limited thereto.

The composition for the prevention and treatment of anxiety disorder of the present invention can additionally include a proper carrier, an excipient and a diluent.

Pharmaceutical formulations of the composition of the present invention for the administration can include the form of a pharmaceutically acceptable salt, the compound itself, or a complex compound mixed with other pharmaceutically active compounds or other proper combinations .

The composition of the present invention can be formulated for oral administration, for example powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, and for parenteral administration, for example external use, suppositories and sterile injections, etc. Possible suitable formulations are oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, preparations for external use, suppositories and sterile injections. The carriers, excipients and diluents are exemplified by lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silcate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Formulations can be prepared by using generally used excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant.

The composition of the present invention can be administered orally or parenterally . The parenteral administration is preferably performed by systemic or local administration, in particular injection into mediodorsal thalamus (MD) is more preferred, but not always limited thereto .

Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, lyophilized preparations, suppositories and injections. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

The effective dosage of the composition for the prevention and treatment of anxiety disorder of the present invention can be determined according to weight and condition of a patient, severity of a disease, preparation of a drug, administration pathway and frequency. The administration frequency can be once a day or a few times a day. The above dosage cannot limit the scope of the invention in any way.

The administration method can be any of those oral administration, rectal administration, intravenous injection, intramuscular injection, hypodermic injection, intrauterine injection or intracerebroventricular injection.

The composition containing the said T-type calcium channel blocker is preferably the compound capable of passing through blood-brain barrier (BBB) , but if the compound itself is not capable of passing through blood- brain barrier, it is preferably delivered by a carrier that can pass through BBB.

The compound capable of passing through blood-brain barrier is exemplified by 1, 4-dihydropyridine. 1,4- dihydropyridine derivatives can be effectively used for the screening of a specific derivative specifically binding to a T-type calcium channel blocker by the same manner as the screening method of T-type calcium channel blocker. As mentioned above, if the compound is not capable of passing through blood-brain barrier on its own, the composition can be delivered to MD by a carrier that can pass through blood-brain barrier. The carrier is exemplified in the descriptions of WO 2002/89776 and WO 2004/1050062. However, the carrier of the present invention is not limited thereto.

The anxiety disorder is preferably selected from the group consisting of phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder, but not always limited thereto.

As explained hereinbefore, the phospholipaseβ4 (PLCβ4) knock-out mouse exhibited normal learning ability, long-term memory and living activity, but had significantly reduced fear-memory extinction, so that it could be used effectively as an anxiety disorder animal model.

To construct the phospholipase β4 (PLCβ4) knock-out mouse, the present inventors constructed PLCβ4 deleted vector and then introduced the vector into embryonic stem cells of a mouse. After culturing the PLCβ4 deleted embryonic stem cell clone, the clone was injected into blastocoel of the blastocyst, followed by mating with a male which was vesactomized. Then, the blastocyst was transplanted into the uterus of a surrogate mother mouse to induce the development of a chimera mouse. The chimera mouse was mated with a wild-type mouse to generate a heterozygote mouse having the genotype of PLCβ4 +/-. The female and male heterozygote mice were mated each other to generate a mutant mouse having the genotype of PLCβ4-/- (PLCβ4 knock-out mouse) . In this invention, the PLCβ4 knock-out mouse constructed in the above was used as an anxiety disorder animal model and the embryo of the PLCβ4 knock-out mouse was deposited at KCTC (Korean Collection for Type Cultures) on December 6, 2007 (Accession No: KCTC 11247BP) .

The present inventors inserted a cannula in mediodorsal thalamus (MD) of the PLCβ4 knock-out mice. The mice were divided into two groups. Learning ability of each group was investigated by freezing response against a specific sound. As a result, there was no response observed in the first attempt, but in the second and third attempts, as being conditioned, response against the specific sound increased in both groups (see Figure 1) . Therefore, it was suggested that the two groups which would be respectively treated with mibefradil and saline had no difference in fear memory learning before the treatment.

To investigate the effect of a T-type calcium channel blocker on learning ability of the PLCβ4 knock-out mice, the present inventors injected the T-type calcium channel blocker mibefradil through the cannula into the PLCβ4 knock-out mice and then freezing response against the conditioned sound was examined in both the mibefradil treated experimental group and the control group treated with saline. As a result, memory was reduced in the experimental group treated with mibefradil, compared with the control group (see Figures 2 and 3) . The above result indicates that mibefradil can restore the reduced fear- memory extinction. Therefore, the administration of a T- type calcium channel blocker into MD leads to the prevention or treatment of anxiety disorder by restoring the memory extinction capacity.

The present inventors also investigated the effect of mibefradil in the wild type mice by the same manner as described above. The wild type mice were randomly divided into two groups and learning ability of each group was investigated by observing freezing response against a specific sound. As a result, there was no significant difference in learning ability in both groups (consistent with the above result from the experiment using the PLCβ4 knock-out mice) (see Figure 4) . The above result suggested that the two groups which would be treated with mibefradil and saline respectively had no difference in their fear memory learning ability before the treatment.

To investigate the effect of the said T-type calcium channel blocker on learning ability of the wild type mouse, the present inventors administered the representative T- type calcium channel blocker mibefradil (experimental group) and saline (control group) respectively to the mice, followed by observation of freezing response against a specific sound. As a result, memory to the conditioned sound was lost in both experimental group treated with mibefradil and control group, but the memory extinction was faster in the experimental group treated with mibefradil (see Figures 6 and 7). Therefore, it is suggested that a T-type calcium channel blocker promotes memory extinction and suppresses restoration of the extinct memory, so that it can be effectively used for the prevention and treatment of anxiety disorder. The present inventors investigated learning ability of the two wild type mouse groups by observing freezing response against a specific sound. As a result, there was no difference in learning ability in the two wild type groups (see Figure 5) . That is, the two groups which would be treated with efonidipine and 50% DMSO respectively had no difference in fear memory learning ability before the treatment .

To investigate whether or not another T-type calcium channel blocker had memory extinction accelerating effect, the present inventors administered another T-type calcium channel blocker efonidipine into the mice through cannula by the same manner as described above and administered 50% DMSO into the control, followed by observation of freezing response against a specific sound. As a result, memory extinction was faster in the experimental group treated with efonidipine, which was consistent with the result of the experimental group treated with mibefradil, than in the control (see Figures 8 and 9) . The above result indicates that various T-type calcium channel blockers can be effectively used for a composition for the prevention and treatment of anxiety disorder.

The present invention also provides a method for the prevention and treatment of anxiety disorder containing the step of administering a pharmaceutically effective dose of a T-type calcium channel blocker to a subject, and a use of a T-type calcium channel blocker for the development of a drug containing a T-type calcium channel blocker for the prevention and treatment of anxiety disorder.

The pharmaceutically effective dose herein indicates the amount of the composition that can relieve the symptoms of anxiety disorder or interrupt the progress of the disorder, and more preferably the amount that can prevent the disease from being developed or cure the disease. It is also important for the effective dose not to be excessive to have an adverse effect. The effective dose can be determined after testing with test animals such as mouse, rat, dog or pig, and this test method is well known to those in the art.

In this invention, the "subject" indicates a random living thing, preferably an animal, more preferably a mammal and most preferably a human. The subject herein is supposed to have anxiety disorder or risk of anxiety disorder.

As explained hereinbefore, the composition of the present invention restores memory extinction capacity significantly, so that it can be effectively used for the production of a drug for the prevention and treatment of anxiety disorder. [Advantageous Effect]

When the composition of the invention containing a T- type calcium channel blocker is administered into mediodorsal thalamus (MD) , fear memory is lost fast and recall of the extinct fear memory is retarded, so this composition can be effectively used for the prevention and treatment of anxiety disorder.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a graph illustrating the learning ability of each PLCβ4 knock-out mouse group before the administration of the T-type calcium channel inhibitor mibefradii and saline:

-/- saline: PLCβ4 knock-out mouse supposed to be administered with saline into MD; and

-/- mibefradii: PLCβ4 knock-out mouse supposed to be administered with mibefradil into MD.

Figure 2 is a graph illustrating the fear memory extinction learning capacity of the PLCβ4 knock-out mouse administered with mibefradil to a specific conditioned sound, investigated 24 hours after the sound conditioning:

-/- saline: PLCβ4 knock-out mouse supposed to be administered with saline into MD; -/- mibefradil: PLCβ4 knock-out mouse supposed to be administered with mibefradil into MD; and

1 hour: the time point of mibefradil administration, one hour later the conditioned sound was given.

Figure 3 is a graph illustrating the recalling of the extinct fear memory of the PLCβ4 knock-out mouse administered with mibefradil to a specific conditioned sound, investigated 24 hours after the fear memory extinction learning: -/- saline: PLCβ4 knock-out mouse supposed to be administered with saline into MD;

-/- mibefradil: PLCβ4 knock-out mouse supposed to be administered with mibefradil into MD; and

Figure 4 is a graph illustrating the comparison of learning ability between the wild type groups respectively treated with the T-type calcium channel blocker mibefradil and saline: +/+ saline: wild type mouse supposed to be administered with saline into MD; and

+/+ mibefradii : wild type mouse supposed to be administered with mibefradil into MD.

Figure 5 is a graph illustrating the comparison of learning ability between the wild type groups respectively treated with the T-type calcium channel blocker efonidipine and 50% DMSO: +/+ 50% DMSO: wild type mouse supposed to be administered with 50% DMSO into MD; and

+/+ efonidipine: wild type mouse supposed to be administered with efonidipine into MD.

Figure 6 is a graph illustrating the fear memory extinction learning capacity of the wild type mouse administered with mibefradil to a specific conditioned sound, investigated 24 hours after the sound conditioning:

+/+ saline: wild type mouse supposed to be administered with saline into MD; and

+/+ mibefradil: wild type mouse supposed to be administered with mibefradil into MD.

Figure 7 is a graph illustrating the recalling of the extinct fear memory of the wild type mouse administered with mibefradil to a specific conditioned sound, investigated 24 hours after the fear memory extinction learning:

Figure 8 is a graph illustrating the fear memory extinction learning capacity of the wild type mouse administered with efonidipine to a specific conditioned sound, investigated 24 hours after the sound conditioning:

+/+ 50% DMSO: wild type mouse supposed to be administered with 50% DMSO into MD; and +/+ efonidipine: wild type mouse supposed to be administered with efonidipine into MD.

Figure 9 is a graph illustrating the recalling of the extinct fear memory of the wild type mouse administered with efonidipine to a specific conditioned sound, investigated 24 hours after the fear memory extinction learning

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Construction of phospholipaseβ4 (PLCβ4) knockout mouse <!-!> Construction of PLCβ4 deleted vector To separate PLCβ4 gene from mouse genome, the certain region of PLCβ4 cDNA (SEQ. ID. NO: 1) ranging from the starting codon to 226-369^th nucleotides was isolated by RT- PCR, which was used as a probe for hybridization to 129/svJae mouse genome DNA library (LamdaFixII, Stratagene Inc., USA). After selecting genomic clone phage having PLCβ4 gene, it was examined whether or not the gene was PLCβ4 by using restriction enzyme map, Southern blotting and nucleotide sequencing.

To construct PLCβ4 deleted vector, a part of X domain of PLCβ4 protein was eliminated from the PLCβ4 gene clone, which was cloned into PSK-plasmid vector (Stratagene Inc., USA) . To increase targeting efficiency, thymidine kinase gene cassette and negative selection marker were inserted into the 3' homologous fragment of the targeting vector containing deleted PLCβ4 gene.

<l-2> Cell culture

Jl embryonic stem cells were used as a host cell line for the transfection with the above targeting vector constructed in Example <1-1>. Jl embryonic stem cells (provided by R. Jeanisch, MIT, USA) were inoculated into ES medium prepared by supplementing 15% fetal bovine serum (Hyclone Co., USA), l^χ penicillin-streptomycin, l^χ nonessential amino acids (Gibco Co., USA), and 0.1 mM 2- mercaptoethanol to DMEM (Gibco Co., USA), followed by culture at 37 ^°C for 2 - 3 days. The embryonic cells prepared from the culture were treated with 1 mM EDTA solution containing 0.25% trypsin to separate as single cells .

<l-3> Introduction of PLCβ4 deleted vector into cells

Electroporation was performed to transfect the embryonic stem cells separated as single cells in Example

<l-2> with the targeting vector constructed in Example <1- 1>. Particularly, the embryonic stem cells diluted at the concentration of 2^χlO⁷ cells/ml were mixed with 25 βg of the targeting vector DNA prepared in Example <1-1>, followed by electric shock at 270 V/500 μF. The embryonic stem cells were cultured in ES medium supplemented with 0.3 mg/mβ of G418 and 2 μM of ganciclovir for 5 - 7 days. The embryonic stem cell clone where PLCβ4 gene was precisely targeted by the targeting vector by homologous recombination was selected by Southern blotting.

<l-4> Generation of PLCβ4+/- mouse

To generate a chimera mouse having the genotype of PLCβ4 +/-, the embryonic stem cell clone selected in Example <l-3> was micro-injected into the fertilized blastocyst . Particularly, C57BL/6J female and male mice (Jackson Laboratory, USA) were mated. The 3.5 p.c. female mouse was sacrificed by cervical dislocation. The uterus was extracted from the sacrificed female mouse and the bottom of the uterus was cut by scissors. 1 ml of injection solution comprising 20 mM HEPES, 10% fetal calf serum, 0.1 mM 2-mercaptoethanol and DMEM was refluxed using a 1 mi syringe. The blastocyst was separated from the uterus tissues by using a micro glass tube under the dissecting microscope. The separated blastocyst was placed in a drop of the injection solution pre-dropped on a 35 mm Petri-dish, which was used for the following experiment.

To introduce the embryonic stem cell clone selected in Example <l-3> into the separated blastocyst, 10 - 15 embryonic stem cell clones were sucked in an injecting pipette, which was inserted into blastocoel of the blastocyst with giving negative pressure to the direction of inner cell mass of the blastocyst by using a holding pipette and then the embryonic stem cell clones were injected into blastocoel of the blastocyst with a microinjector (Zeiss Inc., USA) by changing the pressure to positive. The blastocyst harboring the clones was mated with the vesactomized male mouse, which was transplanted into the uterus of the 2.5 p.c. pseudopregnant surrogate mother mouse to induce a chimera mouse, a kind of cross- bred hybrid, from the embryonic stem cell clone (Jl) and the blastocyst of a C57BL/6J mouse. At this time, for the transplantation into the uterus, the abdomen of the surrogate mother anesthetized with avertine (1 mg/kg) was cut 1 cm; the upper part of the uterus was pulled about 2 cm up to the outside of the body with a pincette; a hole was made by an injection needle, and through this hole a micro glass tube was inserted, through which the blastocyst was injected; peritoneal membrane was sewed two stitches with a suture; and the outer skin was sealed with a medical clip. The blastocyst inserted with the embryonic stem cell clone was transplanted into the uterus of the surrogate mother mouse, followed by culture for approximately 19 days, leading to the fusion of embryonic stem cell originated cells and blastocyst originated cells, resulting in the construction of a chimera mouse having the genotype of PLCβ4 +/-.

<l-5> Generation of PLCβ4 knock-out mouse

Each chimera mouse was mated respectively with the C57BL/6J and 129sv mice more than 20 times, from which C57BL/6J-PLCβ4+/- and 129sv-PLCβ4+/- mice were generated. The produced mice were mated each other to generate Fl generations ^λPLCβ4+/+ and PLCβ4-/-' , which were used for the following experiments. The genotype was confirmed by PCR. Primers used for the PCR were Kl (5¹- CTCCACACTCTGCAACCTAC-3 ' ; SEQ. ID. NO: 2), K9 (5'- AGTTACTTCTGGATTTTCAGCC-3' ; SEQ. ID. NO: 3) and PFK22 (5'- CTGACTAGGGGAGGAGTAGAAG-3' ; SEQ. ID. NO: 4) and PCR was performed as follows: 94 ^"C for 30 seconds, 58 ^"C for 30 seconds, and 72^°C for 30 seconds (40 cycles) . Kl and K9 primers were the primer set to confirm the genotype of the normal mouse and Kl and PFK22 were the primer set to confirm the genotype of the mutant mouse. Bands corresponding to each PCR product were confirmed on 1.5% EtBr/aragose gel (PLCβ4+/+: 190 bp, PLCβ4+/-: 250/190 bp and PLCβ 4 -/- : 250 bp ) .

Example 2: Mouse raising and organization

The mice were raised in SPF (specific pathogen free) environment where temperature was maintained at 22 "C and humidity was regulated at 55% and water and feed were provided freely under the light cycle of 12 hour light/12 hour dark. The mice for the experiment were all male and 6 - 18 mice in total. T-test and repeated two-way ANOVA test were performed for the statistical treatment.

Cannulas were implanted in the wild type and PLCβ4 knock-out mice, through which the drug could be administered into mediodorsal thalamus (MD) . The implantation was performed in Kopf (small animal stereotaxic frame) . Particularly, the mouse was anesthetized by the injection of 2% avertine (20 βt per 1 g of body weight) , followed by fixation at stereotaxic. Then, a hole was made on skull of the mouse at the coordinate (AP -1.7 mm, lateral 0.2 mm, depth 3.0 mm) of MD (mediodorsal thalamus), determined by using stereotaxic map. Guide cannula (Plastics One Inc., C315G, tubing length: 3 mm) was inserted through the hole and fixed in skull using dental cement. The operated mouse was recovered in an isolated cage, at least for one week, and then used for the following experiments. Experimental Example 1: Investigation of fear memory learning ability of PLCβ4 knock-out mouse

The mice recovered from the operation in Example 2 were randomly divided into two groups, followed by investigation of learning ability by observing freezing response. After one week from the implantation of the cannula in MD, the PLCβ4 knock-out mice were randomly divided into two groups, 10 - 13 mice per each group. In the conditioning chamber shutting out the outside sound, the mice were forced to listen to a specific unusual sound

(2900 Hz, 100 db) for 30 seconds. Electric shock (0.5 mA) was given to the sole of the foot for 1 second, which was the cause of freezing response. The shock was given at 120 seconds intervals three times and it was arranged to end the shock at the same time with the end of sound.

As a result, through the above learning, in the first trial, freezing response was 0%, but as conditioning went on, trial by trial, freezing response to the sound was increased in both group mice (Figure 1) . Therefore, it was confirmed that the two groups which would be respectively treated with mibefradil and saline had no difference in fear memory learning before the treatment.

Experimental Example 2: Investigation of memory extinction of PLCβ4 knock-out mouse

The present inventors administered mibefradii (10 nmol, 0.5 μi) into MD of the one of the two groups divided in Experimental Example 1 (11 mice) through the implanted cannula, and administered saline (0.9% NaCl, 0.5 βi) to the other group (control, 8 mice) . One hour after the administration of mibefradil and saline, sound conditioning was induced in both group mice by the same manner as described in Experimental Example 3. 24 hours later, the mice were forced to listen to the sound (30 seconds) 20 times in a conditioning chamber (extinction day 1) . The interval between the sound was determined as 5 seconds. 24 hours later, mice were forced again to listen to the sound 6 times (extinction day 2). As a result, the PLCβ4 knock-out mice administered with saline in MD (referred as "control" hereinafter) demonstrated that their fear memory of the sound conditioned by 20 times of trials was not extinct. In the meantime, the PLCβ4 knock-out mice administered mibefradil (referred as "experimental group" hereinafter) demonstrated that their fear memory against the sound was significantly lost (Figure 2). So, it was confirmed that mibefradil accelerated fear memory extinction of the PLCβ4 knock-out mice. 48 hours after the sound conditioning, fear memory of the control was not lost after 6 times of trials. But, freezing response was significantly reduced in the experimental group administered with mibefradii (Figure 3). So, it was confirmed that mibefradil reduced recalling of fear memory in the PLCβ4 knock-out mice.

Experimental Example 3: Investigation of fear memory learning ability of wild type mouse

One week after the implantation of the cannula in MD of the wild type mouse prepared in Example 2, learning ability of the mouse was investigated by the same manner as described in Experimental Example 1.

The result was consistent with that of the above experiment with the PLCβ4 knock-out mouse. In the first trial, freezing response was observed 0%. But as being conditioned, in the second and the third trials, freezing response against the conditioned sound was increased in both groups (Figures 4 and 5). Therefore, it was confirmed that the two groups which would be respectively treated with mibefradil and saline in Experimental Example 4 had no difference in fear memory learning before the treatment.

Experimental Example 4 : Investigation of memory extinction of wild type mouse <4-l> Effect of mibefradil on memory extinction The present inventors investigated fear memory extinction and fear memory recall of the wild type mice administered with mibefradil or saline into MD through cannula by the same manner as described in Experimental Example 2.

As a result, fear memory was lost in both wild type mouse groups administered respectively with mibefradil and saline in their MD, and the fear memory extinction of the wild type mice administered with mibefradil was faster (Figure 6). Therefore, it was confirmed that mibefradil accelerated fear memory extinction of the wild type mice.

In the meantime, regarding fear memory recall, fear memory of the conditioned sound was not lost after 6 times of trials in the control, while freezing response was significantly reduced in the experimental group treated with mibefradil (Figure 7). Therefore, it was confirmed that mibefradil reduced fear memory recall in the wild type mice.

<4-2> Effect of efonidipine on memory extinction

The present inventors administered efonidipine (10 nmol, 0.5 βi) into MD of the one mouse group (7 mice) through the implanted cannula, and administered 50% DMSO (dimethyl sulfoxide) (0.9% NaCl, 0.5 μJL) to the other mouse group (control, 6 mice) through the cannula. Fear memory extinction and fear memory recall were investigated in the two groups by the same manner as described in Experimental Example 2, except that efonidipine and 50% DMSO were administered . As a result, fear memory was lost in both the wild type mouse group administered with 50% DMSO in their MD and in the wild type mouse group administered with efonidipine. And, fear memory extinction was faster in the wild type mouse group treated with efonidipine (Figure 8). Therefore, it was confirmed that efonidipine accelerated fear memory extinction of the wild type mice.

In the meantime, regarding fear memory recall, fear memory of the conditioned sound was not lost after 6 times of trials in the control, while freezing response was significantly reduced in the experimental group treated with mibefradil (Figure 9) . Therefore, it was confirmed that efonidipine significantly reduced fear memory recall of the wild type mice.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims,

Claims

[CLAIMS]

[Claim l]

A composition for the prevention and treatment of anxiety disorder containing a T-type calcium channel blocker.

[Claim 2]

The composition according to claim 1, wherein the T- type calcium channel blocker is selected from the group consisting of mibefradii, tetramethrin, ethosuximide, SUN- N8075, efonidipine, triply charged ions such as Ni²⁺, Y³⁺, La³⁺, Ce³⁺, Nd³⁺, Gd³⁺, Ho³⁺, Er³⁺ and Yb³⁺, ϋ-92032 (7-[[4- [bis (4-fluorophenyl) methyl] -l-piperazinyl]methyl] -2- [ (2- hydroxyethyl) amino] 4- (1-methylethyl) -2,4,6- cycloheptatrien-1-one) , penfluridol, fluspirilene and valproate.

[Claim 3]

The composition according to claim 1, wherein the anxiety disorder is selected from the group consisting of phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder

[Claim 4]

The composition according to claim 1, wherein the composition additionally includes a carrier capable of passing through blood-brain barrier.

[Claim 5]

The composition according to claim 1, wherein the T- type calcium channel blocker is a compound capable of passing through blood-brain barrier.

[Claim 6]

The composition according to claim 1, wherein the composition can be administered orally or parenterally.

[Claim 7]

The composition according to claim 6, wherein the parenteral administration is systemic or local administration .

[Claim 8]

The composition according to claim 7, wherein the local administration is performed into mediodorsal thalamus (MD) .

[Claim 9] The composition according to claim 1, wherein the composition can prevent and treat anxiety disorder by restoring fear memory extinction ability significantly.

[Claim 10]

A method for treating anxiety disorder containing the step of administering a pharmaceutically effective dose of a T-type calcium channel blocker to a subject with anxiety disorder.

[Claim 11]

A method for preventing anxiety disorder containing the step of administering a pharmaceutically effective dose of a T-type calcium channel blocker to a subject with anxiety disorder.

[Claim 12]

A use of a T-type calcium channel blocker for the development of a drug for the prevention and treatment of anxiety disorder.