KR20090081307A - A method for treatment of anxiety disorder by regulating t-type calcium channel - Google Patents

Abstract

A method for treating anxiety disorder by suppressing T-type calcium channel is provided to quickly eliminate phobia memory by administering a composition inside thalamus and reduce reminiscence to phobia memory. A method for treating anxiety disorder by suppressing T-type calcium channel comprises a step of administering in pharmaceutically active amount of inhibitor for T-type calcium channel to subject. The anxiety disorder is phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder.

Description

A method for treatment of anxiety disorder by regulating T-type calcium channel

The present invention relates to a composition for preventing and treating anxiety disorders containing T-type calcium channel inhibitors, and a method for preventing and treating anxiety disorders by administering the composition to the medial dorsal medial nucleus (mediodorsal thalamus, MD).

Calcium channels play an important role in the excitatory-constriction linking mechanisms in skeletal, vascular, and heart muscle cells, and in the regulation of neurotransmitter secretion in the central and peripheral nervous systems. These calcium channels are classified into high voltage dependent L-type calcium channels and low voltage dependent T-type calcium channels according to physiological and molecular biological characteristics. L-type calcium channel plays a major role in the transport of calcium ions across cell membranes such as vascular, skeletal, and smooth muscle, regulating the contraction of these muscles, and nerves from endocrine glands and nervous tissues. It is also involved in transporter secretion. In addition, the T-type calcium channel is present in many cardiac cells such as central muscle cells, adrenal glands, and parietal nodules. It plays an important role in producing beats and is known to be involved in calcium ion influx into muscle cells due to small potential difference. Calcium channel antagonists are expected to be able to control the proliferation of blood cells, blood pressure, abnormal neuronal secretion, etc., and many research subjects have many calcium channel antagonists in the development of drugs for the treatment of heart disease and widespread psychomotor disorders. This has been.

Anxiety disorders include phobic disorders, generalized anxiety disorders, obsessive compulsive disorders, post-traumatic stress disorders, somatoform disorders, and dissociative disorders ( dissociative disorders or factitious disorders. A fear disorder is a disorder in which a person feels irrational fear in a particular subject or situation and continually avoids it because of social phobia, acrophogia, claustrophobia, or antiexpectation. There are many types, depending on their targets. A general anxiety disorder is one in which a person feels widespread and persistent anxiety in everyday situations, and an obsessive compulsive disorder usually involves a certain amount of compulsive thoughts or actions, regardless of his will. Although obsessive-compulsive symptoms can interfere with your life or bother you, you will be diagnosed with OCD. Usually they appear to be a characteristic of compulsive personalities such as accuracy, perfection, and principleism. Post-traumatic stress disorder can be re-experienced by experiencing extremely traumatic events (war, plane accidents, rapes, Sampoong Department Store accidents, etc.) that people cannot experience in general, and then repeatedly thinking about such terrible accidents or appearing in dreams. Refers to disorders that are insensitive to the outside and accompanied by symptoms of the autonomic nervous system. Somatoform disorders are cases in which physical symptoms are suspected of physical illness, but in reality there are no physical abnormalities and physical symptoms are caused by psychological conflicts or factors. Psychological conflicts or factors often cause physical symptoms or illnesses, which are called psychophysiological disorders. The expression of psychological conflicts as physical symptoms is generally thought to occur on an unconscious level. In other words, it is different from malingering, in which the patient is consciously and deliberately expresses physical symptoms. Dissociative disorders are rare, as in the case of Dr. Jekyll and Hyde, where suddenly part of the mental function is temporarily lost.

The dorsal medial nucleus (mediodorsal thalamus, MD) is part of the limbic system and is connected to the frontal lobe, the amygdala and the hippocampus, which are related to emotion. However, the mechanism by which MD is involved in the act of emotion is not well known. On the other hand, phospholipase β4 (Phospholipase β4, PLCβ4) is expressed in MD, but rarely expressed in the frontal lobe or amygdala, which are important for fear disappearance. The inventors of the present invention prepared a mouse knocked-out of the PLCβ4 gene expressed in MD, and the PLCβ4 knock-out mouse exhibited a severe lack of memory extinction ability. It was confirmed that it can be used as.

Therefore, the inventors of the present invention, when the administration of mibefradil, a representative T-type calcium channel inhibitor to the MD of PLCβ4 knock-out mice, returns to the normal memory loss ability, which was lacking in the mice, mibepradil and epony The present invention was completed by confirming that administration of diffine (efonidipine) to MD of normal mice quickly eliminates fear memory and decreases the recall ability for the expired fear memory.

An object of the present invention is to provide a composition for preventing and treating anxiety disorders containing T-type calcium channel inhibitor, and a method for preventing and treating anxiety disorders using the composition.

In order to achieve the above object, the present invention provides a composition for preventing and treating anxiety disorder containing T-type calcium channel inhibitor.

In addition, the present invention provides a method for treating anxiety disorder comprising administering a pharmaceutically effective amount of a T-type calcium channel inhibitor to a subject having anxiety disorder.

The present invention also provides a method for preventing anxiety disorder comprising administering to a subject a pharmaceutically effective amount of a T-type calcium channel inhibitor.

In addition, the present invention provides a use for the production of T-type calcium channel inhibitors for the prevention and treatment of anxiety disorders.

When the composition containing the T-type calcium channel inhibitor of the present invention is administered to the medial dorsal medial nucleus (mediodorsal thalamus, MD), the fear memory is quickly extinguished, and the anxiety disorder is prevented and treated by reducing the recall ability for fear memory annihilation. This can be useful for.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing and treating anxiety disorders containing T-type calcium channel inhibitors.

As used herein, "T-type calcium channel inhibitor" refers to a substance that selectively inhibits the function of T-type calcium ion channels, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts , Plant extracts, animal tissue extracts or plasma, and the like may be novel compounds or well-known compounds. Such a substance may form a salt. Salts of candidate substances include salts such as physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids, etc.), and among these, physiologically acceptable acid addition salts are preferable. Such salts include, for example, salts of inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid). , Malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

Preferred examples of such T-type calcium channel inhibitors are mibefradil, tetramethrin (Sumitomo, Japan), ethosuximide (Sigma, USA), SUN-N8075 (Daiichi Suntory Biomedical Research Co Ltd, Japan), eponidipine, Masumiya et al ., Life Sci ., 2000, 68 (3): 345-51), divalent ions of nickel (Ni ^{2 +} ) and Y ^{3 +} (trivalent ions of yttrium), La ^{3 +} (trivalent ions of lanthanum), Ce ^{3 +} a (three of cerium ions), Nd ^{3 +} (3 the neodymium ion), Gd ^{3 +} (3 ion of gadolinium), Ho ^{3 +} (3 ion of holmium), Er ^{3 +} (3 of erbium Ions) and trivalent metal ions such as Yb ³⁺ (trivalent ions of ytterbium) (Mlinar et al ., J. Physiol ., 1993, 469: 639-52), U-92032 (7-[[4- [bis (4-fluorophenyl) methyl] -1-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), penpeul Metalurgs stone ( penfluridol) and fluspirilene (Enyeart et) al ., Mol. Pharmacol ., 1992, 42 (2): 364-72), valproate, Macdonald and Kelly, Epilepsia , 1994, 35 Suppl 4: S41-50), but is not limited thereto.

In addition, the T-type calcium channel inhibitor may have cross-reactivity with sub-types of other calcium channels, but selectively only T-type calcium channels without reactivity to other subtypes. It is preferable that it is a substance which can inhibit.

The T-type calcium channel inhibitors used in the present invention are in addition to the compounds listed above. It is possible to screen by methods known in the art. Examples of such methods include those described in Song et al . ( Br . J. Pharmacol . , 129 (5): 893-900, 2000), Korean Patent Laid-Open Publication No. 2006-94776 or Korean Patent Publication No. 2006-128148. It is not limited to this.

The composition for preventing and treating anxiety disorders of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of the composition.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients, and diluents that may be included in compositions containing wood vinegar include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

The composition of the present invention can be oral or parenteral, the parenteral administration is most preferably administered systemically or topically, in particular into the dorsal medial medial nucleus (MD), but is not particularly limited thereto. no.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the herbal compound extract. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, injections. 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.

The preferred dosage of the composition for preventing and treating anxiety disorders of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous or intracerebroventricular injection.

In addition, the composition containing the T-type calcium channel inhibitor is preferably a compound that can cross the blood-brain barrier (BBB), if the compound itself is a compound that cannot cross the cerebrovascular barrier The carrier may be used to cross the cerebrovascular barrier.

Compounds that can cross the cerebrovascular barrier include 1,4-dihydropyridine, and the 1,4-dihydropyridine derivatives are prepared to specifically bind to T-type calcium channel inhibitors. It is possible to screen derivatives using the above-described method for screening T-type calcium channel inhibitors.

On the other hand, if it does not cross the cerebrovascular barrier itself, the composition can be delivered to the MD using a carrier that can cross the cerebrovascular barrier. Examples of such carriers are disclosed in WO 2002/89776 and WO 2004/1050062. However, the carrier of the present invention is not limited to the specific carrier.

The anxiety disorders include phobic disorders, generalized anxiety disorders, obsessive compulsive disorders, post-traumatic stress disorders, somatoform disorders, and dissociative disorders. It is preferred to be selected from the group consisting of dissociative disorders and factitious disorders, but is not particularly limited thereto.

The present inventors found that mice with knock-out genes of phospholipase β4 (PLCβ4), which are highly expressed in the medial medial nucleus of the thalamus (MD), are capable of learning, long-term memory, and activity. Although normal, the memory extinction ability was found to be significantly lowered. This means that PLCβ4 knock-out mice can be used as animal models of anxiety disorders.

In order to prepare PLCβ4 knock-out mice, the inventors first prepared a vector lacking the PLCβ4 gene and introduced it into mouse embryonic stem cells. Next, embryonic stem cell clones in which the PLCβ4 gene deletion was confirmed were cultured, injected into the blastocyst of the blastocyst, crossed with males subjected to vas deferens, and transplanted into the uterus of surrogate mice to induce the development of chimeric mice. The chimeric mice were bred with normal mice to obtain heterozygote mice having the PLCβ4 +/- genotype, and again the females and males of the heterozygous mouse were transgenic mice having the PLCβ4-/-genotype (PLCβ4). Knock-out mice). In the present invention, the PLCβ4 knock-out mouse prepared as described above was used as an animal model of anxiety disorder, and the fertilized egg of PLCβ4 knock-out mouse was deposited in KCTC (Korean Collection for Type Cultures) on December 6, 2007 (consignment). Number: KCTC 11247BP).

We performed cannula injection into the medial medial nucleus (MD) of the thalamus of PLCβ4 knock-out mice, and then randomly divided the two groups through a freezing reaction to a specific sound. We examined whether there was a difference in learning ability. As a result, there was little response in the first trial but similar trends in both groups of mice tended to increase in response to specific sounds in the second and third trials as conditioned (see FIG. 1). Therefore, it can be seen that the two groups to be administered mibefradil and saline do not differ from each other in fear memory learning the day before administration.

In order to determine the effect of T-type calcium channel inhibitors on the learning ability of PLCβ4 knock-out mice, the present inventors examined the control group and control group of mibepradil, which is a T-type calcium channel inhibitor, through a kennurer of PLCβ4 knock-out mice. In the saline solution, the freezing response to the specific sound was investigated. As a result, the mibepradil administration group confirmed that the memory gradually disappeared compared to the control group (see FIGS. 2 and 3). This means that mibepradil can reverse the loss of memory normally. Therefore, it can be seen that administration of T-type calcium channel inhibitor to MD significantly prevents or treats anxiety disorders by significantly restoring memory extinction ability.

In addition, the present inventors examined the efficacy of mibepradil in the same manner as in the normal mouse. Normal mice were randomly divided into two groups to examine whether there was a difference in learning ability between two groups by freezing responses to specific sounds. The results were similar in both groups of normal mice, as in PLCβ4 knock-out mice (see FIG. 4). Therefore, it can be seen that the two groups to be administered mibepradil and saline do not differ from each other in the fear memory learning the day before administration.

In order to determine the effect of the representative T-type calcium channel inhibitors on the learning ability of normal mice, the present inventors froze the specific sound in the mibepradil-administered group and the control group (saline administration) through the kenuler as described above. The reaction was investigated. As a result, it was confirmed that the memory for a specific sound disappeared in both the mibepradil administration group and the control group, but disappeared more quickly in the mibepradil administration group (see FIGS. 6 and 7). Therefore, it can be seen that T-type calcium channel inhibitors can prevent or treat anxiety disorders by increasing memory annihilation ability and decreasing the ability to recover the extinct memory.

In addition, the present inventors randomly divided the normal mice into two groups and examined whether there was a difference in learning ability of the two groups through a freezing response to a specific sound. The results were similar in both groups of normal mice (see FIG. 5). Therefore, it was confirmed that the two groups to be administered eponidipine and 50% DMSO did not differ from each other in fear memory learning the day before administration.

The inventors of the present invention to determine whether the amnesia effect is also applied to other T-type calcium channel inhibitors, as described above through the Kenuler T-type calcium channel inhibitors eponidipine administration group and control group (50% DMSO administration) We investigated the freezing response to a specific sound at. As a result, similar to the mibepradyl, it was confirmed that the eponidipine administration group disappears faster than the control group (see FIGS. 8 and 9). Therefore, it can be seen that various T-type calcium channel inhibitors can be used in the composition for preventing or treating anxiety disorder.

The present invention also provides a method for preventing and treating anxiety disorders comprising administering to a subject a pharmaceutically effective amount of a T-type calcium channel inhibitor, and a T-type calcium channel inhibitor for preventing and treating anxiety disorders. It provides a use in the manufacture of a pharmaceutical.

The therapeutically effective amount is an amount that alleviates the symptoms of the anxiety disorder or prevents progression, and is preferably an amount that prevents or cures the onset of the anxiety disorder. In addition, the amount which does not produce the adverse effect beyond the benefit by administration is preferable. Such amounts can be appropriately determined by tests on animal models such as mice, rats, dogs or pigs and such assays are well known to those skilled in the art.

The term "subject" in the context of the present invention means any biological subject, preferably an animal, more preferably a mammal, most preferably a human subject. The subject refers to a subject having anxiety disorder or at risk of anxiety disorder.

As described above, since the composition of the present invention significantly restores the memory extinction ability, it may be usefully used in the manufacture of a medicament for the prevention and treatment of anxiety disorders.

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

However, the following Examples and Experimental Examples are only for illustrating the present invention and do not limit the present invention.

< Example  1> Phospholipase β4 ( Phospholipase β4) gene knock-out ( knock - out ) mouse( PLCβ4 knock-out mouse)  Produce

<1-1> PLC Preparation of β4 Gene Deletion Vectors

To isolate the PLCβ4 gene from the mouse genome, 226 to 369 sites from the start codon of the cDNA (SEQ ID NO: 1) of the PLCβ4 gene were isolated by RT-PCR and used as a probe. Hybridization reactions were performed on svJae mouse genomic DNA library (LamdaFixII, Stratagene Inc., USA). From this, genome clone phage having PLCβ4 gene was selected, and it was confirmed that the phage clone was PLCβ4 gene by restriction enzyme map, Southern blot analysis and sequencing.

To prepare a vector lacking the PLCβ4 gene, a portion of the X domain of the PLCβ4 protein was removed from the PLCβ4 gene clone and cloned into a PSK-plasmid vector (Stratagene Inc., USA). A thymidine kinase gene cassette and a negative selection marker were inserted at the 3 'homologous end of the hit vector including a part of the PLCβ4 gene deleted.

<1-2> cell culture

J1 embroinic liver cells were used as a cell line for transducing the hit vector prepared in Example <1-1>. J1 embryonic stem cells (received from R. Jeanisch, Massachusetts Institute of Technology, USA) were placed in DMEM medium (Gibco Co., USA) in 15% fetal bovine serum, Hyclone Co., USA, 1 × penicillin -Inoculated into ES medium with phenicillin-streptomycin, 1x non-essential amino acid (Gibco Co., USA), 0.1 mM 2-mercaptoethanol and then at 37 ° C. for 2-3 days Incubated. The germ cell group obtained from the culture was treated with 1 mM EDTA solution containing 0.25% trypsin to isolate single cells.

<1-3> into cells PLC Introduction of β4 Gene Deletion Vectors

Electroporation was performed to transfect the hit vector prepared in Example <1-1> to embryonic stem cells separated into single cells in Example <1-2>. Specifically, 25 μg of the hit vector DNA prepared in Example <1-1> was added to the embryonic hepatocytes diluted to a concentration of 2 × 10 ⁷ cells / ml and mixed, followed by electric shock at 270 V / 500 μF. Was added. The embryonic hepatocytes were cultured in ES medium containing 0.3 mg / ml G418 and 2 μM hepatocyclovir for 5 to 7 days, and the PLCβ4 gene in embryonic hepatocytes was correctly hit by a hit vector by homologous recombination. Embryonic stem cell clones were selected and maintained by Southern blot.

<1-4> PLC Preparation of β4 +/− Mice

To prepare chimeric mice with genotype PLCβ4 +/−, fertilized embryonic cells were microinjected with the embryonic hepatocyte clones selected in Example 1-1 above.

Specifically, females and males of C57BL / 6J mice (Jackson Laboratory, USA) were crossed, and 3.5 days (3.5 p.c.) of females were sacrificed by cervical dislocation. After removal of the uterus from the sacrificed females and excision of the terminal part of the uterus with scissors, 1 ml injection solution containing 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM was added using a 1 ml syringe. Perfusion. The blastocyst was separated from the uterine tissue under a dissecting microscope using a micro glass tube, and the separated blastocyst was transferred to a drop of injection solution previously dropped on a 35 mm Petri dish and used for the following introduction process.

In order to introduce the embryonic stem cell clones selected in Example <1-3> to the isolated embryos as described above, internal cell masses of the embryos were held by a holding pipette using a microinjector (Zeiss Inc., USA). An injection pipette with 10-15 embryonic stem cell clones inhaled with negative pressure in the direction of (inner cell mass) was inserted into the blastocyst of the blastocyst and injected into the blastocyst of the blastocyst under positive pressure. The clone-implanted blastocysts were mated with a vas deferens male, and then transplanted into the uterus of 2.5 pcs of fertility surrogate mice to form heterologous cell hybrids formed from embryonic stem cell clones (J1) and blastocysts of C57BL / 6J mice. Induction of in chimeric mice was induced. At this time, uterine transplantation is excised the abdomen of the surrogate mother anesthetized with avertine (1 mg / kg per body weight) by about 1 cm; Pick the upper uterus with tweezers and pull it out of the body about 2 cm; Puncturing the uterus with a needle to inject the blastocyst into the microglass tube through the hole; The inner peritoneal membrane was sewn with two stitches, and the outer skin was sealed with a medical clip. As described above, blastocysts injected with embryonic stem cells were transplanted into the uterus of surrogate mice and cultured for about 19 days, thereby culturing cells derived from embryonic stem cells and cells derived from blastocysts and having a genotype of PLCβ4 +/- in the genome. Secured.

<1-5> PLC Preparation of β4 Knock-out Mice

The chimeric mice were maintained at least 20 crossings with C57BL / 6J and 129sv mice, respectively, and the resulting C57BL / 6J-PLCβ4 + // and 129sv-PLCβ4 +/- mice were crossed to PLCβ4 + / + and PLCβ4- / at the F1 stage. Mice were prepared and used in the following behavioral experiments. Genotyping was performed using polymerase chain reaction (PCR). Primers were prepared using K1 (5'-CTCCACACTCTGCAACCTAC-3 '; SEQ ID NO: 2), K9 (5'-AGTTACTTCTGGATTTTCAGCC-3'; SEQ ID NO: 3) and PGK22 (5'-CTGACTAGGGGAGGAGTAGAAG-3 '; SEQ ID NO: 4). The reaction was carried out 40 times in 30 seconds at 30 캜, 30 seconds at 58 캜, and 30 seconds at 72 캜. The primer K1 and K9 was used as a primer pair for identifying genotypes of normal mice, and primers K1 and PFK22 were used as primer pairs for identifying genotypes of mutant mice. The PCR product thus obtained identified each band in 1.5% EtBr / agarose gel. (PLCβ4 + / +: 190 bp, PLCβ4 +/-: 250/190 bp and PLCβ4-/-: 250 bp)

< Example  2> Breeding and composition of mouse

The mice were bred in a SPF (specific pathogen free) environment in which water and feed were given freely under a 12-hour bright, 12-hour dark-illuminated circadian and maintained at a temperature of 22 ° C. and a humidity of 55%. The mice used in the experiment were all males and 6-18 animals were used. Statistical tests were performed using T-test and repeated two-way ANOVA.

A cannula capable of microinjecting drugs was planted in the dorsal medial nucleus (MD) of the normal mouse and PLCβ4 knock-out mouse thalamus. Surgery was performed in a small animal stereotaxic frame (Koff). Mice were anesthetized by injecting 2% aberyne (averine, 20 μl / g body weight) and fixed to stereotaxic. Thereafter, a hole was drilled in the skull of the mouse at the coordinates (AP -1.7 mm, lateral 0.2 mm, depth 3.0 mm) of the MD (mediodorsal thalamus) determined using the stereotaxic map of the mouse. A cannula guide (guide cannula, Plastics One Inc., C315G, tubing length: 3 mm) was injected into the hole and fixed to the skull using dental cement. The surgical mice were transferred to an isolated cage for recovery, and were recovered for at least one week before being used in the following experimental example.

< Experimental Example  1> PLC Fear Memory Learning Ability in β4 Knock-out Mice

The present inventors measured the freezing response in order to investigate whether there is a difference in learning ability of the two groups after dividing the two groups at random after the recovery of the surgery of Example 2. One week after the cannula injection into the MD of PLCβ4 knock-out mice, the mice were randomly divided into two groups of 10 to 13 animals. Each of the two groups of mice listened to a specific sound (2900 Hz, 100 db) for 30 seconds in a specific conditioning chamber where external sound was cut off and caused a freezing reaction. Sole electrical stimulation (0.5 mA) was given for 1 second, but stimulation was done three times at 120 second intervals to end simultaneously with a specific sound.

As a result, the freezing response was almost 0% in the first trial through the above learning, but as the condition became condition, the freezing response to the specific sound gradually increased in the second and third trials. It appeared similar in all mice (FIG. 1). Therefore, it can be seen that the group to which mibefradil is administered and the group to which saline is administered in Experimental Example 2 do not differ from the previous day in fear memory learning.

< Experimental Example  2> PLC Investigation of Memory Extinction Capacity in β4 Knock-out Mice

The inventors of the present invention in two groups of <Experimental Example 1>, one group (11 animals) to the mibepradil (10 nmol, 0.5 μl) through a knul Administered to MD, In the other group (8), saline (0.9% NaCl, 0.5 μl) was administered to the MD through the cannula as a control. 1 hour after the administration of mibepradyl and saline, to mice as shown in <Experiment 1> After 24 hours of conditioning, a specific sound (30 seconds) was heard 20 times in a specific sound memory test box (extinction day1). The specific sound and the specific sound interval were 5 seconds. 24 hours later, the sound was repeated six times (extinction day2).

As a result, in a PLCβ4 knock-out mouse administered with saline to the MD (hereinafter referred to as the "control"), 20 attempts did not disappear the fear memory for the sound, while the PLCβ4 knock-administered with MD in the MD In out mice (hereinafter referred to as "experimental groups"), fear memory was significantly extinguished (FIG. 2). Thus, it can be seen that mibepradil kills fear memory more quickly in PLCβ4 knock-out mice.

In addition, after 48 hours of conditioning of a particular sound, the control group did not lose the fear memory for that particular sound in 6 trials, whereas in the mibepradil-administered group, the freezing response was significantly reduced compared to the control group (Fig. 3). Thus, it can be seen that mibepradil further reduces the recall ability for fear amnesia in PLCβ4 knock-out mice.

< Experimental Example  3> Fear Memory Learning in Normal Mice

The present inventors examined the learning ability of the normal mouse as in <Experimental Example 1> one week after injecting the cannula into the MD of the normal mouse prepared in <Example 2>.

As a result, the normal mouse group was similar to the PLCβ4 knock-out mouse group, with a freezing response of almost 0% on the first trial, but as a condition, the frozen response to a specific sound on the second and third trials. This increasing trend was similar in both groups of mice (FIGS. 4 and 5). Thus, in Experimental Example 4, it can be seen that the two groups to be administered mibepradil and saline are not different from the previous day's fear memory learning.

< Experimental Example  4> Investigation of Amnesia in Normal Mice

<4-1> Mibepradil  Memory extinction ability

The present inventors examined the fear memory utterance ability and recall ability of fear memory shedding of normal mice injected with a cannula in MD in the same manner as in <Experimental Example 2>.

As a result of the fear memory extinction ability, the fear memory was extinguished in both normal mice treated with saline and MD-administered MD, and faster in normal mice treated with mibepradil. (FIG. 6). Thus, it can be seen that mibepradil kills fear memory more quickly in normal mice.

In addition, as a result of the recall ability of fear memory extinction, 6 trials in the control group did not disappear the fear memory for a specific sound, while in the mibepradil administration group, the freezing response was significantly reduced compared to the control group (Fig. 7). Thus, it can be seen that mibepradil further reduces the recall ability for fear memory in normal mice.

<4-2> Eponidipine  Memory extinction ability

In the present inventors, one group (7 mice) received efonidipine (10 nmol, 0.5 μl) through a Kenuler. Administered to MD, The other group (6 animals) received 50% dimethyl sulfoxide (DMSO) (0.9% NaCl, 0.5 μl) as a control group to MD via cannula. Except for administration of the eponidipine and 50% DMSO, the fear memory and ability of recall of normal memory in which cannulated MD was injected were examined in the same manner as in <Experiment 2>.

As a result of the fear memory extinction ability, fear memory was extinguished in both normal mice treated with saline and MD-administered eponidipine, and was faster in normal mice treated with eponidipine. (FIG. 8). Therefore, it can be seen that in normal mice, eponidipine destroys fear memory more quickly.

In addition, as a result of the recall ability of fear memory extinction, 6 trials in the control group did not disappear the fear memory for a particular sound, while in the eponidipine administration group, the freezing response was significantly reduced compared to the control group (Fig. 9). Thus, it can be seen that in normal mice, eponidipine further reduces the recall ability for fear amnesia.

1 is a graph comparing the learning ability of PLCβ4 knock-out mice to be administered T-type calcium channel inhibitors mibefradil and saline to MD:

     -/-Saline: PLCβ4 knock-out mice to administer saline to MD; And

    Mibepradil: PLCβ4 knock-out mice to be administered mibepradil to MD.

FIG. 2 is a graph showing the horror memory erasing learning ability of PLCβ4 knock-out mice administered mibepradyl for specific conditioned sounds after 24 hours after conditioning:

     -/-Saline: PLCβ4 knock-out mice administered saline to MD;

     Mibepradil: PLCβ4 knock-out mice administered with mibepradil to MD; And

    1 hour: time of administration and one hour after administration, sound stimulation is applied.

FIG. 3 is a graph showing the fear memory recall recall ability of PLCβ4 knock-out mice administered mibepradil for a specific conditioned sound following 24 hours after fear memory annihilation learning:

     -/-Saline: PLCβ4 knock-out mice administered saline to MD;

     Mibepradil: PLCβ4 knock-out mice administered with mibepradil to MD; And

    1 hour: time of administration and one hour after administration, sound stimulation is applied.

Figure 4 is a graph comparing the learning ability of normal mice to administer T-type calcium channel inhibitors mibepradil and saline to MD:

     + / + Saline: normal mice to be given saline to MD; And

     + / + Miberadil: Normal mice to receive mibepradil at MD.

FIG. 5 is a graph comparing the learning ability of normal mice to be administered T-type calcium channel inhibitors, eponidipine and 50% DMSO to MD:

     + / + 50% DMSO: normal mice to receive 50% DMSO in MD; And

     + / + Eponidipine: Normal mice to receive eponidipine in MD.

FIG. 6 is a graph showing the fear memory decay learning ability of normal mice administered mibepradil to a specific conditioned sound after 24 hours after conditioning:

     + / + Saline: normal mice receiving saline on MD; And

     + / + Miberadil: Normal mice administered Mibepradil to MD.

FIG. 7 is a graph showing the fear memory recall recall ability of normal mice administered mibepradil to a specific conditioned sound following 24 hours after fear memory loss learning:

     + / + Saline: normal mice receiving saline on MD; And

     + / + Miberadil: Normal mice administered Mibepradil to MD.

FIG. 8 is a graph showing the fear memory decay learning ability of normal mice administered eponidipine for specific conditioned sounds following 24 hours after conditioning:

     + / + 50% DMSO: normal mice administered 50% DMSO to MD; And

     + / + Eponidipine: Normal mice receiving eponidipine from MD.

FIG. 9 is a graph showing the fear memory recall recall ability of normal mice administered with eponidipine for specific conditioned sounds following 24 hours after fear memory loss learning:

     + / + 50% DMSO: normal mice administered 50% DMSO to MD; And

     + / + Eponidipine: Normal mice receiving eponidipine from MD.

<110> Korea Institute of Science and Technology <120> A Method for treatment of anxiety disorder by regulating T-type          calcium channel <130> 8p-04-02 <150> KR1020080007203 <151> 2008-01-23 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 3528 <212> DNA <213> Mus musculus <400> 1 atggccaaac cttacgaatt taactggcag aaggaagtgc cctctttctt gcaagaagga 60 gcagtttttg acagatacga agaggaatct tttgtgtttg agcccaactg cctcttcaaa 120 gtagatgaat tcggcttctt cctgacgtgg aagagtgaag gcaaggaagg acaagtgcta 180 gaatgttccc tcatcaacag tattcgccaa gcagccatac caaaggatcc caaaatcctg 240 gctgctctcg aagctgttgg aaaatctgaa aatgatctgg aagggcggat attgtgtgtc 300 tgcagcggca cggatctggt gaatatcggc ttcacttaca tggtggctga aaatccagaa 360 gtaactaagc aatgggtaga aggcctgaga tcgatcattc acaacttcag ggcaaacaac 420 gtcagtccga tgacatgcct caagaaacac tggatgaaac tggcctttct gaccaacaca 480 actggtaaaa tcccagtgag gagtatcact agaaccttcg catcagggaa aacagaaaag 540 gtgatctttc aagccctcaa ggaactaggt cttcccagtg gaaagaatga tgaaattgaa 600 cctgctgcat ttacttatga aaagttctat gaactgacac aaaagatttg tcctcggaca 660 gatatagaag atctttttaa aaaaatcaat ggagacaaaa ctgattattt aacggtagac 720 caattagtga gctttctaaa cgaacatcag cgagatcctc ggctgaatga aattttattc 780 ccattttatg atgctaaaag agcaatgcag atcattgaaa tgtatgagcc tgatgaagag 840 ctgaagaaaa aaggcctcat atccagtgat ggattctgca gatatctgat gtcagatgaa 900 aatgcccctg tcttcttaga tcgcttagaa ctttaccagg agatggacca cccgctggct 960 cattacttca tcagttcctc ccacaacacc tatctcactg gccggcaatt tggaggaaag 1020 tcttcagtgg aaatgtacag acaagttctc ctggctggtt gcaggtgtgt tgaacttgac 1080 tgttgggatg gaaaaggtga agatcaggaa ccgataataa ctcacggaaa agcaatgtgt 1140 acagacatcc tttttaagga tgtaatccag gccatcaagg aaacggcgtt tgtcacatca 1200 gaataccctg tcattctctc cttcgaaaac cactgcagca aatatcaaca gtacaagatg 1260 tccaagtatt gtgaagatct atttggggat ctcctgttga aacaagcact tgagtcgcat 1320 ccacttgaac caggaaggcc cttgccgtct cctaatgacc tcaaaagaaa aatactcatc 1380 aagaataaga ggctgaagcc tgaagttgaa aagaaacagc ttgaagcttt gaaaagcatg 1440 atggaagctg gagagtcagc cgccccagct agcatcttgg aagacgacaa tgaagaggaa 1500 atagaaagtg ctgatcaaga ggaagaagcc caccctgaat acaaatttgg aaatgaactt 1560 tctgccgatg actacagtca caaggaagcg gttgcaaaca gcgtcaagaa gggcctggtc 1620 accgtagagg atgagcaagc atggatggca tcttataaat acgtaggtgc taccacgaac 1680 atccatccgt acttgtccac gatgatcaac tatgcccagc ccgtgaagtt tcaaggtttc 1740 cacgtggctg aagagcgcaa tattcactat aacatgtctt cttttaacga gtcggttggc 1800 cttggctact tgaagacgca cgcgattgag tttgtaaatt acaataagcg acaaatgagc 1860 cgcatttacc ccaagggagg ccgagttgat tccagtaatt acatgcctca gattttctgg 1920 aacgctggtt gccagatggt ttcactgaac tatcaaaccc cagatttagc gatgcaattg 1980 aatcaaggaa aatttgagta taatggatca tgcgggtacc ttctcaagcc agatttcatg 2040 aggcggcctg atcggacatt tgaccccttc tctgaaaccc ctgtggacgg ggttattgca 2100 gccacgtgct cagtgcaggt tatatcaggg cagttcctct cagataagaa gatcgggaca 2160 tacgtggaag tcgatatgta cgggctgccc accgacacca tacggaaaga gttccgaacc 2220 cgcatggtta tgaacaatgg actcaaccca gtgtataatg aagaatcgtt tgtgtttcga 2280 aaggtgatcc tgcctgacct agctgtcctg agaatcgcag tctacgatga caacaacaag 2340 ctaattggcc agaggatcct tcctttggat ggtctccaag caggctaccg acacatctcc 2400 ctgagaaacg agggaaacaa accattatca ctgccaacaa ttttctgcaa tattgttctt 2460 aaaacatacg tgcctgatgg atttggagat attgtggatg ctttatccga tccaaagaaa 2520 tttctttcaa tcacagagaa gagagcagac caaatgagag caatgggcat tgaaactagt 2580 gacatagcag atgtgcccag tgacacttcc aaaaatgaca agaaaggcaa ggccaaccca 2640 gccaaagcga acgtgacccc tcagagcagc tctgagctca gaccaaccac cacagccgcc 2700 ctgggctctg gccaggaagc caagaaaggt attgaactta tccctcaagt gaggatagaa 2760 gatttaaagc aaatgaaggc ttacttgaag catttaaaga aacaacagaa ggagttaaac 2820 tctttaaaga agaaacatgc aaaggagcac agtaccatgc agaagttaca ctgcacacaa 2880 gttgacaaaa tcgtggccca gtatgacaaa gagaagtcga ctcatgagaa aatcctagag 2940 aaggcgatga agaagaaggg gggaagtaat tgtcttgaaa taaaaaaaga aacagaaatt 3000 aaaattcaga ccctgacaac ggatcacaaa tctaaggtca aagagattgt ggcccagcac 3060 acaaaggagt ggtcagaaat gatcaacact cacagtgcgg aggagcagga aatccgggat 3120 ctgcacctga gccagcagtg tgagctgctg agaaagctgc tcatcaatgc tcatgagcag 3180 cagacccagc agctgaaact ctcccatgac agggaaagca aggagatgag agcccatcag 3240 gctaagattt ctatggaaaa tagcaaggcc atcagtcaag ataaatctat caagaacaag 3300 gcagaacggg aaaggcgagt cagggagttg aacagcagca acactaagaa gttcctagaa 3360 gaaagaaaga gactggcgat gaagcagtca aaagaaatgg atcagttgaa aaaagtccag 3420 ctggagcacc tagaattcct agagaaacag aacgagcagg cgaaggagat gcagcagatg 3480 gtgaaattgg aagccgagat ggaccgcaga ccagcaacag tagtatga 3528 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer k1 <400> 2 ctccacactc tgcaacctac 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer k9 <400> 3 agttacttct ggattttcag cc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer PFK22 <400> 4 ctgactaggg gaggagtaga ag 22  

Claims (12)

Composition for the prevention and treatment of anxiety disorders containing T-type calcium channel inhibitor. The method of claim 1, wherein the T-type calcium channel inhibitor is mibefradil, tetramethrin, ethosuximide, SUN-N8075, eponidipine and Y ^{3 +} , ^{3 +} La, Ce ^{+ 3,} Nd ^{+ 3,} Gd ^{+ 3,} Ho ^{+ 3,} Er ^{3 +} 3 and the metal ion is selected from the group consisting of ^{Yb 3+, 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) And penfluridol, fluspirilene and valproate. The method of claim 1, wherein the anxiety disorder is a phobic disorder, a generalized anxiety disorder, an obsessive compulsive disorder, a post-traumatic stress disorder, or a somatoform disorder. disorders, dissociative disorders, and factitious disorders. The composition of claim 1, wherein the composition additionally contains a carrier capable of crossing the cerebrovascular barrier. The composition of claim 1, wherein the T-type calcium channel inhibitor is a compound capable of crossing the cerebrovascular barrier. The composition of claim 1, which is administrable orally or parenterally. 7. The composition of claim 6, wherein the parenteral administration is systemic or topical administration. 8. The composition of claim 7, wherein the topical administration is administration into the dorsal medial medial nucleus (MD). 2. The composition of claim 1, wherein the composition is capable of preventing and treating anxiety disorder by significantly restoring the memory extinction ability. A method of treating anxiety disorder comprising administering a pharmaceutically effective amount of a T-type calcium channel inhibitor to a subject with anxiety disorder. A method of preventing anxiety disorder comprising administering to a subject a pharmaceutically effective amount of a T-type calcium channel inhibitor. Use in the manufacture of a medicament containing a T-type calcium channel inhibitor for the prevention and treatment of anxiety disorders.

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