KR100861696B1 - Method for screening of anti-depression agents - Google Patents

Abstract

The present invention relates to a method for screening an antidepressant using an N-type calcium channel, more specifically, to determine whether the test compound or composition specifically inhibits the N-type calcium channel; And administering a test compound or composition which is found to specifically block an N-type calcium channel to an experimental animal to determine whether the symptoms of depression are reduced. The method of the present invention can be usefully used for the screening of new mechanisms for treating depression.

Description

Method for screening of anti-depression agents

1 is a schematic diagram showing the α1B locus, the hit vector and the hit locus,

A: normal α1B locus,

B: a hit vector containing an IS3 moiety substituted with GFP-Neo and containing the tk gene,

* C: locus hit by the hit vector,

□: section to be used as a probe,

* A of FIG. 2 is the result of PCR confirmed the calcium ion channel α1B − / − genotype for the genetically modified mouse of the present invention,

Lane 1: marker, lane 2: normal mouse (+ / +),

Lane 3: heterozygous mouse (+/-), lane 4: homozygous mouse (-/-),

FIG calcium channel α1B for B are transgenic mice of the present invention at 2 - / - genotype, and the results confirmed by Southern blot analysis (southern blot analysis),

Lanes 1 and 2: normal mice (+ / +), lanes 3 to 5: heterozygous mice (+/-),

Lanes 6 and 7: homozygous mice (-/-),

C of Figure 2 shows the results confirmed by Western blot analysis (western blot analysis) a not a calcium ion channel α1B protein expressed in the brain tissues of transgenic mice of the present invention,

Lane 1: cerebellum of normal mice, lane 2: cerebellum of genetically modified mice,

Lane 3: cerebral of normal mouse, lane 4: cerebral of genetically modified mouse,

3 is a graph showing the results of forced swimming test of α1B gene mutant mice and normal mice in which N-type calcium channel activity was inhibited.

4 is a graph showing tail suspension test results of α1B mutant mice and normal mice in which N-type calcium channel activity was inhibited.

The present invention relates to a method of screening for an antidepressant.

Voltage-dependent calcium channels play an important role in regulating neurotransmitter secretion, membrane excitability and gene expression, and several types of voltage-dependent calcium channels have been identified to date (Catterall, Cell). Calcium , 1998 , 24, 307-323). Voltage-dependent calcium channels have a multi-subunit structure consisting of subunits of α1, α2, β and γ (Dunlap et al ., Trends Neurosci ., 1995 , 18, 89-98; De Waard et al ., Ion Channels, 1996, 4, 41-87 ;.... Hofmann et al, Rev. Physiol Biochem Pharmacol, 1999, 139, 33-87), L- type according to the electrical physiological or pharmaceutical properties, N- type , P-type, Q-type, R-type and T-type (Tsien et al., Soc . Gen. Physiol . Ser . 1987 , 41, 167-187; Randall and Tsien, J. Neusci . , 1995 , 15, 2995-3012). In general, L-type, N-type, P / Q-type, and R-type have the characteristic of opening the passage at high voltage, and of the T-type having the characteristic of opening the passage at low voltage. Among the subunits of voltage-dependent calcium channels, the α1 subunit plays an essential role in the functioning of the calcium pathway and determines the nature of the pathway, and to date six different genes (α1A-α1F) that encode several subgroups of the α1 subunit. (Chin., Exp . Mol . Med ., 1998 , 30, 123-130; Ertel et al ., Neuron , 2000 , 25, 533-535; Hofmann et al ., Rev. Physiol . Pharmacol ., 1999 , 139, 33-87).

The presence of α1 subunits in the rat brain through in situ hybridization shows that α1B subunits are expressed throughout the central nervous system, especially the dorsal raphe and the auditory tract. (locus coeruleus) sites are also known (TanaKa et al ., Brain Res. Mol . Brain Res ., 1995 , 30, 1-16). At the intracellular level, α1B subunits are primarily located at the ends of the presynapse of neurons and play a physiological function that regulates the release of various neurotransmitters (Miller, Science , 1987 , 235, 46-52; Hirning et al ., Science , 1988 , 239, 57-61; Westenbroek et al ., J. Neurosci ., 1995 , 15, 6403-18), as well as serotonin and norepinephrine receptors at presynaptic or postsynapse It is known to modulate intrasynaptic neurotransmission by participating in the inhibitory regulatory function of (Okada, et al ., J. Neurosci . , 2001 , 21, 628-640; Rittenhous AR, J. Neurobiol . , 1999 , 40, 137-148; Sun, QQ, et al ., J. Physiol . , 1998 , 510, 103-20).

Recently, several genetics have been identified to determine the exact role of N-type calcium channels in the processing of nociceptor information in vivo using mutant mice that lack the α1B subunit of N-type calcium channels as a gene targeting method. Results of studies have been reported (Kim et al ., Mol . Cellular Neuroscience , 2001 , 18, 235-245; Saegusa, H. et al , EMBO J. , 2001 , 15, 2349-56; Hatakeyama, S. et al , Neuroreport , 2001 , 8, 2423-7). According to the above documents, α1B mutant mice showed significantly reduced pain response to mechanical pain, radiant fever and inflammatory pain than normal mice, and also hypersensitivity to pain and heat for harmless stimuli caused by nerve damage. Pain was remarkably reduced compared to normal mice. Thus, it can be seen that N-type calcium channels play an important role in recognizing pain at the level of spinal cord and spinal cord abnormalities. In addition, the elevated plus maze test reported that the α1B mutant mice had less anxiety than normal mice (Saegusa, H. et al , EMBO J. , 2001 , 15, 2349-56). ). However, no pharmacological or genetic studies have yet been reported to elucidate the precise role of N-type calcium channels in emotional disorders such as depression.

In this regard, the present inventors performed various depression-related animal behavioral experiments to investigate the association between depression, which is a representative emotional disorder, and the physiological function of N-type calcium channel, using a mutant mouse targeting the N-type calcium channel α1B gene. The present invention has been completed by finding that depression of N-type calcium channel activity is reduced and depression symptoms are reduced.

It is an object of the present invention to provide a method for screening antidepressants.

In order to achieve the above object, the present invention provides a method for screening antidepressants using N-type calcium channel.

Hereinafter, the present invention will be described in detail.

I. The present invention provides a method of reducing depression by inhibiting the activity of N-type calcium channels.

In the above method, the method of inhibiting the activity of the N-type calcium channel is administered to a compound that specifically inhibits the activity by acting on the N-type calcium channel, or an antibody that specifically binds to the N-type calcium channel Or a method of inhibiting transcription of a gene encoding an N-type calcium channel, or inhibiting translation of a transcribed N-type calcium channel gene, but is not necessarily limited thereto. Any method of inhibiting the activity of the N-type calcium channel can be used. In a preferred embodiment of the present invention, the correlation between the inhibitory activity of the N-type calcium channel and antidepression using mice that do not express the N-type calcium channel due to the α1B gene encoding the N-type calcium channel (See FIGS . 3 and 4 ).

To determine whether N-type calcium channels are associated with depression, a representative emotional disorder, the present inventors used a forced swimming test, a representative depression-related animal behavior experiment, using α1B transgenic mice (Korean Patent Application No. 2002-2343). The forced swimming test and the tail suspension test showed that for the forced swimming test, the mice were swimmed for 15 minutes in a beaker of water. (immobile floating position), while α1B mutant mice continued to swim for 15 minutes, indicating that the floating time on water was significantly shorter than normal mice, indicating that α1B mutant mice were significantly more depressed than normal mice. It was confirmed to show a reduced response (see FIG. 3 ). In addition, in the case of the tail suspension test, α1B mutant mice showed a significantly reduced immobilization time than the normal mice, similarly to the forced swimming test (see FIG. 4 ). As described above, α1B mutant mice that were hit with genes encoding N-type calcium channels showed antidepressant responses in two depression-related behavioral experiments.

Recently, abnormalities of monoamines such as serotonin and noreponephrine or the substance P (substance P), which are peptides, are associated with depression and pathologically physiologically. It is currently being developed as a treatment for depression (Okada, et al ., J. Neurosci . , 2001 , 21, 628-640; Rittenhous AR, J. Neurobiol . , 1999 , 40, 137-148; Sun, QQ, et al . , J. Physiol . , 1998 , 510, 103-20). Α1B mutant mice with inhibitory N-type calcium channel activity include selective serotonin reuptake inhibitors (SSRI), norepinephrine reuptake inhibitors (tricyclics), primary amine oxidase inhibitors, substance P (substance P) or NK-1 receptor inhibitors, such as when treated with antidepressant treatment shows the same behavioral response. Serotonin 1A receptor mutant mice are known to have increased anxiety and reduced depression than normal mice, and norepinephrine transporter mutant mice have a reduced depression behavioral response than normal mice (Ramboz, S. et al ., Proc . Natl . Acad . Sci . USA , 1998 , 95, 14476-81; Xu, F. et al ., Nature Neuroscience , 2000 , 3, 465-471).

In addition, the inhibition of substance P (substance P), which is known to be secreted by N-type calcium channels, and its receptor, NK-1 receptor, associated with pain at the spinal cord level, reduces depression or anxiety. Genetic studies have been reported (Santarelli, L. et al ., PNAS USA , 2001 , 98, 1912-17). However, to date, no studies have shown that N-type calcium channels are associated with depression. Therefore, the present inventors found that depression of the N-type calcium channel was reduced through the animal behavior experiments related to depression using genetically modified mice for the first time.

Other studies of calcium channels related to mood regulation have shown that nimodepine, an L-type calcium channel inhibitor, is an ultra-rapid-cycling bipolar among bipolar disorders. And the ability to relieve simple recurrent depression, and the results suggest that the calcium channel α2-delta inhibitors, gabapentin and lithium, may also be candidates for treating these emotional disorders. (Post, RM, et al ., Bipolar Disord ., 2000 , 2, 305-15; Goodnick, PJ, Biopolar Disord ., 2000 , 2, 165-73). However, no in vivo function of calcium channels related to depression has been reported at all except for the results of the present invention, and the inventors for the first time found that inhibition of N-type calcium channel activity is associated with depression reduction.

In conclusion, inhibition of N-type calcium channel activity by inhibiting the expression of α1B protein results in an antidepressant response. Therefore, N-type calcium channel, which is a substance that inhibits the activity of N-type calcium channel gene or a product of the gene, is inhibited. It can be seen that a substance that inhibits the activity of can be used as a therapeutic agent for depression.

II. The present invention provides a method for screening antidepressants using the α1B gene of the N-type calcium channel or a protein thereof.

If you look for a substance that inhibits the activity of α1B, it can be used as an antidepressant. Therefore, the substance which suppresses the activity of (alpha) 1B can be screened by the well-known method of screening the substance which inhibits the activity of a protein. The general method of screening for substances that inhibit the activity of α1B is

1) obtaining a transformant by introducing a vector containing the α1B structural gene and a reporter gene into a host cell;

2) culturing the transformant and the sample to be screened together; And

3) measuring the expression level of the reporter gene.

LacZ, GFP, luciferase and the like may be used as a gene that can be used as a reporter gene in the above, but it is obvious to those skilled in the art that is not necessarily limited thereto.

In addition, as another embodiment,

The present invention comprises the steps of determining whether the test compound or composition specifically inhibits N-type calcium channels; And administering a test compound or a composition identified to specifically block an N-type calcium channel to an experimental animal to determine whether the symptoms of depression are reduced.

At this time, whether the specific compound or composition inhibits the N-type calcium channel can be confirmed by the conventional method, it is preferable to use a method of measuring the flow of current through the channel, such as a patch clamp, but is not limited thereto. .

Depression of the experimental animals is preferably confirmed through a forced swimming test or tail hanging test, but is not limited thereto.

III. The present invention provides an antidepressant containing an inhibitor of N-type calcium channel as an active ingredient.

In the above, the N-type calcium channel inhibitor is a compound that specifically inhibits activity by acting on the N-type calcium channel, an antibody specifically binding to the N-type calcium channel, a gene encoding the N-type calcium channel There is a substance that inhibits the transcription of, a substance that inhibits the translation of the transcribed N-type calcium channel gene, etc., but is not necessarily limited thereto, and all substances that inhibit the activity of the N-type calcium channel in addition to the substances listed above This can be used.

The inhibitor of the N-type calcium channel can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. Preferred pharmaceutical preparations include oral preparations such as tablets, hard or soft capsules, solutions, suspensions and the like, and parenteral injections such as injectable solutions or suspensions, which are pharmaceutically acceptable. Conventional carriers, for example oral preparations, excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives or extenders, etc., in the case of injections are commonly used fillers, extenders, binders, wetting agents And diluents such as disintegrants and surfactants, or excipients. In addition, preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the inhibitor of the N-type calcium channel may be used in admixture with a number of carriers (pharmaceutically acceptable) such as physiological saline or organic solvents, such as glucose, sucrose or dextran to increase the stability or absorption Antioxidants such as carbohydrates, ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

In the pharmaceutical composition of the present invention, the total effective amount of the inhibitor of N-type calcium channel may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may also be administered by long-term fractionated treatment protocols. Since the concentration of the inhibitor of the N-type calcium channel is determined in consideration of various factors such as the age and health condition of the patient, as well as the route of administration and the number of treatments of the drug, in view of this point, Those skilled in the art will be able to determine appropriate effective dosages for the particular use of the apisidine derivatives and pharmaceutically acceptable salts thereof as pharmaceutical compositions.

The preferred effective dose of the inhibitor of the N-type calcium channel is 0.014 to 0.14 μg / kg for injection, 0.14 to 1.4 μg / kg for oral administration and may be administered 1 to 6 times per day (Richard DP and Judith AP Pain , 2000 , 85, 291-296).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Production Example  1> Preparation of α1B Gene Mutant Mice

<1-1> Calculation of Hit Vector Deleting Calcium Ion Channel α1B Gene

The present inventors used a commonly used gene targeting method to prepare genetically modified mice lacking the calcium ion channel α1B gene. First, a bacteriophage lamda FIX II (Stratagene) library was randomly inserted with 129 / sv mouse genomic DNA fragments to obtain mouse genomic DNA containing the α1B gene. The α1B rat cDNA (AUG to 720 bp, Dr. Screened using Jin HM (NIH) as a probe. Through the screening, a bacteriophage clone having an 18.4 kb genomic DNA containing the α1B gene intervening segment (IS) 1-3 coding region was obtained. The restriction enzyme map was completed using various restriction enzymes, and then used to generate a hit vector. . As the base vector, pBluescript II KS (+/-) (Stratagene) was used as the base vector, and the exon (amino acid 158) was obtained by cutting the GFP (green fluorescent protein) gene containing SV40-polyA from pEGFP-1 (Clonetech) vector. Fusion to BamH I-tag IS2 comprising. The PGK-neo gene was cut from the pLNT vector (obtained from Sugitaui Noda) and linked to the lower part of the GFP gene.

As a result, a fragment containing the IS3 moiety was deleted, and a hit vector prepared with a GFP-NEO cassette for positive selection was prepared ( FIG. 1 ). For negative selection, the HSB-tk gene was linked to the 5 'end of the 5'-homologous region.

<1-2> Into embryonic stem cells  Transduction

J1 embryonic stem cell line was used as a cell line for transducing the hit vector prepared from Preparation Example <1-1>. Embryonic period cell culture and embryo manipulation were performed in the same manner as in the previous literature (Kim et al ., Nature , 1997, 389: 290-293). J1 embryonic stem cells (available from R. Jeanisch, MIT, USA) were cultured in ES medium [15% fetal bovine serum, Hyclone Co., 1 × penicillin-streptomycin (Gibco Co.), 1 × non-essential amino acid (Gibco Co.), 0.1 mM 2-mercapto tanol (mercaptoethanol), DMEM medium (Gibco Co.)] and incubated for 2 to 3 days at 37 ℃. To the germ cell group obtained from the culture was added 1 mM EDTA solution containing 0.25% trypsin was isolated into single cells.

Electroporation was performed to transfect the target vector prepared in Preparation Example <1-1> into embryonic stem cells separated into single cells. Specifically, 2 × 10 ⁷ isolated single embryonic stem cells and 25 μg of the hit vector DNA of Preparation Example <1-1> were added and mixed, followed by electroporation under conditions of 270 V / 500 microF. Embryonic stem cells into which the vector was introduced were cultured for 5 to 7 days in ES medium containing 0.3 mg / ml G418 and 2 μM of gancyclovior, and the calcium ion channel α1B gene in embryonic stem cells was correctly hit by the vector. Embryonic stem cell clones were screened and maintained. Embryonic stem cell clones identified with the deletion of the calcium ion channel α1B gene were further aliquoted into ES medium, cultured for 18 to 22 hours, then treated with trypsin, isolated into single cells, and only surviving cells were used for microinjection.

<1-3> chimera (chimera) Preparation of Mice

In order to prepare chimeric mice with genotypes of calcium ion channel α1B +/−, fertilized embryonic cells were microinjected with the embryonic stem cell clones selected in Preparation Example <1-2>. Specifically, females and males were mated with C57BL / 6J mice (Jackson Laboratory, USA), and females 3.5 days (3.5 p.c.) after mating were sacrificed by cervical dislocation. The uterus is removed from the sacrificed females, and the distal end of the uterus is cut with scissors, then a 1 ml syringe is used to perfuse 1 ml of an injection solution containing 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM. I was. The blastocysts were isolated from the uterine tissue using a micro glass tube under a dissecting microscope, and the separated blastocysts were transferred to a drop of injection solution previously dropped on a 35 mm Petri dish and used for subsequent introduction. In order to introduce the embryonic stem cell clones selected in Preparation Example <1-3> to the isolated embryos, the inner cell mass of the embryos was held by a holding pipette using a microinjector (Zeiss). The injection pipette in which 10-15 embryonic stem cell clones were aspirated with negative pressure) was inserted into the blastocyst of the blastocyst, and the embryonic stem cell clone was injected into the blastocyst of the blastocyst under positive pressure. The clone-implanted blastocysts were mated with a male orthopedic male and transplanted into the uterus of 2.5 pcs of fertility surrogate mice, which is a heterologous cell hybrid type formed from embryonic stem cell clones (J1) and blastocysts of C57BL / 6J mice. The development of chimeric mice was induced. At this time, the uterine transplant is excised by about 1 cm of the abdomen of the surrogate mother anesthetized with avertine (average, 1 mg / kg body weight); 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. The embryos injected with embryonic stem cells were transplanted into the uterus of surrogate mice and cultured for about 19 days, whereby cells derived from embryonic stem cells and cells derived from blastocysts were fused to have a genotype of calcium ion pathway α1B +/- in the genome. Mice were secured.

<1-4> Preparation of Heterozygous Mice with Calcium Ion Channel α1B +/- Genotype

Male chimeric mice obtained from Preparation Example <1-3> were crossed with C57BL / 6J female mice to obtain progeny. In order to select heterozygous mice having a genotype of the double calcium ion channel α1B +/-, PCR (polymerase chain reaction) was performed as follows.

Specifically, in order to extract genomic DNA from mouse offspring, the tail of the mouse was cut to a size of about 1.5 cm, and then the tissue was lysed in lysis buffer (100 mM Tris (pH 8.0), 5 mM EDTA, 200 mM sodium chloride and 0.2% SDS). ) Was added to 0.4 ml, and the proteinase K was added so as to be 0.1 mg / ml and treated at 55 ° C. for 5 hours. 75 µl of 8 M potassium acetate and 0.4 ml of chloroform were added to the reaction solution, followed by shaking for 10 minutes at 4 ° C. The reaction was centrifuged at 15,000 rpm. 0.4 ml of the supernatant was added to 1 ml of ethanol to precipitate genomic DNA, washed with 70% ethanol, dried, and then dissolved in 50 µl of distilled water.

A PCR using the primer 3 is the mouse genomic DNA using a 1 ㎕ as a template and the substrate using the primer 1, SEQ ID NO: 2 Primer 2 SEQ ID NO: 3 is described as described in SEQ ID NO: 1, extracted as described above with each 10 pmol Was performed. At this time, primers 1 and 2 were designed to amplify a 190 bp region of the normal calcium ion channel α1B gene, and primers 1 and 3 were designed to amplify a 320 bp region of the targeted calcium ion channel α1B gene. As the conditions of PCR, the reaction was repeated 40 times for 30 seconds at 94 ° C, 30 seconds at 58 ° C, and 30 seconds at 72 ° C. PCR products obtained therefrom were electrophoresed on 1.5% agarose gel and observed by staining with ethidium bromide (EtBr).

As a result, mice showing two bands of 190 bp size and 320 bp size were selected as heterozygous mice having genotype of calcium ion channel α1B +/− (see lane A of FIG. 2 ).

<1-5> Preparation of Homozygous Mutant Mice with Calcium Ion Channel α1B-/-Genotype

Heterozygote Mice Having a Genotype of Calcium Ion Channel α1B +/- Selected in Preparation Example <1-3> Females and males were crossed to obtain homozygous gene mutants having a calcium ion channel α1B − / − genotype. Southern blot analysis and PCR were performed to confirm that the genetically modified mice had a calcium ion channel α1B-/-genotype in the genome, and Western blot was used to confirm that the calcium ion channel α1B protein was not expressed in the genetically modified mice. The analysis was performed.

First, genomic DNA was isolated from the tail tissue of the transgenic mice in the same manner as in Preparation Example <1-4> for Southern bloy analysis, and then digested with restriction enzyme EcoR I. Hybridization was performed on the mouse DNA using a box portion (□) fragment ( C of FIG. 1 ) obtained from the DNA of Preparation Example <1-1> as a probe. At this time, genomic DNA extracted from normal mice and genomic DNA extracted from heterozygous genetically modified mice having genotypes of calcium ion channel α1B +/- were used.

The result is the same as B of FIG . In the figure, lanes 1 and 2 are control mice (+ / +), lanes 3 to 5 are heterozygous mice (+/-), and lanes 6 and 7 are homozygous mice (-/-). The 8.0 kb band is derived from the normal calcium ion channel α1B gene, and the 14.5 kb band is derived from the targeted calcium ion channel α1B gene. As shown in the figure, the prepared mutated mice showed only 14.5 kb band, from which it was confirmed that they have a genotype of calcium ion channel α1B-/-.

Next, in order to perform a polymerase chain reaction (PCR) analysis, the present inventors separated genomic DNA from the tail tissue of a transgenic mouse in the same manner as in Production Example <1-4>, and then 1 μl of the genomic DNA was used as a template. PCR was carried out using the same primers as in Preparation Example <1-4>.

As a result, only one band of 190 bp was observed in normal mice (+ / +) and two bands of 190 and 320 bp were observed in heterozygous mice (+/-). In addition, in the genetically modified mouse of the present invention, only one 320 bp band was observed, and it was confirmed that the genetically modified mouse had a genotype of calcium ion channel α1B − / − ( FIG . 2A ).

Next, Western blot analysis was performed to confirm that the calcium mutant α1B gene was not actually expressed in the transgenic mice having the genotype of the calcium ion channel α1B − / − gene. . Specifically, cerebral (cerebrum) was isolated by dissecting the skull after the sacrifice of normal and homozygous genetically modified mice by cervical dislocation method. Isolated cerebral tissues were lysed with proteolytic inhibitors (50 mM Tris (pH 7.4), 1 mM EGTA, 1 mM DTT and 1 mM PMSF, proteolytic inhibitor mixture (Boehringer Mannheim), calpine inhibitors I and II) In the ground and ground. The tissue solution was centrifuged at 1,000 rpm for 5 minutes to separate supernatant and deposits, and then only the supernatant was taken and centrifuged at 28,000 rpm for 15 minutes to obtain an approximate cell membrane protein. The cell membrane proteins were electrophoresed on 8-16% acrylamide gels and transferred to nitrocellulose membranes (PROTROL, Schleicher & Schuell). The nitrocellulose membrane was treated with TTBS buffer (10 mM Tris buffer (pH 7.5), 150 mM sodium chloride, 0.05% tween 20) containing 5% skim milk. Subsequently, a hybrid reaction was performed by treating monoantibody α1B (CW21, Vance et al ., J. Biol . Chem ., 1998 , 273, 14495-502) against calcium ion channel α1B. In order to detect a single antibody bound to the calcium ion channel α1B protein, anti-mouse IgG antibody bound to peroxidase was added, followed by color reaction using an ECL kit (Amersham) as a substrate.

The result is the same as C of FIG . In the figure, lane 1 is a cerebellar protein of a normal mouse, lane 2 is a genetically modified mouse, lane 3 is a normal mouse, lane 4 is a cerebral protein of a genetically modified mouse. As shown in the figure, it was confirmed that the calcium ion channel α1B protein was not produced in the transgenic mice. The present inventors deposited the fertilized eggs of the genetically modified mice having the calcium ion channel α1B +/- genotype with the Korea Biotechnology Research Institute Gene Bank on Jan. 8, 2002 (Accession No .; KCTC 10158BP).

Example 1 Measurement of the Effect of N-type Calcium Channel on Depression

In order to observe the response to depression-related behavioral experiments in α1B mutant mice (Accession No .; KCTC 10158BP) with N-type calcium channel removed, the present inventors used a forced swimming test and a tail suspension test using the mice and control mice. Was performed. All animals had free access to food and water in a controlled temperature and humidity environment, and were bred under daytime conditions at 8 am with a 12 hour night / day cycle. Both females and males of the F1 mice were used in the experiment, and mice of 8 to 10 weeks old were used. All behavioral experiments were conducted according to a blind test. All depression-related behavioral tests were conducted between 9 and noon to minimize the effects of the 24-hour cycle, and all mice were used only once for each experiment.

<1-1> Reduction of immobility in forced swimming test

Forced swimming tests were performed by some modified methods with reference to Forsault et al . (Porsolt, RD et al ., Eur. J. Pharmacol ., 1978 , 51 (3): 291-294). Specifically, the present inventors filled 15 l of water in a 4 l plastic beaker (13 cm x 25 cm) containing water and forced the α1B mutant and control mice to swim to avoid inevitable stress depression. And floating time, expressed as a depressive behavioral response, was measured. Measurements were made for a total of 15 minutes, and data were aggregated at 5 minute intervals and the average was used for analysis.

As a result, in the forced swimming test, when mice were swimming in a beaker of water for 15 minutes, normal mice showed an immobile floating position, which was depressive from the initial 5 minutes, whereas α1B mutant mice had a 15 minute duration. The swimming behavior continued, resulting in a significantly shorter overall floating time on the water than normal mice. From the above facts, it was confirmed that α1B mutant mice showed a decreased response to depression than normal mice (see FIG. 3 ).

<1-2> tail hanging test

Tail suspension testing was performed with reference to Steru et al. (Steru, L. et al., Psychopharmacology , 1985 , 85, 367-370). Specifically, the animals were wound with a tape wrapped around the tail of the mouse head at a height of 25 cm from the bottom in a barrel of 35 cm × 40 cm each, and then suspended for 7 minutes in the center of the metal rod. The dead time was measured by. During the initial 1 minute, the violent behavior was shown as a period of intense behavior, and the average value was used for the analysis of the immobility time expressed as the depressive behavioral response for 6 minutes except the initial 1 minute.

As a result, in the case of the tail hanging test, α1B mutant mice showed a significantly reduced immobilization time than the normal mice, similarly to the forced swimming test (see FIG. 4 ). From the above results, it was confirmed that the α1B gene mutant mice exhibited a significantly reduced immobilization reaction compared to the normal mice, thereby significantly reducing depression.

As described above, gene-mutated mice with N-type calcium channel inhibition show a significantly reduced antidepressant response than normal mice, and therefore, whether a specific compound or composition inhibits N-type calcium channel activity. Substances that inhibit the function of the product N-type calcium channel can be usefully used as a therapeutic agent for depression.

<110> KIST <120> Method for screening of anti-depression agents <130> 7p-05-13 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 1 cagtcaggag ctgtcaagcc ac 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 2 ccacgaagtc catgacattc c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 3 cgggcagctt gccggtggtg c 21  

Claims (2)

Determining whether the test compound or composition specifically inhibits the alpha 1B subunit of the N-type calcium channel; And A method for screening a depression drug comprising administering to a test animal a test compound or a composition identified to specifically block the alpha 1B subunit of an N-type calcium channel to determine whether symptoms of depression are reduced. The method of claim 1, wherein the depression of the experimental animal is confirmed by a forced swimming test or a tail hanging test.

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