KR102546560B1 - Pharmaceutical composition for preventing or treating anxiety disorder comprising inhibitor of Slitrk3 and Nlgn2 - Google Patents

Abstract

The present invention relates to a technology capable of treating anxiety disorders by targeting Slitrk3 and Nlgn2, and more particularly, to a pharmaceutical composition for preventing or treating anxiety disorders comprising Slitrk3 and Nlgn2 inhibitors as active ingredients, and a pharmaceutical composition comprising the composition. It's about drugs. According to the present invention, knowledge about neural circuits involved in anxiety symptoms is provided by selectively controlling the properties of inhibitory synapses that regulate the structure and activity of prefrontal neural circuits, and the Slitrk3 and Nlgn2 genes control anxiety symptoms and behaviors. It is expected to be useful as a therapeutic target for the development of controllable new drugs.

Description

Pharmaceutical composition for preventing or treating anxiety disorders comprising inhibitors of Slitrk3 and Nlgn2

The present invention relates to a technology capable of treating anxiety disorders by targeting Slitrk3 and Nlgn2, and more particularly, to a pharmaceutical composition for preventing or treating anxiety disorders comprising Slitrk3 and Nlgn2 inhibitors as active ingredients, and a pharmaceutical composition comprising the composition. It's about drugs.

Anxiety disorder (anxiety disorder) is a collective term for mental disorders that cause difficulties in daily life due to various forms of abnormal and pathological anxiety and fear. Anxiety and fear are normal emotional responses as warning signs of imminent danger, but if they are excessive, they make it more difficult to properly cope with the situation and cause mental pain and physical symptoms. Anxiety stimulates the sympathetic nervous system, resulting in physical symptoms such as headache, increased heart rate, increased respiratory rate, and gastrointestinal abnormalities, causing discomfort, and anxiety, worry, or physical symptoms are associated with daily activities such as work life, interpersonal relationships, and schoolwork. An anxiety disorder can be diagnosed if it causes difficulties. Specifically, anxiety disorders include phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, panic disorder, These include agoraphobia, separation anxiety disorder, and selective mutism. Phobic disorder, a typical anxiety disorder, refers to a disorder in which a person feels irrational fear in a specific object or situation and continuously avoids the object or situation because of this. There are many types depending on the object, such as expectation anxiety. Generalized Anxiety Disorder refers to a case where you feel widespread and persistent anxiety in everyday situations, while Obsessive-Compulsive Disorder is when you keep repeating certain thoughts or actions regardless of your will. There may be. Therefore, obsessive-compulsive disorder is diagnosed when life is disturbed by obsessive-compulsive symptoms or when the mind and body are distressed. Post-traumatic stress disorder is a psychological response that occurs after experiencing life-threatening extreme stress, such as experiencing or witnessing a shocking and frightening event, and continuing to experience the traumatic experience at the time even after the trauma has passed. Memories come up, avoid activities or places that remind you of the trauma, lose control over what will happen in the future, may feel fearful, and show symptoms accompanying autonomic nervous system symptoms. In addition, panic disorder is one of the anxiety disorders in which a severe anxiety attack and various physical symptoms accompanying it suddenly occur without any notice.

Currently, drug treatment using antidepressants and antianxiety drugs is often used in the treatment of anxiety disorders. Anxiolytics are used to immediately relieve anxiety symptoms. For example, the most representative target protein of anxiolytics is the GABA-benzodiazepine receptor of the GABA (γ-aminobutyric acid) system. Benzodiazepines bind to GABA-benzodiazepine receptors to increase the affinity of GABA and increase the influx of chloride ions to act as anti-anxiety. However, caution is needed because benzodiazepines have various side effects such as excessive muscle relaxation, sedation, and dependence.

Anxiety disorders include many mental disorders of different personalities, so it is difficult to define a single cause, but generally, lack or excess of neurotransmitters in the brain circuits responsible for emotional parts such as anxiety or depression, genetically inherited Social psychological aspects, including predisposition, functional or structural changes in the brain revealed in brain imaging studies, and cognitive-behavioral aspects of interpreting and judging past experiences and current received information are the causes of pathological anxiety. It can be. In terms of neuroanatomy, anxiety disorder is presumed to be a function problem of the frontal lobe and limbic system, but it is mainly thought to be the result of dysfunction of emotional centers such as the limbic system rather than higher cognitive function centers such as the frontal lobe. there is. The prefrontal cortex, which is responsible for higher functions, is an important part of planning, making decisions, predicting potential behavioral outcomes, and understanding and regulating social behavior. It is known that the orbitofrontal cortex is responsible for information, impulse control, and mood regulation, and the ventromedial prefrontal cortex is responsible for compensation processes and visceral responses to emotions. In this regard, it is known that post-traumatic stress disorder is mediated by the limbic system including the nucleus accumbens and the amygdala through fear conditioning learning, which is widely used as an animal model thereof. Recently, the involvement of the prefrontal cortex in fear memory recurrence has emerged (Moussawi et al., Nat. Neurosci. 2009 (2):182-9), and it is known that changes in glutamate secretion and receptor expression in the amygdala and prefrontal cortex are important for the maintenance of fear memory. there is.

However, since there is still a lack of understanding of the related neural circuits that regulate anxiety symptoms, their mechanism of action, and the factors that regulate them, further research on the neural circuits involved in anxiety symptoms should be conducted. Based on this, anxiety symptoms And there is a need to develop a treatment that can effectively control related behaviors.

Under the above background, the present inventors analyzed antianxiety behavioral characteristics using 3 mice in which each or both of the Slitrk3 and Nlgn2 genes known to regulate inhibitory synapses were specifically knocked out in the prefrontal cortex region. As a result, in the case of knockout mice in which each gene was deleted, anxiety symptoms were abnormal (abnormal reduction in anxiety symptoms), and when both genes were deleted simultaneously, it was confirmed that abnormal anxiety-related behaviors were restored to normal. The present invention was completed by developing a technique that can be used to treat disorders.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating anxiety disorders, which contains inhibitors of Slitrk3 (SLIT and NTRK like family member 3) and Nlgn2 (Nuroligin 2) as active ingredients.

In addition, another object of the present invention is to provide a pharmaceutical preparation for preventing or treating anxiety disorders, including the pharmaceutical composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating anxiety disorders, comprising inhibitors of Slitrk3 (SLIT and NTRK like family member 3) and Nlgn2 (Nuroligin 2) as active ingredients. to provide.

In one embodiment of the present invention, the inhibitor may be an expression inhibitor of Slitrk3 and Nlgn2 genes or an activity inhibitor of proteins encoded by the respective genes.

In another embodiment of the present invention, the Slitrk3 gene may consist of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the Nlgn2 gene may consist of the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the expression inhibitor is an antisense oligonucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA) that complementarily binds to the mRNA of the Slitrk3 and Nlgn2 genes, respectively. ), it may be any one selected from the group consisting of ribozyme and CRISPR/Cas9.

In another embodiment of the present invention, the activity inhibitor may be any one selected from the group consisting of antibodies, peptides, peptide analogs, aptamers, and compounds that specifically bind to Slitrk3 and Nlgn2 proteins, respectively.

In another embodiment of the present invention, the anxiety disorder is phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, panic It may be any one selected from the group consisting of panic disorder, agoraphobia, separation anxiety disorder, and selective mutism.

In one embodiment of the present invention, the pharmaceutical composition may be for topical administration to the brain.

In another embodiment of the present invention, the local administration may be administered to the prefrontal cortex region of the brain.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating anxiety disorders, including the pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical formulation may be for local administration to the brain.

In another embodiment of the present invention, the local administration may be administered to the prefrontal cortex region of the brain.

In addition, the present invention provides a method for preventing or treating anxiety disorders, comprising administering to a subject a pharmaceutical composition containing Slitrk3 and Nlgn2 inhibitors as active ingredients.

In addition, the present invention provides a use of the pharmaceutical composition for preventing or treating anxiety disorders.

In the present invention, mice in which the Slitrk3 or Nlgn2 genes, which are inhibitory synaptic adhesive proteins, were specifically knocked out in the prefrontal cortex region, and double knockout mice in which all of these genes were deleted were prepared, and the anxiety symptoms in the single knockout mice were abnormally changed. In mice in which both genes were simultaneously knocked out, it was confirmed that these changes were restored to normal levels. Accordingly, according to the present invention, knowledge about neural circuits involved in anxiety symptoms is provided by selectively controlling the properties of inhibitory synapses that regulate the structure and activity of prefrontal neural circuits, and the Slitrk3 and Nlgn2 genes are associated with abnormal anxiety symptoms and It is expected to be useful as a therapeutic target for the development of new drugs that can control behavior.

Figure 1 shows the results of constructing medial prefrontal cortex (mPFC ⁾ -specific ^Slitrk3 conditional knockout (cKO) mice. ^/f Results showing adeno-associated virus (AAV)-mediated delivery and expression of EGFP-tagged active and inactive Cre-recombinant enzymes via topical administration into the mPFC in mice (ACC: anterior cingulate cortex; IL: infralimbic cortex; PL: prelimbic cortex), Figure 1c shows the PCR genotyping results of WT, Slitrk3 ^f/+ (Het) and Slitrk3 ^f/f mice, Figure 1d Illustrates the position of amplification using forward and reverse primers used for genotyping in FIG. 1c.
Figure 2 shows quantitative immunoblot results using brain tissue samples from each knockout mouse according to the present invention, and Figures 2a to 2c show mPFC-specific Slitrk3 ^f/f (Figure 2a) and Nlgn2 ^f/f (Figure 2a), respectively. 2b) or Slitrk3/Nlgn2 ^f/f (FIG. 2c) shows the results of immunoblot analysis of the expression levels of synapse-related proteins in mice and their quantitative results.
Figure 3 is a result confirming that knockout (cKO) of Slitrk3 in the mPFC induces loss of inhibitory synapses, Figures 3a to 3d are the anterior precingulate cortex (ACC), limbic subcortex (IL) and ΔCre and Cre mice Expression of excitatory or inhibitory synaptic markers, GAD65 (Fig. 3a), GABAAγ2 (Fig. 3b), GABAAα2 (Fig. 3b), and VGLUT1 (Fig. 3d), respectively, in the prelimbic cortex (PL) was analyzed by immunohistochemical staining. and the results of quantifying the puncta density and size of each marker protein (*** p <0.001).
Figure 4 is a result of confirming that knockout of Nlgn2 in the mPFC induces loss of inhibitory synapses, Figures 4a to 4d are the anterior precingulate cortex (ACC), limbic subcortex (IL) and prelimbic cortex of ΔCre and Cre mice The expression of GAD65 (Fig. 4a), VGLUT1 (Fig. 4b), GABAAγ2 (Fig. 4c) and GABAAα2 (Fig. 4d) in (PL) was analyzed by immunohistochemical staining method, and puncta density and size of each marker protein were analyzed. The quantified results are shown (* p <0.05, *** p <0.001, **** p <0.0001).
Figure 5 is a result confirming that Slitrk3 / Nlgn2 double knockout does not affect inhibitory synapses in the mPFC, and Figures 5a to 5d show the anterior precingulate cortex (ACC), limbic subcortex (IL) and The expression of VGLUT1 (Fig. 5a), GAD65 (Fig. 5b), GABAAα2 (Fig. 5c) and GABAAγ2 (Fig. 5d) in the prelimbic cortex (PL) was analyzed by immunohistochemical staining, respectively, and the puncta density of each marker protein. And it shows the result of quantifying the size.
Figure 6 briefly shows the implementation strategy for the behavioral experiments of each of the three types of knockout mice according to the present invention.
Figure 7 is a result confirming that instinctive anxiety-related behaviors are reduced in mPFC-specific Slitrk3 knockout mice, Figure 7a is a heat map image of open-field experiment results in ΔCre and Cre mice and the total distance recorded in each mouse (Total distance), velocity (Velocity), frequency of moving to the center area (Frequency), and time spent in the center (Time in center) are quantitative results, and FIG. Map image and quantitative results for open arm entries and time in open arm, and FIG. 7c is a heat map image of Y-maze test results and spontaneous alternation (Spontaneous Alternation) in ΔCre and Cre mice. alternation) and total arm entries, and FIG. 7d is a schematic diagram of the progress of the contextual fear conditioning experiment and the results of quantifying the percentage of freezing of mice in each condition. FIG. 7e Shows the heat map image of the 3-chamber test results, and the quantitative results of socialability and social recognition time (**p < 0.01, ***p < 0.001).
Figure 8 is a result confirming that instinctive anxiety-related behaviors are reduced in mPFC-specific Nlgn2 knockout mice, Figure 8a is a heat map image of open-field experiment results in ΔCre and Cre mice and the total distance recorded in each mouse , Quantitative results for speed, frequency of moving to the central region and time staying in the center, and FIG. 8b is a heat map image of the results of the cross-type elevated maze experiment in ΔCre and Cre mice, and quantitative results for the number of entries into the open arm and the time spent staying Figure 8c is a heat map image of Y-maze test results in ΔCre and Cre mice and quantitative results for voluntary alternation and total number of arm entries, and Figure 8d is a schematic diagram of the progress of the contextual fear conditioning experiment and the mice in each condition The freezing response was quantified as a percentage, and FIG. 8e shows the heat map image of the 3-chamber test results, and the quantitative results of sociability and social cognition time (**p < 0.01, ***p < 0.001).
9 is a result of confirming the recovery effect of instinctive anxiety-related behaviors to a normal level in mPFC-specific Slitrk3/Nlgn2 double knockout mice. FIG. 9a is a heat map image of open-field experiment results in ΔCre and Cre mice. Quantitative results for the total distance, speed, frequency of entry into the center, and time of stay in the center recorded in each mouse, and FIG. Figure 9c is a heat map image of Y-maze test results in ΔCre and Cre mice and quantitative results for voluntary alternation and total number of arm entries. Figure 9d is a schematic diagram of the progress of the contextual fear conditioning experiment and 9e shows the heat map image of the 3-chamber test result, and the quantitative results of sociality and social cognition time (**p < 0.01, *** p < 0.001).

The present inventors constructed three types of conditional knockout (cKO) mice specific to the prefrontal cortex region, that is, Slitrk3 cKO mice, Nlgn2 cKO mice, and Slitrk3/Nlgn2 double cKO mice, and used them to treat anxiety symptoms by knockout of Slitrk3 and Nlgn2. A normal recovery effect was confirmed, and based on this, the present invention was completed.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating anxiety disorders, comprising inhibitors of Slitrk3 (SLIT and NTRK like family member 3) and Nlgn2 (Nuroligin 2) as active ingredients.

The present inventors confirmed through in vivo experiments that the effect of restoring anxiety symptoms to normal appears when Slitrk3 and Nlgn2 genes are suppressed together through specific examples below.

Specifically, in one embodiment of the present invention, a prefrontal lobe-specific conditional knockout (cKO) mouse was prepared, specifically, a mouse in which Slitrk3 and Nlgn2, GABA synaptic adhesive proteins, were specifically knocked out in the medial prefrontal cortex, and a Slitrk3/Nlgn2 double Knockout mice were prepared (see Example 2), and immunoblot was performed on the brain tissue samples of each knockout mouse to confirm that the target gene was knocked out, thereby confirming that the knockout mice were well prepared (see Example 3).

In another embodiment of the present invention, changes in inhibitory synaptic and excitatory synaptic markers were confirmed as a result of knockout of each target gene using brain tissue sections of the three knockout mice (see Example 4). Various mouse behavior experiments were conducted using each of the three types of knockout mice, and it was confirmed in detail that there was a therapeutic effect of recovering anxiety symptoms in double cKO mice in which both Slitrk3 and Nlgn2 were knocked out (see Example 5). The gene or each protein encoded by the gene has been identified as a target for treating anxiety disorders.

In the present invention, the inhibitor may be an expression inhibitor of Slitrk3 and Nlgn2 genes or an activity inhibitor of proteins encoded by the respective genes.

As used in the present invention, the term "expression inhibitor" means to cause a decrease in protein expression of a target gene. In the present invention, expression inhibitors include suppressing the expression of Slitrk3 and Nlgn2 genes as proteins, and specifically, antisense oligonucleotides and small interfering RNAs that complementarily bind to mRNAs of Slitrk3 and Nlgn2 genes; siRNA), short hairpin RNA (shRNA), ribozyme, and CRISPR/Cas9, but may be any one selected from the group consisting of, but is not limited thereto.

The "antisense oligonucleotide" refers to an oligonucleotide capable of controlling the expression of a target gene by hybridizing to a target nucleic acid, in particular, to an adjacent sequence on the target nucleic acid, and differs from siRNA and shRNA in that it is not essentially double-stranded. there is.

The "small interfering RNA (siRNA)" refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or a gene therapy method. In the present invention, the target genes include Slitrk3 and Nlgn2.

The "short hairpin RNA (shRNA)" means a single strand consisting of 50-60 nucleotides, and forms a stem-loop structure in vivo . That is, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference. Long RNAs of 15-30 nucleotides complementary to both sides of the loop of 5-10 nucleotides are base-paired to form a double-stranded stem. shRNAs are transduced into cells via a vector containing a U6 promoter to ensure constitutive expression and are usually passed on to daughter cells to allow inheritance of gene repression. The shRNA hairpin structure is cleaved by intracellular mechanisms to become siRNA, which then binds to RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.

The term “CRISPR/Cas9” refers to a technology that induces gene editing by cleaving a target gene using sgRNA (single-guide RNA) that recognizes the DNA of a specific gene and Cas9 (CRISPR associated protein 9), a nuclease. . The target gene cleaved by Cas9 nuclease is recovered by the cell's DNA repair system, typically through the non-homologous end joining (NHEJ) pathway. Deletion (frame shift) phenomenon due to the generation of indels causes gene knock-out (knock-out) results.

As used herein, the term “activation inhibitor” refers to causing a decrease in the function of a target protein, and preferably means that the function of the target protein becomes undetectable or present at an insignificant level. In the present invention, activity inhibitor means to inhibit the inherent intracellular function of Slitrk3 and Nlgn2 proteins, and specifically, antibodies, peptides, peptide analogs, aptamers and compounds that specifically bind to Slitrk3 and Nlgn2 proteins It may be any one selected from the group consisting of, but is not limited thereto.

The “antibody” includes an immunoglobulin molecule immunologically reactive with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies), heterologous antibodies (e.g., bispecific antibodies), and bispecific antibodies. . In the present invention, an antibody is a fragment having antigen-binding ability. Includes antigen-binding forms of antibodies, including, for example, Fab', F(ab')2, Fab, Fv alc rIgG. The term also refers to recombinant single chain Fv fragments (scFv) and includes bivalent or bispecific molecules, diabodies, triibodies and tetrabodies.

The "aptamer" is a single-stranded DNA or RNA molecule, and is high in specific chemical or biological molecules by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). Oligomers that bind with affinity and selectivity can be separated and obtained. An aptamer can specifically bind to a target and modulate the activity of the target, such as blocking the target's ability to function through binding.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of anxiety disorders by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any activity that improves or beneficially changes the symptoms of an anxiety disorder by the administration of the pharmaceutical composition according to the present invention.

In the present invention, the Slitrk3 gene and the Nlgn2 gene may be composed of the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. At this time, at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95, 96, 97, 98, 99% or more of the nucleotide sequence represented by SEQ ID NO: 1 or 2 It may contain nucleotide sequences having the same identity.

In the present invention, proteins encoded by the Slitrk3 and Nlgn2 genes include Slitrk3 protein, Nlgn2 protein, and functional equivalents of the amino acid sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 3 or 4 as a result of addition, substitution or deletion of amino acids. Preferably, it refers to a protein having a sequence homology of 95, 96, 97, 98, or 99% or more, and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 3 or 4. "Substantially homogeneous physiological activity" refers to a function in the prefrontal cortex of a mammal.

In the present invention, anxiety disorders refer to mental disorders in which anxiety appears at an abnormal level different from normal, and the treatment of anxiety disorders according to the present invention controls, that is, reduces or increases the abnormal anxiety to restore it to a normal level. include that

Specifically, anxiety disorders include phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, panic disorder, It may be any one disease selected from the group consisting of agoraphobia, separation anxiety disorder, and selective mutism, but is not limited to the above diseases.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating anxiety disorders, including the pharmaceutical composition.

The pharmaceutical composition according to the present invention includes Slitrk3 and Nlgn2 inhibitors as active ingredients, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. It is not, and if necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. are additionally added to form injectable formulations such as aqueous solutions, suspensions, and emulsions, injections such as infusion bags, sprays such as aerosol formulations, pills, capsules, granules, or tablets. can be formulated. Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection, an injection, a spray, a liquid formulation, or an external skin preparation.

The pharmaceutical composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, nasally or locally, including the brain) depending on the purpose, and the dosage depends on the patient's condition, body weight and disease. Depending on the degree, drug form, administration route and time, it can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level is the type of disease, severity, drug activity, drug sensitivity, administration time, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivity rate and excretion rate of the active ingredient in the body, disease type, and concomitant drugs.

In addition, the present invention provides a method for preventing or treating anxiety disorders comprising administering the pharmaceutical composition to a subject.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. means mammals.

In addition, the present invention provides a use of the pharmaceutical composition for preventing or treating anxiety disorders.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Test materials and test methods

1-1. laboratory animal

The use and management of laboratory animals is performed in a standard temperature-controlled laboratory in accordance with guidelines and protocols (DGIST-IACUC-19051401-00, DGIST-IACUC-19121706-01) approved by the Animal Experimentation Ethics Committee of DIGIST. did

Slitrk3 conditional knockout mice were manufactured by Biocytogen Co., Ltd. (Beijing, China), and Neuroligin-2 (Nlgn2) conditional knockout mice were provided by Dr. Thomas C. Sudhof (Stanford University, USA). Mice were maintained in a 12:12 light/dark cycle environment (lights on at 9 am) and fed with water and food. Floxed Slitrk3 (Slitrk3 ^f/f ) was prepared so that loxP sites were located on both sides of exon 2. In addition, Slitrk3/Nlgn2 double floxed mice were prepared by crossing Nlgn2 floxed mice and Slitrk3 ^f/f mice, and the genetic background of the mice was maintained as C57BL/6 N.

1-2. Mouse behavior experiment

Male mice (12-13 weeks of age) were injected with the corresponding AAV and used for all behavioral experiments. The behavioral experiments were conducted in the following order: open-field, Y-maze, three-chamber, elevated-plus maze, and contexture fear conditioning test. Methods for each behavioral experiment were described in detail below.

1-2-1. Open-field test

The mice were placed in a white acrylic open field box (40 × 40 × 40 cm) and allowed to freely explore the environment for 30 minutes in a dark place (0 lux). The movement distance and time required for movement in the central region of the freely moving mouse were recorded with a top-view infrared camera and analyzed using EthoVision XT 10 software (Noldus).

1-2-2. Elevated plus-maze test

The cross-shaped high maze experiment is a cross-shaped white acrylic maze consisting of two open arms (30 × 5 × 0.5 cm) and two closed arms (30 × 5 × 30 cm) located 75 cm above the floor. For the test, the mouse was placed in the center of the cross-shaped maze and allowed to move freely for 5 minutes. All behaviors were recorded with a top-view infrared camera, and the dwell time and number of crossings on each arm were measured and analyzed with EthoVision XT 10 software (Noldus).

1-2-3. Y-maze test

A Y-shaped white acrylic maze with three arms 40 cm long at an angle of 120° was used. Mice were placed in the center of the maze and allowed to move freely for 8 minutes. The number of crossings was measured when all four limbs of the mouse were in the arm, and the movement of the mouse was recorded with a top-view infrared camera and analyzed using EthoVision XT 10 software (Noldus).

1-2-4. Social interaction test

After the mice were placed in the central chamber for 10 min for habituation, both chambers (the social chamber with the new mouse and the non-social chamber with the plastic object) were left open. The test mice were allowed to move freely between the three chambers for 10 minutes, and the time spent in each chamber was recorded and analyzed using EthoVision XT 10 software (Noldus).

1-2-5. Contextual fear conditioning test

A fear conditioning test was performed using a fear conditioning chamber. During training, mice were given a 2-minute search time, followed by sound and electrical stimulation (55 mA) three times at 1-minute intervals. After 24 hours of training, a context test was conducted, and after 48 hours of training, an altered context and a sound stimulus were given, and shown as an experimental method in Fig. 7d.

1-3. recombinant virus production

HEK293T cells were co-transfected with the indicated AAV vectors, pHelper and AAV1.0 (serotype 2/9) capsid vectors. After 72 hours, the transfected HEK293T cells were collected and resuspended in PBS, followed by freezing in ethanol/dry ice (7 minutes each) and thawing in a water bath at 37°C (5 minutes each) 4 times to obtain cells. After dissolution, the dissolved sample was centrifuged, and the supernatant was collected and incubated for 1 hour with 40% polyethylene glycol in 2.5M NaCl solution. It was then centrifuged at 2000 rcf for 30 min, and the pellet was resuspended in HEPES buffer (20 mM HEPES pH 8.0, 115 mM NaCl, 1.2 mM CaCl ₂ , 1.2 mM MgCl ₂ , 2.4 mM MKH ₂ PO ₄ ), followed by an equal volume of Chloroform was added. After the mixture was centrifuged at 400 rcf for 10 minutes, the supernatant was collected in a new EP tube and concentrated using an Amicon Ultra Centrifugal filter. Finally, the titer of the virus produced through RT-PCR was measured, and a concentration higher than 1X10 ¹⁰ infectious units/ul was used.

1-4. virus infection

Mice (~12 weeks of age) were anesthetized with isoflurane and then the viral solution was injected using a Nanoliter 2010 injector (World Precision Instruments) containing a NanoFil syringe and 33 gauge needle at a flow rate of 50 nl/min. (injection volume, 500 nl). Coordinates used for stereotaxic injection targeting the medial prefrontal cortex were anterior-posterior (AP) + 1.8 mm from bregma; medial-lateral (ML), ± 0.4 mm; and dorso-abdominal (DV), -2.1 mm. Meanwhile, immunohistochemical analysis was performed after 5 weeks.

1-5. antibody

A Slitrk3-specific polyclonal antibody was prepared using the Slitrk3 peptide sequence (aa 263-281: TPFHFHGKDLREIRKTELC, SEQ ID NO: 5). After immunization of rabbits with the above immunogen, affinity purification of the Slitrk3-specific antibody JK054 was performed using a Sulfolink column (Pierce) immobilized with the same peptide. In addition, the following commercially available antibodies were used in this example: rabbit polyclonal anti-Slitrk3 (Milipore); rabbit polyclonal anti-Slitrk1 (ProSci incorporated); rabbit polyclonal anti-Slitrk2 (ProSci incorporated); rabbit polyclonal anti-Slitrk5 (ProSci incorporated); mouse monoclonal anti-Nlgn1 (clone N97A/31; NeuroMab); rabbit polyclonal anti-Nlgn2 (Synaptic Systems); rabbit polyclonal anti-Nlgn3 (Synaptic Systems); guinea pig polyclonal anti-VGLUT1 (Millipore); mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore); rabbit polyclonal anti-GABAARα2 (Synaptic Systems); rabbit polyclonal anti-GABAARΥ2 (Synaptic Systems); rabbit polyclonal anti-Nrxn1 (Millipore); mouse monoclonal anti-gephyrin (clone 3B11; Synaptic Systems); mouse monoclonal anti-PTPσ (clone 17G7.2; MediMabs); rabbit polyclonal anti-IgSF9b (Sigma); mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology); and rabbit polyclonal anti-TrkC (Cell Signaling).

1-6. Immunohistochemical staining

First, the mice were sequentially perfused with 1x PBS and 4% paraformaldehyde by transcardial perfusion, and the brains were excised and fixed overnight, and then the brain tissue was slowly sliced to a thickness of 40 μm using a vibratome (VT1200S; Leica). and washed with 1x PBS. Subsequently, brain tissue sections were treated with a blocking solution containing 10% house serum, 0.2% bovine serum albumin, and 0.2% Triton X-100, and reacted at room temperature for 1 hour, followed by GAD65 (1:100), GABAAα2 (Synaptic Systems ; 1:500), GABAAγ2 (Synaptic Systems; 1:500), and VGLUT1 (Synaptic Systems; 1:500), and incubated overnight at 4°C. Thereafter, the tissue sections were washed three times and incubated with Cy3-conjugated secondary antibody (Jackson ImmunoResearch, PA, USA, PA) for 1 hour at room temperature. Next, the tissue sections were washed, and slide glass was mounted using Vectashield mounting medium (Vector Laboratories). Images were obtained using a confocal microscope (LSM700, Carl Zeiss) with a 40× objective, and all images were kept constant with settings.

1-7. statistical analysis

All data were expressed as the mean ± standard error (means ± SEM), and the statistical significance of the data was assessed using the Mann-Whitney U test. N values are described in the individual figure descriptions. A P-value <0.05 was considered statistically significant. Prism7.0 (GraphPad Software) was used for data analysis and bar graph creation.

Example 2. Construction of prefrontal lobe-specific conditional knockout (cKO) mice

The inventors of the present invention attempted to construct a mouse in which Slitrk3 and Neuroligin-2 (Nlgn2), which are GABA synaptic adhesive proteins, were specifically knocked out in the medial prefrontal cortex (hereinafter mPFC), and a double knockout mouse in which both Slitrk3 and Nlgn2 were knocked out, A more detailed description of this was described in Example 1-1.

In more detail, the Cre/loxP system was used to construct mice in which Slitrk3 was specifically knocked out in the mPFC, and a gene targeting strategy for constructing Slitrk3 conditional knockout mice using the Cre/loxP system was shown in FIG. 1A. In addition, Cre recombinase recognizing the loxP was delivered to the mPFC through an adeno-associated virus (AAV) vector. Specifically, the AAV vector expressing the Cre recombinase produced by the method of Example 1-3 was stereotactically injected into the mPFC of the mouse by the method of Example 1-4, and the Cre recombinase was well delivered to the target site. As a result of injecting an AAV vector expressing Cre recombinase conjugated with EGFP to confirm whether it was expressed, as shown in Figure 1b, the main subregion of the mPFC, the anterior cingulate cortex (ACC), under the limbic It was confirmed that EGFP was expressed in the infralimbic cortex (IL) and prelimbic cortex (PL). Through this, it was found that the Cre recombinase was well delivered and expressed in the mPFC region through the AAV vector. In addition, as a result of analyzing the genotype of Slitrk3 conditional knockout mice (Slitrk3 ^f/f ) prepared by the above method, as shown in FIGS. 1c and 1d, Slitrk3 ^f/f and Slitrk3 ^f/+ (Hetero) mice Bands of -bp 5' loxP and 327-bp 3' loxP specific PCR products were observed.

Example 3. Confirmation of knockout of target genes in mPFC-specific conditional knockout (cKO) mice

The present inventors performed immunoblot using brain tissue lysates extracted from the three types of mPFC-specific knockout mice (Slitrk3 cKO, Nlgn2 cKO, and Slitrk3/Nlgn2 cKO) obtained in Example 2 to determine whether target genes and related genes were present. The expression level of the encoded protein was analyzed. At this time, the expression level of each protein was compared in mice expressing the inactive (ΔCre) and active (Cre) Cre recombinase.

As a result, as shown in FIGS. 2a to 2c, when Cre was expressed compared to the case where ΔCre was expressed, the expression of Slitrk3 and Nlgn2 proteins was significantly reduced in Slitrk3 cKO and Nlgn2 cKO mice, respectively, and it was confirmed that they were hardly expressed. , It was confirmed that both Slitrk3 and Nlgn2 proteins were hardly expressed in Slitrk3/Nlgn2 cKO mice. Therefore, it was found that each knockout mouse was well produced through the lack of expression of the target gene in each mouse.

Example 4. Analysis of the effect of mPFC-specific target gene knockout (cKO) on inhibitory synapses

The Slitrk3 and Nlgn2 genes according to the present invention are GABA synaptic adhesive proteins and are known to promote the development of inhibitory synapses. Therefore, excitatory or inhibitory synaptic markers (GAD65, GABAAα2, GABAAγ2, and VGLUT1) were detected in the anterior cingulate cortex (ACC), sublimbic cortex (IL), and prelimbic cortex (PL), which are sub-regions of the mPFC of each knockout mouse according to the present invention. The expression level of was analyzed by immunohistochemical staining to analyze the effect of knockout of each target gene on inhibitory synapses.

First, as a result of analyzing the expression changes of the above markers according to Slitrk3 gene knockout in Slitrk3 cKO mice, as shown in Figs. It was found that the density of GAD65 puncta was significantly decreased in the ACC and PL regions. These results suggest that Slitrk3 knockout induces loss of inhibitory synapses in some areas of the mPFC.

Next, as a result of analyzing the expression changes of the above markers according to Nlgn2 gene knockout in Nlgn2 cKO mice, as shown in FIGS. 4A to 4D, inhibitory synaptic markers in mice expressing Cre compared to cases expressing ΔCre The densities of GAD65, GABAAα2, and GABAAγ2 puncta were significantly decreased in all three subregions of the mPFC. These results suggest that Nlgn2 knockout induces loss of inhibitory synapses in the mPFC.

Finally, the expression changes of the above markers following knockout of Slitrk3 and Nlgn2 genes in Slitrk3/Nlgn2 double cKO mice were analyzed. As a result, as shown in FIGS. 5A to 5D , it was found that the expression of all four markers was not changed in the double knockout mice when compared to the case where ΔCre and Cre were respectively expressed. In particular, the expression level of GAD65, which was decreased in both the Slitrk3 cKO and Nlgn2 cKO mice, was recovered in the double mutant. Through these results, it was found that Slitrk3 and Nlgn2 regulate inhibitory synapses in the prefrontal cortex, and when Slitrk3 and Nlgn2 were double knocked out, unlike single knockout, it was found that inhibitory synapses were not affected.

Example 5. Behavioral analysis in mPFC-specific knockout mice

The present inventors conducted a behavioral experiment using each of the three types of conditional knockout mice prepared in the above example to analyze whether behaviors related to instinctive anxiety of mice are reduced. To this end, behavioral experiments were conducted according to the process shown in FIG. 6 using Slitrk3 cKO, Nlgn2 cKO, and Slitrk3/Nlgn2 double cKO mice, respectively, and the details of each experiment were described in Examples 1-2 above.

5-1. open field experiments

First, an open field experiment was performed on each cKO mouse expressing ΔCre and Cre according to the method of Example 1-2-1. This is a simple test to observe the movement of the mouse in an open space. Experimental mice are instinctively timid and do not rush to the middle of a wide box. level of anxiety can be indirectly assessed. Therefore, the test was performed on mice expressing ΔCre and Cre, respectively, and the total distance, speed, frequency, and staying time each moved to the box center area were quantitatively compared, and the movement change was shown as a heat map image.

As a result of the experiment, in the case of Slitrk3 cKO mice, as shown in FIG. 7a, it can be observed that the total movement and movement to the central region increased through the heat map image in the Cre-expressing mouse, and the total movement distance through the quantitative results. , it was confirmed that the frequency and length of stay were slightly increased, although not statistically significant.

In the case of Nlgn2 cKO mice, as shown in FIG. 8a , the total moving distance and speed significantly increased in Cre-expressing mice, and the frequency of moving to the central region and the staying time also slightly increased.

In addition, in the case of Slitrk3/Nlgn2 double cKO mice, as shown in FIG. 9a, it was confirmed that the total moving distance, speed, and frequency increased slightly, although not statistically significant.

5-2. criss-cross high maze experiment

The cross-shaped high maze experiment is a cross-shaped maze that exists in high places and is one of the behavioral experiments that can determine the level of anxiety of mice. The maze consists of two open arms and two closed arms blocked by high walls. When the mouse is placed in the middle of the cross-shaped maze, the mouse is instinctively scared and moves into the safe closed arms blocked by the wall, and enters the open arm passage. Whether or not the level of anxiety has decreased can be evaluated through the entry and stay time.

As a result of the experiment, in the case of Slitrk3 cKO mice, as shown in FIG. 7B, the number of entries into the open arm passage slightly increased and the staying time increased statistically significantly. Through the heat map results, ΔCre mice entered the open arm passage There was no movement, but movement to the open arm passage was found in Cre mice.

In the case of Nlgn2 cKO mice, as shown in FIG. 8B, the number of entries into the open arm passage slightly increased and the staying time increased statistically significantly. confirmed an increase.

In the case of Slitrk3/Nlgn2 double cKO mice, as shown in FIG. 9B, there was no difference between ΔCre and Cre mice in the number of entries and retention time in the open arm passage, and no difference was confirmed in the heat map image. Through this, it was found that in the case of Slitrk3/Nlgn2 double cKO mice, the abnormal decrease in anxiety symptoms seen in the single cKO mice was restored to the instinctive anxiety level in wild-type mice.

5-3. Y-maze experiment

The Y-maze experiment is a behavioral experiment that can evaluate working memory. Working memory requires the ability to quickly form memories based on specific events and quickly distinguish current valid information from unnecessary information. It is considered to be a phenomenon in which behavioral flexibility is reduced or repetitive behaviors are reinforced. Experiments were conducted according to the method of Example 1-2-3, and ΔCre and Cre mice were compared by measuring spontaneous alternations (%) and frequency. Voluntary turnover rate refers to the case in which the mouse selects a new passage that has not been explored before among the next two passages when entering and exiting one passage. It is an experimental method using the habit of doing. Therefore, it was evaluated that the working memory decreased as the subjects reentered the previously entered passage.

As a result, in the case of Slitrk3 cKO mice, as shown in FIG. 7c, it was confirmed that there was no significant difference in spontaneous alternation rate and frequency between ΔCre and Cre mice as a result of quantitative analysis.

Even in the case of Nlgn2 cKO mice, as shown in FIG. 8c , it was confirmed that there was no significant difference between ΔCre and Cre mice as a result of quantitative analysis.

In addition, in the case of Slitrk3/Nlgn2 double cKO mice, as shown in FIG. 9c, it was confirmed that there was no significant difference between ΔCre and Cre mice as a result of quantitative analysis.

Through the above results, it was found that there were no side effects related to memory ability due to gene knockout in Slitrk3 cKO, Nlgn2 cKO, and Slitrk3/Nlgn2 double cKO mice.

5-4. Contextual Fear Conditioning Experiment

As an experiment to measure memory by learning about fear, the experiment was conducted according to the method of Example 1-2-6. The experimental results are the freezing response (Context) shown when the mouse is placed in the same chamber again after the training process of putting the mouse in the chamber and giving sound and electric shock stimulation, and the freezing response (Altered context) when the mouse is placed in a new chamber. And when a sound stimulus was given, the freezing response (Tone) was measured, respectively.

As a result of the experiment, in the case of Slitrk3 cKO mice, as shown in Fig. 7d, it was found that the freezing response of Cre mice was significantly reduced in all three cases.

In the case of Nlgn2 cKO mice, as shown in FIG. 8D, the freezing response in the Context and Tone phases was statistically significantly reduced, and the freezing response in the case of the Altered context was slightly reduced, although not significantly.

In the case of Slitrk3/Nlgn2 double cKO mice, as shown in FIG. 9d, Context and Altered context in Cre mice showed a slightly reduced but not statistically significant decrease, and a statistically significant reduction in freezing response in Tone. did

5-5. social interaction experiment

Finally, the present inventors performed a 3-chamber experiment according to the method of Examples 1-2-4 as a social interaction experiment of the mouse, and then showed the movement of the mouse as a heat map image, and showed the sociability and social Social recognition was quantified and graphed.

As a result of the experiment, in the case of Slitrk3 cKO mice, as shown in FIG. 7e, the social interaction index was slightly increased and the social novelty index was slightly decreased in Cre mice, but no statistically significant difference was found.

In the case of Nlgn2 cKO mice, as shown in FIG. 8e, there was no difference in the social interaction index between ΔCre and Cre mice, and the social novelty index decreased.

In addition, in the case of Slitrk3/Nlgn2 double cKO mice, as shown in FIG. 9e, the social interaction index increased slightly, but no statistically significant difference was found, and the social novelty index decreased statistically significantly. did In other words, looking at the degree of movement to S2 in search of social novelty in the latency result, Slitrk3/Nlgn2 double cKO mice showed a decrease in the social novelty index through the disappearance of the difference between S1 and S2.

These results indicate that the social novelty effect does not recover in Slitrk3/Nlgn2 double cKO mice, and that the difference in social novelty behavior is a behavioral change that appears only in Nlgn2 and occurs independently of Slitrk3.

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Pharmaceutical composition for preventing or treating anxiety disorder comprising inhibitors of Slitrk3 and Nlgn2 <130> PD20-182 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 2934 <212> DNA <213> Slitrk3 <400> 1 atgaaacctt ccatagctga gatgcttcac agaggaagga tgttgtggat aattcttcta 60 agcacaattg ctctaggatg gactaccccg attcccctaa tagaggactc agaggaaata 120 gatgagccct gttttgatcc atgctactgt gaagttaaag aaagcctctt tcatatacat 180 tgtgacagta aaggatttac aaatattagt cagattaccg agttctggtc aagacctttt 240 aaactgtatc tgcagaggaa ttctatgagg aaattatata ccaacagttt tcttcatttg 300 aataatgctg tgtctattaa tctt gggaac aatgcattgc aggacattca gactggagct 360 ttcaatggtc ttaagatttt aaagagacta tatctacatg aaaacaaact agatgtcttc 420 agaaatgaca ccttccttgg cttggaaagt ctagaatatc tgcaggcaga ttacaatgtc 480 attaaacgta ttgagagtgg ggcatttcgg aacctaagta aattgagggt tctgatttta 540 aatgataatc tcatccccat gcttccaacc aatttattta aggctgtctc tttaacccat 600 ttggacctac gtggaaatag gttaaaggtt cttttttacc gaggaatgct agatcacatt 660 ggcagaagcc tgatggagct ccagctggaa gaaaaccctt ggaactgtac atgtgaaatt 720 gtacaactga agagttggct ggaacgcatt ccttatactg ccctggtggg agacattacc 780 tgtgagaccc ctttccactt ccatggaaag gacctacgag aaatcaggaa gacagaactc 840 tgtcccttgt tgtctgactc tgaggtagag gctagtttgg gaattccaca ctcgtcatca 900 agtaaggaga atgcatggcc aactaagcct tcctca atgc tatcctctgt tcattttact 960 gcttcttctg tcgaatacaa gtcctcaaat aaacagccta agccacccaa acagcctcga 1020 acaccaaggc caccctccac ctcccaagct ttatatcctg gtccaaacca gcctcccatt 1080 gctccttatc agaccagacc accaatcccc attatatgcc ccactgggtg tacctgtaat 1140 ttgcacatca atgaccttgg cttgactgtc aactgcaaag agcgaggatt taataacatt 1200 tctgaacttc ttccaaggcc cttgaatgcc aagaaactgt atctgagtag caatctgatt 1260 cagaaaatat accgttctga tttttggaat ttttcttcct tggatctctt gcatctgggg 1320 aacaatcgta tttcctatgt ccaagatggg gccttta tca acttgcccaa cttaaagagc 1380 ctcttcctta atggcaacga tatagagaag ctgacaccag gcatgttccg aggcctacag 1440 agtttgcact acttgtactt tgagttcaat gtcatccggg aaatccagcc tgcagccttc 1500 agcctcatgc ccaacttgaa gctgctattc ct caataata acttactgag gactctgcca 1560 acagacgcct ttgctggcac atccctggcc cggctcaacc tgaggaagaa ctacttcctc 1740 agctcagtca gtgtggttgg tgatgtgctt tgcaggagcc ct gagaacct cacgcaccgt 1800 gatgtgcgca ctattgagct ggaagttctt tgcccagaga tgctgcacgt tgcaccagct 1860 ggagaatccc cagcccagcc tggagattct caccttattg ggggacccaac cagtgcatca 1920 ccttatgagt tttctcctcc tgggggcc ct gtgccacttt ctgtgttaat tctcagcctg 1980 ctggttctgt ttttctcagc agtctttgtt gctgcaggcc tctttgccta cgtgctccga 2040 aggcgtcgaa agaagctgcc cttcagaagc aagcggcagg aaggtgtgga ccttactggc 2100 atccaaatgc aatgccacag gctgtttgag gatggtggag gtggtggtgg cggaagtggg 2160 ggtggtggtc gaccaactct ttcctctcca gagaaggccc ctcccgtggg tcatgtgtat 2220 gagtacatcc cccacccggt tacccaaatg tgcaacaacc ccatctacaa gcctcgtgag 2280 gaggaggagg tggctgtttc atcagcccaa gaagcaggga gtgcagaacg tgggggtcca 2340 gggacacaac caccgggaat gggtgaggct ctcctaggaa gtga gcagtt tgctgagaca 2400 cccaaggaga accatagtaa ctaccggacc ttgctggaaa aagagaagga gtgggcccta 2460 gcagtgtcca gctcccagct taacaccata gtgacggtga atcaccatca ccctcaccac 2520 ccagcagttg gtggggtttc aggagtagtt gggggaactg ggggagactt ggcagggttc 2580 cgccaccatg agaaaaatgg tggggtggtg ctgtttcctc ctgggggagg ctgtggtagt 26 40 ggcagtatgc tactagatcg agagaggcca cagcctgccc cctgcacagt gggatttgtg 2700 gactgtctct atggaacagt gcccaaatta aaggaactgc acgtgcaccc tcctggcatg 2760 caatacccag acttacagca ggatgccagg ctcaaagaaa cccttctctt ctcggct gga 2820 aagggcttca cagaccacca aacccaaaaa agtgattacc tcgagttaag ggccaaactt 2880 caaaccaagc cggattacct cgaagtcctg gagaagacaa catacaggtt ctaa 2934 <210> 2 <211> 2508 <212> DNA <213> Nlgn2 <400> 2 atgtggctcc tggcgctgtg tctggtgggg ctggcggggg ctcaacgcgg gggagggggt 60 cccggcggcg gcgccccggg c ggccccggc ctgggcctcg gcagcctcgg cgaggagcgc 120 ttcccggtgg tgaacacggc ctacgggcga gtgcgcggtg tgcggcgcga gctcaacaac 180 gagatcctgg gccccgtcgt gcagttcttg ggcgtgccct acgccacgcc gcccctgggc 240 gcccgccgct tccagccgcc tgaggcgccc gcctcgtggc ccggcgtgcg caacgccacc 300 accctgccgc ccgcctgccc gcagaacctg cacggggcgc tgcccgccat catgctgcct 360 gtgtggttca ccgacaact t ggaggcggcc gccacctacg tgcagaacca gagcgaggac 420 tgcctgtacc tcaacctcta cgtgcccacc gaggacggtc cgctcacaaa aaaacgtgac 480 gaggcgacgc tcaatccgcc agacacagat atccgtgacc ctgggaagaa gcctgtgatg 540 ctgtt tctcc atggcggctc ctacatggag gggaccggaa acatgttcga tggctcagtc 600 ctggctgcct atggcaacgt cattgtagcc acgctcaact accgtcttgg ggtgctcggt 660 tttctcagca ccggggacca ggctgcaaaa ggcaactatg ggctcctgga ccagatccag 720 gccctgcgct ggctcagtga aaacatcgcc cactttgggg gcgaccccga gcgtatcacc 780 atctttggtt ccggggcagg ggcctcctgc gt caaccttc tgatcctctc ccaccattca 840 gaagggctgt tccagaaggc catcgcccag agtggcaccg ccatttccag ctggtctgtc 900 aactaccagc cgctcaagta cacgcggctg ctggcagcca aggtgggctg tgaccgagag 960 gacagcgctg aagctgtgga gt gtctgcgc cggaagccct cccgggagct ggtggaccag 1020 gacgtgcagc ctgcccgcta ccacatcgcc tttgggcccg tggtggatgg cgacgtggtc 1080 cccgatgacc ctgagatcct catgcagcag ggagaattcc tcaactacga catgctcatc 1140 ggcgtcaacc agggagaggg cctcaagttc gtggaggact ctgcagagag cgaggacggt 1200 gtgtctgcca gcgcctttga cttcactgtc tccaactttg tgg acaacct gtatggctac 1260 ccggaaggca aggatgtgct tcgggagacc atcaagttta tgtacacaga ctgggccgac 1320 cgggacaatg gcgaaatgcg ccgcaaaacc ctgctggcgc tctttactga ccaccaatgg 1380 gtggcaccag ctgtggccac tgccaagct g cacgccgact accagtctcc cgtctacttt 1440 tacaccttct accaccactg ccaggcggag ggccggcctg agtgggcaga tgcggcgcac 1500 ggggatgaac tgccctatgt ctttggcgtg cccatggtgg gtgccaccga cctcttcccc 1560 tgtaacttct ccaagaatga cgtcatgctc agtgccgtgg tcatgaccta ctggaccaac 1620 ttcgccaaga ctggggaccc caaccagccg gtgccgcagg ataccaagtt catcc acacc 1680 aagcccaatc gcttcgagga ggtggtgtgg agcaaattca acagcaagga gaagcagtat 1740 ctgcacatag gcctgaagcc acgcgtgcgt gacaactacc gcgccaacaa ggtggccttc 1800 tggctggagc tcgtgcccca cctgcacaac ctgcacacgg ag ctcttcac caccaccacg 1860 cgcctgcctc cctacgccac gcgctggccg cctcgtcccc ccgctggcgc cccgggcaca 1920 cgccggcccc cgccgcctgc caccctgcct cccgagcccg agcccgagcc cggcccaagg 1980 gcctatgacc gcttccccgg ggactcacgg gactactcca cggagctgag cgtcaccgtg 2040 gccgtgggtg cctccctcct cttcctcaac atcctggcct ttgctgccct ctactacaag 210 0 cgggaccggc ggcaggagct gcggtgcagg cggcttagcc cacctggcgg ctcaggctct 2160 ggcgtgcctg gtgggggccc cctgctcccc gccgcgggcc gtgagctgcc accagaggag 2220 gagctggtgt cactgcagct gaagcggggt ggtggcgtcg gggcggaccc tgcc gaggct 2280 ctgcgccctg cctgcccgcc cgactacacc ctggccctgc gccgggcacc ggacgatgtg 2340 cctctcttgg cccccggggc cctgaccctg ctgcccagtg gcctggggcc accgccaccc 2400 ccaccgcccc cctcccttca tcccttcggg cccttccccc cgccccctcc caccgccacc 2460 agccacaaca acacgctacc ccacccccac tccaccactc gggtatag 2508 <210> 3 <211> 97 7 <212> PRT <213> Slitrk3 <400> 3 Met Lys Pro Ser Ile Ala Glu Met Leu His Arg Gly Arg Met Leu Trp 1 5 10 15 Ile Ile Leu Leu Ser Thr Ile Ala Leu Gly Trp Thr Thr Pro Ile Pro 20 25 30 Leu Ile Glu Asp Ser Glu Glu Ile Asp Glu Pro Cys Phe Asp Pro Cys 35 40 45 Tyr Cys Glu Val Lys Glu Ser Leu Phe His Ile His Cys Asp Ser Lys 50 55 60 Gly Phe Thr Asn Ile Ser Gln Ile Thr Glu Phe Trp Ser Arg Pro Phe 65 70 75 80 Lys Leu Tyr Leu Gln Arg Asn Ser Met Arg Lys Leu Tyr Thr Asn Ser 85 90 95 Phe Leu His Leu Asn Asn Ala Val Ser Ile Asn Leu Gly Asn Asn Ala 100 105 110 Leu Gln Asp Ile Gln Thr Gly Ala Phe Asn Gly Leu Lys Ile Leu Lys 115 120 125 Arg Leu Tyr Leu His Glu Asn Lys Leu Asp Val Phe Arg Asn Asp Thr 130 135 140 Phe Leu Gly Leu Glu Ser Leu Glu Tyr Leu Gln Ala Asp Tyr Asn Val 145 150 155 160 Ile Lys Arg Ile Glu Ser Gly Ala Phe Arg Asn Leu Ser Lys Leu Arg 165 170 175 Val Leu Ile Leu Asn Asp Asn Leu Ile Pro Met Leu Pro Thr Asn Leu 180 185 190 Phe Lys Ala Val Ser Leu Thr His Leu Asp Leu Arg Gly Asn Arg Leu 195 200 205 Lys Val Leu Phe Tyr Arg Gly Met Leu Asp His Ile Gly Arg Ser Leu 210 215 220 Met Glu Leu Gln Leu Glu Glu Asn Pro Trp Asn Cys Thr Cys Glu Ile 225 230 235 240 Val Gln Leu Lys Ser Trp Leu Glu Arg Ile Pro Thr Ala Leu Val 245 250 255 Gly Asp Ile Thr Cys Glu Thr Pro Phe His Phe His Gly Lys Asp Leu 260 265 270 Arg Glu Ile Arg Lys Thr Glu Leu Cys Pro Leu Leu Ser Asp Ser Glu 275 280 285 Val Glu Ala Ser Leu Gly Ile Pro His Ser Ser Ser Ser Lys Glu Asn 290 295 300 Ala Trp Pro Thr Lys Pro Ser Ser Met Leu Ser Ser Val His Phe Thr 305 310 315 320 Ala Ser Ser Val Glu Tyr Lys Ser Ser Asn Lys Gln Pro Lys Pro Thr 325 330 335 Lys Gln Pro Arg Thr Pro Arg Pro Pro Ser Thr Ser Gln Ala Leu Tyr 340 345 350 Pro Gly Pro Asn Gln Pro Pro Ile Ala Pro Tyr Gln Thr Arg Pro Pro 355 360 365 Ile Pro Ile Ile Cys Pro Thr Gly Cys Thr Cys Asn Leu His Ile Asn 370 375 380 Asp Leu Gly Leu Thr Val Asn Cys Lys Glu Arg Gly Phe Asn Asn Ile 385 390 395 400 Ser Glu Leu Leu Pro Arg Pro Leu Asn Ala Lys Lys Leu Tyr Leu Ser 405 410 415 Ser Asn Leu Ile Gln Lys Ile Tyr Arg Ser Asp Phe Trp Asn Phe Ser 420 425 430 Ser Leu Asp Leu Leu His Leu Gly Asn Asn Arg Ile Ser Tyr Val Gln 435 440 445 Asp Gly Ala Phe Ile Asn Leu Pro Asn Leu Lys Ser Leu Phe Leu Asn 450 455 460 Gly Asn Asp Ile Glu Lys Leu Thr Pro Gly Met Phe Arg Gly Leu Gln 465 470 475 480 Ser Leu His Tyr Leu Tyr Phe Glu Phe Asn Val Ile Arg Glu Ile Gln 485 490 495 Pro Ala Ala Phe Ser Leu Met Pro Asn Leu Lys Leu Leu Phe Leu Asn 500 505 510 Asn Asn Leu Leu Arg Thr Leu Pro Thr Asp Ala Phe Ala Gly Thr Ser 515 520 525 Leu Ala Arg Leu Asn Leu Arg Lys Asn Tyr Phe Leu Tyr Leu Pro Val 530 535 540 Ala Gly Val Leu Glu His Leu Asn Ala Ile Val Gln Ile Asp Leu Asn 545 550 555 560 Glu Asn Pro Trp Asp Cys Thr Cys Asp Leu Val Pro Phe Lys Gln Trp 565 570 575 Ile Glu Thr Ile Ser Ser Val Ser Val Val Val Gly Asp Val Leu Cys Arg 580 585 590 Ser Pro Glu Asn Leu Thr His Arg Asp Val Arg Thr Ile Glu Leu Glu 595 600 605 Val Leu Cys Pro Glu Met Leu His Val Ala Pro Ala Gly Glu Ser Pro 610 615 620 Ala Gln Pro Gly Asp Ser His Leu Ile Gly Ala Pro Thr Ser Ala Ser 625 630 635 640 Pro Tyr Glu Phe Ser Pro Pro Gly Gly Pro Val Pro Leu Ser Val Leu 645 650 655 Ile Leu Ser Leu Leu Leu Val Leu Phe Phe Ser Ala Val Phe Val Ala Ala 660 665 670 Gly Leu Phe Ala Tyr Val Leu Arg Arg Arg Arg Lys Lys Leu Pro Phe 675 680 685 Arg Ser Lys Arg Gln Glu Gly Val Asp Leu Thr Gly Ile Gln Met Gln 690 695 700 Cys His Arg Leu Phe Glu Asp Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly 705 710 715 720 Gly Gly Gly Arg Pro Thr Leu Ser Ser Pro Glu Lys Ala Pro Pro Val 725 730 735 Gly His Val Tyr Glu Tyr Ile Pro His Pro Val Thr Gln Met Cys Asn 740 745 750 Asn Pro Ile Tyr Lys Pro Arg Glu Glu Glu Glu Val Ala Val Ser Ser 755 760 765 Ala Gln Glu Ala Gly Ser Ala Glu Arg Gly Gly Pro Gly Thr Gln Pro 770 775 780 Pro Gly Met Gly Glu Ala Leu Leu Gly Ser Glu Gln Phe Ala Glu Thr 785 790 795 800 Pro Lys Glu Asn His Ser Asn Tyr Arg Thr Leu Leu Glu Lys Glu Lys 805 810 815 Glu Trp Ala Leu Ala Val Ser Ser Ser Gln Leu Asn Thr Ile Val Thr 820 825 830 Val Asn His His His Pro His Pro His Pro Ala Val Gly Gly Val Ser Gly 835 840 845 Val Val Gly Gly Thr Gly Gly Asp Leu Ala Gly Phe Arg His Glu 850 855 860 Lys Asn Gly Gly Val Val Leu Phe Pro Pro Gly Gly Gly Cys Gly Ser 865 870 875 880 Gly Ser Met Leu Leu Asp Arg Glu Arg Pro Gln Pro Ala Pro Cys Thr 885 890 895 Val Gly Phe Val Asp Cys Leu Tyr Gly Thr Val Pro Lys Leu Lys Glu 900 905 910 Leu His Val His Pro Pro Gly Met Gln Tyr Pro Asp Leu Gln Gln Asp 915 920 925 Ala Arg Leu Lys Glu Thr Leu Leu Phe Ser Ala Gly Lys Gly Phe Thr 930 935 940 Asp His Gln Thr Gln Lys Ser Asp Tyr Leu Glu Leu Arg Ala Lys Leu 945 950 955 960 Gln Thr Lys Pro Asp Tyr Leu Glu Val Leu Glu Lys Thr Thr Tyr Arg 965 970 975 Phe <210> 4 <211> 835 <212> PRT <213> Nlgn2 <400> 4 Met Trp Leu Leu Ala Leu Cys Leu Val Gly Leu Ala Gly Ala Gln Arg 1 5 10 15 Gly Gly Gly Gly Pro Gly Gly Gly Ala Pro Gly Gly Pro Gly Leu Gly 20 25 30 Leu Gly Ser Leu Gly Glu Glu Arg Phe Pro Val Val Asn Thr Ala Tyr 35 40 45 Gly Arg Val Arg Gly Val Arg Arg Glu Leu Asn Asn Glu Ile Leu Gly 50 55 60 Pro Val Val Gln Phe Leu Gly Val Pro Tyr Ala Thr Pro Pro Leu Gly 65 70 75 80 Ala Arg Arg Phe Gln Pro Pro Glu Ala Pro Ala Ser Trp Pro Gly Val 85 90 95 Arg Asn Ala Thr Thr Leu Pro Pro Ala Cys Pro Gln Asn Leu His Gly 100 105 110 Ala Leu Pro Ala Ile Met Leu Pro Val Trp Phe Thr Asp Asn Leu Glu 115 120 125 Ala Ala Ala Thr Tyr Val Gln Asn Gln Ser Glu Asp Cys Leu Tyr Leu 130 135 140 Asn Leu Tyr Val Pro Thr Glu Asp Gly Pro Leu Thr Lys Lys Arg Asp 145 150 155 160 Glu Ala Thr Leu Asn Pro Pro Asp Thr Asp Ile Arg Asp Pro Gly Lys 165 170 175 Lys Pro Val Met Leu Phe Leu His Gly Gly Ser Tyr Met Glu Gly Thr 180 185 190 Gly Asn Met Phe Asp Gly Ser Val Leu Ala Ala Tyr Gly Asn Val Ile 195 200 205 Val Ala Thr Leu Asn Tyr Arg Leu Gly Val Leu Gly Phe Leu Ser Thr 210 215 220 Gly Asp Gln Ala Ala Lys Gly Asn Tyr Gly Leu Leu Asp Gln Ile Gln 225 230 235 240 Ala Leu Arg Trp Leu Ser Glu Asn Ile Ala His Phe Gly Gly Asp Pro 245 250 255 Glu Arg Ile Thr Ile Phe Gly Ser Gly Ala Gly Ala Ser Cys Val Asn 260 265 270 Leu Leu Ile Leu Ser His His Ser Glu Gly Leu Phe Gln Lys Ala Ile 275 280 285 Ala Gln Ser Gly Thr Ala Ile Ser Ser Trp Ser Val Asn Tyr Gln Pro 290 295 300 Leu Lys Tyr Thr Arg Leu Leu Ala Ala Lys Val Gly Cys Asp Arg Glu 305 310 315 320 Asp Ser Ala Glu Ala Val Glu Cys Leu Arg Arg Lys Pro Ser Arg Glu 325 330 335 Leu Val Asp Gln Asp Val Gln Pro Ala Arg Tyr His Ile Ala Phe Gly 340 345 350 Pro Val Val Asp Gly Asp Val Val Pro Asp Asp Pro Glu Ile Leu Met 355 360 365 Gln Gln Gly Glu Phe Leu Asn Tyr Asp Met Leu Ile Gly Val Asn Gln 370 375 380 Gly Glu Gly Leu Lys Phe Val Glu Asp Ser Ala Glu Ser Glu Asp Gly 385 390 395 400 Val Ser Ala Ser Ala Phe Asp Phe Thr Val Ser Asn Phe Val Asp Asn 405 410 415 Leu Tyr Gly Tyr Pro Glu Gly Lys Asp Val Leu Arg Glu Thr Ile Lys 420 425 430 Phe Met Tyr Thr Asp Trp Ala Asp Arg Asp Asn Gly Glu Met Arg Arg 435 440 445 Lys Thr Leu Leu Ala Leu Phe Thr Asp His Gln Trp Val Ala Pro Ala 450 455 460 Val Ala Thr Ala Lys Leu His Ala Asp Tyr Gln Ser Pro Val Tyr Phe 465 470 475 480 Tyr Thr Phe Tyr His His Cys Gln Ala Glu Gly Arg Pro Glu Trp Ala 485 490 495 Asp Ala Ala His Gly Asp Glu Leu Pro Tyr Val Phe Gly Val Pro Met 500 505 510 Val Gly Ala Thr Asp Leu Phe Pro Cys Asn Phe Ser Lys Asn Asp Val 515 520 525 Met Leu Ser Ala Val Val Met Thr Tyr Trp Thr Asn Phe Ala Lys Thr 530 535 540 Gly Asp Pro Asn Gln Pro Val Pro Gln Asp Thr Lys Phe Ile His Thr 545 550 555 560 Lys Pro Asn Arg Phe Glu Glu Val Val Trp Ser Lys Phe Asn Ser Lys 565 570 575 Glu Lys Gln Tyr Leu His Ile Gly Leu Lys Pro Arg Val Arg Asp Asn 580 585 590 Tyr Arg Ala Asn Lys Val Ala Phe Trp Leu Glu Leu Val Pro His Leu 595 600 605 His Asn Leu His Thr Glu Leu Phe Thr Thr Thr Thr Arg Leu Pro Pro 610 615 620 Tyr Ala Thr Arg Trp Pro Pro Arg Pro Pro Ala Gly Ala Pro Gly Thr 625 630 635 640 Arg Arg Pro Pro Pro Pro Ala Thr Leu Pro Pro Glu Pro Glu Pro Glu 645 650 655 Pro Gly Pro Arg Ala Tyr Asp Arg Phe Pro Gly Asp Ser Arg Asp Tyr 660 665 670 Ser Thr Glu Leu Ser Val Thr Val Thr Val Ala Val Gly Ala Ser Leu Leu Phe 675 680 685 Leu Asn Ile Leu Ala Phe Ala Ala Leu Tyr Tyr Lys Arg Asp Arg Arg 690 695 700 Gln Glu Leu Arg Cys Arg Arg Leu Ser Pro Gly Gly Ser Gly Ser 705 710 715 720 Gly Val Pro Gly Gly Gly Pro Leu Leu Pro Ala Ala Gly Arg Glu Leu 725 730 735 Pro Pro Glu Glu Glu Leu Val Ser Leu Gln Leu Lys Arg Gly Gly Gly 740 745 750 Val Gly Ala Asp Pro Ala Glu Ala Leu Arg Pro Ala Cys Pro Pro Asp 755 760 765 Tyr Thr Leu Ala Leu Arg Arg Ala Pro Asp Asp Val Pro Leu Leu Ala 770 775 780 Pro Gly Ala Leu Thr Leu Leu Pro Ser Gly Leu Gly Pro Pro Pro Pro 785 790 795 800 Pro Pro Pro Ser Leu His Pro Phe Gly Pro Phe Pro Pro Pro Pro 805 810 815 Pro Thr Ala Thr Ser His Asn Asn Thr Leu Pro His Pro His Ser Thr 820 825 830 Thr Arg Val 835 <210> 5 <211> 19 <212> PRT <213> Slitrk3 peptide <400> 5 Thr Pro Phe His Phe His Gly Lys Asp Leu Arg Glu Ile Arg Lys Thr 1 5 10 15 Glu Leu Cys

Claims (13)

A pharmaceutical composition for preventing or treating anxiety disorders, comprising a dual inhibitor of Slitrk3 (SLIT and NTRK like family member 3) and Nlgn2 (Nuroligin 2) as an active ingredient,
The dual inhibitor is a dual expression inhibitor of Slitrk3 and Nlgn2 genes,
Antisense oligonucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozymes, CRISPR/Cas9 and vectors that complementarily bind to mRNAs of Slitrk3 and Nlgn2 genes, respectively Characterized in that any one selected from the group consisting of, a pharmaceutical composition.
According to claim 1,
The pharmaceutical composition, characterized in that the Slitrk3 gene consists of the nucleotide sequence of SEQ ID NO: 1.
According to claim 1,
The Nlgn2 gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, a pharmaceutical composition.
delete According to claim 1,
The vector is characterized in that the adeno-associated virus (AAV), the pharmaceutical composition.
delete According to claim 1,
The anxiety disorders include phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, panic disorder, agoraphobia ( agoraphobia), separation anxiety disorder (separation anxiety disorder) and selective mutism (selective mutism) characterized in that any one selected from the group consisting of, a pharmaceutical composition.
According to claim 1,
The pharmaceutical composition is characterized in that for topical administration to the brain, a pharmaceutical composition.
According to claim 8,
The local administration is characterized in that the administration to the prefrontal cortex (prefrontal cortex) region of the brain, the pharmaceutical composition.
A pharmaceutical formulation for preventing or treating anxiety disorders, comprising the composition of claim 1.
According to claim 10,
The pharmaceutical formulation is characterized in that for brain topical administration, pharmaceutical formulation.
According to claim 11,
The local administration is characterized in that it is administered to the prefrontal cortex (prefrontal cortex) region of the brain, the pharmaceutical formulation.
According to claim 10,
The pharmaceutical formulation is a pharmaceutical formulation, characterized in that the injection formulation, injection formulation or liquid formulation.

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