Antidepressant and anxiolytic uses of polypeptides and complexes

Abstract

The present invention relates to antidepressant and anti-anxiety uses of polypeptides, providing the use of a polypeptide having the sequence shown in SEQ ID NO: 1 in the preparation of a medicament for the treatment and / or prevention of depression or anxiety. Furthermore, the present invention relates to a complex comprising said polypeptide.

Description

Technical Field

[0001] The present invention relates to the fields of antidepressant and antianxiety, and more specifically, to the use of polypeptides in antidepressant and antianxiety.

Background Art

[0002] Depression is a mental disorder disease caused by various reasons, with the main clinical symptom being a depressed mood. Depression is characterized by a high incidence rate, a high rate of ability decline, a high suicide rate, etc. The pathogenesis of depression has not been fully elucidated, and most of the antidepressants commonly used in clinical practice are developed based on the "monoamine neurotransmitter hypothesis". In this hypothesis, it is suggested that the deficiency of monoamine neurotransmitters such as dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NA) in the brain is related to the occurrence of depression, and antidepressants can block the reuptake of monoamine neurotransmitters by inhibiting the functions of 5-HT transporter and NA transporter, and increase the concentration of monoamine neurotransmitters in the synaptic cleft, thereby improving the symptoms of depression.

[0003] Antidepressants commonly used in clinical practice are classified into categories such as first-generation antidepressants (monoamine oxidase inhibitors (MAOI) and tricyclic antidepressants), second-generation antidepressants (selective serotonin reuptake inhibitors (SSRI)), and third-generation antidepressants (dual reuptake inhibitors of 5-hydroxytryptamine and norepinephrine, norepinephrine reuptake inhibitors (SNRI), and norepinephrine-related serotoninergic antidepressants) according to their mechanism of action and development time.

[0004] These drugs, developed based on the "monoamine neurotransmitter hypothesis," have clear therapeutic effects, but their efficacy rate is only 60%, and they take time to show antidepressant effects (usually 2-3 weeks after taking the antidepressant). Furthermore, these antidepressants have drawbacks such as causing significant harmful side effects with long-term use and a high likelihood of relapse after discontinuation of medication.

[0005] In recent years, detailed studies of the pathogenesis of depression have led to the discovery of several new molecular targets for antidepressants based on non-monoamine neurotransmitters, such as NMDA receptors, CRF1 receptors, δ receptors, κ receptors, GABAB receptors, M-choline receptors, IDO, CysLT1R, PDE4, PPARγ, PPARδ, and NOS, in addition to the classic "monoamine neurotransmitter hypothesis" for depression. Several of these new targets have been identified as having antidepressant activity, and some of them are progressing to clinical trials.

[0006] In order to provide more effective treatment strategies and to further elucidate the pathogenesis of depression, there is still a strong need in the field of depression to explore and discover more important pathogenesis mechanisms and therapeutic targets. [Overview of the project] [Means for solving the problem]

[0007] To achieve the above objective, the inventors conducted numerous studies based on molecular changes in a model of depression, discovered a new mechanism of the onset of depression, and completed the present invention.

[0008] Specifically, 5-HT receptors are a group consisting of G protein-coupled receptors and ligand-gated ion channels, and are found in the central nervous system and peripheral nervous system. 5HT receptors can be classified into seven subfamilies: 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. The 5-HT2 receptor has three subtypes: A, B, and C, namely 5-HT2A receptor protein, 5-HT2B receptor protein, and 5-HT2C receptor protein. Of these, the 5-HT2A receptor (5-HT2AR) is an excitatory 5-HT receptor and is widely expressed in the mammalian brain.

[0009] Dopamine (DA) receptors are a type of receptor found in living organisms that exerts its effects through corresponding cell membrane receptors. DA receptors can be classified into five types: D1, D2, D3, D4, and D5. D1 receptors (D1R) are widely expressed in the brain.

[0010] The inventors have found that 5-HT2AR not only regulates neurotransmitters in its standalone form, but also in the form of complexes such as the 5-HT1AR / 5-HT2AR complex and the 5-HT2AR / oxytocin receptor (OXTR) complex.

[0011] To date, it has not been reported that 5-HT2AR can form a protein complex with D1R in vivo, and there have been no previous reports clarifying the relationship between the 5-HT2AR / D1R complex and depression. Unexpectedly, the inventors discovered that 5-HT2AR forms a protein complex with D1R via its carboxyl terminus, and that this complex has depressive pathogenicity. Further research revealed that the interaction between 5-HT2AR and D1R can be reduced by certain polypeptides, thereby alleviating symptoms of depression and anxiety.

[0012] Accordingly, in a first embodiment, the present invention provides a polypeptide having the amino acid sequence shown in Sequence ID No. 1 (SKDNSDGVNEKVSCV). The present invention also provides the use of the polypeptide of the present invention in the preparation of pharmaceuticals for the treatment and / or prevention of depression or anxiety disorders.

[0013] In some embodiments, the length of the polypeptide of the present invention may be 15 to 90 aa, and in particular, 15 to 50 aa, 15 to 30 aa, or 15 to 20 aa. For example, the length of the polypeptide of the present invention may be 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38 aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48 aa, or 49 aa.

[0014] In some embodiments, the polypeptide of the present invention may be derived from 5-HT2AR. The 5-HT2AR from which the polypeptide of the present invention is derived can be extracted from a bioinformation database (e.g., Genbank, EMI, DDBJ, etc.), and it can be confirmed that the polypeptide is derived from 5-HT2AR based on the information extracted from such a database. For example, if the polypeptide is derived from 5-HT2AR (Genbank accession number: NP_001365853), then this polypeptide is the sequence shown in SEQ ID NO: 1 (i.e., the amino acid sequence shown in NP_001365853) (Amino acid sequence shown in Sequence ID No. 9) It includes (positions 457-471) and may also contain one or more amino acid residues at the N-terminus.

[0015] In one exemplary embodiment, the polypeptide of the present invention is As shown in Sequence ID 9 It consists of amino acid residues 442 to 471.

[0016] In one exemplary embodiment, the polypeptide of the present invention is As shown in Sequence ID 9 It consists of amino acid residues 385 to 471.

[0017] In some embodiments, 5-HT2AR is derived from primates.

[0018] In one preferred embodiment, 5-HT2AR is derived from human.

[0019] In this specification, “to treat” means to produce a desired or beneficial effect in a patient, and may include a reduction in the frequency or severity of one or more symptoms of a disease, or the suppression or prevention of further development of a disease, illness or disorder.

[0020] In this specification, “prevent” means to block or delay the onset of a disease, or to block the onset of its clinical or subclinical symptoms.

[0021] The polypeptide of the present invention exerts antidepressant effects through a novel pathological mechanism of depression (5-HT2AR / D1R complex), and possesses the characteristics of immediate effect, good activity, and minimal side effects, making it highly valuable for clinical development. Furthermore, the polypeptide of the present invention can also provide anxiolytic effects.

[0022] As confirmed in Examples 4 and 7 described later, and in Figures 5, 6, 9, and 10, it should be noted that the polypeptide of the present invention disrupts the interaction of the 5-HT2AR / D1R complex, but does not significantly affect the expression of 5-HT2AR and D1R. In other words, the polypeptide of the present invention exerts its function by specifically disrupting the interaction of the 5-HT2AR / D1R complex.

[0023] In a second aspect, the present invention provides a nucleic acid molecule encoding the polypeptide of the present invention. Furthermore, the present invention provides the use of the nucleic acid molecule of the present invention in the preparation of pharmaceuticals for the treatment and / or prevention of depression or anxiety disorders.

[0024] Using the nucleic acid molecule of the present invention, the antigenic peptide of the present invention is produced, and the sequence of the nucleic acid molecule of the present invention can be appropriately adjusted by those skilled in the art according to the expression system to be used.

[0025] In an exemplary embodiment, the nucleic acid molecule has the sequence shown in SEQ ID NO: 2.

[0026] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention. Furthermore, the present invention provides the use of the expression vector of the present invention in the preparation of a medicament for the treatment and / or prevention of depression or anxiety disorder.

[0027] The nucleic acid sequence of the present invention can be inserted into an expression vector using various known methods. For example, the nucleic acid molecule of the present invention can be inserted into an appropriate restriction endonuclease site. Also, standard techniques for cloning, isolation, amplification and purification, enzymatic reactions using DNA ligase, DNA polymerase or restriction endonucleases, and various separation techniques during the operation belong to the commonly used techniques known to those skilled in the art.

[0028] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector of the present invention. Also, the present invention provides the use of the host cell of the present invention in the preparation of a medicament for the treatment and / or prevention of depression or anxiety disorder.

[0029] The polypeptides of the present invention may be produced using an expression vector and host cells in various expression systems, such as prokaryotic and eukaryotic expression systems. Next, a mammalian expression system is described below as an example. Examples of host cells include the COS-7 cell line of monkey renal fibroblasts and other cell lines capable of expressing a suitable vector, such as the C127 cell line, 3T3 cell line, CHO cell line, Hela cell line, and BHK cell line. The mammalian expression vector must contain an origin of replication, a suitable promoter and enhancer, and, if necessary, a ribosome binding site, a polyadenylation site, a splicing donor site and a splicing acceptor site, a transcription termination sequence and a 5' adjacent non-transcription sequence. For example, the DNA sequences derived from the splicing site and polyadenylation site of SV40 can be used to provide the desired non-transcription gene element. The expression vector may be introduced into host cells by various methods well known to those skilled in the art, including, but not limited to, calcium phosphate transfection, DEAE-glucan transfection, or electroporation.

[0030] In a fifth aspect, the present invention provides a complex comprising the polypeptide of the present invention and a (transport) carrier bound to the polypeptide for permeability across the blood-brain barrier.

[0031] In one exemplary embodiment, the carrier used to cross the blood-brain barrier is HIV-1 Tat protein, insulin, cationized albumin, monoclonal antibody against rat transferrin receptor (OX26), mouse-derived monoclonal antibody against human insulin receptor (HIRMAb), penetratin, transduction domain of Tat protein, Pep-1 peptide, S4 13- It may be one or more of PV, magainin 2, and buforin 2. For example, the transduction domain of TAT having the amino acid YGRKKRRQRRR (shown in SEQ ID NO: 3) can be transduced into the cell by passing through the cell membrane.

[0032] The polypeptide of the present invention can be bound to a carrier for crossing the blood-brain barrier by appropriate linking techniques. Examples of linking techniques include avidin-biotin technology, polyethylene glycol (PEG) spacer arm technology, and fusion protein technology. For example, when using the transduction domain of the HIV-1 Tat protein as the carrier for crossing the blood-brain barrier, the polypeptide of the present invention can be directly linked to the transduction domain of the Tat protein by fusion protein technology.

[0033] In some embodiments, when using fusion protein technology, the polypeptides of the present invention can also be linked to a carrier for crossing the blood-brain barrier using a linker. Exemplary linkers may be flexible linkers containing glycine, such as G, GSG, and GSGGSG. (Sequence No. 4) , GSGGSGG (Sequence ID 5) , GSGGSGGG (Sequence ID 6) , GGGGSGGG (Sequence ID 7) , GGGGS (Sequence 8) Examples include SGG.

[0034] In a sixth embodiment, the present invention provides a method for treating depression in a subject suffering from depression or at risk of depression, comprising the step of administering an effective amount of the polypeptide or complex of the present invention to the subject.

[0035] The polypeptide or complex of the present invention treats depression in the subject by reducing the interaction between 5-HT2AR and D1R.

[0036] Furthermore, the present invention provides a method for treating anxiety disorder in a subject suffering from or at risk of developing an anxiety disorder, comprising the step of administering an effective amount of the polypeptide or complex of the present invention to the subject.

[0037] The polypeptide or complex of the present invention treats anxiety disorders in the subject by reducing the interaction between 5-HT2AR and D1R.

[0038] The “effective dose” or “therapeutic dose” refers to the amount of active agent sufficient to induce a desired biological outcome. This desired biological outcome may be a reduction in the signs, symptoms, or causes of the disease, or other desired changes in the biological system. In this specification, the term “therapeutic dose” is used to mean the amount of formulation that, when administered repeatedly to a lesioned area for a certain period of time, can substantially improve the state of the disease. The therapeutic dose varies considerably depending on the disease condition being treated, the stage of the disease, and the type and concentration of the formulation used. Those skilled in the art may determine an appropriate dose by performing routine experiments.

[0039] The terms “subject,” “individual,” and “patient” are used herein to mean the same thing, and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rats, monkeys, humans, livestock, and companion animals. This also includes biological tissues and biological cells obtained or cultured in vitro, as well as their derivatives.

[0040] In a seventh embodiment, the present invention provides polypeptides or complexes of the present invention used as drugs.

[0041] In some embodiments, use as a drug means use to treat or prevent depression or anxiety disorders. [Brief explanation of the drawing]

[0042] [Figure 1] The results of co-immunoprecipitation Western blots of hippocampal tissue obtained from mice are shown. [Figure 2] The results of co-immunoprecipitation Western blots in CRS mouse models, CMS mouse models, and CSDS mouse models are shown. [Figure 3] The positions of each fragment used in Example 3 on 5-HT2AR are shown. [Figure 4] The results of Western blot GST pulldown analysis of mouse hippocampal tissue using each fragment from Example 3 are shown. [Figure 5] The results of co-immunoprecipitation Western blots of hippocampal tissue obtained from polypeptide-treated mice are shown. [Figure 6] The results of Western blotting of hippocampal tissue obtained from mice treated with polypeptides are shown. [Figure 7] The results of OFT, FST, and TST analyses of polypeptide-treated mice are shown. *: P<0.05; **: P<0.01. [Figure 8] The results of FST, TST, and SPT analyses of polypeptide-treated CRS, CMS, and CSDS mice are shown. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 9] The results of co-immunoprecipitation Western blots of hippocampal tissue obtained from polypeptide-treated CRS mice, CMS mice, and CSDS mice are shown. [Figure 10] The results of Western blotting of hippocampal tissue obtained from polypeptide-treated CRS mice are shown. [Figure 11] The results of OFT analysis (measurement of time spent in the central compartment) of polypeptide-treated mice are shown. **: P<0.01. [Figure 12] The results of EPM analysis of polypeptide-treated mice are shown. *: P<0.05; **: P<0.01. [Modes for carrying out the invention]

[0043] Throughout this specification, unless otherwise stated, terms used herein are understood to have their meanings as commonly understood in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the meanings as commonly understood by those skilled in the art to which this invention pertains. In the event of any conflict, the provisions of this application shall prevail.

[0044] Embodiments of the present invention will be described in more detail below with reference to examples, but the advantages and various effects of the present invention are described more clearly in the following examples. Those skilled in the art will understand that these specific embodiments and examples are intended to illustrate the present invention and are not intended to limit it. [Examples]

[0045] Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall be followed. Furthermore, unless otherwise specified, the reagents or equipment used shall be conventional commercially available products.

[0046] Unless otherwise stated, the experimental animals used in the examples were adult C57BL / 6J male mice (12-14 weeks old) reared at 18-22°C under a 12-hour light / 12-hour dark cycle. Throughout the entire experimental process, the animals were given free access to food and tap water.

[0047] In the statistical analysis performed in the examples, a one-way analysis of variance (ANOVA) was conducted, followed by a post-hoc comparison to evaluate the difference between means. Unless otherwise specified, an independent sample t-test was used to compare the differences between two groups.

[0048] Example 1. Confirmation of 5-HT2AR / D1R complex formation in vivo. Co-immunoprecipitation was performed using protein samples (100-500 μg) obtained from mouse hippocampal tissue. This immunoprecipitation was carried out using anti-5-HT2AR mouse antibody (Santa Cruz Biotechnology, sc-166775) and 25 μl of Protein A / G PLUS-Agarose bead slurry (Santa Cruz Biotechnology, sc-2001).

[0049] Western blot analysis was performed after immunoprecipitation. Specifically, denatured proteins were separated on an 8% SDS-PAGE gel, transferred to a nitrocellulose membrane, and blocked with TBST. Next, anti-D1R mouse antibody (Santa Cruz Biotechnology, sc-33660) and anti-D2R mouse antibody (Santa Cruz Biotechnology, sc-5303) were added and incubated overnight at 4°C. Then, the samples were treated with HRP-labeled secondary antibody for 1 hour, the signal was detected using a SuperSignal ECL chemiluminescence kit, and the band density was analyzed using Image Lab software. The results are shown in Figure 1.

[0050] As can be seen from Figure 1, the anti-5-HT2AR antibody can precipitate D1R but not D2R, indicating that 5-HT2AR can form a complex with D1R in vivo but not with D2R.

[0051] Example 2.5 - Verification of the pathological significance of the HT2AR / D1R complex To investigate the pathological significance of the 5-HT2AR / D1R complex, various depression models were constructed, including chronic restraint stress (CRS), chronic mild stress (CMS), and chronic social defeat stress (CSDS). The construction methods for each model are described below.

[0052] Construction of the CRS mouse model: Mice were fixed horizontally in a cylindrical, flat-bottomed acrylic restraint (25 x 90 mm) for 6 hours a day (10:00 to 16:00), and this restraint stress was applied for two weeks. The filter had several slots to firmly restrain the mouse according to its size and to suppress the physical movement of the mouse's limbs without causing pain. After the restraint period ended, the mice were immediately returned to their cages. Mice that were not restrained (controls) were housed in their home cages without CRS procedures, and neither control mice nor CRS mice were given food or water during the CRS stress period.

[0053] Construction of the CMS mouse model: Mice were subjected to various stresses, including restraint (4 hours), cage tilting (45 times, 12 hours each), light-dark cycle reversal (1 time), flashing light (12 hours), and a dirty cage (2 times, 14 hours each). This program was continued for 6 weeks. Control mice were not subjected to these stresses.

[0054] Construction of the CSDS mouse model: Mice were exposed to highly aggressive CD1 mice (target mice) for 10 minutes each day, with a different CD1 mouse used daily for 10 days. After each 10-minute exposure, the experimental mice were separated from the CD1 mice using a perforated plastic partition. Furthermore, the experimental mice were subjected to a 24-hour continuous stress load via a partition placed in the center of the cage. For control mice, the same procedure (without social stress) was performed using mice of the same strain instead of the highly aggressive mice.

[0055] The interaction between D1R and 5-HT2AR in the constructed CRS mouse models, CMS mouse model, and CSDS mouse model was investigated by co-immunoprecipitation and Western blotting, respectively, in the same manner as in Example 1. The results are shown in Figure 2.

[0056] As can be seen in Figure 2, a significant increase in the 5-HT2AR / D1R complex was observed in the CRS model, CMS model, and CSDS model, confirming that the 5-HT2AR / D1R complex is pathogenic in depression.

[0057] Example 3.5 - Identification of the mutual binding sites of the HT2AR / D1R complex To identify the mutual binding sites of the 5-HT2AR / D1R complex, a full-length 5-HT2AR cDNA clone (Genbank accession number: NM_001378924) was first amplified to obtain cDNA fragments of the CT region (K385~V471) and the third intracellular loop (IL3) region (F255~V324) of 5-HT2AR. These fragments were subcloned into the BamH1 / EcoR1 or BamH1 / Xho1 site of the pGEX-4T-3 plasmid (YouBio, No: VT1255). The initial methionine residue and stop codon were incorporated as appropriate. All constructs were resequenced to confirm that splicing and fusion were performed correctly. GST fusion proteins containing the third intracellular loop (IL3) of 5-HT2AR (GST-5HT2AR-IL3) and GST fusion proteins containing the CT region of 5-HT2AR (GST-5HT2AR-CT) were expressed in live E. coli BL21 cells (AlpalifeBio, No.: KTSM104L) and purified from bacterial lysates. The specific locations of the coding sequences of the IL3 region and CT region on 5-HT2AR are shown in Figure 3.

[0058] 500 μg of lysed mouse hippocampal tissue extract was diluted with 1×PBS / 1% Triton X-100, and then incubated overnight at 4°C with 20 μl of a solution saturated with protein-GST resin containing either GST protein or 15 μg of GST fusion protein. The beads were washed 1 to 8 times with 1×PBS / 1% Triton X-100. The bound proteins were eluted with 2× loading buffer, separated by SDS-PAGE, and Western blotting was performed using each antibody. The results are shown in Figure 4A.

[0059] As can be seen from Figure 4A, the CT region of 5-HT2AR allows for the pulldown of D1R.

[0060] Next, to further investigate the precise interaction sequence / site between 5-HT2AR and D1R, the CT region was divided into the KV region (K385~V411), the NN region (N412~N441), and the DV region (D442~V471) of 5-HT2AR. The specific locations of these regions on 5-HT2AR are shown in Figure 3.

[0061] According to the method described in this embodiment, pull-down analysis was performed using a GST fusion protein containing the KV region of 5-HT2AR (GST-5HT2AR-KV), a GST fusion protein containing the NN region of 5-HT2AR (GST-5HT2AR-NN), or a GST fusion protein containing the DV region of 5-HT2AR (GST-5HT2AR-DV), and the results of the Western blot are shown in Figure 4B.

[0062] As can be seen from Figure 4B, the DV region of 5-HT2AR can be used to pull down D1R.

[0063] Furthermore, the DV region was divided into the DA region (D442~A456) and the SV region (D442~V471) of 5-HT2AR. The specific locations of these regions on 5-HT2AR are shown in Figure 3.

[0064] According to the method described in this embodiment, pull-down analysis was performed using a GST fusion protein containing the DA region of 5-HT2AR (GST-5HT2AR-DA) or a GST fusion protein containing the SV region of 5-HT2AR (GST-5HT2AR-SV), and the results of the Western blot are shown in Figure 4C.

[0065] As can be seen in Figure 4C, the 5HT2AR-SV polypeptide (S457~V471) has affinity for D1R derived from mouse hippocampal tissue, indicating that 5-HT2AR can interact with D1R via its carboxyl-terminal tail.

[0066] Example 4. Confirmation of polypeptide disruption activity against the 5-HT2AR / D1R complex in vivo. To confirm the effect of the 5-HT2AR carboxyl-terminal polypeptide on the 5-HT2AR / D1R complex in vivo, the C-terminus of the SV region (S457~V471) of 5-HT2AR was fused to the N-terminus of the transduction domain of the HIV-1 type Tat protein (shown in SEQ ID NO: 3; hereinafter referred to as TAT) to obtain a fusion protein capable of crossing the blood-brain barrier. This fusion protein was named TAT-5HT2AR-SV. Similarly, the DV region (D442~V471) of 5-HT2AR was fused with TAT to obtain TAT-5HT2AR-DV. Furthermore, the CT region (K385~V471) of 5-HT2AR was fused with TAT to obtain TAT-5HT2AR-CT.

[0067] Mice were treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, or TAT-5HT2AR-CT, and mice treated with TAT alone were used as controls (all mice were treated with a single intraperitoneal dose; 3 nmol / g). One hour after treatment, the mice used in the experiment were analyzed by co-immunoprecipitation in the same manner as in Example 1. The results are shown in Figure 5.

[0068] As can be seen in Figure 5, we successfully disrupted the interaction between 5HT2AR and D1R in mouse hippocampal tissue using 5HT2AR-SV.

[0069] Furthermore, hippocampal tissue from mice treated with TAT-5HT2AR-SV and mice treated with TAT was directly analyzed by Western blotting. As can be seen in Figure 6, the expression levels of 5-HT2AR and D1R did not change significantly after treatment.

[0070] On the other hand, treatment with TAT-5HT2AR-DV and TAT-5HT2AR-CT also successfully disrupted the interaction between 5-HT2AR and D1R, and no significant changes were observed in the expression levels of 5-HT2AR and D1R.

[0071] Example 5. Evaluation of the antidepressant-like effect of the polypeptide of the present invention. To evaluate the antidepressant-like effects of the polypeptides of the present invention, mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, or TAT-5HT2AR-CT (all mice were treated with a single intraperitoneal dose; 3 nmol / g) were subjected to the open field test (OFT), forced swim test (FST), and tail suspension test (TST) one hour after treatment. Mice treated with TAT alone were used as controls. Each test method is described below.

[0072] Open Field Test (OFT): Mice were acclimatized to the experimental environment for one hour and placed in a 45 x 45 x 30 cm chamber. A 5-minute video recording was made to observe the mice's motor activity. The total distance the mice moved was measured in millimeters and analyzed.

[0073] Forced Swimming Test (FST): Mice were placed in a cylindrical plexiglass container (70 cm high, 30 cm in diameter) filled with water (water temperature 23 ± 1°C) to a depth of more than 30 cm. The mice were videotaped for 5 minutes and analyzed to record the length of immobility. Immobility was defined as the mouse floating motionless or having only its nose above the water's surface. Horizontal movement relative to the entire cylindrical container was defined as swimming, and vertical movement relative to the walls of the cylindrical container was defined as climbing.

[0074] Tail suspension test (TST): Mice were suspended upside down with adhesive tape 40 cm above the floor of a rectangular chamber (55 cm long x 20 cm wide x 11.5 cm deep). Video was recorded for 5 minutes, and the time the mouse remained motionless was recorded. Video recording and analysis were performed using EthoVision XT software.

[0075] Some of the test results are shown in Figure 7. From the OFT results shown on the left side of the figure, it was found that there was no significant difference in the total distance traveled between mice treated with TAT-5HT2AR-SV and mice treated with TAT, indicating that this polypeptide had no effect on the mice's mobility. Furthermore, from the FST results shown in the center of the figure and the TST results shown on the right side of the figure, it can be seen that the immobility time in the TAT-5HT2AR-SV treated group was significantly shorter compared to the control group (Tat only). The results for the TAT-5HT2AR-DV treated group and the TAT-5HT2AR-CT treated group were similar to those for the TAT-5HT2AR-SV treated group. From the above results, the antidepressant effect of the present invention was confirmed.

[0076] Example 6. Evaluation of the antidepressant activity of the polypeptide of the present invention using a drug evaluation model. To further evaluate the antidepressant activity of the polypeptide of the present invention, CRS mouse models, CMS mouse models, and CSDS mouse models were constructed according to the method described in Example 2. One hour after construction of the mouse models, each mouse model was treated with 10 μM of TAT-5HT2AR-SV, TAT-5HT2AR-DV, TAT-5HT2AR-CT, or TAT (all mice were treated with a single intraperitoneal dose; 3 nmol / g). Next, the CRS mice underwent FST and TST, and the CMS mice and CSDS mice underwent FST, TST, and sucrose preference tests (SPT), respectively.

[0077] The experimental procedures for FST and TST were carried out in the same manner as described in Example 6, and the specific experimental procedure for SPT was performed as follows.

[0078] SPT was performed using a two-bottle free-choice paradigm. Mice were acclimatized to a 1% sucrose solution for 3 days and then randomly divided into groups. To evaluate the sucrose intake of each individual, the mice were subjected to a 24-hour fasting and dehydration period within 3 days of acclimatization. The following day, two water bottles were prepared, one containing sucrose and the other containing water, and each mouse was given free access to both. After 2.5 hours, the positions of the water bottle and the sucrose bottle were swapped, and the test was conducted for a total of 5 hours. At the end of the test, the amount of water consumed and the amount of sucrose solution consumed were recorded and calculated according to the following formula (I).

number

[0079] As can be seen from the analysis results shown in Figure 8, in all three drug evaluation models, mice treated with TAT-5HT2AR-SV showed a significantly reduced prolongation of stress-induced immobility (A-C, FST and TST) and a significantly enhanced sucrose preference (A-C, SPT) compared with mice treated with TAT. The results for the TAT-5HT2AR-DV and TAT-5HT2AR-CT treatment groups were similar to those for the TAT-5HT2AR-SV treatment group.

[0080] Example 7.5 - Validation of the efficacy of the polypeptide of the present invention in disrupting HT2AR / D1R complex interactions in an animal model. The CRS mice, CMS mice, and CSDS mice (TAT-5HT2AR-SV, TAT-5HT2AR-DV, TAT-5HT2AR-CT, or TAT) from Example 6 that underwent the treatment were analyzed by co-immunoprecipitation in the same manner as in Example 1, and the results of Western blotting are shown in Figure 9.

[0081] As expected, the binding of 5-HT2AR to D1R was significantly increased in all three mouse models, and the interaction between 5-HT2AR and D1R in the hippocampal tissue of stressed mice was significantly reduced in the TAT-5HT2AR-SV treated group compared to the TAT treated group (Figure 9A-C).

[0082] Furthermore, hippocampal tissue from treated CRS mice was directly analyzed by Western blotting to examine the expression levels of D1R and 5-HT2AR. The results are shown in Figure 10. Interestingly, no significant changes in D1R and 5-HT2AR expression levels were observed between the TAT-5HT2A-SV treated group and the TAT treated group. This result indicates that the polypeptide of the present invention has specificity for the binding of D1R and 5-HT2AR.

[0083] Furthermore, CRS mice, CMS mice, and CSDS mice treated with TAT-5HT2AR-DV or TAT-5HT2AR-CT were tested, respectively. The results showed that in the TAT-5HT2AR-DV and TAT-5HT2AR-CT groups, the interaction between 5-HT2AR and D1R was significantly reduced in the hippocampal tissue of stressed mice, but there were no significant changes in the expression levels of D1R or 5-HT2AR.

[0084] Example 8. Evaluation of the anxiolytic-like effect of the polypeptide of the present invention. To evaluate the anxiolytic-like effects of the polypeptides of the present invention, mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, or TAT-5HT2AR-CT (all mice were treated with a single intraperitoneal administration; 3 nmol / g) were subjected to an open-field test (OFT) with measurement of time spent in the central compartment and an elevated cross maze test (EPM) one hour after treatment. Mice treated with TAT were used as a control. Each test method is described below.

[0085] Open Field Test (OFT) (Measurement of time spent in the central compartment): The open field floor was divided into 25 squares of equal area, and the central 9 squares were used as the central compartment. One hour after treating mice with the polypeptide of the present invention, the mice were placed in the central compartment and recorded for 5 minutes to record the time the mice spent in the central compartment.

[0086] Elevated Cross Maze Test (EPM): The elevated cross maze consists of two open arms (25cm x 8cm) and two closed arms (25cm x 8cm). The intersection of these arms is used as the central compartment (8cm x 8cm), and the maze is set up 40cm above the floor. One hour after treating mice with the polypeptide of the present invention, the mice were placed in the central compartment facing the open arms and allowed to move freely for 5 minutes, during which time was recorded. After each mouse was tested, the apparatus was wiped clean with 70% alcohol. The time spent in the open arms, the time spent in the closed arms, and the total distance traveled by the mice were recorded.

[0087] Some of the OFT results are shown in Figure 11. The TAT-5HT2A-SR polypeptide significantly extended the time mice spent in the central compartment, and similarly, the TAT-5HT2AR-DV polypeptide and TAT-5HT2AR-CT polypeptide also significantly extended the time mice spent in the central compartment (P<0.01), indicating that these polypeptides effectively reduced anxiety.

[0088] Figure 12 shows some of the EPM results. The TAT-5HT2A-SR polypeptide significantly extended the time spent in the open arm (A) and significantly shortened the time spent in the closed arm (B), but had no effect on the mouse's total mobility (C). Similarly, the TAT-5HT2AR-DV polypeptide and the TAT-5HT2AR-CT polypeptide significantly extended the time spent in the open arm (P<0.01) and significantly shortened the time spent in the closed arm (P<0.05), but had no effect on the mouse's total mobility. These results demonstrate that the polypeptides of the present invention effectively reduced anxiety.

Claims

1. The use of a complex comprising a polypeptide and a carrier which is a TAT transduction domain shown in SEQ ID NO: 3, conjugated to the polypeptide, in the preparation of a pharmaceutical for the treatment and / or prevention of depression or anxiety disorders, The polypeptide described above, (i) A polypeptide consisting of residues 457 to 471 of the amino acid sequence shown in SEQ ID NO: 9 (ii) A polypeptide consisting of residues 442 to 471 of the amino acid sequence shown in SEQ ID NO: 9, and (iii) Any one selected from polypeptides consisting of residues 385 to 471 of the amino acid sequence shown in Sequence ID No. 9, The C-terminus of the polypeptide is fused to the N-terminus of the carrier.

2. Use of a nucleic acid molecule encoding the complex according to claim 1 in the preparation of a pharmaceutical product for the treatment and / or prevention of depression or anxiety disorder.

3. Use of an expression vector comprising the nucleic acid molecule according to claim 2 in the preparation of a pharmaceutical product for the treatment and / or prevention of depression or anxiety disorder.

4. Use of a host cell comprising the nucleic acid molecule according to claim 2 or the expression vector according to claim 3 in the preparation of a pharmaceutical product for the treatment and / or prevention of depression or anxiety disorder.

5. A therapeutic and / or prophylactic agent for depression or anxiety disorder comprising the complex described in claim 1.

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