Polypeptide and its use

Abstract

The present application provides a polypeptide and its use. The use of the polypeptide in the preparation of a medicament for treating and / or preventing PTSD, depression, or an anxiety disorder is provided, whereby a treatment strategy for PTSD, depression, or an anxiety disorder is provided. A polypeptide, a complex, a nucleic acid molecule, an expression vector, and a host cell are provided.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical research and development, and more specifically, to the medical use of polypeptides.

Background Art

[0002] Post-traumatic stress disorder (PTSD) refers to a delayed and long-lasting mental disorder in an individual who has experienced, witnessed, or encountered one or more of actual death, threat of death, severe injury, or threat to physical integrity, including the individual himself or herself or others. Studies have reported that PTSD is induced by a failure of the response to stress during trauma, resulting in a series of changes including hormonal and neurochemical changes and structural defects in the brain. According to the hypothesis, cortisol dysregulation during trauma may delay the effect of norepinephrine on synapses in the peripheral or central nervous system and affect the enhancement of memory of the event. In contrast, adrenergic activation promotes learning at low cortisol levels. Furthermore, physical and chemical dysregulation in PTSD may affect serotonergic and dopaminergic mechanisms.

Summary of the Invention

Problems to be Solved by the Invention

[0003] There is still a strong need to further explore and identify the etiology and treatment targets of PTSD in order to provide a more abundant and better treatment strategy and to better elucidate the etiology.

Means for Solving the Problems

[0004] To achieve the above object, the present inventors conducted research and found that there is a dimerization interaction between the D2 receptor in the dopamine (DA) receptor and the 5-HT2CR in the 5-hydroxytryptamine (5-HT) receptor. Further results indicate that the dimer may be useful as a target for treating PTSD, thereby achieving the present invention. In particular, DA receptors are receptors that are localized within an organism and function via their corresponding membrane receptors. They can be classified into five types: D1, D2, D3, D4, and D5. The D2 receptor (D2R) is widely expressed in the brain. 5-HT receptors are a group of G protein-coupled receptors and ligand-gated ion channels that are present in the central part of the central nervous system and the peripheral regions of the peripheral nervous system. They 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 includes three subtypes, A, B, and C, namely, 5-HT2A, 5-HT2B, and 5-HT2C receptor proteins. Among them, 5-HT2CR is mainly widely expressed at the presynaptic position throughout the brain, including the limbic-midlimbic cortex and regions of the striatum, such as the VTA, NAc, PFC, amygdala, hippocampus, and dorsal striatum. Having recognized the existence of the D2R / 5-HT2CR dimer, the inventors continued their research and further found that D2R can interact with 5-HT2CR, for example, via its K226-L240 region (hereinafter referred to as the "KL peptide", and its sequence is represented by SEQ ID NO: 1). Subsequently, using an animal model, it was confirmed that the interaction of the D2R / 5-HT2CR complex is inhibited by the KL peptide, and thus the KL peptide has an effect of treating PTSD.

[0005] Accordingly, in a first aspect, the present invention provides a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. Furthermore, the use of the polypeptide of the present invention in the preparation of a medicament for treating and / or preventing PTSD is also provided. The length of the polypeptide of the present invention is 15 - 30 aa, and may be, for example, 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, or 29 aa. In one embodiment, the polypeptide of the present invention may be derived from D2R. The D2R from which the polypeptide is derived can be examined by searching a bioinformatics database (e.g., Genbank, EMI, DDBJ, etc.), and based on this, the sequence of the polypeptide can be confirmed. For example, when the polypeptide is derived from D2R (Genbank accession number CAB56463), in addition to the sequence represented by SEQ ID NO: 1 (i.e., positions 226 to 240 of the amino acid sequence of CAB56463), the polypeptide may further contain one or more additional amino acid residues. For example, the polypeptide sequence at positions 225 to 240 of CAB56463 has 16 amino acid residues. In one embodiment, D2R is derived from a primate. In a particular embodiment, D2R is derived from a human.

[0006] In one embodiment, the polypeptide of the present invention consists of up to 30 consecutive amino acid residues derived from human D2R and includes the sequence represented by SEQ ID NO: 1. In an exemplary embodiment, the polypeptide of the present invention consists of up to 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 consecutive amino acid residues derived from human D2R and includes the sequence represented by SEQ ID NO: 1. In a specific embodiment, the amino acid sequence of D2R derived from a human is represented by SEQ ID NO: 2.

[0007] As used herein, the term "treat" may include inducing a desired or beneficial effect in a patient, which may include reducing the frequency or severity of one or more symptoms of a disease, or suppressing or inhibiting further progression of a disease, condition, or disorder. As used herein, the term "prevent" refers to preventing or delaying the onset of a disease or its clinical or subclinical symptom(s).

[0008] Furthermore, the inventors have found through further research that the polypeptide of the present invention is also useful for controlling depression and anxiety disorders. Accordingly, the use of the polypeptide of the present invention in the preparation of a medicament for treating and / or preventing depression is provided. The use of the polypeptide of the present invention in the preparation of a medicament for treating and / or preventing anxiety disorders is also provided. As used herein, the term "depression" mainly refers to a depressed mental state that may be inappropriate for a person's situation, ranging from emotional decline to extreme sadness; and in certain cases, further to the onset of nausea. As used herein, the term "anxiety disorder" mainly refers to a neurosis characterized mainly by anxious emotions. The polypeptide of the present invention exerts an anti-PTSD effect by acting through a new etiological mechanism (destroying the D2R / 5-HT2CR complex) and shows excellent clinical development value. In addition, the polypeptide of the present invention can also exert antidepressant and anxiolytic effects.

[0009] As demonstrated by the following Examples 4 and 5, and Figures 6, 10 and 11, it is noteworthy that the polypeptide of the present invention disrupted the interaction of the D2R / 5-HT2CR complex without significantly affecting the expression of DA or 5-HT. In other words, the polypeptide of the present invention acts by specifically disrupting the interaction of the D2R / 5-HT2CR complex.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the polypeptide of the present invention. In an alternative embodiment, the nucleic acid molecule of the present invention is the reverse complementary sequence of the coding sequence of the polypeptide of the present invention. In an exemplary embodiment, the nucleic acid molecule of the present invention is represented by SEQ ID NO: 4. Furthermore, the use of the nucleic acid molecule of the present invention in the preparation of a medicament for treating and / or preventing PTSD, depression and / or anxiety disorders is also provided. The nucleic acid molecule of the present invention may be used to produce the polypeptide of the present invention, and those skilled in the art can appropriately adjust the sequence of the nucleic acid molecule based on, for example, the codon preference of the selected expression system according to the expression system used.

[0011] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention. Further provided is the use of the expression vector of the present invention in the preparation of a medicament for treating and / or preventing PTSD, depression and / or anxiety disorder. To insert the nucleic acid molecule of the present invention into an expression vector, various known methods can be used. For example, the nucleic acid molecule can be inserted into appropriate restriction endonuclease sites. Standard techniques for cloning, isolation, amplification and purification, as well as enzymatic reactions including DNA ligase, DNA polymerase, restriction endonucleases, etc., and various separation techniques in manipulation are known to those skilled in the art and are generally used.

[0012] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector of the present invention. Further provided is the use of the host cell of the present invention in the preparation of a medicament for treating and / or preventing PTSD, depression and / or anxiety disorder. The polypeptide of the present invention can be produced using various expression vectors and host cells from expression systems such as prokaryotic and eukaryotic expression systems. In the following description, a mammalian expression system is exemplified, but host cells may include the COS-7 cell line of monkey kidney fibroblasts, and other cell lines capable of expressing compatible vectors such as C127, 3T3, CHO, Hela, and BHK cell lines. Mammalian expression vectors should include an origin of replication, appropriate promoters and enhancers, any necessary ribosome binding sites, polyadenylation sites, splice donor sites and splice acceptor sites, transcription terminator sequences, and 5' non-transcribed flanking sequences. For example, DNA sequences derived from the SV40 splicing and polyadenylation sites can be used to provide the necessary non-transcribed genetic elements. The expression vector can be introduced into host cells by various methods known to those skilled in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation.

[0013] In a fifth aspect, the present invention provides a complex comprising the polypeptide of the present invention and a (transport) carrier for permeating the blood-brain barrier, which is linked to the polypeptide. It is intended that the complex of the present invention makes it more suitable to deliver the polypeptide of the present invention to humans or animals for treating and / or preventing PTSD, depression and / or anxiety disorders. In an exemplary embodiment, the carrier used for permeating the blood-brain barrier may be one or more of HIV-1 Tat protein, insulin, cationized albumin, monoclonal antibody (mAb) against rat transferrin receptor (OX26), human insulin receptor mouse mAb (HIRMAb), penetratin, transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, magainin 2, and buforin 2. For example, the TAT transduction domain can be transduced into cells across the membrane, and its amino acids are YGRKKRRQRRR (represented by SEQ ID NO: 5). The polypeptide of the present invention can be linked to a carrier for permeating the blood-brain barrier by an appropriate linking technique. Exemplary linking techniques may include avidin-biotin technique, polyethylene glycol (PEG)-based spacer arm technique, and fusion protein technique. For example, when the transduction domain of HIV-1 Tat protein is used as a carrier for permeating the blood-brain barrier, the polypeptide of the present invention can be directly linked to the transduction domain of Tat protein using the fusion protein technique. In one embodiment, when using the fusion protein technique, a linker can be used to link the polypeptide of the present invention to a carrier for permeating the blood-brain barrier. Exemplary linkers may be flexible linkers containing glycine, such as G, GSG, GSGGSG (SEQ ID NO: 6), GSGGSGG (SEQ ID NO: 7), GSGGSGGG (SEQ ID NO: 8), GGGGSGGG (SEQ ID NO: 9), GGGGS (SEQ ID NO: 10), and SGG.

[0014] In a sixth aspect, the present invention is a method for treating or preventing PTSD, depression and / or anxiety disorder in a subject, the method comprising administering to the subject an effective amount of the polypeptide or complex of the present invention. The above method is achieved via the polypeptide of the present invention that specifically disrupts the interaction of the D2R / 5-HT2CR complex.

[0015] The term "effective amount" or "therapeutically effective amount" refers to an amount of an active agent sufficient to induce a desired biological result. The result may be a reduction in the signs, symptoms, or causes of a disease, or any other desired change in a biological system. As used herein, the term "therapeutically effective amount" refers to any amount of a preparation that, when administered repeatedly to an affected part over a period of time, induces a substantial alleviation of the disease. This amount may vary depending on the condition being treated, the progression of the condition, and the type and concentration of the preparation used. One of ordinary skill in the art can determine an appropriate amount from conventional experimentation. The terms "subject", "individual", and "patient" as used herein are used interchangeably with respect to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, livestock, and pets. Also included are tissues, cells, and progeny of biological entities obtained or cultured in vitro. In a seventh aspect, there is provided the polypeptide or complex of the present invention for use in the treatment and / or prevention of PTSD, depression and / or anxiety disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

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Modes for Carrying Out the Invention

[0017] Throughout this specification, unless otherwise specified, the terms used herein should be construed in the meaning generally used in the art. Therefore, unless otherwise defined, all technical or scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs, and in case of conflict, the description in this specification shall prevail. Hereinafter, embodiments of the present invention will be described in detail with reference to examples, which will make the advantages and various effects of the present invention clearer. Those skilled in the art should understand that these embodiments and examples are used for illustration rather than limiting the present invention. When specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or equipment used without manufacturer's instructions were commercially available conventional products.

[0018] Experimental animals Balb / c mice were purchased from the Guangdong Provincial Medical Experimental Animal Center in China. They were male mice at 12 - 14 weeks of age and were housed at 18 - 22°C under a 12 - hour light / 12 - hour dark cycle. These animals had free access to food and tap water throughout this process. Adult C57BL / 6J mice (male, 12 - 14 weeks of age) were housed at 18 - 22°C under a 12 - hour light / 12 - hour dark cycle. These animals had free access to food and tap water throughout this process.

[0019] Statistical analysis The results were expressed as mean ± s.e.m. Statistical analysis was performed using Graphpad Prism 8 software. One - way analysis of variance (ANOVA) and post - hoc tests were used to evaluate potential differences between the mean values. Unless otherwise specified, an independent samples t - test was used to compare any two given groups throughout the study. Unless otherwise specified, a test was considered significant when P < 0.05.

Example

[0020] Confirmation of the formation of D2R / 5-HT2CR complex in vivo Co-immunoprecipitation was performed using protein samples from the hippocampal tissues of Balb / c mice (containing 100 - 500 μg of protein). Mouse monoclonal antibodies against 5-HT2CR and D2R, D2R, or 5-HT2CR were used for precipitation with 25 μl of a slurry of protein A / G+ agarose beads (SantaCruz Biotechnology, sc-2001). Western blotting analysis after precipitation: Denatured (100 °C for 10 minutes) proteins were separated on an 8% SDS-PAGE gel and then transferred to a nitrocellulose membrane, which was blocked with skim milk in TBST (Tris-buffered saline containing 0.1% Tween 20). Next, it was incubated overnight at 4 °C with the primary antibody. Finally, after treatment with an HRP-conjugated secondary antibody for 1 hour, signals were detected using an ECL UltraSignal chemiluminescence kit (4A Biotech, cat.no.4AW011-500), and the density of the bands was analyzed using Image Lab software. The results are shown in Figure 1. As can be seen from Figure 1, the antibody against 5-HT2CR pulled down D2R in the hippocampal tissue, confirming the conjugation of D2R to 5-HT2CR.

Example

[0021] Verification of the pathological importance of the complex in PTSD The inventors established a single prolonged stress (SPS) model as follows to determine whether such conjugation is associated with PTSD. The SPS model was established using male Balb / c mice. A series of stress exposures: 2 hours of restraint stress (inside a 50 mL plastic tube with several air holes), 3 minutes of rest, 12 minutes of forced swimming in a transparent glass tank (about 24 °C), 15 minutes of rest (drying under a heating lamp), 20 minutes of odor exposure to a predator (rat), and then ether anesthesia (until the mouse completely lost consciousness) were included using a modified SPS protocol. Then, the mice were returned to a cage with a fresh bedding. Control mice were housed in groups and not given stress exposure. After model establishment, all mice were bred quietly for one week. Using the established SPS mouse model, the interaction between D2R and 5-HT2CR in this model was analyzed by co-immunoprecipitation and Western blotting as described in Example 1, and the results are shown in Figure 2. As seen in Figure 2, a significant interaction between D2R and 5-HT2CR was found in the SPS model, thereby confirming their dimerization.

Example

[0022] Identification of the binding site of the complex To identify the binding site of the D2R / 5-HT2CR complex, the full-length D2R cDNA clone (Genbank accession number X51645) was first amplified to obtain cDNA fragments of the CT region of D2R (D2R-CT, T428 - C443) and the third intracellular loop (IL3) region of D2R (D2R-IL3, K211 - Q373). These fragments were subcloned into the BamH1 / EcoR1 or BamH1 / Xho1 sites of the pGEX-4T-3 plasmid (YouBio No. VT1255). The start methionine residue and stop codon were incorporated when appropriate. All constructs were re-sequenced to confirm that splicing fusion was properly achieved. Escherichia coli BL21 live cells (KangTi Life Technology No. KTSM104L) were used for expression and purification to obtain a GST fusion protein containing the IL3 region of D2R (GST-D2R-IL3) and a GST fusion protein containing the CT region of D2R (GST-D2R-CT) from bacterial lysates. The specific positions of the coding sequences of the CT region and IL3 region on D2R were as shown in Figure 3. The lysed mouse hippocampal tissue extract (containing 500 μg of protein) was diluted with 1×PBS / 1% Triton X-100 and incubated overnight at 4°C with 20 μl of protein-GST-resin saturated with only GST protein or 15 μg of the above GST fusion protein. The beads were washed 1 - 8 times with 1×PBS / 1% Triton X-100. The bound protein was eluted with SDS-PAGE Protein Loading Buffer (YeSen, no: 20315ES05), separated by SDS-PAGE, and Western blotting was performed with each antibody. The results were as shown in Panel A of Figure 4. As can be seen from Panel A of Figure 4, the IL3 region of D2R was able to pull down 5-HT2CR.

[0023] Next, in order to further explore the sequences / regions for the interaction between D2R and 5-HT2CR, the IL3 region was divided into the KVC (K211-V270) region of D2R, the ES (E271-S321) region of D2R, and the PQ (P322-Q373) region of D2R, and their specific positions on D2R are shown in Figure 3. According to the method of this example, pull-down assays were performed using GST fusion proteins containing the KVC region of D2R (GST-D2R-KVC), GST fusion proteins containing the ES region of D2R (GST-D2R-ES), and GST fusion proteins containing the PQ region of D2R (GST-D2R-PQ), and the results of their Western blotting are shown in Panel B of Figure 4. As can be seen from Panel B of Figure 4, the KVC region of D2R was able to pull down 5-HT2CR. Furthermore, the KVC region was divided into the KT (K211-T225) region of D2R, the KL (K226-L240) region of D2R, the KV (K241-V255) region of D2R, and the IV (I256-V270) region of D2R, and the specific positions on D2R are shown in Figure 3. According to the method of this example, pull-down assays were performed using GST fusion proteins containing the KT region of D2R (GST-D2R-KT), GST fusion proteins containing the KL region of D2R (GST-D2R-KL), GST fusion proteins containing the KV region of D2R (GST-D2R-KV), and GST fusion proteins containing the IV region of D2R (GST-D2R-IV), and the results of their Western blotting are shown in Panel C of Figure 4. As can be seen from Panel C of Figure 4, D2R was able to associate with 5-HT2CR with affinity from mouse hippocampal tissue via the KL polypeptide (K226-L240), indicating that D2R can interact with 5-HT2CR via its K226-L240 region.

Example

[0024] Confirmation of the decrease in D2R / 5-HT2CR dimerization by polypeptide To investigate whether the deconjugation of 5HT2CR from D2R by the competing KL polypeptide can reverse their dimerization, the C-terminus of the KL (K226-L240) region of D2R was fused to the transduction domain of the HIV-1 Tat protein (shown in SEQ ID NO: 5, hereinafter referred to as "TAT") to obtain a fusion protein named TAT-D2R-KL that can penetrate the blood-brain barrier. Next, 293T cells were treated with TAT-D2R-KL in the presence of 5-HT2CR and D2R agonists, and those treated with TAT were used as a control. The specific process is shown in Figure 5. Here, the human 293T cell line was cultured in high-glucose modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were maintained in an atmosphere of 95% air and 5% carbon dioxide in an incubator at 37°C. When the cells grew to a density of 70%, transfection was performed using the 5-HT2CR and D2R plasmids at a ratio of 1:1 (purchased from Addgene and Vigenebio (Cat#66411 and CH805293)). The treated cells were analyzed according to the method of Example 1, and the results are shown in Figure 6. It was shown that the interaction between 5-HT2CR and D2R was significantly reduced by treatment with TAT-D2R-KL.

Example

[0025] Evaluation of the anti-PTSD effect of the polypeptide The SPS mouse model was established according to Example 2 to evaluate the anti-PTSD effect of the polypeptide of the present invention. Fear conditioning, safety learning, and recall tests were conducted in two situations (fear conditioning and safety learning tests were conducted in situation A, and the recall test was conducted in situation B). The shapes and scents provided in situations A and B were different. Before the test, the indoor floor was cleaned with 1% acetic acid or 75% ethanol. A camera was placed above the experimental test chamber to record the freezing behavior. After the experiment was completed, the freezing level was manually calculated based on the recorded video. As a criterion for freezing behavior, the case where no movement other than the normal breathing of the mouse was detected within 2 seconds was regarded as the freezing behavior of the mouse. The experimental mice were given CS+ and CS stimuli (30 seconds, 60 dB; CS+: 50 ms pip tone (3 kHz); CS-: white noise) 4 times. On the second day, a fear conditioning test was conducted using CS+ and US (0.8 mA, 2-second foot shock) in combination (3 pairings of CS+ / US with a 90-second inter-trial interval (ITI)). On the third day, the mice were tested in situation B using 5 or 15 CS+ and CS- each. Implementation of safety learning On the ninth day, the SPS mice were treated with TAT-D2R-KL for 1.5 hours (intraperitoneal administration of 100 μl / 10 g mouse of 3 nM TAT peptide), and then CS+ and CS were given in a pseudo-random manner in situation A (ITI: 90 seconds). However, CS+ was paired with US, while CS- was not reinforced with foot shock. On the tenth day, the polypeptide-treated mice were retested in situation B. Interestingly, when the interaction between 5-HT2CR and D2R decreased, the freezing level of SPS mice decreased due to TAT-D2R-KL treatment (Figure 7). Furthermore, TAT-D2R-KL significantly reduced the increase in corticosterone levels in the hippocampus of SPS mice (Figure 8). However, even with TAT-D2R-KL, the levels of cortisol (Figure 9) and DA (Figure 10) in the hippocampus and cortex of SPS mice did not change significantly, and the upregulation of 5-HT in the hippocampus of SPS mice did not decrease (Figure 11). From the above results, it was confirmed that the polypeptide of the present invention achieved an anti-PTSD effect by decoupling the D2R / 5-HT2CR complex.

Example

[0026] Evaluation of the antidepressant effect of the polypeptide using a drug evaluation model CRS and CMS mouse models were established as follows to further evaluate the function of the polypeptide as an antidepressant. CRS: C57BL / 6J mice were horizontally fixed in a first acrylic restraint device with a cylindrical and flat bottom (25×90 mm) for 6 hours daily (10:00 - 16:00) for 2 weeks. The filter has multiple slots that can be adjusted according to the size of the mouse, firmly restraining the mouse and suppressing body movements of the limbs and torso without causing pain. Immediately after restraint, the mice were returned to the cage. When the CRS procedure was not performed, the unrestrained mice (controls) remained in the cage. Both control mice and CRS mice were unable to ingest food and water during CRS exposure. CMS: C57BL / 6J mice were subjected to various stresses such as restraint (4 hours), cage tilt (45 times, 12 hours each), light - dark cycle reversal (1 time), flash (12 hours), and dirty cage (2 times, 14 hours each). The protocol was continued for 6 weeks. Control mice were not subjected to the corresponding stresses. The mice were treated with TAT or TAT - D2R - KL (both single intraperitoneal administrations at 3 nmol / g). One hour after treatment, the open - field test (OFT), forced swim test (FST), tail suspension test (TST), and sucrose preference test (SPT) were performed. The test methods are as follows. OFT (total distance test): The mice were allowed to adapt to the experimental environment for 1 hour and then placed in a chamber of 45 cm×45 cm×30 cm. A video was recorded for 5 minutes to observe the locomotor activity of the mice. The total distance the mice moved was measured and analyzed and expressed in millimeters. FST: The mice were placed in an organic glass cylinder (height 70 cm, diameter 30 cm), filled with water (water temperature 23±1°C) to a water level of 30 cm or more from the bottom. The mice were recorded on video for 5 minutes and the video was analyzed to record the immobility time. Immobility was defined as the mouse floating in the water or not moving with its nose above the water surface. Swimming was defined as horizontal movement across the entire cylinder. Climbing was defined as vertical movement against the wall of the cylinder. TST: The mouse was taped and suspended at a position 40 cm above the floor within a rectangular compartment (length 55 cm × width 20 cm × depth 11.5 cm). A total of 5 minutes of video was recorded to record the immobility time. EthoVision XT software was used for recording and analysis. SPT (Sucrose preference test): A two-bottle choice paradigm was adopted. The mice were acclimated with 1% sucrose solution for 3 days and then randomly grouped. To evaluate the sucrose intake of individuals, the mice were not given water and food for 24 hours over 3 days. The next day, each mouse was allowed to freely use two bottles containing sucrose or water. After 2.5 hours, the positions of the bottles containing water or sucrose were changed. The test was conducted for a total of 5 hours. Finally, the volume of water or sucrose solution consumed was recorded and calculations were performed using the following formula (I).

[0027] [Number] Compound of (I). From the analysis results in Figure 12, it was found that the TAT-D2R-KL treatment did not affect the overall motor ability of the mice. Compared with the TAT-treated mice, the immobility time of the TAT-D2R-KL-treated mice was significantly reduced, and the sucrose preference was significantly enhanced, demonstrating a significant antidepressant effect.

Example

[0028] Evaluation of the anxiolytic effect of the polypeptide using a drug evaluation model Using the method of Example 6, a CRS mouse model was established to evaluate the anxiolytic-like effect of the polypeptide of the present invention. The mice were treated with TAT or TAT-D2R-KL (both at a single intraperitoneal injection of 3 nmol / g), and 1 hour later, the open field test (OFT) and elevated plus maze (EPM) test were performed to test the time spent in the central area. The test methods are as follows. OFT (test for examining the residence time in the central area): The bottom of the open field was divided into 25 equal-area squares, and the central 9 squares were defined as the central area. One hour after the polypeptide treatment, the mice were placed in the central area and recorded for 5 minutes, and the time the mice spent in the central area was recorded. EPM: The apparatus consisted of two open arms (25 cm × 8 cm) and two closed arms (25 cm × 8 cm), with the intersection as the central area (8 cm × 8 cm), and was lifted to a height of 40 cm from the ground. One hour after the polypeptide treatment, the mice were placed in the central area facing the open arms, and the free movement of the mice was recorded for 5 minutes. After each animal was tested, the apparatus was wiped with 70% alcohol. The time the mice spent in the open arms, the time spent in the closed arms, and the total moving distance were recorded. The results of the OFT are shown in Figure 13. The residence time of the mice in the central area was significantly increased by the TAT-D2R-KL peptide. The results of the EPM are shown in Figure 14. The time the mice spent in the open arms was significantly increased by the TAT-D2R-KL peptide, demonstrating that the polypeptide of the present invention effectively reduced the anxious emotion.

Claims

1. Use of a polypeptide in the preparation of a pharmaceutical for the treatment and / or prevention of PTSD, depression, or anxiety disorder, wherein the polypeptide comprises up to 30 consecutive amino acid residues derived from human D2R and includes the sequence represented by Sequence ID No.

1.

2. The use according to claim 1, wherein the polypeptide consists of up to 25 consecutive amino acid residues derived from human D2R.

3. The use according to claim 1 or 2, wherein the amino acid sequence of human D2R is represented by Sequence ID No.

2.

4. A polypeptide consisting of up to 30 consecutive amino acid residues derived from human D2R, and containing the sequence represented by Sequence ID No.

1.

5. The polypeptide according to claim 4, comprising up to 25 consecutive amino acid residues derived from human D2R.

6. The polypeptide according to claim 4, wherein the amino acid sequence of human D2R is represented by Sequence ID No.

2.

7. A complex comprising a polypeptide according to any one of claims 4 to 6 and a blood-brain barrier permeable carrier linked to the polypeptide, wherein the blood-brain barrier permeable carrier comprises HIV-1 Tat protein, insulin, cationized albumin, mAb for rat transferrin receptor, human insulin receptor mouse mAb, penetratin, transduction domain of Tat protein, Pep-1 peptide, S4 １３ - A complex comprising one or more compounds selected from the group consisting of PV, magainin 2, and buforin 2.

8. below: a) A polypeptide according to any one of claims 4 to 6, or a complex comprising the polypeptide and a blood-brain barrier permeable carrier linked to the polypeptide, wherein the blood-brain barrier permeable carrier is one or more selected from the group consisting of HIV-1 Tat protein, insulin, cationized albumin, mAb for rat transferrin receptor, human insulin receptor mouse mAb, penetratin, transduction domain of Tat protein, Pep-1 peptide, S413-PV, magainin 2, and buforin 2, encoding the complex; b) being inversely complementary to the molecule described in a) above; or c) Represented by Sequence ID 4; Nucleic acid molecule.

9. An expression vector comprising the nucleic acid molecule described in claim 8.

10. A host cell comprising the nucleic acid molecule described in claim 8 or an expression vector containing the nucleic acid molecule.

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