Use of polypeptides in combating addiction and its relapse, as well as complexes and polypeptides - Patents.com

Abstract

The present invention relates to the use of a polypeptide in resisting addiction and its relapse.The present invention provides the use of a polypeptide in preparing a medicament for treating and / or preventing substance addiction and its relapse.The polypeptide consists of at least 11 consecutive amino acid residues of the sequence shown in SEQ ID NO:1 and comprises the sequence shown in SEQ ID NO:2.Furthermore, the present invention relates to the use of said polypeptide, the use of a nucleic acid molecule, the use of an expression vector and the use of a host cell, as well as a complex comprising said polypeptide.

Description

[Technical Field]

[0001] The present invention relates to the field of substance addiction, and in particular to the use of polypeptides in the prevention or treatment of addiction or its relapse. [Background technology]

[0002] Substance addiction, as defined by the World Health Organization (WHO), is the repeated use of a substance despite awareness and prior experience of its harmful effects. Substance addiction is a chronic, relapsing disorder characterized by uncontrollable substance use, compulsive substance seeking and craving, continued use despite harmful effects, and physical and / or psychological substance dependence. Substance addiction generally follows a progression of tolerance, withdrawal, compulsive drug-using behavior, drug-seeking behavior, addictive behavior, and relapse.

[0003] Substance addiction has become a global medical and social problem, with significant social and economic impacts for both addicts and society. For example, substance addiction is often closely associated with violent crime and the spread of infectious diseases. Another example is that substance addiction can result in addicts losing all or part of their workforce, thereby negatively impacting individuals, families, and society. In particular, as one of the most commonly abused substances in the world, alcoholism can cause serious liver and cardiovascular diseases, severe mental disorders, social problems, and other negative consequences, such as family breakdown, tragic accidents, and reduced work efficiency.

[0004] Although several withdrawal medications are often used in drug detoxification or withdrawal treatment for addicts, these medications still have limitations.

[0005] For example, common methods used in opioid addiction include (i) substitution therapy (currently, methadone substitution is the most commonly used method for drug detoxification); (ii) opioid receptor antagonists, naltrexone or naloxone, to facilitate withdrawal; and (c) non-opioid treatments using clonidine and lofexidine to suppress withdrawal symptoms. While these methods can prevent addicts from experiencing physical discomfort (chills, vomiting, tremors, etc.) after ceasing use of the addictive substance, research has shown that these medications do not reduce the addict's psychological dependence (or psychological dependency).

[0006] As another example, the potential therapeutic effects of various types of medications (e.g., naltrexone, acamprosate, ondansetron, disulfiram, gamma-hydroxybutyric acid (GHB), and topiramate) are being tested for alcohol addiction. Among these medications, naltrexone, acamprosate, and disulfiram have been confirmed to have some efficacy and are approved for the treatment of alcoholism. Furthermore, among these medications, naltrexone is currently considered a good pharmacological option. However, although these medications (including naltrexone) have shown some promising results, they are not sufficiently effective against alcoholism, and the prognosis remains poor.

[0007] Therefore, there is a strong need in the field of substance addiction treatment to develop safe and effective pharmaceuticals to provide a wider range of better treatment or prevention strategies for substance addiction and its relapse. Summary of the Invention [Means for solving the problem]

[0008] To achieve the above object, the present inventors have conducted numerous studies on the molecular mechanisms of addiction and have found that a new polypeptide can disrupt the interaction between D2 receptors and NMDA receptors.

[0009] Specifically, dopamine (DA) receptors are a type of receptor present in living organisms and exert their effects via corresponding cell membrane receptors. DA receptors can be classified into five types: D1, D2, D3, D4, and D5. D2 receptors (D2R) are widely expressed in the brain.

[0010] NMDA receptors (NMDARs) play important physiological roles in the development of the nervous system, including regulating neuronal survival, regulating the development of neuronal dendrites and axons, and participating in the formation of synaptic plasticity. They also play important roles in the formation of neural circuits and the pathogenesis of various neuropsychiatric disorders. Data suggest that NMDA receptors are one of the receptors crucial for learning and memory processes. NMDARs are composed of three main subunits: NR1, NR2, and NR3. The NR2 subunit has four subunits: NR2A, NR2B, NR2C, and NR2D.

[0011] The present inventors have confirmed that the polypeptide of the present invention is effective in treating addiction to an addictive substance and in preventing or treating relapse, and have completed the present invention.

[0012] Thus, in a first aspect, the present invention provides a polypeptide consisting of at least 11 consecutive amino acid residues of the sequence set out in SEQ ID NO: 1 and comprising the sequence set out in SEQ ID NO: 2. The present invention also provides the use of a polypeptide of the invention in the preparation of a medicament for the prevention and / or treatment of substance addiction.

[0013] As used herein, "treatment" refers to producing 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 inhibiting or preventing further development of a disease, condition, or disorder.

[0014] As used herein, "prevention" means preventing or delaying the onset of a disease, or preventing the onset of its clinical or subclinical symptoms.

[0015] As used herein, "substance addiction" refers to a chronic, relapsing disorder characterized by uncontrollable substance use, compulsive substance seeking and craving, persistent use despite harmful effects, and physical and / or psychological dependence on the substance. Substance addiction may be, for example, an addiction that arises upon initial use of the substance, or may refer to the formation of a substance addiction and / or the manifestation of a substance addiction.

[0016] In an exemplary embodiment, a polypeptide of the invention consists of at least 11 contiguous amino acid residues, at least 12 contiguous amino acid residues, at least 13 contiguous amino acid residues, at least 14 contiguous amino acid residues, or at least 15 contiguous amino acid residues of the sequence set forth in SEQ ID NO:1.

[0017] In an exemplary embodiment, the sequence of the polypeptide of the present invention consists of positions 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14, 2 to 13, 3 to 15, 3 to 14, or 3 to 13 of SEQ ID NO: 1 (KIYIVLRRRRKRVNT).

[0018] In a specific embodiment, the sequence of a polypeptide of the invention is set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4.

[0019] In some embodiments, the addictive substance may be selected from the group consisting of, for example, morphine, nicotine, alcohol, cocaine, codeine, dihydrocodeine, hydromorphone, oxycodone, methadone, morphine, fentanyl, and pethidine.

[0020] In a second aspect, the present invention provides the use of a polypeptide of the invention in the preparation of a medicament for the prevention and / or treatment of relapse to a substance addiction.

[0021] As used herein, "relapse," also known in some contexts as "drug relapse," refers to an addict's return to use of a previously used addictive substance after withdrawal. Relapse can be triggered by environmental factors (events), addictive substance factors, and / or stressors (stress).

[0022] In an exemplary embodiment, the recurrence in the present invention is caused by an environmental factor.

[0023] In an exemplary embodiment, relapse in the present invention is caused by an addictive substance trigger.

[0024] In an exemplary embodiment, a relapse in accordance with the present invention is caused by a stressor.

[0025] The present inventors have unexpectedly discovered that the polypeptides of the present invention can inhibit the binding that results in the formation of the D2R / NR2B complex, thereby eliminating the formation of addictive substances.

[0026] Furthermore, the polypeptides of the present invention are highly valuable for clinical development because they can eliminate relapse of addiction caused by environmental factors, addictive substance factors, and stress factors, and they exhibit statistically significant effects even under conditions induced by high / low doses of addictive substances.

[0027] In a third aspect, the present invention provides a nucleic acid molecule encoding a polypeptide of the present invention. Furthermore, the present invention provides the use of a nucleic acid molecule of the present invention in the preparation of a medicament for the treatment and / or prevention of substance addiction, or the use of a nucleic acid molecule of the present invention in the preparation of a medicament for the treatment and / or prevention of relapse to a substance addiction.

[0028] The polypeptide of the present invention is produced using the nucleic acid molecule of the present invention, and the sequence of the nucleic acid molecule of the present invention can be appropriately adjusted by those skilled in the art depending on the expression system used.

[0029] In an exemplary embodiment, the nucleic acid molecule has the sequence shown in SEQ ID NO:5.

[0030] In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule of the present invention. Furthermore, the present invention provides the use of an expression vector of the present invention in the preparation of a medicament for the treatment and / or prevention of substance addiction, or the use of an expression vector of the present invention in the preparation of a medicament for the treatment and / or prevention of relapse to a substance addiction.

[0031] The nucleic acid sequence of the present invention can be inserted into expression vector by various known methods.For example, the nucleic acid molecule of the present invention can be inserted into suitable restriction endonuclease site.In addition, standard techniques of cloning, isolation, amplification and purification, enzyme reaction using DNA ligase, DNA polymerase or restriction endonuclease, and various separation techniques during operation belong to the commonly used techniques known to those skilled in the art.

[0032] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector of the invention. Furthermore, the present invention provides the use of the host cell of the invention in the preparation of a medicament for the treatment and / or prevention of substance addiction, or the use of the host cell of the invention in the preparation of a medicament for the treatment and / or prevention of relapse to a substance addiction.

[0033] The polypeptides of the present invention may be produced using expression vectors and host cells in various expression systems, including prokaryotic and eukaryotic expression systems. An example of a mammalian expression system is described below. Host cells include the monkey kidney fibroblast COS-7 cell line and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors must contain an origin of replication, an appropriate promoter and enhancer, and, if necessary, a ribosome binding site, a polyadenylation site, splice donor and acceptor sites, a transcription termination sequence, and a 5'-flanking nontranscribed sequence. For example, DNA sequences derived from the SV40 splice and polyadenylation sites, respectively, can be used to provide the desired nontranscribed genetic elements. The expression vector may be introduced into the host cell by various methods well known to those skilled in the art, including, but not limited to, calcium phosphate transfection, DEAE-glucan-mediated transfection, or electroporation.

[0034] In a sixth aspect, the present invention provides a complex comprising a polypeptide of the present invention and a (transport) carrier bound to said polypeptide for permeating the blood-brain barrier.

[0035] In an exemplary embodiment, the carrier used to penetrate the blood-brain barrier is selected from the group consisting of HIV-1 Tat protein, insulin, cationized albumin, a monoclonal antibody against the rat transferrin receptor (OX26), a murine monoclonal antibody against the human insulin receptor (HIRMAb), Penetratin, the transduction domain of the Tat protein, Pep-1 peptide, S4 13The transduction domain may be one or more of -PV, magainin 2, and buforin 2. For example, the transduction domain of TAT, whose amino acids are YGRKKRRQRRR (shown in SEQ ID NO: 6), can cross the cell membrane and be transduced into cells.

[0036] 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 include avidin-biotin technology, polyethylene glycol (PEG)-based spacer arm technology, fusion protein technology, etc. For example, when the transduction domain of the 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 the Tat protein by the fusion protein technique.

[0037] In some embodiments, when using fusion protein technology, the polypeptides of the present invention can be linked to carriers for penetration through the blood-brain barrier using a linker. Exemplary linkers can be flexible linkers containing glycine, such as G, GSG, GSGGSG. (SEQ ID NO: 9) , GSGGSGG (SEQ ID NO: 10) ,GSGGSGGG (SEQ ID NO: 11) ,GGGGSGGG (SEQ ID NO: 12) , GGGGS (SEQ ID NO: 13) , SGG, etc.

[0038] In a seventh aspect, the present invention provides a method of treating or preventing a substance addiction in a subject, comprising: The present invention provides a method comprising the step of administering to a subject an effective amount of a polypeptide or complex of the present invention.

[0039] The present invention further provides a method for preventing relapse to a substance addiction in a subject, comprising: The present invention provides a method comprising the step of administering to a subject an effective amount of a polypeptide or complex of the present invention.

[0040] The polypeptides or complexes of the present invention accomplish this method by decreasing the interaction between D2R and NR2B.

[0041] "Effective amount" or "therapeutically effective amount" refers to the amount of an active agent sufficient to induce a desired biological result. This desired biological result may be a reduction in the signs, symptoms, or causes of a disease, or other desired changes in a biological system. As used herein, the term "therapeutically effective amount" refers to an amount of a formulation that can substantially improve the disease state when repeatedly administered to the affected area over a certain period of time. The therapeutically effective amount varies depending on the condition being treated, the progression of the condition, and the type and concentration of the formulation used. Those skilled in the art will be able to determine the appropriate amount by performing routine experimentation.

[0042] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rats, monkeys, humans, livestock, and pets. Also included are biological tissues and cells obtained or cultured in vitro, and the progeny thereof.

[0043] In an eighth aspect, the present invention provides a polypeptide or complex of the present invention for use as a medicament.

[0044] In some embodiments, medicinal use refers to use to treat or prevent substance addiction.

[0045] In some embodiments, medicinal use refers to use to prevent relapse of a substance addiction. [Brief explanation of the drawings]

[0046] [Figure 1] The results of co-immunoprecipitation of hippocampal tissue from mice are shown. [Figure 2]The positions on D2R of each fragment used in Example 3 are shown. [Figure 3] 1 shows the results of a GST pull-down experiment for each fragment from Example 3. [Figure 4] 1 shows the results of co-immunoprecipitation of hippocampal tissue from place-preference (CPP) and behaviorally sensitized (BS) mice treated with KT polypeptide. [Figure 5] 1 shows the experimental scheme design of Example 5. [Figure 6] 1 shows the results of CPP scores in Example 5. [Figure 7] 1 shows the experimental scheme design of Example 6. [Figure 8] 1 shows the results of CPP scores in Example 6. A: Measurement results for each group on day 10; B: Comparison of results for the TAT-D2R-KT group on days 10 and 11. [Figure 9] 1 shows the experimental scheme design of Example 7. [Figure 10] 1 shows the results of CPP measurement on the 10th day in Example 7. [Figure 11] 1 shows the results of CPP measurement in Test 1 of Example 7. [Figure 12] 1 shows the results of CPP measurement in Test 2 of Example 7. [Figure 13] 1 shows the results of CPP measurement in Test 3 of Example 7. [Figure 14] 1 shows the results of CPP measurement in Test 4 of Example 7. [Figure 15] 1 shows the experimental scheme design of Example 8. [Figure 16] 1 shows the measurement results of BS in Test 1 of Example 8. A: Results of the distance traveled by BS mice in Test 1; B: Results of the distance traveled by BS mice in Test 2; C: Results of the distance traveled by BS mice in Test 3. [Figure 17] The effects of various polypeptides prepared in Example 9 on morphine-induced CPP in mice (n=12) are shown. ##: P<0.01 vs. saline group; **: P<0.01 vs. morphine group. [Figure 18]This shows that the various polypeptides prepared in Example 9 have a negative effect on cocaine-induced behavioral sensitization in mice (n=10). *: P<0.05 vs. vehicle group; **: P<0.01 vs. vehicle group. [Figure 19] This shows that various polypeptides prepared in Example 9 have a negative effect on alcohol-induced behavioral sensitization in mice (n=10). ##: P<0.01 vs. saline group; **: P<0.01 vs. alcohol group. DETAILED DESCRIPTION OF THE INVENTION

[0047] To avoid the enormous burden that substance addiction places on individuals, families, and society, how to eliminate substance addiction and how to prevent relapse are currently attracting global attention as medical and social issues. Interestingly, Liu et al. (Modulation of D2R-NR2B Interactions in Response to Cocaine, Neuron 52, 897-909, Decree 7, 2006) reported that stimulation of dopamine by cocaine in the neostriatum in vivo enhanced the formation of a heteroreceptor complex between the D2R and the NR2B subunit of the NMDA receptor. Furthermore, Liu et al.'s investigation of the interaction between D2R and NR2B revealed that the region responsible for the formation of the D2R-NR2B complex is located in the third intracellular loop (IL3) of the D2R. Further competition experiments by Liu et al. revealed that the 10 residues (TKRSSRAFRA, T225-A234) of IL3 were involved in the formation of the heteroreceptor complex. , SEQ ID NO: 14 ) was shown to be important for blocking the binding of D2R to NR2B.

[0048] Liu et al. also reported that they tested various IL3 polypeptides derived from D2R and obtained the following results (see Figures 3M and 3N in the above reference). Specifically, IL3 polypeptides derived from D2R inhibited the binding of D2R to NR2B only when they contained the motif T225-A234 (P1, P2, P3, and P5), whereas polypeptides lacking this motif (P4, I210-K226) failed to inhibit the binding of D2R to NR2B. Furthermore, the control peptide P6, obtained by scrambling the sequence of peptide P5 containing this motif, also failed to inhibit the binding of D2R to NR2B. Therefore, Liu et al. suggested that the binding of D2R to NR2B may be sequence-specific and that TKRSSRAFRA may be the binding motif.

[0049] However, as a result of our research, we unexpectedly found that, contrary to the report by Liu et al., the polypeptide K211-T225 in the IL3 region of D2R (which, compared to polypeptide P4, which is thought not to inhibit the binding of D2R to NR2B, lacks one residue at the N-terminus and one residue at the C-terminus and does not contain the important motif TKRSSRAFRA) was able to reduce the interaction between D2R and NR2B.

[0050] Further investigation by the inventors has concluded that the polypeptide K211-T225 in the IL3 region of D2R and its variants with N- and C-terminal deletions can reverse altered addictive behavior in an addiction model.

[0051] Throughout this specification, unless otherwise specified, it is understood that the terms used herein have the meanings commonly used in the art.Therefore, unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs.In the event of any discrepancy, the description of this application shall prevail.

[0052] The embodiments of the present invention will be described in more detail below with reference to examples, and the advantages and various effects of the present invention will be more clearly described 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 the present invention. [Example]

[0053] Unless specific conditions are described in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Furthermore, unless the manufacturer of the reagents or equipment used is described, the reagents or equipment used are conventional, commercially available products.

[0054] Unless otherwise stated, the experimental animals used in the examples were adult C57 / BL6J male mice (8 weeks old, weighing 25-30 g) purchased from the Guangzhou Medical Laboratory Animal Center (China). The mice were housed under a 12-hour light / 12-hour dark cycle and had free access to food and water throughout the experimental procedure. All mice were acclimated in the animal room for 1 week before experimental manipulation.

[0055] In the examples, band and morphological data were analyzed using ImageJ and Image Lab software (ImageJ 1.30), and statistical analysis was performed using GraphPad Prism 8 software. Sample amounts were selected according to previous reports (Arifin and Zahiruddin, 2017; Mead et al., 2012). All experiments were performed independently in triplicate. Randomization / blinding was not used. Data are presented as mean ± SEM. One-way or two-way ANOVA was performed for various groups, followed by post-hoc testing with Tukey's multiple comparison test. A p<0.05 was considered statistically significant. *: p<0.05, **: p<0.001, ***: p<0.0001, and ****: p<0.000001, unless otherwise stated.

[0056] Example 1. Confirmation of D2R / NR2B complex formation in vivo Immunoprecipitation procedure: Co-immunoprecipitation was performed using protein samples (100–500 μg) obtained from hippocampal tissue. NR2B and D1R or D2R were precipitated using anti-NR2B and anti-D2R antibodies, respectively, and 25 μl of Protein A / G PLUS-Agarose bead slurry (Santa Cruz Biotechnology, Inc., sc-2001).

[0057] Proteins were then separated on an 8% SDS-PAGE gel and detected with mouse anti-NR2B and mouse anti-D2R antibodies. Proteins were detected using HRP-conjugated secondary antibodies and enhanced chemiluminescence. Band density analysis was performed using Image Lab software.

[0058] To assess whether D2R and NR2B can form a protein complex, we first performed the co-immunoprecipitation assay described above to examine the possibility of an interaction between D2R and NR2B in the hippocampus of normal mice. The results are shown in Figure 1.

[0059] As can be seen in Figure 1, NR2B could be precipitated from the hippocampal protein mixture using the D2R antibody, but not using the D1R antibody, indicating that the D2R and NR2B proteins bind to each other.

[0060] Example 2. Verification of the pathological significance of the D2R / NR2B complex Next, to examine whether the interaction state between D2R and NR2B is related to substance addiction, we performed immunoprecipitation experiments on hippocampal tissue samples obtained from mice with morphine-induced place preference (CPP) or behavioral sensitization (BS) following the method described in Example 1. The results are shown in Figure 1.

[0061] As can be seen in Figure 1, the binding of D2R to NR2B was significantly increased in hippocampal tissue from both CPP and BS mice, confirming that the formation of the D2R / NR2B complex may play a role in the pathogenesis of substance addiction.

[0062] Example 3. Exploration of the mutual binding site of the D2R / NR2B complex To explore the physical interaction site between D2R and NR2B, we first isolated a cDNA fragment from the D2R CT region (T428-C443) and a cDNA fragment from the D2R third intracellular loop (IL3) region (K211-Q373) from the full-length D2R cDNA clone (GenBank accession number: M29066.1). These fragments were subcloned into the BamH1 / EcoR1 or BamH1 / Xho1 sites of the pGEX-4T-3 plasmid (YouBio, catalog number: VT1255). A methionine residue as the initiation codon and a stop codon were added as appropriate. All constructs were resequenced to confirm proper splicing fusion. Each construct was expressed in live Escherichia coli BL21 cells (AlpalifeBio, Catalog No. KTSM104L), and the expressed proteins were purified from bacterial lysates to obtain a GST fusion protein containing the IL3 domain of D2R (GST-D2R-IL3) and a GST fusion protein containing the CT domain of D2R (GST-D2R-CT). The specific locations of the coding sequences for the IL3 and CT domains on D2R are shown in Figure 2.

[0063] 500 μg of the dissolved hippocampal extract was diluted with 1x PBS / 0.1% Triton X-100 and incubated overnight at 4°C with 20 μl of GST resin for protein purification (TransGen Biotech, Catalog No. N21220, Beijing, China) saturated with GST protein alone or 20 μl of GST resin for protein purification saturated with 15 μg of the prepared GST fusion protein. The beads were washed 1 to 8 times with 1x PBS / 0.1% Triton X-100. Bound proteins were eluted with 2x loading buffer, separated by SDS-PAGE, and Western blotted with each antibody. The results are shown in Figure 3A.

[0064] As can be seen in Figure 3A, NR2B was affinity precipitated by the IL3 domain of D2R, indicating that the IL3 domain of D2R is involved in the interaction.

[0065] To further investigate the interaction sequences / sites between D2R and NR2B, the IL3 region was divided into the KVC region of D2R (K211-V270), the ES region of D2R (E271-S321), and the PQ region of D2R (P322-Q373). The specific locations of these regions on D2R are shown in Figure 2.

[0066] According to the method described in this Example, pull-down analysis was performed using a GST fusion protein containing the KVC region of D2R (GST-D2R-KVC), a GST fusion protein containing the ES region of D2R (GST-D2R-ES), or a GST fusion protein containing the PQ region of D2R (GST-D2R-PQ). The results of Western blot analysis are shown in Figure 3B.

[0067] As can be seen in Figure 3B, NR2B was affinity precipitated by the KVC region of D2R, indicating that the KVC region of D2R is involved in the interaction.

[0068] The KVC region was further divided into the D2R KT region (K211-T225), the D2R KL region (K226-L240), the D2R KV region (K241-V255), and the D2R IV region (I256-V270). The specific locations of these regions on the D2R are shown in Figure 2.

[0069] According to the method described in this Example, pull-down analysis was performed using a GST fusion protein containing the KT region of D2R (GST-D2R-KT), a GST fusion protein containing the KL region of D2R (GST-D2R-KL), a GST fusion protein containing the KV region of D2R (GST-D2R-KV), or a GST fusion protein containing the IV region of D2R (GST-D2R-IV). The results of Western blot analysis are shown in Figure 3C.

[0070] Surprisingly, as shown in Figure 3C, the KL region of D2R (K226-L240) was unable to precipitate NR2B, but the KT region of D2R (K211-T225; hereafter, this region is referred to as the "KT polypeptide") affinity-precipitated NR2B.

[0071] Example 4. Inhibition of complex formation by polypeptides in vivo To verify the effect of the D2R KT polypeptide on the D2R / NR2B complex in vivo, the C-terminus of the region (K211-T225) was fused to the N-terminus of the transduction domain of the HIV-1 Tat protein (SEQ ID NO: 6; hereafter referred to as "TAT") to obtain a fusion protein capable of penetrating the blood-brain barrier. This fusion protein was designated "TAT-D2R-KT."

[0072] CPP mice and BS mice were treated with TAT-D2R-KT, respectively, and mice treated with TAT alone served as controls (all mice were treated by a single intraperitoneal injection; 10 ml / kg body weight; drug concentration: 3 nmol / g). One hour after treatment, analysis was performed by co-immunoprecipitation as described in Example 1. The results are shown in Figure 4.

[0073] As can be seen from FIG. 4, the KT polypeptide of the present invention competitively disrupted the D2R / NR2B interaction in the hippocampal tissues of CPP mice and BS mice.

[0074] Example 5. Blockade of morphine-induced CPP formation by polypeptides To evaluate whether the polypeptide of the present invention has an effect on morphine addiction, mice were treated with TAT-D2R-KT or TAT at the stage of morphine-induced CPP formation, and the CPP score was measured.

[0075] The CPP equipment and test system are as follows: The CPP apparatus used in the experiment consisted of two boxes (15 cm × 15 cm × 37 cm) of equal volume, with a movable door between them. Mice were allowed to move freely between the boxes during testing, but were confined to one box during training. One box had a black interior and a striped floor, while the other box had a white interior and a checkered floor. All behavioral tests, including activity distance and activity time, were recorded with a camera and calculated using the SMART Video Tracking system (version 2.5; Panlab Technology for Bioresearch, Spain).

[0076] The experimental design is shown in Figure 5. On day 1, mice were allowed to freely move between the two boxes for 15 min without any treatment (pre-test). On days 2, 4, 6, and 8, the control group (saline group) received saline (10 mL / kg), and the model group (morphine group) received morphine (10 mg / kg). The TAT group (3 nmol / g) and TAT-D2R-KT group (3 nmol / g) received an intraperitoneal injection (10 mL / kg body weight) 1 h before the morphine injection. After the injection, all mice were trained in the white box for 40 min. On days 3, 5, 7, and 9, all mice were administered saline and immediately placed in the black box for 40 min. On day 10, all mice were allowed to freely move between the two boxes for 15 min, and the test was terminated. The CPP scores (time spent by mice) are shown in Figure 6.

[0077] As can be seen in Figure 6, treatment with TAT-D2R-KT significantly reduced CPP scores compared to subjects treated with TAT, indicating that TAT-D2R-KT can interfere with the formation of morphine-induced CPPs.

[0078] Example 6. Effect of polypeptides on the expression stage of morphine-induced CPP To further evaluate the specific effect of the polypeptide of the present invention on the developmental stage of morphine-induced CPP, a test was conducted using the CPP apparatus of Example 5 according to the experimental design shown in Figure 7. In this test, the model group (morphine group) was injected with saline on day 10, and the CPP mice in the TAT-D2R-KT group (morphine + TAT-D2R-KT) were treated with TAT-D2R-KT (single administration) on day 10, and the CPP scores were measured 1 hour later. The experimental results are shown in Figure 8A.

[0079] As shown in Figure 8A, treatment with TAT-D2R-KT on day 10 did not significantly reduce the CPP score.

[0080] However, when the CPP test was repeated on day 11, it was found that the CPP scores of the TAT-D2R-KT group on day 11 were significantly lower than those on day 10 (Fig. 8B), suggesting that when morphine addiction memory recurred on day 10, the memory associated with this memory was eliminated by TAT-D2R-KT.

[0081] Example 7. Elimination of morphine-induced drug relapse by polypeptides To verify that the polypeptide of the present invention can eliminate memories associated with the recurrence of addictive memory and to test the long-term effect of the polypeptide of the present invention on drug relapse in morphine-induced CPP, experiments were designed to induce natural withdrawal and drug relapse in CPP mice, as shown in Figure 9, and multiple tests were carried out. In these tests, a saline group was used as a control.

[0082] CPP was successfully induced in mice on day 10 by administering morphine according to the conditions shown in Figure 9. The results are shown in Figure 10.

[0083] The mice were then returned to their cages and housed for 3 weeks. On day 32, the mice in the TAT and TAT-D2R-KT groups were treated with TAT or TAT-D2R-KT (3 nmol / g), respectively. One hour later, the mice were allowed to move freely for 15 minutes and then test 1 was performed. The results are shown in Figure 11.

[0084] As can be seen from Figure 11, the CPP scores of mice in the TAT-D2R-KT group were significantly lower than those of mice in the TAT group, indicating that the polypeptide of the present invention can eliminate environmentally induced drug relapse.

[0085] To further investigate the potential effect of the polypeptide of the present invention on drug relapse induced by addictive drugs, after Test 1, each group was returned to its cage and housed for 3 weeks. On day 54, the TAT and TAT-D2R-KT groups were injected with morphine (5 mg / kg). The mice were immediately placed in the experimental box and allowed to move freely for 15 minutes, after which Test 2 was performed. The results are shown in Figure 12.

[0086] As can be seen from FIG. 12, in this test as well, the CPP scores of the mice in the TAT-D2R-KT group were significantly lower than those of the mice in the TAT group.

[0087] To rule out a dose effect, after Test 2, the groups were returned to their cages for 2 weeks. On Day 69, the mice in the TAT and TAT-D2R-KT groups were injected with a high dose of morphine (15 mg / kg). Next, all mice were allowed to move freely for 15 minutes, and Test 3 was performed. The results are shown in Figure 13.

[0088] Surprisingly, even high doses of morphine could not reverse the significant decrease in CPP scores induced by TAT-D2R-KT, indicating that TAT-D2R-KT could eliminate drug relapse induced by low / high doses of addictive substances.

[0089] To further investigate the effect of TAT-D2R-KT on drug relapse induced by stress, after Test 3, each group was returned to its cage and housed for one week. On Day 77, yohimbine (α2 adrenergic receptor antagonist, 2 mg / kg, purchased from MCE, Catalog No. 18735) was administered to the TAT and TAT-D2R-KT mice, and a 15-minute test was performed after administration as Test 4. The results are shown in Figure 14.

[0090] As can be seen from Figure 14, no significant difference was observed in the CPP scores between the TAT-D2R-KT group and the control group, indicating that stress-induced drug relapse could be prevented by the polypeptide of the present invention.

[0091] Example 8. Verification of resistance to addiction-related behaviors using a behavioral sensitization model To further verify the effect of the polypeptide of the present invention on morphine addiction-related behavior, the above results were verified using a morphine-induced behaviorally sensitized mouse (BS) model.

[0092] The experiment was carried out according to the scheme shown in Figure 15. On day 1, all mice were allowed to adapt to the experimental box for 1 hour. Then, the mice were administered saline (control group) or 5 mg / kg of morphine (TAT group and TAT-D2R-KT group) and placed in the experimental box (consisting of a white surface and a flat floor, measuring 50 cm x 50 cm x 35 cm). 3 The mice were placed in a box (box) and their locomotor activity was immediately recorded for 60 minutes. The effect of the drug was evaluated using the total walking distance of the mice as an index (hereinafter referred to as "Test 1"), and the results are shown in Figure 16A.

[0093] Next, all mice were returned to their cages and housed for 1 week. On the 9th day, mice were administered TAT or TAT-D2R-KT (3 nmol / g), followed by morphine (1 mg / kg) 1 hour later. All mice were placed in an experimental box and a test to evaluate the drug effects was performed (hereinafter referred to as "Test 2"). The results shown in Figure 16B indicated that the locomotor activity of mice in the TAT-D2R-KT group was significantly reduced over a 60-minute period.

[0094] After Test 2, all mice were returned to their cages and housed for 1 week (withdrawal), after which they were returned to the experimental box and tested again (hereinafter referred to as "Test 3"). The results shown in Figure 16C showed that the TAT-D2R-KT group still showed a significant decrease in locomotor activity even after 1 week of withdrawal, further demonstrating that the polypeptide of the present invention was able to eliminate morphine-induced drug relapse.

[0095] Example 9. Design and preparation of polypeptide variants As shown in detail in Table 1 below, based on the KT polypeptide (hereinafter referred to as "Pep1"), we designed and synthesized a variant Pep2 with an N-terminus deleted, a variant Pep3 with a C-terminus deleted, a variant Pep4 in which the middle segment RRR is mutated to GGG, and a variant Pep5 in which the middle segment RKR is mutated to GGG.

[0096] [Table 1]

[0097] Next, the C-terminus of Pep1 to Pep5 was fused to the N-terminus of TAT, respectively, to obtain fusion peptides capable of penetrating the blood-brain barrier.

[0098] Example 10. Examination of the effects of various polypeptides on morphine-induced CPP in mice Mice were administered morphine (1 mg / kg) or saline for 8 days, and then on the 10th day, various polypeptides (3 nmol / g) described in Example 9 were administered to the mice via intraperitoneal injection (10 ml / kg). Two days after administration, the onset of morphine-induced CPP was recorded according to the CPP experimental method described in Example 5, and the results are shown in Figure 17.

[0099] As can be seen from Figure 17, Pep1 and the truncated forms Pep2 and Pep3 significantly reduced the activity distance of morphine-CPP mice, whereas Pep4 and Pep5, which have substituted middle segments, were unable to significantly reduce the activity distance, suggesting that the important motif of the polypeptide of the present invention is present in the middle segment of Pep1.

[0100] Example 11. Examination of the effects of various polypeptides on cocaine-induced BS in mice To verify that the polypeptide of the present invention is also effective against cocaine-induced addiction, the following test was carried out.

[0101] Cocaine (15 mg / kg) or saline was administered to mice, followed by a 30-minute test. This was repeated for five days, followed by a five-day washout period. On the sixth washout day, mice were given various polypeptides (3 nmol / g) as described in Example 9 via intraperitoneal injection (10 ml / kg) one hour before cocaine administration to induce the drug-induced test. The results are shown in Figure 18.

[0102] As can be seen from Figure 18, as expected, Pep1 and the truncated forms Pep2 and Pep3 significantly suppressed the expression of sensitized behavior, whereas Pep4 and Pep5, which had a replacement in the middle segment, failed to suppress the expression of sensitized behavior.

[0103] Example 12. Examination of the effects of various polypeptides on alcohol-induced BS in mice In order to verify that the polypeptide of the present invention is also effective against alcohol-induced addiction, the following test was carried out.

[0104] During the training period, mice were administered alcohol (2.2 g / kg) or saline every other day, followed immediately by a 15-minute test. This training was carried out for 10 days. On the third day after training, the mice were intraperitoneally injected (10 ml / kg) with various polypeptides (3 nmol / g) described in Example 9 one hour before alcohol administration to induce drug induction, and then the test was carried out. The results are shown in Figure 19.

[0105] As can be seen from Figure 19, as expected, Pep1 and the truncated forms Pep2 and Pep3 significantly suppressed the expression of sensitized behavior, whereas Pep4 and Pep5, which had a replacement in the middle segment, failed to suppress the expression of sensitized behavior.

Claims

1. Use of a polypeptide in the preparation of a medicament for the treatment and / or prevention of substance addiction or its relapse, wherein the polypeptide consists of at least 11 consecutive amino acid residues of the sequence shown in SEQ ID NO: 1 and comprises the sequence shown in SEQ ID NO: 2, and the addiction is induced by a substance selected from one or more of the group consisting of morphine, alcohol and cocaine.

2. The use according to claim 1, wherein the relapse is caused by an environmental factor, an addictive substance factor, or a stress factor.

3. The use according to claim 1, wherein the amino acid sequence of the polypeptide is selected from the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in SEQ ID NO:

4.

4. The use of claim 1, wherein the pharmaceutical agent further comprises a carrier for penetrating the blood-brain barrier, the carrier for penetrating the blood-brain barrier being linked to the polypeptide, and the carrier for penetrating the blood-brain barrier is selected from the group consisting of HIV-1 Tat protein, insulin, cationized albumin, a monoclonal antibody against the rat transferrin receptor, a mouse-derived monoclonal antibody against the human insulin receptor, Penetratin, the transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, magainin 2 and buforin 2.

5. 10. Use of a nucleic acid molecule, an expression vector or a host cell in the preparation of a medicament for the treatment and / or prevention of substance addiction or its relapse, wherein the nucleic acid molecule encodes a polypeptide according to any one of claims 1 to 4, the expression vector comprises the nucleic acid molecule, and the host cell comprises the nucleic acid molecule or the expression vector, and the addiction is induced by a substance selected from one or more of the group consisting of morphine, alcohol and cocaine.

6. A pharmaceutical for treating and / or preventing substance addiction or its recurrence, comprising a polypeptide, a nucleic acid molecule encoding said polypeptide, an expression vector containing said nucleic acid molecule, or a host cell containing said nucleic acid molecule or said expression vector, wherein said polypeptide consists of at least 11 consecutive amino acid residues of the sequence shown in SEQ ID NO: 1 and comprises the sequence shown in SEQ ID NO: 2, and said addiction is induced by a substance selected from the group consisting of one or more of morphine, alcohol and cocaine.

7. The pharmaceutical described in claim 6, wherein the relapse is caused by environmental factors, addictive substance factors, or stress factors.

8. The pharmaceutical described in claim 6, wherein the amino acid sequence of the polypeptide is selected from the sequence shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 3 and the sequence shown in SEQ ID NO:

4.

9. The pharmaceutical described in claim 6, further comprising a carrier for penetrating the blood-brain barrier, wherein the carrier for penetrating the blood-brain barrier is linked to the polypeptide.

10. The pharmaceutical product of claim 9, wherein the carrier for penetrating the blood-brain barrier is selected from the group consisting of HIV-1 Tat protein, insulin, cationized albumin, a monoclonal antibody against rat transferrin receptor, a mouse-derived monoclonal antibody against human insulin receptor, Penetratin, the transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, magainin 2, and buforin 2.

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