Neuropeptide B and W receptors as targets for treating mood disorders and / or chronic stress

Abstract

Pharmaceutical compositions comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease are described. Furthermore, pharmaceutical compositions comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorders, preferably anorexia or bulimia nervosa, are described. Furthermore, methods for evaluating the activity of candidate molecules that appear to be NPBWR1 antagonists / inhibitors or agonists / activators are described.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease. Furthermore, the present invention relates to a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorders, preferably anorexia nervosa or bulimia nervosa. Furthermore, the present invention relates to a method for evaluating the activity of candidate molecules that appear to be NPBWR1 antagonists / inhibitors or agonists / activators. [Background technology]

[0002] Chronic stress is a major risk factor for mental illnesses, including major depressive disorder (depression, MDD), which is a leading cause of disorder and suicide. 1,2 Chronic stress and related disorders such as anxiety disorders are on the rise. 3-9 , included in the largest category of medical expenses 10,11 The neural signature of chronic stress can be reliably modeled in mice. 12,13 Furthermore, it has been observed that caffeine rapidly affects mood-related behaviors. This effect has been shown to be independent of changes in spontaneous movement. 14 In the nucleus accumbens (NAc), the center of the brain's reward system, caffeine altered the binding of the CLOCK / BMAL transcription factor complex to chromatin. This mechanism has been shown to be dependent on the Thr75 phosphorylation of DARPP-32 and occurs circadianally. 14 However, it remains unclear whether the affected genes are functionally related to mood and related disorders. Furthermore, little is understood about how chronic stress and depression can be improved by rapid environmental interventions. Moreover, among the growing number of antidepressants, only the control agent ketamine has a rapid-acting mechanism.

[0003] In light of the prior art, there exists a need for providing additional means and methods for treating, ameliorating or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders.

[0004] The present invention addresses this need and is based on the surprising finding that neuropeptide B and W receptor (Npbwr1, also called GPR7) is a key mediator of depression and stress responses, and that corresponding antagonists such as CYM50769 are potential compounds that rapidly modulate this pathway in a beneficial manner, thereby identifying a novel fast-acting pathway that rapidly ameliorates symptoms of stress and depression.

[0005] More specifically, as exemplified in the accompanying examples below, transcriptome signatures that are rapidly altered by caffeine in the NAc in a T75-DARPP-32- and light period-dependent manner were determined. Surprisingly, the present inventors identified neuropeptide B and W receptor (Npbwr1, also called GPR7) as a key mediator of mood-related states in mice.

[0006] Npbwr1 is a G protein-coupled receptor for neuropeptide B (NPB) and W (NPW), which has been scarcely studied, making this finding of the present invention even more surprising 15 . Npbwr1 is expressed in various brain regions including the NAc 16 . Incidental studies on neuropeptide B suggest its involvement in sleep regulation, affect and eating behavior 17 . Npbwr1- / - mice have no circadian abnormalities 18 or altered locomotor activity 18 . Both NPB and NPW are produced by brain regions projecting to the NAc, including the ventral tegmental area and the dorsal raphe nucleus 19,20 .

[0007] As illustrated in the attached examples below, Npbwr1 was shown to decrease 24 hours after caffeine injection and increase with chronic variable stress (CVS). A causal relationship between Npbwr1 and stress-related phenotypes was demonstrated using viral-mediated gene transfer. Furthermore, NPBWR1 was shown to alter postmortem in the NAc of depressed patients. RNA sequencing after viral Npbwr1 overexpression provided an association with Bdnf, a key gene in antidepressant response. 21 Microinjection of the synthetic Npbwr1 antagonist CYM5069 into NAc reversed the effects of chronic stress on behavioral and altered Bdnf levels from 24 hours to up to 7 days after a single dose. Conversely, microinjection of the natural agonist NPB mimicked the effects of chronic stress and had the opposite effect on Bdnf.

[0008] Furthermore, the remarkable discovery of this invention opens the way for neuropeptide B and W receptor antagonists to be offered as potential compounds for treating Parkinson's disease (PD), the second most common neurodegenerative disorder. PD is characterized by the progressive loss of dopaminergic circuits.

[0009] In fact, caffeine, a well-known adenosine receptor antagonist, has demonstrated neuroprotective properties and has been associated with a reduced risk of developing Parkinson's disease in at least six prospective epidemiological studies and animal models (Ren & Chen, Front Neurosci. 14:602697 (2020)). This neuroprotective effect of caffeine is thought to be triggered downstream of the adenosine 2 receptor, which is highly expressed in dopaminergic neurons in the midbrain, including NAc. Several diverse mechanisms, including mitochondrial regulation and anti-inflammatory pathways, contribute to the PD-protective effect of caffeine.

[0010] As outlined above, and further illustrated below in this invention, Npbwr1 has been identified as a caffeine-regulated transcript that is altered via the adenosine 2 receptor-DARPP-32:CLOCK signaling cascade (see experimental data below and Trautmann et al., Neuropharmacology 172, 108133 (2020)), a pathway that has been shown to be associated with mood-related behaviors. Importantly, mood disorders, including depression, often coexist with PD, suggesting a common cell type or signaling event. Therefore, inhibition of Npbwr1 not only provides mood-enhancing effects similar to those seen after caffeine administration, but also exerts neuroprotective effects on PD-affected cell types.

[0011] Therefore, in summary, the present invention is based on the remarkable identification of a previously unknown pathway with rapid effects on stress and depression-related behaviors, in which neuropeptide B and W receptors (Npbwr1) are key mediators, and opens the way for further means and methods to treat, improve or prevent mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease by antagonistizing neuropeptide B and W receptors (Npbwr1). [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Ren&Chen,Front Neurosci.14:602697(2020) [Non-Patent Document 2] Trautmann et al., Neuropharmacology 172, 108133(2020) [Overview of the project] [Problems that the invention aims to solve]

[0013] Considering the prior art, the underlying technical problem of the present invention is to provide further means and methods for treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease. [Means for solving the problem]

[0014] This technical problem is solved by providing embodiments characterized by the claims.

[0015] Accordingly, in a first aspect, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0016] In preferred embodiments, in connection with the first aspect of the present invention, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, The above-mentioned neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors are not agents that modulate the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (with amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (with amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0017] In another preferred embodiment, the antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) described above does not include, or consist of, an agent that modulates the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)) or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0018] In another preferred embodiment, the above antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) is an antagonist / inhibitor as defined above and below, excluding antagonist / inhibitors that modulate the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)) or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0019] Polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)) are disclosed as SEQ ID NOs. 1, 3, 5, and 7 in International Publication No. 03 / 082907 and are described as agonists of GRP7 / NPBWR1.

[0020] The neuropeptide B / W receptor (NPBWR1) is known in the prior art and is a member of the G protein-coupled receptor superfamily of endogenous membrane proteins that bind to neuropeptides B and W. This receptor is known to be mainly expressed in the CNS and has several functions, including the regulation of cortisol secretion.

[0021] More specifically, NPBWR1 is known to have the following functions and activities, among others: NPBWR1 is known to regulate / inhibit Bdnf expression. NPBWR1 is known to bind to neuropeptides B / W, and NPBWR1 is known to bind to G proteins.

[0022] These functional capabilities of NPBWR1 can be readily evaluated by those skilled in the art by utilizing routine methods and assays known in the art, and as further illustrated below.

[0023] The neuropeptide B / W receptor (NPBWR1) is highly conserved across vertebrates, with 66-100% homology from rodents to humans, and shares structural and functional similarities.

[0024] The coding region of the neuropeptide B / W receptor (NPBWR1) is known, particularly in the field of human origin; see, for example, UniProt accession number P48145. NPBWR1 is a classic seven-transmembrane receptor.

[0025] An exemplary neuropeptide B / W receptor (NPBWR1) may be the human neuropeptide B / W receptor (NPBWR1). The amino acid sequence of the human neuropeptide B / W receptor (NPBWR1) is shown below and in Sequence ID No. 1, which can be retrieved from corresponding databases such as NCBI, EMBL, and Uniprot: MDNASFSEPWPANASGPDPALSCSNASTLAPLPAPLAVAVPVVYAVICAVGLAGNSAVLYVLLRAPRMKTVTNLFILNLAIADELFTLVLPINIADFLLRQWPFGELMCKLIVAIDQYNTFSSLYFLTVMSADRYLVVLATAESRRVAGRTYSAARAVSLAVWG IVTLVVLPFAVFARLDDEQGRRQCVLVFPQPEAFWWRASRLYTLVLGFAIPVSTICVLYTTLLCRLHAMRLDSHAKALERAKKRVTFLVVAILAVCLLCWTPYHLSTVVALTTDLPQTPLVIAISYFITSLSYANSCLNPFLYAFLDASFRRNLRQLITCRAAA

[0026] The above sequence number 1 was retrieved from UniProt with accession number P48145. The term “neuropeptide B / W receptor (NPBWR1)” as used herein also includes its variants, as well as orthologues, such as mouse neuropeptide B / W receptor (NPBWR1). The corresponding nucleotide sequences can also be retrieved from the corresponding databases.

[0027] Therefore, in particular, the wild-type human neuropeptide B / W receptor (NPBWR1), encoded by the nucleic acid sequence represented by Sequence ID No. 2, can be used in accordance with the present invention.

[0028] In this specification, the patient / subject is preferably human. Therefore, the neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist is preferably a human neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist, especially when the patient / subject is human.

[0029] Accordingly, the neuropeptide B / W receptor (NPBWR1) molecules used in the context of the present invention include, but are not limited to, molecules encoded by the nucleic acid molecules described herein. Neuropeptide B / W receptor (NPBWR1) orthologues that are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence shown in Sequence ID No. 2 are also conceivable. These neuropeptide B / W receptor (NPBWR1) molecules referred to herein are defined herein as molecules capable of acting as neuropeptide B / W receptor (NPBWR1) as described above and below. These functions and activities include, among other things, the ability to have neuropeptide B / W receptor (NPBWR1) activity as described above and below herein, i.e., the ability to regulate / inhibit Bdnf expression, the ability to bind to neuropeptide B / W, and / or the ability to bind to G proteins.

[0030] To test the activity of the functional neuropeptide B / W receptor (NPBWR1), assays provided herein and illustrated in the accompanying examples may be used.

[0031] Regarding the ability of NPBWR1 to regulate / inhibit Bdnf expression, for example, quantitative PCR can be used to assay Bdnf expression.

[0032] Regarding the ability of NPBWR1 to bind to neuropeptides B / W, for example, FRAP (fluorescence bleed recovery) can be used, in which neuropeptide B or neuropeptide W is used as a substrate to evaluate binding to NBWBR1.

[0033] Regarding the ability of NPBWR1 to bind to G proteins, for example, FRAP (fluorescence fading recovery) can be used, in which the G protein subunit is used as a substrate to evaluate binding to NBWBR1.

[0034] The corresponding assay is known in the art and can be easily carried out by those skilled in the art using routine methods.

[0035] While not strictly theoretical, Npbwr1 activity is often measured indirectly through the regulation / activation of the downstream protein brain-derived neurotrophic factor (Bdnf). Bdnf RNA levels can be assessed using quantitative PCR. Increased Bdnf expression is an indicator of Npbwr1 inhibition. Conversely, decreased Bdnf expression is an indicator of Npbwr1 activation.

[0036] Alternatively, activity assays could be designed based on the G protein coupling of Npbwr1. This would translate the activity changes into differential binding to G proteins. Using Förster resonance energy transfer (FRET), changes in the interaction between Npbwr1 and its "G protein" can be visualized using the fusion of their components with fluorophores. Activation of a low-wavelength fluorophore bound to Npbwr1 emits wavelengths that excite the G protein-bound fluorophore if the Npbwr1-bound fluorophore and the G protein-bound fluorophore are bound to each other and in close proximity. An increase in fluorescence is an indicator of NPBWR1's ability to bind to G proteins.

[0037] Furthermore, neuropeptide B / W receptor (NPBWR1) orthologues are envisioned that are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in Sequence ID No. 1, and that can act as neuropeptide B / W receptor (NPBWR1) as described above and below in this specification.

[0038] To determine whether a nucleic acid sequence has some degree of identity with respect to the nucleic acid encoding a neuropeptide B / W receptor (NPBWR1) or the amino acids of the neuropeptide B / W receptor (NPBWR1), those skilled in the art can use means and methods well known in the art, such as alignment, manually or using computer programs as described below herein.

[0039] According to the present invention, the terms “identical” or “percent identical” in the context of two or more nucleic acids or amino acid sequences mean that, when compared across a comparison window or across a specified region to be measured using a sequence comparison algorithm known in the art, or by manual alignment and visual inspection, and aligned to obtain the greatest correspondence, two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity, with the nucleic acid sequence of, for example, SEQ ID NO: 2 or the amino acid sequence of, for example, SEQ ID NO: 1, which can act as a functional neuropeptide B / W receptor (NPBWR1) as described above and below herein. For example, sequences having 60%-95% or more sequence identity are considered substantially identical. Such definitions also apply to complements of test sequences. Preferably, the described identity exists over a region of at least about 15-25 amino acids or nucleotides, more preferably over a region of about 50-100 amino acids or nucleotides. Those skilled in the art will know of methods for determining the percentage identity between sequences using algorithms such as the CLUSTALW computer program (Thompson Nucl.Acids Res.2 (1994), 4673-4680) or FASTDB (Brutlag Comp.App.Biosci.6 (1990), 237-245), which are known in the art.

[0040] The FASTDB algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in its calculations, although this can be manually corrected to avoid overestimation of % identity. However, CLUSTALW does consider sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410) are also available to those skilled in the art. The BLASTN program for nucleic acid sequences uses a word length (W) of 11, an expected value (E) of 10, M=5, N=4, and a comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3 and an expected value (E) of 10 as defaults. The BLOSUM62 score matrix (Henikoff (1989) PNAS 89:10915) uses 50 alignments (B), 10 expected values ​​(E), M=5, N=4, and a comparison of both strands.

[0041] In one aspect, the present invention relates to the inhibition / antagonization of neuropeptide B / W receptor (NPBWR1) activity in mechanisms that block / inhibit the downstream pathway of brain-derived neurotrophic factor (BDNF) signaling, for example, and to pave the way for the use of neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0042] The present invention provides inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0043] In this specification, it is assumed that these inhibitors / antagonists may be used as pharmaceuticals or pharmaceutical compositions, that is, that the neuropeptide B / W receptor (NPBWR1) inhibitors provided and described herein are for medical use (e.g., for use in the therapy / treatment / improvement / prevention of diseases, in particular mood / affective disorders and / or chronic stress and / or anxiety and / or Parkinson's disease, and other mood / affective and / or chronic stress and / or anxiety and / or Parkinson's disease-related disorders). The terms “pharmaceutical” and “pharmaceutical composition” are used interchangeably herein. Accordingly, the definitions and descriptions provided herein in relation to “pharmaceutical composition” shall apply mutatis mutandis to the term “pharmaceutical,” with necessary modifications.

[0044] The terms “antagonist” and “inhibitor” are used interchangeably herein. These terms are known in the Art and relate to compounds / substances that can completely or partially block or reduce the physiological activity of (one or more) specific proteins. Accordingly, in the context of the present invention, the antagonist / inhibitor may block, reduce, inhibit, or inactivate the physiological activity of a protein, such as a neuropeptide B / W receptor (NPBWR1), when the compound / substance binds to the protein. The binding of the “antagonist / inhibitor” to a given protein, for example, a neuropeptide B / W receptor (NPBWR1), may compete with or block the binding of an endogenous activating molecule that binds to the protein. Therefore, as used herein, the term “antagonist” also encompasses competitive antagonists, (reversible) non-competitive antagonists, or irreversible antagonists, as described in particular in Mutschler's “Arzneimittelwirkungen” (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. However, in addition to that, “antagonists” or “inhibitors” of neuropeptide B / W receptor (NPBWR1) in the context of the present invention may also be able to inhibit the function of the said protein by blocking / reducing the expression of a nucleic acid molecule encoding a given protein such as neuropeptide B / W receptor (NPBWR1). Therefore, antagonists / inhibitors of the neuropeptide B / W receptor (NPBWR1) may result in a decrease in the expression level of the neuropeptide B / W receptor (NPBWR1) (e.g., a decrease in the levels of neuropeptide B / W receptor (NPBWR1) mRNA and neuropeptide B / W receptor (NPBWR1) protein), which is reflected in a decrease in the activity of the neuropeptide B / W receptor (NPBWR1). This decreased activity is known in the art and can be measured / detected by the methods described herein.Therefore, inhibitors of the neuropeptide B / W receptor (NPBWR1) in the context of the present invention may also include transcriptional repressors of the expression of the neuropeptide B / W receptor (NPBWR1) that can reduce the function of the neuropeptide B / W receptor (NPBWR1). As described in detail below herein, reduction in the expression and / or activity of the neuropeptide B / W receptor (NPBWR1) by an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) results in a decrease in the activity (and / or expression) of the neuropeptide B / W receptor (NPBWR1), thereby reducing the functional capacity of the neuropeptide B / W receptor (NPBWR1).

[0045] In accordance with the theoretical basis of this invention, a decrease in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have various medical significances.

[0046] As described below, a decrease in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have medical significance, namely, it is expected to affect mood / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, and other mood / affective and / or chronic stress and / or anxiety and / or Parkinson's disease-related disorders / disorders.

[0047] The terms “inhibitor” and / or “antagonist” do not imply any specific mechanism of biological action and are considered to expressly include and encompass all possible pharmacological, physiological, and biochemical interactions with the neuropeptide B / W receptor (NPBWR1) (signaling), whether direct or indirect. For the purposes of this disclosure, the terms “inhibitor” and / or “antagonist” shall be clearly understood to encompass all previously identified terms, names, and functional states and characteristics to which the neuropeptide B / W receptor (NPBWR1) itself is substantially inactivated, reduced or neutralized to any degree of any significance (e.g., at least 5%, 10%, 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, 500%, or 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or 1000x) by the biological activity of the neuropeptide B / W receptor (NPBWR1) (including, but not limited to, its ability to modulate / inhibit Bdnf expression, its ability to bind to neuropeptide B / W, and / or its ability to bind to G proteins), or the results of its biological activity. Inhibitors / antagonists reduce abnormal levels of the biological activity of the neuropeptide B / W receptor (NPBWR1), which can cause adverse effects and / or disease in a subject, to levels corresponding to those in a healthy subject, thereby preventing adverse effects and / or disease, halting their progression, and / or curing them.

[0048] Accordingly, the present invention relates in one aspect to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor reduces the biological activity of the neuropeptide B / W receptor (NPBWR1) by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 1000-fold.

[0049] Accordingly, the present invention relates in one aspect to an inhibitor / antagonist of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, the inhibitor / antagonist reducing at least a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 1000-fold abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse effects and / or disease.

[0050] Accordingly, the present invention relates in one aspect to an inhibitor / antagonist of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor / antagonist reduces at least threefold an abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse effects and / or disease.

[0051] Accordingly, the present invention relates in one aspect to an inhibitor / antagonist of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor / antagonist reduces abnormal levels of the biological activity of a neuropeptide B / W receptor (NPBWR1) that causes and / or promotes mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0052] Accordingly, the present invention relates in one aspect to an inhibitor / antagonist of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor reduces an abnormal level of biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease in a subject to a level comparable to that of a healthy subject.

[0053] According to the present invention, the term “inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1)” also means a compound or substance that can completely or partially block or reduce the physiological activity of neuropeptide B / W receptor (NPBWR1). Therefore, in connection with the present invention, the inhibitor can, for example, block, reduce, inhibit or inactivate the physiological activity of neuropeptide B / W receptor (NPBWR1) when the compound / substance (i.e., inhibitor / antagonist) binds to the neuropeptide B / W receptor (NPBWR1).

[0054] As used herein, the term “inhibitor” also encompasses inhibitors that result in reversible inhibition, such as competitive inhibition, uncompetitive inhibition, non-competitive inhibition, mixed inhibition, or irreversible inhibition of the neuropeptide B / W receptor (NPBWR1), such as inhibition by covalent interaction.

[0055] Neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists can also inhibit the function of neuropeptide B / W receptor (NPBWR1) by blocking / reducing the expression of the nucleic acid molecule encoding the neuropeptide B / W receptor (NPBWR1). Therefore, neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists can result in a decrease in the expression level of the neuropeptide B / W receptor (NPBWR1) gene product, for example, a decrease in the levels of neuropeptide B / W receptor (NPBWR1) mRNA and / or neuropeptide B / W receptor (NPBWR1) protein.

[0056] Inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) can reduce the abnormal expression levels of the neuropeptide B / W receptor (NPBWR1) that may cause and / or promote adverse effects and / or diseases associated with mood / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, as outlined above and below herein, to levels corresponding to those in healthy subjects, thereby preventing, halting the progression of, or curing adverse effects and / or diseases in subjects. This is reflected in a decrease in the expression of the neuropeptide B / W receptor (NPBWR1) and / or a decrease in the expression of abnormal neuropeptide B / W receptor (NPBWR1), thereby restoring healthy levels of neuropeptide B / W receptor (NPBWR1) expression. The expression level of the neuropeptide B / W receptor (NPBWR1) may correlate to some extent with the activity of the neuropeptide B / W receptor (NPBWR1) until saturation of the translation mechanism and / or substances binding to the neuropeptide B / W receptor (NPBWR1) is achieved. The expression level of the neuropeptide B / W receptor (NPBWR1) can be measured / detected by methods known in the art.

[0057] Accordingly, in one aspect, the present invention relates to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or disorders associated with Parkinson's disease, wherein the inhibitor reduces the expression level of the neuropeptide B / W receptor (NPBWR1) gene product.

[0058] Accordingly, in one aspect, the present invention relates to a neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist for use in the treatment, improvement or prevention of mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or disorders associated with Parkinson's disease, wherein the inhibitor / antagonist reduces the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes adverse effects and / or disease / disorder.

[0059] Accordingly, in one aspect, the present invention relates to a neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist for use in the treatment, improvement or prevention of disorders associated with mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor / antagonist reduces the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes the mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0060] Accordingly, in one aspect, the present invention relates to a neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist for use in the treatment, improvement or prevention of disorders associated with mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the inhibitor / antagonist reduces the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease in a subject to an expression level comparable to that of a healthy subject.

[0061] Neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists may exert their inhibitory function by directly interacting with any part of the neuropeptide B / W receptor (NPBWR1) protein, i.e., the extracellular domain, transmembrane domain, and / or cytoplasmic domain of the neuropeptide B / W receptor (NPBWR1). Neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists may also exert any inhibitory effect on the function of neuropeptide B / W receptor (NPBWR1) by blocking, reducing, inhibiting, or inactivating any upstream or downstream pathway component that essentially contributes to the function of neuropeptide B / W receptor (NPBWR1). Neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists may exert any indirect inhibitory effect on any neuropeptide B / W receptor (NPBWR1) activating molecule, such as nucleic acids, ribonucleic acid (RNA), double-stranded ribonucleic acid (dsRNA), chromatin leader proteins, and / or ligands.

[0062] The efficacy of neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists can be described using the IC50 (International Coefficient of Use) value. In the sense of the present invention, neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists preferably achieve low IC50 values. The IC50 values ​​of neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists may be less than 100 μM, less than 90 μM, less than 80 μM, less than 70 μM, less than 60 μM, less than 50 μM, less than 40 μM, less than 30 μM, less than 20 μM, or less than 10 μM, with lower values ​​being preferable to higher values. Preferably, the IC50 values ​​of neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists may be less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, or less than 4 μM. Preferably, the IC50 value of the neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist may be less than 4 μM.

[0063] Accordingly, the present invention relates to a neuropeptide B / W receptor (NPBWR1) inhibitor / antagonist for use in the treatment, improvement or prevention of mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the efficacy of the median inhibitory concentration (IC50) is less than 100 μM, less than 90 μM, less than 80 μM, less than 70 μM, less than 60 μM, less than 50 μM, less than 40 μM, less than 30 μM, less than 20 μM or less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM or less than 4 μM, preferably less than 4 μM.

[0064] Those skilled in the art are aware of methods for determining the IC50 values ​​of inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1). This specification assumes that inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) may embody additional IC50 values, and / or other inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) having other IC50 values ​​will be identified.

[0065] Mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease are known disorders or syndromes in the art and are medical indications classified in the ICD system, i.e., the well-known and established medical classification list of the World Health Organization (WHO). ICD stands for the International Statistical Classification of Diseases and Related Health Problems. Hereafter, when referring to more specifically defined disorders or diseases, the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), i.e., "ICD 10," will be referred to.

[0066] The present invention is not limited to any specific mood / affective disorder and / or chronic stress and / or anxiety disorder. Any mood / affective disorder and / or chronic stress and / or anxiety disorder and / or Parkinson's disease can be treated, ameliorated and / or prevented by neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors according to the present invention.

[0067] While not bound by theory, in particularly preferred embodiments, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease are as follows:

[0068] In preferred embodiments, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in a method of treating, improving or preventing mood disorders / affective disorders, wherein the mood disorder / affective disorder is selected from the group consisting of: (ii) Bipolar affective disorder (ICD-10 F31), preferably bipolar affective disorder during the hypomanic phase (ICD-10 F31), (iii) Depressive episode (ICD-10 F32), (iv) Recurrent depressive disorder (ICD-10 F33), (v) Persistent mood / affective disorder (ICD-10 F34), (vi) Other mood / affective disorders (ICD-10 F38), and (vii) Mood / affective disorder of unknown origin (ICD-10 F39).

[0069] The mood / affective disorders of this invention, in a broad sense, refer to disorders in which the underlying disorder is a change in mood or feeling, either to depression (with or without associated anxiety) or to elevated mood. The mood changes are usually accompanied by a change in overall activity levels, and most other symptoms are secondary to or readily understood in the context of the mood and activity changes. Most of these disorders tend to recur, and the onset of individual episodes may often be associated with a stressful event or situation.

[0070] More specific mood / affective disorders and their corresponding subcategories relating to the present invention are described in more detail below.

[0071] Manic episode (ICD-10 F30): All sub-classifications within this category should be used for a single episode only. A hypomanic or manic episode in an individual with one or more previous emotional episodes (depressive, hypomanic, manic, or mixed) should be coded as bipolar affective disorder (F31.-).

[0072] The detailed classification of manic episodes (ICD-10 F30) is described below: F30.0: Hypomania: A disorder characterized by a persistent, mild elevation of mood, increased energy and activity, and a sense of well-being on a daily basis, as well as a sense of both physical and mental efficiency. Increased sociability, talkativeness, hypersociality, increased sexual energy, and decreased need for sleep are often present, though not to the extent of causing serious work disruption or social rejection. Irritability, overconfidence, and crude behavior may replace the more normal euphoric sociability. The mood and behavioral disorders are not accompanied by hallucinations or delusions. F30.1: Mania without psychotic symptoms: Mood can be elevated to an extent inconsistent with the patient's situation, ranging from carefree amusement to almost uncontrollable excitement. This elevation is accompanied by increased energy, leading to hyperactivity, speech pressure, and a decreased need for sleep. Attention is often difficult to maintain, and there is frequently marked distractibility. Often, self-esteem descends into overconfident thinking and overconfidence. The absence of normal social constraints can lead to unruly, impulsive, or inappropriate behavior that deviates from one's character. F30.2: Mania with psychotic symptoms: In addition to the clinical features described in F30.1, there may be delusions (usually grandiosity) or hallucinations (usually voices speaking directly to the patient), or extreme agitation, hyperactivity, and avoidance of thought, and the subject may be unable to understand or access normal communication. Mania with mood-congruent or mood-congruent psychotic symptoms manic stupor F30.8: Other manic episodes: F30.9: Manic episode, details unknown Manic NOS

[0073] Bipolar affective disorder (ICD-10 F31), preferably bipolar affective disorder during the hypomanic phase (ICD-10 F31): Bipolar disorder is characterized by two or more episodes in which the patient's mood and activity levels are significantly disturbed, with the disturbance sometimes consisting of elevated mood and increased energy and activity (hypomania or mania), and sometimes of depressed mood and decreased energy and activity (depression). Recurrent episodes consisting of only hypomania or mania are classified as bipolar disorder. Includes the following: Bipolar disorder Bipolar disorder: ·disease ·Psychosis ·reaction Exclude: Bipolar disorder, single manic episode (F30.-) Cyclothymic disorder (F34.0)

[0074] The following describes the subclassification of bipolar affective disorder (ICD-10 F31). F31.0: Bipolar affective disorder, currently experiencing a hypomanic episode. The patient is currently in a hypomanic state and has a history of at least one other emotional episode (hypomania, mania, depression, or a mixed type). F31.1: Bipolar affective disorder, currently experiencing a manic episode without psychotic symptoms: The patient is currently manic, without psychotic symptoms (as in F30.1), and has a history of at least one other emotional episode (hypomanic, manic, depressive, or mixed). F31.2: Bipolar affective disorder, currently experiencing a manic episode with psychotic symptoms: The patient is currently manic, exhibits psychotic symptoms (similar to those in F30.2), and has a history of at least one other emotional episode (hypomanic, manic, depressive, or mixed). F31.3: Bipolar affective disorder, currently experiencing a mild or moderate depressive episode: The patient currently has depression as a depressive episode of mild or moderate severity (F32.0 or F32.1) and has a history of at least one confirmed hypomanic, manic, or mixed-type affective episode. F31.4: Bipolar affective disorder, currently a severe depressive episode without psychotic symptoms: The patient is currently experiencing depression as a severe depressive episode without psychotic symptoms (F32.2) and has a history of at least one confirmed hypomanic, manic, or mixed-type affective episode. F31.5: Bipolar affective disorder, currently experiencing a severe depressive episode with psychotic symptoms: The patient is currently diagnosed with depression as a severe depressive episode with psychotic symptoms (F32.3) and has a history of at least one confirmed hypomanic, manic, or mixed-type affective episode. F31.6: Bipolar affective disorder, currently experiencing a mixed episode: The patient has a history of at least one confirmed episode of hypomania, mania, depression, or mixed affective disorder, and is currently experiencing either a mixed or rapidly shifting set of manic and depressive symptoms. Exclude: A single mixed-affect episode (F38.0) F31.7: Bipolar Affective Disorder, currently in remission: The patient has a history of at least one confirmed hypomanic, manic, or mixed-type mood episode, and at least one other mood episode (hypomanic, manic, depressive, or mixed-type), but is currently free from any significant mood disorder, and has not been for several months. The remission period during prophylactic treatment should be coded here. F31.8: Other Bipolar Affective Disorders Bipolar disorder type II Recurrent Manic Episode NOS F31.9: Bipolar affective disorder, details unknown Bipolar disorder NOS

[0075] Depressive episode (ICD-10 F32): In a typical mild, moderate, or severe depressive episode, the patient suffers from low mood, low energy, and reduced activity. The ability to enjoy, be interested in, and concentrate is diminished, and even minimal effort commonly leads to significant fatigue. Sleep is usually disrupted, and appetite is reduced. Self-esteem and confidence are almost always low, and even in milder forms, there is some degree of guilt or worthlessness. The depressed mood may be little to no variation from day to day, unresponsive to situations, loss of interest and emotional response, morning awakening several hours earlier than usual, morning depression being the worst, marked psychomotor retardation, agitation, loss of appetite, weight loss, and decreased libido—so-called “physical” symptoms. Depending on the number and severity of symptoms, a depressive episode may be classified as mild, moderate, or severe. Includes the following: Single episode: • Depressive reactions Psychogenic depression Reactive uterine depression Exclude: Adjustment disorder (F43.2) Recurrent depressive disorder (F33.-) In cases related to behavioral disorders in F91 - (F92.0)

[0076] The following describes the subclassification of depressive episodes (ICD-10 F32): F32.0: Mild depressive episode: Two or three of the above symptoms are usually present. Patients typically experience distress due to these symptoms, but are likely able to continue most activities. F32.1: Moderate depressive episode: If four or more of the above symptoms are present, the patient is likely to have great difficulty continuing normal activities. F32.2: Severe depressive episode without psychotic symptoms: A depressive episode in which some of the above symptoms are prominent and distressing, typically involving loss of self-esteem and a concept of worthlessness or self-blame. Suicidal thoughts and actions are common, and many "physical" symptoms are usually present. • Agitated depression • Major depression • Vital depression • Single episode without psychotic symptoms F32.3: Severe depressive episode with psychotic symptoms: This is a depressive episode as described in F32.2, but with hallucinations, delusions, psychomotor retardation, or severe stupor that prevents normal social activity, and there may be a risk to life due to suicide, dehydration, or starvation. Hallucinations and delusions may or may not be mood-congruent. Single episode: • Major depression with psychotic symptoms Psychogenic depressive psychosis • Psychotic depression • Reactive depressive psychosis F32.8: Other depressive episodes: Atypical depression A single episode of "masked" depression NOS F32.9: Depressive episode, details unknown: Depression NOS Depressive disorder NOS

[0077] Recurrent depressive disorder (ICD-10 F33): Recurrent depressive disorder is characterized by recurrent episodes of depression, such as those described in relation to depressive episodes (F32.-), without a history of separate episodes of elevated mood and increased energy (mania). However, short episodes of mild elevation and hyperactivity (hypomania) may occur immediately following a depressive episode, and these can be induced by antidepressant treatment. More severe forms of recurrent depressive disorder (F33.2 and F33.3) largely overlap with previous concepts, such as manic-depressive depression, depression, vitalistic depression, and endogenous depression. The initial episode can occur at any age from childhood to old age, the onset can be either acute or insidious, and the duration varies from weeks to months. No matter how many depressive episodes a patient has experienced, the risk of having a manic episode in a patient with recurrent depressive disorder is never completely eliminated. If such an episode occurs, the diagnosis should be changed to bipolar affective disorder (F31.-). Includes the following: Recurrent episodes: • Depressive reactions Psychogenic depression Reactive uterine depression Seasonal Depressive Disorder Exclude: Recurrent short-term depressive episodes (F38.1)

[0078] The following is a detailed explanation of the subclassification of recurrent depressive disorder (ICD-10 F33). F33.0: Recurrent depressive disorder, currently experiencing a mild episode: A disorder characterized by recurrent depressive episodes, where the current episode is mild and there is no history of mania, as in the case of F32.0. F33.1: Recurrent depressive disorder, currently experiencing a moderate episode: A disorder characterized by recurrent depressive episodes, where the current episode is of moderate severity, as in the case of F32.1, and there is no history of mania. F33.2: Recurrent depressive disorder, currently a severe episode without psychotic symptoms: A disorder characterized by recurrent depressive episodes, the current episode being severe, without psychotic symptoms as in F32.2, and without a history of mania. Endogenous depression without psychotic symptoms Major depressive disorder, recurrent depression without psychotic symptoms Manic-depressive psychosis, depressive type without psychotic symptoms Vital depression, recurrent depression without psychotic symptoms F33.3: Recurrent depressive disorder, currently a severe episode with psychotic symptoms: A disorder characterized by recurrent depressive episodes, where the current episode is severe and accompanied by psychotic symptoms, as in the case of F32.3, and there are no prior manic episodes. Endogenous depression with psychotic symptoms Manic-depressive psychosis, depressive type with psychotic symptoms Recurrent severe episodes: • Major depression with psychotic symptoms Psychogenic depressive psychosis • Psychotic depression • Reactive depressive psychosis F33.4: Recurrent depressive disorder, currently in remission: The patient had previously experienced two or more depressive episodes, as described in F33.0-F33.3, but had not experienced depressive symptoms for several months. F33.8: Other recurrent depressive disorders: F33.9: Recurrent depressive disorder, details unknown: Unipolar depression NOS

[0079] Persistent mood / affective disorder (ICD-10 F34): Persistent and usually volatile mood disorders, where the majority of individual episodes are not severe enough to justify being described as hypomanic or mild depressive episodes. They can last for many years, sometimes spanning a significant portion of the patient's adulthood, and therefore cause considerable distress and impairment. In some cases, recurrent or single manic or depressive episodes may be superimposed on persistent affective disorder.

[0080] The following describes the subclassification of persistent mood / affective disorders (ICD-10 F34): F34.0: Cycle disorder: This involves numerous depressive periods and persistent mood instability accompanied by mild mood elevation, none of which are severe or long-lasting enough to justify a diagnosis of bipolar affective disorder (F31-) or recurrent depressive disorder (F33-). This disorder is frequently seen in relatives of individuals with bipolar affective disorder. Some individuals with cyclothymic disorder eventually develop bipolar affective disorder. Emotional personality disorder Cycloid personality Cyclothymic personality disorder F34.1: Dysthymia: Chronic mood depression lasting at least several years, but not severe enough to justify a diagnosis of severe, moderate, or mild recurrent depressive disorder (F33-), or where individual episodes are not long enough to warrant such a diagnosis. Depression: • Neurosis Personality disorder Neurotic depression Persistent anxiety depression Exclude: Anxiety-depressive disorder (mild or non-persistent) (F41.2) F34.8: Other persistent mood [affective] disorders F34.9: Persistent mood [affective] disorder, details unknown

[0081] Other mood / affective disorders (ICD-10 F38): Other mood disorders that do not justify classification into F30-F34 due to insufficient severity or duration.

[0082] The following is a detailed description of other mood / affective disorders (ICD-10 F38). F38.0: Other isolated mood [affective] disorders: Mixed-type emotional episodes F38.1: Other recurrent mood [affective] disorders: Recurrent short-term depressive episodes F38.8: Other specified mood [affective] disorders.

[0083] Mood / affective disorder of unknown origin (ICD-10 F39): Includes the following: affective psychosis NOS

[0084] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in a method of treating, improving or preventing chronic stress, wherein the chronic stress is selected from the group consisting of: (i) Reaction to severe stress and adjustment disorder (ICD-10 F43), (ii) Acute stress response (ICD-10 F43.0), (iii) Post-traumatic stress disorder (ICD-10 F43.1) (iv) Adjustment disorder (ICD-10 F43.2) (v) Other severe stress reactions (ICD-10 F43.8), and (vi) Severe stress reaction, details unknown (ICD-10 F43.9).

[0085] Chronic stress as described in this invention is generally understood as a physiological or psychological response triggered by long-term endogenous or exogenous stressors. Stressors, whether physically present or recalled, produce the same effect and trigger a chronic stress response. While a wide range of chronic stressors exist, most involve relatively long-term problems, conflicts, and threats that people encounter routinely.

[0086] More specific chronic stress and its corresponding subclassifications related to the present invention will be described in more detail below.

[0087] Severe stress response and adjustment disorder (ICD-10 F43): This category includes disorders that can be identified not only by their symptoms and course, but also by the presence of one or the other of two causal influences: an exceptionally stressful life event that triggers an acute stress response, or a significant life change that leads to a persistent unpleasant situation resulting in an adjustment disorder. Less severe psychosocial stressors, or "life events," may induce or contribute to the presentation of the very wide range of disorders classified elsewhere in this chapter, but their etiological significance is not always clear and is thought to depend on the individual, often unique, vulnerability in each case; that is, life events are neither necessary nor sufficient to explain the onset and form of the disorder. In contrast, the disorders grouped here are considered to always arise as a direct result of acute severe stress or persistent trauma. The stressful event or persistent unpleasant situation is the primary and dominant factor, and the disorder would not have occurred without their influence. Therefore, the disorders in this section can be seen as maladaptive responses to severe or persistent stress, in that they interfere with appropriate coping mechanisms and thus lead to problems of social functioning.

[0088] The following is a detailed description of responses to severe stress and adjustment disorders (ICD-10 F43): Acute stress response (ICD-10 F43.0): A transient disorder that develops in individuals in response to exceptional physical and mental stress, without any other apparent mental disorder, and usually resolves within hours or days. Individual vulnerability and coping abilities play a role in the onset and severity of the acute stress response. Symptoms typically present a mixed and changing picture, including an initial state of "dazedness" and some narrowing of the scope of consciousness and attention, inability to understand stimuli, and disorientation. This state is followed by either further detachment from the surrounding situation (to the extent of dissociative stupor (F44.2)) or agitation and hyperactivity (fleeing reaction or fugue). Autonomic signs of panic anxiety (tachycardia, sweating, flushing) are commonly present. Symptoms usually appear within minutes of the impact of the stressful stimulus or event and disappear within 2-3 days (often within hours). Partial or complete amnesia (F44.0) for the episode may be present. If symptoms persist, a change of diagnosis should be considered.

[0089] acute: • Reaction to the crisis • Reaction to stress Combat fatigue Crisis psychological shock Post-traumatic stress disorder (ICD-10 F43.1): It arises as a delayed or prolonged reaction to a stressful event or situation (short-term or long-term) of an exceptionally threatening or devastating nature, which can cause widespread distress to most people. Predisposing factors such as personality traits (e.g., obsessive-compulsive, helpless) or a history of neurosis may lower the threshold for the onset of the syndrome or worsen its course, but they are not necessary or sufficient to explain its occurrence. Typical features include intrusive memories ("flashbacks"), episodes in which the traumatic experience is repeatedly replayed through dreams or nightmares, which occur amidst persistent states of "numbness" or emotional blunting, detachment from others, unresponsiveness to the surroundings, anhedonia, and avoidance of activities or situations that evoke the trauma. There is usually hyperarousal of the autonomic nervous system with hypervigilance, heightened startle response, and insomnia. Anxiety and depression are generally associated with the above symptoms and signs, and suicidal ideation is not uncommon. Onset follows the trauma, with a latency period that can range from several weeks to several months. The course of the condition varies, but recovery can be expected in most cases. In a small number of cases, the condition may follow a chronic course over many years, eventually progressing to persistent personality changes (F62.0). Traumatic neurosis Adjustment disorder (ICD-10 F43.2): Adjustment disorder is a subjective state of distress and affective disturbance that occurs during a period of adaptation to a significant life change or stressful life event, typically impairing social functioning and performance. Stressors may have affected the integrity of an individual's social network (bereavement, separation), or the expansion of social support systems and values ​​(migration, shelter life), or represented a major developmental shift or crisis (going to school, becoming a parent, failing to achieve important personal goals, retirement). Individual predispositions or vulnerabilities play a significant role in the risk of developing adjustment disorder and the formation of its symptoms, but it is still assumed that the condition would not have occurred without the stressor. Symptoms vary and may include depressed mood, anxiety or worry (or a combination thereof), a feeling of being unable to cope with, plan for, or persevere in the current situation, and some degree of difficulty in performing daily routines. Behavioral disturbances may be a feature, particularly in adolescents. Key features may include short- or long-term depressive reactions, or other affective or behavioral disturbances. Culture shock Grief response Child hospitalism Exclude: Childhood separation anxiety disorder (F93.0) Other severe stress reactions (ICD-10 F43.8): Severe stress reaction, details unknown (ICD-10 F43.9).

[0090] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor for use in a method of treating, improving or preventing an anxiety disorder, wherein the anxiety disorder is selected from the group consisting of: (i) Phobic anxiety disorder (ICD-10 F40), and (ii) Other anxiety disorders (ICD-10 F41.1).

[0091] The anxiety disorders of this invention are a group of disorders in which anxiety is triggered only in, or predominantly in, specific, well-defined situations that are not currently dangerous. As a result, these situations are characteristically avoided or endured with fear. The patient's concerns may focus on individual symptoms such as palpitations or fainting, and are often associated with secondary fears of dying, losing control, or going insane. Attempting to enter a phobic situation usually produces anticipatory anxiety. Phobic anxiety and depression often coexist. Whether two diagnoses, one for phobic anxiety and one for a depressive episode, are necessary, or only one, is determined by the time course of the two conditions and the therapeutic considerations at the time of examination.

[0092] More specific anxiety disorders and their corresponding subclassifications relating to this invention are described in more detail below:

[0093] Agoraphobia (ICD-10 F40.0): A fairly well-defined cluster of phobias that includes fears of leaving home, entering shops, being in crowds or public places, or traveling alone on trains, buses, or airplanes. Panic disorder is a frequent feature in both current and past episodes. Depressive and obsessive-compulsive symptoms, as well as social phobias, are also commonly present as supporting features. Avoidance of phobic situations is often pronounced, and some agoraphobic individuals experience little anxiety because they can avoid phobic situations. Agoraphobia without a history of panic disorder Panic disorder accompanied by agoraphobia.

[0094] Social phobia (ICD-10 F40.1) A fear of being scrutinized by others, leading to avoidance of social situations. More widespread social phobias are usually associated with low self-esteem and a fear of criticism. They can manifest as flushing, hand tremors, nausea, or complaints of an urgent need to urinate, and patients sometimes become convinced that one of these secondary signs of anxiety is the primary problem. Symptoms can progress to panic attacks. • Social anxiety Social neurosis

[0095] Specific (individual) phobias (ICD-10:F40.2): This phobia is limited to very specific situations such as proximity to certain animals, heights, thunder, darkness, flying, enclosed spaces, defecating in public restrooms, eating certain foods, dental treatment, or seeing blood or wounds. The triggering situations are individual, but contact with them can cause panic, similar to agoraphobia or social phobia. ·fear of heights ·Animal phobia Claustrophobia • Simple phobias Exclude: Dysmorphophobia (non-delusional) (F45.2) Nosophobia (F45.2) F40.8 Other phobic anxiety disorders F40.9 Phobia anxiety disorder, details unknown phobia NOS Phobic state NOS

[0096] Other anxiety disorders (ICD-10 F41): Anxiety is a disorder in which the manifestation of anxiety is the primary symptom and is not limited to any particular environmental situation. While elements of depression, obsessive-compulsive disorder, and even some phobic anxiety may be present, they are clearly secondary or less severe.

[0097] Panic disorder [intermittent paroxysmal anxiety] (ICD-10 F41.0): The essential characteristic is the recurrent episodes of severe anxiety (panic) that are not limited to any particular situation or set of situations and are therefore unpredictable. As with other anxiety disorders, the main symptoms include the sudden onset of palpitations, chest pain, a feeling of tightness, dizziness, and derealization (depersonalization or loss of reality). Secondary fears of dying, losing one's sanity, or going insane are also often present. If the patient has a depressive disorder at the onset of an attack, panic disorder should not be given as the primary diagnosis. In these situations, the panic attack is likely secondary to the depression. panic: ·attack ·situation Exclude: Panic disorder with agoraphobia (F40.0)

[0098] Generalized anxiety disorder (ICD-10 F41.1): Anxiety that is generalized, persistent, but not limited to any particular environmental situation (i.e., "floating"), or even strongly dominant. The main symptoms vary, but include complaints of persistent nervousness, tremors, muscle tension, sweating, lightheadedness, palpitations, dizziness, and epigastric discomfort. A fear that the patient or a relative will soon become ill or have an accident is often expressed. anxiety: • Neurosis ·reaction ·situation Exclude: Nervous breakdown (F48.0)

[0099] Mixed-type anxiety and depressive disorder (ICD-10 F41.2): This category should be used when symptoms of both anxiety and depression are present, but neither is clearly dominant, and neither type of symptom is present to a degree that would justify a diagnosis if considered separately. If both anxiety and depressive symptoms are present and severe enough to justify individual diagnoses, both diagnoses should be recorded, and this category should not be used. Anxiety depression (mild or non-persistent)

[0100] Other mixed anxiety disorders (ICD-10 F41.3): Anxiety symptoms mixed with features of other disorders in F42-F48. Neither type of symptom is severe enough to justify a diagnosis when considered separately.

[0101] Other explicitly identified anxiety disorders (ICD-10 F41.8): Anxiety hysteria Anxiety disorder, details unknown (ICD-10 F41.9): Anxiety NOS

[0102] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing Parkinson's disease.

[0103] Parkinson's disease is known in the art as a chronic degenerative disorder of the central nervous system that affects both the motor and non-motor systems. Symptoms usually develop slowly, and non-motor symptoms become more common as the disease progresses. Early symptoms include tremors, rigidity, slowness of movement, and difficulty walking. Problems can also occur in the cognitive, behavioral, sleep, and sensory systems. Parkinson's dementia becomes common as the disease progresses.

[0104] Parkinson's disease is classified under ICD-10 G20.

[0105] In a more preferred embodiment, the present invention relates to any pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease as described above and below herein, wherein the antagonist / inhibitor is selected from NPBWR1 inhibitory peptides, NPBWR1 inhibitory small molecule binding molecules, RNAi, siRNA, shRNA, aptamers and intramers specifically directed to NPBWR1, and anti-NPBWR1 antisense molecules.

[0106] Furthermore, the neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors used in methods for treating, improving, or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease as described above and below in this specification may also be antibodies.

[0107] Therefore, compounds that can specifically function as "antagonists" or "inhibitors" of the neuropeptide B / W receptor (NPBWR1) may include small molecule (organic) compounds or small molecule binding molecules such as ligands for the neuropeptide B / W receptor (NPBWR1). The term "small molecule" in the context of drug discovery is known in the art and refers to medicinal compounds having a molecular weight of less than 2,500 daltons, preferably less than 1,000 daltons, and more preferably between 50 and 350 daltons. (Small molecule) binding molecules include natural and synthetic compounds. The term "compound" in the context of this invention includes a single substance or a group of substances. The above compounds / binding molecules may be contained, for example, in a sample, such as a cell extract from a plant, animal, or microorganism. Furthermore, the above compounds may be compounds that are known in the art but have not been previously known to affect (negatively) the activity of the neuropeptide B / W receptor (NPBWR1) or to affect the expression of the nucleic acid molecule encoding the neuropeptide B / W receptor (NPBWR1).

[0108] However, in the context of the present invention, it is also conceivable that compounds including peptides, proteins, nucleic acids including cDNA expression libraries, small organic compounds, ligands, PNAs, etc., may be used as antagonists to the function of the neuropeptide B / W receptor (NPBWR1). The above compounds may also be functional derivatives or analogues. Methods for preparing chemical derivatives and analogues are well known to those skilled in the art and are described, for example, in Beilstein's "Handbook of Organic Chemistry," Springer Edition, New York, or in "Organic Synthesis," Wiley, New York.

[0109] Furthermore, the above derivatives and analogs can be tested for their effects, namely their antagonistic effects on the function of neuropeptide B / W receptor (NPBWR1), according to methods known in the art. Additionally, peptide mimes and / or computer-aided designs of suitable antagonists or inhibitors of neuropeptide B / W receptor (NPBWR1) can be used. For example, suitable computer systems for the computer-aided design of proteins and peptides are described in the prior art, e.g., Berry (1994) Biochem. Soc. Trans. 22:1033-1036, Wodak (1987), Ann. NYAcad. Sci. 501:1-13, Pabo (1986), Biochemistry 25:5987-5991. The results obtained from the above computer analysis can be used, for example, in combination with the methods of the present invention to optimize known compounds, substances, or molecules. Suitable compounds can also be identified by the synthesis of peptide mimegmatic combinatorial libraries by sequential chemical modification and testing of the resulting compounds, for example, according to the methods described herein. Methods for constructing and using peptide-mimicking combinatorial libraries are described in prior art, e.g., Ostresh (1996) Methods in Enzymology 267:220-234 and Dorner (1996) Bioorg. Med. Chem. 4:709-715. Furthermore, the three-dimensional and / or crystallographic structures of the neuropeptide B / W receptor (NPBWR1) antagonist can be used in the design of (peptide-mimicking) antagonists of the neuropeptide B / W receptor (NPBWR1).

[0110] RNAi approaches are also envisioned in the context of the present invention to be used in the preparation of pharmaceutical compositions for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0111] The terms "RNA interference" or "inhibitory RNA" (RNAi / iRNA) refer to the use of double-stranded RNA to target specific mRNAs for degradation, thereby silencing their expression. Preferred inhibitory RNA molecules can be selected from the group consisting of double-stranded RNA (dsRNA), RNAi, siRNA, shRNA, and stRNA. dsRNAs that match a gene sequence are synthesized in vitro and introduced into cells. dsRNAs can also be introduced into cells in the form of vectors expressing the target gene sequence in sense and antisense orientation, for example, in the form of hairpin mRNA. Sense and antisense sequences can also be expressed from separate vectors, thereby allowing individual antisense and sense molecules to form double-stranded RNA upon their expression. In some cases, it is known in the art that even the expression of a sense-oriented sequence or promoter sequence is sufficient to generate dsRNA, and subsequently siRNA, due to an internal amplification mechanism within the cell. Therefore, all means and methods that result in reduced activity (which may be reflected in lower expression of neuropeptide B / W receptor (NPBWR1)) should be used in accordance with the present invention, particularly by utilizing neuropeptide B / W receptor (NPBWR1)-specific siRNA (i.e., siRNA that specifically targets neuropeptide B / W receptor (NPBWR1) mRNA or its functional fragments). For example, these siRNAs can be generated / introduced using sense constructs, antisense constructs, hairpin constructs, sense molecules and antisense molecules, and combinations thereof. dsRNA feeds into a natural but partially understood process involving highly conserved nuclease dicers that cleave dsRNA precursor molecules into small interfering RNA (siRNA).Methods for generating and preparing siRNAs, as well as for inhibiting the expression of target genes, are described, in particular, in the international publication pamphlet 02 / 055693, Wei (2000) Dev. Biol. 15:239-255, La Count (2000) Biochem. Paras. 111:67-76, Baker (2000) Curr. Biol. 10:1071-1074, Svoboda (2000) Development 127:4147-4156, or Marie (2000) Curr. Biol. 10:289-292. These constructed siRNAs then become sequence-specific portions of RNA-induced silencing complexes (RISCs) (multiplex nucleases that disrupt messenger RNA homologous to the trigger of silencing). Elbashir (2001) EMBO J.20:6877-6888 demonstrated that 21-nucleotide RNA double helix can be used in cell culture to interfere with gene expression in mammalian cells. While RNAi is already known to be very efficiently mediated by siRNA in mammalian cells, the creation of stable cell lines or non-human transgenic animals has been limited. However, new generation vectors can be used to stably express, for example, short hairpin RNA (shRNA). Stable expression of siRNA in mammalian cells has been demonstrated, in particular, by Brummelkamp (2002) Science 296:550-553. Paul (2002) Nat. Biotechnol.20:505-508 also demonstrated the effective expression of small interfering RNA in human cells. RNA interference by the expression of short interfering RNA and hairpin RNA in mammalian cells has also been demonstrated by Yu (2002) PNAS 99:6047-6052. The shRNA approach for gene silencing is well known in the art and may include the use of st (small temporal) RNA; see, in particular, Paddison (2002) Genes Dev. 16:948-958.These approaches may be vector-based, for example the pSUPER vector or RNA polIII vector can be employed, as described, inter alia, in Yu (2002), loc. cit., Miyagishi (2002), loc. cit. or Brummelkamp (2002), loc. cit. It is envisaged that the regulatory sequences of the present invention are used in a similar manner to systems based on pSUPER or RNA polIII vectors.

[0112] Methods for estimating and constructing siRNAs are publicly known in the art, as described in Elbashir (2002) Methods 26:199-213, and on the internet websites of siRNA commercializers, e.g., Qiagen GmbH (https: / / www1.qiagen.com / GeneGlobe / Default.aspx), Dharmacon (www.dharmacon.com), Xeragon Inc. (http: / / www.dharmacon.com / Default.aspx), and Ambion (www.ambion.com) or the website of Tom Tuschl's research group (http: / / www.rockefeller.edu / labheads / tuschl / sirna.html). Furthermore, programs for estimating siRNAs from a given mRNA sequence are available online (e.g., http: / / www.ambion.com / techlib / misc / siRNA_finder.html or http: / / katahdin.cshl.org:9331 / RNAi / html / rnai.html). The uridine residue in the 2-nt 3' overhang can be substituted with 2' deoxythymidine without loss of activity, which significantly reduces the cost of RNA synthesis and can also enhance the resistance of siRNA double helix when applied to mammalian cells (Elbashir (2001) loc.cit). siRNA can also be enzymatically synthesized using T7 or other RNA polymerases (Donze (2002) Nucleic Acids Res 30:e46). Short RNA double helix mediating effective RNA interference (esiRNA) can also be generated by hydrolysis by E. coli RNase III (Yang (2002) PNAS 99:9942-9947). Furthermore, expression vectors have been developed to express double-stranded siRNA linked by a small hairpin RNA loop in eukaryotic cells (e.g., Brummelkamp (2002) Science 296:550-553). All of these constructs can be developed with the help of the programs described above.Alternatively, a commercially available sequence prediction tool incorporated into a sequence analysis program or sold separately, for example the siRNA Design Tool provided by www.oligoEngine.com (Seattle, Washington), may be used for siRNA sequence prediction.

[0113] Accordingly, specific interfering RNAs can be used according to the present invention as antagonists (inhibitors) of (expression and / or function of) neuropeptide B / W receptor 1 (NPBWR1). These siRNAs are formed by an antisense strand and a sense strand, wherein the antisense / sense strand preferably comprises at least 10 nucleotides, more preferably at least 12 nucleotides, still more preferably at least 14 nucleotides, still more preferably at least 16 nucleotides, still more preferably at least 18 nucleotides, and even more preferably at least 19, 20, 21 or 22 nucleotides.

[0114] As noted above, methods for preparing siRNAs for use in accordance with the present invention are well known in the art. Based on the teachings provided herein, it is clear that a person skilled in the art is not only capable of preparing such siRNAs, but also is positioned to assess whether the siRNA can antagonize / inhibit neuropeptide B / W receptor (NPBWR1). It is envisioned herein that the above-described siRNAs cause degradation of neuropeptide B / W receptor (NPBWR1) mRNA, and thereby cause a decrease in the protein level of neuropeptide B / W receptor (NPBWR1).

[0115] In other words, siRNA results in a significant decrease in the mRNA and / or protein levels of the neuropeptide B / W receptor (NPBWR1) (i.e., decreased expression of the neuropeptide B / W receptor (NPBWR1)). This decrease in expression can be reflected in a decrease in the activity of the neuropeptide B / W receptor (NPBWR1). For example, a neuropeptide B / W receptor (NPBWR1)-specific siRNA can lead to a decrease in the capacity of the neuropeptide B / W receptor (NPBWR1) and inhibit its activity. Therefore, the use of potent antagonists / inhibitors of the neuropeptide B / W receptor (NPBWR1) (such as the siRNAs described herein) will result in lower neuropeptide B / W receptor (NPBWR1) activity.

[0116] As used herein, the term “small interfering RNA” (siRNA) refers to a class of RNA molecules, generally short and double-stranded, that may also be known as short interfering RNA or silencing RNA, and that play a variety of biological roles, and increasingly diverse roles in the treatment of various diseases and conditions. As described above, siRNA is involved in the RNA interference (RNAi) pathway, in which it interferes with the expression of specific genes (see, for example, Zamore Nat Struct Biol 2001,8(9):746-50, Tuschl T. CHEMBIOCHEM.2001,2:239-245, Scherr and Eder, Cell Cycle.2007 Feb;6(4):444-9, Leung and Whittaker, Pharmacol Ther.2005 Aug;107(2):222-39, de Fougerolles et al., Nat. Rev. Drug Discov.2007,6:443-453).

[0117] Such siRNAs are generally 18–27 nt long and typically contain two short (usually 19–21 nt) strands of RNA (dsRNA) with or without 2 nt 3' overhangs at both ends. Each strand may have a 5' phosphate group and a 3' hydroxy(-OH) group, or the phosphate group may be absent from one or both strands. This structure is the result of processing by Dicer, an enzyme that converts either long dsRNA or short hairpin RNA into siRNA.

[0118] siRNA can also be introduced exogenously (artificially) into cells by various transfection methods to result in specific knockdown of a target gene. In this regard, other structures other than those described above are also conceivable if they can interfere with gene expression. Preferably, the double-stranded portion has a length of about 12 to about 50 base pairs, more preferably 16 to 30, more preferably 18 to 25, and more preferably 19 to 21. Most preferably, the double-stranded portion has a length of 19 base pairs. The siRNA of the present invention may have overhang sequences of up to 10 base pairs, preferably 5 base pairs or less, at both ends or one end, or may be blunt ends. It is also preferable that complementarity to the target gene extends along the entire length of the double-stranded portion. The region complementary to the target gene has a length of at least 12 bases, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23 or more base pairs. The siRNA of the present invention may be perfectly complementary to the target gene. Alternatively, siRNA may contain up to 5%, 10%, 20%, or 30% mismatches with respect to the target gene. Furthermore, siRNA and antisense RNA can also be chemically modified on a backbone containing, for example, sugar residues. Preferred modifications of the siRNA molecule of the present invention include linkers that connect the two strands of the siRNA molecule. Chemical modifications help to improve the pharmacological properties of siRNA and antisense RNA, among other things, in vivo stability and / or delivery to target sites in organisms. Those skilled in the art will be familiar with such modified siRNA and means and methods for obtaining them; see, for example, Zhang et al., Curr Top Med Chem. 2006;6(9):893-900, and Manoharan, Curr Opin Chem Biol. 2004 Dec;8(6):570-9.

[0119] Therefore, essentially, any gene with a known sequence can be targeted based on its sequence complementarity with appropriately tailored siRNAs. This has made siRNAs an important tool for gene function and drug target validation studies, as well as for the therapeutic interventions envisioned herein. The siRNAs disclosed herein can reduce or block the expression of the neuropeptide B / W receptor (NPBWR1).

[0120] In further embodiments, antisense molecules are envisioned to inhibit the expression or function of neuropeptide B / W receptor (NPBWR1), particularly human neuropeptide B / W receptor (NPBWR1), and to interact with neuropeptide B / W receptor (NPBWR1) expressed by coding regions, mRNA / cDNA as defined herein above, and neuropeptide B / W receptor (NPBWR1) expressed by isoforms and variants of the neuropeptide B / W receptor (NPBWR1). The isoforms or variants may, in particular, include allelic variants or splice variants. Furthermore, antisense molecules used according to the present invention to affect the expression or function of neuropeptide B / W receptor (NPBWR1) are also envisioned to specifically interfere with regulatory sequences of neuropeptide B / W receptor (NPBWR1) as defined herein below.

[0121] In this context, the term "modified" means that the neuropeptide B / W receptor (NPBWR1) nucleotide sequence and the encoded neuropeptide B / W receptor (NPBWR1) amino acid sequence are different from the distinct sequences available in the above-mentioned GenBank accession numbers due to mutations, such as deletions, additions, substitutions, or inversions.

[0122] Therefore, the antisense molecules used in accordance with the present invention specifically interact / hybridize with one or more nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1). Preferably, the nucleic acid molecules are RNA, i.e., pre-mRNA or mRNA. The term "specifically interact / hybridize with one or more nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1)" in relation to the present invention relates to antisense molecules that can interfere with the expression of the neuropeptide B / W receptor (NPBWR1). However, highly mutated anti-neuropeptide B / W receptor (NPBWR1) antisense constructs that cannot hybridize with or specifically interact with the neuropeptide B / W receptor (NPBWR1) encoding nucleic acid molecules should not be used in the context of the present invention. Whether or not an antisense construct specifically interacts / hybridizes with the neuropeptide B / W receptor (NPBWR1) encoding sequence can be easily inferred by those skilled in the art. These tests include, but are not limited to, hybridization assays, RNAse protection assays, Northern blotting, Northwestern blotting, nuclear magnetic resonance and fluorescence-coupled assays, dot blotting, microarrays and macroarrays, and quantitative PCR. Furthermore, such screening is not limited to the neuropeptide B / W receptor (NPBWR1) mRNA molecule, but may also include the neuropeptide B / W receptor (NPBWR1) mRNA / protein (RNP) complex (Hermann (2000) Angew Chem Int Ed Engl 39:1890-1904, DeJong (2002) Curr Trop Med Chem 2:289-302). In addition, functional tests including Western blotting, immunohistochemistry, immunoprecipitation assays, and bioassays based on neuropeptide B / W receptor (NPBWR1) responsive promoters are envisioned to test whether a particular antisense construct can specifically interact with / hybridize with the nucleic acid molecule encoding neuropeptide B / W receptor (NPBWR1).

[0123] As used herein, the term “antisense molecule” includes, in particular, antisense oligonucleotides. The antisense oligonucleotides may also include modified nucleotides and modified nucleoside bonds, as described, in particular, in U.S. Patent No. 6,159,697.

[0124] Most preferably, the antisense oligonucleotide of the present invention comprises at least 8, more preferably at least 10, more preferably at least 12, more preferably at least 14, and more preferably at least 16 nucleotides. The derivation and preparation of antisense molecules are well known in the art. The derivation of antisense molecules is described, among other things, in Smith, 2000. Conventional methods include "gene walking," RNase H mapping, RNase L mapping (Leaman (1999) Meth Enzymol 18:252-265), combinatorial oligonucleotide arrays on solid supports, determination of secondary structure by computational methods (Walton (2000) Biotechnol Bioeng, 65:1-9), aptamer oligonucleotides targeting structured nucleic acids (aptastruc), tethered oligonucleotide probes, foldback triple-forming oligonucleotides (FTFOs) (Kandimalla (1994) Gene 149:115-121), and selection of sequences with minimized nonspecific binding (Han (1994) Antisense Res Dev 4:53-65).

[0125] Preferably, the antisense molecule of the present invention is stabilized against degradation. Such stabilization methods are known in the art and, in particular, are described in U.S. Patent No. 6,159,697. Further methods described for protecting oligonucleotides from degradation include oligonucleotides crosslinked by linkers (Vorobjev (2001) Antisense Nucleic Acid Drug Dev, 11:77-85), molecules minimally modified according to cellular nuclease activity (Samani (2001) Antisense Nucleic Acid Drug Dev, 11:129-136), 2'O-DMAOE oligonucleotides (Prakash (2001) Nucleosides Nucleotides Nucleic Acids 20:829-832), 3'5'-dipeptidyl oligonucleotides (Schwope (1999) J Org Chem 64:4749-4761), 3'-methylenelentimidine and 5-methyluridine / cytidine h-phosphonates and phosphonamidites (An (2001) J Org Examples include encapsulation using anionic liposomes (De Oliveira (2000) Life Sci 67:1625-1637) or ionizable aminolipids (Semple (2001) Biochim Biophys Acta, 10:152-166).

[0126] In addition, antagonists / inhibitors of the expression or function of the neuropeptide B / W receptor (NPBWR1) may also include intracellular binding partners of the neuropeptide B / W receptor (NPBWR1). As used herein, the term “intracellular binding partner” refers to an intracellular molecule that can block or reduce the activity of the neuropeptide B / W receptor (NPBWR1). Such intracellular binding partners of the neuropeptide B / W receptor (NPBWR1) may, among other things, refer to endogenous inhibitor / repressor proteins of the neuropeptide B / W receptor (NPBWR1). In another embodiment of the present invention, the intracellular binding partner is an intracellular antibody. Intracellular antibodies are known in the art and can be used to modulate or inhibit the functional activity of a target molecule. This therapeutic approach is based on the intracellular expression of either Fab or single-stranded Fv recombinant antibody fragments targeted to a desired cellular compartment using an appropriate targeting sequence (Teillaud (1999) Pathol Biol 47:771-775).

[0127] As described herein, antagonists / inhibitors of the expression or function of the neuropeptide B / W receptor (NPBWR1) may also include aptamers. In relation to the present invention, the term "aptamer" includes nucleic acids such as RNA, ssDNA (ss = single-stranded), modified RNA, modified ssDNA, or PNA that bind to multiple target sequences with high specificity and affinity. Aptamers are well known in the art and, in particular, are described in Famulok (1998) Curr. Op. Chem. Biol. 2:320-327. Preparation of aptamers is well known in the art and, in particular, may include the use of combinatorial RNA libraries to identify binding sites (Gold (1995) Ann. Rev. Biochem. 64:763-797).

[0128] Therefore, aptamers are oligonucleotides derived from an in vitro evolutionary process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). A pool of randomized RNA sequences or single-stranded DNA sequences is selected for a specific target. Sequences that bind more tightly to the target are isolated and amplified. Selection is repeated using the enriched pool obtained from the first selection. Several rounds of this process yield a winning sequence called an "aptamer." Aptamers have evolved to bind to proteins associated with several disease conditions. Using this method, many potent antagonists of such proteins can be found. For these antagonists to function in animal models of disease and in humans, modification of the aptamer is usually necessary. Firstly, glycosphing modification of the nucleoside triphosphate is necessary to make the resulting aptamer resistant to nucleases found in serum. Changing the 2'OH group of ribose to a 2'F or 2'NH2 group yields aptamers that are long-lived in the blood. The relatively low molecular weight (8000–12000) of aptamers results in rapid clearance from the blood. Aptamers can be maintained in circulation for hours to days by conjugating them to high molecular weight vehicles. When modified and conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. Aptamers can be applied systemically to animals and humans to treat organ-specific diseases (Ostendorf (2001) J Am Soc Nephrol. 12:909-918). The first aptamer to advance to Phase I clinical studies was NX-1838, an injectable angiogenic inhibitor that can potentially be used to treat macular degeneration-induced blindness (Sun (2000) Curr Opin Mol Ther 2:100-105). Cytoplasmic expression of aptamers ("intramers") can be used to bind to intracellular targets (Blind (1999) PNAS 96:3606-3610; Mayer (2001) PNAS 98:4961-4965). The above intramers are also intended to be used in the context of the present invention.

[0129] The function of the neuropeptide B / W receptor (NPBWR1) and its neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors can be estimated by methods of the Art. Such methods are described herein and may, in particular, include, methods for testing aggregates of substances for interaction with the neuropeptide B / W receptor (NPBWR1) or one or more fragments thereof, and for further testing substances that have tested positive for interaction in the corresponding readout system for their inhibitory effects on the expression or function of the neuropeptide B / W receptor (NPBWR1) in vivo, in vitro, or in silico.

[0130] The "testing of neuropeptide B / W receptor (NPBWR1) interaction" described above is well known in the art and can be carried out by specific immunological, molecular biological, and / or biochemical assays, including, for example, homogeneous and heterogeneous assays described below herein. Neuropeptide B / W receptor (NPBWR1) ligands that can inhibit the function of neuropeptide B / W receptor (NPBWR1) can be identified by screening a large compound library based on their ability to interact with the neuropeptide B / W receptor (NPBWR1) protein. In preferred embodiments, such antagonists or inhibitors of neuropeptide B / W receptor (NPBWR1) function can bind to the protein-binding domain of neuropeptide B / W receptor (NPBWR1).

[0131] In addition to molecules capable of binding to the neuropeptide B / W receptor (NPBWR1), antagonists or inhibitors of the function of the neuropeptide B / W receptor (NPBWR1) can block / reduce the expression of nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1) protein. Those skilled in the art can easily identify the regulatory sequences of neuropeptide B / W receptor (NPBWR1) expression (e.g., promoter sequences, enhancer sequences, replication origins, and other regulatory elements) by, for example, in silico gene prediction methods and experimental verification of functional sites (Elnitski (2006) Genome Res 16:1455-64).

[0132] As already stated above, the neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors intended for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease as described above and below in this specification may also be antibodies.

[0133] Preferably, the antibody is an antibody against the human neuropeptide B / W receptor (NPBWR1), that is, an inhibitory antibody against the human neuropeptide B / W receptor (NPBWR1). The antibodies used herein (used interchangeably in multiple forms) are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, and polypeptides via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Preferred targets herein are the neuropeptide B / W receptor (NPBWR1), particularly the human neuropeptide B / W receptor (NPBWR1). As used herein, the term “antibody” includes not only intact (i.e., full-length) monoclonal antibodies, but also antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain variable fragment (scFv)), their variants, fusion proteins containing the antibody moiety, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camel or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), as well as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies, and any other modified configurations of immunoglobulin molecules containing an antigen recognition site of the desired specificity. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to be of a specific class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of their heavy chain. Immunoglobulins have five main classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0134] An antibody that "binds specifically" to a target or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular target antigen more frequently, more rapidly, with longer duration, and / or with higher affinity than it reacts with another target. An antibody "binds specifically" to a target antigen if it binds with higher affinity and avidity, more readily, and / or with longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to a neuropeptide B / W receptor 1 (NPBWR1) epitope is an antibody that binds to the NPBWR1 epitope with higher affinity and avidity, more readily, and / or with longer duration than it binds to other NPBWR1 epitopes or non-NPBWR1 epitopes. It is also understood by reading this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0135] Inhibitors of the neuropeptide B / W receptor (NPBWR1) may be anti-neuropeptide B / W receptor (NPBWR1) specific antibodies. Anti-neuropeptide B / W receptor (NPBWR1) antibodies are antibodies that can bind to the neuropeptide B / W receptor (NPBWR1) and can inhibit the biological activity of the neuropeptide B / W receptor (NPBWR1) and / or components of downstream pathways mediated by the neuropeptide B / W receptor (NPBWR1). In some examples, the anti-neuropeptide B / W receptor (NPBWR1) antibodies used in the methods described herein suppress the biological activity of the neuropeptide B / W receptor (NPBWR1) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0136] Accordingly, in one aspect, the present invention relates to an antineuropeptide B / W receptor (NPBWR1) antibody for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease as described above and below herein.

[0137] Antineuropeptide B / W receptor (NPBWR1) antibodies are well known and commercially available in the art. Those skilled in the art can easily select and / or produce a suitable antineuropeptide B / W receptor (NPBWR1) antibody.

[0138] In certain embodiments, the present invention relates to an antibody that specifically binds to a polypeptide or fragment thereof as shown in Sequence ID No. 1, and to the use thereof.

[0139] As described above, in relation to the present invention, the term “antibody” as used herein refers particularly to complete immunoglobulin molecules, as well as portions of such immunoglobulin molecules that substantially retain binding specificity. Furthermore, the term refers to modified and / or modified antibody molecules, e.g., chimeric antibodies and humanized antibodies, CDR-transplanted antibodies, recombinant or synthetically produced / synthesized antibodies, as well as intact antibodies, and their antibody fragments, e.g., separated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2. The term “antibody” also includes bifunctional antibodies, trifunctional antibodies, and antibody constructs such as single-chain Fv(scFv) or antibody fusion proteins. Further “antibody” constructs are known in the art and are included in the present invention.

[0140] Techniques for producing antibodies are well known in the art and are described, for example, in Howard and Bethell (2000) Basic Methods in Antibody Production and Characterization, Crc.Pr.Inc. Antibodies against polypeptides according to the present invention can be obtained, for example, by directly injecting a polypeptide (or a fragment thereof) into an animal, or by administering a polypeptide (or a fragment thereof) to an animal. The antibody thus obtained then binds to the polypeptide (or a fragment thereof) itself. In this way, even a fragment of a polypeptide can be used to produce an antibody that binds to the entire polypeptide, as long as the binding is "specific" as defined above.

[0141] Using the ordinary techniques of those skilled in the art, and through routine methods, those skilled in the art can readily infer from the sequences provided herein the relevant epitopes (functional fragments) of the polypeptides of the present invention that are useful in the production of antibodies such as polyclonal and monoclonal antibodies. However, it is clear that those skilled in the art are also in a position to provide engineered antibodies such as CDR grafted antibodies or humanized and fully human antibodies.

[0142] Particularly preferred in relation to the present invention are monoclonal antibodies. Any technique that provides antibodies produced by continuous culture of cell lines can be used to prepare monoclonal antibodies. Examples of such techniques include hybridoma techniques, trioma techniques, human B-cell hybridoma techniques, and EBV-hybridoma techniques for producing human monoclonal antibodies (Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press; Goding and Goding (1996), Monoclonal Antibodies: Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, Academic Pr Inc, USA).

[0143] Antibody derivatives can also be produced by peptide mimetic bodies. Furthermore, the techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent No. 4,946,778) can be adapted to produce single-chain antibodies that specifically recognize the polypeptides of the present invention. In addition, humanized antibodies against the polypeptides of the present invention can be expressed using transgenic animals.

[0144] The production of specific antibodies against natural polypeptides and recombinant polypeptides is based on immunization of animals such as mice. However, other animals for antibody / antiserum production are also envisioned within the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, etc. The polynucleotide according to the present invention, shown in SEQ ID NO: 2, can be subcloned into a suitable vector, in which case the recombinant polypeptide is expressed in an organism capable of expression, such as bacteria. Thus, the expressed recombinant protein can be injected intraperitoneally into a mouse, and the resulting specific antibody can be obtained, for example, from mouse serum provided by intracardiac blood puncture. The amount of the resulting specific antibody can be quantified using ELISA, which is also described below herein. Further methods for producing antibodies are well known in the art; see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.

[0145] As used herein, the term “specifically bind” refers to a binding reaction that determines the presence of neuropeptide B / W receptor (NPBWR1) protein and antibodies in the presence of a heterogeneous population of proteins and other biologics.

[0146] Therefore, under specified assay conditions, the specified antibody and neuropeptide B / W receptor (NPBWR1) protein bind to each other but not to other components present in the sample in significant amounts. Specific binding to the target analyte under such conditions may require a binding site selected for its specificity to the particular target analyte. Various immunoassay formats can be used to select antibodies that react specifically with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that have specific immunoreactivity with an analyte. Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press, and / or Howard and Bethell (2000), Basic Methods in Antibody Production and Characterization, Crc.Pr.Inc., provide descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction is at least twice the background signal versus noise, and more typically more than 10 to 100 times greater than the background. Those skilled in the art are in a position to provide and generate specific binding molecules for novel polypeptides. In the case of specific binding assays, it can be readily used to avoid undesirable cross-reactivity, for example, by easily purifying polyclonal antibodies and selecting them by known methods (Shepherd and Dean, loc. cit.).

[0147] As used herein, the term “purification or detection” refers to a series of processes intended to isolate or detect a single type of protein from a complex mixture using “specific binding” as defined above, which refers to the binding reaction that determines the presence of the neuropeptide B / W receptor (NPBWR1) protein and antibody in the presence of a heterogeneous population of proteins and other biologics. Purification or detection of proteins is essential for characterizing the function, structure, and interactions of the protein of interest. In non-limiting examples, the starting material may be a biological tissue or a microbial culture. Various steps in the purification or detection process can release the protein from the matrix that confines it, separate the protein-non-protein portions in the mixture, and ultimately separate the desired protein from all other proteins. The separation steps take advantage of differences in protein size, physicochemical properties, and binding affinity.

[0148] In a more preferred embodiment, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the antagonist / inhibitor is an NPBWR1 inhibitory small molecule binding molecule, and is of formula (1): [ka] Formula (1) A chemical structure represented by the formula, where, R 1 It is selected from F, Cl, Br, I, and CN. R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (a heterocycline having) and -(6 to 10 ring atoms and optionally one or more substituents R 2a Selected from carbocyclyl having, R 3 is -(5 to 20 ring atoms and optionally one or more substituents R 3a (a heterocycline having), -(one or more substituents RAlk C is arbitrarily replaced by 1-4 Alkylene)-(5-20 ring atoms and optionally one or more substituents R) 3a (a heterocycline having 6 to 20 ring atoms and optionally one or more substituents R) 3a (Carbocyclyl having), and -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(6-20 ring atoms and optionally one or more substituents R) 3a Selected from carbocyclyl having, During the ceremony, Each R 2a -Halogen, -CN, -CF3, -CHF2, -CH2F, -OCF3, -OCHF2, -OCH2F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -OC 1-6 Independently selected from the group consisting of alkyl groups, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R 3a -Halogen, -CN, -CF3, -CHF2, -CH2F, -OCF3, -OCHF2, -OCH2F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -OC 1-6 Independently selected from the group consisting of alkyl groups, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R Alk This is independently selected from the group consisting of -halogens and -CN. It has a chemical structure or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0149] In a more preferred embodiment, the present invention relates to the above-mentioned pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the antagonist / inhibitor is an NPBWR1 inhibitory small molecule binding molecule having a chemical structure represented by formula (1) as defined above, wherein formula (1) satisfies one or more of the following conditions: a)R 1 This is selected from F, Cl, Br and CN, preferably from F, Cl and CN, more preferably from F and Cl, and even more preferably from Cl. b)R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (Having heteroaryls) and -(6 to 10 ring atoms and optionally one or more substituents R 2a From an aryl (having), preferably -(5 to 7 ring atoms and optionally one or more substituents R 2a (a heteroaryl having) and -(6 or 10 ring atoms and optionally one or more substituents R 2a From aryls having one or more substituents R, more preferably benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine (any of these having one or more substituents R 2a Selected from (which may be optionally substituted with R), more preferably benzene, naphthalene, imidazolidine, oxazole and pyridine (each of which has one or more substituents R 2a More preferably, benzene, imidazolidine, oxazole and pyridine (each of which may be optionally substituted with R) 2a (which may be optionally substituted with) more preferably one or more substituents R 2a More preferably, one or more substituents R from a benzene which may be optionally substituted with 2a From benzene substituted with, most preferably, the 4-position is R 2aSelected from benzenes substituted with c)R 3 -(5 to 14 ring atoms and optionally one or more substituents R 3a (a heterocycline having), -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(5-14 ring atoms and optionally one or more substituents R) 3a (a heterocycline having 6 to 14 ring atoms and optionally one or more substituents R) 3a (Carbocyclyl having), and -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (Carbocyclyl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 to 10 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6-10 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 or 10 ring atoms and optionally one or more substituents R) 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4alkylene)-(aryl having 6 or 10 ring atoms and optionally one or more substituents R 3a , still more preferably selected from -(phenyl having one or more substituents R 3a and -(naphthyl having one or more substituents R Alk optionally substituted C 1-4 alkylene)-(naphthyl optionally having one or more substituents R 3a , still more preferably selected from 2,5-dimethylphenyl or 1-naphthylmethyl, d)C 1-4 alkylene is preferably methylene or ethylene, more preferably methylene, e)substituent R 2a is absent, or is present in 1, 2 or 3 instances, preferably, substituent R 2a is absent, or is present in 1 or 2 instances, more preferably 1 substituent R 2a is present, still more preferably 1 substituent R is present at the 4-position 2a , f)substituent R 3a is absent, or is present in 1, 2 or 3 instances, preferably, substituent R 3a is absent, or is present in 1 or 2 instances, more preferably, when R 3 does not contain a -(C 1-4 alkylene) group, 2 substituents R 3a are present, more preferably, when R 3 contains a -(C 1-4 alkylene) group, 1 substituent R 3a is present or is absent, g)substituent R Alk is absent, or is present in 1, 2 or 3 instances, preferably, substituent R Alk is absent, or is present in 1 or 2 instances, more preferably, substituent R Alk is absent or is present in 1 instance, still more preferably substituent R Alk is absent, h)each R 2a is selected from -halogen, -CN, C 1-6 alkyl, C 1-6haloalkyl, C 1-6 cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-6 alkyl or -O-C 1-6 each R* is independently selected from the group consisting of haloalkyl, wherein each R* is H or C 1-6 alkyl or C 1-6 is independently selected from cycloalkyl, preferably, each R 2a is selected from the group consisting of -halogen, -CN, C 1-3 alkyl, C 1-3 haloalkyl, cyclopropyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-3 alkyl or -O-C 1-3 is independently selected from the group consisting of haloalkyl, wherein each R* is H or C 1-3 is independently selected from alkyl or cyclopropyl, more preferably, each R 2a is selected from the group consisting of -halogen, -CN, C 1-3 alkyl, -NR*R*, -NR*COR*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-3 alkyl or -O-C 1-3 is independently selected from the group consisting of haloalkyl, wherein each R* is H or C 1-3 is independently selected from alkyl or cyclopropyl, even more preferably, each R 2a is selected from the group consisting of -halogen, -CN, C 1-3 alkyl or -O-C 1-3 is independently selected from the group consisting of alkyl, even more preferably, each R 2a is selected from the group consisting of -halogen, -CN or -O-C 1-3 is independently selected from the group consisting of alkyl, even more preferably, each R 2a is independently -O-C 1-3 alkyl, even more preferably, each R 2a is independently methoxy or ethoxy, most preferably methoxy, i) each R 3a-Halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-6 Alkyl or -OC 1-6 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-6 Alkyl or C 3-6 Selected independently from cycloalkyl groups, preferably each R 3a These are independently -halogen, -CN, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -OC 1-6 Alkyl or -OC 1-6 Selected from the group consisting of haloalkyls, more preferably each R 3a -Halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, or C 3-6 Independently selected from the group consisting of cycloalkyls, and more preferably, each R 3a -Halogen, -CN, C 1-3 Alkyl, or C 1-3 Independently selected from the group consisting of haloalkyls, and more preferably each R 3a is -halogen and C 1-3 Independently selected from the group consisting of alkyl, and more preferably, each R 3a C is such as methyl or ethyl. 1-3 Independently selected from the group consisting of alkyls, and more preferably methyl, j) Each R Alk The elements are independently selected from the group consisting of -F and -Cl, preferably each R Alk is -F, and more preferably, each R Alk It does not exist, and / or k) The above NPBWR1 inhibitory small molecule binding molecule has a chemical structure represented by the following formula (1) or a pharmaceutically acceptable salt or solvate thereof.

[0150] In a more preferred embodiment, the present invention relates to the above-mentioned pharmaceutical composition comprising an antagonist / inhibitor of a neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, The above antagonist / inhibitor is an NPBWR1 inhibitory small molecule binding molecule and has one of the following chemical structures represented by formulas (2) to (9): [ka] (In the formula, R 1 , R 2a and R 3 (This expresses the same meaning as above.)

[0151] Therefore, in relation to the present invention, the NPBWR1 inhibitory small molecule binding molecule is preferably a chemical structure represented by formula (1), During the ceremony, R 1 It is selected from F, Cl, Br, I, and CN. R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (a heterocycline having) and -(6 to 10 ring atoms and optionally one or more substituents R 2a Selected from carbocyclyl having, R 3 is -(5 to 20 ring atoms and optionally one or more substituents R 3a (a heterocycline having), -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(5-20 ring atoms and optionally one or more substituents R) 3a (a heterocycline having 6 to 20 ring atoms and optionally one or more substituents R) 3a (Carbocyclyl having), and -(one or more substituents R Alk C is arbitrarily replaced by 1-4Alkylene)-(6-20 ring atoms and optionally one or more substituents R) 3a Selected from carbocyclyl having, During the ceremony, Each R 2a -Halogen, -CN, -CF3, -CHF2, -CH2F, -OCF3, -OCHF2, -OCH2F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -OC 1-6 Independently selected from the group consisting of alkyl groups, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R 3a -Halogen, -CN, -CF3, -CHF2, -CH2F, -OCF3, -OCHF2, -OCH2F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -OC 1-6 Independently selected from the group consisting of alkyl groups, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R Alk This is independently selected from the group consisting of -halogens and -CN. It has a chemical structure or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0152] In equation (1), one or more of the following conditions are met: a)R 1 This is selected from F, Cl, Br and CN, preferably from F, Cl and CN, more preferably from F and Cl, and even more preferably from Cl. b)R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (Having heteroaryls) and -(6 to 10 ring atoms and optionally one or more substituents R 2aFrom an aryl (having), preferably -(5 to 7 ring atoms and optionally one or more substituents R 2a (a heteroaryl having) and -(6 or 10 ring atoms and optionally one or more substituents R 2a From aryls having one or more substituents R, more preferably benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine (any of these having one or more substituents R 2a Selected from (which may be optionally substituted with R), more preferably benzene, naphthalene, imidazolidine, oxazole and pyridine (each of which has one or more substituents R 2a More preferably, benzene, imidazolidine, oxazole and pyridine (each of which may be optionally substituted with R) 2a (which may be optionally substituted with) more preferably one or more substituents R 2a More preferably, one or more substituents R from a benzene which may be optionally substituted with 2a From benzene substituted with, most preferably, the 4-position is R 2a Selected from benzenes substituted with c)R 3 -(5 to 14 ring atoms and optionally one or more substituents R 3a (a heterocycline having), -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(5-14 ring atoms and optionally one or more substituents R) 3a (a heterocycline having 6 to 14 ring atoms and optionally one or more substituents R) 3a (Carbocyclyl having), and -(one or more substituents R Alk C is arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (Carbocyclyl having) and -(one or more substituents R AlkC arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6-14 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 to 10 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6-10 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 or 10 ring atoms and optionally one or more substituents R) 3a (aryl having) and -(one or more substituents R Alk C arbitrarily replaced by 1-4 Alkylene)-(6 or 10 ring atoms and optionally one or more substituents R) 3a Selected from aryls having -(one or more substituents R 3a Phenyl having -(one or more substituents R) Alk C arbitrarily replaced by 1-4 Alkylene)-(Optionally one or more substituents R 3a Selected from naphthyl having, more preferably selected from 2,5-dimethylphenyl or 1-naphthylmethyl, d)C 1-4 The alkylene is preferably methylene or ethylene, and more preferably methylene. e) Substituent R 2a It is either absent, or there are one, two or three substituents R 2a Either none, or one or two, more preferably one substituent R 2a A substituent R is present, and more preferably, one substituent R is present at the 4-position.2a There exists, f) Substituent R 3a It is either absent, or there are one, two or three substituents R 3a Either none exist, or one or two exist, more preferably R 3 ga-(C 1-4 If the alkylene group is not present, there are two substituents R 3a There exists, and more comfortably, R 3 ga-(C 1-4 If an alkylene group is included, one substituent R 3a Whether it exists or does not exist, g) Substituent R Alk It is either absent, or there are one, two or three substituents R Alk Either none exists, or one or two exist, more preferably substituent R Alk There are either none or one substituent R, and more preferably substituent R Alk It does not exist. h) Each R 2a -Halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-6 Alkyl or -OC 1-6 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl groups, preferably each R 2a -Halogen, -CN, C 1-3 Alkyl, C 1-3 Haloalkyl, cyclopropyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-3 Alkyl or -OC 1-3 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-3Independently selected from alkyl or cyclopropyl, more preferably each R 2a -Halogen, -CN, C 1-3 Alkyl, -NR*R*, -NR*COR*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-3 Alkyl or -OC 1-3 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-3 Independently selected from alkyl or cyclopropyl, and more preferably each R 2a -Halogen, -CN, C 1-3 Alkyl or -OC 1-3 Independently selected from the group consisting of alkyl, and more preferably, each R 2a These are -halogen, -CN, or -OC 1-3 Independently selected from the group consisting of alkyl, and more preferably, each R 2a -OC is independent 1-3 Alkyl, and more preferably each R 2a is independently methoxy or ethoxy, most preferably methoxy. i) Each R 3a -Halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O2)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-6 Alkyl or -OC 1-6 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-6 Alkyl or C 3-6 Selected independently from cycloalkyl groups, preferably each R 3a These are independently -halogen, -CN, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -OC 1-6 Alkyl or -OC 1-6 Selected from the group consisting of haloalkyls, more preferably each R3a -Halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, or C 3-6 Independently selected from the group consisting of cycloalkyls, and more preferably, each R 3a -Halogen, -CN, C 1-3 Alkyl, or C 1-3 Independently selected from the group consisting of haloalkyls, and more preferably each R 3a is -halogen and C 1-3 Independently selected from the group consisting of alkyl, and more preferably, each R 3a C is such as methyl or ethyl. 1-3 Independently selected from the group consisting of alkyls, and more preferably methyl, j) Each R Alk The elements are independently selected from the group consisting of -F and -Cl, preferably each R Alk is -F, and more preferably, each R Alk It does not exist, and / or k) The above NPBWR1 inhibitory small molecule binding molecule has a chemical structure represented by the following formula (1) or a pharmaceutically acceptable salt or solvate thereof.

[0153] Furthermore, in relation to the present invention, the NPBWR1 inhibitory small molecule binding molecule more preferably has one of the above chemical structures represented by formulas (2) to (9): In the formula, R 1 , R 2a and R 3 This expresses the same meaning as above.

[0154] As used herein, the term “alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group that may be linear or branched. Therefore, an “alkyl” group does not contain a carbon-carbon double bond or a carbon-carbon triple bond. The term “alkyl” is preferably “C 1-6 It refers to "alkyl". 1-6"Alkyl" refers to an alkyl group having 1 to 6 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl) or butyl (e.g., n-butyl, isobutyl, sec-butyl or tert-butyl). Unless otherwise defined, the term "alkyl" more preferably refers to C 1-4 This refers to alkyl, more preferably methyl or ethyl, and even more preferably methyl.

[0155] "Halogen" refers to F, Cl, Br, and I, more preferably F or Cl, and even more preferably F unless otherwise specified.

[0156] As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms, independently selected from fluoro, chloro, bromo, and iodine, and preferably all being fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available bonding sites and therefore depends on the number of carbon atoms in the alkyl portion of the haloalkyl group. “Haloalkyl” may refer to, for example, -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. When the haloalkyl is a substituent on oxygen, it is preferable that the carbon adjacent to oxygen (of the haloalkyl) does not have a halogen directly bonded to it.

[0157] The term "aryl" preferably refers to an aromatic monocyclic ring containing 5 or 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms, or an aromatic tricyclic ring system containing 14 carbon atoms. Examples include phenyl, naphthyl, or anthracenyl, preferably phenyl.

[0158] The term "heteroaryl" preferably refers to a five-membered or six-membered aromatic ring in which one or more carbon atoms in the ring are substituted with the same or different heteroatoms: one, two, three, or four in the case of a five-membered ring, and one, two, three, four, or five in the case of a six-membered ring. The heteroatoms are preferably selected from O, N, and S. Examples of heteroaryl groups are shown below.

[0159] The term "heterocyclyl" encompasses any monocyclic, bicyclic, or polycyclic ring system containing one or more heteroatoms, the same or different heteroatoms, selected from O, N, and S. Preferably, the ring system contains 3 to 15 ring atoms. More preferably, the ring system is monocyclic or bicyclic and has 5 to 10 ring atoms, and even more preferably, the ring system is monocyclic and has 5 or 6 ring atoms. Typically, the ring system can contain 1 to 4, more typically 1 or 2, heteroatoms in the available positions. The term "heterocyclyl" also encompasses heteroaryl rings. Examples include azetidine, pyrrole, pyrrolidine, oxolane, furan, imidazolidine, imidazole, pyrazole, oxazolidine, oxazole, thiazole, piperidine, pyridine, morpholine, piperazine, and dioxolane.

[0160] The term "carbocykrill" encompasses any monocyclic, bicyclic, or polycyclic ring system that does not contain heteroatoms in the ring. Preferably, the ring system contains 3 to 15 ring atoms. More preferably, the ring system is monocyclic or bicyclic and has 5 to 10 ring atoms, and even more preferably, the ring system is monocyclic and has 5 or 6 ring atoms. The term "carbocyclic ring" also encompasses aryl rings.

[0161] As used herein, the term “cycloalkyl” refers to saturated hydrocarbon ring groups including monocyclic rings, as well as bridging rings, spiro rings, and / or fused ring systems (which may consist of, for example, two or three rings, for example, fused ring systems consisting of two or three fused rings). “Cycloalkyl” may refer to, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or adamantyl. Unless otherwise defined, “cycloalkyl” preferably refers to C 3-11 Refers to cycloalkyl, more preferably C 3-8 This refers to cycloalkyl groups. Particularly preferred "cycloalkyl groups" are monocyclic saturated hydrocarbon rings having 3 to 8 ring members, most preferably 3 ring members.

[0162] Where a compound or part is referred to as "may be substituted," it may, in each case, contain one or more of the substituents shown, which may be the same or different substituents.

[0163] As used herein, unless otherwise expressly indicated or inconsistent with the context, the terms “a,” “an,” and “the” are used interchangeably with “one or more” and “at least one.” Therefore, for example, a composition comprising the compound of the present invention (in particular of formula (1)) (“a” compound) can be interpreted as referring to a composition comprising “one or more” compounds of the present invention.

[0164] As used herein, the terms “comprising” (or “comprise,” “comprises,” “contain,” “contains,” or “containing”) mean “in particular,” i.e., “in addition to any further optional elements,” unless otherwise expressly indicated or inconsistent with the context. In addition, the term also encompasses the narrower meanings of “essentially consisting of” and “consisting of.” For example, the term “A containing B and C” means “A contains, in particular, B and C,” and A may contain any further optional elements (e.g., “A containing B, C and D” is also included), but the term also means “A is essentially consisting of B and C” and “A consists of B and C” (i.e., A does not contain any components other than B and C).

[0165] Furthermore, unless otherwise indicated, references to industry standards, pharmacopoeias, or manufacturers' manuals refer to the latest versions of the corresponding documents that were available on the priority date of this specification (i.e., the earliest filing date).

[0166] The scope of the present invention encompasses all pharmaceutically acceptable salt forms of the compounds provided herein, particularly the compounds of the present invention (especially the compounds of formula (1)) that can be formed, for example, by protonation of an atom having a protonation-protonation-easily protonable lone pair of electrons (e.g., an amino group) with an inorganic or organic acid, or as a salt of an acidic group (e.g., a carboxylic acid group) with a physiologically acceptable cation. Examples of base addition salts include alkali metal salts such as sodium or potassium salts, alkaline earth metal salts (e.g., calcium or magnesium salts), zinc salts, ammonium salts, aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salt, meglumine salt, ethylenediamine salt, and choline salt, aralkylamine salts such as N,N-dibenzylethylenediamine salt, benzathine salt, and benetamine salt, heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, and isoquinoline salt, quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt, and basic amino acid salts (e.g., arginine salt, lysine salt, or histidine salt).Exemplary acid addition salts include, for example, mineral salts (e.g., hydrochloride), hydrobromide, hydroiodide, sulfates (e.g., sulfate or hydrogen sulfate), nitrates, phosphates (e.g., phosphate, hydrogen phosphate, or dihydrogen phosphate), carbonates, bicarbonates, perchlorates, borates, or thiocyanates, acetates, propionates, butyrates, pentanoates, hexanoates, heptanoates, octanoates, cyclopentanepropionates, decanoates, undecanoates, oleates, stearates, lactates, maleates, oxalates, fumarates, tartrates, malates, citrates, succinates, adipates, and glucons. Examples include organic salts such as glycolates, nicotinates, benzoates, salicylates, ascorbic acid, pamoates (embonates), camphorates, glucoheptanoates, and pivalates; sulfonates such as methanesulfonates (mesylates), ethanesulfonates (esylates), 2-hydroxyethanesulfonates (isethionates), benzenesulfonates (besilates), p-toluenesulfonates (tosilates), 2-naphthalenesulfonates (naphthylates), 3-phenylsulfonates, and camphorsulfonates; glycerophosphates; and acidic amino acid salts (e.g., aspartates or glutamates).

[0167] Furthermore, the scope of the present invention encompasses the compounds provided herein, in particular the compounds of the present invention (especially the compounds of formula (1)), in any solvate form, including, for example, solvates with water (i.e., as hydrates) or solvates with organic solvents such as methanol, ethanol, or acetonitrile (i.e., as methanol, ethanol, or acetonitrile), or in any crystalline form (i.e., as any polymorph), or in any amorphous form. It should be understood that such solvates of the compounds provided herein, in particular the compounds of the present invention, also include solvates of pharmaceutically acceptable salts of the corresponding compounds.

[0168] Furthermore, the compounds provided herein, in particular the compounds of formula (1), may exist in the form of different isomers, in particular stereoisomers (e.g., geometric isomers (or cis / trans isomers), enantiomers and diastereomers) or tautomers. All such isomers of the compounds provided herein are considered to be part of the present invention, either in mixtures or in pure or substantially pure forms. With respect to stereoisomers, the present invention encompasses isolated optical isomers of the compounds according to the present invention, as well as any mixtures thereof (in particular, including racemic mixtures / racemates). Racemates can be separated by physical methods, such as fractional crystallization, separation or crystallization of diastereomer derivatives, or separation by chiral column chromatography. Individual optical isomers can also be obtained from racemates by salt formation with optically active acids and subsequent crystallization. The present invention further encompasses any tautomers of the compounds provided herein.

[0169] The scope of the present invention also includes compounds provided herein, particularly those of formula (1), in which one or more atoms are substituted with specific isotopes of the corresponding atoms. For example, the present invention includes compounds in which one or more hydrogen atoms (or, for example, all hydrogen atoms) are replaced with deuterium atoms (i.e., 2 This invention includes compounds of formula (1) substituted with H (also called "D"). Therefore, the present invention also includes compounds of formula (1) enriched with deuterium. Naturally occurring hydrogen is present in a concentration of approximately 99.98 mol% hydrogen-1( 1 H) and about 0.0156 mol% deuterium ( 2It is an isotopic mixture containing H or D. The deuterium content at one or more hydrogen positions in the compound of formula (1) can be increased using deuterating techniques known in the art. For example, the compound of formula (1) or the reactants or precursors used in the synthesis of the compound of formula (1) can be subjected to an H / D exchange reaction using heavy water (D2O), for example. More suitable deuterating techniques are described in Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012, William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010, or Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The deuterium content can be determined, for example, by mass spectrometry or NMR spectroscopy. Unless otherwise specified, it is preferable that the compound of formula (1) is not enriched with deuterium. Therefore, naturally occurring hydrogen atoms in the compound of formula (1) or 1 The presence of hydrogen atoms (H) is preferable.

[0170] The present invention also relates to compounds provided herein, in particular to positron-emitting isotopes of corresponding atoms, for example, 18 F, 11 C, 13 N, 15 O, 76 Br, 77 Br, 120 I and / or 124 The present invention includes compounds of formula (1) in which I is substituted. Such compounds can be used as tracers or imaging probes in positron emission tomography (PET). Therefore, the present invention includes (1) compounds in which one or more fluorine atoms (or, for example, all fluorine atoms) 18 A compound of formula (1) substituted with an F atom, (ii) one or more carbon atoms (or, for example, all carbon atoms) 11 A compound of formula (1) substituted with a C atom, (iii) one or more nitrogen atoms (or, for example, all nitrogen atoms) 13A compound of formula (1) substituted with an N atom, (iv) one or more oxygen atoms (or, for example, all oxygen atoms) 15 Compound of formula (1) substituted with an O atom, (v) one or more bromine atoms (or, for example, all bromine atoms) 76 A compound of formula (1) substituted with a Br atom, (vi) one or more bromine atoms (or, for example, all bromine atoms) 77 Compound of formula (1) substituted with a Br atom, (vii) one or more iodine atoms (or, for example, all iodine atoms) 120 Compounds of formula (1) substituted with an I atom, and (viii) one or more iodine atoms (or, for example, all iodine atoms) 124 This includes compounds of formula (1) substituted with an I atom. In general, it is preferable that none of the atoms in the compound of formula (1) are substituted with any specific isotope.

[0171] The compounds provided herein, in particular the pharmaceutically acceptable prodrugs of the compound of formula (1), are derivatives having chemically or metabolically cleavable groups that, upon solvolysis or physiological conditions, become pharmaceutically active compounds of the present invention in vivo. Prodrugs of the compounds according to the present invention can be formed by conventional methods using functional groups of the compound, such as amino, hydroxy, or carboxyl groups. Prodrug forms often offer advantages in terms of solubility, histocompatibility, or delayed release in mammalian organisms (see Bundgaard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives, such as esters prepared by the reaction of a hydrophilic compound with a suitable alcohol, or amides prepared by the reaction of a hydrophilic compound with a suitable amine. When the compound of the present invention has a carboxyl group, prodrugs include ester derivatives obtained by reacting the carboxyl group with a suitable alcohol, or amide derivatives obtained by reacting the carboxyl group with a suitable amine. Particularly preferred ester derivatives as prodrugs are methyl esters, ethyl esters, n-propyl esters, isopropyl esters, n-butyl esters, isobutyl esters, tert-butyl esters, morpholinoethyl esters, N,N-diethyl glycolamide esters, or α-acetoxyethyl esters. When the compound of the present invention has a hydroxyl group, acyloxy derivatives prepared by reacting the hydroxyl group with a suitable acyl halide or suitable acid anhydride are exemplified as prodrugs. Particularly preferred acyloxy derivatives as prodrugs are -OC(=O)-CH3, -OC(=O)-C2H5, -OC(=O)-(tert-Bu), and -OC(=O)-C 15 H 31These are -OC(=O)-(m-COONa-Ph), -OC(=O)-CH2CH2COONa, -O(C=O)-CH(NH2)CH3, or -OC(=O)-CH2-N(CH3)2. When the compound of the present invention has an amino group, amide derivatives prepared by reacting the amino group with a suitable acid halide or a suitable mixed anhydride are exemplified as prodrugs. Particularly preferred amide derivatives as prodrugs are -NHC(=O)-(CH2)2OCH3 or -NHC(=O)-CH(NH2)CH3.

[0172] In a more preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of repressing the expression of NPBWR1.

[0173] The CRISPR / Cas system is known in the art and can be used to target a highly specific target site of interest in a highly specific manner using a specially designed guide RNA. In this case, the specific target sequence is the genomic sequence of the NBPWR1 locus.

[0174] The human NBPWR1 gene is located on the anterior strand of chromosome 8, between base pairs 52,939,182 and 52,943,734 (Genome Reference Consortium-Human GRCh38 / h38).

[0175] The genomic locus of the NBPWR1 gene is publicly known in the art and can be searched, for example, using the following accession number: ENSG 00000288611.

[0176] Its cytogenetic location is the cell membrane.

[0177] While CRISPR / Cas systems are often used to cleave genomic (target) DNA by the nuclease activity of site-specific Cas endonucleases, cleavage is neither anticipated nor desired in the context of the present invention in relation to the use of neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors. Instead, as described in more detail below, in the context of the present invention, it is desirable that the CRISPR / Cas system be modified to lack its nuclease activity, and that the Cas protein be fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of repressing NPBWR1 expression. Thus, the CRISPR / Cas system used in the context of the present invention utilizes the ability of the CRISPR / Cas system to target a highly specific target site of interest in a highly specific manner by specifically designed guide RNA, but without the cleavage of genomic targets. Instead, the CRISPR / Cas system used in the context of the present invention utilizes the ability of the CRISPR / Cas system to target / bind the CRISPR-Cas endonuclease to a highly specific target site of interest in a highly specific manner by a specially designed guide RNA, in which the CRISPR / Cas system is modified to lack its nuclease activity. The CRISPR / Cas system is then used to exert its function at a specific genomic target (i.e., the CRISPR / Cas system is in this case specifically designed to be directed toward NPBWR1) by an effector domain selected from the group consisting of a transcriptional repressor domain fused to the Cas protein of the CRISPR / Cas system and an epigenetic modification domain capable of repressing the expression of NPBWR1.

[0178] In general, the CRISPR / Cas system is described below, and further, the more specific use of the CRISPR / Cas9 system in the context of the present invention is described in more detail below.

[0179] It is generally known in the art that CRISPR (clustered, regularly arranged, short palindromic sequence repeats) genomic loci can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a type of immune system that helps defend prokaryotes from foreign invaders such as viruses and phages. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

[0180] The CRISPR locus contains several short repetitive sequences called "repeats." Repeats can form hairpin structures and / or contain unstructured single-stranded sequences. Repeats typically occur in clusters and frequently diverge between species. Repeats are regularly spaced with specific intervening sequences called "spacers," resulting in a repeat-spacer-repeat locus structure. Spacers are identical to, or have high homology to, known invading sequences. The spacer-repeat unit encodes crisprRNA (crRNA), which is processed into the mature form of the spacer-repeat unit. The crRNA contains a "seed" or spacer sequence involved in the targeting of the target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the invading nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0181] The CRISPR locus also contains polynucleotide sequences encoding CRISPR-related (Cas) genes. Cas genes encode endonucleases involved in the biosynthesis and interference steps of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0182] Type II CRISPR System crRNA biosynthesis in naturally occurring type II CRISPR systems requires trans-activated CRISPR RNA (tracrRNA). The tracrRNA is modified by endogenous RNase III and then hybridizes to crRNA repeats in a pre-crRNA array. Endogenous RNase III is recruited to cleave the pre-crRNA. The cleaved crRNA is subjected to exoribonuclease trimming to produce a mature crRNA morphology (e.g., 5' trimming). The tracrRNA remains hybridized to the crRNA, and both the tracrRNA and crRNA associate with a site-specific polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex induces the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid activates Cas9 for target nucleic acid cleavage (where Cas9 is enzymatically active is not assumed in the context of this invention, as further described above and below). In type II CRISPR systems, the target nucleic acid is called a protospacer-adjacent motif (PAM). In nature, PAMs are essential for facilitating the binding of site-specific polypeptides (e.g., Cas9) to the target nucleic acid. Type II systems are further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) demonstrate the usefulness of the CRISPR / Cas9 system for RNA programmable genome editing, and International Publication No. 2013 / 176772 provides numerous examples and applications of the CRISPR / Cas endonuclease system for site-specific gene editing.

[0183] Cas gene / polypeptide and protospacer adjacent motif An example of a CRISPR / Cas polypeptide is the Cas9 polypeptide shown in Figure 1 of Fonfara et al., Nucleic Acids Research, 42:2577-2590 (2014). The CRISPR / Cas gene naming system has undergone extensive rewriting since the discovery of the Cas gene. Figure 5 of Fonfara's work above provides PAM sequences of Cas9 polypeptides from various species.

[0184] Site-specific DNA endonucleases Generally, site-specific endonucleases are nucleases used for genome editing to cleave DNA. Site-specific endonucleases can be administered to cells or patients as either one or more polypeptides, or one or more mRNAs encoding polypeptides.

[0185] In relation to the CRISPR / Cas system, site-specific DNA endonucleases can bind to guide RNA, which identifies a site in the target DNA to which the polypeptide is directed.

[0186] DNA endonucleases are generally known to contain multiple nucleic acid cleavage (i.e., nuclease) domains. In fact, the naturally occurring wild-type Cas9 enzyme is known to contain two nuclease domains, namely an HNH nuclease domain and a RuvC domain. In this specification, "Cas9" refers to both naturally occurring Cas9 and recombinant Cas9. The Cas9 enzyme contemplated herein contains an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. The HNH or HNH-like domain contains an McrA-like fold. The HNH or HNH-like domain contains two antiparallel β-chains and an α-helix. The HNH or HNH-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The HNH or HNH-like domain can cleave one strand of a target nucleic acid (e.g., the complementary strand of a crRNA targeting strand). The RuvC or RuvC-like domain contains an RNaseH or RNaseH-like fold. The RuvC / RNaseH domain is involved in a diverse set of nucleic acid-based functions, including acting on both RNA and DNA. The RNaseH domain contains five β-chains surrounded by multiple α-helices. The RuvC / RNaseH or RuvC / RNaseH-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The RuvC / RNaseH or RuvC / RNaseH-like domain can cleave one strand of the target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0187] Generally, DNA endonucleases are known to be able to introduce double-strand breaks (or single-strand breaks) into nucleic acids, such as genomic DNA. Double-strand breaks can stimulate the cell's endogenous DNA repair pathways (e.g., homology-dependent repair (HDR), non-homologous end joining (NHEJ), alternative non-homologous end joining (A-NHEJ), or microhomologous-mediated end joining (MMEJ)). NHEJ can repair the cleaved target nucleic acid without requiring a homologous template. This can occasionally result in small deletions or insertions (indels) in the target nucleic acid at the cleavage site, potentially leading to disruption or alteration of gene expression.

[0188] In some embodiments of the present invention, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity with an exemplary wild-type site-specific polypeptide (e.g., Cas9 from Streptococcus pyogenes, S. pyogenes, SEQ ID NO. 8 of U.S. Patent Application Publication No. 2014 / 0068797, or Sapranauskas et al., Nucleic Acids Res, 39(21):9275-9282(2011)).

[0189] In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity with the nuclease domain of an exemplary wild-type site-specific polypeptide (e.g., Cas9 derived from the above-mentioned Streptococcus pyogenes).

[0190] In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) protein / enzyme, includes a nucleotide sequence encoding an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type site-specific polypeptide (e.g., Cas9 derived from Streptococcus pyogenes as described above) over 10 consecutive amino acids. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, includes a nucleotide sequence encoding an amino acid sequence that has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with the wild-type site-specific polypeptide (e.g., Cas9 derived from Streptococcus pyogenes as described above) across 10 consecutive amino acids in the HNH nuclease domain of the encoded site-specific polypeptide. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, includes a nucleotide sequence that encodes at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to the wild-type site-specific polypeptide (e.g., Cas9 derived from the above-mentioned Streptococcus pyogenes) across 10 consecutive amino acids in the RuvC nuclease domain of the encoded site-specific polypeptide.In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, includes a nucleotide sequence that encodes an amino acid sequence with up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to the wild-type site-specific polypeptide (e.g., Cas9 derived from Streptococcus pyogenes as described above) across 10 consecutive amino acids in the RuvC nuclease domain of the site-specific polypeptide being encoded.

[0191] As already stated above, in the present invention, the DNA endonuclease, preferably Cas (more preferably Cas9) protein / enzyme, is "modified to lack its nuclease activity." This means that the DNA endonuclease, preferably Cas (more preferably Cas9) protein / enzyme, encodes a site-specific polypeptide including a modified form of a wild-type exemplary site-specific polypeptide. The modified form of the wild-type exemplary site-specific polypeptide includes mutations that reduce the nucleic acid cleavage activity of the site-specific polypeptide. In some embodiments, the modified form of the wild-type exemplary site-specific polypeptide has a nucleic acid cleavage activity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type exemplary site-specific polypeptide (e.g., Cas9 derived from Streptococcus pyogenes as described above). In the most preferred embodiment, the DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, in its modified form of site-specific polypeptide, may have substantially no nucleic acid cleavage activity. When the site-specific polypeptide is in a modified form that has substantially no nucleic acid cleavage activity, it is referred to herein as “enzymatically inactive.” The corresponding Cas enzyme is the most preferred embodiment of the Cas protein “modified to lack its nuclease activity” according to the present invention.

[0192] In some embodiments, Cas proteins "modified to lack its nuclease activity" are mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Streptococcus pyogenes-derived Cas9 (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). Other examples of mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. Further examples include mutations in two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or HNH domains) to create mutant Cas9 that substantially lacks all DMA cleavage activity. In some embodiments, the D10A mutation is combined with one or more H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered substantially lacking all DNA cleavage activity if the DNA cleavage activity of the mutant enzyme is about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less than that of its non-mutant form. If the enzyme is not derived from Streptococcus pyogenes (SpCas9), mutations can be made in any or all residues corresponding to positions 10, 762, 840, 854, 863, and / or 986 of SpCas9 (this can be confirmed, for example, by a standard sequence comparison tool). In particular, any or all of the D10A, E762A, H840A, N854A, N863A and / or D986A mutations are preferred in SpCas9, and conserved substitutions for any of the substituted amino acids are also considered. These (or conserved substitutions of these mutations) at their corresponding positions in other Cas9s are also preferred. D10 and H840 in SpCas9 are particularly preferred. However, residues in other Cas9s corresponding to SpCas9 D10 and H840 are also preferred.

[0193] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-specific polypeptide described above, is "modified to lack its nuclease activity," and comprises an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., Streptococcus pyogenes), a nucleic acid-binding domain, and two nucleic acid-cleaving domains (i.e., an HNH domain and a RuvC domain).

[0194] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-specific polypeptide described above, is "modified to lack its nuclease activity," and comprises an amino acid sequence having at least 15% amino acid identity with Cas9 derived from bacteria (e.g., Streptococcus pyogenes) and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

[0195] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-specific polypeptide described above, is "modified to lack its nuclease activity," and comprises an amino acid sequence having at least 15% amino acid identity to bacterial (e.g., Streptococcus pyogenes) Cas9, and two nucleic acid cleavage domains, one or both of which have at least 50% amino acid identity to a nuclease domain derived from bacterial (e.g., Streptococcus pyogenes) Cas9.

[0196] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, that is, a site-specific polypeptide "modified to lack its nuclease activity," comprises an amino acid sequence having at least 15% amino acid identity with Cas9 derived from a bacterium (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), and a non-natural sequence (e.g., a nuclear localization signal), or a linker that anneals the site-specific polypeptide to the non-natural sequence.

[0197] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, "modified to lack its nuclease activity," i.e., the site-specific polypeptide comprises an amino acid sequence having at least 15% amino acid identity to Cas9 derived from a bacterium (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein the site-specific polypeptide comprises a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

[0198] In some embodiments, a Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-specific polypeptide described above, is "modified to lack its nuclease activity," and comprises an amino acid sequence having at least 15% amino acid identity with Cas9 derived from a bacterium (e.g., Streptococcus pyogenes), and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein one of the nuclease domains contains a mutation at aspartic acid 10, and / or the other of the nuclease domains contains a mutation at histidine 840, the mutation reducing the cleavage activity of the nuclease domain by at least 50%.

[0199] In some embodiments, the Cas protein, preferably a DNA endonuclease, preferably a Cas (more preferably a Cas9) protein / enzyme, i.e., the site-specific polypeptide (Cas9 protein) described above, which is "modified to lack its nuclease activity," is derived from S. lugdunensis (SluCas9). In some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, a Cas9 protein suitable for use in this disclosure is one of those disclosed in International Publication No. 2019 / 183150 and International Publication No. 2019 / 118935, each of which is incorporated herein by reference.

[0200] Components of the type II CRISPR / Cas system are derived from type IIA, type IIB, or type IIC systems. This includes Cas9 and its orthologs. Non-exclusive exemplary species from which Cas9 nuclease or other components are derived include: Streptococcus pyogenes, Streptococcus rugdunensis, Streptococcus thermophilus, Streptococcus species, Staphylococcus aureus, Listeria inocua, Lactobacillus gasseruli, Francisella novicida, Worinella succinogenes, Sutterella wadsworthensis, Gammaproteobacteria, Neisseria meningitidis, and Campylobacter jezi. Uni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinea espiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocardarius, Bacillus pseudomycoides, Bacillus serenichireducensis, Eciobacterium sibili Cam, Lactobacillus delbruekii, Lactobacillus salivarius, Lactobacillus bucneri, Treponema denticola, Microsilla marina, Burkholderalles, Polaromonas naphthalenivorans, Polaromonas, Crocospera watsonii, Cyanothece, Microcystis aeroginosa, Synechococcus, Acethalobium alabaticum, Ammonifex degensii, Caldicerulosylpctor bexii, Candidatus desulfordis, Crocospera Stridium botulinum, Clostridium difficile, Finegordia magna, Natranerobius thermophilus, Perotomaculum thermopropionicum, Acidithiobacillus cardus, Acidithiobacillus ferrooxydance, Allochromatium vinosum, Malinobacter, Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanchthys, Ctedonobacter racemifer, Metanohalobium evestigatum,Examples include Anabaena variabilis, Nodularia spumigena, Nostoc spumigena, Althrospira maxima, Althrospira pratensis, the genera Althrospira, Limbia, Microcoleus ctonoplastes, Oscillatoria, Petrotoga mobili, Thermosipho africanus, Streptococcus pasthurianus, Neisseria cinerea, Campylobacter lari, Parvivacalm labmentivorance, Corynebacterium diphtheria, or Acariochloris marina. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is derived from S. lugdunensis (SluCas9). In some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, a Cas9 protein suitable for use in this disclosure is one of those disclosed in International Publication No. 2019 / 183150 and International Publication No. 2019 / 118935, each incorporated herein by reference.

[0201] Guide RNA The guide RNA (or "gRNA") comprises at least a spacer sequence and a CRISPR repeat sequence that hybridize to a target nucleic acid sequence of interest. In the type II system, the gRNA also comprises a tracrRNA sequence. In the type II guide RNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a double helix. In the type V guide RNA, the crRNA forms a double helix. In either system, the double helix binds to the site-specific polypeptide so that the guide RNA and site-specific polypeptide form a complex. The guide RNA provides target specificity to the complex by association with the site-specific polypeptide. Thus, the guide RNA directs the activity of the site-specific polypeptide, i.e., a Cas protein "modified to lack its nuclease activity" in this invention, preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme as described above herein.

[0202] In some embodiments, the guide RNA is double-stranded. The first strand includes an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand includes a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0203] In some embodiments, the guide RNA is a single-stranded guide. In a type II system, the single-molecule guide RNA includes an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-stranded guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence, oriented from 5' to 3'. The optional tracrRNA extension may include elements that contribute to further functionality (e.g., stability) of the guide RNA. The single-stranded guide linker ligates the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension includes one or more hairpins.

[0204] For example, guide RNAs used in CRISPR / Cas systems, or other smaller RNAs, can be readily synthesized by chemical means, as described in this art. While chemical synthesis procedures are constantly evolving, the purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond about 100 nucleotides. One approach used to produce longer RNAs is to create two or more molecules that are ligated together. Much longer RNAs, such as those encoding Cas9 endonucleases, are more readily produced enzymatically. Various types of RNA modifications, such as those described in this art, that enhance stability, reduce the likelihood or degree of innate immune responses, and / or enhance other attributes, can be introduced during or after the chemical synthesis and / or enzymatic production of RNA.

[0205] Generally, the guide sequence is any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target polynucleotide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher, when optimally aligned using a suitable alignment algorithm. The optimal alignment can be determined using any suitable algorithm for aligning sequences, and non-restrictive examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wlieeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies; available at wwnv.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence has a nucleotide length of approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more. In some embodiments, the guide sequence has a nucleotide length of approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or less. The ability of the guide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay.For example, sufficient components of a CRISPR system to form a CRISPR complex containing the guide sequence to be tested can be provided to host cells having the corresponding target sequence, such as by transfection with a vector encoding the components of the CRISPR sequence, and then the preferential cleavage within the target sequence can be evaluated by a Surveyor assay known in the art. Similarly, cleavage of a target polynucleotide sequence can be evaluated in vitro by providing components of a CRISPR complex containing the target sequence, the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the test guide sequence reaction and the control guide sequence reaction. Other assays are also possible and will be recalled by those skilled in the art.

[0206] Therefore, those skilled in the art are in a position to easily design a CRISPR / Cas system that is "specifically directed to NPBWR1" by designing the corresponding guide RNA using methods known in the art. Furthermore, those skilled in the art are in a position to easily test whether the guide RNA designed for each CRISPR / Cas system is "specifically directed to NPBWR1" in that it binds to a specific position in the NPBWR1 target.

[0207] In a preferred embodiment, although not constrained by theory, the guide RNA "specifically directed to NPBWR1" may have, for example, one of the sequences shown in any one of SEQ ID NOs: 3-5.

[0208] The above components of the CRISPR / Cas system (which may form part of the pharmaceutical composition according to the present invention) can be expressed and delivered by means and methods known in the art, as briefly described below.

[0209] Accordingly, this disclosure provides nucleic acids comprising nucleotide sequences encoding one or more guide RNAs and one or more DNA endonucleases.

[0210] In some embodiments, the nucleic acids encoding one or more guide RNAs and DNA endonucleases constitute a vector (e.g., a recombinant expression vector). The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, to which additional nucleic acid segments can be ligated to a viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome.

[0211] In some embodiments, vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred herein as “recombinant expression vectors” or more simply “expression vectors” and perform equivalent functions.

[0212] The term "operably linked" means that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that enables the expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells, and those that direct the expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector may depend on factors such as the selection of target cells and the desired expression level.

[0213] The intended expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., mouse leukemia virus, splenic necrosis virus), as well as vectors derived from retroviruses such as Rous sarcoma virus, Harvey's sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus, and other recombinant vectors. Other vectors intended for eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors may be used as long as they are compatible with the host cells.

[0214] In some embodiments, the vector includes one or more transcriptional and / or translational regulatory elements. Depending on the host / vector system used, any of several suitable transcriptional and translational regulatory elements, including constitutive and inducible promoters, transcriptional enhancer elements, and transcriptional terminators, may be used in the expression vector. In some embodiments, the vector is a self-inactivating vector that inactivates a viral sequence or components of the CRISPR mechanism or other elements.

[0215] A non-limiting example of a suitable eukaryotic promoter (i.e., a promoter that functions in eukaryotic cells) is a hybrid construct containing a cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviral long-terminal repeat sequences (LTRs), human elongation factor-1 promoter (EF1), a cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), a mouse stem cell virus promoter (MSCV), a phosphoglycerate kinase-1 locus promoter (PGK), and a promoter derived from mouse metallothionein-I.

[0216] To express small RNAs, including guide RNAs used in conjunction with Cas endonucleases, various promoters may be advantageous, such as RNA polymerase III promoters containing U6 and H1. Descriptions and parameters for enhancing the use of such promoters are publicly known in the art, and further information and approaches are regularly described; see, for example, Ma, H. et al., Molecular Therapy-Nucleic Acids 3, e161 (2014) doi:10.1038 / mtna.2014.12.

[0217] Expression vectors may also contain ribosome-binding sites and transcription terminators for translation initiation. Expression vectors may also contain appropriate sequences for amplifying expression. Expression vectors may also contain nucleotide sequences encoding non-natural tags (e.g., histidine tags, hemagglutinin tags, green fluorescent protein, etc.) that result in a fusion protein when fused to site-specific polypeptides.

[0218] In some embodiments, the promoter is an inductive promoter (e.g., a heat shock promoter, a tetracycline-regulating promoter, a steroid-regulating promoter, a metal-regulating promoter, an estrogen receptor-regulating promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some embodiments, the promoter is a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.).

[0219] In some embodiments, nucleic acids encoding one or more guide RNAs and / or DNA endonucleases are packaged in or on a delivery vehicle for delivery to cells. Intended delivery vehicles include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. Various targeting moieties can be used to enhance the preferential interaction between such a vehicle and the desired cell type or location.

[0220] The introduction of the complexes, polypeptides, and nucleic acids of this disclosure into cells may occur by means of viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0221] delivery Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) can be delivered by viral or nonviral delivery vehicles known in the art. Alternatively, endonuclease polypeptides can be delivered by nonviral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In further alternative embodiments, DNA endonucleases can be delivered alone or as one or more polypeptides precomplexed with one or more guide RNAs.

[0222] Polynucleotides can be delivered by nonviral delivery vehicles, including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. Several exemplary nonviral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18:1127-1133 (2011) (focusing on nonviral delivery vehicles for siRNA, which are also useful for the delivery of other polynucleotides).

[0223] Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, can be delivered to cells or patients by lipid nanoparticles (LNPs).

[0224] LNPs refer to any particles with a diameter of 1000nm, 500nm, 250nm, 200nm, 150nm, 100nm, 75nm, 50nm, or less than 25nm. Alternatively, nanoparticles may range in size from 1 to 1000nm, 1 to 500nm, 1 to 250nm, 25 to 200nm, 25 to 100nm, 35 to 75nm, or 25 to 60nm.

[0225] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusionogenic phospholipid DOPE or membrane component cholesterol, may be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include insufficient stability and rapid clearance, as well as low potency due to the occurrence of inflammatory or anti-inflammatory reactions.

[0226] LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0227] LNPs can be produced using any lipid or combination of lipids known in the art. Examples of lipids used to produce LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0228] Lipids can be combined in any number of molar ratios to produce LNPs. Furthermore, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0229] As mentioned above, DNA endonucleases and guide RNAs may be administered separately to cells or patients. Alternatively, DNA endonucleases may be pre-complexed with one or more guide RNAs. The pre-complexed material can then be administered to cells or patients. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). RNA can form specific interactions with other RNA or DNA. While this property is utilized in many biological processes, it also carries the risk of chaotic interactions in nucleic acid-rich cellular environments. One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which RNA is pre-complexed with endonucleases. Another advantage of RNPs is that they protect RNA from degradation.

[0230] The DNA endonuclease in RNPs may or may not be modified. Similarly, the gRNA may or may not be modified. Numerous modifications are known in the art and can be used.

[0231] DNA endonucleases and gRNAs can generally be combined in a 1:1 molar ratio. However, a wide range of molar ratios may be used to produce RNPs.

[0232] In some embodiments, AAV vectors are used for delivery. Exemplary AAV serotypes include, but are not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. See also Table 1.

[0233] [Table 1]

[0234] Methods for producing packaging cells involve creating cell lines that stably express all the components necessary for AAV particle production. For example, plasmids (or multiple plasmids) containing an rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and selection markers such as neomycin resistance genes are incorporated into the cell genome. The AAV genome has previously been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0235] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various approaches have been proposed by Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. al., J. Virol., 62:1963 (1988) and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988), Samulski et al. al. (1989, J. Virol., 63:3822-3828), U.S. Patent No. 5,173,414, International Publication No. 95 / 13365 and corresponding U.S. Patent No. 5,658,776, International Publication No. 95 / 13392, International Publication No. 96 / 17947, PCT / US98 / 18600, International Publication No. 97 / 09441 (PCT / US96 / 14423), International Publication No. 97 / 08298 (PCT / US96 / 13872), International Publication No. 97 / 21825 (PCT / US96 / 20777), International Publication No. 97 / 06243 (PCT / FR96 / 01064), International Publication No. 99 / 11764, Perrin et al. (1995) Vaccine It is described in 13:1244-1250, Paul et al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595.

[0236] In addition to adeno-associated virus vectors, other viral vectors may be used in the implementation of the present invention. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpesviruses, Epstein-Barr viruses, papovaviruses, poxviruses, vaccinia viruses, and herpes simplex viruses.

[0237] Options are available to deliver the Cas9 nuclease as a DNA plasmid, mRNA, or protein. Guide RNA can be expressed from the same DNA or delivered as RNA. RNA can be chemically modified to alter or improve its half-life, or to reduce the likelihood or degree of the immune response. Endonuclease proteins can be complexed with gRNA before delivery. Viral vectors allow for efficient delivery, and split versions of Cas9 and smaller orthologues of Cas9 can be packaged in AAVs, as well as HDR donors. Various nonviral delivery methods exist that can deliver each of these components, or nonviral and viral methods can be used in parallel. For example, proteins and guide RNA can be delivered using nanoparticles, while donor DNA can be delivered using AAVs.

[0238] As outlined above, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of NPBWR1 for use in the treatment, improvement and / or prevention of specific diseases. Accordingly, the present invention relates to the use of a pharmaceutical composition comprising an antagonist / inhibitor of NPBWR1 in a medical setting.

[0239] This is particularly true for the CRISPR / Cas system. Accordingly, in this regard, the guide RNA of the present invention can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the specific mode of administration and dosage form. The guide RNA composition is generally formulated to achieve a physiologically suitable pH, ranging from about pH 3 to about pH 11, or about pH 3 to about pH 7, depending on the formulation and route of administration. In alternative embodiments, the pH is adjusted to a range of about pH 5.0 to about pH 8. In some embodiments, the composition comprises a therapeutically effective amount of at least one compound described herein, together with one or more pharmaceutically acceptable excipients. Optionally, the composition may comprise a combination of the compounds described herein, or a second active ingredient useful for the treatment or prevention of bacterial growth (e.g., antimicrobial or antimicrobial agents), or a combination of the reagents of the present invention.

[0240] Suitable excipients include, for example, carrier molecules containing large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (e.g., but not limited to ascorbic acid), chelating agents (e.g., but not limited to EDTA), carbohydrates (e.g., but not limited to dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., but not limited to oil, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, and pH buffers.

[0241] "Administration" means delivering the composition described herein (or a vector comprising a guide ribonucleic acid (gRNA) and one or more DNA endonucleases) to a subject by a method or route that localizes the composition at least partially to a desired site. The composition can be administered by any suitable route that results in an effective treatment in the subject, i.e., administration results in the delivery of at least a portion of the composition to a desired site in the subject to be delivered, and that the composition is delivered to the desired site for a period of time. Modes of administration include injection, infusion, drip administration, or ingestion. "Injection" includes, but is not limited to, injection and infusion into veins, muscles, arteries, spinal cavities, ventricles, capsules, orbits, hearts, skin, peritoneal cavities, trachea, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal cord, cerebrospinal cord, and sternal regions. In some embodiments, the route is intravenous. For cell delivery, administration by injection or infusion is generally preferred.

[0242] In some embodiments, the present invention provides a method that may include delivering one or more polynucleotides, e.g., one or more vectors described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom, to a host cell. In some embodiments, the present invention further provides cells produced by such a method, and animals comprising or that can be produced from such cells. In some embodiments, a CRISPR enzyme combined with (and optionally complexed with) a guide sequence is delivered to the cell. Nucleic acids can be introduced into mammalian cells or target tissues using conventional viral and nonviral-based gene transfer methods. Such methods can be used to administer nucleic acids encoding components of the CRISPR system to cells in culture or in a host organism, and nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses that have either an episome or an integrated genome after delivery to the cell.For a review of gene therapy procedures, see Anderson, Science 256:808813 (1992), Nabel & Feigner, TIBTECH 11:211-217 (1993), Mitani & Caskey, TIBTECH 11:162-166 (1993), Dillon, TIBTECH 11:167-175 (1993), Miller, Nature 357:455-460 (1992), Van Brunt, Biotechnology 6(10):1149-1154 (1988), Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995), Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995), and Haddada et al., in Current See Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995) and Yu et al., Gene Therapy 1:13-26 (1994).

[0243] Nonviral delivery methods for nucleic acids include lipofection, microinjection, bioristics, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virions, and drug-mediated enhancement of DMA uptake. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®), and cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides are described in Feigner's International Publications 91 / 17424 and 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0244] The preparation of lipid nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (e.g., Crystal, Science 270:404-410 (1995), Blaese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al. Bioconjugate Chem. 5:382-389 (1994), Remy et al. Bioconjugate Chem. 5:647-654 (1994), Gao et al. Gene Therapy. 2:710-722 (1995), Ahmad et al. Cancer See Res.52:4817-4820 (1992), U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787.

[0245] The use of RNA or DNA virus-based systems for nucleic acid delivery utilizes highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly to the patient (in vivo), or they can be used to treat cells in vitro, and the modified cells can optionally be administered to the patient (ex vivo). Conventional virus-based systems may include retroviral, lentiviral, adenovirus, adeno-associated virulence, and herpes simplex virulence vectors for gene transfer.

[0246] Integration into the host genome is possible using retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0247] The targeting of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transduction system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeat sequences with the ability to package foreign sequences up to 6–10 kb. A minimum amount of cis-acting LTRs is sufficient for vector replication and packaging, which are then used to incorporate therapeutic genes into target cells and provide permanent transgene expression. Widely used retroviral vectors include those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al. J.Virol.66:2731-2739 (1992), Johann et al. J.Virol.66:1635-1640 (1992), Sommnerfelt et al. Virol.176:58-59 (1990), Wilson et al. J.Virol.63:2374-2378 (1989), Miller et al. J.Virol.65:2220-2224 (1991), and PCT / US94 / 05700).

[0248] As described above, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of suppressing the expression of NPBWR1.

[0249] Therefore, according to the present invention, the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of repressing the expression of NPBWR1.

[0250] Therefore, the CRISPR enzyme, preferably the Cas9 protein, is part of a fusion protein that may contain one or more heterologous protein domains (e.g., the CRISPR enzyme plus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains). The CRISPR enzyme fusion protein may contain any further protein sequences and optionally linker sequences between any two of the domains.

[0251] The heterologous protein domains according to the present invention are transcriptional repressor domains or epigenetic modification domains that can suppress the expression of NPBWR1.

[0252] Examples of protein domains that can be fused to CRISPR enzymes are listed below.

[0253] As explained above, the purpose of an epigenetic modification domain fused to a Cas protein that can suppress the expression of a transcriptional repressor domain or NPBWR1 is to regulate the transcription of a target nucleic acid, namely the transcription of the NPBWR1 gene, by reducing (preferably completely inhibiting) its transcription.

[0254] As outlined, this generally involves contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and guide RNA.

[0255] The corresponding transcriptional regulation can be used for therapeutic applications related to the present invention.

[0256] In preferred embodiments, in some cases, transcriptional regulation, preferably transcriptional repression, provides selective regulation (e.g., reduction or increase (increase is assumed in the context of the agonist / activation arm of the present invention, which is further described below)) of a target nucleic acid, namely the NPBWR1 gene. For example, a “selective” reduction in the transcription of a target nucleic acid reduces the transcription of the target nucleic acid by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the transcription level of the target nucleic acid in the absence of the DNA-targeting RNA / modified Cas9 polypeptide complex. A selective reduction in the transcription of a target nucleic acid reduces the transcription of the target nucleic acid but does not substantially reduce the transcription of non-target nucleic acids, for example, the transcription of non-target nucleic acids is reduced by less than 10%, if any, compared to the transcription level of non-target nucleic acids in the absence of the DNA-targeting RNA / modified Cas9 polypeptide complex.

[0257] As will be further outlined below, the increased transcription is assumed in relation to the agonist / activation arm of the present invention, which is further described below. The “selective” increase in the transcription of target DNA can increase the transcription of target DNA by at least about 1.1 times (e.g., at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12 times, at least about 15 times, or at least about 20 times) compared to the transcription level of target DNA in the absence of the DNA-targeting RNA / modified Cas9 polypeptide complex. The selective increase in the transcription of target DNA increases the transcription of target DNA but does not substantially increase the transcription of non-target DNA. For example, the transcription of non-target DNA increases by at most about 5 times (e.g., less than 4 times, less than 3 times, less than 2 times, less than 1.8 times, less than 1.6 times, less than 1.4 times, less than 1.2 times, or less than 1.1 times) compared to the transcription level of non-target DNA in the absence of the DNA-targeting RNA / modified Cas9 polypeptide complex.

[0258] As a non-limiting example, the increase can be achieved by fusing dCas9 (dead Cas9, i.e., a Cas protein "modified to lack its nuclease activity" as per the present invention) to a heterologous sequence. Suitable fusion partners include, but are not limited to, polypeptides that provide activity to indirectly increase transcription by directly acting on target DNA or polypeptides associated with target DNA (e.g., histones or other DNA-binding proteins). Suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

[0259] Further suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or their fragments, proteins or their fragments that recruit transcription activators, small molecule / drug-responsive transcription regulators, etc.).

[0260] Non-limiting examples of the present invention's methods for using dCas9 fusion proteins to increase transcription in eukaryotes include the fusion of dCas9 to an activation domain (AD) (e.g., GAL4, herpesvirus activating proteins VP16 or VP64, human nuclear factor NF-κB p65 subunit, etc.). To make the system inducible, the expression of the dCas9 fusion protein can be controlled by an inducible promoter (e.g., Tet-ON, Tet-OFF, etc.). DNA-targeted RNAs can be designed to target known transcription response elements (e.g., promoters, enhancers, etc.), known upstream activation sequences (UAS), or sequences of unknown or known function that are thought to be able to control the expression of the target DNA.

[0261] Further Fusion Partners Non-limiting examples of fusion partners for achieving transcriptional increase or decrease include transcription activator domains and transcriptional repressor domains (e.g., Kruppel-associated box (KRAB or SKD), Mad mSIN3 interaction domain (SID), ERF repressor domain (ERD), etc.). In some such cases, the dCas9 fusion protein targets a specific location (i.e., sequence) in target DNA by DNA-targeting RNA, exerting locus-specific regulation such as blocking RNA polymerase binding to the promoter (selectively inhibiting the function of the transcription activator) and / or modifying the local chromatin state (e.g., when a fusion sequence is used that modifies the target DNA or a polypeptide associated with the target DNA). In some cases, the change is transient (e.g., transcriptional repression or activation). In some cases, the change is inheritable (e.g., when epigenetic modification occurs on the target DNA or a protein associated with the target DNA, e.g., nucleosome histone).

[0262] In some embodiments, heterologous sequences can be fused to the C-terminus of the dCas9 polypeptide. In some embodiments, heterologous sequences can be fused to the N-terminus of the dCas9 polypeptide. In some embodiments, heterologous sequences can be fused to an internal portion of the dCas9 polypeptide (i.e., a portion other than the N-terminus or C-terminus).

[0263] The biological effects of methods using the target dCas9 fusion protein can be detected by any convenient method (e.g., gene expression assays, chromatin-based assays, e.g., chromatin immunoprecipitation (ChiP), chromatin in vivo assay (CiA), etc.).

[0264] In preferred embodiments, where the effector domain of the fusion protein is a transcriptional activation domain or a transcriptional repressor domain, the guide RNA directs the fusion protein to a specific chromosomal sequence, and the transcriptional activation domain or transcriptional repressor domain activates or represses the expression of the target chromosomal sequence, respectively.

[0265] In an alternative preferred embodiment, where the effector domain of the fusion protein is an epigenetic modification domain, the guide RNA directs the fusion protein to a specific chromosomal sequence, and the epigenetic modification domain modifies the structure of the target chromosomal sequence. Examples of epigenetic modifications include acetylation, histone protein methylation, and / or nucleotide methylation. In some examples, structural modifications of the chromosomal sequence result in changes in the expression of the chromosomal sequence.

[0266] In a preferred embodiment, the transcriptional activation domain is, for example, VP64. In another embodiment, the transcriptional repressor domain is, for example, a KRAB domain, an SID domain, or an SID4X domain.

[0267] Furthermore, in accordance with the theoretical basis of the present invention and the experimental evidence of the accompanying examples, and as described below, an increase in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have medical significance in certain disorders or diseases.

[0268] Accordingly, the present invention relates not only to the above-mentioned pharmaceutical compositions comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, but also, in different aspects of the present invention, to pharmaceutical compositions comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorders, preferably anorexia nervosa or bulimia nervosa.

[0269] Accordingly, in further embodiments, the present invention also relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorders, preferably anorexia or bulimia.

[0270] In preferred embodiments, in connection with a second aspect of the present invention, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa, The above-mentioned agonists / activators for the neuropeptide B / W receptor (NPBWR1) are not agonists selected from the group consisting of polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0271] In another preferred embodiment, the agonist / activator of the neuropeptide B / W receptor (NPBWR1) is neither a agonist nor composed of an agonist selected from the group consisting of polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0272] In another preferred embodiment, the agonist / activator for the neuropeptide B / W receptor (NPBWR1) is an agonist / activator as defined above and below, provided that agonists / activators selected from the group consisting of polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)) are excluded.

[0273] In another preferred embodiment, in connection with a second aspect of the present invention, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa, The above-mentioned agonists / activators of the neuropeptide B / W receptor (NPBWR1) are not agents that modulate the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0274] In another preferred embodiment, the agonist / activator of the neuropeptide B / W receptor (NPBWR1) described above does not include, or consist of, an agent that modulates the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)).

[0275] In another preferred embodiment, the agonist / activator of the neuropeptide B / W receptor (NPBWR1) is an agonist / activator as defined above and below, provided that agonists / activators that modulate the binding properties between the GPR7 / NPBWR1 polypeptide and polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)) or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)) are excluded.

[0276] Polypeptide ligands corresponding to L7 (amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO: 8)), L7C (having amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO: 9)), L8 (amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO: 10)), or L8C (having amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO: 11)) are disclosed as SEQ ID NOs. 1, 3, 5, and 7 in International Publication No. 03 / 082907 and are described as agonists of GRP7 / NPBWR1.

[0277] Bipolar affective disorder in the manic phase (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, are known disorders or syndromes in the art and are medical indications classified in the ICD system, i.e., the well-established list of medical classifications by the World Health Organization (WHO). ICD stands for the International Statistical Classification of Diseases and Related Health Problems. Hereafter, when referring to more specifically defined disorders or diseases, the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), i.e., "ICD 10," will be referred to.

[0278] The present invention is not limited to manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa. Any of these conditions, whether manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa, can be treated, improved, and / or prevented by the neuropeptide B / W receptor (NPBWR1) agonist / activator according to the present invention.

[0279] While not bound by theory, in certain preferred embodiments, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, wherein the manic bipolar affective disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, is as follows:

[0280] In a preferred embodiment, the present invention relates to bipolar affective disorder (ICD-10 F31) during the manic phase. The term “bipolar affective disorder (ICD-10 F31)” has already been described above. Therefore, the same applies with respect to the definition of this disorder or disease as set forth above, with necessary modifications, except that the agonist / activator according to the present invention is used during the manic phase of the above disorder.

[0281] The terms “agonist” and “activator” are used interchangeably herein. These terms are known in the art and relate to compounds / substances that can completely or partially activate or enhance the physiological activity of (one or more) specific proteins. Accordingly, in the context of the present invention, the agonist / activator may activate or enhance the physiological activity of a protein, such as a neuropeptide B / W receptor (NPBWR1), when the compound / substance binds to the protein. The binding of an “agonist / activator” to a given protein, for example, a neuropeptide B / W receptor (NPBWR1), may enhance the binding of an endogenous activating molecule to the protein. Accordingly, as used herein, the term “agonist” also includes an enhancing agonist.

[0282] However, in addition to the above, a “agonist” or “activator” of the neuropeptide B / W receptor (NPBWR1) in the context of the present invention can also activate or enhance the function of a given protein, for example, a nucleic acid molecule encoding the neuropeptide B / W receptor (NPBWR1), by activating or enhancing the expression of the said protein. Thus, an agonist / activator of the neuropeptide B / W receptor (NPBWR1) may result in an increase in the expression level of the neuropeptide B / W receptor (NPBWR1) (e.g., an increase in the levels of neuropeptide B / W receptor (NPBWR1) mRNA and neuropeptide B / W receptor (NPBWR1) protein), which is reflected in an increase in the activity of the neuropeptide B / W receptor (NPBWR1). This increased activity is known in the art and can be measured / detected by the methods described herein. Thus, an activator of the neuropeptide B / W receptor (NPBWR1) in the context of the present invention may also include a transcriptional activator of the expression of the neuropeptide B / W receptor (NPBWR1) that can increase the function of the neuropeptide B / W receptor (NPBWR1). As described in detail below herein, increasing the expression and / or activity of the neuropeptide B / W receptor (NPBWR1) with an agonist / activator of the neuropeptide B / W receptor (NPBWR1) results in an increase in the activity (and / or expression) of the neuropeptide B / W receptor (NPBWR1), thereby increasing the functional capacity of the neuropeptide B / W receptor (NPBWR1).

[0283] In accordance with the theoretical basis of this invention, an increase in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have various medical significances.

[0284] As described below, increased activity of the functional neuropeptide B / W receptor (NPBWR1) has medical significance and is expected to affect disorders / disorders associated with manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0285] The terms “activator” and / or “agonist” do not imply a specific mechanism of biological action, but are considered to expressly include and encompass all possible pharmacological, physiological, and biochemical interactions with the neuropeptide B / W receptor (NPBWR1) (signaling), whether direct or indirect. For the purposes of this disclosure, the terms “activator” and / or “agonist” shall be clearly understood to encompass all previously identified terms, names, and functional states and characteristics, to the extent that the neuropeptide B / W receptor (NPBWR1) itself, the biological activity of the neuropeptide B / W receptor (NPBWR1) (including, but not limited to, the ability to modulate / inhibit Bdnf expression, the ability to bind to neuropeptide B / W, and / or the ability to bind to G proteins), or the results of such biological activity are substantially increased or enhanced to some degree that has some significance, for example, by at least 5%, 10%, 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, 500%, or 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or 1000x. Activators / agonists can increase the abnormal level of biological activity of the neuropeptide B / W receptor (NPBWR1), which can cause adverse effects and / or disease in a subject, to a level corresponding to that in a healthy subject, thereby preventing adverse effects and / or disease, halting its progression, and / or curing it.

[0286] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the activator increases the biological activity of the neuropeptide B / W receptor (NPBWR1) by at least twofold, threefold, fourfold, fivefold, tenfold, twentyfold, fiftyfold, 100fold or 1000fold.

[0287] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during the manic phase, appetite disorders, preferably anorexia or bulimia, which increases the abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) to an abnormal level that causes and / or promotes adverse effects and / or disease, by at least two-fold, three-fold, four-fold, five-fold, ten-fold, twenty-fold, fifty-fold, 100-fold or 1000-fold.

[0288] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during the manic phase, appetite disorders, preferably anorexia nervosa or bulimia nervosa, the activator / agonist that reduces at least threefold an abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse effects and / or disease.

[0289] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, which increases abnormal levels of the biological activity of the neuropeptide B / W receptor (NPBWR1) that cause and / or promote manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0290] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the inhibitor increases in a subject an abnormal level of biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, to a level comparable to that of a healthy subject.

[0291] According to the present invention, the term “activator / agonist of neuropeptide B / W receptor (NPBWR1)” also means a compound or substance that can increase or enhance the physiological activity of neuropeptide B / W receptor (NPBWR1). Therefore, in connection with the present invention, the activator can, for example, activate or enhance the physiological activity of neuropeptide B / W receptor (NPBWR1) when the compound / substance (i.e., activator / agonist) binds to the neuropeptide B / W receptor (NPBWR1).

[0292] "Activators / agonists of the neuropeptide B / W receptor (NPBWR1)" can also increase or activate the function of the neuropeptide B / W receptor (NPBWR1) by increasing, activating, or enhancing the expression of the nucleic acid molecule encoding the neuropeptide B / W receptor (NPBWR1). Therefore, activators / agonists of the neuropeptide B / W receptor (NPBWR1) can result in increased expression levels of the neuropeptide B / W receptor (NPBWR1) gene product, such as increased levels of neuropeptide B / W receptor (NPBWR1) mRNA and / or neuropeptide B / W receptor (NPBWR1) protein.

[0293] Activators / agonists of the neuropeptide B / W receptor (NPBWR1) can increase or enhance the abnormal expression levels of the neuropeptide B / W receptor (NPBWR1) that may cause and / or promote adverse effects and / or diseases associated with bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, during manic episodes, as outlined above and below herein, to levels corresponding to those in healthy subjects, thereby preventing, halting the progression of, or curing adverse effects and / or diseases in subjects. This is reflected in increased expression of the neuropeptide B / W receptor (NPBWR1) and / or increased expression of abnormal neuropeptide B / W receptor (NPBWR1), thereby restoring the expression of the neuropeptide B / W receptor (NPBWR1) to healthy levels. The expression level of the neuropeptide B / W receptor (NPBWR1) may correlate to some extent with the activity of the neuropeptide B / W receptor (NPBWR1) until saturation of the translation mechanism and / or the substance binding to the neuropeptide B / W receptor (NPBWR1) is achieved. The expression level of the neuropeptide B / W receptor (NPBWR1) can be measured / detected by methods known in the art.

[0294] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of disorders associated with manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa, wherein the activator increases the expression level of the neuropeptide B / W receptor (NPBWR1) gene product.

[0295] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of disorders associated with manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia nervosa or bulimia nervosa, which increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes adverse effects and / or disease / disorder.

[0296] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of a disorder associated with manic bipolar affective disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, which increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes the above-mentioned bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorder, preferably anorexia or bulimia.

[0297] Accordingly, in one aspect, the present invention relates to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of a disorder associated with manic bipolar affective disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, according to the present invention, which increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes manic bipolar affective disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, in a subject to an expression level comparable to that of a healthy subject.

[0298] Activators / agonists of the neuropeptide B / W receptor (NPBWR1) may exert their activating function by directly interacting with any part of the neuropeptide B / W receptor (NPBWR1) protein, i.e., the extracellular domain, transmembrane domain, and / or cytoplasmic domain of the neuropeptide B / W receptor (NPBWR1). Activators / agonists of the neuropeptide B / W receptor (NPBWR1) may also exert any inhibitory effect on the function of the neuropeptide B / W receptor (NPBWR1) by activating, enhancing, or increasing any upstream or downstream pathway components that essentially contribute to the function of the neuropeptide B / W receptor (NPBWR1). Activators / agonists of the neuropeptide B / W receptor (NPBWR1) may also exert any indirect activating effect on any neuropeptide B / W receptor (NPBWR1) activating molecule, such as nucleic acids, ribonucleic acid (RNA), double-stranded ribonucleic acid (dsRNA), chromatin leader proteins, and / or ligands.

[0299] The efficacy of neuropeptide B / W receptor (NPBWR1) activators / agonists can be described using the IC50 (International Coefficient of Use) value. In the sense of the present invention, neuropeptide B / W receptor (NPBWR1) activators / agonists preferably embody a high IC50 value. The IC50 value of a neuropeptide B / W receptor (NPBWR1) activator / agonist may be greater than 100 μM, greater than 90 μM, greater than 80 μM, greater than 70 μM, greater than 60 μM, greater than 50 μM, greater than 40 μM, greater than 30 μM, greater than 20 μM, or greater than 10 μM, with higher values ​​being preferable to lower values.

[0300] Accordingly, the present invention relates to an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, according to the present invention, wherein the efficacy of the median inhibitory concentration (IC50) is greater than 100 μM, greater than 90 μM, greater than 80 μM, greater than 70 μM, greater than 60 μM, greater than 50 μM, greater than 4 μM, or greater than 10 μM, greater than 9 μM, greater than 8 μM, greater than 7 μM, greater than 6 μM, greater than 5 μM, or greater than 4 μM, preferably greater than 4 μM.

[0301] Those skilled in the art are familiar with methods for determining the IC50 values ​​of activators / agonists of the neuropeptide B / W receptor (NPBWR1). This specification assumes that activators / agonists of the neuropeptide B / W receptor (NPBWR1) may embody additional IC50 values, and / or other activators / agonists of the neuropeptide B / W receptor (NPBWR1) having other IC50 values ​​will be identified.

[0302] In preferred embodiments, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing manic bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, as defined above herein, wherein the agonist / activator is selected from an NPBWR1 activating peptide, an NPBWR1 activating small molecule binding molecule, and an NPBWR1 RNA molecule.

[0303] In a preferred embodiment, the agonist / activator is an NPBWR1 RNA molecule encoding NPBWR1. Therefore, the NPBWR1 RNA molecule encoding NPBWR1 is the sense molecule used according to the present invention, which is a nucleic acid molecule encoding the neuropeptide B / W receptor (NPBWR1). Preferably, the nucleic acid molecule is RNA, i.e., pre-mRNA or mRNA.

[0304] In a preferred embodiment, the NPBWR1 RNA molecule encoding NPBWR1 includes not only a coding region encoding the NPBWR1 polypeptide (preferably including a start codon at its 5' end), but also an UTR upstream of the coding sequence.

[0305] The ribonucleic acid (RNA) molecules used in accordance with the present invention relate to polymer molecules assembled as chains of nucleotides called G, A, U, and C. Each nucleotide in RNA contains a ribose sugar having carbons numbered from 1' to 5'. Generally, a nitrogen-containing base of adenine (A), cytosine (C), guanine (G), or uracil (U) is attached to the 1' position. In polymer RNA molecules, phosphate groups are attached to the 3' position of one ribose and the 5' position of the next ribose. Thus, the nucleotides in polymer RNA molecules are covalently bonded to each other, with a phosphate group from one nucleotide attaching to the 3' carbon of the subsequent nucleotide, thereby forming a phosphodiester bond. Thus, the RNA chain has a 5' end and a 3' end, named after the carbons on the ribose ring. By convention, upstream and downstream refer to the 5' to 3' direction in which RNA transcription occurs. Preferably, the RNA molecule is a messenger RNA (mRNA) molecule. mRNA is a large family of RNA molecules that transmit genetic information from DNA to ribosomes, where it identifies the amino acid sequence of protein products during gene expression. After transcription of the primary transcript mRNA (known as premRNA) by RNA polymerase, the processed mature mRNA is translated into a polymer of amino acids, i.e., a protein, as summarized in the central dogma of molecular biology. Like DNA, the genetic information in mRNA resides in a sequence of nucleotides, each of which is arranged in a codon consisting of three bases. Each codon codes for a specific amino acid, with the exception of the stop codon that terminates protein synthesis.

[0306] RNA molecules may also contain a UTR at their 3' end. Therefore, the RNA molecules of the present invention are structurally similar to "normal" mRNA molecules that exist in nature and have a coding region as well as (5' and 3') untranslated regions (UTRs) and optionally a poly-A tail.

[0307] The term “coding region containing a start codon at the 5' end” as used in this invention relates to a sequence consisting of codons that are decoded by ribosomes according to the information provided by the genetic code and translated into a protein. A coding region generally begins with a start codon at its 5' end and ends with a stop codon. Generally, the start codon is an AUG triplet, and the stop codon is UAA, UAG, or UGA. In addition to encoding a protein, a portion of a coding region may function as a regulatory sequence in premRNA, such as an exon splicing enhancer or exon splicing silencer. The coding region of a polypeptide or protein-coding gene used in this invention is also known as a coding sequence or CDS (from a coding DNA sequence) and is a portion of the DNA or RNA of a polypeptide or protein-coding gene, consisting of exons. The coding region in mRNA is adjacent to a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), which are also part of the exons. Furthermore, the mRNA molecule may further include a so-called 5' cap and a poly-A tail. The 5' cap, 5' UTR, 3' UTR, and poly-A tail are regions of mRNA molecules that are not translated into protein.

[0308] The terms “untranslated region” or “UTR” as used in accordance with this invention refer to portions of mRNA upstream of the start codon and downstream of the stop codon, which are not translated and are therefore called 5-prime' untranslated regions (5'UTR) and 3-prime' untranslated regions (3'UTR), respectively. These regions are transcribed together with the coding region and are therefore exons, as they are present in mature mRNA.

[0309] The RNA molecule of the present invention may also contain a poly(A) tail. A poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to the 3' end of pre-mRNA by a process called polyadenylation. This tail facilitates export from the nucleus and translation and protects mRNA from degradation. Polyadenylation is the addition of a poly(A) tail to messenger RNA. A poly(A) tail consists of multiple adenosine monophosphates, in other words, a stretch portion of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation.

[0310] The RNA molecule of the present invention containing the above-mentioned UTR can be generated / synthesized recombinantly (e.g., in vivo or in vitro) or synthetically by methods known to those skilled in the art.

[0311] In vitro transcription of RNA typically requires a linear DNA template containing a double-stranded promoter region to which DNA-dependent RNA polymerase binds and initiates RNA synthesis, with the coding region being either double-stranded or single-stranded. If the linear DNA template contains a single-stranded coding region, the antisense strand of the coding region (i.e., the strand read by the DNA-dependent polymerase) is part of the template. Common DNA-dependent RNA polymerases include T7 polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase.

[0312] Transcription templates for in vitro transcription include, for example, cDNA templates synthesized from RNA precursors, templates generated by PCR, and chemically synthesized oligonucleotides and plasmid constructs. Many widely used plasmid cloning vectors have phage polymerase promoters located on both sides of a multi-cloning site to allow transcription of either strand of the nucleotide sequence inserted into the multi-cloning site. Commonly used cloning vectors include, for example, Invitrogen's pCRII, Promega's pGEM, and Stratagene's pBluescript vector. Ambion's pTRIPLEscript family of vectors contains all three phage polymerase promoters in tandem (on the same side of the multi-cloning site), allowing the use of any of the three polymerases, SP6, T7, or T3.

[0313] The RNA molecule of the present invention can be recombinantly produced in vivo by methods known to those skilled in the art.

[0314] Alternatively, the RNA molecules of the present invention may be produced in vitro, for example, using an in vitro transcription system. In vitro transcription systems are generally known and typically require a purified linear DNA template containing a DNA sequence that "encodes" the RNA molecule, the DNA sequence being under the control of a suitable promoter. Furthermore, in vitro transcription systems also generally require a buffer system containing ribonucleoside triphosphate, DTT, and magnesium ions, as well as a suitable RNA polymerase that provides enzymatic activity for in vitro transcription of the DNA sequence into the corresponding RNA molecule of the present invention.

[0315] Furthermore, RNA molecules can be chemically synthesized, for example, by conventional chemical synthesis using a solid-phase support and standard techniques in an automated nucleotide sequence synthesizer, or by chemical synthesis of their respective DNA sequences and subsequent in vitro or in vivo transcription.

[0316] Furthermore, in a more preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in a method of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during manic episodes, appetite disorders, preferably anorexia nervosa or bulimia nervosa, wherein the agonist / activator is neuropeptide B or neuropeptide W.

[0317] Neuropeptide B is known in the art and is a short, biologically active peptide whose precursor in humans is encoded by the NBP gene. Neuropeptide B is a ligand for the neuropeptide B / W receptor (collectively referred to as neuropeptide B / W receptor (NPBWR1) in the context of this invention), which are two G protein-coupled receptors called NPBW1 and NPBW2, encoded by the genes NPBWR1 and NPBWR2, respectively, and acts through their binding. Neuropeptide B has been described in the art as being involved in the regulation of feeding, the neuroendocrine system, memory, learning, and afferent pain pathways. It is expressed at high levels throughout the CNS in the substantia nigra, hypothalamus, hippocampus, and spinal cord.

[0318] The amino acid sequence of human neuropeptide B can be searched using UniProt accession number: Q8NG41.

[0319] The amino acid sequence of human neuropeptide B is as follows: MARSATLAAAALALCLLLAPPGLAWYKPAAGHSSYSVGRAAGLLSGLRRSPYARRSQPYRGAEPPGGAGA SPELQLHPRLRSLAVCVQDVAPNLQRCERLPDGRGTYQCKANVFLSLRAADCLAA (Sequence ID 6).

[0320] Neuropeptide W is a short human neuropeptide. Neuropeptide W acts as a ligand for two neuropeptide B / W receptors, NPBWR1 and NPBWR2 (collectively referred to as neuropeptide B / W receptor (NPBWR1) in the context of this invention), which are incorporated into the GPCR family of alpha-helix transmembrane proteins.

[0321] The amino acid sequence of neuropeptide W can be searched using UniProt accession number: Q8N729.

[0322] The amino acid sequence of human neuropeptide W is as follows: MAWRPGERGAPASRPRLALLLLLLLLPLPSGAWYKHVASPRYHTVGRAAGLLMGLRRSPYLWRRALRAAAGPLARDTLSPEPAAREAPLLLPSWVQELWETRRRSSQAGIPVRAPRSPRAPEPALEPESLDFSGAGQRLR RDVSRPAVDPAANRLGLPCLAPGPF (Sequence ID 7).

[0323] However, the neuropeptides B and W used in the present invention are not particularly limited to the specific amino acid sequences described above, and may be modified versions of the amino acid sequences of the above neuropeptides B and W, as long as they can bind to and / or activate the neuropeptide B / W receptor (NPBWR1) and thus transmit its signal comparable to that of unmodified neuropeptides B and W.

[0324] Therefore, in preferred embodiments, neuropeptides B and W used in the present invention are neuropeptides comprising sequences showing 1 to 8 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 7 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 6 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 5 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 4 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 3 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The neuropeptide sequences may also comprise sequences showing 1 to 2 substitutions, deletions, or insertions compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Most preferably, the neuropeptide sequence may also include a sequence that shows one substitution, deletion, or insertion compared to SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0325] Preferably, the amino acid substitutions, deletions, or insertions described above, compared to SEQ ID NO: 6 and SEQ ID NO: 7, are performed at positions that are less conserved in the sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, compared to the corresponding neuropeptide B and W sequences from non-human organisms or species.

[0326] To determine whether a particular amino acid position is not well conserved and therefore preferably undergoes the modifications outlined above, those skilled in the art can use means and methods well known in the art, such as alignment, manually or using computer programs well known to those skilled in the art. Such alignment can be performed, for example, using means and methods well known to those skilled in the art, such as the Lipman-Pearson method (Science 227 (1985), 1435) or known computer algorithms such as the CLUSTAL algorithm. In such alignment, it is preferable that maximum homology is assigned to conserved amino acid residues present in the amino acid sequence. Preferably, ClustalW2 is used for amino acid sequence comparison. For pairwise comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. For multiple comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62, gap open: 10, gap extension: 0.2, gap distance: 5, no end gap.

[0327] Positions found to be “identical” (i.e., “conserved”) are preferably not subjected to substitution, deletion, or insertion. According to the present invention, the terms “identical” or “percent identical” in the context of two or more nucleic acids or amino acid sequences refer to two or more sequences or subsequences that are identical to SEQ ID NOs. 6 and SEQ ID NOs. 7, respectively, or that have a specific proportion of amino acid residues or nucleotides that are identical to SEQ ID NOs. 6 and SEQ ID NOs. 7. Those skilled in the art will be aware of methods for determining the percentage identicalness between sequences using algorithms such as the CLUSTALW computer program known in the art (Thompson Nucl.Acids Res.2 (1994), 4673-4680) or based on FASTDB (Brutlag Comp.App.Biosci.6 (1990), 237-245).

[0328] The FASTDB algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in its calculations, although this can be manually corrected to avoid overestimation of % identity. However, CLUSTALW does consider sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410) are also available to those skilled in the art. The BLASTN program for nucleic acid sequences uses a word length (W) of 11, an expected value (E) of 10, M=5, N=4, and a comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3 and an expected value (E) of 10 as defaults. The BLOSUM62 score matrix (Henikoff (1989) PNAS 89:10915) uses 50 alignments (B), 10 expected values ​​(E), M=5, N=4, and a comparison of both strands.

[0329] Preferably, the above-mentioned 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions are each conserved amino acid substitutions with respect to the sequences of SEQ ID NO: 6 and SEQ ID NO: 7.

[0330] These “conservative amino acid substitutions” refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation, and rigidity) so that the protein can be frequently modified without altering its biological activity. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, for example, Watson Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. 4th Ed. (1987), 224). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to impair biological activity. In the context of the present invention, the neuropeptide sequences of the present invention include polypeptide chains having sequences containing up to 0 (unchanged), 1, 2, 3, 4, 5, 6, 7, or 8 conservative amino acid substitutions when compared to specific amino acid sequences disclosed herein, e.g., SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0331] Such exemplary substitutions are preferably carried out according to those shown in Table 1 below:

[0332] [Table 1]

[0333] Neuropeptide sequences having one or more of the above substitutions, deletions, or insertions compared to SEQ ID NOs: 6 and SEQ ID NOs: 7, respectively, may result in neuropeptide sequences having similar capabilities to SEQ ID NOs: 6 and SEQ ID NOs: 7, respectively, preferably higher capabilities than SEQ ID NOs: 6 and SEQ ID NOs: 7, respectively (in terms of the ability to bind to and / or activate the neuropeptide B / W receptor (NPBWR1), and thus transmit its signal comparable to that of unmodified neuropeptides B and W). The properties / capabilities of a given modified neuropeptide sequence compared to SEQ ID NOs: 6 and SEQ ID NOs: 7, respectively, can be readily determined by methods known in the art to those skilled in the art. Thus, the properties / capabilities of a given modified neuropeptide sequence compared to SEQ ID NOs: 6 and SEQ ID NOs: 7 are related to the activity of the respective sequences that have the ability to mediate binding to and / or activation of the neuropeptide B / W receptor (NPBWR1), and thus transmit its signal comparable to that of unmodified neuropeptides B and W. The "ability to bind to and / or activate the neuropeptide B / W receptor (NPBWR1), and thus the ability to transmit its signal in a manner comparable to that of unmodified neuropeptides B and W," can be determined, for example, by the methods described in the attached examples and outlined below.

[0334] Regarding the ability of neuropeptides B or W to bind to and / or activate NPBWR1, quantitative PCR for assaying Bdnf expression can be used, for example, as already described above.

[0335] In short, regarding the ability of NPBWR1 to bind to neuropeptides B / W, for example, FRAP (fluorescence bleed recovery) can be used, in which neuropeptide B or neuropeptide W is used as a substrate to evaluate binding to NBWBR1.

[0336] The corresponding assay is known in the art and can be easily carried out by those skilled in the art using routine methods.

[0337] While not theoretically bound, the ability of neuropeptides B or W to bind to and / or activate NPBWR1 is often measured indirectly through the regulation / activation of the expression of the downstream protein brain-derived neurotrophic factor (Bdnf). Bdnf RNA levels can be assessed using quantitative PCR. Increased Bdnf expression is an indicator of NPBWR1 inhibition. Conversely, decreased Bdnf expression is an indicator of NPBWR1 activation.

[0338] However, the (one or more) neuropeptide sequences used in the present invention are not particularly limited to the specific sequences and deletions, substitutions, or insertions described above, and may also relate to neuropeptide sequences that include sequences showing (one or more) amino acid additions compared to SEQ ID NOs: 6 and SEQ ID NOs: 7, respectively. The addition of amino acids may be adjacent or scattered. Thus, additional amino acids may be added to the N-terminus and / or C-terminus of the (one or more) neuropeptide sequences of the present invention. Alternatively, or in addition to these adjacent further amino acids, additional amino acids may also be present in the amino acid sequence of the (one or more) neuropeptide sequences of the present invention. The additional amino acids may comprise a polypeptide chain of up to 0 (unchanged), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably up to 20 amino acids, or more preferably up to 30 amino acids. In light of the rational basis that the addition of amino acids is unlikely to alter the above-mentioned functional properties of the neuropeptide sequences of the present invention, the addition of amino acids may also result in sequences having lengths of up to 40, 50, 60, 70, 80, 90, or even 100 amino acids, or even more, up to 200, 300, 400, or 500 amino acids, as long as these sequences have similar capabilities to, preferably higher than, those defined above, SEQ ID NOs: 6 and SEQ ID NOs: 7 (in that they can bind to and / or activate the neuropeptide B / W receptor (NPBWR1), and thus transmit its signal comparable to that of unmodified neuropeptides B and W).

[0339] The neuropeptide sequences of the present invention can be generated / synthesized recombinantly or synthetically by methods known to those skilled in the art. More specifically, as further described below, the neuropeptides of the present invention can be produced recombinantly by methods known to those skilled in the art, or can be readily synthesized, for example, in an automated peptide synthesizer using standard techniques of solid-phase support and repeated orthogonal deprotection and coupling. Free amino groups in the peptide that will later be used for conjugation with part or other agents are favorably blocked with standard protecting groups such as Boc groups, while the N-terminal residue can be acetylated to increase serum stability. Such protecting groups are well known to those skilled in the art (see Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, NY)). When the peptide is prepared for later use in the construct of the present invention, the peptide is favorably cleaved from the resin to produce the corresponding C-terminal amide to inhibit carboxypeptidase activity in vivo.

[0340] In a more preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in a method of treating, improving or preventing the manic phase of bipolar affective disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the agonist / activator is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcription activator domain and an epigenetic modification domain capable of activating the expression of NPBWR1.

[0341] The feature "a CRISPR / Cas system specifically directed to NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional activator domain and an epigenetic modification domain" has already been described above in relation to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically directed to NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of suppressing the expression of NPBWR1.

[0342] The above-described agonist / activator is a CRISPR / Cas system specifically directed toward NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcription activator domain and an epigenetic modification domain capable of activating NPBWR1 expression, as described herein for manic bipolar disorder (ICD-10 F31) The only difference in the context of a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) (for use in methods of treating, improving or preventing appetite disorders, preferably anorexia or bulimia) is that the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional activator domain and an epigenetic modification domain capable of activating the expression of NPBWR1 (in contrast, in the further context above of a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1), the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of repressing the expression of NPBWR1).

[0343] Therefore, with the exception of this sole difference, the same applies to the agonist / activator arm as defined herein in the medical environment as described above, with respect to this feature and preferred embodiments, with necessary modifications, to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of repressing the expression of NPBWR1.

[0344] In particular, appropriate effector domains selected from the group consisting of transcriptional activator domains and epigenetic modification domains capable of activating NPBWR1 expression, which are used in connection with agonist / activation arms for medical use, are also defined above.

[0345] As outlined above, pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above, and pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above, are particularly useful in medical settings.

[0346] Accordingly, in preferred embodiments, the present invention relates to a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, and a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, and at least one pharmaceutically acceptable excipient.

[0347] Accordingly, in preferred embodiments, the present invention relates to pharmaceutical compositions for use as drugs, comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, and a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above.

[0348] Terms such as “treatment” and / or “prevention” are used herein to generally mean obtaining a desired pharmaceutical and / or physiological effect. Therefore, the treatments of the present invention may relate to the treatment of an (acute) state of a particular disease / disorder, but may also relate to preventive treatment in terms of completely or partially preventing the disease / disorder or its symptoms. Preferably, the term “treatment” should be understood as therapeutic in relation to partially or completely curing the disease / disorder and / or adverse effects and / or symptoms resulting from the disease / disorder. “Acute” in this regard means that the subject exhibits symptoms of the disease or disorder. In other words, the subject being treated actually needs treatment, and the term “acute treatment” in the context of the present invention relates to measures taken to actually treat the disease / disorder after its onset or outbreak. Treatment may also be prophylactic or preventive treatment, i.e., measures taken for the prevention of the disease / disorder.

[0349] The pharmaceutical composition or drug of the present invention may be administered by a wide class of dosage forms known to those skilled in the art. Administration may be systemic, topical, oral, or aerosol, and is not limited to, but may include administration by tablet, needle injection, inhaler, cream, foam, gel, lotion, and ointment.

[0350] Preferably, a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, and a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, should be administered intravenously, topically, intradermally, subcutaneously, intra-cutanously, intramuscularly, and / or intrathecally, respectively.

[0351] These routes of administration, namely intravenous, topical, intradermal, subcutaneous, intra-cutanously, intramuscular, and / or intrathecal routes, are known to those skilled in the art.

[0352] Excipients or carriers are inert substances formulated together with the active ingredient, i.e., the pharmaceutical composition containing the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above, and the pharmaceutical composition containing the neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above, for the purpose of increasing the volume of formulations containing the potent active ingredient. Excipients are often called “volume extenders,” “fillers,” or “diluents.” Volume extension allows for the convenient and accurate dispensing of the active ingredient when manufacturing dosage forms. They can also serve various therapeutic enhancement purposes, such as promoting drug absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process to assist in handling the relevant active ingredient, such as by promoting powder flowability or non-stick properties, in addition to aiding in vitro stability, such as preventing degradation over the expected shelf life. The selection of an appropriate excipient also depends on the route of administration and dosage form, as well as the active ingredient and other factors.

[0353] Accordingly, the pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor of the present invention as defined above, and the pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention as defined above, may each be in solid, liquid, or gaseous form, and in particular may be in powder, tablet, solution, or aerosol form. The pharmaceutical compositions preferably optionally contain a pharmaceutically acceptable carrier and / or diluent.

[0354] These pharmaceutical compositions can be administered to a target in an appropriate dose. Administration of an appropriate composition can be carried out by various methods, e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration. The above administration is particularly preferred to be carried out by injection and / or delivery, for example, to a site in the pulmonary artery, or directly to the lungs. The compositions of the present invention can also be administered directly to the target site, for example, by bioristic delivery to an external or internal target site. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health, and other drugs being administered concurrently. Protein-based pharmaceutically active substances may be present in doses of 1 ng to 10 mg / kg body weight / administered. However, doses below or above this exemplary range are conceivable, particularly considering the aforementioned factors. If the regimen is a continuous infusion, it should also be within the range of 1 μg to 10 mg units per kilogram of body weight per minute.

[0355] Examples of suitable pharmaceutical carriers, excipients, and / or diluents are well known in the art and include phosphate-buffered saline, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in an appropriate dose, i.e., an "effective dose" that can be readily determined by methods known in the art to those skilled in the art. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any one patient depends on many factors, including the size of the patient or subject, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health status, and other drugs being administered simultaneously.

[0356] Therefore, preferably, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor and the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention as defined above are included in effective amounts. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is administered. According to the above, the content of the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor and the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention in the pharmaceutical composition is not limited as long as it is useful for the above treatment, but preferably contains 0.0000001 to 10% by weight of the total composition. Furthermore, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor and the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention as defined above are preferably used in a carrier. Generally, a suitable amount of pharmaceutically acceptable salt is used as a carrier to make the composition isotonic. Examples of carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution.Preferably, acceptable excipients, carriers, or stabilizers are nontoxic at the dosage and concentration used and include, for example, buffers (e.g., citrates, phosphates, and other organic acids), salt-forming counterions (e.g., sodium and potassium), low molecular weight (>10 amino acid residues) polypeptides, proteins (e.g., serum albumin or gelatin), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids such as histidine, glutamine, lysine, asparagine, arginine, and glycine, carbohydrates such as glucose, mannose, or dextrin, monosaccharides, disaccharides, and other sugars (e.g., sucrose, mannitol, trehalose). Examples include sulfite or sorbitol, chelating agents (e.g., EDTA), nonionic surfactants (e.g., Tween, Pluronics, or polyethylene glycol), antioxidants such as methionine, ascorbic acid, and tocopherol, and / or preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol). Suitable carriers and their formulations are described in detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0357] The progress of treatment can be monitored through regular evaluations.

[0358] The pharmaceutical compositions / drugs of the present invention may be present in sterile aqueous or non-aqueous solutions, suspensions, and emulsions, as well as in creams and suppositories. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. Furthermore, the pharmaceutical compositions of the present invention may include further agents depending on the intended use of the pharmaceutical composition. These agents may include, for example, polyoxyethylene sorbitan monolaurate, propylene glycol, EDTA, citric acid, sucrose, and other agents well known to those skilled in the art that are suitable for the intended use of the pharmaceutical composition.

[0359] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient.

[0360] The present invention also relates to a method for treating or preventing a disorder or disease as defined herein in a subject, comprising administering to the subject, preferably in a therapeutically effective amount as defined above, a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above, and a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) of the present invention as defined above.

[0361] With regard to preferred embodiments of the treatment method, the same applies mutatis mutandis to antibodies or pharmaceutical compositions for use as defined above, with necessary modifications.

[0362] In the present invention, the subject is, in preferred embodiments, mammals such as dogs, cats, pigs, cattle, sheep, horses, rodents such as rats, mice, and guinea pigs, or primates such as gorillas, chimpanzees, and humans. In the most preferred embodiment, the subject is humans.

[0363] Other aspects and advantages of the present invention are described in the following examples, which are provided for illustrative purposes only and not for limitation. Each publication, patent, patent application or other document cited herein is incorporated herein by reference in its entirety. [Brief explanation of the drawing]

[0364] [Figure 1]Acute caffeine injection reverses CVS-induced changes in depressive-like behavior and dendritic spine changes in females. A) Experimental overview. For dendritic spine analysis, GFP-expressing AAV was stereotactically injected into subgroups of mice. B-E, H, I) Statistics: Two-way ANOVA Bonferroni post-hoc test. B-E) Stress-induced behavioral changes are reversed 24 hours after caffeine injection. B) Forced swimming test. n=9-10 / group, effect of stress: F(1,35)=10.38, P<0.01, effect of drug: F(1,35)=23.36, P<0.0001, post-hoc test: effect of Caf in naive mice: *P<0.05, in CVS: ***P<0.001, effect of stress in saline mice: **P<0.01, in Caf: P>0.05. C) Novel suppressed feeding test. n=14-15 / group, effect of stress: F(1,54)=6.21, P<0.05, effect of drug: F(1,54)=6.17, P<0.05, interaction: F(1,54)=4.28, P<0.05, post-hoc test: effect of drug within naive: P>0.05, within CVS:**P<0.01, effect of stress within Sal:**P<0.01, within Caf: P>0.05. D) Splash test. n=7-8 / group, effect of stress: F(1,26)=1.05, P=0.32, effect of drug: F(1,26)=0.41, P=0.53, interaction: F(1,26)=0.57, P=0.46, post-hoc test: all comparisons: P>0.05. E) Sucrose preference test. n=10~12 / group, effect of stress: F(1,42)=2.91, P=0.10, effect of drug: F(1,42)=0.72, P=0.40, interaction: F(1,42)=1.61, P=0.21, post-hoc test: all comparisons: P>0.05. F~N) CVS-induced changes in NAc dendritic spines are reversed by caffeine. F) Overview image of GFP-labeled neurons. G) Representative dendrites. Scale bar 10 μm. H) Stubby spine. n=39-58 dendrites from mice of 4-6 mice / group, two-way ANOVA: effect of stress: F(1,193)=2.90, P=0.09; effect of drug: F(1,193)=5.62, P<0.05; interaction: F(1,193)=0.11, P=0.73; post-hoc test: all comparisons: P>0.05. I) Spines with necks.Dendrites from 4-6 mice / group (n=40-58), two-way ANOVA: Effect of stress: F(1,194)=3.34, P=0.06; Effect of drug: F(1,194)=4.34, P<0.05; Interaction: F(1,194)=3.24, P=0.07; Post-hoc test: Effect of drug in naive mice: P>0.05; Effect of drug in CVS: P<0.05; Effect of stress in Sal mice: P<0.05; Effect of stress in Sal mice: P>0.05; Effect of caffeine mice: P>0.05. J-M) Cumulative head diameter increased with CVS and reversed with caffeine, statistics: Gehan-Breslow-Wilcoxon test. J) Effect of drug in naive mice: χ²=65.36, degrees of freedom (DF)=1, ***P<0.0001. K) Effect of drug in CVS: χ²=51.74, DF=1, ***P<0.0001. L) Effect of stress in physiological saline: χ²=141.90, DF=1, ***P<0.0001. M) Effect of stress in caffeine: χ²=7.04, DF=1, **P<0.01. B~E, H, I) Independent data points are plotted, showing mean ± sem. Sketches were made using biorender.com. [Figure 2]CVS induces subtle spine changes in males, which are partially reversed by caffeine. A) Representative dendrites. Scale bar 10 μm. B) Stump-shaped spines. n=31-40 dendrites from 3-4 mice / group, two-way ANOVA: effect of stress: F(1,136)=1.77, P=0.19, effect of drug: F(1,136)=6.68, P<0.05, interaction: F(1,136)=0.66, P=0.42, Bonferroni post-hoc test: effect of drug in CVS: *P<0.05, all other comparisons: P>0.05. C) Spines with necks. Dendrites from 3-4 mice / group (n=32-40), two-way ANOVA: Effect of stress: F(1,139)=0.01, P=0.91; Effect of drug: F(1,139)=0.13, P=0.72; Interaction: F(1,139)=2.53, P=0.11; Bonferroni post-hoc test: P>0.05 for all comparisons. D-G) Cumulative head diameter decreased with CVS and reversed with caffeine, statistics: Gehan-Breslow-Wilcoxon test. D) Effect of drug in naive: χ²=3.61, degrees of freedom (DF)=1, P=0.06. E) Effect of drug in CVS: χ²=6.56, DF=1, *P<0.05. F) Effect of stress in saline: χ²=23.17, DF=1, ***P<0.0001. G) Effect of stress on caffeine: χ² = 1.48, DF = 1, P = 0.22. B, C) Independent data points are plotted, showing mean ± sem. [Figure 3]RNA sequencing quality control markers and read count distribution. A) Pretreatment information regarding RNA quality. All samples achieved at least 90% alignment. B, C) Time-course distribution of Npbwr1 read counts (normalized to WT ZT18). B~K) n=4 / group, statistics: two-way ANOVA and Bonferroni post-hoc test. B) WT, drug effect: F(1,24)=1.38, P<0.05, effect of time: F(3,24)=0.71, P=0.25, interaction: F(3,24)=3.68, P=0.55, post-hoc test: effect of caffeine in ZT18: *P<0.05, in all other ZTs: P>0.05. Sal: ZT18 vs ZT0: P<0.001, ZT18 vs ZT6, 12: P<0.01, all others: P>0.05. C) T75A, drug effect: F(1,24)=0.76, P=0.39, time effect: F(3,24)=0.57, P=0.63, interaction: F(3,24)=0.36, P=0.78, post-hoc tests: caffeine effect within all ZT: overall P>0.05, time point effect within sal and caf: all P>0.05. D~K) Normalized read counts (RC) for circadian genes. Circadian changes are consistent with the literature. Caffeine, rather than the T75A mutation, may slightly flatten the amplitude of circadian oscillations. DG) WT. D) Per1, effect of drug: F(1,24)=0.47, P=0.50, effect of time: F(3,24)=5.05, P<0.01, interaction: F(3,24)=2.25, P=0.11, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal: ZT18 vs ZT0: P<0.01, ZT0 vs ZT12: P<0.05, Caf: ZT0 vs ZT6: P<0.05, all others: P>0.05. E) Per2, effect of drug: F(1,24)=5.35, P<0.05, effect of time: F(3,24)=23.82, P<0.0001, interaction: F(3,24)=1.27, P=0.31, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal: ZT18 vs ZT0, ZT0 vs ZT12, ZT6 vs ZT12: P<0.001, ZT18 vs ZT12: P<0.05, ZT0 vs ZT6: P<0.01, Caf: ZT18 vs 12: P<0.05, ZT0 vs ZT6: P<0.01, ZT0 vs ZT12: P<0.001, all others: P>0.05.F) Cry, effect of drug: F(1,24)=0.03, P=0.87, effect of time: F(3,24)=13.19, P<0.0001, interaction: F(3,24)=1.41, P=0.27, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal: ZT0 vs ZT6: P<0.01, ZT0 vs ZT6, ZT6 vs ZT12: P<0.05, Caf: ZT0 vs ZT12: P<0.05, ZT0 vs ZT12: P<0.001, ZT6 vs ZT12: P<0.01, all others: P>0.05. G) Bmal, effect of drug: F(1,24)=1.09, P=0.31, effect of time: F(3,24)=3.48, P<0.05, interaction: F(3,24)=0.23, P=0.88, post-hoc test: effect of caffeine in all ZT: P>0.05, Caf:ZT0 vs ZT6: P<0.05, all others: P>0.05. HK) T75A. H) Per1, effect of drug: F(1,24)=0.40, P=0.53, effect of time: F(3,24)=5.14, P<0.01, interaction: F(3,24)=0.08, P=0.97, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal, Caf: ZT0 vs ZT6: P<0.05, all others: P>0.05. I) Per2, effect of drug: F(1,24)=4.9, P<0.05, effect of time: F(3,24)=26.49, P<0.0001, interaction: F(3,24)=0.88, P=0.47, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal: ZT18 vs ZT0: P<0.05, ZT18 vs ZT12, ZT0 vs ZT6, ZT0 vs ZT12: P<0.001, Caf: ZT18 vs ZT12, ZT0 vs ZT6: P<0.01, ZT0 vs ZT12: P<0.0001, all others: P>0.05. J) Cry, two-way ANOVA: effect of drug: F(1,24)=0.17, P=0.67, effect of time: F(3,24)=13.76, P<0.0001, interaction: F(3,24)=0.75, P=0.53, post-hoc test: effect of caffeine in all ZT: P>0.05, Sal: ZT18 vs ZT0, ZT6 vs ZT12: P<0.01, ZT0 vs ZT12: P<0.001, Caf: ZT0 vs ZT12: P<0.01, ZT6 vs ZT12: P<0.05, all others: P>0.05.K) Bmal, effect of drug: F(1,24)<0.01, P=0.99, effect of time: F(3,24)=5.73, P<0.01, interaction: F(3,24)=0.45, P=0.72, post-hoc test: effect of caffeine in all ZT: P>0.05, Caf: ZT0 vs ZT6, ZT0 vs ZT12: P<0.05, all others: P>0.05. B~K) Mean ± sem shown. [Figure 4]RNA sequencing identifies Npbwr1 as a gene product circadianally regulated by the DARPP-32 pathway. A-C) RNA sequencing for NAc in wild-type (WT) and DARPP-32-T75A mutant (T75A) mice 2 hours after caffeine injection, during the active and inactive phases of the photoperiod. A) Venn diagram of significant (Padj<0.05, log2FC 0.5<>-0.5) gene expression changes across various conditions. These data support that the maximum effect of caffeine occurs in WT during the active phase. B) Heatmap comparing transcriptional changes (log(FC)) across each phase of the photoperiod and genotype. C) Annotated pathway pie chart in the WT group (dark phase). D-F) Validation in different cohorts by qPCR. D) 2 hours after caffeine, dark phase. n=16-22 / group, two-way ANOVA: Genotype effect: F(1,73)=0.91, P=0.34, Drug effect: F(1,73)=1.53, P=0.22, Interaction: F(1,73)=4.57, P<0.05, Bonferroni post-hoc test: Drug effect within WT: *P<0.05, within T75A: P>0.05, Genotype effect within Sal: P>0.05, within Caf: *P<0.05. E) 2 hours after caffeine, light phase. n=12-16 / group, two-way ANOVA: Genotype effect: F(1,53)=0.05, P=0.82, Drug effect: F(1,53)=0.93, P=0.34, Interaction: F(1,53)=0.06, P=0.80. Bonferroni post-hoc tests: all P>0.05. F) 24 hours after caffeine, during the dark period. n=4~10 / group, two-way ANOVA: genotype: effect of F(1,24) <0.01, P=0.98, drug effect: F(1,24)=2.02, P=0.17, interaction: F(1,24)=1.53, P=0.23, Bonferroni post-hoc tests: drug effect within WT: *P<0.05, within T75A: P>0.05, genotype effect within Sal: P>0.05, within Caf: P>0.05. G, H) Npbwr1 levels increase after chronic variable stress (CVS). G) Female: n=8 / group, t-test: t=9.71, df=14, ***P<0.0001. H) Male: n=8 / group, t-test: t=2.27 df=14, *P<0.05.I) Npbwr1 protein levels increased after CVS: n=6 / group, t-test: t=2.27, df=10, *P<0.05. J) NPBWR1 increased in depressed patients (MDD) compared to controls (Nil): n=30-32 / group, t-test: t=2.36, df=60, *P<0.05. D-J) Plot independent data points showing mean ± sem. [Figure 5] Demographic and causal separation of post-mortem specimens. A) Patient information. B) Analysis separated by causative and diagnostic criteria. n=11-21 / group, two-way ANOVA: effect of depr.: F(1,65)=5.45, #P<0.05, effect of causative: F(1,65)=0.38, P=0.36, interaction: F(1,65)=0.66, P=0.42, Bonferroni post-hoc test: P>0.05 for all comparisons. Independent data points are plotted, showing mean ± sem. Nil: control. Depr.: depression. [Figure 6]Overexpression of Npbwr1 mimics behavior and stress effects on dendritic spines. A) Schematic diagram of OE-Npbwr1-GFP AAV. B) Experimental design. C) Schematic image of virus injection into NAc. D) qPCR. n=7-8 / group, Student's t-test: t13=2.71, *P<0.05. EI) Overexpression of Npbwr1 (OE) increases depressive-like behavior, statistics: Student's t-test. E) Tail suspension test. n=14 / group, t26=4.17, ***P<0.001. F) Forced swim test. n=13-15 / group, t26=3.00, **P<0.01. G) Novel suppression feeding test. n=14 / group, t26=1.21, P=0.24. H) Splash test. n=14 / group. t26=2.68, *P<0.05. I) Sucrose preference test. n=13~15 / group, t26=2.15, P>0.05. J~M) Dendritic spines in NAc are altered by OE-Npbwr1 in a manner reminiscent of CVS. J) Representative dendrites. Scale bar 10μm. K) Stump-type spines. n=46~57, 5~6 mice / group, t98=1.13, P=0.26. L) Spines with necks. n=48~59, 5~6 mice / group, t102=2.76, **P<0.01. M) Cumulative head diameter. Gehan-Breslow-Wilcoxon test: χ²=7.83, DF=1, **P<0.01. DI, K, L) Independent data points are plotted, showing mean ± sem. I created the sketch using biorender.com. [Figure 7]Knockdown of Npbwr1 blocks the effects of CVS on behavior and dendritic spines. A) Schematic diagram of KD-Npbwr1-GFP AAV. B) Experimental design. C) Schematic image of virus injection into NAc. D) qPCR (naive mice): n=6 / group, Student's t-test: t10=2.33, *P<0.05. E~H) Depression-like behavior is partially blocked by KD-Npbwr1. E) Forced swim test. n=10 / group, two-way ANOVA: Effect of stress: F(1,36)=3.41, P=0.07, Effect of AAV: F(1,36)=8.40, P<0.01, Interaction: F(1,36)=6.42, P<0.05, Bonferroni post-hoc test: Effect of AAV within naive groups: P>0.05, within CVS groups: ***P<0.001. Effect of stress within GFP groups: **P<0.01, within KD groups: P>0.05. F) Novel suppression feeding test. n=10 / group, two-way ANOVA: Effect of stress: F(1,36)=4.51, P<0.05, Effect of AAV: F(1,36)=0.03, P=0.87, Interaction: F(1,36)=0.45, P=0.51, Bonferroni post-hoc test: All comparisons: P>0.05. G) Splash test. n=10 / group. Two-way ANOVA: Effect of stress: F(1,36)=0.37, P=0.55, Effect of AAV: F(1,36)=5.13, P<0.05, Interaction: F(1,36)=0.62, P=0.43, Bonferroni post-hoc test: All comparisons: P>0.05. H) Sucrose preference test. n=10 / group, two-way ANOVA: Effect of stress: F(1,36)=0.24, P=0.55, Effect of AAV: F(1,36)=3.71, P=0.06, Interaction: F(1,36)=1.55, P=0.22, Bonferroni post-hoc test: All comparisons: P>0.05. I~O) Stress-induced spine changes are reversed by KD-Npbwr1. I) Representative dendrites. Scale bar 10μm. J) Stump-shaped spines. n=3-4 mice / group, 30-39 dendrites, two-way ANOVA: Effect of stress: F(1,152)=2.24, P=0.14, Effect of AAV: F(1,152)=3.38, P=0.21, Interaction: F(1,152)=2.24, P=0.07, Bonferroni post-hoc test: All comparisons: P>0.05.K) Spines with necks. n=28-37 dendrites from 3-4 mice / group, two-way ANOVA: Effect of stress: F(1,132)=6.10, P<0.05, Effect of AAV: F(1,132)=3.70, P=0.06, Interaction: F(1,132)=23.11, P<0.0001, Bonferroni post-hoc test: Effect of AAV in naive: P>0.05, in CVS: ***P>0.001, Effect of stress in GFP: ***P<0.001, in KD: P>0.05. LO) Cumulative head diameter reduced by CVS and rescued by KD-Npbwr1, statistics: Gehan-Breslow-Wilcoxon test. L) Effect of AAV in naive: χ²<0.01, DF=1, P=0.96. M) Effect of AAV in CVS: χ²=7.71, DF=1, **P<0.01. N) Effect of stress in GFP: χ²=20.05, DF=1, ***P<0.0001. O) Effect of stress in KD: χ²=4.58, DF=1, *P<0.05. EH, J, K) Independent data points are plotted, showing mean ± sem. Sketches were made on biorender.com. [Figure 8]Escape behavior is altered in naive WT mice 24 hours after acute administration of caffeine. A-C) Naive WT and T75A mutants were administered 7.5 mg / kg of caffeine versus saline during the dark period and tested 24 hours later. A) Forced swim test: n=5-7 / group, two-way ANOVA: drug effect: F(1,18)=0.83, P=0.37, genotype effect: F(1,18)=2.49, P=0.13, interaction: F(1,18)=11.73, P<0.01, Bonferroni post-hoc test: effect of Caf in WT:**P<0.01, in T75A:P>0.05. genotype effect in Sal:**P<0.01, in Caf:P>0.05. B) Novel suppression feeding test: n=3-5 / group, two-way ANOVA: drug effect: F(1,13)=0.18, P=0.68, genotype effect: F(1,13)=1.40, P=0.26, interaction: F(1,13)=0.13, P=0.73, Bonferroni post-hoc test: all P>0.05. C) Tail suspension test: n=4-6 / group, two-way ANOVA: drug effect: F(1,14)=0.12, P=0.73, genotype effect: F(1,14)=0.02, P=0.89, interaction: F(1,14)=5.73, #P<0.05, Bonferroni post-hoc test: all P>0.05. A-C) Independent data points are plotted, showing mean ± sem. [Figure 9] Genes differentially expressed after Npbwr1 overexpression. A, B) NAc from AAV-infected mice expressing GFP or OE-Npbwr1-GFP was subjected to RNA sequencing. A) Heatmap of significantly altered gene products. B) Table of the above gene products sorted by adjusted P-value (Padj) and log2-fold change (log2FC). [Figure 10]Npbwr1 ligands alter Bdnf signaling and depression-like behaviors. A) Overview of ligand binding of agonist neuropeptide B (NPB) and antagonist CYM50769 to Npbwr1. B) 1 nM NPB, or C, D) 1 μM CYM50769, or microinjections were performed, and tissues were collected 24 hours (B, C) or 7 days later (D) and analyzed by qPCR. Statistical: Student's t-test. B-D) n=6-7. B) NPB reduces Bdnf. P<0.01**. C) CYM50769 increases Bdnf. P<0.05*. C) The effect of CYM50769 on Bdnf persists at 7 days. P<0.01**. E-G) Mice were subjected to CVS (vs. naive controls), and after the final stress induction, a small amount of NPB or CYM50769 was injected at the end of the active phase. Depression-like behavior tests were conducted approximately 24 hours later during the dark phase. E) Forced swimming test. NPB increased immobility time in naive mice, while CYM50769 blocked the CVS effect. n=8-9 / group, two-way ANOVA: CVS effect: F(1,51)=8.57, P=0.01, ligand effect: F(2,51)=26.96, P<0.01, interaction: F(2,51)=4.70, P=0.01, Bonferroni post-hoc test: CVS effect in ACSF: ***P<0.001, NPB effect in naive: **P<0.01, effect of CYM50769 in CVS: ***P<0.001, all others ns. F) Splash test. NPB may reduce grooming in naive mice, but CYM50769 blocks the CVS effect. n=8-9 / group, two-way ANOVA: interaction: F(2,46)=3.17, P=0.05. G) Sucrose palatability test. NPB and CVS reduce the consumption rate of sucrose solution, but CYM50769 mitigates the effect of CVS. n=9~10 / group, two-way ANOVA: Ligand effect: F(2,52)=13.68, P<0.0001, Interaction: F(2,52)=9.29, P<0.01, Bonferroni post-hoc test: CVS effect in ACSF: ***P<0.001, NPB effect in naive: **P<0.01, CYM50769 effect in CVS: ***P<0.001, all others ns.B-G) Independent data points are plotted, showing the mean ± sem. The sketch was created using biorender.com. [Figure 11] Acute microinjection of Npbwr1 ligand is selective and nontoxic. A-C) 1 nM NPB was microinjected into NAc. D-F) 1 μM CYM50769 was microinjected into NAc. Tissue was collected 24 hours later and analyzed by qPCR. Statistics: Student's t-test. n=6-7. Mean ± sem shown. A) No effect of NPB on Bcl2. P=0.882. B) NPB does not alter Casp3. P=0.66. C) Per2 is not affected by NPB. P=0.38. D) No effect on Bcl2. P=0.58. E) CYM50769 does not alter Casp3. P=0.42. F) Per2 is not affected by CYM50769. P=0.63. [Modes for carrying out the invention] [Examples]

[0365] material and method Animals and Licensing. Mice were housed in accordance with the ethical guidelines of the Thuringian Consumer Protection Agency (Thuringer Landesamt fur Verbraucherschutz, TLV). Experiments were conducted in accordance with animal licenses UKJ-18-036 and UKJ-21-12 (Germany), in accordance with EU Directive 2010 / 63 / EU guidelines for animal experimentation. Experiments using genetically modified organisms were conducted in accordance with the S1 regulation according to GenTAufzV. C57Bl / 6J mice were housed in the animal facility (FZL) of the University Hospital of Jena (Universitatsklinikum Jena, Germany) and purchased from Janvier labs (Saint Berthevin Cedex). Both sexes were used as described. Mice were housed in a 12L:12D photoperiod.

[0366] Drugs and chemicals. Mice were injected intraperitoneally (ip) with 7.5 mg / kg of caffeine (#, C0750, Sigma) or saline at a dose of 10 ml / kg body weight and tested 2 or 24 hours later. NPB (#CSB-MP015971HU-100, Cusabio) was injected into the NAc at doses of 1, 3, and 10 nM. CYM50769 (#1067-25 mg, Sigma Aldrich) was injected into the NAc at doses of 0.1 μM to 10 μM.

[0367] RNA purification and quantification. RNA was purified by resuspension in Trizol and chloroform precipitation. RNA was washed with isopropanol and 75% ethanol. After cDNA conversion using the GoScript® reverse transcriptase kit (#A5001, Promega), quantitative real-time PCR was performed on a Bio-rad CFX96 real-time system. Primer sequences are listed in the supplementary materials. The quantitative PCR results were processed as described in the literature. 22 .

[0368] AAV, stereotactic surgery, and microinjection. Bilateral stereotactic surgery to NAc was performed essentially as described in the literature. 13 The following three viruses were used: pAAV.1-CAG-GFP (#37825, Addgene), OE-Npbwr1-GFP:pAAV-CAG-GFP-P2A-Npbwr1-WPRE1, and KD-Npbwr1-GFP:pAAV-U6-shRNA-Npbwr1#1-CAG-GFP-P2A-WPR3 (NPBWR1-AAV custom-made by the Charite virus vector core in Berlin, Germany).

[0369] Behavioral tests and CVS. Acute behavioral tests, forced swimming tests, tail suspension tests, sucrose preference tests, splash tests, and novel suppression feeding tests were performed as described in the literature. 13,23Since tail suspension is part of the stress induction protocol after CVS, the tail suspension test was not performed. For the CVS group, caffeine was injected immediately before the stressor on the final day of CVS (day 21). The CVS protocol was carried out as described in the literature. 13 In short, mice were subjected to 21 days of stress, with one of three stressors presented in a roughly random order, and the same stressor was not used for two consecutive days. The stressors consisted of one hour of either tube restraint, tail suspension, or 100 mild electric random food shocks. In the presence of only female experimenters, used male t-shirts were wrapped in clean protective clothing from the animal unit and brought into the laboratory to avoid variability due to the scientist's sex-specific scent. 24 Previous experiment 22 To enable comparability, all experiments were conducted during the light phase of the photoperiod.

[0370] Dendritic spine analysis. The analysis was based on the detection of GFP fluorophores in AAV. Photographs of 40 μm PFA-fixed brain sections were acquired using the AiryScan method with a Zeiss LSM 880 confocal microscope. Maximum intensity projections were obtained using Zen Black and Zen Blue software and analyzed at NeuronStudio (CNIC, Mount Sinai School of Medicine). Total spine density, the proportion of slender, mushroom-shaped, and stump-shaped spines, and cumulative neck length were measured using Graph Prism. Neck-containing mushroom density refers to the sum of the densities of slender and mushroom-shaped spines.

[0371] Next-generation RNA sequencing. We applied our in-house RNA sequencing analysis pipeline, as described in the literature. 25 After artificially depleting ribosomal RNA (rRNA) transcripts from the dataset, over 90% of the reads were aligned to the reference genome GRCm38, demonstrating excellent sample quality. Only changes reaching adjusted P-values ​​<0.05 and log2FC 0.5 <> -0.5 were further considered.

[0372] Postmortem specimens. The experiment was conducted in accordance with the ethics committee of Jena University Hospital (Germany) (registration Nr. 2020-1862-Material). The groups were balanced in terms of age and postmortem interval. Specimens under 22 years old and over 80 years old, as well as those with a postmortem interval of more than 130 hours, were excluded.

[0373] Statistics. Statistical analysis was performed using GraphPrism. A two-sided Student's t-test was used to compare the two groups. When the two factors were varied, a two-way ANOVA was used. In addition, Bonferroni's post-hoc test was used. The cumulative head diameter of dendritic spines was analyzed using the Gehan-Breslow-Wilcoxon test. 26 The analysis was performed using [specific method / tool]. Data points that were more than two standard deviations away from the mean were removed as outliers. The experimenters were not informed of the group during the behavioral tests and dendritic spine analysis.

[0374] Example 1: Caffeine rapidly improves the effects of chronic stress. The rapid mood-enhancing effect of caffeine has been previously observed in naive mice. 14 Therefore, it was hypothesized that caffeine could also alleviate symptoms induced by chronic variable stress (CVS) in mice, which is an excellent model of sex-specific transcriptional changes in MDD. 12,13 Acute administration of caffeine 24 hours prior to the study was observed to improve stress-induced behavioral changes (Figure 1).

[0375] Furthermore, to evaluate the morphology of NAc dendritic spines, mice were stereotactically injected with GFP-expressing AAV (Figure 1). Consistent with the literature, CVS increased the density and cumulative head diameter of necked spines in NAc in female mice (Figure 1). 27Male mice showed only slight stress-induced changes in neuromorphology (Figure 2). Importantly, 24 hours after injection, stress-induced spine changes were restored to naive levels by caffeine (Figure 1). This suggests that caffeine can rapidly reverse the stress effects on behavior and neuromorphology.

[0376] Example 2: Npbwr1 is altered circadianally and in a T75-DARPP-32-dependent manner by caffeine. Recently, a caffeine-induced circadian signaling cascade in NAc has been elucidated. Caffeine directly binds to the CLOCK / BMAL1 transcription complex, thereby altering transcription via Thr75-DARPP-32, which regulates gene expression. 14 Therefore, the mood-enhancing effect of caffeine occurs only during the active (dark) phase in both sexes and is blocked by the T75A-DARPP-32 knock-in mutation (T75A). 14 They started at the earliest measurement point (45 minutes) and lasted for at least 24 hours. 14 Therefore, the described pathway was hypothesized to mediate the rapid mood-enhancing effect of caffeine.

[0377] To determine caffeine-regulated transcripts, next-generation RNA sequencing was performed two hours after caffeine injection into the NAc of wild-type (WT) and T75A mice. Tissues were collected at four time points aligned with the circadian rhythm. 14 As expected, significant changes in circadian markers (Per1, Per2, Cry1, Bmal) oscillated in the expected manner across four time points measured were observed in both WT mice and T75A-DARPP-32 mutant mice (Figure 3). Consistent with previous data, most caffeine-induced transcriptional changes were observed to occur in WT mice during the active (dark) phase (Figures 4A, 4B). The altered transcripts belonged to a variety of cellular signaling pathways, including synaptic transmission, transcriptional regulation, and hormonal signaling (Figure 4C).

[0378] Npbwr1 levels increased with caffeine at the end of the dark period in WT mice, but not in the T75A mutant (Figure 3). This was confirmed in an independent cohort (Figures 4D, 4E). Surprisingly, caffeine increased Npbwr1 expression 2 hours after injection, but Npbwr1 levels decreased after 24 hours (Figure 4F). This suggests a specific time-dependent correlation between caffeine, mood, and Npbwr1.

[0379] Next, we evaluated whether Npbwr1 is affected by chronic stress. Npbwr1 expression increased after CVS in both sexes. This effect was more pronounced in females (Figure 4G, Figure 4H). Furthermore, Npbwr1 protein levels increased after CVS (Figure 4I, Figure 2). In addition, NPBWR1 transcription was increased in NAc of postmortem tissue from depressed patients of both sexes (Figure 4J, Figure 5). These data suggest that Npbwr1 is associated with mood and MDD and can be altered using the CVS model.

[0380] Example 3: Npbwr1 causally mediates the symptoms of stress. To investigate whether Npbwr1 is causally related to the morphological and behavioral consequences of stress, genetically modified AAVs were obtained in which Npbwr1 was overexpressed (OE) or knocked down (KD) in NAc mice. AAVs induce stable and long-term changes in the gene product, allowing for manipulation of Npbwr1 throughout the entire CVS time course. Considering a stronger effect size, the experiment was continued only in female mice.

[0381] OE in Npbwr1 mimicked the effects of chronic stress on behavior and dendritic spines (Figure 6). Specifically, OE-Npbwr1 reduced escape behavior in tail suspension and forced swimming tests (Figures 6D, 6E), and reduced the time spent grooming in the splash test (Figure 6G) and the amount of sucrose solution consumed relative to the water (Figure 6H). The density of spines with necks increased, but the density of stump-type spines did not (Figures 6J-6K). Cumulative head diameter decreased (Figure 6L), suggesting an increase in spines with necks that have smaller head diameters ("slender" spines).

[0382] Next, KD of Npbwr1 blocked specific outcomes of CVS on behavior and dendritic spines (Figure 7). In this cohort, the forced swim test was the most sensitive to CVS, and this was reversed by KD of Npbwr1 (Figures 7D–7G). Furthermore, KD reversed the effects of CVS on spines with necks and cumulative head diameter (Figures 7H–7N), consistent with observations from Figure 2. OE or KD of Npbwr1 did not affect the measurement of mouse body weight or anxiety and motor skills (Figure 8).

[0383] Next, we evaluated Npbwr1 downstream signaling, which is virtually unknown. For this purpose, we performed RNA sequencing on NAc tissue injected with OE-Npbwr1 AAV or GFP expression controls. Surprisingly, only seven genes were significantly altered (Figure 9). However, among them was brain-derived neurotrophic factor (Bdnf), which is highly associated with MDD and antidepressant response. 21 In summary, these data demonstrate a causal relationship between Npbwr1 and stress symptoms. However, AAV-based experimental approaches cannot reflect the rapid onset of Npbwr1 changes, such as those observed after caffeine administration.

[0384] Example 4: Microinjection of Npbwr1 ligand rapidly affects depressive-like behaviors. To address the rapidly acting phase of the Npbwr1 pathway, Npbwr1-activating (NPB) or inhibitory (CYM50769) ligands were microinjected into NAc. While NPB is a naturally occurring neurotransmitter, CYM50769 is a synthetic ligand that has not been tested in vivo. Therefore, administration, selectivity, and toxicity were evaluated first. Mice were injected with 0.1 μM, 1 μM, and 10 μM doses of CYM50769 and scored for health markers for 7 days. No effects were observed on body weight, hair condition, respiration, posture, stereotypic behavior, or movement (data are not shown, and all mice scored "0").

[0385] Both CYM50769 and NPB altered Bdnf levels, but in opposite directions as expected. Further testing of ligand concentrations with the clearest effect on Bdnf was conducted (1 μm CYM50769, 1 nM NPB, Figures 10A–10D). Neither NPB nor CYM50769 affected the apoptosis markers Bcl2 and Casp3 (Figure 11). Furthermore, they did not alter the levels of the circadian gene Per2 (Figure 11), suggesting selectivity for the Bdnf pathway.

[0386] Next, we tested the rapid effect of Npbwr1 activity on depressive-like behaviors in vivo. For this purpose, mice were administered CVS and microinjected with 1 nM NPB and 1 μm CYM50769 after the final session of stress induction. Behavioral tests were performed 24 hours later. NPB induced depressive-like behaviors in naive mice in forced swimming, splash, and sucrose preference tests, but did not further enhance the behavioral effects of CVS (Figures 10E-10G). In contrast, CYM50769 had no effect in naive mice. However, it rescued the CVS effect in all tests (Figures 10E-10G). These data are consistent with the effects of virus-mediated OE and KD on Npbwr1 levels (Figures 6, 7) and point to the pro-depressant effect of Npbwr1 that is improved when receptor levels or activity are reduced. Importantly, the Npbwr1 ligand showed a rapid effect on behavior, while the antagonist CYM50769 showed promise in rapidly reversing the effects of chronic stress.

[0387] Summary and Discussion This study describes a previously unknown pathway mediating rapid effects of Npbwr1 on depression-like behaviors and stress responses. Furthermore, selective alterations in Bdnf and dendritic spine morphology levels were demonstrated. Viral-mediated gene transfer stably altered Npbwr1 levels. Npbwr1 overexpression mimicked depression-like symptoms, while knockdown prevented chronic stress effects. This bifurcated effect can be explained by relatively low baseline levels of Npbwr1 in NAc, as observed by Cq values ​​of Npbwr1 in qPCRs exceeding 25 in naive mice. Therefore, the effect on Npbwr1 regulation may only be evident if Npbwr1 levels are upregulated, for example, after CVS. Consistently, modulation of Npbwr1 activity by microinjection of ligands altered depression-related symptoms. Stimulation of Npbwr1 via NPB increased depressive-like behaviors, while inhibition by CYM50769 blocked depressive-like behaviors in the CVS cohort. In summary, these data provide evidence that Npbwr1 signaling causally and rapidly alters depressive-like symptoms.

[0388] The Npbwr1 pathway is rapidly modulated by caffeine, which has an acute resilience-enhancing effect in mice. However, caffeine is already widely used in human populations and may have adverse health effects in some individuals; therefore, it was used as a research tool in the above experiment. The seemingly contradictory observed effects of caffeine on Npbwr1 at 2 hours versus 24 hours are consistent with reports that intracerebroventricular injection of NPB into the mouse brain induced hyperphagia at 2 hours, followed by decreased appetite. 15 .

[0389] Consistent with previous findings, the caffeine effect on Npbwr1 signaling occurred in a circadian pattern. 14 This is consistent with similar reports on the diurnal effects of NPW on feeding behavior, which indicate that the effect occurs only during the active phase. 29 .

[0390] CYM50769 has been developed in vitro as a selective antagonist of Npbwr1. In this example, this compound was further investigated for the first time in an in vivo model. Acute administration of this drug has been shown to be selective and well-tolerated.

[0391] In summary, this study demonstrates that Npbwr1 is a key mediator of depression and stress responses, and that corresponding antagonists, such as CYM50769, are potential compounds that rapidly modulate this pathway in a beneficial manner.

[0392] literature 1. Bebbington, P. The World Health Report 2001-Mental Health: New Understanding, New Hope. Soc. Psychiatry Psychiatr. Epidemiol. 36, 473-4 (2001). 2.Vos, T. et al.Global, regional, and national incidence,prevalence,and years lived with disability for 328 diseases and injuries for 195 countries,1990-2016:A systematic analysis for the Global Burden of Disease Study 2016.Lancet 390,1211-59(2017). 3. Galea, S., Merchant, RM & Lurie, N. The Mental Health Consequences of COVID-19 and Physical Distancing: The Need for Prevention and Early Intervention. JAMA Intern Med Epub ahead, (2020). 4.Torales,J.,O’Higgins,M.,Castaldelli-Mala,J.&Ventriglio,A.The outbreak of COVID-19 coronavirus and its impact on global mental health.Int J Soc Psychiatry 2076402091,(2020). 5.Sanchez-Roige,S.&Palmer,A.A.Emerging phenotyping strategies will advance our understanding of psychiatric genetics.Nat.Neurosci.23,475-80(2020). 6.Pappa,S.et al.Prevalence of depression,anxiety,and insomnia among healthcare workers during the COVID-19 pandemic:A systematic review and meta-analysis.Brain.Behav.Immun.88,901-7(2020). 7.Vindegaard,N.&Eriksen Benros,M.COVID-19 pandemic and mental health consequences:Systematic review of the current evidence.Brain.Behav.Immun.89,531-42(2020). 8.Dubey,S.et al.Psychosocial impact of COVID-19.Diabetes Metab.Syndr.Clin.Res.Rev.14,779-88(2020). 9.Rajkumar,R.P.COVID-19 and mental health:A review of the existing literature.Asian J.Psychiatr.52,102066(2020). 10.Tomonaga,Y.et al.The economic burden of depression in Switzerland.Pharmacoeconomics 31,237-50(2013). 11.Greenberg,P.E.,Fournier,A.A.,Sisitsky,T.,Pike,C.T.&Kessler,R.C.The economic burden of adults with major depressive disorder in the United States(2005 and 2010).J.Clin.Psychiatry 76,155-62(2015). 12.Scarpa,J.et al.Shared Transcriptional Signatures in Major Depressive Disorder and Mouse Chronic Stress Models.Biol.Psychiatry(2020). 13.Labonte,B.et al.Sex-Specific Transcriptional Signatures in Human Depression.Nat.Med.23,1102-1111(2017). 14.Trautmann,C.,Burek,D.,Huebner,C.,Girault,J.-A.&Engmann,O.A regulatory pathway linking caffeine action,mood and the diurnal clock.Neuropharmacology 172,108133(2020). 15.Tanaka,H.et al.Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8.Proc.Natl.Acad.Sci.U.S.A.100,6251-6(2003). 16.Eipper-Mains,J.E.et al.Effects of cocaine and withdrawal on the mouse nucleus accumbens transcriptome.Genes,Brain Behav.(2013).doi:10.1111 / j.1601-183X.2012.00873.x 17.Dvorakova,M.C.Distribution and function of neuropeptides W / B signaling system.Front.Physiol.9,981(2018). 18.Uchio,N.et al.Circadian characteristics of mice depleted with GPR7.Biomed.Res.(2009).doi:10.2220 / biomedres.30.357 19.Dun,S.L.et al.Neuropeptide B immunoreactivity in the central nervous system of the rat.Brain Res.(2005).doi:10.1016 / j.brainres.2005.03.024 20.Singh,G.,Maguire,J.J.,Kuc,R.E.,Fidock,M.&Davenport,A.P.Identification and cellular localisation of NPW1(GPR7)receptors for the no...

Claims

1. A pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor for use in methods of treating, improving, or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

2. The aforementioned mood / affective disorders (ii) Bipolar affective disorder during the hypomanic phase (ICD-10 F31), (iii) Depressive episode (ICD-10 F32), (iv) Recurrent depressive disorder (ICD-10 F33), (v) Persistent mood / affective disorder (ICD-10 F34), (vi) Other mood / affective disorders (ICD-10 F38), and (vii) Mood / affective disorder of unknown origin (ICD-10 F39) A pharmaceutical composition for use according to claim 1, selected from the group consisting of the following.

3. The aforementioned chronic stress (i) Reaction to severe stress and adjustment disorder (ICD-10 F43), (ii) Acute stress response (ICD-10 F43.0), (iii) Post-traumatic stress disorder (ICD-10 F43.1), (iv) Adjustment disorder (ICD-10 F43.2), (v) Other severe stress reactions (ICD-10 F43.8); and (vi) Severe stress reaction, details unknown (ICD-10 F43.9) A pharmaceutical composition for use according to claim 1, selected from the group consisting of the following.

4. The aforementioned anxiety disorder (i) Phobic anxiety disorder (ICD-10 F40); and (ii) Other anxiety disorders (ICD-10 F41.1) A pharmaceutical composition for use according to claim 1, selected from the group consisting of the following.

5. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the antagonist / inhibitor is selected from an NPBWR1 inhibitory peptide, an NPBWR1 inhibitory small molecule binding molecule, RNAi, siRNA, shRNA, and aptamers and intramers specifically directed toward NPBWR1, an anti-NPBWR1 antisense molecule.

6. The aforementioned antagonist / inhibitor is an NPBWR1 inhibitory small molecule binding molecule, and formula (1): 【Chemistry 1】 Formula (1) A chemical structure represented by the formula, where, R 1 It is selected from F, Cl, Br, I, and CN. R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (a heterocycline having) and -(6 to 10 ring atoms and optionally one or more substituents R 2a Selected from carbocyclyl having, R 3 is selected from -(heterocyclyl having 5 to 20 ring atoms and optionally one or more substituents R 3a ), -(C Alk alkylene optionally substituted with one or more substituents R 1-4 )-(heterocyclyl having 5 to 20 ring atoms and optionally one or more substituents R 3a ), -(carbocyclyl having 6 to 20 ring atoms and optionally one or more substituents R 3a ), and -(C Alk alkylene optionally substituted with one or more substituents R 1-4 )-(carbocyclyl having 6 to 20 ring atoms and optionally one or more substituents R 3a ), During the ceremony, Each R 2a -Halogen, -CN, -CF 3 ,-CHF 2 ien-CH 2 F, -OCF 3 , -OCHF 2 , -OCH 2 F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O 2 )NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -O-C 1-6 Independently selected from the group consisting of alkyls, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R 3a -Halogen, -CN, -CF 3 ,-CHF 2 ien-CH 2 F, -OCF 3 , -OCHF 2 , -OCH 2 F, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O 2 )NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, or -O-C 1-6 Independently selected from the group consisting of alkyls, each R* is either H or C 1-6 Alkyl or C 1-6 Selected independently from cycloalkyl, Each R Alk This is independently selected from the group consisting of -halogens and -CN. A pharmaceutical composition for use according to any one of claims 1 to 5, having a chemical structure or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

7. In equation (1), the following applies: a) R 1 This is selected from F, Cl, Br and CN, preferably from F, Cl and CN, more preferably from F and Cl, and even more preferably from Cl. b) R 2 is -(5 to 10 ring atoms and optionally one or more substituents R 2a (a heteroaryl having) and -(6 to 10 ring atoms and optionally one or more substituents R 2a From an aryl having (5 to 7 ring atoms and optionally one or more substituents R), preferably - (5 to 7 ring atoms and optionally one or more substituents R 2a (a heteroaryl having) and -(6 or 10 ring atoms and optionally one or more substituents R 2a From aryls having one or more substituents R, more preferably benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine (all of which have one or more substituents R 2a Selected from (which may be optionally substituted with R), more preferably benzene, naphthalene, imidazolidine, oxazole and pyridine (each of which has one or more substituents R 2a More preferably, benzene, imidazolidine, oxazole and pyridine (each of which may be optionally substituted with R) 2a (which may be optionally substituted with) more preferably one or more substituents R 2a More preferably, one or more substituents R from a benzene which may be optionally substituted. 2a From benzene substituted with, most preferably, the 4-position is R 2a Selected from benzenes substituted with c) R 3 is -(5 to 14 ring atoms and optionally one or more substituents R 3a (a heterocycline having), - (one or more substituents R Alk C is arbitrarily substituted with 1-4 Alkylene)-(5 to 14 ring atoms and optionally one or more substituents R) 3a A heterocycline having (6 to 14 ring atoms and optionally one or more substituents R) 3a (Carbocyclyl having), and - (one or more substituents R Alk C is arbitrarily substituted with 1-4 Alkylene)-(6 to 14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (Carbocyclyl having) and - (one or more substituents R Alk C arbitrarily substituted with 1-4 Alkylene)-(6 to 14 ring atoms and optionally one or more substituents R) 3a Selected from a carbocyclyl having (6 to 14 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily substituted with 1-4 Alkylene)-(6 to 14 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 to 10 ring atoms and optionally one or more substituents R 3a (aryl having) and -(one or more substituents R Alk C arbitrarily substituted with 1-4 Alkylene)-(6 to 10 ring atoms and optionally one or more substituents R) 3a Selected from aryls having (6 or 10 ring atoms and optionally one or more substituents R) 3a (aryl having) and -(one or more substituents R Alk C arbitrarily substituted with 1-4 Alkylene)-(6 or 10 ring atoms and optionally one or more substituents R) 3a Selected from aryls having -(one or more substituents R 3a Phenyl having (one or more substituents R) and -(phenyl) Alk C arbitrarily substituted with 1-4 Alkylene)-(one or more substituents R) 3a Selected from naphthyl having, and more preferably selected from 2,5-dimethylphenyl or 1-naphthylmethyl, d) C 1-4 The alkylene is preferably methylene or ethylene, and more preferably methylene. e) Substituent R 2a Either none, or one, two, or three substituents R are present, preferably 2a Either none, or one or two substituents R are present, more preferably one substituent R 2a A substituent R is present, and more preferably, one substituent R is present at the 4-position. 2a There exists, f) Substituent R 3a is absent, or 1, 2 or 3 are present; preferably, substituent R 3a is absent, or 1 or 2 are present; more preferably, when R 3 does not contain a -(C 1-4 alkylene) group, there are two substituents R 3a present; more preferably, when R 3 contains a -(C 1-4 alkylene) group, one substituent R 3a is present or absent, g) Substituent R Alk Either none, or one, two, or three substituents R are present, preferably Alk Either none, or one or two exist, more preferably substituent R Alk There are either no substituents or one substituent exists, and more preferably substituent R Alk It does not exist. h) each R 2a is independently selected from the group consisting of -halogen, -CN, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O 2 )NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-6 alkyl or -O-C 1-6 haloalkyl, wherein each R* is independently selected from H or C 1-6 alkyl or C 1-6 cycloalkyl; preferably, each R 2a is independently selected from the group consisting of -halogen, -CN, C 1-3 alkyl, C 1-3 haloalkyl, cyclopropyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-3 alkyl or -O-C 1-3 haloalkyl, wherein each R* is independently selected from H or C 1-3 alkyl or cyclopropyl; more preferably, each R 2a is independently selected from the group consisting of -halogen, -CN, C 1-3 alkyl, -NR*R*, -NR*COR*, -C(O)OR*, -C(O)NR*R*, -OH, -O-C 1-3 alkyl or -O-C 1-3 haloalkyl, wherein each R* is independently selected from H or C 1-3 alkyl or cyclopropyl; even more preferably, each R 2a is independently selected from the group consisting of -halogen, -CN, C 1-3 alkyl or -O-C 1-3 alkyl; even more preferably, each R 2a is independently selected from the group consisting of -halogen, -CN or -O-C 1-3 alkyl; even more preferably, each R 2a is independently -O-C 1-3 alkyl; even more preferably, each R 2a is independently methoxy or ethoxy, most preferably methoxy. i) Each R 3a is -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -NR*R*, -NR*COR*, -NR*C(O)NR*R*, -NR*S(O 2 )NR*R*, -C(O)OR*, -C(O)NR*R*, -OH, -OC 1-6 Alkyl or -O-C 1-6 Independently selected from the group consisting of haloalkyls, where each R* is H or C 1-6 Alkyl or C 3-6 Independently selected from cycloalkyl groups, preferably each R 3a These are independently -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, -O-C 1-6 Alkyl or -O-C 1-6 Selected from the group consisting of haloalkyls, more preferably each R 3a is -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, or C 3-6 Independently selected from the group consisting of cycloalkyls, and more preferably each R 3a is -halogen, -CN, C 1-3 Alkyl, or C 1-3 Independently selected from the group consisting of haloalkyls, and more preferably each R 3a is -halogen and C 1-3 Independently selected from the group consisting of alkyls, and more preferably each R 3a C is such as methyl or ethyl. 1-3 Independently selected from the group consisting of alkyls, and more preferably methyl, j) Each R Alk The elements are independently selected from the group consisting of -F and -Cl, preferably each R Alk is -F, and more preferably each R Alk It does not exist, and / or k) The NPBWR1 inhibitory small molecule binding molecule has a chemical structure represented by the following formula (1) or a pharmaceutically acceptable salt or solvate thereof. A pharmaceutical composition for use according to claim 6, wherein one or more of the following conditions are met.

8. The aforementioned antagonist / inhibitor is an NPBWR1 inhibitory small molecule binding molecule, and has the following chemical structure represented by formulas (2) to (9): 【Chemistry 2】 (In the formula, R 1 , R 2a and R 3 (This is as described in claim 6 or 7.) A pharmaceutical composition for use according to any one of claims 1 to 7, having one of the following:

9. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcriptional repressor domain and an epigenetic modification domain capable of suppressing the expression of NPBWR1.

10. A pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for use in methods of treating, improving or preventing bipolar affective disorder (ICD-10 F31) during the manic phase, appetite disorders, preferably anorexia nervosa or bulimia nervosa.

11. The pharmaceutical composition for use according to claim 10, wherein the agonist / activator is selected from an NPBWR1 activating peptide, an NPBWR1 activating small molecule binding molecule, and an NPBWR1 RNA molecule.

12. The pharmaceutical composition for use according to claim 10 or 11, wherein the agonist / activator is neuropeptide B or neuropeptide W.

13. A pharmaceutical composition for use according to any one of claims 9 to 12, wherein the agonist / activator is a CRISPR / Cas system specifically directed to NPBWR1, the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused to an effector domain selected from the group consisting of a transcription activator domain and an epigenetic modification domain capable of activating the expression of NPBWR1.

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