Neuropeptide b and w receptors as targets for the treatment of mood disorders and / or chronic stress

Abstract

Pharmaceutical compositions comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor are described for use in methods of treating, ameliorating or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease. Furthermore, pharmaceutical compositions comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator are described for use in methods of treating, ameliorating or preventing bipolar disorder manic (ICD-10 F31), appetite disorders, preferably anorexia or bulimia. Furthermore, a method of assessing the activity of a candidate molecule suspected to be a NPBWR1 antagonist / inhibitor or agonist / activator is described.

Description

[0001] This invention relates to pharmaceutical compositions comprising 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. Furthermore, this invention relates to pharmaceutical compositions comprising neuropeptide B / W receptor (NPBWR1) agonists / activators, used in methods for treating, improving, or preventing manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia. Additionally, this invention relates to a method for evaluating the activity of candidate molecules suspected to be NPBWR1 antagonists / inhibitors or agonists / activators.

[0002] Chronic stress is a major risk factor for mental illness, including major depressive disorder (MDD), and is also a leading cause of disability and suicide. 1,2 Chronic stress and related illnesses such as anxiety disorders are on the rise. 3–9 And it is one of the largest categories of healthcare spending. 10,11 The neural characteristics of chronic stress can be reliably modeled in mice. 12,13 Furthermore, it has been previously observed that caffeine rapidly affects mood-related behaviors. This effect shows that it is independent of changes in physical activity. 14 In the nucleus accumbens (NAc), at the heart of the brain's reward system, caffeine alters the binding of the CLOCK / BMAL transcription factor complex to chromatin. This mechanism has been shown to depend on Thr75 phosphorylation of DARPP-32 and occur in a circadian rhythm. 14 However, whether the affected genes are related to mood and related functional disorders remains unknown. Furthermore, little is known about how to improve chronic stress and depression through rapid environmental interventions. Moreover, among the increasing number of antidepressants, only the controlled substance ketamine has a rapid onset of action.

[0003] Given the existing technology, there is a need to provide further means and methods for treating, improving or preventing mood disorders and / or chronic stress and / or anxiety disorders.

[0004] This invention addresses this need and is based on the following surprising discovery: neuropeptide B and W receptors ( Npbwr1 GPR7 (also known as GPR7) is a key mediator of depression and stress response, and corresponding antagonists such as CYM50769 are potential compounds that rapidly modulate this pathway in a beneficial manner, thus identifying a new, rapidly acting pathway that can quickly improve stress and depressive symptoms.

[0005] More specifically, as shown in the examples below, transcriptomic features were identified that were rapidly altered in NAc in a T75-DARPP-32 and light-phase-dependent manner by caffeine. Surprisingly, neuropeptide B and W receptors ( Npbwr1 GPR7 (also known as GPR7) has been identified as a key mediator of emotion-related states in mice.

[0006] This discovery is even more surprising because Npbwr1 is a little-studied G protein-coupled receptor for neuropeptides B (NPB) and W (NPW). 15 . Npbwr1 Expressed in multiple brain regions, including NAc 16 Incidental research on neuropeptide B has shown that it is associated with sleep regulation, mood, and eating behavior. 17 Npbwr1- / - mice did not exhibit circadian rhythm abnormalities. 18 Or changes in exercise activities 18 Both NPB and NPW are generated by brain regions projecting to the NAc, including the ventral tegmental region and the dorsal raphe nucleus. 19,20 .

[0007] As demonstrated in the following appended embodiments, it has been shown that Npbwr1 Caffeine levels decrease 24 hours after injection, but increase with chronic variable stress (CVS). This has been demonstrated using viral-mediated gene transfer. Npbwr1 A causal link between stress-related phenotypes and [other factors]. Furthermore, post-mortem NAc levels in patients with depression have been confirmed. NPBWR1 Changes will occur. RNA sequencing of viral Npbwr1 overexpression revealed key genes involved in the antidepressant response. Bdnf The connection between 21 Using synthetic Npbwr1 - The antagonist CYM5069, microinjected into the NAC, can reverse the effects of chronic stress on behavior, and changes begin to occur 24 hours after a single dose. Bdnf The level lasted for up to 7 days. On the other hand, microinjection of the natural agonist NPB mimicked the effects of chronic stress and had a significant impact on... Bdnf It had the opposite effect.

[0008] Furthermore, the remarkable findings of this invention pave the way for providing neuropeptide B and W receptor antagonists as potential compounds for the treatment of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is characterized by the progressive loss of dopaminergic circuits.

[0009] In fact, caffeine, a well-known adenosine receptor antagonist, has been shown to have neuroprotective effects and has been associated with a reduced risk of developing Parkinson's disease (PD) 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 mediated downstream of the adenosine 2 receptor, which is highly expressed in dopaminergic neurons in the midbrain (including NAc). The PD-protective effect of caffeine is facilitated by several different mechanisms, including mitochondrial regulation and anti-inflammatory pathways.

[0010] As described above and further illustrated below, 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)), and this pathway is associated with mood-related behaviors. Importantly, mood disorders, including depression, are often co-occurring with PD, suggesting a shared cellular type or signaling event. Therefore, inhibiting Npbwr1 not only produces a mood-enhancing effect similar to that following caffeine administration but also exerts a neuroprotective effect on cellular types affected by PD.

[0011] Therefore, in summary, this invention, based on the surprising discovery of previously unknown pathways that have a rapid impact on stress- and depression-related behaviors, with neuropeptide B and W receptors (Npbwr1) being key mediators, paves the way for further means and methods to treat, improve, or prevent mood disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease by antagonizing neuropeptide B and W receptors (Npbwr1).

[0012] In view of the prior art, the technical problem of the present invention is to provide an alternative means and method for treating, improving or preventing mood disorders / emotional disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0013] This technical problem is solved by providing the embodiments described in the claims.

[0014] Therefore, in a first aspect, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in methods for treating, improving or preventing mood disorders / emotional disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0015] In a preferred embodiment, within the context of the first aspect of the invention, the invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in a method for treating, improving, or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein said neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor is not an active agent that modulates the binding properties between a GPR7 / NPBWR1 peptide and a peptide ligand corresponding to: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0016] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor does not contain or is not composed of an active agent that modulates the binding properties between the GPR7 / NPBWR1 peptide and a peptide ligand corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0017] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor is an antagonist / inhibitor as defined above and below, provided that it excludes an active agent that modulates the binding properties between the GPR7 / NPBWR1 peptide and a peptide ligand corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0018] The following peptide ligands are disclosed in WO 03 / 082907 as SEQ ID NO:1, 3, 5 and 7 and are described as agonists of GRP7 / NPBWR1: peptide ligands of L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0019] The neuropeptide B / W receptor (NPBWR1) is known in the art and is a member of the G protein-coupled receptor superfamily that integrates membrane proteins and binds neuropeptides B and W. This receptor is known to be primarily expressed in the CNS and is known to have numerous functions, including regulating cortisol secretion.

[0020] More specifically, NPBWR1 is known to have the following functions and activities:

[0021] It is known that NPBWR1 regulates / inhibits Bdnf expression;

[0022] NPBWR1 is known to bind to neuropeptide B / W; and

[0023] NPBWR1 is known to bind to G proteins.

[0024] These functional capabilities of the NPBWR1 can be readily assessed by a skilled technician using conventional methods and tests known in the art, as further illustrated below.

[0025] The neuropeptide B / W receptor (NPBWR1) is highly conserved in vertebrates, with 66 to 100% homology between rodents and humans, and they share structural and functional similarities.

[0026] The coding region of the neuropeptide B / W receptor (NPBWR1) is known in the art, particularly from humans; see, for example, UniProt accession number P48145. NPBWR1 is a classic 7-transmembrane receptor.

[0027] 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 SEQ ID NO:1, and can be retrieved from relevant databases such as NCBI, EMBL, Uniprot, etc.:

[0028] MDNASFSEPWPANANASGPDPALSCSNASTLAPLPAPLAVAVPVVYAVICAVGLAGNSAVLYVLLRAPRMKTVTNLFILNLAIADELFTLVLPINIADFLLRQWPGELMCKLIVAIDQYNTFSSLYFLTVMSADRYLVVLATAESRRVAGRTYSAARAVSLAVWG IVTLVVLPFAVFARLDDEQGRRQCVLVFPQPEAFWWRASRLYTLVLGFAIPVSTICVLYTTLLCRLHAMRLDSHAKALERAKKRVTFLVVAILAVCLLCWTPYHLSTVVALTTDLPQTPLVIAISYFITSLSYANSCLNPFLYAFLDASFRRNLRQLITCRAAA

[0029] The above sequence, SEQ ID NO:1, was retrieved from UniProt, accession number P48145. The term "neuropeptide B / W receptor (NPBWR1)" as used herein also includes its variants and orthologs, such as the mouse neuropeptide B / W receptor (NPBWR1). The corresponding nucleotide sequences can also be retrieved from the relevant databases.

[0030] Therefore, in particular, the wild-type human neuropeptide B / W receptor (NPBWR1) encoded by the nucleic acid sequence represented by SEQ ID NO:2 can be used according to the present invention.

[0031] This study preferably uses human patients / subjects. Therefore, the preferred neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists are human neuropeptide B / W receptor (NPBWR1) inhibitors / antagonists, especially when the patient / subject is human.

[0032] Therefore, the neuropeptide B / W receptor (NPBWR1) molecules used in the context of this invention include, but are not limited to, molecules encoded by the nucleic acid molecules described herein. Orthologs of the neuropeptide B / W receptor (NPBWR1) are also contemplated, which are at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence shown in SEQ ID NO:2. These neuropeptide B / W receptor (NPBWR1) molecules referred to herein are defined as molecules capable of functioning as the neuropeptide B / W receptor (NPBWR1) as described in the context. These functions and activities particularly include the ability to have the neuropeptide B / W receptor (NPBWR1) activities as described above and below, namely, the ability to regulate / inhibit Bdnf expression; the ability to bind neuropeptide B / W; and / or the ability to bind G proteins.

[0033] To test the activity of the functional neuropeptide B / W receptor (NPBWR1), the assay methods provided below and illustrated in the accompanying examples can be used.

[0034] The ability of NPBWR1 to regulate / inhibit Bdnf expression can be assessed using methods such as quantitative PCR to determine Bdnf expression.

[0035] To assess the ability of NPBWR1 to bind neuropeptide B / W, for example, FRAP (fluorescence recovery after photobleaching) can be used, where neuropeptide B or neuropeptide W is used as a substrate to evaluate the binding of NBWBR1.

[0036] To assess the ability of NPBWR1 to bind to G proteins, one can use, for example, FRAP (fluorescence recovery after photobleaching), where G protein subunits are used as substrates to evaluate the binding of NBWBR1.

[0037] The corresponding testing methods are known in the art and can be easily performed by technicians using conventional methods.

[0038] Unbound by theoretical constraints, Npbwr1 activity is often indirectly measured by regulating / activating the expression of the downstream protein brain-derived neurotrophic factor (Bdnf). Quantitative PCR can be used to assess Bdnf RNA levels. Increased Bdnf expression indicates Npbwr1 inhibition, while decreased Bdnf expression indicates Npbwr1 activation.

[0039] Alternatively, an activity assay based on the G protein coupling properties of Npbwr1 can be envisioned. Therefore, changes in activity are translated into differential binding with the G protein. Using Förster resonance energy transfer (FRET), changes in the interaction between Npbwr1 and its G protein can be visualized by fusing these components with a fluorophore. If the two are sufficiently close due to mutual binding, activation of the fluorophore attached to Npbwr1, which has a low wavelength, will emit fluorescence at the wavelength that excites the fluorophore attached to the G protein. The increase in fluorescence indicates the ability of NPBWR1 to bind to the G protein.

[0040] Furthermore, it is envisioned that a neuropeptide B / W receptor (NPBWR1) ortholog is identical to the amino acid sequence shown in SEQ ID NO:1 by at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, and is capable of functioning as the neuropeptide B / W receptor (NPBWR1) as described in the context.

[0041] To determine whether a nucleic acid sequence is identical to a nucleic acid encoding a neuropeptide B / W receptor (NPBWR1) or to the amino acids of the neuropeptide B / W receptor (NPBWR1), a person skilled in the art may use means and methods known in the art, such as manual comparison or comparison using computer programs (such as those mentioned below).

[0042] According to the present invention, in the context of two or more nucleic acid or amino acid sequences, the term "identity" or "percentage of identity" means that two or more sequences or subsequences are identical, or have a specified percentage of identical amino acid residues or nucleotides (e.g., 60% or 65% identity with, for example, the nucleic acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:1, preferably 70-95% identity, more preferably at least 95% identity, and capable of functioning as the functional neuropeptide B / W receptor (NPBWR1) as described above and below) when compared and aligned within a comparison window or a specified region to obtain maximum correspondence using sequence comparison algorithms known in the art or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or higher sequence identity are considered substantially identical. This definition also applies to complementary sequences of the test sequence. Preferably, the identity is present in a region of at least about 15 to 25 amino acids or nucleotides in length, more preferably in a region of about 50 to 100 amino acids or nucleotides in length. Those skilled in the art know how to determine the percentage of identity between (two or more) sequences using algorithms such as those based on the CLUSTALW computer program (Thompson Nucl. AcidsRes. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6. (1990), 237-245), as is known in the art.

[0043] Although the FASTDB algorithm typically does not account for internal mismatches, deletions, or additions (vacancy) in the sequence during its calculations, this can be manually corrected to avoid overestimating the sequence identity percentage. However, CLUSTALW considers sequence vacancy in its identity calculations. Those skilled in the art can also utilize 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). The BLASTN program (for nucleic acid sequences) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W) and an expected value of 10 (E). The BLOSUM62 rating matrix (Henikoff (1989) PNAS 89:10915) uses a comparison of 50 (B), an expected value of 10 (E), M=5, N=4, and a comparison of two chains.

[0044] On the one hand, the present invention relates to inhibiting / antagonizing the activity of neuropeptide B / W receptor (NPBWR1) by blocking / inhibiting downstream pathways of neuropeptide B / W receptor (NPBWR1) (e.g., brain-derived neurotrophic factor (BDNF) signaling), paving the way for the use of inhibitors / antagonists of neuropeptide B / W receptor (NPBWR1) in the methods of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0045] This invention provides an inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1) for use in methods of treating, improving or preventing mood disorders / emotional disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0046] This document envisions these inhibitors / antagonists as being used as medicines or pharmaceutical compositions, specifically the neuropeptide B / W receptor (NPBWR1) inhibitors provided and described herein for medicinal purposes (e.g., for the therapy / treatment / improvement / prevention of diseases, particularly those associated with mood / emotional disorders and / or chronic stress and / or anxiety and / or Parkinson's disease). The terms "medicine" and "pharmaceutical composition" are used interchangeably herein. Therefore, the definitions and interpretations of "pharmaceutical composition" provided herein, with necessary modifications, apply to the term "medicine."

[0047] The terms “antagonist” or “inhibitor” are used interchangeably herein. These terms are known in the art and refer to compounds / substances capable of completely or partially preventing or reducing the physiological activity of one or more specific proteins. Thus, in the context of this invention, the antagonist / inhibitor can prevent or reduce, inhibit or inactivate the physiological activity of a protein, such as the neuropeptide B / W receptor (NPBWR1), when the compound / substance binds to it. The binding of an “antagonist / inhibitor” to a given protein, such as the neuropeptide B / W receptor (NPBWR1), can compete for or prevent the binding of endogenous activating molecules that bind to said protein. Thus, as used herein, the term “antagonist” also encompasses competitive antagonists, (reversible) non-competitive antagonists, or irreversible antagonists, especially as described in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. However, in addition to this, in the context of the present invention, an “antagonist” or “inhibitor” of the neuropeptide B / W receptor (NPBWR1) can also prevent the function of said protein by blocking / reducing the expression of nucleic acid molecules encoding a given protein such as the neuropeptide B / W receptor (NPBWR1). Therefore, neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors can lead to 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 decrease in activity can be measured / detected by methods known in the art and described herein. Therefore, in the context of the present invention, neuropeptide B / W receptor (NPBWR1) inhibitors can also encompass transcriptional repressors capable of reducing the function of the neuropeptide B / W receptor (NPBWR1) and its expression. As described in detail below, the reduction of expression and / or activity of neuropeptide B / W receptor (NPBWR1) by neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors leads to a decrease in the activity (and / or expression) of neuropeptide B / W receptor (NPBWR1), thereby reducing the functional capacity of neuropeptide B / W receptor (NPBWR1).

[0048] Consistent with the basic principles of this invention, the reduction in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have multiple medical implications.

[0049] As explained below, a decrease in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have medical significance, namely, an impact on diseases / disorders associated with mood / affect and / or chronic stress and / or anxiety and / or Parkinson's disease, such as mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0050] The terms “inhibitor” and / or “antagonist” do not imply any specific biological mechanism of action and are considered to explicitly include and cover 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” should be clearly understood to encompass all previously identified terms, names, and functional states and characteristics whereby the neuropeptide B / W receptor (NPBWR1) itself, the biological activity of the neuropeptide B / W receptor (NPBWR1) (including, but not limited to, its ability to regulate / inhibit Bdnf expression; its ability to bind neuropeptide B / W; and / or its ability to bind G proteins), or the result of its biological activity, is significantly eliminated, reduced, or neutralized to any meaningful extent, such meaningful extent as at least 5%, 10%, 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, 500%, or 2, 3, 4, 5, 10, 20, 50, 100, or 1000 times. Inhibitors / antagonists can reduce abnormal levels of the biological activity of the neuropeptide B / W receptor (NPBWR1), which can cause adverse reactions and / or disease in subjects, to levels corresponding to those in healthy subjects, thereby preventing, halting the progression and / or curing adverse reactions and / or disease.

[0051] Therefore, one aspect of the present invention relates to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) in a method for treating, improving or preventing mood disorders / emotional 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, 3, 4, 5, 10, 20, 50, 100 or 1000 times.

[0052] Therefore, one aspect of the present invention relates to an inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1) in a method for 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 the abnormal level of biological activity of neuropeptide B / W receptor (NPBWR1) that causes / or promotes adverse reactions and / or disease by at least 2, 3, 4, 5, 10, 20, 50, 100, or 1000 times.

[0053] Therefore, one aspect of the present invention relates to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) in a method for 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 the abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse reactions and / or disease by at least 3-fold.

[0054] Therefore, one aspect of the present invention relates to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) in a method for 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 the abnormal level of biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and promotes mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0055] Therefore, one aspect of the present invention relates to an inhibitor / antagonist of the neuropeptide B / W receptor (NPBWR1) in a method for 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 abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes / 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.

[0056] According to the present invention, the term "inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1)" also refers to a compound or substance capable of completely or partially preventing or reducing the physiological activity of neuropeptide B / W receptor (NPBWR1). In the context of the present invention, the inhibitor can therefore prevent, reduce, inhibit, or inactivate the physiological activity of neuropeptide B / W receptor (NPBWR1), for example, when the compound / substance (i.e., the inhibitor / antagonist) binds to neuropeptide B / W receptor (NPBWR1).

[0057] As used herein, the term “inhibitor” also encompasses inhibitors that result in reversible inhibition (e.g., competitive inhibition, uncompetitive inhibition, noncompetitive inhibition, or mixed inhibition of the neuropeptide B / W receptor (NPBWR1)) or irreversible inhibition (e.g., inhibition through covalent interactions).

[0058] Inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) can also inhibit the function of the neuropeptide B / W receptor (NPBWR1) by blocking / reducing the expression of nucleic acid molecules encoding NPBWR1. Therefore, inhibitors / antagonists of NPBWR1 can lead to decreased expression levels of NPBWR1 gene products, such as reduced levels of NPBWR1 mRNA and / or NPBWR1 protein.

[0059] Inhibitors / antagonists of neuropeptide B / W receptor (NPBWR1) can reduce the abnormal expression levels of neuropeptide B / W receptor (NPBWR1) that can cause and / or promote adverse reactions and / or diseases / conditions associated with mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, as outlined above and below, to levels corresponding to those in healthy subjects, thereby preventing, halting, or curing the progression of adverse reactions and / or diseases / conditions in subjects. This can be reflected in decreased neuropeptide B / W receptor (NPBWR1) expression and / or aberrant reduction in neuropeptide B / W receptor (NPBWR1) expression, thereby restoring healthy levels of neuropeptide B / W receptor (NPBWR1) expression. The expression level of neuropeptide B / W receptor (NPBWR1) is correlated to some extent with neuropeptide B / W receptor (NPBWR1) activity until the translation mechanism and / or the substance binding to neuropeptide B / W receptor (NPBWR1) reaches saturation. The expression level of neuropeptide B / W receptor (NPBWR1) can be measured / detected using methods known in the art.

[0060] Therefore, one aspect of the present invention relates to inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) for 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 reduces the expression level of the neuropeptide B / W receptor (NPBWR1) gene product.

[0061] Therefore, one aspect of the present invention relates to inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) for 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 inhibitors / antagonists reduce the abnormal expression levels of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes adverse reactions and / or diseases / disorders.

[0062] Therefore, one aspect of the present invention relates to inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) for treating, improving, or preventing disorders associated with mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease according to the present invention, wherein the inhibitor / antagonist reduces the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes the aforementioned mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0063] Therefore, one aspect of the present invention relates to inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) for treating, improving, or preventing disorders associated with mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease according to the present invention, 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 subjects to an expression level comparable to that of healthy subjects.

[0064] Inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) can exert their inhibitory function by interacting directly with the NPBWR1 protein, i.e., by interacting with any part of the NPBWR1 protein, such as its extracellular domain, transmembrane domain, and / or cytoplasmic domain. Inhibitors / antagonists of NPBWR1 can also exert any inhibitory effect on NPBWR1 function by blocking, reducing, inhibiting, or inactivating any upstream or downstream pathway components that substantially contribute to NPBWR1 function. Inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) can exert any indirect inhibitory effect on any neuropeptide B / W receptor (NPBWR1) activating molecules such as nucleic acids, ribonucleic acid (RNA), double-stranded ribonucleic acid (dsRNA), chromatin reader protein and / or ligands.

[0065] The efficacy of inhibitors / antagonists of neuropeptide B / W receptor (NPBWR1) can be described using the half-maximal inhibitory concentration (IC50) value. For the purposes of this invention, inhibitors / antagonists of neuropeptide B / W receptor (NPBWR1) preferably exhibit low IC50 values. The IC50 value of an NPBWR1 inhibitor / antagonist can be below 100 µM, below 90 µM, below 80 µM, below 70 µM, below 60 µM, below 50 µM, below 40 µM, below 30 µM, below 20 µM, or below 10 µM, wherein lower values ​​are preferred over higher values. Preferably, the IC50 value of an NPBWR1 inhibitor / antagonist can be below 10 µM, below 9 µM, below 8 µM, below 7 µM, below 6 µM, below 5 µM, or below 4 µM. Preferably, the IC50 value of the inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1) can be less than 4 μM.

[0066] Therefore, the present invention relates to an inhibitor / antagonist of neuropeptide B / W receptor (NPBWR1) for 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 half-maximal inhibitory concentration (IC50) efficacy 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.

[0067] Technicians know how to determine the IC50 value of inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1). This paper hypothesizes that inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) may exhibit additional IC50 values ​​and / or identify other inhibitors / antagonists of the neuropeptide B / W receptor (NPBWR1) with other IC50 values.

[0068] Mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease are disorders or syndromes known in the art and are medical indications classified in the ICD system (i.e., the well-known list of medical classifications established by the World Health Organization (WHO)). ICD stands for International Statistical Classification of Diseases and Related Health Problems. In the following text, when referring to a more specifically defined disorder or disease, reference is made to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), namely “ICD 10”.

[0069] This invention is not limited to any specific mood / emotional disorder and / or chronic stress and / or anxiety disorder. Any mood / emotional disorder and / or chronic stress and / or anxiety disorder and / or Parkinson's disease can be treated, improved, and / or prevented by the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor according to the present invention.

[0070] Without being bound by theory, in a particularly preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in a method for 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.

[0071] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in a method for treating, improving, or preventing mood disorders / affective disorders, wherein the mood disorder / affective disorder is selected from:

[0072] (ii) Bipolar disorder (ICD-10 F31), preferably hypomanic bipolar disorder (ICD-10 F31);

[0073] (iii) Depressive episode (ICD-10 F32);

[0074] (iv) Recurrent depressive disorder (ICD-10 F33);

[0075] (v) Persistent affective disorder (ICD-10 F34);

[0076] (vi) Other mood and affective disorders (ICD-10 F38); and

[0077] (vii) Undefined mood / affective disorder (ICD-10 F39).

[0078] The mood / affective disorders of this invention generally refer to disorders in which the underlying disturbance is a change in mood or affect towards depression (with or without associated anxiety) or elevated mood. Mood changes are usually accompanied by changes in overall activity levels; most other symptoms are secondary to, or readily understood within, the context of changes in mood and activity. Most of these disorders tend to be recurrent, and the onset of an individual's episode is usually associated with a stressful event or situation.

[0079] More specific mood / affect disorder and its corresponding subdivision according to the present invention will be described in more detail below:

[0080] Manic episode (ICD-10 F30):

[0081] All subdivisions in this category apply only to single episodes. Hypomanic or manic episodes in individuals who have had one or more previous mood episodes (depression, hypomania, mania, or a combination of both) should be coded as bipolar disorder (F31.-).

[0082] The detailed explanation of manic episode (ICD-10 F30) is as follows:

[0083] F30.0: Hypomania:

[0084] A disorder characterized by persistent, mild euphoria, increased energy and activity, often accompanied by significant feelings of well-being and psychosocial efficiency. Common symptoms include increased social skills, talkativeness, excessive intimacy, increased libido, and decreased sleep needs, but these do not severely impact work or lead to social withdrawal. Irritability, arrogance, and rudeness may replace the more common euphoric socialization. The emotional and behavioral disturbances are not accompanied by hallucinations or delusions.

[0085] F30.1: Mania without psychotic symptoms:

[0086] Elevated mood, disproportionate to the patient's current situation, can range from carefree joy to almost uncontrollable excitement. This elevated mood is accompanied by increased energy, leading to hyperactivity, rapid speech, and reduced sleep needs. Attention cannot be sustained, and significant distractibility is common. Inflated ego is frequently present, accompanied by exaggerated ideas and overconfidence. Loss of normal social inhibition can result in reckless, impulsive, and socially inappropriate or inconsistent behavior.

[0087] F30.2: Mania with psychotic symptoms:

[0088] In addition to the clinical manifestations described in F30.1, there may be delusions (usually grandiose delusions) or hallucinations (usually voices speaking directly to the patient), or agitation, hypermotor activity, and flight of ideas that are so severe that the patient cannot be understood or communicate normally.

[0089] Mania accompanied by symptoms of both consistent and inconsistent psychosis.

[0090] Manic stupor

[0091] F30.8: Other manic episodes:

[0092] F30.9: Undefined manic episode

[0093] Manic NOS

[0094] Bipolar disorder (ICD-10 F31), preferably hypomanic bipolar disorder (ICD-10 F31):

[0095] A disorder characterized by two or more episodes that severely disturb the patient's mood and activity levels, sometimes manifesting as elevated mood, increased energy and activity (hypomania or mania), and sometimes as depressed mood, decreased energy and activity (depression). Recurrent episodes of only hypomania or mania are classified as bipolar disorder.

[0096] including:

[0097] Manic-depressive disorder

[0098] Bipolar disorder:

[0099] disease

[0100] mental illness

[0101] reaction

[0102] exclude:

[0103] Bipolar disorder, with a single manic episode (F30 -)

[0104] Cyclic affective disorder (F34.0)

[0105] The breakdown of bipolar disorder (ICD-10 F31) is explained below:

[0106] F31.0: Bipolar disorder, currently experiencing a hypomanic episode.

[0107] The patient is currently in a hypomanic state and has had at least one other emotional episode in the past (hypomania, mania, depression, or a combination thereof).

[0108] F31.1: Bipolar disorder, current episode of mania, no psychotic symptoms:

[0109] The patient is currently in a manic state, without psychotic symptoms (as described in F30.1), and has a history of at least one other emotional episode (hypomania, mania, depression, or a combination thereof).

[0110] F31.2: Bipolar disorder, current episode of mania with psychotic symptoms:

[0111] The patient is currently in a manic state with psychotic symptoms (as described in F30.2) and has a history of at least one other emotional episode (hypomania, mania, depression, or a combination thereof).

[0112] F31.3: Bipolar disorder, currently experiencing mild to moderate depression:

[0113] The patient is currently in a depressive state, such as a mild or moderate depressive episode (F32.0 or F32.1), and has had at least one confirmed hypomanic, manic, or mixed affective episode in the past.

[0114] F31.4: Bipolar disorder, current episode of severe depression, without psychotic symptoms:

[0115] The patient is currently in a depressive state, such as a major depressive episode without psychotic symptoms (F32.2), and has had at least one confirmed hypomanic, manic, or mixed affective episode in the past.

[0116] F31.5: Bipolar disorder, current episode of major depressive disorder with psychotic symptoms:

[0117] The patient is currently in a depressive state, such as a major depressive episode with psychotic symptoms (F32.3), and has had at least one confirmed hypomanic, manic, or mixed affective episode in the past.

[0118] F31.6: Bipolar disorder, current episode mixed type:

[0119] The patient has a history of at least one confirmed hypomanic, manic, depressive, or mixed affective episode and currently exhibits a mixture or rapid alternation of manic and depressive symptoms.

[0120] exclude:

[0121] A single mixed emotional episode (F38.0)

[0122] F31.7: Bipolar disorder, currently in remission:

[0123] The patient has a history of at least one confirmed hypomanic, manic, or mixed-type mood episode, in addition to at least one other mood episode (hypomanic, manic, depressive, or mixed), but is currently without any significant mood disturbances, which have persisted for several months. Remission periods during preventative treatment should be coded here.

[0124] F31.8: Other Bipolar Disorders

[0125] Bipolar disorder II

[0126] Recurrent manic episodes (NOS)

[0127] F31.9: Undetermined bipolar disorder

[0128] Bipolar disorder (NOS)

[0129] Depressive episode (ICD-10 F32):

[0130] In a typical mild, moderate, or severe depressive episode, patients experience symptoms such as depressed mood, decreased energy, and reduced activity. Their ability to enjoy life, interest, and attention diminishes, and they often feel noticeably tired even with minimal effort. Sleep is usually disturbed, and appetite decreases. Self-esteem and confidence almost always decline, and even in mild depression, feelings of guilt or worthlessness are common. The degree of depressed mood does not vary much from day to day, and there is no response to the environment. It may be accompanied by so-called "physical" symptoms, such as loss of interest and pleasure, waking up several hours earlier than usual in the morning (with the depression being most severe in the morning), marked psychomotor retardation, agitation, loss of appetite, weight loss, and decreased libido. Depressive episodes can be classified as mild, moderate, or severe based on the number and severity of symptoms.

[0131] including:

[0132] Single seizure:

[0133] Depressive reaction

[0134] Psychogenic depression

[0135] reactive depression

[0136] exclude:

[0137] Adaptability disorder (F43.2)

[0138] Recurrent depressive disorder (F33.-)

[0139] When related to conduct disorder in F91.-(F92.0)

[0140] The detailed explanation of depressive episode (ICD-10 F32) is as follows:

[0141] F32.0: Mild depressive episode:

[0142] Two or three of the above symptoms usually occur. Patients often experience distress due to these symptoms, but may still be able to continue most activities.

[0143] F32.1: Moderate depressive episode:

[0144] There are usually four or more of the above symptoms, and patients may find it difficult to continue their daily activities.

[0145] F32.2: Major depressive episode without psychotic symptoms:

[0146] A depressive episode is characterized by several of the aforementioned symptoms being pronounced and distressing, often manifesting as loss of self-esteem, feelings of worthlessness, or guilt. Suicidal thoughts and behaviors are common and are usually accompanied by a variety of physical symptoms.

[0147] Agitated depression

[0148] Major depression

[0149] Biological depression (Vital depression)

[0150] A single episode without psychotic symptoms

[0151] F32.3: Major depressive episode with psychotic symptoms:

[0152] A depressive episode that meets the description of F32.2, but is accompanied by hallucinations, delusions, psychomotor retardation, or stupor, severe enough to prevent normal social activities; it may be life-threatening due to suicide, dehydration, or starvation. Hallucinations and delusions may or may not be consistent with the mood.

[0153] Single seizure:

[0154] Major depressive disorder with psychotic symptoms

[0155] Psychogenic depressive psychosis

[0156] psychotic depression

[0157] Reactive depressive psychosis

[0158] F32.8: Other depressive episodes:

[0159] Atypical depression

[0160] Single-episode "hidden" depression (NOS)

[0161] F32.9: Undetermined depressive episode:

[0162] Depression NOS

[0163] Depressive Disorders (NOS)

[0164] Recurrent depressive disorder (ICD-10 F33):

[0165] A disorder characterized by recurrent depressive episodes that fit the description of a depressive episode (F32.-) without any history of independent elevating and hyperactive (manic) episodes. However, brief episodes of mild elevating and hyperactive (hypomanic) episodes may occur immediately after a depressive episode, sometimes induced by antidepressant medication. The more severe forms of recurrent depression (F33.2 and F33.3) share many similarities with earlier concepts such as bipolar disorder, melancholia, vital depression, and endogenous depression. The first episode can occur at any age, from childhood to old age, and the onset can be acute or insidious, lasting from weeks to months. The risk of a manic episode is not completely eliminated in patients with recurrent depression, regardless of the number of depressive episodes they have experienced. If a manic episode does occur, the diagnosis should be changed to bipolar disorder (F31.-).

[0166] including:

[0167] The following are recurring symptoms:

[0168] Depressive reaction

[0169] Psychogenic depression

[0170] reactive depression

[0171] Seasonal Depression

[0172] exclude:

[0173] Recurrent transient depressive episodes (F38.1)

[0174] The breakdown of recurrent depressive disorder (ICD-10 F33) is explained below:

[0175] F33.0: Recurrent depressive disorder, current episode mild:

[0176] A disorder characterized by recurrent depressive episodes, currently mild as described in F32.0, and without any history of mania.

[0177] F33.1: Recurrent depressive disorder, current episode moderate:

[0178] A disorder characterized by recurrent depressive episodes, with the current episode being of moderate severity, as described in F32.1, and without any history of mania.

[0179] F33.2: Recurrent depressive disorder, current episode severe, no psychotic symptoms:

[0180] A disorder characterized by recurrent depressive episodes, with the current episode being severe, without psychotic symptoms as described in F32.2, and without any history of mania.

[0181] Endogenous depression without psychotic symptoms

[0182] Recurrent major depressive disorder, without psychotic symptoms

[0183] Bipolar disorder, a type of depression without psychotic symptoms

[0184] Recurrent biological depression without psychotic symptoms

[0185] F33.3: Recurrent depressive disorder, current episode severe, with psychotic symptoms:

[0186] A disorder characterized by recurrent depressive episodes, with the current episode being severe, accompanied by psychotic symptoms as described in F32.3, and without a history of manic episodes.

[0187] Endogenous depression with psychotic symptoms

[0188] Depressive-manic psychosis with psychotic symptoms

[0189] Recurrent severe seizures:

[0190] Major depressive disorder with psychotic symptoms

[0191] Psychogenic depressive psychosis

[0192] psychotic depression

[0193] Reactive depressive psychosis

[0194] F33.4: Recurrent depressive disorder, currently in remission:

[0195] The patient has had two or more depressive episodes as described in F33.0-F33.3 in the past, but has not experienced depressive symptoms for several months.

[0196] F33.8: Other recurrent depressive disorders:

[0197] F33.9: Undiagnosed recurrent depressive disorder:

[0198] Unipolar depression (NOS)

[0199] Persistent affective disorder (ICD-10 F34):

[0200] Persistent and often fluctuating mood disorders, in most individuals, whose episodes are not severe enough to be described as hypomanic or mild depressive episodes. Because these symptoms can persist for years, sometimes even for much of adulthood, they can cause considerable distress and disability. In some cases, recurrent or isolated manic or depressive episodes may superimpose with persistent mood disorders. The subdivisions of persistent mood disorders (ICD-10F34) are explained below:

[0201] F34.0: Cyclothymic syndrome:

[0202] A persistent mood instability involving multiple episodes of depression and mild elevation, but neither of these moods is severe or lasting enough to be diagnosed as bipolar disorder (F31.-) or recurrent depressive disorder (F33.-). This condition is common in relatives of people with bipolar disorder. Some people with cyclothymia eventually develop bipolar disorder.

[0203] Emotional Personality Disorder

[0204] Cycloid personality

[0205] Cyclothymic personality

[0206] F34.1: Depressed mood:

[0207] A chronic mood depression that lasts for at least several years but is not severe enough or lasts long enough to be diagnosed as major, moderate or mild recurrent depressive disorder (F33.-).

[0208] Depression:

[0209] Neurosis

[0210] Personality Disorder

[0211] Neurotic depression

[0212] Persistent anxiety and depression

[0213] exclude:

[0214] Anxiety and depression (mild or non-persistent) (F41.2)

[0215] F34.8: Other persistent mood / affective disorders

[0216] F34.9: Undefined persistent mood / affective disorder

[0217] Other mood and affective disorders (ICD-10 F38):

[0218] Any other mood disorder that is not classified into F30-F34 due to insufficient severity or duration.

[0219] The breakdown of other mood and affective disorders (ICD-10 F38) is as follows:

[0220] F38.0: Other single-occurrence emotional [affective] disorders:

[0221] Mixed affective episodes

[0222] F38.1: Other recurrent mood / affective disorders:

[0223] Recurrent transient depressive episodes

[0224] F38.8: Other clearly defined mood / affective disorders.

[0225] Unspecified mood / affective disorder (ICD-10 F39):

[0226] including:

[0227] Affective Disorders (NOS)

[0228] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, for use in treating, improving, or preventing chronic stress, wherein the chronic stress is selected from:

[0229] (i) Response to severe stress and maladaptive disorder (ICD-10 F43);

[0230] (ii) Acute stress response (ICD-10 F43.0);

[0231] (iii) Post-traumatic stress disorder (ICD-10 F43.1);

[0232] (iv) Adaptation disorder (ICD-10 F43.2);

[0233] (v) Other responses to severe stress (ICD-10 F43.8); and

[0234] (vi) Undefined response to severe stress (ICD-10 F43.9).

[0235] In this invention, chronic stress is generally understood as a physiological or psychological response caused by long-term internal or external stressors. Whether the stressor is actual or recalled, it produces the same effect and triggers a chronic stress response. Chronic stressors are diverse, but most involve relatively long-term problems, conflicts, and threats encountered in daily life.

[0236] More specific details of chronic stress and its corresponding subdivisions according to the present invention will be described below in more detail:

[0237] Response to severe stress and adaptation disorder (ICD-10 F43);

[0238] This category includes disorders that can be identified not only by symptoms and course of illness, but also by the presence of one of two causative factors: an unusually stressful life event that triggers an acute stress response, or a significant life change that leads to a persistently unpleasant environment, thus triggering an adjustment disorder. While milder psychosocial stress (“life events”) may induce or contribute to the development of a variety of disorders categorized in other sections of this chapter, their etiological importance is not always clear, and in each case, it is found to depend on individual (often trait-based) vulnerability, meaning that life events are neither necessary nor sufficient to explain the occurrence and form of the disorder. In contrast, the disorders summarized here are considered to always be a direct result of acute severe stress or persistent trauma. Stressful events or persistent unpleasant environments are the primary and most important factors leading to the disorder; without their influence, the disorder would not occur. Therefore, the disorders in this section can be viewed as maladaptive responses to severe or persistent stress because they interfere with successful coping mechanisms, leading to problems with social functioning.

[0239] The breakdown of severe stress / response and adaptation disorders to severe stress (ICD-10 F43) is explained as follows:

[0240] Acute stress response (ICD-10 F43.0):

[0241] A transient disorder that occurs in individuals without any other obvious mental disorders, arising in response to unusual psychosomatic stress, and typically resolves within hours or days. An individual's vulnerability and coping abilities play a role in the occurrence and severity of the acute stress response. Symptoms are usually a mixed and fluctuating state, including an initial "confusion" state accompanied by a narrowing of consciousness and attention, inability to comprehend stimuli, and disorientation. Following this state, the patient may further detach from their surroundings (reaching the level of dissociative stupor - F44.2) or exhibit agitation and hyperactivity (escape response or fugue state). Autonomic signs of panic and anxiety (tachycardia, sweating, flushing) are common. Symptoms usually appear within minutes of the stressor or event and disappear within two to three days (usually within hours). Partial or complete amnesia related to the episode may occur (F44.0). If symptoms persist, a change in diagnosis should be considered.

[0242] acute:

[0243] Crisis Response

[0244] stress response

[0245] Combat fatigue

[0246] crisis

[0247] Psychological shock

[0248] Post-traumatic stress disorder (ICD-10 F43.1):

[0249] Post-traumatic stress disorder (PTSD) occurs as a delayed or prolonged response to a highly threatening or catastrophic stressful event or situation (of varying duration) and can cause widespread distress to almost anyone. Some predisposing factors, such as personality traits (e.g., obsessive-compulsive disorder, asthenia) or a history of neurosis, may lower the threshold for the onset of the syndrome or exacerbate its course, but these are neither necessary nor sufficient conditions to explain its occurrence. Typical features include: recurrent recurrence of the trauma in intrusive memories (“flashbacks”), dreams, or nightmares, accompanied by a persistent feeling of numbness and emotional dullness, social isolation, sluggishness of response to the surrounding environment, anhedonia, and avoidance of trauma-related activities and situations. A state of hypervigilance with autonomic hyperactivity, increased startle reflex, and insomnia are often present. Anxiety and depression are usually associated with the above symptoms and signs, and suicidal ideation is not uncommon. PTSD has a latency period that can range from weeks to months. The course of the illness is fluctuating, but recovery is expected in most cases. In a small percentage of cases, the condition may undergo a chronic process over many years, eventually transforming into a persistent personality change (F62.0).

[0250] Traumatic neurosis

[0251] Adjustment disorder (ICD-10F43.2):

[0252] Subjective distress and mood disturbances typically interfere with social functioning and performance, occurring during periods of adjustment to significant life changes or stressful life events. Stressors may affect the integrity of an individual's social network (e.g., bereavement, separation experience), or broader social support and value systems (e.g., immigration, refugee status), or represent a major developmental shift or crisis (e.g., starting school, becoming a parent, failure to achieve cherished personal goals, retirement). An individual's susceptibility or vulnerability plays a significant role in the risk of developing and the manifestation of adjustment disorders, but despite this, we still believe that this condition would not occur without the stressor. Its manifestations are diverse, including depressed mood, anxiety or worry (or a combination of these symptoms), feelings of inability to cope, inability to plan ahead or continue in the current situation, and some degree of impairment in daily life performance. Conduct disorder may be a relevant feature, particularly in adolescents. The primary feature may be a transient or prolonged depressive reaction, or other emotional and behavioral disturbances.

[0253] Culture shock

[0254] grief response

[0255] Pediatric hospitalization

[0256] exclude:

[0257] Childhood separation anxiety disorder (F93.0)

[0258] Other responses to severe stress (ICD-10F43.8):

[0259] Undefined response to severe stress (ICD-10F43.9).

[0260] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in a method for treating, improving, or preventing anxiety disorders, wherein the anxiety disorder is selected from:

[0261] (i) Phobic anxiety disorder (ICD-10 F40); and

[0262] (ii) Other anxiety disorders (ICD-10 F41.1).

[0263] Anxiety disorders according to the present invention are a group of disorders in which anxiety is induced only or primarily in certain well-defined, currently non-dangerous situations. Therefore, these situations are typically avoided or endured with fear. The patient's anxieties may focus on individual symptoms, such as palpitations or dizziness, and are often accompanied by secondary fears of death, loss of control, or madness. Imagining entering the fearful situation usually induces anticipatory anxiety. Phobic anxiety and depression often coexist. Whether both diagnoses (phobic anxiety and depressive episodes) are necessary, or only one, depends on the time course of both conditions and the treatment considerations at the time of consultation.

[0264] More specific anxiety disorders and their corresponding subdivisions according to the invention are described in more detail below:

[0265] Agoraphobia (ICD-10 F40.0):

[0266] Panic disorder is a relatively well-defined group of phobias, including the fear of leaving home, entering shops, crowds and public places, or traveling alone by train, bus or plane. Panic disorder is a common feature in both current and past episodes. Depression, obsessive-compulsive symptoms, and social phobia are also often present as secondary features. Avoidance of the feared situation is usually prominent; some people with agoraphobia experience little to no anxiety because they are able to avoid the feared situation.

[0267] agoraphobia without a history of panic disorder

[0268] Panic disorder accompanied by agoraphobia.

[0269] Social phobia (ICD-10 F40.1)

[0270] Fear of being judged by others leads to avoidance of social situations. More commonly, social phobia is often associated with low self-esteem and fear of criticism. Symptoms may include blushing, hand tremors, nausea, or urinary urgency; sometimes, patients believe these secondary symptoms of anxiety are the primary problem. Symptoms can develop into panic attacks.

[0271] Crowd terror

[0272] Social neurosis

[0273] Specific (isolation) phobia (ICD-10: F40.2):

[0274] Phobias are limited to highly specific situations, such as approaching a particular animal, heights, thunder, darkness, flight, enclosed spaces, urinating or defecating in a public toilet, eating a particular food, dental treatment, seeing blood, or being injured. Although the triggering situations are discrete, exposure to these situations can induce panic, similar to agoraphobia or social phobia.

[0275] Acrophobia

[0276] animal phobia

[0277] Claustrophobia

[0278] Simple phobia

[0279] exclude:

[0280] Teratophobia (non-delusional) (F45.2)

[0281] Phobia of disease (F45.2)

[0282] F40.8 Other phobic anxiety disorders

[0283] F40.9 Undefined phobic anxiety disorder

[0284] Phobia NOS

[0285] Fear State (NOS)

[0286] Other anxiety disorders (ICD-10 F41):

[0287] Anxiety is the primary symptom and is not limited to any specific environmental situation. Depression and obsessive-compulsive symptoms may also occur, and even elements of phobic anxiety may be present, provided that these symptoms are clearly secondary or less severe.

[0288] Panic disorder [paroxysmal anxiety disorder] (ICD-10 F41.0):

[0289] Its main characteristic is recurrent episodes of severe anxiety (panic), which are not limited to any specific situation or environment and are therefore difficult to predict. Like other anxiety disorders, its main symptoms include sudden palpitations, chest pain, a feeling of suffocation, dizziness, and a sense of unreality (depersonalization or derealization). Secondary fears of death, loss of control, or madness are often also present. If the patient has a depressive disorder at the time of the episode, panic disorder should not be considered as the primary diagnosis; in this case, the panic attack is likely a secondary symptom of depression.

[0290] terror:

[0291] Attack

[0292] State

[0293] exclude:

[0294] Panic disorder with agoraphobia (F40.0)

[0295] Generalized anxiety disorder (ICD-10 F41.1):

[0296] A widespread and persistent anxiety, but not limited to any specific environmental situation, and not even dominant in any particular environment (i.e., "free-floating"). Main symptoms are varied, but include complaints of persistent nervousness, tremors, muscle tension, sweating, dizziness, palpitations, vertigo, and upper abdominal discomfort. Patients often worry that they or their relatives will soon become ill or have an accident.

[0297] anxiety:

[0298] Neurosis

[0299] reaction

[0300] state

[0301] exclude:

[0302] Neurasthenia (F48.0)

[0303] Mixed anxiety and depression disorder (ICD-10 F41.2):

[0304] This category should be used when anxiety and depressive symptoms coexist, but neither is clearly dominant, and neither symptom alone is severe enough to warrant a definitive diagnosis. When anxiety and depressive symptoms coexist and are severe enough to warrant a definitive diagnosis alone, both diagnoses should be recorded, and this category should not be used.

[0305] Anxiety and depression (mild or non-persistent)

[0306] Other mixed anxiety disorders (ICD-10 F41.3):

[0307] The anxiety symptoms are mixed with features of other disorders in F42-F48. Neither of these symptoms alone is severe enough to warrant a diagnosis.

[0308] Other clearly defined anxiety disorders (ICD-10 F41.8):

[0309] Anxiety hysteria

[0310] Undiagnosed anxiety disorder (ICD-10 F41.9):

[0311] Anxiety NOS

[0312] In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, used in a method for treating, improving, or preventing Parkinson's disease.

[0313] Parkinson's disease is known in the field as a chronic degenerative disorder of the central nervous system that affects both the motor and non-motor systems. Symptoms typically develop slowly, with non-motor symptoms becoming more common as the disease progresses. Early symptoms include tremor, rigidity, bradykinesia, and difficulty walking. Problems may also occur in the cognitive, behavioral, sleep, and sensory systems. Dementia becomes common in the later stages of the disease.

[0314] Parkinson's disease is classified as ICD-10 G20.

[0315] In a further 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 in the context, wherein said antagonist / inhibitor is selected from NPBWR1 inhibitory peptides, NPBWR1 inhibitory small binding molecules, RNAi, siRNA, shRNA, aptamers and intramers specifically targeting NPBWR1, and anti-NPBWR1 antisense molecules.

[0316] Furthermore, it is envisioned that the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor 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 in the context, is an antibody.

[0317] Therefore, compounds that can act as specific "antagonists" or "inhibitors" of the neuropeptide B / W receptor (NPBWR1) may include small binding molecules, such as small (organic) compounds or ligands of the neuropeptide B / W receptor (NPBWR1). In the context of drug discovery, the term "small molecule" is known in the art and refers to medical compounds with a molecular weight of less than 2,500 Daltons, preferably less than 1,000 Daltons, and more preferably 50 to 350 Daltons. (Small) binding molecules include both natural and synthetic compounds. In the context of this invention, the term "compound" includes a single substance or multiple substances. The compound / binding molecule may be contained, for example, in a sample, such as a cell extract from, for example, a plant, animal, or microorganism. Furthermore, the compound may be known in the art but has not yet been found to (negatively) affect 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).

[0318] However, within the context of this invention, compounds, including, in particular, peptides, proteins, nucleic acids (including cDNA expression libraries), small organic compounds, ligands, PNAs, etc., are also contemplated as antagonists of neuropeptide B / W receptor (NPBWR1) function. These compounds may also be functional derivatives or analogs. Methods for preparing chemical derivatives and analogs are well known to those skilled in the art and are described, for example, in Beilstein, "Handbook of Organic Chemistry", Springer Edition, New York, or in "Organic Synthesis", Wiley, New York.

[0319] Furthermore, the effects of the said derivatives and analogs, namely their antagonistic effects on the neuropeptide B / W receptor (NPBWR1) function, can be tested according to methods known in the art. Additionally, suitable neuropeptide B / W receptor (NPBWR1) antagonists or inhibitors can be designed using peptide mimics and / or computer-aided design. Suitable computer systems for computer-aided design of, for example, proteins and peptides have been described in the prior art, for example in Berry (1994) Biochem.Soc.Trans.22:1033-1036; Wodak (1987), Ann.NY Acad.Sci.501:1-13; Pabo (1986), Biochemistry 25:5987-5991. The results obtained from the above computer analysis can be used in conjunction with the methods of the present invention, for example, for optimizing known compounds, substances, or molecules. Suitable compounds can also be synthesized by sequential chemical modification to create a library of peptide mimics and the resulting compounds can be identified, for example, according to the methods described herein. Methods for generating and using peptide mimicry combinatorial libraries have been described in the prior art, for example in Ostresh (1996) Methods in Enzymology 267:220-234 and Dorner (1996) Bioorg.Med.Chem.4:709-715. Furthermore, the three-dimensional and / or crystal structures of neuropeptide B / W receptor (NPBWR1) antagonists can be used to design (peptide mimicry) antagonists of neuropeptide B / W receptor (NPBWR1).

[0320] In the context of this invention, RNAi methods are also envisioned for the preparation of pharmaceutical compositions used in methods for treating, improving, or preventing mood disorders / emotional disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease.

[0321] The terms "RNA interference" or "repressive RNA" (RNAi / iRNA) describe the use of double-stranded RNA to target and degrade specific mRNAs, thereby silencing their expression. Preferred repressive RNA molecules can be selected from double-stranded RNA (dsRNA), RNAi, siRNA, shRNA, and stRNA. dsRNAs matching the gene sequence are synthesized in vitro and introduced into cells. dsRNAs can also be introduced into cells in the form of vectors expressing target gene sequences in both sense and antisense directions, for example, as hairpin mRNAs. Sense and antisense sequences can also be expressed from separate vectors, whereby individual antisense and sense molecules form a double-stranded RNA upon their expression. It is known in the art that in some cases, expression of the sense-oriented sequence or even the promoter sequence is sufficient to generate dsRNA, subsequently producing siRNA, due to internal amplification mechanisms within the cell. Therefore, according to the present invention, all means and methods leading to reduced activity (which can be reflected in lower expression of the neuropeptide B / W receptor (NPBWR1)) can be used, particularly by utilizing neuropeptide B / W receptor (NPBWR1)-specific siRNAs (i.e., siRNAs that specifically target neuropeptide B / W receptor (NPBWR1) mRNA or functional fragments thereof). For example, sense constructs, antisense constructs, hairpin constructs, sense and antisense molecules, and combinations thereof can be used to generate / introduce these siRNAs. dsRNAs are involved in a natural, but currently poorly understood, process involving the highly conserved nuclease Dicer, which cleaves dsRNA precursor molecules into short interfering RNAs (siRNAs). The generation and preparation of siRNAs, as well as methods for inhibiting target gene expression, are described in particular in WO 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 siRNAs then construct sequence-specific portions of RNA-induced silencing complexes (RISCs), which are multi-complex nucleases that disrupt messenger RNA homologous to silencing triggers. Elbashir (2001) EMBO J. 20:6877-6888 demonstrates that 21-nucleotide double strands of RNA can be used in cell culture to interfere with gene expression in mammalian cells. siRNA is known to mediate RNAi very effectively in mammalian cells, but the generation of stable cell lines or non-human transgenic animals is limited.However, for stable expression of, for example, short hairpin RNA (shRNA), next-generation vectors can be used. Stable expression of siRNA in mammalian cells is particularly demonstrated in Brummelkamp (2002) Science 296:550-553. Furthermore, Paul (2002) Nat. Biotechnol. 20:505-508 demonstrated efficient expression of small interfering RNA in human cells. Yu (2002) PNAS 99:6047-6052 also demonstrated RNA interference in mammalian cells by expressing both short interfering RNA and hairpin RNA. The shRNA approach for gene silencing is well-known in the art and can include the use of st (small temporal) RNA; see, in particular, Paddison (2002) Genes Dev. 16:948-958. These methods can be vector-based, for example, using pSUPER or RNA polIII vectors, particularly as described by Yu (2002) (cited above); Miyagishi (2002) (cited above); or Brummelkamp (2002) (cited above). It is conceivable that the regulatory sequences of the present invention can be used in a manner similar to those of systems based on pSUPER or RNA polIII vectors.

[0322] Methods for deriving and constructing siRNA are known in the art and described in Elbashir (2002) Methods 26:199-213, on the websites of commercial siRNA vendors, such as 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, online programs are available to derive siRNA from a given mRNA sequence (e.g., http: / / www.ambion.com / techlib / misc / siRNA_finder.html or http: / / katahdin.cshl.org:9331 / RNAi / html / rnai.html). The uridine residues at the 3' overhang of the 2-nt RNA can be substituted with 2'-deoxythymidine without loss of activity, which significantly reduces the cost of RNA synthesis and enhances the resistance of siRNA duplexes when applied to mammalian cells (Elbashir (2001), cited above). siRNA can also be synthesized enzymatically using T7 or other RNA polymerases (Donze (2002) Nucleic Acids Res 30:e46). Short RNA duplexes mediating effective RNA interference (esiRNA) can also be produced by hydrolysis using E. coli RNase III (Yang (2002) PNAS 99:9942-9947). In addition, expression vectors have been developed to express double-stranded siRNAs linked by small hairpin RNA loops in eukaryotic cells (e.g., Brummelkamp (2002) Science 296:550-553). All these constructs can be developed with the aid of the procedures described above. Furthermore, commercially available sequence prediction tools can be integrated into sequence analysis programs or sold separately, such as the siRNA design tool for siRNA sequence prediction available at www.oligoEngine.com (Seattle, WA).

[0323] Therefore, according to the present invention, specific interfering RNA can be used as an antagonist (inhibitor) of the neuropeptide B / W receptor (NPBWR1) (expression and / or function). These siRNAs are formed of an antisense strand and a sense strand, wherein the antisense / sense strand preferably contains at least 10, more preferably at least 12, more preferably at least 14, more preferably at least 16, more preferably at least 18, more preferably at least 19, 20, 21 or 22 nucleotides.

[0324] As described above, the methods for preparing the siRNA used in this invention are well known in the art. Based on the teachings provided herein, those skilled in the art can not only easily prepare such siRNA, but also easily assess whether the siRNA can antagonize / inhibit the neuropeptide B / W receptor (NPBWR1). It is envisioned that the above-mentioned siRNAs lead to the degradation of neuropeptide B / W receptor (NPBWR1) mRNA, and thus reduce the protein level of neuropeptide B / W receptor (NPBWR1).

[0325] In other words, siRNA leads to a significant reduction in the mRNA and / or protein levels of the neuropeptide B / W receptor (NPBWR1) (i.e., resulting in decreased expression of NPBWR1). This reduction in expression may be reflected in decreased NPBWR1 activity. For example, NPBWR1-specific siRNAs may lead to reduced NPBWR1 capacity and inhibit NPBWR1 activity. Therefore, the use of effective antagonists / inhibitors of NPBWR1 (such as the siRNAs described herein) will result in lower NPBWR1 activity.

[0326] As used in this article, the term “small interfering RNA” (siRNA), sometimes referred to as short interfering RNA or silent RNA, refers to a class of typically short, double-stranded RNA molecules that play a variety of roles in biology and are playing an increasingly important role in the treatment of a variety of diseases and conditions. As mentioned above, siRNA participates in the RNA interference (RNAi) pathway, in which siRNA 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 etal., Nat. Rev. Drug Discov.2007, 6: 443-453).

[0327] These siRNAs are typically 18-27 nt long and usually contain short (usually 19-21 nt) double-stranded RNAs (dsRNAs) with or without a 2-nt 3' overhang at either end. Each strand may have a 5' phosphate group and a 3' hydroxyl (-OH) group, or neither strand may have a phosphate group. This structure is the result of Dicer processing, an enzyme that converts long dsRNAs or small hairpin RNAs into siRNAs.

[0328] siRNA can also be exogenously (artificially) introduced into cells via various transfection methods to induce specific knockout of the target gene. In this case, other structures besides those described above are also conceivable, as long as they can interfere with gene expression. Preferably, the length of the double-stranded portion is about 12 to about 50 base pairs, more preferably 16 to 30, more preferably 18 to 25, and even more preferably 19 to 21. Most preferably, the length of the double-stranded portion is 19 base pairs. The siRNA of the present invention may have a protruding sequence of up to 10 base pairs, preferably no more than 5 base pairs, at either end or one end, or it may be blunt-ended. It is also preferred that the complementarity with the target gene extends along the entire length of the double-stranded portion. The length of the region complementary to the target gene is at least 12 base pairs, preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or more base pairs. The siRNA of the present invention can be completely complementary to the target gene. Alternatively, the siRNA may contain up to 5%, 10%, 20%, or 30% mismatch with the target gene. Furthermore, the siRNA and antisense RNA may be chemically modified, for example, on a backbone comprising sugar residues. Preferred modifications to the siRNA molecules of the present invention include linkers connecting the two strands of the siRNA molecule. Chemical modifications are particularly used to improve the pharmacological properties of the siRNA and antisense RNA, such as in vivo stability and / or delivery to target sites in organisms. Those skilled in the art are aware of such modified siRNAs and the means and methods for obtaining them, see, for example, Zhang et al., Curr Top Med Chem. 2006;6(9):893-900; Manoharan, Curr Opin Chem Biol. 2004 Dec;8(6):570-9.

[0329] Therefore, virtually any gene with a known sequence can be targeted using appropriately customized siRNAs based on sequence complementarity. This makes siRNAs an important tool for gene function and drug target validation studies, as well as the therapeutic interventions envisioned in this paper. The siRNAs disclosed in this paper can reduce or block the expression of the neuropeptide B / W receptor (NPBWR1).

[0330] In a further aspect, it is envisioned that antisense molecules inhibit the expression or function of the neuropeptide B / W receptor (NPBWR1), particularly the human neuropeptide B / W receptor (NPBWR1), and interact with neuropeptide B / W receptors (NPBWR1) expressed as their coding regions, as defined above, and with neuropeptide B / W receptors (NPBWR1) expressed as isotypes and variants thereof. The isotypes or variants may in particular comprise allelic variants or splice variants. Furthermore, it is envisioned that antisense molecules targeting the expression or function of the neuropeptide B / W receptor (NPBWR1) according to the present invention specifically interfere with the regulatory sequences of the neuropeptide B / W receptor (NPBWR1) as defined below.

[0331] In this paper, the term "variant" refers to the nucleotide sequence of the neuropeptide B / W receptor (NPBWR1) and the amino acid sequence encoding the neuropeptide B / W receptor (NPBWR1) that are different from the sequences available under the aforementioned GenBank accession number through mutations (e.g., deletion, addition, substitution, inversion, etc.).

[0332] Therefore, the antisense molecules used according to 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. In the context of the present invention, the term "specifically interacts / hybridizes with one or more nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1)" refers to antisense molecules capable of interfering with the expression of the neuropeptide B / W receptor (NPBWR1). However, in the context of the present invention, highly mutated anti-neuropeptide B / W receptor (NPBWR1) antisense constructs are not used, as they cannot hybridize or specifically interact with the nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1). Those skilled in the art can readily infer whether an antisense construct specifically interacts / hybridizes with the coding sequence of the neuropeptide B / W receptor (NPBWR1). These tests include, but are not limited to, hybridization assays, RNase protection assays, Northern blotting, Northern-Western blotting, NMR and fluorescence binding assays, dot blots, microarrays and macroarrays, and quantitative PCR. Furthermore, this screening is not limited to neuropeptide B / W receptor (NPBWR1) mRNA molecules, but may also include neuropeptide B / W receptor (NPBWR1) mRNA / protein (RNP) complexes (Hermann (2000) Angew Chem Int Ed Engl 39:1890-1904; DeJong (2002) Curr Trop Med Chem2:289-302). In addition, functional assays based on the neuropeptide B / W receptor (NPBWR1) reactive promoters, including Western blotting, immunohistochemistry, immunoprecipitation assays, and bioassays, are envisioned to test whether specific antisense constructs can specifically interact / hybridize with the nucleic acid molecule encoding neuropeptide B / W receptor (NPBWR1).

[0333] As used herein, the term "antisense molecule" specifically includes antisense oligonucleotides. These antisense oligonucleotides may also contain modified nucleotides and modified nucleoside bonds, particularly as described in US 6,159,697.

[0334] 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 particularly described in Smith, 2000. Commonly used methods include gene walking, RNase H mapping, RNase L mapping (Leaman (1999) Meth Enzymol 18:252-265), combinatorial oligonucleotide arrays on solid supports, secondary structure analysis determined by computational methods (Walton (2000) Biotechnol Bioeng, 65:1-9), aptastruc targeting structured nucleic acids, thetered oligonucleotide probe, foldback triplex-forming oligonucleotides (FTFO) (Kandimalla (1994) Gene 149:115-121), and selection of sequences with minimal nonspecific binding (Han (1994) Antisense Res Dev 4:53-65).

[0335] Preferably, the antisense molecule of the present invention is stabilized to resist degradation. Such stabilization methods are known in the art and are particularly described in US 6,159,697. Further methods described for protecting oligonucleotides from degradation include oligonucleotides bridged 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'-dipeptide oligonucleotides (Schwope (1999) J Org Chem 64:4749-4761), 3'-methylene thymidine and 5-methyluridine / cytidine h-phosphonates and phosphonamides (An (2001) J Org Chem, 66:2789-2801), and anionic liposomes (De Oliveira). (2000) Life Sci67:1625-1637) or encapsulated with ionizable amino lipids (Semple (2001) Biochim Biophys Acta, 10:152-166).

[0336] In addition, antagonists / inhibitors of neuropeptide B / W receptor (NPBWR1) expression or function may also include intracellular binding mates of neuropeptide B / W receptor (NPBWR1). As used herein, the term "intracellular binding mate" refers to an intracellular molecule capable of blocking or reducing the activity of neuropeptide B / W receptor (NPBWR1). Such intracellular binding mates of neuropeptide B / W receptor (NPBWR1) may particularly involve endogenous inhibitors / repressors of neuropeptide B / W receptor (NPBWR1). In another embodiment of the invention, the intracellular binding mate is an intracellular antibody. Intracellular antibodies are known in the art and can be used to modulate or inhibit the functional activity of target molecules. This therapeutic approach is based on the intracellular expression of a recombinant antibody fragment, which is Fab or a single-chain Fv, targeted to a desired cellular compartment using an appropriate targeting sequence (Teillaud (1999) PatholBiol 47:771-775).

[0337] As described above, antagonists / inhibitors of neuropeptide B / W receptor (NPBWR1) expression or function may also include aptamers. In the context of this invention, the term "aptamer" includes nucleic acids, such as RNA, ssDNA (ss = single-stranded), modified RNA, modified ssDNA, or PNA, which bind to multiple target sequences with high specificity and affinity. Aptamers are well known in the art and are particularly described in Famulok (1998) Curr. Op. Chem. Biol. 2:320-327. The preparation of aptamers is well known in the art and may particularly include the use of combinatorial RNA libraries to identify binding sites (Gold (1995) Ann. Rev. Biochem. 64:763-797).

[0338] Therefore, aptamers are oligonucleotides derived from an in vitro evolutionary process called SELEX (systematic evolution of ligands with exponential enrichment). Pools of random RNA or single-stranded DNA sequences are screened for a specific target. Sequences that bind more tightly to the target are isolated and amplified. Screening is repeated using the enrichment pool obtained from the first round. Several rounds of this process produce winning sequences called "aptamers." Aptamers have evolved to bind to proteins associated with a variety of disease states. Using this method, many effective antagonists of these proteins can be discovered. To make these antagonists work in animal models of disease and in humans, aptamers often need to be modified. First, the nucleoside triphosphate needs to be sugar-modified to make the resulting aptamers resistant to nucleases present in serum. Changing the 2'OH group of the ribose to a 2'F or 2'NH2 group produces aptamers that persist in the blood. The relatively low molecular weight (8000-12000) of the aptamers leads to rapid clearance from the blood. Aptamers can be retained in the bloodstream for hours to days by conjugating them to higher molecular weight vehicles. When modified conjugated aptamers are injected into animals, they can inhibit physiological functions known to be associated with their target proteins. Aptamers can be applied systemically in animals and humans to treat organ-specific diseases (Ostendorf (2001) J Am Soc Nephrol. 12:909-918). The first aptamer to enter Phase I clinical trials was NX-1838, an injectable angiogenesis inhibitor that holds promise for treating macular degeneration-related blindness (Sun (2000) Curr Opin Mol Ther 2:100-105). Cytoplasmic expression of aptamers (“cohesives”) can be used to bind intracellular targets (Blind (1999) PNAS 96:3606-3610; Mayer (2001) PNAS 98:4961-4965). The term "cohesive" is also contemplated for use in the context of this invention.

[0339] Neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors can be derived using methods in the art. These methods are described herein, and may include, but are not limited to, methods in which a set of substances is tested for interaction with neuropeptide B / W receptor (NPBWR1) or fragments thereof, and in which substances testing positive for interaction in a corresponding readout system are further tested in vivo, in vitro, or via computer simulation. in the stone The inhibitory effect of this drug on the expression or function of neuropeptide B / W receptor (NPBWR1) was tested in [the study].

[0340] The "assay for neuropeptide B / W receptor (NPBWR1) interaction" described above can be performed using specific immunological, molecular biological, and / or biochemical assays well known in the art, including, for example, homogeneous and heterogeneous assays as described below. Neuropeptide B / W receptor (NPBWR1) ligands capable of inhibiting neuropeptide B / W receptor (NPBWR1) function can be identified by screening large compound libraries based on their ability to interact with the neuropeptide B / W receptor (NPBWR1) protein. In a preferred embodiment, such antagonists or inhibitors of neuropeptide B / W receptor (NPBWR1) function are capable of binding to the protein-binding domain of the neuropeptide B / W receptor (NPBWR1).

[0341] In addition to molecules that can bind to the neuropeptide B / W receptor (NPBWR1), antagonists or inhibitors of NPBWR1 function can also prevent / reduce the expression of nucleic acid molecules encoding the NPBWR1 protein. Technicians can readily identify regulatory sequences (such as promoter sequences, enhancer sequences, origin of replication, and other regulatory elements) of NPBWR1 expression, for example, through computer-simulated gene prediction methods and experimental validation of functional sites (Elnitski (2006) Genome Res 16:1455-64).

[0342] As mentioned above, it is also conceivable that the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor in the methods described in the context for treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease is an antibody.

[0343] Preferably, the antibody is an antibody against the human neuropeptide B / W receptor (NPBWR1), that is, the antibody is an inhibitory antibody against the human neuropeptide B / W receptor (NPBWR1). Antibodies as used herein (which may be used interchangeably in various forms) are immunoglobulin molecules capable of specifically binding to a target (e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Preferred targets herein are neuropeptide B / W receptors (NPBWR1), particularly human neuropeptide B / W receptors (NPBWR1). As used herein, the term "antibody" encompasses not only complete (i.e., full-length) monoclonal antibodies, but also antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain variable region fragments (scFv)), their mutants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camel or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of immunoglobulin molecules containing the desired specific antigen recognition site, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies do not need to be of any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domain. Immunoglobulins are mainly classified into five classes: IgA, IgD, IgE, IgG, and IgM. Several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well-known.

[0344] The term “specifically binds” to a target or epitope (used interchangeably herein) is a well-known term in the art, and methods for measuring 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, for a longer duration, and / or with a higher affinity than it reacts or binds with other targets. An antibody is “specifically bound” to a target antigen if its binding to a target antigen has a higher affinity, greater affinity, easier binding, and / or longer duration than its binding to other substances. For example, an antibody that specifically (or preferentially) binds to the neuropeptide B / W receptor (NPBWR1) epitope is an antibody that binds to that neuropeptide B / W receptor (NPBWR1) epitope with a higher affinity, greater affinity, easier binding, and / or longer duration than its binding to other neuropeptide B / W receptor (NPBWR1) epitopes or non-neuropeptide B / W receptor (NPBWR1) epitopes. It can also be understood from 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 "preferred binding" does not necessarily require (although it can include) exclusive binding. Usually, but not necessarily, mentioning binding implies preferred binding.

[0345] Inhibitors of the neuropeptide B / W receptor (NPBWR1) can be anti-NPBWR1 specific antibodies. Anti-NPBWR1 antibodies are antibodies capable of binding to the neuropeptide B / W receptor (NPBWR1), which 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 instances, the anti-NPBWR1 antibodies used in the methods described herein inhibit 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.

[0346] Therefore, the present invention relates in one aspect to an anti-neuropeptide B / W receptor (NPBWR1) antibody, and a method for treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, as described in the context.

[0347] Anti-neuropeptide B / W receptor (NPBWR1) antibodies are well known in the art and are commercially available. Those skilled in the art can readily select and / or generate suitable anti-neuropeptide B / W receptor (NPBWR1) antibodies.

[0348] In a specific embodiment, the present invention relates to antibodies that specifically bind to the polypeptide or fragment thereof shown in SEQ ID NO:1 and their uses.

[0349] As described above, in the context of this invention, the term "antibody" as used herein specifically refers to complete immunoglobulin molecules and portions of such immunoglobulin molecules that substantially retain their binding specificity. Furthermore, the term refers to modified and / or altered antibody molecules, such as chimeric and humanized antibodies, CDR-transplanted antibodies, recombinant or synthetically produced / synthetic antibodies, and to complete antibodies and their antibody fragments, such as isolated 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 Fvs (scFv) or antibody fusion proteins. Additional "antibody" constructs are known in the art and are included in this invention.

[0350] Antibody production techniques 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 the polypeptides of the present invention can be obtained, for example, by directly injecting the polypeptide (or a fragment thereof) into an animal or by administering the polypeptide (or a fragment thereof) to an animal. The antibody thus obtained will then bind to the polypeptide (or a fragment thereof) itself. In this way, even fragments of the polypeptide can be used to generate antibodies that bind to the complete polypeptide, provided that the binding is “specific” as defined above.

[0351] Using normal skills and conventional methods, those skilled in the art can readily deduce the relevant epitopes (also functional fragments) of the polypeptides of the present invention from the sequences provided herein, which can be used to generate antibodies, such as polyclonal and monoclonal antibodies. However, those skilled in the art can also readily provide engineered antibodies, such as CDR transplanted antibodies or humanized and fully human antibodies.

[0352] In the context of this invention, monoclonal antibodies are particularly preferred. To prepare monoclonal antibodies, any technique that provides antibodies produced from continuous cell line cultures can be used. Examples of such techniques include hybridoma technology, three-source hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology to produce 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).

[0353] Antibody derivatives can also be produced using peptide mimics. Furthermore, the techniques described for producing single-chain antibodies (see, in particular, U.S. Patent 4,946,778) can be applied to produce single-chain antibodies that specifically recognize the polypeptides of the present invention. Similarly, transgenic animals can be used to express humanized antibodies against the polypeptides of the present invention.

[0354] The generation of specific antibodies against natural and recombinant peptides is based on, for example, the immunization of animals (e.g., mice). However, the present invention also contemplates other animals for generating antibodies / antiserum. For example, monoclonal and polyclonal antibodies can be generated from rabbits, mice, goats, donkeys, etc. The polynucleotide of the present invention, as shown in SEQ ID NO:2, can be subcloned into a suitable vector, wherein the recombinant peptide will be expressed in an organism capable of expression, such as bacteria. Thus, the expressed recombinant protein can be injected intraperitoneally into mice, and the resulting specific antibodies can be obtained, for example, from mouse serum provided by intracardiac bloodletting. The amount of specific antibodies obtained can be quantified using an ELISA, which is also described below. Other methods for generating antibodies are well known in the art; see, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

[0355] The term "specific binding" as used in this article refers to the binding reaction between the neuropeptide B / W receptor (NPBWR1) protein and the antibody in the presence of heterogeneous groups of proteins and other biological agents.

[0356] Therefore, under specified assay conditions, specific antibodies and neuropeptide B / W receptor (NPBWR1) proteins bind to each other and do not bind to other components present in the sample in significant amounts. Specific binding to the target analyte under such conditions may require selection of the binding moiety specific to that particular target analyte. Various immunoassay formats can be used to select antibodies that specifically react with a specific antigen. For example, solid-phase ELISA is routinely used to select monoclonal antibodies that specifically react with the analyte. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see 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. Typically, a specific or selective reaction will be at least twice the background signal-to-noise ratio, and more often 10 to 100 times higher than the background. Those skilled in the art can provide and generate specific binding molecules against novel peptides. For specific binding assays, it can be readily used to avoid unwanted cross-reactivity, for example, polyclonal antibodies can be readily purified and selected by known methods (see Shepherd and Dean, in the above citation).

[0357] As used herein, the term "purification or detection" refers to a series of methods designed to isolate or detect a single type of protein from a complex mixture using "specific binding" as defined above, where "specific binding" refers to the binding reaction that identifies the presence of a neuropeptide B / W receptor (NPBWR1) protein and an antibody in the presence of a heterogeneous population of proteins and other biological agents. Protein purification or detection is crucial for characterizing the function, structure, and interactions of target proteins. As a non-limiting example, starting materials can be biological tissues or microbial cultures. The various steps in the purification or detection process can release proteins from a protein-limiting matrix, separate protein and non-protein fractions of the mixture, and ultimately isolate the desired protein from all other proteins. These separation steps utilize differences in protein size, physicochemical properties, and binding affinity.

[0358] In a further preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, said pharmaceutical composition being 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, wherein said antagonist / inhibitor is an NPBWR1 inhibitory small binding molecule and has a chemical structure represented by the following formula (1).

[0359]

[0360] Equation (1)

[0361] in:

[0362] R 1 Selected from F, Cl, Br, I and CN;

[0363] R 2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heterocyclic groups) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a (of carbon cyclic groups);

[0364] R 3 Selected from - (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a (carbon cyclic group)

[0365] in:

[0366] Each R 2a Independently selected from: -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 Alkyl, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl;

[0367] Each R 3a Independently selected from: -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 Alkyl, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl;

[0368] Each R Alk Independently selected from -halogen and -CN;

[0369] Or a pharmaceutically acceptable salt, solvate, or prodrug.

[0370] In a further preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, the pharmaceutical composition being used in a method for 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 binding molecule having a chemical structure as defined above in formula (1), and formula (1) satisfies one or more of the following:

[0371] a)R 1 Selected from F, Cl, Br, and CN; preferably selected from F, Cl, and CN; more preferably selected from F and Cl; even more preferably selected from Cl;

[0372] b)R2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heteroaryl) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a Aryl); preferably selected from - (having 5 to 7 ring atoms and optionally having one or more substituents R 2a (heteroaryl) and - (having 6 or 10 ring atoms and optionally having one or more substituents R) 2a (aryl); more preferably selected from benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine, any of which may optionally be substituented by one or more R groups. 2a Substitution; or more preferably selected from benzene, naphthalene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R. 2a Substitution; still more preferably selected from benzene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R 2a Substitution; or more preferably selected from benzene, which may optionally be replaced by one or more substituents R 2a Substitution; still more preferably selected from benzene, which is substituted with one or more substituents R 2a Substitution; the most preferred choice is benzene, which is R at position 4. 2a replace;

[0373] c)R 3 Selected from - (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbonyl group); preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbon cyclo group); more preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a(aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having 6 to 10 ring atoms and optionally having one or more substituents R). 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 10 ring atoms and optionally having one or more substituents R) 3a (aryl); still more preferably selected from - (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having one or more substituents R) 3a phenyl) and - (optionally substituents R) Alk Replacement C 1-4 alkylene)-(with one or more substituents R) 3a (naphthyl); or even more preferably 2,5-dimethylphenyl or 1-naphthylmethyl;

[0374] d)C 1-4 The alkylene group is preferably methylene or ethylene, more preferably methylene;

[0375] e) No, one, two, or three substituents R 2a Preferably, there are no, one, or two substituents R. 2a More preferably, it has a substituent R. 2a Even more preferably, there is a substituent R at position 4. 2a ;

[0376] f) No, one, two, or three substituents R 3a Preferably, there is no, one, or two substituents R. 3a If R 3 Excluding -(C 1-4 The alkylene group is more preferably composed of two substituents R. 3a If R 3 Contains -(C 1-4 The alkylene group is more preferably a substituent R. 3a Or without substituent R 3a ;

[0377] g) No, one, two, or three substituents R Alk Preferably, there is no, one, or two substituents R. Alk More preferably, there is no or one substituent R. Alk Even better, no substitution base R Alk ;

[0378] h) Each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl; preferably each R 2a Independently selected from -halogen, -CN, C 1-3 Alkyl, C 1-3 Halogenated alkyl, 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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-3 Alkyl or cyclopropyl; more preferably each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-3 Alkyl or cyclopropyl; or even more preferably each R 2a Independently selected from -halogen, -CN, C 1-3 Alkyl or -OC 1-3 Alkyl; still more preferably each R 2a Independently selected from -halogen, -CN, or -OC 1-3 Alkyl; or even more preferably each R 2a Independently for -OC 1-3 Alkyl; or even more preferably each R 2a It can be methoxy or ethoxy independently, with methoxy being the most preferred.

[0379] i) Each R 3a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 3-6 cycloalkyl; preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 cycloalkyl, -OC 1-6 Alkyl or -OC 1-6 Halogenated alkyl; more preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, or C3-6 cycloalkyl; or even more preferably each R 3a Independently selected from -halogen, -CN, C 1-3 Alkyl, or C 1-3 Halogenated alkyl; still more preferably each R 3a Independently selected from -halogen and C 1-3 Alkyl; or even more preferably each R 3a Selected independently from C 1-3 Alkyl groups, such as methyl or ethyl, more preferably methyl;

[0380] j) Each R Alk Independently selected from –F and -Cl; preferably each R Alk For –F; more preferably each R Alk Does not exist; and / or

[0381] k) The NPBWR1 inhibitory small binding molecule has the chemical structure shown in formula (1) or a pharmaceutically acceptable salt or solvate thereof.

[0382] In a further or even more preferred embodiment, the present invention relates to the aforementioned pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, said pharmaceutical composition being 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.

[0383] The antagonist / inhibitor is an NPBWR1 inhibitory small binding molecule and has one of the following chemical structures represented by formulas (2) to (9):

[0384] (twenty three)

[0386] (4) (5)

[0388] (6) (7)

[0390] and (8) (9)

[0392] Where R 1 R 2a and R 3 As defined above.

[0393] Therefore, in the context of this invention, the NPBWR1 inhibitory small binding molecule preferably has the chemical structure represented by the above formula (1).

[0394] in:

[0395] R 1 Selected from F, Cl, Br, I and CN;

[0396] R 2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heterocyclic groups) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a (of carbon cyclic groups);

[0397] R 3 Selected from - (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a (carbon cyclic group)

[0398] in:

[0399] Each R 2a Independently selected from: -halogen, -CN, -CF 3、- 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 Alkyl, wherein each R Independently selected from H or C 1-6Alkyl or C 1-6 cycloalkyl;

[0400] Each R 3a Independently selected from: -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 Alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl;

[0401] Each R Alk Independently selected from -halogen and -CN;

[0402] Or a pharmaceutically acceptable salt, solvate, or prodrug.

[0403] Equation (1) satisfies one or more of the following:

[0404] a)R 1 Selected from F, Cl, Br, and CN; preferably selected from F, Cl, and CN; more preferably selected from F and Cl; even more preferably selected from Cl;

[0405] b)R 2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heteroaryl) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a Aryl); preferably selected from - (having 5 to 7 ring atoms and optionally having one or more substituents R 2a (heteroaryl) and - (having 6 or 10 ring atoms and optionally having one or more substituents R) 2a (aryl); more preferably selected from benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine, any of which may optionally be substituented by one or more R groups. 2a Substitution; or more preferably selected from benzene, naphthalene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R. 2aSubstitution; still more preferably selected from benzene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R 2a Substitution; or more preferably selected from benzene, which may optionally be replaced by one or more substituents R 2a Substitution; or more preferably selected from benzene, which is substituted by one or more substituents R 2a Substitution; the most preferred choice is benzene, which is R at position 4. 2a replace;

[0406] c)R 3 Selected from - (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbonyl group); preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbon cyclo group); more preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having 6 to 10 ring atoms and optionally having one or more substituents R). 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 10 ring atoms and optionally having one or more substituents R) 3a (aryl); still more preferably selected from - (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl) and - (optionally substituents R)Alk Replacement C 1-4 (alkylene)- (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having one or more substituents R) 3a phenyl) and - (optionally substituents R) Alk Replacement C 1-4 alkylene)-(with one or more substituents R) 3a (naphthyl); or even more preferably 2,5-dimethylphenyl or 1-naphthylmethyl;

[0407] d)C 1-4 The alkylene group is preferably methylene or ethylene, more preferably methylene;

[0408] e) No, one, two, or three substituents R 2a Preferably, there is no, one, or two substituents R. 2a More preferably, it has a substituent R. 2a Even more preferably, there is a substituent R at position 4. 2a ;

[0409] f) No, one, two, or three substituents R 3a Preferably, there is no, one, or two substituents R. 3a If R 3 Excluding -(C 1-4 The alkylene group is more preferably composed of two substituents R. 3a If R 3 Contains -(C 1-4 The alkylene group is more preferably a substituent R. 3a Or without substituent R 3a ;

[0410] g) No, one, two, or three substituents R Alk Preferably, there is no, one, or two substituents R. Alk More preferably, there is no or one substituent R. Alk Even better, no substitution base R Alk ;

[0411] h) Each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl; preferably R 2a Each is independently selected from -halogen, -CN, C 1-3 Alkyl, C 1-3 Halogenated alkyl, 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 Haloalkyl, wherein R Each is independently selected from H or C 1-3 Alkyl or cyclopropyl; more preferably each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-3 Alkyl or cyclopropyl; or even more preferably each R 2a Independently selected from -halogen, -CN, C 1-3 Alkyl or -OC 1-3 Alkyl; still more preferably each R 2a Independently selected from -halogen, -CN, or -OC 1-3 Alkyl; or even more preferably each R 2a Independently for -OC1-3 Alkyl; or even more preferably each R 2a It can be methoxy or ethoxy independently, with methoxy being the most preferred.

[0412] i) Each R 3a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 3-6 cycloalkyl; preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 cycloalkyl, -OC 1-6 Alkyl or -OC 1-6 Halogenated alkyl; more preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 3-6 cycloalkyl; or even more preferably each R 3a Independently selected from -halogen, -CN, C 1-3 Alkyl or C 1-3 Halogenated alkyl; still more preferably each R 3a Independently selected from -halogen and C 1-3 Alkyl; or even more preferably each R 3a Selected independently from C 1-3 Alkyl groups, such as methyl or ethyl, more preferably methyl;

[0413] j) Each R Alk Independently selected from –F and -Cl; preferably each R Alk For –F; more preferably each R Alk Does not exist; and / or

[0414] k) The NPBWR1 inhibitory small binding molecule has the chemical structure shown in formula (1) or a pharmaceutically acceptable salt or solvate thereof.

[0415] Furthermore, in the context of this invention, the NPBWR1 inhibitory small binding molecule more preferably has one of the chemical structures shown in formulas (2) to (9) above:

[0416] Where R 1 R 2a and R 3 As defined above.

[0417] As used herein, the term "alkyl" refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group, which may be straight-chain or branched. Therefore, an "alkyl" group does not contain any carbon-carbon double or triple bonds. The term "alkyl" preferably refers to a "C" group. 1-6 Alkyl group. "C" 1-6 "alkyl" refers to an alkyl group having 1-6 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 C14. 1-4 Alkyl, more preferably methyl or ethyl, even more preferably methyl.

[0418] "Halogens represent F, Cl, Br and I, more preferably F or Cl, and even more preferably F unless otherwise stated."

[0419] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (preferably 1-6, more preferably 1-3) halogen atoms, said halogen atoms being independently selected from fluorine, chlorine, bromine, and iodine, preferably all fluorine atoms. It should 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 contained in the alkyl portion of the haloalkyl group. "Haloalkyl" can refer, for example, to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. When the haloalkyl group is a substituent on an oxygen atom, the carbon atom adjacent to the oxygen atom (in the haloalkyl group) is preferably not directly bonded to a halogen.

[0420] The term "aryl" preferably refers to an aromatic monocyclic system containing 5 or 6 carbon atoms, an aromatic bicyclic system containing 10 carbon atoms, or an aromatic tricyclic system containing 14 carbon atoms. Examples are phenyl, naphthyl, or anthracene, with phenyl being preferred.

[0421] The term "heteroaryl" preferably refers to a five- or six-membered aromatic ring, wherein one or more carbon atoms in the ring are replaced by 1, 2, 3, or 4 (for five-membered rings) or 1, 2, 3, 4, or 5 (for six-membered rings) identical or different heteroatoms. The heteroatoms are preferably selected from O, N, and S, and examples of heteroaryl groups are as follows.

[0422] The term "heterocyclic group" encompasses any monocyclic, bicyclic, or polycyclic system containing one or more heteroatoms, wherein the heteroatoms are identical or different and selected from O, N, and S. Preferably, the ring system comprises 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 may contain 1 to 4, more usually 1 or 2 heteroatoms at available positions. The term "heterocyclic group" also encompasses heteroaryl rings. Examples include nitrogen-containing heterocyclic butanes, pyrroles, pyrrolidines, oxacyclopentanes, furans, imidazolides, imidazolium, pyrazoles, oxazolidines, oxazoles, thiazoles, piperidines, pyridines, morpholine, piperazines, and dioxopentane.

[0423] The term "carbocyclic" encompasses any monocyclic, bicyclic, or polycyclic system that does not contain heteroatoms in the ring. Preferably, the ring system comprises 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" also encompasses aryl rings.

[0424] As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon cyclic group, including monocyclic and bridged, spirocyclic, and / or fused ring systems (which may consist of, for example, two or three rings; for example, a fused ring system 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 C14. 3-11 cycloalkyl, more preferably C 3-8 Cycloalkyl. Particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3-8 ring members, most preferably 3 ring members.

[0425] If a compound or part is referred to as “optionally substituted”, it may in each case include one or more specified substituents, which may be the same or different.

[0426] As used herein, unless otherwise expressly indicated or contradicted by the context, the terms “a,” “an,” and “the” are used interchangeably with “one or more” and “at least one.” Thus, for example, a composition comprising a “one” compound of the present invention (particularly formula (1)) can be interpreted as a composition comprising “one or more” compounds of the present invention.

[0427] As used herein, unless otherwise explicitly stated or contradicted by the context, the term “comprising” (or its various grammatical parts of speech or “contain” and its various grammatical parts of speech) has the meaning of “especially containing,” that is, “containing… in addition to other optional elements.” In addition, the term also includes the narrower meanings of “consisting essentially of” and “consisting of.” For example, the term “A comprises B and C” means “A especially contains B and C,” where A may contain other optional elements (e.g., “A contains B, C, and D” would also be included), but the term also includes the meanings of “A is essentially composed of B and C” and “A is composed of B and C” (i.e., A does not contain any components other than B and C).

[0428] Furthermore, unless otherwise specified, any reference to industry standards, pharmacopoeias, or manufacturer's manuals refers to the latest version of the relevant standard available on the priority date of this specification (i.e., the earliest submission date).

[0429] The scope of this invention includes all pharmaceutically acceptable salt forms of the compounds provided herein, particularly the compounds of this invention (especially formula (1)), which can be formed, for example, by protonating an atom (e.g., an amino group) carrying an easily protonated lone pair of electrons with an inorganic or organic acid, or as a salt of an acid group (e.g., a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts include, for example: alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as 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, or choline salt; aralkylamine salts, such as N,N-dibenzylethylenediamine salt, benzylamine salt, phenethylamine salt; heterocyclic aromatic amine salts, such as pyridine salts, methylpyridine salts, quinoline salts, or isoquinoline salts; quaternary ammonium salts, such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, or tetrabutylammonium salt; and basic amino acid salts, such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts include, for example: mineral salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate (e.g., sulfate or hydrogen sulfate), nitrate, phosphate (e.g., phosphate, hydrogen phosphate, or dihydrogen phosphate), carbonate, bicarbonate, perchlorate, borate, or thiocyanate; organic salts, such as acetate, propionate, butyrate, valerate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (emboate), camphorate, gluconate, or... Pivalatesalt; sulfonates, such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-naphthalenesulfonate, 3-phenylsulfonate or camphorsulfonate; glycerophosphates; and acidic amino acid salts, such as aspartate or glutamate.

[0430] Furthermore, the scope of this invention includes the compounds provided herein, particularly the compounds of this invention (especially formula (1)), which are in any solvated form, including, for example, solvates with water (i.e., as hydrates) or solvates with organic solvents (e.g., methanol, ethanol, or acetonitrile) (i.e., as methanol salts, ethanol salts, or acetonitrile salts), or in any crystalline form (i.e., as any polymorph), or in an amorphous form. It should be understood that such solvates of the compounds provided herein, particularly the compounds of this invention, also include solvates of pharmaceutically acceptable salts of the respective compounds.

[0431] Furthermore, the compounds provided herein, particularly those of formula (1), may exist in various isomers, especially stereoisomers (including, for example, geometric isomers (or cis / trans isomers), enantiomers and diastereomers) or tautomers. All such isomers of the compounds provided herein, whether in mixture form or in pure or substantially pure form, are considered part of this invention. As for stereoisomers, this invention includes isolated optical isomers of the compounds of this invention and any mixtures thereof (including, in particular, 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 salting with an optically active acid and then crystallizing. This invention also covers any tautomers of the compounds provided herein.

[0432] The scope of this invention also includes compounds provided herein, particularly compounds of formula (1), wherein one or more atoms are replaced by specific isotopes of the corresponding atoms. For example, this invention covers compounds of formula (1) wherein one or more hydrogen atoms (or, for example, all hydrogen atoms) are replaced by deuterium atoms (i.e., 2 H; also referred to as "D") is substituted. Therefore, the present invention also includes deuterium-rich compounds of formula (1). Naturally occurring hydrogen is an isotopic mixture containing about 99.98 mol-% hydrogen-1 ( 1 H) and approximately 0.0156 mol-% deuterium ( 2 The deuterium content at one or more hydrogen sites in a compound of formula (1) can be increased using deuteration techniques known in the art. For example, a compound of formula (1) or a reactant or precursor used to synthesize a compound of formula (1) can be subjected to an H / D exchange reaction using, for example, heavy water (D2O). Other suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chemistry , 20(18), 5658-5667, 2012; William JS et al., Journal of Labeled 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, compounds of formula (1) are preferably not rich in deuterium. Therefore, naturally occurring hydrogen atoms or 1 H hydrogen atom.

[0433] The invention also includes compounds provided herein, particularly compounds of formula (1), wherein one or more atoms are replaced by positron-emitting isotopes of the respective atoms, for example... 18 F, 11 C 13 N、 15 O、 76 Br、 77 Br、 120 I and / or 124 I. These compounds can be used as tracers or imaging probes in positron emission tomography (PET). Therefore, the present invention comprises: (i) compounds of formula (1) wherein one or more fluorine atoms (or, for example, all fluorine atoms) are... 18 (ii) compounds of formula (1) wherein one or more carbon atoms (or, for example, all carbon atoms) are replaced by F atoms. 11 (iii) A compound of formula (1) in which one or more nitrogen atoms (or, for example, all nitrogen atoms) are replaced by C atoms. 13 (iv) A compound of formula (1) wherein one or more oxygen atoms (or, for example, all oxygen atoms) are replaced by N atoms. 15 O atoms are replaced; (v) compounds of formula (1), wherein one or more bromine atoms (or, for example, all bromine atoms) are replaced. 76 (vi) Compounds of formula (1) wherein one or more bromine atoms (or, for example, all bromine atoms) are replaced by Br atoms. 77 Br atom substitution; (vii) compounds of formula (1), wherein one or more iodine atoms (or, for example, all iodine atoms) are replaced. 120 I atoms are replaced; and (viii) compounds of formula (1), wherein one or more iodine atoms (or, for example, all iodine atoms) are replaced. 124 I atoms are substituted. Generally, atoms in the preferred compound (1) are not substituted by specific isotopes.

[0434] The pharmaceutically acceptable prodrugs of the compounds provided herein, particularly those of formula (1), are derivatives having chemically or metabolically cleavable groups and are converted into pharmaceutically active compounds of the present invention by solvent decomposition or under physiological conditions. The prodrugs of the compounds of the present invention can be formed in a conventional manner with functional groups of the compound, such as amino, hydroxyl, or carboxyl groups. Prodrug forms generally have advantages in terms of solubility, tissue compatibility, 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 reacting a parent acidic compound with a suitable alcohol, or amides prepared by reacting a parent acidic compound with a suitable amine. If the compounds of the present invention have a carboxyl group, ester derivatives prepared by reacting the carboxyl group with a suitable alcohol or amide derivatives prepared by reacting the carboxyl group with a suitable amine can serve as examples of prodrugs. Particularly preferred ester derivatives as prodrugs are methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n-butyl ester, isobutyl ester, tert-butyl ester, morpholinoethyl ester, N,N-diethylglycolamidoester, or α-acetoxyethyl ester. If the compound of the present invention has a hydroxyl group, examples of acyloxy derivatives prepared by reacting the hydroxyl group with a suitable acyl halide or a suitable acid anhydride can be used 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 31 The compounds of the present invention may contain amino groups, such as -OC(=O)-(m-COONa-Ph), -OC(=O)-CH2CH2COONa, -O(C=O)-CH(NH2)CH3, or -OC(=O)-CH2-N(CH3)2. If the compounds of the present invention have an amino group, examples of amide derivatives prepared by reacting the amino group with a suitable acyl halide or a suitable mixed anhydride are provided as prodrugs. Particularly preferred amide derivatives as prodrugs are -NHC(=O)-(CH2)2OCH3 or -NHC(=O)-CH(NH2)CH3.

[0435] In a further preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, said pharmaceutical composition being used in a method of treating, improving or preventing mood disorders / affective disorders and / or chronic stress and / or anxiety disorders and / or Parkinson's disease, wherein said antagonist / inhibitor is a CRISPR / Cas system specifically targeting NPBWR1, wherein said CRISPR / Cas system Cas protein is modified to lack its nuclease activity, and said Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression.

[0436] The CRISPR / Cas system is known in the art and can be used to target CRISPR-Cas endonucleases to very specific target sites in a highly specific manner using specially designed guide RNAs. In the case of this invention, the specific target sequence is the genomic sequence of the NBPWR1 locus.

[0437] The human NBPWR1 gene is located on the positive strand of chromosome 8, from base pair 52,939,182 to base pair 52,943,734 (Genome Reference Consortium – Human GRCh38 / h38).

[0438] The genomic locus of the NBPWR1 gene is known in the art and can be retrieved, for example, by the following accession number: ENSG00000288611.

[0439] Its cytogenetic location is on the cell membrane.

[0440] Although CRISPR / Cas systems are frequently used to cleave genomic (target) DNA by site-directed nuclease activity of the Cas endonuclease, cleavage is not contemplated or desired in the context of this invention, particularly with respect to antagonists / inhibitors of the neuropeptide B / W receptor (NPBWR1). Instead, as will be described in further detail below, in the context of this invention, it is desirable for the CRISPR / Cas system to be modified to lack its nuclease activity, and for the Cas protein to be fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression. Therefore, while the CRISPR / Cas system used in the context of this invention utilizes the ability of the CRISPR / Cas system to target very specific target sites with CRISPR-Cas endonucleases in a highly specific manner via specifically designed guide RNAs, cleavage of the genomic target is undesirable. Conversely, the CRISPR / Cas system used in the context of this invention utilizes the ability of the CRISPR / Cas system to target / bind CRISPR-Cas endonucleases to very specific target sites in a highly specific manner via specifically designed guide RNAs, wherein the CRISPR / Cas system is modified to lack its nuclease activity. The CRISPR / Cas system then functions on specific genomic targets through effector domains (i.e., the CRISPR / Cas system is specifically designed to target NPBWR1 in this application), said effector domains being selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression of Cas proteins fused to the CRISPR / Cas system.

[0441] In general, the CRISPR / Cas system will be described below, and the more specific use of the CRISPR / Cas9 system in this invention will be described in more detail below.

[0442] Typically, CRISPR (clustered, regularly spaced short palindromic repeats) genomic loci are known in the art to 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 to help protect prokaryotes from foreign invaders (e.g., viruses and bacteriophages). Five types of CRISPR systems have been identified (e.g., type I, type II, type III, type U, and type V).

[0443] CRISPR loci comprise numerous short repetitive sequences called “repeat”. These repetitive sequences can form hairpin structures and / or contain unstructured single-stranded sequences. Repetitive sequences typically occur in clusters and often vary between species. These repetitive sequences are regularly spaced by unique intercalation sequences called “spacers,” creating a repetitive-spacer-repetitive locus structure. Spacers are identical to or highly homologous to known foreign invader sequences. Spacer-repetitive sequence units encode crprRNA (crRNA), which is processed into its mature form. crRNA contains a “seed” or spacer sequence that participates in targeting target nucleic acids (in prokaryotes, the spacer sequence targets foreign invader nucleic acids in a naturally occurring form). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0444] The CRISPR locus also contains polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and interference phases of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0445] CRISPR system type II

[0446] In nature, crRNA biogenesis in type II CRISPR systems requires trans-activated CRISPR RNA (tracrRNA). tracrRNA is modified with endogenous RNase III and then hybridizes to a repetitive crRNA sequence in a precursor crRNA array. The endogenous RNase III is recruited to cleave the precursor crRNA. The cleaved crRNA is pruned by an exonuclease to produce a mature crRNA form (e.g., 5' pruning). tracrRNA remains hybridized to crRNA, and both tracrRNA and crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex guides the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA with the target nucleic acid activates Cas9 for cleavage of the target nucleic acid (where Cas9 is enzymatically active, as explained above and further below, but not contemplated in the context of this invention). The target nucleic acid in a type II CRISPR system is called a protospacer neighbor motif (PAM). In nature, PAMs are essential for facilitating the binding of site-directed polypeptides (e.g., Cas9) to target nucleic acids. The Type II system is further subdivided into Type II-A (CASS4) and Type II-B (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) shows that the CRISPR / Cas9 system can be used for RNA-programmable genome editing, and international patent application publication number WO2013 / 176772 provides many examples and applications of the CRISPR / Cas endonuclease system for site-specific gene editing.

[0447] Cas gene / peptide and adjacent motifs of the original spacer sequence

[0448] Exemplary CRISPR / Cas peptides include those from Fonfara et al. Nucleic Acids Research, 42 :2577-2590 (2014) Figure 1 The Cas9 polypeptide. Since the discovery of the Cas gene, the CRISPR / Cas gene nomenclature system has undergone extensive rewriting. Fonfara (ibid.) Figure 5 PAM sequences of Cas9 peptides from different species were provided.

[0449] Site-directed DNA endonuclease

[0450] Generally, site-directed endonucleases are nucleases used in genome editing to cut DNA. Site-directed endonucleases can be administered to cells or patients in one of the following forms: one or more polypeptides, or one or more mRNAs encoding said polypeptides.

[0451] In the case of the CRISPR / Cas system, site-directed DNA endonucleases can bind to guide RNA, which in turn specifies the site in the target DNA that the polypeptide targets.

[0452] DNA endonucleases are generally known to contain multiple cleavage (i.e., nuclease) domains. In fact, naturally occurring wild-type Cas9 enzymes are known to contain two nuclease domains: the HNH nuclease domain and the RuvC domain. In this article, "Cas9" refers to both naturally occurring and recombinant Cas9. The Cas9 enzymes considered in this article contain an HNH or HNH-like nuclease domain and / or a RuvC-like nuclease domain. The HNH or HNH-like domain contains McrA-like folds. The HNH or HNH-like domain contains two antiparallel β strands 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 the target nucleic acid (e.g., the complementary strand of the crRNA target 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 action on RNA and DNA. The RNaseH domain contains five β strands 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).

[0453] Typically, DNA endonucleases are known to introduce double-strand breaks (or single-strand breaks) into nucleic acids such as genomic DNA. Double-strand breaks can stimulate endogenous DNA repair pathways in the cell (e.g., homology-dependent repair (HDR), non-homologous end joining (NHEJ), alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining (MMEJ)). NHEJ can repair cleaved target nucleic acids without requiring a homologous template. This can sometimes lead to small deletions or indels at the cleavage site of the target nucleic acid, resulting in disruption or alteration of gene expression.

[0454] In some embodiments of the invention, the DNA endonuclease, preferably a Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding the following amino acid sequence, which is oriented at a wild-type exemplary site-directed polypeptide [e.g., from Streptococcus pyogenes (Streptococcus pyogenes)]. S. pyogenes Cas9, US2014 / 0068797, Serial ID No. 8, or Sapranauskas et al. Nucleic Acids Res, 39[(21):9275-9282 (2011)] and a variety of other site-directed peptides 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.

[0455] In some embodiments, the DNA endonuclease, preferably a 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 a wild-type exemplary site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above).

[0456] In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) protein / enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) over 10 consecutive amino acids. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) over 10 consecutive amino acids. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) at 10 consecutive amino acids in the HNH nuclease domain of the encoded site-directed polypeptide. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) at 10 consecutive amino acids in the HNH nuclease domain of the encoded site-directed polypeptide. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) at 10 consecutive amino acids in the RuvC nuclease domain of the encoded site-directed polypeptide. In some embodiments, the DNA endonuclease, preferably Cas (more preferably Cas9) enzyme, comprises a nucleotide sequence encoding an amino acid sequence having at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above) at 10 consecutive amino acids in the RuvC nuclease domain of the encoded site-directed polypeptide.

[0457] As described above, in this 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-directed polypeptide, which comprises a modified form of the wild-type exemplary site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide comprises a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. In some embodiments, the modified form of the wild-type exemplary site-directed 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 that of the wild-type exemplary site-directed polypeptide (e.g., Cas9 from Streptococcus pyogenes, as above). In the most preferred embodiment, the DNA endonuclease, preferably Cas (more preferably Cas9) protein / enzyme, may not have substantial nucleic acid cleavage activity in its site-directed polypeptide modified form. When a site-directed polypeptide is a modified form that does not possess substantial 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" in this invention.

[0458] In some embodiments, the Cas protein "modified to lack its nuclease activity" is mutated relative to the corresponding wild-type enzyme, such that the mutated 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 (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9, which is a nuclease that cleaves two strands, into a nicking enzyme (cleaving a single strand). Other examples of mutations that make Cas9 a nicking enzyme include, but are not limited to, H840A, N854A, and N863A. As other examples, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or the HNH domain) can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the 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 in all DNA cleavage activity when the DNA cleaving activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or lower relative to its non-mutated form. When the enzyme is not derived from Streptococcus pyogenes Cas9 (SpCas9), mutations can be made at any or all residues corresponding to positions 10, 762, 840, 854, 863, and / or 986 of SpCas9 (this can be determined, for example, by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A, and / or D986A; and conserved substitutions for any alternative amino acids are also conceivable. The same mutations (or conserved substitutions of these mutations) at corresponding positions in other Cas9s are also preferred. D10 and H840 in SpCas9 are particularly preferred. However, in other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred.

[0459] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-directed polypeptide contains an amino acid sequence with at least 15% amino acid identity to Cas9 from bacteria (e.g., Streptococcus pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain).

[0460] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-directed polypeptide contains an amino acid sequence with at least 15% amino acid identity to Cas9 from bacteria (e.g., Streptococcus pyogenes) and two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain).

[0461] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, wherein the site-directed polypeptide comprises an amino acid sequence that is at least 15% amino acid identical to Cas9 from bacteria (e.g., Streptococcus pyogenes) and two nucleic acid cleavage domains, wherein one or both of the nucleic acid cleavage domains are at least 50% amino acid identical to the nuclease domain of Cas9 from bacteria (e.g., Streptococcus pyogenes).

[0462] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, wherein the site-directed polypeptide comprises an amino acid sequence that is at least 15% identical to Cas9 from bacteria (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., HNH domain and RuvC domain), and a non-natural sequence (e.g., nuclear localization signal) or a linker that connects the site-directed polypeptide to the non-natural sequence.

[0463] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, wherein the site-directed polypeptide comprises an amino acid sequence that is at least 15% identical to Cas9 from bacteria (e.g., Streptococcus pyogenes), two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain), wherein the site-directed polypeptide contains a mutation in one or both nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

[0464] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, wherein the site-directed polypeptide comprises an amino acid sequence with at least 15% amino acid identity to Cas9 from bacteria (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 of aspartic acid 10, and / or wherein one of the nuclease domains contains a mutation of histidine 840, and wherein the mutation reduces the cleavage activity of the nuclease(one or more) nuclease domain(s) by at least 50%.

[0465] In some embodiments, the Cas protein "modified to lack its nuclease activity" is preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme, i.e., the site-directed polypeptide (Cas9 protein) is derived from Staphylococcus ludenbergii ( S. from Lugdunum (SluCas9). In some implementations, the Cas9 protein is derived from Staphylococcus aureus (SluCas9). Staphylococcus aureus (SaCas9). In some embodiments, the suitable Cas9 protein for use in this disclosure is either one disclosed in WO2019 / 183150 and WO2019 / 118935, each of which is incorporated herein by reference.

[0466] Type II CRISPR / Cas system components are derived from Type IIA, IIB, or IIC systems. Cas9 and its orthologs are included. Non-limiting exemplary species from which the Cas9 nuclease or other components originate include: *Streptococcus pyogenes* (…). Streptococcus pyogenes Staphylococcus ludensii ( Streptococcus lugdunensis Streptococcus thermophilus ( Streptococcus thermophilus ), species of the genus Streptococcus ( Streptococcus sp. Staphylococcus aureus Staphylococcus aureus Listeria monocytogenes ( ), harmless Listeria monocytogenes ( Harmless Listeria Lactobacillus gasseri (), Lactobacillus gasseri ), the new culprit, Francisella ( Francisella novicida ), succinic acid-producing Worlian bacteria ( Wolinella succinogenes Waldsart bacteria () Sutterella wadsworthensis ), Gamma Proteus ( Gamma proteobacterium ), Neisseria meningitidis ( Neisseria meningitidis ), Campylobacter jejuni ( Campylobacter jejuni Pasteurella multocida ( ) Pasteurella multocida ), Succinic acid-producing filamentous bacteria ( Fibrobacter succinogene ), Rhodospirillum rubrum ( Rhodospirillum rubrum ), Nocardia dassonvillei ( Nocardiopsis dassonvillei) Streptomyces simulans (Streptomyces pristinespiralis ), green color-producing Streptomyces ( Streptomyces viridochromogenes ), green color-producing Streptomyces ( Streptomyces greenish-yellow ), Rosacea ( Streptosporangium roseum ), Rosacea ( Streptosporangium roseum ), Acidic cyclophosphamide ( Alicyclobacillus acid-caloric ), Bacillus pseudomycosis ( Bacillus pseudomycoides ), selenium-reducing Bacillus ( Bacillus selenite reducing ), Siberian microbacteria ( Exiguobacterium sibiricum Lactobacillus delbrueckii (), Lactobacillus delbrueckii ), Lactobacillus salivarius ( Lactobacillus salivarius Lactobacillus buchneri, and Treponema pallidum ( Treponema denticola Marine microoscillator bacteria ( Microscilla marina) Burkholderia order bacteria (Burkholderiales bacterium) Naphthylazine (Polaromonas naphthalenivorans) Species of the genus *Paramonas* ( Polaromonas sp. ), Coccidioides warbury (Crocosphaera watsonii) Species of the genus Cyanobacteria Cyanothece sp. Microcystis aeruginosa ( Microcystis aeruginosa ), species of the genus Synechococcus ( Synechococcus sp.) , Arabinose ( Acetohalobium arabicum ), Ammonite of the Degens , Caldicellosiruptor becscii , Desulfurous Candidate Clostridium botulinum ( Clostridium botulinum ), Clostridium difficile (C lost difficult ), Griffon's bacterium ( Finegoldia magna ), Natranaerobius thermophilus , Pelotomaculum thermopropionicum Thermophilic acidophilic thiobacillus ( Acidithiobacillus caldus) Acidophilic iron-oxidizing sulfur bacteria ( Acidithiobacillus ferrooxidans ), Allochromatium vinous Species of the genus *Hymenobacter* ( Marinobacter sp. ), nitrosophilic cocci ( Nitrosococcus halophilus ), Nitrosococcus watsoni Pseudomonas alterniflora ( Pseudoalteromonas haloplanktis ), Ktedonobacter racemifer、Methanohalobium evestigatum Anabaena ( Anabaena variable ), Foamy Glomerula ( Foamy nodularia ), species of the genus Nostoc ( Nostoc sp. ), Spirulina macrophylla ( Arthrospira maxima Spirulina platensis () Arthrospira platensis) Arthrospira ( ) Arthrospira sp. ), species of the genus *Lyngbya*, Microcoleus chthonoplastesSpecies of the genus *Oscillatoria* Mobile Petrotoga African thermocline bacteria ( Thermosipho africanus Pasteurella multocida ( Streptococcus pasteurianus ), Neisseria griseus ( Neisseria cinerea ), Campylobacter gull ( Campylobacter lari ), Small wash-eating stick Corynebacterium diphtheriae ( Corynebacterium diphtheria )or Acaryochloris marina In some implementations, the Cas9 protein originates from Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes (SpCas9). In some implementations, the Cas9 protein is derived from Staphylococcus ludensii (SpCas9). S. lugdunensis (SluCas9). In some implementations, the Cas9 protein is derived from Staphylococcus aureus (SluCas9). Staphylococcus aureus (SaCas9). In some embodiments, the suitable Cas9 protein for use in this disclosure is either one disclosed in WO2019 / 183150 and WO2019 / 118935, each of which is incorporated herein by reference.

[0467] Guide RNA

[0468] Guide RNA (or "gRNA") comprises at least one spacer sequence that hybridizes to a target nucleic acid sequence and a CRISPR repeat sequence. In type II systems, the gRNA also comprises a tracrRNA sequence. In type II guide RNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize to form a double strand. In type V guide RNA, the crRNA forms a double strand. In both systems, the double strand binds to a site-directed polypeptide, causing the guide RNA and the site-directed polypeptide to form a complex. The guide RNA provides target specificity to the complex through its association with the site-directed polypeptide. Therefore, the guide RNA directs the activity of the site-directed polypeptide, i.e., in this invention, a Cas protein "modified to lack its nuclease activity," preferably a DNA endonuclease, preferably a Cas (more preferably Cas9) protein / enzyme as described above.

[0469] In some implementations, the guide RNA is double-stranded. The first strand contains an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand contains a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0470] In some implementations, the guide RNA is a single-stranded guide. In the type II system, the single-molecule guide RNA comprises, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-stranded guide adapter, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may contain elements that provide additional functionality (e.g., stability) to the guide RNA. The single-stranded guide adapter connects the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension contains one or more hairpins.

[0471] As illustrated, guide RNAs or other smaller RNAs used in CRISPR / Cas systems can be readily synthesized chemically. While chemical synthesis methods are constantly expanding, purifying these RNAs by methods such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) often becomes more challenging as the polynucleotide length increases significantly beyond about one hundred nucleotides. One approach to producing longer RNAs is to generate two or more molecules linked together. Longer RNAs, such as those encoding the Cas9 endonuclease, are more readily produced enzymatically. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, such as modifications to enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties, as described in the art.

[0472] Typically, a guide sequence is a polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to bind sequence-specifically to the target sequence. In some embodiments, the complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher when optimal alignment is performed using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for sequence alignment, and non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wlieer transform (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 is about 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 nucleotides long. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides long. The ability of the guide sequence to direct the sequence-specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having the corresponding target sequence, for example by transfection with a vector encoding the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence, for example by a Surveyor assay known in the art. Similarly, the cleavage of the target polynucleotide sequence can be assessed in a test tube by providing the target sequence, components of the CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reactions of the test and control guide sequences. Other assays are also possible and will be apparent to those skilled in the art.

[0473] Therefore, those skilled in the art can easily design CRISPR / Cas systems that are "specifically targeted at NPBWR1" by designing corresponding guide RNAs using methods known in the art. Furthermore, it is easy for those skilled in the art to test whether the separately designed guide RNAs of the CRISPR / Cas system are "specifically targeted at NPBWR1," i.e., whether they bind to a specific location within the NPBWR1 target.

[0474] In a preferred embodiment, without being theoretically limited, the guide RNA “specifically targeting NPBWR1” may, for example, have one of the sequences shown in any one of SEQ ID NO:3 to 5.

[0475] The components of the CRISPR / Cas system described above (which can form part of the pharmaceutical composition according to the invention) can be expressed and delivered by means and methods known in the art, as briefly described below.

[0476] Therefore, this disclosure provides a nucleic acid comprising a nucleotide sequence encoding one or more guide RNAs and one or more DNA endonucleases.

[0477] In some implementations, the nucleic acid encoding one or more guide RNAs and DNA endonucleases comprises a vector (e.g., a recombinant expression vector). The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional nucleic acid fragments can be linked. Another type of vector is a viral vector, in which additional nucleic acid fragments can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome.

[0478] In some implementation methods, the vector can direct the expression of nucleic acids operatively linked to it. Such vectors are referred to herein as "recombinant expression vectors," or simply "expression vectors," and all have the same function.

[0479] The term "operably linked" refers to the connection of a target nucleotide sequence to a regulatory sequence in a manner that allows the expression of that nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct nucleotide sequence expression only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of target cells and the desired expression level.

[0480] The expression vectors considered 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., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other vectors considered for use in eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors may be used, provided they are compatible with the host cells.

[0481] In some embodiments, the vector contains one or more transcriptional and / or translational control elements. Depending on the host / vector system used, any element from a wide range of suitable transcriptional and translational control elements can be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc. In some embodiments, the vector is a self-inactivating vector containing inactivated viral sequences or components or other elements of the CRISPR mechanism.

[0482] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that are functional in eukaryotic cells) include: the immediate early promoter of cytomegalovirus (CMV), thymidine kinase of herpes simplex virus (HSV), early and late SV40, long terminal repeat (LTR) sequences of retroviruses, human elongation factor-1 promoter (EF1), hybrid constructs of cytomegalovirus (CMV) enhancers containing fusion with chicken β-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and promoters of mouse metallothionein-I.

[0483] For the expression of small RNAs, including guide RNAs used in conjunction with Cas endonucleases, various promoters such as RNA polymerase III promoters, including, for example, U6 and H1, can be advantageous. Descriptions and parameters enhancing the use of such promoters are known in the art, and additional information and methods are regularly described; see, for example, Ma, H. et al. Molecular Therapy - Nucleic Acids 3 , e161 (2014) doi:10.1038 / mtna.2014.12.

[0484] Expression vectors may also contain ribosome binding sites for translation initiation and transcription terminators. Expression vectors may also include suitable sequences for amplifying expression. Expression vectors may further include nucleotide sequences encoding non-natural tags (e.g., histidine tags, hemagglutinin tags, green fluorescent protein, etc.), which are fused with site-directed polypeptides to produce fusion proteins.

[0485] In some embodiments, the promoter is an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated 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 time-restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.).

[0486] In some embodiments, nucleic acids encoding one or more guide RNAs and / or DNA endonucleases are packaged into or on a delivery medium for delivery to cells. Considered delivery media include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. Various targeting portions can be used to enhance the preferential interaction of such media with desired cell types or locations.

[0487] The complexes, peptides, and nucleic acids of this invention can be introduced into cells through viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipid transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0488] deliver

[0489] Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) can be delivered using viral or non-viral delivery media known in the art. Alternatively, endonuclease peptides can be delivered using non-viral delivery media known in the art (e.g., electroporation or lipid nanoparticles). In other alternative embodiments, DNA endonucleases can be delivered alone as one or more peptides or pre-complexed with one or more guide RNAs.

[0490] Polynucleotides can be delivered via non-viral delivery media, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. Some exemplary non-viral delivery media are described in Peer and Lieberman, Gene Therapy, 18: 1127–1133 (2011) (which focuses on non-viral delivery media for siRNA, but is also used to deliver other polynucleotides).

[0491] Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, can be delivered to cells or patients via lipid nanoparticles (LNPs).

[0492] LNP refers to any particle with a diameter less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size range of the nanoparticles can be 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0493] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as fusogenic phospholipids (DOPE) or membrane component cholesterol, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficiency due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.

[0494] LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0495] Any lipid or combination of lipids known in the art can be used to produce LNPs. 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.

[0496] Lipids can be combined in any molar ratio to produce LNPs. Furthermore, polynucleotides can be combined with (one or more) lipids in a wide range of molar ratios to produce LNPs.

[0497] As previously mentioned, DNA endonucleases and guide RNAs can be administered separately to cells or patients. Alternatively, DNA endonucleases can 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 called ribonucleoprotein particles (RNPs).

[0498] RNA can form specific interactions with either RNA or DNA. While this property is utilized in many biological processes, it also carries the risk of promiscuous interactions in nucleic acid-rich cellular environments. One approach to address this is the formation of ribonucleoprotein particles (RNPs), in which RNA is pre-complexed with endonucleases. Another benefit of RNPs is their protection of RNA from degradation.

[0499] The DNA endonuclease in the RNP can be modified or unmodified. Similarly, the gRNA can be modified or unmodified. Many modifications are known in the art and can be used.

[0500] DNA endonucleases and gRNA can typically be combined in a 1:1 molar ratio. However, RNPs can be prepared using a wide range of molar ratios.

[0501] In some implementations, AAV vectors are used for delivery. Typical 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.

[0502] Table 1

[0503]

[0504] A method for producing packaging cells involves generating cell lines that stably express all the necessary components required for AAV particle production. For example, a plasmid (or multiple plasmids) is integrated into the cell genome containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and optional markers (e.g., neomycin resistance genes). The AAV genome has been introduced into bacterial plasmids via steps such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), adding synthetic adapters 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). The packaging cell lines are then infected with helper viruses such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale rAAV production. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0505] The general principles of rAAV production are summarized in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Several methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988); Samulski et al., (1989, J. Virol., 63:3822-3828); U.S. Patent Nos. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent Nos. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 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.

[0506] In addition to adeno-associated virus vectors, other viral vectors can also be used in the practice of this invention. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, plague viruses, baculoviruses, herpesviruses, Epstein-Barr virus, papillomaviruses, poxviruses, vaccinia virus, and herpes simplex virus.

[0507] Cas9 nuclease can be delivered 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 extent of an immune response. Endonuclease proteins can be complexed with gRNA prior to delivery. Viral vectors allow for efficient delivery; fissuring forms of Cas9 and smaller Cas9 orthologs can be packaged in AAVs, as can be used as donors for HDR. A range of nonviral delivery methods exist that can deliver each of these components, or nonviral and viral methods can be used in tandem. For example, nanoparticles can be used to deliver proteins and guide RNA, while AAVs can be used to deliver donor DNA.

[0508] As described above, this invention relates to pharmaceutical compositions comprising an antagonist / inhibitor of NPBWR1, said pharmaceutical compositions for treating, improving, and / or preventing certain diseases. Therefore, this invention relates to the use of pharmaceutical compositions comprising an antagonist / inhibitor of NPBWR1 in a medical setting.

[0509] This is particularly applicable to CRISPR / Cas systems. Therefore, in this context, the guide RNA of the present invention can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the specific administration route and dosage form. Guide RNA compositions are typically formulated to achieve a physiologically compatible pH, and the pH range is from about 3 to about 11, or from about 3 to about 7, depending on the formulation and route of administration. In alternative embodiments, the pH is adjusted to a range from about pH 5.0 to about pH 8. In some embodiments, the composition comprises a therapeutically effective amount of at least one of the compounds described herein, and one or more pharmaceutically acceptable excipients. Optionally, the composition comprises a combination of the compounds described herein, or may include a second active ingredient for treating or preventing bacterial growth (e.g., but not limited to, antibacterial or antimicrobial agents), or may include a combination of the reagents of the present invention.

[0510] Suitable excipients include, for example, carrier molecules, which include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral 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, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, fluids (e.g., but not limited to oils, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffers, etc.

[0511] "Administration" means delivering the composition described herein, comprising guide ribonucleic acid (gRNA) and one or more DNA endonucleases (or a carrier comprising a polynucleotide encoding gRNA and one or more DNA endonucleases), to a subject by a method or route that causes the composition to be at least partially located at the desired site. The composition may be administered via any suitable route that produces an effective treatment in the subject, i.e., administration results in delivery to the desired location in the subject, at least a portion of the delivered composition is delivered to the desired site, and this is sustained for a period of time. Administration methods include injection, infusion, drip, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarticular, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For cell delivery, administration by injection or infusion is generally preferred.

[0512] In some aspects, the present invention provides methods that may comprise the delivery of one or more polynucleotides, such as, or one or more vectors as described herein, one or more transcripts thereof, and / or one or more proteins transcribed therefrom, to a host cell. In some aspects, the present invention also provides cells produced by such methods, and animals that may contain or 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. Conventional viral and nonviral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the CRISPR system to cultured cells or a host organism. 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 medium (e.g., liposomes). Viral vector delivery systems include DNA and RNA viruses that have an episomal or integrated genome upon delivery to the cell. For reviews on gene therapy, 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); Haddadada et al., Current Topics in Microbiology and Immunology, Doerfler and Bohm (ed.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0513] Non-viral delivery methods for nucleic acids include lipid transfection, microinjection, bio-ballistic delivery, virions, liposomes, immunoliposomes, polycationic or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DMA absorption. Lipid transfection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipid transfection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for effective receptor recognition of polynucleotides include those in Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be made to cells (e.g., in vitro or ex vivo) or to target tissues (in vivo).

[0514] The preparation of lipid-nucleic acid complexes (including targeted liposomes such as immunoliposome complexes) is well known to those skilled in the art (see, for example, 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 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).

[0515] The use of RNA or DNA virus-based systems for nucleic acid delivery leverages a highly evolved process of targeting viruses to specific cells within the body and transporting the viral payload to the cell nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to process cells in vitro, with the modified cells optionally administered to patients (ex vivo). Conventional virus-based systems can include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer.

[0516] It is possible to integrate the inserted transgene into the host genome using retroviral, lentiviral, and adeno-associated virus gene transfer methods, typically leading to long-term expression of the inserted transgene. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

[0517] Retroviral tropism can be altered by incorporating exogenous envelope proteins, thereby amplifying the potential target population of target cells. Lentiviral vectors are a type of retroviral vector capable of transducing or infecting non-dividing cells, typically producing high viral titers. Therefore, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats (LTRs) with a packaging capacity of up to 6–10 kb of exogenous sequence. The smallest cis-acting LTR is sufficient to replicate and package the vector, which is then used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simmon 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); PCT / US94 / 05700).

[0518] As described above, the present invention relates to pharmaceutical compositions comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, said pharmaceutical composition being 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, wherein said antagonist / inhibitor is a CRISPR / Cas system specifically targeting NPBWR1, wherein said CRISPR / Cas system Cas protein is modified to lack its nuclease activity, and said Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression.

[0519] Therefore, according to the present invention, the Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression.

[0520] 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., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may contain any additional protein sequence and optionally include a linker sequence between any two domains.

[0521] According to the present invention, the heterologous protein domain is a transcriptional repression domain or an epigenetic modification domain, which can repress the expression of NPBWR1.

[0522] Examples of protein domains that can be fused with CRISPR enzymes are described below.

[0523] As described above, the purpose of the transcriptional repression domain or epigenetic modification domain fused with the Cas protein that can repress NPBWR1 expression is to regulate the transcription of the target nucleic acid, i.e., the transcription of the NPBWR1 gene, by reducing (preferably completely repressing) the transcription of the target nucleic acid.

[0524] As outlined, this typically involves contacting the target nucleic acid with a Cas9 peptide and guide RNA that inactivate the enzyme.

[0525] The corresponding transcriptional regulation has therapeutic applications in this invention.

[0526] In preferred embodiments, in some cases, transcriptional regulation, preferably transcriptional repression, provides selective regulation of the target nucleic acid, namely the NPBWR1 gene (e.g., reduction or increase (enlargement is contemplated in the context of the activating / activating arms of the invention, further described below)). For example, "selective" reduction of the transcription of the target nucleic acid, compared to the transcriptional level of the target nucleic acid in the absence of the RNA / variant Cas9 polypeptide complex targeting DNA, results in a reduction of 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 greater than 90%. Selective reduction of the transcription of the target nucleic acid may reduce the transcription of the target nucleic acid but not substantially reduce the transcription of non-target nucleic acids; for example, the transcription of non-target nucleic acids may be reduced, if any, by less than 10% compared to the transcriptional level of non-target nucleic acids in the absence of the RNA / variant Cas9 polypeptide complex targeting DNA.

[0527] As further outlined below, in the case of the agonist / activator arm of the invention further described below, an increase in transcription is envisioned. The “selective” increase in target DNA transcription, compared to the level of target DNA transcription in the absence of the RNA / variant Cas9 polypeptide complex targeting the DNA, can result in an increase in target DNA transcription of at least about 1.1-fold (e.g., at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, or at least about 20-fold). Selective increase in transcription of target DNA may increase the transcription of target DNA, but not substantially increase the transcription of non-target DNA. For example, there may be an increase in the transcription of non-target DNA, if any, but less than about 5-fold (e.g., less than about 4-fold, less than about 3-fold, less than about 2-fold, less than about 1.8-fold, less than about 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, or less than about 1.1-fold) compared to the level of transcription of non-target DNA in the absence of RNA / variant Cas9 polypeptide complexes.

[0528] As a non-limiting example, an increase can be achieved by fusing dCas9 (inactivated Cas9, i.e., a Cas protein "modified to lack its nuclease activity" for the purposes of this invention) with a heterologous sequence. Suitable fusion couplers include, but are not limited to, peptides that provide indirect increased transcriptional activity by acting directly on the target DNA or on peptides associated with the target DNA (e.g., histones or other DNA-binding proteins). Suitable fusion couplers include, but are not limited to, peptides that provide the following activities: methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristylation activity, or demyristylation activity.

[0529] Other suitable fusion couplers include, but are not limited to, peptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcription regulators, etc.).

[0530] Non-limiting examples of methods for enhancing transcription in eukaryotes using dCas9 fusion proteins include the fusion of dCas9 with activation domains (ADs) (e.g., GAL4, herpesvirus activators 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.). RNA targeting DNA can be engineered to target known transcriptional response elements (e.g., promoters, enhancers, etc.), known upstream activating sequences (UAS), or sequences with unknown or known functions that are suspected of controlling target DNA expression.

[0531] Other fusion couples

[0532] Non-limiting examples of fusion couplers that achieve increased or decreased transcription include transcriptional activators and transcriptional repression domains (e.g., Kruppel-associated boxes (KRAB or SKD); Mad mSIN3 interaction domains (SID); ERF repression domains (ERD), etc.). In some such cases, the dCas9 fusion protein is targeted by RNA to a specific location (i.e., sequence) in the target DNA and exerts locus-specific regulation, such as blocking the binding of RNA polymerase to a promoter (which selectively represses the transcriptional activator function), and / or altering the local chromatin state (e.g., when the target DNA is modified with the fusion sequence or when a polypeptide associated with the target DNA is modified). In some cases, the changes are transient (e.g., transcriptional repression or activation). In some cases, the changes are heritable (e.g., when epigenetic modifications are made to the target DNA or to proteins associated with the target DNA, such as nucleosome histones).

[0533] In some embodiments, the heterologous sequence may be fused to the C-terminus of the dCas9 peptide. In some embodiments, the heterologous sequence may be fused to the N-terminus of the dCas9 peptide. In some embodiments, the heterologous sequence may be fused to an internal portion of the dCas9 peptide (i.e., a portion other than the N- or C-terminus).

[0534] The biological effects of the method using the tested dCas9 fusion protein of the present invention can be detected by any convenient method (e.g., gene expression assay; chromatin-based assays, such as chromatin immunoprecipitation (ChiP), chromatin in vivo assay (CiA); etc.).

[0535] In a preferred embodiment where the effector domain of the fusion protein is a transcriptional activation domain or a transcriptional repression domain, the guide RNA directs the fusion protein to a specific chromosomal sequence, wherein the transcriptional activation domain or the transcriptional repression domain activates or represses the expression of the target chromosomal sequence, respectively.

[0536] 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, wherein the epigenetic modification domain modifies the structure of the targeted chromosomal sequence. Epigenetic modifications include histone acetylation, methylation, and / or nucleotide methylation. In some cases, structural modifications to the chromosomal sequence lead to changes in chromosomal sequence expression.

[0537] In a preferred embodiment, the transcriptional activation domain is, for example, VP64. Alternatively, a transcriptional repression domain may be used, such as a KRAB domain, a SID domain, or a SID4X domain.

[0538] Furthermore, based on the fundamental principles of the invention and experimental evidence from the appended embodiments, as explained below, an increase in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have medical significance, particularly in certain specific conditions or diseases.

[0539] Therefore, the present invention relates not only to the above-mentioned pharmaceutical compositions comprising 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, but also, in different aspects of the present invention, to pharmaceutical compositions comprising neuropeptide B / W receptor (NPBWR1) agonists / activators, used in methods for treating, improving or preventing manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0540] Therefore, in a further embodiment, the present invention also relates to a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of manic bipolar disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia.

[0541] In a preferred embodiment, within the context of the second aspect of the invention, the invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator, used in methods for treating, improving, or preventing manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0542] The neuropeptide B / W receptor (NPBWR1) agonist / activator described herein is not an agonist selected from the polypeptide ligands corresponding to the following: L7 (with the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (with the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (with the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (with the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0543] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) agonist / activator does not contain or is not composed of an agonist selected from or consisting of a polypeptide ligand corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0544] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) agonist / activator is an agonist / activator as defined above and below, provided that agonists / activators selected from polypeptide ligands corresponding to the following are excluded: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0545] In another preferred embodiment, within the context of the second aspect of the invention, the invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator, used in methods for treating, improving, or preventing manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0546] The neuropeptide B / W receptor (NPBWR1) agonist / activator described herein is not an active agent that regulates the binding properties between the GPR7 / NPBWR1 peptide and a peptide ligand corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0547] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) agonist / activator does not contain or is not composed of an active agent that modulates the binding properties between the GPR7 / NPBWR1 peptide and a peptide ligand corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0548] In another preferred embodiment, the neuropeptide B / W receptor (NPBWR1) agonist / activator is an agonist / activator as defined above and below, provided that activators that regulate the binding properties between the GPR7 / NPBWR1 peptide and peptide ligands corresponding to the following: L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0549] The following peptide ligands are disclosed in WO 03 / 082907 as SEQ ID NO:1, 3, 5 and 7 and are described as agonists of GRP7 / NPBWR1: peptide ligands of L7 (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGL (SEQ ID NO:8)), L7C (having the amino acid sequence WYKPAAGHSSYSVGRAAGLLSGLRRSPYA (SEQ ID NO:9)), L8 (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGL (SEQ ID NO:10)) or L8C (having the amino acid sequence WYKHVASPRYHTVGRAAGLLMGLRRSPYLW (SEQ ID NO:11)).

[0550] Bipolar disorder in manic phase (ICD-10 F31), and appetite disorders, preferably anorexia or bulimia, are diseases or syndromes known in the art and are medical indications classified in the ICD system (i.e., the well-known list of medical classifications established by the World Health Organization (WHO)). ICD stands for International Statistical Classification of Diseases and Related Health Problems (ICD). In the following text, when referring to a more specifically defined condition or disease, reference is made to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), namely “ICD10”.

[0551] This invention is not limited to manic bipolar disorder (ICD-10 F31) or appetite disorders, preferably anorexia or bulimia. Any manic bipolar disorder (ICD-10 F31) or appetite disorder, preferably anorexia or bulimia, can be treated, improved, and / or prevented by the agonist / activator of the neuropeptide B / W receptor (NPBWR1) of this invention.

[0552] Without being bound by theory, in a particularly preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for the treatment, improvement or prevention of manic bipolar disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, wherein the manic bipolar disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia, is as follows.

[0553] In a preferred embodiment, the present invention relates to bipolar disorder (ICD-10 F31) in the manic phase. The term "bipolar disorder (ICD-10 F31)" has been described above. Therefore, the definition of this condition or disorder, as stated above, also applies except that the agonist / activator in this invention is used during the manic phase of said disorder.

[0554] The terms “agonist” or “activator” are used interchangeably herein. These terms are known in the art and refer to compounds / substances capable of fully or partially activating or enhancing the physiological activity of one or more specific proteins. Therefore, in the context of this invention, the agonist / activator activates or enhances the physiological activity of a protein, such as the neuropeptide B / W receptor (NPBWR1), upon binding. The binding of an “agonist / activator” to a given protein, such as the neuropeptide B / W receptor (NPBWR1), enhances the binding of endogenous activating molecules bound to said protein. Therefore, the term “agonist” as used herein also encompasses enhancing agonists.

[0555] However, in addition to this, in the context of the present invention, an "agonist" or "activator" of the neuropeptide B / W receptor (NPBWR1) can also activate or enhance the function of said protein by activating or enhancing the expression of nucleic acid molecules encoding a given protein such as the neuropeptide B / W receptor (NPBWR1). Therefore, an agonist / activator of the neuropeptide B / W receptor (NPBWR1) can lead to 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 increase in activity can be measured / detected by methods known in the art and described herein. Therefore, in the context of the present invention, an activator of the neuropeptide B / W receptor (NPBWR1) may also include a transcriptional activator capable of increasing the function of the neuropeptide B / W receptor (NPBWR1) and its expression. As described in detail below, increased expression and / or activity of neuropeptide B / W receptor (NPBWR1) by agonists / activators of NPBWR1 leads to increased activity (and / or expression) of NPBWR1, thereby enhancing the functional capacity of NPBWR1.

[0556] Consistent with the basic principles of this invention, the anticipated increase in the activity of the functional neuropeptide B / W receptor (NPBWR1) is expected to have multiple medical implications.

[0557] As explained below, the expected increase in the activity of the functional neuropeptide B / W receptor (NPBWR1) is medically significant, namely its impact on manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia-related diseases / disorders.

[0558] The terms “activator” and / or “agonist” do not imply any specific biological mechanism of action and are considered to explicitly 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, it should be clearly understood that the terms “activator” and / or “agonist” encompass all previously identified terms, names, and functional states and characteristics whereby the neuropeptide B / W receptor (NPBWR1) itself, the biological activity of the neuropeptide B / W receptor (NPBWR1) (including, but not limited to, its ability to regulate / inhibit Bdnf expression; its ability to bind neuropeptide B / W; and / or its ability to bind G proteins) or the result of biological activity is significantly increased or enhanced to any meaningful extent, for example, at least 5%, 10%, 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, 500%, or 2, 3, 4, 5, 10, 20, 50, 100, or 1000 times. Activators / agonists can increase abnormal levels of the biological activity of the neuropeptide B / W receptor (NPBWR1), which can cause adverse reactions and / or disease in subjects, to levels corresponding to those in healthy subjects, thereby preventing, halting the progression and / or curing adverse reactions and / or disease.

[0559] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement or prevention of bipolar disorder (ICD-10 F31) in manic phase, anorexia, preferably anorexia or bulimia, wherein the activator increases the biological activity of the neuropeptide B / W receptor (NPBWR1) by at least 2, 3, 4, 5, 10, 20, 50, 100 or 1000 times.

[0560] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of bipolar disorder (ICD-10 F31) in manic phase, anorexia, preferably anorexia or bulimia, wherein the activator / agonist increases the abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse reactions and / or disease by at least 2, 3, 4, 5, 10, 20, 50, 100, or 1000 times.

[0561] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) in a method for treating, improving, or preventing bipolar disorder (ICD-10 F31) in a manic phase, anorexia, preferably anorexia, or bulimia, wherein the activator / agonist reduces the abnormal level of the biological activity of the neuropeptide B / W receptor (NPBWR1) that causes and / or promotes adverse reactions and / or disease by at least 3-fold.

[0562] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the activator / agonist causes an abnormal increase in the bioactivity of the neuropeptide B / W receptor (NPBWR1) that causes / or promotes manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0563] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the inhibitor increases the abnormal level of biological activity of the neuropeptide B / W receptor (NPBWR1) in a subject that induces or promotes manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, to a level comparable to that of a healthy subject.

[0564] According to the present invention, the term "activator / agonist of neuropeptide B / W receptor (NPBWR1)" also refers to a compound or substance capable of increasing or enhancing the physiological activity of neuropeptide B / W receptor (NPBWR1). In the context of the present invention, the activator can therefore, for example, activate or enhance the physiological activity of neuropeptide B / W receptor (NPBWR1) when the compound / substance (i.e., the activator / agonist) binds to neuropeptide B / W receptor (NPBWR1).

[0565] "Neopeptide B / W receptor (NPBWR1) activators / agonists" can also increase or activate the function of the neuropeptide B / W receptor (NPBWR1) by increasing or activating or enhancing the expression of nucleic acid molecules encoding the neuropeptide B / W receptor (NPBWR1). Therefore, activators / agonists of the neuropeptide B / W receptor (NPBWR1) can lead to 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.

[0566] Activators / agonists of the neuropeptide B / W receptor (NPBWR1) can increase or enhance the aberrant expression level of NPBWR1 to levels corresponding to those in healthy subjects, thereby preventing, halting, or curing adverse reactions and / or diseases / conditions in subjects whose aberrant expression levels can cause and / or promote adverse reactions and / or diseases / conditions associated with manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, as outlined above and below. This can be reflected in increased and / or aberrant NPBWR1 expression, thereby restoring healthy NPBWR1 expression levels. NPBWR1 expression levels are correlated to some extent with NPBWR1 activity until translational mechanisms and / or substances binding to NPBWR1 reach saturation. The expression level of neuropeptide B / W receptor (NPBWR1) can be measured / detected by methods known in the art.

[0567] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement or prevention of bipolar disorder (ICD-10 F31) and appetite disorders, preferably anorexia or bulimia, according to the present invention, wherein the activator increases the expression level of the neuropeptide B / W receptor (NPBWR1) gene product.

[0568] Therefore, one aspect of the present invention relates to activators / agonists of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement or prevention of disorders associated with manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, according to the present invention, wherein the activator / agonist increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes adverse reactions and / or diseases / disorders.

[0569] Therefore, the present invention relates, in one aspect, to activators / agonists of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of disorders associated with manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, according to the present invention, wherein the activator / agonist increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that causes and / or promotes the aforementioned manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia.

[0570] Therefore, the present invention relates, in one aspect, to an activator / agonist of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of disorders associated with manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, according to the present invention, wherein the activator / agonist increases the abnormal expression level of the neuropeptide B / W receptor (NPBWR1) gene product that induces and / or promotes manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, in subjects to an expression level comparable to that of healthy subjects.

[0571] Activators / agonists of the neuropeptide B / W receptor (NPBWR1) can exert their activating function by directly interacting with the NPBWR1 protein, i.e., by interacting with any part of the NPBWR1 protein, such as its extracellular domain, transmembrane domain, and / or cytoplasmic domain. Activators / agonists of NPBWR1 can also exert any inhibitory effect on NPBWR1 function by activating or enhancing or increasing any upstream or downstream pathway components that substantially contribute to NPBWR1 function. Activators / agonists of NPBWR1 can also exert any indirect activating effect on any NPBWR1 activating molecule, such as nucleic acids, ribonucleic acid (RNA), double-stranded ribonucleic acid (dsRNA), chromatin reader proteins, and / or ligands.

[0572] The efficacy of activators / agonists of the neuropeptide B / W receptor (NPBWR1) can be described using the half-maximal inhibitory concentration (IC50) value. For the purposes of this invention, activators / agonists of the neuropeptide B / W receptor (NPBWR1) preferably exhibit high IC50 values. The IC50 values ​​of neuropeptide B / W receptor (NPBWR1) activators / agonists can be higher than 100 µM, higher than 90 µM, higher than 80 µM, higher than 70 µM, higher than 60 µM, higher than 50 µM, higher than 40 µM, higher than 30 µM, higher than 20 µM, or higher than 10 µM, with higher values ​​being preferred over lower values.

[0573] Therefore, the present invention relates to agonists / activators of neuropeptide B / W receptor (NPBWR1), which, according to the present invention, are used to treat, improve or prevent bipolar disorder (ICD-10 F31) in manic phase, appetite disorders, preferably anorexia or bulimia, wherein the half-maximal inhibitory concentration (IC50) efficacy 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 40 μM, greater than 30 μM, greater than 20 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.

[0574] Technicians know how to determine the IC50 value of activators / agonists of the neuropeptide B / W receptor (NPBWR1). This paper hypothesizes that activators / agonists of the neuropeptide B / W receptor (NPBWR1) may exhibit additional IC50 values ​​and / or identify other activators / agonists of the neuropeptide B / W receptor (NPBWR1) with different IC50 values.

[0575] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for use in the treatment, improvement or prevention of manic bipolar disorder (ICD-10 F31), appetite disorder, preferably anorexia or bulimia as defined above, wherein the agonist / activator is selected from NPBWR1 activating peptides, NPBWR1 activating small binding molecules and NPBWR1 RNA molecules.

[0576] In a preferred embodiment, the agonist / activator is an NPBWR1 RNA molecule encoding NPBWR1. Therefore, the NPBWR1 RNA molecule encoding NPBWR1 is a sense molecule used according to the present invention, and 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.

[0577] In a preferred embodiment, the NPBWR1 RNA molecule encoding NPBWR1 contains not only the coding region encoding the NPBWR1 polypeptide (preferably including its 5' start codon), but also the UTR upstream of the coding sequence.

[0578] The ribonucleic acid (RNA) molecule used in this invention relates to a polymeric molecule assembled into a chain of nucleotides called G, A, U, and C. Each nucleotide in the RNA contains a ribose sugar, with carbon atoms numbered 1' to 5'. A nitrogenous base is attached to the 1' position, typically adenine (A), cytosine (C), guanine (G), or uracil (U). In the polyRNA molecule, a phosphate group is attached to the 3' position of one ribose and the 5' position of the next ribose. Thus, the nucleotides in the polyRNA molecule are covalently linked to each other, with the phosphate group of one nucleotide binding 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 in relation to 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 class of RNA molecules that transmit genetic information from DNA to the ribosome, where the amino acid sequence of the protein product expressed by the gene is determined. After RNA polymerase transcribes the primary transcript mRNA (called precursor mRNA), the processed mature mRNA is translated into amino acid polymers: proteins, as illustrated by the central dogma of molecular biology. As in DNA, the genetic information of mRNA is contained in nucleotide sequences arranged as codons, each consisting of three bases. Each codon encodes a specific amino acid, except for stop codons, which terminate protein synthesis.

[0579] RNA molecules may also include their 3' end UTR. Thus, the RNA molecules of the present invention are structurally similar to naturally occurring "normal" mRNA molecules, having a coding region and (5' and 3') untranslated regions (UTRs) and optional poly-A tails.

[0580] The term "coding region including a start codon at its 5' end" as used in this invention refers to a sequence of codons that is decoded by ribosomes and translated into a protein based on information provided by the genetic code. A coding region typically begins with a start codon at its 5' end and ends with a stop codon. Typically, the start codon is an AUG triplet, and the stop codon is UAA, UAG, or UGA. In addition to encoding proteins, portions of the coding region can serve as regulatory sequences in precursor mRNA, such as exon splicing enhancers or exon splicing silencers. The gene coding region encoding a polypeptide or protein as used in this invention is also called a coding sequence or CDS (from the coding DNA sequence), and it is a portion of the gene DNA or RNA composed of exons that encodes the polypeptide or protein. The coding region in mRNA is flanked by a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), which are also parts of an exon. Furthermore, the mRNA molecule may also 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 that are not translated into protein molecules.

[0581] The term "untranslated region" or "UTR" as used in this invention refers to the untranslated mRNA segments upstream of the start codon and downstream of the stop codon, and are therefore referred to as the 5'UTR and 3'UTR, respectively. These regions are transcribed along with the coding regions and are therefore exons, as they are present in the mature mRNA.

[0582] The RNA molecule of this invention may also contain a poly-A tail. A poly-A tail is a long string of adenine nucleotides (typically several hundred) added to the 3' end of the precursor mRNA through a process called polyadenylation. This tail facilitates export from the nucleus and translation, and protects the 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, it is a segment of RNA containing only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation.

[0583] The RNA molecules of the present invention containing the above-mentioned UTRs can be generated / synthesized by recombination (e.g., in vivo or in vitro systems) or synthesized by methods known to those skilled in the art.

[0584] In vitro transcription of RNA typically requires a linear DNA template containing a double-stranded promoter region, where a DNA-dependent RNA polymerase binds and initiates RNA synthesis. The coding region can be 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. Commonly used DNA-dependent RNA polymerases are T7 polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase.

[0585] Transcription templates for in vitro transcription include, for example, cDNA templates synthesized from RNA precursors, PCR-generated templates, chemically synthesized oligonucleotides, and plasmid constructs. Many widely used plasmid cloning vectors contain phage polymerase promoters located on each side of the multiple cloning site to allow transcription of either strand of the nucleotide sequence inserted at the multiple 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 tandem phage polymerase promoters (on the same side of the multiple cloning site), allowing the use of any of the three polymerases SP6, T7, or T3.

[0586] The RNA molecules of the present invention can be recombined in vivo using methods known to those skilled in the art.

[0587] Alternatively, the RNA molecules of the present invention can be produced in an in vitro system using, for example, an in vitro transcription system. In vitro transcription systems are well known and typically require a purified linear DNA template containing a DNA sequence “encoding” the RNA molecule, wherein the DNA sequence is under the control of a suitable promoter. Furthermore, the in vitro transcription system typically requires nucleoside triphosphates, a buffer system containing DTT and magnesium ions, and a suitable RNA polymerase that provides the enzymatic activity to transcribe the DNA sequence in vitro into the corresponding RNA molecules of the present invention.

[0588] In addition, RNA molecules can be chemically synthesized, for example, through routine chemical synthesis on an automated nucleotide sequencer using a solid support and standard techniques, or by chemically synthesizing their respective DNA sequences and subsequently transcribing those DNA sequences in vitro or in vivo.

[0589] In a further preferred embodiment, the present invention relates to a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator for the treatment, improvement or prevention of bipolar disorder (ICD-10 F31) in manic phase, appetite disorder, preferably anorexia or bulimia.

[0590] Neuropeptide B is known in the art and is a short, bioactive peptide whose precursor is found in the human body by… NBP The gene encodes neuropeptide B, which is a ligand for two G protein-coupled receptors. It functions by binding to two G protein-coupled receptors, neuropeptide B / W receptors (collectively referred to as neuropeptide B / W receptors (NPBWR1) in this invention), namely NPBW1 and NPBW2, which are encoded by the gene. NPBWR1 and NPBWR2 Encoding. Neuropeptide B is 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 tract.

[0591] The amino acid sequence of human neuropeptide B can be retrieved using UniProt accession number Q8NG41.

[0592] The amino acid sequence of human neuropeptide B is as follows:

[0593] MARSATLAAAALALCLLLAPPGLAWYKPAAGHSSYSVGRAAGLLSGLRRSPYARRSQPYRGAEPPGGAGA SPELQLHPRLRSLAVCVQDVAPNLQRCERLPDGRGTYQCKANVFLSLRAADCLAA (SEQ ID NO: 6).

[0594] Neuropeptide W is a short human neuropeptide. Neuropeptide W acts as a ligand for two neuropeptide B / W receptors (collectively referred to as neuropeptide B / W receptor (NPBWR1) in the context of this invention) NPBWR1 and NPBWR2, which are integrated into the GPCR family of α-helical transmembrane proteins.

[0595] The amino acid sequence of neuropeptide W can be retrieved using UniProt accession number Q8N729.

[0596] The amino acid sequence of human neuropeptide W is as follows:

[0597] MAWRPGERGAPASRPRLALLLLLLPLPSGAWYKHVASPRYHTVGRAAGLLMGLRRSPYLWRRALRAAAGPLARDTLSPEPAAREAPLLLPSWVQELWETRRRSSQAGIPVRAPRSPRAPEPALEPESLDFSGAGQRLRRDVSRPAVDPAANRLGLPCLAPGPF (SEQ ID NO: 7).

[0598] However, the neuropeptide B and neuropeptide W used in this invention are not particularly limited to the specific amino acid sequences described above, but can be modified forms of the amino acid sequences of the neuropeptide B and neuropeptide W, as long as they can bind to and / or activate the neuropeptide B / W receptor (NPBWR1) and thus transduce their signals in the same way as unmodified neuropeptide B and neuropeptide W.

[0599] Therefore, in a preferred embodiment, the neuropeptide B and neuropeptide W used in this invention are neuropeptides comprising 1 to 8 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising 1 to 7 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising 1 to 6 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising 1 to 5 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising 1 to 4 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising 1 to 3 substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The neuropeptide sequence may also be a sequence comprising one or two substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. Most preferably, the neuropeptide sequence may also be a sequence comprising one substitution, deletion, or insertion compared to SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0600] Preferably, the amino acid substitutions, deletions, or insertions compared to SEQ ID NO:6 and SEQ ID NO:7 are performed at non-conservative positions in the sequences of SEQ ID NO:6 and SEQ ID NO:7 compared to the corresponding neuropeptide B and W sequences from organisms or species other than humans.

[0601] To determine whether a particular amino acid position is less conserved and therefore preferably modified as described above, those skilled in the art can use means and methods well known in the art, for example, manual or by using computer programs known to those skilled in the art. Such alignment can be performed, for example, by means and methods known to those skilled in the art, such as using known computer algorithms like the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. Preferably, in such alignment, the maximum homology is assigned to conserved amino acid residues present in the amino acid sequence. Preferably, ClustalW2 is used for amino acid sequence comparison. In the case of pairwise comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; vacancy open: 10; vacancy extension: 0.1. In the case of multiple comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; vacancy open: 10; vacancy extension: 0.2; vacancy distance: 5; no end vacancies.

[0602] Those positions that are proven to be "identical" (i.e., "conservative") are preferably not substituted, deleted, or inserted. According to the invention, in the context of two or more nucleic acid or amino acid sequences, the term "identical" or "percentage identity" means that two or more sequences or subsequences are identical, or have a specified percentage of identical amino acid residues or nucleotides with SEQ ID NO:6 and SEQ ID NO:7, respectively. Those skilled in the art know how to determine the percentage of identity between two or more sequences using algorithms known in the art, such as those based on the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (BrutlagComp. App. Biosci. 6. (1990), 237. (245)).

[0603] Although the FASTDB algorithm typically does not account for internal non-matching deletions or additions, i.e., vacancies, in its calculations, this can be manually corrected to avoid overestimating the identity percentage. However, CLUSTALW considers sequence vacancies in its identity calculations. Those skilled in the art can also utilize 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). The BLASTN program (for nucleic acid sequences) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=4, and a comparison of two strands. For amino acid sequences, the BLASTP program uses a word length of 3 (W) and an expected value of 10 (E). The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses a word length of 50 (B), an expected value of 10 (E), M=5, N=4, and a comparison of two chains by default.

[0604] Preferably, the above-mentioned substitutions of up to 1, 2, 3, 4, 5, 6, 7 or 8 amino acids are conservative amino acid substitutions referring to the sequences of SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0605] These “conserved amino acid substitutions” refer to the substitution of an amino acid in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent changes without altering the protein’s biological activity. Those skilled in the art will recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. 4th Edition, (1987), 224). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. In the context of this invention, the neuropeptide sequences of this invention comprise polypeptide chains having sequences having, compared to specific amino acid sequences disclosed herein (e.g., with SEQ ID NO:6 and SEQ ID NO:7, respectively), up to 0 (no change), 1, 2, 3, 4, 5, 6, 7, or 8 conserved amino acid substitutions.

[0606] Preferred examples of replacement are those listed in Table 1 below:

[0607] Table 1 Exemplary Conserved Amino Acid Substitutions

[0608]

[0609] Neuropeptide sequences having one or more of the aforementioned substitutions, deletions, or insertions, compared to SEQ ID NO:6 and SEQ ID NO:7, respectively, can produce neuropeptide sequences having similar capabilities to SEQ ID NO:6 and SEQ ID NO:77 (in terms of the ability to bind and / or activate the neuropeptide B / W receptor (NPBWR1), and thus be able to transduce their signals in the same manner as unmodified neuropeptide B and neuropeptide W), preferably with higher capabilities than SEQ ID NO:6 and SEQ ID NO:7, respectively. Those skilled in the art can readily determine the properties / capabilities of a given modified neuropeptide sequence compared to SEQ ID NO:6 and SEQ ID NO:7 using methods known in the art. Therefore, the properties / capabilities of a given modified neuropeptide sequence compared to SEQ ID NO:6 and SEQ ID NO:7 relate to the activity of a particular sequence to have the ability to mediate the binding and / or activation of the neuropeptide B / W receptor (NPBWR1), and thus be able to transduce their signals in the same manner as unmodified neuropeptide B and neuropeptide W. The ability to bind to and / or activate the neuropeptide B / W receptor (NPBWR1), and therefore to transduce its signal in the same manner as unmodified neuropeptide B and neuropeptide W, can be determined, for example, by the methods described in the appended examples and the methods outlined below.

[0610] Regarding the ability of neuropeptides B or W to bind to and / or activate NPBWR1, as described above, the expression of Bdnf can be determined using, for example, quantitative PCR.

[0611] In short, the ability of NPBWR1 to bind neuropeptide B / W can be assessed using, for example, FRAP (fluorescence recovery after bleaching), where neuropeptide B or neuropeptide W is used as a substrate to evaluate binding to NBWBR1.

[0612] The corresponding testing methods are known in the art and can be easily performed by technicians using conventional methods.

[0613] Unbound by theory, the ability of neuropeptides B or W to bind to and / or activate NPBWR1 is often indirectly measured by regulating / activating the expression of the downstream protein brain-derived neurotrophic factor (Bdnf). Quantitative PCR can be used to assess Bdnf RNA levels. Increased Bdnf expression indicates Npbwr1 inhibition. Correspondingly, decreased Bdnf expression indicates Npbwr1 activation.

[0614] However, the neuropeptide sequences used in this invention are not particularly limited to the specific sequences and deletions, substitutions, or insertions described above, but may also involve neuropeptide sequences comprising sequences showing amino acid additions compared to SEQ ID NO:6 and SEQ ID NO:7, respectively. The addition of amino acids can be flanking or diffuse. Therefore, additional amino acids can be added to the N- and / or C-termini of the neuropeptide sequences of this invention. Alternatively, or in addition to these flanking additional amino acids, the additional amino acids can also be internal to the amino acid sequence of the neuropeptide sequences of this invention. The additional amino acids comprise polypeptide chains 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 even more preferably up to 30 amino acids. Given the fundamental principle that adding amino acids may not alter the aforementioned functional properties of the neuropeptide sequences of the present invention, the length of the added amino acids may be 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 capabilities similar to SEQ ID NO:6 and SEQ ID NO:7 respectively (in terms of being able to bind and / or activate the neuropeptide B / W receptor (NPBWR1), and thus be able to transduce its signal as equivalent to unmodified neuropeptide B and neuropeptide W), preferably higher than those of SEQ ID NO:6 and SEQ ID NO:7 respectively.

[0615] The neuropeptide sequences of the present invention can be recombinantly or synthetically generated / synthesized using methods known to those skilled in the art. More specifically, as further described below, the neuropeptides of the present invention can be recombinantly generated using methods known to those skilled in the art, or can be conveniently synthesized on an automated peptide synthesizer, for example, using a solid support and standard techniques of repetitive orthogonal deprotection and coupling. The free amino groups in the peptides, which are subsequently used for conjugation to partial or other active agents, are advantageously blocked with standard protecting groups such as Boc groups, while the N-terminal residues can be acetylated to increase serum stability. These 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 preparing peptides for subsequent use in the constructs of the present invention, they are advantageously cleaved from the resin to produce the corresponding C-terminal amides to inhibit in vivo carboxypeptidase activity.

[0616] In a further preferred embodiment, the present invention relates to a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) for the treatment, improvement, or prevention of the aforementioned manic phase of bipolar disorder (ICD-10F31), appetite disorder, preferably anorexia or bulimia, wherein the agonist / activator is a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused with an effector domain selected from a transcriptional activation domain and an epigenetic modification domain capable of activating NPBWR1 expression.

[0617] The feature “a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity and the Cas protein is fused with an effector domain selected from transcriptional activation domains and epigenetic modification domains” has been described in the context of pharmaceutical compositions comprising neuropeptide B / W receptor (NPBWR1) antagonists / inhibitors for use in the treatment, improvement or prevention of mood disorders / affective disorders and / or chronic stress and / or anxiety disorders, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically targeting NPR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity and the Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPR1 expression.

[0618] The pharmaceutical composition now described, comprising a neuropeptide B / W receptor (NPBWR1) agonist / activator (which, as described above, is used to treat, improve, or prevent manic bipolar disorder (ICD-10 F31), appetite disorders, preferably anorexia or bulimia, wherein the agonist / activator is a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused with an effector domain selected from transcriptional activation domains and epigenetically modified domains capable of activating NPBWR1 expression), differs only in the fact that the Cas protein is fused with an effector domain selected from transcriptional activation domains and epigenetically modified domains capable of activating NPBWR1 expression (in contrast, in the context of the pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor described above, the Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetically modified domains capable of repressing NPBWR1 expression).

[0619] Therefore, apart from this single difference, this feature and preferred embodiment, with necessary modifications, are equally applicable to the agonist / activator arm defined herein in the context of a pharmaceutical composition comprising a neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor, 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, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and the Cas protein is fused with an effector domain selected from a transcriptional repression domain and an epigenetically modified domain capable of repressing the expression of NPBWR1.

[0620] In particular, suitable effector domains selected from transcriptional activation domains and epigenetic modification domains capable of activating NPBWR1 expression have also been defined above, in the context of agonist / activator arms for medical use.

[0621] As described above, pharmaceutical compositions comprising the present invention’s neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above and pharmaceutical compositions comprising the present invention’s neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above are particularly useful in medical settings.

[0622] Therefore, in preferred embodiments, the present invention relates to pharmaceutical compositions comprising, as defined above, the antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) of the present invention, and pharmaceutical compositions comprising, as defined above, the agonist / activator of the neuropeptide B / W receptor (NPBWR1) of the present invention, and at least one pharmaceutically acceptable excipient.

[0623] Therefore, in preferred embodiments, the present invention relates to 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, which are used as pharmaceuticals.

[0624] As used herein, the terms “treatment” and / or “prevention” generally refer to achieving the desired pharmacological and / or physiological effects. Therefore, the treatment of the present invention can involve the treatment of an (acute) state of a disease / disorder, but can also involve preventative treatment with regard to the complete or partial prevention of a disease / disorder or its symptoms. Preferably, the term “treatment” should be understood as therapeutic in relation to the partial or complete cure of a disease / disorder and / or adverse reactions and / or symptoms caused by the disease / disorder. In this respect, “acute” means that the subject exhibits symptoms of a disease or disorder. In other words, the subject to be treated actually requires treatment, and in the context of the present invention, the term “acute treatment” refers to measures taken to actually treat a disease / disorder after its onset or outbreak. Treatment can also be prophylactic or preventive treatment, i.e., taking measures for the prevention of a disease / symptom.

[0625] The pharmaceutical compositions or drugs of the present invention can be administered in a variety of forms of administration known to those skilled in the art. Administration can be systemic, local, oral, via aerosol (including but not limited to tablets), injection, inhaler, cream, foam, gel, lotion, and ointment.

[0626] Preferably, pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor of the present invention as defined above and pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention as defined above are administered intravenously, locally, intradermally, subcutaneously, intradermally, intramuscularly, and / or intrathecally.

[0627] These routes of administration, namely intravenous, local, intradermal, subcutaneous, intradermal, intramuscular, and / or intrathecal, are known to those skilled in the art.

[0628] Excipients or carriers are inactive substances formulated together with an active ingredient, namely, pharmaceutical compositions comprising, as defined above, antagonists / inhibitors of the neuropeptide B / W receptor (NPBWR1) of the present invention, and pharmaceutical compositions comprising, as defined above, agonists / activators of the neuropeptide B / W receptor (NPBWR1) of the present invention, for the purpose of compatibilizing a formulation containing the effective active ingredient. Excipients are commonly referred to as “companants,” “fillers,” or “diluents.” When manufacturing dosage forms, compatibilization allows for convenient and precise dispensing of pharmaceutical substances. They can also be used for various therapeutic purposes, such as promoting drug absorption or dissolution, or other pharmacokinetic considerations. Excipients can also be used in the manufacturing process to facilitate handling of the associated active substance, for example by promoting powder flowability or non-stickiness, and to contribute to in vitro stability, such as preventing denaturation within the expected shelf life. The selection of a suitable excipient also depends on the route of administration and dosage form, as well as the active ingredient and other factors.

[0629] Therefore, pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor of the present invention as defined above, and pharmaceutical compositions comprising the neuropeptide B / W receptor (NPBWR1) agonist / activator of the present invention as defined above, may be in solid, fluid, or gaseous form, particularly in the form of powder, tablet, solution, or aerosol. Preferably, the pharmaceutical compositions optionally comprise a pharmaceutically acceptable carrier and / or diluent.

[0630] These pharmaceutical compositions can be administered to subjects at appropriate doses. The administration of appropriate compositions can be influenced by various methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, local, intradermal, intranasal, or intrabronchial administration. Particularly preferred is administration by injection and / or delivery to a site such as the pulmonary artery or directly into the lungs. The compositions of the present invention can also be administered directly to target sites, for example, via biological ballistic delivery to external or internal target sites. Dosing regimens will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any patient depends on many factors, including the patient's body size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and other concurrently administered medications. Protein pharmaceutical active substances can be present in amounts from 1 ng to 10 mg / kg body weight per dose; however, doses below or above this exemplary range are conceivable, especially taking into account the factors described above. If the regimen is a continuous infusion, the range should also be from 1 µg to 10 mg units / kg body weight / minute.

[0631] Examples of suitable drug carriers, excipients, and / or diluents are well known in the art, including phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing these carriers can be formulated using known conventional methods. These pharmaceutical compositions can be administered to subjects at a suitable dose, i.e., an “effective amount” that can be readily determined by those skilled in the art using methods known in the art. The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any patient depends on many factors, including the patient's or subject's body size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and other medications administered concurrently.

[0632] Therefore, it is preferable to include, respectively, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above and the neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above in effective amounts. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in a subject to which the pharmaceutical composition is administered. As mentioned above, the content of the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above and the neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above in the pharmaceutical composition is not limited, provided it is suitable for the treatment described above, but preferably contains 0.0000001-10% by weight per total composition. Furthermore, it is preferable to use, respectively, the neuropeptide B / W receptor (NPBWR1) antagonist / inhibitor as defined above and the neuropeptide B / W receptor (NPBWR1) agonist / activator as defined above in the carrier. Typically, a suitable amount of pharmaceutically acceptable salt is used in the carrier to make the composition isotonic. Examples of carriers include, but are not limited to, saline, Ringer's solution, and glucose solution. Preferably, the acceptable excipients, carriers, or stabilizers are non-toxic at the dosage and concentration used, including buffers such as citrates, phosphates, and other organic acids; salt-forming counterions such as sodium and potassium; and low molecular weight (>) (10 amino acid residues) polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates, including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars, such as sucrose, mannitol, trehalose, or sorbitol; chelating agents, such as EDTA; nonionic surfactants, such as Tween, Pluronics, or polyethylene glycol; antioxidants, including methionine, ascorbic acid, and tocopherol; and / or preservatives, such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Co.

[0633] Treatment progress can be monitored through regular assessments.

[0634] The pharmaceutical compositions / pharmaceuticals of the present invention can be sterile aqueous or non-aqueous solutions, suspensions, emulsions, creams, and suppositories. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including 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 contain other active agents, depending on the intended use of the pharmaceutical composition. These active agents may be, for example, commercially available products under the trade name Tween, polyoxyethylene sorbitan monolaurate, propylene glycol, EDTA, citrate, sucrose, and other active agents well known to those skilled in the art suitable for the intended use of the pharmaceutical composition.

[0635] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient.

[0636] The present invention also relates to a method for treating or preventing a condition or disorder in a subject as defined above, wherein a pharmaceutical composition comprising an antagonist / inhibitor of the neuropeptide B / W receptor (NPBWR1) as defined above and a pharmaceutical composition comprising an agonist / activator of the neuropeptide B / W receptor (NPBWR1) as defined above are administered to the subject, preferably at a therapeutically effective amount as defined above.

[0637] The preferred embodiments of the treatment methods, with necessary modifications, are equally applicable to the content described above in the context of antibody or pharmaceutical compositions.

[0638] In this invention, in a preferred embodiment, the subject is a mammal, such as a dog, cat, pig, cow, sheep, horse, rodent, such as a rat, mouse, and guinea pig, or a primate, such as a gorilla, chimpanzee, and human. In the most preferred embodiment, the subject is a human.

[0639] Other aspects and advantages of the invention will be described in the following embodiments, which are for illustrative purposes only and not for limiting purposes. Every publication, patent, patent application, or other document cited in this application is incorporated herein by reference in its entirety.

[0640] Figure 1 Acute caffeine injection reversed CVS-induced depressive-like behavioral changes and dendritic spine changes in females.

[0641] A) Experimental Overview. Stereotactic injection of GFP-expressing AAV into a subgroup of mice was performed, followed by dendritic spine analysis. BE, H, I) Statistical Analysis: Two-way ANOVA with Bonferroni post-hoc test. BE) Reversal of stress-induced behavioral changes 24 hours after caffeine injection. B) Forced Swimming Test. n = 9-10 / group; Stress Effect: F(1,35) = 10.38, P < 0.01; Drug Effect: F(1,35) = 23.36, P < 0.0001; Post-hoc test: Effect of Caf in untreated (naive) mice: P < 0.05, within CVS: P < 0.001; Stress effects in Sal: P < 0.01, within Caf: P > 0.05. C) Novelty inhibition feeding test. n = 14-15 / group; Stress effect: F(1,54) = 6.21, P < 0.05; Drug effect: F(1,54) = 6.17, P < 0.05; Interaction: F(1,54) = 4.28, P < 0.05; Post-hoc test: Drug effect in untreated mice: P > 0.05, within CVS: P < 0.01; Stress effects in Sal: P < 0.01, within Caf: P > 0.05. D) Splash test. n = 7-8 / group; Stress effect: F(1,26) = 1.05, P = 0.32; Drug effect: 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; Stress effect: F(1,42) = 2.91, P = 0.10; Drug effect: 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. FN) Caffeine reverses CVS-induced changes in NAC dendritic spines. F) Overview image of GFP-labeled neurons. G) Representative dendrites. Scale bar 10 μm. H) Short, thick dendritic spines. n = 39–58 dendrites from 4–6 mice per group; two-way ANOVA: Stress effect: F(1,193) = 2.90, P = 0.09; Drug effect: 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) Dendritic spines with necks. n = 40-58 dendrites from 4-6 mice per group; Two-way ANOVA: Stress effect: F(1,194) = 3.34, P = 0.06; Drug effect: F(1,194) = 4.34, P < 0.05; Interaction: F(1,194) = 3.24, P = 0.07; Post-hoc test: Drug effect in untreated mice: P > 0.05, within CVS: P < 0.05; Stress effects in Sal: P < 0.05, within Caf: P > 0.05. JM) Increased cumulative head diameter by CVS and reversed cumulative head diameter by caffeine; Statistical analysis: Gehan-Breslow-Wilcoxon test. J) Drug effects in untreated mice: χ² 2 = 65.36, Degrees of freedom (DF) = 1, P < 0.0001. Drug effects in K)CVS: χ² 2 = 51.74, DF = 1, P < 0.0001. Stress effects in the L) saline group: χ² 2 = 141.90, DF = 1, P < 0.0001. Stress effects in the caffeine group (M): χ² 2 = 7.04, DF = 1, P < 0.01. BE, H, I) are plotted as independent data points, and the mean ± sem is shown. The diagram was created using biorender.com.

[0642] Figure 2 CVS induces subtle changes in dendritic spines in males, which are partially reversed by caffeine.

[0643] A) Representative dendrites. Scale bar 10 μm. B) Short, thick dendritic spines. n = 31-40 dendrites from 3-4 mice per group; Two-way ANOVA: Stress effect: F(1,136) = 1.77, P = 0.19; Drug effect: F(1,136) = 6.68, P < 0.05; Interaction: F(1,136) = 0.66, P = 0.42; Bonferroni post-hoc test: Drug effect in CVS: P < 0.05, all other comparisons: P > 0.05. C) Dendritic spines in the neck. n = 32-40 dendrites from 3-4 mice per group; two-way ANOVA: stress effect: F(1,139) = 0.01, P = 0.91; drug effect: F(1,139) = 0.13, P = 0.72; interaction: F(1,139) = 2.53, P = 0.11; Bonferroni post-hoc test: all comparisons P > 0.05. DG) Reduced cumulative head diameter by CVS and reversed cumulative head diameter by caffeine; statistics: Gehan-Breslow-Wilcoxon test. D) Drug effect in untreated mice: χ² 2 = 3.61, degrees of freedom (DF) = 1, P = 0.06. E) Drug effects in CVS: χ² 2 = 6.56, DF = 1, P < 0.05. F) Stress effects in the saline group: χ² 2 = 23.17, DF = 1, P < 0.0001.G) Stress effects of caffeine: χ² 2 = 1.48, DF = 1, P = 0.22. B, C) Plot independent data points and show the mean ± sem.

[0644] Figure 3 Quality control markers and read count distribution for RNA sequencing.

[0645] A) RNA quality pretreatment information. All samples achieved at least 90% alignment. B, C) Npbwr1 Distribution of segment counts (standardized for WT ZT18) over time. BK) n = 4 / group; Statistics: Two-way ANOVA & Bonferroni post-hoc test. B) WT; Drug effect: F(1,24) = 1.38, P < 0.05; Time effect: 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; 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 test: caffeine effect in all ZTs: all P > 0.05; time point effect in sal and caf: all P > 0.05. DK) Read counts (RC) normalized to circadian rhythm genes. Circadian rhythm alterations were consistent with the literature. Caffeine, but not the T75A mutation, slightly flattened the amplitude of the circadian rhythm oscillations. DG) WT. D) By1 Drug effect: F(1,24) = 0.47, P = 0.50; Time effect: F(3,24) = 5.05, P < 0.01; Interaction: F(3,24) = 2.25, P = 0.11; Post-hoc test: Caffeine effect in all ZTs: 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) Per2Drug effect: F(1,24) = 5.35, P < 0.05; Time effect: F(3,24) = 23.82, P < 0.0001; Interaction: F(3,24) = 1.27, P = 0.31; Post-hoc test: Caffeine effect in all ZTs: 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. ZT12: P < 0.05; ZT0 vs. ZT6: P < 0.01; ZT0 vs. ZT12: P < 0.001; All others: P > 0.05. F) Cry Drug effect: F(1,24) = 0.03, P = 0.87; Time effect: F(3,24) = 13.19, P < 0.0001; Interaction: F(3,24) = 1.41, P = 0.27; Post-hoc test: Caffeine effect in all ZTs: 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 Drug effect: F(1,24) = 1.09, P = 0.31; Time effect: F(3,24) = 3.48, P < 0.05; Interaction: F(3,24) = 0.23, P = 0.88; Post-hoc test: Caffeine effect in all ZTs: P > 0.05; Caf: ZT0 vs. ZT6: P < 0.05; All others: P > 0.05. HK) T75A. H) Per1 Drug effect: F(1,24) = 0.40, P = 0.53; Time effect: F(3,24) = 5.14, P < 0.01; Interaction: F(3,24) = 0.08, P = 0.97; Post-hoc test: Caffeine effect in all ZTs: P > 0.05; Sal, Caf: ZT0 vs. ZT6: P < 0.05; All others: P > 0.05. I) Per2Drug effect: F(1,24) = 4.9, P < 0.05; Time effect: F(3,24) = 26.49, P < 0.0001; Interaction: F(3,24) = 0.88, P = 0.47; Post-hoc test: Caffeine effect in all ZTs: 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; Drug effect: F(1,24) = 0.17, P = 0.67; Time effect: F(3,24) = 13.76, P < 0.0001; Interaction: F(3,24) = 0.75, P = 0.53; Post-hoc test: Caffeine effect in all ZTs: 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 Drug effect: F(1,24) < 0.01, P = 0.99; Time effect: F(3,24) = 5.73, P < 0.01; Interaction: F(3,24) = 0.45, P = 0.72; Post-hoc test: Caffeine effect in all ZTs: P > 0.05; Caf: ZT0 vs. ZT6, ZT0 vs. ZT12: P < 0.05; All others: P > 0.05. (BK) shows the mean ± sem.

[0646] Figure 4 RNA sequencing identified Npbwr1 as a gene product regulated diurnalally by the DARPP-32 pathway.

[0647] AC) RNA sequencing of NAc in wild-type (WT) and DARPP-32-T75A mutant mice (T75A) during the active and inactive phases of the light cycle 2 hours after caffeine injection. A) Venn diagrams showing significant (Padj < 0.05, log2FC 0.5 < > -0.5) gene expression changes across multiple conditions. These data confirm that the maximum effect of caffeine occurs during the active phase of the WT. B) Heatmap comparing transcriptional changes (log(FC)) between light-phase and genotype. C) Pie chart of annotated pathways in the WT group (dark phase). DF) Validation by qPCR in different cohorts. D) 2 hours after caffeine injection, 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 in WT: P < 0.05, within T75A: P > 0.05; Genotype effect in Sal: P > 0.05, within Caf: P < 0.05. E) 2 hours after caffeine administration, during the light period. 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 test: All P > 0.05. F) 24 hours after caffeine administration, during the dark period. n = 4-10 / group; Two-way ANOVA: Genotype effect: 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 test: Drug effect in WT: P < 0.05, within T75A: P > 0.05; Genotype effect in Sal: P > 0.05, within Caf: P > 0.05. (G, H) Npbwr1 Levels increased after chronic variable stress (CVS). G) Females: 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) Increased Npbwr1 protein levels after CVS: n = 6 / group; t-test: t = 2.27, df = 10; P < 0.05. (J) NPBWR1 Increased incidence in patients with depression (MDD) compared to controls (Nil): n = 30-32 / group; t-test: t = 2.36, df = 60; P < 0.05. (DJ) plotted independent data points and showed the mean ± sem.

[0648] Figure 5 Demographic characteristics of autopsy samples and classification by cause of death.

[0649] A) Patient information. B) Analysis by cause of death and diagnosis. n = 11-21 / group; two-way ANOVA: effect of depr: F(1,65) = 5.45. # P < 0.05; Effect of cause of death: 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 and the mean ± sem is shown. Nil: Control. Depr.: Depression.

[0650] Figure 6 : Npbwr1 Overexpression of the simulated stress effects on behavior and dendritic spines.

[0651] A) Schematic diagram of OE-Npbwr1-GFP AAV. B) Experimental protocol. C) Overview image of virus injection into NAc. D) qPCR. n = 7-8 / group; Student's t-test: t 13 = 2.71, P < 0.05. (EI) Depressive-like behavior via Npbwr1 Increased overexpression (OE); Statistical analysis: Student's t-test. (E) Tail suspension test. n = 14 / group; t 26 = 4.17, P < 0.001. F) Forced swimming test. n = 13-15 / set; t 26 = 3.00, P < 0.01. G) Novelty inhibition of feeding test. n = 14 / group; t 26 = 1.21, P = 0.24. H) Splash test. n = 14 / group; t 26 = 2.68, P < 0.05. I) Sucrose preference test. n = 13-15 / group; t 26 = 2.15, P > 0.05. Dendritic spines in JM) NAc were altered by OE-Npbwr1 in a CVS-like manner. J) Representative dendrites. Scale bar 10 μm. K) Short, thick dendritic spines. n = 46–57, from 5–6 mice per group; t 98 = 1.13, P = 0.26. L) contains cervical dendritic spines. n = 48-59, from 5-6 mice per group; t 102 =2.76, P < 0.01. (M) Cumulative head diameter. Gehan-Breslow-Wilcoxon test: χ² 2 = 7.83, DF = 1, P < 0.01. Independent data points (DI, K, L) are plotted, and the mean ± sem is shown. The diagram was created using biorender.com.

[0652] Figure 7 Knockdown of Npbwr1 blocks the effects of CVS on behavior and dendritic spines.

[0653] A) Schematic diagram of KD-Npbwr1-GFP AAV. B) Experimental protocol. C) Overview image of virus injection into NAc. D) qPCR (untreated mice): n = 6 / group; Student's t-test: t 10 = 2.33, P < 0.05. EH) Depressive-like behavior was partially blocked by KD-Npbwr1. E) Forced swimming test. n = 10 / group; Two-way ANOVA: Stress effect: F(1,36) = 3.41, P = 0.07; AAV effect: F(1,36) = 8.40, P < 0.01; Interaction: F(1,36) = 6.42, P < 0.05; Bonferroni post-hoc test: AAV effect in untreated mice: P > 0.05, within CVS: P < 0.001; Stress effects in GFP: P < 0.01, KD: P > 0.05. F) Novelty Inhibition Feeding Test. n = 10 / group; Two-way ANOVA: Stress effect: F(1,36) = 4.51, P < 0.05; AAV effect: 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: Stress effect: F(1,36) = 0.37, P = 0.55; AAV effect: 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: Stress effect: F(1,36) = 0.24, P = 0.55; AAV effect: 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. IO) Reversal of stress-induced dendritic spine changes by KD-Npbwr1. I) Representative dendrites. Scale bar 10 μm. J) Short, thick dendritic spines. n = 30–39 dendrites, from 3–4 mice per group; two-way ANOVA: stress effect: F(1,152) = 2.24, P = 0.14; AAV effect: 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) Dendritic spines with necks. n = 28-37 dendrites, from 3-4 mice per group; Two-way ANOVA: Stress effect: F(1,132) = 6.10, P < 0.05; AAV effect: F(1,132) = 3.70, P = 0.06; Interaction: F(1,132) = 23.11, P < 0.0001; Bonferroni post-hoc test: AAV effect in untreated mice: P > 0.05, within CVS: P > 0.001; Stress effects in GFP: P < 0.001, within KD: P > 0.05. LO) Cumulative head diameter, reduced by CVS and salvaged by KD-Npbwr1; statistical: Gehan-Breslow-Wilcoxon test. L) Effect of AAV in untreated mice: χ²2 < 0.01, DF = 1, P = 0.96. AAV effect in M)CVS: χ² 2 = 7.71, DF = 1, P < 0.01. Stress effects in N)GFP: χ² 2 = 20.05, DF = 1, P < 0.0001. Stress effects in O)KD: χ² 2 =4.58, DF = 1, P < 0.05. Independent data points (EH, J, K) are plotted, and the mean ± sem is shown. The diagram was created using biorender.com.

[0654] Figure 8 24 hours after an acute dose of caffeine, escape behavior was altered in untreated WT mice.

[0655] AC) Untreated WT and T75A mutants were administered 7.5 mg / kg caffeine vs. saline during the dark period and tested 24 hours later. A) Forced swimming 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: Caf effect in WT: P < 0.01, within T75A: P > 0.05; Genotype influence in Sal: P < 0.01, within Caf: P > 0.05. B) Novelty Inhibition 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) Plot independent data points and show the mean ± sem.

[0656] Figure 9 Genes that are differentially expressed after Npbwr1 overexpression.

[0657] A, B) RNA sequencing of NAc from AAV-infected mice expressing GFP or OE-Npbwr1-GFP. A) Heatmap of significantly altered gene products. B) List of identical gene products sorted by corrected P-value (Padj) and log 2 fold difference (log 2FC).

[0658] Figure 10 Npbwr1 ligands alter Bdnf signaling and depressive-like behavior.

[0659] A) Overview of ligand binding of the agonist neuropeptide B (NPB) and the antagonist CYM50769 to Npbwr1. B) Microinjection of 1 nmolar NPB or C, D) 1 μmollar CYM50769, followed by tissue collection 24 hours (B, C) or 7 days (D) and analysis by qPCR. Statistical analysis: Student's t-test. B) n = 6–7. B) NPB reduction Bdnf P < 0.01 C)CYM50769 added Bdnf P < 0.05 CYM50769 Bdnf The effect persisted for 7 days. P < 0.01 EG) Mice underwent CVS (vs. untreated controls) and were microinjected with NPB or CYM50769 at the end of the active phase following the last stress induction. Depressive-like behavior tests were performed during the dark phase approximately 24 hours later. E) Forced swimming test. NPB increased immobility time in untreated mice, while CYM50769 blocked the effect of CVS. 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 within ACSF: P < 0.001; Effect of NPB on untreated mice: P < 0.01; Effect of CYM50769 in CVS: P < 0.001; all other ns F) Splash test. NPB reduced grooming behavior in untreated mice, while CYM50769 blocked the effect of CVS. n = 8-9 / group; two-way ANOVA: interaction: F(2,46) = 3.17, P = 0.05. G) Sucrose preference test. NPB and CVS reduced the proportion of sucrose solution consumed, while CYM50769 rescued 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 within ACSF: P < 0.001; Effect of NPB on untreated mice: P < 0.01; Effect of CYM50769 in CVS: P < 0.001; all other ns (BG) plotted independent data points and showed the mean ± sem. The schematic was created using biorender.com.

[0660] Figure 11 Acute microinjection of Npbwr1 ligand is selective and non-toxic.

[0661] 1 nmol NPB (AC) and 1 μmol CYM50769 (DF) were microinjected into NAc. Tissue was collected 24 hours later and analyzed by qPCR. Statistical analysis: Student's t-test. n = 6–7. Mean + / - sem is shown. A) NPB versus Bcl2 No effect. P = 0.882. B) NPB remains unchanged. Casp3 P = 0.66. (C) Per2 Unaffected by NPB. P = 0.38. D) For Bcl2 No effect. P = 0.58. E)CYM50769 remains unchanged. Casp3 P = 0.42. (F) Per2 Unaffected by CYM50769. P = 0.63.

[0662] Example

[0663] Materials and methods

[0664] Animals and licenses.Mice were housed in accordance with the ethical guidelines of the Thüringer Landesamt für Verbraucherschutz (TLV). Experiments were conducted under animal licenses UKJ-18-036 and UKJ-21-12 (Germany), in accordance with EU Directive 2010 / 63 / EU on animal experiments. Transgenic experiments were conducted according to the S1 regulation of GenTAufzV. C57Bl / 6J mice were housed in the animal facility (FZL) of the University Hospital Jena, Germany, and were purchased from Janvier Laboratories (Saint Berthevin Cedex, France). Two sexes were used as described. Mice were housed in a 12L:12D photocycle.

[0665] Drugs and chemicals. Mice were intraperitoneally (ip) injected with 7.5 mg / kg caffeine (#, C0750, Sigma) or saline at a volume of 10 ml / kg body weight, and tested after 2 or 24 hours. 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 0.1–10 μM.

[0666] RNA purification and quantification. RNA was purified by resuspending in Trizol and precipitating with chloroform. RNA was washed with isopropanol and 75% ethanol. Quantitative real-time PCR was performed on a Bio-rad CFX96 real-time system after cDNA transformation using the GoScript™ Reverse Transcriptase Kit (#A5001, Promega). Primer sequences are listed in the supplementary materials. Quantitative PCR results were processed as described. 22 .

[0667] AAV, stereotactic surgery, and microinjection. Bilateral stereotactic surgery to enter the NAc was basically performed as described. 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, customized by Charitè viral vector core in Berlin, Germany).

[0668] Behavioral testing and CVS.Acute behavioral tests, as described, include the forced swimming test, tail suspension test, sucrose preference test, splash test, and novelty inhibition feeding test. 13,23 After CVS, tail suspension testing was not performed because tail suspension was part of the stress induction protocol. For the CVS group, caffeine was injected immediately before the stressor on the last day of CVS (day 21). The CVS protocol was performed as described above. 13 In short, mice were subjected to 21 days of stress, with one of three stressors presented in a semi-random order, meaning the same stressor did not occur on consecutive days. The stressors consisted of one hour of tube restraint, tail suspension, or 100 mild, random electric shocks to food. If only female researchers were present, used male T-shirts were wrapped in clean protective suits from the animal facility and placed in the laboratory to avoid differences caused by the scientists' gender-specific odors. 24 All experiments were conducted during the photoperiod of a photocycle in order to allow for comparison with previous experiments. 22 .

[0669] Dendritic spine analysis. Analysis was based on the detection of GFP-fluorophores for AAV. Brain slices fixed with 40 μm PFA were photographed using the AiryScan method with a Zeiss LSM880 confocal microscope. Maximum intensity projections were obtained using Zen Black and Zen Blue software and analyzed in NeuronStudio (CNIC, Mount Sinai School of Medicine). The total density of dendritic spines, the ratio of fine spines, mushroom-shaped spines, and short, thick spines, and the cumulative neck length were determined in Graph Prism. Neck-containing mushroom density refers to the sum of the dendritic spine and mushroom-shaped spine densities.

[0670] Next-generation RNA sequencing. Apply the internal RNA sequencing analysis workflow as described. 25 After ribosomal RNA (rRNA) transcripts were manually removed from the dataset, over 90% of the reads aligned with the reference genome GRCm38, demonstrating excellent sample quality. Further consideration was given only to changes in corrected P-values ​​< 0.05 and log2FC 0.5 < > -0.5.

[0671] Autopsy samples. The experiment was conducted in accordance with the requirements of the Ethics Committee of Jena University Hospital, Germany (Reg.-Nr. 2020-1862-Material). Age and autopsy intervals were balanced across groups. Samples aged <22-80 years and with autopsy intervals >130 hours were excluded.

[0672] statistics.Statistical analyses were performed using GraphPrism. Two-tailed Student's t-tests were used to compare the two groups. Two-way ANOVA was used when both factors were varied. Additionally, Bonferroni post-hoc tests were performed. The Gehan-Breslow-Wilcoxon test was used to analyze the cumulative head diameter of dendritic spines. 26 Outliers were removed when data points deviated from the mean by more than two standard deviations. Participants were unaware of their group assignments during behavioral testing and dendritic spine analysis.

[0673] Example 1: Caffeine can rapidly improve the effects of chronic stress.

[0674] The rapid mood-enhancing effect of caffeine was previously observed in untreated mice. 14 Therefore, it is hypothesized that caffeine can also improve symptoms induced by chronic variable stress (CVS) in mice, making it an excellent model for sex-specific transcriptional changes in MDD. 12,13 Observations showed that administering an acute dose of caffeine 24 hours before testing could improve stress-induced behavioral changes. Figure 1 ).

[0675] In addition, mice were stereotactically injected with AAV expressing GFP to assess the morphology of NAc dendritic spines. Figure 1 Consistent with the literature, CVS increased the density of neck-containing spines and the cumulative head diameter in the NAc of female mice. Figure 1 ) 27 Male mice showed only minor stress-induced changes in neuromorphology. Figure 2 Importantly, 24 hours after injection, caffeine restored stress-induced dendritic spine changes to their original levels. Figure 1 This suggests that caffeine can rapidly reverse the effects of stress on behavior and neuromorphology.

[0676] Example 2: Npbwr1 It is altered by caffeine in a way that alters circadian rhythms and T75-DARPP-32 dependence.

[0677] Recent findings have revealed a caffeine-induced diurnal signaling cascade in NAc. Caffeine alters transcription via Thr75-DARPP-32, which directly binds to the CLOCK / BMAL1 transcriptional complex, thereby regulating 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). 14They begin at the earliest measurement point (45 minutes) and continue for at least 24 hours. 14 Therefore, it is hypothesized that the aforementioned pathway mediates the rapid mood-enhancing effect of caffeine.

[0678] To identify caffeine-regulated transcripts, next-generation RNA sequencing was performed 2 hours after caffeine injection into the NAc of wild-type (WT) and T75A mice. Tissue samples were collected at four time points along the circadian rhythm. 14 As expected, significant changes in circadian rhythm markers were observed in both WT and T75A-DARPP-32 mutant mice, which occurred in the expected manner at all four measurement time points ( Per1 , Per2 , Cry1 , Bmal )oscillation( Figure 3 Consistent with previous data, most caffeine-induced transcriptional changes have been observed to occur during the active (dark) phase of the WT. Figure 4 A, B). The altered transcripts belong to multiple cell signaling pathways, including synaptic transmission, transcriptional regulation, and hormone signaling. Figure 4 C).

[0679] At the end of the dark period in WT mice, caffeine, but not the T75A mutant, increased [the level of] caffeine. Npbwr1 ( Figure 3 This was confirmed in independent queues. Figure 4 (D, E). Surprisingly, although caffeine increased 2 hours after injection... Npbwr1 Expression, 24 hours later Npbwr1 Levels decreased ( Figure 4 F). This demonstrates a time-process-specific correlation between caffeine, mood, and Npbwr1.

[0680] Next, an assessment Npbwr1 Whether it is affected by chronic stress. After CVS in both sexes. Npbwr1 The expression of [something] increases. This effect is more pronounced in females. Figure 4 G, H). Additionally, Npbwr1 protein levels increased after CVS ( Figure 4 I, Figure 2 ).also, NPBWR1 Transcription was increased in NAc from autopsy tissues of depressed patients in both sexes. Figure 4 J, Figure 5 These data indicate Npbwr1 It is related to mood and MDD, and can be altered using a CVS model.

[0681] Example 3: Npbwr1 causally mediates stress symptoms.

[0682] To investigate whether Npbwr1 is causally related to the morphological and behavioral consequences of stress, we obtained transgenic adeno-associated virus (AAV) for overexpression (OE) or knockdown (KD) of Npbwr1 in NAc. AAV induces long-term stable changes in the gene product, thus allowing for the regulation of Npbwr1 throughout the CVS process. Given the greater impact on female mice, the experiments were conducted only in female mice.

[0683] The OE of Npbwr1 simulates the effects of chronic stress on behavior and dendritic spines. Figure 6 Specifically, OE-Npbwr1 reduced escape behavior in mice during tail suspension and forced swimming tests. Figure 6 (D, E) reduced the time spent combing hair during the water splash test. Figure 6 G), and reduced the amount of sucrose solution consumed compared to water. Figure 6 H). Increased density of ridge-bearing dendritic spines rather than short, thick dendritic spines ( Figure 6 JK). Cumulative head diameter reduction ( Figure 6 L) indicates an increase in necked dendritic spines (“fine” spines) with small head diameter.

[0684] Conversely, the KD of Npbwr1 prevents some of the effects of CVS on behavior and dendritic spines. Figure 7 In this queue, the forced swimming test is most sensitive to CVS and shows a reversal via KD of Npbwr1 (). Figure 7 DG). Furthermore, KD reversed the effects of CVS on the neck-bearing dendritic spines and cumulative head diameter (DG). Figure 7 HN), and Figure 2 The observations were consistent. Npbwr1 OE or KD did not affect mouse body weight or measures of anxiety and motor skill. Figure 8 ).

[0685] Next, the downstream signaling pathways of Npbwr1 were evaluated, which were essentially unknown. For this purpose, RNA sequencing was performed on NAc tissues injected with OE-Npbwr1AAV or GFP-expression controls. Surprisingly, only 7 genes were significantly altered ( Figure 9 However, it contains brain-derived neurotrophic factor (BDNF), which is highly correlated with MDD and antidepressant response. 21 In summary, these data demonstrate a causal link between Npbwr1 and stress symptoms. However, AAV-based experimental methods cannot reflect the rapid-acting nature of Npbwr1 changes observed, such as after caffeine administration.

[0686] Example 4: Microinjection of Npbwr1 ligand can rapidly influence depressive-like behavior.

[0687] To address the rapid action of the Npbwr1 pathway, ligands activating (NPB) or inhibiting (CYM50769) Npbwr1 were microinjected into NAc. NPB is a natural neurotransmitter, while CYM50769 is a synthetic ligand that has not been tested in vivo. Therefore, dose, selectivity, and toxicity were evaluated first. Mice were injected with doses of 0.1 μM, 1 μM, and 10 μM of CYM50769, and health markers were scored for 7 days. No effects on body weight, fur condition, respiration, posture, stereotyped behavior, or movement were observed (data not shown, all mice scored "0").

[0688] Both CYM50769 and NPB have changed. Bdnf The horizontal plane, although as expected, acts in the opposite direction. Further testing is needed. Bdnf The ligand concentrations with the most significant effect (1 μm CYM50769, 1 nM NPB); Figure 10 AD). Neither NPB nor CYM50769 affected apoptosis markers. Bcl2 and Casp3 ( Figure 11 Furthermore, they do not alter circadian rhythm genes. Per2 level ( Figure 11 This indicates selectivity for the Bdnf pathway.

[0689] Next, the rapid effects of Npbwr1 activity on depressive-like behavior were tested in vivo. Mice underwent CVS and were microinjected with 1 nM NPB and 1 μM CYM50769 after the last stress induction period. Behavioral tests were performed 24 hours later. It was observed that NPB induced depressive-like behavior in untreated mice during forced swimming, splashing, and sucrose preference tests without further enhancing the behavioral effects of CVS. Figure 10 EG). Conversely, CYM50769 had no effect in untreated mice. However, it rescued the CVS effect in all tests: ( Figure 10 EG). These data are related to virus-mediated... Npbwr1 The effects of level OE and KD are consistent. Figure 6 ,7), pointing to Npbwr1 The prodepressant effect of [the substance] is improved when the level or activity of the receptor is reduced. Importantly, Npbwr1 The ligands exhibit rapid effects on behavior, while the antagonist CYM50769 shows promise for rapidly reversing the effects of chronic stress.

[0690] Summary and Discussion

[0691] This study describes a previously unknown pathway that rapidly influences depressive-like behavior and stress response via Npbwr1. Furthermore, it shows... BdnfSelective alterations in horizontal and dendritic spine morphology. Virus-mediated gene transfer stably alters... Npbwr1 The level. Despite Npbwr1 Overexpression of [a substance] mimics depression-like symptoms, but knockdown prevents the effects of chronic stress. This dichotomy can be observed in NAc [a specific component]. Npbwr1 The relatively low baseline levels can be explained by the fact that, as observed in untreated mice, the Cq value of Npbwr1 exceeded 25 during qPCR. Therefore, the effects on Npbwr1 regulation may only be apparent when Npbwr1 levels are upregulated, for example, after CVS. Consistently, modulating Npbwr1 activity via microinjection of ligands alters depression-related symptoms. In the CVS cohort, Npbwr1 stimulation with NPB increased depressive-like behaviors, while inhibition with CYM50769 blocked these behaviors. In summary, these data demonstrate that Npbwr1 signaling can rapidly alter depressive-like symptoms, and that this alteration is causally related.

[0692] Npbwr1 The pathway is rapidly regulated by caffeine, which promotes acute psychological resilience in mice. However, caffeine was used only as a research tool in the above experiment because it is widely used in humans and may have adverse effects on the health of some individuals. The seemingly contradictory effects of caffeine on Npbwr1 observed at 2 hours and 24 hours are consistent with a report that intraventricular injection of NPB into mice induced euphagia 2 hours later, followed by dyspepsia. 15 .

[0693] Consistent with previous findings, caffeine has the effect of Npbwr1 The effects of signal transduction occur in a circadian rhythm. 14 This is consistent with literature reports that describe the circadian rhythm effects of NPW on feeding behavior, with the effects occurring only during active periods. 29 .

[0694] CYM50769 has been developed as a selective antagonist of Npbwr1 in vitro. This paper presents the first in vivo study of this compound. Acute doses of the drug have been shown to be selective and well-tolerated.

[0695] In summary, this study proves Npbwr1 As a key mediator of depression and stress response, and with corresponding antagonists such as CYM50769 being potential compounds that can rapidly modulate this pathway in a beneficial manner.

[0696] literature

[0697] 1.Bebbington, P. The World Health Report 2001 - Mental Health: NewUnderstanding, New Hope. Soc. Psychiatry Psychiatr. Epidemiol. 36, 473–4(2001).

[0698] 2.Vos, T. et al. Global, regional, and national incidence,prevalence, and years lived with disability for 328 diseases and injuries for195 countries, 1990-2016: A systematic analysis for the Global Burden ofDisease Study 2016. Lancet 390, 1211–59 (2017).

[0699] 3.Galea, S., Merchant, R. M. & Lurie, N. The Mental HealthConsequences of COVID-19 and Physical Distancing: The Need for Prevention andEarly Intervention. JAMA Intern Med Epub ahead, (2020).

[0700] 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).

[0701] 5.Sanchez-Roige, S. & Palmer, A. A. Emerging phenotyping strategieswill advance our understanding of psychiatric genetics. Nat. Neurosci.23,475–80 (2020).

[0702] 6.Pappa, S. et al. Prevalence of depression, anxiety, and insomniaamong healthcare workers during the COVID-19 pandemic: A systematic reviewand meta-analysis. Brain. Behav. Immun. 88, 901–7 (2020).

[0703] 7.Vindegaard, N. & Eriksen Benros, M. COVID-19 pandemic and mentalhealth consequences: Systematic review of the current evidence. Brain. Behav. Immun. 89, 531–42 (2020).

[0704] 8.Dubey, S. et al. Psychosocial impact of COVID-19. Diabetes Metab. Syndr. Clin. Res. Rev. 14, 779–88 (2020).

[0705] 9.Rajkumar, R. P. COVID-19 and mental health: A review of theexisting literature. Asian Journal of Psychiatr. 52, 102066 (2020).

[0706] 10.Tomonaga, Y. et al. The economic burden of depression inSwitzerland. Pharmacoeconomics 31, 237–50 (2013).

[0707] 11.Greenberg, P. E., Fournier, A. A., Sisitsky, T., Pike, C. T. &Kessler, R. C. The economic burden of adults with major depressive disorderin the United States (2005 and 2010). J. Clin. Psychiatry 76, 155–62 (2015).

[0708] 12.Scarpa, J. et al. Shared Transcriptional Signatures in MajorDepressive Disorder and Mouse Chronic Stress Models. Biol. Psychiatry (2020).

[0709] 13.Labonte, B. et al. Sex-Specific Transcriptional Signatures inHuman Depression. Nat. Med. 23, 1102–1111 (2017).

[0710] 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).

[0711] 15.Tanaka, H. et al. Characterization of a family of endogenousneuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proc. Natl. Acad. Sci. USA 100, 6251–6 (2003).

[0712] 16.Eipper-Mains, J. E. et al.Effects of cocaine and withdrawal onthe mouse nucleus accumbens transcriptome. Genes, Brain Behav. (2013). doi:10.1111 / j.1601-183X.2012.00873.x

[0713] 17.Dvorakova, M. C. Distribution and function of neuropeptides W / Bsignaling system. Front. Physiol. 9, 981 (2018).

[0714] 18.Uchio, N. et al. Circadian characteristics of mice depleted withGPR7. Biomed. Res. (2009). doi:10.2220 / biomedres.30.357

[0715] 19.Dun, S. L. et al. Neuropeptide B immunoreactivity in the centralnervous system of the rat. Brain Res. (2005). doi:10.1016 / j.brainres.2005.03.024

[0716] 20.Singh, G., Maguire, J. J., Kuc, R. E., Fidock, M. & Davenport, A.P. Identification and cellular localisation of NPW1 (GPR7) receptors for thenovel neuropeptide W-23 by [125I]-NPW radioligand binding andimmunocytochemistry. Brain Res. (2004). doi:10.1016 / j.brainres.2004.03.079

[0717] 21.Cubillos, S., Engmann, O. & Brancato, A. BDNF as a Mediator ofAntidepressant Response: Recent Advances and Lifestyle Interactions. Int. J. Mol. Sci. 23, (2022).

[0718] 22.Trautmann, C., Bock, A., Urbach, A., Hübner, A. & Engmann, O.Acute vitamin B12 supplementation evokes antidepressant response and altersNtrk-2. Neuropharmacology 171, 108112 (2020).

[0719] 23.Scott, M. M. et al. Hcrtr1 and 2 signaling differentiallyregulates depression-like behaviors....

Claims

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

2. The pharmaceutical composition according to claim 1, wherein the mood disorder / emotional disorder is selected from: (ii) Bipolar disorder with hypomanic phase (ICD-10 F31); (iii) Depressive episode (ICD-10 F32); (iv) Recurrent depressive disorder (ICD-10 F33); (v) Persistent affective disorder (ICD-10 F34); (vi) Other mood and affective disorders (ICD-10 F38); and (vii) Undefined mood / affective disorder (ICD-10 F39).

3. The pharmaceutical composition according to claim 1, wherein the chronic stress is selected from: (i) Response to severe stress and maladaptive disorder (ICD-10 F43); (ii) Acute stress response (ICD-10 F43.0); (iii) Post-traumatic stress disorder (ICD-10 F43.1); (iv) Adaptation disorder (ICD-10 F43.2); (v) Other responses to severe stress (ICD-10 F43.8); and (vi) Undefined response to severe stress (ICD-10 F43.9).

4. The pharmaceutical composition according to claim 1, wherein the anxiety disorder is selected from: (i) Phobic anxiety disorder (ICD-10 F40); and (ii) Other anxiety disorders (ICD-10 F41.1).

5. The pharmaceutical composition used according to any one of claims 1 to 4, wherein, The antagonist / inhibitor is selected from NPBWR1 inhibitory peptides, NPBWR1 inhibitory small binding molecules, RNAi, siRNA, shRNA, aptamers and cohesives specifically targeting NPBWR1, and anti-NPBWR1 antisense molecules.

6. The pharmaceutical composition used according to any one of claims 1 to 5, wherein, The antagonist / inhibitor is an NPBWR1 inhibitory small binding molecule and has the following chemical structure represented by formula (1). Equation (1) in: R 1 Selected from F, Cl, Br, I and CN; R 2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heterocyclic groups) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a (of carbon cyclic groups); R 3 Selected from - (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 20 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 20 ring atoms and optionally having one or more substituents R) 3a (carbon cyclic group) in: Each R 2a Independently selected from: -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 Alkyl, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl; Each R 3a Independently selected from: -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 Alkyl, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl; Each R Alk Independently selected from -halogen and -CN; Or a pharmaceutically acceptable salt, solvate, or prodrug.

7. The pharmaceutical composition according to claim 6, wherein formula (1) satisfies one or more of the following: a)R 1 Selected from F, Cl, Br, and CN; preferably selected from F, Cl, and CN; more preferably selected from F and Cl; even more preferably selected from Cl; b)R 2 Selected from - (having 5 to 10 ring atoms and optionally having one or more substituents R) 2a (heteroaryl) and - (having 6 to 10 ring atoms and optionally having one or more substituents R) 2a Aryl); preferably selected from - (having 5 to 7 ring atoms and optionally having one or more substituents R 2a (heteroaryl) and - (having 6 or 10 ring atoms and optionally having one or more substituents R) 2a (aryl); more preferably selected from benzene, naphthalene, pyrrole, furan, imidazole, pyrazole, oxazole, thiazole and pyridine, any of which may optionally be substituented by one or more R groups. 2a Substitution; or more preferably selected from benzene, naphthalene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R. 2a Substitution; still more preferably selected from benzene, imidazoline, oxazole and pyridine, any of which may optionally be replaced by one or more substituents R 2a Substitution; or even more preferably selected from benzene, which may optionally be replaced by one or more substituents R 2a Substitution; still or even more preferably selected from benzene, which is substituted by one or more substituents R 2a Substitution; the most preferred choice is benzene, which is R at position 4. 2a replace; c)R 3 Selected from - (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic group), - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 5 to 14 ring atoms and optionally having one or more substituents R) 3a (heterocyclic groups), - (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbonyl group); preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a The carbocyclic group) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (a carbon cyclo group); more preferably selected from - (having 6 to 14 ring atoms and optionally having one or more substituents R). 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 14 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having 6 to 10 ring atoms and optionally having one or more substituents R). 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 to 10 ring atoms and optionally having one or more substituents R) 3a (aryl); still more preferably selected from - (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl) and - (optionally substituents R) Alk Replacement C 1-4 (alkylene)- (having 6 or 10 ring atoms and optionally having one or more substituents R) 3a (aryl); or even more preferably selected from - (having one or more substituents R) 3a phenyl) and - (optionally substituents R) Alk Replacement C 1-4 alkylene)-(with one or more substituents R) 3a (naphthyl); or even more preferably 2,5-dimethylphenyl or 1-naphthylmethyl; d)C 1-4 The alkylene group is preferably methylene or ethylene, more preferably methylene; e) No, one, two, or three substituents R 2a Preferably, there is no, one, or two substituents R. 2a More preferably, it has a substituent R. 2a Even more preferably, there is a substituent R at position 4. 2a ; f) No, one, two, or three substituents R 3a Preferably, there is no, one, or two substituents R. 3a If R 3 Excluding -(C 1-4 The alkylene group is more preferably composed of two substituents R. 3a If R 3 Contains -(C 1-4 The alkylene group is more preferably a substituent R. 3a Or without substituent R 3a ; g) No, one, two, or three substituents R Alk Preferably, there is no, one, or two substituents R. Alk More preferably, there is no or one substituent R. Alk Even better, no substitution base R Alk ; h) Each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 1-6 cycloalkyl; preferably each R 2a Independently selected from -halogen, -CN, C 1-3 Alkyl, C 1-3 Halogenated alkyl, 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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-3 Alkyl or cyclopropyl; more preferably each R 2a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-3 Alkyl or cyclopropyl; or even more preferably each R 2a Independently selected from -halogen, -CN, C 1-3 Alkyl or -OC 1-3 Alkyl; still more preferably each R 2a Independently selected from -halogen, -CN, or -OC 1-3 Alkyl; or even more preferably each R 2a Independently for -OC 1-3 Alkyl; still, or even more preferably, each R 2a It can be methoxy or ethoxy independently, with methoxy being the most preferred. i) Each R 3a Independently selected from -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 Halogenated alkyl groups, wherein each R Independently selected from H or C 1-6 Alkyl or C 3-6 cycloalkyl; preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 cycloalkyl, -OC 1-6 Alkyl or -OC 1-6 Halogenated alkyl; more preferably each R 3a Independently selected from -halogen, -CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 3-6 cycloalkyl; or even more preferably each R 3a Independently selected from -halogen, -CN, C 1-3 Alkyl or C 1-3 Halogenated alkyl; still more preferably each R 3a Independently selected from -halogen and C 1-3 Alkyl; or even more preferably each R 3a Selected independently from C 1-3 Alkyl groups, such as methyl or ethyl, more preferably methyl; j) Each R Alk Independently selected from –F and -Cl; preferably each R Alk For –F; more preferably each R Alk Does not exist; and / or k) The NPBWR1 inhibitory small binding molecule has the chemical structure shown in formula (1) or a pharmaceutically acceptable salt or solvate thereof.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the antagonist / inhibitor is an NPBWR1 inhibitory small binding molecule and has one of the following chemical structures represented by formulas (2) to (9): (2) (3) (4) (5) (6) (7) and (8) (9) Where R 1 R 2a and R 3 As defined in claim 6 or 7.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antagonist / inhibitor is a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and wherein the Cas protein is fused with an effector domain selected from transcriptional repression domains and epigenetic modification domains capable of repressing NPBWR1 expression.

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

11. The pharmaceutical composition according to claim 10, wherein, The agonist / activator is selected from NPBWR1 activating peptides, NPBWR1 activating small binding molecules, and NPBWR1 RNA molecules.

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

13. The pharmaceutical composition according to any one of claims 9 to 12; wherein the agonist / activator is a CRISPR / Cas system specifically targeting NPBWR1, wherein the Cas protein of the CRISPR / Cas system is modified to lack its nuclease activity, and wherein the Cas protein is fused with an effector domain selected from transcriptional activation domains and epigenetic modification domains capable of activating NPBWR1 expression.

Images