Anti-anxiety therapy

Abstract

A method for treating anxiety disorders in subjects requiring treatment includes administering to the subject a therapeutically effective dose of an anxiolytic that inhibits signaling or activity of asprosin-mediated protein tyrosine phosphatase receptor δ (PTPRD).

Description

[Technical Field]

[0001] Related applications This application claims priority under U.S. Provisional Application No. 63 / 501,474, filed on 11 May 2023. The entire contents of that application are incorporated herein by reference.

[0002] Sequence List This application includes an electronically submitted sequence listing in XML format; the sequence listing is incorporated herein by reference in its entirety. The filename of the XML copy created on 10 May 2024 is UH-032751WO ORD.st.26, and its size is 18,468 bytes. [Background technology]

[0003] Anxiety is a group of disorders characterized by excessive and irrational fear and dread. Anxiety disorders include panic disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), social anxiety disorder / social phobia, separation anxiety disorder, phobias, and generalized anxiety disorder.

[0004] Anxiety disorders are often classified based on having a duration of at least six months. Stress and fear triggered by highly stressful events can develop into anxiety disorders over time if left untreated.

[0005] Anxiety disorders affect approximately 40 million Americans each year, representing about 18% of the population (see National Institutes of Health (2009): Anxiety Disorders; Patent Publication No. 093879). In many patients, anxiety disorders develop in conjunction with other mental or physical illnesses, or with alcohol or drug abuse. Anxiety disorders, such as panic disorder, can leave individuals feeling helpless, and patients may exhibit symptoms such as a strong fear of daily isolation.

[0006] Anxiety disorders can lead to excessive worry and tension that disrupts sleep and concentration, and can also cause physical symptoms such as headaches, nausea, difficulty swallowing, and dizziness. They can also lead to difficulty performing simple daily tasks and are often associated with substance abuse or depression.

[0007] Currently, anxiety disorders are sometimes diagnosed based on a patient's medical history, which can include various assessment scales (Ebell MH (2008) Am Fam Physician. 78(4):501-502). For pediatric anxiety, standard scales (such as the Child Anxiety Rating Scale (PARS)) can be used to assess the effectiveness of treatment response and remission; the PARS is a clinician assessment scale validated for symptom severity and associated disorders and has been used to assess the treatment of generalized anxiety disorder, social phobia, and separation anxiety disorder (see Caporino NE (2013) J Am Acad Child Adolesc Psychiatry 52(1):57-67). Assessment scales that can be used for related or comorbid conditions in pediatric patients include the Child Depression Rating Scale and the Yale-Brown Child Obsessive-Compulsive Disorder Severity Rating Scale.

[0008] People suffering from anxiety disorders can be treated with medication and / or psychotherapy. However, while the administration of medications that treat anxiety disorders, such as antidepressants, anxiolytics, and beta-blockers, may be beneficial in suppressing some physical symptoms, it cannot cure the disorder. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram of the open-field assay used to evaluate anxiety-like behavior. [Figure 2] This is a schematic diagram of the elevated cusp maze assay used to evaluate anxiety-like behaviors. [Figure 3] This is a schematic diagram of the light-dark assay used to evaluate anxiety-like behavior. [Figure 4]The graphs in Figures 1-3 show that, when determined using the assays, genetic knockout of asprosin leads to a reduction in anxiety in animal models of neonatal progeria syndrome. [Figure 5] This graph shows that anxiety is reduced by knocking out the asprosin receptor (Ptprd) gene, as determined using an elevated cross maze. [Figure 6] This graph shows that anxiety is reduced by pharmacological neutralization of asprosin receptors. [Figure 7] This is a schematic diagram of an assay used to evaluate the asprosin-neutralizing effect of anti-asprosin antibodies used to treat anxiety in animal models. [Figure 8] This plot shows that neutralization of asprosin by administering anti-asprosin antibodies to mice leads to weight loss. [Figure 9] These graphs show that, when measured using the assays in Figures 1-3, asprosin neutralization by anti-asprosin antibodies administered to mice does not cause any changes in locomotion or general movement. [Figure 10] The graphs in Figures 1-3 show that asprosin neutralization by anti-asprosin antibodies administered to diet-induced obesity (DIO) mice leads to a reduction in anxiety, as measured using the assays shown. [Figure 11] These graphs and plots show that asprosin neutralization by administering anti-asprosin antibodies to non-obese mice did not result in any difference in weight loss or locomotion. [Figure 12] These graphs, measured using the assays shown in Figures 1-3, demonstrate that asprosin neutralization by anti-asprosin antibodies administered to non-obese mice leads to a reduction in anxiety. [Figure 13] This graph shows that overexpression of asprosin causes increased anxiety in mice. [Figure 14] These plots and graphs show that asprosin overexpression leads to increased anxiety, as measured by body weight and the assays shown in Figures 1-3. [Figure 15]It is a schematic diagram showing that five different types of asprosin manipulation methods in mice indicate a direct relationship between asprosin and anxiety. [Figure 16] It shows a schematic diagram of a foot shock model that induces anxiety. [Figure 17] It shows that anxiety is reduced by asprosin neutralization of anti-asprosin antibody administered to a mouse-induced anxiety model. [Figure 18] It is a graph showing that anxiety is reduced by asprosin neutralization of anti-asprosin antibody administered to a mouse-induced anxiety model when measured using the assays of FIGS. 1 to 3. [Figure 19] It is a graph showing that serum asprosin levels increase by inducing anxiety. [Figure 20] It shows a schematic diagram of a predator scent stress (PSS) assay used to induce anxiety. [Figure 21] It shows a schematic diagram of a mouse in which the assay of FIG. 20 is performed by administering an anti-asprosin antibody at various time points and then tested using the assays of FIGS. 1 to 3. [Figure 22] It is a graph showing that anxiety is reduced by asprosin neutralization of anti-asprosin antibody administered to PSS mice when measured using the assays of FIGS. 1 to 3. [Figure 23] [[ID=E24]]It is a graph showing that serum asprosin levels increase by inducing anxiety with PSS. [Figure 24] It is a graph showing that asprosin does not mediate anxiety through AgRP neurons. [Figure 25] It is an image showing that Ptprd is highly expressed in the cerebellum. [Figure 26] It is a graph showing that asprosin does not mediate anxiety through Purkinje neurons. [Figure 27] It is a graph showing the possibility that asprosin mediates anxiety through the amygdala. [Figure 28]These are images of the cerebellum of subjects pretreated with asprosin and GFP. [Modes for carrying out the invention]

[0010] Unless otherwise specified, the scientific and technical terms used herein have meanings that are ordinarily understood by those skilled in the art. Furthermore, unless the context specifically requires singular / plural designation, singular terms include plurals, and plural terms include singulars. In general, the terms used herein in relation to cell culture and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligo- or polynucleotides, as well as in the art described herein, have meanings that are well known in the art.

[0011] For convenience, a summary of the specific terms used in this specification, the examples, and the accompanying "Claims" is provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains.

[0012] In this specification, the articles "a" and "an" are used to mean one or more (i.e., "at least one") in relation to the grammatically unmodifying article. For example, "an element" means one or more elements.

[0013] The terms “comprise,” “comprising,” “include,” “including,” “have,” and “having” are used in a comprehensive, non-restrictive sense, meaning they may include additional elements. The terms “such as” and “for example” as used herein are non-restrictive and intended for illustrative purposes only. “Including” and “including but not limited to” are used as synonyms.

[0014] In this specification, the term "or" should be understood to mean "and / or" unless explicitly stated otherwise by the context.

[0015] In this specification, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length that exhibits variation of approximately 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of quantities, levels, values, numbers, frequencies, percentages, sizes, amounts, weights, or lengths that are ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0016] In this specification, "one or more of a, b, and c" means a, b, c, ab, ac, bc, or abc. In this specification, "or" is an inclusive "or".

[0017] In this specification, the terms “chimeric protein,” “fusion protein,” “fusion polypeptide,” and “chimeric polypeptide” are used synonymously and refer to the fusion between a polypeptide encoding a first amino acid sequence and a second amino acid sequence (e.g., a polypeptide portion) defining an exogenous or non-homologous domain that is not substantially homologous to the domain of the first polypeptide. A chimeric protein may also represent an exogenous domain (of a different protein) present in the same organism as the organism expressing the first protein, or it may represent the fusion of protein structures expressed by different species, such as “interspecies” or “intergene.”

[0018] The term "expression" refers to the process by which nucleic acids are translated into peptides or transcribed into RNA; RNA can be translated into peptides, polypeptides, or proteins, for example. If the nucleic acid originates from genomic DNA, expression may involve mRNA splicing, provided that a suitable eukaryotic host cell or eukaryote is selected. To express non-homologous nucleic acids in host cells, they must first be introduced into the aforementioned cells and then finally localized to the nucleus after introduction into the cells.

[0019] The term “genetherapy” and its grammatical variations (e.g., “gene therapy”) involve introducing non-homologous DNA into mammalian cells, particularly human cells with a disorder or condition requiring treatment or diagnosis. The non-homologous DNA is introduced into selected target cells so that it is expressed and produces the therapeutic product it encodes. Alternatively, the non-homologous DNA may mediate the expression of the DNA encoding the therapeutic product; it may encode the product (such as a peptide or RNA) in a manner that directly or indirectly mediates the expression of the therapeutic product. Genetherapy can also be used when introducing nucleic acids encoding a gene product for the purpose of replacing a deficient gene or supplementing the gene product produced by the introduced mammal or cell. The non-homologous DNA encoding the therapeutic product may be modified before being introduced into the affected host cells for the purpose of increasing or altering the product or its expression.

[0020] The term "gene" or "recombinant gene" refers to a nucleic acid that contains an open reading frame encoding a polypeptide, and which includes both exons and (optionally) intron sequences.

[0021] The term “non-homologous nucleic acid sequence” typically refers to DNA that is not normally produced in vivo in the cell expressing it, and which encodes RNA and proteins; or DNA that mediates or encodes a mediator that alters the expression of endogenous DNA by affecting transcription, translation, or other regulatory biochemical processes. Non-homologous nucleic acid sequences may also be called exogenous DNA. In this specification, non-homologous DNA includes any DNA that a person skilled in the art would recognize or consider to be non-homologous or exogenous to the cell expressing it. Examples of non-homologous DNA include, but are not limited to, DNA encoding traceable marker proteins (such as proteins that confer drug resistance); DNA encoding therapeutically effective substances; and DNA encoding other types of proteins (such as antibodies). Antibodies encoded by non-homologous DNA may be secreted by the cell into which the non-homologous DNA has been introduced, or they may be expressed on the surface of the cell.

[0022] In this specification, the terms "parenteral administration" and "administer parenterally" refer to a mode of administration other than enteral and topical administration, usually by injection. Non-limiting examples of such administration include intravenous, intramuscular, intraarterial, intraarachnoid, intraventricular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, and intrasternal injections and infusions.

[0023] In this specification, the terms “systemic administration,” “administer systemically,” “administer peripherally,” and “administer peripherally” refer to the administration of a compound, drug, or other substance other than direct delivery into a target tissue, thereby introducing the compound, drug, or other substance into the animal’s system, and thus undergoing metabolism and other similar processes (e.g., subcutaneous administration).

[0024] The terms “polynucleotide sequence,” “nucleotide sequence,” and “nucleic acid sequence” are also used synonymously in this specification.

[0025] The terms "peptide" and "polypeptide" are used herein as synonyms and refer to compounds consisting of approximately 2 to 90 amino acid residues, wherein throughout the compound, the amino group of one amino acid is sequentially linked to the carboxyl group of another amino acid via a peptide bond. Peptides can be obtained or isolated, for example, from natural proteins by enzymatic or chemical cleavage, or they can be prepared using conventional peptide synthesis techniques (e.g., solid-phase synthesis) or molecular biological techniques (see Sambrook et al., Molecular Cloning: LAB.MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989)). A "peptide" may contain any suitable L- and / or D-amino acids; for example, common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., P-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statines), and atypical amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). The amino groups, carboxyl groups, and / or other functional groups in a peptide may be free groups (e.g., unmodified) or may be protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, as well as means for adding or removing protecting groups, are known in the art. See, for example, Green and Wuts, "Protecting Groups in Organic Synthesis" (John Wiley & Sons, 1991). The functional groups of peptides can be derivatized (e.g., alkylated) using methods known in the art.

[0026] Peptides can be synthesized to construct a library containing a considerable number of different molecular species. Such libraries can be prepared using well-known combinatorial chemistry methods; they can also be screened using the methods described herein or other methods suitable for determining whether the library contains peptides capable of inhibiting asprosin. Such peptides can then be isolated by suitable means.

[0027] The term "peptide mimetic" refers to a protein-like molecule designed to mimic a peptide. Peptide mimetic molecules are typically obtained either by modifying existing peptides or by designing similar systems that mimic peptides (such as peptoids and β-peptides). Regardless of the approach, the modified chemical structure is designed to favorably tune molecular properties (such as stability or biological activity). These modifications include introducing changes into the peptide that do not occur naturally (such as alterations to the backbone and insertion of unnatural amino acids).

[0028] When referring to polypeptides, the terms “part,” “fragment,” “variant,” “derivative,” and “analog” include any polypeptide that retains at least some of the biological activity described herein (e.g., inhibition of interactions such as binding). Polypeptides described herein may, insofar as the polypeptide performs its function, include, non-limiting, parts, fragments, variants, or derivative molecules. Polypeptides or parts thereof of the present invention may include proteolytic fragments, deletion fragments, and, in particular, or fragments that are more readily accessible to the site of action when delivered to animals.

[0029] In some embodiments, a “partial” polypeptide or “fragment” polypeptide (including a domain) is a shortened polypeptide compared to a reference polypeptide (for example, shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues, respectively, compared to the reference polypeptide (e.g., a peptide)) and is identical to or It would be understood to mean short polypeptides consisting of essentially the same and / or the same, each containing polypeptides of consecutive residues that are nearly identical (for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical).

[0030] In this specification, different nucleic acids or proteins that have homology are referred to as “homologous.” The term “homologous” includes homologous sequences and orthologous sequences derived from the same and other species. “Homologous” refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acid sequences and / or amino acid sequences with respect to positional identity (%). Homology also refers to the concept that different nucleic acids or proteins have similar functional properties. Accordingly, the compositions and methods described herein further include homologous to the nucleotide sequences and polypeptides of the present invention. In this specification, “orthologous” and “ortholog” refer to homologous nucleotide sequences and / or amino acid sequences in different species that have derived from a common ancestral gene in speciation. Homogenetics or orthologs of the nucleotide sequences of the present invention have substantial sequence identity to the nucleotide sequences described herein (for example, at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%).

[0031] The term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences remain invariant throughout the entire alignment framework of their components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods, including (but not limited to) those described in the following references: "Computational Molecular Biology" (edited by Lesk, AM), Oxford University Press, New York (1988); "Biocomputing: Informatics and Genome Projects" (edited by Smith, DW), Academic Press, New York (1993); "Computer Analysis of Sequence Data, Part I" (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); "Sequence Analysis in Molecular Biology" (edited by von Heinje, G.), Academic Press (1987); and "Sequence Analysis Primer" (edited by Gribskov, M. and Develeux, J.), Stockton Press, New York (1991).

[0032] The term “sequence identity (%)” or “identity (%)” refers to the percentage of identical nucleotides in the linear polynucleotide sequence of the reference ("search query") polynucleotide molecule (or its complementary chain) compared to the test ("target") polynucleotide molecule (or its complementary chain), when the sequences of the test ("target") polynucleotide molecule (or its complementary chain) and the reference ("search query") polynucleotide molecule are optimally aligned. In some embodiments, “identity (%)” may refer to the percentage of identical amino acids in the amino acid sequence compared to the reference polypeptide.

[0033] The expression "substantially identical" or "substantially identical" for two nucleic acid molecules, nucleotide sequences, polypeptide sequences, or protein sequences refers to two or more sequences or subsequences that, when evaluated and compared using the sequence comparison algorithms described below or by visual inspection and aligned to the maximum correspondence, have at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% nucleotide identity or amino acid residue identity. In some embodiments, there is substantial identity in a continuous nucleotide region within the nucleotide sequence of the present invention, where the continuous nucleotide region is approximately 10 to 20 nucleotides, approximately 10 to 25 nucleotides, approximately 10 to 30 nucleotides, approximately 15 to 25 nucleotides, approximately 30 to 40 nucleotides, approximately 50 to 60 nucleotides, approximately 70 to 80 nucleotides, approximately 90 to 100 nucleotides or more in length, and any smaller range within that range, up to the entire length of the sequence. In some embodiments, the nucleotide sequence may be substantially identical over at least approximately 20 nucleotides (e.g., approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides). In some embodiments, substantially identical nucleotide sequences or protein sequences perform substantially identical functions to nucleotides (or protein sequences encoded therein) that are substantially identical thereto.

[0034] The polynucleotides and / or recombinant nucleic acid constructs described herein can optimize codons with respect to expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors described herein (including / encoding, for example, fusion proteins or chimeric proteins or polypeptides) may optimize codons with respect to expression in a particular organism (e.g., an animal, plant, fungus, archaea, or bacterium). In some embodiments, codon-optimized nucleic acid constructs, polynucleotides, expression cassettes, and / or vectors of the present invention have about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) or more identity with reference nucleic acid constructs, polynucleotides, expression cassettes, and / or vectors that are not codon-optimized.

[0035] In any of the embodiments described herein, the polynucleotide or nucleic acid construct described herein may be controllably ligated to various promoters and / or other regulatory elements relating to expression in the organism or cell. Thus, in some embodiments, the polynucleotide or nucleic acid construct described herein may further include one or more promoters, introns, enhancers, and / or controllably ligated terminators ligated to one or more nucleotide sequences. In some embodiments, a promoter may be controllably ligated to an intron. In some embodiments, the promoter ligated to an intron may also be referred to as a “promoter region.”

[0036] When an expression regulatory sequence modulates and controls the transcription and translation of a polynucleotide sequence (DNA, RNA), the polynucleotide sequence is said to be “operatively linked” to the expression regulatory sequence. The term “operatively linked” includes having an appropriate start signal for expression (e.g., ATG) before the polynucleotide sequence to be expressed, and maintaining a precise reading frame that enables the expression of the polynucleotide sequence and the production of the desired polypeptide encoded by the polynucleotide sequence under the control of the expression regulatory sequence.

[0037] The terms "linked" or "fused" in relation to polypeptides refer to one polypeptide being linked to another polypeptide. Polypeptides may be linked or fused to another polypeptide (at the N-terminus or C-terminus) directly (e.g., via peptide bonds) or via linkers (e.g., peptide linkers).

[0038] The term "linker" in relation to polypeptides is recognized in the art and refers to a chemical group or molecule that links two molecules or parts (e.g., two domains of a fusion polypeptide protein). A linker may consist of a single linking molecule (e.g., a single amino acid) or may contain two or more linking molecules. In some embodiments, the linker may be an organic molecule, group, polymer, or chemical part (such as a divalent organic part). In some embodiments, the linker may be an amino acid or a peptide. In some embodiments, the linker is a peptide.

[0039] A “promoter” is a nucleotide sequence that prepares or controls the transcription of a nucleotide sequence (e.g., a coding sequence) that is controllably ligated to the promoter. The coding sequence regulated or controlled by the promoter may encode a polypeptide and / or functional RNA. Typically, a “promoter” refers to a nucleotide sequence that contains an RNA polymerase II binding site and instructs transcription initiation. Generally, the promoter is located 5′ or upstream of the start of the coding region of the corresponding coding sequence.

[0040] Promoters may include, for example, constitutive, inducible, temporally controlled, developmentally controlled, chemically controlled, tissue-selective, and / or tissue-specific promoters used in the preparation of recombinant nucleic acid molecules, such as "synthetic nucleic acid constructs" or "protein / RNA complexes." Various types of these promoters are known in the art.

[0041] The choice of promoter may vary depending on the temporal and spatial requirements of expression; it may also vary depending on the host cell being transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge in the art, it is possible to select a promoter appropriate for a specific target host organism. For example, details are known about upstream promoters of highly constitutively expressed genes in model organisms, and such knowledge is readily accessible and can be applied to other systems as needed.

[0042] As used herein, the term "recombinant" means a protein derived from a prokaryotic or eukaryotic expression system.

[0043] The term "therapeutably effective" means that the amount of composition used is sufficient to improve one or more causes, symptoms, or complications of a disease or disorder. Such improvement does not necessarily require the elimination of the cause, symptoms, or complications of the disease or disorder, but merely alleviates or alters them.

[0044] The term “treatment” refers to the medical management of a patient with the intention of curing, improving, stabilizing, or preventing a disease, condition, or disorder. This term includes active treatment, i.e., treatment aimed in particular to improve the disease, condition, or disorder, and also causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. Furthermore, this term includes temporary palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing, or partially or completely preventing, the cause of the associated disease, condition, or disorder; and supportive care, i.e., treatment used to support other specific therapies aimed at improving the associated disease, condition, or disorder.

[0045] The term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids to which it is linked. A preferred vector is one capable of one or more of the self-replication and expression of the linked nucleic acid. A vector capable of controlling the expression of a linked gene is referred to herein as an "expression vector."

[0046] The term “wild type” (or “WT”) refers to a protein or a portion thereof, or a protein sequence or a portion thereof, as it normally exists in vivo, and to the natural polynucleotide sequence that codes for each. The term “nucleic acid” as used herein refers to polynucleotides (such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA)). This term should also be understood to include equivalents, analogues, of either RNA or DNA made from nucleotide analogs, and single-stranded (sense or antisense) and double-stranded polynucleotides as may apply to the embodiments described.

[0047] The drugs, compounds, compositions, polypeptides, proteins, etc., used in the methods described herein are considered to have been purified and / or isolated before their use. The resulting substance is typically in a “substantially pure” state, meaning that the nucleic acid, polypeptide or fragment thereof, or other molecule has been separated from its normally associated components. Typically, a polypeptide is substantially pure if it is freed from its normally associated proteins and other organic molecules by weight, to the extent of at least 60%, 70%, 80%, 90%, 95%, and even 99%. For example, a substantially pure polypeptide may be obtained by extraction from a natural source by expressing recombinant nucleic acid in cells that do not normally express that protein, or by chemical synthesis. “Isolated substance” is taken out of its natural site or environment of existence. In the case of an isolated or purified domain or protein fragment, the domain or fragment is substantially separated from the amino acid sequence adjacent to its protein in its natural sequence. The term “isolated DNA” means DNA that is substantially separated from the genes adjacent to a given DNA in the natural genome. Therefore, the term "isolated DNA" includes, for example, cDNA, cloned genomic DNA, and synthetic DNA.

[0048] In this specification, “administer to the subject” means providing, implementing, or using a medicine, drug, or treatment to the subject for the purpose of alleviating, curing, or improving symptoms associated with a disease, disorder, or medical condition (e.g., pathological condition). Oral administration is one method of administering the compound to the subject.

[0049] Anxiety disorders are defined in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), 5th Edition (DSM-5 2013), as a group of disorders sharing the characteristics of persistent, excessive fear and anxiety. While anxiety disorders typically last for at least six months, in some embodiments herein, anxiety disorders are diagnosed after less than six months of duration. In children, anxiety disorders are typically diagnosed using one or more assessment scales, such as the Child Anxiety Rating Scale (PARS), the Child Depression Rating Scale, and the Yale-Brown Child Obsessive-Compulsive Disorder Severity Rating Scale.

[0050] Anxiety disorders include, but are not limited to, "generalized anxiety disorder," "social anxiety disorder," "social phobia," "panic attacks," "panic disorder," "post-traumatic stress disorder," "agoraphobia," "separation anxiety disorder," "anxiety disorders induced by substances / medications or other conditions," and "selective mutism."

[0051] Generalized anxiety disorder is an anxiety disorder characterized by excessive anxiety about a variety of everyday problems, with more anxious days than anxious days for at least six months. Social anxiety disorder is a marked fear or anxiety of one or more social situations and is synonymous with social phobia. A panic attack is a sudden onset of intense fear or discomfort, accompanied by symptoms such as palpitations, sweating, dizziness, or nausea. Panic disorder is diagnosed in patients who experience recurrent, unexpected panic attacks. Post-traumatic stress disorder (or "PTSD") is a condition that can develop in people who have faced real or threatening death, serious injury, or sexual violence, in which, triggered by an event, the individual experiences recurrent painful memories, flashbacks, psychological distress, and / or physiological reactions. Agoraphobia is a condition in which an individual experiences marked fear of situations such as being in public places, in crowds, or being alone outside their home. "Separation anxiety disorder" is a developmentally disproportionate and excessive fear or anxiety of an individual about being separated from a person with whom they have an attachment. "Selective mutism" is a condition characterized by the inability to speak in specific social situations in which speaking is expected (e.g., at school), despite being able to speak in other situations.

[0052] Patients with anxiety disorders may also exhibit inattention, hyperactivity, anxiety, mood disorders, and sleep disturbances.

[0053] In some embodiments, symptoms of anxiety include, but are not limited to, emotional distress, palpitations, hyperventilation, excessive sweating, muscle spasms, weakness, fatigue, insomnia, nausea, repetitive behaviors, or any combination thereof.

[0054] The “anxiety rating scales” listed herein are known to those skilled in the art. For example, the Beck Anxiety Rating Scale (BAI) is a 21-item anxiety scale, which is summed up to obtain a total score of 0 to 63; A score of 0-9 is generally considered to mean normal or no anxiety; A score of 10-18 is generally considered to indicate mild to moderate anxiety. A score of 19-29 is generally considered to indicate moderate to severe anxiety. and A score of 30–63 is generally considered to indicate severe anxiety (Julian 2011). Another anxiety assessment scale is the Hospital Anxiety and Depression Rating Scale–Anxiety (HADS-A), a seven-item scale (Julian 2011). This scale can be used to assess the general level of anxiety and to detect and quantify anxiety symptoms (Julian 2011). The total score of the HADS-A can range from 0 to 21, but the following guidelines are recommended for score interpretation: 0-7 means normal or no anxiety; 8-10 indicates mild anxiety; 11-14 indicate moderate anxiety; and 12-21: Severe anxiety, (Julian, 2011). Other anxiety rating scales are described in Hamilton, 1959; Leary, 1983; and Connor, 2000. In this specification, “anxiety reduced by at least 1 point” means that the patient’s anxiety is reduced when assessed using at least one of the specific anxiety rating scales. For example, the STAI is an anxiety rating scale consisting of two subtests: a state anxiety scale and a trait anxiety scale (trait anxiety) (Julian, 2011). The score range for each subtest is 20–80, with higher scores indicating greater anxiety (Julian, 2011). Therefore, subjects obtain a score of 40–160 after completing the STAI. If a subject’s STAI score decreases by 1 point or more, their anxiety is reduced by 1 point. A patient’s anxiety can also be assessed using one of the following anxiety rating scales: State-Trait Anxiety Scale (STAI), Fear Rating Scale, Beck Anxiety Rating Scale (BAI), Abbreviated Fear of Negative Evaluation Scale - BFNE, Clinical Diagnostic Interview Scale for PTSD (CAPS), Symptom-Anxiety Daily Assessment, Generalized Anxiety Disorder 7 (GAD-7), Hamilton's Anxiety Rating Scale (HAM-A), Hospital Anxiety and Depression Rating Scale (HADS-A), Leibovitz Social Anxiety Scale (LSAS), Generalized Anxiety Severity and Functional Impairment Scale (OASIS), Panic and Agoraphobia Rating Scale (PAS), Panic Disorder Severity Rating Scale (PDSS), PTSD Severity Rating Scale - Self-Report, Social Anxiety Disorder Rating Scale (SPIN), Trauma Screening Questionnaire, Yale-Brown Obsessive-Compulsive Scale (Y-BOCS), and Zung's Self-Rating Depression Scale, Depression Rating Scale.

[0055] The terms "subject" and "patient" are used as synonyms for human beings. The terms "pediatric subject" or "pediatric patient" are used as synonyms for human beings under 18 years of age. "Adult patient" refers to human beings 18 years of age or older. "Adolescent patient" or "adolescent subject" is typically a subject between approximately 12 and 18 years of age (e.g., 12-17 or 13-18).

[0056] Embodiments described herein relate to compositions and methods for treating anxiety disorders in subjects requiring such treatment, and more particularly to compositions and methods for treating or preventing one or more of the following: generalized anxiety disorder, phobias, social anxiety disorder, social phobia, panic disorder, panic attacks, post-traumatic stress disorder, separation anxiety disorder, selective mutism, agoraphobia, or anxiety disorders induced by substances / therapeutic agents or conditions.

[0057] The inventors identified the protein tyrosine phosphatase receptor δ (Ptprd), a membrane-bound phosphatase receptor, as the asprosin receptor and revealed that asprosin-mediated Ptprd signaling can induce and / or exacerbate anxiety in subjects. As schematically shown in the examples, the inventors found that in mice, asprosin neutralization and / or genetic ablation of Ptprd reduces anxiety, particularly in asprosin-overexpressing mice. Therefore, in some embodiments, methods for alleviating anxiety in subjects may include administering a therapeutically effective dose of an anxiolytic that inhibits asprosin-mediated Ptprd signaling or asprosin activity to the subject.

[0058] The anxiolytic may be an antibody or an antigen-binding fragment thereof. In other embodiments, the anxiolytic is not an antibody. For example, the anxiolytic may be one or more small molecules, one or more aptamers, one or more non-antibody phage display-derived peptides, or a combination thereof.

[0059] In some embodiments, the anxiolytic specifically binds to and inactivates asprosin binding to Ptprd and / or asprosin-mediated Ptprd signaling and / or asprosin activity.

[0060] In other embodiments, the anxiolytic specifically inhibits the expression of asprosin and / or Ptprd, or reduces their functional activity.

[0061] In some embodiments, the antibody or its antigen-binding fragment may be any immunoconjugate (such as IgG, IgM, IgA, IgD, and IgE). Generally, IgG and / or IgM are preferred; this is because they are the most common antibodies in physiological situations and are the easiest to prepare using laboratory equipment. The term “antibody” is used to refer to any antibody-like molecule having an antigen-binding domain, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single-domain antibodies (DAB), Fv, and scFv (single-stranded Fv). Techniques for preparing and utilizing various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (see, for example, “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, 1988; this reference is incorporated herein by reference). The antibodies of this disclosure may bind specifically to their targets. The expressions "specifically bind" to a target or "specifically immunoreactive" to a target refer to a binding reaction that determines the presence of the molecule in the presence of a heterogeneous population of other biological molecules. Therefore, under specified immunoassay conditions, the identified molecule selectively binds to a specific target, but no significant binding occurs to other biological molecules present in the sample. Specific binding of an antibody to a target under such conditions requires that the antibody be selected based on its specificity to that target. Various immunoassay forms can be used to select antibodies that are specifically immunoreactive to a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive to a certain protein. For a description of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Press, 1988.

[0062] In some embodiments, the anxiolytic may be a monoclonal antibody. Monoclonal antibodies can be prepared and used as asprosin inhibitors for use in individuals. In some cases, the monoclonal antibody is used in methods for treating anxiety. The immunogen of the monoclonal antibody may be the entire asprosin polypeptide or a fragment thereof. An example of a sequence for preparing a monoclonal antibody of asprosin is HuFbn1 2838:2865 KKKELNQLEDKYDKDYLSGELGDNLKMK (Sequence ID: 1).

[0063] In some embodiments, the antibody binds to an epitope of the amino acid sequence of SEQ ID NO: 1. The epitope may be the entire amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. In some embodiments, the epitope is a continuous sequence of amino acids, but in some cases, the epitope binds to a three-dimensional structure of an amino acid sequence that may be continuous or not continuous in morphology. In some cases, the epitope has a length of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-20 amino acids, 8-15 amino acids, 8-10 amino acids, 10-20 amino acids, or 10-15 amino acids. The epitope may consist of, or essentially consist of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids of SEQ ID NO: 1, in some embodiments the amino acids are continuous in SEQ ID NO: 1, but in other embodiments the amino acids are discontinuous in SEQ ID NO: 1.

[0064] In some embodiments, the antibody is an isolated antibody or antigen-binding moiety that specifically binds to a peptide consisting of, or essentially comprising, SEQ ID NO: 1, or SEQ ID NO: 1. For example, an isolated antibody or antigen-binding moiety that specifically binds to the peptide of SEQ ID NO: 1 may be produced by a hybridoma cell line deposited in the American Type Culture Collection as accession number ATCC PTA-123085. In certain embodiments, the antibody or its antigen-binding fragment contains the same heavy-chain and light-chain polypeptide sequence CDRs as the antibody produced by the hybridoma of deposit accession number ATCC PTA-123085. In some embodiments, the antibody or its antigen-binding fragment may contain a heavy-chain variable region and / or a light-chain variable region containing three heavy-chain CDRs and / or three light-chain CDRs of the antibody from the hybridoma of deposit accession number ATCC PTA-123085.

[0065] This disclosure also includes one or more isolated cells of a hybridoma with deposit accession number ATCC PTA-123085, and further includes a hybridoma cell line with deposit accession number ATCC PTA-123085. Antibodies (including humanized versions) produced by any of the cell lines of this disclosure are included herein. Certain embodiments include isolated and purified monoclonal antibodies produced by a persistent hybridoma cell line having deposit accession number PTA-123085.

[0066] Monoclonal antibodies may be obtained from a substantially homogeneous population of antibodies, that is, from individual antibodies containing such a population that are identical except for naturally occurring latent variants, which may be present in small amounts. Therefore, the modifier "monoclonal" indicates the nature of the antibody as a mixture of individual antibodies.

[0067] For example, the anti-asprosin monoclonal antibody may be produced using the hybridoma method first reported by Milstein and Kohler (Kohler & Milstein, Nature 256:495 (1975)), or using the recombinant DNA method [Cabilly et al., U.S. Patent No. 4,816,567]. In the hybridoma method, mice or other suitable host animals (such as hamsters) are immunized to produce or generate lymphocytes capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Then, to form hybridoma cells, the lymphocytes are fused with myeloma cells using a suitable fusion agent (such as polyethylene glycol) (Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-103 (Academic Press, 1986)).

[0068] The hybridoma cells thus prepared are seeded and grown in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fused parent myeloma cells. For example, if the parent myeloma cells are deficient in the enzyme hypoxanthine guanine ribosyltransferase (HGPRT or HPRT), the medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium) to prevent the proliferation of HGPRT-deficient cells.

[0069] Preferred myeloma cells are those that exhibit excellent fusion efficiency, support stable high-level antibody expression by selective antibody-producing cells, and are sensitive to culture media (such as HAT medium). Particularly preferred myeloma cell lines include mouse myeloma lines such as cells derived from mouse tumor MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center (San Diego, California, USA), and SP-2 cells, available from the American Type Culture Collection (Rockville, Maryland, USA).

[0070] Regarding the production of monoclonal antibodies targeting asprosin, the culture medium in which hybridoma cells are proliferating is assayed. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0071] The binding affinity of monoclonal antibodies can be evaluated, for example, by scatchard analysis (Munson & Pollard, Anal. Biochem. 107:220 (1980)).

[0072] After confirming that hybridoma cells produce antibodies with the desired specificity, affinity, and / or activity, their clones may be subcloned by limiting dilution and grown by standard methods (Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-104 (Academic Press, 1986)). Examples of culture media for this purpose include Dulbecco's Modified Eagle Medium or RPMI-1640 culture medium. Furthermore, the hybridoma cells may be grown in vivo as ascites tumors in animals.

[0073] The monoclonal antibodies secreted by the subclone can be isolated from culture media, ascites fluid, or serum by conventional immunoglobulin purification methods (e.g., protein A-Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0074] The DNA encoding the monoclonal antibody of the present invention is readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells can be a source of such DNA. Once isolated, the DNA may be inserted into an expression vector, and then recombinant host cells obtained by transforming host cells that do not produce immunoglobulin proteins (e.g., Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) are used to synthesize the monoclonal antibody. The DNA may also be modified, for example, by substituting homologous mouse sequences with sequences encoding the constant domains of the human heavy and light chains (Morrison et al., Proc. Nat. Acad. Sci. 81, 6851 (1984)); or by covalently linking the entire or partial coding sequence of a non-immunoglobulin polypeptide to the coding sequence of the immunoglobulin. By such a method, a “chimeric” or “hybrid” antibody having binding specificity to the anti-asprosin monoclonal antibody of this specification is prepared.

[0075] Typically, a chimeric bivalent antibody is created by using such a non-immunoglobulin polypeptide to replace the constant domain of an antibody, or by using such a non-immunoglobulin polypeptide to replace the variable domain of one antigen-binding site of an antibody, thereby containing one antigen-binding site specific to asprosin and another antigen-binding site specific to a different antigen.

[0076] Chimeric or hybrid antibodies may also be prepared in vitro using methods known in synthetic protein chemistry, such methods including those involving crosslinking agents. For example, immunotoxins may be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyryl imidate.

[0077] In certain embodiments, antibodies against asprosin are humanized. Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “imported” residues and typically originate from “imported” variable domains. Humanization can be carried out by substituting rodent CDR sequences (plural or singular) for corresponding sequences in a human antibody, essentially following the methods of Winter and his collaborators (Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332, 323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)). Thus, such “humanized” antibodies are chimeric antibodies in which the corresponding sequences of a non-human species are substituted for a portion substantially shorter than the intact human variable domain. In practice, humanized antibodies are typically human antibodies in which some CDR residues and, if possible, some FR residues are replaced with residues derived from their equivalent sites in rodent antibodies.

[0078] It is important to humanize antibodies while maintaining high affinity for antigens and other desirable biological properties. To achieve this objective, humanized antibodies are prepared according to preferred methods through a process involving the analysis of parental sequences and the analysis of various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and known to those skilled in the art. Computer programs are available that show and display the putative three-dimensional structure of selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the potential role of the residues in the functional performance of the candidate immunoglobulin sequence; that is, it is possible to analyze the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this method, FR residues can be selected and combined from the consensus sequence and imported sequence to obtain the desired properties of the antibody (such as enhanced affinity for the target antigen). In general, CDR residues directly and most substantially affect antigen binding. For further details, please refer to U.S. Patent Application No. 07 / 934,373, filed on 21 August 1992, which is a continuation of part of U.S. Patent Application No. 07 / 715,272, filed on 14 June 1991.

[0079] Human monoclonal antibodies can be produced by the hybridoma method described above. Human myeloma cell lines and mouse / human heteromyeloma cell lines for human monoclonal antibody production are described, for example, Kozbor, J. Immunol. 133, 3001 (1984); and Brodeur et al., "Monoclonal Antibody Production Techniques and Applications," pp. 51-63 (Marcel Dekker, New York, 1987).

[0080] It is now possible to create transgenic animals (e.g., mice) that can produce a human antibody repertoire in the absence of endogenous immunoglobulin production upon immunization. For example, in chimeric mice and germline mutant mice, the antibody heavy chain linkage region (J H It has been reported that homozygous deletion of the gene completely inhibits endogenous antibody production. When a human germline immunoglobulin gene array is transplanted into such germline mutant mice, human antibodies are produced upon antigen addition. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al., Nature 362, 255-258 (1993).

[0081] Alternatively, phage display technology (McCafferty et al., Nature 348, 552-553

[1990] ) can be used to in vitro produce human antibodies and antibody fragments from the immunoglobulin variable (V) domain gene repertoire of non-immune donors. According to this technology, the antibody V domain gene is cloned in-frame into either the major or minor coat protein gene of a linear bacteriophage (such as M13 or fd) and expressed as a functional antibody fragment on the surface of the phage particle.

[0082] In other embodiments, the antibody against asprosin may be a bispecific antibody. The bispecific antibody is monoclonal and preferably a human antibody or humanized antibody having binding specificity to at least two different antigens. In this case, one of the binding specificities is against asprosin and the other is against any other antigen (preferably against another receptor or a subunit of another receptor). For example, a bispecific antibody that specifically binds to asprosin and the asprosin receptor, or specifically binds to two different asprosin receptors, is within the scope of the present invention.

[0083] Methods for producing bispecific antibodies are known in the art. Conventionally, recombinant bispecific antibodies have been produced based on the co-expression of two immunoglobulin heavy / light chain pairs, the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)). Because the heavy and light chains of the immunoglobulins can be in any combination, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the exact bispecific structure. Purification of this exact molecule is usually carried out by affinity chromatography, but this is difficult to handle and yields a low product. Similar methods are disclosed in PCT application publication WO93 / 08829 (published May 13, 1993); and Traunecker et al., EMBO 10, 3655-3659 (1991).

[0084] An antibody variable domain having the desired binding specificity (antibody / antigen binding site) is fused to the constant domain sequence of an immunoglobulin according to a different, more preferred approach. This fusion is preferably with the constant domain of the immunoglobulin heavy chain, which includes at least a portion of the hinge, CH2 region, and CH3 region. Preferably, at least one of the fusions has a first constant heavy chain region (CH1) that includes the site necessary for binding to the light chain. The immunoglobulin heavy chain fusions and, if necessary, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors, and a suitable host organism is simultaneously transformed. This provides a high degree of flexibility in adjusting the relative ratios of the three polypeptide chains in the embodiment in which the optimal yield is obtained by unequal ratios of the three polypeptide chains used in construction. However, if a high yield can be obtained by expressing at least two polypeptide chains in equal ratios, or if the above ratios are not particularly significant, it is also possible to introduce the coding sequences of two or all three polypeptide chains into a single expression vector. In a preferred embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity present in one arm and a hybrid immunoglobulin heavy / light chain pair (providing a second binding specificity) present in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations; this is because the presence of immunoglobulin light chains in only half of the bispecific molecule provides a simple separation method. This approach is disclosed in concurrent application 07 / 931,811, filed on 17 August 1992.

[0085] For further details on the creation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121, 210 (1986).

[0086] In other embodiments, the anxiolytic may comprise an anxiolytic peptide that is a peptide mimetic of the asprosin ligand-binding domain of Ptprd (i.e., Ptprd-lbd). The anxiolytic peptide or peptide mimetic of the asprosin ligand-binding domain of Ptprd may have an amino acid sequence substantially identical to the extracellular portion of the amino acid sequence of Ptprd that binds to asprosin. When the Ptprd-lbd peptide mimetic is ectopically introduced into the blood circulation of the subject requiring it, it inhibits asprosin-inducible or mediated Ptprd signaling in neurons, thereby resulting in the corresponding reduction of anxiety. In some embodiments, the peptide mimetic or anxiolytic peptide can bind to and inhibit asprosin in the blood circulation of the subject. In some embodiments, the anti-anxiety peptide is present in an amount of at least about 70%, at least about 71%, at least about 72%, at least about 73%, relative to 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, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least 1000 amino acids of TEQ:2. They could have amino acid sequences that were identical by at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0087] For example, the anti-anxiety peptide is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, and at least about 76% of the following amino acids in sequence number 2: approximately 10-1240, approximately 20-1220, approximately 30-1210, approximately 40-1200, approximately 50-1190, approximately 60-1180, approximately 70-1170, approximately 80-1160, approximately 90-1180, approximately 100-1150, approximately 200-1100, approximately 300-1000, approximately 400-900, or approximately 500-800. They could have at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical amino acid sequences.

[0088] In other embodiments, the anti-anxiety peptide may have an amino acid sequence that is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:2.

[0089] In other embodiments, the anti-anxiety peptide is at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, and at least about 76% of the consecutive amino acids of 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, at least about 100, at least about 150, and at least about 200 amino acids of the extracellular Ig Domain of Ptprd. They could have had the same amino acid sequence in approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0090] In one example, the anti-anxiety peptide is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, and at least 77% of the consecutive amino acids of SEQ ID NO: 3, with respect to 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, at least about 100, at least about 150, and at least 200 They could have had the same amino acid sequence in approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0091] In another example, the anti-anxiety peptide may have an amino acid sequence identical to 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, or at least about 80 consecutive amino acids of SEQ ID NO: 4 by at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0092] In another example, the anti-anxiety peptide is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, and at least 79% of the consecutive amino acids of SEQ ID NO: 5. They could have had at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical amino acid sequences.

[0093] In another example, the anti-anxiety peptide is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, and at least 79% of the consecutive amino acids of SEQ ID NO: 6. They could have had at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical amino acid sequences.

[0094] In some embodiments, the anti-anxiety peptide has a binding affinity KD of less than about 10 μM, less than about 1 μM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, or less than about 500 pM relative to asprosin.

[0095] The anxiolytic peptides described herein are subject to various other modifications, substitutions, insertions, and deletions, and such modifications may provide specific advantages in the use of the peptides. In this regard, anxiolytic peptides having an amino acid sequence substantially identical to the extracellular portion of the amino acid sequence of Ptprd that binds to asprosin are not identical to, but rather may correspond to, or be substantially homologous to, the sequences of enumerated polypeptides that have undergone one or more modifications but retain the ability to inhibit or reduce one or more of the activities, signaling, and / or functions related to asprosin-mediated anxiety.

[0096] The anti-anxiety peptide may be a polypeptide derivative in various forms, including amides, complexes with proteins, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides, and similar derivatives.

[0097] The anxiolytic peptide may also include conserved substitutions of amino acid residues. If the peptide obtained by such conservative substitutions exhibits essential binding activity, it will be understood that such conservative substitutions may also include the use of chemically derivatized residues as substitutes for non-derivativeated residues.

[0098] A "chemical derivative" refers to a target peptide having one or more residues that have been chemically derivatized by reacting functional side chain groups. Examples of such derivatized molecules include those in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, or formyl group. A free carboxyl group may be derivatized for the purpose of forming a salt, methyl ester, ethyl ester, or other type of ester or hydrazide. A free hydroxyl group may be derivatized for the purpose of forming an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine may be derivatized for the purpose of forming N-imu-benzylhistidine. Polypeptides containing one or more natural amino acid derivatives of 20 standard amino acids are also included in the category of chemical derivatives. For example: Proline may also be substituted with 4-hydroxyproline; Lysine may also be substituted with 5-hydroxylysine; Histidine may be substituted with 3-methylhistidine; It is also acceptable for serine to be replaced with homoserine; and Lysine may be substituted with ornithine. The polypeptides described herein may also include any polypeptide having one or more residue additions and / or deletions from the sequence of the polypeptide having the sequence shown herein, as long as the essential activity is maintained.

[0099] One or more of the anxiolytic peptides described herein may also be modified by natural processes (such as post-translational processing) and / or by chemical modification techniques known in the art. Modifications may occur within the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminus. It will be understood that the same type of modification may exist at multiple sites within a given peptide, either identically or to varying degrees. Examples of modifications include: Acetylation, acylation, and addition of acetamidomethyl (Acm) group; ADP-ribosylation; amidation; Covalent linkage to flavin; Covalent bonding to the heme portion; Covalent linkage to nucleotides or nucleotide derivatives; Covalent linkage to lipids or lipid derivatives; Covalent linkage to phosphatidylinositol; Crosslinking; cyclization; disulfide bond formation; demethylation; covalent crosslinking; cystine formation; pyroglutamic acid formation; formylation; gamma-carboxylation; glycosylation; hydroxylation; iodization; methylation; myristoylation; oxidation; proteolytic processing; phosphorylation; prenylation; racemization; selenoylation; sulfation; RNA-mediated addition of amino acids to proteins (such as arginylation and ubiquitination) (Reference: "Protein-structure and molecular properties, 2nd Ed.," TECreighton, WH Freeman and Company, New York, 1993). These are some examples, but are not limited to them.

[0100] The peptides and / or proteins described herein may also include, for example, biologically active mutants, variants, fragments, chimeras, and analogs. A fragment contains an amino acid sequence having a truncation of one or more amino acids, where the truncation may be at the amino terminus (N-terminus), the carboxyl terminus (C-terminus), or within the protein. Analogs of the present invention include insertions or substitutions of one or more amino acids. Variants, mutants, fragments, chimeras, and analogs may act as inhibitors of asprosin-mediated anxiety (but are not limited to these examples).

[0101] The anxiolytic polypeptides described herein may be prepared by methods known to those skilled in the art. The peptides and / or proteins may also be prepared using recombinant DNA. For example, one preparation may involve culturing a host cell (bacteria or eukaryote) under conditions that allow the peptide and / or protein to be expressed intracellularly.

[0102] The polypeptide may be purified by affinity-based methods, ion-exchange chromatography, size exclusion chromatography, hydrophobicity, or other purification techniques commonly used for protein purification. This purification process can be carried out under non-denaturing conditions. Alternatively, if a denaturation step is necessary, the protein may be regenerated using techniques known in the art.

[0103] In some embodiments, the anxiolytic peptide is an exogenous peptide created by recombinant technology and can be systemically administered to a subject, for example, by parenteral or intravenous administration.

[0104] In some embodiments, the anxiolytic peptide comprises at least one non-homologous or exogenous moiety (such as a non-homologous polypeptide or an exogenous polypeptide). The at least one non-homologous polypeptide may be, for example, an antibody or its antigen-binding fragment, a glucagon-like peptide 1 receptor (GLP-1R) agonist, an Fc moiety of an immunoglobulin, an albumin peptide, an albumin-binding domain (ABD), a signaling peptide, or a combination thereof.

[0105] In some embodiments, the non-homologous moiety is fused to the N-terminus or C-terminus of the anxiolytic peptide. In other embodiments, the non-homologous moiety is inserted between two amino acids in the anxiolytic peptide.

[0106] In other embodiments, the anxiolytic peptide may further comprise two, three, four, five, six, seven, or eight non-homologous sequences. In some embodiments, all of the non-homologous sequences are identical. In some embodiments, at least one non-homologous portion is different from the other non-homologous portions. In some embodiments, the disclosure may comprise two, three, four, five, six, or seven or more non-homologous portions in tandem.

[0107] In some embodiments, the non-homologous moiety extends the half-life of the anxiolytic peptide ("half-life extender").

[0108] In some embodiments, the non-homologous moiety is a peptide or polypeptide having either unstructured or structured properties that, when inserted into the anxiolytic peptide, are associated with an extension of the in vivo half-life. Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, C-terminal peptides (CTPs) of the β-subunit of human chorionic gonadotropins, HAP sequences, XTEN sequences, transferrin or fragments thereof, PAS polypeptides, polyglycine linkers, polyserine linkers, albumin-binding moieties, or fragments, derivatives, variants, or combinations of these polypeptides.

[0109] In a particular embodiment, the non-homologous polypeptide may be an immunoglobulin constant region or a portion thereof, transferrin, albumin, or a PAS sequence. In some aspects, the non-homologous portion includes von Willebrand factor or a fragment thereof. In other relevant aspects, the non-homologous polypeptide may include a linking site for the non-polypeptide portion (e.g., a cysteine ​​amino acid) (such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination thereof). In some aspects, the non-homologous portion includes a cysteine ​​amino acid that functions as a linking site for the non-polypeptide portion (such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination thereof).

[0110] In a particular embodiment, the first non-homologous polypeptide is a half-life extension molecule known in the art, and the second non-homologous moiety is a half-life extension molecule known in the art. In a particular embodiment, the first non-homologous polypeptide (e.g., the first Fc polypeptide) and the second non-homologous polypeptide (e.g., the second Fc polypeptide) associate with each other to form a dimer. In a particular embodiment, the second non-homologous polypeptide is the second Fc polypeptide, which is linked to or conjugated to the first non-homologous polypeptide (e.g., the first Fc polypeptide). For example, the second non-homologous polypeptide (e.g., the second Fc polypeptide) can be linked to the first non-homologous moiety (e.g., the first Fc polypeptide) via a linker; or it can be conjugated to the first non-homologous moiety by a covalent or non-covalent bond.

[0111] In some embodiments, the non-homologous polypeptide comprises, or comprises, at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2500, at least about 3000, or at least about 4000 amino acids, essentially the same as above. In other embodiments, the non-homologous polypeptide comprises, or comprises, approximately 100 to approximately 200 amino acids, approximately 200 to approximately 300 amino acids, approximately 300 to approximately 400 amino acids, approximately 400 to approximately 500 amino acids, approximately 500 to approximately 600 amino acids, approximately 600 to approximately 700 amino acids, approximately 700 to approximately 800 amino acids, approximately 800 to approximately 900 amino acids, or approximately 900 to approximately 1000 amino acids, essentially the same as above.

[0112] In certain embodiments, non-homologous polypeptides improve one or more pharmacokinetic properties of the anxiolytic peptide without significantly affecting its biological activity or function.

[0113] In certain embodiments, the non-homologous polypeptide extends the in vivo and / or in vitro half-life of the anxiolytic peptide. In other embodiments, the non-homologous polypeptide facilitates the visualization or localization of the anxiolytic peptide. The visualization and / or localization of the anxiolytic peptide may be in vivo, in vitro, ex vivo, or a combination thereof.

[0114] In other embodiments, non-homologous polypeptides enhance the stability of the anxiolytic peptide. The term “stability” as used herein refers to a measure of the maintenance of one or more physical properties of the anxiolytic peptide in response to environmental conditions (e.g., an increase or decrease in temperature), as recognized in the art. In certain aspects, the physical property may be the maintenance of the covalent structure of the anxiolytic peptide (e.g., no proteolytic cleavage, unwanted oxidation, or deamidation). In other aspects, the physical property may also be the presence of the anxiolytic peptide in a properly folded state (e.g., no soluble aggregates, insoluble aggregates, or precipitates). In one aspect, the stability of the anxiolytic peptide is assessed by assaying its biophysical properties (e.g., thermal stability, pH denaturation profile, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to proteins, receptors, or ligands)) and / or combinations thereof. In another aspect, biochemical function is indicated by the binding affinity of interactions. In one aspect, protein stability is measured by thermal stability, i.e., resistance to thermal stress. Stability can be measured using methods known in the art (such as HPLC (high-performance liquid chromatography), SEC (size exclusion chromatography), and DLS (dynamic light scattering)). Methods for measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), differential scanning fluorescence (DSF), circular dichroism (CD), and thermal stress assays.

[0115] In some embodiments, the non-homologous moiety or non-homologous polypeptide may include the Fc moiety of an immunoglobulin. The Fc moiety of an immunoglobulin can be linked to the anxiolytic peptide to form a fusion or chimeric polypeptide or protein. Fusion or chimeric polypeptides or proteins are known in which the Fc region of IgG can be combined with one or more domains of another protein (such as various cytokines and soluble receptors). These chimeric proteins may be fusions of a human Fc region and a human domain of another protein. These chimeric proteins become "humanized Fc chimeras" that are advantageous for human therapy. (See, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, (1996); U.S. Patents 5,116,964 and 5,541,087). The fusion polypeptide may be a homodimeric protein linked via a cysteine ​​residue in the hinge region of IgG Fc, thereby yielding an IgG molecule-like molecule that does not contain the CH1 domain and light chain. Due to structural homology, such Fc fusion proteins exhibit a pharmacokinetic profile similar to that of human IgG with similar isotypes in vivo. This approach has been applied to several therapeutically important cytokines (such as IL-2 and IFN-α) and soluble receptors (such as TNF-Rc and IL-5-Rc) (see, for example, U.S. Patents 5,349,053, 6,224,867, and 7,250,493).

[0116] In some embodiments, the anxiolytic peptide-Fc fusion polypeptide or chimeric molecule is a chimeric molecule comprising a human sequence encoding the extracellular portion of Ptprd fused to a human Fc fragment.

[0117] In other embodiments, the non-homologous polypeptide may be a glucagon-like peptide 1 receptor (GLP-1R) agonist, to which the above-mentioned anxiolytic peptide is linked to form a fusion polypeptide or fusion protein. The GLP-1R agonist may include a peptide that binds to and activates GLP-1 receptor-like GLP-1 (glucagon-like peptide 1). The physiological effects of GLP-1 and / or GLP-1R agonists are described, for example, in Nauck, MA et al. (1997) Exp. Clin. Endocrinol. Diabetes, 105, 187-195. These physiological effects in normal subjects, particularly humans, include, for example, glucose-dependent stimulation of insulin secretion, inhibition of glucagon secretion, stimulation of (pro)insulin biosynthesis, decreased food intake, slowed gastric emptying, and / or insulin sensitivity at a pending level.

[0118] Assays that can be used for drug discovery of GLP-1R agonists are described, for example, in Thorkildsen, Chr. et al. (2003), Journal of Pharmacology and Experimental Therapeutics, 307, 490-496; Knudsen, LB et al. (2007), PNAS, 104, 937-942, No.3; Chen, D. et al. (2007), PNAS, 104, 943-948, No.3; or US2006 / 0003417A1 (see, for example, Example 8). In summary, in the "receptor binding assay," for example, purified membrane fractions of eukaryotic cells containing human recombinant GLP-1 receptors (e.g., CHO, BHK, or HEK293 cells) are used to bind, for example, human GLP-1, for example, GLP-1(7-36), for example, 125 The test compound(s) are incubated with an amide labeled with I (e.g., 80 kBq / pmol). Typically, different concentrations of the test compound(s) are used to determine the IC50 concentration that reduces the specific binding of human GLP-1. 50 To decide.

[0119] In some embodiments, a GLP-1R agonist is selected from, for example, Drucker, DJ (2006) Cell Metabolism, 3, 153-165; Thorkildsen, Chr. (2003; cited above); Chen, D. et al. (2007; cited above); Knudsen, L. et al. (2007; cited above); Liu, J. et al. (2007) Neurochem Int., 51, 361-369, No. 6-7; Christensen, M. et al. (2009) Drugs, 12, 503-513; Maida, A. et al. (2008) Endocrinology, 149, 5670-5678, No. 11; and a bioactive GLP-1, GLP-1 analogue or GLP-1 substitute as described in U.S. Patent No. 2006 / 0003417. Examples of GLP-1R agonists include GLP-1(7-37), GLP-1(7-36) amide, exendin-4, liraglutide, CJC-1131, albugon, albiglutide, exenatide, exenatide-LAR, oxytomodulin, lixisenatide, geniposide, short peptides and / or small organic compounds having GLP-1R agonistic activity.

[0120] Human GLP-1(7-37) has the amino acid sequence of SEQ ID NO: 7. Human GLP-1(7-36)amide has the amino acid sequence of SEQ ID NO: 8.

[0121] Other peptides with GLP-1R agonizing activity are disclosed in U.S. Patents 2006 / 0003417 and 2019 / 0085043; small organic compounds with GLP-1R agonizing activity are disclosed in Chen et al., 2007, PNAS, 104, 943-948, No. 3, or Knudsen et al., 2007, PNAS, 104, 937-942.

[0122] The anti-anxiety peptide can be directly linked to a non-homologous peptide, or indirectly linked to a non-homologous polypeptide via a linker. The linker may include a structural unit that is inserted between two or more other units (e.g., between two or more peptides, or between two or more polypeptides, or between two or more proteins, or between one peptide and one protein, or between one polypeptide and one protein, or between one peptide and one polypeptide) to form a single molecule, and which couples these two or more units together. The coupling of the two units is preferably by covalent bonding. As used herein, “linker” also refers to a structural unit that can be linked to the N-terminus or C-terminus of two or more other units (e.g., two or more peptides, or two or more polypeptides, or two or more proteins, or between one peptide and one protein, and between one polypeptide and one protein, and between one peptide and one polypeptide), where the two or more other units are directly linked together. As used herein, “linker” also refers to a combination of the definitions given above; that is, one structural unit is inserted between two or more other units (e.g., between two or more peptides, or between two or more polypeptides, or between two or more proteins, or between one peptide and one protein, or between one polypeptide and one protein, or between one peptide and one polypeptide), and one or more further structural units are linked to the N-terminus or C-terminus of two or more other units (e.g., two or more peptides, or two or more polypeptides, or two or more proteins, or between one peptide and one protein, and between one polypeptide and one protein, and between one peptide and one polypeptide). The linking of the structural unit to the N-terminus or C-terminus of the two or more other units is preferably by covalent bond.

[0123] In some embodiments, the linker may include further residues that may be added to any end of an anti-anxiety peptide, for example, for the purpose of conveniently linking other polypeptides, proteins, or other molecules (such as detectable portions, labels, solid matrices, or carriers).

[0124] While amino acid linkers typically consist of at least one residue, linkers of two or more residues are also possible, often involving amino acids with lengths ranging from approximately 2 to over 1000 residues. For example, lengths of approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (for example, amino acids with a length of approximately 2-40, approximately 2-50, approximately 2-60, approximately 4-40, approximately 4-50, approximately 4-60, approximately 5-40, approximately 5-50, approximately 5-60, approximately 9-40, approximately 9-50, approximately 9-60, approximately 10-40, approximately 10 ~Approximately 50, approximately 10~60, or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids ~Approximately 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or approximately 1000 amino acid residues (for example, amino acids with a length of approximately 105, 110, 115, 120, 130, 140, 150 residues or longer).

[0125] In some embodiments, the linker may be a flexible peptide linker that links the anti-anxiety peptide to other polypeptides, proteins, and / or molecules (such as a detectable moiety, label, solid matrix, or carrier). The flexible peptide linker may be an amino acid with a length of about 20 residues or less. For example, the peptide linker may be an amino acid with about 12 residues or less, e.g., amino acids 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Typical amino acid residues used for linking include glycine, serine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid. In some cases, the peptide linker may contain two or more of the following amino acids: Glycine, serine, alanine, and threonine.

[0126] In some embodiments, the peptide linker may be a GS linker. In some embodiments, the peptide linker is (GGS) n The peptide linker may include the amino acid sequences GS, SG, GSSG (SEQ ID NO: 9), S(GGS)n (SEQ ID NO: 10), SGGS (SEQ ID NO: 11), or (GGGGS)n (SEQ ID NO: 12), where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the peptide linker may include the amino acid sequence SGGSGGSGGS (SEQ ID NO: 11). In some embodiments, the peptide linker may also include the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 13), which is also called the XTEN linker. In some embodiments, the peptide linker may include the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (Sequence ID: 14), which is also called the GS-XTEN-GS linker.

[0127] In one embodiment of the fusion polypeptide or protein described herein, the peptide linker may include a functional moiety that imparts one or more additional functions in addition to linking the anxiolytic peptide with the non-homologous moiety or the non-homologous polypeptide.

[0128] The linker can be added for the purpose of improving or independent folding in one or both of the polypeptides forming the fusion polypeptide or fusion protein, and / or to avoid steric hindrance and / or to introduce further desired functionality (e.g., an introduction site for covalently linking the addition, a tag for protein purification, a protease cleavage site, protein stabilization and / or extension of the protein's half-life). The linker may contain 0 or 1 to 1000 amino acids. The linker may also be absent (i.e., 0 amino acids).

[0129] Typical linker types can be, for example, helix or non-helix. Here, helix linkers are thought to act as rigid spacers separating two domains, while non-helix linkers, being proline-containing / proline-rich, are structurally rigid and also separate the linker from the connecting domain. This means that both linker types are likely to act as a framework to prevent undesirable interactions between folded domains.

[0130] In some embodiments, the linker may be a portion that imparts improved stability and / or an extended half-life to the fusion polypeptide; such portions include: (a) XTENylation, rPEG, PAS, or HESylation sequences or elastin-like polypeptides (ELPs); (b) an introduction site for covalent modification of the fusion protein (such as a cysteine ​​residue or a lysine residue); (c) A portion having intracellular or extracellular target function (such as a protein-binding skeleton (antibody, antigen-binding fragment, or other protein-non-antibody-binding skeleton), nucleic acids (aptamers, PNA, DNA, etc.)); (d) Protease cleavage sites (such as factor Xa cleavage sites or other (preferably extracellular) protease cleavage sites); or (e) Albumin-binding domain (ABD); or (f) an amino acid sequence containing one or more histidine amino acids (His linker, abbreviated as "His"), for example, HAHGHGHAH (SEQ ID NO: 15). The linker may also contain one or more amino acid residues that do not impart further functionality to the linker and the functionalization portion.

[0131] In some embodiments, the linker may include a factor Xa cleavage site containing or consisting of the sequence IEGR (sequence number: 16), or a protease cleavage site containing or consisting of at least one arginine residue (such as GGGRR (sequence number: 17)).

[0132] In some embodiments, the anxiolytic peptide or fusion polypeptide containing the anxiolytic polypeptide may be modified to include a signal peptide for promoting the secretion of the expressed anxiolytic peptide or fusion polypeptide by the cell. The properties of the signal peptide are well known in the art, and the signal peptide typically has 16 to 30 amino acids, although the number of amino acid residues may be greater or less. Conventional signal peptides consist of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The signal peptide may include, for example, the signal peptide IL2 or IL15.

[0133] In some embodiments, the anxiolytic peptide or a fusion polypeptide comprising the anxiolytic peptide and a non-homologous polypeptide can be expressed in vivo or ex vivo in cells using a vector containing a nucleic acid encoding the anxiolytic peptide or fusion polypeptide. The polynucleotide to be introduced may contain the coding sequence of interest in gene therapy. Examples of vectors include viral vectors (such as adenoviruses (Ad), adeno-associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating the introduction of polynucleotides into target cells.

[0134] In some embodiments, the vector may include an expression cassette. The expression cassette may include a nucleic acid molecule and a promoter containing the coding sequence of the anxiolytic peptide or fusion polypeptide (e.g., the coding sequence of the anxiolytic peptide, the non-homologous polypeptide, and the optional linker), but may also include other regulatory sequences relating to the above; the cassette may be engineered to be modified into a genetic element and / or packaged in the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for creating a viral vector includes the anxiolytic peptide and non-homologous polypeptide sequences described herein, flanked by packaging signals and other expression regulatory sequences of the viral genome (such as the sequences described herein). Any expression regulatory sequence can be optimized for a specific species using techniques known in the art (e.g., including codon optimization as described herein).

[0135] The expression cassette typically includes a promoter sequence as part of the expression regulatory sequence. For example, the promoter may include a liver-specific promoter for thyroxine-binding globulin (TBG). In other examples, the vectors described herein may include a CB7 promoter, which is a chicken β-actin promoter with a cytomegalovirus enhancer element. Alternatively, other liver-specific promoters may be used, such as the TTR minimal enhancer / promoter, the α-antitrypsin promoter, or LSP(845nt)25 (requiring intron-free cAAV). Less frequently, other promoters such as viral promoters, constitutive promoters, controllable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), or promoters responsive to physiological cues may be used in the vectors described herein.

[0136] The expression cassette and / or vector may also include other appropriate regulatory sequences in addition to the promoter; such regulatory sequences may include: Transcription start sequences, transcription termination sequences, enhancer sequences, and efficient RNA processing signals (such as splicing and polyadenylation (polyA) signals); Sequences that stabilize cytoplasmic mRNA; Sequences that improve translation efficiency (i.e., Kozak consensus sequences); Sequences that improve protein stability; and If necessary, a sequence that promotes the secretion of the encoded product, Examples of suitable polyA sequences include, for example, the polyA sequences of SV40, bovine growth hormone (bGH), and TK. Examples of enhancers include, for example, the alpha-fetoprotein enhancer, the TTR minimal promoter / enhancer, and the LSP (TH-binding globulin promoter / alpha-1-microglobulin / bikunin enhancer). These regulatory sequences can be controllably ligated to the anxiolytic peptide sequence.

[0137] In some embodiments, the nucleic acid encoding the therapeutic peptide or fusion polypeptide may be modified to include a signal sequence that promotes the cellular secretion of the expressed therapeutic peptide. For example, the nucleic acid may include a cDNA encoding the extracellular domain of Ptprd and the IL2 signal sequence. Several such modifications are known in the art and can be used by those skilled in the art.

[0138] The vector may also contain other components or functionalities that further regulate gene transfer and / or gene expression, or confer beneficial properties to the target cells. Such other components may include, for example: Components that affect cell binding or cell targeting (including components that mediate cell-type specific binding or tissue-specific binding); Components that affect the uptake of vector nucleic acids by cells; Components that affect the intracellular localization of polynucleotides after uptake (such as chemicals that mediate nuclear localization); and Components that affect the expression of the polynucleotide (such as one or more transcriptional regulatory sequences), Examples include the following. Such components may also include markers, such as detectable and / or selectable markers, which can be used for the purpose of detecting or selecting cells that take up and express the introduced nucleic acid via the vector. Such components may be provided as having the original characteristics of the vector (for example, by using a component that mediates binding and uptake or a specific viral vector with functionality); or the vector may be modified to confer such functionality.

[0139] Selectable markers can be positively selective, negatively selective, or biselective. Positively selective markers allow for the selection of cells containing the marker, while negatively selective markers allow for the selective removal of cells containing the marker. Reports exist on various such marker genes (including biselective (i.e., positively selective / negatively selective) markers) (see, for example, Lupton, S., WO92 / 08796, published May 29, 1992; and Lupton, S., WO94 / 28143, published December 8, 1994). Such marker genes may provide further control mechanisms that could be advantageous in gene therapy. Various such vectors are known and generally available in the art.

[0140] In some embodiments, the vector may include an adeno-associated virus (AAV) viral vector. The AAV viral vector is a DNase-resistant AAV particle having an AAV protein capsid that packages a nucleic acid sequence for introduction into target cells. The AAV capsid consists of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, arranged in an icosahedral symmetry with a ratio of approximately 1:1:10 to 1:1:20, depending on the selected AAV. AAV serotypes may be selected as the capsid source for AAV viral vectors (DNase-resistant viral particles); such serotypes include, for example, AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh74, AAVrh10, AAV5, AAV7, AAVS3, AAVHSC, AAV2.7m8, AAV-LK03, AAV8 / Olig001, AAV2i8, AAVhu37, and AAV2tY. Examples include F, AAVh1, AAVhu68, AAVrh.8, AAVrh9, AAV.PHP.B., AAV.PHP.eB, AAV.PHP.S, AAV / BBB, AAV-DJ, AAVr3.45, AAV-sh10, AAV2(Y444F), AAV4, AAV-RPF2, AAV3b, AAVrh64R1, or any known or mentioned AAV variant, or any AAV variant to be discovered in the future. See, for example, U.S. Patent Application No. 2007-0036760A1; U.S. Patent Application No. 2009-0197338A1; EP1310571. See also WO2003 / 042397 (AAV7 and other Simian AAVs), U.S. Patent Nos. 7,790,449 and 7,282,199 (AAV8), WO2005 / 033321, and 7,906,111 (AAV9), and WO2006 / 110689, and WO2003 / 042397 (rh.10). Alternatively, recombinant AAVs based on any of the listed AAVs may be used as AAV capsid sources. These documents also contain descriptions of other AAVs that may be selected for the purpose of creating AAVs; these documents are incorporated herein by reference.In some embodiments, the AAV cap used in the viral vector can be created by mutagenesis (i.e., insertion, deletion, or substitution) in one of the aforementioned AAV caps or the nucleic acids encoding it. In some embodiments, the AAV capsid is a chimera containing two, three, or four or more domains of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers of two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition contains two or more of the aforementioned caps.

[0141] The only AAV component required in cis form in the same construct as the gene for packaging the expression cassette within the viral particle is the ITR. In one embodiment, for the purpose of creating the AAV vector, the coding sequences for replication (rep) and / or capsid (cap) are removed from the AAV genome and supplied instead in trans form, or supplied by a packaging cell line. For example, as described above, the pseudotyped AAV may contain ITRs derived from a different source than the AAV capsid source. Additionally, or alternatively, chimeric AAV capsids may be utilized. Further other AAV components may be selected. Sources of such AAV sequences are also described herein, but may be isolated or obtained from research institution sources, commercial sources, or public sources (e.g., American Type Culture Collection, USA, Virginia, Manassas). Alternatively, the AAV sequences may be obtained by synthesis or other preferred means by referencing publicly available sequences (such as those available from literature or databases (e.g., GenBank, PubMed, etc.)).

[0142] Methods for isolating and preparing AAV virus vectors that can be used for introduction into a target are known in the art. See, for example, U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; WO2003 / 042397; WO2005 / 033321, WO2006 / 110689; and U.S. Patent Publication No. 7,588,772B2. In one specific system, a producer cell line is transiently transformed with a construct encoding a transgene flanked by an ITR, as well as constructs encoding rep and cap. In a second system, a packaging cell line that provides a stable supply of rep and cap is transiently transformed with a construct encoding a transgene flanked by an ITR. In each of these systems, AAV virus particles are produced in response to infection with a helper adenovirus or herpesvirus, but it is necessary to isolate rAAV from the contaminating virus. More recently, systems have been developed that do not require helper virus infection for AAV recovery; the necessary helper functions (i.e., adenoviruses E1, E2a, VA, and E4, or herpesviruses UL5, UL8, UL52, and UL29, as well as herpesvirus polymerases) are also supplied to the trans from this system. In these newer systems, the helper functions can be provided by transiently transforming the cells with constructs encoding the necessary helper functions; or the cells can be engineered to stably contain genes encoding helper functions that can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgenes and rep / cap genes flanked by the ITR are introduced into insect cells by infecting them with a baculovirus-based vector.For a general overview of these production systems, see, for example, Zhang et al., 2009, “Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production,” Human Gene Therapy, 20:922–929; the entire contents of each of these references are incorporated herein by reference. Methods for creating and utilizing these AAV production systems and other AAV production systems are also described in the following U.S. patent documents: U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065; These references are incorporated herein by reference in their entirety. Generally, for example, Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv.Biochem.Engin / Biotechnol.99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J.Gene Med. 10:717-733. See also; the entire contents of the references cited above are incorporated herein by reference. Methods used to construct any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation, and such methods include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, New York, USA, (2012). Similarly, methods for producing rAAV particles are also well known, and the selection of a preferred method is not limited to the present invention. See, for example, K. Fisher et al., (1993) J. Virol., 70:520-532; and U.S. Patent No. 5,478,745.

[0143] Optionally, the anxiolytic peptides or fusion polypeptides described herein may be introduced via viral vectors other than rAAV. For example, other viral vectors available herein include herpes simplex virus (HSV)-based vectors. HSV vectors deleting one or more pre-initial genes (IEs) are advantageous because they are generally non-cytotoxic and maintain a latent-like state in target cells, providing efficient transduction into target cells. Recombinant HSV vectors can accommodate approximately 30 kb of non-homologous nucleic acids.

[0144] Retroviruses (such as type C retrovirus and lentivirus) can also be used in this invention. For example, the retroviral vector may be based on mouse leukemia virus (MLV). See, for example, Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000; and Fong et al., Crit. Rev. Ther. Drug Carrier Syst., 17:1-60, 2000. MLV-based vectors may contain up to 8kb of non-homologous (therapeutic) DNA as a substitute for the viral gene. The non-homologous DNA may contain a tissue-specific promoter and nucleic acid encoding the anxiolytic peptide. In the method of introduction into nerve cells, the non-homologous DNA may also encode a ligand for a tissue-specific receptor.

[0145] Further retroviral vectors that can be used include vectors based on replication-deficient alphavirus vectors and vectors based on human immunodeficiency virus (HIV). See, for example, Vigna and Naldini, J. Gene Med., 5:308-316, 2000; and Miyoshi et al., J. Virol. 72:8150-8157, 1998. Lentiviral vectors have the advantage of being able to infect both actively dividing and non-dividing cells.

[0146] The lentiviral vector used in this application may be derived from human lentiviruses and non-human lentiviruses (including SIV). The lentiviral vector includes, for example, a nucleic acid sequence necessary for vector amplification and a tissue-specific promoter controllably linked to a nucleic acid encoding an anxiolytic peptide. The former may include a viral LTR, a primer binding site, a polypurine tract, an att site, and a capsid-forming site.

[0147] In some aspects, lentiviral vectors can be used. Lentiviruses have been shown to be transductionable into different types of central nervous system neurons (Azzouz et al., (2002) J Neurosci. 22:10302-12) and possess large-scale cloning capabilities, so they can be used in some embodiments.

[0148] Lentiviral vectors can be packaged in lentiviral capsids. Substituting one of the viral particle proteins with a protein from a different virus is called "pseudotyping." The vector capsid may contain viral envelope proteins derived from other viruses, including mouse leukemia virus (MLV) or varicella stomatitis virus (VSV). The use of VSV G-proteins results in higher vector titers and consequently, greater stability of the vector viral particles.

[0149] Alphavirus-based vectors (such as those created from Semryki Forest Fever virus (SFV) and Sindbis virus (SIN)) are also available for use in this invention. The use of alphaviruses is described in Lundstrom, K., Intervirology, 43:247-257, 2000; and Perri et al., Journal of Virology, 74:9802-9807, 2000.

[0150] Recombinant replication-deficient alphavirus vectors are advantageous because they can express non-homologous (therapeutic) genes at high levels and infect a wide range of target cells. Alphavirus replicons can target specific cell types by expressing functional non-homologous ligands on their viral particle surface, or by expressing binding domains that enable selective binding to target cells expressing homogeneous binding partners. Alphavirus replicons can remain latent in target cells and express non-homologous nucleic acids over long periods. The replicons can also transiently express non-homologous nucleic acids in the target cells.

[0151] Many viral vectors conforming to the method of this application may include two or more promoters so that two or more non-homologous genes can be expressed by the vector. Furthermore, the vector may include sequences encoding signal peptides or other portions that promote the expression of the anxiolytic peptide in target cells.

[0152] Hybrid viral vectors can be used to combine the advantageous properties of two viral vector systems when introducing nucleic acids encoding anxiolytic peptides into target neurons, cells, or tissues. Standard techniques for constructing hybrid vectors are well known to those skilled in the art. Such techniques are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, New York, USA; or in numerous laboratory manuals that consider recombinant DNA techniques. A double-stranded AAV genome in an adenovirus capsid containing a combination of AAV and adenovirus ITR may be used for cell transformation. Alternatively, the AAV vector may be inserted into a "gutless," "helper-dependent," or "high-capacity" adenovirus vector. Adenovirus / AAV hybrid vectors are described in Lieber et al., J. Virol., 73:9314-9324, 1999. Retrovirus / adenovirus hybrid vectors are described in Zheng et al., Nature Biotechnol. 18:176-186, 2000. The retroviral genome contained within the adenovirus can be integrated into the genome of target cells and stably express genes.

[0153] Other nucleotide sequence elements that facilitate the expression of the anxiolytic peptide and the cloning of the vector are also intended. For example, expression may be enhanced by the presence of an enhancer upstream of the promoter or a terminator downstream of the coding region.

[0154] In another embodiment, a tissue-specific promoter can be fused to a nucleotide encoding the anti-anxiety peptide described herein. By fusing such a tissue-specific promoter into an adenovirus construct, transgene expression is restricted to a specific tissue. The recombinant adenovirus system can be used to evaluate the degree of efficacy and specificity of gene expression provided by the tissue-specific promoter.

[0155] In addition to viral vector-based methods, non-viral methods can also be used to introduce nucleic acids encoding anxiolytic peptides into target cells. A review of non-viral gene transfer methods is available from Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. In one example of a non-viral gene transfer method according to this application, plasmid DNA is used to introduce nucleic acids encoding anxiolytic peptides into cells. Plasmid-based gene transfer methods are generally well known in the art.

[0156] Synthetic gene transfer molecules can be designed to form multiple molecular aggregates of plasmid DNA. These aggregates can be designed to bind to target cells. Cationic amphiphilic substances, including lipopolyamines and cationic lipids, can be used to provide receptor-independent nucleic acid transport into target cells.

[0157] Furthermore, pre-formed cationic liposomes or cationic lipids may be mixed with plasmid DNA to form a cell transformation complex. Reviews of methods involving cationic lipid preparations include Felgner et al., Ann. NYAcad. Sci. 772:126-139, 1995; and Lasic and Templeton, Adv. Drug Delivery Rev. 20:221-266, 1996. For gene transfer purposes, DNA may be coupled to an amphiphilic cationic peptide (Fominaya et al., J. Gene Med. 2:455-464, 2000).

[0158] In accordance with this application, methods can be used that include both virus-derived and non-virus-derived components. For example, plasmids derived from Epstein-Barr virus (EBV) for therapeutic gene delivery are described by Cui et al., Gene Therapy 8:1508-1513, 2001. Furthermore, a method involving adenovirus-bound DNA / ligand / polycationic additives is described by Curiel, DT, Nat.Immun.13:141-164, 1994.

[0159] Furthermore, by transforming target cells using electroporation technology, the nucleic acid encoding the anxiolytic peptide of the fusion polypeptide can be introduced into the target cells. Electroporation technology is well known and can be used to promote the transformation of cells by plasmid DNA.

[0160] The vector encoding the expression of the anxiolytic peptide can be introduced in vivo into target cells in the form of an injectable preparation containing, if necessary, a pharmaceutically acceptable carrier (such as saline). Other pharmaceutical carriers, formulations, and dosage forms can also be used in accordance with this application.

[0161] The anxiolytic peptide can be expressed in the target cell for any suitable duration (including transient expression and long-term stable expression).

[0162] Anxiolytics described herein, such as anti-asprosin antibodies, anxiolytic peptides, fusion polypeptides, or vectors, may be formulated with one or more pharmaceutically acceptable carriers or excipients for the purpose of providing them in pharmaceutical compositions. Anxiolytics described herein may be combined with a pharmaceutically acceptable buffer, a pH adjusted to obtain acceptable stability, and a pH acceptable with respect to administration (e.g., parenteral administration). Optionally, one or more pharmaceutically acceptable antimicrobial agents may be added. Metacresol and phenol are preferred pharmaceutically acceptable antimicrobial agents. One or more pharmaceutically acceptable salts may be added to adjust ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin is an example of an isotonic adjusting additive. A pharmaceutically acceptable means suitable for administration to humans or other animals is free from toxic elements and undesirable contaminants and does not inhibit the activity of the active compound contained therein.

[0163] In some embodiments, the anxiolytics described herein may be formulated as a solution formulation or as a lyophilized powder that can be reconstituted with a suitable diluent. Regardless of whether or not the reconstituted formulation has buffering capacity to maintain the pH of the solution during the intended post-opening shelf life, the lyophilized form is a stable form for the anxiolytic. Preferably, the solution containing the anxiolytic before lyophilization is substantially isotonic so that an isotonic solution can be formed after the reconstitution of the anxiolytics described herein.

[0164] The anxiolytics described herein may also be provided in pharmaceutically acceptable salt forms. Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Preferred acid addition salts are those formed by mineral acids such as hydrochloric acid and hydrobromic acid.

[0165] Examples of base addition salts include salts derived from inorganic bases such as ammonium or alkali metal or alkaline earth metal hydroxides, carbonates, and bicarbonates. Examples of bases useful for preparing such salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassium carbonate.

[0166] The anxiolytic can be delivered to the subject by any preferred route, such as topical and / or systemic administration. Systemic administration may include parenteral administration, such as intramuscular, intravenous, intra-articular, intra-arterial, subarachnoid, subcutaneous, or intraperitoneal administration. The drug can also be administered orally, transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation, or nasal spray) or rectally. In some embodiments, the anxiolytic can be administered to the subject by intravenous administration using an infusion pump for delivery in daily, weekly, or multiple doses.

[0167] A pharmaceutically acceptable formulation of the anxiolytic can be suspended in an aqueous carrier and introduced using a conventional subcutaneous injection needle or an infusion pump.

[0168] For injection, the anxiolytics described herein can be formulated in solutions, typically physiologically compatible buffers such as Hanks' solution or Ringer's solution. Furthermore, the anxiolytics may be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms are also included. The injection may be, for example, a bolus injection or a continuous infusion (e.g., using an infusion pump).

[0169] In the treatments disclosed herein, a therapeutically effective dose of the anxiolytic is administered to a subject receiving treatment for an anxiety disorder. The anxiety disorder may include one or more of the following: generalized anxiety disorder, phobias, social anxiety disorder, social phobia, panic disorder, panic attacks, post-traumatic stress disorder, separation anxiety disorder, selective mutism, agoraphobia, or anxiety disorders induced by a substance / therapeutic agent or condition.

[0170] In another embodiment, the subject experiences at least one type of anxiety symptom, wherein the at least one type of symptom includes emotional instability, palpitations, hyperventilation, excessive sweating, muscle spasms, weakness, fatigue, insomnia, nausea, repetitive behaviors, or any combination thereof. In one embodiment, the subject is diagnosed with depression alone. In another embodiment, the subject experiences at least one type of depressive symptom, wherein the at least one type of depressive symptom includes depressed mood, anhedonia, low energy levels, guilt, psychomotor stagnation, tremors, suicidal ideation, difficulty concentrating and indecisiveness, or any combination thereof.

[0171] In some embodiments, the subject has an anxiety disorder but does not have any of the following conditions: ADHD, ODD, conduct disorder, autism, Tourette syndrome, phobias, obsessive-compulsive disorder (OCD), anger management difficulties, destructive behavior symptoms, dermatoprophagia, developmental disorders, mood disorders, motor disorders, or depression; In other embodiments, the subject has an anxiety disorder and at least one of the following: ADHD, ODD, autism, conduct disorder, Tourette syndrome, phobia, obsessive-compulsive disorder (OCD), anger management difficulties, destructive behavioral symptoms, dermatopruritic disorder, developmental disorder, mood disorder, motor disorder, or depression.

[0172] In each treatment described herein, the anxiety disorder may be generalized anxiety disorder, phobia, social anxiety disorder, social phobia, panic disorder, panic attacks, post-traumatic stress disorder, separation anxiety disorder, selective mutism, agoraphobia, or an anxiety disorder induced by a substance / therapeutic agent or another medical condition. In each treatment described herein, the subject may have an anxiety disorder but not have one or more neuropsychological abnormalities such as ADHD, ODD, conduct disorder, autism, Tourette syndrome, phobia, obsessive-compulsive disorder (OCD), anger management difficulties, destructive behavior symptoms, dermatologic picking disorder, developmental disorders, mood disorders, motor disorders, and depression.

[0173] In one embodiment, the method reduces the anxiety of the subject. In one embodiment, anxiety is assessed using the State-Trait Anxiety Inventory (STAI), Fear Rating Scale, Beck Anxiety Rating Scale (BAI), Abbreviated Fear of Negative Rating Scale-BFNE, Clinical Diagnostic Interview Scale for PTSD (CAPS), Daily Assessment of Symptoms-Anxiety, Generalized Anxiety Disorder 7 (GAD-7), Hamilton's Anxiety Rating Scale (HAM-A), Hospital Anxiety and Depression Rating Scale (HADS-A), Leibovitz Social Anxiety Scale (LSAS), Generalized Anxiety Severity and Functional Impairment Scale (OASIS), Panic and Agoraphobia Rating Scale (PAS), Panic Disorder Severity Rating Scale (PDSS), PTSD Severity Rating Scale-Self-Report, Social Anxiety Disorder Rating Scale (SPIN), Trauma Screening Questionnaire, Yale-Brown Obsessive-Compulsive Scale (Y-BOCS), or Zung's Self-Rating Depression Scale. For example, anxiety is reduced by at least 1 unit.

[0174] The present invention will be further illustrated by the following examples, but this is not intended to limit the scope of the claims.

[0175] Examples Mouse model Mice were housed in a cabinet with a small ventilation system in an animal facility maintained at 20-25°C and 40-60% humidity, using a 12-hour light cycle (6 AM to 6 PM). Mice were continuously fed with water and standard solid feed. Animal housing, management, experiments, and euthanasia were carried out according to an animal protocol approved by the Case Western Reserve University Animal Research Board (protocol number 2018-0042). The overall health of the mice was monitored by the CWRU Animal Resource Center.

[0176] Wild-type mouse Wild-type C57BL / 6 mice (WT mice; Jackson Laboratory, JAX#:000664) with diet-induced obesity (DIO mice) Diet-induced obesity mice were created by feeding 4-week-old C57BL / 6 mice a high-fat diet (HFD). These mice experienced rapid weight gain and decreased glucose tolerance. Ultimately, they developed type 2 diabetes and metabolic syndrome. By 6 months, these mice were nearly 50g overweight compared to identical age and sex control mice, which typically weighed 28-30g. This is a commonly used non-genetic obesity model.

[0177] NPS mouse strain In 2016, at the Baylor College of Medicine Mouse ES Cell Core Facility, Fbn1 was created using the Crispr-Cas9 approach. NPS / + A mouse strain was created. This mouse was re-established embryonically at Case Western Reserve University and has been maintained in-house since 2016. For all mouse strains, littermates from in-house mating were used.

[0178] PTPRD whole body KO In this study, Ptprd mice were used to maintain a heterozygous state. B6;129-Ptprd <tm1yiw>The mice were purchased from the RIKEN BioResource Research Center.

[0179] AgRP Ptprd knockout series Homozygous phloxed mice that function under specific conditions (Ptprd tm2c(KOMP)Wtsi) were crossed with AgRP-IRES-Cre (C57BL / 6-Agrptm1(cre)Lowl); the objective was to achieve AgRP neuron-specific Ptprd knockout (AgRPcre;Ptprd Flox / Flox The task was to create ).

[0180] Pcp2 Ptprd knockout series Homozygous Ptprd-floxed mice that function under specific conditions (Ptprd tm2c(KOMP)Wtsi) were crossed with Pcp2-cre mice; the purpose was to achieve Purkine neuron-specific Ptprd knockout (Pcp2-cre; Ptprd Flox / Flox The task was to create ).

[0181] Preparation and injection of anti-asprosin antibodies The study was conducted using mouse mAb(M1). Using conventional hybridoma technology, M1 was created by immunizing mice with a 28-amino acid peptide located near the C-terminus of asprosin.

[0182] To evaluate the effects of the mAb on anxiety, 150 μg / mouse (approximately 3-4 mg / kg) of the mAb (or control IgG) was administered intraperitoneally to 6-month-old DIO or non-obese mice using 200 μl of US Pharmacopeia-grade saline; repeated administrations were performed for up to 30 days. Injections were administered between 9:00 AM and 10:00 AM, and behavioral tests were conducted approximately 4-5 hours after injection.

[0183] Viral vector Adenovirus (Ad5) For the purpose of evaluating the overexpression of asprosin in anxiety, adenovirus (Ad5) dissolved in 150 μl of United States Pharmacopeia grade sterile physiological saline was intravenously injected into 12-week-old C57Bl / 6J mice via the tail vein. As a control for the experimental mice injected with Ad5-FBN1 virus (3.6 × 10 9 pfu / mouse) containing the coding region of human FBN1 under the control of the CMV promoter, mice injected with empty Ad5 (3.6 × 10 9 pfu / mouse) were used.

[0184] Adeno-associated virus serotype 8 (AAV8) For the purpose of evaluating the overexpression of asprosin in anxiety, adeno-associated virus serotype 8 (AAV8) dissolved in 150 μl of United States Pharmacopeia grade sterile physiological saline was intravenously injected into 12-week-old C57Bl / 6J via the tail vein. As a control for the experimental mice injected with AAV8-IL2-asprosin (1 × 10 12 GC / mouse) containing the coding region of human asprosin with a his tag added to the N-terminus following the IL2 signal peptide under the control of the EF1 promoter, mice injected with empty AAV8 (1 × 1012 GC / mouse) were used. For the purpose of evaluating weight gain as an indicator of asprosin overexpression, body weight measurement was started 4 weeks after injection and performed weekly.

[0185] Injection of 7BIA 50 μg of 7-BIA (7-butoxyildaric acid analog) dissolved in dimethyl sulfoxide (DMSO) saline, or the same vehicle, was intraperitoneally injected into 4-month-old mice daily.

[0186] DMSO saline The total injection volume was 100 μL. Mice were injected for 7 consecutive days. Body weight was measured on the 1st and 7th days. On the 4th day, an open field test was performed. On the 5th day, a light-dark test, and on the 6th day, an elevated plus maze test was performed.

[0187] Serum asprosin ELISA To quantify serum asprosin levels in non-obese mice induced with induced anxiety, we used a previously reported custom sandwich ELISA. In this assay, asprosin was captured in 25 μL of plasma containing asprosin using a fully human anti-asprosin monoclonal antibody. This antibody was obtained from a naive human phage display antibody library by panning against recombinant full-length human asprosin (Texas Therapeutic Research Institute, University of Texas Health Science Center, Houston). The mouse anti-asprosin monoclonal antibody used as the capture antibody targeted amino acids 106-134 of human asprosin (corresponding to amino acids 2838-2865 of human profibrillin). An HRP-labeled anti-mouse secondary antibody was used to obtain the signal. A standard curve was created using recombinant asprosin produced by mammalian cells (AdipoGen AG-40B-0174T-C010). The necessary materials for blocking, coating, substrate, and stop solution were obtained from SeraCare.

[0188] Induced Anxiety Model Electric Foot Shock Model All animals were brought into the laboratory 30 minutes before training. Each mouse was individually placed in a chamber with a grid floor connected to a shock generator. After being placed in the chamber, each mouse was randomly given five 1-second foot shocks (0.6 mA) within a 120-second period; this was repeated daily for 13 days. Foot shocks were administered in the morning (9-11 am). In the control group, mice were placed in the chambers at the same time, but no foot shocks were administered. After training, the mice were returned to their home cages. The training chambers were cleaned with 70% ethanol before and after each experiment.

[0189] Scent stress of predators All animals were brought into the laboratory 30 minutes before training. For one hour each day, from 9:00 AM to 10:00 AM, mice were individually placed in a chamber containing a cage with a dirty rat bedding. In the control group, mice were placed in a clean mouse cage with a clean bedding for the same amount of time. After training, the mice were returned to their home cages. This was repeated for 10 consecutive days.

[0190] Behavioral assay Open field An open-field test, a method commonly used to evaluate the exploratory behavior and overall activity of animals, was conducted in a soundproofed, darkened room. Mice were placed in the center of a white plastic open-field arena (50cm x 50cm x 50cm) and allowed to explore freely for 15 minutes. An overhead video camera was used to monitor the movement of each animal, and the data was recorded using computer software (Any-maze video tracking software) that tracked the total distance traveled and the time spent in the center of the chamber compared to the areas at the edges.

[0191] The tracking device used infrared lasers and sensors to determine the mouse's center of gravity. The longer the mouse spent in the edge of the box, and the shorter the time spent in the center, the more anxious the behavior was interpreted as. Before each test, the mice were acclimatized to the environment for 30 minutes; the arena was cleaned with 70% ethanol after each test.

[0192] elevated cross maze A four-arm maze, characterized by two open arms without walls and two closed arms with walls (25 cm long, 5 cm wide), was used. Mice were placed in the center of the maze, which was 60 cm above the floor. At the start of the experiment, the mice were positioned in the center, facing the open arms. Data recorded by a video camera was processed using video tracking software Any-maze, and the ratio of time spent in the closed arms to time spent in the open arms was tracked for each mouse during a 5-minute session. It was inferred that longer time spent in the open arms compared to the closed arms indicated increased anxiety. Longer time spent in the open arms compared to the closed arms indicates stronger anxiety. Before the experiment, the mice were acclimatized to the space for 30 minutes; the arena was cleaned with 70% ethanol after each experiment.

[0193] Light and darkness Mice were placed in a two-chamber device connected by a small opening, consisting of one brightly lit section and another dark section. Mice were allowed to freely explore both chambers, and the time spent in each chamber over a 5-minute period was recorded. An increase in time spent in the dark section was interpreted as an increase in anxiety-like behavior, while a decrease in that time suggested a decrease in anxiety levels. A longer time spent in the dark section indicated increased anxiety. Mice were acclimatized to the environment for 30 minutes before the experiment; the arena was cleaned with 70% ethanol after each experiment.

[0194] result Figure 1 is a schematic diagram of the open-field assay used to evaluate anxiety-like behavior. Figure 2 is a schematic diagram of the elevated cusp maze assay used to evaluate anxiety-like behavior. Figure 3 is a schematic diagram of the light-dark assay used to evaluate anxiety-like behavior. Figure 4 is a graph showing that, when determined using the assays in Figures 1-3, genetic knockout of asprosin leads to a reduction in anxiety in an animal model of neonatal progeria syndrome. Figure 5 is a graph showing that anxiety is reduced by knocking out the asprosin receptor (Ptprd) gene, as determined using an elevated cross maze. Figure 6 is a graph showing that pharmacological neutralization of asprosin receptors leads to anxiety reduction. Figure 7 is a schematic diagram of an assay used to evaluate the asprosin-neutralizing effect of anti-asprosin antibodies used to treat anxiety in an animal model. Figure 8 is a plot showing that asprosin neutralization by anti-asprosin antibodies administered to mice leads to weight loss. Figure 9 is a graph showing that, when measured using the assays in Figures 1-3, asprosin neutralization by anti-asprosin antibodies administered to mice does not cause any changes in locomotion or general movement. Figure 10 is a graph showing that, when measured using the assays in Figures 1-3, asprosin neutralization by anti-asprosin antibodies administered to diet-induced obesity (DIO) mice leads to a reduction in anxiety. Figure 11 shows graphs and plots demonstrating that asprosin neutralization by anti-asprosin antibodies administered to non-obese mice did not result in any difference in weight loss or locomotion. Figure 12 is a graph showing that, when measured using the assays in Figures 1-3, asprosin neutralization by anti-asprosin antibodies administered to non-obese mice leads to a reduction in anxiety. Figure 13 is a graph showing that overexpression of asprosin causes increased anxiety in mice. Figure 14 shows plots and graphs illustrating that asprosin overexpression leads to increased anxiety, as measured by body weight and the assays shown in Figures 1-3. Figure 15 is a schematic diagram illustrating the direct relationship between asprosin and anxiety in mice, using five different methods of asprosin manipulation. Figure 16 shows a schematic diagram of the foot shock model that induces anxiety. Figure 17 shows that asprosin neutralization by an anti-asprosin antibody administered to a mouse model of induced anxiety reduces anxiety. Figure 18 is a graph showing that anxiety is reduced in a mouse-induced anxiety model by asprosin neutralization of anti-asprosin antibodies administered using the assays shown in Figures 1-3. Figure 19 is a graph showing that serum asprosin levels increase when anxiety is induced. Figure 20 shows a schematic diagram of the predator odor stress (PSS) assay used to induce anxiety. Figure 21 shows a schematic diagram of mice that undergo the assay shown in Figure 20, administered with anti-asprosin antibodies at various time points, and then tested using the assays shown in Figures 1-3. Figure 22 is a graph showing that anxiety is reduced in PSS mice by asprosin neutralization of anti-asprosin antibodies administered to them, as measured using the assays in Figures 1-3. Figure 23 is a graph showing that serum asprosin levels increase when anxiety is induced with PSS. Figure 24 is a graph showing that asprosin does not mediate anxiety through AgRP neurons. Figure 25 is an image showing that Ptprd is highly expressed in the cerebellum. Figure 26 is a graph showing that asprosin does not mediate anxiety through Purkinje neurons. Figure 27 is a graph showing the potential of asprosin to mediate anxiety through the amygdala. Figure 28 shows images of the cerebellum of subjects pretreated with asprosin and GFP.

[0195] Those skilled in the art will be able to conceive of improvements, changes, and modifications from the above description of the present invention. Such improvements, changes, and modifications within the scope of the art are intended to be included in the attached "Claims." All references, publications, and patents cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for treating anxiety disorders in individuals requiring treatment; The method is: This includes administering to the subject a therapeutically effective dose of an anxiolytic that inhibits the signaling or activity of asprosin-mediated protein tyrosine phosphatase receptor δ (PTPRD), method.

2. The method according to claim 1; Herein, the anxiolytic comprises at least one of a small molecule, nucleic acid, peptide, protein, or antibody that inhibits asprosin binding to PTPRD or PTPRD signaling or activity. method.

3. The method according to claim 1 or 2; Here, the anxiolytic comprises an antibody or antigen-binding fragment that specifically binds to a peptide having the amino acid sequence KKKELNQLEDRYDKDYLSGELGDNLKMK (SEQ ID NO: 1), method.

4. The method according to claim 3; Here, the antibody or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region containing three heavy chain CDRs and / or three light chain CDRs of an antibody produced by a hybridoma cell line deposited in the American Type Culture Collection as accession number ATCC PTA-123085, method.

5. The method according to claim 3 or 4; Here, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. method.

6. The method according to any one of claims 3 to 5; Here, the antibody or its antigen-binding fragment is a monospecific or bispecific antibody or its antigen-binding fragment. method.

7. The method according to claim 1 or 2; Here, the anxiolytic is an anxiolytic peptide containing an amino acid sequence substantially identical to the extracellular portion of the amino acid sequence of PTPRD that binds to asprosin. method.

8. The method according to claim 7; Here is the amino acid sequence of the anti-anxiety peptide: Re-enactment: For 2 consecutive amino acids of 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, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least 1000 amino acids, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 7 4%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. method.

9. The method according to claim 7; Here, the amino acid sequence of the anti-anxiety peptide is as follows, relative to SEQ ID NO: 2: At least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical. method.

10. The method according to claim 7; Here is the amino acid sequence of the anti-anxiety peptide: Sequence number: For three consecutive amino acids of 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, at least about 100, at least about 150, and at least about 200, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, and at least about 78% at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical. method.

11. The method according to any one of claims 8 to 10; Here, the KD of the binding affinity of the anti-anxiety peptide to asprosin is less than approximately 10 μM, less than approximately 1 μM, less than approximately 500 nM, less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, less than approximately 100 nM, less than approximately 10 nM, less than approximately 1 nM, or less than approximately 500 pM. method.

12. The method according to any one of claims 8 to 10; Here, the anti-anxiety peptide is linked to a non-homologous polypeptide. method.

13. The method according to claim 12; Here, the at least one non-homologous polypeptide comprises an antibody or its antigen-binding fragment, a glucagon-like peptide 1 receptor (GLP-1R) agonist, an Fc portion of an immunoglobulin, an albumin peptide, an albumin-binding domain (ABD), a peptide linker, a signal peptide, or a combination thereof. method.

14. A method according to any one of claims 1 to 13; Here, the anxiety disorder is one or more of the following: generalized anxiety disorder, phobia, social anxiety disorder, social phobia, panic disorder, panic attack, post-traumatic stress disorder, separation anxiety disorder, selective mutism, agoraphobia, or anxiety disorders or conditions induced by substances / medicines. method.

15. A method according to any one of claims 1 to 16; Here, the subject suffers from an anxiety disorder and also suffers from one or more of the following: attention deficit hyperactivity disorder (ADHD), oppositional conduct disorder (ODD), conduct disorder, Tourette syndrome, phobias, obsessive-compulsive disorder (OCD), difficulty controlling anger, destructive behavior, skin picking disorder, developmental disorders, mood disorders, motor disorders, or depression. method.

16. An anxiolytic containing an antibody or antigen-binding fragment that specifically binds to a peptide having the amino acid sequence KKKELNQLEDRYDKDYLSGELGDNLKMK (SEQ ID NO: 1).

17. An anxiolytic according to claim 16; Here, the antibody or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region containing three heavy chain CDRs and / or three light chain CDRs of an antibody produced by a hybridoma cell line deposited in the American Type Culture Collection as accession number ATCC PTA-123085, Anxiolytic.

18. An anxiolytic according to claim 16 or 17; Here, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. Anxiolytic.

19. An anxiolytic according to any one of claims 16 to 18; Here, the antibody or its antigen-binding fragment is a monospecific or bispecific antibody or its antigen-binding fragment. Anxiolytic.

20. An anxiolytic containing an anxiolytic peptide having an amino acid sequence substantially identical to the extracellular portion of the amino acid sequence of PTPRD that binds to asprosin.

21. An anxiolytic according to claim 20; Here is the amino acid sequence of the anti-anxiety peptide: Re-enactment: For 2 consecutive amino acids of 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, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least 1000 amino acids, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 7 4%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. Anxiolytic.

22. An anxiolytic according to claim 21; Here, the amino acid sequence of the anti-anxiety peptide is as follows, relative to SEQ ID NO: 2: At least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical. Anxiolytic.

23. An anxiolytic according to claim 22; Here is the amino acid sequence of the anti-anxiety peptide: Sequence number: For three consecutive amino acids of 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, at least about 100, at least about 150, and at least about 200, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, and at least about 78% at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical. Anxiolytic.

24. An anxiolytic according to any one of claims 20 to 23; Here, the KD of the binding affinity of the anti-anxiety peptide to asprosin is less than approximately 10 μM, less than approximately 1 μM, less than approximately 500 nM, less than approximately 400 nM, less than approximately 300 nM, less than approximately 200 nM, less than approximately 100 nM, less than approximately 10 nM, less than approximately 1 nM, or less than approximately 500 pM. Anxiolytic.

25. An anxiolytic according to any one of claims 20 to 24; Here, the therapeutic peptide is linked to a non-homologous polypeptide. Anxiolytic.

26. An anxiolytic according to claim 25; Here, the at least one non-homologous polypeptide comprises an antibody or its antigen-binding fragment, a glucagon-like peptide 1 receptor (GLP-1R) agonist, an Fc portion of an immunoglobulin, an albumin peptide, an albumin-binding domain (ABD), a peptide linker, a signal peptide, or a combination thereof. Anxiolytic.

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