Protein tyrosine phosphatase wedge domain peptide dimers for nervous system repair

Abstract

The present disclosure relates to compositions and methods for nervous system repair. The composition comprises a peptide which is a dimer comprising a first monomer covalently crosslinked with a second monomer, each monomer comprising a domain comprising an amino acid sequence derived from a cytoplasmic wedge domain of receptor protein tyrosine phosphatase (PTPR). The present disclosure also relates to methods and compositions for treating diseases, disorders and / or conditions associated with inhibition of chondroitin sulfate proteoglycans (CSPG) on neural system repair or repression of LAR family phosphatase enzymes, such as PTPRD, PTPRF and PTPRS, on neural repair.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 493,912, filed April 3, 2023, the entire contents of which are hereby incorporated by reference.

[0003] References to sequence lists

[0004] This application contains a sequence list, which is submitted electronically in XML format and hereby incorporated in its entirety by reference. The XML copy was created on March 21, 2024, is named 753221 NGT-001PC.xml, and has a size of 17,882 bytes. Background Technology

[0005] Spinal cord injury and other conditions associated with damage to the central nervous system (CNS) or peripheral nervous system (PNS) can lead to permanent disability or loss of motor, sensory, and / or cognitive function. Limited recovery after CNS injury has fueled considerable interest in potential strategies to overcome this challenge. A fundamental obstacle to efforts to improve neurological function after injury is the non-regenerative nature of the adult CNS.

[0006] Two well-known classes of regeneration inhibitors are myelin-related inhibitors (e.g., MAG, Nogo, and OMGP) and inhibitors in scar tissue formed by glial cells at the site of injury (e.g., chondroitin sulfate proteoglycans (CSPG)). CSPG deposition, which leads to the inhibition of axonal and synaptic repair at sites of neurological injury, has been considered not only a key contributing factor to the pathogenesis of traumatic CNS injuries but also a key contributing factor to the progression of various neurodegenerative and neuroinflammatory diseases.

[0007] CSPGs impair axonal regeneration through several inhibitory mechanisms. The inhibitory effect of CSPGs is manifested not only in the formation of non-regenerating, malnourished axonal retraction spheres through damage, but also in the limited collateral sprouting capacity of remaining fibers. Although sulfated proteoglycans have been recognized over the past two decades as a major contributing factor to the repulsive nature of glial scars, the precise inhibitory mechanisms remain poorly understood.

[0008] Protein tyrosine phosphatase (PTP) plays an important role in dephosphorylation (the process of removing phosphoryl groups from proteins containing phosphotyrosine) (Jing-Fei Huang, Molecular Biology and Evolution(Volume 20, Issue 5, May 2003, pp. 815–820). Receptor-type protein tyrosine phosphatases (PTPRs) are a subgroup of PTPs, sharing transmembrane domains and therefore exhibiting similarities in function and target specificity (Du Y, Grandis JR). Chin J Cancer. 2015;34(2):61-69).

[0009] The leukocyte common antigen-associated (LAR) subfamily PTPR consists of three members: LAR (PTPRF), receptor protein tyrosine phosphatase σ (PTPRS), and receptor protein tyrosine phosphatase δ (PTPD). PTPRS and PTPF have been identified as receptors for CSPGs, which are major inhibitory components of glial scarring and the perineurial network. The glycosidic side chains of CSPGs can bind to PTPRF and PTPRS expressed by cells such as neurons, and inhibit neuronal growth, plasticity, regeneration, and germination failure.

[0010] It has been found that PTPRF-deficient neurons exhibit reduced sensitivity to CSPG-mediated inhibition in various cell-based assays and show increased regeneration after nerve injury (such as spinal cord injury and optic nerve compression). The results of PTPRF knockout are inconclusive, with both increased and decreased regenerative phenotypes observed. Since CSPG is a major barrier to regeneration and plasticity in the injured adult nervous system, regulators of LAR family protein tyrosine phosphatase function could serve as therapeutic agents to promote neural plasticity, regeneration, and ultimately repair of damaged nervous systems.

[0011] In light of the above, there remains an urgent need for compositions that modulate and attenuate the function of inhibitory CSPGs. Furthermore, there remains a need for compositions that can mitigate CSPG-induced cellular and neural dysfunction associated with the function of LAR family protein tyrosine phosphatases. Summary of the Invention

[0012] This disclosure provides wedge-domain peptide dimers for the repair and treatment of neurological deficits related to the function of LAR family protein tyrosine phosphatases. Therefore, wedge-domain peptide dimers can be used to treat diseases, conditions, and / or symptoms associated with CSPG-mediated inhibition of neurological repair.

[0013] Wedge-shaped domain peptide dimers can also be used to treat related diseases and conditions in subjects who need them.

[0014] In one aspect, this application relates to a pharmaceutical composition comprising a peptide, wherein the peptide is a dimer comprising a first monomer covalently cross-linked with a second monomer, each monomer comprising a domain containing an amino acid sequence of a cytoplasmic wedge domain derived from a receptor-type protein tyrosine phosphatase (PTPR); and wherein the mass ratio of the cross-linked dimer to the free monomer in the pharmaceutical composition is greater than 1:20.

[0015] In one aspect, this application relates to a pharmaceutical composition comprising a peptide dimer, wherein the peptide dimer comprises an amino acid sequence containing two monomeric subunits, each monomeric subunit comprising a peptide domain comprising an amino acid sequence independently selected from a group consisting of a PTPRF wedge domain, a PTPRD wedge domain, and a PTPRS wedge domain, and variants having at least 70% identity with them; and wherein the first monomeric subunit is bound to the second monomeric subunit.

[0016] On the other hand, this article provides a method for treating neurological symptoms, diseases, or conditions selected from the group consisting of nerve injury, inflammatory or autoimmune diseases of the nervous system, and neurodegenerative diseases in a subject in need, the method comprising administering to the subject an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises a peptide dimer or a pharmaceutically acceptable salt or solvate thereof, wherein the peptide domain comprises an amino acid sequence independently selected from the group consisting of a PTPRF wedge domain, a PTPRD wedge domain, and a PTPRS wedge domain, and variants having at least 70% identity with them.

[0017] In one aspect, this application relates to the use of a peptide dimer or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for treating a neurological symptom, disease, or condition selected from the group consisting of nerve injury, inflammatory or autoimmune diseases of the nervous system, and neurodegenerative diseases, wherein the peptide dimer comprises a peptide domain independently selected from the wedge domain of leukocyte antigen-associated (LAR) family phosphatases or a variant thereof having at least 70% homology with it.

[0018] In one aspect, this application relates to a method for preparing a peptide dimer or a pharmaceutically acceptable salt or solvate thereof, the method comprising mixing a first monomer and a second monomer in water, and a) adding an oxidant and / or b) oxygenating the solution to form a peptide dimer, wherein each peptide monomer comprises a peptide domain, a transport moiety, and a cysteine ​​residue or a peptide linker comprising a cysteine ​​residue, independently selected from a leukocyte antigen-associated (LAR) family phosphatase wedge domain or a variant having at least 65% homology therewith, and connecting the peptide domain and the transport moiety.

[0019] In another aspect, this application relates to a method for preparing the peptide dimer provided herein or a pharmaceutically acceptable salt or solvate thereof, wherein the method comprises mixing identical peptide monomers in a solvent containing copper sulfate to allow the formation of a non-covalent bond between the two peptide monomers, wherein each peptide monomer comprises a peptide domain, a transport moiety, and a cysteine ​​residue or a peptide linker comprising a cysteine ​​residue, independently selected from a leukocyte antigen-associated (LAR) family phosphatase wedge domain or a variant having at least 70% homology thereto, and connecting the peptide domain and the transport moiety. Attached Figure Description

[0020] The accompanying drawings, which are incorporated herein and form part of this specification, illustrate exemplary embodiments of the present disclosure and, together with the general description given above and the detailed description given below, serve to explain the features of the invention.

[0021] Figure 1 This is a chromatogram of purified acetate of compound 8 (TAT-Cys-PTPRS wedge-shaped domain dimer) obtained by oxidative dimerization, using copper sulfate as the oxidant.

[0022] Figure 2 The BBB scores of rats receiving compound 8 on day 7 after SCI were plotted in graph form compared to those receiving saline, as detailed in Example 3.

[0023] Figure 3 The BBB scores of rats receiving compound 8 at week 7 after SCI were plotted in graph form compared to those receiving saline, as detailed in Example 3.

[0024] Figure 4 The BBB scores of rats receiving compound 8 at week 12 after SCI were plotted in graphical form compared to those receiving saline, as detailed in Example 3.

[0025] Figure 5 The estimated weekly mean of urinary retention in SCI rats treated with saline solution of compound 8 and SCI rats treated with the mediator control (saline) during the study is depicted in graphical form.

[0026] Figure 6 The recovery of occasional walking in SCI rats treated with compound 8 was depicted in graphical form compared with that in SCI rats treated with the mediator control.

[0027] Figure 7 The recovery of frequent walking in SCI rats treated with compound 8 was depicted in graphical form compared with that in SCI rats treated with the mediator control.

[0028] Figure 8AThe effects of compound 8 compared to compound 4 and the mediator control in improving the recovery of hind limb interdigital space, paw position, trunk stability, and tail position in SCI rats are illustrated graphically, as measured by BBB subscale scores. Data are presented as mean ± SEM. N = 12–13 / group.

[0029] Figure 8B Weekly BBB subscores in SCI rats treated with compound 8 were plotted graphically compared to those treated with compound 4 and saline. Data are presented as the distribution of weekly mean BBB subscores. N = 12–13 / group.

[0030] Figure 9 The percentage of SCI rats in the test group treated with compound 8 achieving a BBB sub-score of 1 or better (higher) is depicted in a graph compared with the control group of SCI rats treated with saline alone and the group of SCI rats treated with compound 4.

[0031] Figure 10 The pharmacokinetic characteristics of compound 7 in rat plasma after intravenous or subcutaneous administration at doses of 1.75 or 10.5 mg / kg are depicted in graphical form.

[0032] Figure 11 The weekly BBB scores of SCI rats in the group treated with compound 8 compared to those treated with compound 4 and saline are depicted in graphical form according to embodiments of the present disclosure.

[0033] Figure 12 The percentage of SCI rats with a BBB score of 10 or higher in the groups treated with compound 8, compound 4, and saline alone, according to embodiments of the present disclosure, is depicted in graphical form.

[0034] Figure 13 The percentage of SCI rats with a BBB score of 11 in the groups treated with compound 8, compound 4, and saline alone, according to embodiments of the present disclosure, is depicted in graphical form.

[0035] Figure 14 The mean weekly bladder scores of rat groups treated with compound 8, compound 4, and saline alone, according to embodiments of this disclosure, are depicted in graphical form.

[0036] Figure 15 The percentage of SCI rats with a weekly bladder score of 2 or lower (better) is depicted in graph form in the rat groups treated with compound 8, compound 4 and saline alone, according to embodiments of the present disclosure.

[0037] Figure 16The percentage of rats that achieved a BBB score of 10 or higher and / or a bladder score of 2 or lower at the end of the study disclosed in the embodiments of this application is depicted in graphical form.

[0038] Figure 17 The percentage of rats that achieved a BBB score of 11 and / or a bladder score of 2 or lower at the end of the study, according to embodiments of this disclosure, is depicted in graphical form.

[0039] Figure 18 The stability of compound 7 in physiological buffer (HBSS) compared to compound 3 according to embodiments of the present disclosure is illustrated in graphical form.

[0040] Figure 19 , Figure 20 and Figure 21 The stability of compound 7 in rat plasma, dog plasma, and human plasma, compared to compound 3, is illustrated in graphical form according to embodiments of the present disclosure.

[0041] Figure 22 These are a set of transmission electron microscopy images according to embodiments of the present disclosure, illustrating different self-assembly modes of compounds 7 and 3 in water and isotonic saline. Detailed Implementation

[0042] Embodiments of this disclosure relate to peptide dimers for repairing the nervous system of a subject and compositions comprising peptide dimers.

[0043] Embodiments of this disclosure also provide pharmaceutical compositions comprising the peptide dimers disclosed herein, and methods for repairing the nervous system of a subject using the peptide dimers and / or pharmaceutical compositions disclosed herein.

[0044] In one embodiment, the pharmaceutical composition provided herein further comprises a pharmaceutically acceptable carrier.

[0045] Embodiments of this disclosure also provide methods for treating diseases, conditions and / or symptoms associated with activation and signaling of LAR family phosphatases, said methods comprising administering the disclosed peptide dimer and / or pharmaceutical composition to a subject in need.

[0046] definition

[0047] The following lists definitions of various terms used to describe the compounds and compositions disclosed herein. Unless otherwise limited individually or as part of a larger group in particular cases, these definitions apply to the terminology used in this specification and claims.

[0048] Unless otherwise defined, all scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms. Generally, the nomenclature used in conjunction with the cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization techniques described herein, as well as the techniques used in these fields, are well-known and commonly used in the art.

[0049] As used in this article, "one or more of a, b, and c" means a, b, c, ab, ac, bc, or abc. The use of "or" in this article is inclusive.

[0050] Furthermore, the use of the term "including" and other forms such as "include", "includes", and "included" is not restrictive.

[0051] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent depending on the context of its use. As used herein, when referring to measurable values ​​(such as quantity, duration, etc.), the term “about” is intended to cover variations of ±20% or ±10% relative to a specified value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0052] Chemical

[0053] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon. For example, an alkyl group can have 1 to 12 carbon atoms (i.e., (C1-C1)). 12 Alkyl groups are alkyl groups with 1 to 6 carbon atoms (i.e., (C1-C6)alkyl), 1 to 4 carbon atoms (i.e., (C1-C4)alkyl), or 1 to 3 carbon atoms (i.e., (C1-C3)alkyl). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (... n -Pr, n-propyl, -CH2CH2CH3), isopropyl ( i -Pr, isopropyl, -CH(CH3)2), 1 - Butyl ( n- bu, n-butyl, -CH2CH2CH2CH3), 2-butyl ( s- bu, sec-butyl, -CH(CH3)CH2CH3), tert-butyl ( t-bu, tert-butyl, -CH(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), neopentyl (-CH2C(CH3)3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), heptyl (-(CH2)6CH3), octyl (-(CH2)7CH3), 2,2,4-trimethylpentyl (-CH2C(CH3)2CH2CH(CH3)2), nonyl (-(CH2)8CH3), decyl (-(CH2)9CH3), undecyl (-(CH2) 10 CH3) and dodecyl (-(CH2)) 11 CH3). In one implementation, alkyl refers to C (1-6) Alkyl. In another embodiment, alkyl refers to C10. (1-4) Alkyl. In another embodiment, alkyl refers to C10. (1-3) alkyl.

[0054] As used herein, the term "alkylene" refers to a divalent alkyl group. For example, an alkylene group can have 1 to 12 carbon atoms (i.e., (C1-C1)). 12 Alkylenes (C1-C6), alkylenes (C1-C2), or alkylenes (C1-C6), having 1 to 6 carbon atoms. Examples of alkylenes include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), and n-butylene (-CH2CH2CH2CH2-).

[0055] Nucleic acid

[0056] As used herein, the terms “polynucleotide sequence” and “nucleotide sequence” may be used interchangeably.

[0057] As used herein, the term "wild-type" refers to naturally occurring polynucleotide sequences, or protein sequences, or portions thereof, that encode proteins or portions thereof, as they are commonly found in the body. As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, RNA or DNA analogs prepared from nucleotide analogs, and, where applicable to the described embodiments, single-stranded (sense or antisense) and double-stranded polynucleotides.

[0058] As used herein, the term "recombination" refers to a protein derived from a prokaryotic or eukaryotic expression system. As used herein, the term "recombination" refers to genetic material formed through a gene recombination process. "Recombinant protein" is produced through genetic engineering. Recombinant proteins are encoded by artificially generated DNA sequences. Recombinant proteins are proteins encoded by recombinant nucleic acid sequences. Recombinant nucleic acid sequences have sequences from two or more sources incorporated into a single molecule.

[0059] As used herein, the term "expression cassette" refers to a portion of vector DNA used for cloning and transformation. In each successful transformation, the expression cassette directs the cellular machinery to produce a polypeptide. Some expression cassettes are designed for modular cloning of protein-coding sequences, making it easy to modify the same cassette to produce different proteins. An expression cassette can also refer to a recombinant nucleic acid molecule capable of expressing a gene sequence in a cell. Expression cassettes typically include regulatory regions, such as promoters (allowing transcription initiation) and sequences encoding one or more proteins or RNAs. Optionally, expression cassettes may include transcription enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals. The sequence that controls gene expression (i.e., transcription and translation of its transcriptional products) is often referred to as a regulatory unit. The majority of the regulatory unit is located upstream of and operatively linked to the coding sequence of the heterologous gene. The expression cassette may also contain a downstream 3' untranslated region containing a polyadenylation site. The regulatory units of the present invention are directly linked to the gene to be expressed (i.e., the transcription unit) or separated from it by intervening DNA (e.g., the 5' untranslated region of the heterologous gene). Preferably, the expression cassette has one or more suitable restraint sites on its side to allow insertion into and / or removal from the vector. Therefore, the expression cassette according to the invention can be used to construct expression vectors, particularly mammalian expression vectors.

[0060] As used herein, the term "expression vector," also known as an expression construct, refers to a plasmid or virus designed for protein expression in cells. Vectors are used to introduce a specific gene into target cells and can command the cell's protein synthesis mechanisms to produce the protein encoded by that gene. Plasmids are engineered to contain regulatory sequences that act as enhancer and promoter regions and induce efficient transcription of the gene carried on the expression vector. The goal of carefully designed expression vectors is to produce large quantities of stable messenger RNA, and thus, the protein.

[0061] As used herein, the terms “host cell” and “host” refer to 1) cells carrying foreign molecules, viruses, etc.; and 2) cells that have been introduced with DNA or RNA, such as bacterial cells that act as host cells for DNA isolated from bacteriophages.

[0062] As used herein, a "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences typically found in individual proteins can be incorporated into a fusion polypeptide, or amino acid sequences typically found in the same protein can be arranged in a new way within the fusion polypeptide, for example, the fusion of a PTPR wedge domain sequence with a transport moiety sequence. Fusion proteins can be produced, for example, through chemical synthesis or by generating and translating polynucleotides in which peptide regions are encoded in a desired relationship. Chimeric proteins may also comprise a second amino acid sequence associated with the first amino acid sequence via covalent, non-peptide, or non-covalent bonds.

[0063] As used herein, the terms “modification” and “mutation” when referring to a gene or gene product refer to a gene or gene product that exhibits a modification (i.e., altered characteristics) in sequence and / or functional properties compared to the wild-type gene or gene product.

[0064] polypeptide

[0065] As used herein, the term "amino acid" includes alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (lie or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional amino acids are also within the scope of this disclosure and include leucine, ornithine, valine, homoserine, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, procedures from Noren et al., Science 244:182 (1989), and Ellman et al. (ibid.) can be used. In simple terms, these procedures involve chemically activating repressed tRNA with non-naturally occurring amino acid residues, followed by in vitro transcription and translation of the RNA. The introduction of non-traditional amino acids can also be achieved using peptide chemistry known in the art. As used herein, the term "polar amino acid" includes amino acids with a net charge of zero but with a non-zero partial charge in different portions of their side chains (e.g., M, F, W, S, Y, N, Q, C). These amino acids can participate in both hydrophobic and electrostatic interactions. As used herein, the term "charged amino acid" includes amino acids (e.g., R, K, H, E, D) that can have a non-zero net charge on their side chains. These amino acids can participate in hydrophobic and electrostatic interactions.

[0066] As used herein, the terms “peptide” or “polypeptide” are used interchangeably and refer to compounds consisting of about 2 to about 90 amino acid residues, wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid via a peptide bond. Peptides can be derived from or removed from natural proteins, for example, by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid-phase synthesis) or molecular biology 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, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., P-alanine, 4-aminobutyric acid, 6-aminohexanoic acid, sarcosine, pepsin), and uncommon amino acids (e.g., citrulline, homocitrulline, homoserine, ortholeucine, orthovaline, ornithine). The amino, carboxyl, and / or other functional groups on a peptide can be free (e.g., unmodified) or protected with suitable protecting groups. Protecting groups suitable for amino and carboxyl groups, and the means for adding or removing protecting groups, are known in the art. See, for example, Green & Wuts, Protecting Grops in Organic Synthesis (John Wiley & Sons, 1991). The functional groups of a peptide can also be derivatized (e.g., alkylated) using methods known in the art.

[0067] As will be apparent to those skilled in the art, the peptide sequences disclosed herein are shown from left to right, wherein the left end of the sequence is the N-terminus of the peptide and the right end of the sequence is the C-terminus of the peptide. The sequences disclosed herein include those with a “Hy-” moiety incorporated at the amino terminus (N-terminus) and a “-OH” or “-NH2” moiety incorporated at the carboxyl terminus (C-terminus). In such cases, unless otherwise indicated, the “Hy-” moiety at the N-terminus of the sequence in question represents a hydrogen atom, corresponding to the presence of a free primary or secondary amino group at the N-terminus, while the “-OH” or “-NH2” moiety at the C-terminus represents a hydroxyl or amino group, respectively, corresponding to the presence of an amide group (CONH2) at the C-terminus. In each sequence of the invention, the C-terminal “-OH” moiety may be substituted with the C-terminal “-NH2” moiety, and vice versa.

[0068] Peptides can be synthesized and assembled into libraries comprising many discrete molecular species. Such libraries can be prepared using well-known combinatorial chemistry methods and screened, as described herein or using other suitable methods, to determine whether the library contains the peptide of interest. These peptides can then be isolated using appropriate means.

[0069] As used herein, the term "peptide mimic" refers to a protein-like molecule designed to mimic peptides. Peptides mimic are typically generated by modifying existing peptides or by designing peptide-like systems, such as peptide-like molecules and β-peptides. Regardless of the method used, the altered chemical structure is designed to advantageously tune molecular properties, such as stability or biological activity. These modifications involve unnatural changes to the peptide (such as altering the backbone and incorporating non-natural amino acids).

[0070] As used herein, the term “monomer” or “peptide monomer” refers to a peptide molecule that can chemically combine with other molecules, such as another peptide molecule, to form a polymer.

[0071] As used herein, the term "peptide dimer" broadly refers to a peptide molecule comprising two monomeric subunits that may be identical or different. Therefore, the dimers of this invention include homodimers and heterodimers.

[0072] As used herein, the term "subunit" refers to a single polypeptide chain that constitutes a protein consisting of two or more polypeptide chains linked together. In a protein molecule composed of more than one subunit, each subunit can form its own stable folded structure. The amino acid sequences of protein or polypeptide subunits can be the same, similar, or completely different.

[0073] As used herein, the term "NH2" may refer to a free amino group present at the amino terminus of a peptide. As used herein, the term "OH" may refer to a free carboxyl group present at the carboxyl terminus of a peptide. Furthermore, as used herein, the term "Ac" refers to acetyl protection achieved by acylation of the C-terminus or N-terminus of a peptide. In some peptides shown herein, NH2 at the C-terminus of the peptide represents an amino group.

[0074] As used herein, the terms "linker" and "peptide linker" are used interchangeably and refer to short peptide sequences that exist between and connect functional protein domains. Linkers designed by researchers are generally classified into three categories based on their structure: flexible linkers, rigid linkers, and in vivo cleavable linkers. Flexible linkers are typically composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Linkers can also play a role in the in vivo release of free functional domains (as in in vivo cleavable linkers). Linkers can provide many other advantages for the generation of fusion proteins, such as improved biological activity, increased expression yield, and achievement of desired pharmacokinetic characteristics. The composition and length of linkers can be determined according to methods well known in the art, and their efficacy can be tested. The length of a linker can be from about 3 to about 15 amino acids. In some embodiments of the invention, the length of the linker can be from about 5 to about 10 amino acids; however, longer linkers may be used in embodiments of the invention.

[0075] As used herein, the terms “part,” “fragment,” “variant,” “derivative,” and “analyte” when referring to the polypeptides of the present invention include any polypeptide that retains at least some of the biological activities mentioned herein (e.g., inhibition of interactions such as binding). The polypeptides described herein may include, but are not limited to, parts, fragments, variants, or derivative molecules, as long as the polypeptide still functions. The polypeptides or parts thereof of the present invention may include proteolytic fragments, deleted fragments, and, in particular, fragments that are more readily accessible to the site of action when delivered to animals.

[0076] As used herein, the term "protein purification" refers to a series of processes aimed at isolating one or more proteins or peptides from complex mixtures, such as cell culture media, cells, tissues, or whole organisms. Typically, a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography involves passing a solution containing the protein through a column packed with various materials. Different proteins interact differently with the column materials, and therefore separation can be achieved based on the time required for passage through the column or the conditions required for elution. Many purification strategies exist. For example, proteins can be engineered to be tagged with antigenic peptides and purified using antibodies against those tags. Typically, during purification, the protein tagged with the antigenic peptide can be added to a column loaded with resin coated with the desired antibody, or incubated with loose resin coated with immobilized antibodies. This specific procedure is called immunoprecipitation. Immunoprecipitation is quite capable of producing highly specific interactions, which typically result in the binding of only the desired protein. The purified tagged protein can then be easily separated from other proteins in solution and subsequently eluted back into a clean solution.

[0077] In some embodiments, the dimers disclosed herein are substantially separated. "Substantially separated" means that the dimer is at least partially or substantially separated from the environment in which it is formed or detected. Partial separation may include, for example, dimers rich in the compounds of the present invention. Substantially separated may include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the dimer by weight.

[0078] As used herein, the term "inhibitor" refers to a molecule, compound, or agent that reduces or inhibits at least one activity, signaling, or function of proteoglycan-induced leukocyte common antigen-associated (LAR) family phosphatases, reduces the activity, signaling, and / or function of chondroitin sulfate proteoglycan (CSPG), and / or the interaction between CSPG and LAR family phosphatases. In some embodiments, "inhibitor" also refers to a molecule, compound, or agent that eliminates the inhibitory effect of CSPG on CSPG-activated neurons. In various embodiments, the inhibitors disclosed herein are peptide dimers comprising an amino acid sequence of a cytoplasmic wedge domain derived from a receptor-type protein tyrosine phosphatase (PTPR).

[0079] homology

[0080] As used herein, the terms “homology” and “identity” are used synonymously throughout and refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in the sequences that can be aligned for comparative purposes. When positions in the compared sequences are occupied by the same bases or amino acids, then the molecules are homologous or identical at that position. The degree of homology or identity between sequences varies with the number of shared matching or homologous positions.

[0081] As used herein, the terms "analogue" and "analog" refer to one of a group of compounds that have structural and / or functional similarities but differ in elemental composition. A structural analog is a compound that has a structure similar to another compound but differs in one or more components, such as one or more atoms, functional groups, or substructures. A functional analog is a compound that has similar physical, chemical, biochemical, or pharmacological properties. A functional analog is not necessarily a structural analog with a similar chemical structure.

[0082] As used herein, the terms “sequence identity,” “percentage of identity,” “percentage of homology,” or, for example, “80% identical sequences,” refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid on a comparison window. Therefore, the “percentage of sequence identity” can be calculated as follows: compare two best-aligned sequences on a comparison window, determine the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to produce the percentage of sequence identity.

[0083] The calculation of sequence similarity or sequence identity (these terms are used interchangeably herein) between sequences can be performed as follows. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, sequences can be aligned for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% of the length of the reference sequence, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position.

[0084] The percentage of identity between two sequences varies with the number of common positions shared by the sequences, taking into account the number of gaps that need to be introduced to achieve the best alignment of the two sequences and the length of each gap.

[0085] Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. In some embodiments, the percentage of identity between two amino acid sequences is determined using the Needleman and Wunsch (1970, J. Mol. Biol. 48: 444-453) algorithm from the GAP program, which has been incorporated into the GCG software package, using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix with vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Another set of exemplary parameters includes a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy expansion penalty is 4, and the frameshift vacancy penalty is 5. The percentage of identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4:11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.

[0086] For example, the peptide sequences described herein can be used as "query sequences" to search public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, J. Mol. Biol, 215: 403-10). A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. For obtaining gap-filled alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997), can be used. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0087] Compositions and Formulations

[0088] As used herein, the term “pharmaceutical acceptable” means a compound or drug that is approved or permitted by a federal or state regulatory agency and is listed or permitted in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, including humans.

[0089] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound wherein the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic acid or organic acid salts having basic residues such as amines; alkali metal salts or organic salts having acidic residues such as carboxylic acids; etc. Pharmaceutically acceptable salts of this disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of this disclosure can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods. Typically, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of both; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. The phrase "pharmaceutically acceptable salt" is not limited to a single salt or a 1:1 salt. For example, "pharmaceutically acceptable salt" also includes disalts, such as dihydrochlorides. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0090] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound that can be used in this disclosure with a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of the compound to a subject. Various techniques for administering compounds exist in the art, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0091] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that participates in the delivery or transport of a compound that may be used in this disclosure to a subject, enabling it to perform its intended function. Typically, such constructs are delivered or transported from one organ or body part to another. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation, including compounds that may be used in this disclosure and are harmless to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0092] As used herein, "pharmaceuticalally acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, and absorption delay agents that are compatible with the activity of compounds that may be used in this disclosure and are physiologically acceptable to a subject. Complementary active compounds may also be incorporated into the composition. "Pharmaceuticalally acceptable carrier" may also include pharmaceutically acceptable salts of the compounds disclosed herein. Other additional components that may be included in the pharmaceutical composition are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., MackPublishing Co., 1985, Easton, PA), references which are incorporated herein by reference.

[0093] As used herein, the terms "pharmaceutical formulation" and "drug formulation" refer to mixtures or structures of different chemical substances, including an active pharmaceutical ingredient, that form the final drug product. Examples include sterile products, solutions, powders, emulsions, capsules, tablets, granules, topical preparations, and non-conventional products such as semi-solid or sustained-release formulations, liquids, etc. Pharmaceutical formulations are prepared according to a specific procedure (i.e., a "formulation"). The resulting drug varies depending on the route of administration.

[0094] Medical intervention

[0095] As used herein, the term "dose" refers to the amount of a specific drug taken at one time. "Daily dose" refers to the total amount of drug administered to an individual over a 24-hour period.

[0096] As used herein, the term “mg / kg” refers to the dose of a substance administered to an individual, expressed in milligrams per kilogram of individual body weight.

[0097] As used in this article, the term "dosage" refers to a specific amount, number of times, and frequency of medication administered over a specific period of time. Dosage implies duration. A "dosage regimen" is a treatment plan for administering medication over a period of time.

[0098] As used herein, the phrases “parenteral administration” and “administered parenterally” mean administration other than via the intestines, surface, or digestive tract, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intracerebral injection and infusion.

[0099] As used herein, the phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” mean the administration of a compound, drug, or other material in a manner other than direct application to a target tissue (e.g., the nervous system), such that the compound, drug, or other material enters the animal’s system and is thus metabolized and undergoes other similar processes, such as subcutaneous administration.

[0100] As used herein, the terms “patient” or “subject” or “animal” or “host” refer to any mammal. A subject can be a human, but can also be a mammal requiring veterinary treatment, such as domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, poultry, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0101] As used herein, the term "administration to a patient" includes dispensing, delivering, or applying an active compound of a pharmaceutical preparation to a subject via any suitable route to deliver the active compound to a desired location in the subject (e.g., thereby contacting desired cells, such as desired neurons), including administration into cerebrospinal fluid or across the blood-brain barrier, via parenteral or oral routes, intramuscular injection, subcutaneous or intradermal injection, intravenous injection, buccal administration, dermal delivery, and administration via rectal, colonic, vaginal, intranasal, or respiratory routes. The agent may be administered intravenously, for example, to a comatose, anesthetized, or paralyzed subject, or intravenously to a pregnant subject to stimulate axonal growth in the fetus. Specific routes of administration may include topical application (such as eye drops, creams, or corrosive preparations placed under the eyelid, intraocularly injected into the aqueous humor or vitreous humor, or injected into the outer layer of the eye (such as via subconjunctival or subfascial injection), parenteral administration, or oral administration.

[0102] The term “treat, treated, treating, or treatment” includes reducing or alleviating at least one symptom associated with or caused by the state, condition, or disease being treated. As used herein, the term “treatment” also includes: (1) suppressing the disease or condition, i.e., preventing the development or progression of the disease or condition; (2) alleviating the disease or condition, i.e., causing the condition to subside; (3) stopping the symptoms of the disease; and / or (4) enhancing the desired state.

[0103] As used herein, the term "prevent" means the absence of the development of a symptom or disease (if the development of said symptom or disease has not yet occurred), or the absence of further development of the symptom or disease (if said symptom or disease has already developed). The ability to prevent some or all of the symptoms associated with the symptom or disease is also considered.

[0104] As used herein, the “effective amount” of the agent or therapeutic peptide dimer disclosed herein is an amount sufficient to achieve the desired therapeutic or pharmacological effect, such as an amount capable of activating neuronal growth. The effective amount of an agent as defined herein can vary depending on a variety of factors, such as the subject’s disease state, age, and weight, and the agent’s ability to elicit the desired response in the subject. Dosage regimens can be adjusted to provide the optimal therapeutic response. An effective amount is also the amount by which the therapeutically beneficial effect of an active compound outweighs any toxic or adverse effects. As used herein, the term “therapeutic effective amount” refers to an amount that, at the prescribed dosage and for the required time period, effectively achieves the desired therapeutic outcome. The therapeutic outcome can be, for example, symptom relief, prolonged survival, improved mobility, etc. A therapeutic outcome is not necessarily a “cure.”

[0105] As used herein, the term “improve,” “improving,” or “improvement,” used in relation to behavior, or its grammatical variations, refers to the ability to achieve a measurable improvement in performance on a task used to test subjects (including humans or non-human animals) regarding these behaviors.

[0106] Central nervous system

[0107] As used in this article, the term “central nervous system (CNS) neuron” includes neurons in the brain, cranial nerves, and spinal cord.

[0108] As used herein, the term "peripheral nervous system (PNS) neuron" includes neurons located outside or extending into the CNS. The PNS is intended to include neurons generally understood to be classified as belonging to the peripheral nervous system, including sensory neurons and motor neurons.

[0109] As used herein, the terms "contact neuron" or "therapeutic neuron" refer to any manner of agent delivery or "application," whether to cells or the whole organism, in which the agent is able to exert its pharmacological effect in neurons. "Contact neuron" includes in vivo and in vitro methods of bringing the agents of the present invention into proximity to neurons. Those skilled in the art can determine suitable methods of application, and such methods of application can vary between agents. For example, when stimulating axonal growth in ex vivo neurons, the agent can be applied, for example, by transfection, lipid transfection, electroporation, viral vector infection, or by addition to a growth medium.

[0110] As used herein, the term “neurological disorder” includes diseases, conditions, or symptoms that directly or indirectly affect the normal functioning or anatomical structure of a subject’s nervous system. The term “stroke” is recognized and includes a sudden reduction or loss of consciousness, sensation, and voluntary motor function due to the rupture or blockage of a cerebral artery (e.g., by a blood clot). “Traumatic brain injury” is recognized and includes cases where a traumatic blow to the head results in damage to the brain or the spinal cord, whether or not it penetrates the skull. Typically, the initial trauma can lead to hematoma expansion, subarachnoid hemorrhage, cerebral edema, increased intracranial pressure, and cerebral hypoxia, which can then lead to serious secondary events due to low cerebral blood flow.

[0111] As used herein, the term axonal “growth” or “outgrowth” (also referred to herein as “neuronal outgrowth”) encompasses the process of axons or dendrites extending from neurons. Outgrowth can result in the extension of new neuritic ridges or previously existing cellular processes. Axonal outgrowth can include the linear extension of an axonal process by five cell diameters or more. Neuronal growth processes, including neurite formation, can be demonstrated by GAP-43 expression detected via methods such as immunostaining. “Stimulating axonal growth” means promoting axonal outgrowth.

[0112] As used in this article, the term "dieback" refers to axonal retraction caused by axonal trauma.

[0113] As used in this article, the term "retraction" refers to the axon retracting away from the site of injury (such as where glial scars form). At this point, the ends of the regenerating axon stop extending and become malnourished. These malnourished ends can then retract further away from the glial scars and the site of injury.

[0114] As used in this article, the term “neuronal migration” refers to the ability of neurons to migrate or neuronal processes to migrate (such as axons or dendrites).

[0115] peptide dimers

[0116] This application relates to compositions and methods for repairing the nervous system of a subject in need. This application also relates to methods and compositions for treating diseases, conditions, and / or symptoms related to the function of LAR family phosphatases.

[0117] In one aspect, this document provides a pharmaceutical composition comprising a peptide, the peptide being a dimer comprising a first monomer covalently cross-linked with a second monomer, each monomer comprising a domain containing an amino acid sequence of a cytoplasmic wedge domain derived from a receptor-type protein tyrosine phosphatase (PTPR), and wherein the mass ratio of the dimer to the free monomer in the pharmaceutical composition is greater than 1:20.

[0118] In one embodiment, the pharmaceutical composition provided herein further comprises a dimer comprising a transport portion of a domain connected via a cysteine ​​residue or a peptide linker containing a cysteine ​​residue.

[0119] In one embodiment, the pharmaceutical composition provided herein further comprises a dimer, wherein the dimer improves nerve cell repair.

[0120] In yet another embodiment, the pharmaceutical composition provided herein further comprises a first monomer and a second monomer, each of the first and second monomers independently comprising: a first domain comprising an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% identical to the amino acid sequence of SEQ ID NO: 5, 6, or 7; a second domain comprising an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11; and a cysteine ​​residue; wherein the dimer comprises a chemical linker or bond between the cysteine ​​residue of the first monomer and the cysteine ​​residue of the second monomer.

[0121] In one embodiment, the pharmaceutical composition provided herein further comprises a first monomer and a second monomer, each of the first and second monomers independently comprising at least 70% of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In another embodiment, the pharmaceutical composition provided herein further comprises a first monomer and a second monomer, each of the first and second monomers independently comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In yet another embodiment, the pharmaceutical composition provided herein further comprises a dimer containing the same monomers. In still another embodiment, the pharmaceutical composition provided herein further comprises dimers containing different monomers, and the ratio is calculated based on the total amount of free monomers. In yet another embodiment, the pharmaceutical composition provided herein further comprises a first monomer and a second monomer with different C-terminal modifications to dimers.

[0122] In another embodiment, this document provides a medicament for repairing the nervous system of a subject, the medicament comprising a peptide dimer or a pharmaceutically acceptable salt or solvate thereof, the peptide dimer comprising two subunits, each subunit comprising a peptide domain independently selected from a receptor-type protein tyrosine phosphatase (PTPR) wedge domain or a variant having at least 70% homology thereto. In one embodiment, the medicament provided herein further comprises a peptide dimer or a pharmaceutically acceptable salt or solvate thereof, the peptide dimer comprising two subunits, each subunit comprising a transport portion connected to the peptide domain via a cysteine ​​residue or a peptide linker containing a cysteine ​​residue, wherein the two subunits are covalently cross-linked via a chemical linker between cysteine ​​residues on each subunit. In another embodiment, the medicament provided herein further comprises a chemical linker selected from disulfide bonds, thioether bonds, or thioester bonds. In yet another embodiment, the medicament provided herein further comprises an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11.

[0123] In another embodiment, the pharmaceutical preparation provided herein further comprises a transport portion comprising an amino acid sequence having at least 65% identity with the wild-type HIV TAT. In yet another embodiment, the pharmaceutical preparation provided herein further comprises a TAT sequence comprising an amino acid sequence that is at least 65% identical to the amino acid sequence of SEQ ID NO: 5, 6, or 7. In still another embodiment, the pharmaceutical preparation provided herein further comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4.

[0124] In one embodiment, the pharmaceutical agent provided herein further comprises subunits containing different C-terminal modifications. In another embodiment, the pharmaceutical agent provided herein further comprises a peptide dimer containing two different subunits. In yet another embodiment, the pharmaceutical agent provided herein further comprises a peptide dimer containing two identical monomeric subunits. In still another embodiment, the pharmaceutical agent provided herein further comprises a peptide dimer with a purity of at least 90% or a pharmaceutically acceptable salt or solvate thereof.

[0125] In one embodiment, the pharmaceutical composition provided herein further comprises the pharmaceutical agent provided herein. In another embodiment, the pharmaceutical composition does not comprise DMSO. In yet another aspect, this document provides for the use of the pharmaceutical agent provided herein in the manufacture of a medicament for repairing and / or treating neurological symptoms, diseases, or conditions selected from the group consisting of nerve damage, neurological diseases caused by inflammation or autoimmunity, and neurodegenerative diseases.

[0126] On the other hand, this article provides a method for repairing the nervous system and / or treating neurological symptoms, diseases, or conditions selected from the group consisting of nerve damage, neurological diseases caused by inflammation or autoimmunity, and neurodegenerative diseases in subjects in need, the method comprising administering an effective amount of the agent provided herein to the subject in need.

[0127] In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is greater than 1:19, greater than 1:18, greater than 1:17, greater than 1:16, greater than 1:15, greater than 1:14, greater than 1:13, greater than 1:12, greater than 1:11, greater than 1:10, greater than 1:9, greater than 1:8, greater than 1:7, greater than 1:6, greater than 1:5, greater than 1:4, greater than 1:3, greater than 1:2, or greater than 1:1. In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is greater than 1:1.

[0128] In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is greater than 2:1, greater than 3:1, greater than 4:1, greater than 5:1, greater than 6:1, greater than 7:1, greater than 8:1, greater than 9:1, greater than 10:1, greater than 11:1, greater than 12:1, greater than 13:1, greater than 14:1, greater than 15:1, greater than 16:1, greater than 17:1, greater than 18:1, greater than 19:1, or greater than 20:1. In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is greater than 4:1. In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is a maximum of 10:1, a maximum of 15:1, a maximum of 20:1, a maximum of 25:1, or a maximum of 27:1. In some embodiments, the ratio of dimer to free monomer in the pharmaceutical composition is greater than 10:1 (a maximum of 27:1).

[0129] In another implementation, the ratio is calculated based on the total amount of free monomers.

[0130] The first and second monomers of the dimer can be linked by any method known in the art. In some embodiments, the first and second monomers are linked by a bond. In some embodiments, the first and second monomers are linked by a cysteine ​​bridge. In some embodiments, the dimer includes a linker between the first and second monomers. In some embodiments, the dimer includes a disulfide bond between the first and second monomers.

[0131] In some embodiments, the dimer includes a transport moiety that promotes cellular uptake of the dimer. In some embodiments, the transport moiety may be an HIV TAT transport moiety (i.e., a TAT sequence). The transport moiety may be repeated more than once in the dimer. Repeating the transport moiety may affect (e.g., increase) the desired cellular uptake of the dimer. In either or both of the first and second monomers, the transport moiety may be located in an N-terminal region or a C-terminal region or both regions. In one embodiment, the transport moiety is located in the N-terminal region of the monomer.

[0132] In some embodiments, the transport portion is connected via a peptide linker. In some embodiments, the transport portion is connected via two peptide linkers.

[0133] In some embodiments, the transport portion may include at least one transport peptide sequence that allows the dimer to penetrate into the cell via a receptor-independent mechanism. In some embodiments, the dimer is a synthetic peptide containing a TAT-mediated protein delivery sequence.

[0134] Other known examples of transport components, subdomains, etc., are described in, for example, Canadian Patent Application No. 2,301,157 (conjugates containing homeodomain of antennapedia), PCT International Publication No. WO 99 / 11809, and U.S. Patent Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, 5,804,604, and Bruno P. Meloni et al. Frontiers in Neurology. 2020; 11 (Article 108): 1-28, all of which are incorporated herein by reference in their entirety. Therefore, in some embodiments, the transport portion is the HIV TAT peptide; the herpes simplex virus-1 DNA-binding protein VP22 peptide; the amino acid region of the third α-helix of the antennal foot homologous domain; a histidine tag with a length ranging from 4 to 30 histidine repeats; a variant derivative or homolog thereof capable of promoting the uptake of the active cargo portion through a receptor-independent process; or a cationic arginine-rich peptide (CARP). In some embodiments, the transport moiety may be selected from a neuroprotective CARP having the following characteristics: (i) a size ranging from 4 to 40 amino acids; (ii) a positive net charge ≥ +2 to +20; (iii) one or more positively charged arginine residues comprising between 20% and 100% of the peptide; (iv) other positively charged amino acids, namely lysine and histidine; (v) amphiphilicity due to the presence of hydrophilic (e.g., arginine, lysine) and hydrophobic (e.g., tryptophan, phenylalanine, tyrosine) amino acids; and (vi) endocytic and / or non-endocytic cell membrane transmembrane properties, including the ability to cross the blood-brain barrier and blood-spinal barrier (BBB / BSCB). In some embodiments, the transport moiety may be a cationic arginine-rich peptide fused to TAT.

[0135] Furthermore, the transport region may include a polypeptide having a region rich in basic amino acids. As used herein, the term "region rich in basic amino acids" refers to a region of a protein or peptide that has a high content of basic amino acids (such as arginine, histidine, asparagine, glutamine, lysine). A "region rich in basic amino acids" may have, for example, 15% or more of basic amino acids. In some cases, a "region rich in basic amino acids" may have less than 15% of basic amino acids and still function as a transporter region. In other cases, the basic amino acid region will have 30% or more of basic amino acids.

[0136] The transporter region may also include a proline-rich region. A proline-rich region refers to a region in the polypeptide containing more proline than is typically observed in naturally occurring proteins (e.g., proteins encoded by the human genome). As used herein, the term proline-rich region refers to a region in the polypeptide having 5% or more (up to 100%) proline in its sequence. In some cases, a proline-rich region may have between 5% and 15% proline. The proline-rich region of this application can function as a transporter region.

[0137] Therefore, in some embodiments, the dimer comprises a transport moiety and cysteine ​​residues. In some embodiments, the dimer comprises a transport moiety and cysteine ​​residues, said transport moiety comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the wild-type HIV TAT sequence.

[0138] In some embodiments, the transport portion comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:5, 6, or 7 as shown in Table 1 below. In one embodiment, the transport portion included in the first monomer unit and the second monomer is identical to the amino acid sequence of SEQ ID NO:5.

[0139] Table 1. TAT sequences

[0140] In some embodiments, the dimer comprises a domain selected from the group consisting of a PTPRF wedge domain, a PTPRD wedge domain, and a PTPRS wedge domain, as well as variants having at least 70% identity with them. In some embodiments, the dimer comprises a domain selected from the group consisting of a PTPRF wedge domain, a PTPRD wedge domain, and a PTPRS wedge domain, as well as variants having at least about 65% identity with them, at least about 70% identity with them, at least about 75% identity with them, at least about 80% identity with them, at least about 85% identity with them, at least about 90% identity with them, at least about 95% identity with them, or at least about 97% identity with them. In some embodiments, the domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11 as listed in Table 2. In some embodiments, the domain comprises an amino acid sequence that is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 97% identical to the amino acid sequence of SEQ ID NO:8, 9, 10, or 11 listed in Table 2.

[0141] Table 2: Wedge domain sequences of LAR family phosphatases

[0142] In some embodiments, the first monomer and the second monomer each independently comprise: a first domain containing at least 70% of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 5, 6, or 7; a second domain containing at least 70% of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11; and a cysteine ​​residue; wherein the dimer comprises a disulfide bond between the cysteine ​​residues of the first monomer and the cysteine ​​residues of the second monomer. In one embodiment, the first domain contains an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 5, 6, or 7. In one embodiment, the transport moiety included in the first monomer unit and the second monomer is identical to the amino acid sequence of SEQ ID NO: 5. In one embodiment, the second domain contains an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11.

[0143] In some embodiments, the peptide dimer disclosed herein has the following structure: .

[0144] In some implementations, the "first domain" corresponds to the transport portion described above and the "second domain" corresponds to the peptide domain described above.

[0145] In some embodiments, the peptide dimer disclosed herein has the following structure: , Where X is a bond or chemical linker between two cysteine ​​residues.

[0146] In some embodiments, X is a bond, for example, a disulfide bond between the thiol groups of two cysteine ​​residues. In some embodiments, X is a chemical linker between two cysteine ​​residues. The chemical linker may, for example, comprise a covalent bond (e.g., a disulfide bond, a thioether bond, or a thioester bond) with each thiol group of the cysteine ​​residue. In some embodiments, the length of the chemical linker is between 5 Å and 50 Å. In some embodiments, the length of the chemical linker is between 5 Å and 35 Å. In some embodiments, the length of the chemical linker is between 10 Å and 25 Å. In some embodiments, the chemical linker consists of atoms selected from C, N, S, O, and H. In some embodiments, the chemical linker comprises atoms selected from C, N, S, O, and H. In some embodiments, the chemical linker consists of atoms selected from C, O, and H. In some embodiments, the chemical linker comprises atoms selected from C, O, and H. In some embodiments, the chemical linker comprises between 1 and 8 carbon atoms. In some embodiments, the chemical linker comprises an alkylene chain (e.g., C...). (1-12) Alkylene, C (1-6) Alkylene or C (1-3) Alkylene). In some embodiments, the chemical connector comprises an alkylene chain in which one or more carbon atoms are replaced by oxygen (e.g., a divalent polyethylene glycol chain).

[0147] In some embodiments, the first monomer and the second monomer each independently comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In one embodiment, the first monomer and the second monomer each independently comprise an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4.

[0148] In one embodiment, the dimer comprises the same monomer. In another embodiment, the dimer comprises different monomers.

[0149] In one embodiment, the first and second monomers of the dimer have different C-terminal modifications.

[0150] One potential mechanism for regulating, modulating, and / or inhibiting LAR family phosphatases involves the dimerization of the intracellular portion of the phosphatase. Several protein tyrosine phosphatases (PTPs) have been shown to be inactive as dimers and active as monomers, compared to receptor tyrosine kinases which are active as dimers but inactive as monomers. PTPα, PTP1B, and CD45, crystallized in both forms, have been shown to be active as monomers but inactive as dimers. Since PTPRF exhibits homophilic binding under specific oxidative conditions, and PTPRS dimers in response to ligand binding, it suggests that ligands of LAR family phosphatases can guide the activation state of PTPRF and PTPRS. Therefore, mimicking dimerization through intracellular targeted therapy could directly inactivate LAR family phosphatases without altering the extracellular matrix or other ligands.

[0151] When delivered to nerve cells, peptide mimics of the intracellular portion of LAR family phosphatases can inhibit and / or reduce CSPG-induced LAR activity or function. It has been found that inhibiting LAR family activity, signal transduction, and / or function in response to CSPG activation promotes nerve cell growth, including restoring growth cone movement, process extension, budding, promoting nerve cell survival and plasticity, and inhibiting apical dieback.

[0152] In some embodiments, the function of LAR family phosphatases is inhibited or reduced by peptides or small molecule therapeutic agents that bind to and / or are complexed with the intracellular domain of at least one LAR family phosphatase. In some embodiments, one or more activities and signal transductions of LAR family phosphatases are inhibited or reduced by peptides or small molecule therapeutic agents that bind to and / or are complexed with the intracellular domain of at least one LAR family phosphatase. Therefore, therapeutic peptides or small molecules that bind to and / or are complexed with the intracellular domain of at least one LAR family phosphatase in nerve cells can be used to promote cell growth, motility, survival, and plasticity of these cells.

[0153] In some implementations, the therapeutic agent is a peptide mimic of the wedge-shaped domain (i.e., the wedge domain) of LAR family phosphatases. Structural and sequence analyses have revealed that all members of the LAR family contain a conserved 24-amino acid wedge-shaped helical-loop-helical motif in their first intracellular catalytic domain, which could potentially mediate homophilic / heterophilic receptor interactions.

[0154] Table 3 lists the amino acid sequences of the intracellular portions of LAR family phosphatase members containing wedge-shaped domains. The 24-amino acid wedge-shaped domains of these intracellular portions of LAR family phosphatases are identified by underlining. Although the specific structure of the wedge-shaped domain is conserved in most LAR family wedge-shaped domains, the exact amino acids constituting the wedge-shaped domain vary between individual proteins and subfamilies.

[0155] As shown in Table 3, the wedge-shaped domain is highly conserved among LAR family members. For example, the wedge-shaped domain sequence of PTPRS is highly conserved in mammals, with only a single amino acid change (threonine at position 6 becomes methionine) in mice and rats.

[0156] Table 3: Wedge-shaped domains of LAR family phosphatases

[0157] Preferred embodiments are listed in Tables 4, 5, and 6. This document provides monomeric compounds 1, 2, 3, and 4, and methods for treating subjects suffering from any of the indications provided herein by administering compound 1, 2, 3, or 4 to a subject.

[0158] Table 4: Monomers

[0159] Table 5: Homodimer

[0160] “ "Represents Cys-Cys disulfide bond".

[0161] Table 6: Heteridimers

[0162] “ "Represents Cys-Cys disulfide bond".

[0163] In some embodiments, amino acid residue 17T in SEQ ID NO:3 may be substituted with M, thereby producing rat and mouse PTPRS variants.

[0164] In some embodiments, the peptide dimer disclosed herein may comprise a peptide domain that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% identical to about 10 consecutive amino acids of the wedge-shaped domain of a LAR family phosphatase. In some embodiments, the peptide dimer disclosed herein may comprise a peptide domain that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% identical to about 15 consecutive amino acids of the wedge-shaped domain of a LAR family phosphatase. In some embodiments, the peptide dimer disclosed herein may comprise a peptide domain that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% identical to about 20 consecutive amino acids of the wedge-shaped domain of a LAR family phosphatase. The peptide dimer may modulate the signaling and / or function of LAR family phosphatases in cells (such as nerve cells) expressing LAR family phosphatases.

[0165] In some embodiments, the peptide dimer comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% of the same peptide domain as the wedge-shaped domain of PTPRS, and the peptide dimer can promote cell viability, morphogenesis, or differentiation by undergoing the inhibitory effect of CSPG on cells (e.g., nerve cells).

[0166] In some embodiments, the peptide dimers disclosed herein include a peptide linker. In some embodiments, the peptide linker is located between the monomer subunit that connects the transport moiety and the first or second peptide domain. In some embodiments, each peptide monomer includes a peptide linker having a cysteine ​​residue, such that the first and second monomer subunits are crosslinked via a disulfide bond between the two cysteine ​​residues in the first and second monomer subunits.

[0167] In some embodiments, the monomeric subunit of the peptide dimer may have one or more C-terminal modifications. These modifications may be the same or different. In one embodiment, the monomeric subunit has a C-terminal amide terminus, wherein the charge is removed from the C-terminus of the peptide by amidation, particularly when the peptide monomer is chemically synthesized. The uncharged C-terminal amide terminus more closely mimics the native protein, thus increasing the peptide's biological activity.

[0168] In some embodiments, the monomeric subunit of the peptide dimer may include additional residues at the C-terminus or N-terminus. In other embodiments, the monomeric subunit of the peptide dimer may include a peptide tag at the C-terminus or N-terminus. In some embodiments, the tag may be an affinity tag, such as a His tag, a Flag tag, a Twin-Strep tag, etc.

[0169] The peptides described herein may also include, for example, bioactive mutants, variants, fragments, chimeras, and analogs. The term "fragment" encompasses an amino acid sequence truncated from the amino-terminus (N-terminus), carboxyl-terminus (C-terminus), or within the peptide. Analogs of the present invention are peptides with inserted or substituted amino acids. Variants, mutants, fragments, chimeras, and analogs can act as inhibitors to eliminate the inhibitory effect of CSPG on CSPG-activated nerve cells (not limited to the embodiments of the present invention).

[0170] In various non-limiting embodiments, the peptide dimers disclosed herein can be used as therapeutic agents to promote cell growth, motility, survival, and plasticity of these cells.

[0171] Preparation of peptide dimers

[0172] The embodiments disclosed herein provide methods for preparing peptide monomers for preparing peptide dimers and methods for preparing peptide dimers disclosed herein.

[0173] According to various embodiments, the peptide monomers used to prepare peptide dimers can be prepared by methods known to those skilled in the art. For example, conventional peptide synthesis techniques (e.g., solid-phase synthesis) or molecular biology techniques can be used to prepare peptide monomers.

[0174] In some embodiments, the transport moiety polypeptide and the peptide domain described above can be synthesized and purified separately, and then non-covalently linked using a non-covalently linked polypeptide transducer (such as the polypeptide transducer provided in the Chariot protein delivery system) (see U.S. Patent No. 6,841,535). J Biol Chem 274(35):24941-24946; and Nature Biotec. 19:1173-1176, all references are incorporated into this paper in their entirety by way of citation.

[0175] In some embodiments, the peptide monomers used to form the peptide dimers disclosed herein can be generated using recombinant DNA through genetic engineering. For example, the recombinant DNA can be engineered to encode a fusion peptide for preparing the peptide dimers disclosed herein. The fusion peptide may include a peptide domain comprising an amino acid sequence selected from the wedge domains of leukocyte antigen-associated (LAR) family phosphatases or variants having at least 65% homology thereto, and the peptide domain may be linked to the transport moiety disclosed herein via a cysteine ​​residue or a peptide linker containing one cysteine ​​residue.

[0176] The recombinant DNA can be inserted into the expression cassette of an expression vector and operatively linked to a regulatory region. The regulatory region typically contains a promoter to regulate the expression of the peptide monomer in the cell carrying the vector. In some embodiments, the promoter is a constitutive promoter, such as a CMV, allowing the peptide monomer to be persistently expressed in the cell carrying the vector. In some embodiments, the promoter is an inducible promoter and can induce the expression of the peptide monomer as needed.

[0177] In some implementations, the vector is a plasmid vector that can be transformed into bacteria for preservation or amplification, and can be transfected into mammalian cells to express recombinant peptides.

[0178] In some embodiments, the preparation disclosed herein may include culturing host cells (bacteria or eukaryotic cells) under conditions that provide intracellular expression of peptides and / or proteins.

[0179] Peptide monomers expressed in host cells can be purified using affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, or other purification techniques commonly used for protein purification. The purification steps can be performed under non-denaturing conditions. Alternatively, if a denaturing step is required, the protein can be renatured using techniques known in the art.

[0180] In some embodiments, the peptide monomers described herein may include additional residues that can be added to either end of the polypeptide to provide a “connector” by which the polypeptide can be readily linked and / or attached to other polypeptides, proteins, detectable moieties, tags, solid matrices, or carriers.

[0181] Amino acid linkers typically consist of at least one residue and can have 40 or more residues, but are more commonly 1 to 10 residues. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, among others. Furthermore, the subject peptide can vary depending on the sequence modified by terminal NH2 acylation (e.g., acetylation or thioacetate amidation), terminal carboxyl amidation (e.g., terminal modification with ammonia, methylamine, etc.). Terminal modifications are known to reduce susceptibility to protease digestion and thus to prolong the half-life of peptides in solution (particularly in biological fluids where proteases may be present). In this regard, peptide cyclization is also a useful terminal modification and is particularly preferred due to the stable structure formed by cyclization and in view of the biological activities observed for such cyclic peptides as described herein.

[0182] In some embodiments, the linker may be a flexible peptide linker that links the therapeutic peptide to other polypeptides, proteins, and / or molecules, such as detectable moieties, labels, solid matrices, or carriers. The length of the flexible peptide linker may be about 20 or fewer amino acids. For example, the peptide linker may contain about 12 or fewer amino acid residues, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some cases, the peptide linker contains two or more of the following amino acids: glycine, serine, alanine, and threonine.

[0183] The peptide dimers disclosed herein can be prepared by dimerization of the peptide monomers as described above, wherein the peptide monomers form monomeric subunits in the peptide dimer. The peptide monomers used to prepare the dimer can be the same or different, thereby producing homodimers or heterodimers, respectively.

[0184] In some embodiments, peptide dimers are prepared by oxidizing a first peptide monomer and a second peptide monomer, such that cysteine ​​residues in the first and second peptide monomers form disulfide bonds that link the two peptide monomers together.

[0185] In one embodiment, the dimerization method includes mixing a first peptide monomer and a second monomer in water, and a) adding an oxidant and / or b) oxygenating the solution to allow the formation of a covalently cross-linked dimer. The first peptide monomer and the second peptide monomer may be the same or different. In one embodiment, the combined concentration of the first and second peptide monomers in water may be at least 20 mg / mL or at least 40 mg / mL. In one embodiment, the combination of the first peptide monomer and the second monomer in water is maintained at a temperature of about 20°C to about 25°C. In one embodiment, DMSO is added to the water before, during, or after the addition of the first peptide monomer and the second monomer.

[0186] The oxidant used in the dimerization process can be selected from any common oxidant. Non-limiting examples of oxidants may include bromates, chloroxygenates, chromates, hypoiodates, iodoalkanes, iodates, interhalogen compounds, manganese compounds, nitrates, oxidizing acids, ozone, periodate, permanganate, peroxyacids, persulfates, and rocket oxidants. In some embodiments, the oxidant may be selected from hydrogen peroxide, potassium dichromate, sodium hypochlorite or calcium hypochlorite, nitric acid, oxygen, ozone, potassium perchlorate, potassium chlorate, potassium permanganate, ammonium persulfate or sodium persulfate, or combinations thereof. In some embodiments, the oxidant is selected from copper sulfate, iodides, hydrogen peroxide, trans-3,4-dihydroxyselenocyclopentane oxide (DHS), supported methionine sulfoxide, N-chlorosuccinimide (NCS), or combinations thereof.

[0187] In one embodiment, the oxidant is iodine, and the method employs microwave-assisted oxidation.

[0188] In some embodiments, a method for preparing the peptide dimer disclosed herein includes mixing a first monomer and a second monomer in a solvent containing copper sulfate to generate a dimer. The solvent may also contain purified water (PWP) for purification. In one embodiment, the solvent may also contain ethanol. In one embodiment, the pH of the mixture (i.e., the dimerization reaction mixture) is maintained between about 8.5 and about 9.5.

[0189] An exemplary embodiment for preparing peptide dimers disclosed herein may include the following: 1) adding PWP and ethanol to an oxidation beaker and stirring for 15 minutes to homogenize the mixture, the amount of PWP and ethanol required depending on the amount of peptide monomer; 2) after stirring for 15 minutes, slowly adding the corresponding amount of pure compound 3 to the beaker and mixing until all the powder is dissolved to form a clear solution, i.e., the reaction mixture. 1) Stir the reaction mixture for 15 minutes; 2) After stirring for 15 minutes, check the initial pH of the reaction mixture and adjust it to approximately 9 ± 0.5 with a dilute ammonia solution, which is prepared by taking 100 mL of ammonia solution (25%) and diluting it to 1 L with PWP; 3) Monitor the pH of the reaction mixture every hour; if the pH is below 9 ± 0.5, adjust the reaction mixture by adding more dilute ammonia solution; 4) After about 1 hour, add copper sulfate to the oxidation reaction mixture periodically; 5) Sample every hour and analyze the reaction progress by HPLC, and continue the oxidation reaction until the free peptide monomer compound 3 is detected by HPLC to be below 4 ± 0.5%; 6) Once the presence of compound 3 is confirmed by HPLC to be below 4 ± 0.5%, adjust the pH of the reaction mixture by adding acetic acid until the pH is approximately 4.5 ± 0.5; 7) After oxidation, adjust the reaction mixture to have a pH of approximately 4.5 ± 0.5. 9) The pH was adjusted to 0.5, and the oxidative reaction mixture was then filtered through 5-micron filter paper under vacuum into a receiving container for receiving the filtered solution; 10) The filtered solution was collected and purified by preparative HPLC; 11) The peak eluent from the HPLC was freeze-dried, containing pure freeze-dried peptide dimer compounds.

[0190] preparation

[0191] Non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances, such as phosphates, glycine, sorbic acid, or potassium sorbate; mixtures of metaglycerides of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate; disodium hydrogen phosphate; potassium hydrogen phosphate; sodium chloride; zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene polyoxypropylene block polymers; lanolin; sugars, such as lactose, glucose, and... Sucrose; starch, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solutions.

[0192] In addition, non-toxic and compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, anti-sticking agents, coating agents, sweeteners, flavoring agents and aromatizers, preservatives and antioxidants may also be present in the composition, depending on the formulator's judgment.

[0193] According to various embodiments, the disclosed pharmaceutical compositions can be formulated into any suitable form for delivery to a subject in need at a fixed or non-fixed dose. For example, the pharmaceutical compositions may be suitable for oral or parenteral administration and can be administered to a subject in dosage forms such as tablets, sugar-coated tablets, capsules, delayed-release hard capsules, soft gels, chewable tablets, gummies, sacs, powders, granules, syrups, aerosols, inhalers, suppositories, solutions, suspensions, catheters containing the composition, syringes containing the composition, implants containing the composition, and transdermal patches.

[0194] In some embodiments, the pharmaceutical composition is formulated in a liquid solution, typically in a physiologically compatible buffer such as Hank's solution or Ringer's solution for injection. In some embodiments, therapeutic agents comprising the peptide dimer or its pharmaceutically acceptable analogues, salts, or solvates described herein may be formulated in a solid form and immediately reconstituted or suspended in a pharmaceutically acceptable solvent prior to use. For example, the peptide dimer or its pharmaceutically acceptable analogues, salts, or solvates may be in a lyophilized form, which can be dissolved at the time of use to obtain the final formulation for administration to a subject. Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) may be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as in the form of a 1,3-butanediol solution. Among acceptable media and solvents, water, Ringer's solution USP, and isotonic sodium chloride solutions may be used. In addition, sterile, fixed oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid are used in the preparation of injectables.

[0195] In some implementations, the pharmaceutical composition does not contain DMSO.

[0196] To prolong the effect of a drug, it is often necessary to slow down the absorption of subcutaneously (SC) or intramuscularly injected drugs. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of a drug depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Absorption of parenterally administered drug forms can also be delayed by dissolving or suspending the drug in an oil-based medium.

[0197] In some other embodiments, the pharmaceutical compositions disclosed herein can be formulated into dosage forms such as tablets, soft gels, capsules, pouches, polypills, chewable tablets, gummies, hard capsules, transdermal patches, etc.

[0198] In some other embodiments, the pharmaceutical compositions disclosed herein can be formulated into a liquid form for oral administration, wherein the pharmaceutical composition, together with the therapeutic agent (or active compound), comprises a pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup, and elixir. In addition to the active compound, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol; and mixtures thereof. In addition to inert diluents, the oral composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0199] Similar solid compositions can also be used as fillers in soft and hard filled gelatin capsules, using excipients such as lactose (or milk sugar) and high molecular weight polyethylene glycol.

[0200] Active ingredients comprising peptide dimers or their pharmaceutically acceptable analogs, salts, or solvates may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules may be formulated with coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings well known in the field of pharmaceutical formulation. In such solid dosage forms, the active compound may be blended with at least one inert diluent (such as sucrose, lactose, or starch). Conventionally, such dosage forms may contain additional substances besides inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage form may also contain a buffer.

[0201] Pharmaceutical compositions may be formulated for topical or transdermal application, wherein said forms may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. Where necessary, the active ingredient may be blended under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives or buffers. Ophthalmic preparations, ear drops, ophthalmic ointments, powders, and solutions are also covered within the scope of this disclosure.

[0202] Ointments, pastes, creams, and gels may contain excipients in addition to the active compounds disclosed herein, such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth gum, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0203] The powders and sprays may contain excipients in addition to the compounds disclosed herein, such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powders or mixtures thereof. Sprays may also contain conventional propellants, such as chlorofluorocarbons.

[0204] Transdermal patches offer the added advantage of controlled delivery of compounds into the body. These dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0205] Treatment

[0206] Peptide dimers or their pharmaceutically acceptable analogues, salts or solvates, when delivered to cells (such as nerve cells), can promote cell growth, movement, survival and plasticity of the cells (e.g. nerve cells).

[0207] Therefore, peptide dimers or their pharmaceutically acceptable analogs, salts or solvates, and / or compositions containing peptide dimers or their pharmaceutically acceptable analogs, salts or solvates may be used to eliminate the inhibitory effect of CSPG on CSPG-activated nerve cells and to promote cell growth, movement and survival, and to treat diseases, conditions and / or symptoms associated with CSPG accumulation or with the activation and signal transduction of LAR family phosphatases.

[0208] Therefore, embodiments of this disclosure provide a method for treating diseases, symptoms, and / or conditions associated with the accumulation of CSPG or with the activation and signaling of LAR family phosphatases using the peptide dimers disclosed herein or compositions containing peptide dimers.

[0209] In various embodiments, a method is provided that includes treating a subject in need of a neurological condition, disease, or symptom selected from the group consisting of nerve injury, neurological disease caused by inflammation or autoimmunity, neurodegenerative disease, and neurological symptoms, said method comprising administering to the subject an effective amount of the pharmaceutical composition provided herein.

[0210] In some implementations, nerve injury is selected from the group consisting of acute nerve injury, traumatic brain injury (TBI), spinal cord injury, concussion, and stroke (including ischemic stroke, hemorrhagic stroke, and chronic stroke).

[0211] In some implementation schemes, neurological symptoms, diseases, or conditions are selected from the group consisting of Alzheimer's disease, Alzheimer's-related dementia, Lewy diffuse body diseases, senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis (MS), optic neuritis, Huntington's disease, Tourette's syndrome, hereditary motor and sensory neuropathy, diabetic neuropathy, progressive supranuclear palsy, Jakob-Creutzfeldt disease, epilepsy, and infectious diseases.

[0212] Various embodiments also provide for the use of peptide dimers or pharmaceutically acceptable analogs, salts or solvates thereof in the manufacture of medicaments for the treatment of neurological symptoms, diseases or conditions selected from the group consisting of nerve damage, neurological diseases caused by inflammation or autoimmunity, neurodegenerative diseases, neurological symptoms or combinations thereof.

[0213] Application and dosage

[0214] Generally, the pharmaceutical compositions disclosed herein can be administered to subjects of need via any suitable route, including, for example, oral (e.g., in capsule, suspension, or tablet form), systemic, or parenteral administration. Non-limiting exemplary routes include subcutaneous, intramuscular, intravenous, dermal, intranasal, rectal, ocular, surface, sublingual, and buccal administration.

[0215] In one embodiment, the pharmaceutical composition may be administered to the brain of a subject via lateral ventricle injection, typically within 100 hours of the occurrence of the injury (resulting in symptoms characterized by abnormal axonal growth of neurons in the central nervous system) (e.g., within 6, 12, 24, or 100 hours from the time of injury). The injection may be performed, for example, by drilling a hole in the subject's skull. In another embodiment, the therapeutic agent may be administered into the ventricles of the subject via a surgically inserted shunt, typically within 100 hours of the occurrence of the injury (e.g., within 6, 12, or 24 hours from the time of injury). For example, the injection may be made into the larger lateral ventricles, although injection may also be made into the smaller third and fourth ventricles. In yet another embodiment, the therapeutic agent may be administered by injection into the cisterna magna or lumbar region of the subject within 100 hours of the occurrence of the injury (e.g., within 6, 12, or 24 hours from the time of injury).

[0216] In another embodiment, the pharmaceutical composition may be administered to the subject at or near the site of injury, typically within 100 hours of the injury (e.g., within 6, 12, or 24 hours from the time of injury). Such administration may optionally be subcutaneous.

[0217] In another embodiment, the drug composition may be administered to the subject more than 100 hours after the injury. In some cases, administration may take place one week, several weeks, months, or years after the injury.

[0218] In some embodiments, the therapeutic dose or amount of the therapeutic agent or composition disclosed herein may range from about 0.1 mg / kg to about 500 mg / kg body weight. In some embodiments, the therapeutic dose or amount of the therapeutic agent or composition disclosed herein may range from about 1 to about 50 mg / kg. Generally, a treatment regimen according to this disclosure includes administering about 10 mg to about 1000 mg of the peptide dimer of this disclosure or its pharmaceutically acceptable analogues, salts, or solvates thereof to a subject requiring such treatment daily in a single or multiple dose. The therapeutic dose or amount will also vary depending on the route of administration and the possibility of co-administration with other agents.

[0219] However, it should be understood that the total daily dosage of the compounds and compositions disclosed herein will be determined by the attending physician within the bounds of reasonable medical judgment. The specific inhibitory dosage for any particular subject will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific therapeutic agent or composition used; the subject's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or in combination with the specific compound used; and similar factors well known in the medical field.

[0220] Reagent test kit

[0221] One aspect of this disclosure relates to a kit comprising a therapeutic agent containing a peptide dimer or a pharmaceutically acceptable salt or solvation thereof, or any composition containing the aforementioned peptide dimer or a pharmaceutically acceptable analogue, salt, or solvation thereof.

[0222] In some embodiments, the kit comprises one or more individual dosage forms, each containing a pharmaceutical composition comprising an effective amount or a single dose of the peptide dimer disclosed herein or a pharmaceutically acceptable salt, analogue, or solvation thereof for treating diseases, conditions, and / or symptoms associated with inhibition of neurological repair by chondroitin sulfate proteoglycan (CSPG) or with inhibition of neurological repair by LAR family phosphatases (such as PTPRD, PTPRF, and PTPRS).

[0223] In some embodiments, the kit may include a first container containing a peptide dimer or a pharmaceutically acceptable analogue, salt, or solvation according to the present disclosure in free form (such as lyophilized powder), and optionally, a second container containing a pharmaceutically acceptable solvent for dissolving the analogue, salt, or solvation. In use, the peptide dimer or a pharmaceutically acceptable analogue, salt, or solvation according to the present disclosure in free form may be mixed with the solvent to produce a final formulation for administration to a subject in need.

[0224] In some embodiments, the kit may also include instructions for use of the therapeutic agent or composition contained in the kit to treat diseases, conditions, and / or symptoms associated with the inhibition of neuronal repair by chondroitin sulfate proteoglycan (CSPG) or the inhibitory effect of LAR family phosphatases (such as PTPRD, PTPRF, and PTPRS) on neuronal repair.

[0225] It should be understood that wherever values ​​and ranges are provided herein, all values ​​and ranges covered by such values ​​and ranges are intended to be covered within the scope of this disclosure. Furthermore, all values ​​falling within these ranges, as well as the upper or lower limits of the ranges of values, are also covered by this application.

[0226] The embodiments described herein are not limited to specific methods, schemes, and reagents, and are therefore subject to variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit its scope. Except in the operational examples or as otherwise stated, all figures used herein to indicate the amount of components or reaction conditions should in all cases be understood to be modified by the term "about".

[0227] All identified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methods that may be used in conjunction with the present invention, as described in such publications. These publications are provided only for their disclosure prior to the filing date of this application. In this regard, nothing shall be construed as an admission that the inventor had no prior right to such disclosure by virtue of prior invention or for any other reason. All statements regarding the dates of these documents or representations regarding their content are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or content of these documents.

[0228] The following embodiments further illustrate various aspects of this disclosure. However, they are in no way intended to limit the teachings of this disclosure. It should be understood that these embodiments are given by way of illustration only. From the foregoing discussion and these embodiments, those skilled in the art can determine the essential features of embodiments of the invention. Various changes and modifications can be made to the invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. All publications, including patent and non-patent literature, referenced in this specification are expressly incorporated herein by reference.

[0229] Example

[0230] Unless otherwise indicated, the practice of this disclosure will use conventional techniques of organic synthesis, cell biology, cell culture and molecular biology, which are within the scope of the art.

[0231] Example 1: Synthesis and purification of peptide dimer compound 7

[0232] Compound 7: [Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala- 15 Glu-His-Thr-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser- 35 Ile-NH2]2 ( 11 Cys- 11’ Cys disulfide bond)

[0233] In this embodiment, a peptide dimer compound 7 containing a human PTPRS wedge domain was prepared by oxidative dimerization of compound 3 (the peptide monomer of SEQ ID NO:3 (TAT-Cys-PTPRS human variant)) according to the following raw materials and procedures. The average molecular weight of compound 7 is 8575.65 Da.

[0234] raw materials

[0235] Compound 3 (a peptide monomer containing the amino acid sequence of SEQ ID NO:3, “TAT-Cys-PTPRS”): 11.5 gm

[0236] Purified water (PWP) for purification: 1800 ml

[0237] Ethanol (AR): 200 ml

[0238] Ammonia solution: 21 ml, pH 9.9 ± 0.5

[0239] Acetic acid (HPLC grade): 13 ml, pH 4.5 ± 0.5

[0240] Preparation and purification procedure for peptide dimers (copper sulfate protocol)

[0241] 1) Add PWP and ethanol to the oxidation beaker and stir for 15 minutes to make the mixture homogeneous. The amount of PWP and ethanol required depends on the amount of peptide monomer.

[0242] 2) After stirring for 15 minutes, slowly add the corresponding amount of pure compound 3 to the beaker and mix until all the powder is dissolved to form a clear solution. Stir the reaction mixture for 15 minutes.

[0243] 3) After stirring for 15 minutes, check the initial pH of the reaction and adjust the pH to about 9 ± 0.5 with dilute ammonia solution, which is prepared by taking 100 mL of ammonia solution (25%) and diluting it to 1 L with PWP.

[0244] 4) Monitor the pH of the reaction mixture every hour. If the pH is below 9 ± 0.5, adjust the reaction mixture by adding additional dilute ammonia solution.

[0245] 5) After about 1 hour, add copper sulfate to the oxidation reaction mixture periodically.

[0246] 6) Take samples every hour and analyze the reaction progress by HPLC, and continue the oxidation reaction until the free peptide monomer compound 3 is detected by HPLC to be less than 4 ± 0.5%.

[0247] 7) Once the presence of compound 3 is confirmed by HPLC to be less than 4 ± 0.5%, the pH of the reaction mixture is adjusted by adding acetic acid until the pH is about 4.5 ± 0.5.

[0248] 8) After oxidation, adjust the reaction mixture to a pH of about 4.5 ± 0.5, and filter the oxidation reaction mixture through 5-micron filter paper under vacuum into a receiving container for receiving the filtered solution.

[0249] 9) Collect the filtered solution and purify it by preparative HPLC.

[0250] 10) Freeze-drying of the peak eluent containing pure compound 7 from HPLC to obtain freeze-dried compound 7.

[0251] The resulting compound is the acetate of homodimer compound 7, which has the same monomeric subunit containing the amino acid sequence of SEQ ID NO:3.

[0252] The obtained peptide dimers were highly purified, with a purity greater than 96.3%.

[0253] Example 2: Preparation of peptide dimer compound 8

[0254] Compound 8: [Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala- 15 Glu-His-Met-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser- 35 Ile-NH2]2 ( 11 Cys- 11’ Cys disulfide bond)

[0255] In this embodiment, following the procedure described above in Example 1, a peptide dimer compound 8 containing rat and mouse PTPRS wedge domains was prepared by oxidative dimerization of compound 4 (the peptide monomer of SEQ ID NO:4) (TAT-Cys-PTPRS rat variant), except that the oxidation reaction continued until the free peptide monomer compound 4 was detected by HPLC to be less than 8 ± 0.5%. The average molecular weight of compound 8 is approximately 8635.84 Da.

[0256] raw materials

[0257] Compound 4 (a peptide monomer containing the amino acid sequence of SEQ ID NO:4, “TAT-Cys-PTPRS (rat)”): 1.1 gm

[0258] Ethanol (AR): 200 ml

[0259] Dilute ammonia solution: 440 ml, pH 9.9 ± 0.5

[0260] Acetic acid (HPLC grade): 13 ml, pH 4.5 ± 0.5

[0261] The resulting compound was a homodimer of the acetate salt of compound 8, having the same monomeric subunits containing the amino acid sequence of SEQ ID NO:4. The purity of the purified salt of compound 8 was examined by chromatography. Figure 1 This is an example of a chromatogram showing the purity of the obtained product. For example... Figure 1 As can be seen, the obtained peptide dimer is highly purified, with a single peak corresponding to the purified peptide dimer and a purity greater than 96.3%.

[0262] Example 3: Efficacy evaluation of compound 8 in treating spinal cord injury

[0263] Target

[0264] The aim of this study was to test the efficacy of the novel peptide dimer (intracellular σ-blocking peptide (ISP) dimer) according to this disclosure for behavioral changes in a rat model of thoracic traumatic spinal cord injury (SCI).

[0265] Materials and methods

[0266] animal

[0267] Adult female Lewis rats (~160 g upon arrival) from Envigo were used as subjects receiving treatment in the study. Each rat was assigned a unique identification number (PGI ID and tail tag) and housed in ventilated cages. All rats were examined, treated, and weighed prior to the start of the study to ensure they were adequately healthy and fit. A 12 / 12 light / dark cycle was maintained throughout the study. Room temperature was maintained between 20°C and 23°C, and relative humidity was maintained at approximately 50%. random Food and water were provided. Rats were randomly assigned to treatment groups. Body weight was measured twice weekly during the study period.

[0268] Formulation and dosage

[0269] The lyophilized compound 8 was dissolved in saline and injected subcutaneously into the animals once daily at a volume of 500 µl from day 1 to day 49 following spinal cord injury.

[0270] The dosage concentration was 0.8 mg / ml, corresponding to 400 µg / rat. After reconstitution, the solution was aliquoted and stored at -20°C until administration.

[0271] Treatment group

[0272] The following two groups each used 12-13 rats: 1. Control group: SCI + saline 2. Test group: SCI + compound 8 salt solution.

[0273] Spinal cord injury and postoperative care

[0274] Spinal cord injury (SCI) was surgically induced in rats using the Infinite Horizon (IH) impactor (200 kDyn force) described by Scheff et al. (2003). The surgery was performed under deep anesthesia in a designated area using an aseptic procedure. Isoflurane (4% for induction, 2% for maintenance) and O2 (300 cm⁻¹) were used. 3 Rats were anesthetized with a mixture of [unspecified substance] / min. Hair was removed from the surgical site with scissors, and the skin was cleaned with a disinfectant. To maintain body temperature, the animals were placed on a thermostatic blanket system. The T8 vertebra of the rat was positioned externally and incised to expose the spine from T6 to T11. A laminectomy was performed using a surgical microscope to expose the dorsal spinal cord at the level of the thoracic (T8) vertebra. The spine was stabilized by clamping the nearest rostral and caudal vertebral processes, and an injury was inflicted using an IH impactor device with a force of 200 kDyn. After each injury, the IH plot and impact data were reviewed and recorded. Rats showing an impact force more than 10% higher or lower than expected and / or any abnormalities on the force / displacement impact plot were excluded from the study. Additionally, animals scoring higher than 1 on the Basso, Beattie, and Bresnahan (BBB) ​​scale 1 day after SCI were also excluded from the study.

[0275] Following the injury, the muscles were closed layer by layer using 4-0 Ethicon vicryl sutures, and the skin was closed with a wound clip, which was removed 7-10 days after SCI.

[0276] Postoperatively, the animals were kept in warm cages with easy access to water and food. Rats received postoperative care including antibiotics (amoxicillin administered with diet for 7–10 days); analgesia (buprenorphine 0.03 mg / kg, SC, for 2 days); and fluids (6–8 cc lactated Ringer's solution, SC, twice daily for 3 days). The bladder was squeezed twice daily until spontaneous urination occurred.

[0277] Bladder compression and weight

[0278] Starting the day after injury, the animals were observed twice daily, and the bladder was manually squeezed using gentle intra-abdominal pressure. Any abnormal urinary appearance (i.e., cloudy urine, hematuria) was recorded, and bladder size was estimated by trained researchers to obtain a classification score (bladder score) (empty bladder = 0; microbladder = 1; small bladder = 2; medium bladder = 3; large bladder = 4). Urinary complications (hematuria, foul-smelling urine, urine stains on fur) were reported and treated twice daily with SC fluid, and antibiotics were administered if the problems persisted. Bladder squeezing continued until spontaneous urination was observed (bladder emptying for 3 consecutive days).

[0279] During the first 7 weeks following the injury, measure the animal's weight twice weekly, then once weekly until the endpoint. Evaluate the animal's health and survival during bladder compression, weight, and behavioral tests, report any health problems, and discuss them with a veterinarian as necessary.

[0280] Basso, Beattie, and Bresnahan (BBB) ​​Exercise Rating Scale

[0281] Motor changes were assessed using the open field movement test (the Ohio BBB Motor Rating Scale, developed by Basso et al., 1995). During each test, rats were observed moving around an empty shallow pool for 4 minutes. Hind limb joint movement, weight-bearing, and interlimb coordination were scored according to the BBB scale. This scale tracks the progressive recovery of hind limb function after thoracic SCI. The scale is divided into three sections reflecting different stages of recovery. Scores of 0 to 7 represent the early stage of recovery, in which independent movement of the three joints (hip, knee, and ankle) is restored; scores of 8 to 13 describe the intermediate stage of recovery, in which paw placement, stepping, and forelimb-hindlimb coordination are restored; and scores of 14 to 21 assess the late stage of recovery, in which interdigital distance, primary paw position, trunk stability, and tail position are restored during the stepping phase. The BBB was assessed on days 1, 4, and 7 post-injury, and then weekly until the study was completed.

[0282] The BBB score obtained on day 1 post-SCI was used to maintain concordance with injury. Prior to injury, all animals exhibited normal movement (21 points). On day 1 post-SCI, only animals with a BBB score of 0 (no observable hind limb movement) or 1 (slight movement of one or two hind limb joints) were included in the study and assigned to the treatment group. The BBB scores obtained on day 1 post-SCI were used to balance the treatment groups.

[0283] Statistical analysis

[0284] The data were analyzed using analysis of variance (ANOVA), followed by post-hoc comparisons where appropriate. If p A value < 0.05 is considered significant. Data are expressed as the mean and the standard error of the mean (SEM).

[0285] result

[0286] weight

[0287] The effect of compound 8 on the body weight of rats in the control and test groups after SCI was measured twice weekly for the first 7 weeks following injury, then weekly until week 13. Results showed no significant difference in body weight between the two groups and during the treatment period. These results indicate that the test compound had no adverse effect on body weight during the study.

[0288] Basso, Beattie, and Bresnahan (BBB) ​​Exercise Rating Scale

[0289] The effect of compound 8 on open field motion performance was evaluated using the BBB scale. Figures 2 to 4 As shown in the figure, Figure 2 The BBB scores of rats in the test group compared to the control group were plotted on day 7 after SCI. Figure 3 The BBB scores of rats in the test group compared to the control group were depicted at week 7 after SCI, and Figure 4 Describe the BBB scores of rats in the test group compared to the control group at week 12 after SCI. Figure 2 As can be seen, on day 7 post-SCI, the BBB scores of all 13 rats in the test group treated with compound 8 ranged from 2.5 to 7, with 6 rats having BBB scores of 6 to 7. In contrast, on day 7 post-SCI, 5 of the 12 rats in the control group treated with saline alone had a BBB score of 4, and 2 of these 12 rats had a BBB score of approximately 0.5.

[0290] like Figure 3 As shown, at week 7 after SCI, in the test group, 6 rats had a BBB score of 10 and 2 rats had a BBB score of 11, while in the control group, most rats had a BBB score of 8 to 9, and only 3 rats reached a BBB score of 10. In the control group, no rats reached a BBB score of 11.

[0291] like Figure 4 As shown, at week 12 after SCI, most rats in the test group treated with compound 8 (8 out of 13) had a BBB score of approximately 10 or 11, while most rats in the control group treated with saline alone (8 out of 12) had a BBB score of approximately 8 to 9.

[0292] The results showed that compound 8 promoted the recovery of hindlimb joint movement, weight support, and interlimb coordination in SCI rats.

[0293] Bladder function data

[0294] Following injury, the animals exhibited bladder dysfunction requiring manual bladder compression. During bladder compression, trained researchers estimated the size of each bladder and obtained a semi-quantitative score. By summing the bladder size scores from morning and afternoon bladder compressions, an estimate of daily urinary retention was obtained. Figure 5 The average weekly urinary retention of rats in the test group treated with saline solution of compound 8 is shown compared to that of rats in the control group treated with the medium control (saline).

[0295] Rats treated with saline solution of compound 8 were observed to exhibit a tendency to have smaller bladders during daily examinations, resulting in less urinary retention compared to rats treated with the medium control. Figure 5 As shown, this trend became apparent starting in week 4 post-SCI. Throughout the study period, most animals required bladder compression once or twice a day, and only four rats (one from each experimental group) were able to resume spontaneous urination between days 29 and 34 post-SCI.

[0296] Urinary tract complications were monitored, including hematuria, foul-smelling urine, and urine stains on the fur. A recurrent complication was considered to occur when new complications were observed after the resolution of a previous event. If complications persisted after another cycle of antibiotics, or if the bladder became uncompressible or a general deterioration in health was observed, the animal was euthanized.

[0297] Observe, record, and treat urinary tract complications as needed, such as recurrent hematuria, urine stains on fur, and urinary tract infections. Table 7 summarizes the percentage of complications observed in each experimental group.

[0298] Table 7. Urinary system complications identified during the study.

[0299] As shown in Table 7, the frequency of urinary tract complications was lower in rats receiving compound 8 / saline than in rats receiving the mediator control.

[0300] Summarize

[0301] During the study, acute administration of compound 8 had no adverse effect on the body weight of rats. Animals receiving compound 8 experienced a lower frequency of bladder complications compared to the control group. Animals receiving compound 8 showed a reduction in estimated bladder size, which was particularly pronounced between weeks 8 and 13.

[0302] Example 4 – Effect of Compound 8 on Walking Recovery in SCI Animals

[0303] In this experimental example, the effect of compound 8 on walking recovery in an animal model of spinal cord injury was evaluated.

[0304] Materials and methods

[0305] animal

[0306] Adult female Lewis rats (~160 g upon arrival) from Envigo were used as subjects receiving treatment in the study. Each rat was assigned a unique identification number (PGI ID and tail tag) and housed in ventilated cages. All rats were examined, treated, and weighed prior to the start of the study to ensure they were adequately healthy and fit. A 12 / 12 light / dark cycle was maintained throughout the study. Room temperature was maintained between 20°C and 23°C, and relative humidity was maintained at approximately 50%. random Food and water were provided. Rats were randomly assigned to treatment groups. Body weight was measured twice weekly during the study period.

[0307] Inflict spinal cord injury (SCI) on the animal and provide postoperative care according to the method described in Example 3.

[0308] Formulation and dosage

[0309] The lyophilized compound 8 was dissolved in saline and injected subcutaneously into the animals once daily at a volume of 500 µl from day 1 to day 49 following spinal cord injury.

[0310] The target dose concentration is 0.8 mg / ml. After reconstitution, the solution is aliquoted and kept at -20°C until administration.

[0311] Treatment group

[0312] The following two groups each used 12-13 rats: 1) Control group: SCI + saline 2) Test group: SCI + compound 8 salt solution.

[0313] Evaluation of occasional walking versus frequent walking

[0314] The inverse Kaplan-Meier plot was generated based on the Basso, Beattie, and Bresnaham (BBB) ​​open field movement assessment described above in Example 3. The Kaplan-Meier plot is a statistical method for estimating the gain (threshold) of a certain behavior as a percentage of the population over a given time period. Animals reaching the threshold (as defined below) are represented as an upward step, where the Y-axis represents the cumulative percentage of the group reaching the threshold, and the X-axis indicates the time since the SCI.

[0315] The Body Body Scale (BBS) is an ordinal scale ranging from 0 to 21, where 0 indicates complete loss of motor function in the hind limb and 21 indicates normal motor function. The BBS assesses the recovery of various motor actions, including the ability to bear weight, stand, walk, properly position the foot, and coordinate. A step is defined as when the hind limb paw makes plantar contact with a weight-bearing support, followed by the hind limb being propelled forward and re-establishing plantar contact with the weight-bearing support.

[0316] Resumption of Occasional Walking: Occasional walking is defined as a success rate of less than or equal half of the attempted steps, corresponding to a BBB score of 10. Record the week following the animal achieving an SCI of 10 or higher and plot it on a Kaplan-Meier chart as defined above.

[0317] Reinstatement of frequent walking: Frequent walking was defined as a success rate of more than half of the attempted steps, corresponding to a BBB score of 11. Animals were recorded for one week after achieving a SCI of 11 or higher, and plotted on a Kaplan-Meier plot as defined above.

[0318] Statistical methods: Kaplan-Meier curves were analyzed using the log-rank test. The statistical significance of the effects of compounds 4 and 8 on motor and bladder function was assessed by repeated measures ANOVA (RM-ANOVA) with Geisser-Greenhouse correction for non-sphericity of the data and Tukey correction for multiple comparisons, or by nonparametric Friedman's test with Dunn correction for multiple comparisons. The choice of RM ANOVA or Friedman's test for analyzing each type of data (BBB score, BBB sub-score, and bladder score) was determined by testing the normality of the data distribution for each functional score across all treatment cohorts. RM ANOVA was chosen when all three treatment cohorts of SCI rats (saline, compound 4, and compound 8 alone) passed the normality of the data distribution test. The Friedman's test was used when at least one treatment cohort failed the normality of the data distribution test. Additional quantitative evaluation of the therapeutic effects of compounds 4 and 8 was performed by calculating and comparing the percentage of animals achieving predetermined thresholds of functional improvement in BBB, BBB sub-scores, and bladder scores.

[0319] result

[0320] Compound 8 improves the recovery of occasional walking in SCI animals

[0321] Figure 6 This is a graph depicting the recovery of occasional walking in SCI rats treated with compound 8 compared to SCI rats treated with the control drug. (See figure.) Figure 6As can be seen, during the first two weeks following spinal cord injury, neither the test nor control groups of SCI rats were able to walk, even occasionally. Some degree of recovery of occasional walking (less than 10%) was observed in both groups of SCI rats. From the fourth week post-injury, sustained recovery of occasional walking was detected in SCI rats treated with compound 8, with the recovery rate reaching 50% in less than 6 weeks and approaching 80% by week 12. In contrast, the recovery of occasional walking in the control group of SCI rats was significantly slower than that observed in SCI rats treated with compound 8, and did not even reach 50% by week 12.

[0322] The results showed that compound 8 provides an effective treatment for spinal cord injury and significantly improves the recovery of occasional walking in animals with spinal cord injury.

[0323] Compound 8 improves the recovery from frequent walking in SCI animals

[0324] Figure 7 This is a graph depicting the recovery of frequent walking in SCI rats treated with compound 8 compared to SCI rats treated with the control drug. (See figure.) Figure 7 As can be seen, frequent walking was not detected in SCI rats during the first three weeks following spinal cord injury. Recovery of frequent walking was detected in SCI rats treated with compound 8 three weeks post-injury, with the recovery continuing and reaching nearly 50% by week 12. In contrast, frequent walking was not detected in SCI rats treated with the mediator control until week 9 post-injury, and only a slight improvement (10%) was observed even after 12 weeks.

[0325] The results showed that compound 8 effectively improved the recovery of frequent walking in animals with spinal cord injury.

[0326] Example 5 – Comparison of BBB sub-scores in animals treated with compound 8 and compound 4

[0327] In this embodiment, the effect of compound 8 on improving the recovery of toe gaps, claw position, trunk stability and tail use, which are independent of forelimb-hindlimb coordination, was evaluated by comparing it with compound 4 (a monomer of the TAT-Cys-PTPRS rat variant) and saline.

[0328] Compound 4: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala- 15 Glu-His-Met-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser- 35 Ile-NH2

[0329] Materials and methods

[0330] Compound 4 was synthesized and purified by HPLC, with a purity greater than 95%.

[0331] Animals were prepared according to the method described above in Example 3.

[0332] The lyophilized compounds 8 and 4 were dissolved in saline solution and injected subcutaneously into the animals once daily at a volume of 500 µl from day 1 to day 49 after spinal cord injury.

[0333] The target dose concentration is 0.8 mg / ml. After reconstitution, the solution is aliquoted and kept at -20°C until administration.

[0334] The following three groups each used 12-13 rats: 1) Control group: SCI + saline alone 2) Test group: SCI + compound 4 salt solution 3) Test group: SCI + compound 8 salt solution.

[0335] Inflict spinal cord injury (SCI) on the animal and provide postoperative care to the animal according to the method described in Example 3.

[0336] BBB sub-score measurement

[0337] The BBB subscale quantifies the recovery of toe spacing, paw position, trunk stability, and tail use, independent of forelimb-hindlimb coordination. The BBB subscale is calculated using observations recorded in the BBB record sheet. During the BBB test, hind paw primary position (parallel vs. rotation), toe spacing, trunk stability, and tail position (upper, lower, middle) are assessed in animals with a BBB score of 10 or higher. Animals with a BBB score below 10 are considered to have a BBB subscale score of "0". A maximum subscale score of 13 can be achieved per rat using the scales shown in Table 8.

[0338] Table 8: Motion characteristics considered in BBB sub-score calculation

[0339] result

[0340] As described above in Example 3, high scores (14-21) on the BBB scale quantify fine motor movements related to hind limb movement, including toe spacing, primary paw position, trunk stability, and tail position. These fine motor movements are only considered when the animal exhibits sustained coordination. BBB sub-scores are applied to quantify these improvements unrelated to coordination.

[0341] Figure 8A and Figure 8B The effects of compound 8, compared with compound 4 and saline control, on improving the recovery of fine motor movements related to hind limb toe spacing, claw position, trunk stability, and tail position in SCI animals were described, as measured by BBB sub-scores. Figure 8A Data is presented as mean ± SEM. N = 12-13 / group. For example... Figure 8A As can be seen, after SCI, all rats exhibited a BBB sub-score of 0 during the first 14 days of behavioral assessment, indicating no recovery during this period. Treatment with compound 8 resulted in an accelerated onset of functional improvement compared to the mediator control (saline alone) and the corresponding effects of compound 4. Functional improvement was observed on day 21 in the compound 8 cohort, day 42 in the compound 4 cohort, and day 35 in the control (saline alone) cohort. The sustained trend of functional improvement was evident in the rat cohort treated with compound 8 but not in the control (saline alone) or compound 4 cohorts.

[0342] Figure 8B A violin plot depicts the distribution of weekly mean BBB subscores for individual saline, compound 4, and compound 8 treatment cohorts in SCI rats. Each data point represents the weekly mean BBB subscore for the individual saline (control), compound 4, and compound 8 treatment cohorts, as labeled on the X-axis. The numbers next to the data points indicate the number of days after SCI for calculating the weekly mean BBB subscore for individuals and treatment cohorts. Statistical comparisons of treatment effects were performed using the Friedman test (repeated measures ANOVA for nonparametric values) with Dunn correction for multiple comparisons, and a p-value less than 0.05 was defined as statistical significance. Figure 8B As can be seen, treatment with compound 8 in SCI rats had a statistically significant effect on BBB subscale scores compared to the effect of the control medium (saline alone) (p=0.0008) or the effect of compound 4 (p=0.0201). No statistically significant difference was observed between the cohort treated with compound 4 and the cohort treated with saline alone (control) (p>0.9999).

[0343] Figure 9 The percentage of SCI rats treated with compound 8 achieving a BBB score of 1 or higher was further depicted compared to SCI rats treated with compound 4 or saline alone. Solid bars represent data corresponding to the time period of saline, compound 4, or compound 8 treatment. Striped bars represent data collected after treatment was discontinued. Figure 9As can be seen, at each time point after SCI, e.g., days 28, 42, 63, 70, 77, and 84, the percentage of rats achieving a BBB subscore of 1 or better (the percentage of achievers) was higher in the group treated with compound 8 compared to the group treated with compound 4 or saline alone. Treatment with compound 8 resulted in continued functional improvement even after cessation of compound 8 administration. In contrast, no similar effect was observed in the cohort of SCI rats treated with compound 4 or saline alone.

[0344] Example 6 – Pharmacokinetic characteristics of compound 7

[0345] Using compound 7 as an example, the pharmacokinetic characteristics of the Tat-Cys-PTPRS wedge-domain dimer in rat plasma after intravenous or subcutaneous administration were investigated. In this study, saline solutions of compound 7 prepared according to Example 3 were administered intravenously to three rats at a dose of 1.75 mg / kg (rat body weight), and subcutaneously to another three rats at a dose of 10.5 mg / kg (rat body weight). The concentration of compound 7 in rat plasma was measured after administration. These results are illustrated in... Figure 10 The figure shows the pharmacokinetic characteristics of the Tat-Cys-PTPRS wedge domain dimer (compound 7) in rat plasma after intravenous or subcutaneous administration at doses of 1.75 or 10.5 mg / kg.

[0346] Example 7 – Comparison of BBB and bladder scores in animals treated with compound 8 and compound 4

[0347] In this study, the effect of compound 8 on improving open field mobility and bladder function recovery in SCI rats was further evaluated by assessing BBB and bladder scores according to the method described in Example 3, compared with compound 4.

[0348] In this assessment, each of the following groups included 12–13 rats: (1) Control group 1: SCI + saline alone; (2) Control group 2: SCI + saline solution of compound 4; (3) Test group: SCI + compound 8. Treatment began on day 1 post-injury and continued until day 49 post-injury (after administration of the last dose). The observation period after treatment began on day 50 post-injury and continued until the end of the study on day 84 post-injury.

[0349] The results of the BBB score assessment are in Figure 11 , Figure 12 and Figure 13 As shown in the image. Figure 11A violin plot depicting the distribution of weekly mean BBB scores for each of the three treatment cohorts is shown, as labeled on the X-axis. Each data point represents the weekly mean BBB score for the individual saline (control), compound 4, and compound 8 treatment cohorts, as labeled on the X-axis. The numbers next to the data points indicate the number of days after SCI calculation of the weekly mean BBB score for individuals and treatment cohorts. Statistical comparisons of treatment effects were performed using repeated measures ANOVA with Tukey correction for multiple comparisons, and a p-value less than 0.05 was defined as statistical significance.

[0350] The results of the BBB score assessment are in Figure 11 , Figure 12 and Figure 13 As shown in the image. Figure 11 A violin plot depicting the distribution of weekly mean BBB scores for each of the three treatment cohorts is shown, as labeled on the X-axis. Each data point represents the weekly mean BBB score for the individual saline (control), compound 4, and compound 8 treatment cohorts, as labeled on the X-axis. The numbers next to the data points indicate the number of days after SCI calculation of the weekly mean BBB score for individuals and treatment cohorts. Statistical comparisons of treatment effects were performed using repeated measures ANOVA with Tukey correction for multiple comparisons, and a p-value less than 0.05 was defined as statistical significance.

[0351] like Figure 11 As can be seen, rats treated with compounds 4 and 8 exhibited higher BBB scores compared to rats treated with saline alone. Both compounds 4 and 8 resulted in significant improvements in motor performance as measured by BBB scores. The corresponding p-values ​​are as follows: for the comparison of compound 4 with the SCI rat cohort treated with saline alone, p < 0.0001; for the comparison of compound 8 with the SCI rat cohort treated with saline alone, p < 0.0001. Furthermore, compound 8 was significantly more effective than compound 4 in improving BBB scores (p = 0.0017). Figure 12The percentage of rats in each group who achieved a BBB score of 10 or higher after SCI was depicted. Solid bars represent data corresponding to the time period of administration of the test product (saline alone, compound 4, or compound 8). Striped bars represent data collected during the observation period after discontinuation of aggressive treatment with saline alone, compound 4, or compound 8. The results showed that less than 20% of SCI rats treated with saline alone had a BBB score of 10 or higher during the period from day 28 to day 84 after SCI treatment, compared with approximately 30% to approximately 60% of SCI rats treated with compounds 4 and 8. The overall percentage of rats achieving a BBB score of 10 or higher during treatment was higher in group (3) compared with rats in group (2). More than half of the rats in group (3) achieved a BBB score of 10 or higher 70 days after SCI.

[0352] Figure 13 The percentage of rats in each group who achieved a BBB score of 11 after SCI is depicted. Solid bars represent data corresponding to the time period of administration of the test product (saline alone, compound 4, or compound 8). Striped bars represent data collected during the observation period after discontinuation of aggressive treatment with saline alone, compound 4, or compound 8. Figure 13 As can be seen, over 20% and approximately 50% of rats treated with compound 8 achieved a BBB score of 11 on days 77 and 84, respectively. These results were not observed in rats treated with saline alone or with compound 4, suggesting that compound 8 is more effective than compound 4 in promoting the recovery of motor function as assessed using the BBB scoring index.

[0353] Examples of bladder scoring assessment results are shown in Figure 14 and Figure 15 middle. Figure 14 A violin plot depicting the distribution of weekly mean bladder scores for each of the three treatment cohorts in SCI rats is shown, as labeled on the X-axis. Each data point represents the weekly mean bladder score for the individual saline (control), compound 4, and compound 8 treatment cohorts, as labeled on the X-axis. The numbers next to the data points indicate the number of days after SCI for calculating the weekly mean bladder score for individuals and treatment cohorts. Statistical comparisons of the effects of compound 4 and compound 8 treatments were performed using repeated measures ANOVA with Tukey correction for multiple comparisons, and a p-value less than 0.05 was defined as statistical significance. Figure 14 As can be seen, the average weekly bladder score of rats treated with compound 8 was significantly lower than that of rats treated with compound 4 or saline alone. Figure 14(p=0.0001 between the saline and compound 8 treatment groups, and p=0.0085 between the compound 8 and compound 4 treatment groups). This result indicates that rats treated with compound 8 tended to exhibit smaller bladders during daily examinations, and therefore had less urinary retention compared to rats treated with compound 4 or saline alone.

[0354] The percentage of rats achieving a bladder score of 2 or lower (better) during treatment after SCI is illustrated in [example]. Figure 15 Solid bars represent data corresponding to the time period of administration of the test product (saline alone, compound 4, or compound 8). Striped bars represent data collected during the observation period after discontinuation of aggressive treatment with saline alone, compound 4, or compound 8. Results showed that, overall, more rats in the compound 8-treated group exhibited better bladder scores compared to rats treated with saline alone or compound 4 alone. Starting at week 6 post-SCI, approximately 25% of rats treated with compound 8 showed a bladder score of 2 or better. Bladder scores in compound 8-treated rats continued to improve from week 8 to week 13, with over 40% of rats achieving a score of 2 or better between weeks 10 and 13. These results were not observed in rats treated with saline alone or compound 4 alone.

[0355] At the end of the study, the performance of compound 8 compared with compound 4 and saline alone was evaluated by calculating the percentage of animals that reached a predetermined threshold of functional improvement in the following categories: (i) simultaneous improvement in BBB and bladder scores, (ii) improvement in motor function alone (BBB score) but no improvement in bladder score, and (iii) improvement in bladder score alone but no improvement in BBB score. Figure 16 The percentage of rats in each of the three categories is plotted as shown on the X-axis. Another group (labeled "combinations") represents the percentage of rats in each treatment cohort (saline, compound 4, and compound 8 alone) who achieved improvement in any of the first three categories. At the end of the study (day 84), the predetermined threshold for functional improvement was set at a BBB score of at least 10 and a bladder score of 2 or lower.

[0356] like Figure 16As shown, at the end of the study, nearly 50% of the SCI rats treated with compound 8 achieved a BBB score of at least 10 and a bladder score of 2 or lower, while only about 17% of the rats treated with saline or compound 4 alone achieved this combined improvement. Improvement in BBB score only (no improvement in bladder score) was observed in about 40% of the rats treated with compound 4, compared to about 17% of the rats treated with saline alone and about 15% of the rats treated with compound 8. Furthermore, about 8% of the rats treated with compound 8 showed improvement in bladder score only (a decrease in bladder score). In contrast, rats treated with compound 4 did not show improvement in bladder score only compared to rats treated with compound 8. The combined functional improvement rates (the percentages of simultaneous improvement in BBB and bladder scores, BBB score only, and bladder score only) in SCI rats treated with saline, compound 4, or compound 8 alone were about 33%, about 60%, and about 70%, respectively.

[0357] Figure 17 The percentage of rats achieving a BBB score of 11 and / or a bladder score of 2 or lower at the end of the study was depicted. These results further indicate that compound 8, rather than compound 4 alone, primarily caused simultaneous improvement in both BBB and bladder scores. At the end of the study, approximately 40% of SCI rats treated with compound 8 achieved simultaneous improvement at the level of a BBB score of 11 and a bladder score of 2 or lower, compared to only approximately 8% in each of the saline and compound 4 cohorts alone. In the saline and compound 4-only cohorts of SCI rats, no SCI rats achieved a BBB score of 11 alone, compared to approximately 8% in the compound 8 cohort. The combined functional improvement rate (the combined percentage of SCI rats achieving a BBB score of 11 alone, a bladder score of 2 or lower alone, and a combined percentage of achieving both a BBB score of 11 and a bladder score of 2 or lower) was approximately 60% / such a functional improvement rate was not observed in rats treated with saline or compound 4 alone.

[0358] Overall, Figure 16 and Figure 17 The results shown indicate that compound 8 is more effective than compound 4 in improving both motor function (BBB score) and bladder function (bladder score), while the effect of compound 4 is mainly limited to improvement in motor function only, and the magnitude is low (the maximum BBB score in the compound 4 cohort is 10, compared to 11 in the compound 8 cohort).

[0359] Example 8 – In vitro stability of compounds 3 and 7 in simulated biological matrices (buffered saline and plasma)

[0360] The stability of compound 7 (human variant) in physiological buffer (Hanke's balanced salt solution, HBSS) and plasma was evaluated compared with that of compound 3.

[0361] Compound stability was defined as the percentage recovery of the compound after incubation in the presence of the corresponding plasma for 5 min, 15 min, and 30 min.

[0362] Materials and methods

[0363] 200 µL of a 1.6 µM solution of compounds 3 and 7 were incubated in a matrix (Hank's equilibrium salt solution or plasma) at 37 °C for 0, 5, 15, or 30 min. After incubation, 200 µL of acetonitrile was added, vortexed, and then acidified with 45 µL of pure acetic acid. After vortexing again, the resulting mixture was centrifuged at 14,000 rpm for 5 min, and the supernatant was collected for analysis.

[0364] The levels of compound 3 or compound 7 in the supernatant sample prepared as described above were determined using LC-MS / MS as follows: 40 µL of a 5 µM aqueous solution of compound 7 in 10% acetic acid was added to 160 µL of supernatant. 20 µL of a 5 µM aqueous solution of compound 3 in 10% acetic acid was added to 180 µL of supernatant. The treated sample (20 µL) was injected onto an ACEExcel 2 C18 column (3.0 mm x 75 mm, 2.0 µm).

[0365] The percentage of recovery of compounds 3 and 7 after incubation time points of 0 minutes (at the start of the experiment), 5 minutes, 15 minutes, and 30 minutes was calculated by dividing the corresponding peak area by the peak area obtained from the sample prepared without incubation (incubation time of 0 minutes). The percentage of recovery at time 0 was defined as 100% recovery.

[0366] The presence of compound 3 or compound 7 in the chromatographic peak fraction was verified using an Agilent 1200 high-performance liquid chromatography (HPLC) system coupled with a Sciex API 3200 triple quadrupole mass spectrometer.

[0367] Figure 18 The stability of compound 7 in physiological buffer (HBSS) compared to compound 3 was depicted. It can be seen that compound 7 is highly stable in HBSS, with a recovery of 90% after incubation with HBSS for at least 30 minutes. In contrast, compound 3 is less stable in HBSS, with a recovery of approximately 42% after 30 minutes.

[0368] result

[0369] Figure 19 , Figure 20 and Figure 21 The stability of compound 7 in rat, dog, and human plasma compared to compound 3 was described. The results showed that compound 7 was highly stable in plasma, with 92% recovery in rat plasma, 100% recovery in dog plasma, and 99% recovery in human plasma after 30 minutes of incubation. In contrast, compound 3 exhibited lower stability in plasma.

[0370] Example 9 - Different colloidal behaviors of compounds 7 and 3

[0371] The self-assembly modes of compounds 7 and 3 were analyzed using negative staining transmission electron microscopy.

[0372] Materials and methods

[0373] Compounds 3 and 7 were dissolved in sterile water or a sterile aqueous solution of 0.9% (w / v) sodium chloride to obtain a 20 mg / mL solution. A Formvar / carbon-coated grid was exposed to a 15 mL drop of either compound 3 or compound 7 solution for approximately 1 minute, blotted dry, then exposed to a 15 mL drop of sterile water for 10 seconds, blotted dry again, and then stained with a 2% (w / v) uranyl acetate aqueous solution for 30 seconds. The stained sample was then exposed to a 15 mL drop of sterile water for 10 seconds, blotted dry, and examined by transmission electron microscopy.

[0374] result

[0375] Figure 22 A set of transmission electron microscopy (TEM) images is presented, showing the colloidal structures formed by the self-assembly of compounds 3 (Figs. a and c) and 7 (Figs. b and d) in water (Figs. a and b) and isotonic saline (Figs. c and d). The images shown in Figs. a and b indicate that both compounds 3 and 7 form similar rod-like nanostructures. The images shown in Figs. c and d show that compound 3 forms an amyloid-like fibrous network, while compound 7 forms distinct, larger helical colloidal particles, which were not observed in compound 3. These data illustrate the different physicochemical behavior of compounds 3 and 7 in a simulated biological matrix (isotonic saline).

[0376] The scope of the disclosed subject matter is not limited to the specific embodiments and examples described herein. In fact, various modifications to this disclosure, in addition to those described, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0377] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by full reference and for all purposes, as if each individual reference (e.g., publications, patents, or patent applications) were specifically and individually indicated to be incorporated herein by full reference for all purposes. Other embodiments are set forth in the following claims.

Claims

1. A pharmaceutical composition comprising a peptide, the peptide being a dimer comprising a first monomer covalently cross-linked with a second monomer, each monomer comprising a domain containing an amino acid sequence of a cytoplasmic wedge domain derived from a receptor-type protein tyrosine phosphatase (PTPR), and wherein the mass ratio of the dimer to the free monomer in the pharmaceutical composition is greater than 1:

20.

2. The pharmaceutical composition of claim 1, wherein the ratio is greater than 1:

1.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the ratio is greater than 4:

1.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the ratio is greater than 10:

1.

5. The pharmaceutical composition of any one of claims 1-4, wherein the dimer comprises a transport portion connected to the domain via a cysteine ​​residue or a peptide linker containing a cysteine ​​residue.

6. The pharmaceutical composition of claim 5, wherein the transport portion comprises a TAT sequence.

7. The pharmaceutical composition of claim 6, wherein the TAT sequence comprises at least 70% identical amino acid sequences to the amino acid sequences of SEQ ID NO: 5, 6 or 7.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the dimer improves nerve cell repair.

9. The pharmaceutical composition of any one of claims 1-7, wherein the domain is selected from the group consisting of the PTPRF wedge domain, the PTPRD wedge domain, and the PTPRS wedge domain, and variants having at least 70% identity with them.

10. The pharmaceutical composition of any one of claims 1-9, wherein the domain comprises at least 70% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:8, 9, 10 or 11.

11. The pharmaceutical composition of any one of claims 1-10, wherein the first monomer and the second monomer each independently comprise: The first domain contains at least 70% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:5, 6 or 7; A second structural domain comprising at least 70% of an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 8, 9, 10, or 11; and Cysteine ​​residues; The dimer contains a chemical linker or bond between the cysteine ​​residues of the first monomer and the cysteine ​​residues of the second monomer.

12. The pharmaceutical composition of claim 11, wherein the first domain comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 5, 6 or 7.

13. The pharmaceutical composition of claim 11 or claim 12, wherein the first domain comprises the same amino acid sequence as the amino acid sequence of SEQ ID NO:

5.

14. The pharmaceutical composition of any one of claims 11-13, wherein the second domain comprises an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 8, 9, 10 or 11.

15. The pharmaceutical composition according to any one of claims 11-14, wherein the dimer has the following structure: ， Where X is the bond or chemical linker between the two cysteine ​​residues.

16. The pharmaceutical composition of claim 15, wherein X is a chemical linker selected from disulfide bonds, thioether bonds, or thioester bonds.

17. The pharmaceutical composition of claim 15 or claim 16, wherein X is a chemical linker composed of atoms selected from C, N, S, O and / or H.

18. The pharmaceutical composition of any one of claims 15-17, wherein X is a chemical linker comprising between 1 and 8 carbon atoms.

19. The pharmaceutical composition of any one of claims 15-18, wherein X is a chemical linker comprising an alkylene chain, wherein, Optionally, one or more carbon atoms of the alkylene chain are replaced by oxygen.

20. The pharmaceutical composition of any one of claims 11-19, wherein the dimer has the following structure: 。 21. The pharmaceutical composition of any one of claims 1-20, wherein the first monomer and the second monomer each independently comprise an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

22. The pharmaceutical composition of any one of claims 1-21, wherein the first monomer and the second monomer each independently comprise an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

23. The pharmaceutical composition of any one of claims 1-22, wherein the dimer comprises the same monomer.

24. The pharmaceutical composition of any one of claims 1-23, wherein the dimer comprises different monomers and the ratio is calculated based on the total amount of free monomers.

25. The pharmaceutical composition of claim 24, wherein the first monomer and the second monomer of the dimer have different C-terminal modifications.

26. The pharmaceutical composition of any one of claims 1-25, wherein the dimer has a structure selected from the group consisting of: 。 27. A method for repairing the nervous system and / or treating neurological symptoms, diseases, or conditions selected from the group consisting of nerve damage, neurological diseases caused by inflammation or autoimmunity, and neurodegenerative diseases in a subject in need, said method comprising administering to the subject in need an effective amount of the pharmaceutical composition as described in any one of claims 1-26.

28. The method of claim 27, wherein the neurological injury is selected from the group consisting of acute neurological injury; traumatic brain injury (TBI); spinal cord injury; concussion; stroke, including ischemic stroke, hemorrhagic stroke and chronic stroke; aneurysm; cerebral hemorrhage; thrombosis; and embolism.

29. The method of claim 27, wherein the neurological symptom, disease, or condition is selected from the group consisting of Alzheimer's disease, Alzheimer's-related dementia, diffuse Lewy body disease, senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis (MS), optic neuritis, Huntington's disease, Tourette syndrome, hereditary motor and sensory neuropathy, diabetic neuropathy, progressive supranuclear palsy, Creutzfeldt-Jakob disease, epilepsy, and infectious diseases.

30. Use of the pharmaceutical composition of any one of claims 1-26 in the manufacture of a medicament for treating a neurological symptom, disease, or condition selected from the group consisting of nerve injury, neurological diseases caused by inflammation or autoimmunity, and neurodegenerative diseases.

31. A method for preparing a pharmaceutical composition according to any one of claims 1-26, the method comprising mixing the first monomer and the second monomer in water, and a) adding an oxidant and / or b) oxygenating the solution to form the dimer.

32. The method of claim 31, wherein the combined concentration of the first monomer and the second monomer in water is at least 20 mg / mL.

33. The method of claim 31 or claim 32, wherein the combined concentration of the first monomer and the second monomer in water is at least 40 mg / mL.

34. The method of any one of claims 31-33, wherein the combination of the first monomer and the second monomer in water is maintained at a temperature of about 20°C to about 25°C.

35. The method of any one of claims 31-34, further comprising adding DMSO to the first monomer, the second monomer, and water.

36. The method of any one of claims 31-35, wherein the oxidant is selected from copper sulfate, iodide, hydrogen peroxide, trans-3,4-dihydroxyselenocyclopentane oxide (DHS), supported methionine sulfoxide, and N-chlorosuccinimide (NCS).

37. The method of any one of claims 31-36, wherein the oxidant is iodine (I2), and the method employs microwave-assisted oxidation.

38. A method for preparing a pharmaceutical composition according to any one of claims 1-23 or 26, the method comprising mixing the first monomer and the second monomer in a solvent having copper sulfate to form a mixture, thereby producing the dimer; The first monomer and the second monomer are the same.

39. The method of claim 38, wherein the solvent comprises purified water (PWP) for purification.

40. The method of claim 39, wherein the solvent further comprises ethanol.

41. The method of any one of claims 38-40, wherein the pH of the mixture is maintained between about 8.5 and about 9.

5.

42. A medicament for repairing the nervous system of a subject, the medicament comprising a peptide dimer or a pharmaceutically acceptable salt or solvate thereof, the peptide dimer comprising two subunits, wherein each subunit comprises a peptide domain independently selected from a receptor-type protein tyrosine phosphatase (PTPR) wedge domain or a variant having at least 70% homology with it.

43. The pharmaceutical preparation of claim 42, wherein each subunit comprises a transport portion connected to the peptide domain via a peptide linker containing a cysteine ​​residue or a peptide linker containing a cysteine ​​residue, wherein the two subunits are covalently cross-linked via a chemical linker between the cysteine ​​residues on each subunit.

44. The agent of claim 43, wherein the chemical linker is selected from disulfide bonds, thioether bonds, or thioester bonds.

45. The agent of claim 43, wherein the chemical connector comprises between 1 and 8 carbon atoms.

46. ​​The pharmaceutical preparation of any one of claims 42-45, wherein the peptide domain of each subunit independently comprises an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 8, 9, 10 or 11.

47. The pharmaceutical preparation of any one of claims 43-46, wherein the transport portion is selected from the group consisting of: HIV TAT peptide, herpes simplex virus-1 DNA binding protein VP22 peptide, amino acid region of the third α-helix of the antennal foot homologous domain, histidine tag with a length ranging from 4 to 30 histidine repeats, variant derivatives or homologs thereof capable of promoting the uptake of the active cargo portion through a receptor-independent process, peptides rich in cationic arginine, and combinations thereof.

48. The pharmaceutical preparation of any one of claims 43-47, wherein the transport portion comprises an amino acid sequence having at least 65% identity with wild-type HIV TAT.

49. The pharmaceutical preparation of claim 48, wherein the TAT sequence comprises at least 65% identical amino acid sequences to the amino acid sequences of SEQ ID NO: 5, 6 or 7.

50. The pharmaceutical preparation according to any one of claims 42-49, wherein each subunit independently comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

51. The pharmaceutical preparation according to any one of claims 42-50, wherein the subunit has a different C-terminal modification.

52. The pharmaceutical preparation according to any one of claims 42-51, wherein the peptide dimer comprises two distinct subunits.

53. The pharmaceutical preparation of any one of claims 42-51, wherein the peptide dimer comprises two identical monomeric subunits.

54. The pharmaceutical preparation according to any one of claims 42-53, wherein the peptide dimer or a pharmaceutically acceptable salt or solvate thereof has a purity of at least 90%.

55. A pharmaceutical composition comprising the agent as described in any one of claims 42-54.

56. The pharmaceutical composition of claim 55, wherein the pharmaceutical composition does not contain DMSO.

57. The pharmaceutical composition according to any one of claims 1-26, wherein it does not contain DMSO.

58. Use of the pharmaceutical agent as described in any one of claims 42-54 in the manufacture of a medicament for repairing the nervous system and / or treating symptoms, diseases, or conditions of the nervous system selected from the group consisting of nerve damage, nervous system diseases caused by inflammation or autoimmunity, and neurodegenerative diseases.

59. A method for repairing the nervous system and / or treating neurological symptoms, diseases, or conditions selected from the group consisting of nerve damage, neurological diseases caused by inflammation or autoimmunity, and neurodegenerative diseases in a subject in need, said method comprising administering to the subject in need an effective amount of the agent as described in any one of claims 42-54.

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