Short-chain peptides, pharmaceutical compositions containing them, and their use in the treatment of neurological disorders

Abstract

This specification discloses short-chain peptides having an amino acid sequence at least 85% identical to SEQ ID NO: 1. According to some embodiments of this disclosure, these short-chain peptides have an amino acid sequence 100% identical to SEQ ID NO: 1, 2, 3, or 4. According to certain embodiments of this disclosure, these short-chain peptides are useful for treating neurological disorders by enhancing neurite outgrowth. Accordingly, this specification also discloses pharmaceutical compositions containing these short-chain peptides and their use for treating neurological disorders.

Description

Technical Field

[0001] The present disclosure generally relates to the field of disease treatment. More particularly, the present disclosure relates to novel peptides and their use in the treatment of neurological diseases.

Background Art

[0002] Neurological diseases refer to any diseases caused by or causing dysfunction in an individual's central nervous system (CNS) and / or peripheral nervous system (PNS). This can cause a wide range of symptoms, including headache, dizziness, memory impairment, visual impairment, speech impairment, cognitive difficulties, muscle weakness, paralysis, seizures, pain, fainting, and loss of consciousness. There are more than 600 neurological diseases. Neurological diseases can be classified into hereditary neuropathies (such as muscular dystrophy and neurofibromatosis), neurodegenerative diseases (such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease), vascular-related disorders (such as stroke), immune-related disorders (such as multiple sclerosis and myasthenia gravis), brain or spinal cord injuries, problems in the development of the nervous system (such as split spine), seizure disorders (such as epilepsy), brain tumors, infectious diseases (such as meningitis and HIV neuropathy), etc., according to the main affected location, the main types of dysfunction involved, or the main types of causes. However, in clinical practice, many patients have mixed symptoms and medical histories, resulting in many overlaps and difficulties in classification.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The treatment of neurological diseases usually varies depending on the disease state and severity. Unfortunately, most pharmaceuticals only improve symptoms or slow the progression of symptoms without curing the underlying disease. The management and treatment of neurological diseases are one of the greatest challenges facing medicine today. Therefore, there is a continuing interest in the development of new drugs and methods for treating neurological diseases.

Means for Solving the Problems

[0004] The following is a simplified summary of this disclosure to provide readers with a basic understanding. This summary is not a broad overview of this disclosure and does not identify key / important elements of the invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as an introduction to the more detailed description below.

[0005] A first aspect of this disclosure relates to a short-chain peptide having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1 (SEQ ID NO: 1) "WEAFARGTKALMDEVV".

[0006] According to some embodiments of this disclosure, the short-chain peptide has an amino acid sequence that is 100% identical to SEQ ID NO: 1.

[0007] In certain embodiments, at least one mutation is present in the amino acid sequence of SEQ ID NO: 1.

[0008] In one embodiment, there is a single mutation in the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue alanine (A) at position 10 of SEQ ID NO: 1 is replaced with the amino acid residue proline (P). In this embodiment, the short-chain peptide has 100% the same amino acid sequence as "WEAFARGTKPLMDEVV" of SEQ ID NO: 2.

[0009] In one embodiment, there is a single mutation in the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue lysine (K) at position 9 of SEQ ID NO: 1 is replaced with the amino acid residue arginine (R). In this embodiment, the short-chain peptide has 100% the same amino acid sequence as "WEAFARGTRALMDEVV" of SEQ ID NO: 3.

[0010] In another embodiment, the amino acid sequence of SEQ ID NO: 1 contains two mutations: the amino acid residue aspartic acid (D) at position 13 of SEQ ID NO: 1 is replaced with the amino acid residue arginine (R), and the amino acid residue glutamic acid (E) at position 14 of SEQ ID NO: 1 is replaced with the amino acid residue glutamine (Q). In this embodiment, the short-chain peptide has 100% the same amino acid sequence as "WEAFARGTKALMRQVV" of SEQ ID NO: 4.

[0011] Optionally, the short-chain peptide is acetylated at its N-terminus and / or amidated at its C-terminus.

[0012] A second aspect of the present disclosure relates to a pharmaceutical composition for treating neurological disorders. The pharmaceutical composition comprises at least one of the short-chain peptides of the present disclosure (i.e., short-chain peptides of SEQ ID NO: 1, 2, 3, or 4) and a pharmaceutically acceptable carrier.

[0013] Furthermore, this specification also discloses a method for treating a target neurological disorder. The method comprises administering an effective amount of the short-chain peptide or pharmaceutical composition of this disclosure to the target.

[0014] Examples of neurological disorders treatable by the methods of this disclosure include, but are not limited to, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), frontotemporal lobar degeneration (FTLD), ataxia Friedreich, age-related macular degeneration, and Creutzfeldt-Jakob disease.

[0015] Generally, the subject is a mammal, preferably a human.

[0016] Many of the additional features and benefits of this disclosure will be better understood by referring to the following detailed description, which is considered in conjunction with the attached drawings.

[0017] This specification will be better understood from the following detailed description, which should be read in conjunction with the attached drawings. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows the effect of the short-chain peptide of this disclosure on neurite outgrowth, according to Example 1 of this disclosure. Panel (A): Quantification of neurite outgrowth length. Panel (B): Expression protein level of phosphorylated cofilin (p-cofilin). Sol8 CM: Conditioned medium (culture medium) obtained by culturing wild-type Sol8 cells. Sol-NogoA CM: Conditioned medium (culture medium) obtained by culturing Sol8 cells overexpressing NogoA. *P<0.05;**P<0.01;***P<0.001. [Figure 2] Figure 2 shows the effect of the short-chain peptide of this disclosure on axonal branching of motor neurons in zebrafish embryos, according to Example 2 of this disclosure. **P<0.01;***P<0.001. [Figure 3] Figure 3 shows the effect of the short-chain peptide of this disclosure on delaying denervation of the neuromuscular junction (NMJ) in ALS mice, according to Example 2 of this disclosure. ***P<0.001. [Figure 4] Figure 4 shows the effects of the short-chain peptides of this disclosure on improving motor function (Panel A) and extending lifespan (Panel B) in ALS mice. Panel (A): Total distance traveled averaged for ALS mice treated with the indicated specific treatment. Panel (B): Cumulative survival rate for each group of ALS mice treated with the indicated specific treatment. *P<0.05; ***P<0.001. [Modes for carrying out the invention]

[0019] The detailed description provided below in relation to the attached drawings is intended to describe embodiments of the present disclosure and is not intended to represent the only forms in which embodiments of the present disclosure may be constructed or utilized. This description presents the function of the embodiments and a set of steps for constructing and operating the embodiments. However, the same or equivalent functions and steps may also be achieved by different embodiments.

[0020] I. Definition For convenience, the terms used herein, in the examples, and in the appended claims are set forth herein. Unless otherwise defined herein, the scientific and technical terms used herein have the meanings commonly understood and used by those skilled in the art. Unless otherwise required in context, singular terms are understood to include the plural form of the same thing, and plural terms are understood to include the singular form. Specifically, as used herein and in the claims, the singular "a" and "an" include the plural form unless otherwise clearly indicated in context. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0021] Although the numerical ranges and parameters defining the broad scope of the present invention are approximate values, the numerical values defined in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors that inevitably result from the standard deviation found in each of the test measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the acceptable standard error of the mean value when considered by a person skilled in the art. Unless otherwise specified, except for the operations / examples, all numerical ranges, amounts, values, and ratios disclosed herein, such as amounts of materials, periods of time, temperatures, operating conditions, ratios of amounts, etc., should be understood to be modified in all cases by the term "about". Accordingly, unless otherwise indicated, the numerical parameters defined in the present disclosure and the appended claims are approximate values that can vary as desired. At a minimum, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0022] As used herein, the term "short-chain peptide" refers to a polymer having less than 20 amino acid (a.a.) residues. Preferably, the short-chain peptide has a length of 5 to 20 amino acid residues, and more preferably, a length of 8 to 20 amino acid residues. According to some embodiments of the present disclosure, the short-chain peptide has a length of 16 amino acid residues. When an amino acid sequence is provided herein, the L-, D-, or beta amino acid version of the sequence is also contemplated. Peptides include amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. Further, this term applies to amino acids linked by a peptide bond or other "modified bond" (e.g., when the peptide bond is replaced by an alpha-ester, beta-ester, thioamide, phosphonamide, carbamate, hydroxylate, etc.).

[0023] In certain embodiments, conservative substitutions of amino acid residues that include any of the sequences described herein are contemplated. In various embodiments, 1, 2, 3, 4, or 5 different residues are substituted. The term "conservative substitution" is used to reflect an amino acid substitution that does not substantially change the activity of the molecule (e.g., biological or functional activity and / or specificity). Typically, a conservative amino acid substitution involves the substitution of one amino acid residue with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity). Certain conservative substitutions include "analog substitutions" in which a standard amino acid is replaced with a non-standard (e.g., rare, synthetic, etc.) amino acid that differs minimally from the original residue. Amino acid analogs are considered to be synthetically derived from standard amino acids, isomers, or metabolic precursors that do not give rise to substantial changes in the original structure.

[0024] As used herein, the term "neural disease" refers to neuropathy, neurodegenerative disease, and / or other neuron-related diseases caused by mechanical injury (e.g., trauma), chemical injury (e.g., neurotoxin, immunosuppression due to treatment or side effects), immune response (e.g., inflammation or autoimmunity), or infection. Exemplary neural diseases include, but are not limited to, ALS, SMA, AD, PD, HD, FTLD, Friedreich's ataxia, age-related macular degeneration, and Creutzfeldt-Jakob disease. Neuron-related diseases may include eye diseases caused by nerve damage (e.g., glaucoma), Bell's palsy, or other forms of local paralysis, nerve-based impotence (e.g., caused by nerve damage after radical prostatectomy), or other symptoms. In the present disclosure, the term "neural disease" can be any of a central nervous system (CNS) disease, a peripheral nervous system (PNS) disease, a sympathetic nervous system disease, or a parasympathetic nervous system disease.

[0025] As used herein, the term “neurite outgrowth” refers to the process by which axons or dendrites grow from the cell body of a neuron. Generally, neurite outgrowth plays a vital role in neuronal development and regeneration. It improves neural connectivity, thereby promoting synapse formation or remodeling synapses.

[0026] The “percentage of sequence identity (%)” for peptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given peptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. Alignment for determining the percentage of sequence identity can be achieved in various ways within the scope of the art of the art, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. In this specification, sequence comparison between two peptide sequences was performed using the computer program BLASTP (protein-protein BLAST), provided online by the National Center for Biotechnology Information (NCBI). The percentage of sequence identity from a given peptide sequence A to a given peptide sequence B (or, alternatively, the expression that a given peptide sequence A has a specific percentage of peptide sequence identity with a given peptide sequence B) is calculated by the following formula: JPEG2026512427000002.jpg1626 Here, X is the number of amino acid residues scored as identical by the sequence alignment program BLAST in the alignment of A and B in that program, and Y is the total number of amino acid residues of the shorter of A or B.

[0027] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally considered safe." That is, they are physiologically acceptable and, when administered to humans, do not typically cause allergic reactions or similar adverse reactions such as stomach upset or dizziness. Preferably, as used herein, the term "pharmaceutically acceptable" means that it is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals, more specifically in humans.

[0028] The terms “to be administered,” “to administer,” or “administer” are used interchangeably herein to refer to modes of delivery of the agents of the present invention (e.g., short-chain peptides, variant peptides, or pharmaceutical compositions), including but not limited to intravenous, intra-arterial, intraperitoneal, intracerebral, or intrathecal delivery.

[0029] As used herein, the terms “to treat,” “to treat,” and “treatment” are interchangeable and encompass the partial or complete prevention, improvement, alleviation, and / or management of symptoms, secondary disorders, or conditions associated with neuronal dysfunction. As used herein, “to treat” means applying or administering one or more of the short-chain peptides, variant peptides, or pharmaceutical compositions of this disclosure to a subject having symptoms, secondary disorders, or conditions associated with neuronal dysfunction, for the purpose of partially or completely reducing, improving, alleviating, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms, secondary disorders, or characteristics associated with neuronal dysfunction. Symptoms, secondary disorders, and / or conditions associated with neuronal dysfunction include, but are not limited to, headaches, dizziness, memory impairment, visual impairment, speech impairment, cognitive difficulties, muscle weakness, paralysis, seizures, pain, syncope, and loss of consciousness. Treatment may be administered to a subject showing only early signs of such symptoms, disorders, and / or conditions, for the purpose of reducing the risk of developing symptoms, secondary disorders, and / or conditions associated with neuronal dysfunction. Treatment is generally considered “effective” if one or more symptoms or clinical markers are reduced as defined herein; or if the progression of a symptom, disorder, or condition is reduced or halted.

[0030] As used herein, the term “effective dose” specifies an amount of an ingredient sufficient to produce a desired response. For therapeutic purposes, an effective dose is also such that any toxic or adverse effects of the ingredient outweigh the therapeutically beneficial effects. An effective dose of an agent is not required to cure a disease or condition, but to provide treatment for a disease or condition such that the onset of the disease or condition is delayed, prevented, or prevented, or the symptoms of the disease or condition are improved. An effective dose may be divided into one, two, or more doses in a form suitable for administration over one, two, or more times over a specified period. A particular effective dose or sufficient dose will vary depending on factors such as the specific condition being treated, the patient’s physical condition (e.g., weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concomitant therapies, and the structure or derivatives of the specific formulation and compound used. An effective dose may be expressed, for example, in grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg). Those skilled in the art can calculate the human equivalent dose (HED) of a pharmaceutical product (e.g., the short-chain peptides, variant peptides, or pharmaceutical compositions of this disclosure) based on doses determined from animal models. For example, the maximum safe dose for use in human subjects can be estimated in accordance with the industry guidance issued by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0031] The term "subject" refers to mammals, including human species, that can be treated with the short-chain peptides, mutant peptides, pharmaceutical compositions, and / or methods of the present invention. Unless otherwise specified, the term "subject" is intended to refer to both males and females.

[0032] II. Description of the Invention This disclosure is based, at least in part, on the finding that short-chain peptides or their variants are useful in enhancing neurite outgrowth, and therefore, such short-chain peptides or their variants may be useful as drug development candidates for treating neurological disorders, in particular neurological disorders associated with and / or caused by neuronal dysfunction.

[0033] Accordingly, a first aspect of this disclosure relates to short-chain peptides or their variants (i.e., mutant peptides of this disclosure). According to some embodiments of this disclosure, a short-chain peptide designated as “FD1 peptide” has an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to “WEAFARGTKALMDEVV” (N-terminus to C-terminus) of SEQ ID NO: 1. Preferably, the FD1 peptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. More preferably, the FD1 peptide has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In one particular embodiment, the FD1 peptide has the amino acid sequence of SEQ ID NO: 1 (i.e., has an amino acid sequence that is 100% identical to SEQ ID NO: 1).

[0034] According to some embodiments, the mutant peptide is designated as the "FD2 peptide," which has an amino acid sequence that is at least 85% identical to "WEAFARGTKPLMDEVV" (N-terminus to C-terminus) of SEQ ID NO: 2. Preferably, it is at least 90% identical to SEQ ID NO: 2, and more preferably, at least 95% identical to SEQ ID NO: 2. In one particular embodiment, the FD2 peptide has the amino acid sequence of SEQ ID NO: 2 (i.e., it has an amino acid sequence that is 100% identical to SEQ ID NO: 2).

[0035] According to an alternative embodiment, the mutant peptide is designated as “FD3 peptide,” which has an amino acid sequence that is at least 85% identical to “WEAFARGTRALMDEVV” (N-terminus to C-terminus) of SEQ ID NO: 3. Preferably, it is at least 90% identical to SEQ ID NO: 3, and more preferably, at least 95% identical to SEQ ID NO: 3. In one particular embodiment, the FD3 peptide has the amino acid sequence of SEQ ID NO: 3 (i.e., it has an amino acid sequence that is 100% identical to SEQ ID NO: 3).

[0036] According to an alternative embodiment, the mutant peptide is designated as the "FD4 peptide," which has an amino acid sequence that is at least 85% identical to "WEAFARGTKALMRQVV" (N-terminus to C-terminus) of SEQ ID NO: 4. Preferably, it is at least 90% identical to SEQ ID NO: 4, and more preferably, at least 95% identical to SEQ ID NO: 4. In one particular embodiment, the FD4 peptide has the amino acid sequence of SEQ ID NO: 4 (i.e., it has an amino acid sequence that is 100% identical to SEQ ID NO: 4).

[0037] Optionally, the short-chain peptides of this disclosure may be modified at their N-terminus or C-terminus. Examples of N-terminal modifications include, but are not limited to, N-glycosylation, N-alkylation, and N-acetylation of amino acids. Terminal modifications may include pegylation. An example of C-terminal modification is an amino acid with an amidated C-terminus. Alternatively, one or more peptide bonds may be replaced with non-peptide bonds, and individual amino acid portions may be modified by treatment with agents that can react with selected side chain or terminal residues.

[0038] Various functional groups may be added to various points of the short-chain peptides of this disclosure that are susceptible to chemical modification. Functional groups may also be added to the ends of the short-chain peptides. In some embodiments, functional groups improve the activity of the short-chain peptides with respect to one or more properties, such as improved stability, efficacy, or selectivity of the short-chain peptides, improved penetration of the short-chain peptides across cell membranes and / or tissue barriers, reduced toxicity or clearance, and improved resistance to efflux by cell pumps. Non-limiting examples of suitable functional groups are those that facilitate the intracellular transport of peptides to which they are attached, for example, by reducing the hydrophilicity and increasing the lipophilicity of the peptides, and these functional groups can optionally, and preferably, be hydrolyzed or enzymatically cleaved in vivo within cells. Hydroxy protecting groups include ester, carbonate, and carbamate protecting groups. Amine protecting groups include alkoxy and aryloxycarbonyl groups. Carboxylic acid protecting groups include aliphatic, benzyl, and aryl esters.

[0039] According to some embodiments of the present disclosure, each short-chain peptide (i.e., FD1 peptide) and mutant peptide (i.e., FD2, FD3, and FD4 peptides) is useful for enhancing the extension of neurites in neurons. Accordingly, a second aspect of the present disclosure relates to a pharmaceutical composition or drug for treating neurological disorders associated with and / or caused by neuronal dysfunction. The pharmaceutical composition or drug of the present disclosure comprises the short-chain peptide (i.e., FD1 peptide) or mutant peptide (i.e., FD2, FD3, or FD4 peptide) of the present disclosure, as well as a pharmaceutically acceptable carrier.

[0040] Depending on the desired purpose, the pharmaceutically acceptable carrier may be liposomes, nanoparticles, diluents, dispersions, buffers, stabilizers, or any solution or substance compatible with the administration of the drug.

[0041] The peptide of the present invention (i.e., short-chain peptide or mutant peptide) is present at a level of about 0.1% to 99% by weight based on the total weight of the pharmaceutical composition or drug. In some embodiments, the peptide of the present invention is present at a level of at least 1% by weight based on the total weight of the pharmaceutical composition or drug. In certain embodiments, the peptide of the present invention is present at a level of at least 5% by weight based on the total weight of the pharmaceutical composition or drug. In yet another embodiment, the peptide of the present invention is present at a level of at least 10% by weight based on the total weight of the pharmaceutical composition or drug. In yet another embodiment, the peptide of the present invention is present at a level of at least 25% by weight based on the total weight of the pharmaceutical composition or drug.

[0042] In some embodiments, the pharmaceutical composition or drug is formulated in a liquid dosage form suitable for parenteral administration (e.g., intravenous, intra-arterial, intracerebral, or intrathecal). For this purpose, the pharmaceutical composition or drug may be formulated as an isotonic suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulations such as suspending agents, stabilizers, or dispersants. The preparation of such liquid dosage forms with due consideration to pH, isotonicity, stability, etc., is within the scope of the art for those skilled in the art.

[0043] Preferred pharmaceutical compositions or medicinal products for parenteral injection should contain, in addition to the peptides of this disclosure, an isotonic vehicle such as sodium chloride injection, Ringer's solution, glucose injection, glucose and sodium chloride injection, lactated Ringer's solution, or other vehicles known in the art. The pharmaceutical compositions of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art.

[0044] A third aspect of the present disclosure provides a method for treating a neurological disorder in question. This method involves administering an effective amount to a subject a short-chain peptide (i.e., an FD1 peptide), a mutant peptide (i.e., an FD2, FD3, or FD4 peptide), or a pharmaceutical composition according to any embodiment of the present disclosure.

[0045] Examples of neurological disorders treatable by the methods of this disclosure include, but are not limited to, ALS, SMA, AD, PD, HD, FTLD, Friedreich's ataxia, age-related macular degeneration, and Creutzfeldt-Jakob disease.

[0046] Furthermore, this specification also discloses a method for enhancing the extension of neuronal neurites in a subject. This method involves administering an effective amount to a subject a short-chain peptide (i.e., FD1 peptide), a mutant peptide (i.e., FD2, FD3, and FD4 peptides), or a pharmaceutical composition according to any embodiment of this disclosure.

[0047] The subjects treatable by the methods disclosed herein are mammals, such as humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, cattle, goats, sheep, monkeys, and horses. Preferably, the subject is a human.

[0048] According to some embodiments, the subject suffers from a neurological disorder. In these embodiments, the neurological disorder is caused by dysfunction of neurites. For example, the subject may suffer from neuronal damage or neuropathy caused by mechanical damage (e.g., trauma), chemical damage (e.g., neurotoxins, immunosuppression due to treatment, or side effects of treatment), and / or biological damage (e.g., infection, inflammation, autoimmunity, aging, disease, metabolic disorder, or abnormal protein expression) that interferes with the transmission of electrochemical stimuli in the subject. The methods of the present disclosure are useful for enhancing neurite extension in a subject in order to treat a neurological disorder.

[0049] Depending on the desired purpose, the short-chain peptides, mutant peptides, or pharmaceutical compositions of this disclosure (the present invention) may be administered to a subject via any suitable route, such as intravenous, intra-arterial, intraperitoneal, intracerebral, or intrathecal injection.

[0050] According to some embodiments, the subject is a mouse, and the short-chain peptides, mutant peptides, or pharmaceutical compositions of this disclosure are administered to the subject in doses of approximately 0.1 mg to 1,000 mg per kg of body weight (approximately 0.1 mg / kg to 1,000 mg / kg). For example, doses of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 ,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,1 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 59 The effective dose is 0, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 mg / kg. Preferably, the effective dose is about 1 mg / kg to 100 mg / kg. More preferably, the effective dose is about 5 mg / kg to 50 mg / kg. According to one example, approximately 10 mg / kg of the peptide, mutant peptide, or pharmaceutical composition of the present disclosure is sufficient to produce a therapeutic effect on a subject (e.g., enhancement of neurite outgrowth and / or treatment of neurological disorders).

[0051] Those skilled in the art can calculate the human equivalent dose (HED) of the short-chain peptides, mutant peptides, or pharmaceutical compositions of this disclosure based on doses determined from animal models. Therefore, the effective HED of the short-chain peptides, mutant peptides, or pharmaceutical compositions of this disclosure in humans is approximately 10 μg to 100 mg (10 μg / kg to 100 mg / kg) per dose. In other words, the effective HED of the short-chain peptides, mutant peptides or pharmaceutical compositions of this disclosure is, in humans, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, It may be any of the following: 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 μg / kg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0052] Depending on the specific condition (e.g., the patient's physical condition, the type of neurological disorder, and the severity of the neurological disorder), the effective amount of the short-chain peptide, mutant peptide, or pharmaceutical composition of this disclosure may be divided into one or more doses over a specified period to enhance neurite outgrowth in the subject and / or treat the neurological disorder. For example, the effective amount of the short-chain peptide, mutant peptide, or pharmaceutical composition of this disclosure may be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses. The period between two consecutive doses may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. Practitioners may adjust the administration procedure according to the desired effect.

[0053] To ensure your understanding, the methods described herein can be applied to a subject alone or in combination with additional therapies (e.g., neuroprotective or neurotherapeutic agents) that have some beneficial effects on the prevention or treatment of neurological disorders. Depending on the intended / therapeutic purpose, the methods described herein can be applied to a subject before, during, or after the administration of additional therapies.

[0054] This disclosure also provides the use of the short-chain peptides of this disclosure (i.e., FD1 peptide) or their variants (i.e., FD2, FD3, or FD4 peptide) in the manufacture of pharmaceuticals used for the treatment of neurological disorders (e.g., ALS, SMA, AD, PD, HD, FTLD, Friedreich's ataxia, age-related macular degeneration, Creutzfeldt-Jakob disease, or combinations thereof).

[0055] Furthermore, this specification also discloses short-chain peptides of the present disclosure (i.e., FD1 peptide) or variants thereof (i.e., FD2, FD3, or FD4 peptide) for use in the treatment of neurological diseases (e.g., ALS, SMA, AD, PD, HD, FTLD, Friedreich's ataxia, age-related macular degeneration, Creutzfeldt-Jakob disease, or combinations thereof).

[0056] The following examples are provided to illustrate specific aspects of the present invention and to assist those skilled in the art in carrying it out. These examples should not be considered to limit the scope of the present invention in any way. Without further explanation, those skilled in the art will be able to make the most of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. [Examples]

[0057] Materials and methods

[0058] Peptide synthesis and preparation This study provided four peptides, including a short-chain peptide derived from human PGK1 and three variants of that short-chain peptide. These peptides were produced by Fmoc solid-phase synthesis, which starts from the carboxyl terminus (C-terminus) of the peptide and proceeds toward the amino terminus (N-terminus).

[0059] The peptides produced in this manner were purified using a column and designated as "FD1 peptide," "FD2 peptide," "FD3 peptide," and "FD4 peptide," respectively. The amino acid sequences of these peptides are summarized in Table 1 below.

[0060] [Table 1]

[0061] Preparation of Sol8 cells overexpressing Nogo A (Sol8-NogoA cells)

[0062] A doxycycline-inducible lentiviral plasmid (pAS4.1w.Ppuro-aOn) carrying the human Nogo A gene was co-transfected into HEK-293T cells via LIPOFECTAMINE® 2000 along with a Gag expression plasmid (pCMV△R8.91) and a VSV-G expression plasmid (pMD.G). After 24–48 hours, the supernatant containing the virus particles was collected, centrifuged at 1,250 rpm, and subsequently added to Sol8 cells (ATCC,CRL-2174) pretreated with 8 μg / ml POLYBRENE®. After 24 hours, fresh DMEM medium containing 10% fetal bovine serum (FBS), 100 units / ml penicillin, 1% streptomycin, and 4 μg / ml puromycin was added to the Sol8 cells to obtain Sol8 cells overexpressing Nogo A.

[0063] Preparation of conditioned medium (CM) obtained by culturing wild-type Sol8 cells (Sol8 CM) or Sol8 cells overexpressing NogoA (Sol8-NogoA CM).

[0064] Wild-type Sol8 cells and Sol8 cells overexpressing NogoA (Sol8-NogoA cells) were cultured and differentiated in DMEM medium containing 10% FBS, 100 units / ml penicillin, and 1% streptomycin, respectively. After 2 days, 1 μg / μl doxycycline was added to the medium to induce the expression of the target gene. The medium was collected, and fresh medium containing doxycycline was added to the Sol8-NogoA cells every 24 hours. The collected medium was then centrifuged at 3,000 rpm at room temperature for 5 minutes, and the supernatant was collected and used as CM for the following assays.

[0065] Treatment of NSC34 cells NSC34 cells (mouse neuroblastoma × spinal cord-34 cell line) (RRID:CVCL_D356) were cultured in DMEM medium containing 10% FBS, 100 units / ml penicillin, and 1% streptomycin. To investigate the effects of the peptides disclosed herein (including FD1, FD2, FD3, and FD4) on neurons, NSC34 cells were cultured for 24 hours in differentiation medium (DMEM medium containing 2.5% FBS, 100 units / ml penicillin, and 1% streptomycin), and then cultured in CMs (Sol8-NogoA CMs) obtained by culturing Sol8-NogoA cells with or without the peptides disclosed herein (i.e., FD1, FD2, FD3, or FD4) at a concentration of 66 ng / ml. The CMs containing or without the peptides disclosed herein were replaced daily. Cells were fixed with paraformaldehyde (PFA) for 48 hours, and then the length of neurite outgrowth was measured using software based on the neurites shown in the microscopic images. Each data was averaged from three independent experiments, and the length shown at each time was averaged by counting more than 100 cells. The effect of the peptide of this disclosure on improving neurite outgrowth was determined by comparing the length of neurites in NSC34 cells treated with the peptide of this disclosure in Sol8-NogoA CM with the length of neurites in NSC34 cells treated with phosphate-buffered saline (PBS; used as a control group) in Sol8-NogoA CM.

[0066] Western blot analysis Total proteins extracted from treated NSC34 cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel. Western blotting was performed after electrophoresis. Protein levels of phosphorylated cofilin (p-cofilin) ​​and α-tubulin in NSC34 cells cultured in medium supplemented with either PBS (used as a control) or one of the peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4) were determined using antibodies specific to p-cofilin and α-tubulin, respectively. Intensities shown on the blots were quantified by software. Each data was averaged from three independent experiments. Relative intensities between groups were based on comparison with the intensity of the control group, set to 1.

[0067] Transgenic Zebrafish Transgenic zebrafish Tg(mnx1:GFP) strains exhibiting GFP-tagged motor neurons were purchased from the International Zebrafish Resource Center (ZIRC, USA). Branched primary motor neurons (PMNs) derived from caudal primary (CaP) motor neurons located in segments 11 through 20 of Tg(mnx1:GFP) zebrafish embryos 30 hours post-fertilization (hpf) were examined. Untreated embryos were used as a control group, and embryos injected with any 16 non-functional amino acid residues were used as a mock control group. The experimental group consisted of embryos injected with FD1, FD2, FD3, or FD4. After injection, the proportion of zebrafish embryos exhibiting branched PMNs was quantified from the 20 embryos examined. Each data was averaged from three independent experiments.

[0068] PBS and the peptides of the present invention (i.e., FD1, FD2, FD3, and FD4) were microinjected into the ventricles of 20 hpf transgenic (Tg) (mnx1:GFP) embryos. After 10 hours, control embryos, mock control embryos, and experimental embryos were monitored using a dissecting microscope equipped with a fluorescence system and a digital camera.

[0069] Mouse research Congenic ALS strain carrying human SOD1 with the pathogenic G93A mutation G93A Transgenic mice (mouse models of ALS that exhibit a typical phenotype similar to that of ALS patients) were purchased from Jackson Laboratory (JAX). The peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4) were introduced into 60-day-old ALS SOD1 mice. G93A Mice were started with intravenous injections of 250 μg (approximately 10 mg / kg). Injections were administered weekly. PBS injections were used as a control group. Treated mice were observed daily and analyzed via video tracking software.

[0070] To study denervation of NMJs, 75-day-old ALS SOD1s treated with either PBS (control) or one of the peptides disclosed herein (FD1, FD2, FD3, or FD4) were used. G93AGastrocnemius muscle was extracted from mice and analyzed by immunostaining. Briefly, gastrocnemius muscle fixed with paraformaldehyde (PFA) was placed on a glass slide and treated with 2% TRITON® for 1 hour. After blocking with PBS containing 5% bovine serum albumin (BSA) for 2 hours, an antibody against either synaptic vesicle protein (synapsin) or α-bungarotoxin (α-BTX) was added to the slide, followed by incubation at 4°C for 16 hours. The slide was washed three times with PBS containing 2% TRITON®, and then Cy2-labeled anti-rabbit antibody and ALEXA FUOR® 647-labeled anti-mouse antibody were added to the slide, followed by incubation at 4°C for 16 hours. The slide was washed three times with PBS containing 2% TRITON®. Motor neuron axon terminals were detected via synapsin labeled with a green fluorescent signal, while acetylcholine receptors on the motor endplate were detected via α-BTX labeled with a red fluorescent signal. Immunoblots were observed using a colorimetric system. For muscle samples from SOD1-G93A ALS mice injected with PBS and peptides, the proportion of yellow signals (where green signals overlapped with red signals) among all red signals shown in a 600x magnified image area (400x400 pixels) was calculated using software. Each experiment was calculated from 13–15 images.

[0071] The motor skills of ALS model mice (SOD1-G93A) injected with PBS or the peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4) were evaluated by measuring the movement trajectory of each treated mouse at 124 days postnativity.

[0072] Cumulative survival rate The initial total number of ALS mice in each group (7 samples in the control group and 5 samples in the FD-treated group) was set as the 100% survival rate. During the experiment, if an ALS mouse died, the number of deaths and the number of days of survival were recorded, and the cumulative survival rate was calculated. If all ALS mice in the experimental group died, the cumulative survival rate was considered to be 0%. If a mouse's weight loss exceeded 20% of its original weight, or if the mouse was unable to eat or drink normally, it was euthanized and considered a non-surviving sample. The number of days of survival for each group was averaged from 7 samples for the PBS-infused control group and from 5 samples for the FD-infused experimental group.

[0073] statistical analysis Data from at least three independent experiments under identical conditions were expressed as mean ± standard error of the mean (SEM). Student's t-test was used to analyze differences between groups. Statistical analysis was performed using software. Significance probabilities (p) were expressed as *p<0.05, **p<0.01, and ***p<0.001.

[0074] Example 1. The peptides of this disclosure enhanced neurite outgrowth in vitro. In this example, the effects of the peptides of the Disclosure (FD1, FD2, FD3, and FD4) on neurite outgrowth were investigated. As described in Materials and Methods, motor neuron NSC34 cells were treated for 48 hours with or without the peptides of the Disclosure (i.e., FD1, FD2, FD3, or FD4) using conditioned medium (CM) recovered from skeletal muscle cells Sol8 (i.e., wild-type (Sol8) or a strain overexpressing NogoA (Sol8-NogoA)).

[0075] The data in Figure 1 demonstrate that extracellular addition of the peptides of this disclosure in CM can improve neurite outgrowth in motor neuron cells. Compared to the mean neurite length of NSC34 cells cultured in Sol8 CM containing PBS (99.4 μm) (used as a control group), the neurite length of NSC34 cells cultured in Sol8-NogoA CM containing PBS decreased to 53.6 μm (Panel (A) in Figure 1). However, the neurite lengths of NSC34 cells cultured in Sol8-NogoA CM supplemented with peptides FD1, FD2, FD3, or FD4 were significantly improved to 89.2 μm, 66.6 μm, 88.2 μm, and 95.8 μm, respectively (Panel (A) in Figure 1). These results demonstrate that extracellular addition of each peptide (i.e., FD1, FD2, FD3, or FD4) has the ability to improve neurite outgrowth in motor neuron cells.

[0076] Next, we detected the expression levels of phosphorylated cofilin (p-cofilin) ​​protein, a growth cone collapse marker, and α-tubulin, which was used as an internal loading control. p-cofilin levels in NSC34 cells cultured in Sol8 CM overexpressing NogoA containing PBS (used as the control group) were normalized to 1. Based on a constant intensity of the α-tubulin internal control used, p-cofilin protein levels in NSC34 cells cultured in Sol8 CM overexpressing NogoA containing the peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4) were significantly lower compared to the PBS control (Panel (B) in Figure 1). This evidence suggests that the reduction in p-cofilin mediated by each administered peptide (i.e., FD1, FD2, FD3, or FD4) was favorable for improved neurite outgrowth, as shown in Panel (A) in Figure 1.

[0077] The data from this example showed that neurite outgrowth was significantly increased in NSC34 cultured with Sol8-NogoA CM, but with the addition of the peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4), compared to treatment with Sol8-NogoA CM, which inhibits neurite outgrowth by increasing the phosphorylation level of cofilin. Therefore, in vitro studies have shown that each of the peptides of this disclosure (including FD1, FD2, FD3, and FD4) is effective in enhancing neurite outgrowth in motor neurons.

[0078] Example 2. The peptides of this disclosure enhanced neurite outgrowth in vivo. In this disclosure, two animal models, including a zebrafish model and a mouse ALS model, were used to evaluate the in vivo effects of the peptides of this disclosure on increased axonal branching of motor neurons in zebrafish embryos and delayed denervation of the neuromuscular junction (NMJ) in the mouse ALS model.

[0079] The data in Figure 2 showed that extracellular addition of the peptides of this disclosure increased axonal branching of motor neurons in zebrafish embryos. The number of branched primary motor neurons (PMNs) was examined in vivo in zebrafish embryos. The proportion of zebrafish embryos showing branched PMNs was quantified out of 20 embryos. Compared to an untreated control group and a control group injected with any non-functional peptide having the amino acid sequence "MSLSNKLTLDKLDVKG" (SEQ ID NO: 5), the proportion of zebrafish embryos showing branched motor neurons was significantly increased in those injected with FD1, FD2, FD3, or FD4 peptides (Figure 2). This evidence indicates that extracellular administration of each peptide (i.e., FD1, FD2, FD3, or FD4) can increase axonal branching of motor neurons in zebrafish embryos.

[0080] To examine NMJ integrity, motor neuron axonal terminals were detected via synapsin labeled with a green fluorescent signal, while acetylcholine receptors on the motor endplate were detected via α-BTX labeled with a red fluorescent signal. The proportion of yellow signals (where green signals overlapped with red signals) among all red signals observed in muscle samples dissected from PBS and peptide-injected SOD1-G93A ALS mice was calculated. The results showed that the proportion of colocalized synapsin-1 and α-BTX in SOD1-G93A ALS mice injected with each peptide was significantly higher than that in mice injected with PBS (Figure 3). This evidence indicates that injection of the peptides of this disclosure (i.e., FD1, FD2, FD3, or FD4) enabled SOD1-G93A ALS to maintain NMJ integrity for longer than control mice, resulting in delayed NMJ denervation.

[0081] The motility of mice administered with PBS or the peptides of this disclosure was measured by recording their movement trajectories. The results showed that the distance traveled by ALS mice injected with FD1, FD2, FD3, or FD4 was significantly improved compared to control ALS mice injected with PBS (Panel (A) in Figure 4). The mean survival days for ALS mice injected with PBS, FD1, FD2, FD3, and FD4 were 130.0, 140.0, 147.0, 130.8, and 139.0 days, respectively (Panel (B) in Figure 4). These results revealed that although the cumulative survival rate of ALS mice injected with FD3 did not differ from that of control mice, the cumulative survival rate of ALS mice injected with one of the peptides FD1, FD2, or FD4 (5 samples per group) showed a longer survival time than that of control mice injected with PBS (7 samples). This evidence shows that intravenous injection of peptides FD1, FD2, or FD4 significantly extends the lifespan of ALS mice.

[0082] The data from this example showed that administration of the peptides disclosed (i.e., FD1, FD2, FD3, or FD4 peptides) significantly enhanced motor neuron axonal branching in zebrafish compared to an untreated control group and a control group with any 16 non-functional amino acid residues (MSLSNKLTLDKLDVKG; SEQ ID NO: 5) (Figure 2). In a mouse model, denervation of the neuromuscular junction (NMJ) occurred in ALS mice, and intravenous injection of the peptides disclosed (i.e., FD1, FD2, FD3, or FD4 peptides) significantly reduced motor neuron denervation in ALS mice (Figure 3). Furthermore, compared to a PBS control group, the peptides disclosed (i.e., FD1, FD2, FD3, or FD4 peptides) improved motor function and extended survival time in ALS mice (Figure 4).

[0083] In conclusion, this disclosure provides novel peptides, including short-chain peptides (i.e., FD1 peptide) and its variants (i.e., FD2, FD3, and FD4 peptides), each of which has the ability to enhance neurite outgrowth / branching and reduce neuronal denervation. Therefore, each of the peptides in this disclosure may be a potential candidate for drug development to treat neurological diseases.

[0084] The above description of embodiments is given for illustrative purposes only, and those skilled in the art should understand that various modifications can be made. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Various embodiments of the present invention are described above with some specificity or by reference to one or more individual embodiments, but those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A short-chain peptide having at least 85% identical amino acid sequence to "WEAFARGTKALMDEVV" (SEQ ID NO: 1).

2. The short-chain peptide according to claim 1, wherein the short-chain peptide has 100% the same amino acid sequence as "WEAFARGTKALMDEVV" with SEQ ID NO:

1.

3. The short-chain peptide according to claim 1, wherein the N-terminus of the short-chain peptide is acetylated and the C-terminus is amidated.

4. The short-chain peptide according to claim 1, wherein at least one mutation exists in the amino acid sequence of SEQ ID NO:

1.

5. The short-chain peptide according to claim 4, wherein the short-chain peptide has 100% the same amino acid sequence as "WEAFARGTKPLMDEVV" with SEQ ID NO: 2, "WEAFARGTTRALMDEVV" with SEQ ID NO: 3, or "WEAFARGTKALMRQVV" with SEQ ID NO:

4.

6. A pharmaceutical composition comprising the short-chain peptide described in claim 1 and a pharmaceutically acceptable carrier.

7. A method for treating a target neurological disorder, comprising administering to the target in an effective amount the short-chain peptide described in claim 1 or the pharmaceutical composition described in claim 6.

8. The method according to claim 7, wherein the neurological disease is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), frontotemporal lobar degeneration (FTLD), Friedreich's ataxia, age-related macular degeneration, or Creutzfeldt-Jakob disease.

9. The method according to claim 7, wherein the subject is a human.

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