WO2024080440A1 - Composition for preventing or treating neurodegenerative disorders, comprising humanin peptide as active ingredient - Google Patents

Abstract

The purpose of the present invention is to provide a composition for preventing or treating neurodegenerative disorders, comprising humanin peptide as an active ingredient. When the humanin peptide of the present invention was administered nasally to a mouse model of Parkinson's disease, it was confirmed that the humanin peptide was effectively delivered and distributed to the brain. In addition, it was confirmed that dopamine neuron expression increased and neuron cell damage was suppressed. Further, it was confirmed that the humanin peptide suppresses neuron cell damage in a Parkinson's disease cell model, thus being effective in treating Parkinson's disease, and as a result can be utilized in related businesses.

Description

Composition for preventing or treating degenerative brain diseases containing humanin peptide as an active ingredient

The purpose of the present invention is to provide a composition for preventing or treating degenerative brain diseases containing humanin peptide as an active ingredient.

Neurodegenerative disorders are diseases that cause various symptoms as degenerative changes occur in nerve cells of the central nervous system. Representative degenerative brain diseases include Alzheimer's disease, Parkinson's disease, and progressive supranuclear disease. Paralysis (Progressive supranuclear palsy), Multiple system strophy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration , Huntington's disease, amyotrophic lateral sclerosis (ALS), Essential tremor, Cortico-basal ganlionic degeneration, Diffuse Lewy body disease ), Parkinson-ALS-dementia complex of Guam, Pick's disease, etc.

Among the above diseases, Parkinson's disease is a degenerative neurological disorder characterized by loss of dopaminergic neurons in the substantia nigra. Existing treatment methods for Parkinson's disease include oral treatment using levodopa drugs, dopamine agonists, anticholinergics, COMT, and MAO-B enzyme inhibitors. These are drugs designed to supplement dopamine substances or mimic the action of dopamine. They cannot prevent the disease from worsening and cause serious problems such as dyskinesia, nausea, and delusions, so the development of a treatment for Parkinson's disease is urgent.

A key hurdle in developing treatments for degenerative brain diseases such as Parkinson's disease is the difficulty in drug delivery due to the existence of the blood brain barrier (BBB). Existing therapeutic drugs cannot be efficiently delivered to the brain through the blood-brain barrier using systemic delivery. The method of delivering drugs to the brain by bypassing the blood-brain barrier without systemic injection is known to deliver drugs to the brain via the trigeminal nerve through the nasal cavity, and is attracting attention as a new method of administering drugs to treat brain diseases. . Research on nasal-brain delivery through nasal injection of relatively small-sized peptides such as insulin has recently been conducted (PMID: 29970188), and such nasal-brain injection 1) enables efficient brain delivery of drugs, and 2) whole body. It is known to have the therapeutic effect of preventing the decomposition of drugs due to injection, and 3) reducing the side effects of drugs due to systemic injection.

Humanin is the first mitochondria-derived peptide (MDP) discovered. Humanin, first reported in 2001, was cloned from a cDNA library derived from surviving neurons of Alzheimer's patients and mapped to the mitochondrial genomic 16S ribosomal RNA locus (PMID: 11371646). Afterwards, through various cell and animal experiments, 1) reduction of cytotoxicity through amyloid beta binding (PMID: 28282805), 2) prevention of myocardial ischemia (PMID: 20651283), 3) inhibition of cytotoxicity through IGFBP3 binding (PMID: 14561895), The biological functions of humanin have been revealed: 4) inhibition of cytotoxicity through BAX binding (PMID: 12732850), 5) improvement of endothelial function and prevention of atherosclerosis (PMID: 21763658).

Although the therapeutic mechanism of Humanin is not yet clearly known, the receptors expressed on the cell surface are trimeric gp130, WSX1, and CNTF receptor (PMID: 19386761) and G protein-coupled formylpeptide receptor-like-1 (PMID: 15153530). ), etc., it is known that ligand-receptor binding can occur, thereby activating important signaling pathways within cells.

The fundamental mechanisms causing Parkinson's disease are not yet known, and treatment options are still very limited. Therefore, the present inventor found that the mitochondria-derived peptide Humanin peptide passes through the blood-brain barrier through the trigeminal nerve due to nasal inhalation. In addition, it was confirmed that dopaminergic neurodegeneration was suppressed by increasing the expression of oxidative stress defense genes.

In addition, the present invention was completed by confirming the therapeutic effect by injecting the mitochondria-derived peptide humanin of the present invention into a Parkinson's mouse model.

The purpose of the present invention is to provide a composition for preventing or treating degenerative brain diseases containing humanin peptide as an active ingredient.

In addition, an object of the present invention is to provide a method for preventing and treating degenerative brain diseases comprising administering to an individual a pharmaceutical composition of humanin peptide in a pharmaceutically effective amount.

The present invention provides a composition for preventing or treating degenerative brain diseases containing humanin peptide as an active ingredient.

In addition, the present invention provides a method for preventing and treating degenerative brain diseases comprising administering a pharmaceutical composition of humanin peptide in a pharmaceutically effective amount to a subject.

When the humanin peptide of the present invention was administered nasally to a mouse model of Parkinson's disease, it was confirmed that it was effectively delivered and distributed to the brain. In addition, it was confirmed that dopamine neuron expression increased and neuron damage was suppressed. In addition, it was confirmed to suppress nerve cell damage in a Parkinson's disease cell model, making it effective in treating Parkinson's disease, so it can be usefully used in related projects.

Figure 1 is a diagram showing a schedule for intranasal administration of the mouse model of the present invention.

Figure 2 is a diagram measuring the time for the mouse model to change direction to come down the bar by intranasally administering Humanin peptide to the neurotoxin MPTP Parkinson's disease mouse model.

Figure 3 is a diagram showing whether humanin peptide, a neurotoxin, was administered intranasally to a mouse model of Parkinson's disease, a neurotoxin MPTP, and the total time to descend the bar was measured to confirm whether or not there was improvement in movement.

Figure 4 shows fluorescent staining of Lectin, a vascular marker in the trigeminal nerve, after intranasal administration of FITC fluorescent Humanin peptide to a genetically modified mouse model overexpressing alpha-synuclein. It's a degree.

Figure 5 shows fluorescence staining of α-SMA, a marker for blood vessel wall cells in the trigeminal nerve, after intranasal administration of FITC fluorescent humanin peptide to a genetically modified mouse model overexpressing α-synuclein. It's a degree.

Figure 6 shows the area around the ventral tegmental area (VTA) where dopaminergic neurons are located and the cerebral aqueduct (AQ) after intranasal administration of FITC fluorescent Humanin peptide to a genetically modified mouse model overexpressing α-synuclein. ) This is a diagram confirming the distribution of the Humanin peptide discovered in ).

Figure 7 is a diagram confirming that there is no significant difference in the amount of circulating humanin peptide between normal patients and Parkinson's disease patients.

Figure 8 shows the inhibition of tyrosine hydroxylase (TH) enzyme damage to substantia nigra dopamine neurons after humanin peptide was intranasally injected for 8 days into a Parkinson's disease mouse model injected with the neurotoxin MPTP. This diagram shows the results of immunochemical staining.

Figure 9 shows tyrosine hydride with increased expression of cerebrostriatal dopamine neurons after intranasal injection of Humanin peptide (0.1 mg/kg, 5 mg/kg) for 8 days in a Parkinson's disease mouse model injected with the neurotoxin MPTP. This is a diagram showing the tyrosine hydroxylase (TH) protein confirmed by immunoblot.

Figure 10 is a diagram showing neuronal damage through MTT assay after administration of Humanin peptide to a Parkinson's disease cell model induced with 6-OHDA neurotoxin.

Figure 11 is a diagram showing neuronal damage through trypan blue exclusion assay after administration of Humanin peptide to a Parkinson's disease cell model induced with 6-OHDA neurotoxin.

Figure 12 is a diagram showing the confirmation of neurons by administering Humanin peptide to a Parkinson's disease cell model induced by 6-OHDA or MPP+ neurotoxin in dopaminergic neurons derived from fetal midbrain undifferentiated neurons.

Figure 13 is a diagram showing the length of nerve cell protrusions by administering Humanin peptide to a Parkinson's disease cell model in which 6-OHDA or MPP+ neurotoxin was induced in dopaminergic neurons derived from fetal midbrain undifferentiated neurons.

Figure 14 is a diagram confirming the number of nerve cell projections by administering Humanin peptide to a Parkinson's disease cell model in which 6-OHDA or MPP+ neurotoxin was induced in dopaminergic neurons derived from fetal midbrain undifferentiated neurons.

Figure 15 shows that after intranasally injecting Humanin peptide (0.1 mg/kg, 5 mg/kg) for 8 days into a Parkinson's disease mouse model injected with the neurotoxin MPTP, the cerebrostriatal antioxidant enzyme NQO1 (Quinone-Oxidoreductase-1) ) This is a diagram showing the expression.

Figure 16 shows the cerebrostriatal antioxidant enzyme GSR (glutathione-disulfide reductase) after intranasal injection of Humanin peptide (0.1 mg/kg, 5 mg/kg) for 8 days in a Parkinson's disease mouse model injected with the neurotoxin MPTP. This is a diagram showing manifestation.

Figure 17 shows the level of cerebrostriatal antioxidant enzyme SOD1 (Superoxide dismutase-1) after intranasal injection of Humanin peptide (0.1 mg/kg, 5 mg/kg) for 8 days in a Parkinson's disease mouse model injected with the neurotoxin MPTP. This is a diagram showing manifestation.

Hereinafter, the present invention will be described in detail as an example of implementation of the present invention with reference to the attached drawings. However, the following implementation examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited by this, and the present invention is capable of various modifications and applications within the description of the claims described later and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and feeds are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.

As used in the present invention, the term “prevention” may mean any act of suppressing or delaying the onset of a degenerative brain disease by administering the pharmaceutical composition for preventing or treating a degenerative brain disease according to the present invention to an individual.

As used in the present invention, the term “treatment” may refer to any act of administering the composition of the present invention to a subject suspected of developing a degenerative brain disease to improve or benefit the symptoms of the degenerative brain disease.

As used herein, the term “improvement” may mean any action that reduces at least the degree of a parameter related to the condition being treated, such as a symptom.

When alpha-synuclein (α-syn) protein is abnormally formed, alpha-synuclein, the main causative gene for Parkinson's disease, binds and aggregates into a toxic oligomer and fibril form. When alpha-synuclein (α-syn) is overproduced or in the presence of specific ligands, it forms oligomers and aggregates into mature fibrils.

The present invention provides a composition for preventing or treating degenerative brain diseases containing Humanin peptide as an active ingredient.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase immunity.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.

Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

Additionally, the injectable preparation may use fatty acids such as oleic acid.

The humanin peptide may be represented by SEQ ID NO: 1.

In one embodiment, the method of administering the Humanin peptide may be one or more of the following: nasal administration, oral administration, transdermal administration, subcutaneous administration, sublingual administration, intradermal administration, oral administration, topical administration, and ophthalmic administration. However, it is specifically a nasal administration.

In one embodiment, the humanin peptide may include SEQ ID NO: 1.

In one embodiment, the composition may inhibit damage to substantia nigra dopamine neurons, but is not limited thereto.

In one embodiment, the composition may, but is not limited to, increase striatal dopamine neuron expression.

In one embodiment, the composition may be used to restore nerve cell damage, but is not limited thereto.

In one embodiment, the composition may increase the length or number of nerve cell projections, but is not limited thereto.

In one embodiment, the composition increases the expression of antioxidant enzymes in the cerebrostriatum, but is not limited thereto.

The antioxidant enzymes include Quinone-Oxidoreductase-1 (NQO1), glutathione-disulfide reductase (GSR), and Superoxide dismutase-1 (SOD1), but are not limited thereto.

In one embodiment, the degenerative brain disease includes Parkinson's disease (PD), Alzheimer's disease, progressive supranuclear palsy, multiple system atrophy, and olivary nucleus-pontine-cerebellar atrophy. (Olivopontocerebellar atrophy; OPCA), Shy-Drager syndrome, Striatonigral degeneration, Huntington's disease, Amyotrophic lateral sclerosis (ALS), essential tremor (Essential tremor), Cortico-basal ganlionic degeneration, Diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, Pick's disease ( It may be one or more selected from the group consisting of Pick's disease, but is not limited thereto.

Alpha-synuclein, the main causative gene for Parkinson's disease, is a toxic oligomer that binds and aggregates when the alpha-synuclein protein is formed abnormally. , it converts into a fibril form, and overproduction of α-synuclein or the presence of a specific ligand forms oligomers and aggregates into mature fibrils.

Example 1. Mitochondrial-derived peptide Humanin peptide synthesis and intranasal administration process

Example 1-1. Mitochondrial-derived peptide Humanin peptide synthesis sequence

The mitochondria-derived peptide Humanin peptide of the present invention was synthesized in the following steps. One). Humanin peptide was weighed with 10 g of Fmoc-Ala-2-CTC resin (loading 0.3 mmol/g), added to the reaction column, and swollen with DCM for 30 minutes. 2). Fmoc was deprotected with 20% Piperidine/DMF, mixed for 10 minutes, washed with DMF, and the washing step was repeated. 3). 6 mmol Fmoc-Arg(Pbf)-OH, 6 mmol HOBT, 6 mmol HBTU, and 6 mmol DIEA were added to the resin and coupled for 40 min at room temperature. 4). After coupling is completed, wash the resin 1-2 times with DMF. 5). Steps 2) through 4) were repeated to increase the length of the peptide chain until all amino acids were sequentially bound to the chain. 6). After the last amino acid was attached to the chain, the peptide was deprotected, the resin was washed three times with MeOH, and the resin was dried. 7). Put the resin in a tube, add an appropriate amount of cleavage solution, incubate at 35 degrees for 2.5 hours, and add ether to precipitate the solution. 8) The peptide sample was air-dried and then freeze-dried to synthesize the Humanin peptide of the present invention. The humanin peptide is included in SEQ ID NO: 1.

Example 1-2. Process of intranasal administration of Humanin peptide, a mitochondria-derived peptide

In the case of intranasal administration of the Humanin peptide of the present invention, Humanin is a composition containing 24 amino acids, and the final concentration (0.1 mg/kg, 5 mg/kg) is obtained by adding RNase-free water to the dried powder. 40 μl of the solution was injected into the mouse, 20 μl each, into the left and right nostrils for 3 minutes using a micropipette. To help absorb the injected reagent, the mouse head was kept in a downward position for 3 minutes after injection.

As shown in Figure 1, to verify the efficacy of preventing or treating Parkinson's disease neurodegeneration, the neurotoxin MPTP was administered intranasally once a day for a total of 8 days, starting 3 days before injection and 4 days after MPTP injection.

Example 2. Production of Parkinson's disease-induced mouse model

Example 2-1. Preparation of experimental animals

As experimental animals, 7-8 week old C57 Black 6 (C57BL/6) male mice (DBL Co., Ltd., Korea) weighing approximately 25 g were bred in the animal laboratory of the College of Life and Resources Sciences at Dong-A University. Water and feed were allowed to be consumed freely, and temperature (23 ± 2°C), humidity (55 ± 10%), and light/dark cycle (12 hours) were automatically controlled. All animals were acclimatized for 7 days before drug administration. All experiments were conducted in accordance with the approval of Dong-A University Animal Experiment Ethics Committee (DIACUC-Approval-20-25).

Example 2-2. Preparation of MPTP-induced Parkinson's animal model

Animal model was created by intraperitoneally administering 20 mg/kg of MPTP hydrochloride (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride, Sigma-Aldrich, USA) to experimental animals at 2-hour intervals four times a day. was prepared, and the control group was administered the same volume of sterile saline solution in the same manner.

Example 2-3. Alpha-synuclein gene animal model

An alpha-synuclein gene animal model was used as a genetically modified model for Parkinson's disease. The C57BL/6-Tg(NSE-hαSyn)Korl model is a mouse that has been genetically modified to overexpress α-synuclein, the causative agent of Parkinson's disease, only in brain tissue. Genetically modified mice were purchased from the Korea Food and Drug Safety Evaluation Institute (registration number 18-NIFDS-M-NE-014), and 12-month-old mice were used in the experiment.

Example 2-4. 6-OHDA toxicity cell model using human neuroblastoma SH-SY5Y

A Parkinson's disease cell model was created by treating SH-SY5Y cells, a human neuroblastoma cell, with 6-OHDA (6-Hydroxydopamine hydrobromide, Sigma-Aldrich, USA) neurotoxin. 100 μM of 6-OHDA was treated for 24 hours, and cell damage was measured using the MTT Assay Kit (abcam, USA).

Example 2-5. MPP+, 6-OHDA toxicity cell model using primary midbrain dopaminergic neurons

Neurons were obtained by dissecting brain tissue from the cortex of mouse (C57BL6) embryos on day 13.5 (E13.5) of embryogenesis. To induce differentiation, embryonic cells were treated with 0.025% trypsin/EDTA and then attached to a glass coverslip coated with poly-L-ornithine (PLO, Sigma-Aldrich, USA). Cell culture medium for neuronal differentiation is Neurobasal medium containing 50 U/ml penicillin and streptomycin, 1x B-27 supplement, 5% (vol/vol) FBS, 0.36% D-(+)-Glucose (wt/vol), and 0.25% Prepared by adding BSA (wt/vol). It was cultured in a 5% CO2 incubator for 9 days and then used in the experiment.

Example 3. Confirmation of motor improvement ability in MPTP Parkinson's disease mouse model upon intranasal administration of Humanin peptide

When the humanin peptide of the present invention is administered intranasally to the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model, the time and bar for which the mouse model changes direction by raising and lowering the bar The ability to improve exercise was confirmed by measuring the total time to come down.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a prodrug of the neurotoxic MPP ⁺ , which causes permanent symptoms of Parkinson's disease by destroying dopaminergic neurons in the substantia nigra of the brain. .

Therefore, it was confirmed that when the Humanin peptide (0.1 mg/kg, 5 mg/kg) of the present invention was administered intranasally to the MPTP-injected Parkinson's mouse model, the time to turn downward to lower the bar was shortened. (Figure 2), it was confirmed that the total time to come down the bar was shortened (Figure 3).

Example 4. Confirmation of intranasal administration and brain delivery effect of Humanin peptide to mouse model overexpressing alpha-synuclein

In a mouse model overexpressing alpha-synuclein of the humanin peptide of the present invention To confirm successful delivery when administered intranasally, 5 mg/kg of FITC fluorescent Humanin peptide was administered intranasally and Lectin, a vascular marker within the trigeminal nerve in an alpha-synuclein overexpressing mouse model, was administered. The presence of markers, vascular wall cell markers within the trigeminal nerve, and humanin peptide around the ventral tegmental area (VTA) and cerebral aqueduct (AQ) were confirmed.

A key hurdle in the development of treatments for degenerative brain diseases such as Parkinson's disease is the presence of the blood brain barrier (BBB), which makes drug delivery difficult. It is known that drugs are delivered to the brain via the trigeminal nerve through the nasal cavity. there is.

Therefore, after intranasal administration of FITC fluorescent Humanin peptide (5 mg/kg), Lectin, a vascular marker within the trigeminal nerve, was fluorescently stained, and α-SMA, a vascular wall cell marker within the trigeminal nerve, was fluorescently stained. In addition, the distribution of Humanin peptide found around the ventral tegmental area (VTA) where dopaminergic neurons are located and in the cerebral aqueduct (AQ) was confirmed.

As a result, when FITC fluorescent Humanin peptide (5 mg/kg) was administered intranasally to a mouse model overexpressing α-synuclein, Lectin, a vascular marker within the trigeminal nerve, was fluorescently stained (Figure 4), and α-SMA, a vascular wall cell marker within the trigeminal nerve, was fluorescently stained (Figure 5), and the ventral tegmental area where dopaminergic neurons are located. (Figure 6), it was confirmed that the Humanin peptide of the present invention was well delivered to the brain when administered intranasally.

Example 5. Confirmation of the amount of humanin peptide circulating in the body of normal patients and Parkinson's disease patients

The amount of humanin peptide circulating in the body of normal patients and Parkinson's disease patients was confirmed.

Normal patients are those with no current or past clinical history of malignant tumors and no systemic diseases such as diabetes, high blood pressure, viral infections, or tuberculosis.

As a result of quantifying the amount of humanin peptide circulating in the peripheral blood plasma of Parkinson's disease patients using ELISA (enzyme-linked immunosorbent assay), the amount of circulating humanin peptide between normal patients and Parkinson's disease patients was It was confirmed that there was no significant difference (Figure 7, Table 1).

[Table 1]

* BMI, body mass index; UPDRS III, part III of the Movement Disorders Society Unified Parkinson's disease rating scale

Example 6. Confirmation of inhibition of nerve cell damage by the Humanin peptide of the present invention.

Example 6-1. Confirmation of inhibition of nerve cell damage by the Humanin peptide of the present invention (in vivo)

To confirm whether the Humanin peptide of the present invention inhibits nerve cell damage, Humanin peptide (0.1 mg/kg, 5 mg/kg) was intranasally injected into Parkinson's disease mice injected with the neurotoxin MPTP for 8 days. Thus, suppression of damage to substantia nigra dopamine neurons and expression of dopamine neurons in the cerebrostriatum were confirmed.

In the Parkinson's disease mouse model injected with the neurotoxin MPTP, substantia nigra dopamine neurons were damaged, but as a result of intranasal administration of 0.1 mg/kg and 5 mg/kg of the Humanin peptide of the present invention, Humanin peptide As the concentration increased, damage to substantia nigra dopamine neurons was suppressed (Figure 8), and expression of dopamine neurons in the cerebrostriatum was confirmed to increase (Figure 9).

Example 6-2. Confirmation of inhibition of nerve cell damage by the Humanin peptide of the present invention (in vitro)

In order to confirm whether the Humanin peptide of the present invention inhibits neuronal damage, Humanin was applied to SH-SY5Y cells, a human neuroblastoma cell model, in a Parkinson's disease cell model induced by the neurotoxin 6-OHDA. 24 hours have passed since treatment at different peptide concentrations (0.1, 1, 10, 50 um).

The degree of nerve cell damage was analyzed through MTT assay and trypan blue exclusion assay at different humanin peptide concentrations (0.1, 1, 10, and 50 μm).

As a result, according to the MTT assay, the higher the concentration of Humanin peptide, the higher the concentration of Humanin peptide compared to the control group that was not treated with 6-OHDA neurotoxin, the degree of neuronal damage was similar to that of the control group. This was confirmed (Figure 10). In addition, according to the trypan blue exclusion assay, the higher the humanin peptide concentration, the higher the concentration of humanin peptide compared to the control group not treated with 6-OHDA neurotoxin, the degree of neuronal damage in the control group was similar. By confirming this, it was confirmed that the Humanin peptide of the present invention suppressed nerve cell damage in a Parkinson's disease cell model (FIG. 11).

Example 7. Confirmation of changes in nerve cell protrusion length and number when administering the Humanin peptide of the present invention

In order to determine whether administration of the Humanin peptide of the present invention affects the length and number of nerve cell projections, dopaminergic neurons derived from fetal midbrain undifferentiated neurons were treated with the neurotoxin 6-OHDA or MPP ⁺ to treat Parkinson's disease cells. A model was created, and after treatment with 10 μM of the Humanin peptide of the present invention for 24 hours, the length and number of nerve cell protrusions were measured. Confirmed. Humanin peptide was directly treated with neuronal culture medium.

As a result, healthy nerve cells were confirmed in the group treated with the Humanin peptide of the present invention compared to the control group (DMSO) (FIG. 12), and the protrusion length and number of protrusions of nerve cells were higher than those of the control group (DMSO) due to the neurotoxin 6-OHDA or It was confirmed that MPP ⁺ was induced to increase the length of nerve cell projections and the number of projections in the Parkinson's disease cell model (Figures 13 and 14).

Example 8. Analysis of antioxidant enzyme expression in the Humanin peptide Parkinson's disease mouse model of the present invention

In order to analyze the expression of antioxidant enzymes using the Humanin peptide of the present invention, the Humanin peptide of the present invention was administered to a Parkinson's disease mouse model injected with the neurotoxin MPTP to express antioxidant enzymes NQO1, GSR, and SOD1 in the striatum. was analyzed.

Humanin peptide (0.1 mg/kg, 5 mg/kg) was injected intranasally for 8 days into a Parkinson's disease mouse model injected with the neurotoxin MPTP. As a result, Humanin peptide from the Parkinson's disease mouse model was administered intranasally. Upon administration, it was confirmed that the higher the concentration, the higher the expression of NQO1, GSR, and SOD1, confirming that the Humanin peptide of the present invention expresses antioxidant enzymes in the brain (Figures 15, 16, and 17) .

Additionally, the present invention provides a method for preventing and treating degenerative brain diseases comprising administering a pharmaceutically effective amount of the humanin to an individual. The pharmaceutical composition of the present invention is administered in a therapeutically effective or pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (12)

1. A composition for preventing or treating degenerative brain diseases containing humanin peptide as an active ingredient.
2. According to clause 1,

   A composition wherein the humanin peptide includes SEQ ID NO: 1.
3. According to clause 1,

   The method of administration of the humanin peptide is one or more of the following: nasal administration, oral administration, transdermal administration, subcutaneous administration, sublingual administration, intradermal administration, oral administration, topical administration, and ophthalmic administration.
4. According to clause 3,

   The composition, wherein the administration method is nasal administration.
5. According to clause 1,

   The composition inhibits damage to substantia nigra dopamine neurons.
6. According to clause 1,

   The composition increases the expression of cerebrostriatal dopamine neurons.
7. According to clause 1,

   The composition is for recovering nerve cell damage.
8. According to clause 1,

   The composition increases the length or number of nerve cell projections.
9. According to clause 1,

   The composition increases the expression of antioxidant enzymes in the cerebrostriatum.
10. According to clause 9,

    The composition, wherein the antioxidant enzymes are NQO1 (Quinone-Oxidoreductase-1), GSR (glutathione-disulfide reductase), and SOD1 (Superoxide dismutase-1).
11. According to clause 1,

    The degenerative brain diseases include Parkinson's disease (PD), Alzheimer's disease, progressive supranuclear palsy, multiple system atrophy, olivopontocerebellar atrophy; OPCA), Shy-Dragersyndrome, Striatonigral degeneration, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Essential tremor , cortico-basal ganlionic degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, and Pick's disease. A composition selected from the group consisting of one or more.
12. A method for preventing and treating degenerative brain disease comprising administering a pharmaceutically effective amount of the pharmaceutical composition of claim 1 to a subject.

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