KR20180021470A - Anti-infalmmatory pharmaceutical composition containing Atox1 fusion protein - Google Patents

Abstract

The object of the present invention is to provide an effective anti-inflammatory therapeutic agent with negligible or no side effects. To this end, the present invention provides an anti-inflammatory pharmaceutical composition comprising a cell-transducible ATOX1 fusion protein, in which the Atox1 fusion protein penetrated into a cell increased cell viability against the toxicity of hydrogen peroxide and reduced the level of active oxygen species, thereby providing cell protection. In addition, the Atox1 fusion protein significantly reduced the expression levels of LPS-induced COX2 and iNOS protein in a dose-dependent manner. In addition, a Tat-Atox1 fusion protein suppressed the inflammation response induced by high-concentration LPS. Further, the Tat-Atox1 fusion protein significantly inhibited the infiltration of inflammatory cells, such as monocytes, which occurs at an early stage of skin inflammation; significantly reduced the expression level of COX-2 and the production of TNF-α, IL-1β, and IL-6 in an animal model of TPA-induced inflammation; and inhibited TPA-induced phosphorylation of p38, ERK and JNK, and phosphorylation of p65 and IkBα in an animal model of TPA-induced skin inflammation. Accordingly, the transduced Tat-Atox1 fusion protein, by inhibiting an autophagy pathway, was shown to protect cells from hydrogen peroxide-induced cell apoptosis, and has a therapeutic effect against inflammations.

Description

[0001] The present invention relates to an anti-inflammatory pharmaceutical composition containing a cytotoxic Atoxl fusion protein,

The present invention relates to an anti-inflammatory pharmaceutical composition comprising a cell-permeable Atox1 fusion protein.

Reactive oxygen species (ROS) are naturally produced as a by-product of various cellular metabolism processes including oxygen interactions and macromolecular damage. The cells are damaged by changing the structure and function of the cells due to the unbalance of the production and disappearance of reactive oxygen species. When exposed to long-term reactive oxygen species, the production of peroxidized lipids, DNA damage, inflammation and death of cells, , Parkinson's, Huntington, and many other diseases. Active oxygen species that regulate the oxidation of cells play an important role in the immune system, balancing its production and destruction. Many researchers are focusing on inflammation and aging caused by reactive oxygen species.

Atox1 (Antioxidant 1), known as ATX1, is composed of 68 amino acids. Atox1 is a copper chaperone that plays an important role in maintaining copper homeostasis. Copper is essential in all organisms and is important as a common factor for catalysts involved in many biochemical processes such as eukaryotic respiration, antioxidant, and iron metabolism. Atox1 activates cerroloplasmin, an oxidase in the blood that binds to copper, by providing ATPase (adenosine triphosphatase) to the cytoplasmic copper in the late period, and protects the cells against peroxidation and hydrogen peroxide-induced toxicity. And affects the development of disease caused by oxidative stress.

Protein transduction uses protein transduction domains (PTDs) that transfer various exogenous proteins to mammalian cells. Eukaryotic cells are surrounded by a plasma membrane consisting of a lipid bilayer that limits the transfer of macromolecules such as proteins and nucleic acids. Proteins also have limited permeability to tissues or cells due to their size, low permeability and biochemical properties. The major amino acid sequence of the protein transport domain to overcome this limitation is used as a way to transfer macromolecules in many studies because of its ability to penetrate cell membranes. Although exact mechanisms have not been elucidated, fusion proteins covalently linked to protein transport domains are widely used to deliver various proteins into cells in vitro and in vivo . The Tat-peptide is derived from the HIV-1 Tat peptide (RKKRRQRRR; Tat 49-57 aa) and is the most commonly used protein transport domain. The Tat-peptide, which can efficiently be transduced into cells, has an advantage of being excellent in stability, having little toxicity and being insensitive to serum.

The present invention aims at providing therapeutic pharmaceutical compositions useful for inflammatory diseases.

We investigated the function of Tat-Atox1 fusion protein transduced in oxidative stress induced cell death by hydrogen peroxide. Tat-Atox1 fusion protein was prepared by fusing Atox1 and Tat peptide, and it was confirmed that it was efficiently transduced into Raw 264.7 cells. In addition, oxidative stress was induced in Raw 264.7 cells and cytoprotective effect of Tat-Atox1 fusion protein in hydrogen peroxide induced cell death was examined. The expression of anti-apoptotic protein in Raw 264.7 cells induced oxidative stress was found to be inhibited in proportion to the concentration of Tat-Atox1 fusion protein. Thus, Tat-Atox1 fusion protein is well penetrated into Raw 264.7 cells, and it protects cells by inhibiting oxidative stress-induced production of reactive oxygen species and apoptosis. This suggests that the Tat-Atox1 fusion protein transfected into Raw 264.7 cells prevents apoptosis and is useful as a therapeutic agent for Raw 264.7 cell inflammatory diseases due to oxidative stress.

The present invention relates to an inflammatory disease therapeutic composition containing an Atox1 fusion protein in which a protein transport domain is covalently bound to one or more of the N-terminal and C-terminal of an Atox1 (Antioxidant 1) protein.

The present invention also relates to an inflammatory therapeutic composition containing an Atox1 fusion protein, wherein the protein transport domain is an HIV Tat peptide.

In addition, the present invention relates to an inflammatory therapeutic composition comprising an Atox1 fusion protein, wherein the fusion protein is SEQ ID NOS: 4, 6,

The pharmaceutical composition containing the Atox1 fusion protein as an active ingredient can be formulated in oral form or injected form by a conventional method in combination with a carrier which is conventionally acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single daily dose of 0.0001 to 100 mg / kg dividedly in several doses. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

The intracellular delivery of the Atox1 protein molecule according to the present invention is carried out in the form of a hydrophobic domain consisting of 15 to 30 amino acids in Atox1 and containing 5 or more tryptophan, a hydrophilic domain containing 4 or more lysines domain, and a spacer that separates the two domains is covalently linked to the N-terminus of Atox1, the target protein, and / or the transmembrane domain, including Pep-1, Terminus and a covalently linked form of the fusion protein with the C-terminus. Examples of the transport domain of the present invention include Pep-1 peptide and HIV Tat 49 to 57 peptide. However, the protein transport domain of the present invention is not limited to the Pep-1 peptide, the HIV Tat 49-55 residue peptide, and the Pep-1 peptide or HIV Tat Since it is easy for a person skilled in the art to make a peptide having a function similar to that of a peptide, a hydrophobic domain consisting of 15 to 30 amino acids and containing at least 5 tryptophan, , A hydrophilic domain containing four or more lysines, and a spacer for separating the two domains, and a protein transport domain carrying the same or similar protein-transporting function with partial amino acid substitution therefrom Domain, or six to twelve amino acid residues, wherein the arginine or lysine residue An oligolysine transport domain consisting of 6 to 12 lysines, an oligosaccharide transport domain consisting of 6 to 12 arginines, or an oligos (lysine, lysine, or lysine) chain consisting of 6 to 12 lysine or arginine, Arginine) transport domains are also within the scope of the invention.

The definitions of the main terms used in the description of the present invention and the like are as follows.

"Atox1 fusion protein" means a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and Atox1, and a protein transport domain and a cargo molecule (i.e., Atox1 in the present invention). As a specific example in the present specification and drawings, "Tat-Atox1" refers to the binding of the Tat protein transport domain to the N-terminus of Atox1 among the Atox1 fusion proteins.

In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in an embodiment of the present invention, the target cell refers to Raw 264.7 cells.

As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bound to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, or energy. For example, it refers to HIV-1 Tat.

As used herein, the term "target protein" refers to a molecule that is covalently bound to Tat protein transport domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

In the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

In the present invention, the protein transport domain is a protein transport domain composed of 15 to 30 amino acids and composed of a non-hydrophilic domain containing 5 or more tryptophan, a hydrophilic domain containing many lysines, and a spacer separating the two domains, A transport domain comprising 6 to 12 amino acid residues and comprising ¾ or more arginine or a lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoroginalin transport domain consisting of 6 to 12 arginines, Or an oligo (lysine, arginine) transport domain consisting of 12 lysine or arginine. In addition, the target protein (cargo molecule) is Atox1. The protein transport domain and the target protein may be replaced by other amino acid (s) of similar polarity in which one or more amino acids functionally equivalently function in the sequence according to the silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, the hydrophobic amino acid class includes alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Acidic amino acids with negative charge include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a certain range of homology, for example, 85-100%, between the fusion protein of the present invention and the amino acid sequence are included in the scope of the present invention.

The inflammatory diseases defined in the present invention include inflammatory diseases such as skin inflammatory diseases including atopic dermatitis, nerve cell inflammatory diseases such as glioma cells, spondylitis, urethritis, cystitis, nephritis, pyelonephritis, vasculitis, rhinitis, sore throat, tonsillitis, acute pain or inflammatory bowel Disease, etc., and is preferably a skin inflammatory disease, urethritis, cystitis, nephritis, pyelonephritis, rhinitis, sore throat, tonsillitis or inflammatory bowel disease.

The Atox1 fusion protein of the present invention was permeabilized intracellularly in a dose and time-dependent manner, and the permeable Atox1 fusion protein showed a remarkable level in the cells for 13 hours.

The Atoxl fusion protein of the present invention significantly reduced LPS-induced levels of COX2 and iNOS protein expression in a dose-dependent manner. In addition, Atox1 fusion protein inhibited the inflammatory response induced by high concentration of LPS.

Also, Atox1 fusion proteins of the present invention is transmitted to the cell exhibited a cell protective effect by reducing the active oxygen levels in LPS or cell H ₂ O ₂ treatment.

In addition, the Atoxl fusion protein of the present invention significantly inhibited the infiltration of inflammatory cells such as monocyte early on in skin inflammation, and the expression level of COX2 and TNF-alpha, IL- 1β and IL-6 production, and inhibited p38, ERK and JNK phosphorylation and p65 and I k Bα phosphorylation in TPA-induced skin inflammatory animal models.

1 is a diagram showing the construction and purification of a Tat-Atox1 fusion protein.
Figure 2 shows the permeation of Tat-Atoxl fusion protein into Raw 264.7 cells. The cells were cultured in a 60 mm culture dish and Tat-Atox1 fusion protein (0.01-0.3 μM) and control Atox1 protein were added to the culture medium for one hour and analyzed by Western blotting.
FIG. 3 shows Western blot analysis of Tat-Atox1 fusion protein (0.3 μM) and control Atox1 protein added to the culture medium for 15 to 60 minutes into Raw 264.7 cells.
FIG. 4 is a photograph showing the intracellular distribution of a cell-permeable Tat-Atox1 fusion protein observed by a confocal microscope.
Figure 5 shows the stability of the Tat-Atoxl fusion protein permeated into Raw 264.7 cells after various time lapses (1 to 15 hours). Cells were pretreated with 0.3 μM Tat-Atox1 fusion protein for one hour and analyzed by Western blot.
Figure 6 shows the inhibitory effect of Tat-Atoxl fusion protein on inflammatory response induced by LPS in Raw 264.7 cells.
FIG. 7 shows the result of COX-2 and iNOS measurement by western blot analysis by treating Atox1 fusion protein (0.01-0.3 μM) into Raw 264.7 cells for 1 hour and inducing inflammation with LPS.
FIG. 8 shows the result of measuring TNF-α, IL-1β and IL-6 by RT-PCT analysis by treating Atox1 fusion protein (0.01-0.3 μM) into Raw 264.7 cells for 1 hour and inducing inflammation with LPS.
FIG. 9 shows the result of p65, IκBα measurement by western blot analysis by treating Atox1 fusion protein (0.01-0.3 μM) into Raw 264.7 cells for 1 hour and inducing inflammation with LPS.
FIG. 10 shows the result of p38, JNK, and ERK measurement by western blot analysis by treating Atox1 fusion protein (0.01-0.3 μM) into Raw 264.7 cells for 1 hour and inducing inflammation with LPS.
Figure 11 shows the results of Western blot analysis of Akt by treating Atox1 fusion protein (0.01-0.3 [mu] M) into Raw 264.7 cells for 1 hour and inducing inflammation with LPS.
FIG. 12 shows the result of treatment of 0.3 μM of Tat-Atox1 fusion protein and H & E staining by inducing inflammation with TPA in 8-week-old male male ear.
Fig. 13 shows the results of Western blot analysis and RT-PCT of 8-week-old male mice ear treated with 0.3 μM of Tat-Atox1 fusion protein induced by TPA, and COX-2 and iNOS.
FIG. 14 shows the result of RT-PCT analysis of TNF-α, IL-1β, and IL-6 treated with 0.3 μM of Tat-Atox1 fusion protein induced by TPA in 8-week old male ears.
FIG. 15 shows the results of western blot analysis of p65 and IκBα treated with 0.3 μM of Tat-Atox1 fusion protein induced by TPA in 8-week-old male mice ear.
FIG. 16 shows results of western blot analysis of p38, JNK, and ERK treated with 0.3 μM of Tat-Atox1 fusion protein induced by TPA in 8-week old male ears.
FIG. 17 shows the results of Western blot analysis of Akt treated with 0.3 μM of Tat-Atox1 fusion protein induced by TPA in 8-week old male ears.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it should be apparent to those skilled in the art that the scope of the present invention is not limited by the description of the embodiments. Particularly, in the following examples, HIV Tat peptide was used as a protein transport domain and covalently bonded to the N-terminus of the target protein Atox1. However, the Atox1 fusion protein of the present invention is not necessarily limited to the Tat-Atox1 fusion protein It will be apparent to those skilled in the art to which the present invention pertains.

material

12- O -TPA (tetradecanoylphorbol-13- acetate) was purchased from the Sigma-Aldrich (St. Louis, MO, USA) , Ni 2 + Night reel tree ethyl Sepharose Super flow ^{(Ni 2 + - nitrilotri-acetic} acid Sepharose superflow) were purchased from Qiagen (Valencia, CA, USA). DMEM (Dulbecco's modified Eagle's medium) was purchased from Lonza (Walkersville, MD, USA), and FBS and antibiotics were purchased from Gibco BRL. The synthetic Tat peptide was purchased from Peptron (Daejeon, Korea), and the basic antibody and actin were purchased from Cell Signaling Technology (Beverly MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents used the highest quality products available.

Tat-Atox1 plasmid construction

To prepare the cell-permeable Tat-Atox1 fusion protein, the cDNA sequence of human Atox1 and two primers were used. The sense primer comprises a Xho I restriction site with 5'-CTCGAGATGCCGAAGCACG-3 'and the antisense primer contains a Bam HI restriction site with 5'-GGATCCCTACTCAAGGCCAAGG-3'. The resulting PCR product is ligated to TA vector (Novagen, Darmstadt, Germany) using T4 DNA ligase (Promega, Madison, Wis., USA) and the specific region of the plasmid is cut with Xho I and Bam HI. The cleaved fragments were ligated to the Tat expression vector to produce the Tat-Atox1 fusion protein, and the control Atox1 protein was prepared without the Tat peptide.

Tat-Atox1 fusion protein expression and purification

The recombinant Tat-Atoxl fusion protein and the control Atoxl protein plasmid were introduced into E. coli BL21 cells. The transformed cells were cultured in 100 ml of LB medium containing 100 μg / ml of ampicillin at 37 ° C. and 180 rpm for 6 hours, and then 0.5 mM IPTG (isopropyl- β-D-thiogalactoside) rpm for 24 hours. The recovered cells were sonicated and centrifuged. Only the supernatant was separated and purified with Ni ²⁺ -nitrile triacetic acid Sepharose Superfluent Affinity Chromatography (Qiagen, Balencia, CA, USA) and PD-10 column chromatography (Amersham, Braunschweig, Germany). Protein concentration was determined by standardizing bovine serum albumin (BSA) using a protein assay kit (Bio-Rad, Hercules, Calif., USA).

Raw 264.7 cells were transfected with Tat- Atox1  Transfection of fusion protein and hydrogen peroxide treatment

Subcultured Raw 264.7 cells were plated in 60 mm dishes and cultured at 37 ° C, 95% humidity and 5% CO ₂ . Protein was treated at various concentrations (0.001-0.3 μM) for 1 hour to confirm the cytotoxicity of Tat-Atox1 fusion protein and Atox1 protein. Protein (0.3 μM) was treated for 10 to 60 minutes at different treatment times. To determine how long Tat-Atox1 fusion protein remains in Raw 264.7 cells, 0.3 nM Tat-Atox1 fusion protein was treated and cells were harvested after a certain period of time (1 to 15 hours).

In addition, Tat-Atox1 fusion protein and Atox1 protein were treated with Raw 264.7 cells for 1 hour (0.01-0.3 μM) for 1 hour, then 1 mM hydrogen peroxide was added and incubated for 1 hour. The cultured cells were rinsed with PBS and treated with trypsin-EDTA. Protein was extracted from the supernatant by centrifugation (4 ° C, 12,000 rpm, 10 min) after dissolving the precipitate with RIPA lysis buffer (ELPIS BIOTECH, Daejeon, Korea)

Western Blot  analysis

Protein samples were prepared by mixing 30 ~ 50 ㎍ of protein with 5X sample buffer and boiled for 2 minutes. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) of 15% mini gel (Hoefer Scientific, San Francisco, It was separated according to molecular weight. After electrophoresis, the membrane was electro-transferred to nitrocellulose membrane and the membrane was blocked with 5% skim milk powder in TBS buffer containing 0.1% Tween 20 for 1 hour at room temperature. In order to measure protein expression, the primary antibody was diluted 1: 500 in PBS, reacted at room temperature for 3 hours, and then washed with TBS-T. As the secondary antibody, anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase was diluted 1: 10,000 in PBS and reacted at room temperature for 1 hour. After washing with TBS-T, the amount of each protein was detected using a detection reagent.

Confocal fluorescence microscopy observation

Cover glasses were placed in a 24-well plate, cells were placed on the plate, and cultured for 2 hours. 0.1 nM Tat-Atox1 fusion protein and Atox1 protein were transfected into Raw 264.7 cells for 2 hours and then washed 3 times with PBS. 4% paraformaldehyde was added to each well and the cells were fixed for 5 minutes, followed by washing three times with PBS. The cells were blocked with PBS (PBS-BT) containing 3% BSA and 0.1% Triton X-100 for 40 minutes at room temperature and washed three times with PBS-BT. The primary antibody (His-probe, Santa Cruz Biotechnology) was diluted at a ratio of 1: 2,000 and cultured at 4 ° C for 12 hours. The secondary antibody (Alexa Fluor 488) was diluted 1: 10,000, darkened, reacted for 1 hour, and washed 3 times with PBS-BT for 5 minutes. The cover glass was removed from the wells to remove moisture from the edges and mounted. Confocal fluorescence microscopy was used to observe fluorescence expression using an image analyzer.

Animal model manufacturing

ICR mice (male, 8 weeks old) were bred at a temperature of 23 ° C, 60% humidity and 12 hours of light / 12 hours dark cycle, where food and water were freely available. All experimental procedures, including animal care, were conducted in accordance with the guidelines for the management and use of laboratory animals by the National Veterinary Research and Quarantine Service at the Animal Care and Use Committee of the Hallym Medical Center.

Statistical analysis

All data were the mean values of the test results, and statistical differences were compared using the Student't-test method and were statistically significant at p <0.05.

Results 1: Tat- Atox1 Fusion protein  Produce

To prepare the cell-permeable Tat-Atox1 fusion protein, the human Atox1 gene was obtained by PCR and cloned into the pET vector system. The resulting protein expression gene was transformed into BL21. The transformed proteins were overexpressed and purified (Fig. 1).

Result 2: Raw 264.7 cells were transfected with Tat- Atox1 Of the fusion protein permeability  Confirm

The permeability of Tat-Atox1 fusion protein was confirmed by Raw 264.7 cells. Raw 264.7 cells were treated with various concentrations of Atox1 fusion protein and, as a result, were confirmed to be infiltrated in a concentration dependent manner. This was confirmed by western blot analysis using His Ab (Fig. 2).

In addition, the Atox1 fusion protein was infiltrated into Raw 264.7 cells in a time-dependent manner when the concentration of Tat-Atox1 fusion protein was fixed to 0.3 μM and treated with Raw 264.7 cells for various times. This was confirmed by Western blot analysis (FIG. 3) and fluorescence microscopy (FIG. 4) using His Ab.

Result 3: Tat- Atox1 Of the fusion protein  Raw 264.7 Confirm intracellular stability

The cell-permeable Tat-Atox1 fusion protein was fixed at 0.3 μM, treated with Raw 264.7 cells, and cultured for 1 hour. The stability of the Tat-Atox1 fusion protein infiltrated was confirmed and it was confirmed that the Tat-Atox1 fusion protein stably remained until 13 hours after the treatment (FIG. 5).

Result 4: Tat- Atox1 Of the fusion protein  Raw 264.7 cell protection efficacy

The cell-permeable Tat-Atox1 fusion protein was fixed at 0.3 μM, treated with Raw 264.7 cells, and cultured for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of the Tat-Atox1 fusion protein was confirmed using DCF-DA. As a result, it was confirmed that the group treated with the cell-permeable Tat-Atox1 fusion protein had an anti-inflammatory protective effect (Fig. 6).

The cell-permeable Tat-Atox1 fusion protein was treated with 0.01 μM to 0.3 μM Raw 264.7 cells and incubated for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of cell-permeable Tat-Atox1 fusion protein was confirmed by Western blot analysis using COX2 and iNOS. In addition, mRNA levels were also confirmed by RT-PCR. As a result, it was confirmed that the group treated with the Tat-Atoxl fusion protein had a cytoprotective effect (Fig. 7).

The cell-permeable Tat-Atox1 fusion protein was treated with 0.01 μM to 0.3 μM Raw 264.7 cells and incubated for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of the cell-permeable Tat-Atox1 fusion protein was confirmed by RT-PCR at the mRNA level using TNF-α, IL-1β, and IL-6. As a result, it was confirmed that the group treated with the cell-permeable Tat-Atox1 fusion protein had a protective effect (Fig. 8).

The cell-permeable Tat-Atox1 fusion protein was treated with 0.01 μM to 0.3 μM Raw 264.7 cells and incubated for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of the cell-permeable Tat-Atox1 fusion protein was confirmed by western blot analysis using p65 and IκBα. As a result, it was confirmed that the group treated with the permeable Atox1 fusion protein had a protective effect (Fig. 9).

The cell permeable Atox1 fusion protein was treated with 0.01 μM to 0.3 μM Raw 264.7 cells and cultured for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of the cell-permeable Tat-Atox1 fusion protein was confirmed by western blot analysis using p38, JNK, and ERK. As a result, it was confirmed that the group treated with the cell permeable Tat-Atox1 fusion protein had a protective effect (Fig. 10).

The cell permeable Atox1 fusion protein was treated with 0.01 μM to 0.3 μM Raw 264.7 cells and cultured for 1 hour. LPS was then treated to induce inflammation in the cells. At this time, the efficacy of the cell-permeable Tat-Atox1 fusion protein was confirmed by western blot analysis using Akt. As a result, it was confirmed that the group treated with the cell permeable Tat-Atox1 fusion protein had a protective effect (Fig. 11).

Result 5: Studies on inflammation through animal experiments

8 weeks old ICR mice were fed with TPA to induce inflammation. The TAt-Atox1 fusion protein was then treated with 0.3 [mu] M and the efficacy was assessed. The tissues were cut and H & E staining was performed. As a result, it was confirmed that the group treated with the cell-permeable Tat-Atox1 fusion protein had a protective effect (Fig. 12).

In addition, TPA was applied to the ears of 8 - week - old ICR mice and induced inflammation. The TAt-Atox1 fusion protein was then treated with 0.3 [mu] M and the efficacy was assessed. Tissue was cut and Western blot analysis was performed with COX2 and iNOS. In addition, mRNA levels were confirmed simultaneously by RT-PCR. (1: control, 2: TPA, 3: Atox1 protein, and 4: Tat-Atox1 fusion protein) were treated with the cell permeable Tat-Atox1 fusion protein. (Fig. 13).

In addition, TPA was applied to the ears of 8 - week - old ICR mice and induced inflammation. Then, 0.3 μM of the TAt-Atox1 fusion protein was treated and the efficacy was evaluated. Tissue was cut and the changes in mRNA levels of TNF-α, IL-1β, and IL-6 were confirmed by RT-PCR. (1: control, 2: TPA, 3: Atox1 protein, and 4: Tat-Atox1 fusion protein) were treated with the cell permeable Tat-Atox1 fusion protein. (Fig. 14).

In addition, TPA was applied to the ears of 8 - week - old ICR mice and induced inflammation. Then, 0.3 μM of permeable Atox1 fusion protein was treated and the efficacy was evaluated. The tissue was cut and Western blot analysis was performed using p65, IκBα. As a result, it was confirmed that the cell-permeable Atox1 fusion protein-treated group had a protective effect (1: control group, 2: TPA treatment group, 3: Atox1 protein treatment group, 4: Tat- 15).

In addition, TPA was applied to the ears of 8 - week - old ICR mice and induced inflammation. Then, 0.3 μM of permeable Atox1 fusion protein was treated and the efficacy was evaluated. Western blot analysis using p38, JNK, and ERK was performed. As a result, it was confirmed that the cell-permeable Atox1 fusion protein-treated group had a protective effect (1: control group, 2: TPA treatment group, 3: Atox1 protein treatment group, 4: Tat- 16).

In addition, TPA was applied to the ears of 8 - week - old ICR mice and induced inflammation. Then, 0.3 μM of permeable Atox1 fusion protein was treated and the efficacy was evaluated. Tissues were cut and Western blot analysis was performed using Akt. As a result, it was confirmed that the group treated with the permeable Atox1 fusion protein had a protective effect (1: control group, 2: TPA treatment group, 3: Atox1 protein treatment group, 4: Tat-Atox1 fusion protein treatment group) ).

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating Parkinson's disease          containing cell-transducible ATOX1 fusion protein <130> HallymU-syCHOI-Atox1-PD <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ctcgagatgc cgaagcacg 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatccctac tcaaggccaa gg 22 <210> 3 <211> 270 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Atox1 fusion protein <400> 3 agcagcggcc tggtgccgcg cggcagccat aggaagaagc ggagacagcg acgaagactc 60 gagatgccga agcacgagtt ctctgtggac atgacctgtg gaggctgtgc tgaagctgtc 120 tctcgggtcc tcaataagct tggaggagtt aagtatgaca ttgacctgcc caacaagaag 180 gtctgcattg aatctgagca cagcatggac actctgcttg caaccctgaa gaaaacagga 240 aagactgttt cctaccttgg ccttgagtag 270 <210> 4 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> Tat-Atox1 fusion protein <400> 4 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Pro Lys His Glu   1 5 10 15 Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala Glu Ala Val Ser Arg              20 25 30 Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp Ile Asp Leu Pro Asn          35 40 45 Lys Lys Val Cys Ile Glu Ser Glu His Ser Met Asp Thr Leu Leu Ala      50 55 60 Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr Leu Gly Leu Glu  65 70 75 <210> 5 <211> 278 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Atox1-Tat fusion protein <400> 5 agcagcggcc tggtgccggc ggcagccact cgagatgccg aagcacgagt tctctgtgga 60 catgacctgt ggaggctgtg ctgaagctgt ctctcgggtc ctcaataagc ttggaggagt 120 taagtatgac attgacctgc ccaacaagaa ggtctgcatt gaatctgagc acagcatgga 180 cactctgctt gcaaccctga agaaaacagg aaagactgtt tcctaccttg gccttgagta 240 gggatcctag gaagaagcgg agacagcgac gaagatag 278 <210> 6 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> Atox1-Tat fusion protein <400> 6 Met Pro Lys His Glu Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala   1 5 10 15 Glu Ala Val Ser Arg Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp              20 25 30 Ile Asp Leu Pro Asn Lys Lys Val Cys Ile Glu Ser Glu His Ser Met          35 40 45 Asp Thr Leu Leu Ala Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr      50 55 60 Leu Gly Leu Glu Arg Lys Lys Arg Arg Gln Arg Arg Arg  65 70 75 <210> 7 <211> 277 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding Tat-Atox1-Tat fusion protein <400> 7 aggaagaagc ggagacagcg acgaagactc gagatgccga agcacgagtt ctctgtggac 60 atgacctgtg gaggctgtgc tgaagctgtc tctcgggtcc tcaataagct tggaggagtt 120 aagtatgaca ttgacctgcc caacaagaag gtctgcattg aatctgagca cagcatggac 180 actctgcttg caaccctgaa gaaaacagga aagactgttt cctaccttgg ccttgagtag 240 ggatcctagg aagaagcgga gacagcgacg aagatag 277 <210> 8 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Tat-Atox1-Tat fusion protein <400> 8 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Pro Lys His Glu   1 5 10 15 Phe Ser Val Asp Met Thr Cys Gly Gly Cys Ala Glu Ala Val Ser Arg              20 25 30 Val Leu Asn Lys Leu Gly Gly Val Lys Tyr Asp Ile Asp Leu Pro Asn          35 40 45 Lys Lys Val Cys Ile Glu Ser Glu His Ser Met Asp Thr Leu Leu Ala      50 55 60 Thr Leu Lys Lys Thr Gly Lys Thr Val Ser Tyr Leu Gly Leu Glu Gly  65 70 75 80 Ser Arg Lys Lys Arg Arg Gln Arg Arg Arg                  85 90

Claims (3)

An inflammatory therapeutic composition comprising an Atox1 fusion protein having a protein transport domain covalently bound to one or more of the N-terminal and C-terminal of an Atox1 (Antioxidant 1) protein.
The method according to claim 1,
Wherein the protein transport domain is an HIV Tat peptide.
The method according to claim 1,
Wherein said fusion protein is SEQ ID NOS: 4, 6, or 8.

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