KR101671197B1 - Pharmaceutical composition for arthritis containing glutaredoxin 1 fusion protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of inflammatory arthritis, and more particularly to a pharmaceutical composition for the prevention or treatment of inflammatory arthritis which comprises glutaredoxin 1 fusion protein capable of penetrating into cartilage tissue as an active ingredient will be.

Description

[0001] The present invention relates to pharmaceutical compositions for arthritis containing glutaredoxin 1 fusion protein,

The present invention relates to a pharmaceutical composition for the prevention and treatment of arthritis, and more particularly, to a pharmaceutical composition for preventing and treating arthritis containing GLRX1 (glutaredoxin 1) fusion protein permeable into cartilage tissue as an active ingredient.

Inflammatory arthritis is an autoimmune disease in which inflammation of the joints occurs due to various factors such as genetic and environmental factors, such as rheumatoid arthritis and ankylosing spondylitis.

Rheumatoid arthritis is a representative chronic disease in which about 1% of the population in Korea is estimated to be a patient. Although the etiology of rheumatoid arthritis varies from bacterial infection to viral infection, the precise mechanism of the disease has not yet been established. Rheumatoid arthritis is an inflammatory reaction caused by activation of synovial cells around the cartilage tissue by T lymphocytes stimulated by cytokine secretion such as tumor necrosis factor in an early stage of an onset with antigen-specific or antigen-nonspecific mechanism.

Ankylosing spondylitis is inflammatory arthritis, which is the main lesion of the spine and the cephalad joint.

Since inflammatory arthritis such as inflammatory arthritis is various, anti-inflammatory agent and anti-rheumatic agent are applied for treatment, but the therapeutic effect is incomplete.

Glutaredoxins (GLRX) is a member of the thioredoxin superfamily, a thiol / disulfide exchange catalyst, and is therefore known as a thiol transferase, and is a protein-SG which provides a reduction equivalent to ribonucleotide reductase 1 It serves as a reducing agent for mixed disulphides. In mammals, GLRX exists in two main forms. GLRX-1 is present in the cytoplasm and GLRX-2 is mainly located in the mitochondria, but also in the nucleus. Both GLRX-1 and GLRX-2 play an important role in redox regulation and protect cells against apoptosis.

In order to transfer a therapeutic drug or protein into a cell, a method of directly delivering the target protein through the cell membrane may be considered. However, proteins are very difficult to pass through cell membranes because of their size and their biochemical nature. In general, substances with a molecular weight of 600 Daltons or more are known to be almost impossible to pass through cell membranes.

In recent years, it has been found that one of the methods of protein transport is to use a PEP-1 peptide to carry a naturally occurring heterologous protein into a cell. The PEP-1 peptide consists of 21 amino acids (KETWWETWWTEW SQP KKKRKV) and has three domains (hydrophobic domain, spacer, hydrophilic domain). Until now, studies using PEP-1 peptides have revealed that when the PEP-1 peptide and the external protein are simultaneously administered to the cells, the proteins can be transported into the cells in a natural state. In addition, PEP-1 peptide has several advantages as a protein therapeutic agent compared to HIV-1 Tat protein, namely, it penetrates the protein very efficiently into cells, exhibits stability in physiological buffer solution and lack of sensitivity to serum. However, PEP-1 peptides must be administered at a constant rate with external proteins, such as green fluorescent protein (GFP), beta-galactosidase, and beta -Gal, Respectively. However, it remains unclear whether all proteins, including therapeutic proteins, can be delivered into cells by PEP-1 peptides.

In view of the above prior art, development into a formulation that allows a new protein component therapeutic agent, which has less adverse effects than conventional immunosuppressants, to penetrate into the cartilage is very important in the field of inflammatory arthritis treatment. Accordingly, the present invention aims to provide a pharmaceutical composition effective for inflammatory arthritis by using GLRX1 protein to facilitate cell infiltration.

In order to achieve the above object, the present inventors have studied to develop a protein, which is an immunosuppressive drug, as a form of enhancing cartilage penetration ability, and have found that when a GLRX1 fusion protein is treated with cartilage cells, The decrease of cytokine expression was confirmed at the protein level and the mRNA level, and the effect of treating GLRX1 fusion protein with inflammatory arthritis was confirmed, thereby completing the present invention.

The present invention relates to a pharmaceutical composition for preventing and treating arthritis, which contains a PEP-1-GLRX1 fusion protein in which the protein transport domain PEP-1 is covalently bonded to the N-terminus of a glutaredoxin 1 (GLRX1) protein.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating arthritis, which comprises the glutaredoxin 1 fusion protein, wherein the fusion protein is SEQ ID NO: 4.

The pharmaceutical composition containing the glutaredoxin 1 fusion protein as an active ingredient can be formulated together with a carrier which is conventionally acceptable in the pharmaceutical field and can be formulated into an oral, external or injection form by a conventional method. 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 dose of 0.01 to 100 mg / kg per day. 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.

Furthermore, the present invention provides a pharmaceutical composition useful for the prevention and treatment of arthritis, which comprises the GLRX1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The present invention also provides a method for efficiently delivering a GLRX1 protein into a cell. The intracellular delivery of the GLRX1 protein molecule according to the present invention is carried out by constructing a fusion protein in which the protein transport domain including the PEP-1 peptide is covalently bonded. An example of the transport domain of the present invention is a PEP-1 peptide. However, the protein transport domain of the present invention is not limited to the PEP-1 peptide, and it is possible to produce a peptide having a function similar to the PEP-1 peptide due to partial substitution, addition or deletion of the amino acid sequence of PEP-1 It is possible to use a protein transport domain which is composed of 7 to 15 amino acids and contains 4 or more lysine or arginine and which carries the same or similar protein transport function with partial amino acid substitution therefrom The fusion protein using the protein transport domain also belongs to the scope of the present invention.

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

"GLRX1 fusion protein" refers to a covalent bond complex formed by genetic fusion or chemical bonding of a protein transport domain and a GLRX1 protein and a transport domain and a target protein (i. E., The target protein in the present invention means "GLRX1 protein & . In this specification, "PEP-1-GLRX1" and "PEP-1-GLRX1 fusion protein" were used in combination.

"Target protein" is a molecule which is not originally able to enter the target cell, or which is not a transport domain or a fragment thereof that can not enter the target cell at an inherently useful speed, as a molecule itself before being fused with the transport domain, Means the target protein portion. The target protein includes a polypeptide, a protein, and a peptide. In the present invention, "GLRX1 protein"

"Fusion protein" means a complex comprising a transport domain and one or more target protein fragments, formed by genetic fusion or chemical bonding of the transport domain and the target protein.

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 target protein is delivered by a transport domain, and the target cell refers to a cell in the body or in vitro. That is, the target cell is meant to include a body cell, that is, a living animal, or a cell or living organism of a human organ or tissue, or a microorganism found in a human being. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism.

The term "protein transport domain" in the present invention refers to a protein transport domain that is covalently bonded to a polymer organic compound such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide to introduce the organic compound into cells without requiring additional receptor, It can be said.

Also, in the present specification, the terms "transport", "penetration", "transport", "delivery", "permeation" and "passage" are used in combination with the expression "introduction" of proteins, peptides and organic compounds into cells.

The GLRX1 fusion protein of the present invention refers to a GLRX1 fusion protein composed of 9 to 15 amino acid residues and having a transporting domain containing 3/4 or more of arginine or lysine residues covalently bonded to at least one terminal of GLRX1 to improve cell penetration efficiency. Also, the transport domain refers to at least one of HIV Tat 49-57 residue, PEP-1 peptide, oligo lysine, oligoarginine or oligo (lysine, arginine).

In addition, the GLRX1 fusion protein amino acid sequence of the present invention includes SEQ ID NO: 4. The fusion protein of various sequences can be obtained according to the selection of the restriction site sequence in the production of the GLRX1 fusion protein, and this is apparent to a person having ordinary skill in the art. It is obvious that the above amino acid sequence is only exemplary and that the amino acid sequence of the GLRX1 fusion protein is not limited to the above sequence.

In addition, the present invention provides a pharmaceutical composition comprising a GLRX1 fusion protein as an active ingredient and a pharmaceutically acceptable carrier, for the prevention and treatment of arthritis.

The present invention also provides a health functional food composition comprising the GLRX1 fusion protein as an active ingredient and having an arthritis prevention and improvement effect.

The present invention relates to a cell-transducing GLRX1 fusion protein comprising 9 to 15 amino acids, wherein a protein transport domain comprising four or more lysine or arginine is covalently bonded to at least one end of the GLRX1 protein. Also, depending on the silent change, one or more amino acids within the sequence may be replaced with other amino acid (s) of similar polarity functionally equivalent. 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. Also included within the scope of the invention are fragments or derivatives thereof having homologous homology, for example within the range of 85-100%, between the fusion protein of the present invention and the amino acid sequence, having the same similar biological activity.

The PEP-1-GLRX1 fusion protein according to the present invention penetrated chondrocytes in a time-dependent and dose-dependent manner.

In addition, the PEP-1-GLRX1 fusion protein according to the present invention penetrated into chondrocytes and stably maintained for 15 hours.

In addition, the PEP-1-GLRX1 fusion protein according to the present invention decreased protein and mRNA expression levels of inflammatory markers in inflammatory chondrocytes.

In addition, the PEP-1-GLRX1 fusion protein according to the present invention lowered the expression of inflammatory cytokines in inflammatory chondrocytes.

Therefore, the PEP-1-GLRX1 fusion protein according to the present invention is expected to be useful as a pharmaceutical composition for the prophylaxis or treatment of inflammatory arthritis.

Brief Description of the Drawings Figure 1 is a gel photograph showing the preparation and purification of the cell permeable PEP-1-GLRX1 fusion protein vector.
FIG. 2 is a photograph of SW1353 chondrocyte treated with 2 μM of PEP-1-GLRX1 fusion protein for 15 to 60 minutes followed by Western blotting. A GLRX1 protein without a protein transduction domain binding was used as a control. "C" refers to a negative control without treatment of PEP-1-GLRX1 fusion protein or GLRX1 protein.
FIG. 3 is a photograph of Western blotting after treatment of SW1353 chondrocytes with 0.25 to 2 .mu.M of PEP-1-GLRX1 fusion protein at different concentrations for one hour. A GLRX1 protein without a protein transduction domain binding was used as a control. "C" refers to a negative control without treatment of PEP-1-GLRX1 fusion protein or GLRX1 protein.
FIG. 4 is a photograph of SW1353 chondrocytes treated with 2 μM of PEP-1-GLRX1 fusion protein for one hour, followed by Western blotting with time. The fusion protein was stably maintained until 15 hours.
FIG. 5 shows the anti-inflammatory function of PEP-1-GLRX1 fusion protein by Western blotting after treatment with 2 μM of PEP-1-GLRX1 fusion protein after inducing inflammation by adding TNF-α to SW1353 chondrocytes. COX2 and iNOS are representative inflammation markers (lane 1: TNF-α inflammation induction group, lane 2: TNF-α + GLRX1 0.5 μM treated group, lane 3: TNF-α + GLRX1 treated group 1 μM, lane 4: TNF-α-PEP-1-GLRX1 treated with 0.5 μM TNF-α + PEP-1-GLRX1 and lane 7 treated with 1 μM TNF- 8: TNF-α + PEP-1-GLRX1 treated at 2 μM).
FIG. 6 shows the anti-inflammatory activity of PEP-1-GLRX1 fusion protein by RT-PCR after treatment with 2 μM of PEP-1-GLRX1 fusion protein after TNF-α was added to SW1353 chondrocytes to induce inflammation. COX2 and iNOS are representative inflammation markers (lane 1: TNF-α inflammation induction group, lane 2: TNF-α + GLRX1 0.5 μM treated group, lane 3: TNF-α + GLRX1 treated group 1 μM, lane 4: TNF-α-PEP-1-GLRX1 treated with 0.5 μM TNF-α + PEP-1-GLRX1 and lane 7 treated with 1 μM TNF- 8: TNF-α + PEP-1-GLRX1 treated at 2 μM).
FIG. 7 is a photograph showing that anti-inflammatory function of PEP-1-GLRX1 fusion protein is inhibited by inflammatory cytokine expression by treating TNF-α with TNF-α and then treating with 2 μM of PEP-1-GLRX1 fusion protein TNF-α + GLRX1 treated with 2 μM Lane 5: TNF-α + GLRX1 treated with 0.5 μM Lane 3: TNF-α + GLRX1 treated with 1 μM Lane 1: TNF- Α-PEP-1-GLRX1 treated group 1 μM, lane 8: TNF-α-PEP-1-GLPX-treated group, TNF-α-induced inflammatory group, lane 6: treated with 0.5 μM TNF- -GLRX1 2 [mu] M treated group).

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

material

TPA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni ^{2 +} -nitrile trichlorosilicate Sepharose Superflow was purchased from Qiagen (Valencia, CA, USA). FBS and antibiotics are Gibco BRL products. Synthetic PEP-1 peptides were purchased from PEPTRON (Daejeon, Korea), and all other chemicals used top-grade products available.

PEP-1-GLRX1 fusion protein expression and purification

PEP-1 expression vector was prepared [Eum, WS et al., Free Radic. Biol. Med. 37: 1656-1669; 2004]. The human GLRX1 cDNA sequence was amplified by PCR using the sense primer 5'-CTCGAGGCAGAGCCAGACCC-3 'and the antisense primer 5'-GGATCCTCACT GGGGTTTCTCC-3'. The resulting PCR product was subcloned into the TA cloning vector and ligated into the PEP-1 expression vector to produce the PEP-1-GLRX1 protein. A vector expressing the control GLRX1 protein without PEP-1 was constructed in a similar manner. The PEP-1-GLRX1 plasmid was transformed into E. coli BL21 cells and induced with 0.5 mM IPTG (isopropyl- beta -D-thio-galactoside; Duchefa, Haarlem, Netherlands) for 6 hours at 37 ° C. The harvested cells were lysed and the PEP-1-GLRX1 fusion protein was purified with Ni ^{2 +} -nitrilohexanoic acid sepharose superflow affinity chromatography and PD-10 chromatography (Amersham, Braunschweig, Germany). Protein concentration was determined by the Bradford method using bovine serum albumin as a standard [Bradford, MA Anal. Biochem. 72: 248-254; 1976].

Experimental Example 1: Intracellular penetration efficiency of GLRX1 fusion protein according to time and dose

1-1. Cartilage cell

Human chondrosarcoma cell line SW1353 cells, also referred to as HTB94 cells, were cultured in DMEM medium containing 10% fetal calf serum and used in the experiments.

1-2. Cell penetration efficiency by treatment time

2 μM GLRX1 fusion protein was treated with SW1353 cells for 15, 30, 45, and 60 minutes, respectively, and the intracellular penetration efficiency was confirmed by Western blotting.

As a result, as shown in Fig. 2, it was confirmed that the GLRX1 fusion protein infiltrated into SW1353 cells in a time-dependent manner. In contrast, the GLRX1 protein that did not bind the transport domain did not penetrate into the cells.

1-3. Cell Penetration Efficiency by Treatment Concentration

GLRX1 fusion proteins were treated with SWI 533 cells at concentrations of 0.25, 0.5, 1 and 2 μM, respectively, for one hour, and then the intracellular penetration efficiency was confirmed by Western blotting.

The results confirmed that the GLRX1 fusion protein infiltrated into SW1353 cells in a concentration-dependent manner, as shown in Fig. In contrast, the GLRX1 protein that did not bind the transport domain did not penetrate into the cells.

Experimental Example 2: Stability of GLRX1 fusion protein penetrated into cells

SW1353 cells were treated with 2 μM GLRX1 fusion protein for one hour. Western blotting was carried out after 1, 3, 6, 9, 12, 15, and 18 hours, respectively, for each GLRX1 fusion protein. As a result, as shown in Fig. 4, the fusion protein was stably maintained up to 15 hours.

Experimental Example  3: In inflammatory cartilage cells GLRX1 Fusion protein  by Inflammation-reducing effect

TNF-α was added to SW1353 cells to induce inflammation. After treatment of 2 μM GLRX1 fusion protein with inflammation-induced SW1353 cells, protein and mRNA expression of inflammation markers COX2 and iNOS was confirmed by Western blotting and RT-PCR. TNF-α + GLRX1 treated with 0.5 μM, lane 3: treated with 1 μM TNF-α + GLRX1, and lane 4: treated with TNF- α + GLRX1 treated with 2 μM, lane 5: TNF-α induced with inflammation, lane 6 treated with 0.5 μM TNF-α + PEP-1-GLRX1, lane 7 treated with 1 μM TNF- Lane 8: TNF-α + PEP-1-GLRX1 treated at 2 μM). The expression of COX2 and iNOS protein and mRNA expression were decreased when PEP-1-GLRX1 fusion protein was treated as compared with TNF-a treatment-induced inflammation-induced group. On the other hand, there was no change in the treatment of GLRX1 protein.

In addition, when TNF-α is added to SW1353 cells to induce inflammation, the expression of inflammatory cytokines is increased. At this time, 2 μM GLRX1 fusion protein was first treated, and inflammation was induced, and the change in the expression of inflammatory cytokines in the cells was observed by Western blotting.

The results are shown in FIG. 7 (lane 1: TNF-α inflammation induction group, lane 2: TNF-α + GLRX1 0.5 μM treatment group, lane 3: TNF- TNF-α-PEP-1-GLRX1 treated with 0.5 μM TNF-α + PEP-1-GLRX1, lane 7 treated with 1 μM TNF- : Treated with TNF-α + PEP-1-GLRX1 2 μM). Compared with TNF-α-treated inflammation-inducing group, the expression of inflammatory cytokines IL-6 and IL-1β was decreased when PEP-1-GLRX1 fusion protein was treated. On the other hand, there was no change in the treatment of GLRX1 protein.

From the above experimental results, it can be seen that, when GLRX1 fusion protein is treated on chondrocytes and cartilage tissues, GLRX1 fusion protein smoothly penetrates into cells or tissues, exhibits stability for at least 15 hours, inhibits the expression of inflammatory cytokines, And thus GLRX1 fusion proteins are expected to be available as pharmaceutical compositions for the prevention or treatment of inflammatory arthritis.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating arthritis containing          glutaredoxin 1 fusion protein <130> HallymU-sychoi-Glrx-arthritis <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer of glutaredoxin <400> 1 ctcgagggca acgcgcag 18 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of glutaredoxin <400> 2 ggatcctcag gaatcttcgg actc 24 <210> 3 <211> 398 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding PEP-1-GLRX fusion protein <400> 3 taaaagaaac ctggtgggaa acctggtgga ccgaatggtc tcagccgaaa aaaaaacgta 60 aagtgctcga gatggctcaa gagtttgtga actgcaaaat ccagcctggg aaggtggttg 120 tgttcatcaa gcccacctgc ccgtactgca ggagggccca agagatcctc agtcaattgc 180 ccatcaaaca agggcttctg gaatttgtcg atatcacagc caccaaccac actaacgaga 240 ttcaagatta tttgcaacag ctcacgggag caagaacggt gcctcgagtc tttattggta 300 aagattgtat aggcggatgc agtgatctag tctctttgca acagagtggg gaactgctga 360 cgcggctaaa gcagattgga gctctgcagt aaggatcc 398 <210> 4 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> PEP-1-GLRX fusion protein <400> 4 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Gln Glu Phe Val Asn Cys Lys              20 25 30 Ile Gln Pro Gly Lys Val Val Val Phe Ile Lys Pro Thr Cys Pro Tyr          35 40 45 Cys Arg Arg Ala Gln Glu Ile Leu Ser Gln Leu Pro Ile Lys Gln Gly      50 55 60 Leu Leu Glu Phe Val Asp Ile Thr Ala Thr Asn His Thr Asn Glu Ile  65 70 75 80 Gln Asp Tyr Leu Gln Gln Leu Thr Gly Ala Arg Thr Val Pro Arg Val                  85 90 95 Phe Ile Gly Lys Asp Cys Ile Gly Gly Cys Ser Asp Leu Val Ser Leu             100 105 110 Gln Gln Ser Gly Glu Leu Leu Thr Arg Leu Lys Gln Ile Gly Ala Leu         115 120 125 Gln    

Claims (2)

A pharmaceutical composition for the prevention and treatment of arthritis, which contains glutaredoxin 1 fusion protein in which PEP-1 peptide is covalently bonded to the N-terminus of Glutaredoxin 1.
The method according to claim 1,
Wherein the glutaredoxin 1 fusion protein is SEQ ID NO: 4, wherein the glutaredoxin 1 fusion protein is SEQ ID NO: 4.

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