Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/495—Transforming growth factor [TGF]

Definitions

• the present invention relates to novel use of ⁇ ig- h3 fragments. More particularly, the present invention relates to novel use of a ⁇ ig-h3 fragment for preventing and treating rheumatoid arthritis, which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• Rheumatoid arthritis is a chronic inflammation lasting for 6 weeks or longer occurring at the synovial membrane of the joints. Once rheumatoid arthritis onsets, various inflammatory cells from the blood form a lump in the synovial tissue called "pannus,” which impairs the cartilage and bone and results in the deformities of the joints. As a result of the arthritis, the joints become painful, swollen and warm and the movement of the joints becomes restricted because of pain. This disease is caused by an abnormality in the immune system of the body. To put in simplicity, it is a condition in which the immune system, which normally protects our body from external materials such as germs, attacks the body itself, for some unknown reasons.
• autoimmune causes chronic inflammation at the joints as well as other organs, such as muscles, lungs, skin, blood vessels, nervous system, eyes, and the like.
• Rheumatoid arthritis occur in men and women of all ages, but it occurs frequently in people of 30' s and 40' s and more frequently in women than in men.
• rheumatoid arthritis may be associated with disorders in organs other than the joints, which causes fever, skin eruption, weight loss, weariness, inflammatory changes in the lungs, heart and eyes, and the like.
• anti-rheumatic drugs such as aspirin & non-steroidal antiphlogistics, low-dose oral steroid, antimalarial drugs, sulfasalazine, gold compounds, penicillanmine and immunosuppressive drugs (methotrexate (MTX) and Immuran etc.), steroid injection at the joints, biological drugs such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab) , interleukin-1 receptor antagonists (anakinra) , anti-CD20 antibodies (rituximab) , and the like are used.
• tumor necrosis factor inhibitors etanercept, infliximab, adalimumab
• interleukin-1 receptor antagonists anakinra
• anti-CD20 antibodies rituximab
• TGF- ⁇ - inducible gene-h3 which is induced by the transforming growth factor- ⁇ (TGF- ⁇ )
• TGF- ⁇ transforming growth factor- ⁇
• ⁇ ig-h3 is expressed a lot in human rheumatoid arthritis tissues and plays an important role in controlling adhesion and migration of synoviocytes isolated from rheumatoid arthritis patients [Nam EJ et al . , Up-Regulated Transforming Growth Factor ⁇ -Inducible Gene h3 in Rheumatoid Arthritis Mediates Adhesion and Migration of Synoviocytes Through ⁇ v ⁇ 3 Integrin. Arthritis Rheum. Vol. 54, No. 9, September 2006, pp. 2734-2744].
• the inventors of the present invention found out that a fragment obtained by removing the Hl and H2 regions from the fourth (4th) fas-1 domain of ⁇ ig-h3 not only inhibits adhesion and migration of synoviocytes isolated from rheumatoid arthritis tissues but also is outstandingly effective in treating rheumatoid arthritis in mouse collagen-induced arthritis (CIA) model of rheumatoid arthritis. Therefore, the present invention provides a pharmaceutical composition for prevention and treatment of rheumatoid arthritis comprising the fragment as an active ingredient.
• an objection of the present invention is to provide a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising a peptide as an effective component, wherein the peptide is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• Another objection of the present invention is to provide a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising a vector which includes a promoter and a polynucleotide which is operatively linked to the promoter, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• Another objection of the present invention is to provide use of a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3 for the preparation of a therapeutic agent for rheumatoid arthritis.
• Another objection of the present invention is to provide a method for preventing or treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a peptide which is obtained by deleting Hl and H2 regions in the fourth fas- 1 domain of ⁇ ig-h3.
• Another objection of the present invention is to provide use of a vector comprising a promoter and a polynucleotide which is operatively linked to the promoter for the preparation of a therapeutic agent for rheumatoid arthritis, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• Another objection of the present invention is to provide a method for preventing or treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a vector comprising a promoter and a polynucleotide which is operatively linked to the promoter, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the present invention provides a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising a peptide as an effective component, wherein the peptide is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the present invention provides a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising a vector which includes a promoter and a polynucleotide which is operatively linked to the promoter, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the present invention provides use of a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3 for the preparation of a therapeutic agent for rheumatoid arthritis.
• the present invention provides use of a vector comprising a promoter and a polynucleotide which is operatively linked to the promoter for the preparation of a therapeutic agent for rheumatoid arthritis, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the present invention provides a method for preventing or treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a vector comprising a promoter and a polynucleotide which is operatively linked to the promoter, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the inventive composition is characterized by comprising a ⁇ ig-h3 fragment as an effective component.
• the inventive ⁇ ig-h3 fragment is a peptide obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the inventive ⁇ ig-h3 fragment may have the amino acid sequence of SEQ ID NO: 1.
• ⁇ ig-h3 as used in the present invention may be any known ⁇ ig-h3. However, preferably, it may be human ⁇ ig-h3 such as those of Genbank Accession No. NP_000349, Q15582, AAH04972, AAH00097, AAH26352, AAC08449 and AAA6116.
• the ⁇ ig-h3 of the present invention may have nucleotide sequence represented by SEQ ID NO: 3 or an amino acid sequence represented by SEQ ID NO: 4.
• Human ⁇ ig-h3 protein consists of 683 amino acids, and includes a ligand recognizing RGD motif (Arg-Gly-Asp) and four internal repeated domains (fas-1 domains) (see Fig. 1).
• the fas-1 domain consists of 110 to 140 amino acids.
• the four fas-1 domains of the ⁇ ig-h3 protein comprise a first fas-1 domain D-I which includes 133rd to 236th amino acids of the ⁇ ig-h3 protein, a second fas-1 domain D-II which includes 242nd to 372nd amino acids, a third fas-1 domain D-III which includes 373rd to 501st amino acids, and a fourth fas-1 domain D-IV which includes 502nd to 632nd amino acids.
• the fourth domain (D-IV) has EPDIM, which is a motif interacting with ⁇ 3 ⁇ l integrin (Kim JE et al . , J. Biol. Chem. , 275, 30907-30915, 2000), a tyrosine-histidine amino acid sequence, and YH, which is a motif mediating fibroblast adhesion (Kim, JE et al . , J. Biol. Chem., 277, 46159-46165, 2002).
• the 4th fas-1 domain has Hl and H2 regions comprising about 10 amino acids with very high homology.
• polypeptide fragment includes functional equivalents of polypeptide having the amino acid sequence of SEQ ID NO: 1.
• the functional equivalents may be polypeptides having a sequence homology (i.e., identity) of at least 60%, preferably at least 70%, and more preferably at least 80% to the amino acid sequence set forth in SEQ ID NO: 1.
• the functional equivalents may include polypeptides having a sequence homology of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% to the amino acid sequence of SEQ ID NO: 1.
• the functional equivalents include amino acid sequence variants with addition, substitution or deletion of some of the amino acid sequence of SEQ ID NO: 1.
• the substitutions of the amino acid sequence are conservative substitutions.
• conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (GIy, Ala, Pro) , hydrophobic amino acids (lie, Leu, VaI), aromatic amino acids (Phe, Tyr, Trp) , acidic amino acids (Asp, GIu) , basic amino acids (His, Lys, Arg, GIn, Asn) and sulfur-containing amino acids (Cys, Met).
• the functional equivalents also include variants with deletion of some of the amino acid sequence of the polypeptide.
• deletion or substitution of the amino acid may happen at regions irrelative to physiological activity of the inventive polypeptide directly.
• the functional equivalents also include variants with addition of several amino acids in both terminal ends of the amino acid sequence or in the sequence.
• inventive functional equivalents also include polypeptide derivatives which have modification of some of the chemical structure of the inventive polypeptide while maintaining the fundamental backbone and physiological activity of the inventive polypeptide. Examples of this modification include structural modifications for changing the stability, storage, volatility or solubility of the inventive polypeptide.
• sequence identity or homology is defined as the percentage of amino acid residues in the candidate sequence that are identical with amino acid sequence of SEQ ID NO: 1, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions (as described above) as part of the sequence identity. None of N-terminal, C- terminal, or internal extensions, deletions, or insertions into the amino acid sequence of SEQ ID NO: 1 shall be construed as affecting sequence identity or homology. Thus, sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides.
• two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a predetermined portion of one or both sequences) .
• the programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used in conjunction with the computer program.
• PAM 250 a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)
• the percent identity can be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences.
• the peptide according to the present invention can be prepared by a genetic engineering method using the expression of recombinant nucleic acid encoding the same.
• the inventive peptide can be prepared by a genetic engineering method comprising the steps of: inserting a nucleic acid sequence or its fragment encoding the inventive peptide into a recombinant vector comprising one or more expression control sequences which are operatively linked to the nucleic acid sequence to control the expression of the nucleic acid sequence; transforming a host cell with the resulting recombinant expression vector; culturing the transformed cell in a medium and condition suitable to express the nucleic acid sequence; and isolating and purifying a substantially pure protein from the culture medium.
• the inventive peptide can be chemically synthesized according to any technique known in the art (Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., NY 1983). Namely, the inventive peptide can be prepared by conventional liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989) .
• the recombinant peptide prepared by the genetic engineering method or the chemically synthesizing can be isolated and purified according to methods known in the art, including extraction, recrystallization, various chromatographic techniques (e.g., gel filtration, ion exchange, precipitation, adsorption, reverse phase, etc.), electrophoresis and counter current distribution.
• the therapeutic effect of the ⁇ hFas-1 fragment for rheumatoid arthritis and its mechanism were investigated.
• the administration of ⁇ hFas-1 inhibited or alleviated arthritic symptoms in the arthritis animal model.
• the dosage was increased to 30 mg/kg, the severity of arthritis did not increase, and the effect was maintained after the end of treatment.
• infiltration of T cells and B cells in the rheumatoid arthritis area reduced obviously as compared to the control group. Only slight inflammations were observed, and joint destruction was not observed. And, the transcripts of each inflammation-mediating substance decreased in the ⁇ hFas-1 treatment group in a dose- dependent manner.
• the present invention provides a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising the ⁇ ig-h3 fragment as an effective component, wherein the ⁇ ig-h3 fragment is obtained by deleting Hl and H2 regions in the fourth fas- 1 domain of ⁇ ig-h3.
• the ⁇ ig-h3 fragment according to the present invention is a fragment prepared by removing Hl and H2 regions from the 4th fas-1 domain (D- IV) .
• it includes an amino acid sequence represented by SEQ ID NO: 1, and includes its functional equivalents.
• Use of the ⁇ ig-h3 fragment of the present invention is advantageous in manipulation during drug making and in drug delivery, because the proteins aggregate less to form lumps due to relatively smaller fragment size.
• drug stability because it can be better included in biopolymer, etc., its concentration can be more stably maintained until transfer to the target area. Also, the improved stability results in better long-term storage as compared to the full-length ⁇ ig-h3.
• a disease that can be prevented or treated by the present inventive compound is rheumatoid arthritis.
• the inventive ⁇ ig-h3 fragment can be used for inflammatory diseases.
• Inflammatory diseases may include, although not limited thereto, inflammatory skin diseases, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, peritonitis, osteomyelitis, phlegmon, meningitis, encephalitis, pancreatitis, traumatogenic shock, bronchial asthma, allergic rhinitis, cystic fibrosis, stroke, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, Reiter' s syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis
• the inventive pharmaceutical composition includes the ⁇ ig-h3 fragment alone or may include more than one of pharmaceutically acceptable carriers, excipients or diluents additionally.
• the pharmaceutically acceptable carriers may include, for example, oral carrier, and parenteral administration carrier.
• the oral carrier may include lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid.
• the oral carrier may include diverse substances to deliver drugs, which are used for oral administration of peptide agents.
• the parenteral carrier may include water, suitable oil, saline solution, aqueous glucose and glycol.
• the composition according to the present invention may further comprise stabilizer and preservative. Suitable stabilizer includes antioxidant, such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservative includes benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
• the inventive pharmaceutical composition can be administered to human beings and animals, for example by oral route or by parenteral route.
• Parenteral administration methods are not limited, but include intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, hypodermic, intraperitoneal, intranasal, enteral, sublingual, intrarectal administration or local administration.
• composition of the present invention may be formulated into various forms for oral or parenteral administration according to the administration routes.
• the composition of the present invention may be formulated into powder, granule, tablet, pill, troche, capsule, elixir, gel, syrup, slurry, suspension and the like by conventional methods known to one skilled in the art.
• the oral preparations may be obtained as tablets or sugar- coated tablets by blending the active ingredient with a solid excipient, crushing the blend, adding suitable adjuvants and then processing the mixture into a granular mixture.
• excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; starches including corn starch, wheat starch, rice starch and potato starch; celluloses including cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl cellulose; and fillers including gelatin and polyvinylpyrrolidone.
• the pharmaceutical composition of the present invention may, if necessary, contain a disintegrant, such as crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate.
• the pharmaceutical composition may further comprise an anticoagulant, a lubricant, a wetting agent, a perfumery, an emulsifier, and a preservative.
• parenteral preparations they can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalers by any method known in the art. These preparations are described in the following formulary known to all pharmaceutical chemists : Remington ' s Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour.
• the total effective amount of ⁇ ig-h3 fragments of the present invention can be administered to a subject as a single dose, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time.
• the content of the active ingredient in the pharmaceutical composition of the present invention can be varied depending on the severity of disease.
• the total dose of ⁇ ig-h3 fragments of the present invention is 0.01 ⁇ g/kg/day to 1,000 mg/kg/day, and more preferably 0.1 ⁇ g/kg/day to 100 mg/kg/day.
• the concentration of ⁇ ig-h3 fragments of the present invention required to obtain an effective dose in a subject depends on many factors including the age, bodyweight, health condition, disease severity, diet and excretion of the subject, the route of administration, the number of treatments to be administered, and so forth. In view of these factors, any person skilled in the art can determine the suitable effective dose of ⁇ ig- h3 fragments for preventing or treating inflammatory diseases. No particular limitation is imposed on the formulation, administration route and administration mode of the pharmaceutical composition according to the present invention, as long as the composition shows the effects of the present invention.
• the present invention provides a pharmaceutical composition for preventing and treating rheumatoid arthritis comprising a vector which includes a promoter and a polynucleotide which is operatively linked to the promoter, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the peptide may have the amino acid sequence of SEQ ID NO: 1. If coding a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3, the polynucleotide may be used without limitations.
• the polynucleotide may have the nucleotide sequence of SEQ ID NO: 2. The polynucleotide is operatively linked to a promoter to express the ⁇ ig-h3 fragment.
• the ⁇ promoter' means a DNA sequence regulating the expression of nucleic acid sequence operatively linked to the promoter in a specific host cell, and the term ⁇ operatively linked' means that one nucleic acid fragment is linked to other nucleic acid fragment so that the function or expression thereof is affected by the other nucleic acid fragment.
• the promoter may include an operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome-binding site, and sequences controlling the termination transcription and translation.
• constitutive promoter which constitutively induces the expression of a target gene
• inducible promoter which induces the expression of a target gene at a specific site and a specific time
• examples thereof include U6 promoter, CMV (cytomegalovirus) promoter, a SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991; Monahan et al . , Gene Therapy, 7:24-30, 2000), CaMV 35S promoter (Odell et al., Nature 313:810- 812, 1985), Rsyn7 promoter (US Patent Application No.
• the inventive vectors are not limited, but may include plasmid vectors, cosmid vectors, bacteriophage vectors, virus vectors and so on.
• a suitable vector may include elements regulating expression such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, as expression vector.
• the vectors may be prepared with a variety of methods for a purpose.
• the present invention provides use of a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3 for preparation of a therapeutic agent for rheumatoid arthritis.
• the peptide can be represented by an amino acid sequence having at least 60% sequence homology to SEQ ID NO: 1. Most preferably, the peptide can have the amino acid sequence of SEQ ID NO: 1.
• the therapeutic agent for rheumatoid arthritis may also be formulated for oral or parenteral administration by administration routes as stated above.
• the present invention provides a method for preventing or treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of the peptide.
• the peptide can be represented by an amino acid sequence having at least 60% sequence homology to SEQ ID NO: 1. Most preferably, the peptide can have the amino acid sequence of SEQ ID NO: 1.
• the present invention provides use of a vector comprising a promoter and a polynucleotide which is operatively linked to the promoter for preparation of a therapeutic agent for rheumatoid arthritis, wherein the polynucleotide codes for a peptide which is obtained by deleting Hl and H2 regions in the fourth fas-1 domain of ⁇ ig-h3.
• the peptide can have the amino acid sequence of SEQ ID NO: 1 and the polynucleotide can have the nucleotide sequence of SEQ ID NO: 2.
• the therapeutic agent for rheumatoid arthritis may also be formulated for oral or parenteral administration by administration routes as stated above.
• the present invention provides a method for preventing or treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of the vector.
• the peptide can have the amino acid sequence of SEQ ID NO: 1 and the polynucleotide can have the nucleotide sequence of SEQ ID NO: 2.
• the term ⁇ subject in need' means mammals in need of preventing and treating rheumatoid arthritis, and may preferably be human beings.
• the term ⁇ effective amount' refers to administering to a subject as a single dose, or administering using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time. It can be varied depending on the severity of disease.
• the total dose is 0.01 ⁇ g/kg/day to 1,000 mg/kg/day, and more preferably 0.1 ⁇ g/kg/day to 100 mg/kg/day.
• concentration required to obtain an effective dose in a subject depends on many factors including the age, bodyweight, health condition, disease severity, diet and excretion of the subject, the route of administration, the number of treatments to be administered, and so forth.
• any person skilled in the art can determine the suitable effective dose for preventing or treating rheumatoid arthritis.
• No particular limitation is imposed on the formulation, administration route and administration mode according to the present invention, as long as the composition shows the effects of the present invention.
• the present invention provides a new use of a peptide fragment of ⁇ ig-h3 or a vector including a polynucleotide encoding the same for the prevention and treatment of rheumatoid arthritis.
• the peptide fragment of ⁇ ig-h3 or the vector including a polynucleotide encoding the same inhibit inflammation at the rheumatoid arthritis site and, thus, can be used to prevent and treat rheumatoid arthritis and other various inflammatory diseases .
• FIG. 1 schematically illustrates the structure of ⁇ ig-h3 according to the present invention.
• Fig. 2 shows the inhibition effects of the human 4th fas-1 domain on adhesion (A) and migration (C) and of the human ⁇ hFas-1 on adhesion (B), in human synoviocytes.
• Fig. 3 shows the inhibition effects of the mouse 4th fas-1 domain on adhesion (A) and migration (C) and of the mouse ⁇ hFas-1 on adhesion (B) , in mouse cells (NIH3T3) .
• Fig. 4 shows the arthritis therapeutic effect of mouse ⁇ hFas-1.
• Fig. 5 shows the body weight change of mice before and after the administration of mouse ⁇ hFas-1.
• Fig. 6 shows the distribution of inflammation mediator in the hind paw tissues in mouse CIA model, after the administration of ⁇ hFas-1 (CD3: T cell marker;
• B220 B cell marker
• CD31 endothelial cell marker
• Human ⁇ ig-h3 gene (NM_000358, SEQ ID NO: 3) was cloned in pET29b expressing vector, and transformed into E. coli BL21DE. The transformed E. coli was cultured to express ⁇ ig-h3 protein.
• Ni-NTA agarose beads (Qiagen) were mixed well and washed twice with resuspension buffer after moving 1 mL to the tube. The supernatant was added to the washed 50% suspension of Ni- NTA agarose beads. After binding for 1 hour at 4 °C using an agitator, and centrifuging at 4 °C, 1500 rpm for 3 minutes, the supernatant was separated. Ni-NTA agarose beads were mixed and washed well while adding 10 times the volume of bead washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole) . Through centrifuge at 4 °C, 1500 rpm for 3 minutes, non-specifically bound proteins were removed. This procedure was repeated for 10 times. His- tag ⁇ ig-h3 protein was extracted using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole) .
• Mouse ⁇ ig-h3 gene (NM_009369, SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6) was cloned in pET29b expressing vector, and transformed into E. coli BL21DE. The transformed E. coli was cultured to express ⁇ ig-h3 protein. The protein was isolated by adhesion using Ni- NTA resin column, in the same manner as in Example ⁇ 1-1>. LPS was also removed in the same manner as in Example ⁇ 1- 1> .
• human fas-1 domain fragment A gene which encodes the 4th fas-1 domain of the fas-1 domains of human ⁇ ig-h3 (hereinafter, referred to as human fas-1 domain fragment) was amplified by PCR using human ⁇ ig-h3 (NM_000358) as template and using primer of SEQ ID NO: 7 (forward 5'- atggagatatcgctgaccccccca-3' ) and primer of SEQ ID NO: 8 (reverse 5' -tcctgctcgaggttggctggaggc-3' ) .
• the amplified PCR product was cloned in pET29b expressing vector, and transformed into E. coli BL21DE. The transformed E.
• Example coli was cultured to express the human fas-1 domain fragment.
• the fragment was isolated by adhesion using Ni-NTA resin column, in the same manner as in Example ⁇ 1-1>.
• LPS was also removed in the same manner as in Example ⁇ 1-1>.
• ⁇ l-4> Preparation and isolation of fragment including mouse ⁇ ig-h3 fas-1 domain
• mouse fas-1 domain fragment A gene which encodes the 2nd and 4th fas-1 domains of the fas-1 domains of mouse ⁇ ig-h3 (hereinafter, referred to as mouse fas-1 domain fragment) was amplified by PCR using mouse ⁇ ig-h3 (NM_009369) as template and using primer of SEQ ID NO: 9 (forward 5'- tatgatatcccaagccatgcc-3' ) and primer of SEQ ID NO: 10 (reverse 5' -tcaatctcgagcatgatgtc-3' ) • The fragment was isolated by adhesion using Ni-NTA resin column, in the same manner as in Example ⁇ 1-1>. LPS was also removed in the same manner as in Example ⁇ 1-1>.
• a gene which encodes a peptide obtained by removing Hl and H2 regions from the 4th fas-1 domain of human ⁇ ig- h3 ( ⁇ hFas-1) was amplified by PCR using human ⁇ ig-h3
• Example ⁇ 1-1> The fragment was isolated by adhesion using Ni-NTA resin column, in the same manner as in Example ⁇ 1-1>. LPS was also removed in the same manner as in Example ⁇ 1-1>.
• a gene which encodes a peptide obtained by removing Hl and H2 regions from the 4th fas-1 domain of mouse ⁇ ig- h3 ( ⁇ hFas-1) was amplified by PCR using mouse ⁇ ig-h3
• Synovial membrane tissues obtained during joint replacement surgery of rheumatoid arthritis patients were treated with 1 mg/mL collagenase I (Sigma) at 37 °C for 3 hours in a culture medium (DMEM + 10% FBS (fetal bovine serum) + 2% penicillin, streptomycin) .
• the tissues were filtered through sterilized gauze and cultured in a 5% C02 incubator at 37 0 C.
• Synoviocytes sub-cultured to 3rd through 8th generations were subjected to experiments. ⁇ 2-2> Effect on adhesion of human synoviocytes
• BSA bovine serum albumin
• Human ⁇ ig-h3 was coated on the lower filter surface of a transwell having 8 ⁇ m-sized pores, through which cells can migrate, by incubating at 4 °C for 12 to 14hours.
• cultured cells were inoculated at the upper chamber of a filter and cultured for 7 hours at 37 0 C. Then, after fixing the cells that migrated to the lower surface using paraformaldehyde, the cells were stained with crystal violet. Cells remaining at the upper chamber without migration were wiped out with sterilized swab, and the number of cells that migrated to the lower surface was counted.
• human fas-1 domain fragment did not have significant effect on migration of synoviocytes.
• mice fibroblasts NASH3T3, Korean Cell Line Bank, KCLB21658
• mouse fas-1 domain fragment instead of human fas-1 domain fragment
• mouse ⁇ hFas-1 instead of human ⁇ hFas-1.
• mouse fas-1 domain fragment did not inhibit adhesion (Fig. 3A) and migration (Fig. 3B) of fibroblasts. This result is consistent with that for human fas-1 domain fragment.
• mouse ⁇ hFas-1 inhibited ⁇ ig-h3 mediated cell adhesion (Fig. 3C) and migration (Fig. 3D) in a concentration-dependent manner.
• CIA mouse model with characteristics very similar to human rheumatoid arthritis was constructed as follows according to the literature [Protocol for the successful induction of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in mice. Chondrex, Redmond, WA] .
• Bovine type 2 collagen (100 ⁇ g) was subcutaneously inoculated as mixed with Freund' s complete adjuvant at the tail of a mouse. 3 weeks later, bovine type 2 collagen (100 ⁇ g) was subcutaneously inoculated again as mixed with Freund' s incomplete adjuvant at the tail of the mouse.
• mouse ⁇ hFas-1 was injected to the mouse intra-peritoneal at a dose of 10 mg/kg body weight of 30 mg/kg body weight, every day for 4 weeks to investigate arthritis therapautic effect in CIA model.
• the mouse ⁇ hFas-1 used for therapeutic purpose included LPS less than 1 EU per mouse.
• the same amount of PBS was administered intra-peritoneal.
• ⁇ hFas-1 30 mg/kg administration group showed lasting arthritis therapeutic effect even after stopping the administration (data not shown) .
• ⁇ 3-3> Toxicity of ⁇ hFas-1 in mouse CIA model In order to evaluate toxicity of mouse ⁇ hFas-1 in mice, body weight of 5 mice was measured before (on day 22) and after (on day 52) the administration of ⁇ hFas-1.
• Tissue samples were prepared from leg tissue of mouse model in order to examine inflammation in synovial membrane tissues, change of infiltrated inflammatory cells and angiogenesis, and distribution of T cells, B cells, endothelial cells and ICAM-I was investigated as follows.
• the tissue was stained with hematoxylin and subjected to microscopy after mounting with Eukitt mounting media (Fluka, Germany) .
• the paw tissue slices of the control group showed significant destruction of joints as well as severe infiltration of inflammatory cells.
• the ⁇ hFas-1 10 mg/kg administration group did not show significantly decreased infiltration of T cells and B cells as compared to the control group.
• the 30 mg/kg administration group showed significantly decreased infiltration of T cells and B cells as compared to the control group.
• the administration of 30 mg/kg ⁇ hFas-1 resulted in significantly decreased angiogenesis .
• the 30 mg/kg ⁇ hFas-1 treatment group showed only slight inflammation and almost no joint destruction.
• RNA was extracted using an extraction reagent (Easy-spinTM, Intron) . After quantification, reverse transcription was carried out using 7 ⁇ g of RNA, AMV reverse transcriptase (Roche, 0.5 ⁇ L) , oligo-dT (Roche, 1 ⁇ L) , 10 mM dNTP (Takara, 2 ul) and RNase inhibitor (Roche, 0.1 ⁇ L) .
• the primers for PCR were as follows: mouse IL-I ⁇ (SEQ ID NO: 15: forward 5'- tgtaatgaaagacggcacacc-3' , SEQ ID NO: 16: reverse 5'- tcttctttgggtattgcttgg-3' ) , IL-6 (SEQ ID NO: 17: forward 5' -gagaaaagagttgtgcaatggc-3' , SEQ ID NO: 18: reverse 5'- ccagtttggtagcatccatca-3' ) , TNF- ⁇ (SEQ ID NO: 19: forward 5' -ctgtagcccacgtcgtagc-3' , SEQ ID NO: 20: reverse 5'- ttgagatccatgccgttg-3' ) , MMP-I (SEQ ID NO: 21: forward 5'- tgtgttcacaacggagacc 3', SEQ ID NO: 22
• the transcripts of each inflammation-mediating substance decreased in a therapeutic dose-dependent manner, in the ⁇ hFas-1 administration groups.
• the 30 mg/kg ⁇ hFas- 1 administration group exhibited a remarkable decrease.
• the present invention provides a novel use of a ⁇ ig-h3 fragment or a vector including a polynucleotide encoding the same for the prevention and treatment of rheumatoid arthritis.
• the ⁇ ig-h3 fragment or the vector including a polynucleotide encoding the same inhibit inflammation at the rheumatoid arthritis site and, thus, can be used to prevent and treat rheumatoid arthritis and other various inflammatory diseases .

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