WO2024025261A1 - Pharmaceutical composition for preventing or treating osteoarthritis - Google Patents

Pharmaceutical composition for preventing or treating osteoarthritis Download PDF

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WO2024025261A1
WO2024025261A1 PCT/KR2023/010545 KR2023010545W WO2024025261A1 WO 2024025261 A1 WO2024025261 A1 WO 2024025261A1 KR 2023010545 W KR2023010545 W KR 2023010545W WO 2024025261 A1 WO2024025261 A1 WO 2024025261A1
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pharmaceutical composition
preventing
rapamycin
glycero
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French (fr)
Korean (ko)
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김병수
손희수
조미라
전주연
최정원
나현식
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서울대학교산학협력단
가톨릭대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating osteoarthritis comprising a type 2 collagen peptide and a carrier carrying rapamycin.
  • Osteoarthritis along with rheumatoid arthritis, is known to be the most representative joint disease. Osteoarthritis is accompanied by excessive formation of bone around joints, joint deformation and pain, and progressive movement disorders as the disease progresses.
  • Treatment methods for osteoarthritis known to date include anti-inflammatory drug administration, non-drug therapy to improve lifestyle and eating habits, and outpatient treatment that involves invasive joint surgery.
  • Non-steroidal anti-inflammatory drugs are effective in reducing pain through inflammation relief, but may cause gastrointestinal side effects, and COX-2 inhibitors, which are used as alternatives, may cause cardiovascular side effects and gastrointestinal damage.
  • COX-2 inhibitors which are used as alternatives, may cause cardiovascular side effects and gastrointestinal damage.
  • the cause of pain can be effectively removed, but there is a burden of the possibility of reoperation depending on the condition of the joint and artificial joint.
  • osteoarthritis In general, osteoarthritis is known to be caused by physical factors, but as it has become known through many recent papers that the immune system is involved in osteoarthritis, understanding the function of immune cells in osteoarthritis and immunomodulation based on this have become important.
  • the biggest characteristics of osteoarthritis are cartilage cell death and cartilage wear.
  • cartilage wear begins due to physical causes, disease, environment, and genetic factors, the number of inflammatory T cell groups such as type 1, type 2, and type 17 and type 1 macrophages in the synovium increases, while the number of anti-inflammatory regulatory T cells increases. The number of cells (regulatory T cells, Tregs) and type 2 macrophages decreases.
  • inflammatory cells secrete inflammatory cytokines such as interleukin 1 ⁇ (IL-1 ⁇ ), interferon ⁇ (IFN- ⁇ ), and tumor necrosis factor ⁇ (TNF- ⁇ ).
  • IL-1 ⁇ interleukin 1 ⁇
  • IFN- ⁇ interferon ⁇
  • TNF- ⁇ tumor necrosis factor ⁇
  • the secreted cytokines act on surrounding T cell groups to promote the activation of inflammatory cells, secreting more inflammatory cytokines, and at the same time act on cartilage cells to produce extracellular matrix decomposing enzymes such as Matrix Metalloproteinase 13 (MMP-13). Promotes secretion.
  • MMP-13 causes cartilage wear by decomposing type 2 collagen protein, the most representative extracellular matrix component of cartilage.
  • Inflammatory immune cells and cytokines are relatively superior to anti-inflammatory immune cells and cytokines, and if the imbalance between inflammatory and anti-inflammatory immune cells and substances is not corrected, an inflammatory environment is continuously maintained within the joint.
  • Celebrex a representative inflammation-relieving substance currently commercially available, can relieve inflammation, but it only relieves ongoing pain and is not a fundamental solution, and surgically operating the joint not only places a physical burden on the patient, Because there is a possibility of future reoperation depending on the condition of the joint, it is difficult to say that this method is a fundamental treatment method.
  • regulatory T cells can suppress immune responses, research and clinical trials are being conducted as a treatment for various inflammatory diseases, and it has been found that they can suppress excessive immune responses in autoimmune diseases, allergic diseases, organ transplant rejection, etc.
  • regulatory T cell transplantation (adoptive Treg cell transfer) has the disadvantage of being expensive and complicated in manufacturing the treatment because it requires the step of isolating cells from the patient and then proliferating/cultivating them in vitro.
  • regulatory T cells of various clones that bind to and act on various antigens are cultured and then transplanted to the patient, immune system is suppressed systemically, which may lead to side effects such as infection and cancer development.
  • a therapeutic agent that induces regulatory T cells against a single antigen in the body needs to be developed.
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating osteoarthritis.
  • the purpose is to provide a composition that can reduce the inflammatory response caused by inflammatory immune cells occurring in bone joints, alleviate cartilage destruction and pain accompanying the inflammatory response, and further regenerate cartilage.
  • a pharmaceutical composition for preventing or treating osteoarthritis comprising:
  • composition for preventing or treating osteoarthritis according to 1 above, further comprising dendritic cells (DC).
  • DC dendritic cells
  • type 2 collagen peptide and a pharmaceutical composition for preventing or treating osteoarthritis, wherein rapamycin or a derivative thereof is supported on a carrier.
  • the carrier includes viral carriers, virus-like particles (VLP), positively charged polymers, liposomes, lipid nanoparticles, gold, and semiconductor nanocrystal particles (quantum dots).
  • VLP virus-like particles
  • Quantum dots semiconductor nanocrystal particles
  • the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene) glycol)] (PEG PE) and a pharmaceutical composition for preventing or treating osteoarthritis, which are lipid nanoparticles containing cholesterol.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DOPE dioleoylphosphatidylethanolamine
  • PEG PE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene) glycol)]
  • a pharmaceutical composition for preventing or treating osteoarthritis which are lipid nanoparticles containing cholesterol.
  • the present invention can provide a pharmaceutical composition for preventing or treating osteoarthritis, including a type 2 collagen peptide and a carrier carrying rapamycin. This can promote the prevention or treatment of osteoarthritis by minimizing the inflammatory response caused by type 2 collagen peptides exposed in osteoarthritis articular cartilage. Furthermore, in addition to osteoarthritis, carrying other disease-related antigens instead of type 2 collagen peptide can be used as a treatment for inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal damage, or autoimmune diseases such as atopy, rheumatoid arthritis, type 1 diabetes, Crohn's disease, and lupus. It can be used as.
  • inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal damage
  • autoimmune diseases such as atopy, rheumatoid arthritis, type 1 diabetes, Crohn's disease, and lupus. It can be used as.
  • Figure 1 shows data analyzing the manufacturing method of lipid nanoparticles and their characteristics. It was confirmed that lipid nanoparticles can normally support type 2 collagen peptide and rapamycin.
  • Figure 2 shows data confirming that lipid nanoparticles carrying type 2 collagen peptide and rapamycin can induce differentiation of undifferentiated dendritic cells into tolerogenic dendritic cells.
  • Figure 3 shows data confirming that lipid nanoparticles carrying type 2 collagen peptide and rapamycin were distributed in lymph nodes (LN) near the injection site when injected into an in vivo model.
  • LN lymph nodes
  • Figure 4 shows that type 2 collagen peptides or peptides with amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles loaded with rapamycin can activate type 2 collagen-specific regulatory T cells in vivo. This is data that confirmed .
  • Figure 5 shows data confirming that type 2 collagen peptides or peptides with amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles loaded with rapamycin are effective in alleviating and/or improving symptoms in osteoarthritis-induced model mice. am.
  • Figure 6 is a schematic diagram of the lipid nanoparticle-based osteoarthritis treatment mechanism of the present invention.
  • the present invention relates to a pharmaceutical composition for preventing or treating osteoarthritis, comprising type 2 collagen peptide and rapamycin or a derivative thereof.
  • the present invention induces differentiation of immature dendritic cells into tolerant dendritic cells by using type 2 collagen peptide together with rapamycin or its derivatives, and adds a type 2 collagen peptide epitope to the MHC molecule of tolerant dendritic cells. You can present it.
  • the dendritic cells induce a large amount of regulatory T cells specific for type 2 collagen peptide, and the regulatory T cells can alleviate and/or treat osteoarthritis symptoms by suppressing inflammatory immune cells at the site of joint inflammation.
  • the type 2 collagen peptide may be a peptide containing at least some amino acid sequences of type 2 collagen protein.
  • Type 2 collagen proteins have epitopes that bind to MHC molecules on dendritic cells. At least a portion of the epitope may include a sequence that can be recognized as the epitope.
  • a sequence that can be recognized as an epitope refers to a sequence that functions as an epitope even if it consists of the entire epitope sequence, some amino acids are added to both ends of the sequence, or some amino acids are removed from both ends of the sequence.
  • the epitope may be the amino acid sequence of SEQ ID NO: 1, and the sequence that can be recognized as the epitope may include it or may be a sequence that does not include 1 to 3 amino acids at both ends.
  • any sequence that can be recognized as the epitope may be included without limitation, and a known epitope sequence may be used, or it may include a fragment of type 2 collagen protein cut with collagenase. If at least part of the above is included, the type/length of the sequence added to both ends is not limited, and the entire type 2 collagen protein can be used.
  • the length of the type 2 collagen peptide is not limited as long as it can be recognized as a type 2 collagen peptide epitope by binding to an MHC molecule, for example, 0.5 kDa to 120 kDa, 1 kDa to 100 kDa, It may be 1.5 kDa to 50 kDa, 2 kDa to 30 kDa, 3 kDa to 20 kDa, etc.
  • Type 2 collagen peptide may fall within the scope of the present invention regardless of which species the collagen peptide originates from.
  • Type 2 collagen is a gene with a high degree of conservation across species.
  • type 2 collagen peptides derived from species selected from the group including humans, mice, cattle, goats, horses, cats, and dogs are also included in the present invention. may fall within the range.
  • the type 2 collagen peptide sequence contains some sequences that are inconsistent between species, the effect of the present invention can be exerted. For example, even if one or two amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted with different amino acids, the effect of the present invention can be achieved.
  • the 8th amino acid in SEQ ID NO: 1 may be aspartic acid or glutamic acid, and the 15th amino acid in SEQ ID NO: 1 may be threonine or proline. However, it is not limited to this.
  • rapamycin or a derivative thereof may be included in the scope of the present invention regardless of its type.
  • rapamycin can inhibit the growth of lymphocytes, promote differentiation, and induce regulatory T cells specific for type 2 collagen peptides, and any derivative of rapamycin that can perform these functions can be used without limitation. It may be included in the scope of the invention. Examples include benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus, and ridaporolimus. However, it is not limited to this.
  • the composition of the present invention may further include dendritic cells (DC).
  • the dendritic cells may be exposed to a solution containing type 2 collagen peptide and/or rapamycin, and in this case, rapamycin is introduced into the dendritic cells by the type 2 collagen peptide and/or rapamycin, thereby producing a Type 2 collagen peptides can be presented to MHC molecules on the cell surface.
  • DC dendritic cells
  • osteoarthritis is a disease caused by gradual/irreversible degeneration of cartilage tissue, and includes degenerative joint disease, osteoarthrosis, hypertrophic arthritis, or degenerative arthritis ( It is a disease also known as degenerative arthritis, and is a term referring to the same disease as the names above.
  • Symptoms of osteoarthritis include a stage in which cartilage loses its elasticity and becomes more easily damaged by injury or use; A stage in which wear and tear of the cartilage causes changes to the underlying bone, which may include bone thickening, cysts, and bone growths called spurs or osteophytes on the ends of bones in the affected joint.
  • the composition reduces the number of inflammatory immune cells, such as macrophages and type 1 helper T cells, that enter the cartilage through the synovium for osteoarthritis, or reduces the number of inflammatory cytokines caused by inflammatory immune cells, such as
  • the inflammatory response in cartilage can be alleviated by suppressing the secretion of interleukin 1 ⁇ , TNF- ⁇ , or IFN- ⁇ .
  • it can suppress the destruction of proteins that make up the cartilage tissue and/or extracellular matrix that accompany the inflammatory response and relieve pain.
  • the weight ratio of the type 2 collagen peptide of the present invention and rapamycin may be included within the scope of the present invention without limitation.
  • it may be 1 to 0.1 to 10, 1 to 0.2 to 8, 1 to 0.3 to 5, or 1 to 0.5 to 3.
  • it is not limited to this.
  • the type 2 collagen peptide of the present invention may be delivered while being carried on a carrier.
  • the delivery vehicle is type 2 collagen peptide; And it greatly improves the delivery efficiency of rapamycin or its derivatives to dendritic cells, resulting in the induction efficiency of tolerant dendritic cells that present type 2 collagen peptides to type 2 MHC molecules and type 2 collagen peptide-specific regulatory T cells.
  • the induction efficiency can be greatly increased.
  • the delivery vehicle has a type 2 collagen peptide inside; and rapamycin or a derivative thereof, and can be selected without limitation by those skilled in the art as long as it can be delivered to the target cell and release the substance therein.
  • rapamycin or a derivative thereof selected from the group including viral carriers, virus-like particles (VLP), positively charged polymers, liposomes, lipid nanoparticles, gold, and semiconductor nanocrystal particles (quantum dots). It could be. However, it is not limited to this.
  • lipid nanoparticles can be selected and used by those skilled in the art regardless of their components.
  • neutral lipids, anionic and/or cationic phospholipids can all be used, for example, L- ⁇ -phosphatidylcholine, PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn- Glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoylsn) -glycero-3-phophocholine, DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) There may be at least one
  • the anionic lipids include L- ⁇ -phosphatidic acid, L- ⁇ -phosphatidyl-DL-glycerol, cardiolipin, L - ⁇ -phosphatidylinositol (L- ⁇ -phosphatidylinositol), L- ⁇ -phosphatidylserine, 1,2-dilauroyl-sn-glycero-3-[phospho-rac-( 1-glycerol)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3-[ phospho-L-serine](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1 ,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dim
  • cationic lipid examples include 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) 3-Trimethylammonium-propane (1,2-dioleoyl-3-trimethylammoniumpropane, DOTAP), dioleoyl glutamide, distearoylglutamide, dipalmitoyl glutamide glutamide), dioleoylaspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), ß-[N-(N ',N'-dimethylaminoethane-carbamoyl], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), methyldiochtadecylammonium bromide
  • the lipid particle may have its phospholipid surface PEGylated with polyethylene glycol (PEG), etc. to improve the fluidity, loading efficiency, and/or stability of the particle, and may be PEGylated with cholesterol, 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) may be further included.
  • PEG polyethylene glycol
  • DOTAP 1,2-Dioleoyl-3-trimethylammoniumpropane
  • DOPE dioleoylphosphatidylethanolamine
  • the average diameter of the carrier is within the scope of the present invention without limitation, and may be selected by a person skilled in the art depending on conditions such as the amount of peptide and drug to be carried.
  • the average diameter may be 50 nm to 1,000 nm.
  • the lower limit of the average particle diameter could be, for example, 50 nm, 60 nm, 70 nm, 80 nm or 90 nm
  • the upper limit could be, for example, 1,000 nm, 900 nm, 800 nm, 700 nm, or 600 nm. there is. However, it is not limited to this.
  • composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar function in relation to the prevention or treatment of osteoarthritis, or a compound that maintains/increases the solubility and/or absorption of the active ingredient.
  • the appropriate dosage of the composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can. However, it is not limited to this.
  • concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient's condition, necessary period, etc., and is not limited to a specific concentration range.
  • the composition of the present invention is administered in a pharmaceutically effective amount.
  • the effective dosage level depends on factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. can be decided.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the dosage range of the composition of the present invention varies greatly depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
  • the appropriate dosage can be determined by, for example, the patient. It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the delivery vehicle of the present invention used. For example, it may be 0.01 ⁇ g to 1 g per kg of body weight, and may be administered in divided doses, such as daily, weekly, monthly, or yearly units, once or several times per unit period, or administered continuously over a long period of time using an infusion pump. It can be. The number of repeated doses is determined by considering the time the drug stays in the body and the concentration of the drug in the body. Even after treatment, depending on the course of disease treatment, the composition may be administered for recurrence.
  • Osteoarthritis-specific T cell-inducing lipid nanoparticles were prepared. 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)(DOTAP), 1,2-dioleoyl-sn-glycero-e-phosphethanolamine (DOPE), 1,2-distearyl-sn-glycero-3-phosphoethanolamine- After preparing N-[methoxy(polyehylene glycol)-2000] (ammonium salt) (PEG2000 PE) and cholesterol at concentrations of 10 mg/mL, 25 mg/mL, 10 mg/mL, and 25 mg/mL, respectively, Each lipid component was added to the vial at a molar ratio of 0.475:0.15:0.05:0.12.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DOPE 1,2-dioleoyl-sn-glycero-e-phosphethanol
  • rapamycin prepared at 10 mg/mL
  • the glass vial was rotated to blow away the organic solvent to create a thin lipid film, and the organic solvent was additionally evaporated for 1 hour with a stirrer set at 40°C.
  • 100 ⁇ g of type 2 collagen peptide treated with collagenase III enzyme or type 2 collagen peptide of SEQ ID NO: 1 and 1 mL of phosphate-buffered saline were added to a glass vial, and then stirred for 2 hours on a stirrer set at 40°C.
  • Stirring was performed for a time to primarily produce nanoparticles loaded with type 2 collagen and rapamycin (LNP-Col II-R or LNP-Col II259-273-R).
  • final lipid nanoparticles with a size of 200 nm were manufactured through a continuous extrusion process using an extruder.
  • the size of the nanoparticles prepared by the method of Preparation Example 1 above was measured using Dynamic Light Scattering, and the shape was confirmed using Transmission Electron Microscopy (FIG. 1a).
  • the average diameter of the prepared lipid nanoparticles was 215.4 ⁇ 53.8 nm, and it was confirmed that they had a spherical shape.
  • High Performance Liquid Chromatography and Differential Scanning Calorimetry were used to confirm the drug loaded on the nanoparticles, and as a result, it was confirmed that rapamycin was loaded with a loading efficiency of 65.9% ( Figures 1b, 1c). Additionally, it was confirmed through confocal microscopy that type 2 collagen was supported within the nanoparticles (Figure 1d).
  • the nanoparticles were placed in a liquid containing 50% (v/v) serum, and the change in size of the nanoparticles was monitored every day using a dynamic light scattering device (Figure 1e). As a result, it was confirmed that there was almost no change in the size of the nanoparticles until the third day, and it was judged that this was sufficient time for the nanoparticles to be absorbed by dendritic cells in the body after injection.
  • dendritic cells were isolated from the bone marrow of 6-week-old C57BL/6J mice ordered from Orient Bio. Different types of lipid nanoparticles were produced, cells laid out at a density of 1 ⁇ 10 6 dendritic cells/well were treated with nanoparticles at a concentration of 10 ⁇ g antigen/well, and after 2 days, 200 ng/ml LPS was treated for 24 hours.
  • the cells were scraped with a cell scraper, and the CD40, CD80, and CD86 phenotypes, known as mature dendritic cell markers, were compared and analyzed between groups using flow cytometry, and the expression of inflammatory (TNF- ⁇ ) and anti-inflammatory (TGF- ⁇ ) cytokines was analyzed. The levels were compared and analyzed between groups using polymerase chain reaction. As a result, it was confirmed that lipid nanoparticles containing rapamycin induced immature dendritic cells more effectively than nanoparticles not containing rapamycin, and that they mainly secreted anti-inflammatory cytokines ( Figures 2a1 to 2a3 and Figure 2b).
  • Nanoparticles carrying FITC-labeled Col II peptide were co-cultured with dendritic cells laid out under the same conditions and analyzed by flow cytometry 24 hours later. As a result, approximately 93% of dendritic cells were able to co-culture nanoparticles carrying Col II peptide. It was absorbed, and as confirmed by confocal microscopy, the absorbed Col II peptide appeared on the surface of dendritic cells (Figure 2c2; for free Col II, it is the left peak and the value corresponding to 2.2%, and for LNP-Col II-R The case is the right peak and the value corresponding to 93.3%, Figure 2c3).
  • Col II peptide acts as an antigen and exists on the MHC II molecule of dendritic cells
  • Col II peptide was replaced with E ⁇ peptide in the nanoparticle prepared in Preparation Example 1 above and co-cultured with dendritic cells. 24 hours later, dendritic cells were stained with an antibody that simultaneously recognizes E ⁇ peptide and MHC II and analyzed by flow cytometry and confocal microscopy.
  • the surgery was performed carefully to avoid damaging the surrounding cartilage, ligaments, and bone tissue.
  • the internal tissue and external skin tissue were sutured with absorbable and non-absorbable sutures, respectively, and povidone-iodine was applied to the surgical site to disinfect the wound area to complete the induction of osteoarthritis.
  • an osteoarthritis treatment experiment was conducted by intradermally injecting lipid nanoparticles (LNP-Col II-R) loaded with type 2 collagen peptide and a regulatory T cell inducer (rapamycin).
  • the control group was injected with ovalbumin and rapamycin-loaded nanoparticles (LNP-OVA-R).
  • LNP-antigen-R nanoparticles are injected intradermally into mice, and after a certain period of time, isolated spleen cells are treated with different antigens to determine CD4+CD25+Foxp3+ by antigen. Stimulation and proliferation of regulatory T cells were compared and analyzed between groups.
  • the experimental method was performed in the same manner as above, and as a result, it was confirmed that the T cells of the osteoarthritis mice also reacted specifically to the type 2 collagen 259-273 antigen, although to a lesser extent than to the type 2 collagen ( Figure 4b). .
  • Example 4 To confirm the therapeutic effect of the lipid nanoparticles prepared in Preparation Example 1 on osteoarthritis, the DMM surgery of Example 4 was performed on experimental animals.
  • the prepared nanoparticles were injected intradermally near the right groin according to the experimental plan shown in Figure 5a, and 8 weeks after surgery, the experimental animals were sacrificed, the right joint area was collected, and a paraffin block was produced through fixation and decalcification.
  • the tissue was cut to a thickness of 3 ⁇ m using a thin slice cutter, and the therapeutic effect of nanoparticles was compared and analyzed between groups using various tissue staining techniques.
  • the nanoparticles prepared through the process of Preparation Example 1 have a therapeutic effect on osteoarthritis by suppressing joint wear through type 2 collagen-specific immunomodulation (immune tolerance) and simultaneously regenerating cartilage extracellular matrix.
  • the lipid nanoparticles (LNP-Col II259-273-R) prepared in Preparation Example 1 were intradermally injected into experimental rats that had undergone DMM surgery. As a control group, lipid nanoparticles containing only rapamycin were injected.

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Abstract

The present invention may provide a pharmaceutical composition for preventing or treating osteoarthritis, the composition comprising a carrier containing type 2 collagen peptide and rapamycin. Ideally, the composition may reduce inflammatory responses that occur in joints due to inflammatory immune cells, alleviate cartilage destruction and pain that accompany inflammatory responses and, further, may regenerate cartilage. Furthermore, the composition of the present invention contains antigens associated with other diseases in addition to osteoarthritis and thus may be utilized as a therapeutic agent for inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal cord injuries, or autoimmune diseases such as atopy, rheumatoid arthritis, type 1 diabetes, Crohn's disease, and lupus.

Description

골관절염 예방 또는 치료용 약학 조성물Pharmaceutical composition for preventing or treating osteoarthritis
본 발명은 제 2형 콜라겐 펩타이드 및 라파마이신이 담지된 전달체를 포함하는 골관절염 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating osteoarthritis comprising a type 2 collagen peptide and a carrier carrying rapamycin.
골관절염(osteoarthritis)은 류머티즘 관절염과 함께 가장 대표적인 관절질환으로 알려져 있다. 골관절염은 병증진행과 함께 관절 주변 골의 과잉 형성, 관절 변형과 통증 및 점진적 운동 장애를 동반한다. 현재 전 세계 55세 이상 인구의 약 80%와 75세 이상 고령 인구의 거의 대부분이 퇴행성관절염 소견을 보이는 것으로 알려져 있다. 우리나라의 경우, 사회의 고령화가 급속도로 진행됨에 따라 골관절염으로 인한 통증을 호소하는 인구가 점차 증가하고 있으며, 노령인구에서뿐 아니라 젊은 인구 층에서도 환경적, 유전적 요인으로 인해 골관절염 발생이 증가하면서 골관절염으로 인한 의료비 지출 역시 빠른 속도로 증가하고 있는 추세이다.Osteoarthritis, along with rheumatoid arthritis, is known to be the most representative joint disease. Osteoarthritis is accompanied by excessive formation of bone around joints, joint deformation and pain, and progressive movement disorders as the disease progresses. Currently, approximately 80% of the world's population over 55 years of age and almost all of the elderly population over 75 years of age are known to show signs of degenerative arthritis. In Korea, as society rapidly ages, the number of people complaining of pain due to osteoarthritis is gradually increasing, and the incidence of osteoarthritis is increasing not only in the elderly population but also in the younger population due to environmental and genetic factors. Medical expenses are also increasing at a rapid rate.
현재까지 알려진 골관절염 치료방법으로는 항 염증성 약물투여, 생활 및 식습관을 개선하는 비약물요법 그리고 침습적 방법으로 관절을 수술하는 외래적 치료방법이 있다. 비스테로이드성 항염증 약물의 경우, 염증완화를 통한 통증 감소에는 효과적이나 위장관 부작용이 발생할 수 있고, 이 대안으로 사용된 약물인 COX-2 저해제의 경우는 심혈관계 부작용 및 위장관 손상을 일으킬 수 있다. 또한 외래적 수술방법의 경우, 효과적으로 통증의 원인을 제거할 수 있으나, 관절의 상태와 인공관절의 상태에 따라 재수술 가능성의 부담이 존재한다.Treatment methods for osteoarthritis known to date include anti-inflammatory drug administration, non-drug therapy to improve lifestyle and eating habits, and outpatient treatment that involves invasive joint surgery. Non-steroidal anti-inflammatory drugs are effective in reducing pain through inflammation relief, but may cause gastrointestinal side effects, and COX-2 inhibitors, which are used as alternatives, may cause cardiovascular side effects and gastrointestinal damage. In addition, in the case of outpatient surgical methods, the cause of pain can be effectively removed, but there is a burden of the possibility of reoperation depending on the condition of the joint and artificial joint.
대개, 골관절염은 물리적 요인이 원인인 것으로 알려져 있으나, 최근 다수 논문들을 통해 골관절염에 면역체계가 관여하는 것이 알려지면서, 골관절염에서의 면역세포 작용의 이해 및 이를 바탕으로 한 면역조절이 중요성을 갖게 되었다. 골관절염의 가장 큰 특징은 연골세포 사멸과 연골마모이다. 물리적 원인을 비롯하여 질병, 환경 및 유전적 요인 등으로 인해 연골 마모가 시작될 경우 활막 내 염증성 1형, 2형, 17형 등의 T 세포군 및 1형 대식세포의 세포 수는 증가하는 반면 항 염증성 조절 T 세포 (regulatory T cell, Treg) 및 2형 대식세포의 수는 감소한다. 이때, 염증성 세포들은 인터류킨 1β(interleukin 1 β, IL-1 β), 인터페론 γ(interferon γ, IFN- γ), 종양괴사인자 α(Tumor necrosis factor α, TNF- α) 등의 염증성 사이토카인을 분비하는데, 분비된 사이토카인들은 주변 T 세포군에 작용하여 염증성 세포의 활성화를 촉진시켜 보다 많은 염증성 사이토카인을 분비시키고, 동시에 연골세포에 작용하여 Matrix Metalloproteinase 13(MMP-13) 과 같은 세포 외 기질 분해효소 분비를 촉진시킨다. MMP-13은 가장 대표적인 연골에서의 세포 외 기질 구성요소인 제 2형 콜라젠 단백질을 분해하여 연골 마모를 유발한다. 염증성 면역세포 및 사이토카인은 항 염증성 면역세포 및 사이토카인에 비해 상대적으로 우세하고, 이러한 염증성과 항 염증성 면역세포와 물질 간 불균형이 바로잡히지 않을 경우, 지속적으로 관절 내에는 염증성 환경이 유지된다. 현재 상용되고 있는 대표적 염증완화물질인 세레브렉스는 염증을 완화시킬 수는 있지만, 이는 진행 중인 통증을 완화시킬 뿐 근본적인 해결책이 되기 어렵고, 외과적 방법으로 관절을 수술하는 것은 환자에게 육체적 부담이 될뿐더러, 관절의 상태에 따라 추후 재수술의 가능성이 존재하기 때문에 이 방법 역시 근본적인 치료방법이라고 하기 어렵다.In general, osteoarthritis is known to be caused by physical factors, but as it has become known through many recent papers that the immune system is involved in osteoarthritis, understanding the function of immune cells in osteoarthritis and immunomodulation based on this have become important. The biggest characteristics of osteoarthritis are cartilage cell death and cartilage wear. When cartilage wear begins due to physical causes, disease, environment, and genetic factors, the number of inflammatory T cell groups such as type 1, type 2, and type 17 and type 1 macrophages in the synovium increases, while the number of anti-inflammatory regulatory T cells increases. The number of cells (regulatory T cells, Tregs) and type 2 macrophages decreases. At this time, inflammatory cells secrete inflammatory cytokines such as interleukin 1 β (IL-1 β), interferon γ (IFN- γ), and tumor necrosis factor α (TNF- α). The secreted cytokines act on surrounding T cell groups to promote the activation of inflammatory cells, secreting more inflammatory cytokines, and at the same time act on cartilage cells to produce extracellular matrix decomposing enzymes such as Matrix Metalloproteinase 13 (MMP-13). Promotes secretion. MMP-13 causes cartilage wear by decomposing type 2 collagen protein, the most representative extracellular matrix component of cartilage. Inflammatory immune cells and cytokines are relatively superior to anti-inflammatory immune cells and cytokines, and if the imbalance between inflammatory and anti-inflammatory immune cells and substances is not corrected, an inflammatory environment is continuously maintained within the joint. Celebrex, a representative inflammation-relieving substance currently commercially available, can relieve inflammation, but it only relieves ongoing pain and is not a fundamental solution, and surgically operating the joint not only places a physical burden on the patient, Because there is a possibility of future reoperation depending on the condition of the joint, it is difficult to say that this method is a fundamental treatment method.
조절 T 세포는 면역반응을 억제할 수 있으므로 다양한 염증질환의 치료제로서 연구 및 임상시험 실시 중이고, 자가면역질환, 알레르기질환, 장기이식 거부반응 등에서 과도한 면역 반응을 억제할 수 있음이 밝혀졌다. 하지만, 조절 T 세포 이식술(adoptive Treg cell transfer)은 환자로부터 세포를 분리한 후 체외에서 증식/배양하는 단계가 필요하므로 치료비가 비싸고 치료제 제조가 복잡하다는 단점이 있다. 또한, 여러 가지 항원에 결합하여 작용하는 다양한 클론의 조절 T 세포가 배양된 후 환자에 이식되므로 전신적으로 면역이 억제되어 감염, 암 발생 등의 부작용이 발생할 수 있다. 이러한 문제를 해결하기 위해서는 체내에서 단일 항원에 대한 조절 T 세포를 유도하는 치료제가 개발될 필요가 있다.Because regulatory T cells can suppress immune responses, research and clinical trials are being conducted as a treatment for various inflammatory diseases, and it has been found that they can suppress excessive immune responses in autoimmune diseases, allergic diseases, organ transplant rejection, etc. However, regulatory T cell transplantation (adoptive Treg cell transfer) has the disadvantage of being expensive and complicated in manufacturing the treatment because it requires the step of isolating cells from the patient and then proliferating/cultivating them in vitro. In addition, since regulatory T cells of various clones that bind to and act on various antigens are cultured and then transplanted to the patient, immune system is suppressed systemically, which may lead to side effects such as infection and cancer development. To solve this problem, a therapeutic agent that induces regulatory T cells against a single antigen in the body needs to be developed.
본 발명은 골관절염의 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다. The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating osteoarthritis.
바람직하게는 골 관절에서 발생하는 염증성 면역세포에 의한 염증반응을 감소시키면서, 염증반응으로 수반되는 연골 파괴 및 통증을 완화시키고, 더 나아가서는 연골을 재생시킬 수 있는 조성물을 제공하는 것을 목적으로 한다.Preferably, the purpose is to provide a composition that can reduce the inflammatory response caused by inflammatory immune cells occurring in bone joints, alleviate cartilage destruction and pain accompanying the inflammatory response, and further regenerate cartilage.
1. 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체; 를 포함하는 골관절염 예방 또는 치료용 약학 조성물.1. Type 2 collagen peptide; and rapamycin or its derivatives; A pharmaceutical composition for preventing or treating osteoarthritis, comprising:
2. 위 1에 있어서, 상기 제 2형 콜라겐 펩타이드는 서열번호 1의 아미노산 서열을 포함하는 골관절염 예방 또는 치료용 약학 조성물.2. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, wherein the type 2 collagen peptide contains the amino acid sequence of SEQ ID NO: 1.
3. 위 1에 있어서, 상기 제 2형 콜라겐 펩타이드 및 라파마이신의 중량비는 1 대 0.1 내지 10인 골관절염 예방 또는 치료용 약학 조성물.3. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, wherein the weight ratio of the type 2 collagen peptide and rapamycin is 1 to 0.1 to 10.
4. 위 1에 있어서, 수지상세포(dendritic cell, DC)를 더 포함하는 것인 골관절염 예방 또는 치료용 약학 조성물.4. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, further comprising dendritic cells (DC).
5. 위 1에 있어서, 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체는 전달체에 담지된 것인 골관절염 예방 또는 치료용 약학 조성물.5. In 1 above, type 2 collagen peptide; and a pharmaceutical composition for preventing or treating osteoarthritis, wherein rapamycin or a derivative thereof is supported on a carrier.
6. 위 5에 있어서, 상기 전달체는 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 및 반도체 나노결정 입자(quantum dot)를 포함하는 군에서 선택되는 것인 골관절염 예방 또는 치료용 약학 조성물.6. In item 5 above, the carrier includes viral carriers, virus-like particles (VLP), positively charged polymers, liposomes, lipid nanoparticles, gold, and semiconductor nanocrystal particles (quantum dots). A pharmaceutical composition for preventing or treating osteoarthritis selected from the group comprising:
7. 위 5에 있어서, 상기 전달체는 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), dioleoylphosphatidylethanolamine(DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)](PEG PE) 및 콜레스테롤을 포함하는 지질나노입자인 골관절염 예방 또는 치료용 약학 조성물.7. In item 5 above, the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene) glycol)] (PEG PE) and a pharmaceutical composition for preventing or treating osteoarthritis, which are lipid nanoparticles containing cholesterol.
8. 위 6에 있어서, 상기 지질나노입자의 직경은 50 nm 내지 1,000 nm인 골관절염 예방 또는 치료용 약학 조성물.8. The pharmaceutical composition for preventing or treating osteoarthritis according to item 6 above, wherein the lipid nanoparticles have a diameter of 50 nm to 1,000 nm.
본 발명은 제 2형 콜라겐 펩타이드 및 라파마이신을 담지하는 전달체를 포함하는 골관절염의 예방 또는 치료용 약학 조성물을 제공할 수 있다. 이는 골관절염 관절연골에서 노출된 제 2형 콜라겐 펩타이드로 인한 염증반응을 최소화하여 골관절염의 예방 또는 치료 효과를 도모할 수 있다. 더 나아가 골관절염뿐 아니라, 제 2형 콜라겐 펩타이드 대신 다른 질병 관련 항원을 담지하면 심근경색, 뇌경색, 척추손상 등의 염증성 질환 또는 아토피, 류마티스관절염, 1형 당뇨병, 크론병, 루푸스 등 자가면역질환의 치료제로서 활용할 수 있다.The present invention can provide a pharmaceutical composition for preventing or treating osteoarthritis, including a type 2 collagen peptide and a carrier carrying rapamycin. This can promote the prevention or treatment of osteoarthritis by minimizing the inflammatory response caused by type 2 collagen peptides exposed in osteoarthritis articular cartilage. Furthermore, in addition to osteoarthritis, carrying other disease-related antigens instead of type 2 collagen peptide can be used as a treatment for inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal damage, or autoimmune diseases such as atopy, rheumatoid arthritis, type 1 diabetes, Crohn's disease, and lupus. It can be used as.
도 1은 지질나노입자의 제조 방법 및 그 특성을 분석한 데이터이다. 지질나노입자가 제 2형 콜라겐 펩타이드 및 라파마이신을 정상적으로 담지할 수 있음을 확인한 것이다.Figure 1 shows data analyzing the manufacturing method of lipid nanoparticles and their characteristics. It was confirmed that lipid nanoparticles can normally support type 2 collagen peptide and rapamycin.
도 2는 제 2형 콜라겐 펩타이드 및 라파마이신을 담지한 지질나노입자가 미분화 수지상세포를 관용 (tolerogenic) 수지상세포로 분화를 유도할 수 있음을 확인한 데이터이다.Figure 2 shows data confirming that lipid nanoparticles carrying type 2 collagen peptide and rapamycin can induce differentiation of undifferentiated dendritic cells into tolerogenic dendritic cells.
도 3은 제 2형 콜라겐 펩타이드 및 라파마이신을 담지한 지질나노입자가 in vivo 모델에 주사하였을 때 주사 부위 근처의 림프절(lymph node, LN)에서 분포되는 것을 확인한 데이터이다.Figure 3 shows data confirming that lipid nanoparticles carrying type 2 collagen peptide and rapamycin were distributed in lymph nodes (LN) near the injection site when injected into an in vivo model.
도 4는 제 2형 콜라겐 펩타이드 또는 제 2형 콜라겐의 259 - 273번째 아미노산 서열을 가진 펩타이드 및 라파마이신을 담지한 지질나노입자가 in vivo 에서 제 2형 콜라겐 특이적인 조절 T 세포를 활성화 시킬 수 있음을 확인한 데이터이다.Figure 4 shows that type 2 collagen peptides or peptides with amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles loaded with rapamycin can activate type 2 collagen-specific regulatory T cells in vivo. This is data that confirmed .
도 5는 제 2형 콜라겐 펩타이드 또는 제 2형 콜라겐의 259 - 273번째 아미노산 서열을 가진 펩타이드 및 라파마이신을 담지한 지질나노입자가 골관절염 유도 모델 마우스에서 증상 완화 및/또는 개선 효과가 있음을 확인한 데이터이다.Figure 5 shows data confirming that type 2 collagen peptides or peptides with amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles loaded with rapamycin are effective in alleviating and/or improving symptoms in osteoarthritis-induced model mice. am.
도 6은 본 발명의 지질나노입자 기반의 골관절염 치료 기전에 대한 모식도이다.Figure 6 is a schematic diagram of the lipid nanoparticle-based osteoarthritis treatment mechanism of the present invention.
이하 본 발명을 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적인 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 의미를 따른다.Hereinafter, the present invention will be described in detail. Unless otherwise specified, all terms in this specification have the same general meaning as understood by a person skilled in the art to which the present invention pertains, and if there is a conflict with the meaning of the terms used in this specification, this specification Follow the meaning used in the specification.
본 발명은 제 2형 콜라겐 펩타이드;및 라파마이신 또는 그 유도체;를 포함하는 골관절염 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating osteoarthritis, comprising type 2 collagen peptide and rapamycin or a derivative thereof.
본 발명은 제2형 콜라겐 펩타이드를 라파마이신 또는 그 유도체와 함께 사용함으로써, 미성숙 수지상세포의 관용 수지상세포로의 분화를 유도하고, 관용 수지상세포의 MHC 분자에 제 2형 콜라겐 펩타이드 에피토프(epitope)를 제시하도록 할 수 있다. 상기 수지상세포는 제 2형 콜라겐 펩타이드에 특이적인 조절 T 세포를 다량 유도하고, 상기 조절 T 세포는 관절 염증 부위에서 염증성 면역세포를 억제하여 골관절염 증상을 완화 및/또는 치료할 수 있다.The present invention induces differentiation of immature dendritic cells into tolerant dendritic cells by using type 2 collagen peptide together with rapamycin or its derivatives, and adds a type 2 collagen peptide epitope to the MHC molecule of tolerant dendritic cells. You can present it. The dendritic cells induce a large amount of regulatory T cells specific for type 2 collagen peptide, and the regulatory T cells can alleviate and/or treat osteoarthritis symptoms by suppressing inflammatory immune cells at the site of joint inflammation.
본 발명에서 상기 제 2형 콜라겐 펩타이드는 제 2형 콜라겐 단백질 중 적어도 일부 아미노산 서열을 포함하는 펩타이드일 수 있다. 제2형 콜라겐 단백질은 수지상 세포의 MHC 분자에 결합하는 에피토프를 갖는다. 상기 적어도 일부는 상기 에피토프로서 인식될 수 있는 서열을 포함하는 것일 수 있다. In the present invention, the type 2 collagen peptide may be a peptide containing at least some amino acid sequences of type 2 collagen protein. Type 2 collagen proteins have epitopes that bind to MHC molecules on dendritic cells. At least a portion of the epitope may include a sequence that can be recognized as the epitope.
에피토프로서 인식될 수 있는 서열은 에피토프 서열 전체로 이루어지거나, 그 서열의 양 말단에 일부 아미노산이 추가되거나, 양 말단에서 일부 아미노산이 제거되어도 에피토프로서 작동하는 서열을 의미한다.A sequence that can be recognized as an epitope refers to a sequence that functions as an epitope even if it consists of the entire epitope sequence, some amino acids are added to both ends of the sequence, or some amino acids are removed from both ends of the sequence.
상기 에피토프는 서열번호 1의 아미노산 서열일 수 있고, 에피토프로서 인식될 수 있는 서열은 이를 포함하거나, 그 양 말단의 1 내지 3개의 아미노산을 포함하지 않는 서열일 수 있다. 서열번호 1의 아미노산 서열 외에도 상기 에피토프로서 인식될 수 있는 서열이라면 제한 없이 포함될 수 있고 공지된 에피토프 서열을 사용할 수도 있고, 제2형 콜라겐 단백질을 콜라게네이즈로 절단한 절편을 포함할 수도 있다. 상기 적어도 일부를 포함한다면 그 양 말단에 추가되는 서열의 종류/길이 등은 제한되지 않고, 제2형 콜라겐 단백질 전체를 사용할 수도 있다.The epitope may be the amino acid sequence of SEQ ID NO: 1, and the sequence that can be recognized as the epitope may include it or may be a sequence that does not include 1 to 3 amino acids at both ends. In addition to the amino acid sequence of SEQ ID NO: 1, any sequence that can be recognized as the epitope may be included without limitation, and a known epitope sequence may be used, or it may include a fragment of type 2 collagen protein cut with collagenase. If at least part of the above is included, the type/length of the sequence added to both ends is not limited, and the entire type 2 collagen protein can be used.
상기 제 2형 콜라겐 펩타이드의 길이는 앞서 언급하였듯, MHC 분자에 결합하여 제 2형 콜라겐 펩타이드 에피토프로 인식될 수 있는 것이라면 제한되지 않으며, 예를 들면 0.5 kDa 내지 120 kDa, 1 kDa 내지 100 kDa, 1.5 kDa 내지 50 kDa, 2 kDa 내지 30 kDA, 3 kDa 내지 20 kDa 등일 수 있다.As mentioned above, the length of the type 2 collagen peptide is not limited as long as it can be recognized as a type 2 collagen peptide epitope by binding to an MHC molecule, for example, 0.5 kDa to 120 kDa, 1 kDa to 100 kDa, It may be 1.5 kDa to 50 kDa, 2 kDa to 30 kDa, 3 kDa to 20 kDa, etc.
상기 제 2형 콜라겐 펩타이드는 어느 종에서 유래한 콜라겐 펩타이드인지와 무관하게 본 발명의 범위에 속할 수 있다. 제 2형 콜라겐은 종 간 보존도가 높은 유전자로서, 예를 들면 인간, 마우스, 소, 염소, 말, 고양이 및 개를 포함하는 군에서 선택되는 종에서 유래한 제 2형 콜라겐 펩타이드도 본 발명의 범위에 속할 수 있다. 제 2형 콜라겐 펩타이드 서열 중에서 종 간 일치하지 않는 서열을 일부 포함하더라도, 본 발명의 효과가 발휘될 수 있는데, 예를 들면 서열번호 1의 아미노산 서열에서 1개 또는 2개가 다른 아미노산으로 치환되더라도, 관용 수지상세포의 MHC 분자에 제 2형 콜라겐 펩타이드 에피토프로 인식되는 한 본 발명의 효과가 발휘될 수 있다. 더욱 구체적으로 서열번호 1에서 8번째 아미노산이 아스파라트산 또는 글루탐산일 수 있고, 서열번호 1에서 15번째 아미노산이 트레오닌 또는 프롤린일 수 있다. 다만 이에 제한되는 것은 아니다.The type 2 collagen peptide may fall within the scope of the present invention regardless of which species the collagen peptide originates from. Type 2 collagen is a gene with a high degree of conservation across species. For example, type 2 collagen peptides derived from species selected from the group including humans, mice, cattle, goats, horses, cats, and dogs are also included in the present invention. may fall within the range. Even if the type 2 collagen peptide sequence contains some sequences that are inconsistent between species, the effect of the present invention can be exerted. For example, even if one or two amino acids in the amino acid sequence of SEQ ID NO: 1 are substituted with different amino acids, the effect of the present invention can be achieved. As long as it is recognized as a type 2 collagen peptide epitope by the MHC molecule of dendritic cells, the effect of the present invention can be exerted. More specifically, the 8th amino acid in SEQ ID NO: 1 may be aspartic acid or glutamic acid, and the 15th amino acid in SEQ ID NO: 1 may be threonine or proline. However, it is not limited to this.
본 발명에서 상기 라파마이신 또는 그 유도체는 그 종류에 무관하게 본 발명의 범위에 포함될 수 있다. 본 발명에서 라파마이신은 림프구의 성장을 억제하고, 분화를 촉진하여 제 2형 콜라겐 펩타이드에 대해 특이적인 조절 T세포를 유도할 수 있고, 이러한 기능을 수행할 수 있는 라파마이신의 유도체라면 제한 없이 본 발명의 범위에 포함될 수 있다. 그 예를 들면, 벤조일 라파마이신, 템시로리무스, 에베로리무스, 조타롤리무스, 바이오리무스, 피메크로리무스, 피메크로리무스, 타크로리무스, 리다포로리무스 등을 들 수 있다. 다만, 이에 제한되는 것은 아니다.In the present invention, rapamycin or a derivative thereof may be included in the scope of the present invention regardless of its type. In the present invention, rapamycin can inhibit the growth of lymphocytes, promote differentiation, and induce regulatory T cells specific for type 2 collagen peptides, and any derivative of rapamycin that can perform these functions can be used without limitation. It may be included in the scope of the invention. Examples include benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus, and ridaporolimus. However, it is not limited to this.
본 발명의 상기 조성물은 수지상세포(dendritic cell, DC)를 더 포함할 수 있다. 상기 수지상세포는 제 2형 콜라겐 펩타이드 및/또는 라파마이신을 포함하는 용액에 노출되어 있을 수 있고, 그 경우, 수지상세포 내부로 제 2형 콜라겐 펩타이드 및/또는 라파마이신에 의하여 라파마이신이 유입됨으로써 제 2형 콜라겐 펩타이드를 세포 표면의 MHC 분자에 제시할 수 있다. 다만 이에 제한되는 것은 아니다.The composition of the present invention may further include dendritic cells (DC). The dendritic cells may be exposed to a solution containing type 2 collagen peptide and/or rapamycin, and in this case, rapamycin is introduced into the dendritic cells by the type 2 collagen peptide and/or rapamycin, thereby producing a Type 2 collagen peptides can be presented to MHC molecules on the cell surface. However, it is not limited to this.
본 발명에서 상기 골관절염(osteoarthritis)는 연골조직의 점진적/비가역적인 퇴행에 의하여 유발되는 질병으로서, 퇴행성 경관절 질환(degenerative joint disease), 변형성 관절증(ostoarthrosis), 비대성 관절염(hypertrophic arthritis) 또는 퇴행성관절염 (degenerative arthritis)으로도 알려져 있는 질병으로서, 상기 명칭들과 동일한 질병을 지칭하는 용어에 해당한다. 골관절염의 증상은 연골이 탄력성을 잃어 상처나 이용에 의해 보다 더 쉽게 상처받는 단계; 연골의 마모가 밑에 있는 뼈에 변화를 야기시키는 단계로, 뼈가 비후화되고 낭종이 발생할 수 있으며, 돌기(spurs) 또는 골증식체(osteophytes)로 불리는 뼈의 증식이 영향 받은 관절에 있는 뼈 말단에서 일어나 가려움과 고통을 야기하는 단계; 뼈 또는 연골의 조각이 관절강(joint space)에 느슨하게 부유하는 단계; 및 연골 파괴(breakdown)로 인해 관절막 또는 활액막에 염증을 일으키는 단계로, 연골에 손상을 입히는 사이토카인 및 효소를 추가적으로 야기시키는 단계로서 네 단계로 분류할 수 있다. 이는 자가면역질환 혹은 전신성 염증반응으로 인하여 급격한 연골 조직의 손상 및/또는 파괴를 야기시키는 류마티스성 관절염(rheumatoid arthritis)와는 구별되는 질병에 해당한다.In the present invention, osteoarthritis is a disease caused by gradual/irreversible degeneration of cartilage tissue, and includes degenerative joint disease, osteoarthrosis, hypertrophic arthritis, or degenerative arthritis ( It is a disease also known as degenerative arthritis, and is a term referring to the same disease as the names above. Symptoms of osteoarthritis include a stage in which cartilage loses its elasticity and becomes more easily damaged by injury or use; A stage in which wear and tear of the cartilage causes changes to the underlying bone, which may include bone thickening, cysts, and bone growths called spurs or osteophytes on the ends of bones in the affected joint. Occurs in the stage causing itching and pain; a step in which a piece of bone or cartilage floats loosely in the joint space; and a stage that causes inflammation in the joint membrane or synovium due to cartilage breakdown, and a stage that additionally causes cytokines and enzymes that damage cartilage, and can be classified into four stages. This is a disease distinct from rheumatoid arthritis, which causes rapid damage and/or destruction of cartilage tissue due to an autoimmune disease or systemic inflammatory response.
본 발명에서 상기 조성물은 상기 골관절염에 대해서 활막을 통해 연골에 유입되는 염증성 면역세포, 예를 들면 대식세포, 제 1형 helper T 세포의 수를 감소시키거나, 염증성 면역세포에 의한 염증성 시토카인, 예를 들면 인터루킨 1β, TNF-α 또는 IFN-γ의 분비를 억제시킴으로써 연골에서에 염증반응을 완화시킬 수 있다. 또한 염증반응으로 수반되는 연골조직 및/또는 세포외기질을 구성하는 단백질의 파괴를 억제하고, 통증을 완화시킬 수 있다. In the present invention, the composition reduces the number of inflammatory immune cells, such as macrophages and type 1 helper T cells, that enter the cartilage through the synovium for osteoarthritis, or reduces the number of inflammatory cytokines caused by inflammatory immune cells, such as For example, the inflammatory response in cartilage can be alleviated by suppressing the secretion of interleukin 1β, TNF-α, or IFN-γ. In addition, it can suppress the destruction of proteins that make up the cartilage tissue and/or extracellular matrix that accompany the inflammatory response and relieve pain.
본 발명의 제 2형 콜라겐 펩타이드 및 라파마이신의 중량비는 제한 없이 본 발명의 범위에 포함될 수 있다. 예를 들면 1 대 0.1 내지 10, 1대 0.2 내지 8, 1대 0.3 내지 5, 1대 0.5 내지 3 일 수 있다. 다만 이에 제한되는 것은 아니다.The weight ratio of the type 2 collagen peptide of the present invention and rapamycin may be included within the scope of the present invention without limitation. For example, it may be 1 to 0.1 to 10, 1 to 0.2 to 8, 1 to 0.3 to 5, or 1 to 0.5 to 3. However, it is not limited to this.
본 발명의 상기 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체는 전달체에 담지되어 전달될 수 있다. The type 2 collagen peptide of the present invention; And rapamycin or a derivative thereof may be delivered while being carried on a carrier.
상기 전달체는 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체의 수지상세포에 전달효율을 크게 향상시키므로, 결과적으로 제 2형 콜라겐 펩타이드를 제 2형 MHC 분자에 제시하는 관용 수지상세포의 유도 효율과 제 2형 콜라겐 펩타이드 특이적 조절 T 세포의 유도 효율을 크게 높일 수 있다.The delivery vehicle is type 2 collagen peptide; And it greatly improves the delivery efficiency of rapamycin or its derivatives to dendritic cells, resulting in the induction efficiency of tolerant dendritic cells that present type 2 collagen peptides to type 2 MHC molecules and type 2 collagen peptide-specific regulatory T cells. The induction efficiency can be greatly increased.
상기 전달체는 내부에 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체를 담지하고, 타깃 세포에 전달하여 내부의 물질을 방출할 수 있는 것이라면, 당업자에 의하여 제한 없이 선택될 수 있다. 예를 들면, 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 및 반도체 나노결정 입자(quantum dot) 를 포함하는 군에서 선택되는 것일 수 있다. 다만 이에 제한되는 것은 아니다.The delivery vehicle has a type 2 collagen peptide inside; and rapamycin or a derivative thereof, and can be selected without limitation by those skilled in the art as long as it can be delivered to the target cell and release the substance therein. For example, selected from the group including viral carriers, virus-like particles (VLP), positively charged polymers, liposomes, lipid nanoparticles, gold, and semiconductor nanocrystal particles (quantum dots). It could be. However, it is not limited to this.
상기 지질나노입자는 그 성분에 무관하게 당업자에 의하여 선택하여 사용할 수 있다. 지질나노입자를 구성하는 인지질 분자로서, 중성지질, 음이온성 및/또는 양이온성 인지질 모두가 사용이 가능하고, 예를 들면, 예를 들면 중성지질로서는 L-α-포스파티딜콜린(L-α-phosphatidylcholine, PC), 1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoylsn-glycero-3-phophocholine, DPPC), 1,2-디미리스토일-sn-글리세로-3-포스포콜린(1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC)으로 이루어진 군에서 선택된 적어도 하나일 수 있고, 상기 PC는 콩, 계란, 수소화된 콩 또는 계란으로부터 유래된 PC일 수 있으나, 이로 제한되지 않는다. The lipid nanoparticles can be selected and used by those skilled in the art regardless of their components. As phospholipid molecules constituting lipid nanoparticles, neutral lipids, anionic and/or cationic phospholipids can all be used, for example, L-α-phosphatidylcholine, PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn- Glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phophocholine, DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoylsn) -glycero-3-phophocholine, DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) There may be at least one selected, and the PC may be PC derived from soybeans, eggs, hydrogenated soybeans or eggs, but is not limited thereto.
상기 음이온성 지질로는 L-α-포스파티딕산(L-α-phosphatidic acid), L-α-포스파티딜-DL-글리세롤(L-α-phosphatidyl-DL-glycerol), 카디오리핀(cardiolipin), L-α-포스파티딜이노시톨(L-α-phosphatidylinositol), L-α-포스파티딜세린(L-α-phosphatidylserine), 1,2-디라우로일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-디라우로일-sn-글리세로-3-[포스포-L-세린](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-디라우로일-sn-글리세로-3-포스페이트(1,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-디미리스토일-sn-글리세로-3-[포스포-rac-(1-글리세롤)]1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPG), 1,2-디미리스토일-sn-글리세로-3-[포스포-L-세린] (1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine], DMPS), 1,2-디미리스토일-sn-글리세로-3-포스페이트(1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA), 1,2-디올레오일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG), 1,2-디올레오일-sn-글리세로-3-[포스포-L-세린](1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS), 1,2-디올레오일-sn-글리세로-3-포스페이트(1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-디팔미토일-sn-글리세로-3-[포스포-L-세린](1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine], DPPS), 1,2-디팔미토일-sn-글리세로-3-포스페이트(1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-디스테로일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DSPG), 1,2-디스테로일-sn-글리세로-3-[포스포-L-세린](1,2-distearoyl-sn-glycero-3-[phospho-L-serine], DSPS), 1,2-디스테로일-sn-글리세로-3-포스페이트(1,2-distearoyl-sn-glycero-3-phosphate, DSPA), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[카복시(폴리에틸렌글리콜2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000]), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[말레이미드(폴리에틸렌글리콜)2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]), 1,2-디스테로일-sn-글리세로-3-포스포에탄올아민-N-[PDP(폴리에틸렌글리콜)2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000]), 1-팔미토일-2-올레일-sn-글리세로-3-[포스포-rac-(1-글리세롤)](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], POPG), 1-팔미토일-2-올레일-sn-글리세로-3-[포스포-L-세린](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], POPS), 1-팔미토일-2-올레일-sn-글리세로-3-포스페이트(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, POPA) 및 올레산(oleicacid)으로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 이로 제한되지는 않는다.The anionic lipids include L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, cardiolipin, L -α-phosphatidylinositol (L-α-phosphatidylinositol), L-α-phosphatidylserine, 1,2-dilauroyl-sn-glycero-3-[phospho-rac-( 1-glycerol)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3-[ phospho-L-serine](1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1 ,2-dilauroyl-sn-glycero-3-phosphate, DLPA), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]1,2-dimyristoyl- sn-glycero-3-[phospho-rac-(1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] (1,2- dimyristoyl-sn-glycero-3-[phospho-L-serine], DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (1,2-dimyristoyl-sn-glycero-3-phosphate , DMPA), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1,2-dioleoyl-sn-glycero-3-[phospho-rac-( 1-glycerol)], DOPG), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](1,2-dioleoyl-sn-glycero-3-[phospho-L- serine], DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (1,2-dioleoyl-sn-glycero-3-phosphate, DOPA), 1,2-dipalmitoyl-sn- Glycero-3-[phospho-L-serine](1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine], DPPS), 1,2-dipalmitoyl-sn-glycero- 3-phosphate (1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-disteroyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1 ,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DSPG), 1,2-distearoyl-sn-glycero-3-[phospho-L-serine]( 1,2-distearoyl-sn-glycero-3-[phospho-L-serine], DSPS), 1,2-distearoyl-sn-glycero-3-phosphate (1,2-distearoyl-sn-glycero- 3-phosphate, DSPA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy (polyethylene glycol 2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine -N-[carboxy(polyethylene glycol)2000]), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) 2000](1,2-disteroyl -sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)2000]), 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000 ](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000]), 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac -(1-glycerol)](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], POPG), 1-palmitoyl-2-oleoyl-sn-glycero ro-3-[phospho-L-serine](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], POPS), 1-palmitoyl-2-oleoyl-sn- It may be at least one selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and oleic acid, but is not limited thereto.
상기 양이온성 지질로서는 1,2-디올레오일-sn-글리세로-3-에틸포스포콜린(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EDOPC), 1,2-디올레오일-3-트리메틸암모늄-프로판(1,2-dioleoyl-3-trimethylammoniumpropane, DOTAP), 디올레오일 글루타마이드(dioleoyl glutamide), 디스테아로일 글루타마이드(distearoylglutamide), 디팔미토일 글루타마이드(dipalmitoyl glutamide), 디올레오일 아스파르타마이드(dioleoylaspartamide), 1,2-디올레오일-3-디메틸암모늄-프로판(1,2-dioleoyl-3-dimethylammonium-propane, DODAP), ß-[N-(N',N'-디메틸아미노에탄-카바모일], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), 메틸디옥타데실암모늄 브로마이드 (dimethyldiochtadecylammonium bromide, DDAB), 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디스테아로일-sn-글리세로-3-포스포에탄올아민(1,2-distearoyl-sn-glycero-3-phophoethanolamine, DSPE) 및 1,2-디팔미토일-sn-글리세로-3-포스포에탄올아민 (1,2-dipalmitoyl-sn-glycero-3-phophoethanolamine, DPPE)으로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 이로 제한되지는 않는다.Examples of the cationic lipid include 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) 3-Trimethylammonium-propane (1,2-dioleoyl-3-trimethylammoniumpropane, DOTAP), dioleoyl glutamide, distearoylglutamide, dipalmitoyl glutamide glutamide), dioleoylaspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), ß-[N-(N ',N'-dimethylaminoethane-carbamoyl], (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), methyldiochtadecylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-distearoyl-sn-glycero -3-phosphoethanolamine (1,2-distearoyl-sn-glycero-3-phophoethanolamine, DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl) -sn-glycero-3-phophoethanolamine, DPPE), but is not limited thereto.
상기 지질입자는 입자의 유동성, 담지 효율 및/또는 안정성 등을 개선하기 위하여 추가적으로 폴리에틸렌글리콜(polyethylene glycol, PEG) 등으로 인지질 표면이 PEGylation 될 수 있고, 콜레스테롤, 1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP) 또는 dioleoylphosphatidylethanolamine(DOPE) 등을 더 포함할 수도 있다. 다만 이에 제한되는 것은 아니다.The lipid particle may have its phospholipid surface PEGylated with polyethylene glycol (PEG), etc. to improve the fluidity, loading efficiency, and/or stability of the particle, and may be PEGylated with cholesterol, 1,2-Dioleoyl-3-trimethylammoniumpropane ( DOTAP) or dioleoylphosphatidylethanolamine (DOPE) may be further included. However, it is not limited to this.
본 발명에서 상기 전달체의 평균 직경은 제한 없이 본 발명의 범위에 포함되고, 담지되는 펩타이드 및 약물의 양 등의 조건에 따라 당업자에 의해 선택될 수 있다. 전달체의 직경이 클수록 더 많은 양의 물질의 담지가 가능하나, 전달체의 혈중 안정성, 혈중 이동속도 등에 영향을 줄 수 있어 적절한 범위의 평균 직경을 선택할 수 있다. 평균 직경의 예를 들면, 50 nm 내지 1,000 nm일 수 있다. 평균 입자 직경의 하한의 예를 들면, 50 nm, 60 nm, 70 nm, 80 nm 또는 90 nm 일 수 있고, 상한의 예를 들면, 1,000 nm, 900 nm, 800 nm, 700 nm, 600 nm 일 수 있다. 다만, 이에 제한되는 것은 아니다.In the present invention, the average diameter of the carrier is within the scope of the present invention without limitation, and may be selected by a person skilled in the art depending on conditions such as the amount of peptide and drug to be carried. The larger the diameter of the delivery vehicle, the more it can carry a larger amount of material, but it can affect the stability of the delivery vehicle in the blood and the speed of movement through the blood, so an average diameter within an appropriate range can be selected. For example, the average diameter may be 50 nm to 1,000 nm. The lower limit of the average particle diameter could be, for example, 50 nm, 60 nm, 70 nm, 80 nm or 90 nm, and the upper limit could be, for example, 1,000 nm, 900 nm, 800 nm, 700 nm, or 600 nm. there is. However, it is not limited to this.
본 발명의 상기 조성물은 상기 골관절염의 예방 또는 치료와 관련하여 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 또는 유효성분의 용해성 및/또는 흡수성을 유지/증가 시키는 화합물을 추가로 함유할 수 있다.The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar function in relation to the prevention or treatment of osteoarthritis, or a compound that maintains/increases the solubility and/or absorption of the active ingredient.
본 발명의 상기 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 다만, 이에 제한되지 않는다. 또한, 본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며, 특정 범위의 농도로 한정되지 않는다. The appropriate dosage of the composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can. However, it is not limited to this. In addition, the concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient's condition, necessary period, etc., and is not limited to a specific concentration range.
본 발명의 상기 조성물은 약학적으로 유효한 양으로 투여한다. 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. The effective dosage level depends on factors including the type and severity of the patient's disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. can be decided. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 상기 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 매우 다양하며, 적정한 투여량은 예를 들면 환자의 체내에 축적된 약물의 양 및/또는 사용되는 본 발명의 전달체의 구체적 효능정도에 따라 달라질 수 있다. 예를 들면 체중 1 kg당 0.01 μg 내지 1 g 일 수 있으며, 일별, 주별, 월별 또는 연별의 단위 기간으로, 단위 기간 당 일회 내지 수회 나누어 투여될 수 있으며, 또는 인퓨전 펌프를 이용하여 장기간 연속적으로 투여될 수 있다. 반복투여 횟수는 약물이 체내 머무는 시간, 체내 약물 농도 등을 고려하여 결정된다. 질환 치료 경과에 따라 치료가 된 후라도, 재발을 위해 조성물이 투여될 수 있다.The dosage range of the composition of the present invention varies greatly depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The appropriate dosage can be determined by, for example, the patient. It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the delivery vehicle of the present invention used. For example, it may be 0.01 μg to 1 g per kg of body weight, and may be administered in divided doses, such as daily, weekly, monthly, or yearly units, once or several times per unit period, or administered continuously over a long period of time using an infusion pump. It can be. The number of repeated doses is determined by considering the time the drug stays in the body and the concentration of the drug in the body. Even after treatment, depending on the course of disease treatment, the composition may be administered for recurrence.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in detail with reference to examples.
제조예 1. 지질 나노입자 제조Preparation Example 1. Preparation of lipid nanoparticles
골관절염 특이적 T 세포 유도 리피드 나노입자를 제조하였다. 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)(DOTAP), 1,2-dioleoyl-sn-glycero-e-phosphethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyehylene glycol)-2000] (ammonium salt) (PEG2000 PE) 및 콜레스테롤을 각각 10 mg/mL, 25 mg/mL, 10 mg/mL, 25 mg/mL의 농도로 제조한 후, 유리 바이알에 0.475:0.15:0.05:0.12 의 몰비율에 맞춰 각 지질구성물질들을 넣었다. 10 mg/mL로 제조한 라파마이신 70 μg을 추가한 후, 유리 바이알을 회전시키며 유기용매를 날려주어 얇은 지질막을 생성하고, 40 ℃로 설정된 교반기 위해서 1 시간 동안 추가적으로 유기용매를 증발시켰다. 이후, 유리 바이알에 콜라게네이즈 Ⅲ 효소로 처리된 제2형 콜라겐 펩타이드 또는 서열번호 1의 제 2형 콜라겐 펩타이드 100 μg 과 인산완충생리식염수를 1 mL를 추가한 후, 40 ℃로 설정된 교반기에서 2 시간 교반을 진행하여 1차적으로 제2형 콜라젠과 라파마이신이 담지된 나노입자를 생성하였다 (LNP-Col II-R 또는 LNP-Col II259-273-R). 이후, 압출기를 이용하여 연속적 압출과정을 통해 200 nm 크기의 최종 리피드 나노입자를 제조하였다. Osteoarthritis-specific T cell-inducing lipid nanoparticles were prepared. 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)(DOTAP), 1,2-dioleoyl-sn-glycero-e-phosphethanolamine (DOPE), 1,2-distearyl-sn-glycero-3-phosphoethanolamine- After preparing N-[methoxy(polyehylene glycol)-2000] (ammonium salt) (PEG2000 PE) and cholesterol at concentrations of 10 mg/mL, 25 mg/mL, 10 mg/mL, and 25 mg/mL, respectively, Each lipid component was added to the vial at a molar ratio of 0.475:0.15:0.05:0.12. After adding 70 μg of rapamycin prepared at 10 mg/mL, the glass vial was rotated to blow away the organic solvent to create a thin lipid film, and the organic solvent was additionally evaporated for 1 hour with a stirrer set at 40°C. Thereafter, 100 μg of type 2 collagen peptide treated with collagenase III enzyme or type 2 collagen peptide of SEQ ID NO: 1 and 1 mL of phosphate-buffered saline were added to a glass vial, and then stirred for 2 hours on a stirrer set at 40°C. Stirring was performed for a time to primarily produce nanoparticles loaded with type 2 collagen and rapamycin (LNP-Col II-R or LNP-Col II259-273-R). Afterwards, final lipid nanoparticles with a size of 200 nm were manufactured through a continuous extrusion process using an extruder.
실시예 1. 지질 나노입자의 특성 확인Example 1. Confirmation of properties of lipid nanoparticles
위 제조예 1의 방법으로 제조한 나노입자의 크기는 동적광산란기기(Dynamic Light Scattering)를 이용하여 측정하고, 투과전자현미경 (Transmission Electron Microscopy)으로 형태를 확인하였다(도 1a). 제조된 리피드 나노입자의 평균지름은 215.4 ± 53.8 nm였고, 구의 형태를 띄고 있는 것을 확인하였다. 나노입자에 담지된 약물을 확인하기 위해 고성능 액체 크로마토그라피 (High Performance Liquid Chromatography)와 시차주사열량계 (Differential Scanning Calorimetry)를 이용하였고, 그 결과 65.9%의 담지 효율로 라파마이신이 담지된 것을 확인하였다(도 1b, 1c). 또한 공초점 현미경 (Confocal microscopy)를 통해 나노입자 내에 제 2형 콜라젠이 담지되었음을 확인하였다(도 1d). 리피드 나노입자의 안정성을 확인하기 위해 50% (v/v) 혈청이 포함된 액체에 나노입자를 두고 매일 동적광산란기기로 나노입자의 크기변화를 추적 관찰하였다(도 1e). 그 결과, 3일차까지 나노입자의 크기변화가 거의 없는 것을 확인하였고, 이는 나노입자 주사 후 체내에서 수지상세포에 의해 나노입자가 이물 흡수되기에 충분한 시간이라고 판단하였다.The size of the nanoparticles prepared by the method of Preparation Example 1 above was measured using Dynamic Light Scattering, and the shape was confirmed using Transmission Electron Microscopy (FIG. 1a). The average diameter of the prepared lipid nanoparticles was 215.4 ± 53.8 nm, and it was confirmed that they had a spherical shape. High Performance Liquid Chromatography and Differential Scanning Calorimetry were used to confirm the drug loaded on the nanoparticles, and as a result, it was confirmed that rapamycin was loaded with a loading efficiency of 65.9% ( Figures 1b, 1c). Additionally, it was confirmed through confocal microscopy that type 2 collagen was supported within the nanoparticles (Figure 1d). To confirm the stability of the lipid nanoparticles, the nanoparticles were placed in a liquid containing 50% (v/v) serum, and the change in size of the nanoparticles was monitored every day using a dynamic light scattering device (Figure 1e). As a result, it was confirmed that there was almost no change in the size of the nanoparticles until the third day, and it was judged that this was sufficient time for the nanoparticles to be absorbed by dendritic cells in the body after injection.
실시예 2. 지질 나노입자의 항염증성 세포 유도능 확인Example 2. Confirmation of anti-inflammatory cell-inducing ability of lipid nanoparticles
위 제조예 1에서 제조한 나노입자의 항 염증성 세포 유도를 확인하기 위해 오리엔트 바이오에서 주문한 6주령 C57BL/6J 실험 쥐의 골수에서 수지상 세포를 분리하였다. 각기 다른 종류의 리피드 나노입자를 제작하여, 1×106개 수지상 세포/well의 밀도로 깔아놓은 세포에 10 μg 항원/well의 농도에 맞춰 나노입자를 처리하고, 2일 뒤 200 ng/ml의 LPS를 24시간 처리하였다. 이후 세포 스크레이퍼로 세포들을 긁어모아 성숙한 수지상세포 마커로 알려진 CD40, CD80과 CD86 표현형을 유세포분석기 (Flow cytometry)로 군간 비교분석하고, 염증성 (TNF-α) 및 항 염증성 (TGF-β) 사이토카인 발현 수준을 중합효소연쇄반응 (Polymerase Chain Reaction)으로 군간 비교분석하였다. 그 결과, 라파마이신을 포함한 리피드 나노입자는 라파마이신을 포함하고 있지 않은 나노입자에 비해 효과적으로 미성숙 수지상세포를 유도시켰으며, 이들은 주로 항 염증성 사이토카인을 분비하는 것을 확인하였다 (도 2a1 내지 도 2a3 및 도 2b). 위 실험을 진행하기 앞서 수지상 세포의 나노입자 흡수실험을 진행하였다. 1×106개 수지상 세포/well의 밀도로 깔아놓은 세포와 위 제조예 1에서 제조한 나노입자를 DiI의 형광물질로 표지 하여 공 배양하였다. 24시간 뒤, 수지상 세포를 DAPI로 염색하여 그 결과를 공 초점 현미경으로 분석한 결과, 약 92%의 수지상 세포가 나노입자를 흡수하였다(도 2c1; No treatment의 경우는 왼쪽 피크 및 0.36%에 해당하는 값이며, LNP의 경우 오른쪽 피크 및 0.36%에 해당하는 값이다). 동일한 조건으로 깔아놓은 수지상 세포에 FITC로 표지한 Col II 펩타이드를 담지한 나노입자를 공 배양하여 24시간 뒤 유세포 분석기로 분석한 결과, 약 93%의 수지상 세포가 Col II 펩타이드를 담지한 나노입자를 흡수하였고, 공 초점 현미경으로 확인한 결과, 흡수된 Col II 펩타이드는 수지상 세포의 표면에 나타났다(도 2c2; Free Col II의 경우는 왼쪽 피크 및 2.2 %에 해당하는 값이며, LNP-Col II-R의 경우는 오른쪽 피크 및 93.3 %에 해당하는 값이다, 도 2c3). Col II 펩타이드가 항원으로 작용하여 수지상 세포의 MHC II 분자 위에 존재함 것을 증명하기 위해, 위 제조예 1에서 제조한 나노입자에 Col II 펩타이드를 Eα펩타이드로 치환하여 수지상 세포와 공 배양하였다. 24시간 뒤, Eα펩타이와 MHC II를 동시에 인지하는 항체로 수지상 세포를 염색하여 유세포 분석기와 공 초점 현미경으로 분석한 결과, 약 82%의 수지상 세포가 Eα펩타이드를 담지한 나노입자를 흡수하였고, 이들은 MHC II 분자 위에 해당 항원을 나타냈다 (도 2c4; No treatment의 경우는 가장 왼쪽 피크 및 0.6%에 해당하는 값이며, Free Eα의 경우는 가운데 피크 및 17.1 %에 해당하는 값이며, LNP-Eα-R의 경우는 가장 오른쪽 피크 및 82.0%에 해당하는 값이다, 도 2c5).To confirm the induction of anti-inflammatory cells by the nanoparticles prepared in Preparation Example 1 above, dendritic cells were isolated from the bone marrow of 6-week-old C57BL/6J mice ordered from Orient Bio. Different types of lipid nanoparticles were produced, cells laid out at a density of 1 × 10 6 dendritic cells/well were treated with nanoparticles at a concentration of 10 μg antigen/well, and after 2 days, 200 ng/ml LPS was treated for 24 hours. Afterwards, the cells were scraped with a cell scraper, and the CD40, CD80, and CD86 phenotypes, known as mature dendritic cell markers, were compared and analyzed between groups using flow cytometry, and the expression of inflammatory (TNF-α) and anti-inflammatory (TGF-β) cytokines was analyzed. The levels were compared and analyzed between groups using polymerase chain reaction. As a result, it was confirmed that lipid nanoparticles containing rapamycin induced immature dendritic cells more effectively than nanoparticles not containing rapamycin, and that they mainly secreted anti-inflammatory cytokines (Figures 2a1 to 2a3 and Figure 2b). Before proceeding with the above experiment, a nanoparticle uptake experiment by dendritic cells was conducted. Cells laid out at a density of 1 × 10 6 dendritic cells/well and the nanoparticles prepared in Preparation Example 1 above were labeled with DiI fluorescent substance and co-cultured. 24 hours later, dendritic cells were stained with DAPI and the results were analyzed by confocal microscopy. As a result, approximately 92% of dendritic cells absorbed the nanoparticles (Figure 2c1; for No Treatment, the left peak corresponds to 0.36%. This is the value corresponding to the right peak and 0.36% in the case of LNP). Nanoparticles carrying FITC-labeled Col II peptide were co-cultured with dendritic cells laid out under the same conditions and analyzed by flow cytometry 24 hours later. As a result, approximately 93% of dendritic cells were able to co-culture nanoparticles carrying Col II peptide. It was absorbed, and as confirmed by confocal microscopy, the absorbed Col II peptide appeared on the surface of dendritic cells (Figure 2c2; for free Col II, it is the left peak and the value corresponding to 2.2%, and for LNP-Col II-R The case is the right peak and the value corresponding to 93.3%, Figure 2c3). To prove that Col II peptide acts as an antigen and exists on the MHC II molecule of dendritic cells, Col II peptide was replaced with Eα peptide in the nanoparticle prepared in Preparation Example 1 above and co-cultured with dendritic cells. 24 hours later, dendritic cells were stained with an antibody that simultaneously recognizes Eα peptide and MHC II and analyzed by flow cytometry and confocal microscopy. As a result, approximately 82% of dendritic cells absorbed nanoparticles carrying Eα peptide, They displayed the corresponding antigen on the MHC II molecule (Figure 2c4; for No Treatment, it is the leftmost peak and the value corresponding to 0.6%; for Free Eα, it is the middle peak and the value corresponding to 17.1%; LNP-Eα- For R, it corresponds to the rightmost peak and 82.0%, Figure 2c5).
실시예 3. 지질 나노입자의 Example 3. Lipid Nanoparticles in vivoin vivo 특성 분석 Characterization
만들어진 리피드 나노입자의 체내에서의 분배양상을 확인하기 위해 2 mg/mL의 농도로 분산시킨 형광물질인 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR)을 LNP:DiR=500:1의 비율로 넣어 형광이 표지된 리피드 나노입자를 제작하였다. 10 주령의 C57BL/6 마우스는 오리엔트바이오에서 주문하였고, 케타민(200 mg/kg)과 럼푼(10 mg/kg)으로 마취를 실시한 실험동물의 오른쪽 서혜부 근처 피내에 제작한 형광표지 나노입자를 주사하였다. 주사 24시간 후, 발광- 형광 전임상 분자영상시스템(IVIS spectrum)을 이용하여, 시간에 따른 나노입자의 분배양상을 추적 관찰하였다. 그 결과, 서혜부 근처 오른쪽 림프절 에서만 형광물질이 발견되고, 타 장기 (심장, 폐, 간, 지라, 서혜부 근처 왼쪽 림프절)에서는 형광물질이 발견되지 않은 것을 통해 주사한 나노입자가 인근 림프절로 이동한 것을 확인하였다(도 3). To check the distribution pattern of the created lipid nanoparticles in the body, 1,1'-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a fluorescent substance dispersed at a concentration of 2 mg/mL, was added at a ratio of LNP:DiR=500:1 to obtain fluorescence. These labeled lipid nanoparticles were produced. 10-week-old C57BL/6 mice were ordered from Orient Bio, and the fluorescently labeled nanoparticles were injected into the skin near the right groin of the experimental animals anesthetized with ketamine (200 mg/kg) and lumpun (10 mg/kg). . 24 hours after injection, the distribution pattern of nanoparticles over time was tracked using a luminescence-fluorescence preclinical molecular imaging system (IVIS spectrum). As a result, fluorescent material was found only in the right lymph node near the groin, but not in other organs (heart, lung, liver, spleen, and left lymph node near the groin), indicating that the injected nanoparticles moved to nearby lymph nodes. Confirmed (Figure 3).
실시예 4. 마우스 DMM 수술 및 지질나노입자의 처리에 따른 면역반응 유도 확인Example 4. Confirmation of induction of immune response following mouse DMM surgery and treatment with lipid nanoparticles
골관절염에서 제2형 콜라젠의 자가항원으로서 작용 가능성을 확인하기 위한 동물실험을 수행하였다. C57BL/6 실험동물은 오리엔트바이오에서 주문하였고, 케타민(200 mg/kg)과 럼푼(10 mg/kg)으로 마취를 실시한 실험동물의 오른쪽 다리에 내측반월판 불안정화 수술로 골관절염을 유도하였다. 정밀한 수술을 위해 실체 현미경을 이용하였다. 마취된 실험동물의 오른쪽 다리를 에탄올로 소독한 후, 11번 블레이드로 슬개건 아래쪽을 열고, 지방을 제거하며 시야를 확보하였다. 반투명색의 내측 반월판을 확인한 후, 내측 반월판 대퇴인대를 블레이드로 조심스럽게 잘라주었다. 이 과정에서 주변 연골과 인대 및 뼈 조직에 손상을 가하지 않도록 조심스럽게 수술을 진행하였다. 내측반월판 불안정화 수술이 마무리되면 내부조직과 외부피부조직을 각각 흡수성 봉합사 및 비흡수성 봉합사로 봉합하고, 수술부위에 포비돈 요오드를 발라주어 상처부위를 소독함으로 골관절염 유도를 마무리하였다.An animal experiment was performed to confirm the possibility of type 2 collagen acting as an autoantigen in osteoarthritis. C57BL/6 experimental animals were ordered from Orient Bio, and osteoarthritis was induced by medial meniscus destabilization surgery on the right leg of the experimental animals anesthetized with ketamine (200 mg/kg) and lumpun (10 mg/kg). A stereomicroscope was used for precise surgery. After disinfecting the right leg of the anesthetized experimental animal with ethanol, the lower part of the patellar tendon was opened with a No. 11 blade, fat was removed, and the field of view was secured. After confirming the translucent medial meniscus, the medial meniscus femoral ligament was carefully cut with a blade. During this process, the surgery was performed carefully to avoid damaging the surrounding cartilage, ligaments, and bone tissue. After the medial meniscus destabilization surgery was completed, the internal tissue and external skin tissue were sutured with absorbable and non-absorbable sutures, respectively, and povidone-iodine was applied to the surgical site to disinfect the wound area to complete the induction of osteoarthritis.
골관절염 유도 후, 제2형 콜라젠 펩타이드와 조절 T세포 유도 물질(라파마이신)을 담지한 리피드 나노입자(LNP-Col II-R)를 피내 주사하여 골관절염 치료실험을 진행하였다. 대조군에는 ovalbumin과 라파마이신이 담지된 나노입자(LNP-OVA-R)를 주사하였다. 항원 특이적 면역조절 T 세포 유도확인을 위해서는 LNP-항원-R 나노입자를 마우스에 피내 주사하고, 일정 기간이 지난 후, 분리한 비장세포에 각기 다른 항원을 처리하여 항원에 의한 CD4+CD25+Foxp3+ 조절 T 세포의 자극 및 증식을 군간 비교 분석하였다. 그 결과 제 2형 콜라젠에 대하여 특이적으로 (면역관용 성질의) 조절 T세포가 활성화됨을 확인하였다(도 4a1 및 도 4a2). 류머티즘은 골관절염보다 염증의 정도는 심하지만 병증을 유발하는 메커니즘과 이에 관여하는 세포가 골관절염과 크게 다르지 않다는 연구결과에 따라, 골관절염에서 류머티즘 항원으로 알려져 있는 제2형 콜라젠 259-273에 대한 항원특이적 반응을 추가적으로 분석하였다. 실험방법은 위와 동일하게 진행하였고, 그 결과 골관절염 실험쥐의 T 세포가 제 2형 콜라젠에 비해서는 미미하지만 제 2형 콜라젠 259-273 항원에 대해서 역시 특이적으로 반응하였음을 확인하였다(도 4b).After inducing osteoarthritis, an osteoarthritis treatment experiment was conducted by intradermally injecting lipid nanoparticles (LNP-Col II-R) loaded with type 2 collagen peptide and a regulatory T cell inducer (rapamycin). The control group was injected with ovalbumin and rapamycin-loaded nanoparticles (LNP-OVA-R). To confirm the induction of antigen-specific immunoregulatory T cells, LNP-antigen-R nanoparticles are injected intradermally into mice, and after a certain period of time, isolated spleen cells are treated with different antigens to determine CD4+CD25+Foxp3+ by antigen. Stimulation and proliferation of regulatory T cells were compared and analyzed between groups. As a result, it was confirmed that regulatory T cells (immunotolerant) were specifically activated against type 2 collagen (Figures 4a1 and 4a2). Although rheumatism has a more severe degree of inflammation than osteoarthritis, research has shown that the mechanisms causing the disease and the cells involved are not much different from those of osteoarthritis. Accordingly, antigen-specific antigens for type 2 collagen 259-273, known as rheumatism antigens, are used in osteoarthritis. The reaction was further analyzed. The experimental method was performed in the same manner as above, and as a result, it was confirmed that the T cells of the osteoarthritis mice also reacted specifically to the type 2 collagen 259-273 antigen, although to a lesser extent than to the type 2 collagen (Figure 4b). .
실시예 5. 마우스 DMM 수술 및 지질나노입자의 처리에 따른 관절염 치료 효과 확인Example 5. Confirmation of arthritis treatment effect according to mouse DMM surgery and lipid nanoparticle treatment
제조예 1에서 제조한 리피드 나노입자의 골관절염에서의 치료효과를 확인하기 위해 실험동물에 앞서 실시예 4의 DMM 수술을 수행하였다. 제조한 나노입자는 도 5a에 도시된 실험계획에 따라 오른쪽 서혜부 근처에 피내 주사하였고, 수술 8주 후 실험동물들을 희생하고 오른쪽 관절 부위를 채취하고, 고정 및 탈회과정을 거쳐 파라핀 블록을 제작하였다. 박편절단기를 이용하여 3μm의 두께로 조직을 절단하였고, 다양한 조직염색기법을 이용하여 나노입자의 치료효과를 군간 비교분석하였다. To confirm the therapeutic effect of the lipid nanoparticles prepared in Preparation Example 1 on osteoarthritis, the DMM surgery of Example 4 was performed on experimental animals. The prepared nanoparticles were injected intradermally near the right groin according to the experimental plan shown in Figure 5a, and 8 weeks after surgery, the experimental animals were sacrificed, the right joint area was collected, and a paraffin block was produced through fixation and decalcification. The tissue was cut to a thickness of 3 μm using a thin slice cutter, and the therapeutic effect of nanoparticles was compared and analyzed between groups using various tissue staining techniques.
사프라닌 오 패스트 그린 염색기법으로 채취한 파라핀 블록에 대해 조직학 검사를 진행한 결과, LNP-Col II-R 그룹에서 관절연골조직이 잘 보존되었으나, LNP-OVA-R 그룹을 포함한 다른 그룹에서는 관절연골조직이 파괴됨을 확인하였다 (도 5a, 5b). 또한 실험시작 8주 후, 관절활막의 면역화학염색 분석 결과, LNP-Col II-R 그룹에서 IL-10을 분비하는 regulatory T 세포의 분포와 활성도가 가장 높았고, 활막에 침투한 제1형 대식세포, IFN-γ를 분비하는 CD4+ Th1 세포 및 IL-17을 분비하는 CD4+ Th17세포 등 염증성 면역세포 수가 가장 많이 감소되었음을 확인하였다(도 5c 내지 도 5g). 염증성 세포 및 물질의 감소와 항염증성 세포 및 물질의 증가는 골관절염으로 인한 통증을 감소시켰으며, 이는 이질통증실험 (allodynia test)와 체중부하평가 (incapacitance test)를 통해 확인하였다 (도 5h). 실험종료 8주 후, 균질기로 균질화시킨 관절조직으로 중합효소연쇄반응실험을 진행하였고, LNP-Col II-R 을 주사한 관절조직에서 제 2형 콜라겐 mRNA의 발현이 다른 실험군들에 비해 증가하는 것을 확인하였다 (도 5i). 제조예 1 의 과정을 통해 제조한 나노입자가 제2형 콜라젠 특이적 면역조절(면역관용)을 통하여 관절의 마모를 억제함과 동시에 연골 세포외 기질을 재생함으로써 골관절염에 치료효능이 있음을 확인하였다. 추가적으로 제 2형 콜라겐 259-273의 골관절염에서의 치료효과를 확인하기 위해 제조예 1에서 제조한 리피드 나노입자 (LNP-Col II259-273-R)를 DMM수술을 실시한 실험 쥐에 피내 주사하였다. 대조군으로는 라파마이신만 담지한 리피드 나노입자를 주사하였다. 실험시작 8주 후, 채취한 오른쪽 다리 관절부위를 사프라닌 오 패스트 그린 염색으로 분석한 결과, LNP-Col II259-273-R군의 연골이 LNP-R군보다 잘 보존된 것을 확인하였다 (도 5j1. 도 5j2). 또한 면역화학염색기법을 통해 분석한 결과, DMM 수술을 진행한 군들 중 LNP-Col II259-273-R군에서 F4/80+iNOS+ M1 대식세포가 가장 감소하고 CD4+Foxp3+ Treg세포가 가장 증가한 것을 확인하였다(도 5k, 5l). 제 2형 콜라겐 259-273이 라파마이신과 함께 담지 되었을 때 골관절염에서도 치료효능이 있음을 확인하였다.As a result of histological examination of paraffin blocks collected using the Safranin O Fast Green staining technique, the articular cartilage tissue was well preserved in the LNP-Col II-R group, but the articular cartilage tissue was well preserved in the LNP-OVA-R group and other groups. It was confirmed that the cartilage tissue was destroyed (Figures 5a and 5b). In addition, 8 weeks after the start of the experiment, immunochemical staining analysis of the joint synovium showed that the LNP-Col II-R group had the highest distribution and activity of regulatory T cells secreting IL-10, and type 1 macrophages infiltrated the synovium. , it was confirmed that the number of inflammatory immune cells, such as CD4+ Th1 cells secreting IFN-γ and CD4+ Th17 cells secreting IL-17, was decreased the most (Figures 5c to 5g). A decrease in inflammatory cells and substances and an increase in anti-inflammatory cells and substances reduced pain caused by osteoarthritis, which was confirmed through the allodynia test and incapacitance test (Figure 5h). 8 weeks after the end of the experiment, a polymerase chain reaction experiment was performed on the joint tissue homogenized with a homogenizer, and it was found that the expression of type 2 collagen mRNA increased compared to the other experimental groups in the joint tissue injected with LNP-Col II-R. This was confirmed (Figure 5i). It was confirmed that the nanoparticles prepared through the process of Preparation Example 1 have a therapeutic effect on osteoarthritis by suppressing joint wear through type 2 collagen-specific immunomodulation (immune tolerance) and simultaneously regenerating cartilage extracellular matrix. . Additionally, to confirm the therapeutic effect of type 2 collagen 259-273 on osteoarthritis, the lipid nanoparticles (LNP-Col II259-273-R) prepared in Preparation Example 1 were intradermally injected into experimental rats that had undergone DMM surgery. As a control group, lipid nanoparticles containing only rapamycin were injected. 8 weeks after the start of the experiment, the collected right leg joint area was analyzed with Safranin O Fast Green staining, and it was confirmed that the cartilage of the LNP-Col II259-273-R group was better preserved than the LNP-R group (Figure 5j1. Fig. 5j2). In addition, as a result of analysis using immunochemical staining, it was confirmed that among the groups that underwent DMM surgery, the LNP-Col II259-273-R group showed the greatest decrease in F4/80+iNOS+ M1 macrophages and the greatest increase in CD4+Foxp3+ Treg cells. (Figures 5k, 5l). It was confirmed that type 2 collagen 259-273 had therapeutic efficacy in osteoarthritis when loaded with rapamycin.

Claims (8)

  1. 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 그 유도체; 를 포함하고,type 2 collagen peptide; and rapamycin or its derivatives; Including,
    상기 라파마이신 유도체는 벤조일 라파마이신, 템시로리무스, 에베로리무스, 조타롤리무스, 바이오리무스, 피메크로리무스, 타크로리무스 및 리다포로리무스로 이루어진 군에서 선택되는 골관절염 예방 또는 치료용 약학 조성물. The rapamycin derivative is a pharmaceutical composition for preventing or treating osteoarthritis selected from the group consisting of benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, tacrolimus and ridaforolimus.
  2. 청구항 1에 있어서, 상기 제 2형 콜라겐 펩타이드는 서열번호 1의 아미노산 서열을 포함하는 골관절염 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, wherein the type 2 collagen peptide comprises the amino acid sequence of SEQ ID NO: 1.
  3. 청구항 1에 있어서, 상기 제 2형 콜라겐 펩타이드 및 라파마이신의 중량비는 1 대 0.1 내지 10인 골관절염 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, wherein the weight ratio of the type 2 collagen peptide and rapamycin is 1 to 0.1 to 10.
  4. 청구항 1에 있어서, 수지상세포(dendritic cell, DC)를 더 포함하는 것인 골관절염 예방 또는 치료용 약학 조성물. The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, further comprising dendritic cells (DC).
  5. 청구항 1에 있어서, 상기 제 2형 콜라겐 펩타이드; 및 라파마이신 또는 라파마이신 유도체는 전달체에 담지된 것인 골관절염 예방 또는 치료용 약학 조성물.The method according to claim 1, wherein the type 2 collagen peptide; and a pharmaceutical composition for preventing or treating osteoarthritis, wherein rapamycin or a rapamycin derivative is supported on a carrier.
  6. 청구항 5에 있어서, 상기 전달체는 바이러스 전달체, 바이러스 유사 입자(virus-like particle, VLP), 양전하성 폴리머, 리포좀, 지질나노입자(lipid nanoparticle), 금 및 반도체 나노결정 입자(quantum dot)를 포함하는 군에서 선택되는 것인 골관절염 예방 또는 치료용 약학 조성물.The method of claim 5, wherein the carrier includes a viral carrier, a virus-like particle (VLP), a positively charged polymer, a liposome, a lipid nanoparticle, gold, and a semiconductor nanocrystal particle (quantum dot). A pharmaceutical composition for preventing or treating osteoarthritis selected from the group.
  7. 청구항 5에 있어서, 상기 전달체는 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP), dioleoylphosphatidylethanolamine(DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2000 PE) 및 콜레스테롤을 포함하는 지질나노입자인 골관절염 예방 또는 치료용 약학 조성물.The method of claim 5, wherein the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) -2000] (PEG2000 PE) and a pharmaceutical composition for preventing or treating osteoarthritis, which is a lipid nanoparticle containing cholesterol.
  8. 청구항 6에 있어서, 상기 지질나노입자의 직경은 50 nm 내지 1,000 nm인 골관절염 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating osteoarthritis according to claim 6, wherein the lipid nanoparticles have a diameter of 50 nm to 1,000 nm.
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