WO2024025261A1 - Composition pharmaceutique pour la prévention ou le traitement de l'arthrose - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de l'arthrose 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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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

La présente invention peut fournir une composition pharmaceutique pour la prévention ou le traitement de l'arthrose, la composition comprenant un support contenant un peptide de collagène de type 2 et de la rapamycine. Idéalement, la composition peut réduire les réponses inflammatoires qui se produisent dans les articulations en raison de cellules immunitaires inflammatoires, atténuer la destruction du cartilage et la douleur qui accompagnent les réponses inflammatoires et, en outre, peut régénérer le cartilage. En outre, la composition de la présente invention contient des antigènes associés à d'autres maladies en plus de l'arthrose et peut ainsi être utilisée en tant qu'agent thérapeutique pour des maladies inflammatoires telles que l'infarctus du myocarde, l'infarctus cérébral et les lésions de la moelle épinière, ou des maladies auto-immunes telles que l'atopie, la polyarthrite rhumatoïde, le diabète de type 1, la maladie de Crohn et le lupus.
PCT/KR2023/010545 2022-07-26 2023-07-21 Composition pharmaceutique pour la prévention ou le traitement de l'arthrose WO2024025261A1 (fr)

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KR102490400B1 (ko) * 2022-07-26 2023-01-25 서울대학교산학협력단 골관절염 예방 또는 치료용 약학 조성물

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