WO2016064202A1 - Thermosensitive pluronic-chitosan oligosaccharide-kartogenin nanocomposite with dual drug release function - Google Patents

Thermosensitive pluronic-chitosan oligosaccharide-kartogenin nanocomposite with dual drug release function Download PDF

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WO2016064202A1
WO2016064202A1 PCT/KR2015/011169 KR2015011169W WO2016064202A1 WO 2016064202 A1 WO2016064202 A1 WO 2016064202A1 KR 2015011169 W KR2015011169 W KR 2015011169W WO 2016064202 A1 WO2016064202 A1 WO 2016064202A1
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pluronic
chitosan oligosaccharide
katogenin
nanoparticles
diclofenac
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PCT/KR2015/011169
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French (fr)
Korean (ko)
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임군일
강미란
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동국대학교 산학협력단
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    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

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  • the present invention is a temperature-sensitive dual drug carrier using a pluronic-chitosan oligosaccharide-katogenin nanocomposite, More specifically, nanocomposites in which pluronics, chitosan oligosaccharides, and katogenin are connected by sequential covalent bonds; And a drug enclosed in the complex.
  • cartilage tissue that forms the joints of vertebrates is very difficult to regenerate in vivo once damaged.
  • cartilage tissues of these joints are damaged, they are severely painful and restricted in daily activities, and when they become chronic, they cause fatal degenerative arthritis and interfere with normal life or professional activities. Will be affected.
  • topical injection therapy that injects steroids or hyaluronic acid directly into the joint is widely used.
  • the anti-inflammatory drugs can reduce the symptoms of the cartilage and cause systemic side effects. Can't regenerate damaged cartilage, which is not a fundamental cure. Therefore, if the drug that can induce cartilage regeneration at the site of osteoarthritis is applied to the topical injection treatment, it will be a more effective treatment.
  • intra-articular injection is the most effective treatment method that can directly inject the drug into the damaged joint.
  • the liquid drug injected into the joint has a very short time to stay in the joint due to rapid diffusion, so the concentration of the drug should be increased or the frequency of administration should be increased, which may cause systemic side effects.
  • the drug therapy of osteoarthritis for cartilage regeneration is not only a drug for inhibiting the progression of cartilage damage and regeneration of damaged cartilage, but also a drug for relieving rapid pain in the inflammatory area, and the pharmacological or pharmacokinetic characteristics are different. There is a fear that the frequency of administration further increases.
  • the present invention has been made to solve the above problems, the present inventors and the immediate release of diclofenac by the temperature change in Pluronic-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing Diclofenac (Diclofenac) and The sustained release of catogenin has been confirmed and the present invention has been completed based on this.
  • Diclofenac Diclofenac
  • an object of the present invention is a nanocomposite in which pluronics, chitosan oligosaccharides, and katogenin are sequentially covalently linked; And a drug encapsulated in the complex, to provide a temperature-sensitive dual drug carrier.
  • Another object of the present invention to provide a pharmaceutical composition for preventing or treating bone diseases, comprising the temperature-sensitive dual drug carrier as an active ingredient.
  • the present invention is a nanocomplex in which pluronics, chitosan oligosaccharides, and catogenin are sequentially covalently linked; And a drug encapsulated in the complex, to provide a temperature sensitive dual drug delivery system.
  • the covalent bond may be an amide bond.
  • the enclosed drug may be an anti-inflammatory analgesic.
  • the anti-inflammatory analgesic may be Diclofenac.
  • the present invention provides a pharmaceutical composition for preventing or treating bone disease, comprising the dual drug carrier as an active ingredient.
  • the bone disease may be osteoarthritis.
  • the composition may be an intraarticular injection preparation.
  • the present invention provides a method for treating a bone disease comprising administering the dual drug carrier to a subject.
  • the present invention provides a therapeutic use of a bone disease of a composition comprising a dual drug delivery.
  • the present invention provides novel uses of dual drug carriers for the preparation of therapeutic agents for bone disease.
  • the present invention relates to a temperature-sensitive dual drug carrier using a Pluronic-Chitosan oligosaccharides-katogenin nano complex, wherein the covalent bonds of catogenin and nanoparticles to the complex by changing temperature It was confirmed that the release rate of the anti-inflammatory analgesic drug encapsulated in the present invention can be controlled, and thus, the continuous cartilage regeneration effect and the rapid anti-inflammatory effect have been confirmed. Therefore, it is expected to be useful as a pharmaceutical composition for the prevention or treatment of bone diseases. do.
  • Figure 1 is a schematic diagram showing the chemical structural formula for the synthesis of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex.
  • Figure 3 shows the covalent bonds of catogenin, Pluronic F127, dicarboxylated Pluronic F127, chitosan oligosaccharide and Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with infrared spectrophotometer (FT-IR; Results obtained through Fourier Transform Infrared Spectrophotometer (COS: chitosan oligosaccharide, KGN: catogenin, Dicaboxylated pluronic F127: COOH-F127-COOH, F127 / COS / KGN: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex) Nanoparticles).
  • FT-IR Fourier Transform Infrared Spectrophotometer
  • Figure 4a is the result of confirming the chitosan oligosaccharides in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
  • Figure 4b is the result of confirming the dicarboxylated Pluronic F127 in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles through a Hydrogen Nuclear Magnetic Resonance spectroscopy ( 1 H-NMR) .
  • Figure 4c is the result of confirming the catogenin in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
  • Figure 4d is the result of confirming the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
  • FIG. 5 is a schematic diagram illustrating the preparation of Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac.
  • Figure 7a is a result of confirming the size change of the Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles according to the temperature change (4 °C ⁇ 37 °C) when the mole fraction of catogenin and Pluronic F127 is changed.
  • FIG. 7B shows that when the mole fraction of Pluronic F127 and chitosan oligosaccharide is fixed to 4: 1 and the mole fraction of catogenin is changed, Pluronic F127-chitosan oligosaccharide-katogenin according to the temperature difference (4 ° C., 37 ° C.) This is the result of checking the change in the size of the nanoparticles.
  • FIG. 7C shows that when the mole fraction of catogenin and chitosan oligosaccharide is fixed at 0.1: 1 and the mole fraction of Pluronic F127 is changed, Pluronic F127-chitosan oligosaccharide-katogenin according to the temperature difference (4 ° C., 37 ° C.) This is the result of checking the change in the size of the nanoparticles.
  • FIG. 7D illustrates nanoparticles (4F127) having a molar fraction of 4: 1: 0.1 and 4: 1: 0.1 and nanoparticles (5F127) having a molar fraction of Pluronic F127: chitosan oligosaccharide: canogenin according to temperature change (4 to 37 ° C.). This is the result of checking the size change.
  • FIG. 8B shows the results of dichlorofenac released from Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac by liquid chromatography (HPLC) (3F127 / COS / KGN DCF w /: Pluronic)
  • Pluronic F127: chitosan oligosaccharides: Pluronic F127-chitosan oligosaccharides-katogenin tri-complexed nanoparticles and cold shock treated with DCF consisting of catogenin 3: 1: 0.1, 4F127 / COS /
  • Figure 9a is a result confirming the increased gene expression of Collagenase type II (COL2A1) by Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles in bone marrow mesenchymal stem cells (KGN: liquid catogenin alone Treated group, F127 / COS / KGN DCF w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing DCF, F127 / COS / KGN DCF w / cs: DCF Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles encapsulated and cold shock treated).
  • KGN liquid catogenin alone Treated group
  • F127 / COS / KGN DCF w / o cs group treated with Pluronic F127-chitosan oligosacc
  • Figure 11a is a result confirming the change in cell viability of chondrocytes according to the amount of catogenin released.
  • Figure 11b is a result of measuring the secretion amount of IL-6 by treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac in chondrocytes (LPS: group treated with LPS (lipopolysaccharide) only , LPS + nanoparticles w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing diclofenac after LPS treatment, LPS + nanoparticles w / cs: diclofenac diclofenac after LPS treatment Sealed Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock treated groups).
  • LPS group treated with LPS (lipopolysaccharide) only
  • LPS + nanoparticles w / o cs group treated
  • Figure 11c is the result of measuring the secretion amount of IL-6 by treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles sealed with diclofenac in U937 cells (LPS: group treated with LPS (lipopolysaccharide) only , LPS + nanoparticles w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing diclofenac after LPS treatment, LPS + nanoparticles w / cs: diclofenac diclofenac after LPS treatment Sealed Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock treated groups).
  • LPS group treated with LPS (lipopolysaccharide) only
  • LPS + nanoparticles w / o cs group treated with Plur
  • Figure 13a shows the cartilage protection effect according to treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac-encapsulated red osteoarthritis model, Safranin-O / fast green staining and collagenase type II (COL2), aggrecan It was confirmed through the immunostaining for (vehicle: PBS administration group, KGN + DCF w / cs: Katogenin and Diclofenac and cold shock treatment, F-127 / COS / KGN DCF W / o CS: Diclofenac F-127 / COS / KGN DCF W / CS: Pluronic F127-chitosan oligosaccharide-catozenine tri-treated with this encapsulated Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticle only. -complex nanoparticles and cold shock treated groups).
  • FIG. 13B is a result of evaluating the cartilage protection effect according to the treatment of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac in the red osteoarthritis model by OARSI score (vehicle: PBS group, KGN + DCF) w / cs: Cold shock treated, catogenin and diclofenac, respectively, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac Bay-treated group, F-127 / COS / KGN DCF W / CS: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac and cold shock treated groups).
  • Figure 14a is a result of confirming the reduction of the relative amount of chondrogenic enzyme (COX-2) RNA in serum following treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac in the red osteoarthritis model ( vehicle: PBS administration group, KGN + DCF w / cs: cold shock treatment with catogenin and diclofenac, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide with diclofenac -Group treated with catogenin tri-complex nanoparticles only, F-127 / COS / KGN DCF W / CS: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac and cold shock ).
  • vehicle PBS administration group
  • Figure 14b is a result of confirming the reduction of the relative amount of COX-2 protein in the total protein in the synovial membrane following treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac in the red osteoarthritis model (vehicle : PBS-administered group, KGN + DCF w / cs: group treated with cold shock and catogenin and diclofenac respectively, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide-containing diclofenac- F-127 / COS / KGN DCF W / CS treated with catogenin tri-complex nanoparticles only, Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac and cold shock treated groups) .
  • the present invention provides a nanocomposite in which pluronics, chitosan oligosaccharides, and katogenin are connected by sequential covalent bonds; And a drug encapsulated in the complex, to provide a temperature sensitive dual drug delivery system.
  • the dual drug released to the damaged joint site is a drug encapsulated in a nanocomposite with cattogenin that promotes regeneration of damaged cartilage.
  • cattogenin triggers the activity of mesenchymal stem cells in cartilage. It is a compound that promotes the regeneration of damaged cartilage by allowing chondrocytes to be produced. In experiments with degenerative arthritis model mice, differentiation from mesenchymal stem cells to chondrocytes has been confirmed.
  • the "encapsulated drug in the nanocomposite” may be an anti-inflammatory analgesic, which is a drug used for the purpose of eliminating or alleviating pain, and for the purpose of the present invention, a drug for alleviating pain in a damaged cartilage area. It is referred to, preferably may be Diclofenac (Diclofenac), but is not limited thereto.
  • Diclofenac Diclofenac
  • a hydrophobic drug catogenin is bound to a hydrophilic chitosan oligosaccharide, and the catogenin-chitosan oligosaccharide complex is sequentially bound to pluronic to prepare a nanocomposite, and the catogenin and chitosan oligosaccharide, chitosan oligosaccharide and Pluronic chemical bonds are covalent bonds, preferably amide bonds.
  • a dual drug delivery carrier having not only the regeneration effect of damaged cartilage of the inflammation site, but also the pain relief effect of the inflammation site was prepared.
  • thermosensitive dual drug carrier of the present invention has a rapid analgesic anti-inflammatory effect and continuous cartilage regeneration effect on the site of cartilage damage by temperature controlled drug release.
  • Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles and diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles were prepared (see Examples 1 to 2). The change in the size of the nanoparticles according to the molar fraction of the pluronic and the catogenin and the temperature change (see Example 3).
  • the sustained release and sustained cartilage regeneration effect of catogenin, rapid release and fast anti-inflammatory effect of diclofenac were confirmed. In the case of showing excellent osteoarthritis treatment effect, it was confirmed that it can be usefully used to treat osteoarthritis (see Examples 4 to 7).
  • the present invention provides a pharmaceutical composition for preventing or treating bone disease, comprising the dual drug carrier as an active ingredient.
  • prevention means any action that inhibits or delays the development of a bone disease by administration of a pharmaceutical composition according to the invention.
  • treatment means any action that improves or advantageously alters the condition for bone disease by administration of the pharmaceutical composition according to the present invention.
  • Bone disease which is the object of the prophylaxis or treatment according to the present invention, refers to a condition or a disease that requires or is required to increase bone mass by promoting activity of osteoblasts, and includes a bone loss disease, preferably osteoarthritis. May be, but is not limited thereto.
  • the pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • it may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative and the like.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, preferably by intraarticular injection. .
  • the composition according to the present invention is capable of immediate release of analgesics encapsulated in nanoparticles and sustained release of covalently bound catogenin according to temperature changes, thereby effectively treating bone diseases. This can be
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • the dosage will vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and time of administration, and may be appropriately selected by those skilled in the art, and the "pharmaceutically effective amount" of the present invention is applicable to medical treatment.
  • compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150mg, preferably 0.01 to 100mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day.
  • the dose may be increased or decreased depending on the route of administration, the severity of the bone disease, sex, weight, age and the like, and the above dosage does not limit the scope of the present invention by any method.
  • the Pluronic-chitosan oligosaccharide-katogenin nanocomposite may preferably be administered intra-articularly at a dose of 0.2 mg per kg of body weight two times at three week intervals. It doesn't happen.
  • the present invention provides a method for treating a bone disease comprising administering the pharmaceutical composition to a subject.
  • subject means a subject in need of treatment for a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse and cow, which is human or non-human. .
  • Pluronic F127 In order to induce covalent linkage between Pluronic F127 and chitosan oligosaccharides, dicarboxylated Pluronic (HOOC-Pluronic-COOH) was prepared in which both ends of Pluronic F127 were substituted with carboxyl groups.
  • Pluronic F127 (63 g), succinic acid (1.25 g), 4 dimethylaminopyridine (1.2 g) and triethylamine (1.4 mL) were dissolved in anhydrous dioxane (200 mL) and stirred for 24 hours.
  • dioxane was evaporated in a rotary evaporator, and diethyl ether was added to precipitate the synthesized HOOC-Pluronic-COOH.
  • the diethyl ether was dried in a vacuum oven to remove white powder of HOOC-Pluronic-COOH. Obtained.
  • the chitosan oligosaccharide-katozenin complex and HOOC-Pluronic-COOH were used to induce covalent bonds between chitosan oligosaccharides and pluronic by emulsification / solvent evaporation.
  • Pluronics chitosan oligosaccharides: pluronic 1: 1, 1: 2, 1: 3, 1: 4, 1: 5 of the appropriate molar ratio with respect to chitosan oligosaccharide and EDC were dissolved in dichloromethane and reacted for 30 minutes.
  • the Pluronic F127 reaction solution prepared in the chitosan oligosaccharide-katogenin complex dissolved in a 1% (v / v) acetic acid solvent was dropped in a drop method and reacted with a high frequency to induce emulsification to prepare nanoparticles.
  • the particles were stirred for 24 hours to induce sufficient covalent bonding of chitosan oligosaccharide and pluronic.
  • dichloromethane was evaporated with a rotary evaporator until the reaction solution became clear, and the prepared nanoparticles were recovered by freeze drying.
  • the mole fraction of katogenin to chitosan oligosaccharide used in this example is 1: 0.02, 1: 0.1, 1: 0.5, and the mole fraction of pluronic to chitosan oligosaccharide is 1: 1, 1: 2, 1: 3, 1: 4, 1: 5.
  • Diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles were prepared using the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles prepared in Example 1, and the prepared nanoparticles were It was observed under a microscope.
  • diclofenac (5 mg) and nanoparticles (10 mg) were suspended in deionized water and stirred slowly at 4 ° C. for 24 hours.
  • the reaction solution was heated at 55 ° C. for 10 minutes, centrifuge at 10,000 rpm, and the supernatant was removed.
  • Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac were prepared.
  • a drop of nanoparticles suspended in deionized water was dropped in a stud and dried at room temperature. After the water was completely removed, platinum was coated and observed with FE-SEM to confirm the shape of the nanoparticles.
  • the size change of the nanoparticles was observed according to the temperature change from 4 ° C to 37 ° C, and at this time, the mole fraction of pluronic: chitosan oligosaccharide: catogenin was 4: 1:
  • the phase shift between the nanoparticles (4F127) of 0.1 and the nanoparticles (5F127) of 5: 1: 0.1 the difference in the phase variation of the nanoparticles according to the mole fraction change of the pluronic was observed.
  • lyophilized F127 / COS / KGN DCF nanoparticles (100 mg) were suspended in PBS and reacted at 37 ° C. 100 rpm shaking incubator. At each sampling time, nanoparticles were precipitated at 10,000 rpm centrifugation and the supernatants were collected and replaced with fresh PBS of the same volume. Cold shock was applied by treatment on ice for 10 minutes at each sampling time, and the contents of diclofenac and catogenin were analyzed by HPLC.
  • cattogenin sustained release was confirmed in Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac.
  • the release rate of catogenin was faster. In this case, it was confirmed that the release was less than 60% until 2 weeks.
  • 4F127 / COS / KGN which has a high molar concentration ratio of Pluronic F127, was slow to release cattogenin and fast to release diclofenac compared to 3F127 / COS / KGN.
  • myeloid mesenchymal stem cells cultured in Pellet form were transferred to a lower well of a trans well plate, and diclofenac-encapsulated pluronac capable of secreting 100 nM of catogenin in an upper well coated with a 0.05 ⁇ m pore-size Membrane.
  • F127-chitosanoligosaccharide-katogenin nanoparticles were treated respectively.
  • TRIzol Invitrogen Co.
  • dT Maxime RT preMix kit oligo
  • GPDH Glyceraldehyde-3-phosphatedehydrogenase
  • collagenase type II The expression levels of (COL2A1), aggrecan, and genes were quantified by real-time polymerase chain reaction and compared with those of house-keeping gene GAPDH.
  • the group treated with Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac showed a COL2A1 gene expression rate higher than 2 to 4 times higher than the group treated with liquid catogenin.
  • Aggrecan showed a gene expression rate of 2 to 2.5 times higher.
  • cartilage (1 ⁇ 10 5 cells / well) and U937 (1 ⁇ 10 5 cells / well) cells were cultured in 6-well plates and lipopolysaccharide (LPS) (1 ⁇ g / ml) induced the inflammatory response. Thereafter, each cell was treated with F127 / COS / KGN DCF nanoparticles capable of secreting DCF 100 nM for 2 days. Cultures were collected at different times and quantified by the enzyme-linked immunoabsorbent assay (ELISA).
  • LPS lipopolysaccharide
  • IL-6 secretion of the Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticle treated group containing diclofenac was significantly reduced in both cartilage and U937 cells.
  • IL-6 secretion was more effectively reduced in the cold shock group than in the cold sock group.
  • Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac and cold shock treated group (F-127 / COS / KGN DCF W / CS) significantly inhibited the progression of arthritis lesions. It was confirmed.
  • the PBS-administered group had a large area of cartilage detachment and destruction, while the COL2 and Aggrecan proteins were markedly reduced, while diclofenac-containing Pluronic F127-chitosan oligosaccharide-katogenin
  • the cartilage surface layer was closer to normal, and the increase of COL2 and Aggrecan proteins was confirmed.
  • This effect was significantly different compared to the group administered with catogenin and diclofenac, respectively, and more pronounced in the group treated with diclofenac-containing Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock.
  • cartilage protection effect was the best in the group treated with diclofenac-filled Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock.
  • the cartilage regeneration effect can be promoted through sustained release of cattogenin and encapsulated by low temperature treatment.
  • the rapid release of diflofenac was induced to effectively inhibit the cartilage matrix degrading enzyme and the inflammatory response according to the animal model, which can significantly increase the osteoarthritis treatment effect.

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Abstract

The present invention relates to a thermosensitive dual drug carrier using pluronic-chitosan oligosaccharide-kartogenin nanocomposite. Since the drug carrier of the present invention has an ongoing cartilage regeneration effect and a rapid anti-cancer effect by controlling the release rate of analgesic drug encapsulated in a composite particle with karogenin, the drug carrier is expected to be capable of preventing or treating bone diseases more effectively.

Description

이중 약물 방출 기능을 가진 온도감응성 플루로닉-키토산올리고당-카토제닌 나노 복합체Temperature-sensitive Pluronic-chitosan oligosaccharide-katogenin nanocomposites with dual drug release
본 발명은 플루로닉-키토산올리고당-카토제닌 나노 복합체를 이용한 온도감응성 이중 약물 전달체로서, 보다 구체적으로는 플루로닉(pluronics), 키토산올리고당(Chitosan oligosaccharides), 및 카토제닌(Kartogenin)이 순차적인 공유 결합으로 연결된 나노 복합체; 및 상기 복합체 내에 봉입된 약물을 포함하는, 이중 약물 전달체에 관한 것이다. The present invention is a temperature-sensitive dual drug carrier using a pluronic-chitosan oligosaccharide-katogenin nanocomposite, More specifically, nanocomposites in which pluronics, chitosan oligosaccharides, and katogenin are connected by sequential covalent bonds; And a drug enclosed in the complex.
일반적으로 척추 동물의 관절을 이루는 연골 조직은 한번 손상되면 정상적으로 생체 내에서 재생이 매우 어렵다. 이러한 관절의 연골 조직이 손상될 경우, 심한 통증과 함께 일상 활동에 제한을 받게 되며, 만성화될 경우, 치명적인 퇴행성 관절염 등이 유발되어 정상적인 생활이나 직업적인 활동에 방해가 되는바, 현대인의 생활에 큰 영향을 미치게 된다.In general, the cartilage tissue that forms the joints of vertebrates is very difficult to regenerate in vivo once damaged. When the cartilage tissues of these joints are damaged, they are severely painful and restricted in daily activities, and when they become chronic, they cause fatal degenerative arthritis and interfere with normal life or professional activities. Will be affected.
이러한 연골손상은 관절 연골조직의 외상성 결손이나 점진적인 파괴를 가져오는 골관절염에 의하여 높은 빈도로 발생하고 있다. 따라서, 골관절염 치료방법에 대한 연구가 진행 중에 있으며 연골성형술(chondroplasty), 골연골 이식술(osteochondral transplantation), 자가유래 연골세포 이식술(autologous chondrocyte transplantation) 등이 시행되고 있으나, 60대 이하의 비교적 젊은 환자에 시술하는 경우는 인공관절의 수명이 문제가 된다.Such cartilage damage is frequently caused by osteoarthritis, which causes traumatic defects or gradual destruction of joint cartilage tissue. Therefore, research on the treatment of osteoarthritis is ongoing, and chondroplasty, osteochondral transplantation, autologous chondrocyte transplantation, etc. are being performed. In the case of the procedure, the life of the artificial joint is a problem.
또한, 스테로이드제제 또는 히알루론산을 관절에 직접 주입하는 국소주사치료법이 널리 이용되고 있으나, 이러한 소염 진통제류의 약물은 증상을 완화시키는 효과가 있는 반면, 연골의 약화와 전신적인 부작용을 초래할 수 있으며, 손상된 연골을 재생할 수 없어서 근본적인 치료법이 되지 못한다. 따라서, 골관절염 부위에서 연골 재생을 유도할 수 있는 약물을 국소 주사 치료에 적용한다면 더욱 효과가 높은 치료법이 될 것이다. In addition, topical injection therapy that injects steroids or hyaluronic acid directly into the joint is widely used. However, the anti-inflammatory drugs can reduce the symptoms of the cartilage and cause systemic side effects. Can't regenerate damaged cartilage, which is not a fundamental cure. Therefore, if the drug that can induce cartilage regeneration at the site of osteoarthritis is applied to the topical injection treatment, it will be a more effective treatment.
한편, 골관절염의 치료를 위한 약물의 투여방법 측면에서, 관절 내 주사법은 손상된 관절에 직접 약물을 주입할 수 있어 기대 효과가 가장 높은 치료법이다. 하지만 관절 내로 주입된 액체 상태의 약물은 빠른 확산으로 관절 내에서 머무를 수 있는 시간이 매우 짧아 약물의 농도를 높이거나 투여 횟수를 증가시켜야만 하는데, 이 경우 오히려 전신 부작용을 초래할 수 있다. 또한, 연골 재생을 위한 골관절염의 약물치료는 단순한 연골 손상의 진행 억제 및 손상된 연골의 재생을 위한 약물뿐만 아니라, 염증부위의 빠른 통증 완화를 위한 약물도 병행하여 투여하고 있으며, 약리 또는 약동학적 특성 차이에 의해 투여 횟수가 더욱 증가하게 될 우려가 있다. On the other hand, in terms of the method of administering the drug for the treatment of osteoarthritis, intra-articular injection is the most effective treatment method that can directly inject the drug into the damaged joint. However, the liquid drug injected into the joint has a very short time to stay in the joint due to rapid diffusion, so the concentration of the drug should be increased or the frequency of administration should be increased, which may cause systemic side effects. In addition, the drug therapy of osteoarthritis for cartilage regeneration is not only a drug for inhibiting the progression of cartilage damage and regeneration of damaged cartilage, but also a drug for relieving rapid pain in the inflammatory area, and the pharmacological or pharmacokinetic characteristics are different. There is a fear that the frequency of administration further increases.
따라서, 이중 약물 전달체를 이용한 치료가 주요한 과제의 대상이 되고 있고, 이에 대한 연구가 이루어지고 있으나(한국 특허공개번호 10-2006-0064048), 아직은 미비한 실정이다. Therefore, treatment with dual drug carriers has been the subject of major challenges, and research on this has been made (Korean Patent Publication No. 10-2006-0064048), which is still inadequate.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 디클로페낙(Diclofenac)이 봉입된 플루로닉-키토산올리고당-카토제닌 tri-complex 나노 입자에서 온도 변화에 의한 디클로페낙의 속방형 방출과 카토제닌의 서방형 방출을 확인하고 이에 기초하여 본 발명을 완성하게 되었다.The present invention has been made to solve the above problems, the present inventors and the immediate release of diclofenac by the temperature change in Pluronic-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing Diclofenac (Diclofenac) and The sustained release of catogenin has been confirmed and the present invention has been completed based on this.
이에, 본 발명의 목적은 플루로닉(pluronics), 키토산올리고당(Chitosan oligosaccharides), 및 카토제닌(Kartogenin)이 순차적으로 공유 결합으로 연결된 나노 복합체; 및 상기 복합체 내에 봉입된 약물을 포함하는, 온도감응성 이중 약물 전달체를 제공하는 것이다.Accordingly, an object of the present invention is a nanocomposite in which pluronics, chitosan oligosaccharides, and katogenin are sequentially covalently linked; And a drug encapsulated in the complex, to provide a temperature-sensitive dual drug carrier.
또한, 본 발명의 다른 목적은 상기 온도감응성 이중 약물 전달체를 유효성분으로 포함하는, 골 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, another object of the present invention to provide a pharmaceutical composition for preventing or treating bone diseases, comprising the temperature-sensitive dual drug carrier as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 플루로닉(pluronics), 키토산올리고당(Chitosan oligosaccharides), 및 카토제닌(Kartogenin)이 순차적으로 공유 결합으로 연결된 나노 복합체; 및 상기 복합체 내에 봉입된 약물을 포함하는, 온도감응성 이중 약물 전달체를 제공한다.In order to achieve the object of the present invention as described above, the present invention is a nanocomplex in which pluronics, chitosan oligosaccharides, and catogenin are sequentially covalently linked; And a drug encapsulated in the complex, to provide a temperature sensitive dual drug delivery system.
본 발명의 일 구현예로서, 상기 공유 결합은 아마이드 결합(amide bond)일 수 있다. In one embodiment of the present invention, the covalent bond may be an amide bond.
본 발명의 다른 구현예로서, 상기 봉입된 약물은 소염 진통제일 수 있다. In another embodiment of the present invention, the enclosed drug may be an anti-inflammatory analgesic.
본 발명의 또 다른 구현예로서, 상기 소염 진통제는 디클로페낙(Diclofenac)일 수 있다.In another embodiment of the present invention, the anti-inflammatory analgesic may be Diclofenac.
본 발명은 상기 이중 약물 전달체를 유효성분으로 포함하는, 골 질환 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating bone disease, comprising the dual drug carrier as an active ingredient.
본 발명의 일 구현예로서, 상기 골질환은 골관절염(osteoarthritis)일 수 있다. In one embodiment of the present invention, the bone disease may be osteoarthritis.
본 발명의 다른 구현예로서, 상기 조성물은 관절 내 주사제 제제일 수 있다. In another embodiment of the invention, the composition may be an intraarticular injection preparation.
본 발명은 상기 이중 약물 전달체를 개체에 투여하는 단계를 포함하는 골 질환의 치료방법을 제공한다.The present invention provides a method for treating a bone disease comprising administering the dual drug carrier to a subject.
본 발명은 이중 약물 전달체를 포함하는 조성물의 골 질환의 치료용도를 제공한다.The present invention provides a therapeutic use of a bone disease of a composition comprising a dual drug delivery.
본 발명은 골 질환 치료제 제조를 위한 이중 약물 전달체의 신규한 용도를 제공한다. The present invention provides novel uses of dual drug carriers for the preparation of therapeutic agents for bone disease.
본 발명은 플루로닉-키토산올리고당-카토제닌 나노 복합체 (Pluronic-Chitosan oligosaccharides-katogenin nano complex)를 이용한 온도감응성 이중 약물 전달체에 관한 것으로, 온도를 변화시킴으로써 상기 복합체에 공유 결합된 카토제닌과 나노 입자에 봉입된 소염 진통제 약물의 방출 속도를 조절할 수 있음을 확인하였고, 이에 따른 지속적인 연골 재생 효과 및 빠른 항염 효과를 확인하였는바, 골 질환의 예방 또는 치료를 위한 약학 조성물로 유용하게 사용될 수 있을 것으로 기대된다.The present invention relates to a temperature-sensitive dual drug carrier using a Pluronic-Chitosan oligosaccharides-katogenin nano complex, wherein the covalent bonds of catogenin and nanoparticles to the complex by changing temperature It was confirmed that the release rate of the anti-inflammatory analgesic drug encapsulated in the present invention can be controlled, and thus, the continuous cartilage regeneration effect and the rapid anti-inflammatory effect have been confirmed. Therefore, it is expected to be useful as a pharmaceutical composition for the prevention or treatment of bone diseases. do.
도 1은 플루로닉 F127-키토산올리고당-카토제닌 tri-complex의 합성 반응에 대한 화학구조식을 나타낸 모식도이다.Figure 1 is a schematic diagram showing the chemical structural formula for the synthesis of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex.
도 2는 카토제닌-키토산올리고당 복합체를 적외선분광광도계(FT-IR; Fourier Transform Infrared spectrophotometer)를 통하여 확인한 결과이다. 2 is a result of confirming the catogenin-chitosan oligosaccharide complex by Fourier Transform Infrared spectrophotometer (FT-IR).
도 3은 카토제닌, 플루로닉 F127, 다이카복실레이티드 플루로닉 F127, 키토산올리고당 및 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자의 공유 결합을 적외선분광광도계(FT-IR; Fourier Transform Infrared spectrophotometer)를 통하여 확인한 결과이다(COS : 키토산올리고당, KGN : 카토제닌, Dicaboxylated pluronic F127 : COOH-F127-COOH, F127/COS/KGN : 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자). Figure 3 shows the covalent bonds of catogenin, Pluronic F127, dicarboxylated Pluronic F127, chitosan oligosaccharide and Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with infrared spectrophotometer (FT-IR; Results obtained through Fourier Transform Infrared Spectrophotometer (COS: chitosan oligosaccharide, KGN: catogenin, Dicaboxylated pluronic F127: COOH-F127-COOH, F127 / COS / KGN: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex) Nanoparticles).
도 4a는 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 내 키토산올리고당을 수소핵자기공명분광기(1H-NMR; Proton Nuclear Magnetic Resonance spectroscopy)를 통하여 확인한 결과이다.Figure 4a is the result of confirming the chitosan oligosaccharides in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
도 4b는 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 내 다이카복실레이티드 플루로닉 F127을 수소핵자기공명분광기(1H-NMR; Proton Nuclear Magnetic Resonance spectroscopy)를 통하여 확인한 결과이다.Figure 4b is the result of confirming the dicarboxylated Pluronic F127 in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles through a Hydrogen Nuclear Magnetic Resonance spectroscopy ( 1 H-NMR) .
도 4c는 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 내 카토제닌을 수소핵자기공명분광기(1H-NMR; Proton Nuclear Magnetic Resonance spectroscopy)를 통하여 확인한 결과이다.Figure 4c is the result of confirming the catogenin in the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
도 4d는 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자를 수소핵자기공명분광기(1H-NMR; Proton Nuclear Magnetic Resonance spectroscopy)를 통하여 확인한 결과이다.Figure 4d is the result of confirming the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR).
도 5는 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 제조과정 모식도이다. FIG. 5 is a schematic diagram illustrating the preparation of Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac. FIG.
도 6은 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 형태를 전자 현미경으로 관찰한 결과이다. 6 shows the results of observing the morphology of Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac under an electron microscope.
도 7a는 카토제닌과 플루로닉 F127의 몰분율을 변화시킨 경우, 온도 변화(4℃~37℃)에 따른 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 크기 변화를 확인한 결과이다. Figure 7a is a result of confirming the size change of the Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles according to the temperature change (4 ℃ ~ 37 ℃) when the mole fraction of catogenin and Pluronic F127 is changed.
도 7b는 플루로닉 F127과 키토산 올리고당의 몰분율을 4:1로 고정시키고, 카토제닌의 몰분율을 변화시킨 경우, 온도 차이(4℃, 37℃)에 따른 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 크기 변화를 확인한 결과이다. FIG. 7B shows that when the mole fraction of Pluronic F127 and chitosan oligosaccharide is fixed to 4: 1 and the mole fraction of catogenin is changed, Pluronic F127-chitosan oligosaccharide-katogenin according to the temperature difference (4 ° C., 37 ° C.) This is the result of checking the change in the size of the nanoparticles.
도 7c는 카토제닌과과 키토산 올리고당의 몰분율을 0.1:1로 고정시키고, 플루로닉 F127의 몰분율을 변화시킨 경우, 온도 차이(4℃, 37℃)에 따른 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 크기 변화를 확인한 결과이다. FIG. 7C shows that when the mole fraction of catogenin and chitosan oligosaccharide is fixed at 0.1: 1 and the mole fraction of Pluronic F127 is changed, Pluronic F127-chitosan oligosaccharide-katogenin according to the temperature difference (4 ° C., 37 ° C.) This is the result of checking the change in the size of the nanoparticles.
도 7d는 온도 변화(4~37℃)에 따른 플루로닉 F127: 키토산 올리고당 : 카노제닌의 몰분율이 4:1:0.1인 나노 입자(4F127)와 5:1:0.1인 나노 입자(5F127)의 크기 변화를 확인한 결과이다. FIG. 7D illustrates nanoparticles (4F127) having a molar fraction of 4: 1: 0.1 and 4: 1: 0.1 and nanoparticles (5F127) having a molar fraction of Pluronic F127: chitosan oligosaccharide: canogenin according to temperature change (4 to 37 ° C.). This is the result of checking the size change.
도 8a는 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자로부터 방출된 카토제닌의 함량을 액체크로마토그래피 (HPLC)로 분석한 결과이다(3F127/COS/KGNDCF w/o cs: 플루로닉 F127:키토산올리고당:카토제닌=3:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자만 처리한 군, 3F127/COS/KGNDCF w/ cs : 플루로닉 F127:키토산올리고당:카토제닌=3:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자 및 cold shock 처리한 군, 4F127/COS/KGNDCF w/o cs: 플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자만 처리한 군, 4F127/COS/KGNDCF w/ cs : 플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자 및 cold shock 처리한 군). Figure 8a is the result of analyzing the content of catogenin released from Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles packed with diclofenac (HPLC) (3F127 / COS / KGN DCF w / o cs: Pluronic F127: chitosan oligosaccharide: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles containing DCF consisting of catogenin = 3: 1: 0.1, 3F127 / COS / KGN DCF w / cs: Pluronic F127: chitosan oligosaccharide: catogenin = 3: 1: 0.1 Pluronic F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles and cold shock treated group, 4F127 / COS / KGN DCF w / o cs: Pluronic F127: Chitosan oligosaccharide: Catogenin = 4: 1: 0.1, treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles encapsulated, 4F127 Pluronic F127:: / COS / KGN DCF w / cs in the DCF is configured to 0.1 encapsulated flu: chitosan oligosaccharide: Kato Janine = 4: 1 F127- chitosan oligosaccharide - Kato Janine tri-complexed nanoparticles and cold shock-treated group).
도 8b는 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자로부터 방출된 디클로페낙의 함량을 액체크로마토그래피 (HPLC)로 분석한 결과이다(3F127/COS/KGNDCF w/: 플루로닉 F127:키토산올리고당:카토제닌=3:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자만 처리한 군, 3F127/COS/KGNDCF w/o cs : 플루로닉 F127:키토산올리고당:카토제닌=3:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자 및 cold shock 처리한 군, 4F127/COS/KGNDCF w/ : 플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자만 처리한 군, 4F127/COS/KGNDCF w/o cs : 플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1로 구성된 DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자 및 cold shock 처리한 군). FIG. 8B shows the results of dichlorofenac released from Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac by liquid chromatography (HPLC) (3F127 / COS / KGN DCF w /: Pluronic) F127: Chitosan oligosaccharide: A group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles containing DCF consisting of catogenin = 3: 1: 0.1, 3F127 / COS / KGN DCF w / o cs: Pluronic F127: chitosan oligosaccharides: Pluronic F127-chitosan oligosaccharides-katogenin tri-complexed nanoparticles and cold shock treated with DCF consisting of catogenin = 3: 1: 0.1, 4F127 / COS / KGN DCF w /: Pluronic F127: chitosan oligosaccharides: Katogenin = 4: 1: 0.1 group treated with Pluronic F127-chitosan oligosaccharides-katogenin tri-complexed nanoparticles only, 4F127 / COS / KGN DCF w / o cs: Pluronic F127: Chitosan oligosaccharide: Catogenin = Pluronic sealed with DCF consisting of 4: 1: 0.1 F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles and cold shock treated group).
도 9a는 골수간엽줄기세포에서 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자에 의한 Collagenase type II (COL2A1)의 유전자 발현 증가를 확인한 결과이다(KGN : 액체상태의 카토제닌을 단독으로 처리한 군, F127/COS/KGNDCF w/o cs : DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만 처리한 군, F127/COS/KGNDCF w/ cs : DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). Figure 9a is a result confirming the increased gene expression of Collagenase type II (COL2A1) by Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles in bone marrow mesenchymal stem cells (KGN: liquid catogenin alone Treated group, F127 / COS / KGN DCF w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing DCF, F127 / COS / KGN DCF w / cs: DCF Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles encapsulated and cold shock treated).
도 9b는 골수간엽줄기세포에서 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자에 의한 Aggrecan의 유전자 발현 증가를 확인한 결과이다(KGN : 액체상태의 카토제닌을 단독으로 처리한 군, F127/COS/KGNDCF w/o cs : DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만 처리한 군, F127/COS/KGNDCF w/ cs : DCF가 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). 9b is a result confirming the gene expression of Aggrecan by Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles in bone marrow mesenchymal stem cells (KGN: group treated with liquid catogenin alone, F127 / COS / KGN DCF w / o cs: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles treated with DCF only, F127 / COS / KGN DCF w / cs: Fluated with DCF Nick F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock treated groups).
도 10은 골수간엽줄기세포에서 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자에 의한 Collagenase type II (COL2A1)과 Aggrecan의 단백질 발현 증가를 확인한 결과이다. 10 is a result confirming the increased protein expression of Collagenase type II (COL2A1) and Aggrecan by Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles in bone marrow mesenchymal stem cells.
도 11a는 방출되는 카토제닌 양에 따른 연골세포의 세포 생존율 변화를 확인한 결과이다.Figure 11a is a result confirming the change in cell viability of chondrocytes according to the amount of catogenin released.
도 11b는 연골세포에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 의한 IL-6의 분비량을 측정한 결과이다(LPS: LPS(lipopolysaccharide)만을 처리한 군, LPS+nanoparticles w/o cs: LPS 처리 후 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, LPS+nanoparticles w/ cs: LPS 처리 후 디클로페낙이 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군).Figure 11b is a result of measuring the secretion amount of IL-6 by treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac in chondrocytes (LPS: group treated with LPS (lipopolysaccharide) only , LPS + nanoparticles w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing diclofenac after LPS treatment, LPS + nanoparticles w / cs: diclofenac diclofenac after LPS treatment Sealed Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock treated groups).
도 11c는 U937 세포에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 의한 IL-6의 분비량을 측정한 결과이다(LPS: LPS(lipopolysaccharide)만을 처리한 군, LPS+nanoparticles w/o cs: LPS 처리 후 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, LPS+nanoparticles w/ cs: LPS 처리 후 디클로페낙이 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군).Figure 11c is the result of measuring the secretion amount of IL-6 by treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles sealed with diclofenac in U937 cells (LPS: group treated with LPS (lipopolysaccharide) only , LPS + nanoparticles w / o cs: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles only containing diclofenac after LPS treatment, LPS + nanoparticles w / cs: diclofenac diclofenac after LPS treatment Sealed Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock treated groups).
도 12는 렛드 골관절염 모델에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 따른 경골하부의 손상 억제 효과를 micro-CT를 통하여 확인한 결과이다(vehicle: PBS 투여군, KGN+DCF w/cs: 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군, F-127/COS/KGNDCF W/o CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, F-127/COS/KGNDCF W/ CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군).12 is a result of confirming the effect of inhibiting damage to the tibia in the lower tibia according to the treatment of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac in a red osteoarthritis model (vehicle: PBS-administered group) , KGN + DCF w / cs: group treated with cold shock and catogenin and diclofenac, respectively, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide-katogenin tri encapsulated with diclofenac group treated with -complex nanoparticles only, F-127 / COS / KGN DCF W / CS: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock with diclofenac).
도 13a는 렛드 골관절염 모델에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 따른 연골 보호 효과를 Safranin-O/fast green 염색 및 collagenase type II (COL2), aggrecan에 대한 면역 염색을 통하여 확인한 결과이다(vehicle: PBS 투여군, KGN+DCF w/cs: 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군, F-127/COS/KGNDCF W/o CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, F-127/COS/KGNDCF W/ CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). Figure 13a shows the cartilage protection effect according to treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac-encapsulated red osteoarthritis model, Safranin-O / fast green staining and collagenase type II (COL2), aggrecan It was confirmed through the immunostaining for (vehicle: PBS administration group, KGN + DCF w / cs: Katogenin and Diclofenac and cold shock treatment, F-127 / COS / KGN DCF W / o CS: Diclofenac F-127 / COS / KGN DCF W / CS: Pluronic F127-chitosan oligosaccharide-catozenine tri-treated with this encapsulated Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticle only. -complex nanoparticles and cold shock treated groups).
도 13b는 렛드 골관절염 모델에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 따른 연골 보호효과를 OARSI score로 평가한 결과이다(vehicle: PBS 투여군, KGN+DCF w/cs: 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군, F-127/COS/KGNDCF W/o CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, F-127/COS/KGNDCF W/ CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). 13B is a result of evaluating the cartilage protection effect according to the treatment of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac in the red osteoarthritis model by OARSI score (vehicle: PBS group, KGN + DCF) w / cs: Cold shock treated, catogenin and diclofenac, respectively, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac Bay-treated group, F-127 / COS / KGN DCF W / CS: Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac and cold shock treated groups).
도 14a는 렛드 골관절염 모델에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 따른 혈청 내 상대적인 연골파괴효소(COX-2) RNA 양의 감소를 확인한 결과이다(vehicle: PBS 투여군, KGN+DCF w/cs: 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군, F-127/COS/KGNDCF W/o CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, F-127/COS/KGNDCF W/ CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). Figure 14a is a result of confirming the reduction of the relative amount of chondrogenic enzyme (COX-2) RNA in serum following treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac in the red osteoarthritis model ( vehicle: PBS administration group, KGN + DCF w / cs: cold shock treatment with catogenin and diclofenac, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide with diclofenac -Group treated with catogenin tri-complex nanoparticles only, F-127 / COS / KGN DCF W / CS: group treated with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac and cold shock ).
도 14b는 렛드 골관절염 모델에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 처리에 따른 synovial membrane 내 전체 단백질 중 상대적인 COX-2 단백질 양의 감소를 확인한 결과이다(vehicle: PBS 투여군, KGN+DCF w/cs: 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군, F-127/COS/KGNDCF W/o CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군, F-127/COS/KGNDCF W/ CS: 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군). Figure 14b is a result of confirming the reduction of the relative amount of COX-2 protein in the total protein in the synovial membrane following treatment with Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles packed with diclofenac in the red osteoarthritis model (vehicle : PBS-administered group, KGN + DCF w / cs: group treated with cold shock and catogenin and diclofenac respectively, F-127 / COS / KGN DCF W / o CS: Pluronic F127-chitosan oligosaccharide-containing diclofenac- F-127 / COS / KGN DCF W / CS treated with catogenin tri-complex nanoparticles only, Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac and cold shock treated groups) .
본 발명자들은 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 복합체 (Pluronic-Chitosan oligosaccharides-kartogenin nano complex)에서, 카토제닌 또는 플루로닉 F127-COOH의 몰분율, 및 온도 변화에 따른 나노 입자의 크기 변화를 확인하였다, 또한, Cold shock 처리를 한 경우, 디클로페낙의 속방형 방출 및 빠른 항염 효과와 공유 결합된 카토제닌의 서방형 방출 및 지속적인 연골 분화 효과를 확인하고, 이에 기초하여 본 발명을 완성하였다. In the Pluronic-Chitosan oligosaccharides-kartogenin nanocomposite containing diclofenac-filled Pluronic-Chitosan oligosaccharides-kartogenin nanocomposite, the present inventors have suggested that the mole fraction of catogenin or Pluronic F127-COOH, and nanoparticles according to temperature change. In addition, in the case of cold shock treatment, the sustained release of diclofenac and the sustained release of cattogenin covalently coupled with the fast anti-inflammatory effect and the sustained cartilage differentiation effect were confirmed. Completed.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 플루로닉(pluronics), 키토산올리고당(Chitosan oligosaccharides), 및 카토제닌(Kartogenin)이 순차적인 공유 결합으로 연결된 나노 복합체; 및 상기 복합체 내에 봉입된 약물을 포함하는, 온도감응성 이중 약물 전달체를 제공한다. The present invention provides a nanocomposite in which pluronics, chitosan oligosaccharides, and katogenin are connected by sequential covalent bonds; And a drug encapsulated in the complex, to provide a temperature sensitive dual drug delivery system.
본 발명에서 손상된 관절 부위로 방출되는 이중 약물은 손상된 연골의 재생을 촉진시키는 카토제닌과 나노 복합체 내 봉입된 약물로서, 본 발명에서, "카토제닌"은 연골에 있는 중간엽 줄기세포의 활동을 촉발시켜 연골세포가 생성되게 함으로써 손상된 연골의 재생을 촉진시키는 화합물이며, 퇴행성 관절염 모델 쥐를 이용한 실험에서, 중간엽 줄기세포로부터 연골세포로의 분화를 확인한바 있다. 또한, 본 발명에서, "나노 복합체 내 봉입된 약물"은 소염 진통제일 수 있으며, 이는 동통을 제거하거나 경감시키는 것을 목적으로 사용하는 약물로서, 본 발명의 목적상 손상된 연골 부위의 동통을 완화시키는 약물을 일컬으며, 바람직하게는 디클로페낙(Diclofenac)일 수 있으나, 이로 제한되는 것은 아니다. In the present invention, the dual drug released to the damaged joint site is a drug encapsulated in a nanocomposite with cattogenin that promotes regeneration of damaged cartilage. In the present invention, "katogenin" triggers the activity of mesenchymal stem cells in cartilage. It is a compound that promotes the regeneration of damaged cartilage by allowing chondrocytes to be produced. In experiments with degenerative arthritis model mice, differentiation from mesenchymal stem cells to chondrocytes has been confirmed. In addition, in the present invention, the "encapsulated drug in the nanocomposite" may be an anti-inflammatory analgesic, which is a drug used for the purpose of eliminating or alleviating pain, and for the purpose of the present invention, a drug for alleviating pain in a damaged cartilage area. It is referred to, preferably may be Diclofenac (Diclofenac), but is not limited thereto.
본 발명에서는 소수성 약물인 카토제닌을 친수성인 키토산 올리고당에 결합시키고, 상기 카토제닌-키토산 올리고당 복합체를 순차적으로 플루로닉에 결합시킴으로써, 나노 복합체를 제조하였으며, 상기 카토제닌과 키토산 올리고당, 키토산 올리고당과 플루로닉간 화학결합은 공유 결합으로서, 바람직하게는 아마이드 결합일 수 있다. 또한, 상기 나노 복합체 내에 소염 진통제를 봉입함으로써, 염증부위의 손상된 연골의 재생 효과뿐만 아니라, 염증 부위의 통증 완화 효과 역시 가지는 이중 약물 전달체를 제조하였다. In the present invention, a hydrophobic drug catogenin is bound to a hydrophilic chitosan oligosaccharide, and the catogenin-chitosan oligosaccharide complex is sequentially bound to pluronic to prepare a nanocomposite, and the catogenin and chitosan oligosaccharide, chitosan oligosaccharide and Pluronic chemical bonds are covalent bonds, preferably amide bonds. In addition, by enclosing the anti-inflammatory analgesic agent in the nanocomposite, a dual drug delivery carrier having not only the regeneration effect of damaged cartilage of the inflammation site, but also the pain relief effect of the inflammation site was prepared.
또한, 본 발명에서, "온도감응성"은 온도에 따라 약물 방출 거동을 조절할 수 있는 능력으로서, 골관절염 환자의 염증 완화를 위한 냉온찜질 요법에 착안하여, 온도변화에 따라 약물 방출을 조절할 수 있도록 온도감응성 폴리머인 플루로닉을 상기 약물 전달체 제조에 적용하여, 복합체 내에 봉입된 진통제의 속방형 방출 및 공유 결합된 카토제닌의 서방형 방출을 유도하였다. 따라서, 본 발명의 온도감응성 이중 약물 전달체는 온도조절에 의한 약물 방출로 연골 손상 부위에 대해 빠른 진통 소염 효과와 지속적인 연골 재생 효과를 갖는다. In addition, in the present invention, "temperature sensitivity" is the ability to adjust the drug release behavior according to the temperature, focusing on cold and cold therapy for the relief of inflammation in osteoarthritis patients, temperature sensitivity to adjust the drug release according to the temperature change Pluronic, a polymer, was applied to the preparation of the drug carrier to induce immediate release of analgesics encapsulated within the complex and sustained release of covalently bound cattogenin. Therefore, the thermosensitive dual drug carrier of the present invention has a rapid analgesic anti-inflammatory effect and continuous cartilage regeneration effect on the site of cartilage damage by temperature controlled drug release.
본 발명의 일 실시예에서는 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자 및 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자를 제조하여(실시예 1 내지 2 참조), 플루로닉, 카토제닌의 몰분율 및 온도 변화에 따른 나노 입자의 크기 변화를 확인하였다(실시예 3 참조). 또한, 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서 cold shock를 준 경우, 카토제닌의 서방출 및 지속적인 연골 재생 효과, 디클로페낙의 속방출 및 빠른 항염효과를 확인하였으며, 이를 동물모델에 적용한 경우에도 우수한 골관절염 치료 효과를 나타내었는 바, 골 관절염 치료에 유용하게 이용될 수 있음을 확인하였다(실시예 4 내지 7 참조). In one embodiment of the present invention, Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles and diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles were prepared (see Examples 1 to 2). The change in the size of the nanoparticles according to the molar fraction of the pluronic and the catogenin and the temperature change (see Example 3). In addition, when cold shock was given in the Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles, the sustained release and sustained cartilage regeneration effect of catogenin, rapid release and fast anti-inflammatory effect of diclofenac were confirmed. In the case of showing excellent osteoarthritis treatment effect, it was confirmed that it can be usefully used to treat osteoarthritis (see Examples 4 to 7).
따라서, 본 발명은 상기 이중 약물 전달체를 유효성분으로 포함하는, 골 질환 예방 또는 치료용 약학적 조성물을 제공한다. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating bone disease, comprising the dual drug carrier as an active ingredient.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 골 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits or delays the development of a bone disease by administration of a pharmaceutical composition according to the invention.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 골 질환에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" means any action that improves or advantageously alters the condition for bone disease by administration of the pharmaceutical composition according to the present invention.
본 발명에 따른 예방 또는 치료의 대상인 "골 질환"은 조골세포의 활성 촉진으로 골량 증가가 필요 또는 요구되는, 상태 또는 질병을 의미하는 것으로 골량 저하 질환을 포함하며, 바람직하게는 골관절염(osteoarthritis)일 수 있으나, 이에 제한되는 것은 아니다. "Bone disease", which is the object of the prophylaxis or treatment according to the present invention, refers to a condition or a disease that requires or is required to increase bone mass by promoting activity of osteoblasts, and includes a bone loss disease, preferably osteoarthritis. May be, but is not limited thereto.
본 발명에 따른 약학적 조성물은 유효성분 이외에 약학적으로 허용되는 담체를 포함할 수 있다. 이때, 약학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세 결정성셀룰로스, 폴리비닐피로리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일등을 포함하나, 이에 한정되는 것은 아니다. 또한, 상기성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, it may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative and the like.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 바람직하게는 관절 내 주사제로 투여할 수 있다. 본 발명에 따른 조성물은 골 질환 치료를 위한 종래의 관절 내 주사제와 달리 온도 변화에 따른 나노 입자에 봉입된 진통제의 속방형 방출 및 공유 결합된 카토제닌의 서방형 방출이 가능하므로 골 질환의 효과적인 치료법이 될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, preferably by intraarticular injection. . Unlike conventional intraarticular injections for the treatment of bone diseases, the composition according to the present invention is capable of immediate release of analgesics encapsulated in nanoparticles and sustained release of covalently bound catogenin according to temperature changes, thereby effectively treating bone diseases. This can be
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있으며, 본 발명의 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 다른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래 의 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 pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The dosage will vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and time of administration, and may be appropriately selected by those skilled in the art, and the "pharmaceutically effective amount" of the present invention is applicable to medical treatment. Means a sufficient amount to treat the disease at a reasonable benefit / risk ratio, and the effective dose level refers to the type of disease, severity, drug activity, drug sensitivity, time of administration, route and rate of administration, duration of treatment, It can be determined according to factors including drugs used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로 본 발명의 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성율 및 배설속도, 질병종류, 병용되는 약물에 따라 달라질 수 있으며, 일반적으로는 체중 1kg 당 0.001 내지 150mg, 바람직하게는 0.01 내지 100mg을 매일 또는 격일 투여하거나, 1일 1내지 3회로 나누어 투여할 수 있다. 또한, 투여 경로, 골 질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150mg, preferably 0.01 to 100mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. In addition, the dose may be increased or decreased depending on the route of administration, the severity of the bone disease, sex, weight, age and the like, and the above dosage does not limit the scope of the present invention by any method.
또한, 상기 약물이 관절내 주사제 제제로서 투여되는 경우, 플루로닉-키토산 올리고당-카토제닌 나노 복합체는 바람직하게는 체중 1kg 당 0.2mg을 3주 간격으로 2회 관절 내로 투여할 수 있으나, 이로 제한되는 것은 아니다. In addition, when the drug is administered as an intra-articular injection preparation, the Pluronic-chitosan oligosaccharide-katogenin nanocomposite may preferably be administered intra-articularly at a dose of 0.2 mg per kg of body weight two times at three week intervals. It doesn't happen.
본 발명의 다른 양태로서, 본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 골 질환의 치료방법을 제공한다. 본 발명에서 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는, 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말 및 소 등의 포유류를 의미한다.In another aspect of the present invention, the present invention provides a method for treating a bone disease comprising administering the pharmaceutical composition to a subject. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse and cow, which is human or non-human. .
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예 1. 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자의 제조 및 확인Example 1 Preparation and Identification of Pluronic F127-chitosanoligosaccharide-katogenin tri-complex Nanoparticles
플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자의 합성을 위하여 먼저, 반응 유도체인 EDC/NHS(ethyl(dimethylaminopropyl)carbodiimide/ N-Hydroxysuccinimide)를 사용하여 카토제닌의 카르복실그룹과 키토산올리고당의 아민 그룹간 공유 결합 형성을 유도하였다. 키토산올리고당에 대한 적정 몰비의 카토제닌 (키토산올리고당:카토제닌, 1:0.02, 1:0.1, 1:0.5)을 EDC/NHS 반응 유도체와 1 시간 동안 25℃에서 반응시킨 후, 1%(v/v) acetic acid 용매에 녹인 키토산올리고당 용액에 첨가하여 24시간 동안 저속으로 교반하였으며, 투석을 통해 결합되지 않은 카토제닌, 키토산올리고당 및 EDC/NHS를 제거하고 동결 건조하여 키토산올리고당-카토제닌 복합체를 제조하였다. For the synthesis of Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles, first, the carboxyl group of chitosan oligosaccharide and ethyl chitosan oligosaccharide were prepared using the reaction derivative EDC / NHS (ethyl (dimethylaminopropyl) carbodiimide / N-Hydroxysuccinimide). Covalent bond formation was induced between amine groups. The appropriate molar ratio of katogenin (chitosan oligosaccharide: katogenin, 1: 0.02, 1: 0.1, 1: 0.5) to chitosan oligosaccharide was reacted with the EDC / NHS reaction derivative at 25 ° C. for 1 hour, followed by 1% (v / v) Chitosan oligosaccharides dissolved in a solvent of acetic acid were added and stirred at low speed for 24 hours, and unbound catogenin, chitosan oligosaccharides and EDC / NHS were removed by dialysis and freeze-dried to prepare chitosan oligosaccharide-katogenin complex. It was.
플루로닉 F127과 키토산올리고당의 공유 결합을 유도하기 위해 플루로닉 F127의 양쪽 말단이 카르복시기로 치환된 dicarboxylated 플루로닉 (HOOC-플루로닉-COOH)을 제조하였다. 먼저, 플루로닉 F127 (63g), 숙신산 (1.25g), 4 dimethylaminopyridine (1.2g), triethylamine (1.4 mL)을 anhydrous dioxane (200 mL)에 녹여 24 시간 동안 교반하였다. 이 후, Dioxane을 회전 증발 농축기로 증발시키고, diethyl ether를 첨가하여 합성된 HOOC-플루로닉-COOH을 침전시킨 후, diethyl ether는 진공 오븐에서 건조시켜서 HOOC-플루로닉-COOH의 하얀색 가루를 수득하였다. In order to induce covalent linkage between Pluronic F127 and chitosan oligosaccharides, dicarboxylated Pluronic (HOOC-Pluronic-COOH) was prepared in which both ends of Pluronic F127 were substituted with carboxyl groups. First, Pluronic F127 (63 g), succinic acid (1.25 g), 4 dimethylaminopyridine (1.2 g) and triethylamine (1.4 mL) were dissolved in anhydrous dioxane (200 mL) and stirred for 24 hours. Subsequently, dioxane was evaporated in a rotary evaporator, and diethyl ether was added to precipitate the synthesized HOOC-Pluronic-COOH. The diethyl ether was dried in a vacuum oven to remove white powder of HOOC-Pluronic-COOH. Obtained.
상기 합성된 키토산올리고당-카토제닌 복합체와 HOOC-플루로닉-COOH을 이용하여 emulsification/solvent evaporation 방법으로 키토산올리고당과 플루로닉의 공유 결합을 유도하였다. 키토산올리고당에 대한 적정 몰비의 플루로닉 (키토산올리고당:플루로닉 1:1, 1:2, 1:3, 1:4, 1:5)과 EDC를 dichloromethane에 녹인 후 30분 동안 반응시켰다. 이 후, 1%(v/v) acetic acid 용매에 녹인 키토산올리고당-카토제닌 복합체에 준비된 플루로닉 F127 반응액을 드롭 방식으로 떨어뜨리고 고주파에 반응시켜 에멀젼화를 유도함으로써 나노 입자를 제조하였다. 상기 입자를 24시간 동안 교반하면서 키토산올리고당과 플루로닉의 충분한 공유 결합을 유도하였으며, 이후, 반응액이 투명해질 때까지 회전 증발기로 dichloromethane을 증발시키고 제조된 나노 입자는 동결 건조하여 회수하였다. 본 실시예에서 사용된 키토산올리고당에 대한 카토제닌의 몰분율은 1:0.02, 1:0.1, 1:0.5이며, 키토산올리고당에 대한 플루로닉의 몰분율은 1:1, 1:2, 1:3, 1:4, 1:5로 합성되었다. The chitosan oligosaccharide-katozenin complex and HOOC-Pluronic-COOH were used to induce covalent bonds between chitosan oligosaccharides and pluronic by emulsification / solvent evaporation. Pluronics (chitosan oligosaccharides: pluronic 1: 1, 1: 2, 1: 3, 1: 4, 1: 5) of the appropriate molar ratio with respect to chitosan oligosaccharide and EDC were dissolved in dichloromethane and reacted for 30 minutes. Thereafter, the Pluronic F127 reaction solution prepared in the chitosan oligosaccharide-katogenin complex dissolved in a 1% (v / v) acetic acid solvent was dropped in a drop method and reacted with a high frequency to induce emulsification to prepare nanoparticles. The particles were stirred for 24 hours to induce sufficient covalent bonding of chitosan oligosaccharide and pluronic. After that, dichloromethane was evaporated with a rotary evaporator until the reaction solution became clear, and the prepared nanoparticles were recovered by freeze drying. The mole fraction of katogenin to chitosan oligosaccharide used in this example is 1: 0.02, 1: 0.1, 1: 0.5, and the mole fraction of pluronic to chitosan oligosaccharide is 1: 1, 1: 2, 1: 3, 1: 4, 1: 5.
또한, 상기 각 합성반응에 있어서, 생성물을 확인하기 위하여, 적외선분광광도계(FT-IR; Fourier Transform Infrared spectrophotometer) 및 수소핵자기공명분광기(1H-NMR; Proton Nuclear Magnetic Resonance spectroscopy) 분석을 실시하였다. In addition, in each of the above synthesis reactions, in order to identify the product, an infrared spectrophotometer (FT-IR) and a hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR) were performed. .
도 2 에 나타낸 바와 같이, 적외선분광광도계(FT-IR)에서, 키토산의 첫 번째 아민 그룹의 강한 N-H 결합에 비해 약한 N-H 결합을 가진 두 번째 아민그룹이 카토제닌의 카르복실기와 공유 결합하면서 고유의 amide 결합을 잃어버린 것을 확인하였다.As shown in FIG. 2, in the infrared spectrophotometer (FT-IR), a second amine group having weak NH bonds compared to the strong NH bonds of the first amine group of chitosan is covalently bonded to the carboxyl group of the cattogenin, and has an intrinsic amide. It was confirmed that the bond was lost.
또한, 도 3 에 나타낸 바와 같이, COOH-플루로닉 F127-COOH에 대한 적외선분광광도계(FT-IR)에서, wavelength 1735nm에서 카르복실기 특이적인 peak가 관찰되었고, 말단기의 카르복실기가 치환됨을 확인하였다. 또한, 플루로닉 F127-키토산올리고당-카토제닌 tri-complex에 대한 적외선분광광도계(FT-IR)에서, 카토제닌의 -CONH-기와 COOH-F127-COOH의 카르복실기 및 C=O 그룹의 작용기가 나타내는 resonance peaks가 관찰되었으며, 이로써, 키토산올리고당의 아민기에 플루로닉 F127-COOH 및 카토제닌이 성공적으로 공유 결합됨을 확인하였다. In addition, as shown in FIG. 3, in the infrared spectrophotometer (FT-IR) for COOH-Pluronic F127-COOH, a carboxyl group specific peak was observed at wavelength 1735 nm, and it was confirmed that the carboxyl group of the terminal group was substituted. In addition, in the infrared spectrophotometer (FT-IR) for the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex, the -CONH- group of the catogenin and the carboxyl group of the COOH-F127-COOH and the functional group of the C = O group are represented. Resonance peaks were observed, thereby confirming the successful covalent binding of Pluronic F127-COOH and Catogenin to the amine group of the chitosan oligosaccharide.
아울러, 도 4에 나타낸 바와 같이, 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자에 대한 수소핵자기공명분광(1H-NMR)에서, 카토제닌 벤젠고리의 수소가 나타내는 reasonance peak (δ7.5-8), 키토산 올리고당의 C2 위치의 메틸(-CH3)/메틸렌(-CH2)기에 있는 수소의 reasonance peak (δ1.9/δ2.7), 및 COOH-F127-COOH의 메틸렌(-CH2-CH2-CONH-)기에 있는 수소의 reasonance peak (δ2.2/δ2.4)을 관찰하였으며, 이를 통해 키토산올리고당의 아민기에 플루로닉 F127-COOH 및 카토제닌이 성공적으로 공유 결합됨을 확인하였다. In addition, as shown in Figure 4, in the hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR) to the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles, the reasonance peak (represented by hydrogen of the catogenin benzene ring) δ 7.5-8), reasonance peak (δ 1.9 / δ 2.7) of hydrogen in the methyl (-CH 3 ) / methylene (-CH 2 ) group at the C2 position of chitosan oligosaccharide, and methylene of COOH-F127-COOH We observed the reasonance peak (δ2.2 / δ2.4) of hydrogen in the (-CH 2 -CH 2 -CONH-) group, which successfully shared Pluronic F127-COOH and catogenin in the amine group of the chitosan oligosaccharide. Confirmed binding.
실시예 2. 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자의 제조 및 확인Example 2. Preparation and Identification of Pluronic F127-chitosanoligosaccharide-katogenin tri-complex Nanoparticles Encapsulated with Diclofenac
실시예 1에서 제조된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자를 이용하여 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자를 제조하였으며, 제조된 나노 입자를 전자현미경으로 관찰하였다. Diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles were prepared using the Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles prepared in Example 1, and the prepared nanoparticles were It was observed under a microscope.
보다 구체적으로 디클로페낙 (5mg)과 나노 입자 (10mg)을 deionized water에 부유하고 4℃에서 24시간 동안 천천히 교반하였으며, 반응액을 55℃에서 10분간 heating한 후, 10,000 rpm에서 centrifuge하고 상층액을 제거하여 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자를 제조하였다. 이후, deionized water에 부유한 나노 입자를 stud에 한 방울 떨어뜨리고 상온에서 건조한 후, 물기가 완전히 없어지면 백금 코팅하고 FE-SEM으로 관찰하여 나노 입자의 형태를 확인하였다. More specifically, diclofenac (5 mg) and nanoparticles (10 mg) were suspended in deionized water and stirred slowly at 4 ° C. for 24 hours. The reaction solution was heated at 55 ° C. for 10 minutes, centrifuge at 10,000 rpm, and the supernatant was removed. Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles encapsulated with diclofenac were prepared. Subsequently, a drop of nanoparticles suspended in deionized water was dropped in a stud and dried at room temperature. After the water was completely removed, platinum was coated and observed with FE-SEM to confirm the shape of the nanoparticles.
도 6에 나타낸 바와 같이, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자의 둥근 형태를 확인하였다. As shown in FIG. 6, the rounded shape of Pluronic F127-chitosan oligosaccharide-katogenin nanoparticles containing diclofenac was confirmed.
실시예 3. 플루로닉 F127 및 카토제닌의 몰분율 변화에 따른 나노 입자 크기 변화 확인Example 3 Confirmation of Nanoparticle Size Variation with Mole Fraction Changes of Pluronic F127 and Catogenin
플루로닉 F127-COOH:키토산올리고당의 몰분율을 4:1로 고정하고, 카토제닌의 몰분율을 변화시킨 경우, 온도(4℃, 37℃)에 따른 나노 입자의 크기 변화 차이를 평가하였고, 키토산올리고당:카토제닌의 몰분율을 1:0.1로 고정하고 플루로닉 F127-COOH의 몰분율을 변화시킨 경우, 온도 변화(4℃, 37℃)에 따른 나노 입자의 크기 변화 차이를 평가하였다. 또한, 상변이가 일어나는 온도 구간을 확인하기 위해 4℃에서 37℃ 까지 온도변화에 따른 나노 입자의 크기변화를 확인하였으며, 이 때, 플루로닉:키토산올리고당:카토제닌의 몰분율이 4:1:0.1인 나노 입자(4F127)와 5:1:0.1인 나노 입자(5F127)간 상변이를 비교함으로써, 플루로닉의 몰분율 변화에 따른 나노 입자의 상변이의 차이를 관찰하였다. When the molar fraction of Pluronic F127-COOH: chitosan oligosaccharide was fixed at 4: 1 and the mole fraction of catogenin was changed, the difference in size change of nanoparticles with temperature (4 ° C., 37 ° C.) was evaluated. : When the mole fraction of catogenin was fixed at 1: 0.1 and the mole fraction of Pluronic F127-COOH was changed, the difference in size change of nanoparticles according to temperature change (4 ° C., 37 ° C.) was evaluated. In addition, in order to confirm the temperature range in which the phase transition occurs, the size change of the nanoparticles was observed according to the temperature change from 4 ° C to 37 ° C, and at this time, the mole fraction of pluronic: chitosan oligosaccharide: catogenin was 4: 1: By comparing the phase shift between the nanoparticles (4F127) of 0.1 and the nanoparticles (5F127) of 5: 1: 0.1, the difference in the phase variation of the nanoparticles according to the mole fraction change of the pluronic was observed.
도 7에 나타낸 바와 같이, 카토제닌의 몰분율이 증가할수록 온도변화에 따른 나노 입자의 크기변화가 줄어들어든 반면, 플루로닉 F127-COOH의 몰분율이 증가할수록 온도변화에 따른 나노 입자의 크기 변화가 증가하였다. 4℃에서 37℃ 까지 온도변화에 따른 나노 입자의 크기변화를 관찰한 결과, 24℃~27℃ 사이에서 급격한 나노 입자의 크기변화가 일어남을 확인하였으며, 저온 상태에서, 나노 입자가 팽창함을 확인하였다. 또한 나노 입자 제조시 플루로닉의 몰분율이 증가할수록 온도 변화에 따른 나노 입자의 상변이 폭이 커짐을 확인하였다. As shown in FIG. 7, as the mole fraction of catogenin increases, the size change of nanoparticles decreases with temperature change, while as the mole fraction of Pluronic F127-COOH increases, the size change of nanoparticles increases with temperature change. It was. As a result of observing the size change of nanoparticles according to the temperature change from 4 ℃ to 37 ℃, it was confirmed that the rapid change of size of nanoparticles occurred between 24 ℃ ~ 27 ℃. It was. In addition, it was confirmed that as the molar fraction of the pluronic nanoparticles increased, the width of the phase change of the nanoparticles increased with temperature change.
상기 결과는 플루로닉 F127, 키토산올리고당 및 카토제닌의 몰분율을 조절함으로써, 온도 변화에 따른 나노 입자의 크기를 조절할 수 있음을 의미한다. The results indicate that by controlling the mole fraction of Pluronic F127, chitosan oligosaccharide and catogenin, the size of the nanoparticles can be adjusted according to temperature change.
실시예 4. 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서 카토제닌의 서방형 방출 및 디클로페낙 속방형 방출의 서방형 방출 확인Example 4 Confirmation of Sustained Release Release of Catogenin and Diclofenac Immediate Release in Pluronic F127-chitosanoligosaccharide-katogenin Composite Nanoparticles Encapsulated with Diclofenac
디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서의 약물 방출을 확인하기 위하여, 시험관 내 약물 방출 실험으로서, 나노 입자로부터 방출된 카토제닌 및 디클로페낙의 함량을 액체크로마토그래피 (HPLC)로 분석하였으며, 5℃ 내지 20℃의 cold shock(F127/COS/KGNDCF w/o cs, F127/COS/KGNDCF w/ cs)에 따른 방출 속도의 차이를 비교하였다. To confirm drug release in Pluronic F127-chitosanoligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac, the content of cattogenin and diclofenac released from nanoparticles was determined by liquid chromatography (HPLC). ), And the difference in release rate according to cold shock (F127 / COS / KGN DCF w / o cs, F127 / COS / KGN DCF w / cs) of 5 ° C. to 20 ° C. was compared.
보다 구체적으로 동결 건조된 F127/COS/KGNDCF 나노 입자 (100mg)을 PBS에 부유하고 37℃ 100rpm shaking incubator에 반응시켰다. 각 sampling time에 10,000rpm centrifugation으로 나노 입자를 침전시키고 상층액을 수거하고 같은 volume의 fresh PBS로 교체하였다. 각 sampling time에 10분 동안 ice에서 처리함으로써 Cold shock을 가하였으며, 디클로페낙 및 카토제닌의 함량은 HPLC로 분석하였다. More specifically, lyophilized F127 / COS / KGN DCF nanoparticles (100 mg) were suspended in PBS and reacted at 37 ° C. 100 rpm shaking incubator. At each sampling time, nanoparticles were precipitated at 10,000 rpm centrifugation and the supernatants were collected and replaced with fresh PBS of the same volume. Cold shock was applied by treatment on ice for 10 minutes at each sampling time, and the contents of diclofenac and catogenin were analyzed by HPLC.
아울러, 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자의 각 구성물질의 몰농도 비율은 3F127/COS/KGN의 경우, 플루로닉 F127:키토산올리고당:카토제닌=3:1:0.1이며, 4F127/COS/KGN의 경우, 플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1이었으며, 이를 통하여, 플루로닉 F127의 몰 농도 비율에 따른 각 물질의 방출 속도 차이를 확인하였다. In addition, the molar concentration ratio of each component of the Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticle is 3F127 / COS / KGN, and Pluronic F127: Chitosan Oligosaccharide: Katozenin = 3: 1: 0.1, In the case of 4F127 / COS / KGN, Pluronic F127: chitosan oligosaccharide: katogenin = 4: 1: 0.1, through which the difference in release rate of each substance according to the molar concentration ratio of Pluronic F127 was confirmed.
도 8에 나타낸 바와 같이, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서의 카토제닌 서방출을 확인하였으며, cold shock를 준 경우, 좀 더 빠른 카토제닌의 방출 속도를 보였으나, 이 경우에도 2주까지 60%이하의 지속적인 방출을 확인하였다. As shown in FIG. 8, cattogenin sustained release was confirmed in Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac. When cold shock was given, the release rate of catogenin was faster. In this case, it was confirmed that the release was less than 60% until 2 weeks.
또한, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서 디클로페닉의 속방출을 확인하였으며, 특히, cold shock를 준 경우, 9시간 이내에 90%에 가까운 빠른 방출 속도를 확인한 반면, cold shock를 주지 않은 경우, 24시간 동안 20% 정도의 상대적으로 느린 방출 속도를 확인하였다. In addition, the rapid release of diclofenic was confirmed in Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac, and particularly, in the case of cold shock, a fast release rate of nearly 90% was confirmed within 9 hours. On the other hand, when the cold shock was not given, a relatively slow release rate of about 20% was confirmed for 24 hours.
아울러, 플루로닉 F127의 몰농도 비율이 높은 4F127/COS/KGN의 경우, 3F127/COS/KGN와 비교하여 카토제닌은 느리게 방출되었으며, 디클로페낙은 빠르게 방출되었다 따라서, 4F127/COS/KGN (플루로닉 F127:키토산올리고당:카토제닌=4:1:0.1 몰농도) 나노 입자에서 최적의 카토제닌 서방형 방출 및 디클로페낙의 속방형 방출 효과를 얻을 수 있음을 확인하였다. In addition, 4F127 / COS / KGN, which has a high molar concentration ratio of Pluronic F127, was slow to release cattogenin and fast to release diclofenac compared to 3F127 / COS / KGN. Thus, 4F127 / COS / KGN (Fluro Nick F127: chitosan oligosaccharide: katogenin = 4: 1: 0.1 molarity) It was confirmed that the optimal release of cattogenin sustained release and immediate release of diclofenac can be obtained from the nanoparticles.
상기 결과는 연골 재생 약물인 카토제닌의 서방형 방출로 국소 부위에서 지속적인 연골 재생 효과를 가질 수 있음을 의미하며, 소염 진통제인 디클로페낙은 온도 변화(cold shock)에 따른 입자의 팽창에 의해 빠른 방출이 유도됨을 의미한다. The results indicate that sustained release of cartogenin, a cartilage regenerative drug, may have a sustained effect of cartilage regeneration at the local site. Diclofenac, an anti-inflammatory analgesic, is rapidly released due to the expansion of particles due to cold shock. Induced.
실시예 5. 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자의 연골 분화 효과 확인Example 5. Confirmation of cartilage differentiation effect of Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles packed with diclofenac
실시예 4의 카토제닌의 서방형 방출에 따른 지속적인 연골 재생 효과를 확인하기 위하여 골수간엽줄기세포에 액체 상태의 카토제닌을 단독으로 처리한 군(KGN), 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자만 처리한 군(F127/COS/KGNDCF w/o cs), 및 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자를 처리한 후 cold shock를 가한 군(F127/COS/KGNDCF w/ cs)간 연골 분화 인자인 Collagenase type II (COL2A1)과 Aggrecan 유전자 및 단백질의 발현율을 평가하였다. In order to confirm the sustained cartilage regeneration effect according to the sustained release of the katogenin of Example 4 group treated with liquid katogenin alone (KGN), Pluronic F127-chitosan sealed with diclofenac The group treated with the oligosaccharide-katogenin complex nanoparticles only (F127 / COS / KGN DCF w / o cs), and the Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac were subjected to cold shock. The expression rates of collagenase type II (COL2A1) and Aggrecan genes and proteins, which are cartilage differentiation factors, between groups (F127 / COS / KGN DCF w / cs) were evaluated.
보다 구체적으로 Pellet 형태로 배양한 골수간엽줄기세포를 Trans well plate의 하층 well로 옮기고, 0.05 μm pore size의 Membrane을 깔아준 상층 well에 100nM의 카토제닌을 분비할 수 있는 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 나노 입자를 각각을 처리하였다. 3주 후, TRIzol (Invitrogen Co.)을 처리하여 RNA를 분리하고, Maxime RT preMix kit oligo(dT) primer (iNtRon biotechnology)를 이용하여 역전사 시켰으며, Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), collagenase type II (COL2A1), aggrecan, 유전자의 발현량은 real-time polymerase chain reaction으로 정량하여 house keeping 유전자인 GAPDH의 발현량과 비교, 상대 정량하였다. More specifically, myeloid mesenchymal stem cells cultured in Pellet form were transferred to a lower well of a trans well plate, and diclofenac-encapsulated pluronac capable of secreting 100 nM of catogenin in an upper well coated with a 0.05 μm pore-size Membrane. F127-chitosanoligosaccharide-katogenin nanoparticles were treated respectively. After 3 weeks, RNA was isolated by treatment with TRIzol (Invitrogen Co.), reverse transcription using Maxime RT preMix kit oligo (dT) primer (iNtRon biotechnology), Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), collagenase type II The expression levels of (COL2A1), aggrecan, and genes were quantified by real-time polymerase chain reaction and compared with those of house-keeping gene GAPDH.
또한, 연골 분화 4주 후 pellet을 수거하고 RIPA lysis buffer (Thermo scientific)로 total protein을 분리하였다. 8 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE)로 단백질을 전개한 후, polyvinylidene fluoride (PVDF) membrane에 blotting하였으며, 0.05%(v/v) tween 20이 포함된 PBS에 5%(v/w) skim milk를 첨가한 후, 단백질이 블로팅된 membrane을 1시간 동안 반응시켰다. SOX9 (SRY (sex determining region Y)-box 9), Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), collagenase type II (COL2A1), aggrecan, 에 대한 Primary antibody를 4℃에서 overnight 반응시키고, horseradish peroxidase가 표지된 secondary antibody를 상온에서 1시간 처리하였다. chemiluminescence (ECL) Western blotting detection reagent (Amersham Biosciences)로 발광된 시그널을 LAS-3000 (Fujifilm)를 통해 이미지를 획득하였다. After 4 weeks of cartilage differentiation, pellets were collected and total protein was isolated by RIPA lysis buffer (Thermo scientific). Proteins were developed by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by blotting on polyvinylidene fluoride (PVDF) membrane and 5% (v / w) in PBS containing 0.05% (v / v) tween 20. After skim milk was added, the protein-blotting membrane was reacted for 1 hour. Primary antibodies against SOX9 (SRY (sex determining region Y) -box 9), Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), collagenase type II (COL2A1), aggrecan, were reacted overnight at 4 ° C, and horseradish peroxidase-labeled secondary The antibody was treated for 1 hour at room temperature. Chemiluminescence (ECL) The signal luminescent with Western blotting detection reagent (Amersham Biosciences) was acquired by LAS-3000 (Fujifilm).
도 9에 나타낸 바와 같이, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자를 처리한 그룹은 액체 상태의 카토제닌을 처리한 그룹보다 2~4배 이상 높은 COL2A1의 유전자 발현율을 보였고, Aggrecan에서도 2~2.5배 이상 높은 유전자 발현율을 확인하였으며, cold shock를 가한 경우, COL2A1 및 Aggrecan의 발현이 더욱 증가하였다. As shown in FIG. 9, the group treated with Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac showed a COL2A1 gene expression rate higher than 2 to 4 times higher than the group treated with liquid catogenin. Aggrecan showed a gene expression rate of 2 to 2.5 times higher. When cold shock was applied, the expression of COL2A1 and Aggrecan was further increased.
또한, 도 10에 나타낸 바와 같이, real-time qPCR 결과와 마찬가지로, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자를 처리한 그룹은 액체 상태의 카토제닌을 처리한 그룹보다 더 높은 COL2A1 및 Aggrecan의 단백질 발현율을 확인하였다.In addition, as shown in FIG. 10, the group treated with Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles encapsulated with diclofenac, as in the real-time qPCR results, was more than the group treated with liquid katogenin. High COL2A1 and Aggrecan protein expression rates were confirmed.
상기 결과를 통해서 나노 입자에서 지속적으로 방출된 카토제닌에 의한 연골 재생 효과의 증가를 확인하였다. Through the above results, it was confirmed that the cartilage-induced cartilage regeneration effect was continuously released from the nanoparticles.
실시예 6. 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complexed 나노 입자에 의한 빠른 항염 효과 확인 Example 6.Improved anti-inflammatory effect by Pluronic F127-chitosan oligosaccharide-katogenin tri-complexed nanoparticles packed with diclofenac
실시예 4의 디클로페낙의 빠른 방출에 따른 항염효과를 확인하기 위하여 연골세포 및 U937 macrophage-like cells에 LPS(lipopolysaccharide)만을 처리한 군(LPS), LPS 처리 후 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군(LPS+nanoparticles w/o cs), 및 LPS 처리 후 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군(LPS+nanoparticles w/ cs)간 함염증성 cytokine인 IL-6의 분비량을 비교하였다.In order to confirm the anti-inflammatory effect of the rapid release of diclofenac of Example 4 group treated only with LPS (lipopolysaccharide) to chondrocytes and U937 macrophage-like cells, Pluronic F127-chitosan sealed with diclofenac after LPS treatment Group treated with oligosaccharide-katogenin tri-complex nanoparticles only (LPS + nanoparticles w / o cs), and Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles sealed with diclofenac after LPS treatment and cold shock treatment The secretion of IL-6, an inflammatory cytokine, was compared between one group (LPS + nanoparticles w / cs).
보다 구체적으로, 연골 (1×105cells/well) 및 U937 (1×105cells/well) 세포를 6-well 플레이트에 배양하고 Lipopolysaccharide (LPS) (1㎍/ml)로 염증 반응을 유도한 뒤, 2일 동안 DCF 100nM을 분비할 수 있는 F127/COS/KGNDCF 나노 입자를 각 세포에 처리하였다. 시간대별로 배양액을 수거하여 분비가 억제된 IL-6를 enzyme-linked immunoabsorbent assay (ELISA)로 정량하였다.More specifically, cartilage (1 × 10 5 cells / well) and U937 (1 × 10 5 cells / well) cells were cultured in 6-well plates and lipopolysaccharide (LPS) (1 μg / ml) induced the inflammatory response. Thereafter, each cell was treated with F127 / COS / KGN DCF nanoparticles capable of secreting DCF 100 nM for 2 days. Cultures were collected at different times and quantified by the enzyme-linked immunoabsorbent assay (ELISA).
도 11에 나타낸 바와 같이, 연골 및 U937 세포 모두에서, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자 처리군의 IL-6 분비량이 유의적으로 감소하였다. 또한, cold sock를 준 그룹과 그렇지 않은 그룹을 비교하였을 때, cold shock를 준 그룹에서 IL-6의 분비량이 더욱 효과적으로 줄어들었다. As shown in FIG. 11, IL-6 secretion of the Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticle treated group containing diclofenac was significantly reduced in both cartilage and U937 cells. In addition, IL-6 secretion was more effectively reduced in the cold shock group than in the cold sock group.
상기 결과는 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에서, 온도 변화(cold shock)에 따른 디클로페낙의 속방출을 통하여 효과적인 항염 효과와 함께 이에 따른 부작용을 최소화시킬 수 있음을 의미한다.The results indicate that diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles can be effectively released through diclofenac with cold shock and minimize side effects. it means.
실시예 7. 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자에 의한 골관절염 치료 효과 확인Example 7 Confirmation of Osteoarthritis Treatment Effect by Pluronic F127-chitosan-oligosaccharide-katogenin Complex Nanoparticles Packed with Diclofenac
디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 복합체 나노 입자의 처리에 의한 골관절염 치료 효과를 확인하기 위하여 렛드에 전방십자인대 절제 및 반월판 제거술을 실시하여 골관절염을 유도하였으며, 상기 골관절염 동물모델을 이용하여 경골하부의 손상 억제 및 연골 보호 효과를 확인하였다. In order to confirm the effect of osteoarthritis treatment by treatment of Pluronic F127-chitosan oligosaccharide-katogenin complex nanoparticles containing diclofenac, an osteoarthritis was induced by anterior cruciate ligament resection and meniscus removal. Inhibition of damage to the lower tibia and cartilage protection effect was confirmed.
7-1. 관절염에 의한 경골하부의 손상 억제효과 확인7-1. Inhibition of damage to the lower tibia due to arthritis
PBS 투여군 (vehicle), 카토제닌 및 디클로페낙을 각각 투여하고 cold shock 처리한 군(KGN+DCF w/cs), 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자만을 처리한 군(F-127/COS/KGNDCF W/o CS), 및 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군(F-127/COS/KGNDCF W/ CS)의 골관절염 진행(6주, 14주)에 따른 경골하부의 손상 및 변형 정도를 micro-CT로 평가하였다. Cold shock treated (KGN + DCF w / cs) and Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles treated with PBS (vehicle), catogenin and diclofenac, respectively, and treated with cold shock (KGN + DCF w / cs). Group (F-127 / COS / KGN DCF W / o CS), and Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles with diclofenac and cold shock treated group (F-127 / COS / KGN) The degree of damage and deformity of the lower tibia according to osteoarthritis progression (6 weeks, 14 weeks) of DCF W / CS) was evaluated by micro-CT.
그 결과, 도 12에 나타낸 바와 같이, 카토제닌 및 디클로페낙을 각각 투여한 군(KGN+DCF w/cs)과 비교하여 볼 때, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자를 처리한 군(F-127/COS/KGNDCF W/o CS, F-127/COS/KGNDCF W/ CS)에서는, 경골하부의 손상 및 변형이 억제되는 경향을 보였다. 특히, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군(F-127/COS/KGNDCF W/ CS)에서는, 관절염 병변의 진행이 현저하게 억제됨을 확인하였다. As a result, as shown in FIG. 12, compared to the group (KGN + DCF w / cs) administered with catogenin and diclofenac, respectively, Pluronic F127-chitosan oligosaccharide-katogenin tri-complex containing diclofenac In the group treated with nanoparticles (F-127 / COS / KGN DCF W / o CS, F-127 / COS / KGN DCF W / CS), the damage and deformation of the tibial area tended to be suppressed. In particular, Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles containing diclofenac and cold shock treated group (F-127 / COS / KGN DCF W / CS) significantly inhibited the progression of arthritis lesions. It was confirmed.
7-2. 연골 보호 효과 확인7-2. Check cartilage protection
상기 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자의 처리에 의한 연골 보호 효과를 확인하고자, 상기 실시예 7-1과 마찬가지로 처리된 렛드를 안락사시키고, 관절 조직을 분리하여 10% paraformaldehyde에 고정한 후, 6% nitric acid를 처리하여 탈칼슘화하였다. 이후, Tissue-Tek O.C.T. compound (Sakura Finetek)에 조직을 embedding하고 10μm로 section하여 슬라이드 글라스에 붙이고 이후, Safranin-O/fast green 염색 및 collagenase type II (COL2), aggrecan에 대한 immunofluorescence를 실시하였으며, 상기 염색된 연골 조직의 손상 정도를 OARSI score로 평가하였다. 아울러, 혈청 내 연골파괴효소(COX-2) RNA 및 synovial membrane 내 전체 단백질 중 COX-2 단백질의 상대적인 양을 비교하였다. To confirm the cartilage protection effect of the diclofenac-encapsulated Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticle, euthanized the treated red as in Example 7-1, and isolating joint tissue Fixed to 10% paraformaldehyde, and then treated with 6% nitric acid for decalcification. Thereafter, Tissue-Tek O.C.T. Tissues were embedded in the compound (Sakura Finetek), sectioned to 10 μm and attached to the slide glass, followed by Safranin-O / fast green staining and immunofluorescence for collagenase type II (COL2), aggrecan, and damage to the stained cartilage tissue. The extent was assessed by the OARSI score. In addition, the relative amounts of COX-2 protein in serum chondrogenic enzyme (COX-2) RNA and total protein in synovial membrane were compared.
그 결과, 도 13에 나타낸 바와 같이, PBS 투여군은 넓은 영역의 연골이 박리 및 파괴되어 있었으며, COL2 및 Aggrecan 단백질이 눈에 띄게 감소하였던 반면, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자를 처리한 군에서는, 보다 정상에 가까운 연골 표면층을 나타내고 있었으며, COL2 및 Aggrecan 단백질의 증가를 확인하였다. 이러한 효과는 카토제닌 및 디클로페낙을 각각 투여한 군과 비교하여 유의적인 차이를 나타냈으며, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군에서 보다 현저하였다. 또한, 상기 결과와 마찬가지로 OARSI score 평가에서도 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군에서 연골 보호 효과가 가장 우수하였다. As a result, as shown in FIG. 13, the PBS-administered group had a large area of cartilage detachment and destruction, while the COL2 and Aggrecan proteins were markedly reduced, while diclofenac-containing Pluronic F127-chitosan oligosaccharide-katogenin In the group treated with tri-complex nanoparticles, the cartilage surface layer was closer to normal, and the increase of COL2 and Aggrecan proteins was confirmed. This effect was significantly different compared to the group administered with catogenin and diclofenac, respectively, and more pronounced in the group treated with diclofenac-containing Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock. It was. In addition, in the OARSI score evaluation as well as the above results, cartilage protection effect was the best in the group treated with diclofenac-filled Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock.
또한, 도 14에 나타낸 바와 같이, 디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자 및 cold shock 처리한 군에서, 연골기질파괴효소(COX-2)를 현저하게 억제시킴을 확인하였다. In addition, as shown in Fig. 14, in the group treated with diclofenac-containing Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles and cold shock, it significantly inhibited cartilage matrix degrading enzyme (COX-2). It was confirmed.
디클로페낙이 봉입된 플루로닉 F127-키토산올리고당-카토제닌 tri-complex 나노 입자를 골 관절염 부위에 투여함으로써, 카토제닌의 서방출을 통해 연골 재생효과를 촉진시킬 수 있을뿐만 아니라, 저온 처리에 의해 봉입된 디플로페낙의 속방출이 유도되어 연골기질파괴효소 및 이에 따른 염증반응을 효과적으로 억제시킬 수 있음을 동물 모델을 통하여 구체적으로 확인하였는바, 이를 통해 골관절염 치료 효과를 현저히 증진시킬 수 있다. By administering Pluronic F127-chitosan oligosaccharide-katogenin tri-complex nanoparticles encapsulated with diclofenac to osteoarthritis sites, the cartilage regeneration effect can be promoted through sustained release of cattogenin and encapsulated by low temperature treatment. The rapid release of diflofenac was induced to effectively inhibit the cartilage matrix degrading enzyme and the inflammatory response according to the animal model, which can significantly increase the osteoarthritis treatment effect.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (9)

  1. 플루로닉(pluronics), 키토산올리고당(Chitosan oligosaccharides), 및 카토제닌(Kartogenin)이 순차적으로 공유 결합으로 연결된 나노 복합체; 및 상기 복합체 내에 봉입된 약물을 포함하는, 온도감응성 이중 약물 전달체.Nanocomposites in which pluronics, chitosan oligosaccharides, and katogenin are sequentially covalently linked; And a drug enclosed in the complex.
  2. 제1항에 있어서,The method of claim 1,
    상기 공유 결합은 아마이드 결합(amide bond)인 것을 특징으로 하는, 이중 약물 전달체.The dual drug carrier, characterized in that the amide bond (amide bond).
  3. 제1항에 있어서,The method of claim 1,
    상기 봉입된 약물은 소염 진통제인 것을 특징으로 하는, 이중 약물 전달체.The enclosed drug is an anti-inflammatory analgesic, characterized in that the dual drug delivery.
  4. 제3항에 있어서,The method of claim 3,
    상기 소염 진통제는 디클로페낙(Diclofenac)인 것을 특징으로 하는 이중 약물 전달체.The anti-inflammatory analgesic is a double drug carrier, characterized in that Diclofenac (Diclofenac).
  5. 제1항의 이중 약물 전달체를 유효성분으로 포함하는, 골 질환 예방 또는 치료용 약학적 조성물.Claim 1, comprising a dual drug carrier as an active ingredient, a pharmaceutical composition for preventing or treating bone diseases.
  6. 제5항에 있어서,The method of claim 5,
    상기 골 질환은 골관절염(osteoarthritis)인 것을 특징으로 하는, 조성물.The bone disease is characterized in that the osteoarthritis (osteoarthritis), composition.
  7. 제5항에 있어서, The method of claim 5,
    상기 조성물은 관절 내 주사제 제제인 것을 특징으로 하는, 조성물.Wherein said composition is an intraarticular injection preparation.
  8. 제1항의 이중 약물 전달체를 개체에 투여하는 단계를 포함하는, 골 질환의 예방 또는 치료방법.A method of preventing or treating bone disease, comprising administering to a subject a dual drug delivery agent of claim 1.
  9. 제1항의 이중 약물 전달체의 골 질환 치료 용도.Use of the dual drug carrier of claim 1 for the treatment of bone diseases.
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