WO2018030579A1 - 루테인의 용출률 및 안정성이 개선된 이중층 구조의 고분자 캡슐, 이의 제조방법 및 이를 함유하는 안과질환 예방 또는 치료용 약제학적 조성물 - Google Patents

루테인의 용출률 및 안정성이 개선된 이중층 구조의 고분자 캡슐, 이의 제조방법 및 이를 함유하는 안과질환 예방 또는 치료용 약제학적 조성물 Download PDF

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WO2018030579A1
WO2018030579A1 PCT/KR2016/011639 KR2016011639W WO2018030579A1 WO 2018030579 A1 WO2018030579 A1 WO 2018030579A1 KR 2016011639 W KR2016011639 W KR 2016011639W WO 2018030579 A1 WO2018030579 A1 WO 2018030579A1
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Prior art keywords
lutein
polymer
poly
polymer capsule
double
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PCT/KR2016/011639
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English (en)
French (fr)
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조정원
전혜숙
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충남대학교산학협력단
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Publication of WO2018030579A1 publication Critical patent/WO2018030579A1/ko

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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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4808—Preparations in capsules, e.g. of gelatin, of chocolate characterised by the form of the capsule or the structure of the filling; Capsules containing small tablets; Capsules with outer layer for immediate drug release
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5015—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A61K9/5057—Gelatin

Definitions

  • the present invention relates to a polymer capsule of a double layer structure with improved dissolution rate and stability of lutein, and to a method of manufacturing the same. More specifically, it is possible to stabilize oxidative-sensitive lutein and to prolong lutein's efficacy in the eye for a long time.
  • the present invention relates to polymer capsules and to compositions for preventing or treating ophthalmic diseases or cosmetic compositions to which the polymer capsules can be applied.
  • Lutein of Formula 1 is the most abundant pigment among xanthophylls of carotenoids (C 4 (i H 56 0 2 , orange crystal, melting point about 193 ° C, absorption band of light). The wavelength is 508, 475, 445 nm), which is the only carotenoid present in this tissue as the major constituent of the cornea, pupil and macula of the eye with Zeaxanthin of Formula 2 .
  • Lutein acts as a filter for high energy blue light absorbed by the eye or by removing photocatalytic radicals or reactive oxygen species, protecting the eyes. Humans do not synthesize carotenoids, so lutein Contains food or
  • Lutein contains an unsaturated structure with 9 or more double bonds, so it is very unstable and easily oxidized in environments such as light, heat, and air (oxygen). Therefore, lutein is easily oxidized under conditions such as shading, low temperature, and vacuum. Lutein is very soluble in water and low in fats and oils. The very low water solubility (1.3xl0 9 M) in particular of lutein shows a drawback in bioavailability.
  • Patent Publication No. 2012-0108170 discloses lutein, phospholipid and oil. And a formulation in which a mixture of surfactants is layered in soft or hard capsules in liquid form to improve the dissolution rate and bioavailability of drugs.
  • WO2016 / 025394 is a nanoparticle comprising a zein protein or polymer.
  • Encapsulated or a composition in the form of a mixture of the nanoparticles and hydrogels is disclosed, but these prior art techniques show both the stability and dissolution rate of lutein. There is a limit to improvement.
  • the new double-layered polymer capsules are made possible, confirming their effectiveness in overcoming the limitations of existing formulations and completing the present invention.
  • An object of the present invention is to provide a double-layered polymer capsule capable of stabilizing lutein, improving the yield of lutein, and a manufacturing method thereof.
  • Another object of the present invention is to provide a pharmaceutical composition or health functional food for preventing or treating ophthalmic diseases, which can exhibit the long-term efficacy of the active substance, containing the above-mentioned polymer capsule.
  • Another object of the present invention is to provide a cosmetic composition having antioxidant activity by containing the above polymer capsule.
  • the present invention relates to a core comprising a lutein or a lutein-containing extract as a physiologically active substance; a biodegradable polymer layer surrounding the core; and a mixed layer of mucoadhesive polymer and emulsifier coated on the biodegradable polymer layer; It is about a polymer layer of a bilayer structure containing a.
  • the lutein extract is based on the total weight of the lutein extract.
  • Lutein can be included in amounts of 10-90% (w / w).
  • the polymer capsule may be lutein based on the total weight of the polymer capsule.
  • It can contain 1.0 to 95.0% (Ww), preferably 3.0 to 90.0% (w / w), and more preferably 5.0 to 75.0% (w / w).
  • the biodegradable polymer layer is poly-L-lactic acid, poly-glycolic acid,
  • Poly-D L-lactic acid-co-glycolic acid, poly-caprolactone, poly-valerolacron,
  • It may be made of one or more selected from the group consisting of poly-hydroxybutyrate and poly-hydroxy valerate.
  • said mucoadhesive polymers include alginates or salts thereof, gelatin,
  • oligochitosan chitooligosaccharide
  • chitosan chitosan
  • glacin glycine
  • arginine arginine
  • lysine lysine
  • cyclodextrin cyclodextrin
  • the emulsifier may be at least one selected from the group consisting of sucrose, polyvinyl pyrrolidone, and poly vinyl alcohol.
  • the microparticle cap [22] According to another aspect of the present invention, the microparticle cap
  • the above manufacturing method is: Lutein, a bioactive substance Or dissolving the lutein-containing extract and the biodegradable polymer in an organic solvent to produce a primary emulsion dog (step 1); dispersing and homogenizing the primary emulsion in an aqueous solution of mucoadhesive polymer and emulsifier. Centrifugation and freeze drying after forming microspheres (step 2);
  • the present invention relates to an ophthalmic disease prevention or therapeutic pharmaceutical composition comprising the double-layered polymer capsule.
  • the present invention relates to an ophthalmic disease preventing or improving dietary supplement comprising the double layered polymer capsule.
  • the present invention relates to a cosmetic composition comprising the polymer capsule of the double layer structure.
  • the present invention is a core-shell double-layered polymer capsule, which primarily encapsulates oxidation-sensitive lutein with biodegradable polymer particles, and provides a mucoadhesive polymer and an emulsifying agent on the outer wall of the secondary capsule.
  • a mixed layer while stabilizing lutein; also, the effect of lutein can be prolonged in the eye for a long time.
  • 'polymer capsule' refers to the encapsulation of lutein or lutein-containing extracts by biodegradable polymers, mucoadhesive polymers and emulsifiers, and the term 'microspheres',' polymeric microspheres, 'or' Also called polymeric microparticle capsule.
  • the method for producing a double-layered polymer capsule of the present invention comprises the steps of: (1) generating a primary emulsion by dissolving a lutein or lutein-containing extract, which is a physiologically active substance, and a biodegradable polymer in an organic solvent; Dispersing the primary emulsion in mucoadhesive polymers and emulsifier-soluble aqueous solution and homogenizing to form microspheres, followed by centrifugation and freeze-drying (step 2).
  • lutein as a physiologically active substance may be a pure substance having a purity of 90% or more separated by lutein itself, but a lutein 10 to 90% extract extracted from a plant or an animal may be used.
  • a lutein 10 to 90% extract extracted from a plant or an animal may be used.
  • 10-40% extract of lutein may be used, wherein the extract may be in the form of a dispersion in vegetable oil, and sunflower seed oil, safflower oil, olive oil, etc. may be used as the vegetable oil.
  • the biodegradable polymer may be used as a normal polymer used in the manufacture of particulates, and does not limit the type of polymer, but in the present invention, poly-L-lactic acid, poly-glycolic acid, poly-D-lactic acid-co- Glycolic acid, poly-L-lactic acid-co-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, poly-caprolactone, poly-valerolactone,
  • One or more species may be used in the group consisting of poly-hydroxybuty: rate and poly-hydroxyvalerate.
  • poly-L-lactic acid, poly-D-lactic acid-co-glycolic acid, poly-L Lactic acid-co-glycolic acid and One or more polymers selected from the group consisting of poly-D, L-lactic acid-co-glycolic acid (PLGA) can be used.
  • L-lactic acid-co-glycolic acid (PLGA) and poly-L-lactic acid alone or two kinds thereof are used in combination.
  • the biodegradable polymer has a weight average molecular weight of 60,000 or less, preferably 60,000 to 5,000. For example, the molecular weight of about 13,000
  • biodegradable polymers for example Boehringer Ingelheim
  • the organic solvent is not particularly limited as long as it is a solvent for biodegradable polymers and a volatile organic solvent having excellent easiness of removal through evaporation, but preferably methylene chloride, ethyl acetate, One or more solvents selected from chloroform, acetone, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone, and acetonitrile Can be used , More
  • methylene chloride ethyl, ethyl acetate, chloroform and
  • One solvent or two or more mixed solvents selected from tetrahydrofuran may be used, most preferably methylene chloride and
  • One or two mixed solvents selected from ethyl acetate may be used.
  • the solubility in water is 20 g / L (20 Methylene chloride was used as low as ° C).
  • the primary emulsion is dispersed in a mucoadhesive polymer and an emulsifier-soluble aqueous solution and homogenized to form microspheres, followed by centrifugation and freeze-drying.
  • step 2 Proceed with step (step 2).
  • the mucoadhesive polymer used here may adhere to the eye mucosa:
  • the alginate or salts thereof, gelatin, ligochitosan, chitooligosaccharide, chitosan, glycine, arginine, lysine, and cyclodextrin.
  • the emulsifier may be a conventional emulsifier used in the preparation of polymer microspheres, preferably a hydrophilic emulsifier dispersed in an aqueous solution, and more preferably sucrose. , At least one emulsifier selected from the group consisting of polyvinylpyrrolidone and polyvinyl alcohol is used.
  • polyvinyl alcohol is used.
  • the polyvinyl alcohol preferably has a molecular weight of 10,000-200,000, and more preferably,
  • the lutein dispersed oil solution, emulsifier and mucoadhesive properties In the second step, the lutein dispersed oil solution, emulsifier and mucoadhesive properties
  • the polymer-soluble aqueous solution can be appropriately adjusted according to the conditions, but is preferably mixed in a volume ratio of preferably 1: 1-50, more preferably ⁇ 1: 5-30, most preferably 1: 5-20.
  • the content of lutein can be appropriately adjusted depending on the dosage and the dosage, preferably 0.01 to 1.0% (w / v), more preferably. Ol lO ⁇ iw / v), most preferably 0.1 to 0.5% (w / v), and the content of biodegradable polymers can be adjusted appropriately depending on the conditions.
  • the content of the mucoadhesive polymer can be properly adjusted according to the conditions, preferably 0.01 to 3.0% (w / v), more preferably 0.01 -1.0% (w / v), most preferably 0.01-0.7% (w / v).
  • the amount of emulsifier can be adjusted appropriately, preferably 0.01-10% (w / v), More preferably, it is 0.1 to 5% (w / v), and most preferably, 5 to 3.0% (w / v).
  • the homogenization in the second step can be performed using a conventional homogenizer, and the degree of homogenization is almost similar to any use.
  • a stirrer, a homogenizer, and an ultrasonic grinder are used. This can be done by selecting from a group consisting of).
  • the microspheres are recovered by centrifugation, and then the microspheres are dried using one or more methods such as freeze drying, vacuum drying, and spray drying. Produce adhesive polymer capsules.
  • the double-layered polymer capsule produced according to the present invention is a microsphere form.
  • the polymer capsules may be used to determine lutein based on the total weight of polymer capsules.
  • the polymer granule capsule of the double layer structure [46] According to another aspect of the present invention, the polymer granule capsule of the double layer structure
  • composition for the prevention or treatment of ophthalmic diseases comprising.
  • the pharmaceutical composition may be applied to the prevention or treatment of eye diseases such as cataract, elderly macular degeneration, glaucoma, diabetic eye disease, dry eye, and low vision.
  • eye diseases such as cataract, elderly macular degeneration, glaucoma, diabetic eye disease, dry eye, and low vision.
  • composition for the prevention or treatment of ophthalmic diseases of the present invention may be administered orally or parenterally during clinical administration, for example, intravenous and intraarterial, intramuscular, subcutaneous, intraperitoneal, mucosal or topical (eg, It can be applied to eye drops, eyes, dermis, etc .:
  • composition may be in an oral dosage form, eg, tablets, troches,
  • lozenges aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, etc.
  • Pharmaceutically acceptable formulations for the formulation of tablets and capsules, etc. Possible carriers or
  • lactose As an additive, lactose, saccharose, solbi, manny, starch, amylopectin,
  • Binding agents such as cellose or gelatin; excipients, such as decal phosphate;
  • Disintegrating systems such as corn starch or sweet potato starch; lubricants such as magnesium stearate, calamate stearate, and stearic fumaric acid: thri may be contained.
  • lubricants such as magnesium stearate, calamate stearate, and stearic fumaric acid: thri may be contained.
  • a liquid carrier such as fatty oil may be contained in addition to the substances mentioned above. .
  • composition of the present invention can be administered parenterally, and parenteral administration is applied to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection method and mucous membrane or topical, dispersant, suppository, powder, aerosol. (Nasal sprays or inhalants), eye drops, gels,
  • Suspensions aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-water suspensions), solutions, etc., in liquid dosage forms suitable for parenteral administration. It can be prepared as a solution by mixing in water together with a topical agent or a complete agent, and can be prepared in the form of a unit dosage ampoule or a multiplex dosage.
  • composition of the present invention in particular in combination with a pharmaceutically acceptable additive,
  • the eye drop is preferably an isotonic aqueous solution or suspension.
  • isotonic agents such as sodium chloride, potassium chloride; aminocapronic acid, sodium hydrogen phosphate, And
  • Buffering agents such as sodium hydrogen phosphate; stabilizing agents such as sodium edate and sodium perborate; PH adjusting agents such as hydrochloric acid, sodium hydroxide, etc.
  • the solvent it is preferable to use sterile purified water or distilled water for injection.
  • the eye drop composition may be added with preservatives and preservatives as necessary.
  • the dosages appropriate for the pharmaceutical compositions of the present invention may be determined by the patient's type of disease, mildness of disease, It may be prescribed by factors such as the type of formulation, age of the patient, sex, weight, health condition, diet, administration time and method of administration of the pharmaceutical treatment, route of administration, rate of excretion and response sensitivity.
  • the dosage of the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg per day on an adult basis
  • Preferably it is a01-80mg / kg (weight), most preferably
  • a lutein-containing double layer polymer capsule is prepared.
  • It provides a health functional food for preventing or improving ophthalmic diseases comprising.
  • the dietary supplement may be prepared in the form of tablets, capsules, pills, or liquids.
  • the dietary supplement may contain ingredients normally added in the manufacture of the food, for example, proteins, carbohydrates, fats, various vitamins, Contains nutrients and seasonings.
  • a cosmetic composition comprising a lutein-containing double layered polymer capsule.
  • the polymer capsule of the present invention is effective in improving skin wrinkles or preventing skin aging due to the antioxidant effect of lutein.
  • stabilization and sustained release of lutein make it useful as a cosmetic composition.
  • the cosmetic composition includes softening cream, nutrient cream, nutrient cream, massage cream,
  • the cosmetic composition may also contain ingredients commonly used in cosmetics. For example, emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers and fragrances may be included. Depending on the formulation or purpose of use, the addition can be selected within a range that does not compromise the effectiveness of the cosmetic.
  • the double-layered polymer capsule of the present invention stabilizes lutein, which is useful for treating ophthalmic diseases and has antioxidant activity, and improves lutein solubility and dissolution rate, thereby exerting long-term effects of lutein. Therefore, the polymer capsule may be usefully applied as a composition for preventing or treating ocular diseases, or as a cosmetic composition for improving skin wrinkles or preventing skin aging.
  • FIG. 1 is a parallax scan for the second bilayer microspheres in Examples 1-7
  • FIG. 2 shows a differential scanning electron microscope for particles prepared in Comparative Examples 1 to 8. Electronic Microscope, SEM).
  • FIG. 3 is a graph showing the solubility comparison test results of the lutein 20% extract and the bilayer microspheres prepared according to Example 2 in Experimental Example 2 (solubility test).
  • This graph shows the measurement of lutein accumulation over time.
  • FIG. 5 shows lutein 20% extract, PLGA, and Example in Experiment 4 (analysis of DSC).
  • FIG. 7 is a diagram showing the stability results showing the residual amount (%) of lutein according to the storage temperature conditions of the double-layered microspheres manufactured according to Example 6 (Stability Test).
  • FIG. 8 is a stability result chart showing the residual amount of lutein (%) according to the storage temperature conditions of the double-layered microspheres manufactured according to Comparative Examples 1 to 6 in Experimental Example 6 (stability test).
  • the oil solution was injected into 92 ml of an aqueous solution of 500 mg to 1000 mg (manufactured by Sigma Co., Ltd.) to form a 0 / W emulsion.
  • an aqueous solution of 500 mg to 1000 mg manufactured by Sigma Co., Ltd.
  • the type and concentration of each component contained in the O / W emulsion dog C3 ⁇ 4 (w / V )) is shown in Table 1 below.
  • microspheres were stirred at room temperature for 6 hours at room temperature and cured while methylene chloride was slowly evaporated as air and centrifuged for washing with distilled water (12000 rpm, 10 minutes, 3 times). It was. Freeze-drying at -70 ° C. followed by freeze-drying for 3 days to prepare lutein-containing microspheres of Examples 1-7 with final good stability.
  • Table 1
  • microspheres were cured while stirring at room temperature for 6 hours at room temperature and under normal pressure while evaporating methylene chloride as air and centrifuged for washing with distilled water (12000 rpm, 10 minutes, 3 cycles). Recovered. Freeze-drying at -70 ° C, followed by freeze-drying for 3 days to prepare lutein-containing microspheres of Comparative Examples 1-8 with final good stability.
  • FIGS. 1 and 2 Observations were made using a scanning electron microscope (SEM), and the measurement results are shown in FIGS. 1 and 2, respectively.
  • SEM scanning electron microscope
  • Lutein 20% Extract and Micropreparative Formulation Prepared by Example 2 as Lutein 1 Purified water at a concentration of mg / mL, dispersed in various pH (1.2, 4.0, 6.8, 7.4), and then ultrasonic extraction (25 ° C, 1 h).
  • FIG. 3 shows the soluble 20% extract of the lutein microspheres in lutein. Significantly improved under various pH conditions.
  • the 20% extract (control) had almost no release for 72 hours, whereas the microspheres according to Examples 1-7 had no initial excess release, maintained zero order release and were continuously released for 72 hours, which is useful as a sustained release agent. It can be used to make sure
  • Lutein 20% extract, PLGA, alginate sodium, and PVA were measured using differential scanning calorimetry (DSC1, Mertoredo, Switzerland). Each sample was accurately weighed 2 mg and placed in an aluminum pan. The heat variance curves were measured at 10 ° C./distillation to 0-600 ° C.
  • FIG. 5A is the DSC thermal curve of 20% lutein extract. It refers to an endothermic reaction at 156.12 ° C, 431.05 o C which is related to the melting point of the foreign matter.
  • FIGS. 5C-5E are the microspheres of Example 1 (FIG. 5C) and the microspheres of Example 2 (FIG. 5d), which is the thermal curve for the microspheres of Example 3 (FIG. 5E). All show phase transition temperatures similar to PLGA, which are lutein-blocked from the external environment and stably encapsulated in the PLGA and exposed to the external environment. Positive ions mean that lutein has been stabilized efficiently.
  • Lutein 20% extract, PLGA, alginate sodium, PVA and Examples 1 to 7 were each measured using powder X-ray diffraction (D8 DISCOVER, Brookker, Germany). °, step size () .02 was obtained as ⁇ .
  • FIG. 6 is 20% lutein extract itself 2theta embodiment, while showing a crystallinity of from 10 ⁇ 30 ° examples 1 to embodiment example 7 eseoui double layer of microspheres powder diffraction pattern similar to PLGA Indicative of mandatory shaping indicates the successful inclusion in PLGA, suggesting that lutein could be effectively isolated from the external environment to ensure stability and to increase solubility due to loss of crystallinity at the particle surface.

Abstract

본 발명은 루테인을 안정화한 이중층 구조의 고분자 캡슐 및 그 제조 방법 에 관한 것으로서 상기 이중층 구조의 고분자 캡슐은 산화에 민감한 루테인을 안정화하면서도 지속적인 루테인 용출을 가능하게 하는 효과가 있어, 안과질환 예방 또는 치료용 조성물 또는 화장료 조성물로 유용하게 적용될 수 있다.

Description

명세서
발명의명칭:루테인의용출률및안정성이 개선된이중층구조의 고분자캡슐，이의제조방법 및:이를함유하는안과질환예방또는 치료용약제학적조성물
기술분야
[1] 본발명은루테인의용출률및안정성이개선된이증층구조의고분자캡슐,및 그제조방법에관한것이다.더욱구체적으로는산화에민감한루테인을 안정화시키고，안구내에서루테인의효능을장기간발휘할수있는고분자 캡슐에관한것이다.또한，상기고분자캡슐을적용할수있는안과질환예방 또는치료용조성물,또는화장료조성물에관한것이다.
배경기술
[2] 하기화학식 1의루테인은카로티노이드 (carotenoids)의잔토필류 (xanthophyll) 중에서가장다량으로존재하는색소이며 (C4(iH5602,주황색결정，융점약 193°C, 빛의흡수극대파장은 508, 475, 445 nm),이의동질이성질체인하기화학식 2의 제아잔틴 (Zeaxanthin)과함께눈의각막,동공및황반의주요구성성분으로서이 조직에존재하는유일한카로티노이드임이최근연구에의해:밝혀졌다.
Figure imgf000003_0001
[5] 루테인은눈에흡수된높은에너지의청색광에대한필터로작용하거나광촉매 기 (radical)나활성산소종을제거함으로써，눈을보호해주는역할을한다.인간의 경우카로티노이드를합성하지못하므로루테인이포함된음식또는
건강보조식품을통해루테인을섭취할수있다.
[6] 루테인을섭취하면황반색소의밀도가증가하여눈질환이 있는환자들에게서 시각기능을개선시킬수있으며，눈의유해활성산소에의한시세포손상에따른 노화과정을효과적으로억제함으로써 백내장이나노인성황반변성 (Age-related macular degeneration)과같은안구질환의발생율을감소시킬수있다 (Bernstein P.S. et al., Ophthalmology, 2002(109), 1780-1787; Kvansakul J. et al., Ophthalmic and Physiological Optics, 2006(26), 362-371).
[7] 그런데，눈은생물학적장벽의여러층에의해분리되어 있기때문에루테인을 황반에국소전달시키는데에 있어효율성올어떻게증진시킬것인지가 문제되고있다.루테인은황반에도달하기위해각막의견고한접합，점막표면, 혈액망막장벽을포함하는장애물을극복해야한다.경구투여가간초회통과 효과 (liver first-pass effect)를피할수없는반면에，투여편리한점안액 (eye drops)은누액과함께소실및배출됨으로써안구에남아서약효를 :발휘하는 양이적어안구생체이용률 (ocular bioavailability)이낮은문제가있고점도를 상승시킨연고제등의경우는점안감이불량하고점안과정이불편한등의 문제가있다.
[8] 또한，루테인은 9개이상의이중결합이 있는불포화구조를포함하고있어빛, 열,공기 (산소)등의환경에매우불안정하며쉽게산화된다.따라서，차광，저은, 진공등의조건에서저장되어야하며보호제를첨가하여산화를방지시켜야 한다ᅳ아울러 ,루테인은물에아주난용성이며，지방및오일에도용해도가낮다. 특하루테인의매우낮은수용해도 (1.3xl09 M)는생체이용률에단점을 나타낸다.
[9] 일반적으로난용성약물은생체내에서의용출속도가느려흡수속도가늦고: 이에따라약효의발현및생체이용률도낮아지므로，난용성약물의
생체어용률을증가시키기위해서는용출률이우수한제제를설계하는것이 요구된다.
[10] 이에따라，루테인，오일및계면활성제를함유하는액상제제를제조하여
루테인의난용성을개선시키고자하는연구들이시도되었으나，용해성이 개선된제제를제조하는것은어려을뿐만아니라，용해성이일부극복되더라도 보관중에층분리현상또는주성분의침전현상이일어나는등일반적으로 알려져있는방법으로는안정성및용출률이우수한루테인약학제제의개발이 쉽지않다.또한，루테인등의카로티노이드는세포재생및항산화능에의해 피부주름개선및노화방지등의우수한효능으로인해화장품에와도입이 시도되어왔지만，공기나수분둥외부자극과의접촉으로쉽게변색및변취 . 현상이일어나고역가가장기적으로떨어져효능이감소하는문제점을가진다.
[11] 이러한문제점들을개선하기위한종래선행기술로서한국공개특허
제 2009-0070161호는카로티노이드를포함하는양이온성고분자캡슬과상기 캡슐의표면에코팅된음이온성고분자층을포함하는고분자캡술을개시하고 있고，한국공개특허제 2012-0108170호는루테인,인지질，오일및계면활성제의 흔합물을액상형태로연질또는경질캡슐에층진시켜 약물의용출률및 생체이용률을제고시킨제제를개시하고있으며 ,국제공개특허
WO2016/025394는제인단백질또는폴리머를포함하는나노입자로
캡슐화하거나，또는상기나노입자와하이드로겔의흔합물형태의조성물을 개시하고있으나，이들선행기술도루테인의안정성및용출률을모두 개선시키는데에한계가있다.
[12] 이에，본발명자들은,루테인을안정화하면서도지속적인루테인용출을
가능하게하는새로운이중층구조의고분자캡슐을제조하여，기존제제의 한계를극복하는.효과가있음을확인하고본발명을완성하게되었다.
발명의상세한설명
기술적과제
[13] 본발명의목적은,루테인을안정화할수있으며,루테인의 ^출률이개선된 이중층구조의고분자캡슐및그제조방법을제공하는것이다.
[14] 또한，본발명의다른목적은상기고분자캡슐을함유하여，활성물질의효능을 장기간발휘할수있는안과질환예방또는치료용약제학적조성물또는 건강기능식품을제공하는것이다.
[15] 본발명의또다른목적은상기고분자캡슐을함유하여，항산화활성을갖는 화장료조성물을제공하는것이다.
과제해결수단
[16] 본발명은，생리활성물질로서루테인또는루테인함유추출물을포함하는 코어 ;상기코어를둘러싸고있는생분해성고분자층;및상기생분해성 고분자층상에코팅된，점막부착성고분자와유화제의흔합층；을포함하는 이중층구조의고분자캡슐에관한것이다.
[17] 상기루테인함유추출물은,루테인함유추출물의총중량을기준으로
루테인을 10~90%(w/w)의양으로포함할수있다.
[18] 상기고분자캡슐은,고분자캡슐총중량을기준으로루테인을
1.0~95.0%(Ww)로함유할수있다.바람직하게는 3.0~90.0%(w/w)로함유할수 있고，보다바람직하게는 5.0~75.0%(w/w)로함유할수있다.
[19] 상기생분해성고분자층은폴리 -L-락트산，폴리 -글리콜산，
폴리 -D-락트산-코 -글리콜산，폴리 -L-락트산—코 -글리콜산,
폴리 -D,L-락트산-코 -글리콜산，폴리-카프로락톤，폴리-발레로락론，
폴리 -하이드록시부티레이트및폴리 -하이드록시발러레이트로구성된 군으로부터선택된 1종이상으로이루어질수있다.
[2이 상기점막부착성고분자는,알지네이트또는그의염 ,젤라틴，
올리고키토산 (chitooligosaccharide),키토산 (chitosan),글라아신 (glycine), 아르기닌 (arginine),라이신 (lysine),및사이클로덱스트린 (cyclodextrin)으로 구성된군에서선택된 1종이상일수있다.
[21] 상기유화제는수크로오스 (sucrose),폴리비닐피를리돈 (polyvinyl pyrrolidone) 및폴리비닐알코올 (poly vinyl alcohol)로구성된군으로부터선택된 1종이상일수 있다.
[22] 본발명의다른양태에따르면，상기이증층구조의고분자미립구캡술의
제조방법에관한것이다.구체적으로，상기제조방법은:，생리활성물질인루테인 또는루테인함유추출물과，생분해성고분자를유기용매에용해시켜， 1차 에멀견을생성하는단계 (1단계);상기 1차에멀견을점막부착성고분자와 유화제가용해된수용액에분산시키고균질화하여미립구를형성한후 원심분리및동결건조하는단계 (2단계)；를포함한다.
[23] 본발명의다른양태에따르면,상기이중층구조의고분자캠슐을포함하는 안과질환예방또는치료용약제학적조성물에관한것이다.
[24] 본발명의다른양태에따르면，상기이중층구조의고분자캡슐을포함하는 안과질환예방또는개선용건강기능식품에관한것이다.
[25] 본발명의또다른양태에따르면,상기이중층구조의고분자캡슐을포함하는 화장료조성물에관한것이다.
[26] 이하，본발명을더상세하게설명한다.
[27] 본발명은코어 -쉘 (Core-shell)형태의이중층구조의고분자캡슐로서 ,산화에 민감한루테인을생분해성고분자입자로일차캡슐화하고,이일차캡슐의 외벽에점막부착성고분자와유화제의흔합층을형성함으로써 ,루테인을 안정화시키면서;도，루테인의효능이안구내에서장기간발휘할수있는효과가 있다.
[28] 본명세서에서， '고분자캡슐'이란루테인또는루테인함유추출물이생분해성 고분자，및점막부착성고분자와유화제에의해캡슐화된것 (encapsulation)을 의미하며， '미립구，, '고분자미립구，또는 '고분자미립구캡슐，이라고도 지칭한다.
[29] 본발명의이중층구조의고분자캡슐의제조방법은，생리활성물질인루테인 또는루테인함유추출물과，생분해성고분자를유기용매에용해시켜, 1차 에멀젼을생성하는단계 (1단계)；상기 1차에멀견을점막부착성고분자와 유화제가용해된수용액에분산시키고균질화하여미립구를형성한후 원심분리및동결건조하는단계 (2단계)；를포함한다.
[30] 상기 1단계에 있어서，생리활성물질로서의루테인은루테인자체로써분리된 순도 90%이상의순수한물질도이용될수있으나，식물또는동물로부터추출한 루테인 10~90%함유추출물이이용될수있다.본발명에 있어서，바람직하게는 루테인 10~40%함유추출물이이용될수있고，이때추출물은식물성오일에 분산시킨형태일수있으며，상기식물성오일로서해바라기씨유，홍화씨유， 올리브유등이이용될수있다.
[31] 상기생분해성고분자는미립구제조시사용되는통상의고분자들이사용될수 있으며，고분자의종류를제한하지는않지만，본발명에서는폴리 -L-락트산， 폴리 -글리콜산,폴리 -D-락트산-코 -글리콜산，폴리 -L-락트산-코 -글리콜산， 폴리 -D,L-락트산-코 -글리콜산，폴리-카프로락톤，폴리-발레로락톤,
폴리 -하이드록시부티:레이트및폴리 -하이드록시발러레이트로이루어진 군에서 1종이상이사용될수있다.또한，바람직하게는폴리 -L-락트산, 폴리 -D-락트산-코 -글리콜산,폴리 -L-락트산-코-글리콜산및 폴리 -D,L-락트산-코-글리콜산 (PLGA)으로이루어지는군으로부터선택되는 1종 이상의고분자를사용할수있다.바람직하게는
폴리 -D,L-락트산-코-글리콜산 (PLGA)및폴리 -L-락트산중에서단독또는 2종을 흔합하여사용한다.
[32] 상기생분해성고분자는중량평균분자량이 60,000이하인것,바람직하게는 60，000~5，000인것이사용된다.예를들면,분자량약 13,000인
폴리 -D,L-락트산-코 -글리콜산 (50:50)，분자량약 33,000인
폴리 -D,L-락트산-코-글리콜산 (50:50)，분자량약 52,000인
폴리 -D,L-락트산-코-글리콜산 (50:50)，분자량약 20,000인
폴리 -D,L-락트산-코-글리콜산 (75:25)，분자량약 16,000인폴리 -L-락트산 (100:0) 등을사용하는것이가능하다.
[33] 이와같은생분해성고분자는，예를들면，베링거인겔하임 (Boehringer
Ingelheim)사의 Resomer L 206S, L 207S, L 209S, L 210， L 210S, LR 704S, LR 706S, LR 708, LR 927S, LG 824S, LG 855S, LG 857S, LC 703S, R 207S, RG 509S, X 206S, R 202S, R 202H, R 203H, R 205S, RG 502, RG 502H, RG 503, RG 503H, RG 504, RG 504H, RG 505, RG 653H, RG 752H, RG 752S, RG 753S, RG 755S, RG 756S, RG 757S, RG 750S, RG 858S일수있다.
[34] 또한，상기 : 1단계에서,유기용매로는생분해성고분자에대한용매성과증발을 통한제거용이성이모두뛰어난휘발성유기용매이면특별히제한되지않으나， 바팜직하게는메틸렌클로라이드,에틸아세테이드,클로로포름，아세톤， 다이메틸설폭사이드，다이메틸포름아마이드， N-메틸피롤리돈,다이옥산, 테트라하이드로퓨란，에틸아세테이트，메틸에틸케톤및아세트니트릴에서 선택되는 1종의용매또는 2종이상의흔합용매가사용될수있으며，더
바람직하게는메틸렌클로라:이드，에틸아세테이트，클로로포름및
테트라하이드로퓨란에서선택되는 1종의용매또는 2종이상의흔합용매가 사용될수있으며,가장바람직하게는메틸렌클로라이드및
에틸아세테이트에서선택되는 1종또는 2종의혼합용매가사용될수있다.이때， 본발명의실시예에서는，생분해성고분자와루테인 20%추출물을용해시키는 유기용매로서，수용해도가 20g/L(20°C)로낮은편인메틸렌클로라이드가 사용되었다.
[35] 다음으로,상기 1차에멀견을점막부착성고분자와유화제가용해된수용액에 분산시키고균질화하여미립구를형성한후원심분리및동결건조하는
단계 (2단계)를진행한다.
[36] 이때사용되는점막부착성고분자는눈점막에부착될수:있는물질이라면
어떤것이라도사용가능하다.바람직하게는알지네이트：또는그의염,젤라틴, 을리고키토산 (chitooligosaccharide),키토산 (chitosan),글라이신 (glycine), 아르기닌 (arginine),라이신 (lysine),및사이클로텍스트린 (cyclodextrin)으로 이루어지는군에서선택되는 1종이상의점막부착성고분자가사용되며，보다 바람직하게는알지네이트，또는그의염이사용되며,가장바람직하게는 알지네이트소디움이사용된다.점막부착성고분자를사용하여미립구를 형성함으로써 ,눈점막에서의체류시간을길게하는효과가있다.
[37] 또한，상기유화제로는고분자미립구를제조할때사용하는통상의유화제를 이용할수있으며，바람직하게는수용액에분산되는친수성유화제 (hydrophilic emulsifier)를사용할수있고，보다바람직하게는수크로오스 (sucrose), 폴리비닐프를리돈 (poly vinyl pyrrolidone)및폴리비닐알코올 (poly vinyl alcohol)로 이루어지는군으로부터 1종이상선택되는유화제가사용되며,가장
바람직하게는폴리비닐알코올을사용한다.또한，상기폴리비닐알코올은 바람직하게는분자량 10,000-200,000인것을사용하며，더바람직하게는
50,000-200,000,가장바람직하게는 100,000~200，000인것을사용한다.
[38] 또한，상기 2단계에서,루테인분산체오일용액과，유화제및점막부착성
고분자가용해된수용액은,조건에따라적절하게조절가능하나，바람직하게는 1:1-50,더바람직하게^ 1:5-30,가장바람직하게는 1:5~20의부피비로흔합되어 분산될수있다.
[39] 상기 2단계에서루테인분산체용액을수용액에분산시킴으로서 OA 형의 이중유화미립구가형성될수있다.
[40] 상기미립구를포함하는 0/W형에멀견용액에 있어서，루테인의함량은용법， 용량에따라적절히조절가능하나,바람직하게는 0.01~1.0%(w/v)이고，더 바람직하게는 O.l l.O^i w/v),가장바람직하게는 0.1~0.5%(w/v)이고,생분해성 고분자의함량은조건에따라적절하게조절가능하나，바람직하게는
0.05~2.5%(w/v),더바람직하게는 0.1~1.0%(w/v),가장바람직하게는
0.25~1.0%(w/v)이다.
[41] 또한,상기 0/W형에멀젼용액에 있어서,점막부착성고분자의함량은조건에 따라적절하게조절가능하나，바람직하게는 0.01~3.0% (w/v),더바람직하게는， 0.01-1.0% (w/v),가장바람직하게는 0.01~0.7% (w/v)이다.아을러，유화제의 함량도적절하게조절가능하나，바람직하게는 0.01~10%(w/v),더바람직하게는 0.1~5%(w/v),가장바람직하게는으 5~3.0%(w/v)이다.
[42] 상기 2단계에서의균질화는통상적인균질기를이용하여수행할수있으며， 어떤것을사용해도균질화정도는거의유사하다.이때，바람직하게는 교반기 (Stirrer),균질기 (Homogenizer)및초음파분쇄기 (Ultrasonicator)로 이루어진군에서선택되는것을이용하여수행할수있다.
[43] 또한，동결건조하여미립구를건조하기위해，원심분리법을통해미립구를 회수한후미립구를동결건조법，진공건조법,분무건조법등한가지이상의 방법을사용하여건조하여，이중층구조의생분해성,점막부착성고분자캡슐을 생산한다.
[44] 본발명에따라생성된이중층구조의고분자캡슐은,미립구형태를
유지하면서내부에루테인을함유하여루테인을안정화시키고，고분자가 서서히분해되면서내부의루테인을지속적으로방출해주는효과가있다.
[45] 상기고분자캡술은,고분자캡슐총중량올기준으로루테인을
1.0~95.0%(w/w)로함유한다.바람직하게는 3.0~90.0%(w/w)로함유하고，보다 바람직하게는 5.0~75.0%(w/w)로함유한다.
[46] 본발명의다른양태에따르면,상기이중층구조의고분자미립구캡슐을
포함하는안과질환예방또는치료용약제학적조성물을제공한다.
[47] 상기약제학적조성물은,백내장,노인성황반변성，녹내장，당뇨병성안과질환, 안구건조증,저시력등의안과질환의예방또는치료에적용될수있다.
[48] 본발명의안과질환예방또는치료용약제학적조성물은임상투여시에경구 또는비경구로투여할수있으며 ,예를들어정맥및동맥내，근육내，피하，복강 내，점막또는국소 (예，점안),안구，경피등에적용될수있다.:
[49] 상기조성물은경구투여용제형 ,예를들면，정제，트로키제 (troches),
로진지 (lozenge),수용성또는유성현탁액，조제분말또는과립，에멀젼，경질 또는연질캡슐,시럽또는엘릭실제 (elixirs)등으로제제화된다.정제및캡슐 등의제형으로제제화하기위해，약제학적으로허용가능한담체또는
첨가제로서,락토오스，사카로오스,솔비를，만니를，전분，아밀로펙틴,
셀를로오스또는젤라틴과같은결합제;디칼슴포스페이트와같은부형제;
옥수수전분또는고구마전분과같은붕괴계;스테아린산마그네슘，스테아린산 칼슴，스테아릴푸마르산나:트륨과같은활택제가함유될수있다.캡술제형의 경우는상기에서언급한물질이외에도지방유와같은액체담체를함유할수 있다.
[50] 또한，본발명의조성물은비경구로투여할수있으며 ,비경구투여는피하주사, 정맥주사，근육내주사,복강내주사주입방식과점막，또는국소에적용되는데， 분산제，좌제，분제,에어로졸 (비강스프레이또는흡입제),점안제，겔，
현탁액제 (수성,또는비수성액상현탁액，수중유에멀견또는유중수에멀견)， 용액제등비경구투여에적합한액상투여형태둥에의한다ᅳ비경구투여용 제형으로제제화하기위해서는상기조성물을안정화제또는완층제등과함께 물에서흔합하여용액으로제조하고，단위투약앰플또는다수화투약형태로 제조할수있다.
[51] 본발명의조성물은，특히 약학적으로허용가능한첨가제와함께배합하여
점안제로제형화할수있고，점안제는통상등장성수용액또는현탁액인것이 바람직하다.상기약학적으로허용가능한첨가제로서，예를들면,염화나트륨， 염화칼륨등의등장화제;아미노카프론산,인산일수소나트륨，및
인산어수소나트륨등의완충제;에데트산나트륨,과붕산나트륨등의안정화제; 염산，수산화나트륨등의 pH조절제등을들수있다.용제로는멸균정제수또는 주사용증류수를사용하는것이바람직하다.상기점안제조성물에는필요에 따라보존제，방부제둥을부가할수있다.
[52] 본발명의약제학적조성물와적합한투여량은환자의질환종류，질환의경증， 제형의종류，환자의연령，성별，체중，건강상태,식이，약제학적치료용 조성물의투여시간및투여방법，투여경로，배설속도및반응감웅성과같은 요인들에의해다양하게처방될수있다.바람직하게는，본발명의약제학적 조성물의투여량은성인기준으로 1일당 0.001~100mg/kg이고，보다
바람직하게는 a01~80mg/kg (체중)이며，가장바람직하게는
0.1~60mg/kg (체중)이다.또한，의사또는약사의판단에따라일정시간간격으로
1일 1회내지수회로분할투여할수도있다.특히，점안투여의경우에는， 0.001 내지 3%(w/v,이하동일),바람직하게는 0.01내지 1%정도의제제를 1일 1회 내지수회점안한다.
[53] 본발명의다른양태에따르면,루테인함유이중층구조의고분자캡슐을
포함하는안과질환예방또는개선용건강기능식품을제공한다.상기
건강기능식품은정제,캡슐제，환제또는액제등의형태로제조될수있다.상기 건강기능식품은식품제조시통상적으로첨가되는성분을포함할수있으며, 예를들어단백질,탄수화물，지방,각종비타민，영양소및조미제를포함한다.
[54] 본발명의또다른양태에따르면,루테인함유이중층구조의고분자캡술을 포함하는화장료조성물을제공한다.본발명의고분자캡슐은,루테인의항산화 효과에의한피부주름개선또는피부노화방지효과를가질뿐만아니라， 루테인의안정화및지속적방출을가능하게하므로，화장료조성물로유용하게 적용될수있다.
[55] 상기화장료조성물은유연화장수,영양화장수，영양크림，맛사지크림,
에센스，아이크림，해어토닉，샴푸，린스，클렌징크림，클렌징폼，클렌징워터，팩， 스프레이및파우더의제형으로제조될수있다.또한,상기화장료조성물은 화장료에통상적으로이용되는성분을포함할수있다.예를들면，유화제, 점증제,유제，계면활성제,윤활제，알코올류，수용성고분자제，겔화제， 안정화제,비타민，무기염류，유화제，향료같은일반적인보조성분을포함할수 있다.상기성분들은제형또는사용목적에따라그첨가량을화장료고유의 효과를손상시키지않는범위내에서선택할수있다.
발명의효과
[56] 본발명의이중층구조의고분자캡슐은，안과질환치료에유용하고항산화한 활성을갖는루테인을안정화하면서，루테인의용해도및용출률을개선시켜， 루테인의효능을장기간발휘시킬수있는효과가있다.따라서，상기고분자 캡슐은，안과질환예방또는치료용조성물，또는피부주름개선또는피부 노화방지를위한화장료조성물로유용하게적용될수있다.
도면의간단한설명
[57] 도 1은실시예 1내지 7에서제 2된이중층미립구에대한시차주사
전자현미경 (Scanning Electronic Microscope, SEM)사진아다.
[58] 도 2는비교예 1내지 8에서제조된입자에대한시차주사전자현미경 (Scanning Electronic Microscope, SEM)사진이다.
[59] 도 3은실험예 2(용해도시험)에 있어서，루테인 20%추출물과실시예 2에따라 제조된이중층미립구의용해도비교시험결과를나타낸그래프이다.
[60] 도 4는실험예 3(In vitro시험관내방출)에 있어서，실시예 1내지 7의시간
경과에따른루테인누적방출량을측정하여나타낸그래프이다.
[61] 도 5는실험예 4(DSC의분석)에 있어서，루테인 20%추출물， PLGA,및실시예
1, 2및 5에따라제조된이중층미립구의 DSC의결과를나타낸도면이다.
[62] 도 6은실험예 5(PXRD의분석)에 있어서,루테인 20%추출물，
알지네이트소디움， PLGA,폴리비닐알코올,및실시예 1내지 7에따라제조된 이중층미립구의 PXRD의결과를나타낸도면이다.
[63] 도 7은실험예 6(안정성시험)에：있어서,실시예 1, 2및 5에따라제조된이중층 미립구의보관온도조건에따른루테인의잔존량 (%)을나타낸안정성결과 도면이다.
[64] 도 8은실험예 6(안정성시험)에 있어서,비교예 1내지 6에따라제조된이중층 미립구의보관온도조건에따른루테인의잔존량 (%)을나타낸안정성결과 도면이다.
발명의실시를위한최선의형태
[65] 이하본발명의바람직한실시예를상세하설명하기로한다.그러나,본발명은 여기서설명되는실시예에한정되지않고다른형태로구체화될수도있다. 오히려，여기서소개되는내용이철저하고완전해지고,당업자에게본발명의 사상을층분히전달하기위해제공하는것이다.
[66] <실시예 1내지 7.본발명에따른미립구의쩨조 (OAV형 )>
[67] ：하기표 1의실시예 1내지 7의성분조성으로미립구제형을제조하기위해， 먼저생분해성고분자 PLGA 250mg~l,000mg(Boehringer Ingelheim)및루테인 20%추출물 500mg (루테인 100mg함유，해바라기씨유에분산된마리골드( Tagetes erecta L.)추출물，프랑스 DSM사제품)을메틸렌클로라이드쎄완전히 용해시켜오일용액 (8ml)을제조하였다.
[68] 알지네이트소디움 100mg~500mg (삼전화학제품)및폴리비닐알코올
500mg~1000mg(Sigma사제품)이용해된수용액 92ml에상기오일용액을 주입하여 0/W형에멀젼을형성하였고，이때각실시예에 있어서 O/W형 에멀견에함유된각성분의종류및농도 C¾(w/V))를정리하면하기표 1과같다.
[69] 이후，초음파분쇄기를사용하여균질화시킨후유화시켜미립구를
형성하였다.이렇게형성된미립구를 6시간동안실온에서 ,상압하에서 교반하여메틸렌클로라이드를공기중으로서서히증발시키면서경화시키고 이를증류수로세척을위해원심분리한후 (12000 rpm, 10분， 3회) pellet을 회수하였다. -70°C에서동결한다음 3일간동결건조하여최종적으로우수한 안정성을지닌,실시예 1내지 7의루테인함유미립구제형을제조하였다. [70] [표 1]
Figure imgf000012_0001
[71] <비교예 1내지 8.미립구의제조 (O/W형)〉
[72] 하기표 2의비교예 1내지 8의성분조성으로미립구제형을제조하기위해, 먼저생분해성고분자 PLGA 250mg~500mg(Boehringer Ingelheim)및루테인 20% 추출물 500mg (루테인 lOOmg함유,해바라기씨유에분산된마리골드 (ragetes erecta L.)추출물,프랑스 DSM사제품)을메틸렌클로라이드에완전히용해시켜 오일용액 (8ml)을제조하였다.점막부착성고분자로서 POLYOX™또는 Tween, 및 /또는유화제로서폴리비닐알코올또는폴록사머가용해된수용액 92ml에 상기오일용액을주입하여 0/W형에멀견을형성하였고，이때각비교예에 있어서 0/W형에멀젼에함유된각성분의종류및농도 (9 (w/v))를정리하면 하기표 2와같다.
[73] 이후,초음파분쇄기를사용하여균질화사킨후유화시켜미립구를
형성하였다.이렇게형성된미립구를 6시간동안실온에서,상압하에서 교반하여메틸렌클로라이드를공기중으로서서히증발시키면서경화시키고 이를증류수로세척을위해원심분리한후 (12000 rpm, 10분， 3희) pellet을 회수하였다. -70°C에서동결한다음 3일간동결건조하여최종적으로우수한 안정성을지닌，비교예 1내지 8의루테인함유미립구제형을제조하였다.
[74] [표 2]
Figure imgf000013_0001
[75] <실험예 1. PLGA함유미립구의표면형태측정 >
[76] 실시예 1내지 7，및비교예 1내지 8에따라제조된미립구의표면을
주사전자현미경 (SEM)을사용하여관찰하고，그측정결과를도 1및도 2에각각 나타내었다.도 1에와하면，본발명의실시예 1내지 7에따라,이중층구조의 표면이매끄러운미립구 (고분자:캡술)가형성된점이확인된다.반면에，비교예 1 내지 8에의해서는，도 2에서：확인되는바와같이미립구가전혀형성되지않고 그물구조의결정성이그대로남아있거나，또는미립구가완전하지않은형태로 형성된상태였다.
[77] <실험예 2.용해도시험 >
[78] 루테인 20%추출물및실시예 2에의해제조된미립구제형을루테인으로써 1 mg/mL의농도로정제수,다양한 pH (1.2, 4.0, 6.8, 7.4)에분산시킨후,초음파 추출 (25°C, 1 h)을하였다. HPLC조건에따라생리활성물질인루테인 20% 추출물및실시예 2에의해제조된미립구제형을루테인으로서용해도를 측정하였다 (n=3).도 3은이증층미립구의용해도가비교군인루테인 20% 추출물에비해다양한 pH조건에서크게개선되었음을나타낸다ᅳ
[79] HPLC분석조건
[80] 하기조건을이용하여용해도시험의샘플 (n=3)을 HPLC분석하였다.
[81] 장비 : Agilent 1 100 series
[82] 검출기 : UV (446 nm)
[83] 컬럼: Prontosil;C18 (5 ra, 4.6 x 150 mm)
[84] 이동상: 0.1%암모늄아세테이트를포함한메탄올：아세토니트릴：
디클로로메탄 = 2:6:2 (V/V/V)
[85] 유속: 1.0 mL/분
[86] 주입량: 10 μ
[87] 유의성평가 : Τ < 0.05， * < 0.01
[88] <실험예 3.제조된미립구의 In vitro시험관내방출 >
[89] 하기의조건에서실시예 1 ~7에서제조된루테인함유미립구의시험관내 (in vitro)루테안방출특성평가를수행하였다.루테인으로써 10 mg의건조된 미됩:구들을 10mL의 PBS (Phosphate buffered saline, pH 7.4, 1M)용액으로 분산시킨후프랜즈다퓨견셀 (Franz Diffusion Cell,랩화인， FCDV-15)의멤브레인 free모델을이용하여 32±0.5°C, 200 rpm조건에서시간경과에따라 lmL샘플을 취하고여과하여약물꾀농도를실험예 2의 HPLC분석조건에따라 HPLC로 측정하여미립구로부터방출되는루테인의양을측정하였다.샘플을취한후 신선한 PBS 7Λ로다시보층하였다. 72시간동안측정된미립구로부터방출되는 루테인의방출량 (%)를도 3에나타내었다 (n=3).도 4를참고하면,루테인
20%추출물 (대조군)은 72시간동안거의방출이일어나지않은반면에，실시예 1 내지 7에따른미립구는초기과다방출이없고， 0차방출을유지하며 72시간 동안지속적으로방출되므로서방형제제로유용하게사용될수있음아 확인된다.
[90] <실험예 4.루테인 20%추출물및제조된미립구의시차주사열량법 (DSC)의 분석 >
[91] 루테인 20%추출물， PLGA,알지네이트소디움, PVA를각각시차주사열량기 (DSC1,메를러토레도,스위스)를이용하여측정하였다.각각의샘플은 2 mg 정확히취해알루미늄팬에올려놓고 0-600oC까지 10oC/분승온시키며열변량 곡선을측정하였다.도 5a는루테인 20%추출물의 DSC열곡선이다. 156.12°C, 431.05oC에서흡열반응을나타내며이것은이물질의녹는점과관계있다.
[92] 도 5b은 PLGA (50:50)의 DSC열곡선이다. 46.81°C, 338.63°C에서흡열반웅을 나타낸다.도 5c내지 5e는각각실시예 1의미립구 (도 5c),실시예 2의미립구 (도 5d),실시예 3의미립구 (도 5e)에대한열곡선이다.모두 PLGA와유사한상전이 온도를나타내는바,이는루테인이외부환경으로부터차단되어 PLGA에 안정적으로봉입되었고，외부환경에노출되는루테인의양이제로화되어 루테인이효율적으로안정화되었음을의미한다ᅳ
[93] <실험예 5.제조된미립구의분말 X-선회절 (PXRD, Powder X-Ray
Diffraction)의분식 >
[94] 루테인 20%추출물， PLGA,알지네이트소디움, PVA및실시예 1내지 7을각각 분말 X-선회절 (D8 DISCOVER,브룩커,독일)을이용하여측정하였다.데이터는 산란각 2theta 5-70°, step size ().02ο으로얻었다.도 6는루테인 20%추출물 자체에는 2theta 10~30°에서결정성을나타낸반면실시예 1내지실시예 7의 이중층미립구에서의분말회절은 PLGA와유사한패턴의무정형을나타내어 PLGA에봉입이성공적으로이루어졌음을나타낸다.이는루테인을효과적으로 외부환경으로부터차단하여안정성을보장하고입자표면의결정성소실로 인해용해도를높일수있었다는점을제시하는것이다.
[95] <실험예 6.제조된미립구의안정성시험 >
[96] (1)루테인 20%추출물,및실시예 1,실시예 2，실시예 5를각각 4°C,실온， 40°C에 저장후시간 (초기， 1주, 2주, 4주)의변화에따른안정성을루테인 보유양으로써시험하였다.초기는동결건조가완료된시점에측정되었고 샘플은모두 3회시험되었다.
[97] 4°C에서비교군인루테인 20%추출물및실시예 1, 2，및 5의이중층미립구가 4주동안안정한결과를확인할수있었다 (도 7a).
[98] 실은에서비교군인루테인 20%추출물은초기에비교해서 4주후 -10.2%의 함량감소를보인반면실시예 1, 2，및 5의이중층미립구의시험결과는각각 -5.1%, -2.7%,및 -2.5%의경시변화를확인할수있었다 (도 7b).
[99] 40°C에서저장한비교군인루테인 20%추출물은초기에비교해서 1주， 2주및 4주후，각각， -16.3%. -25.2%, -40.8%의눈에띄는함량감소 #보여루테인 자체가열에취약하다는사실을재확인할수있었다.그러나，본발명의실시예 1, 2，및 5의이증층미립구의시험결과는 4주후측정결과각각 -4.3%, -6.7%,및 -4.8%의경시변화만을나타내어,본발명이루테인을완전히캡슬화함으로써 온도에 예민한자체의성질을보호함으로써안정:성이현저하개선되었음을 확인할수있었다 (도 7c).
[100] (2)루테인 20%추출물,및비교예 1내지 6을각각 40C,실욘, 40oC에저장후 시간 (초기， 1주및 2주)의변화에따른안정성을루테인보유양으로써시험 하였다.초기는동결건조가완료된시점에측정되었고샘플은모두 3회시험 되었다.
[101] 비교예 1내지 6은,이미실험예 1을통해미립구가전혀형성되지않거나， 불완전한미립구를형성하는것으로확인되었던바，도 8에서와같이, 4°C,실은, 및 40°C에서의 2주안정성시험결과，모든시험조건에서루테인 20%추출물 보다역가가현저히저하되어안정성이확보되지않는것으로나타났다.

Claims

청구범위
[청구항 1] 생리활성물질로서루테인또는루테인함유추출물을포함하는코어;
상기코어를둘러싸고있는생분해성고분자층;
상기생분해성고분자층위에코팅된,점막부착성고분자및유화제함유 코팅층;
을포함하는이증층구조의고분자캡슐.
[청구항 2] 제 1항에 있어서，
상기루테인함유추출물은,루테인을 10~90%(w/w)의양으로포함하는 것을특징으로하는고분자캡슐.
[청구항 3] 제 1항에 있어서， '
상기고분자캡슐은고분자캡술총중량을기준으로루테인을 1.0~ 95.0%(w/w)로함유하는것을특징으로하는고분자캡슐.
[청구항 4] 제 1항에 있어서，
상기생분해성고분자층은폴리 -L-락트산，폴리 -글리콜산， 폴리 -D-락트산-코 -글라콜산，폴리 -L-락트산-코 -글리콜산， 폴리 -D,L-락트산-코 -글리콜산，폴리-카프로락톤，폴리-발레로락톤， 폴리 -하어드록시부티레이트및폴리 -하이드록시발러레이트로구성된 군으로부터선택된 1종이상의생분해성고분자로이루어진것을 특징으로하는이중층구조의고분자캡슐.
[청구항 5] 제 1항에있어서,
상기점막부착성고분자는，알지네이트또는그의염 ,젤라틴, 을리고키토산 (chitooligosaccharide),키토산 (chitosan),글라이신 (glycine), 아르기닌 (arginine),라이신 (lysine),및사이클로덱스트린 (cyclodextrin)으로 구성된군에서선택된 1종이상의점막부착성고분자인것을특징으로 하는이중층구조의고분자캡슐.
[청구항 6] 게 1항에 있어서，
상기유화제는수크로오스 (sucrose),폴리비닐파롤리돈 (polyvinyl pyrrolidone)및폴리비닐알코올 (polyvinyl alcohol)로구성된군으로부터 선택된 1종이상인것을특징으로하는이증층구조의고분자캡슐.
[청구항 7] 제 1항내지제 6항증어느한항에기재된이중층구조의고분자캡슬의 제조방법으로서， ：
생리활성물질인루테인또는루테인함유추출물과，생분해성고분자를 유기용매쎄용해시켜 , 1차에멀젼을생성하는단계 (1단계); 상기 1차에멀젼을점막부착성고분자와유화제가용해된수용액에 분산시키고균질화하여미립구를형성한후원심분리및동결건조하는 단계 (2단계);
를포함 -하는것을특징으로하는이중층구조의고분자캡슐의제조방법.
[청구항 8] 제 1항내지제 6항중어느한항에기재된이증층구조의고분자캡술을 포함하는안과질환예방또는치료용약제학적조성물.
[청구항 9] 제 1항내지제 6항증어느한항에기재된이증층구조의고분자캡슐을 포함하는안과질환예방또는개선용건강기능식품.
[청구항 10] 제 1항내지제 6항증어느한항에기재된이중층구조의고분자캡슐을 포함하는화장료조성물.
PCT/KR2016/011639 2016-08-12 2016-10-17 루테인의 용출률 및 안정성이 개선된 이중층 구조의 고분자 캡슐, 이의 제조방법 및 이를 함유하는 안과질환 예방 또는 치료용 약제학적 조성물 WO2018030579A1 (ko)

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