WO2011142504A1 - Polymeric nanospheres containing hydroxybenzyl alcohol - Google Patents

Polymeric nanospheres containing hydroxybenzyl alcohol Download PDF

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Publication number
WO2011142504A1
WO2011142504A1 PCT/KR2010/005522 KR2010005522W WO2011142504A1 WO 2011142504 A1 WO2011142504 A1 WO 2011142504A1 KR 2010005522 W KR2010005522 W KR 2010005522W WO 2011142504 A1 WO2011142504 A1 WO 2011142504A1
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Prior art keywords
nanoparticles
hba
copolyoxalate
drug
hp0x
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PCT/KR2010/005522
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French (fr)
Korean (ko)
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이동원
박현진
김세호
성경렬
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전북대학교산학협력단
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Publication of WO2011142504A1 publication Critical patent/WO2011142504A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/12—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from polycarboxylic acids and polyhydroxy compounds
    • C08G63/16—Dicarboxylic acids and dihydroxy compounds
    • C08G63/18—Dicarboxylic acids and dihydroxy compounds the acids or hydroxy compounds containing carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/12—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from polycarboxylic acids and polyhydroxy compounds
    • C08G63/16—Dicarboxylic acids and dihydroxy compounds
    • C08G63/18—Dicarboxylic acids and dihydroxy compounds the acids or hydroxy compounds containing carbocyclic rings
    • C08G63/19—Hydroxy compounds containing aromatic rings
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to oxalyl chloride, 1,4-cyclonucleic acid dimethanol
  • polymeric drug delivery systems can be classified as micro and nanoparticulate drug delivery systems or as complex polymeric drugs.
  • Particulate drug delivery systems physically mix drugs and protect them from the environment, including micelles and solid particles.
  • the composite polymer drug has a drug covalently attached to the branch of the polymer main chain through a linker.
  • linker examples are proteins, polysaccharides or synthetic polymers such as dendrimers and polyhydroxypropyl methacryl amides. This approach has led to significant improvements in drug stability and pharmacokinetic properties.
  • polymeric drug carriers containing such small amounts of drug may be a weak point in some applications.
  • One of the new approaches for the delivery of low molecular weight drugs is to combine the low molecular weight drug itself with the polymer main chain to deliver the drug.
  • the best function of these drug carriers is that the polymer itself is a controlled drug delivery system. This is because the degradation product itself is a therapeutic agent, and the polymer is completely degraded due to hydrolytically labile anhydrides and ester bonds in the backbone.
  • Inhalation-type drug delivery technology has recently been in the spotlight, delivering a therapeutic agent through the lungs with a large surface area and excellent permeability.
  • the market expanded from $ 7.5 billion in 1997 to $ 9 billion in 2000.
  • Avonex a treatment for multiple sclerosis (MS)
  • MS was introduced to Europe in 1996 and has posted annual sales of $ 600 million, growing more than 60% annually.
  • Inhaled insulin also starting in Europe, is expected to be $ 618.1 million in 2012 from $ 49.3 million in 2006 alone.
  • the demand for inhalable drug delivery technology which occupies most of the respiratory market, has continued to grow stably for the next five years and the technical demand expands.
  • inhaled insulin such as inhaled insulin
  • inhalation is a major driving force for the future evolution of the field of treatment.
  • inhalation with conventional injectable treatments such as inhaled insulin
  • the present inventors devised biodegradable and biocompatible peroxalate polymers in which hydroxybenzyl alcohol (HBA) was incorporated into the polymer main chain, and prepared them as nanoparticles.
  • HBA hydroxybenzyl alcohol
  • the nanoparticles have also been developed in inhaled drug delivery through the lungs.
  • HBA-containing copolyoxalate oxayl chloride
  • 1,4-cyclohexamethanol 1,4-cyclohexamethanol
  • HBA-containing copolyoxalate HBA- incorporated copolyoxalate, HP0X. More specifically, HBA-containing copolyoxalate can be accelerated by hydrogen peroxide and hydroxy benzyl alcohol (HBA) is released to provide nanoparticles with anti-inflammatory and antioxidant effects.
  • X is an integer.
  • the compound is a copolyoxalate chain containing 1,4-cyclohexanedimethanol, which can be easily removed from the body due to unstable peroxalate esters in the copolyoxalate main chain.
  • Polyoxalate structures undergo ester hydrolysis to break down to oxal ic acid and diol under aqueous conditions.
  • Polyoxalates which contain aromatic peroxalate esters in the backbone, react with hydrogen peroxide for instantaneous decomposition into di and carbon dioxide.
  • Hydrogen peroxide is overproduced by neutrophiles that are stratified at macrophages and inflamed sites and rapidly breaks down aromatic peroxalate esters bonds.
  • the HBA-incorporated copolyoxalate (HPOX) compound of the present invention is a new stimulus responsive biodegradation which promotes degradation by reacting with hydrogen peroxide produced at high concentration in inflammatory cells. It is a polymer.
  • X is an integer.
  • 1,4-cyclohexanedimethanol of step a) was used as the main component of this copolymer because of its excellent biocompatibility.
  • 1,4-cyclonucleic acid dimethanol is an indirect food additive that has been approved for human consumption and has an excellent toxicity profile (LD50: 3200 mg / kg oral intake). In addition, it does not undergo significant enzyme conversion in vivo and is quickly removed from the body.
  • the HBA in step a) is one of the well-known phenolic compounds in various plants including carrot and palm orchid (Gymnadenia conopsea) and is the main active component of the traditional herbal medicine, Gastrodia elata. HBA has been reported to have anti-inflammatory, antioxidant and anti-angiogenic activity. In addition, HBAs have the ability to prevent brain damage through increased expression of antioxidant proteins encoding after transient focal cerebral ischemia.
  • HBAs are aromatic diol compounds in which phenol and methylene units are substituted with alcohols and are therefore suitable for the synthesis of polyoxalates using oxalyl chloride.
  • Therapeutic compounds HBAs are chemically bound into the polymer backbone without attachment as side groups, making them available for polymer degradation.
  • step a) The polymerization of step a) is tetrahydrofuran dried under nitrogen charge.
  • THF Tetrahydrofuran
  • 1,4-cyclohexanedimethanol and 4-hydroxybenzyl alcohol in dried THF and add triethylamine.
  • the triethylamine may act as a catalyst and a base for promoting the reaction by removing HCL generated in the polymer synthesis step as a reagent for making oxalate. This may include adding oxalyl chloride dissolved in dried THF to the mixture.
  • ⁇ 3i> in order to be able to prepare in the form of solid nanoparticles under drying, it is preferable that the molar ratio of 1,4-cyclohexane dimethyl methane and 4-hydroxybenzyl alcohol is 4: 1.
  • the nonpolar solvent of step b) is not limited thereto, but is cold nucleic acid.
  • copolyoxalate (hexane) is preferred, and the copolyoxalate can be obtained after purification through repeated precipitation in cold nucleic acids and drying under high vacuum.
  • the present invention also provides nanoparticles prepared using the copolyoxalate compound represented by the formula (1).
  • X is an integer.
  • the copolyoxalate compound represented by Chemical Formula 1 dissolved in DCM may be added to an emulsifier solution.
  • the emulsifier polyvinyl alcohol
  • Nanoparticles can be prepared by the oil-in-water emulsion method. More specifically, the copolyoxalate mixture added to the emulsifier solution to form an oil / water emulsion is sonicated and homogenized. The emulsion dog is added to a polyvinyl alcohol solution and homogenized. The remaining solvent is removed using a rotary evaporator and nanoparticles can be obtained by centrifuge.
  • the average size of the nanoparticles is preferably 300nm to lum, more preferably 500nm.
  • the present invention also provides a drug delivery agent comprising the nanoparticles. More specifically, HBA-incorporated copolyoxalate (HP0X) nanoparticles release HBA, which reduces production of TNF- ⁇ and inhibits NO production in LPS-activated RAW 264.7 macrophages.
  • HP0X is a very effective drug carrier due to its salient features such as biodegradability, biocompatibility, anti-inflammatory and antioxidant properties. In particular, HP0X may be used to treat inflammatory diseases such as acute liver failure and acute lung injury. Useful drug carriers can be provided.
  • the present invention provides a drug carrier, characterized in that the drug is supported on the nanoparticles.
  • the drug is preferably theophyline, naringenin.
  • the nanoparticles of the present invention can be in the form of inhaled drug delivery through the lungs, which can greatly contribute to the improvement of medical technology of human beings.
  • the drug carrier of the present invention provides a drug carrier, characterized in that the drug carrier for treating lung disease.
  • Nanoparticles of the present invention can act as a drug itself, but can see a synergistic effect through parallel with existing inflammatory therapeutic agents. These nanoparticles are manufactured in various drug delivery forms, such as inhalation and injection, for lung disease, acute / chronic hepatitis,
  • nanoparticles of the present invention will be developed in the form of inhaled drug delivery through the lungs, which will greatly contribute to the improvement of medical technology of human beings.
  • FIG. 2 is the —spectrum of HBA-incorporated copolyoxalate (HPOX) in which HBA in CDC1 3 is synthesized.
  • HPOX HBA-incorporated copolyoxalate
  • FIG. 6 is a ⁇ analysis of copolyoxalate nanoparticles using RAW 264.7 cells.
  • FIG. 7 shows copolyoxalate Na for NO production in LPS-treated RAW 264.7 cells. Inhibitor effect of no microspheres. Each value is the average of four experiments. ( ⁇
  • FIG. 8 shows the results of Western blotting to see if it reduces production of iNOS in LPS-treated RAW 264.7 cells.
  • Figure 9 shows inhibition of TNF-a production by HP0X nanoparticles in LPS treated RAW 264.7 cells. Each value is the average of four experiments. ( ⁇ S.D) ** P ⁇ 0. () 1 is
  • 1,4-Cyclohexanedimethanol (21.96 'ol) and 4-hydroxybenzyl alcohol (5.49' ol) ol well-dried 20 mL under nitrogen Dissolve in tetrahydrofuran (THF) and add triethylamine (60 ⁇ ol) dropwise at 4 ° C.
  • THF tetrahydrofuran
  • triethylamine 60 ⁇ ol
  • oxalyl chloride 27.45 ⁇ ol
  • the reaction product was maintained in the presence of nitrogen at room temperature for 6 hours, and extracted with dichloromethane to produce a polymer and precipitated in cold nucleic acid (hexane) to obtain a polymer.
  • the molecular weight of the polymer is polystyrene -25000 (polydispersity) using polystyrene standards and by gel permeation chromatography (Gel Permeation Chromatography, GPC, Futech, Korea)
  • the hydrolysis rate of copolyoxalate was studied by measuring the molecular weight by gel permeation chromatography (GPC), GPC, Futecs, Korea.
  • the polymer was ground to fine particles and placed in phosphate buffer at 37 ° C. Polymer samples were mixed with gentle stirring and hydro lyzed polymers were collected at specific times. The hydrolyzed molecular weight was measured by time period and measured by GPC to compare the reduced amount by percentage.
  • the structure and size of the polyoxalate nanoparticles were observed by Scanning Electron Microscopy (SEM, Hitachi).
  • HP0X polymer (5mg) contains ImL of PBS (pH 7.4) at 37 ° C. Was added. Test tubes were kept kept in 37 ° C incubation. Prepare test tubes containing the same amount of HP0X polymer, remove the test tubes from the incubation for each time period to be measured, freeze and freeze-dry. The dried samples were dissolved in THF (Tetrahydro furan) and examined by Gel Permeation Chromatography (GPC) to determine the reduced molecular weight over time as a percentage of the initial molecular weight before degradation.
  • THF Tetrahydro furan
  • RAW 264.7 cells were seeded at a concentration of 1 ⁇ 10 6 cells / well in a 24 well plate. The cells were incubated for 24 hours at various concentrations of nanoparticles (lOmg / mL ⁇ 100mg / mL), and then the culture medium was removed. Then, 1 mL of the culture solution was added and 20pL MTT reagent was added and cultured for 4 hours. When purple crystals were produced, 10 mL of dimethyl sulfoxide solution was added thereto, followed by 10 minutes of incubation, and the microphone was measured at 570 nm using a plate reader.
  • RAW 264.7 cells (1 ⁇ 10 6 cells / well in a 24 well plate) were treated at different concentrations of HP0X nanoparticles and then inflamed to determine whether HP0X nanoparticles themselves are involved in anti-inflammatory effects.
  • LPS lipopolysaccharide
  • TNF ⁇ a tumor necrosis factor alpha
  • HPOX was synthesized from a one-step condensation reaction between oxalyl chloride and two diols, cyclohexanedimethanol and HBA. 1 shows the synthesis and degradation of copolyoxalate.
  • the introduction of HBAs other than cyclonucleic acid dimethanol did not cause any synthetic complications and the procedure used for the synthesis of polyoxalates was suitable for the synthesis of copolymers.
  • the polymerization proceeded in dry THF to give the corresponding copolymer under nitrogen gas.
  • the resulting copolyoxalate was purified through repeated precipitation in cold nucleic acids and a yellow solid was obtained after drying under high vacuum.
  • the chemical structure of the polyoxalate was found to be- ⁇ . Methylene protons in the hydroxyl groups of 1,4-cyclonuclear dimethanol appear at 3.5 ppm. Large peaks below 4.2 ppm are due to the methylene of adjacent oxalate ester bond Matched both. Two polyline aromatic quantum peaks were found at 7.2 and 7.5 ppm, and both benzyl groups were found at 5.3 ppm. These results suggest that oxalyl chloride, two diols, and 1,4-cyclohexanedimethanol to generate peroxalate containing peroxalate esters bonds. successful polymerization from the condensation reaction between dimethanol) and HBA (FIG. 2).
  • the molar ratio of 1,4-cyclohexanedimethanol to HBA used for polymer synthesis was 4: 1. Comparing the integration ratio between 2d protons and 2c protons, it was also confirmed that copolymerization yielded a 4: 1 molar ratio of 1,4-cyclohexanedimethanol to HBA.
  • the HP0X obtained from this reaction has a molecular weight (MW) of -25000 which corresponds to the degree of polymerization of ⁇ 100 repeated units with a polydispersity index of 1.8 (FIG. 3).
  • the rate of hydrolysis of HP0X was measured under physiological conditions. Degradation of polyoxalate by hydrolysis of water was investigated by measuring the molecular weight of the polymer by crushing it into fine powder and incubating under aqueous conditions. 4 shows the hydrolysis characteristics of HP0X. It has a half-life of less than 12 hours at pH 7.4 and means that HP0X is hydrolytically degraded. To confirm hydrolytic degradation of HP0X, fine copolymer powder was added at 37 ° C for 24 hours.
  • HP0X was formulated into nanoparticulates using poly (vinyl alcohol) as an emulsifier by an oil-in-water emulsion method. Particle size and structure were examined using a scanning electron microscope (SEM). HP0X Nano Me The lip globules were spherical spheres with a smooth surface and an average diameter of 500 nm or less, which is a form for increasing the exposure area to cells (FIG. 5). The polymer had a half-life of 12 hours of hydrolysis but appeared to have sufficient hydrophobicity to formulate into nanoparticles under aqueous conditions. HP0X nanoparticles in this range may be suitable for the maturation of intracellular and extracellular drug carriers.
  • a drug carrier including phagocytosis by macrophages by the macrophages easily phagocytosis of foreign substances in the range of 0.5 ⁇ 3 / / m.
  • MTT assay was performed using RAW 264.7 cells. Cells were incubated in various amounts of HP0X nanoparticles for 24 hours and the viability of the cells was measured (FIG. 6). HP0X nanoparticles showed minimal cytotoxicity in a dose-dependant manner. No cytotoxicity was observed in the nanoparticles of. Cells treated with 50 and 100 / zg nanoparticles showed a slight decrease in cell viability. This may suggest that copolyoxalate nanoparticles may be acceptable for use as drug carriers.
  • NO is known as a pro-inflammatory mediator in the onset of inflammation and its production is promoted by inducible nitric oxide synthases (iNOS).
  • iNOS inducible nitric oxide synthases
  • results of Western blotting to confirm that HP0X nanoparticles reduce the production of iNOS in LPS-treated RAW 264.7 macrophages are as follows. Stimulation of cells by treatment of LPS significantly increased the production of iNOS, and the addition of HBA completely inhibited the production of iNOS. When the concentration of HP0X was added to the cells, the production of iNOS protein gradually decreased as the concentration of HP0X increased. Referring to FIG. 8, the darker the band, the stronger the protein, the more iNOS is generated. As a result, it was confirmed that the NO inhibitory effect of HBA is due to the inhibition of iNOS production.
  • TNF-a is a cytokine involved in systemic inflammation and is produced by macrophages after stimulation by LPS or interlukin-1 (IL-1).
  • TNF- ⁇ is widely used in previous studies to measure inflammatory responses as a variety of drugs and drug carriers. Macrophages were previously stimulated with LPS and treated with HP0X nanoparticles and levels of TNF- ⁇ were measured at post treatment day 1 post. 9 demonstrates the effect of HP0X on TNF-a production.
  • LPS treatment resulted in a marked increase in TNF-a production by macrophages.
  • HPOX nanoparticulates reduced LPS-induced TNF- ⁇ production in a dose-dependent manner by cells.
  • HP0X nanoparticles less than 50 Ug were not noticeable, but were found to slightly reduce TNF-a production. However, P0X did not inhibit TNF-a production. These results support that HBA released from HP0X nanoparticles exert an anti-inflammatory effect.
  • the present invention provides oxalyl chloride, 1,4-cyclonucleodioxide

Abstract

The present invention relates to obtaining HBA-incorporated copolyoxalate (HPOX) using oxalyl chloride, 1,4-cyclohexamethanol, and HBA. More specifically, the present invention relates to nanospheres exhibiting anti-inflammatory and antioxidant effects as HBA-incorporated copolyoxalate may selectively respond only to hydrogen peroxide and may be decomposed, wherein p-hydroxybenzyl alcohol (HBA) is released. Although nanospheres of the present invention may serve as a drug, synergistic effects may be obtained by combining same with conventional anti-inflammatory agents. These nanospheres may be prepared in the form of various drug delivery vectors, such as inhalation, injection, etc., may be decomposed by hydrogen peroxide excessively produced due to pulmonary diseases, acute/chronic hepatitis, Alzheimer's disease, Parkinson's disease, arthritis, and cardiovascular diseases such as atherosclerosis, etc., in order to release drugs, and thus may be applied as a stimulus-responsive biodegradable polymer that treats diseases.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
하이드록시벤질 알콜을 함유하는 고분자 나노미립구  Polymer nanoparticles containing hydroxybenzyl alcohol
【기술분야】  Technical Field
<ι> 본 발명은 옥사릴 크를라이드 (oxalyl chloride), 1,4-사이클로핵산디메탄올 <ι> The present invention relates to oxalyl chloride, 1,4-cyclonucleic acid dimethanol
( 1 , 4-cyc 1 ohexamet hano 1 ) 및 하이드톡시 벤질 알콜 (hydroxybenzyl alcohol , ΗΒΑ) 을 이용하여 ΗΒΑ를 방출할 수 있는 코폴리옥살레이트 (HBA-incorporated copolyoxalate, HP0X)를 합성하고 이를 나노미립구로 제조하여 약물전달체로 활용 하는 것에 관한 것이다. (1, 4-cyc 1 ohexamet hano 1) and hydroxybenzyl alcohol (ΗΒΑ) were used to synthesize HBA-incorporated copolyoxalate (HP0X) capable of releasing ΗΒΑ and convert it into nanoparticles. It relates to the manufacturing and use as drug delivery.
【배경기술】  Background Art
<2> 임상적으로 사용된 대다수의 약물은 짧은 혈액 순환과 높은 총체적인 대사 속도를 가진 저분자 화합물이다. 약물의 투여에 있어， 이런 작은 분자 약물들은 악성 조직 모두로 빠르게 확산되고 몸 전체로 균등하게 분산된다. 결과적으로 상대 적으로 적은 양의 약물이 타겟 부위에 도달하고 부작용도 발생한다. 이런 한계점을 극복하고 각각의 약물의 잠재력을 향상시키기 위해 약물 전달체의 개발을 위한 많 은 노력이 있었다. 대부분의 약물 전달체는 다양한 구조와 물리화학적 특성을 가진 생체적합성과 생분해성에 기초한다.  The majority of drugs used clinically are low molecular weight compounds with short blood circulation and high overall metabolic rates. In the administration of the drug, these small molecule drugs rapidly spread to all malignant tissues and are evenly distributed throughout the body. As a result, a relatively small amount of drug reaches the target site and side effects occur. Much effort has been made to develop drug delivery vehicles to overcome these limitations and to improve the potential of each drug. Most drug carriers are based on biocompatibility and biodegradability with various structural and physicochemical properties.
<3> 일반적으로 고분자 약물 전달 시스템은 마이크로와 나노미립자 약물 전달 시 스템으로서 또는 복합 고분자 약물로서 분류될 수 있다. 미립자 약물 전달 시스템 은 물리적으로 약물을 흔합하게 하고 미셀과 고체 입자를 포함해서 환경으로부터 보호한다. 한편으로 복합 고분자 약물은 가교제 (linker)를 통해 고분자 주사슬의 가지에 공유결합으로 첨부된 약물을 가지고 있다. 예로는 단백질， 다당류 또는 덴 드리머 (dendrimer)와 폴리하이드록시프로필 메타크릴아미드 (polyhydroxypropyl methacryl amide) 같은 합성 고분자가 있다. 이런 접근은 약물의 안정성과 약동학적 특성의 큰 개선을 이루었다. 그러나 이렇게 적은 양의 약물을 함유한 고분자 약물 전달체는 몇 몇 웅용에 있어 약점이 될 수도 있다.  In general, polymeric drug delivery systems can be classified as micro and nanoparticulate drug delivery systems or as complex polymeric drugs. Particulate drug delivery systems physically mix drugs and protect them from the environment, including micelles and solid particles. On the other hand, the composite polymer drug has a drug covalently attached to the branch of the polymer main chain through a linker. Examples are proteins, polysaccharides or synthetic polymers such as dendrimers and polyhydroxypropyl methacryl amides. This approach has led to significant improvements in drug stability and pharmacokinetic properties. However, polymeric drug carriers containing such small amounts of drug may be a weak point in some applications.
<4> 저분자 약물의 전달을 위한 새로운 접근 방법 중 하나로 저분자 약물자체를 고분자 주사슬에 결합하여 약물을 전달하는 방법이 있다. 이러한 약물전달체가 가 지는 가장 좋은 기능은 고분자 자체가 통제된 약물 전달 시스템이란 것이다. 이것 은 분해 산물 그 자체가 치료제이고， 고분자는 가수 분해성 불안정한 무수물과 백 본내의 에스테르 본드 때문에 완전히 분해되기 때문이다.  One of the new approaches for the delivery of low molecular weight drugs is to combine the low molecular weight drug itself with the polymer main chain to deliver the drug. The best function of these drug carriers is that the polymer itself is a controlled drug delivery system. This is because the degradation product itself is a therapeutic agent, and the polymer is completely degraded due to hydrolytically labile anhydrides and ester bonds in the backbone.
<5> 기존의 생분해성 고분자들은 여러 가지 문제점을 가지고 있다. 미국 식품의 약청 (FDA)의 승인을 받은 생체고분자인 PLGA (Polylact ide-co-glycol ide)와， 나노 미 립구와 미 샐로 제조되어 약물전달체로 많은 연구가 이루어지고 있는 PCL (Polycaprolactone) , Polyorthoester 및 Polyacetal 등은 높은 가격 및 분해 후에 염증을 유발하는 분해산물이 생성되거나 가수분해 속도가 느려 약물의 방출속도가 지 연되는 문제점들을 가지고 있다 . 이 러한 고분자들은 또한 특정부위를 표적하거나 질병부위의 환경에 감응하는 능력 이 없다 . Existing biodegradable polymers have various problems. American food PLGA (Polylact ide-co-glycol ide), a biopolymer approved by the National Drug Administration (FDA), and PCL (Polycaprolactone), Polyorthoester, and Polyacetal, which are made of nanoparticles and US sal After the high price and decomposition, there is a problem in that degradation products that cause inflammation are produced or the release rate of the drug is delayed due to the slow hydrolysis rate. These polymers also lack the ability to target specific sites or to respond to the environment of disease sites.
<6> 흡입 형 약물전달 기술은 최근에 각광받고 있는 기술로 표면적 이 넓고 투과도 가 우수한 폐를 통해 치료제를 전달하는 기술이다. 1997년 75억 달러에서 2000년 90억 달러로 시장이 확대되었다. 다발성 경화증 (MS) 치료제인 아보넥스 (Avonex)는 96년에 유럽에 소개된 이후 99년도 연간 매출액은 6억 달러로 연간 60% 이상의 높 은 성장률을 보이고 있다. 흡입형 인슐린도 유럽에서 시작해 유럽에서만 2006년 4 , 930만 달러에서 2012년 6억 170만 달러로 예상된다 . 현재， 대부분의 호흡기 시장 이 차지하는 흡입형 약물전달 기술의 수요는 향후 5년간 안정 성장을 지속하고 기 술적 인 수요도 확대된다. 흡입형 인슐린 등 기존 주사식 치료에서 흡입 형으로 교체 됨으로서 환자의 수용과 컴플라이 언스 향상과 함께 기술 혁신에 따른 작용 발현 시 간의 단축이 치료 분야의 장래적 진화를 가져온 주요 견인력 이다 . 흡입형 인슬린 등 기존 주사식 치료에서 흡입형으로 교체됨으로서 환자의 수용과 컴플라이 언스 향 상과 함께 기술 혁신에 따른 작용 발현 시간의 단축이 치료 분야의 장래적 진화를 가져온 주요 견인력 이다 .  Inhalation-type drug delivery technology has recently been in the spotlight, delivering a therapeutic agent through the lungs with a large surface area and excellent permeability. The market expanded from $ 7.5 billion in 1997 to $ 9 billion in 2000. Avonex, a treatment for multiple sclerosis (MS), was introduced to Europe in 1996 and has posted annual sales of $ 600 million, growing more than 60% annually. Inhaled insulin, also starting in Europe, is expected to be $ 618.1 million in 2012 from $ 49.3 million in 2006 alone. At present, the demand for inhalable drug delivery technology, which occupies most of the respiratory market, has continued to grow stably for the next five years and the technical demand expands. The replacement of inhaled insulin, such as inhaled insulin, by inhalation, is a major driving force for the future evolution of the field of treatment. By replacing inhalation with conventional injectable treatments such as inhaled insulin, the improvement of patient acceptance and compliance, along with the reduction of the onset of action due to technological innovation, are the main driving forces for the future evolution of the therapeutic field.
<7>  <7>
<8> 이에 , 본 발명자는 고분자 주사슬 안에 하이드록시벤질알콜 (hydroxybenzyl alcohol , HBA)이 합유된 생분해성과 생체적합성을 가진 퍼록사레이트 고분자 (peroxalate polymers)를 고안하여 이를 나노미 립구로 제조하고 , 상기 나노미 립구 또한 폐를 통한 흡입형 약물전달 형 태로 개발하기 이르렀다 .  Therefore, the present inventors devised biodegradable and biocompatible peroxalate polymers in which hydroxybenzyl alcohol (HBA) was incorporated into the polymer main chain, and prepared them as nanoparticles. The nanoparticles have also been developed in inhaled drug delivery through the lungs.
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
<9> 이와 같이 본 발명은 상술한 바와 같은 문제점을 해결하기 위 한 것으로 옥사 릴 크를라이드 (oxalyl chloride) , 1 ,4-cyclohexamethanol 및 HBA을 이용하여 HBA- 함유하는 코폴리옥살레이트 (HBA- incorporated copolyoxalate, HP0X)를 제공함을 목적으로 한다 . 보다 구체적으로 HBA-함유하는 코폴리옥살레이트는 과산화수소에 의해 분해가 촉진될 수 있고 하이드록시 벤질 알콜 ( Hiyroxybenzyl alcohol , HBA) 이 방출되어 항염증， 항산화제 효과를 보이는 나노미 립구를 제공함을 목적으로 한 다. As described above, the present invention solves the problems described above. HBA-containing copolyoxalate (oxayl chloride), 1,4-cyclohexamethanol, and HBA-containing copolyoxalate (HBA- incorporated copolyoxalate, HP0X). More specifically, HBA-containing copolyoxalate can be accelerated by hydrogen peroxide and hydroxy benzyl alcohol (HBA) is released to provide nanoparticles with anti-inflammatory and antioxidant effects. One All.
【기술적 해결방법】  Technical Solution
<ιο> 상기와 같은 본 발명의 목적을 달성하기 위하여， 하기 화학식 1의 구조를 갖 는 코폴리옥살레이트 (HBA- incorporated copolyoxalate, HP0X) 화합물을 제공한다. <ιι> [  In order to achieve the object of the present invention as described above, to provide a copolyoxalate (HBA-incorporated copolyoxalate, HP0X) compound having a structure of formula (1). <ιι> [
Figure imgf000005_0001
Figure imgf000005_0001
<13> 상기 화학식 1에서， X는 정수이다.  In Formula 1, X is an integer.
<14> 상기 화합물은 코폴리옥살레이트 (copolyoxalate) 주사슬 안에서 불안정한 페록사레이트 이스터 (peroxalate esters) 결합 때문에 코폴리옥살레이트는 체내에 서 쉽 게 제거될 수 있는 1, 4-사이클로헥산디메탄올， HBA와 옥살산 및 /또는 이산화 탄소로 완전히 분해된다 (도 1 참조) . 폴리옥살레이트 구조들은 수성 조건하에서 옥살산 (oxal ic acid)과 디올로 분해되기 위해서 에스테르 가수분해를 겪는다 . 백 본 안에 방향족 페록사레이트 이스터 (peroxalate esters) 결합올 포함하고 있는 폴리옥살레이트는 순간적으로 디을과 이산화탄소로 분해되 기 위해서 과산화수소 (hydrogen peroxide)와 반웅한다 .  The compound is a copolyoxalate chain containing 1,4-cyclohexanedimethanol, which can be easily removed from the body due to unstable peroxalate esters in the copolyoxalate main chain. Completely decomposed into HBA and oxalic acid and / or carbon dioxide (see FIG. 1). Polyoxalate structures undergo ester hydrolysis to break down to oxal ic acid and diol under aqueous conditions. Polyoxalates, which contain aromatic peroxalate esters in the backbone, react with hydrogen peroxide for instantaneous decomposition into di and carbon dioxide.
<15> 과산화수소 (hydrogen peroxide)는 대식세포와 염증 부위에서 보층된 호중성 백혈구 (neutrophi les)에 의해서 과잉 생산되며， 방향족 페록사레이트 이스터 (peroxalate esters) 결합을 빠르게 쪼갠다 .  Hydrogen peroxide is overproduced by neutrophiles that are stratified at macrophages and inflamed sites and rapidly breaks down aromatic peroxalate esters bonds.
<16> 즉 , 본 발명의 코폴리옥살레이트 (HBA-incorporated copolyoxal ate , HPOX) 화합물은 염증세포에서 높은 농도로 생산하는 과산화수소에 반웅해서 분해가 촉진 되는 새로운 자극 감응형 (st imulus responsive) 생분해성 고분자인 것이다.  In other words, the HBA-incorporated copolyoxalate (HPOX) compound of the present invention is a new stimulus responsive biodegradation which promotes degradation by reacting with hydrogen peroxide produced at high concentration in inflammatory cells. It is a polymer.
<17>  <17>
<18> 또한, 본 발명은  <18> In addition, the present invention
<i9> a) 1 ,4-사이클로핵산디 메탄올 (l , 4-cyclohexanedimethanol ) , 4-하이드록시벤 질 알콜 (4-hydroxybenzyl alcohol ) 및 옥사릴 크를라이드 (oxalyl chloride)를 중합 시키는 단계 ;  a) polymerizing 1,4-cyclonucleic acid dimethanol (l, 4-cyclohexanedimethanol), 4-hydroxybenzyl alcohol and oxalyl chloride;
<20> b) 상기 a) 단계의 결과 중합체를 비극성 용매를 이용해 침 전시키는 단계를 포함하는 것을 특징으로 하는 화학식 1로 표시되는 코폴리옥살레이트 화합물의 제 조방법을 제공한다 .  B) provides a method for producing a copolyoxalate compound represented by the formula (1) characterized in that it comprises the step of precipitating the polymer using a non-polar solvent.
<21> [화학식 1]
Figure imgf000006_0001
<21> [Formula 1]
Figure imgf000006_0001
<23>  <23>
<24> 상기 화학식에서， X는 정수이다.  In the above formula, X is an integer.
<25> 상기 a) 단계의 1,4-사이클로핵산디메탄올 (1,4— cyclohexanedimethanol)은 뛰어난 생체적합성 때문에 이 공중합체의 주 구성성분으로서 사용되었다. 또한, 1,4-사이클로핵산디메탄올은 간접적인 식품 첨가제로서 인간이 섭취할 수 있는 것 으로 승인되었고 뛰어난 toxicity profile (LD50 ： 3200mg/kg 구강 섭취 양)을 가 지고 있다. 게다가， 이는 생체 내에서 상당한 효소의 변환을 겪지 않으며 몸으로부 터 빠르게 제거된다.  1,4-cyclohexanedimethanol of step a) was used as the main component of this copolymer because of its excellent biocompatibility. In addition, 1,4-cyclonucleic acid dimethanol is an indirect food additive that has been approved for human consumption and has an excellent toxicity profile (LD50: 3200 mg / kg oral intake). In addition, it does not undergo significant enzyme conversion in vivo and is quickly removed from the body.
<26>  <26>
<27> 상기 a) 단계의 HBA는 당근과손바닥 난초 (Gymnadenia conopsea)를 포함한 다양한 식물에 있어 잘 알려진 페놀계 화합물의 하나이고 전통적인 약초의 약품， 천마 (Gastrodia elata)의 주요 활성 구성요소이다. HBA는 항염증， 항산화와 항-혈 관생성 ( ant i -angiogenic) 활성을 가진 것으로 많은 연구가 보고되고 있다. 게다 가， HBA는 일시적인 국소 뇌 허혈 후의 인코딩 하는 항산화 단백질의 증가된 유전 자의 발현을 통해 뇌 손상을 예방하는 능력을 가지고있다.  The HBA in step a) is one of the well-known phenolic compounds in various plants including carrot and palm orchid (Gymnadenia conopsea) and is the main active component of the traditional herbal medicine, Gastrodia elata. HBA has been reported to have anti-inflammatory, antioxidant and anti-angiogenic activity. In addition, HBAs have the ability to prevent brain damage through increased expression of antioxidant proteins encoding after transient focal cerebral ischemia.
<28> HBA는 알콜로 치환된 페놀과 메틸렌 유닛이 화합된 방향족 디올 (diol) 화 합물이고， 따라서 옥사릴 크롤라이드 (oxalyl chloride)를 사용하여 폴리옥살레이 트의 합성에 적합하다. 치료적 화합물 HBA는 결사슬 군 (side group)으로서 첨부되 지 않고 고분자 백본 안으로 화학적으로 결합되고， 고분자 분해로 이용할 수 있게 한다.  HBAs are aromatic diol compounds in which phenol and methylene units are substituted with alcohols and are therefore suitable for the synthesis of polyoxalates using oxalyl chloride. Therapeutic compounds HBAs are chemically bound into the polymer backbone without attachment as side groups, making them available for polymer degradation.
<29>  <29>
<30> 상기 a) 단계의 중합은 질소 충전 하에서 건조된 테트라히드로푸란 The polymerization of step a) is tetrahydrofuran dried under nitrogen charge.
(Tetrahydrofuran, THF)에서 진행되는 것이 바람직하다. 보다 자세하게는 1,4-사이 클로핵산디메탄올 (1,4-cyclohexanedimethanol)와 4-하이드록시벤질 알콜 (4- hydroxybenzyl alcohol)을 건조된 THF에 용해하고 트리에틸아민 (triethylamine)을 첨가하는 것이 바람직하다. 상기 트리에틸아민은 옥살레이트 (oxalate)를 만들기 위한 시약으로서 고분자 합성 단계에서 발생하는 HCL을 제거하여 반응을 촉진하는 촉매제 및 염기 (base)로서 작용할 수 있다. 여기에 건조된 THF에 용해한 옥사릴 크를라이드 (oxalyl chloride)를 흔합물에 첨가하는 것을 포함할수 있다. <3i> 또한, 건조하에 고체나노미립구형태로 제조할 수 있도록 하기 위해 1,4-사이 클로핵산디메탄을과 4-하이드록시벤질 알콜의 몰비는 4:1인 것이 바람직하다. Preferably in (Tetrahydrofuran, THF). More specifically, it is preferable to dissolve 1,4-cyclohexanedimethanol and 4-hydroxybenzyl alcohol in dried THF and add triethylamine. Do. The triethylamine may act as a catalyst and a base for promoting the reaction by removing HCL generated in the polymer synthesis step as a reagent for making oxalate. This may include adding oxalyl chloride dissolved in dried THF to the mixture. <3i> In addition, in order to be able to prepare in the form of solid nanoparticles under drying, it is preferable that the molar ratio of 1,4-cyclohexane dimethyl methane and 4-hydroxybenzyl alcohol is 4: 1.
<32>  <32>
<33> 상기 b) 단계의 비극성 용매는 이에 제한되는 것은 아니나, 차가운 핵산 The nonpolar solvent of step b) is not limited thereto, but is cold nucleic acid.
(hexane)이 바람직하고， 차가운 핵산에서 반복된 침전을 통해 정제되었고 고진공 하에서 건조 시킨 후에 코폴리옥살레이트를 얻을 수 있다. (hexane) is preferred, and the copolyoxalate can be obtained after purification through repeated precipitation in cold nucleic acids and drying under high vacuum.
<34>  <34>
<35> 또한 본 발명은 화학식 1로 표시되는 코폴리옥살레이트 화합물을 이용하여 제조된 나노미립구를 제공한다.  The present invention also provides nanoparticles prepared using the copolyoxalate compound represented by the formula (1).
<36> [ <36> [
Figure imgf000007_0001
Figure imgf000007_0001
<38> 상기 화학식에서， X는 정수이다.  In the above formula, X is an integer.
<39> 상기 나노미립구의 제조는 DCM에 용해된 상기 화학식 1로 표시되는 코폴리옥 살레이트 화합물을 유화제 용액에 첨가할 수 있다. 상기 유화제로는 폴리 (비닐 알 콜) (polyvinyl alcohol))이 바람직하다.  In preparing the nanoparticles, the copolyoxalate compound represented by Chemical Formula 1 dissolved in DCM may be added to an emulsifier solution. As the emulsifier, polyvinyl alcohol) is preferable.
<40> 상기 흔합물을 유 /수 에멀견 법 (oil-in-water emulsion method)에 의해 나 노미립구를 제조할 수 있다. 보다 자세하게는 유 /수 에멀견을 형성하기 위해 유화 제 용액에 첨가된 코폴리옥살레이트 흔합물을 초음파 처리를 하고 균질하게 한다. 상기 에멀견은 폴리비닐알콜 용액으로 첨가되고 균질화한다. 남아있는 용매는 회전 증발기 (rotary evaporator)를 사용하여 제거하고 나노미립구는 원심분리기에 의해 획득할수 있다. Nanoparticles can be prepared by the oil-in-water emulsion method. More specifically, the copolyoxalate mixture added to the emulsifier solution to form an oil / water emulsion is sonicated and homogenized. The emulsion dog is added to a polyvinyl alcohol solution and homogenized. The remaining solvent is removed using a rotary evaporator and nanoparticles can be obtained by centrifuge.
<41> 상기 나노미립구의 평균 크기는 300nm 내지 lum이 바람직하며 , 보다 바람직 하게는 500nm 이다.  The average size of the nanoparticles is preferably 300nm to lum, more preferably 500nm.
<42>  <42>
<43> 본 발명은 또한 상기 나노미립구를 포함하는 약물 전달체를 제공한다. 보다 자세하게는 코폴리옥살레이트 (HBA-incorporated copolyoxalate, HP0X) 나노미립구 는 HBA를 방출하며， 이는 TNF-α의 생산올 감소하게 하고 LPS-활성화 된 RAW 264.7 대식세포 안의 NO 생산을 억제할 수 있다. HP0X는 생분해성, 생체 적합성， 항염증 과 항산화 특성들과 같은 두드러진 특징들에 기인해서 매우 효능 있는 약물 전달체 인 것이다. 특히， HP0X는 급성 간부전과 급성 폐손상과 같은 염증성 질환의 치료에 유용한 약물 전달체를 제공할수 있다. The present invention also provides a drug delivery agent comprising the nanoparticles. More specifically, HBA-incorporated copolyoxalate (HP0X) nanoparticles release HBA, which reduces production of TNF-α and inhibits NO production in LPS-activated RAW 264.7 macrophages. HP0X is a very effective drug carrier due to its salient features such as biodegradability, biocompatibility, anti-inflammatory and antioxidant properties. In particular, HP0X may be used to treat inflammatory diseases such as acute liver failure and acute lung injury. Useful drug carriers can be provided.
<44> 또한 본 발명은 상기 나노미립구에 약물을 담지시킨 것을 특징으로 하는 약 물 전달체를 제공한다. 상기 약물은 테오필린 (theophyline), 나린제닌 (Naringenin)인 것이 바람직하다. 또한 본 발명의 나노미립구는 폐를 통한 흡입형 약물전달형태로 할수 있는바， 인류의 의료기술 향상에 크게 기여할 수 있다.  In another aspect, the present invention provides a drug carrier, characterized in that the drug is supported on the nanoparticles. The drug is preferably theophyline, naringenin. In addition, the nanoparticles of the present invention can be in the form of inhaled drug delivery through the lungs, which can greatly contribute to the improvement of medical technology of human beings.
<45> 또한 본 발명의 상기 약물전달체는 폐 질환 치료용 약물전달체인 것을 특징 으로 하는 약물전달체를 제공한다.  In addition, the drug carrier of the present invention provides a drug carrier, characterized in that the drug carrier for treating lung disease.
【발명의 효과】  【Effects of the Invention】
<46> 본 발명의 나노미립구는 자체가 약물로써 작용할 수 있지만, 기존 염증치료 제와의 병행을 통하여 시너지 효과를 볼 수 있다. 이러한 나노미립구는 흡입형 및 주사제 등 다양한 약물 전달체 형태로 제조되어 폐 질환， 급성 /만성 간염， Nanoparticles of the present invention can act as a drug itself, but can see a synergistic effect through parallel with existing inflammatory therapeutic agents. These nanoparticles are manufactured in various drug delivery forms, such as inhalation and injection, for lung disease, acute / chronic hepatitis,
Alzheimer병， Parkinson병， 관절염 그리고 죽상동맥경화증 등의 심혈관질환 등에 과다하게 생성된 과산화수소에 의해 분해되어 약물을 방출하여 질병을 치료하는 자 극감응형 생분해성 고분자로써 적용될 것이다. It will be applied as an irritant biodegradable polymer that is decomposed by excessive hydrogen peroxide, such as Alzheimer's disease, Parkinson's disease, arthritis and atherosclerosis, to release the drug to treat the disease.
<47> 또한， 본 발명의 나노미립구는 폐를 통한 흡입형 약물전달 형태로 개발하여 인류의 의료기술 향상에 크게 기여할수 있을 것이다.  In addition, the nanoparticles of the present invention will be developed in the form of inhaled drug delivery through the lungs, which will greatly contribute to the improvement of medical technology of human beings.
【도면의 간단한 설명】  [Brief Description of Drawings]
<48> 도 1은 HBA가 합성된 코폴리옥살레이트 (HBA-incorporated copolyoxalate, 1 is a copolyoxalate synthesized with HBA (HBA-incorporated copolyoxalate,
HPOX)의 합성과 분해에 관한 것이다. HPOX) synthesis and degradation.
<49> 도 2는 CDC13 안의 HBA가 합성된 코폴리옥살레이트 (HBA— incorporated copolyoxalate, HPOX)의 —匿스펙트럼이다. FIG. 2 is the —spectrum of HBA-incorporated copolyoxalate (HPOX) in which HBA in CDC1 3 is synthesized.
<50> 도 3은 THF 안의 HBA가 합성된 코폴리옥살레이트 (HBA-incorporated copolyoxalate, HPOX)의 GPC trace이다. 3 is a GPC trace of HBA-incorporated copolyoxalate (HPOX) synthesized with HBA in THF.
<5i> 도 4는 HBA가 합성된 코폴리옥살레이트 (HBA-incorporated copolyoxalate,<5i> Figure 4 is HBA synthesized copolyoxalate (HBA-incorporated copolyoxalate,
HPOX)의 가수분해 특성이다. (A)는 가수분해 속도론이고 (B)는 37°C에서 D20 안에서 가수분해 후 분해 산물의 -NMR스펙트럼이다. HPOX). (A) is the hydrolysis kinetics and (B) is the -NMR spectrum of the degradation product after hydrolysis in D 2 0 at 37 ° C.
<52> 도 5는 HBA가 합성된 코폴리옥살레이트 (HBA-incorporated copolyoxalate,5 shows cobalt oxalate synthesized with HBA (HBA-incorporated copolyoxalate,
HPOX)의 SEM 이미지이다. SEMOX).
<53> 도 6은 RAW 264.7 세포를 사용한 코폴리옥살레이트 나노미립구의 ΜΊΤ분석이 다. FIG. 6 is a ΜΊΤ analysis of copolyoxalate nanoparticles using RAW 264.7 cells.
<54> 도 7은 LPS-처리된 RAW 264.7 세포안의 NO 생산에 대한 코폴리옥살레이트 나 노미립구의 저해제 효과를 나타낸다. 각각의 값들은 4번의 실험의 평균값이다. (土FIG. 7 shows copolyoxalate Na for NO production in LPS-treated RAW 264.7 cells. Inhibitor effect of no microspheres. Each value is the average of four experiments. (土
S.D) ** PO.01는 LPS 처리된 그룹에 비례한다. S.D) ** PO.01 is proportional to the LPS processed group.
<55> 도 8은 LPS-처리된 RAW 264.7 세포에서 iNOS의 생성을 감소시키는지 확인하 기 위한 웨스턴블롯팅 (Western blotting)에 대한 결과이다. FIG. 8 shows the results of Western blotting to see if it reduces production of iNOS in LPS-treated RAW 264.7 cells.
<56> 도 9는 LPS 처리된 RAW 264.7 세포의 HP0X 나노미립구에 의한 TNF-a 생산의 저해를 나타낸다. 각각의 값들은 4번의 실험의 평균값이다. (土 S.D) ** P<0.()1는Figure 9 shows inhibition of TNF-a production by HP0X nanoparticles in LPS treated RAW 264.7 cells. Each value is the average of four experiments. (土 S.D) ** P <0. () 1 is
LPS 처리된 그룹에 비례한다. Proportionate to LPS processed groups.
<57> 도 10은 HBA가 합성된 코폴리옥살레이트 (HBA-incorporated copolyoxalate,10 shows cobalt oxalate synthesized with HBA (HBA-incorporated copolyoxalate,
HP0X)의 과산화수소와의 반응성을 나타낸 그래프이다. It is a graph showing the reactivity of HP0X) with hydrogen peroxide.
【발명의 실시를 위한 형태】  [Form for implementation of invention]
<58> 이하， 본 발명을 실시예를 통하여 상세히 설명하도록 한다. 하기 실시예는 본 발명을 설명하기 위한 일 예에 지나지 않으며， 이에 의하여 본 발명의 범위가 제한되는 것은 아니다.  Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are merely examples for describing the present invention, and the scope of the present invention is not limited thereto.
<59>  <59>
<60> <실시예 >  <60> <Example>
<61> 재료  <61> materials
<62> ；广하이드록시벤질알콜 (/rhydroxybenzyl alcohol), 1,4-사이클로핵산디메탄 올 (1,4-cyclohexanedimethanol) , 옥사릴 크롤라이드 (oxalyl chloride) , 풀리 (비 닐알콜) (丽 13， 000-21， 000)은 시그마 알드 리치사 (Μ0. USA)로부터 구매했다. 테트 라히드로푸란 (Tetrahydrofuran)과 트리에틸아민 (triethylamine)은 Showa (Japan) 로부터 획득했다. 테트라히드로푸란 (Tetrahydrofuran, THF)은 수소 칼슘 하에서 증류되었다. 모든 시약들은 추가 정제 없이 사용되었다.  <62>; hydroxybenzyl alcohol (1,4-cyclohexanedimethanol), oxalyl chloride, pulleys (vinyl alcohol) (丽 13 , 000-21, 000) was purchased from Sigma Aldrich, Μ0.USA. Tetrahydrofuran and triethylamine were obtained from Showa (Japan). Tetrahydrofuran (THF) was distilled under calcium hydrogen. All reagents were used without further purification.
<63>  <63>
<64> 고분자합성  <64> Polymer Synthesis
<65> 1，4-사이클로핵산디메탄올 (l,4-Cyclohexanedimethanol)(21.96誦 ol)과 4-하 이드록시벤질 알콜 (4-hydroxybenzyl alcohol)(5.49隱 ol)올 질소 충전 하에 잘 건 조된 20mL의 테트라히드로푸란 (Tetrahydrofuran, THF)에 용해하고 4°C에서 트리에 틸아민 (triethylamine)(60 隱 ol)을 한 방울씩 첨가한다. 여기에 25 mL의 건조된 THF에 용해한 옥사릴 크롤라이드 (oxalyl chloride)(27.45 隱 ol)를 4°C에서 흔합물 에 한 방울씩 첨가했다. 이 반웅물은 6시간 동안 상온에서 질소 존재 하에 유지되 었고， 디클로로메탄 (Dichloromethane)을 이용하여 생성된 고분자를 추출한 다음 차가운 핵산 (hexane)에서 침전시켜 고분자를 얻었다. 고분자의 분자량은 폴리스티 렌 표준 (polystyrene standards)을 사용하고 겔 투과 크로마토그래피 (Gel Permeation Chromatography, GPC, Futech,Korea)에 의해 -25000 (다분산도1,4-Cyclohexanedimethanol (21.96 'ol) and 4-hydroxybenzyl alcohol (5.49' ol) ol well-dried 20 mL under nitrogen Dissolve in tetrahydrofuran (THF) and add triethylamine (60 隱 ol) dropwise at 4 ° C. To this was added dropwise oxalyl chloride (27.45 隱 ol) in 25 mL of dried THF to the mixture at 4 ° C. The reaction product was maintained in the presence of nitrogen at room temperature for 6 hours, and extracted with dichloromethane to produce a polymer and precipitated in cold nucleic acid (hexane) to obtain a polymer. The molecular weight of the polymer is polystyrene -25000 (polydispersity) using polystyrene standards and by gel permeation chromatography (Gel Permeation Chromatography, GPC, Futech, Korea)
(polydispersity)=1.8)로 측정되었다. 고분자의 화학구조는 400MHz H-NMR 분광계 (J丽 -EX400JE0L)로 식별되었다. 400MHz spectrometer에 의해 중수소 치환된(polydispersity) = 1.8). The chemical structure of the polymer was identified by a 400 MHz H-NMR spectrometer (J-EX400JE0L). Deuterium substituted by 400MHz spectrometer
(deuterated) 클로로포름 (Chloroform)의 -NMR 은 ： 7.2-7.3 (/B,2H,Ar), 7.5-7.6 (m,2H,Ar), 5.3 (m,2HOCH2-PhO) , 4.1-4.3 (ra,4H,C00CH2CH) , 1.8-2.0-NMR of (deuterated) chloroform is: 7.2-7.3 (/ B, 2H, Ar), 7.5-7.6 (m, 2H, Ar), 5.3 (m, 2HOCH 2 -PhO), 4.1-4.3 (ra , 4H, C00CH 2 CH), 1.8-2.0
(m,2H,C(CH2)3H0), 1.0-1.8 (m,8H,Cycl icCH2)이다. (m, 2H, C (CH 2 ) 3 HO), 1.0-1.8 (m, 8H, Cycl icCH 2 ).
<66>  <66>
<67> 입자준비 (Particle preparation)  <67> Particle preparation
<68> 500 uL의 DCM에 용해된 50 밀리그램의 고분자는 5mL의 10 (w/v)% 폴리 (비닐 알콜) 용액에 첨가되었다. 흔합물은 초음파 파쇄기 (Sonicator, Fisher Scientific, Sonic Dismembrator 500)를 사용하여 30초 동안 초음파 처리되었고 정 제한 오일 /물 에멀견을 형성하기 위해 2분 동안 균질화 되었다 (PRO Scientific, PRO 200-호모지나이저). 상기 에멀견은 20 mL PVA (lw/w ) 용액 안으로 첨가 되었 고 1분 동안 균질화되었다. 남아있는 용매는 회전 증발기 (rotary evaporator)를 사용하여 제거 되었다. 나노미립구는 원심분리기에 의해 4°C에서 5분 동안 11,000 xg을 통해 획득했고， 탈이은수 (deionized water)로 두 번 씻고 이를 통해 얻어진 파티클 용액을 동결건조하여 건조된 나노미립구를 얻었다. 50 mg of polymer dissolved in 500 uL of DCM was added to 5 mL of 10 (w / v)% poly (vinyl alcohol) solution. The mixture was sonicated for 30 seconds using an ultrasonic crusher (Sonicator, Fisher Scientific, Sonic Dismembrator 500) and homogenized for 2 minutes to form a static limiting oil / water emulsion (PRO Scientific, PRO 200-homogenizer). ). The emulsion dog was added into a 20 mL PVA (lw / w) solution and homogenized for 1 minute. The remaining solvent was removed using a rotary evaporator. Nanoparticles were obtained through 11,000 xg for 5 minutes at 4 ° C by centrifugal separator, washed twice with deionized water and lyophilized particle solution obtained to obtain dried nanoparticles.
<69>  <69>
<70> 물리화학적 특징 (Phvsicochemical characterization)  Physicochemical characterization
<7i> 코폴리옥살레이트 (copolyoxalate)의 가수분해 속도는 겔 투과 크로마토그래 피 (Gel Permeation Chromatography, GPC, Futecs,Korea)에 의한 분자량을 측정함 으로써 연구되었다. 고분자는 고운 입자로 갈아졌고 37°C에서 인산염완충액 (phosphate buffer)에 두었다. 고분자 샘플은 약하게 교반하여 섞여 졌고 수화된 (hydro lyzed) 고분자는 특정시간에서 수집되었다. 시간대별로 가수분해 되어진 분 자량을 측정하여 감소된 양을 퍼센트 비율로 비교하기 위해 GPC를 통해 측정하였 다. 폴리옥살레이트 나노미립구의 구조와 사이즈는 주사전자현미경 (Scanning Electron Microscopy, SEM, Hitachi)에 의해 관찰되었다. The hydrolysis rate of copolyoxalate was studied by measuring the molecular weight by gel permeation chromatography (GPC), GPC, Futecs, Korea. The polymer was ground to fine particles and placed in phosphate buffer at 37 ° C. Polymer samples were mixed with gentle stirring and hydro lyzed polymers were collected at specific times. The hydrolyzed molecular weight was measured by time period and measured by GPC to compare the reduced amount by percentage. The structure and size of the polyoxalate nanoparticles were observed by Scanning Electron Microscopy (SEM, Hitachi).
<72>  <72>
<73> HP0X분해 역학 (Hvdrolvsis kinetics)  HP0X digestion kinetics (Hvdrolvsis kinetics)
<74> HP0X고분자 (5mg)는 37°C상에서 ImL의 PBS (pH 7.4)를 포함하고 있는 시험관 으로 첨가되었다. 시험관은 37°C 인큐베이션에서 계속적으로 유지 보관 되었다. 동 일한 양의 HP0X 고분자를 넣은 시험관들을 준비한 후 측정 하고자 하는 시간대 별 로 시험관을 인큐베이션에서 꺼내 얼린 후 동결건조 한다. 건조가 된 샘플을 THF (Tetrahydro furan)에 녹여 시간대 별로 감소된 분자량을 분해되기 전 처음의 분자 량에 대한 퍼센트 비율로 알아보기 위해 Gel Permeation Chromatography (GPC)를 통해 조사하였다. HP0X polymer (5mg) contains ImL of PBS (pH 7.4) at 37 ° C. Was added. Test tubes were kept kept in 37 ° C incubation. Prepare test tubes containing the same amount of HP0X polymer, remove the test tubes from the incubation for each time period to be measured, freeze and freeze-dry. The dried samples were dissolved in THF (Tetrahydro furan) and examined by Gel Permeation Chromatography (GPC) to determine the reduced molecular weight over time as a percentage of the initial molecular weight before degradation.
<75>  <75>
<76> 세포독성검사 (cytotoxicity assay)  <76> Cytotoxicity Assay
<77> HP0X 나노 미립구의 세포독성은 3-(4， 5-디메틸티아졸 -2-일) -2， 5-디페닐테트 라 졸리움 브로마이드 (ΜΊΤ) 환원 분석에 의해 RAW 264.7 세포를 이용하여 측정하 였다. RAW 264.7 세포는 24 웰 폴레이트 (well plate)안의 1X106세포 /웰의 농도로 파종하였다. 세포에 나노 미립구를 (lOmg/mL ~ 100mg/mL)의 다양한 농도로 24시간 동안 배양한 후 배양액을 제거 후 lmL의 배양액올 넣어주고 20pL MTT 시약을 넣고 4시간 배양하였다. 보라색 결정이 생성되면 디메틸 설폭사이드 용액을 lmL 씩 넣어 10분 배양후흡광도 570nm에서 마이크를 플레이트 리더를 이용하여 측정하였다.Cytotoxicity of HP0X Nanoparticulates was Measured Using RAW 264.7 Cells by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (ΜΊΤ) Reduction Assay It was. RAW 264.7 cells were seeded at a concentration of 1 × 10 6 cells / well in a 24 well plate. The cells were incubated for 24 hours at various concentrations of nanoparticles (lOmg / mL ~ 100mg / mL), and then the culture medium was removed. Then, 1 mL of the culture solution was added and 20pL MTT reagent was added and cultured for 4 hours. When purple crystals were produced, 10 mL of dimethyl sulfoxide solution was added thereto, followed by 10 minutes of incubation, and the microphone was measured at 570 nm using a plate reader.
<78> <78>
<79> 염증 반응 (inf lammatorv response)  <79> inflammatory response (inf lammatorv response)
<80> RAW 264.7 세포 (24 웰 풀레이트 (well plate)안의 1X106세포 /웰)는 HP0X 나노미립구의 농도별로 처리한 다음, HP0X 나노미립구 자체가 항염증효과에 관여하 는지를 판단하기 위해 염증유발물질인 지질다당류 (LPS, lmg/mL)를 2처리함으로써 염증을 유도한 후 염증반웅을 관여하는 전-염증성 사이토카인인 종양 괴사 인자의 방출량 (tumor necrosis factor alpha, TNF一 a)을 측정하였다. RAW 264.7 cells (1 × 10 6 cells / well in a 24 well plate) were treated at different concentrations of HP0X nanoparticles and then inflamed to determine whether HP0X nanoparticles themselves are involved in anti-inflammatory effects. Induction of inflammation by treatment with lipopolysaccharide (LPS, lmg / mL), a trigger, was measured for tumor necrosis factor alpha (TNF 一 a), a pro-inflammatory cytokine involved in inflammatory reactions. .
<8i> NO 생성량과 TNF-a 방출량을 조사하여 코폴리옥살레이트 나노미립구의 항염 증성 기능의 유무와 정도를 알아보았다. NO의 생성량은 배양 상충액을 모아 griess reagent로 10분간 차광시켜 반응시킨 후에 마이크로플레이트 리더 (microplate reader )(E-Max, Molecular Device Co, US)를 사용하여 흡광도 570 nm에서 비색분석 법 (color imetric assay)을 사용함으로써 측정되었다. N0 표준 곡선은 알려진 아질 산 나트륨 (sodium nitrite)의 농도를 사용함으로써 작도하였다. 세포 안에서 처리 된 TNF-a 함량은 제조회사 (eBioScience, US)에 의해 권장된 ELISA 키트로 측정되 었다. TNF-a 실험은 2번 시행되었고 TNF-a 함량은 표준 키트로부터 얻어진 표준 곡 선을사용하여 pg/mL농도의 관점에서 계산되었다.  <8i> NO production and TNF-a release were investigated to determine the presence and extent of anti-inflammatory function of copolyoxalate nanoparticles. The amount of NO produced was collected by incubating the reaction mixture with a griess reagent for 10 minutes, followed by colorimetric analysis at 570 nm using a microplate reader (E-Max, Molecular Device Co, US). It was measured by using an assay). The N0 standard curve was constructed by using known concentrations of sodium nitrite. TNF-a content treated in cells was measured with an ELISA kit recommended by the manufacturer (eBioScience, US). TNF-a experiments were performed twice and the TNF-a content was calculated in terms of pg / mL concentration using standard curves obtained from the standard kit.
<82> <83> 웨스턴불롯 ¾ (Western blotting) <82> <83> Western blotting
<84> HPOX의 염증 완화작용을 입증하기 위한 것으로， iNOS생성에 관여하는 단백질 인 iNOS (inducieble nitrix oxide synthase) 를 항체로 사용하여 HPOX가 LPS처리 된 세포에서 iNOS의 생성을 감소시키는지를 알아보기 위해 웨스턴블롯팅 (western blotting)하였다. 세포는 RAW 264.7을 passaged에서 이용하였으며， 24 웰 (well)에 세포를 1X106씩 분주 한후， 하루 동안 보관후 medium(DMEM Low)을 갈아주었다. 코 폴리옥살레이트 미립구를 함량별로 각각 넣어주고， 코폴리옥살레이트 미립구 주입 후 2시간 후에 NO 생성을 위해 LPS를 씩 넣어주었다. 24시간 후에 RIPA buffer를 이용하여 lysis하고， BCA 정량법으로 단백질 정량을 하여 western blotting을 실시하였다. NO의 생성정도를 알아보기 위해 Antibody는 iNOS를 이용하 였다 (도 8). <84> To demonstrate the anti-inflammatory effects of HPOX, to investigate whether HPOX reduces iNOS production in LPS-treated cells using inducible nitrix oxide synthase (iNOS), a protein involved in iNOS production. Western blotting. The cells were passaged in RAW 264.7. After dispensing 1 × 10 6 cells into 24 wells, the medium (DMEM Low) was changed after storage for one day. Copolyoxalate microspheres were added to each content, and 2 hours after copolyoxalate microsphere injection, LPS was added for generation of NO. After 24 hours, lysis was performed using RIPA buffer, and Western blotting was performed by quantifying proteins by BCA assay. Antibody used iNOS to determine the production of NO (Fig. 8).
<85>  <85>
<86> 코폴리옥살레이트와과산화수소의 반옹성 검사  <86> Reactivity Test of Copolyoxalate and Hydrogen Peroxide
<87> HP0X나노미립구가 얼마나 과산화수소와 반웅을 하는지 실험을 하였다. 과산 화수소 (¾ ) 10 μΜ 용액에 HP0X 나노미립구를 농도별로 넣고 24시간 동안 37°C에서 인큐베이션에 보관한후 amplex red assay kit를 이용하여 ¾02농도를 측정하였다. <88> 이하， 본 발명의 실시예에 따른 결과를 설명하면 다음과 같다. The experiment was carried out how HP0X nanoparticles react with hydrogen peroxide. Peracid hydrogen (¾) 10 to μΜ solution into the HP0X nano-microspheres at different concentrations and then kept in incubation at 37 ° C for 24 hours using a amplex red assay kit were measured ¾0 2 concentration. Hereinafter, the results according to the embodiment of the present invention will be described.
<89>  <89>
<90> HBA가 합성된 코폼리옥살레이트의 합성과 특징 (Synthesis and character i zation of HBA- incorporated copolyoxalate)  Synthesis and characterization of HBA-incorporated copolyoxalate
<9i> HPOX는 옥사릴 크를라이드 (oxalyl chloride)와 두 개의 디올 (diol), 1,4-사 이클로핵산디메탄올 (cyclohexanedimethanol)과 HBA 사이의 원 스텝 축합반웅으로 부터 합성되었다. 도 1은 코폴리옥살레이트의 합성과 분해를 나타낸다. 사이클로핵 산디메탄올 이외 HBA의 도입은 어떤 합성 합병증에 원인이 되지 않았고 폴리옥살레 이트의 합성에 사용된 절차는 공중합체의 합성에 적합하였다. 중합은 질소 기체 하 에서 상응하는 공중합체를 발생시키기 위해 건조 THF에서 진행되었다. 결과물인 코 폴리옥살레이트 (copolyoxalate)는 차가운 핵산에서 반복된 침전올 통해 정제되었 고 고진공 하에서 건조 시킨 후에 노란 고체를 얻게 되었다. HPOX was synthesized from a one-step condensation reaction between oxalyl chloride and two diols, cyclohexanedimethanol and HBA. 1 shows the synthesis and degradation of copolyoxalate. The introduction of HBAs other than cyclonucleic acid dimethanol did not cause any synthetic complications and the procedure used for the synthesis of polyoxalates was suitable for the synthesis of copolymers. The polymerization proceeded in dry THF to give the corresponding copolymer under nitrogen gas. The resulting copolyoxalate was purified through repeated precipitation in cold nucleic acids and a yellow solid was obtained after drying under high vacuum.
<92> 폴리옥살레이트의 화학적 구조는 -賺로 확인되었다. 1,4-사이클로핵산디 메탄올의 히드록실 그룹 (hydroxyl groups) 에 있는 메틸렌 양자는 3.5 ppm에서 나타난다. 4.2 ppm 이하에서 큰 피크는 인접한 옥살레이트 에스테르 결합의 메틸렌 양자에 일치했다. 두 개의 다중선 방향족 양자 피크는 7.2와 7.5 ppm에서 나타났고 벤질기 양자는 5.3ppm에서 나타났다. 이런 결과들은 페록사레이트 이스터 (peroxalate esters) 결합을 포함하고 있는 페록사레이트를 발생시키기 위한 옥사 릴 크롤라이드 (oxalyl chloride)와 두 개의 디올 (diol), 1,4-사이클로핵산디메탄 올 (cyclohexane dimethanol)과 HBA 사이의 축합 반웅으로부터 성공적인 중합을 의미한다 (도 2). 고분자 합성을 위해 사용된 1,4-사이클로핵산디메탄올과 HBA의 몰비율은 4:1이었다. 2d 양자와 2c 양자의 통합 비율을 비교함으로서 공중합이 1,4-사이클로핵산디메탄올 (cyclohexane dimethanol)대 HBA가 4:1의 몰비율을 나 타내는 것을 또한 확인하였다. 이 반웅으로부터 획득한 HP0X는 1.8의 다분산 지수 (polydispersity index)를 가진 ~100 반복된 유닛의 중합정도와 일치하는 -25000의 분자량 (MW)을 가지고 있다 (도 3). The chemical structure of the polyoxalate was found to be-賺. Methylene protons in the hydroxyl groups of 1,4-cyclonuclear dimethanol appear at 3.5 ppm. Large peaks below 4.2 ppm are due to the methylene of adjacent oxalate ester bond Matched both. Two polyline aromatic quantum peaks were found at 7.2 and 7.5 ppm, and both benzyl groups were found at 5.3 ppm. These results suggest that oxalyl chloride, two diols, and 1,4-cyclohexanedimethanol to generate peroxalate containing peroxalate esters bonds. successful polymerization from the condensation reaction between dimethanol) and HBA (FIG. 2). The molar ratio of 1,4-cyclohexanedimethanol to HBA used for polymer synthesis was 4: 1. Comparing the integration ratio between 2d protons and 2c protons, it was also confirmed that copolymerization yielded a 4: 1 molar ratio of 1,4-cyclohexanedimethanol to HBA. The HP0X obtained from this reaction has a molecular weight (MW) of -25000 which corresponds to the degree of polymerization of ˜100 repeated units with a polydispersity index of 1.8 (FIG. 3).
<93>  <93>
<9 > 공중합체의 가수분해와 HBA의 방출  <9> Hydrolysis of Copolymer and Release of HBA
<95> HP0X의 가수분해 속도는 생리학상 (physiological)의 조건 하에서 측정되었 다. 물의 가수분해에 의한 폴리옥살레이트의 분해는 고분자를 미세분말 (fine powder)로 잘게 부스고 수성조건 하에서 배양하는 동안 분자량을 측정함으로써 조 사되었다. 도 4는 HP0X의 가수분해 특성을 나타낸다. 그것은 pH 7.4에서 12시간 이 하의 반감기를 가지고 있고 HP0X는 가수분해성 분해된다는 것을 의미한다. HP0X의 가수분해성 분해를 확인하기 위해 미세한 공중합체 가루를 37°C에서 24시간 동안The rate of hydrolysis of HP0X was measured under physiological conditions. Degradation of polyoxalate by hydrolysis of water was investigated by measuring the molecular weight of the polymer by crushing it into fine powder and incubating under aqueous conditions. 4 shows the hydrolysis characteristics of HP0X. It has a half-life of less than 12 hours at pH 7.4 and means that HP0X is hydrolytically degraded. To confirm hydrolytic degradation of HP0X, fine copolymer powder was added at 37 ° C for 24 hours.
D20에서 배양하였고 상청액은 ^-NMR 분석을 위해 수집되었다. 인접한 옥살레이트 에스테르 결합의 메틸렌 양자에 일치하는 피크는 거의 사라졌고 1,4-사이클로 핵산 디메탄올 (cyclohexanedi methanol)의 환식의 메틸렌 양자와 일치하는 다중선 피크 는 3.3ppm에서 나타난다. 게다가 HBA의 벤질 양자는 4.6ppm에서 두 개의 방향족 양 자신호는 6.7과 7.1ppm에서 나타난다. 이 결과는 HP0X는 모노머， HBA와 1,4—사이클 로핵산디메탄올을 발생시키기 위해 분명히 가수분해의 분해를 하는 것을 의미하는 것이다. Incubated at D 2 0 and supernatants were collected for ^ -NMR analysis. The peaks coincident with both methylene protons of adjacent oxalate ester bonds are almost gone and the polyline peaks coincident with the cyclic methylene protons of 1,4-cyclohexanedi methanol appear at 3.3 ppm. In addition, the benzyl protons of HBA appear at 4.6 ppm and the two aromatic quantum rings at 6.7 and 7.1 ppm. This result implies that HP0X decomposes hydrolyzed to generate monomers, HBA and 1,4—cyclohexanedimethanol.
<96>  <96>
< 7> 코폼리옥살레이트 나노미립구의 특징 (Characteristics of copolvoxalate nanoparticles)  <7> Characteristic of copolvoxalate nanoparticles
<98> 오일 -물의 에멀견 방법 (an oil-in-water emulsion method)에 의해 유화제로 서 폴리 (비닐 알콜)을 사용하여 HP0X는 나노미립구로 제형화 되었다. 입자 사이즈 와 구조를 SEM (scanning electron microscope)을 사용하여 조사했다. HP0X 나노미 립구는 매끄러운 표면과 세포에 대한 노출 면적을 크게 하기 위한 형태인， 500nm 이하의 평균지름을 가진 등근 구형 이었다 (도 5). 이 고분자는 12시간의 가수분해 의 반감기를 가졌으나， 수성 상태 하에서 나노미립구로 제형화하기 위한 충분한 소 수성 (hydrophobicity)을 가진 것처럼 보였다. 이 범위에서 HP0X 나노미립구는 세 포내 (intracellular)와 세포외 (exracellular) 약물 전달체의 웅용을 위해 적당한 것일지도 모른다. 특별히， 그것은 대식세포는 0.5~3//m 범위의 이물질을 쉽게 식균 작용을 함으로서 대식세포 (Macrophages)에 의한 식균 작용 (phagocytosis)을 포함 한 약물 전달체로서 적합하다. HP0X 나노미립구 세포독성 (cytotoxicity)을 평가하 기 위해서 MTT 분석은 RAW 264.7 세포를 사용함으로서 실행되었다. 세포는 24시간 동안 HP0X 나노미립구의 다양한 양에서 배양되었고， 세포의 생존력을 측정했다 (도 6). HP0X 나노미립구는 용량에 의존한 방식 (dose-dependant manner)에 있어 최소 한의 세포독성을 나타냈다. 의 나노미립구에서 세포독성이 없는 것이 관찰되었 다. 50과 100/zg의 나노미립구로 처리한 세포는 세포 생존력이 약간 감소하는 것으 로 나타났다. 이것은 약물 전달체로서의 웅용에 있어 코폴리옥살레이트 나노미립구 (copolyoxalate nanoparticles)가수용될 수 있다는 것으로 암시될 수 있다. HP0X was formulated into nanoparticulates using poly (vinyl alcohol) as an emulsifier by an oil-in-water emulsion method. Particle size and structure were examined using a scanning electron microscope (SEM). HP0X Nano Me The lip globules were spherical spheres with a smooth surface and an average diameter of 500 nm or less, which is a form for increasing the exposure area to cells (FIG. 5). The polymer had a half-life of 12 hours of hydrolysis but appeared to have sufficient hydrophobicity to formulate into nanoparticles under aqueous conditions. HP0X nanoparticles in this range may be suitable for the maturation of intracellular and extracellular drug carriers. In particular, it is suitable as a drug carrier, including phagocytosis by macrophages by the macrophages easily phagocytosis of foreign substances in the range of 0.5 ~ 3 / / m. To assess HP0X nanoparticulate cytotoxicity, MTT assay was performed using RAW 264.7 cells. Cells were incubated in various amounts of HP0X nanoparticles for 24 hours and the viability of the cells was measured (FIG. 6). HP0X nanoparticles showed minimal cytotoxicity in a dose-dependant manner. No cytotoxicity was observed in the nanoparticles of. Cells treated with 50 and 100 / zg nanoparticles showed a slight decrease in cell viability. This may suggest that copolyoxalate nanoparticles may be acceptable for use as drug carriers.
<99>  <99>
<ιοο> 코폴리옥살레이트나노미립구의 항염증효과 (AnH-i'nflanmiatorv effects of copolyoxalate nanoparticles) <ιοο> Anti-Humanity Effects of Copolyoxalate Nanoparticles (AnH-i ' nflanmiatorv effects of copolyoxalate nanoparticles)
<ioi> NO는 발병의 염증에 있어 친염증성 매개자 (pro- inflammatory mediator)로 알려졌고 그것의 생산은 유도성 질소산화물 신타세스 (inducible nitric oxide synthases, iNOS)에 의해 촉진된다. 코폴리옥살레이트로부터 방출된 HBA의 항염증 효과를 확인하기 위해 LPS로 처리된 RAW 264.7 대식세포의 염증 반응을 HP0X 나노 미립구의 부존재와 존재 하에 조사했다. 첫 번째로 세포안의 LPS-유도된 NO 생산에 있어 HP0X 나노미립구로부터 방출된 HBA의 저해제 효과를 조사했다. 하루 LPS로 배 양되는 동안， 세포는 12 μΜ에 이르기까지 많은 양의 NO를 산출했다. 세포가 HP0X 나노미립구로 처리되었을 때， NO 생산은 두드러지게 약물 농도 의존성 (dose dependent manner)에 따라 저해되었다 (도 7). HP0X 나노미립구의 2¾g의 처리 후， NO 생산은 반으로 감소했고， 50 g의 HP0X 나노미립구는 NO 생산을 완전히 억제했 다 . 50/ig의 HP0X 나노미립구는 0.11^의 1¾ 처럼 같은 억제효과를 발휘했다 . HP0X 로부터 방출된 HBA의 항산화 효과를 추가 확인하기 위해서， HBA를 함유하지 않은 폴리옥살레이트 (P0X)를 또한 테스트 했다. P0X 나노미립구에 의한 NO 생산은 저해 되지 않았고， 이는 HP0X로부터 방출된 HBA는 NO 생산에 있어 억제효과를 발휘하는 것을 증명했다. <ioi> NO is known as a pro-inflammatory mediator in the onset of inflammation and its production is promoted by inducible nitric oxide synthases (iNOS). In order to confirm the anti-inflammatory effect of HBA released from copolyoxalate, the inflammatory response of RAW 264.7 macrophages treated with LPS was examined in the absence and presence of HP0X nanoparticles. First, we investigated the inhibitory effect of HBA released from HP0X nanoparticles on LPS-induced NO production in cells. While cultured in LPS per day, cells yielded large amounts of NO down to 12 μΜ. When cells were treated with HP0X nanoparticulates, NO production was markedly inhibited in a drug dependent manner (FIG. 7). After 2¾ g of HP0X nanoparticles, NO production was halved and 50 g of HP0X nanoparticles completely inhibited NO production. 50 / ig HP0X nanoparticles showed the same inhibitory effect as 1¾ of 0.11 ^. To further confirm the antioxidant effects of HBA released from HP0X, polyoxalate (P0X) containing no HBA was also tested. NO production by P0X nanoparticles was not inhibited. HBA released from HP0X exerts inhibitory effect on NO production. Proved that.
<102>  <102>
<103> 또한， HP0X 나노미립구가 LPS로 처리된 RAW 264.7 대식세포에 iNOS의 생성 을 감소시키는지 확인하기 위한 웨스턴블롯팅 (Western blotting)에 대한 결과는 다음과 같다. LPS를 처리하여 세포를 자극시키면 iNOS의 생성이 현저하게 증가하고 HBA를 투입하면 iNOS의 생성이 완전히 억제되었다. HP0X의 농도를 달리하면서 세포 에 투입하였을 때 HP0X의 농도가 증가할수록 iNOS단백질의 생성이 점차 감소됨을 알 수가 있었다. 도 8을 보면， 검게 나타나는 띠의 세기가 진한 것이 단백질이 많 다는 것으로 iNOS의 발생이 많다는 것이고， 세기가 약한 것은 iNOS의 생성이 감소 된다는 것올 나타낸다. 이 결과로 HBA가 가지는 NO억제 효과는 iNOS의 생성 억제에 기인한다는 것을 확인 할 수 있었다.  In addition, the results of Western blotting to confirm that HP0X nanoparticles reduce the production of iNOS in LPS-treated RAW 264.7 macrophages are as follows. Stimulation of cells by treatment of LPS significantly increased the production of iNOS, and the addition of HBA completely inhibited the production of iNOS. When the concentration of HP0X was added to the cells, the production of iNOS protein gradually decreased as the concentration of HP0X increased. Referring to FIG. 8, the darker the band, the stronger the protein, the more iNOS is generated. As a result, it was confirmed that the NO inhibitory effect of HBA is due to the inhibition of iNOS production.
<104>  <104>
<105> TNF-α의 발현량의 측정에 의해 LPS-처리된 대식세포의 염증 반웅에 있어 Inflammatory reaction of LPS-treated macrophages by measurement of expression level of TNF-α
HP0X 나노미립구의 효과를 추가 조사했다. TNF-a는 전신성 염증 (systemic inflammation)에 관계된 사이토카인 (cytokine)이고 LPS 또는 인터루킨 -1 (interlukin-l)(IL-l)에 의해 자극 후 대식 세포에 의해 생성된다. TNF- α는 다양 한 약물과 약물 전달체로서 염증 반응 측정하기 위해 이전 연구에서 널리 사용되고 있다. 대식 세포는 사전에 LPS로 자극을 받고 HP0X 나노미립구로 처리되었고 TNF- α의 레벨은 1 day 후 처리 (post treatment) 시점에 측정되었다. 도 9은 TNF-a 생산에 있어 HP0X의 효과를 증명한다. LPS 처리는 대식 세포에 의해 TNF-a 생산에 있어 두드러진 증가의 결과를 가져왔다. 세포에 의해 용량에 의존한 방식 (dose- dependant manner)으로 HPOX 나노미립구는 LPS—유도 TNF-α 생산을 감소시켰다. 50 Ug 미만인 HP0X 나노미립구는 두드러지지는 않으나， 조금 TNF-a 생산을 감소시키 는 것으로 나타났다. 그러나 P0X는 TNF- a 생산을 억제하지는 못했다. 이런 결과들 은 HP0X 나노미립구로부터 방출된 HBA는 항염증 효과를 발휘하는 것을 뒷받침 한 다. The effects of HP0X nanoparticles were further investigated. TNF-a is a cytokine involved in systemic inflammation and is produced by macrophages after stimulation by LPS or interlukin-1 (IL-1). TNF-α is widely used in previous studies to measure inflammatory responses as a variety of drugs and drug carriers. Macrophages were previously stimulated with LPS and treated with HP0X nanoparticles and levels of TNF-α were measured at post treatment day 1 post. 9 demonstrates the effect of HP0X on TNF-a production. LPS treatment resulted in a marked increase in TNF-a production by macrophages. HPOX nanoparticulates reduced LPS-induced TNF-α production in a dose-dependent manner by cells. HP0X nanoparticles less than 50 Ug were not noticeable, but were found to slightly reduce TNF-a production. However, P0X did not inhibit TNF-a production. These results support that HBA released from HP0X nanoparticles exert an anti-inflammatory effect.
<106>  <106>
<107> 코폼리옥살레이트의 과산화수소와의 반옹성  <107> Reactivity of Coformryoxalate with Hydrogen Peroxide
<108>  <108>
<109> *처음 10 μΜ 양의 과산화수소가 HP0X를 첨가함으로 인해 과산화수소의 농도 가 줄어드는 것올 확인 할 수 있었다. 또한 HP0X의 양이 증가할수록 줄어드는 농도 가 더 많은 것을 확인할 수 있었다 (도 10). 이러한 실험결과를 바탕으로 본 발명의 코폴리옥살레이트 나노미 립구는 과산화수소 (¾¾)와 반웅하여 ¾02의 양을 감소시키 는 역할을 하게 되어 염증완화역할을 하게 되는 것을 알 수 있었다. * The first 10 μΜ of hydrogen peroxide was found to decrease the concentration of hydrogen peroxide due to the addition of HP0X. In addition, as the amount of HP0X increased it was confirmed that the concentration decreases more (Fig. 10). Based on the experimental results of the present invention Copolyoxalate nanoparticles reacted with hydrogen peroxide (¾¾) to reduce the amount of ¾0 2 was found to be an inflammation-relieving role.
<110>  <110>
<111> 이상 첨부된 도면을 참조하여 본 발명의 실시 예들을 설명하였지만， 본 발명 이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명의 그 기술적 사상이나 필수적 인 특징을 변경하지 않고서 다른 구체적 인 형 태로 실시될 수 있다는 것을 이 해할 수 있을 것이다. 그러므로 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적 이 아닌 것으로 이해해야만 한다.  Although embodiments of the present invention have been described above with reference to the accompanying drawings, a person having ordinary knowledge in the technical field to which the present invention pertains does not change the technical spirit or essential features of the present invention. It can be understood that the present invention can be implemented in the same manner. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
【산업상 이용가능성】  Industrial Applicability
<ι ΐ2> 본 발명은 옥사릴 크롤라이드 (oxalyl chloride) , 1，4-사이클로핵산디 메탄올 <ι ΐ2> The present invention provides oxalyl chloride, 1,4-cyclonucleodioxide
( 1 , 4-cy c 1 ohexame t hano 1 ) 및 하이드록시 벤질 알콜 (hydroxybenzyl alcohol , ΗΒΑ) 을 이용하여 ΗΒΑ를 방출할 수 있는 코폴리옥살레이트 (HBA-incorporated copolyoxalate, HP0X)를 합성하고 이를 나노미 립구로 제조하여 약물전달체로 활용 할 수 있어 산업상 이용가능하다. (1, 4-cy c 1 ohexame t hano 1) and hydroxybenzyl alcohol (ΗΒΑ) were used to synthesize HBA-incorporated copolyoxalate (HP0X) capable of releasing ΗΒΑ. It can be used industrially because it can be manufactured as microspheres and used as drug delivery system.

Claims

【청구의 범위】 [Range of request]
【청구항 11  [Claim 11
하기 화학식 1의 구조를 갖는 코폴리옥살레이트 (copolyoxalate) 화합물.  Copolyoxalate compound having a structure of formula (1).
Figure imgf000017_0001
Figure imgf000017_0001
상기 X는 정수이다.  X is an integer.
【청구항 2]  [Claim 2]
a) 1,4-사이클로핵산디메탄올 (1,4-cyclohexanedi methanol), 4-하이드록시벤 질 알콜 (4-hydroxybenzyl alcohol) 및 옥사릴 크롤라이드 (oxalyl chloride)를 중합 시키는 단계;  a) polymerizing 1,4-cyclohexanedi methanol, 4-hydroxybenzyl alcohol and oxalyl chloride;
b) 상기 a) 단계의 결과 중합체를 비극성 용매를 이용해 침전시키는 단계를 포함하는 것을 특징으로 하는 하기 화학식 1로 표시되는 코폴리옥살레이트 화합물 의 제조방법.  b) A method for producing a copolyoxalate compound represented by the following Chemical Formula 1, comprising the step of a) precipitating the polymer using a nonpolar solvent.
Figure imgf000017_0002
Figure imgf000017_0002
상기 X는 정수이다.  X is an integer.
【청구항 3】  [Claim 3]
제 2항에 있어서，  The method of claim 2,
상기 a) 단계의 중합은 질소층전 하에 건조된 테트라히드로푸란 (Tetrahydrofuran, THF)에서 진행되는 것올 특징으로 하는 방법 .  The polymerization of step a) is characterized in that proceeded in tetrahydrofuran (THF) dried under a nitrogen layer charge.
【청구항 4】  [Claim 4]
제 2항에 있어서，  The method of claim 2,
상기 a) 단계의 1,4-사이클로핵산디메탄올과 4-하이드록시벤질 알콜의 몰비 는 4:1인 것을 특징으로 하는 방법.  The molar ratio of 1,4-cyclonucleodimethanol and 4-hydroxybenzyl alcohol of step a) is 4: 1.
【청구항 5】  [Claim 5]
제 2항에 있어서，  The method of claim 2,
상기 b) 단계의 비극성 용매는 핵산 (hexane)인 것을 특징으로 하는 방법. The non-polar solvent of step b) is characterized in that the nucleic acid (hexane).
【청구항 6] [Claim 6]
제 1항에 따른 코폴리옥살레이트 화합물에 유화제를 첨가하여 제조되는 나노 미립구.  Nanoparticles prepared by adding an emulsifier to the copolyoxalate compound according to claim 1.
【청구항 7]  [Claim 7]
제 6항에 있어서，  The method of claim 6,
상기 유화제는 폴리 (비닐 알콜) (polyvinyl alcohol)인 것을 특징으로 하는 나노미립구.  The emulsifier is a poly (vinyl alcohol) nanoparticles, characterized in that (polyvinyl alcohol).
【청구항 8]  [Claim 8]
제 6항에 있어서,  The method of claim 6,
상기 나노미립구의 평균 크기는 300nm 내지 600nm인 것을 특징으로 하는 나노미립 구. Nanoparticles, characterized in that the average size of the nanoparticles is 300nm to 600nm.
【청구항 9】  [Claim 9]
제 6항에 따른 나노미립구를 포함하는 약물 전달체 .  Drug delivery comprising the nanoparticles according to claim 6.
【청구항 10]  [Claim 10]
제 9항에 있어서，  The method of claim 9,
상기 나노미립구에 약물을 담지시킨 것을 특징으로 하는 약물 전달체 . Drug carrier, characterized in that the drug is supported on the nanoparticles.
【청구항 11]  [Claim 11]
제 10항에 있어서,  The method of claim 10,
상기 약물은 테오필린 (theophyline), 나린제닌 (Naringenin)인 것을 특징으로 하는 약물 전달체 . The drug is a drug carrier, characterized in that theophyline (theophyline), naringenin (Naringenin).
【청구항 12]  [Claim 12]
제 9항 내지 제 11항중 어느 한항에 있어서，  The method according to any one of claims 9 to 11,
상기 약물전달체는 폐 질환 치료용 약물전달체인 것을 특징으로 하는 약물전달체. The drug carrier is a drug carrier, characterized in that the drug carrier for treating lung disease.
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