WO2018080172A1 - Eye drop formulation for glaucoma treatment - Google Patents

Eye drop formulation for glaucoma treatment Download PDF

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Publication number
WO2018080172A1
WO2018080172A1 PCT/KR2017/011864 KR2017011864W WO2018080172A1 WO 2018080172 A1 WO2018080172 A1 WO 2018080172A1 KR 2017011864 W KR2017011864 W KR 2017011864W WO 2018080172 A1 WO2018080172 A1 WO 2018080172A1
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Prior art keywords
drug
albumin
treating glaucoma
nanoparticles
coating layer
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PCT/KR2017/011864
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French (fr)
Korean (ko)
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김현철
이지황
박기호
김영국
이현주
Original Assignee
서강대학교 산학협력단
서울대학교 산학협력단
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Publication of WO2018080172A1 publication Critical patent/WO2018080172A1/en

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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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; 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
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin

Definitions

  • the present invention relates to an eye drops formulation for treating glaucoma, and more particularly, to include a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticles.
  • the present invention relates to an ophthalmic preparation for treating glaucoma, which is biodegraded by an enzyme present, which can increase the medicinal time through continuous release of the drug, and does not release the drug when stored.
  • Glaucoma is a disease in which the intraocular pressure rises and the optic nerve is injured and gradually becomes blind, and drugs are widely used to treat it.
  • Conventional drugs for treating glaucoma treat glaucoma by lowering the pressure in the eye by increasing the outflow of the drug through the cornea and into the anterior water when the drug is prescribed on the surface of the cornea, as in the following patent document. .
  • Patent Publication No. 10-2012-0047851 (2012. 95. 14. published) "phosphate-free pharmaceutical composition for treating glaucoma"
  • the conventional drug for treating glaucoma is present as a single molecule and only 2 to 3% penetrates the cornea and flows into the anterior water. Therefore, the drug is inferior to the amount used, and the drug washed off the surface of the cornea is systemic with tears. In this case, since the drug is moved to other organs of the body, there are problems that accompany various side effects.
  • the present invention has been made to solve the above problems,
  • An object of the present invention is to provide an ophthalmic preparation for treating glaucoma that can increase the retention time by increasing the retention time on the surface of the cornea, thereby increasing the drug efficacy and reducing the amount of drug circulation systemically.
  • an object of the present invention is to provide an ophthalmic preparation for treating glaucoma in which the coating layer on the surface of the nanoparticles is gradually degraded by enzymes present in the tears, thereby releasing the drug in the sustained release form from the nanoparticles.
  • the onset is implemented by the embodiment having the following configuration in order to achieve the above object.
  • the ophthalmic preparation for treating glaucoma comprises a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticles, and administering the ophthalmic preparation
  • the coating layer is characterized in that the biodegradation.
  • the coating layer is characterized in that it is decomposed by the enzyme contained in the tear.
  • the ophthalmic formulation in the ophthalmic preparation for treating glaucoma according to the present invention, is characterized in that it has a spherical form.
  • the ophthalmic preparations for treating glaucoma according to the present invention is characterized in that it has a diameter of 100 to 300nm.
  • the coating layer is characterized by consisting of acetylated or acrylated chitosan.
  • the nanoparticles are formed by disulfide bonds between albumin molecules.
  • the drug in the ophthalmic preparation for treating glaucoma according to the present invention, is characterized in that latanoprost is used.
  • a method of preparing an ophthalmic eye drops preparation for treating glaucoma is a thiolation step of thiolating albumin, and disulfide bond and desolvation using a thiolated albumin and a glaucoma treatment drug.
  • the thiolation step in the preparation method of the eye drops formulation for treating glaucoma according to the present invention is characterized in that it is made by dissolving albumin in a solvent and adding and reacting 2-Iminothiolane hydrochloride.
  • the particle forming step is induced by adding a drug for treating glaucoma to an albumin solution into which a thiol group is introduced and titrating ethanol. It is characterized in that a thiol group is made to make a cystine bond.
  • the particle formation step may be performed by slowly adding a solution in which the drug for treating glaucoma is dissolved in an albumin solution into which a thiol group is introduced, thereby adding hydrophobicity and It is characterized by being separated into two hydrophilic layers and applying energy where the layer separation takes place.
  • the coating layer forming step in the preparation method of eye drops for treating glaucoma according to the present invention is electrostatic by injecting albumin nanoparticles carrying glaucoma drug on the surface of the acetylated chitosan solution After the chitosan reacts to cover the albumin nanoparticles by attraction, the coated nanoparticles are uniformly treated, and are treated for a predetermined time using an ultrasonicator.
  • the onset is implemented by the embodiment having the following configuration in order to achieve the above object.
  • An object of the present invention is to provide an ophthalmic preparation for treating glaucoma that can increase the retention time by increasing the retention time on the surface of the cornea, thereby increasing the drug efficacy and reducing the amount of drug circulation systemically.
  • an object of the present invention is to provide an ophthalmic preparation for treating glaucoma in which the coating layer on the surface of the nanoparticles is gradually degraded by enzymes present in the tears, thereby releasing the drug in the sustained release form from the nanoparticles.
  • 1 is a chart showing the measurement results using the dynamic light scattering of D-HSA-NPs.
  • Figure 2 is a chart showing the measurement results using Dynamic light scattering of D-tHSA-NPs.
  • Figure 3 is a chart showing the measurement results using Dynamic light scattering of C-D-tHSA-NPs.
  • Figure 4 is a chart showing the measurement results using the dynamic light scattering of HSA NPs.
  • 5 is a chart showing the measurement results using dynamic light scattering of Acrylated chi HSA NPs.
  • Example 8 is a chart showing the measurement results using dynamic light scattering of albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in Example (5).
  • FIG. 10 is a chart showing the surface charge value measurement results of HSA NPs and Acrylated chi HSA NPs.
  • FIG. 11 is a chart showing the surface charge value measurement results of HSA NPs and Acrylated chi HSA NPs (2: 1).
  • FIG. 12 is a graph showing the results of measuring surface charge values of HSA NPs and Acrylated chi HSA NPs (1: 2).
  • 13 is an SEM image of D-tHSA-NPs and C-D-tHSA-NPs.
  • 16 is a table showing the results of measuring the surface charge value before and after the reaction with C-D-tHSA-NPs and Lysozyme.
  • FIG. 17 is a diagram showing SEM images before and after the Lysozyme reaction with C-D-tHSA-NPs.
  • FIG. 18 is a chart showing drug release results of HSA NPs, Acrylated chi HSA NPs, and Acrylated chi HSA NPs to which lysozyme was added.
  • An ophthalmic preparation for treating glaucoma includes a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticle, wherein the coating layer is biodegradable upon administration of the ophthalmic preparation. It is characterized by.
  • the ophthalmic preparations have a certain shape and diameter but preferably have a spherical shape and a diameter of 100 to 300 nm.
  • the drug for treating glaucoma is composed of albumin nanoparticles, and various drugs capable of treating glaucoma may be used.
  • various drugs capable of treating glaucoma may be used.
  • latanoprost may be used.
  • the albumin nanoparticles are formed to support a drug for treating glaucoma, and a coating layer is formed on the outside thereof, and may be formed by various methods capable of forming spherical and nano-size diameter particles. It can be formed by disulfide bonds.
  • the coating layer is formed outside the albumin nanoparticles to be biodegraded by enzymes (eg, lysozyme, etc.) present in the tear, and for example, an amine group, which is a polymer that can be biodegraded by an enzyme (lysozyme) contained in the tear, is fixed.
  • enzymes eg, lysozyme, etc.
  • an amine group which is a polymer that can be biodegraded by an enzyme (lysozyme) contained in the tear, is fixed.
  • Ratio acetylation and / or acrylation of chitosan can be used (the rate of degradation can vary if varying the degree of acetylation or acrylation of chitosan).
  • the drug does not exist as a single molecule, but exists in a form supported on albumin nanoparticles, that is, the retention time on the corneal surface of the ophthalmic preparation is increased, thereby continually releasing the drug to increase the drug efficacy time. This reduces the amount of drug circulating throughout the system.
  • the Yanja formulation is formed on the outside of the albumin nanoparticles carrying the drug is a biodegradable coating layer is formed by the enzyme present in the tear, it can increase the period of use do not release the drug during storage.
  • the ophthalmic preparation may vary the thickness of the coating layer along the outer surface of the albumin nanoparticles or vary the components (degrees of acetylation or acrylation of chitosan).
  • the coating layer formed on the outer side of the albumin nanoparticles carrying the same may vary in the rate of decomposition along the outer surface of the albumin nanoparticles so that the drug can be released in a sustained release form from the nanoparticles.
  • the method for producing the eye drops formulation for treating glaucoma is the particle formation step of forming an albumin nanoparticles carrying the drug by reacting the albumin and the drug for treating glaucoma , Forming a coating layer forming a coating layer surrounding the drug-supported albumin nanoparticles.
  • Albumin nanoparticles carrying the drug can be prepared using the effect of aggregation by the polarity difference of the protein, latanoprost having hydrophobic properties is effectively albumin because it has a strong affinity for four hydrophobic sites present in the albumin molecule Can be supported on nanoparticles.
  • the particle forming step is to form an albumin nanoparticles carrying a drug, dissolve the albumin and glaucoma treatment drug in a solvent, titrate and coagulate ethanol, and crosslink the amine group of the albumin bound drug by adding glutaraldehyde Is done.
  • the coating layer forming step injects albumin nanoparticles carrying glaucoma drugs on the surface of the acetylated or acrylated chitosan solution to react chitosan to surround the albumin nanoparticles by electrostatic attraction, and then coated nanoparticles.
  • an ultrasonicator Ultrasonicator
  • the coating layer forming step may further include a step of irradiating ultraviolet rays by additionally adding a photoinitiator after the ultrasonic treatment. That is, the albumin nanoparticles carrying the drug have a negative charge property on the surface, and chitosan is a polymer having a positive charge property, and thus the surface of the albumin nanoparticles can be coated by electrostatic attraction.
  • a method of preparing an ophthalmic preparation for treating glaucoma includes a thiolation step of thiolating albumin, and a drug supported through disulfide bond and desolvation process using a thiolated albumin and a glaucoma treatment drug.
  • Particle forming step of forming albumin nanoparticles, and a coating layer forming step of forming a coating layer surrounding the albumin nanoparticles carrying the drug Since the coating layer forming step is made the same as the coating layer forming step described above, a description thereof will be omitted below.
  • the thiolation step is a step of introducing a thiol group into albumin, by dissolving albumin in a solvent and adding 2-Iminothiolane hydrochloride and reacted.
  • the particle forming step is performed by adding a drug for treating glaucoma to the albumin solution into which the thiol group is introduced, and titrating the ethanol so that the induced thiol group has cystine bonds.
  • the particle forming step may be performed by slowly adding a solution in which a drug for treating glaucoma is dissolved in an albumin solution into which a thiol group is introduced, and separating the hydrophobic and hydrophilic layers into two layers, and using tip sonic where the layer separation occurs. Can be performed by addition.
  • centrifugation was carried out at 13000 rpm for 10 minutes to remove the ungranulated latanoprost and albumin to carry the albumin nanoparticles carrying the glaucoma treatment drug (D-HSA -NPs).
  • centrifugation was performed at 13000 rpm for 10 minutes to remove the ungranulated latanoprost and albumin to carry the albumin nanoparticles carrying the glaucoma treatment drug (D-tHSA -NPs).
  • Albumin nanoparticles carrying glaucoma treatment drug were coated using chitosan (molecular weight: 50 kDa), a polymer in which 30% of the amine groups in the polymer chain were substituted with acetyl groups.
  • chitosan molecular weight: 50 kDa
  • 50 ⁇ L of a 30% acetylated chitosan solution (0.5 mg / mL) was placed in a 200 ⁇ L tube, and then 50 ⁇ L of a D-tHSA-NPs solution (20 mg / mL) was slowly injected onto the surface of the chitosan solution to produce chitosan albumin nanoparticles.
  • the coated nanoparticles After reacting for 30 minutes so as to be covered by electrostatic attraction, the coated nanoparticles are treated for 10 seconds using an Ultrasonicator. Thereafter, centrifugation was performed at 13000 rpm for 10 minutes to remove ungranulated chitosan to obtain albumin nanoparticles (C-D-tHSA-NPs) loaded with chitosan-coated glaucoma treatment drug.
  • Latanoprost drug is dissolved in 1 ml of ACN to form a drug dissolution solution.
  • ACN thiolated albumin
  • centrifugation was performed at 13200 rpm for 10 minutes to remove unlatified latanoprost and albumin, leaving only pellets, and centrifuging at 3000 rpm for 5 minutes to obtain supernatant, which was loaded with albumin.
  • Nanoparticles (HSA NPs) are obtained. 500 ⁇ l of the 30 mg acrylated chitosan aqueous solution of 5 mg / ml on the surface of the above-obtained drug nanoparticles 500 ⁇ l was added dropwise so as not to mix slowly, and then shielded and stored at room temperature for 2 hours for reaction, and after 2 hours.
  • Latanoprost drug is dissolved in 200 ul of chloroform to form a drug dissolution solution.
  • Slowly adding the drug dissolution solution to the thiolated albumin solution separates it into two hydrophobic and hydrophilic layers, applying energy using a tip sonic where the layer separation takes place (at this time, stopping at intervals of three seconds to prevent heat generation). Energy), and quickly blow chloroform using nitrogen gas.
  • 1 shows measurement results of D-HSA-NPs
  • FIG. 2 shows measurement results of D-tHSA-NPs
  • FIG. 3 shows measurement results of CD-tHSA-NPs
  • FIG. 4 shows results of measurement of HSA NPs
  • 5 shows measurement results of Acrylated chi HSA NPs
  • FIG. 6 shows measurement results of Acrylated chi HSA NPs (2: 1) (compared to Acrylated chi HSA NPs prepared in (4) of Example 1). Only the mixing volume ratio of HSA NPs and acrylated chitosan is 2: 1 only.
  • FIG. 1 shows measurement results of D-HSA-NPs
  • FIG. 2 shows measurement results of D-tHSA-NPs
  • FIG. 3 shows measurement results of CD-tHSA-NPs
  • FIG. 4 shows results of measurement of HSA NPs
  • 5 shows measurement results of Acrylated chi HSA NPs
  • FIG. 6 shows measurement results of Acrylated chi HSA NP
  • FIG. 7 shows measurement results of Acrylated chi HSA NPs (1: 2) (Acrylated chi HSA NPs prepared in Example 4 (4)). Compared with HSA NPs and acrylated chitosan, the mixing volume ratio is only 1: 2), and FIG. 8 shows the measurement results of albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in Example (5). Indicates.
  • D-HSA-NPs D-tHSA-NPs, D-tHSA-NPs, HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), Acrylated chi HSA NPs
  • the albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in (1: 2) and Example 1 (5) all have a uniform particle distribution and have a particle diameter of 200 to 300 nm.
  • D-HSA-NPs have a supporting efficiency of 29.31%
  • D-tHSA-NPs have a supporting efficiency of 35.82%
  • HSA-NPs have a supporting efficiency of 76.21%.
  • the nanoparticles of uniform size can be produced by the preparation method, and the albumin nanoparticles carrying the drug cross-linked with the amine group by the albumin nanoparticles carrying the drug formed by disulfide bonds. Compared to the strong binding force can be seen that many drugs can be supported.
  • FIG. 13A is an image of D-tHSA-NPs
  • FIG. 13B is an image of C-D-tHSA-NPs.
  • HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), and Acrylated chi HSA NPs (1: 2) were measured by SEM and the results are shown in FIG. 14.
  • Acrylated chi HSA NPs were measured under a fluorescence microscope and the results are shown in FIG. 15.
  • FIG. 15 (c) shows the synthesis of the images (a) and (b).
  • Albumin has a negative charge property on the surface and chitosan has a positive charge property.
  • D-tHSA-NPs have a negative charge value
  • CD-tHSA-NPs have a positive charge value.
  • HSA NPs have negative charge values and Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), and Acrylated chi HSA NPs (1: 2) have positive charge values. Changes can be confirmed, and red fluorescence and green fluorescence images can be confirmed through FIG. 15. It can be seen that the coating layer was formed on the albumin nanoparticles carrying the glaucoma therapeutic drug.
  • Lysozyme concentration in real human lacrimal fluid is known to be 1.7mg / mL. Therefore, in order to determine the degree of degradation of chitosan coating when CD-tHSA-NPs are used as eye drops, a hypoxic tear solution The degree of degradation was analyzed in (1.7 mg / ml Lysozyme solution). Specifically, Lysozyme solution (3.4 mg / mL) dissolved in the same conditions as CD-tHSA-NPs solution (10 mg / mL) dispersed in pH 7.4 PBS was mixed and reacted at 37 ° C. for 24 hours and 48 hours.
  • FIG. 17 is an image before reaction
  • FIG. 17 (b) is an image after 24 hours of reaction.
  • HSA NPs solution (Sample 1) and Acrylated chi HSA NPs solution (Sample 2) having the same concentration were prepared, and sample 2 was mixed with a virtual tear solution (1.7 mg / ml Lysozyme solution).
  • a virtual tear solution 1.7 mg / ml Lysozyme solution.
  • chitosan-coated nanoparticles (Sample 2) is slower drug release rate compared to the chitosan-coated nanoparticles (Sample 1), chitosan-coated nanoparticles (sample) 3) can be seen that the drug release rate is faster than the chitosan-coated nanoparticles (Sample 2) is not mixed with the lacrimal fluid solution, it can be seen that the lacrimal enzyme has a function of decomposing chitosan.
  • coating chitosan reduces the release effect when present in the formulation rather than uncoating, and when instilled, it will react with the lacrimal enzyme in the tear to remove the coated chitosan and release the supported glaucoma drug quickly.

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Abstract

The present invention relates to an eye drop formulation for glaucoma treatment and, more particularly, to an eye drop formulation for glaucoma treatment, comprising a drug for treating glaucoma, albumin nanoparticles carrying the drug and a coating layer surrounding the nanoparticles, wherein upon administration of the eye drop, the coating layer may be biodegraded by enzymes present in tears, thereby increasing a duration of a medicinal effect through sustained release of the drug, and does not release the drug when stored.

Description

녹내장 치료용 안약 제제Eye Drops for Glaucoma Treatment
본 발명은 녹내장 치료용 안약 제제에 대한 것으로, 더욱 상세하게는 녹내장 치료용 약물과 상기 약물을 담지한 알부민 나노입자와 상기 나노입자를 에워싸는 코팅층을 포함하며, 안약 제제의 투여시 상기 코팅층은 눈물에 존재하는 효소에 의해 생분해되어, 약물의 지속적인 방출을 통한 약효시간을 늘릴 수 있고, 보관시 약물을 방출하지 않는 녹내장 치료용 안약 제제에 대한 것이다.The present invention relates to an eye drops formulation for treating glaucoma, and more particularly, to include a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticles. The present invention relates to an ophthalmic preparation for treating glaucoma, which is biodegraded by an enzyme present, which can increase the medicinal time through continuous release of the drug, and does not release the drug when stored.
녹내장은 안압이 상승하여 시신경이 손상을 입어 서서히 실명이 되는 질병으로, 이를 치료하기 위하여 약물이 널리 이용되고 있다. 종래의 녹내장 치료용 약물은 하기의 특허문헌처럼 약물이 각막의 표면에 처방될 때 약물이 각막을 투과하여 전방수로 유입되고 유리체 내에서 전방수가 빠져나오는 유출량을 늘려 안구 내의 압력을 낮추어 녹내장을 치료한다.Glaucoma is a disease in which the intraocular pressure rises and the optic nerve is injured and gradually becomes blind, and drugs are widely used to treat it. Conventional drugs for treating glaucoma treat glaucoma by lowering the pressure in the eye by increasing the outflow of the drug through the cornea and into the anterior water when the drug is prescribed on the surface of the cornea, as in the following patent document. .
<특허문헌><Patent Documents>
특허공개공보 제10-2012-0047851호(2012. 95. 14. 공개) "녹내장 치료용 인산염 무첨가 약학 조성물"Patent Publication No. 10-2012-0047851 (2012. 95. 14. published) "phosphate-free pharmaceutical composition for treating glaucoma"
하지만, 종래의 녹내장 치료용 약물은 단분자로 존재하여 단지 2 내지 3%만이 각막을 투과하여 전방수에 유입되므로, 사용량에 비해 약효가 떨어지고, 각막의 표면에서 씻겨나간 약물은 눈물과 함께 전신으로 순환하게 되는데 이러할 경우 약물이 신체의 다른 장기로 이동이 되기 때문에 다양한 부작용을 수반하게 되는 문제가 있다.However, the conventional drug for treating glaucoma is present as a single molecule and only 2 to 3% penetrates the cornea and flows into the anterior water. Therefore, the drug is inferior to the amount used, and the drug washed off the surface of the cornea is systemic with tears. In this case, since the drug is moved to other organs of the body, there are problems that accompany various side effects.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로,The present invention has been made to solve the above problems,
본 발명은 각막표면에서 머무름 시간을 증대시켜 약물을 지속적으로 방출하여 약효시간을 늘릴 수 있고 약물이 전신으로 순환하는 양을 줄일 수 있는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.An object of the present invention is to provide an ophthalmic preparation for treating glaucoma that can increase the retention time by increasing the retention time on the surface of the cornea, thereby increasing the drug efficacy and reducing the amount of drug circulation systemically.
또한, 본 발명은 보관시 약물을 방출하지 않는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.It is also an object of the present invention to provide an ophthalmic preparation for treating glaucoma that does not release the drug when stored.
또한, 본 발명은 눈물에 존재하는 효소에 의해 나노입자 표면의 코팅층이 서서히 분해되어 나노입자로부터 약물이 서방형으로 방출될 수 있는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.In addition, an object of the present invention is to provide an ophthalmic preparation for treating glaucoma in which the coating layer on the surface of the nanoparticles is gradually degraded by enzymes present in the tears, thereby releasing the drug in the sustained release form from the nanoparticles.
본 발병은 앞서 본 목적을 달성하기 위하여 다음과 같은 구성을 가진 실시예에 의해 구현된다.The onset is implemented by the embodiment having the following configuration in order to achieve the above object.
본 발명의 일 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제는 녹내장 치료용 약물과, 상기 약물을 담지한 알부민 나노입자와, 상기 나노입자를 에워싸는 코팅층을 포함하며, 상기 안약 제제의 투여시 상기 코팅층은 생분해되는 것을 특징으로 하는 한다.According to an embodiment of the present invention, the ophthalmic preparation for treating glaucoma according to the present invention comprises a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticles, and administering the ophthalmic preparation The coating layer is characterized in that the biodegradation.
본 발명의 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 코팅층은 눈물에 포함되어 있는 효소에 의해 분해되는 것을 특징으로 한다.According to another embodiment of the present invention, in the ophthalmic glaucoma formulation according to the present invention, the coating layer is characterized in that it is decomposed by the enzyme contained in the tear.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 안약제제는 구형의 형태를 가지는 것을 특징으로 한다.According to another embodiment of the present invention, in the ophthalmic preparation for treating glaucoma according to the present invention, the ophthalmic formulation is characterized in that it has a spherical form.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 안약제제는 100 내지 300nm의 직경을 가지는 것을 특징으로 한다.According to another embodiment of the present invention, the ophthalmic preparations for treating glaucoma according to the present invention is characterized in that it has a diameter of 100 to 300nm.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 코팅층은 아세틸화된 또는 아크릴레이션화된 키토산으로 이루어지는 것을 특징으로 한다.According to another embodiment of the present invention, in the ophthalmic glaucoma formulation according to the present invention, the coating layer is characterized by consisting of acetylated or acrylated chitosan.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 나노입자는 알부민 분자 사이의 디설파이드 결합에 의해서 형성되는 것을 특징으로 한다.According to another embodiment of the present invention, in the ophthalmic preparation for treating glaucoma according to the present invention, the nanoparticles are formed by disulfide bonds between albumin molecules.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제에 있어서 상기 약물은 라타노프로스트(latanoprost)가 사용되는 것을 특징으로 한다.According to another embodiment of the present invention, in the ophthalmic preparation for treating glaucoma according to the present invention, the drug is characterized in that latanoprost is used.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제의 제조방법은 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 녹내장 치료용 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자를 에워싸는 코팅층을 형성하는 코팅층 형성단계를 포함하며, 상기 안약 제제의 투여시 상기 코팅층은 눈물에 존재하는 효소에 의해 생분해되는 것을 특징으로 한다.According to another embodiment of the present invention, a method of preparing an ophthalmic eye drops preparation for treating glaucoma according to the present invention is a thiolation step of thiolating albumin, and disulfide bond and desolvation using a thiolated albumin and a glaucoma treatment drug. A particle forming step of forming a drug-supported albumin nanoparticle through a process, and a coating layer forming step of forming a coating layer surrounding the drug-supported albumin nanoparticles, wherein the coating layer is in tears upon administration of the ophthalmic preparation. It is characterized by biodegradation by the enzyme present.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제의 제조방법에 있어서 상기 티올화단계는 용매에 알부민을 녹이고 2-Iminothiolane hydrochloride를 첨가하고 반응시켜 이루어지는 것을 특징으로 한다.According to another embodiment of the present invention, the thiolation step in the preparation method of the eye drops formulation for treating glaucoma according to the present invention is characterized in that it is made by dissolving albumin in a solvent and adding and reacting 2-Iminothiolane hydrochloride.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제의 제조방법에 있어서 상기 입자형성단계는 티올기가 도입된 알부민 용액에 녹내장 치료용 약물을 첨가하고, 에탄올을 적정하여 유도된 티올기가 시스틴 결합을 하도록 하여 이루어지는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for preparing an ophthalmic eye drops preparation for treating glaucoma according to the present invention, the particle forming step is induced by adding a drug for treating glaucoma to an albumin solution into which a thiol group is introduced and titrating ethanol. It is characterized in that a thiol group is made to make a cystine bond.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제의 제조방법에 있어서 상기 입자형성단계는 티올기가 도입된 알부민 용액에 녹내장 치료용 약물이 용해된 용액을 천천히 첨가하여 소수성 및 친수성의 두 층으로 분리하고 층 분리가 일어난 곳에 에너지를 가하여 이루어지는 것을 특징으로 한다.According to another embodiment of the present invention, in the method for preparing an ophthalmic preparation for treating glaucoma according to the present invention, the particle formation step may be performed by slowly adding a solution in which the drug for treating glaucoma is dissolved in an albumin solution into which a thiol group is introduced, thereby adding hydrophobicity and It is characterized by being separated into two hydrophilic layers and applying energy where the layer separation takes place.
본 발명의 또 다른 실시예에 따르면, 본 발명에 따른 녹내장 치료용 안약 제제의 제조방법에 있어서 상기 코팅층형성단계는 아세틸화된 키토산 용액의 표면에 녹내장 약물이 담지된 알부민 나노입자를 주입하여 정전기적 인력에 의해 키토산이 알부민 나노입자를 감싸도록 반응시킨 후, 코팅된 나노입자를 균일화시키기 위하여, 울트라소니케이터(Ultrasonicator)를 이용하여 일정 시간 동안 처리하여 이루어지는 것을 특징으로 한다.According to another embodiment of the present invention, the coating layer forming step in the preparation method of eye drops for treating glaucoma according to the present invention is electrostatic by injecting albumin nanoparticles carrying glaucoma drug on the surface of the acetylated chitosan solution After the chitosan reacts to cover the albumin nanoparticles by attraction, the coated nanoparticles are uniformly treated, and are treated for a predetermined time using an ultrasonicator.
본 발병은 앞서 본 목적을 달성하기 위하여 다음과 같은 구성을 가진 실시예에 의해 구현된다.The onset is implemented by the embodiment having the following configuration in order to achieve the above object.
본 발명은 각막표면에서 머무름 시간을 증대시켜 약물을 지속적으로 방출하여 약효시간을 늘릴 수 있고 약물이 전신으로 순환하는 양을 줄일 수 있는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.An object of the present invention is to provide an ophthalmic preparation for treating glaucoma that can increase the retention time by increasing the retention time on the surface of the cornea, thereby increasing the drug efficacy and reducing the amount of drug circulation systemically.
또한, 본 발명은 보관시 약물을 방출하지 않는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.It is also an object of the present invention to provide an ophthalmic preparation for treating glaucoma that does not release the drug when stored.
또한, 본 발명은 눈물에 존재하는 효소에 의해 나노입자 표면의 코팅층이 서서히 분해되어 나노입자로부터 약물이 서방형으로 방출될 수 있는 녹내장 치료용 안약 제제를 제공하는데 그 목적이 있다.In addition, an object of the present invention is to provide an ophthalmic preparation for treating glaucoma in which the coating layer on the surface of the nanoparticles is gradually degraded by enzymes present in the tears, thereby releasing the drug in the sustained release form from the nanoparticles.
도 1은 D-HSA-NPs의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.1 is a chart showing the measurement results using the dynamic light scattering of D-HSA-NPs.
도 2는 D-tHSA-NPs의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.Figure 2 is a chart showing the measurement results using Dynamic light scattering of D-tHSA-NPs.
도 3은 C-D-tHSA-NPs의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.Figure 3 is a chart showing the measurement results using Dynamic light scattering of C-D-tHSA-NPs.
도 4는 HSA NPs의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.Figure 4 is a chart showing the measurement results using the dynamic light scattering of HSA NPs.
도 5는 Acrylated chi HSA NPs의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.5 is a chart showing the measurement results using dynamic light scattering of Acrylated chi HSA NPs.
도 6은 Acrylated chi HSA NPs(2:1)의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.6 is a chart showing the measurement results using dynamic light scattering of Acrylated chi HSA NPs (2: 1).
도 7은 Acrylated chi HSA NPs(1:2)의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.7 is a chart showing the measurement results using dynamic light scattering of Acrylated chi HSA NPs (1: 2).
도 8은 실시예 1의 (5)에서 제조된 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자의 Dynamic light scattering을 이용한 측정결과를 나타내는 도표.8 is a chart showing the measurement results using dynamic light scattering of albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in Example (5).
도 9는 D-tHSA-NPs 및 C-D-tHSA-NPs의 표면 전하값 측정결과를 나타내는 도표.9 is a table showing the results of surface charge value measurement of D-tHSA-NPs and C-D-tHSA-NPs.
도 10은 HSA NPs 및 Acrylated chi HSA NPs의 표면 전하값 측정결과를 나타내는 도표.10 is a chart showing the surface charge value measurement results of HSA NPs and Acrylated chi HSA NPs.
도 11은 HSA NPs 및 Acrylated chi HSA NPs(2:1)의 표면 전하값 측정결과를 나타내는 도표.11 is a chart showing the surface charge value measurement results of HSA NPs and Acrylated chi HSA NPs (2: 1).
도 12는 HSA NPs 및 Acrylated chi HSA NPs(1:2)의 표면 전하값 측정결과를 나타내는 도표.12 is a graph showing the results of measuring surface charge values of HSA NPs and Acrylated chi HSA NPs (1: 2).
도 13은 D-tHSA-NPs 및 C-D-tHSA-NPs의 SEM 이미지.13 is an SEM image of D-tHSA-NPs and C-D-tHSA-NPs.
도 14는 HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs(2:1) 및 Acrylated chi HSA NPs(1:2)의 SEM 이미지.14 is an SEM image of HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1) and Acrylated chi HSA NPs (1: 2).
도 15는 Acrylated chi HSA NPs의 형광 현미경 이미지.15 is a fluorescence microscopic image of Acrylated chi HSA NPs.
도 16은 C-D-tHSA-NPs와 Lysozyme 반응 전후 표면 전하값 측정결과를 나타내는 도표.16 is a table showing the results of measuring the surface charge value before and after the reaction with C-D-tHSA-NPs and Lysozyme.
도 17은 C-D-tHSA-NPs와 Lysozyme 반응 전후 SEM 이미지를 나타내는 도표.FIG. 17 is a diagram showing SEM images before and after the Lysozyme reaction with C-D-tHSA-NPs. FIG.
도 18은 HSA NPs, Acrylated chi HSA NPs 및 Lysozyme을 가한 Acrylated chi HSA NPs의 약물 방출 결과를 나나태는 도표.FIG. 18 is a chart showing drug release results of HSA NPs, Acrylated chi HSA NPs, and Acrylated chi HSA NPs to which lysozyme was added.
이하에서는 본 발명에 따른 녹내장 치료용 안약 제제를 첨부된 도면을 참조하여 상세히 설명한다. 특별한 정의가 없는 한 본 명세서의 모든 용어는 본 발명이 속하는 기술분야의 통상의 지식을 가진 기술자가 이해하는 당해 용어의 일반적 의미와 동일하고 만약 본 명세서에 사용된 용어의 의미와 충돌하는 경우에는 본 명세서에 사용된 정의에 따른다. 또한, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대해 상세한 설명은 생략한다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다.Hereinafter, the eye drops formulation for treating glaucoma according to the present invention will be described in detail with reference to the accompanying drawings. Unless otherwise defined, all terms in this specification are equivalent to the general meaning of the terms understood by those of ordinary skill in the art to which the present invention pertains and, if they conflict with the meanings of the terms used herein, Follow the definition used in the specification. In addition, detailed description of well-known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted. Throughout the specification, when a part is said to "include" a certain component, it means that it may further include other components, without excluding other components unless specifically stated otherwise.
본 발명의 일 실시예에 따른 녹내장 치료용 안약 제제는 녹내장 치료용 약물과, 상기 약물을 담지한 알부민 나노입자와, 상기 나노입자를 에워싸는 코팅층을 포함하며, 상기 안약 제제의 투여시 상기 코팅층은 생분해되는 것을 특징으로 한다. 상기 안약 제제는 일정 형태 및 직경을 가지나 바람직하게는 구형의 형태 및 100 내지 300nm의 직경을 가진다.An ophthalmic preparation for treating glaucoma according to an embodiment of the present invention includes a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticle, wherein the coating layer is biodegradable upon administration of the ophthalmic preparation. It is characterized by. The ophthalmic preparations have a certain shape and diameter but preferably have a spherical shape and a diameter of 100 to 300 nm.
상기 녹내장 치료용 약물은 알부민 나노입자에 담지되는 구성으로, 녹내장을 치료할 수 있는 다양한 약물이 사용될 수 있으며, 일 예로 라타노프로스트(latanoprost)가 사용될 수 있다. 상기 알부민 나노입자는 녹내장 치료용 약물을 담지하는 구성으로 외측에는 코팅층이 형성되며, 구형 및 나노 사이즈 직경의 입자를 형성할 수 있는 다양한 방법에 의해 형성될 수 있으나 일 예로 티올화된 알부민 분자 사이의 디설파이드 결합에 의해서 형성될 수 있다. 상기 코팅층은 알부민 나노입자 외측에 형성되어 눈물에 존재하는 효소(예컨대, lysozyme 등)에 의해 생분해되는 구성으로, 일 예로 눈물에 포함되어 있는 효소(lysozyme)에 의해 생분해될 수 있는 고분자인 아민기가 일정 비율 acetylation 및/또는 acrylation된 키토산이 사용될 수 있다(키토산의 acetylation 또는 acrylation 정도를 변화시키는 경우 분해되는 속도가 다르도록 할 수 있음). 상기 안약 제제에서는 기존과 같이 약물이 단분자로 존재하지 않고 알부민 나노입자에 담지되는 형태로 존재하여, 즉 안약 제제의 각막표면에서 머무름 시간이 증대되어 약물을 지속적으로 방출하여 약효시간을 늘릴 수 있고 약물이 전신으로 순환하는 양을 줄일 수 있다. 또한, 상기 얀약 제제는 약물을 담지한 알부민 나노입자의 외측에는 눈물에 존재하는 효소에 의해 생분해되는 코팅층이 형성되어, 보관시 약물을 방출하지 않아 사용기간을 증대시킬 수 있다. 또한, 상기 안약 제제는 눈물에 존재하는 효소에 의해 나노입자 표면의 코팅층이 서서히 분해되므로, 알부민 나노입자의 외면을 따라 코팅층의 두께를 달리하거나 성분(키토산의 acetylation 또는 acrylation 정도)을 달리하여 상기 약물을 담지한 알부민 나노입자의 외측에 형성된 코팅층이 알부민 나노입자에 외면을 따라 분해되는 속도를 달리할 수 있어 나노입자로부터 약물이 서방형으로 방출될 수 있도록 할 수 있다.The drug for treating glaucoma is composed of albumin nanoparticles, and various drugs capable of treating glaucoma may be used. For example, latanoprost may be used. The albumin nanoparticles are formed to support a drug for treating glaucoma, and a coating layer is formed on the outside thereof, and may be formed by various methods capable of forming spherical and nano-size diameter particles. It can be formed by disulfide bonds. The coating layer is formed outside the albumin nanoparticles to be biodegraded by enzymes (eg, lysozyme, etc.) present in the tear, and for example, an amine group, which is a polymer that can be biodegraded by an enzyme (lysozyme) contained in the tear, is fixed. Ratio acetylation and / or acrylation of chitosan can be used (the rate of degradation can vary if varying the degree of acetylation or acrylation of chitosan). In the ophthalmic preparation, the drug does not exist as a single molecule, but exists in a form supported on albumin nanoparticles, that is, the retention time on the corneal surface of the ophthalmic preparation is increased, thereby continually releasing the drug to increase the drug efficacy time. This reduces the amount of drug circulating throughout the system. In addition, the Yanja formulation is formed on the outside of the albumin nanoparticles carrying the drug is a biodegradable coating layer is formed by the enzyme present in the tear, it can increase the period of use do not release the drug during storage. In addition, since the coating layer on the surface of the nanoparticles is gradually degraded by enzymes present in the tears, the ophthalmic preparation may vary the thickness of the coating layer along the outer surface of the albumin nanoparticles or vary the components (degrees of acetylation or acrylation of chitosan). The coating layer formed on the outer side of the albumin nanoparticles carrying the same may vary in the rate of decomposition along the outer surface of the albumin nanoparticles so that the drug can be released in a sustained release form from the nanoparticles.
상기와 같은 구성을 가지는 녹내장 치료용 안약 제제의 제조방법을 살펴보면, 상기 녹내장 치료용 안약 제제의 제조방법은 알부민과 녹내장 치료용 약물을 반응시켜 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자를 에워싸는 코팅층을 형성하는 코팅층 형성단계를 포함한다. 상기 약물을 담지한 알부민 나노입자는 단백질의 극성차이에 의하여 응집되는 효과를 이용하여 제조될 수 있고, hydrophobic한 성질을 가지고 있는 latanoprost는 알부민 분자에 존재하는 4개의 hydrophobic site에 친화력이 강하기 때문에 효과적으로 알부민 나노입자에 담지 될 수 있다.Looking at the manufacturing method of the eye drops formulation for treating glaucoma having the configuration as described above, the method for producing the eye drops formulation for treating glaucoma is the particle formation step of forming an albumin nanoparticles carrying the drug by reacting the albumin and the drug for treating glaucoma , Forming a coating layer forming a coating layer surrounding the drug-supported albumin nanoparticles. Albumin nanoparticles carrying the drug can be prepared using the effect of aggregation by the polarity difference of the protein, latanoprost having hydrophobic properties is effectively albumin because it has a strong affinity for four hydrophobic sites present in the albumin molecule Can be supported on nanoparticles.
상기 입자형성단계는 약물이 담지된 알부민 나노입자를 형성하는 단계로, 알부민과 녹내장 치료용 약물을 용매에 녹인 후, 에탄올을 적정하여 응집시키고, glutaraldehyde를 첨가하여 약물이 결합한 알부민의 아민기를 가교시켜 행하여진다.The particle forming step is to form an albumin nanoparticles carrying a drug, dissolve the albumin and glaucoma treatment drug in a solvent, titrate and coagulate ethanol, and crosslink the amine group of the albumin bound drug by adding glutaraldehyde Is done.
상기 코팅층형성단계는 아세틸화된 또는 아크릴레이션화된 키토산 용액의 표면에 녹내장 약물이 담지된 알부민 나노입자를 주입하여 정전기적 인력에 의해 키토산이 알부민 나노입자를 감싸도록 반응시킨 후, 코팅된 나노입자를 균일화시키기 위하여 울트라소니케이터(Ultrasonicator)를 이용하여 일정 시간 동안 처리하여 이루어진다. 상기 코팅층형성단계에서는 초음파 처리 후 추가적으로 광개시제를 넣고 자외선을 조사시키는 단계를 추가로 포함할 수도 있다. 즉, 약물을 담지하는 알부민 나노입자는 표면이 음전하 성질인데, 키토산은 양전하 성질을 띠는 고분자이기 때문에, 알부민 나노입자의 표면에 정전기적 인력을 통한 코팅이 가능하게 된다.The coating layer forming step injects albumin nanoparticles carrying glaucoma drugs on the surface of the acetylated or acrylated chitosan solution to react chitosan to surround the albumin nanoparticles by electrostatic attraction, and then coated nanoparticles. In order to homogenize the treatment by using an ultrasonicator (Ultrasonicator) for a predetermined time. The coating layer forming step may further include a step of irradiating ultraviolet rays by additionally adding a photoinitiator after the ultrasonic treatment. That is, the albumin nanoparticles carrying the drug have a negative charge property on the surface, and chitosan is a polymer having a positive charge property, and thus the surface of the albumin nanoparticles can be coated by electrostatic attraction.
본 발명의 다른 실시예 따른 녹내장 치료용 안약 제제의 제조방법은 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 녹내장 치료용 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자를 에워싸는 코팅층을 형성하는 코팅층 형성단계를 포함한다. 상기 코팅층형성단계는 앞서 설명한 코팅층 형성단계와 동일하게 이루어지므로 이하에서 이에 대한 설명은 생략하기로 한다.According to another embodiment of the present invention, a method of preparing an ophthalmic preparation for treating glaucoma includes a thiolation step of thiolating albumin, and a drug supported through disulfide bond and desolvation process using a thiolated albumin and a glaucoma treatment drug. Particle forming step of forming albumin nanoparticles, and a coating layer forming step of forming a coating layer surrounding the albumin nanoparticles carrying the drug. Since the coating layer forming step is made the same as the coating layer forming step described above, a description thereof will be omitted below.
상기 티올화단계는 알부민에 티올기를 도입하는 단계로, 용매에 알부민을 녹이고 2-Iminothiolane hydrochloride를 첨가하고 반응시켜 이루어진다.The thiolation step is a step of introducing a thiol group into albumin, by dissolving albumin in a solvent and adding 2-Iminothiolane hydrochloride and reacted.
상기 입자형성단계는 티올기가 도입된 알부민 용액에 녹내장 치료용 약물을 첨가하고, 에탄올을 적정하여 유도된 티올기가 시스틴 결합을 하도록 하여 이루어진다. 다른 실시예에 따른 상기 입자형성단계는 티올기가 도입된 알부민 용액에 녹내장 치료용 약물이 용해된 용액을 천천히 첨가하여 소수성과 친수성의 두 층으로 분리하고 층 분리가 일어난 곳에 tip sonic을 이용하여 에너지를 가하여 수행될 수 있다.The particle forming step is performed by adding a drug for treating glaucoma to the albumin solution into which the thiol group is introduced, and titrating the ethanol so that the induced thiol group has cystine bonds. According to another embodiment, the particle forming step may be performed by slowly adding a solution in which a drug for treating glaucoma is dissolved in an albumin solution into which a thiol group is introduced, and separating the hydrophobic and hydrophilic layers into two layers, and using tip sonic where the layer separation occurs. Can be performed by addition.
이하, 실시예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐, 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are only for explaining the present invention in more detail, the scope of the present invention is not limited thereto.
<실시예 1> 코팅되고 녹내장 치료 약물이 담지된 알부민 나노입자의 제조Example 1 Preparation of Albumin Nanoparticles Coated and Carrying Glaucoma Therapeutic Drugs
(1) 20mg의 알부민(human serum albumin)과 1mg의 latanoprost(acetonitrile solution 120μL)를 2mL의 3차 증류수에 녹여 1시간 동안 교반하고, 알부민 및 latanoprost 혼합액에 NaOH를 첨가하여 pH를 약 8로 조절하고, 에탄올을 1mL/minute의 속도로 3mL 넣어준 후, latanoprost가 결합된 알부민의 아민기를 cross-linking시켜 안정화를 유도하기 위하여 8%의 glutaraldehyde를 5μL를 첨가하고 48시간 동안 교반한다. 이후, latanoprost를 용해시키기 위해 사용된 acetonitrile과 에탄올을 기화시킨 후, 13000rpm으로 10분 동안 원심분리를 수행하여 입자화되지 않은 latanoprost 및 알부민을 제거하여 녹내장 치료 약물이 담지된 알부민 나노입자(D-HSA-NPs)를 얻었다.(1) 20 mg of albumin (human serum albumin) and 1 mg of latanoprost (acetonitrile solution 120 μL) are dissolved in 2 mL of tertiary distilled water and stirred for 1 hour.The pH is adjusted to about 8 by adding NaOH to the albumin and latanoprost mixture. After adding 3 mL of ethanol at a rate of 1 mL / minute, in order to induce stabilization by cross-linking an amine group of latanoprost-bound albumin, 5 μL of 8% glutaraldehyde was added and stirred for 48 hours. After evaporating the acetonitrile and ethanol used to dissolve the latanoprost, centrifugation was carried out at 13000 rpm for 10 minutes to remove the ungranulated latanoprost and albumin to carry the albumin nanoparticles carrying the glaucoma treatment drug (D-HSA -NPs).
(2) 3차 증류수에 20mg의 알부민을 녹이고 2-Iminothiolane hydrochloride 4mg을 첨가하여, 상온에서 한 시간 반응시켜 알부민 분자에 티올기를 도입하고, 도입되지 않은 2-Iminothiolane hydrochloride를 제거하고, 티올기가 도입된 알부민 용액에 1mg의 latanoprost(acetonitrile solution 120μL)를 첨가하여 1시간 동안 교반하고, 에탄올을 1mL/minute의 속도로 적정하여 유도된 티올기가 시스틴 결합을 하여 cross-linking이 되도록 48시간 동안 교반한다. 이후, latanoprost를 용해시키기 위해 사용된 acetonitrile과 에탄올을 기화시킨 후, 13000rpm으로 10분 동안 원심분리를 수행하여 입자화되지 않은 latanoprost 및 알부민을 제거하여 녹내장 치료 약물이 담지된 알부민 나노입자(D-tHSA-NPs)를 얻었다.(2) 20 mg of albumin was dissolved in tertiary distilled water, and 4 mg of 2-Iminothiolane hydrochloride was added and reacted at room temperature for one hour to introduce a thiol group into the albumin molecule, to remove unintroduced 2-Iminothiolane hydrochloride, and to introduce a thiol group. 1 mg of latanoprost (acetonitrile solution 120 μL) was added to the albumin solution and stirred for 1 hour, ethanol was titrated at a rate of 1 mL / minute, and stirred for 48 hours so that the induced thiol group was crosslinked by cystine binding. After evaporating the acetonitrile and ethanol used to dissolve the latanoprost, centrifugation was performed at 13000 rpm for 10 minutes to remove the ungranulated latanoprost and albumin to carry the albumin nanoparticles carrying the glaucoma treatment drug (D-tHSA -NPs).
(3) 고분자 체인에 존재하는 아민기의 30%가 아세틸기로 치환된 고분자인 키토산(분자량은 50kDa)을 이용하여 녹내장 치료 약물이 담지된 알부민 나노입자를 코팅하였다. 구체적으로, 30% 아세틸화된 키토산 용액(0.5mg/mL) 50μL를 200μL 튜브에 넣은 후, 키토산 용액의 표면에 D-tHSA-NPs 용액(20mg/mL) 50μL를 서서히 주입하여 키토산이 알부민 나노입자를 정전기적 인력의 의해 감싸도록 30분 반응시킨 후, 코팅된 나노입자를 균일화시키기 위하여, Ultrasonicator를 이용하여 10초간 처리한다. 이후, 13000rpm으로 10분 동안 원심분리를 수행하여 입자화되지 않은 키토산을 제거하여 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자(C-D-tHSA-NPs)를 얻였다.(3) Albumin nanoparticles carrying glaucoma treatment drug were coated using chitosan (molecular weight: 50 kDa), a polymer in which 30% of the amine groups in the polymer chain were substituted with acetyl groups. Specifically, 50 μL of a 30% acetylated chitosan solution (0.5 mg / mL) was placed in a 200 μL tube, and then 50 μL of a D-tHSA-NPs solution (20 mg / mL) was slowly injected onto the surface of the chitosan solution to produce chitosan albumin nanoparticles. After reacting for 30 minutes so as to be covered by electrostatic attraction, the coated nanoparticles are treated for 10 seconds using an Ultrasonicator. Thereafter, centrifugation was performed at 13000 rpm for 10 minutes to remove ungranulated chitosan to obtain albumin nanoparticles (C-D-tHSA-NPs) loaded with chitosan-coated glaucoma treatment drug.
(4) 40mg의 알부민(Human serum albumin)을 3차 증류수 1ml에 녹이고 0.2M NaOH를 첨가하여 pH가 8이 되도록 조절하여 알부민 용액을 형성하고, 2mg의 2-Iminothiolane hydrochloride를 3차 증류수 1ml에 녹여 2-IH 용액을 형성한 후, 알부민 용액과 2-IH 용액을 부피비 1:1로 혼합하여 상온에 한 시간 반응시킨 후 30kDa centricone을 이용하여 4000rpm, 10min centrifuge로 도입되지 않은 2-Iminothiolane hydrochloride를 제거하여 티올화된 알부민 용액을 형성한다. Latanoprost 약물 5mg를 ACN 1ml에 용해시켜 약물 용해 용액을 형성한다. 약물 용해 용액과 티올화된 알부민 용액을 혼합한 후 에탄올을 한 방울씩 떨어뜨리면서 적정하여 유도된 티올기가 시스틴 결합을 하여 cross-linking이 되도록 24시간 교반하고, 24시간동안 latanoprost를 용해시키기 위해 사용된 acetonitrile과 에탄올을 증발시킨 후, 13200rpm으로 10분 동안 원심분리를 하여 입자화되지 않은 latanoprost와 알부민을 제거하여 pellet만 남기고, 3000rpm으로 5분 동안 원심분리를 하여 상층액만 얻어내어 약물이 담지된 알부민 나노입자(HSA NPs)를 얻는다. 5mg/ml의 30% 아크릴레이션화된 키토산 수용액 500ul의 표면 위에 위에서 얻은 약물이 담지된 나노입자 500ul를 천천히 섞이지 않게 한 방울씩 넣어준 후, 차광하여 2시간 정도 상온 보관하여 반응시키고, 2시간 이후 충분히 pipetting 해주고 bath sonication 10s, vortexing 10s를 차례대로 수행한 후, Centrifuge 13200rpm, 10min 진행하고 상층액을 걷어내고 pellet만 남기고, 3차 증류수 1ml을 넣고 pipetting과 bath sonication을 가하여 pellet을 충분하게 풀어주고(Acrylated chitosan이 코팅된 에탄올 적정 방식 nanoparticle 완성), Photoinitiator 1ul를 넣고 254nm 파장대 UV를 1분간 쐬어주어 crosslinking 시켜, 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자(Acrylated chi HSA NPs)를 얻었다.(4) Dissolve 40 mg of albumin (Human serum albumin) in 1 ml of distilled water and adjust the pH to 8 by adding 0.2 M NaOH to form an albumin solution, and dissolve 2 mg of 2-Iminothiolane hydrochloride in 1 ml of distilled water. After the 2-IH solution was formed, the albumin solution and the 2-IH solution were mixed at a volume ratio of 1: 1, and reacted at room temperature for 1 hour. Then, 2-Iminothiolane hydrochloride which was not introduced at 4000 rpm and 10 min centrifuge was removed using 30 kDa centricone. To form a thiolated albumin solution. 5 mg of Latanoprost drug is dissolved in 1 ml of ACN to form a drug dissolution solution. After mixing the drug dissolution solution and the thiolated albumin solution, drop the ethanol dropwise and titrate for 24 hours so that the thiol group derived by titration is crosslinked by cystine bond, and used to dissolve latanoprost for 24 hours. After evaporating acetonitrile and ethanol, centrifugation was performed at 13200 rpm for 10 minutes to remove unlatified latanoprost and albumin, leaving only pellets, and centrifuging at 3000 rpm for 5 minutes to obtain supernatant, which was loaded with albumin. Nanoparticles (HSA NPs) are obtained. 500 μl of the 30 mg acrylated chitosan aqueous solution of 5 mg / ml on the surface of the above-obtained drug nanoparticles 500 μl was added dropwise so as not to mix slowly, and then shielded and stored at room temperature for 2 hours for reaction, and after 2 hours. After sufficient pipetting, bath sonication 10s, vortexing 10s are performed in order, centrifuge 13200rpm, 10min, and the supernatant is removed, leaving only pellet, 1 ml of tertiary distilled water is added, pipetting and bath sonication are added to loosen the pellets sufficiently ( Acrylated chitosan-coated ethanol titration method nanoparticles), 1 ul of Photoinitiator was added and crosslinked by UV light at 254 nm wavelength for 1 minute to obtain albumin nanoparticles (Acrylated chi HSA NPs) coated with chitosan-coated glaucoma treatment drug.
(5) 40mg의 알부민(Human serum albumin)을 3차 증류수 1ml에 녹이고 0.2M NaOH를 첨가하여 pH가 8이 되도록 조절하여 알부민 용액을 형성하고, 2mg의 2-Iminothiolane hydrochloride를 3차 증류수 1ml에 녹여 2-IH 용액을 형성한 후, 알부민 용액과 2-IH 용액을 부피비 1:1로 혼합하여 상온에 한 시간 반응시킨 후 30kDa centricone을 이용하여 4000rpm, 10min centrifuge로 도입되지 않은 2-Iminothiolane hydrochloride를 제거하여 티올화된 알부민 용액을 형성한다. Latanoprost 약물 2mg를 chloroform 200ul에 용해시켜 약물 용해 용액을 형성한다. 약물 용해 용액을 티올화된 알부민 용액에 천천히 첨가하면 소수성과 친수성 두 층으로 분리되게 되며, 층 분리가 일어난 곳에 tip sonic을 이용하여 에너지를 가하고(이때, 열이 발생하지 않도록 3초 간격으로 멈춰가면서 에너지를 가함), 질소 가스를 이용하여 chloroform을 빠르게 날려준다. 이후, 13200rpm, 10min centrifuge를 작동시켜서 상층액을 모두 걷어내고 pellet만 남기고, 3차 증류수 1ml을 첨가하여 pipetting과 bath sonication을 이용하여 pellet을 모두 풀어준 후, 3000rpm, 5min centrifuge를 작동시켜서 micropellet 모두 다운시키고 상층액을 얻어내어 약물이 담지된 나노입자(HSA NPs)로 얻는다. 5mg/ml의 30% 아크릴레이션화된 키토산 수용액 500ul의 표면 위에 위에서 얻은 약물이 담지된 나노입자 500ul를 천천히 섞이지 않게 표면 위에 한 방울씩 넣어준 후, 차광하여 2시간 정도 상온 보관하여 반응시키고, 2시간 이후 충분히 pipetting 해주고 bath sonication 10s, vortexing 10s를 차례대로 수행한 후, Centrifuge 13200rpm, 10min 진행하고 상층액을 걷어내고 pellet만 남기고, 3차 증류수 1ml을 넣고 pipetting과 bath sonication을 가하여 pellet을 충분하게 풀어주고(Acrylated chitosan이 코팅된 emulsion 방식 nanoparticle 완성), Photoinitiator 1ul를 넣고 254nm 파장대 UV를 1분간 쐬어주어 crosslinking 시켜, 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자를 얻었다.(5) Dissolve 40 mg of albumin (Human serum albumin) in 1 ml of distilled water and adjust the pH to 8 by adding 0.2M NaOH to form an albumin solution, and dissolve 2 mg of 2-Iminothiolane hydrochloride in 1 ml of distilled water. After the 2-IH solution was formed, the albumin solution and the 2-IH solution were mixed at a volume ratio of 1: 1, and reacted at room temperature for 1 hour. Then, 2-Iminothiolane hydrochloride which was not introduced at 4000 rpm and 10 min centrifuge was removed using 30 kDa centricone. To form a thiolated albumin solution. 2 mg of Latanoprost drug is dissolved in 200 ul of chloroform to form a drug dissolution solution. Slowly adding the drug dissolution solution to the thiolated albumin solution separates it into two hydrophobic and hydrophilic layers, applying energy using a tip sonic where the layer separation takes place (at this time, stopping at intervals of three seconds to prevent heat generation). Energy), and quickly blow chloroform using nitrogen gas. Then, remove all the supernatant by operating 13200rpm, 10min centrifuge, leaving only the pellet, adding 1ml of tertiary distilled water to release all pellets by pipetting and bath sonication, and then operating the 3000rpm, 5min centrifuge The supernatant is obtained and drugged nanoparticles (HSA NPs) are obtained. On the surface of 500mg of 5mg / ml 30% acrylated chitosan aqueous solution, 500ul of the nanoparticles carrying the drug obtained above were added dropwise onto the surface so as not to mix slowly, and then shielded and stored at room temperature for 2 hours for reaction. After the time, pipetting enough, after performing bath sonication 10s, vortexing 10s in order, centrifuge 13200rpm, 10min proceed, remove the supernatant, leave only pellet, add 1ml of tertiary distilled water, pipetting and bath sonication (Acrylated chitosan-coated emulsion-type nanoparticles were completed), 1 μl of Photoinitiator was added and crosslinking was performed for 1 minute under UV light at 254 nm wavelength to obtain albumin nanoparticles containing chitosan-coated glaucoma treatment drug.
<실시예 2> 알부민 나노입자의 특성 확인Example 2 Characterization of Albumin Nanoparticles
(1) Dynamic light scattering 방법을 이용하여 알부민 나노입자를 측정하고 그 결과를 도 1 내지 8에 나타내었다. 도 1은 D-HSA-NPs의 측정결과를 나타내며, 도 2는 D-tHSA-NPs의 측정 결과를 나타내고, 도 3은 C-D-tHSA-NPs의 측정결과를 나타내며, 도 4는 HSA NPs의 측정결과를 나타내고, 도 5는 Acrylated chi HSA NPs의 측정결과를 나타내며, 도 6은 Acrylated chi HSA NPs(2:1)의 측정결과를 나타내고(실시예 1의 (4)에서 제조된 Acrylated chi HSA NPs에 비하여 HSA NPs와 acrylated chitosan의 섞는 부피비만 2:1인 차이만 있음), 도 7은 Acrylated chi HSA NPs(1:2)의 측정결과를 나타내고(실시예 1의 (4)에서 제조된 Acrylated chi HSA NPs에 비하여 HSA NPs와 acrylated chitosan의 섞는 부피비만 1:2인 차이만 있음), 도 8은 실시예 1의 (5)에서 제조된 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자의 측정결과를 나타낸다.(1) Albumin nanoparticles were measured using a dynamic light scattering method and the results are shown in FIGS. 1 to 8. 1 shows measurement results of D-HSA-NPs, FIG. 2 shows measurement results of D-tHSA-NPs, FIG. 3 shows measurement results of CD-tHSA-NPs, and FIG. 4 shows results of measurement of HSA NPs. 5 shows measurement results of Acrylated chi HSA NPs, and FIG. 6 shows measurement results of Acrylated chi HSA NPs (2: 1) (compared to Acrylated chi HSA NPs prepared in (4) of Example 1). Only the mixing volume ratio of HSA NPs and acrylated chitosan is 2: 1 only. FIG. 7 shows measurement results of Acrylated chi HSA NPs (1: 2) (Acrylated chi HSA NPs prepared in Example 4 (4)). Compared with HSA NPs and acrylated chitosan, the mixing volume ratio is only 1: 2), and FIG. 8 shows the measurement results of albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in Example (5). Indicates.
(2) 약물의 담지 효율은 최초 투여한 latanoprost의 양과 입자의 원심분리 시 얻어진 상층액에서의 담지되지 않은 latanoprost의 차에 의해 산출되었으며 그 결과를 하기의 표 1에 나타내었다.(2) The loading efficiency of the drug was calculated by the difference between the amount of latanoprost initially administered and the amount of unsupported latanoprost in the supernatant obtained at the centrifugation of particles, and the results are shown in Table 1 below.
(3) 도 1 내지 8을 보면, D-HSA-NPs, D-tHSA-NPs, D-tHSA-NPs, HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs(2:1), Acrylated chi HSA NPs(1:2), 실시예 1의 (5)에서 제조된 키토산이 코팅된 녹내장 치료 약물이 담지된 알부민 나노입자 모두 균일한 입자 분포를 가지고, 200 내지 300nm의 입자 직경을 가짐을 알 수 있으며, 표 1을 보면 D-HSA-NPs의 경우 29.31%의 담지 효율을 가지고 D-tHSA-NPs는 35.82%의 담지 효율을 가지며, HSA-NPs이 경우 76.21%의 담지 효율을 가짐을 알 수 있다. 즉, 상기 제조방법에 의해 균일한 나노사이즈의 입자를 제조할 수 있음을 알 수 있고, 디설파이드 결합에 의해 형성한 약물을 담지한 알부민 나노입자가 아민기를 cross-linking한 약물을 담지한 알부민 나노입자에 비해 결합력이 강하여 많은 약물을 담지할 수 있음을 알 수 있다.1 to 8, D-HSA-NPs, D-tHSA-NPs, D-tHSA-NPs, HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), Acrylated chi HSA NPs It can be seen that the albumin nanoparticles carrying the chitosan-coated glaucoma treatment drug prepared in (1: 2) and Example 1 (5) all have a uniform particle distribution and have a particle diameter of 200 to 300 nm. In Table 1, it can be seen that D-HSA-NPs have a supporting efficiency of 29.31%, D-tHSA-NPs have a supporting efficiency of 35.82%, and HSA-NPs have a supporting efficiency of 76.21%. That is, it can be seen that the nanoparticles of uniform size can be produced by the preparation method, and the albumin nanoparticles carrying the drug cross-linked with the amine group by the albumin nanoparticles carrying the drug formed by disulfide bonds. Compared to the strong binding force can be seen that many drugs can be supported.
D-HSA-NPsD-HSA-NPs D-tHSA-NPsD-tHSA-NPs HSA NPsHSA NPs
담지 효율(%)Support Efficiency (%) 29.3129.31 35.8235.82 76.2176.21
<실시예 3> 알부민 나노입자의 코팅층의 확인Example 3 Confirmation of Coating Layer of Albumin Nanoparticles
(1) C-D-tHSA-NPs(원심분리된 C-D-tHSA-NPs)를 phosphate buffer saline(PBS)에 분산 후 측정(C-D-tHSA-NPs wash ×), 용액을 증류수로 교체 후 측정(C-D-tHSA-NPs wash ○)) 및 D-tHSA-NPs에 대하여 표면 전하값을 측정하여 그 결과를 도 9에 나타내었고, HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs(2:1), Acrylated chi HSA NPs(1:2) 각각을 염이 없는 3차 증류수에 넣어 표면 전하값을 측정하여 그 결과를 도 10 내지 12에 나타내었다.(1) Measured after dispersing CD-tHSA-NPs (centrifuged CD-tHSA-NPs) in phosphate buffer saline (PBS) (CD-tHSA-NPs wash ×) and measuring after replacing the solution with distilled water (CD-tHSA -NPs wash ○)) and D-tHSA-NPs were measured and the surface charge values are shown in FIG. 9. HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), Acrylated chi HSA Each of NPs (1: 2) was put in tertiary distilled water without salt to measure the surface charge value, and the results are shown in FIGS. 10 to 12.
(2) D-tHSA-NPs 및 C-D-tHSA-NPs에 대하여 SEM으로 측정하여 그 결과를 13에 나타내었다. 도 13의 (a)는 D-tHSA-NPs의 이미지이고, 도 13의 (b)는 C-D-tHSA-NPs의 이미지이다. HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs(2:1), Acrylated chi HSA NPs(1:2)에 대하여 SEM으로 측정하여 그 결과를 14에 나타내었다. Acrylated chi HSA NPs에 대하여 형광 현미경으로 측정하여 그 결과를 도 15에 나타내었다. Acrylated chi HSA NPs의 제조시 Alexa 555(붉은 형광을 띰)로 염색된 Acrylated chitosan을 사용하고, Alexa 488(녹색 형광을 띰)로 염색된 HSA를 사용하였으며, 도 15의 (a)와 (b)는 서로 다른 필터를 사용한 형광현미경 사진이며, 도 15의 (c)는 (a)와 (b) 이미지를 합성한 것이다.(2) D-tHSA-NPs and C-D-tHSA-NPs were measured by SEM and the results are shown in Table 13. FIG. 13A is an image of D-tHSA-NPs, and FIG. 13B is an image of C-D-tHSA-NPs. HSA NPs, Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), and Acrylated chi HSA NPs (1: 2) were measured by SEM and the results are shown in FIG. 14. Acrylated chi HSA NPs were measured under a fluorescence microscope and the results are shown in FIG. 15. Acrylated chitosan stained with Alexa 555 (red fluorescence) and HSA stained with Alexa 488 (green fluorescence) were used to prepare acrylated chi HSA NPs. Are fluorescence micrographs using different filters, and FIG. 15 (c) shows the synthesis of the images (a) and (b).
(3) 알부민은 표면이 음전하 성질을 가지고 키토산은 양전하 성질을 가지는데, 도 9를 보면 D-tHSA-NPs는 음전하값을 가지고 C-D-tHSA-NPs는 양전하값을 가지며, 도 10 내지 12를 보면 HSA NPs는 음전하값을 가지고 Acrylated chi HSA NPs, Acrylated chi HSA NPs(2:1), Acrylated chi HSA NPs(1:2)는 양전하값을 가지며, 도 13 및 14를 보면 키토산 코팅에 따라 입자 이미지를 변화를 확인할 수 있고, 도 15를 통해 붉은 형광, 녹색 형광 이미지를 확인할 수 있다. 녹내장 치료 약물이 담지된 알부민 나노입자에 코팅층이 형성되었음을 알 수 있다.(3) Albumin has a negative charge property on the surface and chitosan has a positive charge property. Referring to FIG. 9, D-tHSA-NPs have a negative charge value, and CD-tHSA-NPs have a positive charge value. HSA NPs have negative charge values and Acrylated chi HSA NPs, Acrylated chi HSA NPs (2: 1), and Acrylated chi HSA NPs (1: 2) have positive charge values. Changes can be confirmed, and red fluorescence and green fluorescence images can be confirmed through FIG. 15. It can be seen that the coating layer was formed on the albumin nanoparticles carrying the glaucoma therapeutic drug.
<실시예 4> Lysozyme에 의한 알부민 나노입자의 코팅층 분해의 확인Example 4 Confirmation of Coating Layer Degradation of Albumin Nanoparticles by Lysozyme
(1) 실제 사람의 누액에 존재하는 Lysozyme의 농도는 1.7mg/mL로 알려져 있으므로, C-D-tHSA-NPs가 안약 제재로 사용되었을 때 키토산 코팅의 분해 정도를 알아보기를 위하여, 가상의 눈물액 용액(1.7mg/ml Lysozyme solution)에서 분해 정도를 분석하였다. 구체적으로, pH 7.4 PBS에 분산된 C-D-tHSA-NPs 용액(10mg/mL)과 같은 조건에 녹여져 있는 Lysozyme 용액(3.4mg/mL)을 혼합하여, 37℃에서 반응시키고, 24시간, 48시간, 72시간 후에 용액으로부터 Lysozyme과 분해된 chitosan을 제거하기 위하여, 13000rpm으로 10분동안 원심분리를 수행하고 증류수에 재분산시킨다. 이후, 각각의 시간대의 Lysozyme에 의해 분해된 알부민 나노입자의 표면전하값을 측정하여 그 결과를 도 16에 나타내었다. 또한, Lysozyme 용액 반응전과 반응 24시간 후의 용액을 SEM으로 촬영하여 그 결과를 도 17에 나타내었다. 도 17의 (a)는 반응 전 이미지이고, 도 17의 (b)는 반응 24 시간 후 이미지이다.(1) Lysozyme concentration in real human lacrimal fluid is known to be 1.7mg / mL. Therefore, in order to determine the degree of degradation of chitosan coating when CD-tHSA-NPs are used as eye drops, a hypoxic tear solution The degree of degradation was analyzed in (1.7 mg / ml Lysozyme solution). Specifically, Lysozyme solution (3.4 mg / mL) dissolved in the same conditions as CD-tHSA-NPs solution (10 mg / mL) dispersed in pH 7.4 PBS was mixed and reacted at 37 ° C. for 24 hours and 48 hours. To remove Lysozyme and digested chitosan from solution after 72 hours, centrifuge for 10 minutes at 13000 rpm and redisperse in distilled water. Thereafter, the surface charge value of the albumin nanoparticles decomposed by Lysozyme at each time zone was measured and the results are shown in FIG. 16. In addition, the solution before and after the reaction of Lysozyme solution 24 hours by SEM, the results are shown in Figure 17. (A) of FIG. 17 is an image before reaction, and FIG. 17 (b) is an image after 24 hours of reaction.
(2) 또한, 동일 농도의 HSA NPs 용액(시료 1), Acrylated chi HSA NPs 용액(시료 2)을 준비하고, 시료 2에 가상의 눈물액 용액(1.7mg/ml Lysozyme solution)을 혼합하여 시료 3을 형성한 후, 시료 1 내지 3을 각각 dialysis membrane에 삽입하고, 50ml 튜브에 PBS를 10ml씩 넣고 시료가 들어있는 dialysis membrane을 삽입한 후 HLPC 분석을 통해 지남에 따라 각 용액(시료, 1, 2 및 3)의 Latanoprost 농도를 측정하여 그 결과를 도 18에 나타냈었다.(2) In addition, HSA NPs solution (Sample 1) and Acrylated chi HSA NPs solution (Sample 2) having the same concentration were prepared, and sample 2 was mixed with a virtual tear solution (1.7 mg / ml Lysozyme solution). After the formation of the samples, the samples 1 to 3 were inserted into the dialysis membrane, respectively, 10 ml of PBS was added to the 50 ml tube, and the dialysis membrane containing the sample was inserted. And Latanoprost concentration of 3) was measured and the results are shown in Figure 18.
(3) 도 16을 보면 Lysozyme 용액 반응 24시간 후 음전하값이 측정되고, 키토산이 코팅되면서 알부민 나노입자는 연결되게 되는데 도 17을 보면 Lysozyme 용액 반응 24시간 후 연결된 나노입자들이 현저하게 감소하여, 코팅층을 형성하는 키토산이 분해되었음을 알 수 있다. 또한, 도 18을 보면, 키토산이 코팅된 나노입자(시료 2)가 키토산이 코팅되지 않은 나노입자(시료 1)에 비해 약물 방출 속도가 느리며, 누액 용액이 혼합된 키토산이 코팅된 나노입자(시료 3)가 누액 용액이 혼합되지 않은 키토산이 코팅된 나노입자(시료 2)에 비해 약물 방출 속도가 더 빠른 것을 확인할 할 수 있어 누액 효소를 키토산을 분해하는 기능이 있음을 알 수 있다. 따라서, 키토산을 코팅했을 경우 코팅하지 않을 때보다 제형으로 존재할 시 방출효과를 줄이면서, 점안이 되었을 때, 눈물에 있는 누액효소와 반응하여 코팅된 키토산이 제거되면서 빠르게 담지된 녹내장 약물이 방출될 것이다.16, negative charge values are measured after 24 hours of the Lysozyme solution reaction, and albumin nanoparticles are connected as chitosan is coated. Referring to FIG. 17, the nanoparticles are significantly reduced after 24 hours of the Lysozyme solution reaction. It can be seen that the chitosan which forms the compound was decomposed. In addition, referring to Figure 18, chitosan-coated nanoparticles (Sample 2) is slower drug release rate compared to the chitosan-coated nanoparticles (Sample 1), chitosan-coated nanoparticles (sample) 3) can be seen that the drug release rate is faster than the chitosan-coated nanoparticles (Sample 2) is not mixed with the lacrimal fluid solution, it can be seen that the lacrimal enzyme has a function of decomposing chitosan. Thus, coating chitosan reduces the release effect when present in the formulation rather than uncoating, and when instilled, it will react with the lacrimal enzyme in the tear to remove the coated chitosan and release the supported glaucoma drug quickly. .
이상에서, 출원인은 본 발명의 다양한 실시예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며, 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.In the above, the Applicant has described various embodiments of the present invention, but these embodiments are merely one embodiment for implementing the technical idea of the present invention, and any changes or modifications may be made to the present invention as long as the technical idea of the present invention is implemented. It should be interpreted as falling within the scope of.

Claims (12)

  1. 안약 제제에 있어서,In the ophthalmic preparations,
    상기 안약 제제는 녹내장 치료용 약물과, 상기 약물을 담지한 알부민 나노입자와, 상기 나노입자를 에워싸는 코팅층을 포함하며,The ophthalmic preparation includes a drug for treating glaucoma, albumin nanoparticles carrying the drug, and a coating layer surrounding the nanoparticles.
    상기 안약 제제의 투여시 상기 코팅층은 생분해되는 것을 특징으로 하는 녹내장 치료용 안약 제제.The ophthalmic preparation for treating glaucoma, wherein the coating layer is biodegradable upon administration of the ophthalmic preparation.
  2. 제1항에 있어서, 상기 코팅층은The method of claim 1, wherein the coating layer
    눈물에 포함되어 있는 효소에 의해 분해되는 것을 특징으로 하는 녹내장 치료용 안약 제제.An ophthalmic preparation for treating glaucoma, characterized by being degraded by an enzyme contained in the tear.
  3. 제1항에 있어서, 상기 안약 제제는The method of claim 1, wherein the eye drops formulation is
    구형의 형태를 가지는 것을 특징으로 하는 녹내장 치료용 안약 제제.An ophthalmic preparation for treating glaucoma, which has a spherical form.
  4. 제1항에 있어서, 상기 안약 제제는The method of claim 1, wherein the eye drops formulation is
    100 내지 300nm의 직경을 가지는 것을 특징으로 하는 녹내장 치료용 안약 제제.Eye drops formulation for treating glaucoma, characterized in that having a diameter of 100 to 300nm.
  5. 제1항에 있어서, 상기 코팅층은The method of claim 1, wherein the coating layer
    아세틸화된 또는 아크릴레이션화된 키토산으로 이루어지는 것을 특징으로 하는 녹내장 치료용 안약 제제.An ophthalmic preparation for treating glaucoma, characterized by consisting of acetylated or acrylated chitosan.
  6. 제1항에 있어서, 상기 나노입자는The method of claim 1, wherein the nanoparticles
    알부민 분자 사이의 디설파이드 결합에 의해서 형성되는 것을 특징으로 하는 녹내장 치료용 안약 제제.An ophthalmic preparation for treating glaucoma, which is formed by disulfide bonds between albumin molecules.
  7. 제1항에 있어서, 상기 약물은The method of claim 1, wherein the drug
    라타노프로스트(latanoprost)가 사용되는 것을 특징으로 하는 녹내장 치료용 안약 제제.Eye drops formulation for treating glaucoma, characterized in that the use of latanoprost (latanoprost).
  8. 안약 제제의 제조방법에 있어서,In the manufacturing method of the eye drops formulation,
    상기 안약 제제의 제조방법은 알부민을 티올화하는 티올화단계와, 티올화된 알부민과 녹내장 치료용 약물을 이용하여 이황화 결합 및 탈용매화 과정을 통해 약물이 담지된 알부민 나노입자를 형성하는 입자형성단계와, 상기 약물이 담지된 알부민 나노입자를 에워싸는 코팅층을 형성하는 코팅층 형성단계를 포함하며,The preparation method of the ophthalmic preparation is a thiolation step of thiolating albumin, and a particle formation step of forming albumin nanoparticles carrying the drug through disulfide bond and desolvation process using thiolated albumin and glaucoma treatment drug. And a coating layer forming step of forming a coating layer surrounding the drug-supported albumin nanoparticles,
    상기 안약 제제의 투여시 상기 코팅층은 눈물에 존재하는 효소에 의해 생분해되는 것을 특징으로 하는 녹내장 치료용 안약 제제의 제조방법.The method of preparing an ophthalmic preparation for treating glaucoma, wherein the coating layer is biodegraded by an enzyme present in the tear when the ophthalmic preparation is administered.
  9. 제8항에 있어서, 상기 티올화단계는The method of claim 8, wherein the thiolation step is
    용매에 알부민을 녹이고 2-Iminothiolane hydrochloride를 첨가하고 반응시켜 이루어지는 것을 특징으로 하는 녹내장 치료용 안약 제제의 제조방법.A method of producing an ophthalmic preparation for treating glaucoma, comprising dissolving albumin in a solvent, and adding and reacting 2-Iminothiolane hydrochloride.
  10. 제8항에 있어서, 상기 입자형성단계는The method of claim 8, wherein the particle forming step
    티올기가 도입된 알부민 용액에 녹내장 치료용 약물을 첨가하고, 에탄올을 적정하여 유도된 티올기가 시스틴 결합을 하도록 하여 이루어지는 것을 특징으로 하는 녹내장 치료용 안약 제제의 제조방법.A method for producing an ophthalmic preparation for treating glaucoma, comprising adding a drug for treating glaucoma to an albumin solution into which a thiol group is introduced, and titrating ethanol to cause cystine bonds.
  11. 제8항에 있어서, 상기 입자형성단계는The method of claim 8, wherein the particle forming step
    티올기가 도입된 알부민 용액에 녹내장 치료용 약물이 용해된 용액을 천천히 첨가하여 소수성 및 친수성의 두 층으로 분리하고 층 분리가 일어난 곳에 에너지를 가하여 이루어지는 것을 특징으로 하는 녹내장 치료용 안약 제제의 제조방법.A method for producing an ophthalmic preparation for treating glaucoma, characterized in that the solution of the drug for treating glaucoma is slowly added to the albumin solution into which the thiol group is introduced, separated into two hydrophobic and hydrophilic layers, and energy is applied to the layer separation.
  12. 제10항에 있어서, 상기 코팅층형성단계는The method of claim 10, wherein the coating layer forming step
    아세틸화된 키토산 용액의 표면에 녹내장 약물이 담지된 알부민 나노입자를 주입하여 정전기적 인력에 의해 키토산이 알부민 나노입자를 감싸도록 반응시킨 후, 코팅된 나노입자를 균일화시키기 위하여, 울트라소니케이터(Ultrasonicator)를 이용하여 일정 시간 동안 처리하여 이루어지는 것을 특징으로 하는 녹내장 치료용 안약 제제의 제조방법.Injecting albumin nanoparticles carrying glaucoma drugs on the surface of the acetylated chitosan solution to react the chitosan to surround the albumin nanoparticles by electrostatic attraction, and then to make the coated nanoparticles uniform, an ultrasonicator ( Ultrasonicator) for the preparation of eye drops for treating glaucoma, characterized in that the treatment for a predetermined time.
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