WO2005023906A1 - ヒアルロン酸修飾物、及びそれを用いた薬物担体 - Google Patents
ヒアルロン酸修飾物、及びそれを用いた薬物担体 Download PDFInfo
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- WO2005023906A1 WO2005023906A1 PCT/JP2004/013026 JP2004013026W WO2005023906A1 WO 2005023906 A1 WO2005023906 A1 WO 2005023906A1 JP 2004013026 W JP2004013026 W JP 2004013026W WO 2005023906 A1 WO2005023906 A1 WO 2005023906A1
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- hyaluronic acid
- pla
- polymer
- acid
- drug carrier
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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
- C08G85/00—General processes for preparing compounds provided for in this subclass
-
- 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
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- 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
- C08G63/91—Polymers modified by chemical after-treatment
-
- 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
- C08G63/91—Polymers modified by chemical after-treatment
- C08G63/912—Polymers modified by chemical after-treatment derived from hydroxycarboxylic acids
Definitions
- the present invention relates to a novel modified hyaluronic acid, and a drug carrier using the same.
- biodegradable polymers that degrade in the body are widely used because they do not need to be removed after introduction into the body.
- biodegradable polymers that have been put to practical use include polylactic acid (PLA), polyglycolic acid (PGA), and copolymers of these such as lactic acid-glycolic acid copolymer (PLGA).
- Non-Patent Documents 3 and 4 For example, ribosomes (see Non-Patent Documents 3 and 4), albumin microspheres (see Non-Patent Document 5), gelatin Z chondroitin sulfate microspheres (see Non-Patent Document 6), lactic acid 'Glycolic acid copolymer (PLGA) microspheres (see Non-Patent Document 7) have been reported.
- albumin microspheres see Non-Patent Document 5
- gelatin Z chondroitin sulfate microspheres see Non-Patent Document 6
- PLGA lactic acid 'Glycolic acid copolymer
- hyaluronic acid is a biomaterial (polysaccharide) isolated from the vitreous of bovine eyes by K. Meyer in 1934, and has long been known as a main component of the extracellular matrix. I have. HA is a type of darcosamide dalican that also has a disaccharide unit force in which D-glucuronic acid and N-acetyltilcosamine are linked by a j8 (1 ⁇ 3) glycosidic bond. HA, which has no difference in its chemical and physical structure, has a metabolic system even in humans, and is the safest biomaterial in terms of immunity and toxicity.
- HA is one of the main components of synovial fluid and exerts an analgesic effect on joints due to its viscoelastic effect and anti-inflammatory effect.
- HA-based drugs have already been marketed and used for the treatment of arthropathy such as osteoarthritis and rheumatoid arthritis (for example, Smony (trade name): manufacturing and marketing Chugai Pharmaceutical Co., Ltd.) Corporation).
- Examples of the modified HA comprising a combination of HA and a biodegradable polymer include a film in which HA is coated with PLGA (see Patent Document 5) and an injection in which PLGA microspheres and HA are mixed (see Patent Document 6). ), A protein sustained-release formulation in which PLGA microspheres are dispersed in an HA hydrated mouth gel (see Patent Document 7), and a polyproprota using a cationic surfactant.
- HA-coated nanospheres due to the electrostatic interaction of tons (PCL), biomaterials such as HA-PLL (see Non-Patent Document 14) in which HA is bound to poly-L-lysine side chains, and sustained-release drug preparations have also been reported. ing.
- an HA-modified product obtained by grafting HA to PLA, PGA or PLGA which is excellent in safety, biodegradability, and in vivo stability, is not specifically known, and its preparation is not known.
- conventional drug carriers include the encapsulation efficiency of drugs (especially low-molecular drugs), their sustained release periods, dispersibility in aqueous solutions, blood retention periods, or safety (for example, inflammation occurs in vivo). Had a problem. Further, there has been no known drug carrier having excellent biocompatibility, which is composed of injectable fine particles having little aggregation between particles.
- Patent Document 1 International Publication No. W098Z43664
- Patent Document 2 International Publication WO00Z78356
- Patent Document 3 Japanese Patent Publication No. 11-513047
- Patent Document 4 JP-T-2003-525232
- Patent Document 5 JP-A-8-208706
- Patent Document 6 International Publication WO01Z28591
- Patent Document 7 International Publication WO97Z13502
- Patent Document 8 International Publication WO01Z88019
- Non-patent document l Eur.J. Clin.PHArmacol. 42, 301-305 (1992)
- Non-Patent Document 2 Clin.PHArmacol.Ther. 39, 313-317 (1986)
- Non-Patent Document 3 Biochem.J. 158, 473-476 (1976)
- Non-Patent Document 4 PHArm.PHArmaco. 45, 576-578 (1993)
- Non-Patent Document 5 Int.J.PHArmcol. 39, 129-136 (1987)
- Non-Patent Document 6 Arthritis Rhem. 41, 21.85-2195 (1998)
- Non-Patent Document 7 Int.PHArm. 195, 179-188 (2000)
- Non-Patent Document 8 J. Joint Surgery 11, 87-95 (1992)
- Non-Patent Document 9 PHArm.Res. 19, 403-410 (2002)
- Non-Patent Document 10 J. Biomed.Mater.Res. 50, 557-565 (2000)
- Non-Patent Document 11 PHArm.Res. 20, 1284-1292 (2003)
- Non-patent Document 12 Drug Dev.Ind. PHArm. 25 (1), 15-20 (1999)
- Non-Patent Document 13 Intern. J. PHArm. 87, 21-29 (1992)
- Non-Patent Document 14 Bioconj.Chem. 9, 476-481 (1998)
- the problem to be solved is to solve the problems of the conventional drug carrier, to efficiently enclose a low molecular drug, to control the sustained release period for a long period of time, and to control the blood retention.
- An object of the present invention is to provide a drug carrier having good dispersibility in an aqueous solution and having no problem in safety. It is another object of the present invention to provide a drug carrier excellent in biocompatibility, which comprises an injectable fine particle with little aggregation between particles and which solves the problem of the conventional drug carrier.
- hyaluronic acid or a derivative thereof was selected from polylactic acid, polyglycolic acid, or lactic acid-glycolic acid copolymer.
- the modified hyaluronic acid combined with the polymer to be encapsulated efficiently encloses low-molecular-weight drugs, controls the sustained release period over a long period of time, has good dispersibility in aqueous solutions, and has no problem with safety. And that the modified product of hyaluronic acid has less aggregation between particles.
- hyaluronic acid or a derivative thereof and a polylactic acid, a polydalicholic acid, and a lactic acid-glycolic acid copolymer are combined with one or more selected polymers. Modifications are provided.
- the polymer may be grafted as a side chain of hyaluronic acid or a derivative thereof.
- most or all of the polymer is It may be bound to hyaluronic acid or a derivative thereof at only one end.
- the polymer may be bonded to a carboxyl group of hyaluronic acid or a derivative thereof.
- the polymer may be bonded to the carboxyl group of hyaluronic acid or a derivative thereof via an spacer via an amide bond.
- the polymer may be bonded to a carboxyl group of hyaluronic acid or a derivative thereof via a spacer via an amide bond.
- R, R, R and R are each independently a hydrogen atom, a C alkyl group and
- Ra and Rb are each independently selected from a hydrogen atom and a C alkyl group
- Q is a polymer selected from the group consisting of polylactic acid, polydalicholic acid and lactic acid-glycolic acid copolymer.
- the polymer forms an amide bond with a nitrogen atom at a terminal carboxyl group
- n is an integer from 0 to 10.
- Hyaluronic acid modified products containing at least one repeating structure represented by Provided containing at least one repeating structure represented by Provided.
- the C alkyl group is a linear or branched alkyl group having 16 carbon atoms.
- alkylcarbonyl group means an alkylcarbonyl group having the above C alkyl group in the molecule.
- Ra and Rb are both hydrogen atoms, and A is — (CH 2) CH—CH— (O—CH
- the spacer has two or more amino groups which may be independently substituted, and the terminal carboxyl group of the polymer has the amino group. Which forms an amide bond with one of the amino groups described above, and a carboxyl group of hyaluronic acid or a derivative thereof is bonded to another amino group of the spacer by an amide bond, thereby providing a modified hyaluronic acid.
- the substituent of the amino group is not particularly limited, but may be a C alkyl group, a C
- the hyaluronic acid derivative has a formula ( ⁇ ):
- Ra, Rb and A are as defined above
- the hyaluronic acid derivative and Z or the modified hyaluronic acid itself are resistant to degradation by hyaluronan-didase, for example, an average of 18 hours or more in mammals.
- Hyaluronic acid derivatives having a residence time in blood are provided.
- the shape of the drug carrier may be a fine particle such as a microsphere or a nanosphere.
- the drug carrier may have a structure in which the polymer is coated with hyaluronic acid or a derivative thereof in an aqueous solution.
- the particle size is not particularly limited, but is preferably 200 m or less, more preferably 100 m or less, in order to allow the needle to pass through without clogging. In the case of joint administration, the particle diameter is preferably 5 m or less, because friction in the joint is physically caused and this easily induces new inflammation.
- the particle size is more preferably 500 nm or less, more preferably 200 nm or less, in order not to block peripheral blood vessels.
- the drug carrier can have properties such as the ability to target the HA receptor; resistance to hyal-oralase; and mucoadhesion.
- the drug carrier can also be a drug carrier with reduced local irritation.
- a pharmaceutical composition comprising the above drug carrier and a drug.
- the pharmaceutical composition may further comprise one or more polymers selected from polylactic acid, polyglycolic acid or lactic acid 'glycolic acid copolymer.
- the polymer is hyaluronic acid. It is of the same type as the polymer attached to the modifier.
- the drug may be encapsulated by microparticles formed by the hyaluronic acid decoration.
- the fine particles preferably have a shape in which the hyaluronic acid-modified product has a shape in which the polymer portion and Z or the hydrophobic core formed by the polymer are coated with the hyaluronic acid portion.
- the drug is encapsulated in the hydrophobic core.
- a polylactic acid having a carboxyl group at a terminal a polylactic acid Glycolic acid or lactic acid 'Glycolic acid copolymer power
- R 1, R 2, R 3, R 4, Ra, Rb and A are as defined above.
- a method for producing a modified hyaluronic acid comprising a step of reacting with a hyaluronic acid derivative, which comprises at least one repeating structure represented by the following formula:
- the polymer is
- a method for producing the drug carrier by the above-mentioned modified hyaluronic acid by a drying method in an emulsion liquid, a solvent diffusion method or a dialysis method. Provided.
- the method for producing the drug carrier of the present invention is carried out using an emulsion-in-liquid drying method, for example, the following steps:
- step c) mixing the polymer solution obtained in the step a with the modified hyaluronic acid solution or dispersion obtained in the step b to form an emulsion; and d) obtaining the emulsion in the step c. Drying the emulsion in the liquid to form drug carrier fine particles,
- step d) a step of dissolving or dispersing the modified hyaluronic acid in another aqueous phase; and e) a step of dissolving or dispersing the polymer fine particles obtained in the step c) and the modified hyaluronic acid solution or dispersion obtained in the step d. And mixing to form drug carrier fine particles.
- the method for producing the drug carrier of the present invention is performed using a solvent diffusion method, and includes, for example, the following steps;
- a) Polylactic acid, polyglycolic acid, or lactic acid 'glycolic acid copolymer power Dissolves one or more selected polymers in a water-miscible organic phase (but not water) containing one or more organic solvents The step of causing; b) dissolving or dispersing the modified hyaluronic acid in an aqueous phase; and c) dropping the polymer solution obtained in the step a) into the solution or dispersion of the modified hyaluronic acid obtained in the step b.
- a polylactic acid, polydalicholic acid and lactic acid-glycolic acid copolymer are added.
- One or more polymers selected and the hyaluronic acid Dissolving the acid modifier;
- step b) a step of dropping the polymer 'hyaluronic acid modified product solution obtained in the step a) into an aqueous phase to form drug carrier fine particles
- the method for producing the drug carrier of the present invention by a solvent diffusion method comprises the following steps:
- step c) a step of dissolving or dispersing the modified hyaluronic acid in another aqueous phase; and d) the fine polymer particles obtained in the step b) and the solution or dispersion of the modified hyaluronic acid obtained in the step c). And mixing to form drug carrier fine particles.
- the method for producing a drug carrier of the present invention is performed using a dialysis method.
- step b) a step of permeating the polymer 'hyaluronic acid modified product solution obtained in the step a) into an aqueous phase to form fine particles
- step c) a step of dissolving or dispersing the modified hyaluronic acid in another aqueous phase; and d) the fine polymer particles obtained in the step b) and the solution or dispersion of the modified hyaluronic acid obtained in the step c). And mixing to form drug carrier fine particles.
- a method for producing a pharmaceutical composition including the method for producing a drug carrier described above.
- a solvent diffusion method can be used in step b of the preparation method.
- the preparation method includes the following steps:
- a dialysis method can be used in step b of the preparation method.
- the preparation method includes the following steps:
- a method for producing a pharmaceutical composition including the method for preparing the above-mentioned aqueous solution or dispersion of a modified hyaluronic acid.
- R, R, R and R are each independently a hydrogen atom, a C alkyl group and
- Ra, Rb, Rc and Rd are each independently selected from a hydrogen atom and a C alkyl group.
- Q is a polymer selected from polylactic acid, polydalicholic acid, polyproprolataton or a copolymer thereof, wherein the polymer is bonded to a nitrogen atom at a terminal carboxyl group;
- n is an integer from 0-6
- a modified hyaluronic acid comprising at least one repeating structure represented by the following formula in the molecule:
- a polylactic acid having a carboxyl group at a terminal a polyglycolic acid, a polyprolatetaton or a copolymer thereof, Formula (IV)
- R 1, R 2, R 3, R 4, Ra, Rb, Rc, Rd and n are as defined above
- the method for producing the above-mentioned modified hyaluronic acid which comprises a step of reacting with a hyaluronic acid derivative containing at least one repeating structure represented by the following formula in the molecule, is also provided.
- the modified hyaluronic acid of the present invention comprises hyaluronic acid (HA) or a derivative thereof and polylactic acid.
- PLA polyglycolic acid
- PGA polyglycolic acid
- PLGA lactic acid-glycolic acid copolymer
- hyaluronic acid (HA) or a derivative thereof and polylactic acid (PLA), polydalicholic acid (PGA) or lactic acid / glycolic acid copolymer (PLGA) are selected. It is chemically covalently bonded rather than bonded by physical interaction such as electrostatic interaction. For this reason, HA or a derivative thereof will not be desorbed by environmental changes such as changes in salt concentration and pH after administration.
- HA or its derivative When HA or its derivative is bonded to the above-mentioned polymer, it may be bonded via a part of the spacer in order to easily control the bonding. Further, the HA derivative may include a part of a spacer for bonding to the polymer.
- a compound having a plurality of amino groups can be reacted with the carboxyl group of the glucuronic acid moiety in hyaluronic acid to introduce a part of a spacer having a hydrazide group or an amino group.
- the modification rate of the carboxylic acid by the spacer and the modification rate of the spacer by the polymer are independently controlled. can do.
- the site at which the polymer binds to HA or a derivative thereof may be the terminal of HA or a derivative thereof, or may be a site where the polymer is introduced as a side chain of HA or a derivative thereof.
- HA is a very bulky molecule in an aqueous solution
- the thickness of the HA layer is generally increased when biodegradable microparticles are prepared using HA or a derivative thereof having the polymer bonded to the terminal.
- the HA or its derivative having the polymer graft-bonded as a side chain is used, the HA or its derivative is coated on the surface of the biodegradable fine particles in a loop shape, and the thickness of the HA layer can be reduced.
- it is preferable that the polymer is graft-bonded to the side chain of HA or its derivative.
- the position of introduction of the polymer or spacer part into the modified HA or a derivative thereof includes a hydroxyl group and a carboxyl group in the HA molecule.
- a hydroxyl group when introduced into a hydroxyl group, it can be introduced by reacting an activated carboxyl group or the like with the hydroxyl group to form a carbonate ester, or by reacting an isocyanate group or the like with the hydroxyl group or the like. It can also be introduced by forming a rubamic acid ester.
- an amide group, a hydrazide group, a dihydrhydrazide group, or a sulfonic acid ester group is formed to introduce the polymer or a part of the spacer. People can.
- the HA modified product in which the carboxylic acid ester is formed and the polymer or the spacer portion is introduced may be used in an administration solution or in vivo.
- Desorption of the polymer by hydrolysis occurs in a relatively short time. Therefore, in general, it is preferable to modify via a rubamic acid ester having a slow hydrolysis rate, or an amide group, a hydrazide group or the like.
- the polymer is bonded to the carboxyl group of hyaluronic acid or a derivative thereof.
- the polymer is preferably bonded to a hyaluronic acid or a derivative thereof via an amide group or a hydrazide group. It is particularly preferred that the compound is bonded to the carboxyl group of the above by an amide bond.
- Examples of a method for binding a polymer to the terminal of HA or a derivative thereof include, for example, an aldehyde at the reducing end of HA or a derivative thereof, and a hydrazide (HZ) group introduced at the terminal of a part of the polymer or spacer.
- HZ hydrazide
- a Schiff base formed by an aldehyde group such as an amino group (AM) and a reactive functional group is converted to a reducing agent such as sodium borohydride (NaBH).
- a functional group reactive with an active ester such as a hydrazide (HZ) group or an amino (AM) group
- HZ hydrazide
- AM amino
- TAA tetrabutylammonium-palladium
- DMSO dimethyl sulfoxide
- R, R, R, R, Ra, Rb and A are as defined above.
- a terminal carboxyl group of the polymer is activated in advance with EDCZNHS, and the polymer is reacted with diamine, dihydrazide, or the like to synthesize a polymer having amino group or hydrazide group ends, and this is directly synthesized.
- a method of binding to the carboxyl group of HA or a derivative thereof with a condensing agent such as EDCZNHS can be used.
- the polymer having an amino group and a hydrazide group terminal is represented by the following formula (V):
- Ra, Rb, Q and A are as defined above.
- bonded at one end refers to a state of bonding at one of the terminal reactive groups of the polymer.
- the term “most” is not particularly limited, but specifically, at only one end thereof. It refers to a state where the amount of the polymer bound to HA or its derivative is 70% wZw or more, preferably 85% wZw or more based on the total amount of the polymer bound to HA or its derivative. Particularly preferably a state of 95% wZw or more.
- the rate at which both ends of the polymer are introduced into HA or its derivative is 5 mol% or less, preferably 3 mol% or less, per glucuronic acid in HA or its derivative. And particularly preferably 1 mol% or less.
- the modified hyaluronic acid of the present invention may be such that the polymer is composed of only the modified hyaluronic acid having only one end bound to HA or a derivative thereof.
- both ends of the polymer bound to HA or its derivative are introduced into HA or its derivative.
- the hydrazide group in the hydrazide (HZ)-or amino (AM) -modified HA derivative before polymer introduction by polymerizing the amount of the polymer introduced by proton NMR and the amino group Comparability with the actual reduction of the group can be determined.
- the ratio of the total amount of the combined polymer to the total amount of the polymer is l- (HZ) ZZ d-XZY), and the rate at which both ends are introduced into HA or its derivative is as follows: HZ) X (1 XZY).
- Hyaluronic acid (HA) or a derivative thereof used in the present invention refers to HA or a drug thereof. It may be a physiologically acceptable salt or a derivative thereof.
- HA or a pharmaceutically acceptable salt thereof examples include alkali metal salts such as a sodium salt, a potassium salt, and a lithium salt. Particularly preferred salts are widely used as pharmaceuticals. It is a sodium salt.
- HA or a pharmaceutically acceptable salt thereof can be produced by various known methods such as a method of extracting a biological source such as a cockscomb or subcutaneous swine, a biological fermentation method, or a commercially available product. It can also be purchased (for example, from Denki Kagaku Kogyo Co., Ltd., Shiseido Co., Ltd., Seikagaku Kogyo Co., Ltd., etc.).
- Examples of the HA derivative obtained by derivatizing HA or a pharmaceutically acceptable salt thereof include those obtained by modifying the carboxyl group of glucuronic acid in a diagonal manner. At this time, the pharmacokinetics can also be controlled by the modification state of the carboxyl group of the dalc acid.
- HA receptors such as CD44 expressed in large amounts in inflammatory or tumor sites, It can be expected to have a targeting effect on liver and lymphatic tissues, which are the main metabolic systems. For example, targeting to synovial cells inflamed in patients with osteoarthritis and rheumatism, targeting to synovial cells, uptake into cells by receptor-dependent endocytosis, and cure of inflammation by intracellular drug release can be expected.
- the carboxyl group modification rate of the glucuronic acid portion of the HA derivative is high, the binding to the HA receptor is suppressed, and the drug carrier becomes a long-lasting drug particle having a stealth effect in the body. In this case, a targeting effect on tumor cells using the EPR effect can also be expected. Furthermore, if the modification of the carboxyl group is cleaved after a certain period of time and the modification group is released from HA (eg, ester or amide bond), passive targeting to tumor tissue by EPR effect can be achieved. After that, it is advantageous to deliver the drug-containing microparticles of the present invention into tumor cells with high efficiency by active targeting via the HA receptor.
- the method for modifying the carboxyl group of the glucuronic acid portion of HA or its derivative is not particularly limited.
- the carboxyl group of HA or its derivative may be replaced with N, N, monocarbo- Rudiimidazole (CDI), N, N, 1-dicyclohexyl carbodiimide (DCC), N-ethoxycarbol-2-ethoxy-1,2-dihydroquinoline ( EEDQ), 4- (4,6-dimethoxy-1,3,5-triazine) 4-methylmorpholium (DMT-MM), 2-benzotriazole-1,1,3,3-tetramethyl Pum tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODh bt), benzotriazole-1-oxocitris-pyrrolidino phospho-pum hexafluoride Phosphate (PyBOP), benzotriazole
- HA derivative It is reacted with HA derivative, and a method of chemically bonding. Note that HA or its derivative may be modified with a polymer first, and the remaining carboxyl group of the resulting modified hyaluronic acid may be similarly reacted and modified.
- the molecular weight of HA or its derivative used in the present invention is not particularly limited, but the size of HA or its derivative molecule itself in an aqueous solution is much larger than other molecules (for example, viscosity Since HA with an average molecular weight of 2 million daltons has a size of 200-300 nm, small molecular weights (for example, viscosity average molecular weights of 300,000 daltons or less, for example, 5000-100,000) are required for preparing nanospheres with small particle systems. Dalton) HA or a derivative thereof is preferably used.
- Polylactic acid (PLA), polyglycolic acid (PGA) or lactic acid-glycolic acid copolymer (PLGA) used in the present invention The molecular weight of the selected polymer is not particularly limited! From the viewpoint of the reaction efficiency with HA or its derivatives, it is usually preferable to use those having a viscosity average molecular weight of 100,000 daltons or less, for example, those having a viscosity average molecular weight of 1,000 to 50,000 daltons. These polymers can be purchased from Alkermes, BirmingHAM Polymers, Inc, Wako Pure Chemical Industries, Ltd., etc., or a non-catalytic dehydration polycondensation method (Japanese Patent Application Laid-Open No.
- the compound can also be produced by a ring-opening polymerization method using a catalyst of a dangling property (Encyclopedic HAndoook of Biomaterials and Bioengmeermg Part AMatenals, Vol. 2, Marcel Dekker, Inc. (1995)).
- the selection of the polymer used in the present invention may be, for example, a drug for the purpose of targeting.
- a polymer such as PLA having a low decomposition rate (that is, drug release rate) because it is desired to minimize drug release in a living body until reaching the target site.
- the drug release rate can be controlled using these copolymers in order to achieve the desired drug release rate.
- the modified hyaluronic acid in which HA or a derivative thereof is bonded to a polymer selected from PLA, PGA or PLGA and a salt thereof are different in weight from the introduced polymer (PLA, PGA or PLGA).
- a solvent that is soluble in organic solvents such as DMSO, DM Ac, DMF, and mixed solvents with a sufficient ratio of these solvents (eg, tetrahydrofuran, methanol, ethanol, etc.), for example, acetonitrile, acetone Almost insoluble in organic solvents such as methylene chloride and ethyl acetate.
- a covalent bond is used. It is preferable to use a hyaluronic acid decoration which is adjusted so that the weight ratio of the polymer (PLA, PGA or PLGA) to be introduced is 50% or less, preferably 30% or less.
- the modified hyaluronic acid in which HA or a derivative thereof is bound to a polymer selected from PLA, PGA or PLGA and a salt thereof are different in weight from the introduced polymer (PLA, PGA or PLGA).
- an organic solvent such as DMSO, DMAc, or DMF, or a mixed solvent with a solvent that is mixed with a sufficient ratio of these, then the solvent is removed by dialysis or solvent diffusion.
- an organic solvent such as DMSO, DMAc, or DMF, or a mixed solvent with a solvent that is mixed with a sufficient ratio of these, then the solvent is removed by dialysis or solvent diffusion.
- the modified hyaluronic acid spontaneously accumulates a hydrophobic polymer portion as a core portion and exists in water with hydrophilic HA or its derivative portion facing the surface.
- the modified hyaluronic acid of the present invention can be used as a drug carrier.
- the drug may be encapsulated, included, carried and / or dispersed in the modified hyaluronic acid according to a conventional method.
- the drug used in the present invention is not particularly limited as long as it can be carried on a biodegradable polymer. It is not limited, and it is possible to use low molecular compounds, proteins, peptides, and the like.
- low molecular weight compound examples include, for example, anticancer drugs (eg, alkylating agents, antimetabolites, alkaloids, etc.), immunosuppressants, anti-inflammatory drugs (steroid drugs, non-steroidal anti-inflammatory drugs, etc.) And antirheumatic agents, antibacterial agents ( ⁇ -latatam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfa drugs, etc.).
- anticancer drugs eg, alkylating agents, antimetabolites, alkaloids, etc.
- immunosuppressants e.g., anti-inflammatory drugs (steroid drugs, non-steroidal anti-inflammatory drugs, etc.)
- antirheumatic agents antibacterial agents ( ⁇ -latatam antibiotics, aminoglycoside antibiotics, macrolide antibiotics, tetracycline antibiotics, new quinolone antibiotics, sulfa drugs, etc.).
- proteins and peptides include, for example, erythropoietin ( ⁇ ), dalurocytoco-oral stimulating factor (G-CSF), interferon- ⁇ , ⁇ , ⁇ , (INF- ⁇ , j8, ⁇ ) , Thrombopoietin (TPO), serially-eutrophic factor (CNTF), tumer necrosis factor (TNF), tumer necrosis factor binding protein (TNFbp), interleukin 10 (IL-10), FMS-like tyrosine kinase (Fit — 3), growth hormone (GH), insulin, insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), interleukin-1 receptor antagonist (IL-lra), brain-derived euro trophy factor (BDNF), keratinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth factor (MGDF), osteoprotege (OPG), lebutin, parathyroid hormone (PTH), erythrop
- the most preferred embodiment of the present invention is one in which the drug carrier is formed into fine particles.
- the modified hyaluronic acid of the present application has a structure in which the surface of the polymer is covered with HA or its derivative in an aqueous solution, since the HA or its derivative portion is hydrophilic and the polymer is hydrophobic, so that the hyaluronic acid has a fine particle form. Become.
- the drug can be encapsulated, included, carried, and Z or dispersed in the microparticles. Since this drug carrier has a particle surface coated with hydrophilic HA or its derivative, it prevents aggregation and friction of particles in an aqueous solution for administration and in vivo, and has a highly biocompatible HA surface.
- the particle size of the drug carrier of the present invention in the form of fine particles is preferably 200 ⁇ m or less, more preferably 100 ⁇ m or less, in order to allow the needle to pass through without clogging. .
- the particle size is preferably 5 m or less because friction in the joint occurs physically and immediately induces new inflammation.
- the particle diameter is preferably 500 nm or less, more preferably 200 nm or less.
- the fine particles of the present invention have good biocompatibility and no inflammatory properties.
- Hyaluronic acid itself has mucoadhesiveness. Therefore, by controlling the particle size, nasal, pulmonary, oral, etc. It can also be used for non-invasive administration. Mucoadhesiveness can be adjusted by the amount of HA carboxylic acid, and the larger the amount, the better the modification rate with spacer polymer, which is more preferable.
- the method for preparing fine particles of the present invention includes, for example, a method of drying in an emulsion liquid, and a solvent dispersion method (J. Contr. Rel. 83, 365-375 (2002)), a dialysis method and the like.
- the fine particles having polymer power are prepared according to a conventional method, and then the carboxyl groups on the surface of the particles are reduced to 1-ethyl acetate.
- Active esterification with a condensing agent such as 3- (3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS), which is then performed with an active ester such as a hydrazide (HZ) group or an amino (AM) group
- a condensing agent such as 3- (3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS)
- EDC 3- (3-dimethylaminopropyl) carbodiimide
- NHS N-hydroxysuccinimide
- an active ester such as a hydrazide (HZ) group or an amino (AM) group
- a modified hyaluronic acid containing one or more polymers selected from PLA, PGA or PLGA by an in-solution drying method can be prepared. That is, PLA, PGA or PLGA is dissolved in a water-immiscible organic solvent such as methylene chloride or ethyl acetate or a mixed solvent with a water-miscible solvent, while the hyaluronic acid modification of the present invention is carried out. The product is dissolved or dispersed in an aqueous phase, and after the emulsion is prepared, it is dried in a liquid to prepare fine particles coated with the modified hyaluronic acid of the present invention. There is no particular limitation ⁇
- the modified product of hyaluronic acid of the present invention is an amphipathic molecule having a hydrophobic part and a hydrophilic part in one molecule, and thus is considered to exhibit a surfactant effect.
- fine particles coated with a modified hyaluronic acid further comprising one or more polymers selected from PLA, PGA or PLGA can be prepared by a solvent diffusion method. That is, if PLA, PGA or PLGA is dissolved in a water-miscible organic solvent such as acetone or acetonitrile, or a mixed solvent with an organic solvent such as ethanol or methanol mixed in a sufficient ratio to these, and mixed with the aqueous phase.
- a water-miscible organic solvent such as acetone or acetonitrile
- an organic solvent such as ethanol or methanol mixed in a sufficient ratio to these
- Fine particles are generated as the organic solvent is diffused, and the hyaluronic acid-modified product of the present invention can be used by dissolving or dispersing the same in the aqueous phase.When the fine particles are generated, the fine particles are coated with the hyaluronic acid-modified product of the present invention. can do.
- the modified hyaluronic acid of the present invention can be used by dissolving it in an organic phase.
- the modified hyaluronic acid or PLA, PGA or PLGA of the present invention dissolved in the organic phase is mixed with the selected polymer, and the solvent is replaced with water by dialyzing or dripping against the aqueous phase.
- the hydrophilic HA portion is accumulated on the surface of the fine particles, whereby the fine particles can be coated with the modified hyaluronic acid of the present invention.
- the hyaluronic acid-modified The fine particles can be used by dissolving or dispersing them in an aqueous phase, and when the fine particles are formed, the fine particles can be coated with the modified hyaluronic acid of the present invention.
- the surface of the fine particle comprising PLA, PGA or PLGA is hydrophobic. Since these particles are mixed with an aqueous solution in which the modified hyaluronic acid of the present invention is dissolved or dispersed, the particles are adsorbed on the surface of the particles by hydrophobic interaction, and the hydrophilicity of the present invention is improved. Fine particles coated with the modified hyaluronic acid can be prepared.
- the aqueous phase used in the preparation of the fine particles of the present invention may be a mixed solvent with a solvent that is water-only or miscible with water at a sufficient ratio.
- additives usually used in the technical field for example, a surfactant, a pH adjuster, a buffer, and the like may be included, and the pH and the salt concentration may be appropriately adjusted.
- Fine particles containing the modified product of hyaluronic acid can also be formed.
- the microparticles containing the modified hyaluronic acid of the present invention may contain a polymer in which PLA, PGA, or HA whose PLGA force is also selected is bound to the core portion! it can. By adding the additive or the polymer, the particle size of the fine particles and the drug carrying ability can be adjusted.
- the particle size is 5 ⁇ m or less, for example.
- the method for preparing lnm—5 ⁇ m is preferably a dialysis method, a solvent diffusion method, a drying method in emulsion, or the like.
- the modified hyaluronic acid used in this case may have a carboxylic acid modification rate of 10 mol% or less, for example, 0.1 to 10%, in the dalonic acid portion of HA so that the binding to the HA receptor is not suppressed. preferable.
- the particle size is 200 nm.
- a method of preparing the compound having a wavelength of preferably from 1 nm to 200 nm may be a dialysis method, a solvent diffusion method, a drying method in an emulsion liquid, or the like.
- the modified hyaluronic acid used at this time is modified at a carboxylic acid power of the glucuronic acid portion of HA of 30 to 100%, preferably 50 to 90%, so that the binding to the HA receptor is suppressed. It is preferred to use
- the particle size is 200 / zm or less, for example, lnm-200m.
- the dialysis method, the solvent diffusion method and the emulsion drying method can be used.
- the particle size is 200 ⁇ m or less, for example, lnm-
- the preparation method preferably having a thickness of 200 m may be a dialysis method, a solvent diffusion method, or an emulsion drying method.
- the modified hyaluronic acid used has a low modification rate of the carboxylic acid of HA so that it has adhesion to mucous membranes.
- a method of encapsulating the drug in these fine particles a method of encapsulating the drug in a polymer is usually used.
- the drug when the polymer is dissolved in an organic solvent such as DMSO during preparation of the fine particles, the drug may be allowed to coexist, and the fine particles may be prepared as it is. If the drug has high water solubility, the encapsulation efficiency can be increased by changing the drug to a salt with low solubility in water or by using the WZOZW emulsion method.
- freeze-drying may be performed in the presence of a dispersant such as trehalose or mantole.
- the residence time in blood of the modified product of hyaluronic acid of the present invention may be longer than the residence time in blood of HA itself! / ,.
- a hyaluronic acid derivative having an increased retention time in blood can be used as a raw material.
- the blood residence time is calculated from known representative parameters such as average blood residence time (hereinafter also referred to as MRT), blood half-life (hereinafter also referred to as tlZ2), and blood clearance (hereinafter also referred to as C1). It can be used for comparison as appropriate.
- the blood retention time in mammals is preferably an average blood retention time (MRT) of 18 hours or more from a practical viewpoint. More than 30 hours is more preferable.
- MRT average blood retention time
- the modified hyaluronic acid of the present invention can It may be one that is more resistant to degradation by hyal-mouthase than A itself.
- hyaluronic acid which is more resistant to degradation by hyalurodidase than HA itself, as a raw material, hyaluronic acid, which is more resistant to degradation by hyaluronidase than HA itself, is used. Acid derivatives can be used.
- HA itself and hyaluronic acid derivative of the present invention are each degraded by enzymatic degradation of hyaluronic acid by hyalurodase.
- Decomposition rate is slower or decomposition does not proceed more than that.
- HA when treated with hyal-outase for a certain period of time, HA itself has a property that ⁇ degradation does not proceed '' unless the disaccharide degradation peak observed is observed (i.e., Resistance).
- the decomposition rate and decomposition state of A can be observed using a conventional method such as gel permeation chromatography (hereinafter, also referred to as GPC).
- GPC gel permeation chromatography
- a modified hyaluronic acid in which hyaluronic acid or a derivative thereof is bound to one or more polymers selected from polylactic acid, polydalicholic acid or lactic acid-glycolic acid copolymer.
- hyaluronic acid Using this modified product of hyaluronic acid, it is possible to efficiently encapsulate a low-molecular-weight drug, control the sustained-release period for a long period of time, control the retention in blood, and disperse well in an aqueous solution.
- a drug carrier is provided.
- the modified hyaluronic acid of the present invention also provides a drug carrier excellent in biocompatibility, which is composed of fine particles having an inditable power and capable of controlling the particle size with less aggregation between particles.
- FIG. 1 is an example of a photograph taken by a SEM of a microsphere of a modified hyaluronic acid according to the present invention.
- FIG. 2 is an example of a photograph taken by a 3D laser microscope of a microsphere made of the modified product of hyaluronic acid of the present invention.
- FIG. 3 shows an example of a photo taken by SEM of a PLGA microsphere as a comparative example.
- Figure 4 shows a PLGA microsphere as a comparative example taken with a 3D laser microscope. It is an example of a photograph.
- FIG. 5 is an example of the results of measuring the modified hyaluronic acid of the present invention by GPC.
- FIG. 6 is an example of the result of measuring the modified hyaluronic acid of the present invention by NMR.
- FIG. 7 is an example of a photograph of a nanosphere dispersion solution (HA (25 kDalton) —PLA nanosphere) consisting of the modified hyaluronic acid of the present invention after storage at 4 ° C. for one month. is there.
- HA 25 kDalton
- PLA nanosphere a nanosphere dispersion solution
- FIG. 8 is an example of a photograph of a nanosphere dispersion solution (HA (19 Ok Dalton) —PLA nanosphere) composed of the modified hyaluronic acid of the present invention after storage at 4 ° C. for one month. is there.
- HA (19 Ok Dalton) —PLA nanosphere HA (19 Ok Dalton) —PLA nanosphere
- FIG. 9 is an example of a photograph of a PLA nanosphere dispersion solution (comparative example) after being stored at 4 ° C. for one month.
- FIG. 10 is an example of an SEM photograph of a nanosphere dispersion solution (HA (25 kDalton) —PLA nanosphere) comprising the modified hyaluronic acid of the present invention after storage at 4 ° C. for one month. Confuse.
- FIG. 11 is an example of a SEM photograph of a nanosphere dispersion solution (HA (190 kDalton) —PLA nanosphere) comprising the modified hyaluronic acid of the present invention, stored at 4 ° C. for one month. Confuse.
- HA 190 kDalton
- PLA nanosphere a nanosphere dispersion solution comprising the modified hyaluronic acid of the present invention
- FIG. 12 is an example of a SEM photograph of a dispersion solution of PLA nanospheres (Comparative Example) after storing at 4 ° C. for one month.
- FIG. 13 is an example of a proton NMR ⁇ vector in an aqueous solution of a dispersion solution of nanospheres comprising the modified hyaluronic acid of the present invention.
- FIG. 14 is an example of a proton NMR ⁇ vector in an aqueous solution of a dispersion solution of nanospheres comprising the modified hyaluronic acid of the present invention in an aqueous solution.
- FIG. 15 shows an example of the NMR-vector of HA-HZ obtained in Example 4-1.
- FIG. 16 shows the results of the degradation test for HA-HZ obtained in Example 41, and the results of the degradation test on hyaluronan-SD, and the unmodified HA under the same conditions. It is an example of the result in the case of Hyal mouth-Dase SD processing.
- FIG. 17 is an example of the proton NMR spectrum of HA-AM obtained in Example 5-1.
- FIG. 18 shows the results of a degradation test on hyaluronidase SD of HA-AM obtained in Example 5-1-5-3, and the case where unmodified HA was treated with hyaluronidase SD under the same conditions. It is an example of the result of.
- FIG. 19 is an example of a GPC chromatogram of HA-HZ-PLA obtained in Example 6-2.
- FIG. 20 is an example of a proton NMR spectrum of HA—HZ—PLA obtained in Example 6-2.
- FIG. 21 is a GPC chromatogram of HA—HZ—PLA obtained in Example 6-3.
- FIG. 22 is an example of a proton NMR spectrum of HA-HZ-PLA obtained in Example 6-3.
- FIG. 23 is an example of a proton NMR spectrum of HA-AM-PLA obtained in Examples 7-7.
- HA-PLA the combination of HA and PLA is collectively referred to as HA-PLA regardless of the binding mode.
- HA-HZ-PLA When a dihydrazide compound is contained as one spacer molecule, it may be referred to as HA-HZ-PLA, and when it contains a diamine compound, it may be referred to as HA-AM-PLA.
- TNBS tri-trobenzenesulfonic acid
- the NMR measurement was carried out using a JNM-ECA500 (500MHz spectrometer) as an NMR spectrometer under the following conditions (parameters).
- Example 1-1 Synthesis of hyaluronic acid (HA-HZ) into which hydrazide group (HZ) was introduced 100 mg of hyaluronic acid (HA) having a molecular weight of 2.5 ⁇ 10 4 daltons (manufactured by Denki Kagaku Kogyo Co., Ltd.) It was dissolved in distilled water at a concentration of 1%, and the pH was adjusted to 4.7-4.8 with 5N hydrochloric acid.
- HZ hyaluronic acid
- EDC 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide
- ADH adipic dihydrazide
- Hyaluronic acid dialyzed against lOOmM sodium chloride aqueous solution and 25% aqueous ethanol solution (Spectrapore 7, fractional molecular weight (MWCO): 12k-14k dalton), lyophilized and introduced with the title hydrazide group (HZ) 88 mg of the derivative (HA-HZ) was obtained.
- MWCO fractional molecular weight
- HZ N-acetyl group, 2. lppm
- HZ methylene group of adipic acid portion, 1.7 ppm, 2.4 ppm
- PLGA7510 (Wako Pure Chemical Industries, Ltd .: molecular weight 10,000 daltons) was dissolved by adding 10 mL of methylene chloride (Junsei Chemical) to prepare a 10% (w / v) solution. This was mixed with 90 mL of 1% polyvinyl alcohol (87-89% hydrolyzed, molecular weight 13000-26000) aqueous solution (aqueous PVA solution) at a ratio of 1: 9 (v / v), and stirred with a stirrer at about 700 rpm. I got oil-in-water (O / W) emulsion.
- microsphere suspension was placed in 900 mL of a 1% PVA aqueous solution, stirred at about 200 rpm under reduced pressure, and the solvent was distilled off to obtain a microsphere suspension. This suspension is centrifuged at 1000 rpm and 4 ° C for 10 minutes, and the supernatant is removed. After washing sufficiently, the lyophilized product was collected as a microsphere. As a result of particle size measurement by SEM image analysis, microspheres with a particle size of about 30 to 80 m were obtained.
- FIGS. 1 and 2 show SEM and 3D laser microscope photographs of the obtained hyaluronic acid-coated PLGA microsphere.
- SEM photograph dry state
- Fig. 1 it can be seen that the surface of the PLGA microsphere became less smooth with the HA coat.
- FIG. 2 in the aqueous solution, the obtained hyaluronic acid-coated PLGA microsphere has PLGA particles coated on the HA gel-like shell.
- Example 11 HA—HZ100 mg obtained in the same manner as in Example 1 was dissolved in 20 mL of water (0.5% concentration) to form an H-type cation exchange resin (Dowex 50WX8—400, 4.8 meq / g) 4 The mL was allowed to stand at room temperature for 3 days at room temperature with added kato (ion exchange capacity 100 times the carboxyl group of HA). The supernatant was collected, passed through a 0.22 m filter, added with a 1 M TBA-OH (tetra-N-butylammonium hydroxide) solution at 150 / z L, adjusted to pH 8, and lyophilized.
- TBA-OH tetra-N-butylammonium hydroxide
- HA PLA was approximately 1: 9).
- Figure 5 shows the measurement data of gel permeation chromatography (GPC) (solvent: DMSO).
- GPC gel permeation chromatography
- FIG. 6 shows the proton NMR of the obtained HA-PLA.
- HA The PLA incorporation rate in PLA was determined by the protein NMR method (solvent: DMSO d).
- Le 0/0 has been PLA I spoon (HA: N-Asechiru group, 1. 5-1 9ppm, PLA: PLA :. Cal ball - the ⁇ -position of the Le group methine proton (however, Omikuron'ita end is excluded) , 5.0-5.6 ppm).
- Example 2-1 the cloudy solution obtained after dialysis against water was used as a nanosphere dispersion containing HA-PLA from HA at 25K daltons.
- HA-HZ was prepared in the same manner as in Example 1-1 except that HA having a molecular weight of 190 K daltons was dissolved in distilled water at a concentration of 0.5%.
- the reaction solution after DMSO dialysis prepared in the same manner as in 2-1 was dialyzed against water (MWCO: 25000, room temperature, 3 days), and a nanosphere dispersion solution containing HA-PLA derived from HA of 190K daltons was obtained.
- MWCO 25000, room temperature, 3 days
- Example 2-3 Preparation of hyaluronic acid-coated PLA nanospheres by solvent diffusion method Among the ones using 25 kDa HA in Example 2-1, the dispersion solution before purification by acetone reprecipitation (HA-PLA: PLA Approximately 20 mg of each of freeze-dried 1: 9) and HA-PLA after acetone reprecipitation were weighed out and dissolved in 1. OmL of DMSO. This was added dropwise to 4. OmL of water with stirring, and both resulted in a pale white translucent solution. This mixed solution was transferred to a dialysis tube (MWCO: 12000-14000, Spectra / Por4) and dialyzed against water (room temperature, 3 days). DLS measurement (Nicomp370) of the obtained nanosphere dispersion solution revealed that the particle diameter of the nanosphere before purification was 17.6 nm and that after refinement was 15.2 nm. Also, this nanosphere dispersion solution was
- Example 3-1 Preparation method A nanosphere dispersion solution containing HA-PLA derived from HA of 25kDa (HA-PLA: PLA is approximately 1: 9), prepared by the same operation as in Example 2-2, is frozen. 40 mg of the dried product and 10 mg of diclofenac (the product purchased from Sigma, which was ion-exchanged into H form by reprecipitation with hydrochloric acid, washed with pure water, and dried) were weighed and dissolved in 1. OmL of DMSO. Was This was dropped into 4.OmL of water with stirring to give a pale white translucent solution.
- diclofenac the product purchased from Sigma, which was ion-exchanged into H form by reprecipitation with hydrochloric acid, washed with pure water, and dried
- This mixed solution was transferred to a dialysis tube (MWCO: 12000-14000, Spectra / Por 4), and dialyzed against water (3 days, room temperature).
- the obtained aqueous solution is centrifuged (IOOrpm X 10 ), The precipitate and the supernatant were separated, and the supernatant was recovered and freeze-dried.
- a freeze-dried nanosphere dispersion solution (HA-PLA: PLA is approximately 1: 9) containing HA-PLA derived from 25K dalton HA prepared by the same operation as in Example 2-2, 40 mg , and diclofenac lOmg were obtained. Each was weighed and dissolved in 1. OmL of DMSO. This was transferred to a dialysis tube (MWCO: 12000-14000, Spectra / Por 4), and dialyzed against water. Immediately, it became cloudy and a precipitate was formed (3 days, room temperature). The obtained aqueous solution was separated into a precipitate and a supernatant by centrifugation (100 rpm ⁇ 10 minutes), and the supernatant was recovered and freeze-dried.
- MWCO 12000-14000, Spectra / Por 4
- a two-fold dilution series (six rows) of diclofenac, 5.13 mgZmL-acetonitrile was prepared (A).
- a n-heptyl 4-hydroxybenzoate, 2.60 mg / mL-acetonitrile solution was prepared as an internal standard (IS) solution (B).
- B 200 L
- a and 20 ⁇ L ( ⁇ ) were mixed to prepare a standard solution for diclofenac determination (C).
- the diclophanac encapsulation rate was 4.9% by weight by the solvent diffusion method and 1.4% by weight by the dialysis method.
- PLA was dissolved in DMSO (0.316 g / mL DMSO) and dialyzed against water in the same manner as in Example 2-3. A cloudy aqueous solution was obtained, but a precipitate was also observed. The particle size of the dispersion solution was measured to be 273.3 nm. It was stored at 4 ° C for one month, but most of it was aggregated and precipitated one day after storage. A photograph one month after storage is shown in Fig. 9, and the SEM photograph is shown in Fig. 12. It can be seen that the particles are aggregated.
- HA unit: EDC: ADH 1: 4: 40
- the mixture was added to make a molar ratio, and reacted with 5N hydrochloric acid at room temperature for 2 hours while maintaining the pH at 4.7-4.8.
- Hyaluronic acid (HA-HZ) with a hydrazide group introduced by dialysis (MWCO: 12000-14000) against a large excess of lOOmM sodium chloride solution, 25% ethanol solution and distilled water in order 86.6 mg were obtained. NMR of the obtained HA-HZ in D O
- Fig. 15 shows the spectrum measurement results.
- the introduction rate of the HZ group was determined by the proton NMR method as the introduction rate of the ADH, and was 63.0% based on the HA unit (disaccharide) (HA: methyl proton of N-acetyl group, 1.85 ppm, HZ: (4 methylene protons from ADH, 1.5, 2.1 and 2.25 ppm) 0
- Example 41 HA-HZ obtained in Example 1 was dissolved in a 0.1 M phosphate buffer (pH 6.2) to a concentration of 0.5 mg / mL. To 80 L of this solution, 32 L of 0.5 U / mL solution of Hyal Mouth-Dase SD (manufactured by Seikagaku Corporation) was added, and incubated at 37 ° C for 24 hours. As a control, a group without addition of the enzyme was similarly performed (data omitted). Gel permeation chromatography (below) , And GPC) to observe changes in the molecular weight of HA-HZ and the pattern of formation of degradation products. GPC conditions are shown below.
- GPC column Superdex 200 10/300 G Superdex 75 HR 10/30, Superdex Peptide HR 10Z30 (all manufactured by Amersham Biosciences, Inc.)
- Figure 16 shows the GPC profile of the enzymatic degradation product. Under GPC conditions in the study, the peak top of HA-HZ is observed at a retention time of about 75 minutes, and the disaccharide, an enzymatic degradation product, is observed at a retention time of about 130 minutes.
- Example 41 The sample obtained in Example 41 showed that the decrease in molecular weight due to enzymatic degradation was significantly suppressed.
- HA-HZ 38.9 mg obtained in Example 41 was dissolved in 50 mM carbonate buffer (PH 9.0) at a concentration of 2.0 mg ZmL. 0.15 moles of fluorescein isothiocyanate (hereinafter also referred to as FITC) (manufactured by Pierce Co.) as a HA unit was prepared as a 1Z10 volume dimethyl sulfoxide (hereinafter also referred to as DMSO) solution of HA-HZ solution. In addition, the mixture was stirred at room temperature for 1 hour.
- FITC fluorescein isothiocyanate
- DMSO dimethyl sulfoxide
- HA—SUC unit: y nmolZmL (unit with residual HZ treated with succinic anhydride) Formula 1: (379.3 X x) + (635.57 X y) + (924.88 X (FITC concentration)) 2 50mg
- Example 4 A single intravenous injection of the fluorescently labeled HA derivative of 3 at a dose of lOmgZkg in terms of hyaluronic acid was performed before and after administration of 0.25, 1, 2, 4, 6, 8, 10, 12, 24. Blood was collected at 30 and 54 hours (treatment with Henon) and plasma was obtained by centrifugation. This plasma sample was stored frozen at 20 ° C or lower until measurement.
- the standard sample for calibration curve and the sample for measurement are analyzed by GPC. The conditions are shown below.
- ⁇ ⁇ Calibration curve samples Each fluorescently labeled HA derivative was diluted with PBS (pH 7.4), and 1, 5, 10, 50, 100, 500 g / mL and 0 g / mL (control, PBS (pH 7.4) ) Prepare the standard solution of). An equal volume of normal rat plasma was added to this standard solution to prepare a sample for a calibration curve. • Preparation of a sample for measurement: A sample for measurement was prepared by adding an equal volume of PBS (pH 7.4) to a plasma sample of a rat to which the HA modification had been administered.
- Peak area was calculated using analysis software Millenium.
- concentration of the HA-modified product in plasma was calculated from the calibration curve obtained from the peak area of each standard sample.
- Pharmacokinetic parameters were calculated using WinNonlin Ver 3.3 (Pharsight) based on the data on the changes in the blood concentration of the fluorescently labeled HA derivative of Example 43. Model-independent analysis was performed using the data of the last three measurement points of each individual, and the half-life (tlZ2) and mean blood residence time (MRT) were calculated. The calculated pharmacokinetic parameters are shown in Table 1 [Table 1] Table 1. Pharmacokinetic parameters of FITC-labeled HA derivatives
- Example 4 One 3 25 63 4.03 Sat 0.3 32.8 Sat 1.7 25.2 Sat 2.0 According to a pharmacokinetic test using a fluorescently labeled HA derivative, the hydrazide group-introduced HA derivative was compared to unmodified HA in a single rat. It was confirmed that the retention time in blood after intravenous administration was improved. The half-life in blood (tl / 2) of unmodified HA is about several minutes, whereas the introduction of a hydrazide group resulted in a blood half-life of 25.2 ⁇ 2.0 (hours). .
- Example 5 Synthesis of Stealth-Modified Hyaluronic Acid by Introduction of Amino Group 'Function Evaluation (Example 5-1) Synthesis of Hyaluronic Acid (HA-AM) with AM Group 1
- HA—TBA (HA: Denki Kagaku Kogyo Co., Ltd .: viscosity average molecular weight 19 kDalton) using DOWEX 50WX8-400 (manufactured by Aldrich) chlorinated with tetrabutylammonium (TBA) 305 2 mg was dissolved in DMSO at a concentration of 2 mg ZmL.
- HA cut: BOP (manufactured by Wako Pure Chemical Industries): Ethylenediamine (EDA) (manufactured by Sigma Aldrich) 1: 1.1: EDA and BOP are added in order of 50 mole ratio in order of BDA. The reaction was carried out at room temperature.
- Fig. 17 shows the results of NMR measurement of HA-AM in DO.
- the HZ group introduction rate was determined by proton NMR as the AM introduction rate, and was 94.0% based on the HA unit (disaccharide) (HA: methyl proton of N-acetyl group, 1.85 ppm, AM: One methylene proton from EDA, 2.98 ppm).
- the NMR measurement was carried out using a JNM-ECA500 (500 MHZ spectrometer) as an NMR spectrometer under the same conditions (nometer) as in Example 4-1.
- HA-AM obtained in Example 5-1-5-3 was dissolved in water to a concentration of 2 mgZmL.
- This solution contains 132 L of 0.2 M phosphate buffer (pH 6.2) and 77 L of water, and contains a Hyanore mouth Nidase SD (manufactured by Seikagaku Corporation) lUZmL solution (containing 0.01% BSA. 44 L of 05M phosphate buffer (PH6.2) was prepared and incubated at 37 ° C for 24 hours. Subject In the same manner, the group without the enzyme was also used (data omitted). Each was subjected to gel permeation chromatography (hereinafter also referred to as GPC) to observe changes in the molecular weight of HA-AM and the pattern of formation of degradation products. GPC was performed under the same conditions (parameters) as in Example 42.
- GPC gel permeation chromatography
- Figure 18 shows the GPC profile of the enzymatic degradation product. Under the GPC conditions in this study, the enzymatic degradation product disaccharide is observed at a retention time of approximately 130 minutes. The samples obtained in Examples 5-1 to 5-1 showed that the decrease in molecular weight due to enzymatic degradation was significantly suppressed.
- Example 6-1 TBA salinization of hyaluronic acid (HA-HZ) into which HZ group was introduced
- HA-HZ lOOOOmg obtained in the same manner as in Example 4-1 was dissolved in lOOOmL of distilled water (0.1% concentration), and the H-form cation exchange resin (Dowex 50WX8-400, 2.1 meq / g) 37. Add 5mL of syrup (100 times ion exchange capacity of carboxyl group of HA) at room temperature
- Example 6-2 Synthesis of hyaluronic acid (HA-HZ-PLA) into which PLA (viscosity average molecular weight 5 kDalton) was introduced
- TBA chloride HA-HZ prepared in Example 6-1 was dissolved in anhydrous DMSO to a concentration of 5 mg / mL.
- FIG. 19 shows a GPC chromatogram when DMSO was used as a solvent. GPC conditions are as follows.
- Figure 20 shows the MR.
- the introduction rate of PLA in HA—HZ—PLA was quantified by NMR method, as shown in Table 3, assuming that the molecular weight distribution of PLA was the same as that of the raw material, the HA unit (disaccharide) On the other hand, it was 1.0 to 46.1%.
- the weight ratio of PLA in the HA—HZ—PLA molecule was 5.7—73.4% wZw (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: ⁇ -position of carboyl group). Methine proton (excluding the ⁇ -terminal), 5.0-5.6 ppm).
- Example 6-3 Synthesis of hyaluronic acid (HA-HZ-PLA) into which PLA (viscosity average molecular weight 20 kDalton) was introduced
- PLA-0005 of Example 6-2 was changed to PLA-0020 (Wako Pure Chemical Industries, Ltd .: viscosity average molecular weight 20 kDalton, number average molecular weight 8.73 kDalton), TBA chloride HA-HZ solution and anhydrous DMS 0, active ester
- the HA-HZ-PLA white powder was obtained in the same manner as in Example 6-2 except that the mixing ratio of the modified PLA solution was changed to Table 4 (k-t).
- the GPC chromatogram at this time is shown in FIG. 21, and the proton NMR is shown in FIG.
- the PLA introduction rate in HA—HZ—PLA was determined by NMR method (solvent: DMSO—d). As shown in Table 5, the PLA molecular weight distribution was
- the weight ratio of PLA in the HA—HZ—PLA molecule was 13.5—69.9% wZw (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: ⁇ -position of carboxyl group) Methine proton (excluding the ⁇ terminal), 5.0-5.6 ppm).
- Example 6-4 Dispersibility or solubility of HA—HZ—PLA in water and DMSO HA-HZ obtained by the same method as in Example 4-1; Example 6-2; and Example 6— About 3 mg of each of the HA-HZ-PLA white powders synthesized in this manner was weighed, distilled water was added to a volume of 5 mgZmL, and the mixture was stirred at room temperature overnight. Table 6 shows the results of visually confirming the appearance when stirring was stopped and then allowed to stand at room temperature for 3 days. As a result, HA-HZ-PLA is uniformly dispersed in water in the range of 5.7-19.6% w / w of PLA, and is difficult to disperse in water immediately above the PLA weight ratio.
- Example 63 about 5 mg of the HA-HZ obtained in the same manner as in Example 4-1 and about 5 mg of the HA-HZ-PLA white powder synthesized in Example 63 were weighed, respectively.
- DMSO was added to 5 mg / mL, and the mixture was stirred at room temperature overnight. Visualize the solubility at that time Table 6 shows the results. The results suggest that HA-HZ-PLA has solubility in DMSO at least in the range of PLA weight ratio of 5.7-73.4% wZw.
- the results suggest that HA-HZ-PLA has solubility in DMSO at least in the range of PLA weight ratio of 5.7-73.4% wZw.
- Example 6-5 Solubility of HA—HZ—PLA in water by solvent replacement Approximately 5 mg of each of the HA-HZ obtained in the same manner as in Example 4-1 and the HA-HZ-PLA white powder synthesized in Example 6-3 and Example 6-3 were weighed. Was dissolved in DMSO to a concentration of 5 mgZmL. When 0.6 mL of this DMSO solution was mixed with 2.4 mL of distilled water, and further dialyzed against distilled water (MWCO: 12000 to 14000, room temperature, 3 days), the resulting aqueous solution was all a colorless and transparent solution. Was. When this aqueous solution was centrifuged (40000 g, 10 minutes), no precipitate was observed. The results suggest that HA—HZ—PLA can be dissolved in water by dissolving it in DMSO once and then replacing the solvent with water in the range of 5.7 to 73.4% wZw by weight of PLA. Was done.
- HA Denki Kagaku Kogyo Co., Ltd .: viscosity average molecular weight 23 kDalton
- HA unit: EDC: ADH 1: 0.1: 40
- the molar ratio was added, and the mixture was reacted at room temperature for 2 hours while maintaining the pH at 4.7-4.8 with 5N hydrochloric acid. It was dialyzed (MWCO: 12000-14000) against a large excess of 100 mM sodium salt solution, 25% ethanol solution and distilled water in that order. About 10 mL was freeze-dried for measuring the HZ group introduction rate.
- the introduction rate of the HZ group was determined by the proton NMR method (measurement solvent: DO) as the introduction rate of the ADH, it was 9.0% with respect to the HA unit (disaccharide) (HA: N-acetate).
- Methyl proton of tyl group 1.85 ppm, HZ: four methylene protons from ADH, 1.5, 2.1, and 2.25 ppm) 0
- Example 7-1 To the aqueous HA-HZ solution (about 0.1% concentration) prepared in Example 7-1, 50 mL of H-form cation exchange resin (Dowex 50WX8-400, 2. lmeq / g) was added. ⁇ ⁇ I left it. The supernatant was collected and filtered through a 0.22 m filter. The pH was adjusted to 7.0 by adding 1150 L of 40 wt% TBA-OH aqueous solution, and the resulting aqueous solution was freeze-dried to obtain TBA-chlorinated HA-HZ Was obtained as a white powder. The yield was 1113.6 mg.
- H-form cation exchange resin Dowex 50WX8-400, 2. lmeq / g
- Example 7-3 Synthesis of hyaluronic acid (HA-HZ-PLA) with PLA (5 kDalton) -introduced intact HA function
- HA-HZ-PLA hyaluronic acid
- PLA 5 kDalton
- the weight ratio of PLA in the HA—HZ—PLA molecule was 36.7% w / w (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: methine in ⁇ -position of carboxyl group) Proton (however, excluding the ⁇ terminal), 5.0-5.6 ppm).
- Example 7-2 Using the TBA-chlorinated HA-HZ prepared in Example 7-2, the PLA-0005 in Example 6-2 was changed to PLA-0020, and the TBA-chlorinated HA-HZ solution, anhydrous DMSO, and the active esterified PLA solution were used. The mixing ratio was changed to Table 7, and otherwise the same method as in Example 6-2 was carried out to obtain a white powder of HA-HZ-PLA.
- the introduction rate of PLA in HA-HZ-PLA was determined by NMR method (measurement solvent: DMSO-d6), when it was assumed that the molecular weight distribution of PLA was the same as that of the raw material, the HA unit ( 1.9% of disaccharide).
- the weight ratio of PLA in the HA—HZ—PLA molecule was 29.5% wZw (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: methine proton at ⁇ -position of carboyl group ( However, ⁇ -terminal is excluded), 5.0-5.6 ppm).
- Example 6-1 Using TBA Shiridani HA-HZ prepared in the same manner as in Example 6-1, the mixing ratio of the TBA chloride HA-HZ solution of Example 6-2, anhydrous DMSO, and the active esterified PLA solution was tabulated. 7, and in the same manner as in Example 6-2, a white powder of HA—HZ—PLA was obtained.
- the PLA introduction rate in HA HZ-PLA was determined by NMR method (measurement solvent: DMSO-d)
- the weight ratio of PLA in the HA—HZ—PLA molecule was 21.1% WZW (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: Methine proton at the ⁇ - position of the ball group (excluding the ⁇ -terminal), 5.0-5.6 ppm) 0 (Example 7-6) PLA (5 kDalton) was introduced at a high rate and introduction rate Synthesis of stealth hyaluronic acid (HA-HZ-PLA)
- Example 6-1 Using TBA Shiridani HA-HZ prepared in the same manner as in Example 6-1, the mixing ratio of the TBA Shiridani HA-HZ solution of Example 6-2, anhydrous DMSO, and active ester Iridani PLA solution was determined. The procedure was changed to Table 7 and the other conditions were the same as in Example 6-2 to obtain a white powder of HA-HZ-PLA.
- HA HZ— PLA introduction rate in PLA was determined by NMR method (solvent: DMSO—d 6).
- the weight ratio of PLA in the HA—HZ—PLA molecule was 65.1% WZW (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: carie)
- PLA sample molecular weight (PLA / DMSO
- Example 7-7 Synthesis of stealth hyaluronic acid (HA-AM-PLA) in which PLA (5 kDalton) was high and introduced at an introduction rate
- the HA-AM DMSO solution obtained in the same manner as in Example 5-1 was dialyzed four times against 1 L of DMSO (MWCO: 12000-14000, room temperature) to remove unreacted impurities such as EDA and BOP. Removed.
- This active ester solution was added so that the amount of the carboxylic acid was twice as much as the amino group of HA-AM, and the mixture was stirred at room temperature for 1 minute.
- reaction solution was dialyzed against DMSO (MWCO: 12000-14000, room temperature, 2 days) four times and water five times, and the obtained cloudy solution was freeze-dried.
- DMSO MWCO: 12000-14000, room temperature, 2 days
- the lyophilized product was washed with an excess of acetone and then with an excess of ethanol, and the insoluble material was dried under reduced pressure to obtain 365.lmg of HA-AM-PLA white powder.
- Fig. 23 shows proton NMR of the obtained HA-AM-PLA in DMSO-d.
- the weight ratio of PLA in the HA—AM—PLA molecule is 8 6. 1% WZW (HA: methyl proton of N-acetyl group, 1.85 ppm; PLA: methine proton at ⁇ - position of carboyl group (excluding ⁇ terminal), 5.0-5 6 ppm).
- Example 7-3 250 mg of HA-HZ-PLA synthesized in Example 7-3 was weighed, distilled water was added to make up to 40 mgZmL, and the mixture was stirred at room temperature overnight to dissolve. The resulting aqueous solution was a clear solution that was slightly white.
- Example 8-2 Preparation of HA-HZ-PLA aqueous solution by DMSO solvent diffusion method 1 250 mg of HA-HZ-PLA synthesized in Example 7-4 was weighed, and 5.DMSO was added to make OmgZmL. It was mashed and dissolved. This was mixed with distilled water of 4 times the volume of DMSO, and dialyzed against distilled water (MWCO: 10,000, room temperature, 1 day). The resulting aqueous solution was a colorless and transparent solution. This aqueous solution was filtered through a 5.0 m filter, and then concentrated by ultrafiltration (Vivaspin20 MWCO: 10,000, Vivascience). The concentration was determined by freeze-drying 200 L of the obtained concentrated solution and determining the weight of the dried powder.The concentration was 5.7 mgZmL, and 2.Add distilled water to OmgZmL. And mixed well.
- Example 8-3 Preparation of HA-HZ-PLA aqueous solution by DMSO solvent diffusion method 2
- DMSO solvent diffusion method 2 Weigh 150 mg of HA-HZ-PLA synthesized in Example 7-5, and add DMSO to OmgZmL. It was mashed and dissolved. This was mixed with 4 times the volume of distilled water of mashed DMSO, and dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days). The resulting aqueous solution was a colorless and transparent solution.
- This aqueous solution was filtered through a 5.0 m filter, and then concentrated by ultrafiltration (Vivaspin20 MWCO: 10,000, Vivascience). The concentration was determined by freeze-drying 200 L of the obtained concentrated solution and measuring the weight of the dried powder. The concentration was 7.2 mgZmL, and 2.Distilled water was further added to OmgZmL. And mixed well.
- Example 8-4 Preparation of HA—HZ—PLA Aqueous Solution by DMSO Solvent Diffusion Method 3
- HA-HZ-PLA synthesized in Example 7-6 weigh 150 mg of HA-HZ-PLA synthesized in Example 7-6, and adjust to 5.
- OmgZmL. was added with DMSO and dissolved. This was mixed with 4 times the volume of distilled water of mashed DMSO, and dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days). The resulting aqueous solution was a colorless and transparent solution.
- This aqueous solution was filtered through a 5.0 m filter, and then concentrated by ultrafiltration (Vivaspin20 MWCO: 10,000, Vivascience). The concentration was determined by freeze-drying 200 L of the obtained concentrated solution and measuring the weight of the dried powder, which was 6.5 mgZmL. And mixed well.
- HA-AM-PLA synthesized in Examples 7 to 7 20 mg was weighed out, and added with DMSO to a concentration of 5. OmgZmL and dissolved. This was mixed with 4 times the volume of distilled water of mashed DMSO, and dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days). The resulting aqueous solution was a colorless and transparent solution. This aqueous solution was filtered through a 5.0 m filter, and then concentrated by ultrafiltration (Vivaspin20 MWCO: 10,000, Vivascience). The concentration was determined by freeze-drying the obtained concentrated solution 2001 and measuring the weight of the dried powder. The concentration was 10.4 mgZmL, and 2.Add distilled water to make it OmgZmL. And mixed well.
- Example 9 4 Preparation of Hyaluronic Acid-Coated PLA Fine Particles by DMSO Solvent Diffusion Method 3 4 mg of HA-HZ-PLA synthesized in Example 7-4 and 36 mg of PLA-0005 were weighed and dissolved in 1 mL of DMSO. Was. This is added dropwise to 4 mL of distilled water while stirring with a stirrer.
- the resulting mixed solution was dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days), and the dialysate was filtered through a 5.0 m filter to remove aggregates, and then centrifuged (40000 g, Washing operations such as 10 minutes), supernatant removal, and addition of distilled water were repeated about three times, and the precipitate (hyaluronic acid-coated PLA fine particles) was collected and freeze-dried.
- the yield of the resulting white powder was 3.7 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio, 1.89% w / w).
- DLS measurement (Nicomp370) was performed on the dialysate, and the measured value of the particle size was 288.2 nm.
- the yield of the resulting white powder was 20.lmg. This white powder was washed well with excess acetone, centrifuged (10000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA HZ—PLA ratio, 0.49% wZw). DLS measurement (Nicomp370) was performed on the solution after evaporation, and the measured value of the particle size was 336.9 nm.
- Example 9 Preparation of hyaluronic acid-coated PLA fine particles by acetone solvent diffusion method 3 40 mg of PLA-0005 was weighed and dissolved in 1 mL of acetone. This was added dropwise to 4 mL of the aqueous HA—HZ—PLA solution prepared in Example 8-2 under stirring with a stirrer, mixed and stirred for about 15 minutes, and the obtained mixed solution was evaporated to obtain acetone. Was removed. After filtering through the filter in step 5 to remove aggregates, the washing operation such as centrifugation (40,000 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (hyaluronic acid-coated PLA fine particles).
- the washing operation such as centrifugation (40,000 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (hyaluronic acid-coated PLA fine particles).
- the yield of the resulting white powder was 22.9 mg.
- the white powder was washed well with excess acetone, centrifuged (10000g, 10 minutes), the supernatant was removed, and the insoluble material (HA-HZ-PLA) was dried under reduced pressure. HA HZ-PLA ratio of 0.12% wZw).
- DLS measurement (Nicomp370) was performed on the solution after evaporation, and the measured value of the particle size was 328. 1 nm.
- PLGA-7520 (Wako Pure Chemical Industries, Ltd., viscosity average molecular weight: 20 kDalton, Lactide ratio: 75% molZmol) was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of the aqueous solution of HA-HZ-PLA prepared in Example 8-1, and the mixture was mixed with a rotary homogenizer (Polytron). The mixture was emulsified (10000 rpm, 5 minutes) using PT3100). The methylene chloride was removed by stirring the emulsion with an open stirrer in an open system.
- the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect and freeze the precipitate (hyaluronic acid-coated PLGA fine particles). Dried. The yield of the resulting white powder was 62.lmg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio, 1.09% wZw).
- the particle size was measured by re-dispersing white powder of hyaluronic acid-coated PLA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. When the deviation was calculated, it was te at 2.7 ⁇ 1.1 ⁇ m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (10000 rpm, 5 minutes) using a rotary homogenizer (Polytron PT3100). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sieving through a 32 m sieve, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times.
- a rotary homogenizer Polytron PT3100
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 82, and emulsified (10000 rpm, 5 minutes) using a rotary homogenizer (Polytron PT3100). The emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sifting through a 32 m sieve, washing operations such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water were repeated about three times to collect the precipitate (hyaluronic acid-coated PLGA fine particles). Lyophilized.
- the yield of the resulting white powder was 55. Omg. This white powder was washed well with excess acetone, centrifuged (10000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio of 0.46% wZw).
- the particle size was measured by redispersing white powder of hyaluronic acid-coated PLA fine particles in distilled water, extracting 50 fine particles randomly by optical microscopy, and analyzing the image to obtain the average value and standard deviation. When I asked, I got 3.0 fc at 1.2 ⁇ m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (10000 rpm, 5 minutes) using a rotary homogenizer (Polytron PT3100). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sieving through a 32 m sieve, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times.
- a rotary homogenizer Polytron PT3100
- the particle size was measured by redispersing white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. The deviation was found to be 1.7 ⁇ 0.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 18 mL of l% w / v PVA aqueous solution, and emulsified (10000 rpm, 5 minutes) using a rotary homogenizer (Polytron PT3100). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing the aggregates by sieving at 32 / zm, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (PLGA fine particles). Lyophilized. The yield of the resulting white powder was 57.3 mg.
- the particle size is measured by re-dispersing white PLGA microparticles in distilled water, randomly extracting 50 microparticles by optical microscopy, and analyzing the image to obtain the average value and standard deviation. was found to be 1.7 ⁇ 0.3 m.
- Fine particle preparation method PLA Fine 3 ⁇ 4i PLA
- Example 10-1 Preparation of Hyaluronic Acid-Coated PLA Nanosphere by DMSO Solvent Diffusion Method 4 mg of HA-HZ-PLA synthesized in Example 7-6 and 36 mg of PLA-0005 were taken out, and ImL In DMSO. The mixture was added dropwise to 4 mL of distilled water while stirring with a stirrer, and the obtained mixed solution was dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days), and the dialysate was collected to a volume of 5.0 m.
- MWCO 10,000, room temperature, 3 days
- the washing operation such as centrifugation (40,000 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times, and the precipitate (hyaluronic acid-coated PLA fine particles) was collected and frozen. Dried. The yield of the resulting white powder was 27.8 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA—HZ—PLA) was dried under reduced pressure. HA-HZ-PLA ratio, 7.82% w / w). The particle size was measured by DLS measurement (Nicomp370) of the dialysate and found to be 304. Onm.
- the yield of the resulting white powder was 26.6 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. The weight was 841 g (HA in fine particles). — HZ—PLA ratio, 3.16% wZw). The particle size was measured by DLS measurement (Nico mp370) of the dialysate and found to be 198.3 nm.
- Example 8 - 4 dropped and mixed into prepared HA-HZ- PLA solution 4mL in, dialyzed against distilled water and the resulting et a mixed solution (MWCO: 10000, room temperature, 3 days)
- the dialysis solution was filtered through a filter (5) to remove aggregates, and the washing operation such as centrifugation (40,000 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to obtain the precipitate (hyaluronic acid-coated PLA fine particles). ) was recovered and freeze-dried.
- the yield of the resulting white powder was 35.Omg.
- the yield of the resulting white powder was 36 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10000 g, 10 minutes), the supernatant was removed, and the insolubles (HA-HZ-PLA) were dried under reduced pressure. HA— HZ-PLA ratio, 3.83% wZw). DLS measurement (Nicomp370) was performed on the solution after evaporation, and the measured value of the particle size was 195.6 nm.
- Fine particle preparation method Organic phase Weight * Ratio (How to use HA PLA Content
- Example 11-1 Preparation of hyaluronic acid-coated PLGA microsphere by dialysis method 1 1 It was prepared in the same procedure as in Example 9-1.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 8-11 and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (E YEL4, MAZERA Z). The emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After sifting through a 300 ⁇ sieve to remove aggregates, centrifugation (1500 g, 10 minutes), supernatant removal, addition of distilled water, etc.
- the washing operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLGA fine particles) was collected and freeze-dried.
- the yield of the resulting white powder was 52.4 mg.
- This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure.
- HA-HZ-PLA ratio of the sample was 0.05% wZw).
- the particle size is measured by re-dispersing the white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. When the deviation was determined, it was fc at 6.8 ⁇ 3.6 ⁇ m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred in an open system overnight with a stirrer to remove methylene chloride. After sifting through a 300-m sieve to remove agglomerates, the washing operation of centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times, and the PLGA fine particles obtained as a precipitate were used.
- EYEL4, MAZERA Z mechanical stirrer
- Example 8-1 Mix with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 8-1, mix for about 15 minutes, and then wash again by centrifugation (1500 g, 10 minutes), remove supernatant, and add distilled water. The operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLGA fine particles) was collected and freeze-dried. The yield of the resulting white powder was 58.8 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio of 0.32% wZw).
- the particle size was measured by re-dispersing white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. The deviation was found to be 7.4 ⁇ 2.7 m.
- Example 8 80 mg of PLGA-7520 was weighed and dissolved in 2 mL of methylene chloride.
- Example 8 The mixture was mixed with 8 mL of the HA—HZ—PLA aqueous solution prepared in —2, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (E YEL4, MAZERA Z). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sifting through a 300 m sieve, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (hyaluronic acid-coated PLGA fine particles).
- a mechanical stirrer E YEL4, MAZERA Z
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred in an open system overnight with a stirrer to remove methylene chloride. After sifting through a 300-m sieve to remove agglomerates, the washing operation of centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times, and the PLGA fine particles obtained as a precipitate were used.
- EYEL4, MAZERA Z mechanical stirrer
- Example 8-2 Mix with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 8-2, stir for about 15 minutes, and wash again by centrifuging (1500 g, 10 minutes), removing the supernatant, and adding distilled water. The operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLGA fine particles) was collected and freeze-dried. The yield of the resulting white powder was 60.8 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio of 0.14% wZw).
- the particle size was measured by re-dispersing the white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average value and the average value.
- the calculated standard deviation was 7.8 ⁇ 5.4 m.
- Example 11 6 Preparation of hyaluronic acid-coated PLGA microspheres by dialysis method 1 12 mg of HA-HZ-PLA synthesized in Example 7-6 and 108 mg of PLA-0005 were weighed and dissolved in 3 mL of DMSO. I let it. This was dialyzed against distilled water (MWCO: 10,000, room temperature, 3 days), the dialysate was sieved by 32 m to remove aggregates, centrifuged (1500 g, 10 minutes), supernatant was removed, distilled water was added, This washing operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLA fine particles) was recovered and freeze-dried.
- distilled water MWCO: 10,000, room temperature, 3 days
- the yield of the resulting white powder was 91.7 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. Its weight was 2560 g (HA in the fine particles). — HZ—PLA ratio, 2.79% w / w). The particle size was measured by re-dispersing white powder of hyaluronic acid-coated PLA fine particles in distilled water, randomly extracting 50 fine particles by observation with an optical microscope, and analyzing the image. The calculated value and standard deviation were 1.6 ⁇ 0.4 m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 8-3, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (E YEL4, MAZERA Z). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sifting through a 300 m sieve, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (hyaluronic acid-coated PLGA fine particles).
- a mechanical stirrer E YEL4, MAZERA Z
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred in an open system overnight with a stirrer to remove methylene chloride. After sifting through a 300-m sieve to remove agglomerates, the washing operation of centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times, and the PLGA fine particles obtained as a precipitate were used.
- EYEL4, MAZERA Z mechanical stirrer
- Example 8-3 Mix with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 8-3, stir for about 15 minutes, and then wash again by centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water. The operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLGA fine particles) was collected and freeze-dried. The yield of the resulting white powder was 58.6 mg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio of 0.27% wZw).
- the particle size was measured by re-dispersing white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. The deviation was found to be 7.7 ⁇ 5.7 m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of the HA-HZ-PLA aqueous solution prepared in Example 84, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred with an open stirrer in an open system to remove methylene chloride. After removing aggregates by sifting through a 300 m sieve, the washing operation such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times to collect the precipitate (hyaluronic acid-coated PLGA fine particles).
- a mechanical stirrer EYEL4, MAZERA Z
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 8 mL of a 1% w / v PVA aqueous solution, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred in an open system overnight with a stirrer to remove methylene chloride. After sifting through a 300-m sieve to remove agglomerates, the washing operation of centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water was repeated about three times, and the PLGA fine particles obtained as a precipitate were used.
- EYEL4, MAZERA Z mechanical stirrer
- HA-HZ-PLA aqueous solution prepared in Example 8-4, stir for about 15 minutes, then wash again by centrifugation (1500 g, 10 minutes), remove supernatant, and add distilled water. The operation was repeated about three times, and the precipitate (hyaluronic acid-coated PLGA fine particles) was collected and freeze-dried. The yield of the resulting white powder was 65. Omg. This white powder was thoroughly washed with excess acetone, centrifuged (10,000 g, 10 minutes), the supernatant was removed, and the insoluble matter (HA-HZ-PLA) was dried under reduced pressure. HA-HZ-PLA ratio, 0.60% wZw).
- the particle size was measured by re-dispersing white powder of hyaluronic acid-coated PLGA fine particles in distilled water, randomly extracting 50 fine particles by optical microscopy, and analyzing the image to obtain the average and standard values. The deviation was found to be 5.6 ⁇ 2.3 m.
- PLGA-7520 80 mg was weighed and dissolved in 2 mL of methylene chloride. This was mixed with 18 mL of 1% w / v PVA aqueous solution, and emulsified (400 rpm, 15 minutes) using a mechanical stirrer (EYEL4, MAZERA Z). This emulsion was stirred in an open system overnight with a stirrer to remove methylene chloride. After sifting through a 300 m sieve to remove agglomerates, repeat washing operations about 3 times, such as centrifugation (1500 g, 10 minutes), supernatant removal, and addition of distilled water. Then, the precipitate (PLGA fine particles) was recovered and freeze-dried.
- EYEL4, MAZERA Z mechanical stirrer
- the yield of the resulting white powder was 53.9 mg.
- the particle size was measured by redispersing the white PLGA particles in distilled water, randomly extracting 50 particles by optical microscopy, and analyzing the image to obtain the average value and standard deviation.
- Fine particle preparation method Organic phase PLA weight ratio ( ⁇ HZ_PLA Usage Content
- Example 12 Encapsulation of paclitaxel (PTX) in hyaluronic acid-coated PLA nanospheres (Example 12-1) Preparation by acetone solvent diffusion method-1
- paclitaxel (hereinafter also referred to as PTX) (Natural Pharmaceuticals, Inc. Lot No. 150206602-l) was weighed and dissolved in 2 mL of acetone. 9. 9. mg of PLA-0005 was weighed and added to an acetone solution of PTX to dissolve. Stirrer this The HA-HZ-PLA aqueous solution obtained in the same manner as in Examples 7-6 and synthesized in the same manner as in Examples 7-6 was dropped and mixed with 2 mL of the aqueous solution, and the mixture was stirred for about 15 minutes. The acetone was removed by evaporating the mixed solution.
- PTX Paclitaxel
- PTX was quantified by an internal standard (IS) method using reverse phase chromatography (RP-HPLC).
- IS solution an acetonitrile solution of about 0.7 mg / mL n-hexyl-p-hydroxybenzoate was prepared.
- standard solution for preparing the calibration curve a 5-fold dilution series (8 rows) of a 10 mg / mL PTX acetonitrile solution was prepared.
- PTX concentration determination solution 1.5 mg was weighed, added with 1 mL of acetonitrile, stirred well, centrifuged (10000 g, 10 minutes), and the supernatant was used as a PTX concentration determination solution.
- the standard solution and 400 ⁇ L of the PTX concentration determination solution were mixed at a ratio of 25 ⁇ L of the IS solution, and RP-HPLC measurement was performed. As a result, the ⁇ content in the nanosphere was 9.9% wZw.
- the measurement conditions of RP-HPLC are shown below.
- A MeCN
- B Ultrapure water (MilliQ water)
- PTXIOmg was weighed and dissolved in 2 mL of acetone. 9. 9. mg of PLA-0005 was weighed and added to an acetone solution of PTX to dissolve. This was dropped and mixed with 2 mL of distilled water under stirring with a stirrer, and after stirring for about 15 minutes, acetone was removed by evaporating the obtained mixed solution. Then, it was synthesized by the same method as in Example 7-6, dropped and mixed into 2 mL of the HA—HZ—PLA aqueous solution obtained by the same method as in Example 8-4, and stirred for about 15 minutes.
- Hyaluronic acid or its derivative and polylactic acid, polyglycolic acid or lactate / glycolic acid copolymer of the present invention The hyaluronic acid modified product in which the selected polymer is bonded, efficiently encapsulates a low molecular drug, It is possible to provide a drug carrier which can control the sustained release period for a long period of time, can control the retention in blood, has good dispersibility in an aqueous solution, and has no problem in safety. Further, the modified hyaluronic acid of the present invention provides a drug carrier having excellent biocompatibility, which is composed of injectable fine particles with little aggregation between particles.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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US10/571,005 US7767806B2 (en) | 2003-09-08 | 2004-09-08 | Hyaluronic acid modification products and drug carriers using them |
JP2005513709A JP4755496B2 (ja) | 2003-09-08 | 2004-09-08 | ヒアルロン酸修飾物、及びそれを用いた薬物担体 |
KR1020067003168A KR101121403B1 (ko) | 2003-09-08 | 2004-09-08 | 히알루론산 수식물, 및 그것을 사용한 약물 담체 |
EP04787709.7A EP1666518B1 (en) | 2003-09-08 | 2004-09-08 | Hyaluronic acid modification products and drug carriers using them |
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JP2003-315387 | 2003-09-08 | ||
JP2003315387 | 2003-09-08 | ||
JP2003-407681 | 2003-12-05 | ||
JP2003407681 | 2003-12-05 | ||
JP2004-259157 | 2004-09-07 | ||
JP2004259157 | 2004-09-07 |
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WO2005023906A1 true WO2005023906A1 (ja) | 2005-03-17 |
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PCT/JP2004/013026 WO2005023906A1 (ja) | 2003-09-08 | 2004-09-08 | ヒアルロン酸修飾物、及びそれを用いた薬物担体 |
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US (1) | US7767806B2 (ja) |
EP (1) | EP1666518B1 (ja) |
JP (1) | JP4755496B2 (ja) |
KR (1) | KR101121403B1 (ja) |
TW (1) | TW200524959A (ja) |
WO (1) | WO2005023906A1 (ja) |
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- 2004-09-08 JP JP2005513709A patent/JP4755496B2/ja not_active Expired - Fee Related
- 2004-09-08 WO PCT/JP2004/013026 patent/WO2005023906A1/ja active Application Filing
- 2004-09-08 KR KR1020067003168A patent/KR101121403B1/ko active IP Right Grant
- 2004-09-08 EP EP04787709.7A patent/EP1666518B1/en not_active Not-in-force
- 2004-09-08 TW TW093127171A patent/TW200524959A/zh not_active IP Right Cessation
- 2004-09-08 US US10/571,005 patent/US7767806B2/en not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
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TWI378941B (ja) | 2012-12-11 |
KR101121403B1 (ko) | 2012-04-12 |
US7767806B2 (en) | 2010-08-03 |
TW200524959A (en) | 2005-08-01 |
KR20060065689A (ko) | 2006-06-14 |
EP1666518A1 (en) | 2006-06-07 |
JPWO2005023906A1 (ja) | 2007-11-08 |
EP1666518B1 (en) | 2018-03-28 |
JP4755496B2 (ja) | 2011-08-24 |
EP1666518A4 (en) | 2006-11-29 |
US20070031503A1 (en) | 2007-02-08 |
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