WO2009104706A1 - マイクロ粒子およびその医薬品組成物 - Google Patents
マイクロ粒子およびその医薬品組成物 Download PDFInfo
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- WO2009104706A1 WO2009104706A1 PCT/JP2009/052951 JP2009052951W WO2009104706A1 WO 2009104706 A1 WO2009104706 A1 WO 2009104706A1 JP 2009052951 W JP2009052951 W JP 2009052951W WO 2009104706 A1 WO2009104706 A1 WO 2009104706A1
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- Prior art keywords
- microparticles
- polymer
- poly
- hydrophilic active
- hydrophilic
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1682—Processes
- A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
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- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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Definitions
- the present invention relates to a microparticle formed by associating particles containing a hydrophilic active substance and a pharmaceutical composition thereof.
- the present invention relates to a microparticle as a so-called drug delivery system and a pharmaceutical composition thereof. More specifically, the present invention relates to, for example, microparticles that effectively contain protein / peptide drugs, nucleic acid drugs, etc., which are hydrophilic and have a large molecular weight, and pharmaceutical compositions thereof.
- a fine particle formulation in which a drug is encapsulated in a fine particle called nanoparticle, microparticle, nanosphere, microsphere, or microcapsule has been developed and is being studied for use as a sustained-release drug.
- Examples of the fine particle preparation based on a polymer compound include fine particles using biodegradable polylactic acid or poly (lactic acid-glycolic acid).
- biodegradable polylactic acid or poly (lactic acid-glycolic acid) In these fine particle preparations, it is difficult to encapsulate a protein / peptide drug having hydrophilicity and a large molecular weight while maintaining physiological activity.
- a phenomenon called initial burst occurs in a large amount in a short time when administered in vivo.
- Patent Document 1 discloses a microcapsule for carrying a pharmacologically active agent composed of a reaction product of a polyol and polylactic acid. Yes. In this technique, no polysaccharide is used, and there is no disclosure of encapsulation of peptides and proteins. Microcapsules produced by spray drying release 62% of the encapsulated drug in 24 hours. This release rate is too fast to prevent the microcapsule from being used as a drug sustained-release agent.
- Patent Document 2 and Non-Patent Document 1 disclose nanoparticles or microparticles made of a material obtained by grafting a biodegradable polymer to a polysaccharide. However, in these documents, microparticles formed by associating nanoparticles are described. Not mentioned. Patent Document 2 includes a method already described in the literature, for example, a “double emulsion” method, as a method for producing microparticles for encapsulating a hydrophilic active ingredient. However, the encapsulation of the drug in the particles and the release of the drug from the particles have not been realized.
- Non-Patent Document 1 discloses microparticles encapsulating albumin produced by the “double emulsion” method, but the encapsulation rate relative to the charged amount of albumin is 53% or less, and the hydrophilic encapsulation rate is low. However, there is a problem that manufacturing costs are high.
- fine particles comprising an amphiphilic polymer comprising a polysaccharide and an aliphatic polyester, comprising an inner core comprising a polysaccharide, a hydrophobic outer layer comprising an aliphatic polyester, and a surface modifier bonded to the hydrophobic outer layer.
- the particles do not have a structure in which fine particles are associated, and there is no specific example of particles having a micrometer size.
- the encapsulation rate of the hydrophilic active substance is 50% or less, and a low encapsulation rate is a problem as described above.
- Patent Document 4 discloses nanoparticles having an average particle size of less than 300 nm, which is made of a natural derivatized polymer of dextran, but there is no specific example. Moreover, it is not preferable as a sustained-release agent that it does not have a structure in which fine particles are associated and has an average particle diameter of several hundred nanometers because it causes diffusion from the administration site.
- Patent Documents 5 and 6 discuss the use of an amphiphilic block polymer having a hydrophilic part such as polyethylene glycol and a hydrophobic part such as poly (lactic acid-glycolic acid) as a polymer for forming particles. And disclosed.
- Micellar particles using such an amphiphilic block polymer usually have a hydrophobic structure inside and a hydrophilic structure in the outer layer, so they are suitable for inclusion of hydrophobic low molecular weight drugs, but proteins, peptides, etc. It is difficult to encapsulate the hydrophilic active substance.
- Patent Document 7 and Non-Patent Document 2 disclose attempts to encapsulate proteins in particles using amphiphilic block polymers, but there are disadvantages such as a small amount of drug that can be encapsulated or a large initial burst. However, a technique for producing particles having properties suitable as a sustained-release injection of a hydrophilic drug has not yet been established.
- Japanese Patent Publication No.8-19226 Japanese translation of PCT publication No. 2004-521152 WO2006 / 095668 Japanese National Patent Publication No.
- microparticles As described above, the development of microparticles using polymers has been carried out, but the microparticles can efficiently encapsulate hydrophilic active substances, do not show a significant initial burst, and are encapsulated at an appropriate rate. It is an object of the present invention to provide microparticles capable of releasing a drug.
- the present invention comprises hydrophilic active substance-containing particles comprising an amphiphilic polymer comprising a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol and a hydrophilic active substance.
- hydrophilic active substance-containing particles comprising an amphiphilic polymer comprising a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol and a hydrophilic active substance.
- Microparticles specifically, microparticles that have hydrophilic segments of an amphiphilic polymer inside and hydrophilic active substance-containing particles that have an outer layer of a hydrophobic segment of the amphiphilic polymer, a method for producing the same, and About the pharmaceutical composition.
- microparticles of the present invention can efficiently encapsulate a hydrophilic active substance and release the hydrophilic active substance at an appropriate rate in vivo, it can be used as a novel DDS preparation.
- Drug release from human growth hormone-encapsulated microparticles Drug release from human insulin-encapsulated dextran-PLGA microparticles.
- Drug release in buffer of exendin 4 encapsulated microparticles The time-dependent change of the blood drug concentration of the mouse
- mouth which administered human growth hormone inclusion microparticle subcutaneously The time-dependent change of the blood drug density
- the present invention is characterized in that hydrophilic active substance-containing particles containing an amphiphilic polymer and a hydrophilic active substance are associated to form microparticles.
- aggregation means that two or more particles are bonded through interparticle forces or other substances to form an aggregate.
- the interparticle force is not particularly limited, and examples thereof include hydrophobic interaction, hydrogen bond, and van der Waals force.
- the association is not limited to the state in which the particles are in contact with each other, and a substance having an affinity for the particles may exist between the particles, or the particles may be dispersed in the matrix. Polymers are preferable as the substance having affinity for particles and the matrix.
- the association of the hydrophilic active substance-containing particles brings about an effect that the inclusion ratio of the hydrophilic active substance is superior to that of a single particle.
- the particle diameters of the hydrophilic active substance-containing particles to be associated may be different.
- Microparticles are particles having a particle size from submicron to submillimeter.
- the average particle size of the microparticles of the present invention is not particularly limited, but for example, when the microparticles are administered to a living body by injection, it is necessary to use a thicker injection needle that has a greater burden on the patient as the average particle size increases. Therefore, the thickness is preferably 1 ⁇ m to 50 ⁇ m from the viewpoint of reducing the burden on the patient.
- the average particle size of the microparticles can be determined by an image analysis method using a scanning electron microscope.
- the number of associations of the hydrophilic active substance-containing particles constituting the microparticles is preferably in the range of 10 to 10 7, more preferably in the range of 10 5 to 10 7.
- the number of associations can be calculated from the average particle size of the hydrophilic active substance-containing particles and the average particle size of the microparticles.
- the amphiphilic polymer is characterized by comprising a hydrophobic segment of poly (hydroxy acid) and a hydrophilic segment of polysaccharide or polyethylene glycol.
- the amphiphilic property mentioned here means that it has both hydrophilic and hydrophobic properties, and hydrophilicity means that when the solubility in water at any site is higher than that of other segments, The site is said to be hydrophilic.
- the hydrophilic segment is desirably soluble in water. However, even if it is poorly soluble, the hydrophilic segment only needs to have a higher solubility in water than other sites.
- hydrophobic means that a segment is hydrophobic when the solubility in water at any site is lower than at other sites.
- the hydrophobic segment is desirably insoluble in water, but may be soluble as long as it has a lower solubility in water than other sites.
- poly (hydroxy acid) chain of the amphiphilic polymer examples include polyglycolic acid, polylactic acid, poly (2-hydroxybutyric acid), poly (2-hydroxyvaleric acid), and poly (2-hydroxycaproic acid). ), Poly (2-hydroxycapric acid), poly (malic acid), or derivatives and copolymers of these polymer compounds, but the microparticles of the present invention do not have a significant detrimental effect when administered in vivo. Therefore, it is preferable that the amphiphilic polymer poly (hydroxy acid) is also a biocompatible polymer.
- the biocompatible polymer mentioned here refers to a polymer that does not have a significant harmful effect when administered to a living body, and more specifically, when the polymer is orally administered to a rat, the LD50 is 2. , 000mg / kg or more.
- the poly (hydroxy acid) which is a biocompatible polymer is preferably polylactic acid, polyglycolic acid or a poly (lactic acid-glycolic acid) copolymer.
- the poly (hydroxy acid) is poly (lactic acid-glycolic acid)
- the composition ratio of poly (lactic acid-glycolic acid) (lactic acid / glycolic acid) (mol / mol%) is as long as the object of the present invention is achieved.
- those of 100/0 to 30/70 are preferred, and those of 60/40 to 40/60 are particularly preferred.
- hydrophilic segment of the amphiphilic polymer is a polysaccharide
- examples of the polysaccharide include cellulose, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan, and dextran, with dextran being preferred.
- a poly (hydroxy acid) graft chain is preferably graft-polymerized on a polysaccharide main chain.
- the average molecular weight of the polysaccharide main chain is preferably 1,000 to 100,000, more preferably 2,000 to 50,000, and the average molecular weight of the poly (hydroxy acid) is 500 to 500. 100,000 is preferred, and 1,000 to 10,000 is more preferred.
- the average molecular weight of the poly (hydroxy acid) relative to the average molecular weight of the polysaccharide is preferably 0.01 to 100 times, more preferably 0.02 to 10 times, and still more preferably 0.02 times to 1 time.
- the number of poly (hydroxy acid) graft chains bonded to the polysaccharide main chain is preferably 2 to 50.
- the number of graft chains can be determined from the graft type amphiphilic polymer, polysaccharide main chain, and poly (hydroxy acid) graft chain average molecular weight.
- the amphiphilic polymer is preferably a block polymer of polyethylene glycol and poly (hydroxy acid).
- block refers to a part of a polymer molecule, which is composed of at least 5 or more monomer units and is different in chemical structure or configuration from other parts adjacent to the part.
- a polymer formed by linearly connecting the above blocks is called a block polymer.
- Each block itself constituting the block polymer may be a random, alternating, or gradient polymer composed of two or more types of monomer units.
- the hydrophilic segment of the amphiphilic polymer is polyethylene glycol
- the amphiphilic polymer is preferably a block polymer in which polyethylene glycol and polyhydroxy acid are connected one by one.
- the polyethylene glycol to be used includes linear or branched polyethylene glycol or a derivative thereof, preferably a linear polyethylene glycol derivative.
- the polyethylene glycol derivative include polyethylene glycol monoalkyl ether.
- the alkyl group of the polyethylene glycol monoalkyl ether include a linear or branched alkyl group having 1 to 10 carbon atoms, and a linear or branched alkyl group having 1 to 4 carbon atoms is preferable.
- Methyl, ethyl, propyl An iso-propyl group is more preferable.
- the average molecular weight of polyethylene glycol is not particularly limited, but is preferably 2,000 to 15,000, more preferably 2,000 to 12,000, and 4,000 to 12,000. Is more preferably 5,000 to 12,000.
- the average molecular weight of the poly (hydroxy acid) when the hydrophilic segment of the amphiphilic polymer is polyethylene glycol is not particularly limited, but is preferably 5,000 to 200,000, and 15,000 to 150,000. More preferred is 20,000 to 100,000.
- the average molecular weight of the poly (hydroxy acid) relative to the average molecular weight of polyethylene glycol is preferably 1 or more, more preferably 2 or more, still more preferably 4 or more, and particularly preferably. Is 4 times or more and 25 times or less.
- the average molecular weight in the present specification refers to the number average molecular weight unless otherwise specified, and the number average molecular weight is an average molecular weight calculated by a method that does not consider weighting of the molecular size, an amphiphilic polymer,
- the average molecular weight of the polysaccharide and polyethylene glycol can be determined as polystyrene or pullulan-converted molecular weight measured by gel permeation chromatography (GPC).
- GPC gel permeation chromatography
- the average molecular weight of the poly (hydroxy acid) can be determined from the ratio of the peak integrated value of the terminal residue to the peak integrated value of other than the terminal residue by nuclear magnetic resonance ( 1 H-NMR) measurement.
- the amphiphilic polymer comprising a polysaccharide and a poly (hydroxy acid) used in the present invention may be synthesized by a known method, and is not limited by the synthesis method as long as it can form a reverse phase emulsion. It can be produced as in (1), (2) or (3).
- amphiphilic polymer composed of polyethylene glycol and poly (hydroxy acid) used in the present invention may be synthesized by a known method, and is not limited by the synthesis method as long as it can form a reversed phase emulsion.
- a method of producing an amphiphilic block polymer by adding a hydroxy acid-activating monomer to polyethylene glycol in the presence of a tin catalyst and introducing poly (hydroxy acid) [Journal of Controlled Release, 71, 203- 211 (2001)].
- the structure of the hydrophilic active substance-containing particles comprising the above-mentioned amphiphilic polymer and a hydrophilic physiologically active substance is not particularly limited, but has a hydrophilic segment of the amphiphilic polymer inside, and the amphiphilic polymer
- a hydrophilic active substance-containing particle having an outer layer of a hydrophobic segment is preferable because the hydrophilic active substance contained therein can be more stably retained.
- the hydrophilic active substance-containing particle is a particle having a hydrophilic segment of an amphiphilic polymer inside and an outer layer of a hydrophobic segment of the amphiphilic polymer
- a surface modifier is added to the outer layer of the poly (hydroxy acid).
- the bond here may be a non-covalent bond or a covalent bond.
- the non-covalent bond is preferably a hydrophobic interaction, but may be an electrostatic interaction, a hydrogen bond, Juan der Waalska, or a combination of them.
- the hydrophobic outer layer of fine particles containing an amphiphilic polymer and the hydrophobic portion of the surface modifier described later are bonded by a hydrophobic interaction.
- the fine particle dispersion medium is water, a buffer solution, physiological saline, a surface modifier aqueous solution or a fine particle dispersion which is a hydrophilic solvent.
- the surface modifier is preferably one that stabilizes the water-oil interface of the S / O / W type emulsion or the oil-oil emulsion interface of the S / O1 / O2 type emulsion. It is a compound having the property of improving colloidal stability.
- the surface modifier may be one kind or a mixture of plural kinds.
- improving colloidal stability means preventing or delaying aggregation of microparticles in a solvent.
- the surface modifier is preferably an amphiphilic compound or a hydrophilic polymer.
- hydrophilic polymer of the surface modifier in the present invention polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene imine, polyacrylic acid, polymethacrylic acid, poly-1,3-dioxolane, 2-methacryloyloxyethyl phosphorylcholine polymer , Poly-1,3,6-trioxane, polyamino acid, peptide, protein, saccharide or any analog thereof.
- hydrophilic polymer analog examples include, but are not limited to, a surfactant obtained by partially modifying a hydrophilic polymer with a hydrophobic group such as long-chain alkyl.
- Pluronic registered trademark of BASF commercially available from BASF or its equivalent is preferable.
- polyamino acid of the surface modifier in the present invention polyaspartic acid, polyglutamic acid or their analogs are preferable.
- An analog in which a long-chain alkyl group is introduced into a part of polyaspartic acid or polyglutamic acid is particularly preferable.
- the surface modifying agent peptide in the present invention includes basic peptides.
- gelatin, casein or albumin is preferable for improving the dispersibility of the particles.
- An example of a protein is an antibody.
- saccharide of the surface modifier in the present invention monosaccharides, oligosaccharides and polysaccharides are preferable.
- polysaccharide cellulose, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, pullulan or dextran is preferable, and cholesterolated pullulan is particularly preferable for improving the dispersibility of the particles, cellulose, chitin, chitosan, gellan gum, alginic acid, An analogue of either hyaluronic acid, pullulan or dextran is preferred.
- peptides, proteins, and saccharides as surface modifiers are particularly analogs in which hydrophobic groups such as long-chain alkyls are partially modified, and analogs in which the aforementioned hydrophilic polymers and amphiphilic compounds are modified. preferable.
- the amphiphilic compound is a lipid.
- the amphiphilic compound is preferably a surfactant.
- Surfactants include polyoxyethylene polypropylene glycol copolymer, sucrose fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene sorbitan monofatty acid ester, polyoxyethylene sorbitan difatty acid ester, polyoxyethylene glycerin monofatty acid ester, Nonionic active agents such as oxyethylene glycerin difatty acid ester, polyglycerin fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl sulfates such as sodium lauryl sulfate, ammonium lauryl sulfate, sodium stearyl sulfate, or lecithin Is preferred.
- the hydrophilic active substance is exemplified by a low molecular weight compound, protein, peptide, DNA, RNA, or modified nucleic acid. Further, even in the case of a hydrophobic drug, it may be contained in the microparticles of the present invention by making it hydrophilic with a solubilizer or the like. As the solubilizer here, cyclodextrin and its analogs are preferable.
- the protein or peptide used as the hydrophilic active substance in the present invention is not particularly limited, but a physiologically active protein or physiologically active peptide is preferable.
- a physiologically active protein or physiologically active peptide include peptide hormones, cytokines, enzyme proteins, antibodies, etc.
- parathyroid hormone PTH
- calcitonin insulin, insulin-like growth factor, angiotensin, glucagon
- GLP- GLP-1 receptor agonist peptides represented by 1 and exendin 4
- bombesin motilin, gastrin, growth hormone, prolactin (lutein stimulating hormone), gonadotropin (gonadotropic hormone), tropotropic hormone, corticotropin (ACTH) , ACTH derivatives (such as shrimp tide), melanocyte stimulating hormone, follicle stimulating hormone (FSH), sermorelin, vasopressin, oxytocin, protyrelin, luteinizing hormone (LH), cortico Lopin, secretin, somatropin, thyrotropin (thyroid stimulating hormone), somatostatin, gonadotropin releasing hormone (GnRH), G-CSF, erythropoietin (EPO), thrombopoietin (TPO), megacaryocytopotentiator, HGF,
- hydrophilic active substance is DNA, RNA, or modified nucleic acid
- it may be complexed with a cationic surfactant, a cationic lipid, a cationic polymer, and analogs thereof.
- saccharide used as the hydrophilic active substance in the present invention examples include hyaluronic acid, heparin, dextran sulfate, dextran, and FITC-labeled dextran (for example, FD40).
- the present invention is a method for producing microparticles in which the hydrophilic active substance-containing particles are associated, (A) a step of forming a reversed-phase emulsion by mixing an aqueous solvent containing a hydrophilic active substance and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved; (B) removing the solvent from the reverse emulsion to obtain a hydrophilic active ingredient-containing solid content; (C) introducing a hydrophilic active ingredient-containing solid content or a hydrophilic active ingredient-containing solid content dispersion into a liquid phase containing a surface modifier;
- the present invention relates to a method for producing microparticles containing
- the reverse phase emulsion is obtained by changing an aqueous solvent containing a hydrophilic active substance into a water-immiscible organic solvent in which an amphiphilic polymer is dissolved. It is formed by adding and mixing.
- a stirring device such as a magnetic stirrer, a turbine-type stirrer, a homogenizer, a membrane emulsifying device equipped with a porous membrane, or the like may be used.
- the water-immiscible organic solvent in the present invention is an organic solvent having a solubility in water of 30 g (water-immiscible organic solvent) / 100 ml (water) or less, which exceeds the solubility in water described on the left.
- the organic solvent is positioned as a water-miscible organic solvent.
- aqueous solvent in step (a) water or an aqueous solution containing a water-soluble component is used.
- water-soluble component include inorganic salts, saccharides, organic salts, amino acids and the like.
- the nature of the water-immiscible organic solvent in the step (a) is not particularly limited, but the poly (hydroxy acid) that is the hydrophobic segment of the amphiphilic polymer is soluble and the hydrophilic segment is hardly soluble or insoluble. It is preferable that Further, the non-water miscible organic solvent can be volatilized and removed by freeze drying or the like, and more preferably 0.1 g (water immiscible organic solvent) / 100 ml (water) or less. Specific examples of the water-immiscible organic solvent include ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride, and chloroform.
- the ratio of the aqueous solvent to the water-immiscible organic solvent is preferably 1,000: 1 to 1: 1, more preferably 100: 1 to 3: 1.
- the concentration of the amphiphilic polymer in the water-immiscible organic solvent varies depending on the type of the water-immiscible organic solvent and amphiphilic polymer, but is 0.01 to 90% (w / w), more preferably 0. 1 to 50% (w / w), more preferably 1 to 20% (w / w).
- step (a) in the step of forming a reverse emulsion with an aqueous solvent containing a hydrophilic active substance and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, two or more types are used depending on the pharmaceutical purpose.
- a reverse emulsion may be formed with a water-immiscible organic solvent in which the amphiphilic polymer is dissolved.
- the formation of the reverse phase emulsion is assisted, Coagents can be added for the purpose of forming a fine inverse emulsion.
- the coagent is preferably a compound selected from alkyl alcohols having 3 to 6 carbon atoms, alkylamines having 3 to 6 carbon atoms, and alkyl carboxylic acids having 3 to 6 carbon atoms.
- the structure of the alkyl chain of these coagents is not particularly limited, and may be a linear structure or a branched structure, and may be a saturated alkyl or an unsaturated alkyl.
- the coagent is particularly preferably tert-butanol, iso-propanol, or pentanol.
- the average particle size of the reversed-phase emulsion in step (a) is not particularly limited by the desired particle size of the microparticles of the present invention, but microparticles for pharmaceuticals that are one of the uses of the microparticles of the present invention are produced.
- the upper limit of the average particle diameter is preferably 50 ⁇ m, more preferably 5 ⁇ m, still more preferably 500 nm, still more preferably 150 nm, and most preferably 100 nm.
- the lower limit of the average particle size of the reversed phase emulsion is preferably 10 nm, more preferably 50 nm.
- step (b) in which the solvent is removed from the reverse phase emulsion obtained in the step (a) to obtain a hydrophilic active ingredient-containing solid content.
- the method for removing the solvent from the reversed-phase emulsion is not particularly limited.
- heating, reduced-pressure drying, dialysis, lyophilization, centrifugation, filtration, reprecipitation, and combinations thereof Is mentioned.
- the freeze-drying conditions and equipment include a freezing process and a drying step under reduced pressure.
- Pre-freezing which is a conventional freeze-drying method, primary drying under reduced pressure and low temperature, and secondary drying under reduced pressure.
- the process through is particularly preferable. For example, by cooling and freezing below the melting point of the aqueous solvent and the water-immiscible organic solvent constituting the reverse emulsion, freeze-dried solid content containing the hydrophilic active substance can be obtained.
- the pre-freezing temperature may be appropriately determined experimentally from the solvent composition, but is preferably ⁇ 20 ° C. or lower. Further, the degree of reduced pressure in the drying process may be appropriately determined experimentally from the solvent composition, but is preferably 3,000 Pa or less, more preferably 500 Pa or less for shortening the drying time. For freeze-drying, it is preferable to use a laboratory freeze-dryer equipped with a cold trap and connectable to a vacuum pump, or a shelf-type vacuum freeze-dryer used for manufacturing pharmaceuticals. Thereafter, drying under reduced pressure may be performed with a decompression device such as a vacuum pump under cooling or at room temperature.
- a decompression device such as a vacuum pump under cooling or at room temperature.
- the hydrophilic active ingredient-containing solid content obtained in the step (b) is obtained as an aggregate of hydrophilic active substance-containing particles containing an amphiphilic polymer reflecting the structure of the reversed-phase emulsion.
- the aggregate mentioned here is an indeterminate lump in which fine particles are aggregated by interparticle force, and is clearly distinguished from the microparticle of the present invention in terms of shape.
- the average particle size of the hydrophilic active substance-containing fine particles constituting the aggregate is not particularly limited by the desired particle size of the microparticles of the present invention, but the microparticles for pharmaceuticals, which is one of the uses of the microparticles of the present invention.
- the upper limit of the average particle diameter is preferably 50 ⁇ m, more preferably 5 ⁇ m, still more preferably 500 nm, still more preferably 150 nm, and most preferably 100 nm.
- the lower limit of the average particle diameter of the hydrophilic active substance-containing fine particles is preferably 10 nm, more preferably 50 nm.
- the method of introducing the hydrophilic active ingredient-containing solid content or the hydrophilic active ingredient-containing solid content dispersion into the liquid phase containing the surface modifier includes, for example, the hydrophilic active ingredient-containing solid content on the surface.
- a method of adding to a liquid phase containing a modifier or a solid component containing a hydrophilic active ingredient is once dispersed in a dispersion medium, and the resulting dispersion (solid-in-oil (S / O) suspension) is applied to the surface.
- the method of adding to the liquid phase containing a modifier is mentioned.
- the dispersion medium is not particularly limited, but the amphiphilic property reflects the structure of the reversed-phase emulsion constituting the hydrophilic active ingredient-containing solid content.
- the poly (hydroxy acid) is soluble and does not substantially dissolve the hydrophilic segment constituting the amphiphilic polymer.
- the solvent in which poly (hydroxy acid) is soluble and does not substantially dissolve the hydrophilic segment is a solvent having a solubility of the hydrophilic segment in the solvent of 50 mg / ml or less, preferably 10 mg / mL or less. is there.
- the dispersion medium may be a water-immiscible organic solvent or a water-miscible organic solvent as long as it has the above characteristics, but is preferably a water-immiscible organic solvent.
- water-immiscible organic solvents in which the amphiphilic polymer poly (hydroxy acid) is soluble and do not substantially dissolve the hydrophilic segment include ethyl acetate, isopropyl acetate, butyl acetate, and dimethyl carbonate. , Diethyl carbonate, methylene chloride, chloroform, dioxane, toluene, xylene and the like.
- various additives soluble in the dispersion medium for example, for the purpose of controlling the release rate of the hydrophilic active substance by the decomposition or disintegration of the hydrophilic active substance-containing particles, You may contain an acidic compound, a basic compound, an amphiphilic polymer, a biodegradable polymer, etc.
- the liquid phase in the step (c) preferably has a boiling point higher than that of the solid component dispersion medium containing the hydrophilic active ingredient and soluble in the above-described surface modifier, an aqueous solvent, a water-immiscible organic solvent, Any of water-miscible organic solvents may be used.
- an aqueous solvent here, water or an aqueous solution containing a water-soluble component is preferable.
- the water-soluble component include inorganic salts, saccharides, organic salts, amino acids, and the like.
- liquid phase in step (c) is preferably an aqueous solvent or a water-miscible organic solvent.
- the suspension obtained in step (c) is a so-called solid-in-oil-in-water (S / O / W) type emulsion
- the liquid phase is a water-immiscible organic solvent or a water-miscible organic solvent and not miscible with the dispersion medium
- the volume ratio of the liquid phase to the dispersion medium for dispersing the hydrophilic active substance-containing solid content is generally 1,000: 1 to 1: 1,000, more preferably 100: 1 to 1: 100.
- the concentration of the surface modifier in the liquid phase of the present invention varies depending on the type of the surface modifier, but is preferably 0.01 to 90% (w / v), more preferably 0.1 to 50%. (W / v), more preferably 5 to 10% (w / v).
- the surface modifier may be bound to the poly (hydroxy acid) outer layer of the microparticle amphiphilic polymer of the present invention, in which case the binding amount is preferably 0.0001% based on the microparticle weight. To 1%.
- liquid phase in step (c) may contain various additives such as buffers, antioxidants, salts, polymers or sugars depending on the pharmaceutical purpose. good.
- step (c) it is also preferable to add inorganic salts to the liquid phase.
- the inorganic salt is preferably an alkali metal salt or an alkaline earth metal salt, and more preferably sodium chloride.
- the concentration of inorganic salts in the liquid phase is preferably 0 to 1M, more preferably 10 mM to 1M, and even more preferably 10 mM to 100 mM.
- step (c) a solid-in-oil-in-water (S / O / W) type emulsion or solid-in-oil-in-oil (S) is formed for the purpose of producing finer microparticles.
- / O1 / O2) type emulsion may be emulsified.
- the emulsification method is not particularly limited as long as a stable emulsion can be prepared. For example, a method using stirring, a method using a high-pressure homogenizer, a high-speed homomixer, or the like can be given.
- step (c) when the solid component containing the hydrophilic active ingredient is once dispersed in the dispersion medium, and the obtained dispersion liquid is added to the liquid phase containing the surface modifier, the dispersion medium is removed.
- the method for removing the dispersion medium is not particularly limited, and examples thereof include submerged drying, dialysis, lyophilization, centrifugation, filtration, and reprecipitation, and submerged drying and lyophilization are particularly preferable.
- an aqueous solvent is used as the liquid phase, an aqueous dispersion of microparticles is obtained by this step.
- the microparticles of the present invention can be obtained by removing the liquid phase from the microparticle dispersion obtained as described above.
- the method for removing the liquid phase is not particularly limited, but distillation by evaporation, dialysis, lyophilization, centrifugation, and filtration are preferred.
- the application field of the microparticles obtained by the present invention is wide and can be used in various ways, but is preferably used as a pharmaceutical composition.
- it may contain various pharmaceutically useful additives in addition to the microparticle, and the additive that can be added is preferably a buffer, an antioxidant. Agent, salt, polymer or sugar.
- Examples of the administration method when the microparticles of the present invention are used as a pharmaceutical composition include oral administration and parenteral administration, preferably parenteral administration.
- parenteral administration it can be used for subcutaneous administration, intramuscular administration, enteral administration, pulmonary administration, local administration (nasal, dermal, eye), intraperitoneal administration, etc., but as a particularly preferred administration method, subcutaneously or intramuscularly Injection.
- the dose and frequency of administration when the pharmaceutical composition of the present invention is administered to a living body can be appropriately selected depending on the hydrophilic active substance, dosage form, patient age, body weight, and severity of symptoms. It is administered in a range of 0.1 ⁇ g to 100 mg, preferably 1 ⁇ g to 10 mg per day.
- Example 1 Synthesis of Dextran-Polylactic Acid (PLA) 1-1.
- Synthesis of TMS-dextran (compound (1)) Dextran (Nacalai Tesque Co., Ltd., Nacalai standard special grade product, number average molecular weight 13000, 5.0 g) was added to formamide (100 ml) and heated to 80 ° C. To this solution, 1,1,1,3,3,3-hexamethyldisilazane (100 ml) was added dropwise over 20 minutes. After completion of dropping, the mixture was stirred at 80 ° C. for 2 hours. After completion of the reaction, the reaction solution was returned to room temperature, and the two layers were separated using a separatory funnel. The upper layer was concentrated under reduced pressure, methanol (300 ml) was added, and the resulting solid was filtered and dried to obtain TMS-dextran (11.4 g) as a white solid.
- Example 2 Synthesis of Dextran-Poly (lactic acid-glycolic acid) (PLGA) 2-1. Synthesis of TMS-dextran-PLGA (compound (4), compound (5), compound (6)) Compound (1) (0.5 g) and tert-butoxypotassium (35 mg) were dried under reduced pressure for 1 hour, and then tetrahydrofuran ( 10 ml) was added and stirred for 1 hour at room temperature. To this solution, a solution of (DL) -lactide (1.12 g) and glycolide (0.9 g) in tetrahydrofuran (15 ml) was added dropwise and stirred for 5 minutes.
- Dextran-PLGA was obtained from compounds (5) and (6) in a similar manner except that trifluoroacetic acid was used (compound (8) and compound (9)).
- the weight average molecular weight and number average molecular weight of the polymers of compounds (7) to (9) were determined by GPC measurement (column Toso-TSK-gel ⁇ -5000 ⁇ 2, DMF solvent, detector RI, standard pullulan). .
- the average molecular weight of the graft chain and the number of graft chains were determined by 1 H-NMR measurement.
- Compound (7) had a weight average molecular weight of 43,820, a number average molecular weight of 33,422, a graft chain molecular weight of 1,900, and a graft chain number of 7 to 10.
- Compound (8) had a weight average molecular weight of 94,088, a number average molecular weight of 81,250, a graft chain molecular weight of 3,250, and a graft chain number of 21.
- Compound (9) had a weight average molecular weight of 137,695, a number average molecular weight of 109,630, a graft chain molecular weight of 6,442, and a graft chain number of 15.
- Example 3 Preparation Method of Human Growth Hormone (hGH) Encapsulated Microparticles
- Dextran-polylactic acid (PLA) of Example 1 (dextran average molecular weight 13,000, PLA average molecular weight 2,300, PLA graft chain number 10 to 12, compound (3)) or dextran-poly (lactic acid-glycolic acid) (PLGA) of Example 2 (dextran average molecular weight 13,000, PLGA average molecular weight 1,900, PLGA graft chain number 7 to 10, compound (7)) 5 mg
- PHA Dextran-polylactic acid
- PLGA dextran-poly (lactic acid-glycolic acid)
- Example 2 (dextran average molecular weight 13,000, PLGA average molecular weight 1,900, PLGA graft chain number 7 to 10, compound (7)) 5 mg
- 5 mg was dissolved in 100 ⁇ l of dimethyl carbonate to prepare a 50 mg / ml polymer solution.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- the average particle size was calculated by observing the obtained microparticles with a scanning electron microscope (SEM: HITACHI, S-4800), the average particle size of the microparticles was 4.0 ⁇ m.
- Example 4 Measurement of Drug Encapsulation Rate of Human Growth Hormone (hGH) Encapsulated Microparticles Human prepared by the method of Example 3 using dextran-PLA (compound (3)) or dextran-PLGA (compound (7)) polymer 20 mg of growth hormone-encapsulated microparticles were weighed into a 1.5 ml Eppendorf tube, and 1 ml of buffer A (0.1% bovine serum albumin, 0.1% Pluronic F-68 (registered trademark of BASF) and 0.02% azimuth) The solution was dissolved in PBS containing sodium chloride, and the particles (precipitate) and the supernatant were separated by centrifugation at 18,000 ⁇ g for 10 minutes.
- buffer A 0.1% bovine serum albumin, 0.1% Pluronic F-68 (registered trademark of BASF) and 0.02% azimuth
- the particles were resuspended in 1 ml of buffer solution, and the particles were separated from the supernatant by centrifugation under the above conditions. This washing operation was repeated once more (centrifugation three times in total), and the concentration of human growth hormone in the supernatant collected by each centrifugation was measured with an ELISA kit (manufactured by R & D Systems). By subtracting the total amount of hGH in the centrifugal supernatant three times from the amount of hGH charged at the time of particle preparation (per 20 mg of particle weight), the encapsulation rate was calculated by the following formula.
- Encapsulation rate (%) charged hGH amount (ng) ⁇ total hGH amount in supernatant (ng) ⁇ 100 Charged hGH amount (ng).
- the inclusion rate of hGH in dextran-PLA microparticles or dextran-PLGA microparticles is 92.6% for dextran-PLA microparticles and 85.7% for dextran-PLGA microparticles. It was shown that the drug can be included.
- Example 5 Analysis of In Vitro Drug Release Rate from Microparticles Encapsulating Human Growth Hormone (hGH)
- the microparticles washed three times in Example 4 were suspended in 1.2 ml of Buffer A. A part (40 ⁇ l) of this solution was transferred to another tube, particles were precipitated by centrifugation at 18,000 ⁇ g for 10 minutes, and 30 ⁇ l of the supernatant was collected in another tube (0 hour sample). The remaining particle suspension was slowly mixed by inversion in a 1.5 ml Eppendorf tube at a speed of 6 rpm using a rotator in a 37 ° C. incubator. A small amount (40 ⁇ l) was collected from this solution over time, and the supernatant was separated by centrifugation as described above. The concentration of hGH in the supernatant sample collected at each time point was measured with an ELISA kit, and the released amount (%) was calculated by the following formula.
- Released amount (%) hGH concentration in supernatant (ng / ml) ⁇ 1.2 (ml) ⁇ 100 Encapsulated hGH amount in 20 mg particles (ng).
- FIG. 1 shows the time course of drug release from microparticles prepared using dextran-PLA or dextran-PLGA polymer. None of the particles had an initial burst and showed a good profile with the drug released linearly over time. The time required to release 50% of the drug is about 1 month for dextran-PLA microparticles and about 1 week for dextran-PLGA microparticles, and the release rate can be selected by selecting the type of poly (hydroxy acid) Was shown to be adjustable.
- Example 6 Preparation Method of Microparticles Encapsulating Human Insulin Dextran-PLA (Dextran Average Molecular Weight 13,000, PLA Average Molecular Weight 2,300, PLA Graft Chain Number 10-12, Compound (3)) or Dextran-PLGA (Dextran Average Molecular Weight) 13,000, PLGA average molecular weight 1,900, PLGA graft chain number 7 to 10, 5 mg of compound (7)) were dissolved in 100 ⁇ l of dimethyl carbonate to prepare a polymer solution of 50 mg / ml.
- Dextran Average Molecular Weight 13,000, PLA Average Molecular Weight 2,300, PLA Graft Chain Number 10-12, Compound (3) or Dextran-PLGA (Dextran Average Molecular Weight) 13,000, PLGA average molecular weight 1,900, PLGA graft chain number 7 to 10, 5 mg of compound (7)
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000). Human insulin-encapsulated particle powder was obtained.
- the average particle size of the microparticles obtained from the compound (3) was 6.4 ⁇ m
- the compound ( The average particle size of the microparticles obtained from 7) was 5.3 ⁇ m.
- Example 7 Measurement of drug encapsulation rate of human insulin-encapsulated microparticles 20 mg of human insulin-encapsulated microparticles prepared by the method of Example 6 using dextran-PLGA (compound (7)) polymer was weighed into a 1.5 ml Eppendorf tube. 18,800 ⁇ g dissolved in 1 ml of buffer A (0.1% bovine serum albumin, 0.1% Pluronic F-68 (BASF®) and 0.02% sodium azide in PBS) Particles (precipitate) and supernatant were separated by centrifugation for 10 minutes. After the supernatant was collected in another tube, the particles were suspended in 1 ml of buffer solution, and the particles and the supernatant were separated again by centrifugation under the above conditions.
- buffer A 0.1% bovine serum albumin, 0.1% Pluronic F-68 (BASF®) and 0.02% sodium azide in PBS
- Encapsulation rate (%) Amount of prepared insulin (ng) ⁇ Total amount of insulin in supernatant (ng) ⁇ 100 The amount of insulin charged (ng).
- the inclusion rate of human insulin in dextran-PLGA microparticles was 75.7%, indicating that it can be encapsulated with high efficiency.
- Example 8 Analysis of in vitro drug release rate from human insulin-encapsulated microparticles
- the particles washed three times in Example 7 were suspended in 1.2 ml of buffer A. A part (40 ⁇ l) of this solution was transferred to another tube, particles were precipitated by centrifugation at 18,800 ⁇ g for 10 minutes, and 30 ⁇ l of the supernatant was collected in another tube (0 hour sample). The collected sample was stored frozen until measurement. The remaining particle suspension was slowly mixed by inversion in a 1.5 ml Eppendorf tube at a speed of 6 rpm using a rotator in a 37 ° C. incubator. A small amount (40 ⁇ l) was collected from this solution over time, and the supernatant was separated by centrifugation as described above. The concentration of human insulin in the supernatant sample collected at each time point was measured by sandwich ELISA, and the released amount (%) was calculated by the following formula.
- Amount released (%) human insulin concentration in supernatant (ng / ml) ⁇ 1.2 (ml) ⁇ 100 Encapsulated human insulin amount in 20 mg particles (ng).
- the time course of human insulin release is shown in FIG. There was little initial burst and a good profile with drug released linearly over time. The time required to release 50% of the drug was about 6 days.
- Example 9 Change over time of microparticle morphology 5 mg of hGH-encapsulated microparticle powder prepared in Example 3 was weighed into an Eppendorf tube, dispersed in 1 ml of Milli-Q, centrifuged at 13,000 rpm for 30 minutes, and the supernatant was collected. After removal, the microparticles were washed again by dispersing in 1 ml of Milli-Q and centrifuging. In addition, 1 ml of Milli-Q is added to the microparticle suspension solution incubated for a predetermined time and centrifuged at 13,000 rpm for 30 minutes. After removing the supernatant, it is dispersed again in 1 ml of Milli-Q and centrifuged in the same manner.
- microparticles were washed with The obtained microparticles after washing were dispersed in 100 ⁇ l of Milli-Q, 5 ⁇ l of the microparticle dispersion solution was dropped on a silicon substrate, allowed to stand at room temperature for about 10 minutes, and then dried in a desiccator for 3 hours. After that, using ion sputtering (HITACHI, E-1030), platinum was deposited on the sample surface (deposition time 15 seconds), and the acceleration voltage was 1 kV and the probe current with a scanning electron microscope (SEM: HITACHI, S-4800). Was set to high, and the microparticle shape and surface state were observed.
- HITACHI ion sputtering
- the spherical particles with a smooth surface immediately after fabrication were remarkably deformed after incubation at 37 ° C. for 13 days, resulting in an image with many holes, corresponding to the release of the drug. It was shown that particle decomposition was progressing.
- Comparative Example 1 5 mg of polyethylene glycol-poly (epsilon-caprolactone) (average molecular weight of polyethylene glycol 5,000, average molecular weight of poly (epsilon-caprolactone) 37,000) was dissolved in 100 ⁇ l of dimethyl carbonate to prepare a polymer solution of 50 mg / ml. . After adding 20 ⁇ l of tert-butanol to the polymer solution, 20 ⁇ l of 2 mg / ml aqueous hGH solution was added dropwise, and vortexed to produce a reverse phase emulsion.
- polyethylene glycol-poly (epsilon-caprolactone) average molecular weight of polyethylene glycol 5,000, average molecular weight of poly (epsilon-caprolactone) 37,000
- the inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- the obtained solid content was dispersed in 200 ⁇ l of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-encapsulated particle powder was obtained.
- the average particle size was calculated by observing the microparticles with a scanning electron microscope (SEM: HITACHI, S-4800), the average particle size of the obtained microparticles was 8.0 ⁇ m.
- microparticles were washed with The obtained microparticles after washing were dispersed in 100 ⁇ l of Milli-Q, 5 ⁇ l of the microparticle dispersion solution was dropped on a silicon substrate, allowed to stand at room temperature for about 10 minutes, and then dried in a desiccator for 3 hours. After that, using ion sputtering (HITACHI, E-1030), platinum was deposited on the sample surface (deposition time 15 seconds), and the acceleration voltage was set to 1 kV and the probe current was set to high by SEM (HITACHI, S-4800). Then, the microparticle shape and surface state were observed.
- HITACHI ion sputtering
- Example 10 Subcutaneous administration of human growth hormone (hGH) -encapsulated microparticles in mice Dextran-polylactic acid (PLA) (dextran average molecular weight 13,000, PLA average molecular weight 2,300, PLA graft chain number 10 to 12, compound (3) ) Or dextran-poly (lactic acid-glycolic acid) (PLGA) (dextran average molecular weight 13000, PLGA average molecular weight 1,900, PLGA graft chain number 7-10, compound (7)) 25 mg is dissolved in 500 ⁇ l of dimethyl carbonate, and 50 mg A / ml polymer solution was prepared.
- PHA Dextran-polylactic acid
- PLGA dextran-poly (lactic acid-glycolic acid)
- PLGA dextran-poly (lactic acid-glycolic acid)
- 25 mg is dissolved in 500 ⁇ l of dimethyl carbonate, and 50 mg A / ml polymer solution was prepared.
- the S / O suspension was dropped into 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000). An hGH-containing microparticle powder was obtained.
- the average particle diameter of the obtained microparticles was 4.
- the microparticles of 9 ⁇ m and the compound (7) were 4.2 ⁇ m.
- the 300 mg of the microparticles prepared above were suspended in 3 ml of phosphate physiological buffer (PBS), and the microparticles were precipitated by centrifugation at 80 ⁇ g for 5 minutes, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining particles, and the supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove growth hormone not encapsulated in the microparticles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- PBS phosphate physiological buffer
- the amount of growth hormone contained in each of the dextran-PLA microparticles and dextran-PLGA microparticles was calculated by measuring the concentration in the washing solution with an ELISA kit and subtracting it from the charged amount.
- the amount contained in the 300 mg particles was 590 ⁇ g for dextran-PLA microparticles and 536 ⁇ g for dextran-PLGA microparticles.
- This solution was injected and administered subcutaneously at two sites on the back of 10-week-old male Balb / C mice, and blood was collected from the tail vein over time.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml and centrifuged at 5,000 rpm for 5 minutes to collect plasma, and the plasma growth hormone concentration was measured using an ELISA kit.
- mice were subcutaneously administered with a non-particulate human growth hormone protein solution (700 ⁇ g / 0.2 ml).
- the immunosuppressive agent tacrolimus hydrate (Astellas) 26 ⁇ g / mouse was subcutaneously administered 3 days before the particle administration, and then drug administration. At the same time, 13 ⁇ g / mouse was subcutaneously administered after 3 days and 7 days.
- FIG. 5 shows the time course of plasma human growth hormone concentration.
- the blood concentration is very high at 5,000 ng / ml or more after 1 hour of administration, and then rapidly decreases to the level before administration after 1 day. did.
- the transient increase in blood concentration immediately after administration was suppressed to 200 ng / ml or less, and the blood concentration persisted until 7 days later. High value.
- dextran-PLGA microparticles there was no transient increase in concentration after administration, and very good sustained release performance was maintained that maintained a substantially constant blood concentration until 7 days later.
- Example 11 Subcutaneous administration of human growth hormone (hGH) -containing microparticles in mice (pharmacological activity evaluation) 25 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (dextran average molecular weight 13,000, PLGA average molecular weight 1,900, PLGA graft chain number 7 to 10, compound (7)) of Example 2 in 500 ⁇ l of dimethyl carbonate. Dissolved to prepare a 50 mg / ml polymer solution. After adding 100 ⁇ l of tert-butanol to the polymer solution, 250 ⁇ l of 10 mg / ml hGH aqueous solution was dropped, and stirred by vortex to prepare a reverse phase emulsion.
- hGH human growth hormone
- the inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- the obtained solid content was dispersed in 1 ml of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- the average particle size was calculated by observing the microparticles with a scanning electron microscope (SEM: HITACHI, S-4800), the average particle size of the obtained microparticles was 4.1 ⁇ m.
- 300 mg of the microparticles prepared above were suspended and dispersed in 3 ml of phosphate physiological buffer (PBS), and the particles were precipitated by centrifugation at 80 ⁇ g for 5 minutes, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining particles, and the supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove the growth hormone not encapsulated in the particles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- PBS phosphate physiological buffer
- This solution was injected and administered subcutaneously to the back of an 8-week-old hypophysectomized ICR mouse (Japan SLC), and blood was collected from the tail vein over time.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml and centrifuged at 5,000 rpm for 5 minutes to collect plasma, and the plasma growth hormone concentration and mouse IGF-1 concentration were measured using an ELISA method.
- mice were subcutaneously administered with a non-particulate human growth hormone protein solution (700 ⁇ g / 0.2 ml).
- the immunosuppressive agent tacrolimus hydrate (Astellas) 26 ⁇ g / mouse was subcutaneously administered 3 days before the particle administration, and then drug administration. At the same time, 13 ⁇ g / mouse was subcutaneously administered after 3 days and 7 days.
- FIG. 7 shows a change in the weight of the mouse at this time.
- the increase in body weight was suppressed to about 5%, whereas in mice administered with dextran-PLGA microparticles, an increase in body weight of about 20% was confirmed.
- Fig. 8 shows the IGF-1 concentration in plasma.
- the plasma IGF-1 concentration was correlated with the human growth hormone concentration in the blood, and it was confirmed that the mice administered with dextran-PLGA microparticles maintained a high value until 10 days after administration.
- Example 12 Analysis of Release Rate of Drug in Buffer from Exendin 4 (GLP-1 Receptor Agonist)
- Dextran-poly lactic acid-glycolic acid
- PLGA lactic acid-glycolic acid
- PLGA graft chain number 21 PLGA graft chain number 21
- 15 compound (9)
- the S / O suspension was dropped into 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000). Exendin 4 encapsulated microparticle powder was obtained.
- the average particle size of the obtained microparticles was 4.3 ⁇ m for the microparticles of the compound (8).
- the microparticles of compound (9) were 4.5 ⁇ m.
- microparticles were washed three times according to the method of Example 4, and then suspended in 1.2 ml of buffer A. A part (40 ⁇ l) of this solution was transferred to another tube, microparticles were precipitated by centrifugation at 18000 ⁇ g for 10 minutes, and 30 ⁇ l of the supernatant was collected in another tube (0 hour sample). The remaining microparticle suspension was gently inverted by inversion in a 1.5 ml Eppendorf tube at a speed of 6 rpm using a rotator in a 37 ° C. incubator. A small amount (40 ⁇ l) was collected from this solution over time, and the supernatant was separated by centrifugation as described above. The concentration of exendin 4 in the supernatant sample collected at each time point was measured by ELISA method, and the released amount (%) was calculated by the following formula.
- Released amount (%) Exendin 4 concentration in supernatant (ng / ml) ⁇ 1.2 (ml) ⁇ 100 Encapsulated exendin 4 amount (ng) in 20 mg particles.
- Example 13 Subcutaneous administration of exendin 4 (GLP-1 receptor agonist) -encapsulating microparticles in mice 300 mg of the microparticles of Example 12 were suspended in 3 ml of phosphate buffered saline (PBS), 80 ⁇ g, 5 minutes The microparticles were precipitated by centrifugation, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining microparticles, and the supernatant was removed.
- PBS phosphate buffered saline
- the first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove exendin 4 not encapsulated in the microparticles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- This solution was injected subcutaneously into the back of an 8-week-old SCID mouse (CB17 / lcr-Prkdcscid / CrlCrlk) (Claire Japan), and blood was collected over time from the tail vein.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml, centrifuged at 5000 rpm for 5 minutes to collect plasma, and the concentration of exendin 4 in plasma was measured using an ELISA method.
- blood was also collected in the same manner for mice administered subcutaneously with the exendin 4 solution (700 ⁇ g / 0.2 ml) that was not microparticulated.
- the time course of plasma exendin 4 concentration is shown in FIG.
- mice administered with non-microparticulated drug the blood concentration showed a very high value 1 hour after administration, and then rapidly decreased to the level before administration.
- dextran-PLGA microparticles a transient increase in concentration after administration was suppressed to a low level, and a high plasma concentration was maintained until 5 weeks.
- Example 14 Synthesis of Dextran-Poly (lactic acid-glycolic acid) (PLGA) 14-1. Synthesis of TMS-dextran-PLGA (compound (10), compound (11), compound (12), compound (13)) Compound (1) (0.5 g) and tert-butoxypotassium (35 mg) under reduced pressure for 1 hour After drying, tetrahydrofuran (10 ml) was added and stirred at room temperature for 1 hour. To this solution, a solution of (DL) -lactide (0.558 g) and glycolide (0.45 g) in tetrahydrofuran (15 ml) was added dropwise and stirred for 5 minutes. After completion of the reaction, the solvent was concentrated under reduced pressure, and TMS-dextran-PLGA (1.96 g) was obtained as a white solid by reprecipitation purification in a chloroform-methanol system (compound (10)).
- Dextran-PLGA was also obtained from compounds (11), (12) and (13) in a similar manner (compound (15), compound (16) and compound (17)).
- the weight average molecular weight and number average molecular weight of the polymers of compounds (14) to (17) were determined by GPC measurement (column Toso-TSK-gel ⁇ -5000 ⁇ 2, DMF solvent, detector RI, standard pullulan). .
- the average molecular weight of the graft chain and the number of graft chains were determined by 1 H-NMR measurement.
- Compound (14) had a weight average molecular weight of 99,462, a number average molecular weight of 85,101, a graft chain number average molecular weight of 2,167, and a graft chain number of 33.
- Compound (15) had a weight average molecular weight of 107,779, a number average molecular weight of 92,134, a graft chain number average molecular weight of 3,127, and a graft chain number of 25.
- Compound (16) had a weight average molecular weight of 121,281, a number average molecular weight of 101,873, a graft chain number average molecular weight of 3,000, and a graft chain number of 30.
- Compound (17) had a weight average molecular weight of 144,838, a number average molecular weight of 122,151, a graft chain number average molecular weight of 4,864, and a graft chain number of 22.
- Example 15 Preparation Method of Human Growth Hormone (hGH) Encapsulated Microparticles 5 mg of each dextran-poly (lactic acid-glycolic acid) (dextran-PLGA polymer, compounds (14) to (17)) of Example 14 was added to 100 ⁇ l of dimethyl carbonate. Dissolved to prepare a 50 mg / ml polymer solution. After adding 20 ⁇ l of tert-butanol to the polymer solution, 50 ⁇ l of 1 mg / ml hGH aqueous solution was added dropwise and vortexed to produce a reverse phase emulsion.
- hGH Human Growth Hormone
- the inverse emulsion was pre-frozen in liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- the obtained solid content was dispersed in 200 ⁇ l of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF) and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion was pre-frozen with liquid nitrogen, and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- SEM scanning electron microscope
- Example 16 Measurement of Drug Encapsulation Rate of Human Growth Hormone (hGH) -Encapsulated Microparticles 20 mg of growth hormone-encapsulated microparticles prepared by the method of Example 15 using each dextran-PLGA polymer (compounds (14) to (17)) was weighed into a 1.5 ml eppendorf tube and 1 ml of buffer A (PBS containing 0.1% bovine serum albumin, 0.1% Pluronic F-68 (BASF®) and 0.02% sodium azide) 18,000 ⁇ g and centrifugation for 10 minutes to separate particles (precipitate) and supernatant.
- buffer A PBS containing 0.1% bovine serum albumin, 0.1% Pluronic F-68 (BASF®) and 0.02% sodium azide
- the particles were resuspended in 1 ml of buffer solution, and the particles were separated from the supernatant by centrifugation under the above conditions. This washing operation was repeated once more (centrifugation three times in total), and the concentration of human growth hormone in the supernatant collected by each centrifugation was measured with an ELISA kit (manufactured by R & D Systems). By subtracting the total amount of hGH in the centrifugal supernatant three times from the amount of hGH charged at the time of particle preparation (per 20 mg of particle weight), the encapsulation rate was calculated by the following formula.
- Encapsulation rate (%) charged hGH amount (ng) ⁇ total hGH amount in supernatant (ng) ⁇ 100 Charged hGH amount (ng).
- the encapsulation rate of hGH in each dextran-PLGA microparticle was 87.5% for the microparticle of compound (14), 94.2% for the microparticle of compound (15), and 95.7 for the microparticle of compound (16). %, 97.5% of the microparticles of the compound (17), and it was shown that any microparticle can encapsulate the protein drug with high efficiency.
- Example 16 After the sample was lyophilized, 1 mL of water was added to a 50 mg sample to redisperse the particles, yielding non-associated hydrophilic active substance-containing particles. The average particle size of the particles was measured by a dynamic light scattering method using an apparatus ELS-Z (Otsuka Electronics Co., Ltd.), and the drug encapsulation rate was determined in the same manner as in Example 16.
- the average particle diameter of the compound (14) particles was 190.5 nm
- the encapsulation rate was 73%
- the particles of the compound (17) were 197.0 nm
- the encapsulation rate was 70%.
- the encapsulation rate was inferior to that of the microparticles.
- Example 17 Analysis of in vitro drug release rate from human growth hormone (hGH) -containing microparticles
- the particles washed three times in Example 16 were suspended in 1.2 ml of buffer A. A part (40 ⁇ 1) of this solution was transferred to another tube, particles were precipitated by centrifugation at 18000 ⁇ g for 10 minutes, and 30 ⁇ l of the supernatant was collected in another tube (0 hour sample). The remaining particle suspension was slowly mixed by inversion in a 1.5 ml Eppendorf tube at a speed of 6 rpm using a rotator in a 37 ° C. incubator. A small amount (40 ⁇ l) was collected from this solution over time, and the supernatant was separated by centrifugation as described above. The concentration of hGH in the supernatant sample collected at each time point was measured with an ELISA kit, and the released amount (%) was calculated by the following formula.
- Released amount (%) hGH concentration in supernatant (ng / ml) ⁇ 1.2 (ml) ⁇ 100 Encapsulated hGH amount in 20 mg particles (ng).
- the time course of drug release from the microparticles produced in Example 15 is shown in FIG. None of the particles had an initial burst and showed a good profile with the drug released linearly over time.
- the time required for the release of 50% of the drug is about 6 days for microparticles of compound (14), about 9 days for microparticles of compound (15), about 16 days for microparticles of compound (16),
- the microparticles of compound (17) were about one month, and it was shown that the drug release rate can be adjusted by changing the amount of lactide and glycolide charged during TMS-dextran-PLGA synthesis.
- Example 18 Preparation of Fluorescein-Labeled Dextran (FD40) Encapsulated Microparticles with Different Particle Sizes 5 mg of dextran-poly (lactic acid-glycolic acid) (PLGA) (compound (7)) of Example 2 was dissolved in 100 ⁇ l of dimethyl carbonate, and 50 mg A / ml polymer solution was prepared. After adding 20 ⁇ l of tert-butanol to the polymer solution, 20 ⁇ l of a 1 mg / ml FD40 aqueous solution was added dropwise, and the mixture was vortexed to produce a reverse phase emulsion.
- PLGA dextran-poly (lactic acid-glycolic acid)
- the inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- the obtained solid content was dispersed in 50 ⁇ l, 100 ⁇ l, 200 ⁇ l, 350 ⁇ l, 500 ⁇ l, 1 ml, 2 ml and 6 ml of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- the average particle size was calculated by observing the obtained microparticles with a scanning electron microscope (SEM: HITACHI, S-4800).
- FIG. 12 shows the correlation between the average particle diameter and the amount of dimethyl carbonate added when preparing the S / O / W emulsion. Between 50 ⁇ l and 500 ⁇ l, a decrease in average particle size was observed with increasing amount of dimethyl carbonate. Little difference in average particle size was observed between 500 ⁇ l and 6 ml.
- PEG-PLGA polymer PEG2k series
- (DL) -lactide and glycolide were mixed in the amounts shown in Table 1 below, and 140 ° C. Heated. After stirring for 20 minutes, tin
- II tin octylate
- II tin octylate
- Example 22 Preparation Method of FD40-Encapsulated Microparticles 5 mg of the PEG-PLGA polymer prepared in Examples 19 to 21 was dissolved in 100 ⁇ l of dimethyl carbonate to prepare a 50 mg / ml polymer solution. After adding 20 ⁇ l of tert-butanol to the polymer solution, a predetermined amount of 10 mg / ml FD40 aqueous solution shown in Table 4 below was added and stirred to prepare a reverse emulsion solution. The inverse emulsion solution was pre-frozen with liquid nitrogen, and then freeze-dried for 24 hours using a freeze dryer (EYELA, FREEZE DRYER FD-1000) at a trap cooling temperature of ⁇ 45 ° C.
- EYELA FREEZE DRYER FD-1000
- the obtained solid content was dispersed in 200 ⁇ l of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- the microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C.
- FIG. 14 shows an SEM image of a powder prepared from a 5k-10k PEG-PLGA polymer
- FIG. 15 shows an SEM image of a powder prepared from a 5k-61k PEG-PLGA polymer.
- Example 23 Measurement of encapsulation rate of FD40-encapsulated microparticles
- FD40-encapsulated microparticles (5 mg) prepared by the method of Example 22 using PEG-PLGA polymer were weighed into a 1.5 ml Eppendorf tube and Milli-Q (1 ml). After the dispersion, the supernatant containing unencapsulated FD40 and the FD40-encapsulated particles were separated and collected by centrifugation for 30 minutes. The recovered FD40-encapsulated microparticles were disintegrated by dissolving them in N, N-dimethylformamide (250 ⁇ l).
- the supernatant containing unencapsulated FD40 and the N, N-dimethylformamide solution (50 ⁇ l) containing encapsulated FD40 were respectively added to Milli-Q (3 ml), and after stirring well, a fluorescence spectrophotometer (HORIBA, Fluoro MAX-3 FD40 was quantified using an excitation wavelength of 495 nm and a fluorescence wavelength of 520 nm, and the inclusion rate relative to the total recovery amount was calculated.
- HORIBA Fluoro MAX-3 FD40
- FIG. 13 shows the encapsulation rate of FD40 in microparticles made from PEG-PLGA polymer.
- the encapsulation rate tends to increase when the molecular weight of PLGA is high.
- the encapsulation rate was about 90% at 5k-65k.
- the encapsulation rate was about 55 %Met.
- the inclusion rate of FD40 was 48% and the average particle size was 203.8 nm, which was inferior to the microparticles of Example 23.
- Example 24 Analysis of in vitro FD40 release rate from FD40-encapsulated microparticles
- the FD40-encapsulated microparticles prepared in Example 22 were analyzed. Among them, the release behavior of 5k-23k, 5k-32.5k, 5k-47k, and 5k-61k particles was evaluated.
- microparticles were stored at ⁇ 30 ° C. in a lyophilized state and returned to room temperature before use. After weighing 20 mg of particle powder and putting it in a 1.5 ml tube (Eppendorf), assay buffer (0.02% sodium azide, 0.1% Pluronic F-68 (registered trademark of BASF), 0.1% bovine serum 1 ml of an albumin-added PBS solution) was added, and the mixture was suspended by vigorous stirring with a touch mixer.
- assay buffer 0.02% sodium azide, 0.1% Pluronic F-68 (registered trademark of BASF), 0.1% bovine serum 1 ml of an albumin-added PBS solution
- the mixture was centrifuged at 18,900 ⁇ g for 10 minutes using a Hitachi high-speed centrifuge (CF16RX) to remove 950 ⁇ l of the supernatant fraction containing unencapsulated FD40, and then 950 ⁇ l of assay buffer was added again to suspend the suspension. The particle washing with post-centrifugation was repeated for a total of 3 washes.
- C16RX Hitachi high-speed centrifuge
- 950 ⁇ l of assay buffer was added again to the particles that had been washed three times, and the particles were suspended, and then 100 ⁇ l each was dispensed into a 1.5 ml tube.
- 900 ⁇ l of assay buffer was added to make a total of 1 ml, and incubated in a 37 ° C. incubator using a rotator while rotating at 10 rpm.
- Each of the incubated tubes was centrifuged over time at 18900 ⁇ g for 10 minutes, and 950 ⁇ l of the supernatant was collected and stored at 4 ° C. until measurement of the fluorescence intensity.
- the fluorescence intensity of the sampled solution is measured using a 3 ml disposal cuvette (KARTELL) and HORIBA Fluoro MAX-3 at an excitation wavelength of 494 nm and a fluorescence wavelength of 512 nm. It was determined.
- FIG. 16 shows the amount of FD40 released from various microparticles determined by the release evaluation.
- the horizontal axis represents the incubation time, and the vertical axis represents the release ratio relative to the charged amount.
- the vertical axis represents the release ratio relative to the charged amount.
- the initial release amount was reduced, and the initial one-day release amount was 10% or less for the 5k-61k microparticles.
- Example 25 Measurement of drug encapsulation rate of human insulin-encapsulated microparticles Using the PEG-PLGA polymer (5k-61k) produced in Example 20, human insulin-encapsulated microparticles were produced in the same manner as in Example 22. The resulting microparticles (20 mg) were weighed into a 1.5 ml Eppendorf tube and 1 ml of buffer A (0.1% bovine serum albumin, 0.1% Pluronic F-68 (registered trademark of BASF) and 0.02 The solution was dissolved in PBS containing 1% sodium azide, and the particles (precipitate) and the supernatant were separated by centrifugation at 18,800 ⁇ g for 10 minutes.
- buffer A 0.1% bovine serum albumin, 0.1% Pluronic F-68 (registered trademark of BASF) and 0.02
- the solution was dissolved in PBS containing 1% sodium azide, and the particles (precipitate) and the supernatant were separated by centrifugation at 18,800 ⁇ g for 10 minutes.
- the particles were resuspended in 1 ml of buffer A, and the particles and the supernatant were separated again by centrifugation under the above conditions. This washing operation was repeated once more (3 times in total), and the insulin concentration in the supernatant collected by each centrifugation was measured by sandwich ELISA.
- the sandwich ELISA method was performed by the following method.
- An ELISA buffer (0.25% BSA and 0.05% Tween 20) was immobilized on an ELISA plate (Nunc Maxisorp) on which an anti-human insulin monoclonal antibody (clone No. E6E5 manufactured by Fitzgerald) was immobilized at a concentration of 5 ⁇ g / ml.
- the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20) to remove unreacted reagents, and then biotin-labeled anti-human insulin monoclonal antibody (Clone No. D4B8 manufactured by Fitzgerald) 0.5 ⁇ g / ml , And streptavidin-HRP conjugate (Zymed) were sequentially shaken at room temperature for 1 hour and 15 minutes. After each reaction, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20) to remove unreacted reagents. Finally, the HRP enzyme activity of the conjugate that was bound by adding the substrate of HRP was colorimetrically determined, and the insulin concentration in the sample was read based on the calibration curve prepared from the color development of standard insulin.
- a washing solution PBS containing 0.05% Tween 20
- the encapsulation rate was calculated by the following formula by subtracting the total amount of insulin in the centrifugal supernatant three times from the amount of insulin charged at the time of particle production (per 20 mg of particle weight).
- Inclusion rate (%) Amount of prepared insulin (ng) ⁇ Total amount of insulin in supernatant (ng) ⁇ 100 The amount of insulin charged (ng).
- the average particle size of the obtained microparticles was 4.7 ⁇ m. Moreover, the encapsulation rate of human insulin in the microparticles was 86.7%, indicating that the protein drug can be encapsulated with high efficiency.
- Example 26 Analysis of in vitro drug release rate from human insulin-containing microparticles
- the microparticles washed three times in Example 25 were suspended in 1.0 ml of buffer A. Each 0.1 ml of this solution was dispensed into 10 Eppendorf tubes (1.5 ml capacity), and each solution was diluted 10-fold by adding 0.9 ml of Buffer A. Immediately after dilution, one tube was centrifuged at 18800 ⁇ g for 10 minutes to precipitate the particles, and the supernatant was collected in another tube (0 hour sample). The remaining nine tubes were gently mixed by inversion at a speed of 6 rpm using a rotator in a 37 ° C. incubator. Using a single tube over time, the supernatant was separated by centrifugation as described above. The insulin concentration in the supernatant sample collected at each time point was measured by sandwich ELISA, and the amount of insulin released (%) was calculated by the following formula.
- Insulin release amount (%) insulin concentration in supernatant (ng / ml) ⁇ 1 (ml) ⁇ 10 ⁇ 100 Encapsulated insulin amount in 20 mg particles (ng).
- FIG. 17 shows the time course of insulin release. The drug was gradually released over time, and the release rate increased after 30 days, and most of the drug was released in about 60 days.
- Example 27 Subcutaneous administration of human growth hormone (hGH) -encapsulated microparticles in mice 25 mg of PEG-PLGA polymer (5k-61k) was dissolved in 500 ⁇ l of dimethyl carbonate to prepare a 50 mg / ml polymer solution. After adding 100 ⁇ l of tert-butanol to the polymer solution, 250 ⁇ l of 10 mg / ml hGH aqueous solution was dropped, and stirred by vortex to prepare a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C.
- hGH human growth hormone
- microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- the average particle diameter of the obtained microparticles was 6.0 ⁇ m.
- 300 mg of the microparticles prepared above were suspended and dispersed in 3 ml of phosphate physiological buffer (PBS), and the particles were precipitated by centrifugation at 80 ⁇ g for 5 minutes, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining particles, and the supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove the growth hormone not encapsulated in the particles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- PBS phosphate physiological buffer
- the amount of growth hormone encapsulated in the PEG-PLGA particles was calculated by measuring the concentration in the washing solution with an ELISA kit and subtracting it from the charged amount. As a result, it was encapsulated in 300 mg of particles administered per mouse. The amount of PEG-PLGA microparticles was 700 ⁇ g.
- This solution was injected and administered into two subcutaneous sites on the back of 10-week-old male Balb / C mice, and blood was collected from the tail vein over time.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml, centrifuged at 5,000 rpm for 5 minutes to collect plasma, and the plasma growth hormone concentration was measured using an ELISA kit.
- mice were subcutaneously administered with a non-particulate human growth hormone protein solution (700 ⁇ g / 0.2 ml).
- the immunosuppressive agent tacrolimus hydrate (Astellas) 26 ⁇ g / mouse was subcutaneously administered 3 days before the particle administration, and then drug administration. At the same time, 13 ⁇ g / mouse was subcutaneously administered after 3 days and 7 days.
- FIG. 18 shows the time course of plasma human growth hormone concentration.
- the blood concentration shows a very high value of 5,000 ng / ml or more 1 hour after administration, and then rapidly decreases and then decreases to the level before administration after 1 day. did.
- the transient increase in blood concentration immediately after administration was suppressed to 100 ng / ml or less, and the blood concentration continued until 7 days later. High value.
- Example 28 Production of microparticles in which salt was added to the liquid phase in step (c) 20 ⁇ l of tert-butanol was added to 100 ⁇ l of a 50 mg / ml PEG-PLGA polymer (5k-61k) / dimethyl carbonate solution and 10 mg / ml A reverse micelle (W / O emulsion) solution was prepared by adding 20 ⁇ l of FD40 aqueous solution and stirring. The resulting solution was pre-frozen with liquid nitrogen, and then freeze-dried overnight with a freeze dryer to obtain an FD40-containing solid (Solid).
- a reverse micelle (W / O emulsion) solution was prepared by adding 20 ⁇ l of FD40 aqueous solution and stirring. The resulting solution was pre-frozen with liquid nitrogen, and then freeze-dried overnight with a freeze dryer to obtain an FD40-containing solid (Solid).
- the washed microparticles were resuspended in 1 ml of PBS buffer, dispensed 100 ⁇ l each into a 1.5 ml Eppendorf tube, 900 ⁇ l of PBS buffer was added, incubated at 37 ° C., and the sample was collected one day later.
- the collected sample is centrifuged at 14,000 rpm for 10 minutes, and fluorescence measurement of FD40 contained in the supernatant is performed using a fluorescence spectrophotometer (HORIBA, Fluoro MAX-3, excitation wavelength 495 nm, fluorescence wavelength 520 nm). The amount released was calculated.
- the amount of FD40 in the supernatant collected at the time of washing was measured in the same manner, and the encapsulation rate was calculated from the charged amount.
- the encapsulation rate was 73%, 97%, 84%, and 82% under conditions of sodium chloride concentrations of 0 M, 10 mM, 50 mM, and 1 M, respectively. Moreover, the release amount after one day is 14%, 7%, 15%, and 11% under the respective conditions of sodium chloride concentration 0M, 10mM, 50mM, and 1M, and the inclusion rate is highest when 10mM sodium chloride is added. One day later, the release amount (initial burst) was small.
- Example 29 Subcutaneous administration of human growth hormone (hGH) -containing microparticles in mice (pharmacological activity evaluation) 25 mg each of the PEG-PLGA polymer (5k-55k) and PEG-PLGA polymer (5k-105k) of Example 20 were dissolved in 500 ⁇ l of dimethyl carbonate to prepare a polymer solution of 50 mg / ml. After adding 100 ⁇ l of tert-butanol to the polymer solution, 250 ⁇ l of 10 mg / ml hGH aqueous solution was dropped, and stirred by vortex to prepare a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C.
- hGH human growth hormone
- microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- An hGH-containing microparticle powder was obtained.
- the average particle size was calculated by observing the obtained microparticles with a scanning electron microscope (SEM: HITACHI, S-4800), the average particle size of the microparticles was the PEG-PLGA polymer (5k-55k).
- the microparticles (5k-55k microparticles) were 4.2 ⁇ m
- the PEG-PLGA polymer (5k-105k) microparticles (5k-105k microparticles) were 7.5 ⁇ m.
- each 300 mg of the microparticles prepared above was suspended and dispersed in 3 ml of a phosphate physiological buffer solution (PBS), the particles were precipitated by centrifugation at 80 ⁇ g for 5 minutes, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining particles, and the supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove the growth hormone not encapsulated in the particles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- PBS phosphate physiological buffer solution
- This solution was injected and administered subcutaneously to the back of an 8 week-old hypophysectomized ICR mouse (Japan SLC), and blood was collected from the tail vein over time.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml, centrifuged at 5,000 rpm for 5 minutes to collect plasma, and the plasma growth hormone concentration and plasma IGF-1 concentration were measured using an ELISA method. .
- mice were subcutaneously administered with a non-particulate human growth hormone protein solution (700 ⁇ g / 0.2 ml).
- the immunosuppressive agent tacrolimus hydrate (Astellas) 26 ⁇ g / mouse was subcutaneously administered 3 days before the particle administration, and then drug administration. After that time, 13 ⁇ g / mouse was administered subcutaneously at twice weekly intervals.
- FIG. 19 shows the time course of plasma human growth hormone concentration. Mice administered with non-microparticulate drugs showed very high values 1 hour after administration, then decreased rapidly and decreased to pre-dose levels after 1 day. On the other hand, in mice administered with microparticulate drugs prepared using PEG-PLGA polymer, the transient increase in blood concentration immediately after administration is about 1 / 100th that of mice administered with non-microparticulate drugs. The blood concentration was continuously high for 9 days or more after administration.
- the plasma IGF-1 concentration at this time is shown in FIG.
- the plasma IGF-1 concentration rose after administration for both 5k-55k microparticles and 5k-105k microparticles, and increased to 7 days for 5k-55k microparticles and 14 days or more for 5k-105k microparticles. Maintained.
- Example 30 Subcutaneous administration of exendin 4 (GLP-1 receptor agonist) -encapsulating microparticles in mice 25 mg of the PEG-PLGA polymer (5k-61k) of Example 20 was dissolved in 500 ⁇ l of dimethyl carbonate to prepare a polymer solution of 50 mg / ml. did. After adding 100 ⁇ l of tert-butanol to the polymer solution, 250 ⁇ l of 10 mg / ml exendin 4 (synthesized by Sigma Genosys) was added dropwise and stirred by vortex to produce a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C.
- exendin 4 GLP-1 receptor agonist
- microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- Exendin 4 encapsulated microparticle powder was obtained.
- the average particle size was calculated by observing the obtained microparticles with a scanning electron microscope (SEM: HITACHI, S-4800), the average particle size of the microparticles was 6.0 ⁇ m.
- 300 mg of the microparticles prepared above were suspended and dispersed in 3 ml of phosphate physiological buffer (PBS), and the particles were precipitated by centrifugation at 80 ⁇ g for 5 minutes, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 80 ⁇ g for 5 minutes to precipitate the remaining particles, and the supernatant was removed. The first centrifugal precipitation and the second centrifugal precipitation were combined and redispersed in 1 ml of PBS, and the same centrifugal washing operation was repeated three times to remove exendin 4 not encapsulated in the particles. Finally, the precipitate was redispersed in 200 ⁇ l of PBS to obtain a dosing solution.
- PBS phosphate physiological buffer
- This solution was injected and administered subcutaneously to two backs of 10-week-old male Balb / C mice (Japan SLC), and blood was collected over time from the tail vein.
- the collected blood was added with heparin at a final concentration of 3.3 IU / ml, centrifuged at 5,000 rpm for 5 minutes to collect plasma, and the concentration of exendin 4 in plasma was measured using an ELISA method.
- mice subcutaneously administered with exendin 4 solution (700 ⁇ g / 0.2 ml) that had not been granulated.
- exendin 4 which is a heterologous protein for mice
- 26 ⁇ g / mouse of the immunosuppressive agent tacrolimus hydrate (Astellas) was administered subcutaneously 3 days before particle administration, and then at the time of drug administration. Thereafter, 13 ⁇ g / mouse was subcutaneously administered at an interval of twice a week.
- FIG. 21 shows the time course of plasma exendin 4 concentration.
- the blood concentration showed a very high value 1 hour after administration, then rapidly decreased, and decreased to the level before administration 1 day later.
- the transient increase in blood concentration immediately after administration was suppressed to approximately 1/100 or less, and the blood concentration was maintained until 1 month later. The concentration was continuously high.
- Example 31 Preparation of fluorescein-labeled dextran (FD40) -encapsulating microparticles having different particle sizes 5 mg of the PEG-PLGA polymer (5k-55k) of Example 20 was dissolved in 100 ⁇ l of dimethyl carbonate to prepare a 50 mg / ml polymer solution. After adding 20 ⁇ l of tert-butanol to the polymer solution, 20 ⁇ l of 1 mg / ml FD40 aqueous solution was added dropwise and stirred by vortex to produce a reverse phase emulsion. The inverse emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C.
- FD40 fluorescein-labeled dextran
- the obtained solid content was dispersed in 50 ⁇ l, 200 ⁇ l, and 500 ⁇ l of dimethyl carbonate to prepare an S / O suspension.
- the S / O suspension was dropped into 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and stirred and emulsified with a vortex mixer to prepare an S / O / W type emulsion.
- Pluronic F-68 registered trademark of BASF
- a water-immiscible organic solvent was removed from the S / O / W emulsion by submerged drying to obtain a microparticle dispersion.
- microparticle dispersion is pre-frozen with liquid nitrogen and then freeze-dried for 24 hours at a trap cooling temperature of ⁇ 45 ° C. and a vacuum of 20 Pa using a freeze dryer (EYELA, FREEZE DRYER FD-1000).
- FD40 inclusion microparticle powder was obtained.
- the average particle size was calculated by observing the obtained microparticles with a scanning electron microscope (SEM: HITACHI, S-4800).
- FIG. 22 shows the correlation between the average particle diameter and the amount of dimethyl carbonate added when preparing the S / O / W emulsion. A decrease in average particle size was observed with an increase in the amount of dimethyl carbonate between 50 ⁇ l and 500 ⁇ l.
- microparticle of the present invention can release a hydrophilic active substance at an appropriate rate in vivo, it can be used as a DDS preparation.
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Abstract
Description
(1)すず触媒存在下、多糖にヒドロキシ酸活性化モノマーを加えて重合反応を行い、ポリ(ヒドロキシ酸)を導入することでグラフト型両親媒性ポリマーを製造する方法[Macromolecules,31,1032-1039(1998年)]。
(2)水酸基の大部分が置換基で保護された多糖の一部未保護の水酸基を塩基で活性化後、ヒドロキシ酸活性化モノマーを加えてポリ(ヒドロキシ酸)からなるグラフト鎖を導入し、最後に保護基を取り除くことにより、グラフト型両親媒性ポリマーを製造する方法[Polymer,44,3927-3933,(2003年)]。
(3)多糖に対して、ポリ(ヒドロキシ酸)の共重合体を脱水剤および/または官能基の活性化剤を用いて縮合反応させることにより、グラフト型両親媒性ポリマーを製造する方法[Macromolecules,33,3680-3685(2000年)]。
(a)親水性活性物質を含む水系溶媒と両親媒性ポリマーを溶解した水非混和性有機溶媒を混合することにより、逆相エマルジョンを形成する工程、
(b)逆相エマルジョンから溶媒を除去して親水性活性成分含有固形分を得る工程、
(c)親水性活性成分含有固形分または親水性活性成分含有固形分分散液を、表面改質剤を含む液相に導入する工程、
を含むマイクロ粒子の製造方法に関する。
1-1.TMS-デキストラン(化合物(1))の合成
デキストラン(ナカライテスク株式会社、ナカライ規格特級適合品、数平均分子量13000、5.0g)をホルムアミド(100ml)に加え、80℃に加熱した。この溶液に1,1,1,3,3,3-ヘキサメチルジシラザン(100ml)を20分掛けて滴下した。滴下終了後、80℃で2時間攪拌した。反応終了後、反応溶液を室温に戻し、分液漏斗で2層を分離した。上の層を減圧下濃縮した後、メタノール(300ml)を加え、得られた固体をろ過、乾燥し、TMS-デキストラン(11.4g)を白色固体として得た。
化合物(1)(0.5g)とtert-ブトキシカリウム(35mg)を減圧下1時間乾燥後、テトラヒドロフラン(20ml)を加え、1時間室温で攪拌した。この溶液に(L)-ラクチド(4.49g)のテトラヒドロフラン(20ml)溶液を滴下し、5分間攪拌した。反応終了後、溶媒を減圧下濃縮し、クロロフォルム-メタノール系で再沈殿精製を行うことによってTMS-デキストラン-PLA(1.9g)を白色固体として得た。
化合物(2)(1.9g)のクロロフォルム(24ml)溶液に、メタノール(10.8ml)と12N塩酸(1.2ml)を加え、 室温で30分間攪拌した。溶媒を減圧下留去後、残渣をクロロフォルム(10ml)に溶解し、0℃に冷却したジエチルエーテルに滴下することで生成物を析出させた。析出物をろ別後、減圧下濃縮しデキストラン-PLA(1.6g)を得た。本ポリマーの重量平均分子量は48720、数平均分子量は43530であった。(GPCによる測定: カラム 東ソ- TSK-gel α-5000×2、DMF系溶媒、検出器 RI、標準品 プルラン)。本ポリマーの1H-NMR測定により決定されたグラフト鎖の平均分子量は2300であった。グラフト鎖数は10~12であった。
2-1.TMS-デキストラン-PLGA(化合物(4)、化合物(5)、化合物(6))の合成
化合物(1)(0.5g)とtert-ブトキシカリウム(35mg)を減圧下1時間乾燥後、テトラヒドロフラン(10ml)を加え、1時間室温で攪拌した。この溶液に(DL)-ラクチド(1.12g)とグリコリド(0.9g)のテトラヒドロフラン(15ml)溶液を滴下し、5分間攪拌した。反応終了後、溶媒を減圧下濃縮し、クロロフォルム-メタノール系で再沈殿精製を行うことによってTMS-デキストラン-PLGA(1.96g)を白色固体として得た(化合物(4))。同様な方法で、(DL)-ラクチド(0.784g)とグリコリド(0.63g)の仕込量で化合物(5)を、同仕込量(DL)-ラクチド(1.12g)とグリコリド(0.9g)で化合物(6)を合成した。
化合物(4)(1.96g)のクロロフォルム(14ml)溶液に、メタノール(6.3ml)と12N塩酸(0.7ml)を加え、 室温で30分間攪拌した。溶媒を減圧下留去後、残渣をクロロフォルム(10ml)に溶解し、0℃に冷却したジエチルエーテルに滴下することで生成物を析出させた。析出物をろ別後、減圧下濃縮しデキストラン-PLGA(1.25g)を得た(化合物(7))。化合物(5)、(6)からもトリフルオロ酢酸を使用した以外は類似の方法でデキストラン-PLGAを得た(化合物(8)、化合物(9))。化合物(7)~(9)のポリマーの重量平均分子量、数平均分子量はGPC測定(カラム 東ソ- TSK-gel α-5000×2、DMF系溶媒、検出器 RI、標準品 プルラン)によって決定した。グラフト鎖の平均分子量、グラフト鎖数は1H-NMR測定により決定した。
実施例1のデキストラン-ポリ乳酸(PLA)(デキストラン平均分子量13,000、PLA平均分子量2,300、PLAグラフト鎖数10~12、化合物(3))または実施例2のデキストラン-ポリ(乳酸-グリコール酸)(PLGA)(デキストラン平均分子量13,000、PLGA平均分子量1,900、PLGAグラフト鎖数7~10、化合物(7))5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、2mg/mlのhGH水溶液20μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル200μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、マイクロ粒子の平均粒径は4.0μmであった。
デキストラン-PLA(化合物(3))またはデキストラン-PLGA(化合物(7))ポリマーを用いて実施例3の方法で作成したヒト成長ホルモン内包マイクロ粒子20mgを1.5mlエッペンドルフチューブに秤量し、1mlの緩衝液A(0.1%ウシ血清アルブミン、0.1% Pluronic F-68(BASFの登録商標)および0.02%アジ化ナトリウムを含むPBS)に溶解して18,000×g、10分間の遠心により粒子(沈殿)と上清を分離した。上清を別のチューブに回収した後、粒子を1mlの緩衝液に再けん濁し、上記条件での遠心による粒子と上清の分離を再度行った。この洗浄操作をもう一度繰り返し(計3回遠心)、各遠心で回収した上清中のヒト成長ホルモン濃度をELISAキット(R&Dシステムズ社製)で測定した。粒子作製時の仕込みのhGH量(粒子重量20mgあたり)から3回の遠心上清中のhGH量合計を差し引くことで、以下の式により内包率を計算した。
仕込みhGH量(ng)。
実施例4で3回洗浄を行ったマイクロ粒子を1.2mlの緩衝液Aにけん濁分散させた。この溶液から一部(40μ1)を別のチューブに移し、18,000×g10分間の遠心により粒子を沈殿させ、上清30μlを別のチューブに回収した(0時間サンプル)。残りの粒子けん濁液は、1.5mlエッペンドルフチューブ中で、37℃インキュベーター内でローテーターを用いて6rpmの速度でゆっくりと転倒混和させた。この溶液から経時的に少量(40μl)を分取して、上記と同様に遠心による上清分離を行った。各時点で回収した上清サンプル中のhGH濃度をELISAキットで測定し、放出量(%)を以下の式により計算した。
20mg粒子中の内包hGH量(ng)。
デキストラン-PLA(デキストラン平均分子量13,000、PLA平均分子量2,300、PLAグラフト鎖数10~12、化合物(3))もしくはデキストラン-PLGA(デキストラン平均分子量13,000、PLGA平均分子量1,900、PLGAグラフト鎖数7~10、化合物(7))5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、2mg/mlのヒトインスリン水溶液20μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル200μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、ヒトインスリン内包粒子粉末を得た。マイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、化合物(3)から得られたマイクロ粒子の平均粒径は6.4μm、化合物(7)から得られたマイクロ粒子の平均粒径は5.3μmであった。
デキストラン-PLGA(化合物(7))ポリマーを用いて実施例6の方法で作成したヒトインスリン内包マイクロ粒子20mgを1.5mlエッペンドルフチューブに秤量し、1mlの緩衝液A(0.1%ウシ血清アルブミン、0.1% Pluronic F-68(BASFの登録商標)および0.02%アジ化ナトリウムを含むPBS)に溶解して18,800×g、10分間の遠心により粒子(沈殿)と上清を分離した。上清を別のチューブに回収した後、粒子を1mlの緩衝液にけん濁し、上記条件での遠心による粒子と上清の分離を再度行った。この洗浄操作をもう一度繰り返し(計3回遠心)、各遠心で回収した上清中のヒトインスリン濃度をサンドイッチELISA法で測定した。粒子作製時の仕込みのヒトインスリン量(粒子重量20mgあたり)から3回の遠心上清中のヒトインスリン量合計を差し引くことで、以下の式により内包率を計算した。
仕込みインスリン量(ng)。
実施例7で3回洗浄を行った粒子を1.2mlの緩衝液Aにけん濁分散させた。この溶液から一部(40μ1)を別のチューブに移し、18,800×g10分間の遠心により粒子を沈殿させ、上清30μlを別のチューブに回収した(0時間サンプル)。回収後のサンプルは測定まで凍結保存した。残りの粒子けん濁液は、1.5mlエッペンドルフチューブ中で、37℃インキュベーター内でローテーターを用いて6rpmの速度でゆっくりと転倒混和させた。この溶液から経時的に少量(40μl)を分取して、上記と同様に遠心による上清分離を行った。各時点で回収した上清サンプル中のヒトインスリン濃度をサンドイッチELISA法で測定し、放出量(%)を以下の式により計算した。
20mg粒子中の内包ヒトインスリン量(ng)。
実施例3で調製したhGH内包マイクロ粒子粉末5mgをエッペンドルフチューブに秤量し、Milli-Q 1mlに分散後、13,000rpmで30分間遠心分離し、上清を除去後に再度Milli-Q 1mlに分散させ、遠心分離することでマイクロ粒子の洗浄を行った。また、所定時間インキュベートしたマイクロ粒子懸濁溶液には、Milli-Q 1mlを加えて13,000rpmで30分間遠心分離し、上清除去後に再度Milli-Q 1mlに分散させ、同様に遠心分離することでマイクロ粒子の洗浄を行った。得られた洗浄後のマイクロ粒子をMilli-Q 100μlに分散させ、マイクロ粒子分散溶液をシリコン基板上に5μl滴下し、約10分間室温放置したのちに、デシケーター内で3時間乾燥させた。その後、イオンスパッタ(HITACHI、E-1030)を用いて試料表面への白金蒸着(蒸着時間15秒)を行い、走査型電子顕微鏡(SEM:HITACHI、S-4800)により加速電圧を1kV、プローブ電流をhighに設定して、マイクロ粒子形状、表面状態観察を行った。
ポリエチレングリコール-ポリ(イプシロン-カプロラクトン)(ポリエチレングリコールの平均分子量5,000、ポリ(イプシロン-カプロラクトン)の平均分子量37,000)5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、2mg/mlのhGH水溶液20μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル200μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包粒子粉末を得た。マイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、得られたマイクロ粒子の平均粒径は8.0μmであった。
デキストラン-ポリ乳酸(PLA)(デキストラン平均分子量13,000、PLA平均分子量2,300、PLAグラフト鎖数10~12、化合物(3))またはデキストラン-ポリ(乳酸-グリコール酸)(PLGA)(デキストラン平均分子量13000、PLGA平均分子量1,900、PLGAグラフト鎖数7~10、化合物(7))25mgを炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのhGH水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。マイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、得られたマイクロ粒子の平均粒径は、化合物(3)のマイクロ粒子が4.9μm、化合物(7)のマイクロ粒子が4.2μmであった。
実施例2のデキストラン-ポリ(乳酸-グリコール酸)(PLGA)(デキストラン平均分子量13,000、PLGA平均分子量1,900、PLGAグラフト鎖数7~10、化合物(7))25mgを炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのhGH水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。マイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、得られたマイクロ粒子の平均粒径は4.1μmであった。
デキストラン-ポリ(乳酸-グリコール酸)(PLGA)(デキストラン平均分子量13,000、PLGA平均分子量3,250(化合物(8))または6,442(化合物(9))、PLGAグラフト鎖数21(化合物(8))または15(化合物(9)))25mgを炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのエキセンジン4(シグマジェノシス社委託合成)水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、エキセンジン4内包マイクロ粒子粉末を得た。マイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、得られたマイクロ粒子の平均粒径は化合物(8)のマイクロ粒子が4.3μm、化合物(9)のマイクロ粒子が4.5μmであった。
20mg粒子中の内包エキセンジン4量(ng)。
実施例12のマイクロ粒子300mgを3mlのリン酸生理緩衝液(PBS)にけん濁分散させ、80×g、5分間の遠心によりマイクロ粒子を沈殿させ、上清を別チューブに移した。上清は再度80×g、5分間の遠心を行って残存しているマイクロ粒子を沈殿させ、上清は除去した。1回目の遠心沈殿と2回目の遠心沈殿をあわせて1mlのPBSに再分散し、同様の遠心洗浄操作を3回繰り返すことにより、マイクロ粒子に内包されていないエキセンジン4を除去した。最後に沈殿を200μlのPBSに再分散して、投与溶液とした。
14-1.TMS-デキストラン-PLGA(化合物(10)、化合物(11)、化合物(12)、化合物(13))の合成
化合物(1)(0.5g)とtert-ブトキシカリウム(35mg)を減圧下1時間乾燥後、テトラヒドロフラン(10ml)を加え、1時間室温で攪拌した。この溶液に(DL)-ラクチド(0.558g)とグリコリド(0.45g)のテトラヒドロフラン(15ml)溶液を滴下し、5分間攪拌した。反応終了後、溶媒を減圧下濃縮し、クロロフォルム-メタノール系で再沈殿精製を行うことによってTMS-デキストラン-PLGA(1.96g)を白色固体として得た(化合物(10))。
化合物(10)のクロロフォルム溶液(10mL)に、トリフルオロ酢酸(1mL)を加え、 室温で30分間攪拌した。溶媒を減圧下留去後、残渣をクロロフォルム(10ml)に溶解し、0℃に冷却したジエチルエーテルに滴下することで生成物を析出させた。析出物をろ別後、減圧下濃縮しデキストラン-PLGA(0.44g)を得た(化合物(14))。
実施例14の各デキストラン-ポリ(乳酸-グリコール酸)(デキストラン-PLGAポリマー、化合物(14)~(17))5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、1mg/mlのhGH水溶液50μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル200μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察したところ、粒径は1.0~10μmの範囲内であった。
各デキストラン-PLGAポリマー(化合物(14)~(17))を用いて実施例15の方法で作成した成長ホルモン内包マイクロ粒子20mgを1.5mlエッペンドルフチューブに秤量し、1mlの緩衝液A(0.1%ウシ血清アルブミン、0.1% Pluronic F-68(BASFの登録商標)および0.02%アジ化ナトリウムを含むPBS)に溶解して18,000×g、10分間の遠心により粒子(沈殿)と上清を分離した。上清を別のチューブに回収した後、粒子を1mlの緩衝液に再けん濁し、上記条件での遠心による粒子と上清の分離を再度行った。この洗浄操作をもう一度繰り返し(計3回遠心)、各遠心で回収した上清中のヒト成長ホルモン濃度をELISAキット(R&Dシステムズ社製)で測定した。粒子作製時の仕込みのhGH量(粒子重量20mgあたり)から3回の遠心上清中のhGH量合計を差し引くことで、以下の式により内包率を計算した。
仕込みhGH量(ng)。
デキストラン-ポリ(乳酸-グリコール酸)(PLGA)(化合物(14)又は、化合物(17))10mgを酢酸エチル2mLに溶解し、ポリマー溶液を調製した。該ポリマー溶液に、0.5mg/mLの成長ホルモン溶液100μLを滴下し攪拌した。攪拌後、20mLのジオキサンに添加した。溶媒を蒸発させ約2mLまで濃縮した後、該粒子分散液を500mgのPluronic F-68(BASFの登録商標)を含有した水に添加した。試料を凍結乾燥した後、50mgの試料に1mLの水を添加し粒子を再分散させて、会合していない親水性活性物質含有粒子を得た。該粒子の平均粒径は、装置ELS-Z(大塚電子))を使用し、動的光散乱法で測定し、薬剤の内包率は、実施例16と同様に求めた。
実施例16で3回洗浄を行った粒子を1.2mlの緩衝液Aにけん濁分散させた。この溶液から一部(40μ1)を別のチューブに移し、18000×g10分間の遠心により粒子を沈殿させ、上清30μlを別のチューブに回収した(0時間サンプル)。残りの粒子けん濁液は、1.5mlエッペンドルフチューブ中で、37℃インキュベーター内でローテーターを用いて6rpmの速度でゆっくりと転倒混和させた。この溶液から経時的に少量(40μl)を分取して、上記と同様に遠心による上清分離を行った。各時点で回収した上清サンプル中のhGH濃度をELISAキットで測定し、放出量(%)を以下の式により計算した。
20mg粒子中の内包hGH量(ng)。
実施例2のデキストラン-ポリ(乳酸-グリコール酸)(PLGA)(化合物(7))5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、1mg/mlのFD40水溶液20μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル50μl,100μl,200μl,350μl,500μl,1ml,2ml,6mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで、平均粒子径を算出した。
ポリエチレングリコールモノメチルエーテル(日本油脂製、SUNBRIGHT MEH-20H、数平均分子量1,862、Mw/Mn=1.03)、(DL)-ラクチドとグリコリドを下記表1の仕込量で混合し、140℃に加熱した。20分攪拌後、オクチル酸すず(II)(ポリエチレングリコールモノメチルエーテルに対して0.05重量%)を加え、180℃で3時間攪拌した。反応液を室温に戻した後、クロロフォルム(約100mg/ml濃度になるよう)に溶解し、0℃に冷却したジエチルエーテルで再沈殿精製し、得られた固体を濾別、減圧乾燥することでPEG-PLGAポリマーを白色、または薄茶色の固体として得た。本ポリマーの数平均分子量は1H-NMRから求めた(表1)。
ポリエチレングリコールモノメチルエーテル(日本油脂製、SUNBRIGHT MEH-20H、数平均分子量5,128、Mw/Mn=1.02)、(DL)-ラクチドとグリコリドを下記表2の仕込量で混合し、140℃に加熱した。20分攪拌後、オクチル酸すず(II)(ポリエチレングリコールモノメチルエーテルに対して0.05重量%)を加え、180℃で3時間攪拌した。反応液を室温に戻した後、クロロフォルム(約100mg/ml濃度になるよう)に溶解し、0℃に冷却したジエチルエーテルで再沈殿精製し、得られた固体を濾別、減圧乾燥することでPEG-PLGAポリマーを白色、または薄茶色の固体として得た。本ポリマーの数平均分子量は1H-NMRから求めた(表2)。
ポリエチレングリコールモノメチルエーテル(日本油脂製、SUNBRIGHT MEH-10T、数平均分子量9,975、Mw/Mn=1.02)、(DL)-ラクチドとグリコリドを下表3の割合で混合し、140℃に加熱した。20分攪拌後、オクチル酸すず(II)(ポリエチレングリコールモノメチルエーテルに対して0.05重量%)を加え、180℃で3時間攪拌した。反応液を室温に戻した後、クロロフォルム(約100mg/ml濃度になるよう)に溶解し、0℃に冷却したジエチルエーテルで再沈殿精製し、得られた固体を濾別、減圧乾燥することでPEG-PLGAポリマーを白色、または薄茶色の固体として得た。本ポリマーの数平均分子量は1H-NMRから求めた(表3)。
実施例19~21で作製したPEG-PLGAポリマー5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノールを20μl添加後、下表4に示す所定量の10mg/ml FD40水溶液を加え、攪拌することで逆相エマルション溶液を製造した。該逆相エマルジョン溶液を、液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル200μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、FD40内包マイクロ粒子粉末を得て、それらの一部については走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察し、平均粒子径を算出した(表4)。5k-10kのPEG-PLGAポリマーから調製した粉末のSEM像を図14に、5k-61kのPEG-PLGAポリマーから調製した粉末のSEM像を図15に示す。
PEG-PLGAポリマーを用いて実施例22の方法で作成したFD40内包マイクロ粒子(5mg)を1.5mlエッペンドルフチューブに秤量し、Milli-Q(1ml)に分散後、30分間の遠心分離により、未内包FD40を含む上清とFD40内包粒子を分離、回収した。回収したFD40内包マイクロ粒子はN,N-ジメチルホルムアミド(250μl)に溶解させることで粒子を崩壊させた。未内包FD40を含む上清と、内包FD40を含むN,N-ジメチルホルムアミド溶液(50μl)をそれぞれMilli-Q(3ml)に添加し、よく攪拌後、蛍光分光光度計(HORIBA、Fluoro MAX-3、励起波長495nm、蛍光波長520nm)を用いてFD40の定量を行い、全回収量に対する内包率を算出した。
PEG-PLGAポリマー(5k-61k)10mgを酢酸エチル2mLに溶解し、ポリマー溶液を調製した。該ポリマー溶液に、2mg/mLの成長ホルモン溶液100μLを滴下し攪拌した。攪拌後、20mLのジオキサンに添加した。溶媒を蒸発させ約2mLまで濃縮した後、該粒子分散液を500mgのPluronic F-68(BASFの登録商標)を含有した水に添加した。試料を凍結乾燥した後、50mgの試料に1mLの水を添加し粒子を再分散させて、会合していない親水性活性物質含有粒子を得た。該粒子の平均粒径は、装置ELS-Z(大塚電子))を使用し、動的光散乱法で測定し、薬剤の内包率は、実施例23と同様に求めた。
PEG-PLGAポリマー粒子を構成するPLGA鎖の長さと徐放挙動との関係を評価するため、実施例22で作成したFD40内包マイクロ粒子のうち、5k-23k、5k-32.5k、5k-47k、5k-61kの粒子の放出挙動を評価した。
実施例20で作製したPEG-PLGAポリマー(5k-61k)を用いて、実施例22と同様の方法でヒトインスリン内包マイクロ粒子を作製した。得られたマイクロ粒子(20mg)を1.5mlエッペンドルフチューブに秤量し、1mlの緩衝液A(0.1%ウシ血清アルブミン、0.1% Pluronic F-68(BASFの登録商標)および0.02%アジ化ナトリウムを含むPBS)に溶解して18,800×g、10分間の遠心により粒子(沈殿)と上清を分離した。上清を別のチューブに回収した後、粒子を1mlの緩衝液Aに再懸濁し、上記条件での遠心による粒子と上清の分離を再度行った。この洗浄操作をもう一度繰り返し(計3回遠心)、各遠心で回収した上清中のインスリン濃度をサンドイッチELISA法にて測定した。
仕込みインスリン量(ng)。
実施例25で3回洗浄を行ったマイクロ粒子を1.0mlの緩衝液Aにけん濁分散させた。この溶液を0.1mlずつ10本のエッペンドルフチューブ(1.5ml容量)に分注し、それぞれに緩衝液A0.9mlを加えて10倍に希釈した。希釈直後に、1本のチューブを18800×g10分間で遠心して粒子を沈殿させ、上清を別のチューブに回収した(0時間サンプル)。残りの9本のチューブは、37℃インキュベーター内でローテーターを用いて6rpmの速度でゆっくりと転倒混和させた。経時的に1本ずつのチューブを用いて、上記と同様に遠心による上清分離を行った。各時点で回収した上清サンプル中のインスリン濃度をサンドイッチELISAで測定し、インスリン放出量(%)を以下の式により計算した。
20mg粒子中の内包インスリン量(ng)。
PEG-PLGAポリマー(5k-61k)25mgを炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのhGH水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。得られたマイクロ粒子の平均粒径は、6.0μmであった。
50mg/mlのPEG-PLGAポリマー(5k-61k)/炭酸ジメチル溶液100μlにtert-ブタノールを20μl添加し、10mg/mlのFD40水溶液を20μl加え、撹拌することで逆ミセル(W/Oエマルション)溶液を調製した。得られた溶液を、液体窒素で予備凍結した後、凍結乾燥機で一晩凍結乾燥することでFD40含有固形分(Solid)とした。得られたFD40含有固形分に炭酸ジメチル200μlを加えてボルテックスで10秒間撹拌することでS/Oサスペンションとし、所定濃度の塩化ナトリウム(0M、10mM、50mM、1M)を含む10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下して、30秒間ボルテックスで撹拌して乳化させることでS/O/W型エマルション溶液を調製した。得られたS/O/W型エマルション溶液から、エバポレーターを用いて水非混和性有機溶媒を除去(30hPaまで減圧後、5分間減圧留去)することで、FD40封入マイクロ粒子の水分散体とした。FD40封入マイクロ粒子分散水溶液を液体窒素で予備凍結した後、凍結乾燥機で一晩凍結乾燥することで、粉末状のFD40封入マイクロ粒子を調製した。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、いずれの塩化ナトリウム濃度条件下においてもマイクロ粒子の平均粒径は6.5μmであった。
実施例20のPEG-PLGAポリマー(5k-55k)およびPEG-PLGAポリマー(5k-105k)それぞれ25mgを、炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのhGH水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、hGH内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、マイクロ粒子の平均粒径は、PEG-PLGAポリマー(5k-55k)のマイクロ粒子(5k-55kマイクロ粒子)は4.2μm、PEG-PLGAポリマー(5k-105k)のマイクロ粒子(5k-105kマイクロ粒子)は7.5μmであった。
実施例20のPEG-PLGAポリマー(5k-61k)25mgを炭酸ジメチル500μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール100μlを添加後、10mg/mlのエキセンジン4(シグマジェノシス社委託合成)水溶液250μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル1mlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液10mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、エキセンジン4内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで平均粒子径を算出したところ、マイクロ粒子の平均粒径は、6.0μmであった。
実施例20のPEG-PLGAポリマー(5k-55k) 5mgを炭酸ジメチル100μlに溶解し、50mg/mlのポリマー溶液を調製した。該ポリマー溶液にtert-ブタノール20μlを添加後、1mg/mlのFD40水溶液20μlを滴下し、ボルテックスで撹拌して逆相エマルジョンを製造した。該逆相エマルジョンを液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥した。得られた固形分を炭酸ジメチル50μl,200μl,500μlに分散させ、S/Oサスペンションを調製した。該S/Oサスペンションを10% Pluronic F-68(BASFの登録商標)含有水溶液2mlに滴下し、ボルテックスミキサーで撹拌・乳化させてS/O/W型エマルジョンを調製した。該S/O/W型エマルジョンから液中乾燥により水非混和性有機溶媒を除去して、マイクロ粒子分散液とした。該マイクロ粒子分散液を液体窒素で予備凍結した後、凍結乾燥機(EYELA、FREEZE DRYER FD-1000)を用いて、トラップ冷却温度-45℃、真空度20Paにて24時間凍結乾燥することで、FD40内包マイクロ粒子粉末を得た。得られたマイクロ粒子を走査型電子顕微鏡(SEM:HITACHI、S-4800)により観察することで、平均粒子径を算出した。
Claims (15)
- ポリ(ヒドロキシ酸)の疎水性セグメントと多糖またはポリエチレングリコールの親水性セグメントからなる両親媒性ポリマーおよび親水性活性物質を含有してなる親水性活性物質含有粒子が会合してなる、マイクロ粒子。
- 親水性活性物質含有粒子が、内部に両親媒性ポリマーの親水性セグメントを有し、両親媒性ポリマーの疎水性セグメントの外層を有することを特徴とする、請求項1に記載のマイクロ粒子。
- 両親媒性ポリマーが、多糖主鎖およびポリ(ヒドロキシ酸)グラフト鎖からなるグラフト型両親媒性ポリマーであることを特徴とする、請求項1または2に記載のマイクロ粒子。
- 多糖主鎖がデキストランであることを特徴とする、請求項3に記載のマイクロ粒子。
- 両親媒性ポリマーが、ポリエチレングリコールおよびポリ(ヒドロキシ酸)からなるブロックポリマーであることを特徴とする、請求項1または2のマイクロ粒子。
- ポリエチレングリコールの平均分子量が2,000~15,000であることを特徴とする、請求項5に記載のマイクロ粒子。
- ポリエチレングリコールの平均分子量に対するポリ(ヒドロキシ酸)の平均分子量の比が4以上であることを特徴とする、請求項5または6に記載のマイクロ粒子。
- ポリ(ヒドロキシ酸)がポリ(乳酸-グリコール酸)であることを特徴とする、請求項1~7のいずれかに記載のマイクロ粒子。
- 平均粒径が1~50μmであることを特徴とする、請求項1~8のいずれかに記載のマイクロ粒子。
- 親水性活性物質がペプチドまたはタンパク質であることを特徴とする、請求項1~9のいずれかに記載のマイクロ粒子。
- (a)親水性活性物質を含む水系溶媒と両親媒性ポリマーを溶解した水非混和性有機溶媒を混合することにより、逆相エマルジョンを形成する工程、(b)逆相エマルジョンから溶媒を除去して親水性活性成分含有固形分を得る工程、および(c)親水性活性成分含有固形分または親水性活性成分含有固形分分散液を、表面改質剤を含む液相に導入する工程、
を含むことを特徴とする、マイクロ粒子の製造方法。 - 逆相エマルジョンから溶媒を除去する方法が凍結乾燥法であることを特徴とする、請求項11記載のマイクロ粒子の製造方法。
- 親水性活性成分含有固形分分散液の分散媒が、ポリ(ヒドロキシ酸)が可溶で且つ、両親媒性ポリマーを構成する親水性セグメントの溶解度が10mg/mL以下の溶媒であることを特徴とする、請求項11または12に記載のマイクロ粒子の製造方法。
- 液相が水系溶媒または水混和性有機溶媒であることを特徴とする、請求項11~13のいずれかに記載のマイクロ粒子の製造方法。
- 請求項1~10のいずれかに記載のマイクロ粒子を含んでなる、医薬品組成物。
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JP2011140470A (ja) * | 2010-01-08 | 2011-07-21 | Toray Ind Inc | 親水性生理活性物質含有微粒子の製造方法 |
KR101843385B1 (ko) | 2010-08-26 | 2018-03-29 | 도레이 카부시키가이샤 | 면역원성 조성물 |
CN103052402A (zh) * | 2010-08-26 | 2013-04-17 | 东丽株式会社 | 免疫原性组合物 |
CN103052402B (zh) * | 2010-08-26 | 2015-08-12 | 东丽株式会社 | 免疫原性组合物 |
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WO2012026508A1 (ja) * | 2010-08-26 | 2012-03-01 | 東レ株式会社 | 免疫原性組成物 |
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JP2015525245A (ja) * | 2012-04-23 | 2015-09-03 | ナノジェン ファーマシューティカルズ | ポリマーナノ粒子およびその調整のプロセス |
US10092617B2 (en) | 2012-04-23 | 2018-10-09 | Nanoproteagen | Polymeric nanoparticles and a process of preparation thereof |
JP2018162310A (ja) * | 2012-04-23 | 2018-10-18 | ナノプロティアジェン, リミテッド | ポリマーナノ粒子およびその調整のプロセス |
US11246904B2 (en) | 2012-04-23 | 2022-02-15 | Hillstream Biopharma, Inc. | Polymeric nanoparticles and a process of preparation thereof |
JP2022191364A (ja) * | 2012-04-23 | 2022-12-27 | ナノプロティアジェン エルエルシー | ポリマーナノ粒子およびその調整のプロセス |
RU2672861C2 (ru) * | 2014-10-31 | 2018-11-20 | Общество с ограниченной ответственностью "Эксифарм" (ООО "Эксифарм") | Композиция на основе интерферона I или III типа и гамма-D-глутаминил-L-триптофана для профилактики и лечения инфекционных заболеваний и иммунодефицитных состояний (варианты) |
US11484507B2 (en) | 2016-11-02 | 2022-11-01 | Hillstream Biopharma, Inc. | Polymeric nanoparticles providing nucleic acids encoding TNF-α |
Also Published As
Publication number | Publication date |
---|---|
US8431161B2 (en) | 2013-04-30 |
DK2251006T3 (en) | 2017-10-16 |
AU2009216216A1 (en) | 2009-08-27 |
RU2010138925A (ru) | 2012-03-27 |
EP2251006A1 (en) | 2010-11-17 |
PT2251006T (pt) | 2017-10-13 |
US20110003007A1 (en) | 2011-01-06 |
JPWO2009104706A1 (ja) | 2011-06-23 |
KR20100120119A (ko) | 2010-11-12 |
JP5712485B2 (ja) | 2015-05-07 |
RU2490009C2 (ru) | 2013-08-20 |
CN101932311A (zh) | 2010-12-29 |
BRPI0905790A2 (pt) | 2015-07-14 |
AU2009216216B2 (en) | 2013-11-07 |
CA2715665A1 (en) | 2009-08-27 |
KR101542433B1 (ko) | 2015-08-06 |
PL2251006T3 (pl) | 2017-12-29 |
EP2251006A4 (en) | 2013-07-10 |
ES2635515T3 (es) | 2017-10-04 |
CA2715665C (en) | 2016-04-19 |
EP2251006B1 (en) | 2017-07-19 |
HUE035331T2 (en) | 2018-05-02 |
CN101932311B (zh) | 2014-07-16 |
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