WO2011052089A1 - Matériaux vecteurs permettant la libération prolongée in vivo d'un médicament et comprenant un hydrogel réticulé par rayonnement ionisant, et leur procédé de fabrication - Google Patents

Matériaux vecteurs permettant la libération prolongée in vivo d'un médicament et comprenant un hydrogel réticulé par rayonnement ionisant, et leur procédé de fabrication Download PDF

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WO2011052089A1
WO2011052089A1 PCT/JP2009/068754 JP2009068754W WO2011052089A1 WO 2011052089 A1 WO2011052089 A1 WO 2011052089A1 JP 2009068754 W JP2009068754 W JP 2009068754W WO 2011052089 A1 WO2011052089 A1 WO 2011052089A1
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drug
carrier material
crosslinked
sustained release
vivo
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PCT/JP2009/068754
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English (en)
Japanese (ja)
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石黒智之
竹崎彰人
一樹 磯部
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ニチバン株式会社
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Priority to PCT/JP2009/068754 priority Critical patent/WO2011052089A1/fr
Priority to JP2011538202A priority patent/JP5514222B2/ja
Publication of WO2011052089A1 publication Critical patent/WO2011052089A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a carrier material for sustained release of an in vivo drug composed of a hydrogel crosslinked with ionizing radiation mainly used in the medical field, and a method for producing the same.
  • a biodegradable polymer water-soluble substance such as gelatin is a water-soluble polymer material suitable for medical and cosmetic applications because it has high biological safety and exhibits degradability in vivo.
  • Biodegradable polymer water-soluble substances such as gelatin can form a crosslinked product such as crosslinked gelatin by crosslinking.
  • Cross-linked products such as cross-linked gelatin can be formed into various shapes such as plate, columnar, prismatic, sheet, disc, and spherical.
  • Cross-linked products such as cross-linked gelatin absorb water and form hydrogels.
  • cross-linked gelatin is sometimes referred to as cross-linked gelatin gel.
  • Cross-linked products such as cross-linked gelatin are degradable by enzymes present in the living body, such as protease, and the degradation rate can be controlled by the degree of cross-linking.
  • the degree of cross-linking of a cross-linked product such as cross-linked gelatin is almost inversely proportional to its water content. The smaller the moisture content of a crosslinked product such as crosslinked gelatin, the higher the degree of crosslinking.
  • Cross-linked products such as cross-linked gelatin have good biocompatibility and can carry drugs such as bioactive factors.
  • a cross-linked product such as cross-linked gelatin carrying a bioactive factor such as a growth factor
  • the supported bioactive factor is gradually released as the cross-linked product such as cross-linked gelatin is decomposed in vivo.
  • a physiologically active factor for example, basic fibroblast growth factor (bFGF) having an angiogenesis-inducing effect as a growth factor is known.
  • bFGF basic fibroblast growth factor
  • angiogenesis-inducing effect as a growth factor
  • a physiologically active factor is administered as an aqueous solution in a living body such as a human, it is deactivated in a relatively short period of time or eliminated from the living body, and its effect is reduced. Is difficult.
  • a cross-linked product such as cross-linked gelatin carrying a bioactive factor is administered by a method in which it is embedded in the living body or injected into the living body using a syringe, the bioactive factor is gradually released. Therefore, the effect can be maintained over a long period of time compared to the case where the physiologically active factor is administered as an aqueous solution.
  • biodegradable polymeric water-soluble substances include chitin, chitosan, hyaluronic acid, alginic acid, starch and other polysaccharides, gelatin, collagen and other proteins, poly- ⁇ -glutamic acid, poly-L-lysine and other polyamino acids, And their derivatives, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid, polycaprolactone, copolymers of lactide and glycolide, etc.
  • gelatin is an animal protein having collagen as a parent substance. Therefore, the crosslinked gelatin is expected not only for use as a carrier material for sustained drug release but also for use in cosmetics. In addition, the crosslinked gelatin can be used for a wide range of applications such as industrial use. However, the conventional cross-linked gelatin still has important problems to be solved.
  • the biggest problem is that the conventional crosslinking method of biodegradable polymer water-soluble substances such as gelatin is substantially limited to the chemical crosslinking method using a crosslinking agent.
  • the chemical cross-linking method has a problem that in addition to being inferior in productivity and difficult to accurately control the degree of cross-linking, there are concerns about adverse effects on the living body due to residual chemicals such as cross-linking agents and reaction terminators. Have.
  • Patent Document 1 proposes a crosslinked gelatin gel preparation in which bFGF is supported on a crosslinked gelatin obtained by crosslinking gelatin.
  • Patent Document 1 describes that the gelatin can be crosslinked by heat treatment or ultraviolet irradiation in addition to the chemical crosslinking method using a crosslinking agent.
  • the Examples of Patent Document 1 only show a chemical crosslinking method using a crosslinking agent such as glutaraldehyde or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
  • Patent Document 1 describes that a crosslinked gelatin obtained using a crosslinking agent is washed with distilled water or an organic solvent.
  • Example 1 of Patent Document 1 describes that crosslinked gelatin was washed with distilled water at 37 ° C. for 12 hours.
  • a long time of 24 hours is also spent for the crosslinking reaction. Therefore, the method for producing a crosslinked gelatin using a crosslinking agent requires a great amount of time and labor for ensuring a crosslinking reaction and safety.
  • a photoinitiator is used to initiate a cross-linking reaction, and to terminate the cross-linking reaction, a reaction terminator is added to activate the photoinitiator activity. Need to be stopped. Since photoreaction initiators and reaction terminators are chemical substances, they have the same problems as the chemical crosslinking method.
  • the method of crosslinking gelatin by heat treatment requires a long time for crosslinking, and it is difficult to control the degree of crosslinking, and it is difficult to stably obtain a crosslinked gelatin having a desired degree of crosslinking. Therefore, it is difficult to obtain a crosslinked gelatin gel preparation that stably exhibits a desired sustained release property even when a drug such as a physiologically active factor is supported on the crosslinked gelatin obtained by heat treatment.
  • the cross-linked product such as cross-linked gelatin carrying a drug has a particle shape depending on the administration means.
  • cross-linked gelatin particles are suitable for cosmetic applications because they can be easily mixed with other components.
  • a crosslinked product such as crosslinked gelatin that does not contain harmful residual chemical substances and has a desired degree of crosslinking is formed into a particle shape. It was difficult to manufacture efficiently.
  • the method for producing crosslinked gelatin particles using the crosslinking agent disclosed in Patent Document 1 requires a great deal of time and labor to ensure safety by crosslinking reaction and purification, and there is a limit to reducing the product cost. there were. Even if the crosslinked gelatin particles are produced by adopting the crosslinking method by ultraviolet irradiation or heat treatment taught in Patent Document 1, such production method has various problems as described above.
  • Patent Document 2 discloses a layer configuration in which a plurality of crosslinked gelatin gel layers in which gelatin or a gelatin derivative is crosslinked by electron beam irradiation in an oxygen atmosphere are arranged adjacent to each other.
  • a cross-linked gelatin gel multilayer structure having the following and a method for producing the same are disclosed.
  • the focus is on improving the adhesiveness of the two sheets, and this is a method of irradiating an electron beam in an oxygen-containing atmosphere such as the air, so there is still room for improvement in the adsorptivity and sustained release of the drug. .
  • JP-A-2004-339395 discloses that a raw material containing gelatin is mixed with water or a buffer solution to form a mixture having a concentration of 5 to 50%, and pressure-molded to obtain a desired shape. After that, a method for producing a molded body made of gelatin that is irradiated with ionizing radiation at an irradiation dose of 1 kGy or more and less than 200 kGy is described. Patent Document 3 describes that this molded body can be used as a substitute for products conventionally molded from plastic as a film, container, etc. In addition, cell culture carriers, burns, wounds, pressure ulcers, scratches, etc. Alternatively, it is described that it is used as a medical substrate such as a skin defect agent such as a skin ulcer. However, there is no suggestion that this molded body is used as a carrier material for sustained release of an in vivo drug.
  • An object of the present invention is an in vivo drug sustained-release carrier comprising a hydrogel obtained by crosslinking a biodegradable polymer water-soluble substance, which is excellent in safety for a living body and is useful as a carrier for a drug such as a bioactive factor. It is to provide a material and a manufacturing method thereof.
  • an object of the present invention is to provide a carrier material for sustained release of an in vivo drug comprising a cross-linked hydrogel of a biodegradable polymer water-soluble substance capable of obtaining two types of drug release profiles, and a method for producing the same.
  • a carrier material for sustained release of in vivo drugs comprising a cross-linked hydrogel of a biodegradable polymer water-soluble substance capable of obtaining two types of drug release profiles, and a method for producing the same.
  • an initial burst in which a large amount of drug is released in a short time after implantation in the living body, depending on the type of drug such as a physiologically active factor carried.
  • the present invention meets the requirements of these two types of drug release profiles.
  • an object of the present invention is to provide a carrier material for sustained release of an in vivo drug comprising a cross-linked product of a biodegradable polymer water-soluble substance such as cross-linked gelatin, which has an excellent drug adsorbing ability as compared with a chemically crosslinked product It is in providing the manufacturing method.
  • the present inventors have conducted an in vivo drug slowdown comprising a hydrogel crosslinked with a biodegradable polymer water-soluble substance, such as a crosslinked gelatin gel sheet crosslinked with ionizing radiation.
  • a biodegradable polymer water-soluble substance such as a crosslinked gelatin gel sheet crosslinked with ionizing radiation.
  • the release characteristics of the drug in an aqueous solution were confirmed by loading the drug on a release carrier material.
  • the inventors investigated that the eluted substance may be eluted from the cross-linked gelatin gel sheet in an initial stage (a few seconds to several hours depending on the cross-linked state).
  • the cross-linked gelatin gel sheet has an uncrosslinked portion of gelatin having a low molecular weight, and the uncrosslinked portion of the gelatin carries a drug. Therefore, the present inventors control the drug release by utilizing the uncrosslinked portion of the gelatin of the crosslinked gelatin gel to roughly divide the two-stage drug release type including the initial burst (hereinafter referred to as “2”). And a linear release drug release type (hereinafter, also referred to as “linear release”) that is continuously released over a long period of time. It was conceived to obtain a crosslinked hydrogel of a biodegradable polymer water-soluble substance such as a crosslinked gelatin gel.
  • a biodegradable polymer water-soluble substance such as gelatin is irradiated with ionizing radiation in an inert gas atmosphere and crosslinked as a carrier material for sustained release of an in vivo drug.
  • a cross-linked gelatin sheet having a two-stage release property of the loaded drug was conceived even when it was formed as a single layer without laminating sheets of the carrier material for sustained release of in vivo drugs.
  • biodegradable polymer water-soluble substances such as gelatin are irradiated with ionizing radiation in an inert gas atmosphere and the carrier material for sustained release of in vivo drugs consisting of a hydrogel is washed with water.
  • a carrier material for sustained release of an in vivo drug that is substantially free of uncrosslinked portions, and by loading the drug on the carrier material for sustained release of the in vivo drug, a constant amount of the supported drug is obtained over a long period of time.
  • a cross-linked gelatin sheet having a linear release property that can be continuously released has been conceived.
  • An in vivo biodegradable polymer water-soluble substance comprising a hydrogel crosslinked by irradiating with ionizing radiation in an inert gas atmosphere,
  • the hydrogel has an uncrosslinked portion and a crosslinked portion that are soluble in water at 40 ° C. in a mass ratio of 95: 5 to 5:95, and After the carrier material carries the drug and is implanted in the living body, 55% by mass or more of the drug to be carried is released into the body until one day has passed, and the rest of the carried drug is gradually released from the second day onward.
  • In vivo drug sustained release carrier materials are provided.
  • the carrier material for in-vivo drug sustained release exhibiting the two-stage release property is washed with water, There is provided a carrier material for sustained release of an in-vivo drug that exhibits linear release and is composed of a crosslinked hydrogel substantially free of uncrosslinked portions.
  • a method for producing a carrier material for sustained release of an in vivo drug comprising a hydrogel obtained by crosslinking a biodegradable polymer water-soluble substance, While having an ionizing radiation irradiation step of crosslinking by irradiating ionizing radiation in an inert gas atmosphere,
  • the hydrogel has an uncrosslinked portion and a crosslinked portion that are soluble in water at 40 ° C. in a mass ratio of 95: 5 to 5:95, and After the carrier material carries the drug and is implanted in the living body, 55% by mass or more of the drug to be carried is released into the body until one day has passed, and the rest of the carried drug is gradually released from the second day onward.
  • a method for producing a carrier material for sustained release of an in vivo drug and After the ionizing radiation irradiation step, further comprising a washing step with water, There is provided a method for producing a carrier material for sustained release of an in-vivo drug that exhibits linear release and is composed of a crosslinked hydrogel substantially free of uncrosslinked portions.
  • the carrier material for sustained release of an in vivo drug comprising a hydrogel crosslinked with ionizing radiation according to the present invention does not use the harmful crosslinking agent used in the conventional chemical crosslinking method, it is safe for the living body. Therefore, complicated steps that have been performed by chemical crosslinking can be largely omitted.
  • the biomedical sustained release carrier material comprising a hydrogel cross-linked with ionizing radiation of the present invention has superior drug adsorption ability compared to a chemically cross-linked biodegradable polymer water-soluble substance, The controllability of the sustained release of the drug is enhanced, and the formulation design can be performed efficiently.
  • the chemical cross-linking reaction proceeds using the functional group of the side chain. As a result, the functional group that is the binding site of the drug is reduced, so that the drug-carrying adsorption capacity is low. Since the drug release is not caused by the biodegradation of the crosslinked product, the ability of drug release per hour is low.
  • the in vivo drug sustained release carrier material of the present invention is composed of a hydrogel crosslinked by irradiating a biodegradable polymer water-soluble substance with ionizing radiation in an inert gas atmosphere. It is a carrier material for sustained drug release in vivo.
  • Biodegradable polymer water-soluble substance The biodegradable polymer water-soluble substance used in the present invention is not particularly limited as long as it can be cross-linked by irradiation with ionizing radiation in an inert gas atmosphere. Commercially available products may be used, and examples thereof include gelatin, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid, polycaprolactone, and a copolymer of lactide and glycolide.
  • the biodegradable polymer water-soluble substance used in the present invention is usually one that can be dissolved in water at a concentration of 1 to 80% by mass.
  • gelatin is particularly preferable from the viewpoints of safety, solubility in water, cross-linkability by ionizing radiation, suitability for use as a cross-linked gel preparation and cosmetic ingredients, and the like. preferable.
  • gelatin will be mainly described as the biodegradable polymer water-soluble substance, but technical matters applicable to gelatin are other biodegradable highly biodegradable materials that can be cross-linked by irradiation with ionizing radiation. It can also be applied to molecular water-soluble substances.
  • gelatin that is preferably used as the biodegradable polymer water-soluble substance includes alkali-treated gelatin, acid-treated gelatin, and gelatin derivatives.
  • gelatin is mainly produced from cow bone, cow skin, and pig skin.
  • the parent substance that is converted into gelatin is a protein called collagen.
  • collagen which is a poorly soluble substance in water
  • acid or alkali the molecular structure of the triple-stranded helix is broken and separated into three random molecules.
  • Gelatin thus heat-denatured and solubilized is called narrowly defined gelatin.
  • the raw material is pretreated with an inorganic acid such as hydrochloric acid or sulfuric acid or lime (alkali).
  • the former is called acid-treated gelatin and the latter is called alkali-treated gelatin, depending on the raw material pretreatment conditions.
  • amphoteric electrolyte gelatin positive and negative in the molecule are balanced, and with respect to the isoelectric point which is the pH when the charge becomes zero as a whole, for example, alkali-treated gelatin having an isoelectric point of about 5, and isoelectric point Acid-treated gelatin with a point around 9 is included.
  • Gelatin derivatives are modified or modified gelatin side chains, cationized gelatin derivatives obtained by graft-polymerizing ethylenediamine, spermidine, or spermine with carbodiimide on acid-treated gelatin; Derivatives; etc. are typical ones.
  • the biodegradable polymer water-soluble substance at least one gelatin or gelatin derivative selected from the group consisting of alkali-treated gelatin, acid-treated gelatin, cationized gelatin derivatives, and succinylated gelatin derivatives is used. preferable.
  • Examples of commercially available gelatin include alkali-treated gelatin having an isoelectric point of 4.9 or 5.0 manufactured by Nitta Gelatin Co., Ltd .; acid-treated gelatin having an isoelectric point of 9.0 manufactured by Nitta Gelatin Co., Ltd .; And cationized gelatin derivatives (gelatin derivatives obtained by grafting ethylenediamine with carbodiimide to acid-treated gelatin having an isoelectric point of 9.0).
  • Ionizing radiation The present invention is to obtain a crosslinked hydrogel by irradiating a biodegradable polymer water-soluble substance such as gelatin with ionizing radiation in an inert gas atmosphere.
  • Ionizing radiation means ionizing radiation such as electron beam, ⁇ ray, ⁇ ray, and ⁇ ray. Specifically, electron beam or ⁇ ray is usually used for ease of handling. In particular, an electron beam is preferable. Ultraviolet rays are not included in ionizing radiation.
  • Inert Gas nitrogen, helium, argon, krypton, and the like can be used as the inert gas that forms an inert gas atmosphere when ionizing radiation is irradiated. Nitrogen is preferably used.
  • ionizing radiation when ionizing radiation is irradiated in an oxygen atmosphere, the workability of implantation in a living body decreases due to generation of ozone, decrease in the crosslinking reaction efficiency, increase in surface adhesion of the produced carrier material, In the present invention, ionizing radiation is not irradiated in an oxygen atmosphere because it may be difficult to control the proportion of the uncrosslinked portion of the resulting crosslinked gelatin gel.
  • the in vivo drug sustained release carrier material of the present invention can carry a drug, and after the drug is loaded and implanted in the living body, the carried drug is gradually released. If it is a thing, a shape will not be specifically limited. For example, any shape such as a columnar shape, a prismatic shape, a sheet shape, a disk shape, a spherical shape, or a particle shape can be used.
  • a carrier material for sustained release of an in vivo drug such as a crosslinked gelatin gel carrying a physiologically active factor is usually used in a method of being embedded in the living body by surgery, and in particular, a sheet-like material is preferable, but a particulate material Can also be administered by injection.
  • the carrier material for sustained release of an in vivo drug comprising the crosslinked hydrogel of the present invention is in the form of a sheet
  • a two-stage release property can be realized without combining two sheets having different degrees of crosslinking. It is not necessary to have a sheet.
  • a multilayer laminated sheet may be used.
  • Uncrosslinked part soluble in water at 40 ° C. The carrier material for sustained release of an in vivo drug of the present invention has an uncrosslinked part and a crosslinked part soluble in water at 40 ° C.
  • an uncrosslinked portion that is soluble in water at 40 ° C. (hereinafter, simply referred to as “uncrosslinked portion”) is an ionizable substance in a biodegradable polymer water-soluble substance in an inert gas atmosphere. It refers to an uncrosslinked portion in a hydrogel that has been crosslinked by irradiation with radiation and is soluble in water at 40 ° C.
  • the uncrosslinked portion is readily released from a crosslinked hydrogel of a biodegradable polymer water-soluble substance such as a crosslinked gelatin gel at body temperature in vivo.
  • Ratio of uncrosslinked portion soluble in water at 40 ° C. (%) (AB) / A ⁇ 100 (%) (1)
  • substantially free of uncrosslinked portion means that a hydrogel obtained by crosslinking a biodegradable polymer water-soluble substance such as a crosslinked gelatin gel is water at 40 ° C. or physiological saline at a temperature near body temperature. It means a state where there is no or only a slight release of uncrosslinked parts when contacted with a liquid based on water such as water or blood. Specifically, the hydrogel has a temperature of 40 ° C. This means that the uncrosslinked portion dissolved in water is less than 5% by mass based on the total of the uncrosslinked portion and the crosslinked portion.
  • the proportion of the uncrosslinked portion to be released is preferably less than 3% by mass, more preferably less than 1% by mass, and particularly preferably less than 0.5% by mass.
  • Drug The drug to be carried on the in vivo drug sustained release carrier material such as the cross-linked gelatin gel of the present invention is not particularly limited as long as it is desired to be sustained released after being embedded in the living body. Is preferred.
  • a physiologically active factor (also referred to as “physiologically active substance”) is a substance that expresses a physiological action or pharmacological action on a living organism, and is embedded in or administered to a living body while being supported on a carrier material. It can be freely selected within the range of the purpose of sustained release. In this case, even for the same drug, the required drug release profile may differ depending on the expected physiological activity.
  • bFGF basic fibroblast growth factor
  • bFGF is a substance that has been shown to stimulate cell proliferation not only to fibroblasts but also to many types of cells such as vascular endothelial cells, vascular smooth muscle cells, corneal endothelial cells, osteoblasts, and chondrocytes. is there.
  • vascular endothelial cells vascular smooth muscle cells
  • corneal endothelial cells corneal endothelial cells
  • osteoblasts chondrocytes.
  • chondrocytes chondrocytes.
  • TGF- ⁇ 1 transforming growth factor
  • HGF hepatocyte growth factor
  • PDGF-BB platelet-derived growth factor
  • KGF keratinocyte growth factor
  • BMP-2 bone morphogenetic factor
  • VEGF vascular endothelial growth factor
  • an anticancer agent (adriamycin), an angiotensin II receptor antagonist for organ protection and antihypertensive [Telmisartan: Micardis (Boehringer Ingelheim), Candesartan (Takeda Pharmaceutical Co., Ltd.), Valsartan (Novartis Pharma)] And erythropoietin (protein preparation, hematopoietic hormone) and the like.
  • an anticancer agent as adriamycin
  • an angiotensin II receptor antagonist for organ protection and antihypertensive [Telmisartan: Micardis (Boehringer Ingelheim), Candesartan (Takeda Pharmaceutical Co., Ltd.), Valsartan (Novartis Pharma)] And erythropoietin (protein preparation, hematopoietic hormone) and the like.
  • erythropoietin protein preparation, hematopoietic hormone
  • One type or two or more types of physiologically active factors to be carried on the carrier material for sustained drug release in vivo can be selected as necessary.
  • a different physiologically active factor can be carried for each sheet.
  • the carrier material for sustained release of an in vivo drug comprising a cross-linked hydrogel of a biodegradable polymer water-soluble substance such as the cross-linked gelatin gel of the present invention is a drug to be carried.
  • an uncrosslinked portion is used.
  • the carrier material for sustained release of an in vivo drug comprising a hydrogel crosslinked with a biodegradable polymer water-soluble substance such as a crosslinked gelatin gel of the present invention is roughly divided into two stages according to the use of the uncrosslinked portion. Either a releasable drug release or a linear release drug release can be selected. 1. Drug carrying performance
  • the drug-carrying performance is defined as the amount of drug remaining between the initial stage of implantation after the drug is carried on the carrier material for sustained release of the in-vivo drug of the present invention and implanted in the living body. Can be determined by measuring.
  • Crosslinking method that irradiates ionizing radiation because gelatin gel cross-linked by irradiation with ionizing radiation has been found to have higher drug adsorption capacity than chemically cross-linked gelatin gel, and can use the drug more efficiently Was found to be excellent. Although the detailed mechanism is unknown, it is assumed that the chemically cross-linked gelatin gel has reduced functional groups due to cross-linking and reduced adsorption capacity.
  • a carrier material for sustained release of an in vivo drug exhibiting two steps of release is a biodegradable polymer water-soluble substance such as gelatin, which is irradiated with ionizing radiation in an inert gas atmosphere.
  • a carrier material for sustained release of an in vivo drug such as a crosslinked gelatin gel that has been irradiated and crosslinked, wherein the uncrosslinked portion and the crosslinked portion that are soluble in water at 40 ° C. have a mass ratio of 95: 5 to 5:95.
  • a hydrogel having a range It is possible to have a two-step release property by loading the drug on both the uncrosslinked portion and the crosslinked portion by adsorption or the like.
  • the two-stage release property indicates an initial burst, that is, after the drug is supported on the in vivo drug sustained-release carrier material composed of the hydrogel and embedded in the living body, the initial stage (although it is slightly longer or shorter depending on the degree of crosslinking, Usually 55 days or more of the drug carried on the cross-linked gelatin gel is released, preferably 57% or more, more preferably 60% or more by weight. In addition to being released into the body, it means that the rest of the carried drug is sustainedly released in the latter period (after the second day). In the living body, as the uncrosslinked portion carrying the drug is eluted from the crosslinked gelatin gel at body temperature, the drug is delivered from the initial stage to the later stage to the necessary affected part or tissue.
  • the cross-linked gelatin gel cross-linked by irradiation with ionizing radiation can continuously release the drug for a period of up to about 4 weeks.
  • 55% by mass or more of the drug carried by the cross-linked gelatin gel is not released into the body after 1 day (24 hours) after the drug is loaded on the carrier material for sustained release in the living body and embedded in the living body
  • the upper limit of the amount of the drug released in the initial burst is not particularly limited as long as the remaining part of the drug carried in the latter period (after the second day) is sustainedly released, but usually 80% by mass or less, preferably 75 % By mass, more preferably 70% by mass.
  • a crosslinked gelatin gel obtained by electron beam irradiation is in a state containing a crosslinked part and an uncrosslinked part, and by adsorbing and supporting a drug such as a bioactive factor on both the crosslinked part and the uncrosslinked part. Two-stage release is exhibited.
  • the biodegradable polymer water-soluble substance can be crosslinked by irradiating it with ionizing radiation.
  • the obtained cross-linked gel such as a cross-linked gelatin gel having an uncross-linked portion and a cross-linked portion may be washed with water so as not to substantially contain the uncross-linked portion.
  • Linear release means that a drug is supported on a carrier material for sustained release of an in-vivo drug composed of a hydrogel, embedded in the living body, and continuously and substantially linear from the date of implantation in the living body to the seventh day and thereafter. That is, it means that the drug carried from the crosslinked gel of the biodegradable polymer water-soluble substance such as crosslinked gelatin gel is released at a substantially constant rate. That is, in the crosslinked gelatin gel substantially free of uncrosslinked portions, there is no initial burst of the drug to be carried, and the drug to be carried is released mainly due to the degradation of the crosslinked gelatin gel by metabolism in the living body. The release profile is gentle and the drug can be released continuously.
  • a cross-linked gel of a biodegradable polymer water-soluble substance such as a cross-linked gelatin gel that substantially does not contain an uncross-linked portion is embedded in the living body until one day (24 hours) elapses. It has been found that about 10 to 30% by mass of the drug carried is released into the body. This is because some of the drugs to be supported on the crosslinked gelatin gel include those that are not sufficiently adsorbed on the crosslinked gelatin gel, and there are relatively low molecular weight crosslinked portions in the crosslinked portion of the crosslinked gelatin gel. It is presumed that this occurs due to early degradation in the body.
  • the linear release property means that a drug release profile in which the drug release property from the second day onward is almost linear after being implanted in the living body is obtained.
  • the drug release profile has a constant rate portion within a range of preferably 1 to 20% by mass / day, more preferably 2 to 15% by mass / day.
  • the crosslinked gelatin gel substantially free of uncrosslinked portions can release the drug continuously for a period of up to 4 weeks.
  • at least about 55% by mass of the loaded drug is released within two days immediately after the drug is loaded and embedded in the living body. It has been found that this does not result in a certain amount of drug release.
  • a coating layer of an aqueous solution of a biodegradable polymer water-soluble substance such as gelatin or a gelatin derivative is formed (coating). Construction layer formation process). Specifically, an aqueous solution of gelatin or a gelatin derivative is adjusted to a temperature of 15 to 80 ° C., preferably 20 to 70 ° C., more preferably 40 to 60 ° C. The coating layer is formed by casting in a mold (container) with a desired capacity. When the temperature of the aqueous solution is too low, casting becomes difficult, and a coating layer having a uniform thickness may not be formed. If the temperature of the aqueous solution is too high, the volatilization of moisture increases and it becomes difficult to maintain a desired concentration.
  • the mold (container) is preferably a surface material that does not repel the aqueous solution when an aqueous solution of gelatin or a gelatin derivative is cast, and glass, metal, or various plastic materials can be used. Further, the shape of the mold (container) is not particularly limited as long as a coating layer having a desired thickness and a uniform thickness can be formed.
  • the concentration of the aqueous solution of the biodegradable polymer water-soluble substance is generally 1 to 80% by mass, preferably 3 to 60% by mass, more preferably 5 to 50% by mass, still more preferably 7 to 40% by mass, Particularly preferably, it is in the range of 10 to 30% by mass.
  • the concentration of the aqueous solution is preferably within a range suitable for each biodegradable polymer water-soluble substance such as gelatin species. For example, in the case of high molecular weight gelatin having an average molecular weight of 100,000 or more, since the solution viscosity is high, the concentration of the aqueous solution is preferably in the range of 5 to 30% by mass.
  • the concentration of the aqueous solution is preferably 7 to 60% by mass, more preferably 10 to 50% by mass, and particularly preferably 15 to 40% by mass. If the concentration of the aqueous solution is too low, the crosslinking density of the crosslinked gelatin gel is too small, and it may be difficult to provide sufficient drug carrying performance. If the concentration of the aqueous solution is too high, it may be difficult to form a coating layer having a uniform thickness during casting.
  • the thickness of the coating layer is selected in consideration of the dose of ionizing radiation to be irradiated and the ratio of the uncrosslinked portion of the crosslinked gelatin gel, but is usually in the range of 50 ⁇ m to 8 mm, preferably Is 200 ⁇ m to 6 mm, more preferably 300 ⁇ m to 5 mm, particularly preferably 500 ⁇ m to 3 mm. If the coating layer is too thin, the in vivo drug sustained release carrier material crosslinked by irradiation with ionizing radiation will not have sufficient drug carrying capacity, or the ratio between the uncrosslinked part and the crosslinked part will be reduced. It may be difficult to adjust.
  • the thickness of the coating layer is too thick, it may be necessary to increase the acceleration voltage excessively, or it may be difficult to adjust the ratio between the uncrosslinked portion and the crosslinked portion.
  • the thickness of the coating layer is substantially maintained even after crosslinking by irradiating with ionizing radiation.
  • the coating layer is irradiated with ionizing radiation in an inert gas atmosphere to obtain a crosslinked gelatin gel (ionizing radiation irradiation step).
  • ionizing radiation an electron beam is preferably used.
  • a general-purpose electron beam irradiation apparatus can be used to irradiate the coating layer with the electron beam.
  • irradiation with an acceleration voltage upper limit of 800 kV using an electron beam irradiation apparatus “Scanning Electron Irradiation Apparatus EPS-800” manufactured by NHV Corporation can be exemplified, but the present invention is not limited to this apparatus.
  • Electron beam irradiation is performed by irradiating the coating layer with a desired irradiation dose (kGy) defined by the electron current (mA) and the moving speed of the irradiated object (m / min) in the irradiation area, Crosslink biodegradable polymer water-soluble substances.
  • Irradiation with an electron beam may be performed by selecting optimum conditions depending on the type and shape of the biodegradable polymer water-soluble substance, the thickness of the coating layer, and the like.
  • an acceleration voltage of 100 kV to 3 MV is preferable in a nitrogen atmosphere.
  • the attenuation of electron beam energy that may occur in the thickness direction of the coating layer is simple. Since it is not a proportional relationship, it is necessary to adjust the acceleration voltage and the irradiation dose.
  • the electron beam irradiation is usually performed once, but the acceleration voltage can be changed and the electron beam irradiation can be performed in several times.
  • an optimal range may be selected according to the electron beam irradiation.
  • a gelatin or gelatin derivative aqueous solution is cast on the crosslinked sheet-like gelatin gel layer in the same manner as described above, and then irradiated with an electron beam.
  • a crosslinked gelatin gel multilayer structure having a desired number of layers and a total thickness can be obtained.
  • the crosslinked gelatin gel is not usually washed with water such as ultrapure water, distilled water or ion exchange water.
  • a certain amount of uncrosslinked parts can be removed by washing with water.
  • water such as ultrapure water, distilled water or ion exchange water to remove a specific amount of the uncrosslinked portion.
  • the mass ratio of the uncrosslinked portion and the crosslinked portion soluble in water at 40 ° C. is in the range of 95: 5 to 5:95, preferably 95: 5 to 20:80, more preferably 95: 5 to 30:70.
  • the recovered sheet-like carrier material may be stored as it is, but if it is dried under reduced pressure or freeze-dried (freeze-dried), it can be stored for a relatively long period of time.
  • particulate carrier material When producing a spherical, particulate, granular (hereinafter, collectively referred to as “particulate”) crosslinked gelatin gel, an aqueous gelatin solution is dispersed in oil, A method of preparing a W / O type emulsion in which droplets of the gelatin aqueous solution are dispersed (an emulsion in which an aqueous phase is dispersed in an oil phase) can be suitably employed. By adjusting the dispersion size of the aqueous phase in the W / O type emulsion, the size of the obtained particulate crosslinked gelatin gel can be designed within a desired range.
  • the oil used for forming the oil phase is preferably a vegetable oil, and examples thereof include olive oil, soybean oil, and corn oil.
  • Preparation of a W / O type emulsion can be performed using the method conventionally known as an emulsion formation method. For example, after adding the gelatin aqueous solution to the oil, stirring is preferably performed for 1 minute or more, more preferably 5 minutes or more, and even more preferably 10 minutes or more. In this case, a W / O emulsion having a particularly uniform dispersion can be obtained. In terms of uniformity of dispersion, it is preferable that the stirring time in the formation of the emulsion is long. However, if the stirring time is too long, physical properties such as gelatin gelation ability may deteriorate, so the stirring time is 24 hours or less. , Preferably 12 hours or less, more preferably 5 hours or less.
  • a stirring motor and a propeller for stirring made of fluororesin are attached to a three-necked round bottom flask, and an gelatin aqueous solution is put into a device in which these are fixed, and oil such as olive oil is added thereto to about 200 to 600 rpm. It can be made into a W / O type emulsion by stirring at a speed of.
  • the emulsion is irradiated with ionizing radiation such as an electron beam, and then particulate crosslinked gelatin is collected by centrifugation through a filter having a predetermined mesh interval, and washed with acetone, ethyl acetate, IPA, ethanol or the like. As a result, a particulate crosslinked gelatin gel can be obtained.
  • ionizing radiation such as an electron beam
  • the average particle diameter of the particulate crosslinked gelatin gel is in the range of 1 to 500 ⁇ m, preferably in the range of 1.5 to 200 ⁇ m, more preferably in the range of 2 to 100 ⁇ m, especially for handling work using a syringe. Convenient.
  • the average particle diameter is a value measured by a method of detecting laser scattering using a laser diffraction analyzer, for example.
  • ultrasonic irradiation preferably within about 1 minute under cooling
  • the crosslinked gelatin gel is usually not washed with water.
  • the biodegradable polymer water-soluble substance is ionized such as an electron beam in an inert gas atmosphere.
  • the carrier material for sustained release of an in vivo drug is subjected to water such as ultrapure water, distilled water or ion exchange water.
  • a method of eluting uncrosslinked portions by performing a cleaning process may be used (cleaning process step).
  • an in vivo body comprising a crosslinked hydrogel in water at a temperature of 5 to 50 ° C., preferably 10 to 45 ° C., more preferably 15 to 42 ° C.
  • the washing operation in which the carrier material for sustained drug release is immersed for 10 minutes to 2 days, preferably 20 minutes to 1 day, more preferably 30 minutes to 10 hours, is performed 1 to 10 times, preferably 1 to 6 times. More preferred is a method of repeating 2 to 4 times.
  • a washing treatment step with water for eluting uncrosslinked portions can be performed before or after laminating the multilayer structure, In order not to make the process complicated, it is preferable to perform the washing treatment after all the layers have been laminated.
  • the carrier material for sustained release of an in vivo drug according to the present invention is for loading a drug such as a bioactive factor.
  • the method for supporting the drug on the carrier material for sustained release of the in vivo drug of the present invention is not particularly limited.
  • a drug solution a drug dissolved in an aqueous solution
  • the crosslinked gelatin gel is impregnated to impregnate the drug.
  • There are a method of adsorbing and supporting a drug a method of immersing a cross-linked gelatin gel in an aqueous solution of the drug, and adsorbing and supporting the drug by impregnation.
  • the amount of the drug that can be supported on the crosslinked gelatin gel varies depending on the ratio of the uncrosslinked portion and the crosslinked portion of the crosslinked gelatin gel and the moisture content, but is usually 0.1 to 500 ⁇ g per 1 mg of the crosslinked gelatin gel, preferably 1 Up to 450 ⁇ g, more preferably 5 to 400 ⁇ g can be supported.
  • the obtained cross-linked gelatin gel can adsorb the drug solution by impregnation or the like even in a hydrogel state to carry the drug.
  • the cross-linked gelatin gel can be dried under reduced pressure or freeze-dried (freeze-dried) to remove the drug solution. It is possible to impregnate quickly.
  • a cross-linked gelatin gel is frozen under the condition that it is kept in liquid nitrogen for 30 minutes or more or in an ultra-low temperature freezer at -90 ° C to -80 ° C for 1 hour or more.
  • the method of making it a freeze-dried body by taking the process adjusted to 30 degreeC so that it may not dry at the time of taking out and dew condensation at the time of taking out is mentioned.
  • a carrier material for sustained release of an in vivo drug consisting essentially of the crosslinked portion having substantially no uncrosslinked portion, and uncrosslinked After separating into a carrier material for sustained release of an in vivo drug consisting of only a part, each is lyophilized to form a powder.
  • the powdered in vivo drug sustained release carrier material consisting only of the crosslinked part and the in vivo drug sustained release carrier material consisting only of the uncrosslinked part are each adsorbed and supported by impregnation with a chemical solution, and then crosslinked.
  • a carrier material for sustained release of an in vivo drug that exhibits two-stage release properties can be obtained. Since it is in a powder state, it is possible to easily measure the weight of the carrier material for sustained release of an in vivo drug consisting only of a crosslinked portion and the carrier material for sustained release of an in vivo drug consisting of only an uncrosslinked portion. It becomes easier to control the staged release.
  • the sheet-like cross-linked gelatin gel was freeze-dried and the initial weight (A) was measured. Subsequently, after leaving still for one day in 40 degreeC ultrapure water obtained using MILIPORE (use column: SIMAKKRJ) by Yamato Scientific Co., Ltd., after removing the uncrosslinked part in a crosslinked gelatin gel, 50 degreeC And dried for 1 day to measure the weight (B) after removal of the uncrosslinked portion. From the above formula (1), the ratio of the uncrosslinked part in the crosslinked gelatin gel was determined, and the ratio of the crosslinked part was determined as the ratio of 100-uncrosslinked part. Table 1 shows the results of determining the ratio (mass%) between the crosslinked part and the uncrosslinked part.
  • the ratio of the cross-linked portion increases with the increase of the irradiation dose, but even when the irradiation dose exceeds 20 kGy, the ratio of the cross-linking portion does not increase with the increase of the irradiation dose.
  • the acceleration voltage of the electron beam is 800 kV
  • the energy attenuation of the electron beam occurs in the deep part of the uniform coating layer having a thickness of 3 mm
  • the irradiation dose increases to 50 kGy or more
  • the decomposition reaction of gelatin occurs. It is inferred that the number of uncrosslinked parts that are soluble in water at 40 ° C. increases.
  • Example 1 A uniform coating layer having a thickness of 3 mm is cast by casting an aqueous solution (concentration: 10% by mass) of beef bone type I collagen with alkali-treated gelatin (Nitta Gelatin Co., Ltd .; isoelectric point 5.0) in a mold. Formed. Next, without substantially drying the water in the coating layer, an electron beam irradiation system EPS-800 (manufactured by NHV Corporation) is used from above the coating layer in an atmosphere of nitrogen. Was irradiated at an accelerating voltage of 800 kV and an irradiation dose of 20 kGy to form a single-layer sheet-like crosslinked gelatin gel. The proportion of the uncrosslinked portion of the obtained crosslinked gelatin gel was 68% by mass, and the proportion of the crosslinked portion was 32% by mass.
  • EPS-800 electron beam irradiation system
  • bFGF for biochemistry, purchased from Wako Pure Chemical Industries, Ltd.
  • a fluorescent reagent AlexalexFlour 488 (manufactured by invitrogen)
  • a protein labeling kit Alexa Flour 488 Protein Labeling Kit (manufactured by invitrogen)
  • the freeze-dried sheet-like cross-linked gelatin gel 5 mg was impregnated with 100 ⁇ l of 1 mg / ml bFGF aqueous solution, and the drug was adsorbed and supported on the cross-linked gelatin gel.
  • a cross-linked gelatin gel carrying a drug was implanted subcutaneously on the back of a mouse (ddY 4-week-old female).
  • the cross-linked gelatin gel was taken out and treated with 0.05% trypsin (protease, Wako Pure Chemical Industries, Ltd.) in 200 ⁇ l of ultrapure water for 24 hours at 40 ° C.
  • trypsin prote, Wako Pure Chemical Industries, Ltd.
  • the gel was dissolved, and the residual bFGF concentration in the crosslinked gelatin gel was measured by measuring fluorescence at 535 nm with a luminometer (PerkinVOElmer, ARVOMX1420). The obtained results are shown in FIG. 1 with the residual bFGF as a residual rate compared to the initial input amount.
  • the crosslinked gelatin gel of Example 1 comprising a hydrogel crosslinked by irradiating with an electron beam in an inert gas atmosphere has 68% by mass of an uncrosslinked portion and is supported by 1 day after being embedded in the living body. About 69% by mass of the bFGF thus released is released, and then the rest of the supported bFGF is released gradually. Particularly, after the third day, the supported bFGF is gradually released at a substantially constant rate until the 14th day. It shows staged release.
  • Example 1 The residual ratio of the drug was examined in the same manner as in Example 1 except that the thickness of the coating layer of the gelatin aqueous solution before cross-linking was 1 mm. The obtained results are shown in FIG. In addition, the ratio of the uncrosslinked part of the obtained crosslinked gelatin gel was 37 mass%, and the ratio of the crosslinked part was 63 mass%.
  • the crosslinked gelatin gel of Comparative Example 1 composed of electron beam crosslinked hydrogel has 37% by mass of an uncrosslinked portion, but by 1 day after being embedded in the living body, Since about 50% by weight is released, it does not show an initial burst. However, after the second day, the supported bFGF is gradually released until the 14th day at a substantially constant rate.
  • the crosslinked gelatin gel of Comparative Example 1 does not exhibit a two-stage release property.
  • the cross-linked gelatin gel of Comparative Example 2 composed of a chemically cross-linked hydrogel releases about 42% by mass of the supported bFGF until 1 day has passed after being embedded in the living body, By the end of 2 days, about 71% by mass of the supported bFGF has been released, but it is not clear whether the supported bFGF is being released gradually thereafter.
  • the crosslinked gelatin gel of Comparative Example 2 does not exhibit a two-stage release property.
  • the carrier material for sustained release of an in vivo drug comprising a hydrogel crosslinked by irradiation with ionizing radiation of Example 1 showing a two-stage release property contains a large amount of uncrosslinked parts and undergoes initial burst in the initial stage of in vivo implantation.
  • This is a cross-linked gelatin gel with a large amount of drug release and capable of sustained and quantitative sustained drug release after the end of bursting, but it can be said to have excellent angiogenic efficiency.
  • the in vivo drug sustained release carrier material showing a two-stage release profile of the present invention is useful when the in vivo drug sustained release carrier material carrying a growth factor such as bFGF as a drug. I found out.
  • Example 2 20% by mass of pig skin-derived type I collagen acid-treated gelatin (Nitta Gelatin Co., Ltd., isoelectric point 9.0) aqueous solution (hereinafter referred to as “20% PI-9”) is cast into the mold. Thus, a uniform coating layer having a thickness of 1 mm was formed. Next, without substantially drying the water in the coating layer, an electron beam irradiation system EPS-800 (manufactured by NHV Corporation) is used from above the coating layer in an atmosphere of nitrogen. Was irradiated at an accelerating voltage of 800 kV and an irradiation dose of 20 kGy to form a single-layer sheet-like crosslinked gelatin gel.
  • EPS-800 electron beam irradiation system
  • the step of allowing the crosslinked gelatin gel to stand in ultrapure water at 40 ° C. for 4 hours was repeated twice. Thereafter, the crosslinked gelatin gel was freeze-dried to obtain a dried sheet-like crosslinked gelatin gel.
  • the ratio of the uncrosslinked portion dissolved in water at 40 ° C. of the obtained crosslinked gelatin gel was 0.1% by mass with respect to the total of the uncrosslinked portion and the crosslinked portion.
  • Example 3 A dry sheet-like crosslinked gelatin gel was obtained in the same manner as in Example 2 except that the operation of washing with water for eluting uncrosslinked portions was not performed.
  • the ratio of the uncrosslinked portion dissolved in water at 40 ° C. of the obtained crosslinked gelatin gel was 42 mass%, and the ratio of the crosslinked portion was 58 mass%.
  • Example 2 Using 1000 ⁇ l of 1/10 PBS solution of each freeze-dried gel not containing DNA as a control, the amount of DNA released from the crosslinked gelatin gel was calculated from the difference, and the adsorption rate was evaluated.
  • the evaluation results of Example 2 and Comparative Examples 3 and 4 are shown in FIG.
  • Example 2 and Comparative Example 3 using a crosslinked gelatin gel crosslinked by irradiating an electron beam were chemically crosslinked.
  • Comparative Example 4 using the crosslinked gelatin gel thus obtained, it can be seen that the salmon DNA adsorption rate is high and the drug carrying ability is excellent.
  • the chemical cross-linked product has a larger decrease in adsorption rate.
  • Example 3 A sheet-like cross-linked gelatin gel was obtained in the same manner as in Example 2 except that the acceleration voltage of the electron beam was 800 kV, the irradiation dose was 20 kGy, the casting thickness was 500 ⁇ m, and then the salmon DNA was adsorbed and supported. I let you. In addition, the ratio of the uncrosslinked part which melt
  • Example 4 A sheet-like cross-linked gelatin gel was obtained in the same manner as in Example 2 except that the acceleration voltage of the electron beam was 800 kV, the irradiation dose was 50 kGy, and the casting thickness was 500 ⁇ m, and then the salmon DNA was adsorbed and supported. I let you.
  • dissolves in 40 degreeC water of the obtained crosslinked gelatin gel was 0.1 mass% with respect to the sum total of an uncrosslinked part and a crosslinked part.
  • Example 5 A sheet-like cross-linked gelatin gel was prepared in the same manner as in Example 2 except that the acceleration voltage of the electron beam was 800 kV, the irradiation dose was 50 kGy, and no washing operation with water was used to elute uncrosslinked portions. After obtaining, salmon DNA was adsorbed and supported. In addition, the ratio of the non-crosslinked part which melt
  • Example 5 10% by mass of beef bone type I collagen-treated gelatin (Nitta Gelatin Co., Ltd .; isoelectric point 5.0) aqueous solution (hereinafter referred to as “10% PI-5”) was cast to A sheet-like crosslinked gelatin gel was obtained in the same manner as in Example 2 except that the line acceleration voltage was 800 kV and the irradiation dose was 10 kGy, and then salmon DNA was adsorbed and supported. In addition, the ratio of the uncrosslinked part which melt
  • the prepared sheet-like cross-linked gelatin gel was cut into about 5 mg, the initial weight was precisely measured, and this was embedded in the back of the mouse (ddY 4-week-old female). Thereafter, the cross-linked gelatin gels on the 1st to 48th days were taken out of the mouse, the dry weight after freeze-drying was measured, and the residual ratio was obtained by comparing with the initial weight. The results are shown in FIG.
  • the slope of the regression line shows a constant rate release profile for Example 3 at about 5% by weight / day, Example 4 at about 2% by weight / day, and Example 5 at about 10% by weight / day, A linear release of the drug over a predetermined period is expected.
  • Comparative Example 5 in which the washing treatment with water for eluting the uncrosslinked portion was not performed, a large amount of about 70% by mass of the drug was released at the initial stage (one day after the embedding), and thereafter, the constant rate gradually increased. The release is about 1.2% by mass / day, indicating a two-stage release. Since the DNA carried on the cross-linked gelatin gel flows out in a large amount in the initial stage, the DNA release profile is not preferable.
  • Example 6 A sheet-like crosslinked gelatin gel was obtained in the same manner as in Example 2 except that the acceleration voltage of the electron beam was 800 kV and the irradiation dose was 50 kGy. The ratio of the uncrosslinked portion dissolved in water at 40 ° C. of the obtained crosslinked gelatin gel was 0.1% by mass with respect to the total of the uncrosslinked portion and the crosslinked portion.
  • Example 6 A sheet-like cross-linked gelatin gel was prepared in the same manner as in Example 2, except that the electron beam acceleration voltage was 800 kV, the irradiation dose was 50 kGy, and no washing treatment with water was performed to elute uncrosslinked portions. Got. The ratio of the non-crosslinked portion dissolved in water at 40 ° C. of the obtained crosslinked gelatin gel was 29 mass%, and the ratio of the crosslinked portion was 71 mass%.
  • the loading of BMP-2 and the evaluation of bone formation were performed by the following methods.
  • Example 6 30 ⁇ l of PBS solution (pH 7.2-7.4, Nippon Suisan Co., Ltd.) containing 5 ⁇ g of bone formation factor BMP-2 (Bone Morphogenetic Protein-2, for biochemistry, purchased from Wako Pure Chemical Industries, Ltd.) Manufactured in Example 6), and the crosslinked gelatin gel prepared as Comparative Example 6 containing both the uncrosslinked portion and the crosslinked portion was cut into about 5 mg each.
  • the sheet thus obtained is impregnated overnight, and BMP-2 is adsorbed and supported thereon. This is embedded into the mouse dorsal tail subcutaneously from the cut surface of the neck of the mouse (ddY 5-week-old female), and then the cut surface of the epidermis is sutured.
  • mice are sacrificed on days 1, 7, and 14, and the gel and surrounding subcutaneous tissue (2 ⁇ 2 cm) are scraped off with a scalpel and enclosed in a microtube. Immediately freeze using liquid nitrogen and freeze dry. The freeze-dried body is homogenized and 5 mg of the powder tissue is taken out. This is suspended in 1 ml of sample buffer (0.2% IGEPAL CA-630, 10 mM Tris-HCl, 1 mM MgCl 2 , pH 7.5) and centrifuged at 12,000 rpm at 4 ° C. for 15 minutes, and the supernatant is separated from the sample solution. To do. Alkaline phosphatase (ALP) is present in osteoblasts and serves as an index of bone formation.
  • ALP Alkaline phosphatase
  • ALP alkaline fosterase activity
  • Example 6 the crosslinked gelatin gel of Example 6 impregnated with a PBS solution not containing BMP-2 (referred to as “healthy”) and PBS containing BMP-2 without using the crosslinked gelatin gel were used.
  • the mice were sacrificed on the 14th day using the solution injected into the vicinity of the gel implant (referred to as “injection”), and alkaline fosterase activity was evaluated.
  • Example 6 using a crosslinked gelatin gel that was washed with water and was substantially free of uncrosslinked portions, the 14th day after implantation in the living body.
  • the ALP is about 5 times that of the control (healthy).
  • Blood vessels were induced around the sample of Example 6, and according to the X-ray photograph of the blood vessel portion, a dark white portion was present around the biomaterial. When this part was touched, it was found that the bone was formed because it had a hardness not found in gelatin gel.
  • a carrier material for sustained release of an in vivo drug comprising a cross-linked hydrogel of a biodegradable polymer water-soluble substance that is excellent in safety for a living body and is useful as a carrier for a drug such as a bioactive factor, and its production Method, specifically, biodegradation that can obtain two types of drug release profiles, ie, two-stage release or linear release, having an initial burst in vivo, with a bioactive agent drug carried in vivo
  • a carrier material for sustained release of an in vivo drug comprising a crosslinked hydrogel of a water-soluble polymeric water-soluble substance and a method for producing the same are provided.

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Abstract

L'invention concerne un matériau vecteur permettant la libération prolongée in vivo d'un médicament, lequel matériau vecteur est formé d'une substance réticulée hydrosoluble, de poids moléculaire élevé et biodégradable. Ledit matériau vecteur peut présenter deux types de profils de libération de médicament, à savoir un profil de libération linéaire et un profil de libération en deux étapes, et il possède une capacité d'adsorption de médicament supérieure à celle d'une substance réticulée chimiquement, hydrosoluble, de poids moléculaire élevé et biodégradable, ce qui lui permet de produire un effet thérapeutique optimal. L'invention concerne également un procédé de fabrication dudit matériau vecteur. Le matériau vecteur permettant la libération prolongée in vivo d'un médicament selon l'invention comprend un hydrogel préparé par réticulation d'une substance hydrosoluble de poids moléculaire élevé, biodégradable, telle que la gélatine, au moyen d'un rayonnement ionisant dans une atmosphère de gaz inerte (par exemple de l'azote). L'hydrogel comporte une partie non-réticulée hydrosoluble à 40°C et une partie réticulée selon un rapport massique de 95:5 à 5:95, de préférence de 95:5 à 55:45. Ledit matériau vecteur, dont la forme permet de transporter un médicament, présente une capacité de libération en deux étapes après enrobage in vivo. L'invention concerne également un matériau vecteur permettant la libération prolongée in vivo d'un médicament, lequel matériau comprend l'hydrogel précité qui a été lavé avec de l'eau et ne comporte pratiquement pas de partie non-réticulée. L'invention concerne de plus des procédés de fabrication de ces matériaux vecteurs.
PCT/JP2009/068754 2009-11-02 2009-11-02 Matériaux vecteurs permettant la libération prolongée in vivo d'un médicament et comprenant un hydrogel réticulé par rayonnement ionisant, et leur procédé de fabrication WO2011052089A1 (fr)

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