JPWO2011052089A1 - In vivo drug sustained release carrier material comprising hydrogel crosslinked with ionizing radiation and method for producing the same - Google Patents

Abstract

Two types of drug release profiles, linear release and two-stage release, can be controlled, and the drug adsorption capacity is superior to chemically cross-linked biodegradable polymer water-soluble substances. In addition, a carrier material for sustained release of an in vivo drug derived from a biodegradable polymer water-soluble substance and a method for producing the same are provided. A biomaterial sustained release carrier material comprising a hydrogel obtained by crosslinking a biodegradable polymer water-soluble substance such as gelatin in an inert gas atmosphere such as nitrogen with ionizing radiation, the hydrogel having a temperature of 40 ° C. It has an uncrosslinked portion and a crosslinked portion that are soluble in water in a mass ratio of 95: 5 to 5:95, preferably 95: 5 to 55:45, and the carrier material carries a drug. In vivo drug sustained release carrier material that exhibits two-stage release properties after being embedded in the living body, or in vivo drug sustained release carrier material that is substantially free of uncrosslinked portions obtained by washing the hydrogel with water And methods for producing these carrier materials.

Description

  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 biosafety 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. Thus, for example, 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 a drug such as a bioactive factor. When a cross-linked product such as cross-linked gelatin carrying a bioactive factor such as a growth factor is administered in vivo, the supported bioactive factor is gradually released as the cross-linked product such as cross-linked gelatin is decomposed in vivo.

  As a physiologically active factor, for example, basic fibroblast growth factor (bFGF) having an angiogenesis-inducing effect, which is a growth factor, is known. When such 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. In contrast, when 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 polymer water-soluble substances include chitin, chitosan, hyaluronic acid, alginic acid, starch and other polysaccharides, gelatin, collagen and other proteins, poly-γ-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. Among them, 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.

  International Publication No. 1994/27630 (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. However, 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.

  In the chemical crosslinking method of gelatin, it is necessary to add a reaction terminator made of a chemical substance in the reaction system in order to carry out a crosslinking reaction using a crosslinking agent made of a chemical substance and to terminate the crosslinking reaction. Even when a low-toxic crosslinking agent or reaction terminator is used, since these are chemical substances, there is a risk of adverse effects on the living body even in a trace amount. Therefore, it is necessary to pay attention to ensuring safety, for example, by sufficiently purifying the crosslinked gelatin so that no crosslinking agent or reaction terminator remains. Patent Document 1 describes that a crosslinked gelatin obtained using a crosslinking agent is washed with distilled water or an organic solvent. Specifically, Example 1 of Patent Document 1 describes that crosslinked gelatin was washed with distilled water at 37 ° C. for 12 hours. In Example 1 of Patent Document 1, 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.

  On the other hand, in the method of crosslinking by irradiating gelatin with ultraviolet rays, 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.

  In the method of crosslinking gelatin by heat treatment, it takes a long time for crosslinking, and it is difficult to control the degree of crosslinking, and it is difficult to stably obtain 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.

  In addition, the cross-linked product such as cross-linked gelatin carrying a drug may be desirably in the form of particles depending on the administration means. For example, cross-linked gelatin particles are suitable for cosmetic applications because they can be easily mixed with other components. However, in the conventional method for producing a crosslinked product of a biodegradable polymer water-soluble substance such as crosslinked gelatin, 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.

  That is, 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.

  As a method for producing cross-linked gelatin, in addition to chemical cross-linking, ultraviolet irradiation, heat treatment, etc., a method using electron beam cross-linking is known.

  International Publication No. 2008/016163 (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. However, 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. .

  Furthermore, in Japanese Patent Application Laid-Open No. 2004-339395 (Patent Document 3), a raw material containing gelatin and water or a buffer solution are mixed to form a mixture having a concentration of 5 to 50%, and pressure-molded to obtain a required 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.

International Publication No. 1994/27630 International Publication No. 2008/16163 JP 2004-339395 A

  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.

  In particular, 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. There is. In other words, in the carrier material for sustained release of in vivo drugs, 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. There are those that are desired, and those that are desired to continue to release a certain amount of drug over a long period of time after being implanted in a living body, and the present invention meets the requirements of these two types of drug release profiles.

  Furthermore, 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 compared with a chemically cross-linked product. It is in providing the manufacturing method.

  In order to solve the above-mentioned problems, 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. The release characteristics of the drug in an aqueous solution were confirmed by loading the drug on a release carrier material. In this case, 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). As a result, it was found that 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.

  Specifically, 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. By utilizing the uncrosslinked portion, 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. In addition, 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. To obtain 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.

Thus, according to the present invention,
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. Which exhibits a two-stage release property,
In vivo drug sustained release carrier materials are provided.

Moreover, according to the present invention,
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.

Furthermore, according to the present invention,
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. Which exhibits a two-stage release property,
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 that is substantially free of uncrosslinked portions.

  Since 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.

  In addition, 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. In the case of a chemically cross-linked carrier material, 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 figure which shows the sustained release property of the gelatin hydrogel which carry | supported the chemical | medical agent bFGF. The figure which shows the adsorptivity and discharge | release property of salmon DNA. The figure which shows the washing | cleaning removal of an unbridged part, and the in-vivo residual rate. The figure which shows examination of the bone formation by BMP-2 sustained release.

A. In vivo drug sustained release carrier material 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.

1. 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.

  Among these biodegradable polymer water-soluble substances, 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. Thus, hereinafter, 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.

  In the present invention, the gelatin preferably used as the biodegradable polymer water-soluble substance includes alkali-treated gelatin, acid-treated gelatin, and gelatin derivatives.

  That is, gelatin is mainly produced from cow bone, cow skin, and pig skin. Among these raw materials, the parent substance that is converted into gelatin is a protein called collagen. When collagen, which is a poorly soluble substance in water, is pretreated with acid or alkali and then heated, 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. In order to extract gelatin from a collagen raw material, 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. As the 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.

  A gelatin derivative is a modified or modified gelatin side chain, a cationized gelatin derivative obtained by graft polymerization of ethylenediamine, spermidine or spermine with carbodiimide on acid-treated gelatin; Derivatives; etc. are typical ones.

  In the present invention, as 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).

2. 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, α ray, β ray, and γ ray. Specifically, electron beam or γ ray is usually used for ease of handling. In particular, an electron beam is preferable. Ultraviolet rays are not included in ionizing radiation.

3. Inert Gas In the present invention, 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. In addition, 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.

4). In vivo drug sustained release carrier material 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.

  As a drug, 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.

  When 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. However, in particular, when the thickness of the sheet needs to be 6 mm or more, or when it is necessary that the amount of drug released in the initial stage is large, a multilayer laminated sheet may be used.

5. 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. In the present invention, 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.

  The ratio of the uncrosslinked portion that is soluble in water at 40 ° C. is determined by the dry mass ratio. That is, a certain amount of the crosslinked hydrogel after electron beam crosslinking was taken out, dried by freeze-drying, etc., measured for mass (A), then allowed to stand in ultrapure water at 40 ° C. for 1 day for crosslinking. The uncrosslinked portion in the hydrogel is removed and further dried at 50 ° C. for 1 day, and the mass (B) after removal of the uncrosslinked portion is measured. The ratio of the uncrosslinked portion that is soluble in water at 40 ° C. is determined by the following equation (1).

  Ratio of uncrosslinked portion soluble in water at 40 ° C. (%) = (A−B) / A × 100 (%) (1)

  In the present invention, “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, that is, the uncrosslinked portion dissolved in water at 40 ° C., is preferably less than 3% by mass, more preferably less than 1% by mass, and particularly preferably less than 0.5% by mass.

6). 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.

  A representative example of the physiologically active factor is basic fibroblast growth factor (bFGF). 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. For example, when bFGF is supported on a cross-linked gelatin gel to induce angiogenesis, a two-stage release profile may be expected.

  Other physiologically active factors include, for example, transforming growth factor (TGF-β1), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF-BB), keratinocyte growth factor (KGF), bone morphogenetic factor ( BMP-2), vascular endothelial growth factor (VEGF), plasmid DNA encoding these, and the like are included. For example, when BMP-2 is supported on a crosslinked gelatin gel to induce bone formation, a linear release profile in which the drug is stably released without waste for as long as possible may be expected.

  In addition, as a bioactive 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. When these drugs are supported on a cross-linked gelatin gel, as described above, there are cases where two-stage release properties are expected and linear release properties in which drugs are stably released without waste for as long as possible. There are both expected cases.

  As the physiologically active factor to be carried on the in vivo drug sustained release carrier material, one type or two or more types can be selected as necessary. In the case of a multilayer sheet, a different physiologically active factor can be carried for each sheet.

B. Two-stage release drug release and linear release drug release 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. In order to control the release property, 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.

2.2 Step Release In the present invention, 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 an uncrosslinked portion and a 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. As a result, 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. When 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 As a result of lack of an initial burst, the desired drug effect may not be achieved. 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.

  Usually, 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.

3. On the other hand, depending on the type of drug, when linear release that keeps a certain amount of drug released over a long period of time is desired, 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. By supporting the drug on a crosslinked gelatin gel that does not substantially contain an uncrosslinked portion by adsorption or the like, it becomes possible to linearly release the drug to be supported and to exhibit a stable drug effect.

  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.

  However, even 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.

  Therefore, in the present invention, the linear release property means that a drug release profile is obtained in which the drug release property from the second day onward is almost linear after being implanted in the living body.

  For linear release, the drug release profile preferably has a constant rate portion within the range of 1 to 20% by mass / day, more preferably 2 to 15% by mass / day. As a result, the crosslinked gelatin gel substantially free of uncrosslinked portions can release the drug continuously for a period of up to 4 weeks. In addition, in the gelatin gel obtained by chemical crosslinking obtained at present, 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.

C. Method for Producing In vivo Drug Sustained Release Carrier Material The method for producing the in vivo drug sustained release carrier material of the present invention is as follows.

1. Formation of a coating layer In order to produce a sheet-form carrier material for sustained release of an in vivo drug, first, 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, Especially preferably, it exists in the range of 10-30 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. Since gelatin derivatives generally have low solution viscosity, 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 μm to 8 mm, preferably Is 200 μm to 6 mm, more preferably 300 μm to 5 mm, and particularly preferably 500 μm 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. If 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.

2. Irradiation of ionizing radiation Thereafter, the coating layer is irradiated with ionizing radiation in an inert gas atmosphere to obtain a crosslinked gelatin gel (ionizing radiation irradiation step). As the 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. For example, irradiation at 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. For example, in a nitrogen atmosphere, an acceleration voltage of 100 kV to 3 MV is preferable. Is performed within a range of 500 kV to 1 MV, and an irradiation dose of 2 to 500 kGy, preferably 3 to 300 kGy. Generally, when the thickness of the coating layer is large, it is necessary to increase the acceleration voltage or increase the irradiation dose. However, 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.

  In addition, when γ-rays are irradiated as ionizing radiation, an optimal range may be selected according to electron beam irradiation.

  When obtaining a multilayer structure of a sheet-like crosslinked gelatin gel, 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. By repeatedly performing the crosslinking operation, a crosslinked gelatin gel multilayer structure having a desired number of layers and a total thickness can be obtained.

3. Recovery of sheet-like carrier material The cross-linked gelatin gel that has been cross-linked by irradiation with ionizing radiation is taken out of the mold, and the carrier material for sustained release of the in-vivo drug in the body is recovered.

  When recovering the carrier material for sustained release of an in vivo drug in a two-stage release form, the crosslinked gelatin gel is generally not washed with water such as ultrapure water, distilled water or ion exchange water.

  However, in order to control the drug release, a certain amount of uncrosslinked parts can be removed by washing with water. For example, after irradiating with ionizing radiation to crosslink the biodegradable polymer water-soluble substance, it is washed with water such as ultrapure water, distilled water or ion exchange water to remove a specific amount of the uncrosslinked portion. The mass ratio between 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. And, more preferably, the drug release profile in the body can be within the range of the present invention by adjusting the uncrosslinked portion to the range of 95: 5 to 55:45 in which the majority is uncrosslinked.

  The recovered sheet-like carrier material may be stored as it is, but if it is vacuum-dried or freeze-dried (freeze-dried), it can be stored for a relatively long period of time.

4). Production of 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.

  Specifically, a stirring motor and a fluororesin stirring propeller are attached to a three-necked round bottom flask, and a gelatin aqueous solution is put into a device in which these are fixed, and then oil such as olive oil is added to about 200 to 600 rpm. It can be made into a W / O type emulsion by stirring at a speed of.

  Next, 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.

  The average particle size of the particulate crosslinked gelatin gel is in the range of 1 to 500 μm, preferably 1.5 to 200 μm, more preferably 2 to 100 μm, especially for handling work using a syringe. Convenient. Here, the average particle diameter is a value measured by a method of detecting laser scattering using a laser diffraction analyzer, for example.

  When the crosslinked gelatin gel particles are aggregated, for example, ultrasonic irradiation (within cooling, preferably within about 1 minute) may be performed.

  In the case of producing a two-stage release particulate carrier material for sustained release of an in vivo drug, the crosslinked gelatin gel is usually not washed with water. However, in order to control the drug release, it is also possible to adjust the ratio of the uncrosslinked portion to a desired range by removing a certain amount of the uncrosslinked portion by washing with water.

5. Removal of Uncrosslinked Part In the present invention, in order to produce a crosslinked hydrogel substantially free of an uncrosslinked part, the biodegradable polymer water-soluble substance is ionized such as an electron beam in an inert gas atmosphere. After obtaining a carrier material for sustained release of an in vivo drug comprising a crosslinked hydrogel by irradiation with radiation, 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). As a washing treatment step with water for eluting the uncrosslinked portion, for example, an in vivo body comprising a hydrogel crosslinked in water at a temperature of 5 to 50 ° C., preferably 10 to 45 ° C., more preferably 15 to 42 ° C. A washing operation for immersing the carrier material for sustained drug release for 10 minutes to 2 days, preferably 20 minutes to 1 day, more preferably 30 minutes to 10 hours, 1 to 10 times, preferably 1 to 6 times, More preferably, the method of repeating 2-4 times etc. is mentioned. By this washing operation, uncrosslinked portions can be reduced, and a crosslinked gelatin gel substantially free of uncrosslinked portions can be obtained.

  When the in vivo drug sustained release carrier material is a multilayer structure, 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.

D. Loading of Drug The carrier material for sustained release of an in vivo drug of 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. For example, a drug solution (a drug dissolved in an aqueous solution) is dropped and 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 μg per 1 mg of the crosslinked gelatin gel, preferably 1 ˜450 μg, more preferably 5 to 400 μg 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. However, 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.

  As an example of lyophilization, a cross-linked gelatin gel is kept in liquid nitrogen for 30 minutes or more or frozen in a cryogenic freezer at −90 ° C. to −80 ° C. for 1 hour or more. Stored at −40 ° C. in DRC-1000 manufactured by Rika Kikai Co., Ltd., then reduced in pressure to 1 Pa or less with a freeze dryer (FDU-2100 manufactured by Tokyo Rika Kikai Co., Ltd.), and 1 to 3 at −10 ° C. 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. By making the crosslinked gelatin gel of the present invention into a lyophilized product, the storage stability is improved as well as the adsorptivity of the chemical solution.

E. Two-stage release by combination after drug loading As another method for producing a carrier material for sustained release of an in vivo drug that exhibits two-stage release, there is a method in which a crosslinked portion carrying a drug and an uncrosslinked portion are combined. is there.

  That is, by washing a crosslinked gelatin gel having an uncrosslinked portion and a crosslinked portion with water several times, 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. Next, 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. By integrating the part and the uncrosslinked part, 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.

  Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples. However, the present invention is not limited only to these examples. Unless otherwise indicated, the part and% in a following example and a comparative example show a mass part and mass%, respectively.

A. Cross-linked gelatin gel showing a two-stage release profile using uncrosslinked parts

(1) Experiments on the cross-linking conditions of gelatin gel by electron beam irradiation and the ratio of uncrosslinked parts

[Reference Example 1]
In a mold (a glass petri dish having a diameter of 55 mm and a depth of 8 mm was used), an aqueous solution (concentration of 10% by mass) of an alkali-treated gelatin (made by Nitta Gelatin Co., Ltd .; isoelectric point 5.0) of bovine bone-derived type I collagen ) Was cast to form a uniform coating layer having a thickness of 3 mm. Next, without substantially drying the moisture of the coating layer, an electron beam is irradiated from above the coating layer using an electron beam irradiation system EPS-800 (manufactured by NHV Corporation) in a nitrogen atmosphere. Was irradiated under the conditions shown in Table 1 at an accelerating voltage of 800 kV and an irradiation dose in the range of 5 to 100 kGy to form a single-layer sheet-like crosslinked gelatin gel.

  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 previous 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.

  Until the irradiation dose reaches 20 kGy, the ratio of the cross-linked portion increases with the increase of the irradiation dose. However, 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. When 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, and when 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.

(2) Sustained release of crosslinked gelatin hydrogel carrying drug bFGF (two-stage release)
Production of the crosslinked gelatin hydrogel and evaluation of the sustained release were performed by the following methods.

[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 moisture of the coating layer, an electron beam is irradiated from above the coating layer using an electron beam irradiation system EPS-800 (manufactured by NHV Corporation) in a nitrogen atmosphere. 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.

  In addition, a fluorescent reagent (Alexa Flour 488 (manufactured by Invitrogen)) and a protein labeling kit (Alexa Flour 488 Protein Labeling Kit (manufactured by Invitrogen)) are used for bFGF (for biochemistry, purchased from Wako Pure Chemical Industries, Ltd.). Added.

  100 mg of a 1 mg / ml bFGF aqueous solution was impregnated with 5 mg of the freeze-dried sheet-like crosslinked gelatin gel, and the drug was adsorbed and supported on the crosslinked gelatin gel. Next, a cross-linked gelatin gel carrying a drug was implanted subcutaneously on the back of a mouse (ddY 4-week-old female).

  After 1, 3, 7, 10 and 14 days, the cross-linked gelatin gel was taken out and treated with 0.05% trypsin (protease, Wako Pure Chemical Industries, Ltd.) in 200 μl of ultrapure water for 24 hours at 40 ° C. 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 (Perkin Elmer, 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.

[Comparative 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%.

  From the results shown in FIG. 1, 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.

[Comparative Example 2]
The residual rate of the drug was examined in the same manner as in Example 1 except that MedGEL (chemically crosslinked product, thickness: 3 mm, manufactured by Medgel Co., Ltd.) was used as the single-layer sheet-like crosslinked gelatin gel. The obtained results are shown in FIG.

  According to FIG. 1, 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.

(3) Evaluation of angiogenesis by sustained release of bFGF Regarding the sheet-like cross-linked gelatin gel carrying bFGF of Example 1, Comparative Examples 1 and 2 in (2) above, and bFGF solution injection (control), the back of the mouse After being implanted subcutaneously or after injection, the angiogenic state in mice was evaluated from the subcutaneous condition on day 14 (n = 3). The bFGF used here is not fluorescently labeled. The results are shown in Table 2.

  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.

  On the other hand, it is a carrier material for sustained release of an in vivo drug consisting of a hydrogel crosslinked by irradiating with ionizing radiation, but it is excellent in the cross-linked gelatin gel sheet of Comparative Example 1 that does not show two-stage release properties. Angiogenesis efficiency has not been obtained. Further, the chemically crosslinked gelatin gel sheet of Comparative Example 2 cannot be said to have sufficient angiogenic efficiency.

  That is, 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.

B. Cross-linked gelatin gel that removes uncross-linked parts and provides a linear release profile

(4) Experiments on DNA adsorption and release control by elution removal of uncrosslinked parts

[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 moisture of the coating layer, an electron beam is irradiated from above the coating layer using an electron beam irradiation system EPS-800 (manufactured by NHV Corporation) in a nitrogen atmosphere. 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. Next, as a washing treatment operation with water for eluting uncrosslinked portions, 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.

[Comparative 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%.

[Comparative Example 4]
Instead of the dried electron beam crosslinked gelatin gel of Example 2, a crosslinked gelatin dried body (Medgel PI9, manufactured by Medgel Co., Ltd.) by chemical crosslinking was used.

(Evaluation of salmon DNA adsorption and release)
DNA adsorption and release were evaluated by the following methods.

  5 mg of each of the crosslinked gelatin gels of Example 2 and Comparative Examples 3 and 4 was placed in a microtube and adjusted to 5 mg / ml with a PBS solution (pH 7.2 to 7.4, manufactured by Nihon Suisan Co., Ltd.). (Salmon semen-derived 049-17321, purchased from Wako Pure Chemical Industries, Ltd.) 50 μl of the solution was added to the above microtube and allowed to stand in an incubator at 37 ° C. for 18 hours to adsorb and carry DNA on a crosslinked gelatin gel. Next, elution of salmon spam DNA was started with 1000 μl of the PBS solution diluted to 1/10, and the absorbance at 260 nm after 0, 3, 7 and 24 hours was measured. The atmosphere was 20 ° C. for 7 hours from the start of elution, and the atmosphere was 37 ° C. after 7 hours.

  Using 1000 μl 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.

  As a result of the results shown in FIG. 2, in particular, in view of the adsorption rate at the beginning of elution (elution time 0 hour), Example 2 and Comparative Example 3 using a crosslinked gelatin gel crosslinked by irradiating an electron beam were chemically crosslinked. Compared with 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. In addition, it can be seen from the difference in slope between Example 2 and Comparative Example 4 at the beginning of elution (elution time 0 hours to 3 hours) that the chemically crosslinked product has a larger decrease in adsorption rate.

  In the chemically cross-linked gelatin gel, in order to improve DNA carrying ability, cationized gelatin or the like in which a cation group is introduced into the side chain (a cation group equivalent to 50 times the carboxyl group) is prepared. However, according to the results shown in FIG. 2, gelatin gel crosslinked by irradiating with an electron beam has high DNA carrying ability, so that it is possible to maintain good gene carrying ability even if the amount of introduced cationic groups is reduced. It can be said that sex was also shown. Further, since the elimination of the main chains is suppressed due to the reduction of the cationic groups, it is expected that the efficiency of electron beam crosslinking is also increased.

  Incidentally, even in the gelatin gel crosslinked by irradiating with an electron beam, in Comparative Example 3 in which the washing treatment with water was not performed, the initial adsorptivity that the amount of initial adsorbed DNA that is characteristic of electron beam crosslinking is excellent is Although it was the same as that of Example 2, it was a result that the adsorption rate reduction after that was large. This is because until the elution time of 24 hours elapses, the drug adsorbed on the uncrosslinked portion continues to be released simultaneously with the decomposition of the uncrosslinked portion (about 57% has been released after 24 hours have elapsed). Because.)

  From this, by using a cross-linked gelatin gel substantially free of uncrosslinked parts obtained by performing a washing treatment with water after electron beam cross-linking, it is excellent in drug adsorption and suppresses initial bursts, It is possible to build a release profile of the drug that is released almost linearly over time.

(5) Experiments on in vivo survival rate (constant rate sustained release) by washing and removing uncrosslinked parts of electron beam crosslinked body

[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 μm, and then the salmon DNA was adsorbed and supported. I let you. In addition, the ratio of the uncrosslinked part which melt | 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 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 μm, and then the salmon DNA was adsorbed and supported. I let you. In addition, the ratio of the uncrosslinked part which melt | 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.

[Comparative 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 | dissolves in 40 degreeC water of the obtained crosslinked gelatin gel was 29 mass%, and the ratio of the crosslinked part was 71 mass%.

[Example 5]
A 10% by weight bovine bone-derived type I collagen alkali-treated gelatin (Nitta Gelatin Co., Ltd .; isoelectric point 5.0) aqueous solution (hereinafter referred to as “10% PI-5”) was cast to produce electrons. 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 | 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.

  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 gel on the 1st to 48th days was taken out from the mouse, the dry weight after freeze-drying was measured, and the residual ratio was determined by comparison 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. On the other hand, in 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.

(6) Bone formation test when BMP-2 is supported on a cross-linked gelatin gel sheet

[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.

[Comparative 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 carrying | support of BMP-2 and evaluation of bone formation were performed with the following method.

30 μl of PBS solution (pH 7.2-7.4, Nippon Suisan Co., Ltd.) containing 5 μg of 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. The 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 a 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. Based on the method of use, a kit of “alkaline phosphatase B-Test Wako: manufactured by Wako Pure Chemical Industries, Ltd.” is used. Bone formation was evaluated by evaluating alkaline fosterase activity (hereinafter referred to as “ALP”).

  In addition, as a control, 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. 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.

  The evaluation results are shown in FIG.

  As can be seen from FIG. 4, in Example 6 using the crosslinked gelatin gel that was washed with water and contained substantially no uncrosslinked portion, on the 14th day after being embedded 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.

  On the other hand, in the case of injection administration, ALP hardly changed and no bone formation was observed. Therefore, even when a large amount of cell growth factor was administered at a time, the cell growth factor was retained on the spot. If it cannot be sustainedly released, it can be easily considered that bone formation is not performed.

  From this, when cell growth factor is carried as a drug, it can be seen that cell growth factor stays in the body for a certain period of time and that the drug is released by sustained release is good for cell growth. Was confirmed.

  Also in Comparative Example 6 using the crosslinked gelatin gel in which the uncrosslinked portion was not removed by washing, ALP about 3.5 times that of the control (healthy) was shown on the 14th day after being embedded in the living body. . However, in the case where an uncrosslinked portion exists, in order to follow a two-stage release drug release profile in which a large amount of drug is released immediately after implantation in the living body, in contrast to Example 6 in which the uncrosslinked portion was washed away, The results were somewhat inferior in terms of growth factor utilization efficiency.

  That is, when bone formation by BMP-2 is to be performed, it is more effective to select a linear release property capable of gradual release of the drug continuously by approximately equal amounts over a long period of time.

  INDUSTRIAL APPLICABILITY According to the present invention, 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. As a result, it is possible to control the drug release by actively utilizing the uncrosslinked portion while ensuring safety to the living body, thereby increasing the controllability of the sustained release of the drug and efficiently designing the drug product. Therefore, it can be suitably used in a wide range of technical fields including the medical field and cosmetic field.

Claims (14)

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. Which exhibits a two-stage release property,
In vivo drug sustained release carrier material. The carrier material for sustained release of an in-vivo drug according to claim 1, wherein the hydrogel has an uncrosslinked portion and a crosslinked portion in a mass ratio of 95: 5 to 55:45. The biodegradable polymer water-soluble substance is gelatin or a gelatin derivative, and the drug to be carried is a physiologically active factor.
The carrier material for sustained release of an in vivo drug according to claim 1 or 2. The biodegradable polymeric water-soluble substance is gelatin or a gelatin derivative, the drug to be carried is bFGF which is a growth factor, and
Angiogenesis is observed by the 14th day after the carrier material carries bFGF and is implanted in the body of a mouse.
The carrier material for sustained release of an in vivo drug according to any one of claims 1 to 3. The carrier material according to any one of claims 1 to 4, wherein the carrier material for sustained release of an in vivo medicine is loaded with a drug in a state where an uncrosslinked portion and a crosslinked portion are separated from each other. The uncrosslinked portion and the crosslinked portion are combined.
In vivo drug sustained release carrier material. The carrier material for sustained release of an in vivo drug according to any one of claims 1 to 4, is washed with water.
A carrier material for sustained release of an in-vivo drug that exhibits linear release, comprising a crosslinked hydrogel substantially free of uncrosslinked portions. In the hydrogel, the uncrosslinked portion in which the hydrogel is dissolved in water at 40 ° C. is less than 5% by mass based on the total of the uncrosslinked portion and the crosslinked portion
The carrier material for sustained release of an in-vivo drug according to claim 6. After the carrier material carries the drug and is implanted in the living body, the release profile of the drug to be loaded has a constant rate within a range of 2 to 15% by mass / day.
The carrier material for sustained release of an in vivo drug according to claim 6 or 7. The drug to be carried is plasmid DNA.
The carrier material for sustained release of an in vivo drug according to any one of claims 6 to 8. The drug to be carried is BMP-2.
The carrier material for sustained release of an in vivo drug according to any one of claims 6 to 8. 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. Which exhibits a two-stage release property,
A method for producing a carrier material for sustained drug release in vivo. A method for producing a carrier material for sustained release of an in-vivo drug according to claim 11,
After the ionizing radiation irradiation step, further having a washing treatment step with water,
A method for producing a carrier material for sustained release of an in-vivo drug that exhibits linear release, comprising a crosslinked hydrogel substantially free of uncrosslinked portions. Prior to the ionizing radiation irradiation step, the method further comprises a coating layer forming step of casting an aqueous solution of a biodegradable polymer water-soluble substance to form a coating layer.
A method for producing a carrier material for sustained release of an in vivo drug according to claim 11 or 12. The ionizing radiation is an electron beam,
Electron beam irradiation is performed within an acceleration voltage range of 100 kV to 3 MV in an inert gas atmosphere within a dose range of 2 to 500 kGy.
A method for producing a carrier material for sustained release of an in vivo drug according to any one of claims 11 to 13.

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