JP2012082139A - External agent sustained release carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an external agent sustained release carrier having high biocompatibility, high water retention and high strength.SOLUTION: This invention relates to an external agent sustained release carrier using a silk fibroin porous material. The tensile strength of the porous material is 75-400 kPa.

Description

  The present invention relates to an external drug sustained release carrier, and more particularly to an external drug sustained release carrier having high strength, high affinity for skin, and high water retention.

  Conventionally, oral administration is known as a method for administering drugs to patients, but side effects such as gastrointestinal disturbances, inability to bypass the liver and avoid first-passing metabolism, or therapeutic concentration and toxicity It has drawbacks such as difficulty in controlling administration of a drug in a region where the concentration is narrow.

  As a means for solving the above problems, attempts have been made to use drugs as transdermally applied preparations. In such a preparation for transdermal application, in order to stably absorb a medicinal component over a long period of time, the penetration of the skin using a pharmacologically active substance is controlled, and the active substance is a surface active agent or Although other penetrants could be used, many of these actives actually damaged skin tissue and produced undesirable side effects.

  In addition, as a typical means of other conventional methods for controlling the percutaneous penetration of a drug and continuously controlling the release of the medicinal ingredient, the medicinal ingredient is held by a medicinal carrier such as a porous base material. A method for controlling the percutaneous penetration of the skin has been proposed.

As a material for such a drug carrier, a synthetic polymer or a natural polymer gel or a porous material has been used (Patent Document 1). However, synthetic polymers have problems such as low affinity for skin and low water retention.
On the other hand, natural polymers have high affinity for the skin, but have problems such as low strength. For this reason, it has been necessary to ensure the strength of natural polymers by wrapping them with a cross-linked product of a cross-linking agent, use of a strength reinforcing material, gauze, or the like.
However, when a crosslinking agent is used, there is a concern that the remaining crosslinking agent may adversely affect the skin. In addition, when a strength reinforcing material is used, there is a concern that the structure becomes complicated, and a part of the drug carrier remains on the skin when it is peeled off. Furthermore, when the strength is secured by covering with gauze or the like, the conventional coating material such as gauze is in contact with the skin, and a gel or porous body having high biocompatibility and high water retention is applied to the skin. There was a problem that sufficient effects could not be exhibited because they were not in contact.

  By the way, among natural polymers, silk has a high biocompatibility and has been conventionally used for sutures and the like, and is known to have high safety. Silk is formed from sericin and fibroin protein, and various processing methods have been proposed. For example, hydrogels using silk fibroin have been proposed (Patent Documents 2 and 3). However, there is a problem that the strength is weak when used as a drug carrier.

JP-A-8-175981 Japanese Patent No. 3412012 No. 6-94518

  An object of the present invention is to provide a high-strength external drug sustained-release carrier having high biocompatibility and water retention.

As a result of intensive studies, the present inventors have found that a silk fibroin porous material is suitable as the material, and have completed the present invention.
That is, the present invention
(1) An external drug sustained release carrier using a silk fibroin porous material,
(2) The external drug sustained release carrier according to 1 above, wherein the porous body has a tensile strength of 75 to 400 kPa, and (3) a film layer having no pores only on one surface of the porous layer. 3. The external drug sustained release carrier according to 1 or 2 above,
Is to provide.

  The sustained release carrier for external use of the present invention has high strength and is easy to handle, and has high biocompatibility, so it is gentle to the skin, has high water retention, and can efficiently hold the drug. It is very effective for transdermal absorption.

2 is a scanning electron micrograph of a cross section of a porous body produced in Example 1. FIG. 2 is a scanning electron micrograph of a cross section of a porous body produced in Example 2. FIG. 4 is a scanning electron micrograph of a cross section of a porous body produced in Example 3. FIG. 4 is a scanning electron micrograph of a cross section of a porous body produced in Example 4. FIG. 6 is a scanning electron micrograph of the cross section of the porous body produced in Example 5. FIG. 6 is a scanning electron micrograph of a cross section of a porous body produced in Example 6. FIG. 6 is a scanning electron micrograph of the cross section of the porous body produced in Example 7. FIG. 6 is a scanning electron micrograph of the cross section of the porous body produced in Example 8. FIG. 6 is a scanning electron micrograph of the cross section of the porous body produced in Example 9. 4 is a scanning electron micrograph of the cross section of the porous body produced in Example 10. FIG. 2 is a scanning electron micrograph of a cross section of a porous body produced in Example 11. FIG. 4 is a scanning electron micrograph of a cross section of a porous body produced in Example 12. FIG. 2 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 1. 4 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 2. 4 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 3. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 4. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 5. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 6. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 7. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 8. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 9. 4 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 10. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 11. 6 is a scanning electron micrograph of a cross section of a porous body produced in Comparative Example 12.

The extracorporeal drug sustained release carrier of the present invention is characterized by using a silk fibroin porous material.
Here, the silk fibroin porous body means a porous body containing silk fibroin, preferably having an average pore diameter of 10 to 300 μm.

  The tensile strength of the silk fibroin porous material is preferably 75 kPa to 400 kPa. If it is 75 kPa or more, it has sufficient strength, and can be handled easily as an externally applied drug sustained-release carrier, and the remainder of the externally-supplied drug sustained-release carrier on the skin is reduced. On the other hand, if it is 400 kPa or less, the adhesiveness to skin is maintained. From the above viewpoint, the tensile strength is more preferably 80 kPa to 300 kPa, and further preferably 90 kPa to 200 kPa.

Moreover, it is preferable that the extracorporeal drug sustained release carrier of the present invention has a high water retention rate that retains absorbed water. Since the water retention rate is high, the absorbed exudate does not flow out. More specifically, the water retention rate is preferably 85 to 100%. If the water retention rate is 85% or more, the exudate is retained and the leaked liquid hardly flows out. From the above points, the water retention of the silk fibroin porous material is more preferably 87 to 100%, and still more preferably 90 to 100%.
(Calculation method of water retention rate)
The water retention rate was measured as follows. The porous body is molded to 60 × 30 × 20 mm, used as a measurement sample, and the weight of the sample sufficiently immersed in pure water is measured (Wc). The glass plate (MSA coated micro slide glass made by Matsunami Glass, 76 × 52 mm) whose surface was sufficiently dipped in pure water and wetted with pure water was tilted at 45 degrees and placed on top of it. The surface (60 × 30 mm surface) is placed face down, and the lengthwise direction is placed up and down, and allowed to stand for 10 minutes. Thereafter, the weight of the sample is measured (Wd).
Water retention rate (%) = 100− (Wc−Wd) × 100 / (Wc)

The water absorption rate of the porous layer is preferably 0.1 to 1000 μl / s, more preferably 1 to 100 μl / s, and still more preferably 20 to 30 μl / s. When the water absorption rate is 0.1 μl / s or more, the exudate is rapidly absorbed and rarely leaked out of the external drug sustained-release carrier. When the water absorption rate is 1000 μl / s or less, the exudate is prevented from being excessively absorbed by the external medicine sustained-release carrier, and the wet state of the contact portion can be maintained. It is preferable that the evaporation rate is 0.01~0.2g / m ² · s, more preferably from 0.03~0.15g / m ² · s, more preferably 0.06 to 0 .1 g / m ² · s. If the evaporation rate is 0.01 or more, the leakage can be continuously maintained in a state that can be absorbed by the extracorporeal drug sustained release carrier. When the evaporation rate is 0.2 g / m ² · s or less, the extracorporeal drug sustained release carrier of the present invention can be efficiently kept in a wet state. Here, the water absorption speed and the evaporation speed are values obtained as follows. When the water absorption rate and evaporation rate of the porous layer are within the above ranges, the effect of the present invention is good.

(Calculation method of water absorption rate)
100 μl of pure water was dropped on the silk fibroin porous material (porous layer), and the time until absorption was measured. The water absorption rate is a value calculated from the following equation using the measured time. The measurement was performed 5 times, and the average value was taken as the water absorption rate.
Water absorption rate (μl / s) = pure amount of pure water / time required for water absorption (evaporation rate calculation method)
Silk fibroin porous material (porous layer) is immersed in pure water for 48 hours and completely absorbed, and then on a wire mesh in a constant temperature and humidity chamber set at a temperature of 40 ° C. and a relative humidity of 50%. The weight was measured every 1 minute until 10 minutes passed, and every 2 minutes after 10 minutes, and the change was regarded as a change in the evaporation amount of water. The evaporation rate is a value calculated from the following equation from the change in the evaporation amount from 1 minute to 30 minutes after standing.
Evaporation rate (g / m ² · s) = (Change in evaporation) / Porous material surface area

The extracorporeal drug sustained release carrier of the present invention is preferably in the form of a sheet, and the size and shape of the sheet can be arbitrarily set. Also, the thickness of the sheet can be set to any thickness depending on the dose of the drug. The whole sheet can be a porous body.
Further, the silk fibroin porous body in the present invention may have a porous layer and a film layer having no pores on one or both sides thereof, while the silk fibroin porous body is a porous layer. It may consist only of.

The film layer having no pores is a layer having substantially no pores, and is a layer having no pores or having very few pores as compared with the porous layer. By having such a film layer, the speed at which the liquid drug moves through the pores and the speed at which it evaporates or diffuses are slowed down, so that the liquid permeability is lowered.
The film layer preferably has pores with an area ratio of 10% or less on the surface, and the porous layer preferably has a surface with pores with an area ratio of 50 to 98%. The film layer preferably has 20 pores / mm ² or less (more preferably 10 / mm ² or less) pores having a pore diameter of 0.5 μm or more. Here, the area ratio of pores on the surface, the number of pores and the pore diameter were measured by subjecting a scanning electron micrograph to image processing using image analysis software ImageJ (manufactured by National Institutes of Health). It is.
The water absorption rate of the film layer is preferably 0.1 to 3.5 μl / s, more preferably 1.5 to 3.5 μl / s, and still more preferably 2 to 3.5 μl / s. It is.
Furthermore, the evaporation rate of the film layer is preferably 0.03~0.07g / m ² · s, more preferably from 0.03~0.06g / m ² · s, more preferably 0 0.03 to 0.05 g / m ² · s. When the water absorption rate and the evaporation rate are within the above ranges, the effect of the present invention is good.

As the extracorporeal drug sustained release carrier of the present invention, a silk fibroin porous body having a porous layer and a film layer having no pores only on one side thereof is preferable. As the usage method, it is preferable that the porous layer is in contact with the skin and the film layer is provided on the opposite surface opposite to the skin. This is because when such an external drug sustained-release carrier is used, a liquid drug can be efficiently held, and there is an effect of suppressing evaporation and diffusion of the drug.
In addition, the number of pores of the film layer can be controlled, and a film layer having a small amount of pores can be formed as necessary.
On the other hand, since the film layer has very few pores, the surface is smooth. Therefore, the release rate of the drug can be controlled by using the film layer as the skin side and controlling the liquid permeability.

The drug to be included in the external drug sustained-release carrier of the present invention is not particularly limited, and examples thereof include antihypertensive agents such as reserpine and clonidine, antihypertensive diuretics such as hydrothiazide and pendroflunathiazide, nitroglycerin, Antianginal agents such as Sosanilisorbide, vasodilators such as nitroglycerin, nitroglycol, isosorbite dinitrate, erythritol tetranitrate, pentaerythritol tetranitrate, anticonvulsants such as nitrazepam and meprobamate (Ii) agents, tranquilizers such as fluphenazine, thioridazine, diazepam, chlorpromazine, antihistamines such as diphenhydrazine hydrochloride, chlorpheniramine, diphenylimidazole, hydrocortisone, prednisolone, parameterzone, beclos Corticosteroids such as tazone propionate, flumethasone, betamethasone, beclomethasone propionate, dexitazone, triamcinolone, triamcinolone acetonide, fluocinolone, fluocinolone acetonide, fluocinolone acetonide acetate, clobetasol propionate, acetaminophen , Mefenamic acid, flufenamic acid, indomethacin, diclofenac, alclofenac, oxyphenbutazone, phenylbutazone, ibuprofen, flurbibrofen, salicylic acid, methyl salicylate, L-menthol, camphor and other analgesic anti-inflammatory agents, penicillin, oxytetracycline , Fradiomycin sulfate, erythromycin, chloramphenicol, cephalexin, tetracycline, etc. Antibiotic, vitamin A, ergocalciferol, cholecalciferol, vitamin agents such oct thiamine, riboflavin butyric acid ester, benzocaine, lidocaine, and the like anesthetic such as aminoethyl benzoate. The drug that can be contained in the sustained-release drug carrier for external use of the present invention is not limited to these, and if necessary, an absorption aid such as isopropyl myristate, isopropyl palmitate, N- Mixing methylpyrrolidone, N-ethylpyrrolidone, N, N-diethyl-m-triamide, N, N-diethylacetamide, hyaluronic acid, salicylic acid, crotamiton, diethyl sebacate, lauryl alcohol, dimethyl sulfoxide, decmethyl sulfoxide, etc. It can be held by impregnating or coating the porous layer. These drugs can be used alone or in combination of two or more.
These formulations have a function of promoting drug absorption into the barrier membrane by the skin itself and promoting penetration into blood.

The amount of the drug added is appropriately determined depending on the drug to be selected. As a guideline, the drug is added so that about 30 to 300 mg / 12 cm ² of drug is absorbed in the formed sheet. Is done.

  In addition, the solvent used in the formulation of the drug is not harmful to the human body because it needs to be used as a transdermally applied preparation, and is chemically insensitive to the used drug and other components of the transdermally applied preparation. It must be active, but there is no particular limitation as long as it can dissolve or disperse the drug used. Specifically, water, one or two mixed systems of polyhydric alcohols can be used. Water is most preferable from the viewpoint of safety to the human body. Examples of the polyhydric alcohol include glycerin, propylene glycol, and sorbit. In addition, a water-soluble polymer can be added in order to improve the solubility in dissolving the drug in the solvent. Examples of the water-soluble polymer include gelatin, sodium alginate, tragacanth gum, starches, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, sodium polyacrylate, and the like.

  When the extracorporeal drug sustained-release carrier for external use of the present invention is composed of a porous body consisting only of a porous layer, it is assumed that the drug passes through the porous layer and exudes to the opposite side. Since the exudation amount can be suppressed as compared with the case where the external drug sustained-release carrier is not used, the effect of the present invention is sufficiently exhibited when the amount of the drug is small.

The extracorporeal drug sustained-release carrier for external use of the present invention can have a form in which a sheet-like silk fibroin porous body is fixed with a dressing film, a dressing, an adhesive tape, or the like.
Moreover, since the silk fibroin porous body becomes hard and brittle by drying, it is also a preferable aspect to include a moisturizing agent. As the humectant, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, glycerin and the like can be used. In such an embodiment, it is preferable to keep the extracorporeal drug sustained-release carrier of the present invention in a sealed state so as to prevent drying with the amount of water maintained until just before impregnation with the drug.

Next, the manufacturing method of a silk fibroin porous body is demonstrated.
The silk fibroin porous material used in the present invention can be produced by adding a specific additive to a silk fibroin aqueous solution, freezing the aqueous solution, and then thawing it.
The silk fibroin used here may be any silk fibroin as long as it is produced from silkworms such as rabbits, wild silkworms, and tengu. In the production of the silk fibroin porous material in the present invention, it is used as a silk fibroin aqueous solution, but silk fibroin has poor solubility and is difficult to dissolve directly in water. As a method for obtaining a silk fibroin aqueous solution, any known method may be used, but a method in which silk fibroin is dissolved in a high concentration lithium bromide aqueous solution, followed by desalting by dialysis and concentration by air drying is simple.
In the method for producing a silk fibroin porous body, the concentration of silk fibroin is preferably 0.1 to 40% by mass and preferably 0.5 to 20% by mass in a silk fibroin solution to which an aliphatic carboxylic acid is added. It is more preferable, and it is still more preferable that it is 1.0-12 mass%. By setting it within this range, a porous body having sufficient strength can be efficiently produced.

  Examples of the additive include aliphatic carboxylic acids and amino acids. Although there is no restriction | limiting in particular as an additive, A water-soluble thing is preferable and the thing with the high solubility to water is more preferable. Moreover, as an aliphatic carboxylic acid used in manufacture of a silk fibroin porous body, that whose pKa is 5.0 or less is preferable, the thing of 3.0-5.0 is more preferable, and 3.5-5. Even more preferred is zero.

  As the aliphatic carboxylic acid, for example, a saturated or unsaturated monocarboxylic acid having 1 to 6 carbon atoms, dicarboxylic acid, or tricarboxylic acid can be preferably used. For example, formic acid, acetic acid, propionic acid, butyric acid, succinic acid, Examples include lactic acid, acrylic acid, 2-butenoic acid, and 3-butenoic acid. These aliphatic carboxylic acids can be used alone or in combination of two or more.

The amino acid is not particularly limited, and examples thereof include monoaminocarboxylic acids such as valine, leucine, isoleucine, glycine, alanine, serine, threonine, and methionine, and monoaminodicarboxylic acids (acidic amino acids) such as aspartic acid and glutamic acid. Examples include aliphatic amino acids, aromatic amino acids such as phenylalanine, and amino acids having a heterocyclic ring such as hydroxyproline. Among these, acidic amino acids and oxyamino acids such as hydroxyproline, serine, and threonine are preferable from the viewpoint of easy shape adjustment. . From the same viewpoint, monoaminocarboxylic acid is more preferable among acidic amino acids, aspartic acid and glutamic acid are particularly preferable, and hydroxyproline is more preferable among oxyamino acids. These amino acids can be used alone or in combination of two or more.
In addition, there are L-type and D-type optical isomers of amino acids, but when L-type and D-type are used, there is no difference in the resulting porous material, so which amino acid is used. Also good.

  The amount of the water-soluble additive added to the silk fibroin aqueous solution is preferably 0.1 to 18% by mass, more preferably 0.1 to 5.0% by mass, and 0.5 to 4.0. More preferably, it is mass%. By setting within this range, a porous body having sufficient strength can be produced. Moreover, if it is 18.0 mass% or less, when leaving the silk fibroin solution which added the aliphatic carboxylic acid and / or the acidic amino acid to the silk fibroin aqueous solution, this solution is hard to be gelled and is stably high quality silk. A fibroin porous material is obtained. Moreover, it is preferable that it is 1-500 mass% with respect to fibroin, as for the compounding quantity of an amino acid, it is more preferable that it is 5-50 mass%, and it is further more preferable that it is 10-30 mass%.

Next, a silk fibroin porous body is produced by pouring the added silk fibroin aqueous solution to which the above additives are added into a mold or a container, freezing it in a low-temperature thermostatic bath, and then thawing it. The freezing temperature is not particularly limited as long as the silk fibroin solution containing the additive is frozen, but is preferably about −10 to −30 ° C. Further, the freezing time is preferably 4 hours or more at a predetermined freezing temperature so that it can be sufficiently frozen and kept in a frozen state for a certain time.
As a method of freezing, the silk fibroin solution may be frozen to a freezing temperature all at once, but once it is kept at about −5 ° C. for about 2 hours to be in a supercooled state and then lowered to the freezing temperature. Freezing is preferable for obtaining a porous body having high mechanical strength. The structure and strength of the porous body can be controlled to some extent by adjusting the time taken from −5 ° C. to the freezing temperature.
Thereafter, the silk fibroin porous material is obtained by thawing the frozen silk fibroin solution. The melting method is not particularly limited, and examples include natural melting and a method of holding in a thermostatic bath.

  The obtained porous body contains additives, but depending on the application, if it is necessary to remove the additives, the additives can be removed from the porous body by an appropriate method. . For example, the simplest method is to remove the additive by immersing the porous body in pure water. Or it is possible to remove an additive and a water | moisture content simultaneously by freeze-drying a porous body.

  The silk fibroin porous body has a sponge-like porous structure. Normally, this porous body contains water unless it is removed by lyophilization or the like, and is a rigid structure in a water-containing state. In addition, a dried silk fibroin porous material can be obtained by freeze-drying the porous material.

  The silk fibroin porous material can be formed into a shape suitable for the purpose, such as a film shape, a block shape, a tubular shape, a spherical shape, and the like by appropriately selecting a mold and a container for producing the porous material. There are no restrictions on the shape or container of the silk fibroin solution as long as it does not flow out. The material used is a material with high thermal conductivity such as iron, stainless steel, aluminum, gold, silver, or copper. It is preferable from the viewpoint of obtaining a silk fibroin porous body having a uniform structure. In addition, the thickness of the mold or the wall of the container is preferably 0.5 mm or more from the viewpoint of its function and deformation due to expansion during freezing, etc., and is easy to handle and efficient in cooling. More preferably, the thickness is 1 to 3 mm.

Moreover, the mold | type and container used here can provide a sheet | seat in the inner wall surface which contact | connects the silk fibroin solution inside for the purpose of controlling the structure and thickness of the said film layer.
As the sheet, a sheet made of a fluororesin such as polytetrafluoroethylene (PTFE), tetrafluoroethylene / hexafluoropropylene copolymer (FEP) or tetrafluoroethylene / perfluoroalkyl vinyl ether copolymer (PFA), or Preferable examples include release-treated sheets made of polyethylene terephthalate (PET) or polypropylene (PP). When these sheets are used, a smooth film layer with few pores can be obtained. In addition, when it is not desired to provide a film layer, a sheet having a rough surface such as filter paper can be provided. About adoption of these sheets, what is necessary is just to select suitably according to the use of a porous body.
In addition, it is preferable to use a sheet having a thickness of 1 mm or less that hardly inhibits heat conduction.

  This silk fibroin porous body is obtained through a cutting process subsequent to the melting step described above. In the sheet shape suitable as the sustained-release drug carrier for external use of the present invention, in the case of a porous body consisting only of a porous layer, in addition to selecting the material of the container, the surface structure can be obtained by excising the film layer. Can be selected. Thereby, the sheet | seat which consists of a porous body which consists only of a porous layer can be obtained. Specifically, for example, a block-shaped mold or container in which a sheet such as a Teflon sheet is provided on the inner wall surface is taken out from the mold or container, and then the four side film layers are removed to remove the porous layer. It is obtained by cutting a part. Alternatively, a porous body having a film layer only on one surface can be obtained by using a mold or a container in which a sheet such as a Teflon sheet is provided on one inner surface and a filter paper is provided on the other inner surface.

  EXAMPLES The present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
For silk fibroin aqueous solution, fibroin powder (KB Selen, trade name: “Fibroin IM”) is dissolved in 9M lithium bromide aqueous solution, insoluble matter is removed by centrifugation, and dialysis is repeated against ultrapure water. Was obtained by The obtained silk fibroin aqueous solution was air-dried in a dialysis tube and concentrated. A formic acid aqueous solution was added to this concentrated liquid to prepare a silk fibroin solution having a silk fibroin concentration of 5 mass% and a formic acid concentration of 2 mass%.
This silk fibroin solution was poured into a mold (inside size; 80 mm × 40 mm × 4 mm) made of an aluminum plate, and was frozen and stored in a low-temperature thermostatic bath (NCB-3300 manufactured by EYELA).
For freezing, the low-temperature thermostat was cooled to -5 ° C in advance, and a mold containing the silk fibroin solution was put into the low-temperature thermostat and held for 2 hours, and then cooled to -20 ° C and held for 5 hours. The frozen sample was returned to room temperature by natural thawing, then removed from the mold, immersed in ultrapure water, and the used formic acid was removed by exchanging the ultrapure water twice a day for 3 days.

(Measuring method of mechanical properties)
The mechanical properties of the obtained silk fibroin porous material were evaluated using INSTRON Micro Tester Model 5548. A test piece of 40 mm × 4 mm × 4 mm was cut out from the produced silk fibroin porous material, and the maximum breaking strength (tensile strength) and the maximum strain (elongation) when the test piece was pulled at 2 mm / min were measured. In addition, the elastic modulus was obtained from the slope when the strength and strain were graphed. The results are shown in Table 1. In addition, the measurement result produced the test piece of 5 points | pieces from the produced porous body, and also cut out the test piece of 5 points | pieces from the porous body produced on the different day, and measured the average value which measured about those 10 points | pieces Show.

  Moreover, the structure of the obtained silk fibroin porous body was observed using a scanning electron microscope. The scanning electron microscope was measured using a Philips XL30-FEG in a low vacuum non-deposition mode and an acceleration voltage of 10 kV. The structure of the silk fibroin porous body was not the surface of the porous body, but the inside exposed by cutting the porous body. A scanning electron micrograph of the cross section of the obtained porous body is shown in FIG.

Examples 2-12 and Comparative Examples 1-11
In Example 1, a silk fibroin porous material was obtained in the same manner as in Example 1 except that the additives described in Tables 1 to 3 were used in place of formic acid. The evaluation results of the mechanical properties, water retention, water absorption rate and evaporation rate are shown in Tables 1-3. Moreover, the scanning electron micrographs of the cross section of the obtained porous body are shown in FIGS.
The water retention rate, water absorption rate and evaporation rate were measured as described above.

Examples 13-14
In Example 1, a silk fibroin porous material was obtained in the same manner as in Example 1 except that the types of materials described in Table 2 were used. The measurement results of the water absorption rate are shown in Table 2.

Comparative Example 12
In Example 8 of Patent Document 3, an acetic acid solution is added to a silk fibroin aqueous solution to precipitate a gel, and the resulting gel is freeze-dried to obtain a porous body. In the follow-up test as described, no gel was produced, but a gel-like material and a porous material were obtained under the following conditions.
First, 130 mL of a pH 2.65 acetic acid solution was added to 50 mL of a 5% fibroin aqueous solution (pH 3.0 at this stage), and the resulting solution was allowed to stand at 5 ° C. for 40 hours to form a gel. The obtained gel was centrifuged and the supernatant was removed. After freezing at −30 ° C. for 3 hours and freeze-drying for 50 hours, a porous body was obtained. Table 1 shows the results of evaluating the mechanical properties of the porous body. Moreover, the scanning electron micrograph of the cross section of the obtained porous body is shown in FIG.

Comparative Example 13
Using a commercially available polyurethane sponge (manufactured by Sumitomo 3M), the sample was cut into measurement samples (60 mm × 30 mm × 20 mm), the water retention was measured, and the results are shown in Table 1.

[Material of mold]
A: Aluminum plate (with Teflon sheet)
B: Mirror finish acrylic board (without Teflon sheet)
C: Mirror finish polystyrene plate (without Teflon sheet)

From the results shown in Tables 1 to 3, the tensile strength of the silk fibroin porous material of Examples 1 to 12 using an aliphatic carboxylic acid or amino acid is a water-soluble organic solvent, methanol, ethanol, isopropanol, butanol, t -It turns out that it is larger than the tensile strength of any silk fibroin porous body of the comparative examples 1-11 using butanol, glycerol, DMSO, DMF, pyridine, acetonitrile, acetone. Moreover, the silk fibroin porous body of Examples 1-12 has a large maximum strain (elongation). Furthermore, the porous body produced in Examples 1-12 showed high tensile strength when compared with Comparative Example 12.
Moreover, it turns out that the porous layer which comprises the silk fibroin porous body produced in Example 1 has a comparatively thin wall and several tens micrometer pore from FIG. About the cross section (FIGS. 2-12) of the silk fibroin porous body produced in Examples 2-12, the substantially same scanning electron micrograph was obtained.
In addition, although the cross section (FIGS. 13-23) of the silk fibroin porous body of a comparative example had the thing with the same porous structure, and the layered structure, the correlation with those structures and mechanical characteristics is I was not able to admit. In Comparative Example 12, a clear porous structure could not be confirmed.
Moreover, the water retention of the silk fibroin porous material produced in Examples 2, 5, 6 and 11 is substantially the same as the water retention of the porous material produced in Comparative Example 2, and is in the range of 98% to 99%. I found out. In addition, the porous body produced in Comparative Example 12 was very brittle, and the water retention rate could not be calculated.

Examples 15-17
A human patch test (skin sensitization patch test) was conducted as part of a non-clinical test on the safety of silk fibroin porous materials. The skin sensitization patch test is to determine whether silk fibroin porous body induces irritation or allergic contact dermatitis on human skin.
The sheet used was the same as in Example 1 except that the inner size of the mold made of the aluminum plate was 264 mm × 205 mm × 1 mm thick. ) And succinic acid (Example 17), except that a silk fibroin porous sheet was obtained in the same manner as in Example 1. Each of these silk fibroin porous material sheets was cut into 10 mm × 10 mm × 1 mm thicknesses for use in the test, thereby preparing patches.
The test was conducted with 50 healthy Japanese women aged 18 to 65 at the start of the test. A 10 mm × 10 mm size patch used for the test was fixed to the back with a 20 mm × 20 mm hypoallergenic tape. Moreover, each patch was fixed at least 15 mm apart. For comparison, a 10 mm × 10 mm non-woven cotton fabric was fixed with a 20 mm × 20 mm hypoallergenic tape. In the test, the patch was removed 48 hours after attaching the patch, and the skin reaction was observed. Furthermore, a patch was attached to the same place, and observation was performed after 48 hours. A total of 9 times of pasting and observation were repeated. Also, two weeks later, patching at the same place and observation were repeated 4 times every 48 hours.
As a result, during the test, in Examples 15 and 16, the two subjects showed very mild erythema once, but the erythema also disappeared during the test, resulting in sheet-like silk fibroin porous The mass was found to be very hypoallergenic and not allergic.
Moreover, in Example 17, one test subject showed very mild erythema once during the test, but the result was that erythema also disappeared during the test. As a result, the sheet-like silk fibroin porous material was It was found to be very hypoallergenic and not allergic.

Example 18
A silk fibroin porous material was produced in the same manner as in Example 6 except that the size of the mold made of the aluminum plate was 130 mm × 80 mm × 12 mm (inside size).
As part of a non-clinical study on the safety of silk fibroin porous material, a primary skin irritation test and a skin sensitization test were conducted. This study is based on the “Standards for the Reliability of Application Materials” (Regulation No. 43 of the Pharmaceutical Affairs Law). Regarding the “Concept” (Pharmaceutical Proposal No. 0213001 dated February 13, 2003) and “Reference Materials on the Basic Concept of Biological Safety Testing” (Medical Device Examination No. 36, March 19, 2003) went.

(Skin primary irritation test)
The silk fibroin porous material produced in Example 18 was cut into a size of 5 mm × 5 mm × 5 mm to obtain a test piece (fibroin porous material comprising only a porous layer).
Next, a physiological saline solution and a sesame oil solution extract of silk fibroin porous material were applied to rabbits and examined for local irritation (tissue damage and inflammation-inducing properties). Specifically, physiological saline or sesame extract is added to the above test piece cut out from the silk fibroin porous material produced in Example 18, and extracted in an autoclave at 120 ° C. for 1 hour. Liquid. Moreover, only the extraction solvent (physiological saline solution or sesame oil solution) was processed under the same conditions to obtain a target solution. For administration, 6 male rabbits were used per solvent, and 0.5 mL each of the test solution and the target solution was applied to the intact skin and scratched skin on the back, respectively.
In the test solution obtained by physiological saline extraction, very slight or slight erythema was observed in 3 of 6 cases from 1 hour after administration. This erythema was also found in the physiological saline solution, which was the target solution, and was equivalent to the target solution. The primary irritancy index was 0.3, and was judged to be “negligible irritation”.
In the sesame oil extract, 4 out of 6 cases showed very mild erythema from 1 hour after administration. This erythema was also found in the sesame oil solution, which was the target solution, and was equivalent to the target solution. The primary irritation index was 0.1, which was determined to be “ignorable irritation”.

(Skin sensitization test)
With respect to the methanol extract of silk fibroin porous material produced in Example 18, the presence or absence of sensitization to guinea pig skin was examined using the Maximization Test method.
Before the skin sensitization test, the extraction rate was calculated using acetone and methanol in order to determine an appropriate extraction solvent. As a result, since methanol showed a higher extraction rate than acetone, the extraction solvent used for the skin sensitization test was methanol.
To the above test piece cut out from the silk fibroin porous material produced in Example 18, 10 mL of methanol was added and extracted using a constant temperature immersion culture machine at room temperature. The extraction was performed for 24 hours or more. As a control group, a negative control group sensitized with olive oil and a positive control group sensitized with 1-chloro-2,4-dinitrobenzene were provided. Each control group had 5 animals.
In both the test solution administration group and the negative control group, as a result of inducing with 6.25, 12.5, 25, 50, 100% solution of the extract and acetone, at any observation period of 24, 48 and 72 hours after induction No skin reaction was seen.
On the other hand, in the positive control group, an obvious positive reaction was observed 24, 48 and 72 hours after the induction in all 5 cases due to the induction of 0.1% 1-chloro-2,4-dinitrobenzene.
From this test result, it was determined that the silk fibroin porous material produced according to Example 18 did not contain a substance showing skin sensitization.
Thus, the silk fibroin porous material produced according to Example 18 has high safety due to “a negligible skin irritation” and “no substance showing skin sensitization”. It was confirmed that it can be suitably used as a sustained-release carrier for external use.

  According to the present invention, there is provided a sustained release carrier for external use that is strong and easy to handle, has high biocompatibility, is gentle to the skin, has high water retention, and can efficiently hold the drug. can do.

Claims (3)

  An external drug sustained-release carrier using a silk fibroin porous material.   The external drug sustained-release carrier for external use according to claim 1, wherein the porous body has a tensile strength of 75 to 400 kPa.   The sustained release carrier for external use according to claim 1 or 2, which has a film layer having no pores only on one surface of the porous layer.