TW202140524A - Materials for treating fistula - Google Patents

Abstract

A material for treating fistula which comprises gelatinous hydrogel cross-linked gelatin containing basic fibroblast growth factor.

Description

Materials for fistula treatment

The present invention relates to materials for the treatment of fistulas.

In the living body, a fistula sometimes occurs, which is a tube-shaped defect between the luminal organ and the skin, or between the tube and the tube between the luminal organ. The fistula produced in the internal environment of the living body is called the internal fistula, and the fistula that connects the outside of the living body with the tube hole is called the external fistula. Fistulas are related to the following: entero-intestinal fistula that creates a through hole between the intestine and the intestine, rectovesical fistula that creates a through hole between the rectum and the bladder, and rectovaginal fistula that creates a through hole between the rectum and the vagina , Intestinal skin fistula that creates a through hole between the intestine and the skin, or hemorrhoid fistula that creates a through hole between the rectum and anus, etc.

Among fistulas, hemorrhoids are hemorrhoids in which a through hole appears between the skin around the rectum and the anus. It is generally believed that it may be produced as a result of disease or infection. In anal fistula, for example, the anal gland that discharges mucus is blocked, and an abscess is formed on the skin surface from the rectum to the anus. Sometimes the hemorrhoidal fistula occurs. In addition, when coliform bacteria enter the anal glands, there are wounds nearby, or when the resistance is reduced, the infection/suppuration and chronicity will cause the abscess around the anus to progress and produce hemorrhoidal fistula. Among the hemorrhoid fistulas, the hemorrhoid fistulas associated with inflammatory bowel diseases such as Crohn's disease or ulcerative colitis are caused by the passage of rectal ulcers. It is characterized in that if compared with the normal hemorrhoids, most of the fistulas are in a more complicated form, are more complicated than the normal hemorrhoids, and are prone to repeated recurrence.

Fistulas caused by hemorrhoidal fistulas can be treated by surgical treatment such as the seton method. Although the thread-hanging method is a suitable surgical procedure in short-term hospital admissions, it also has the disadvantage that if the fistula is deeper or longer, the treatment period will be long-term. In addition, in the case of hemorrhoids and fistulas related to inflammatory bowel diseases such as Crohn’s disease or ulcerative colitis, if the anal sphincter is severely damaged due to radical resection, it may cause dysfunction such as fecal incontinence for a long time. It is very difficult to completely cure this type of hemorrhoids.

On the other hand, in Patent Document 1, a novel fistula treatment material composed of biodegradable fibers and collagen has been disclosed. In addition, Patent Document 2 discloses a therapeutic agent for hemorrhoids characterized by containing a basic fibroblast growth factor and a pharmaceutically acceptable carrier. Specifically, Patent Document 2 has described the use as a hemorrhoid treatment agent, including the treatment of hemorrhoids by directly administering the hemorrhoid treatment agent to the area of hemorrhoids, or when the effect of hemorrhoid surgery is insufficient, after the operation The hemorrhoids treatment agent is directly administered to the hemorrhoids affected area to perform hemorrhoids treatment. In more detail, it has been described that for patients with hemorrhoidal fistula, after a radical resection of the hemorrhoidal fistula by thread-drawing method or the like, a trafermin spray or a gel containing trafermin is applied to the affected area. Furthermore, in Patent Document 3, a cross-linked gelatin gel containing a basic fibroblast growth factor has been disclosed, and the angiogenesis effect or bone formation effect has been disclosed. [Prior Technical Literature] [Patent Literature]

[Patent Document 1] International Publication No. 2010/110286 [Patent Document 2] Japanese Patent Laid-Open No. 2004-26808 [Patent Document 3] International Publication No. 1994/027630

[The problem to be solved by the invention]

The prior art described in Patent Documents 1 and 2 is to apply (administer) a drug only to the surface of the fistula tract, instead of administering it to the entire fistula for treatment, and does not aim at the complete cure of the fistula. Therefore, the prior art described in Patent Documents 1 and 2 is not sufficiently satisfactory for fistula treatment. Therefore, a novel fistula treatment material that can exert a more excellent therapeutic effect is desired. [Means to solve the problem]

The inventors of the present invention have made repeated efforts to review and found that by using a cross-linked gelatin gel containing basic fibroblast growth factor as a material for fistula treatment, the above-mentioned problems can be solved, and the present invention has been completed.

The present invention is as follows. [1] A material for fistula treatment, which comprises a gelatin hydrogel cross-linked gelatin gel containing basic fibroblast growth factor. [2] The material for fistula treatment as described in [1], wherein the fistula is related to intestinal tract fistula, rectal bladder fistula, rectovaginal fistula, intestinal skin fistula or hemorrhoid fistula. [3] The material for fistula treatment as described in [1] or [2], wherein the fistula is related to hemorrhoids in patients with autoimmune diseases. [4] The fistula treatment material described in any one of [1] to [3] is used as a medicine, quasi-drug or medical device. [5] The fistula treatment material as described in any one of [1] to [4], wherein the shape is granular. [6] The material for fistula treatment according to any one of [1] to [5], wherein the particle size is a mode diameter of 50 to 300 μm. [7] The fistula treatment material as described in any one of [1] to [6], which remains in a mammal for 10 to 21 days. [8] The fistula treatment material as described in any one of [1] to [7], wherein the fistula treatment material is administered into the fistula. [Effects of the invention]

According to the present invention, it is possible to provide a novel fistula treatment material aimed at the complete cure of the fistula. According to the present invention, it is not limited to being applied to the surface of the fistula, but it can also stay directly on the affected area until it is decomposed, so that the effect of the treatment can be improved exceptionally.

The fistula treatment material of the present invention contains cross-linked gelatin gel containing basic fibroblast growth factor. As the cross-linked gelatin gel containing basic fibroblast growth factor, for example, the materials described in International Publication No. 1994/027630 can be utilized and used.

The basic fibroblast growth factor is a substance also known as bFGF. In the present invention, it is not particularly limited, and a substance known as a basic fibroblast growth factor (bFGF) can be used. Basic fibroblast growth factor (bFGF) is extracted from the pituitary gland, brain, omentum, corpus luteum, adrenal gland, kidney, placenta, prostate, thymus and other organs. It can also be produced by genetic engineering techniques such as recombinant DNA technology. In addition, basic fibroblast growth factor (bFGF) includes those that are modified and can act as fibroblast growth factors. As a modified form of basic fibroblast growth factor (bFGF), for example, in the amino acid sequence of basic fibroblast growth factor (bFGF) obtained by extraction or obtained by genetic engineering methods, One or more amino acids have been added, substituted or deleted. In addition, as a modified form of basic fibroblast growth factor (bFGF), for example, there may be an amino acid with a basic fibroblast growth factor (bFGF) obtained by extraction or obtained by genetic engineering techniques. The sequence has more than 70%, preferably more than 80%, more than 90%, or more than 95% identical amino acid sequence. Basic fibroblast growth factor (bFGF) can be used alone or in the form of a mixture.

In the present invention, as a carrier of basic fibroblast growth factor (bFGF), cross-linked gelatin gel is used. The so-called cross-linked gelatin gel is a gel in which gelatin is used as a raw material, and the gelatin has a cross-linked mesh structure through cross-linking treatment. The cross-linked gelatin gel is produced by cross-linking gelatin, which is a natural polymer that can be decomposed and absorbed in the body. Due to the water insolubility caused by the cross-linking treatment, the cross-linked gelatin gel system has good biocompatibility and less irritation to the organism, so it has excellent properties as a sustained-release carrier. The basic fibroblast growth factor (bFGF) is present in the cross-linked gelatin gel to adjust the degree of cross-linking of the gelatin, the water content of the cross-linked gelatin gel, and the properties (isoelectric point, etc.) of the gelatin, etc. The desired sustained release rate of basic fibroblast growth factor (bFGF) from the cross-linked gelatin gel can be achieved.

In the present invention, the gelatin used to make the gelatin hydrogel can be obtained naturally, and can also be obtained by fermentation using microorganisms, chemical synthesis or genetic recombination operations. These materials can also be appropriately mixed and used. Natural gelatin can be obtained by denaturing collagen derived from humans, pigs, cattle, salmon, sea bream, sharks and other fishes through various treatments such as alkaline hydrolysis, acid hydrolysis, and enzyme decomposition. Gelatin is preferably around 5.0 with an isoelectric point.

Gelatin hydrogel can be prepared by, for example, polymerizing/gelling by adding a crosslinking agent such as glutaraldehyde to gelatin obtained by treating bovine bone collagen with calcium hydroxide. An example of a preparation method of the granular material (acidic gelatin hydrogel microspheres (acidic gelatin hydrogel microspheres), hereinafter referred to as "AGHM".)) can be exemplified as described in the pamphlet of International Publication No. 94/27630 (Patent Document 1) The method described is a method of adding an oil agent such as olive oil to a gelatin aqueous solution and stirring it at 200-600 rpm to form a W/O emulsion, and adding a cross-linking agent aqueous solution to it, or, in advance, at 200-600 rpm After the gelatin solution is dropped into the oil obtained by stirring to make it into a W/O emulsion, the gelatin particles are recovered by centrifugal separation, etc., dried, and the dried gelatin particles are suspended in the crosslinking agent aqueous solution to obtain AGHM. Methods etc. The obtained AGHM is dried under reduced pressure, preferably freeze-dried for use. Depending on the manufacturing method or the difference in raw materials, there may be differences in physical properties such as molecular weight and moisture content, and any of them can be used.

The gelatin is not particularly limited, and what is usually available can be used. Examples of gelatin include alkali-treated gelatin with an isoelectric point of 4.9 (manufactured by Nitta Gelatin Co., Ltd.), and acid-treated gelatin with an isoelectric point of 9.0 (manufactured by Nitta Gelatin Co., Ltd.). One type of gelatin may be used, or multiple types of gelatins having different physical properties such as solubility, molecular weight, isoelectric point, and raw materials may be mixed and used.

The crosslinking agent for crosslinking gelatin is not particularly limited as long as it is non-toxic to living organisms. Carboxy-amine crosslinking agents and amine reactive crosslinking agents (imidate crosslinking agents) can be suitably used. ), maleimide active crosslinking agent, carbonyl reactive crosslinking agent, photoreactive crosslinking agent, etc., for example, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride, 1-cyclohexyl-3-(2-N-morpholinoethyl) carbodiimide-methyl p-toluenesulfonate and other water-soluble carbodiimide, diepoxy compounds , Formalin, etc. As the crosslinking agent, glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are preferred. The cross-linking of gelatin is performed by heat treatment or ultraviolet irradiation. The cross-linking reaction conditions are not particularly limited. Generally, the reaction temperature is 0-40°C, and the reaction time is 0.5-48 hours. In the case of using a carboxyl-amine crosslinking agent, it is preferable to perform the crosslinking under the condition of not containing external carbonyl groups and amines. In the case of using an amine-reactive cross-linking agent, it is preferable to perform cross-linking in a buffer such as a phosphate buffer under neutral or weakly alkaline conditions at room temperature or low temperature for 0.5 to several hours. In the case of using a maleimide reactive crosslinking agent and a carbonyl reactive crosslinking agent, it is preferable to perform crosslinking under conditions near neutral (pH 6.5 to 7.5). The concentration of gelatin and cross-linking agent when preparing cross-linked gelatin gel is not particularly limited. Usually, the gelatin concentration is 1-100 w/v% in the solvent, and the cross-linking agent concentration is 0.01-100 w/v% (equivalent to 1～100 w/v%). 5400mM).

The bioabsorbable polymer hydrogel can be insolubilized by forming chemical crosslinks between the molecules of the bioabsorbable polymer by using a variety of chemical crosslinking agents. As a chemical crosslinking agent, for example, glutaraldehyde, water-soluble carbodiimide such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) can be used, For example, a condensing agent that makes chemical bonds between propylene oxide, diepoxy compounds, hydroxyl groups, carboxyl groups, amine groups, thiol groups, and imidazole groups. Preferably it is glutaraldehyde. In addition, the bioabsorbable polymer may also be chemically crosslinked by thermal dehydration treatment, ultraviolet rays, gamma rays, and electron beam irradiation. In addition, these crosslinking treatments can also be used in combination. Furthermore, hydrogels can also be produced by physical cross-linking using salt cross-linking, electrostatic interaction, hydrogen bonding, hydrophobic interaction, and the like.

The shape of the crosslinked gelatin gel is not particularly limited, and examples thereof include a cylindrical shape, a prismatic shape, a sheet shape, a disc shape, a spherical shape, and a particle shape. In the case of cylindrical, prismatic, sheet, and disc shapes, they are mostly used as implants. In the case of spherical and granular shapes, injections can also be performed. If it is considered that the material for fistula treatment of the present invention is filled For fistulas, the shape of the cross-linked gelatin gel is preferably spherical or particulate, and more preferably particulate.

The cross-linked gelatin gel in spherical or particulate shape can be manufactured according to the method described in International Publication No. 1994/027630. The cross-linked gelatin gel system usually has an average particle size of 1 to 1000 μm, and can be crushed with a homogenizer or the like according to the purpose. Furthermore, it can also be sieved to obtain particles of a desired size using a mesh.

The cross-linked gelatin gel can be made into a desired moisture content by changing the concentration of gelatin and a cross-linking agent that are raw materials. To increase the water content, it is only necessary to reduce the gelatin concentration and the crosslinking agent concentration at the same time. Conversely, to reduce the water content, it is only necessary to increase the gelatin concentration and the crosslinking agent concentration at the same time. The water content of the crosslinked gelatin gel is usually 50-99w/w%. The moisture content of the cross-linked gelatin gel means the ratio of the weight of water in the gel to the total weight of the gel when wet. After the basic fibroblast growth factor (bFGF) is supported on the cross-linked gelatin gel, the water content can be set to 50-99w/w%.

The cross-linked gelatin gel obtained in the above manner can also be dried under reduced pressure or freeze-dried. The cross-linked gelatin gel containing basic fibroblast growth factor is obtained by dripping an aqueous solution of basic fibroblast growth factor (bFGF) and impregnating it in the freeze-dried cross-linked gelatin gel, or cross-linking The gelatin gel is obtained by suspending in an aqueous solution of basic fibroblast growth factor (bFGF) and swelling it again. The content of basic fibroblast growth factor (bFGF) in the cross-linked gelatin gel can be appropriately set according to the water content of the cross-linked gelatin gel, etc. Generally, it is 0.1-500 μg per 1 mg of the cross-linked gelatin gel.

The sustained release period of basic fibroblast growth factor (bFGF) from the cross-linked gelatin gel containing basic fibroblast growth factor (bFGF), the release amount of bFGF, etc. can depend on the water content of the cross-linked gelatin gel. Various conditions such as the isoelectric point of the gelatin used, the amount of bFGF supported in the formulation, and the site to be administered, can be appropriately set.

The fistula treatment material of the present invention is used for the treatment of fistulas, and can be used as, for example, pharmaceuticals and quasi-drugs required by laws related to ensuring the quality, effectiveness, and safety of pharmaceuticals, medical equipment, etc. Or medical machines.

Examples of fistulas that are targeted for fistula treatment include tubular defects known as internal fistulas or external fistulas. The fistula is not particularly limited, and examples thereof include fistulas related to intestinal tract fistula, rectovesical fistula, rectovaginal fistula, intestinal skin fistula, or hemorrhoidal fistula. In particular, the material for the treatment of fistulas of the present invention is suitable for the treatment of fistulas related to hemorrhoids. In addition, for example, the term "related to" for fistulas related to hemorrhoidal fistulas is used in the meaning of fistulas that have been evaluated as fistulas and pathological fistulas.

Among patients with autoimmune diseases such as ulcerative colitis and Crohn's disease, up to 17% to 50% of patients will develop hemorrhoid fistula. Most patients with autoimmune disease as a basic disease are accompanied by a decrease in immunity and do not respond to available treatments. Among patients with autoimmune disease, the management of fistulas related to hemorrhoids continues to be extremely Difficult question. The recurrence of fistulas or fistulas associated with hemorrhoidal fistulas is known to be a complication accompanied by extreme pain that greatly reduces the quality of life of patients suffering from autoimmune diseases. The material for the treatment of fistulas of the present invention is suitable for the treatment of fistulas related to hemorrhoids, in particular, the treatment of fistulas related to hemorrhoids in autoimmune diseases.

The fistula treatment material of the present invention is used to seal the fistula produced in two parts of the living body, such as intestinal tract fistula, rectal bladder fistula, rectovaginal fistula, intestinal skin fistula or hemorrhoid fistula. In the present invention, when a fistula treatment material is used to treat a fistula, the following treatment method (administration method) can be used as an example. For example, it can be used by the following method: a method of injecting a preparation in the form of an injection obtained by suspending the fistula treatment material in a suitable medium into the affected area, so that the fistula treatment material is made into a filament, sheet or disc shape The obtained gel preparation is left in the affected area, or the method of injecting the diseased part (inside hemorrhoids or fistula) from the outside of the body using a syringe or the like. As a treatment method (administration method), a method of administering the material for fistula treatment into the fistula is preferably used, and a method of injecting the diseased part (into the fistula or fistula) using a syringe or the like is more preferable. The fistula treatment material of the present invention can be used in a method of administering to a patient in need, and can also be used in a method of administering to a fistula of a patient with a fistula. More specifically, the tube is inserted into the fistula from the outside (outside the body) toward the intestine. Then, the entrance of the fistula inside the intestine is plugged, while the tube is drawn out, the fistula treatment material in the syringe is injected (filled) into the fistula. After the tube is drawn to the outside of the fistula, the outside fistula is plugged. The fistula can be sealed with a medical cell adhesive or surgically. Although it is also affected by the slow release of basic fibroblast growth factor (bFGF) from the fistula treatment material, the fistula treatment material is more suitable for indwelling Or the injection site, or the diseased site of mammals (in the fistula or fistula), it can survive for about 10 days (8 to 12) to 21 days (19 to 23), 10 to 21 days, It is better to survive for about 2 weeks (12 to 16 days). The shape of the tube used here is not particularly limited as long as it is used in the medical field, and it can be appropriately set according to the diameter of the fistula. In addition, the above-mentioned so-called mammals include human or non-human mammals (such as mice, rats, hamsters, guinea pigs, rabbits, pigs, dogs, horses, cows, monkeys, etc.), which means that they are classified as all mammals. animal.

When the fistula treatment material of the present invention is made into an injectable preparation, it is suitably suspended in a medium such as purified water for injection, physiological saline, and buffer. As the buffer solution, a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, and the like can be cited. If necessary, dispersing agents, surfactants, isotonic agents, pH adjusters, soothing agents, stabilizers, preservatives, coloring agents, etc. usually used in the manufacture of injectable preparations may also be appropriately blended. The particle shape can also be adjusted by penetrating the injection needle. The fistula treatment material of the present invention is preferably administered to the diseased site (in the fistula or in the fistula). Therefore, the size of the particles is preferably the size that passes through the tube, as long as it is measured by the mode diameter. Just 1～500μm, 10～300μm, 50～300μm. The method for measuring the size of the particles includes a sieving method, a microscopy method, a sedimentation method, a chromatography method, etc. Any method may be used as long as it is a method for measuring the mode diameter.

The shape of the material for fistula treatment can be trimmed by proper granulation. It is also possible to insert a cross-linked gelatin gel into a tube such as a cylinder, and trim it into a cylindrical shape, and then support the alkalinity. Fibroblast growth factor (bFGF).

The material for the treatment of fistulas of the present invention may also contain other active ingredients commonly used as a therapeutic agent for hemorrhoids as needed. The therapeutic agent for hemorrhoids is not particularly limited, and examples include adrenal cortex hormone agents such as prednisolone acetate, hydrocortisone, or betamethasone, such as lidocaine. Or local anesthetics such as dibucaine, such as antipyretic, analgesic and anti-inflammatory agents such as indometacin, aspirin, and sodium salicylate, such as anti-inflammatory agents such as lysozyme chloride or glycyrrhetinic acid Agents, such as antipruritic agents such as crotamiton, tissue adhesives such as fibrin paste, wound healing agents such as allantoin, such as liver oil, ergocalciferol, riboflavin, pyridoxine hydrochloride, Vitamin agents such as tocopherol or tocopherol acetate, such as sulfisomidine, sulfamethazine sodium, sulfabenzamide (homosulfamine) or sulfadiazine (sulfadiazine) and other sulfa agents, chlorhexidine hydrochloride ), cetyltrimethylammonium bromide (cetrimide), dequalinium chloride (dequalinium chloride) or benzalkonium chloride (benzalkonium chloride) and other fungicides, such as diphenhydramine (diphenhydramine), diphenhydramine hydrochloride , Antihistamines such as chlorpheniramine maleate, antibiotics, etc. As the therapeutic agent for hemorrhoids, one type may be used, or two or more types may be used.

The dosage form used in the present invention can be used in the form of a suspension or a gel. [Example]

Hereinafter, the present invention will be further described in detail with examples, but the present invention is not limited to the following examples. In addition, various changes can be made without departing from the scope of the technical idea of the present invention.

Example 1 <Preparation of cross-linked gelatin gel containing bFGF> To 40,000 μL of an aqueous solution (5.0%) of alkali-treated gelatin (manufactured by Nitta Gelatin Co., Ltd.) at an isoelectric point of 5.0, 80 μL of glutaraldehyde (Nacalai Tesque Co., Ltd.) was added. Co., Ltd.) aqueous solution (25%, equivalent to a final concentration of 0.05%) is poured into a plastic mold, and cross-linking is performed at 4°C for 12 hours. Then, the cross-linked gelatin gel was finely cut into long strips, and the cross-linking reaction of gelatin was stopped by impregnating it in 1 L of 0.1 M glycine aqueous solution at room temperature for 1 hour. Transfer the gelatin gel to 1L of distilled water and wash it at room temperature for 1 hour. The obtained gelatin gel is pulverized using a homogenizer, sieved, and the fraction with a diameter of 106 to 180 μm is freeze-dried to obtain fine particles of the crosslinked gelatin gel. Secondly, 1.3μg of bFGF (Trifomin (gene recombination), R&D Pharmaceutical Co., ^{Ltd.) was prepared with 125} I-labeled bFGF by the chloramine method with an appropriate amount of phosphate-buffered saline (PBS, manufactured by Nissui Pharmaceutical Co., Ltd.) Prepared) and 8.7μg of unlabeled bFGF into a 30μL mixture. The mixture was impregnated in 3.0 mg of fine cross-linked gelatin gel at 4 degrees Celsius for 18 hours to prepare a bFGF containing Cross-linked gelatin gel.

Example 2 ＜bFGF release test (1)＞ To the prepared cross-linked gelatin gel containing bFGF, 750 μL of PBS(+) containing collagenase D (Roch; 4ug/mL) was added. The cross-linked gelatin gel system decomposes over time and completely decomposes within 8 hours. The RI activity in the solution was measured 1, 2, 4, and 8 hours after the addition. The residual amount of bFGF when collagenase D was added was set to 100%, and the residual amount of bFGF after 8 hours was set to 0%, and the result of in vitro bFGF release obtained is shown in FIG. 1.

Example 3 ＜bFGF release test (2)＞ The prepared cross-linked gelatin gel containing bFGF was embedded under the skin of the back of female ddy mice (purchased from Qingshui Experimental Materials Co., Ltd.). By measuring the RI activity of the subcutaneous tissues 1, 3, 7 and 14 days after the administration, the bFGF release ability of the gel in vivo was evaluated. The cross-linked gelatin gel system decomposes over time, and the RI activity of the subcutaneous tissue decreases with time. It was completely decomposed on the 14th day from the administration, and the RI activity in the subcutaneous tissue almost completely disappeared. The RI activity at the time of implantation was set to 100%, and the result of the release of bFGF in vivo is shown in Figure 2.

Reference Example 4 ＜Test of bFGF and cross-linked gelatin gel in rat model＞ To evaluate the effect of bFGF and cross-linked gelatin gel on cecal skin fistula. BFGF was prepared with an appropriate amount of normal saline (Otsuka normal saline injection) and used in a dose of 10 μg (bFGF group). The cross-linked gelatin gel was prepared by impregnating the Otsuka saline injection solution in the fine particles of the cross-linked gelatin gel at 4 degrees Celsius for 18 hours (cross-linked gelatin gel group). As a control group, Otsuka saline injection (control group) was used. 7-week-old male Wistar rats (purchased from CLEA Co., Ltd., Japan) were used after acclimation for 1 week. Under the alternating light and dark cycle of 12 hours, feeding is carried out under the conditions of free bait and drinking water. As an animal model of fistula, a model rat that has formed an intestinal skin fistula in rats is used. The intestinal skin fistula is made by applying a small incision to the cecum exposed by the fistula made in the abdomen and skin of the rat, and suture the open part of the skin. The bFGF group, the cross-linked gelatin gel group and the control group were injected subcutaneously around the intestinal skin fistula at the same time. The intestinal skin fistula was evaluated on the 1st and 14th day after making the intestinal skin fistula. The treatment effect was judged by the complete closure of the cecal skin fistula on the 14th day, which was evaluated as the completion rate of complete closure in each group. The results are shown in Figure 3. In the control group, bFGF group, and cross-linked gelatin gel group, the completion rate of complete closure on the 14th day after intestinal skin fistula production was all 0%, and the therapeutic effect caused by bFGF and cross-linked gelatin gel was not seen .

Example 5 ＜Test of cross-linked gelatin gel containing bFGF in rat model＞ To evaluate the effect of cross-linked gelatin gel containing bFGF on cecal skin fistula. As the drug administration group, the bFGF-containing cross-linked gelatin gel (bFGF-containing gelatin gel group) containing 10 μg of bFGF prepared in Example 1 was used. As a control group, the reference example was used 4 The bFGF-free cross-linked gelatin gel (control group) impregnated with Otsuka saline solution prepared in the same manner was used to evaluate the therapeutic effect of intestinal skin fistula. The cross-linked gelatin gel containing bFGF is prepared by preparing 10 μg of bFGF with an appropriate amount of physiological saline (Otsuka physiological saline injection), and infiltrating 10 mg of the cross-linked gelatin gel under the same conditions as in Example 1. To be modulated. The preparation and evaluation of the rat intestinal cutaneous fistula were performed in the same manner as in Reference Example 4. The results are shown in Figure 4. The completion rate of complete closure on the 14th day after the preparation of the intestinal skin fistula was 0% for the control group, and 67% for the bFGF-containing gelatin gel group. From the results shown in Figure 4, it can be seen that the cross-linked gelatin gel containing bFGF has a significant therapeutic effect on fistulas.

Example 6 <Test of cross-linked gelatin gel containing bFGF and cross-linked gelatin gel in rabbit model> To evaluate the effect of cross-linked gelatin gel containing bFGF and cross-linked gelatin gel on hemorrhoids and fistulas. The cross-linked gelatin gel containing bFGF is prepared by preparing 100 μg of bFGF with an appropriate amount of physiological saline (Otsuka physiological saline injection), and infiltrating it in 5 mg of cross-linked gelatin gel under the same conditions as in Example 1. To be modulated. The cross-linked gelatin gel was prepared by impregnating the Otsuka saline injection solution in fine particles of the cross-linked gelatin gel at 4 degrees Celsius for 18 hours. 10-week-old female Kbl:JW rabbits (purchased from Oriental Yeast Industry Co., Ltd.) were used after acclimation for 1 week. Under the alternating light and dark cycle of 12 hours, feeding is carried out under the conditions of free bait and drinking water. As an animal model of fistula, model rabbits that have formed fistulas in rabbits are used. Hemorrhoidal fistula is produced by piercing the rabbit’s anus with an injection needle toward the skin, and using a band to promote the formation of the fistula for 6 weeks. After 6 weeks, the band was removed and the inside of the hemorrhoids was scratched, the intestinal mucosa of the hemorrhoids was sutured and closed, and cross-linked gelatin gel or cross-linked gelatin gel containing bFGF was administered from the skin side of the hemorrhoids. Then, the skin side of the hemorrhoidal fistula is sutured and closed, and a cross-linked gelatin gel or a cross-linked gelatin gel containing bFGF is applied to the skin side around the sutured part.

[Table 1] Inject into fistula Apply to the skin around the fistula Group 1 Cross-linked gelatin gel Cross-linked gelatin gel Group 2 Cross-linked gelatin gel containing bFGF Cross-linked gelatin gel Group 3 Cross-linked gelatin gel Cross-linked gelatin gel containing bFGF

The fistula area ratios of the second group and the third group were calculated when the fistula area of the first group on the 14th day after the drug administration was set to 100%. The results are shown in Figure 5. Furthermore, the complete closure of the hemorrhoidal fistula of each body is judged. The results are shown in Figure 6. The fistula area rate on the 14th day after the drug administration was 2% for the second group and 32% for the third group. The complete closure of the hemorrhoidal fistula in each body was not confirmed in all individuals in the first and third groups, and in the second group, it was confirmed in 1/3 of the individuals. From the results shown in Figs. 5 and 6, it can be seen that the cross-linked gelatin gel containing bFGF is administered to the skin around the hemorrhoids to promote healing. In addition, it can be seen that by intra-administering the cross-linked gelatin gel containing bFGF, it can be seen that it has a more significant healing promoting effect than the administration of the skin around the fistula, such as complete closure.

[Figure 1] shows the in vitro release results of bFGF in Example 2. [Figure 2] shows the results of in vivo release of bFGF in Example 3. [Figure 3] shows the results of the treatment effect in Example 4. [Figure 4] shows the results of the treatment effect in Example 5. In the control group, it is considered that the intestinal juice leaked from the cecum, so that the surrounding hair was peeled off in the case of rats that were incurable. Fig. 5 shows the fistula area ratios of the second group and the third group when the fistula area of the first group on the 14th day after the drug administration in Example 6 is set to 100%. [Figure 6] shows the result of the complete closure of the fistula in Example 6.

Claims (8)

A material for fistula treatment, which comprises cross-linked gelatin gel containing basic fibroblast growth factor. Such as the material for fistula treatment of claim 1, wherein the fistula system is related to intestinal tract fistula, rectovesical fistula, rectovaginal fistula, intestinal skin fistula or hemorrhoid fistula. The material for fistula treatment according to claim 1 or 2, wherein the fistula system is related to the hemorrhoid fistula of patients with autoimmune diseases. For example, the material for fistula treatment in any one of claims 1 to 3, which is used as a medicine, quasi-drug or medical device. The material for fistula treatment according to any one of claims 1 to 4, wherein the shape is granular. The material for fistula treatment according to claim 5, wherein the size of the particles is 50 to 300 μm in the mode diameter. Such as the fistula treatment material of any one of claims 1 to 6, which remains in the mammalian body for 10 to 21 days. The material for fistula treatment according to any one of claims 1 to 7, wherein the material for fistula treatment is administered into the fistula.

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