WO2021112254A1

WO2021112254A1 - Production method for gelatin sponge for regeneration of eardrum

Info

Publication number: WO2021112254A1
Application number: PCT/JP2020/045357
Authority: WO
Inventors: 眞一金丸; 雄二厚澤; 宜子油田
Original assignee: ノーベルファーマ株式会社; 株式会社ニッピ
Priority date: 2019-12-06
Filing date: 2020-12-05
Publication date: 2021-06-10
Also published as: TW202135853A; JPWO2021112254A1

Abstract

[Problem] To provide a technique of producing a gelatin sponge, capable of improving the healing rate of therapy of a perforated eardrum using a gelatin sponge carrying a basic fibroblast growth factor. [Solution] This production method for a gelatin sponge comprises (1) a step for foaming an aqueous gelatin solution, (2) a step for obtaining a xerogel by lyophilization of the aqueous gelatin solution foamed in step (1), and (3) a step for sterilizing the xerogel obtained in step (2), and is characterized in that the sterilization process in step (3) is performed by a radiation irradiation process. This gelatin sponge is produced by said method.

Description

Manufacturing method of gelatin sponge for eardrum regeneration

The present invention relates to a method for producing a gelatin sponge for tympanic membrane regeneration.

Eardrum perforation is a condition in which the eardrum is perforated due to an infection or injury in the middle ear, and presents with symptoms such as pain, bleeding, hearing loss, tinnitus, and vertigo. In mild perforation of the eardrum, the eardrum may heal spontaneously, but in the case of severe perforation of the eardrum, treatment such as tympanoplasty may be required. Among the treatment methods for tympanoplasty, tympanoplasty has problems such as high invasiveness and hearing loss due to shallowing and thickening of the tympanic membrane, which is complicated in tympanoplasty. There are problems such as the inability to close a large perforation or a large perforation.

On the other hand, as a method for treating less invasive eardrum perforation, a method is disclosed in which a gelatin sponge carrying a basic fibroblast growth factor is placed in an affected area to regenerate the eardrum (Patent Document 1).

International Publication WO2009 / 157558 Pamphlet

The treatment technique for tympanic membrane perforation in which a gelatin sponge carrying a basic fibroblast growth factor is placed in the affected area has an advantage that it is less invasive and can be performed in an outpatient setting. There is no doubt that if this treatment technique can further improve the cure rate, the benefit to the patient will be great and more desirable. An object of the present invention is to provide a technique for producing a gelatin sponge capable of further improving the cure rate of a perforated eardrum treatment using a gelatin sponge carrying a basic fibroblast growth factor.

The present invention is a method for producing a gelatin sponge, which comprises a step of irradiating gelatin xerogel in the production process, and a gelatin sponge produced by the method.

The present invention is also obtained in (1) a step of foaming a gelatin aqueous solution, (2) a step of freeze-drying the foamed gelatin aqueous solution in step (1) to obtain a xerogel, and (3) a step (2). A method for producing a gelatin sponge, which comprises a step of sterilizing the xerogel, which comprises performing a irradiation step after the step (2), and a method for producing a gelatin sponge, which is produced by the method. It is a gelatin sponge. In the present invention, the irradiation step may be performed as a sterilization step in step (3).

In the present invention, various methods of irradiation such as electromagnetic radiation such as gamma rays and particle beams such as electron beams can be used in the irradiation step, and in particular, electron beam irradiation can be preferably used.

According to the present invention, a gelatin sponge suitable for treating perforated eardrum can be obtained.

It is a figure which shows the shape of the swelling sample of the gelatin sponge (xerogel) before electron beam irradiation. It is a figure which shows the shape of the swelling sample of the gelatin sponge after electron beam irradiation. It is a figure which shows the shape of the swelling sample of the commercially available gelatin sponge (electron beam non-irradiation).

Hereinafter, embodiments of the present invention will be described in detail. In the description of the production process in particular in the present specification, the gelatin sponge after freeze-drying and before the irradiation step is referred to as xerogel, and is distinguished from the gelatin sponge after irradiation. In addition, the following description merely describes preferred embodiments, and is not intended to limit the scope of the present invention.

The method for producing a gelatin sponge according to the present invention includes (1) a step of foaming a gelatin aqueous solution, (2) a step of freeze-drying the foamed gelatin aqueous solution to obtain a xerogel, and (3) a step of sterilizing the obtained xerogel. It is a production method including, and includes a step of irradiating the xerogel obtained in the step (2) with radiation. In a preferred embodiment, the production method according to the present invention is characterized in that the sterilization step in the step (3) is performed by a method by irradiation.

The type of gelatin used as a raw material is not particularly limited, and is obtained by partially hydrolyzing or heat-decomposing collagen contained in bones, ligaments, skin, etc. of cows, pigs, chickens, salmon, etc. with acid or alkali. Can be used. Preferably, Japanese Pharmacopoeia gelatin can be used.

In the present invention, steps (1) and (2) can be carried out by a method known to those skilled in the art, for example, the method described in Patent Document 1.

The gelatin aqueous solution used in the step (1) is not particularly limited as long as it has a viscosity sufficient to retain the bubbles generated by the foaming during the freeze-drying step. For example, a gelatin aqueous solution having a concentration of 1 to 30% by weight and / or a gelatin aqueous solution having a viscosity of 1 to 20 mPa · s can be preferably used. By using the gelatin aqueous solution having such properties, the generated bubbles can be retained even during the freeze-drying step, and a gelatin sponge having a porous structure suitable for tympanic membrane perforation treatment can be obtained.

As the foaming step in the step (1), various methods such as a method of stirring an aqueous gelatin solution at high speed to foam (see Patent Document 1) and a method of bubbling with a gas can be used. Of these methods, a method of bubbling using a gas can be preferably used. When bubbling with a gas, the gas to be used is not particularly limited as long as gelatin is not altered, and a rare gas such as nitrogen or argon can be used in addition to air. As the bubbling condition, a normal bubbling method such as blowing a gas while stirring the gelatin aqueous solution can be used. For example, a foamed gelatin aqueous solution can be obtained by pouring a gelatin aqueous solution and air at a constant ratio into a blender for stirring the gelatin aqueous solution and continuously stirring the gelatin aqueous solution. The porosity of the gelatin sponge can be changed by changing the flow rates of the gelatin aqueous solution and air, but if the speed is too low, there is a high possibility of gelation in the middle. The stirring speed of the gelatin aqueous solution and the flow velocity of the added air are appropriately adjusted according to the concentration and viscosity of the gelatin aqueous solution used.

The freeze-drying step of step (2) can be carried out by a method usually used in the production of xerogel. For example, as described in Patent Document 1, an aqueous gelatin solution is poured into a mold of an appropriate size, frozen at about -40 to about -80 ° C for about 30 to about 120 minutes, and then the frozen product is frozen. For example, it can be carried out by a method such as freeze-drying under the condition of about 0.1 Torr.

In a preferred embodiment, the sterilization step of step (3) is performed by irradiation. The greatest feature of the present invention is that this sterilization step is performed by irradiation. By performing the sterilization step by irradiation, it is possible to obtain a gelatin sponge that slightly changes the physical properties of the gelatin sponge and further improves the healing rate of tympanic membrane perforation treatment using a gelatin sponge carrying a basic fibroblast growth factor. It becomes.

As the radiation to be irradiated, various methods of irradiation such as electromagnetic radiation such as gamma rays and particle beams such as electron beams can be used. Of these, an electron beam can be particularly preferably used. Unlike gamma-ray irradiation, which causes sterilization and changes in physical properties due to secondary electron beams, electron beam irradiation can directly cause sterilization and changes in physical properties, so the dose can be set high and the irradiation time can be set short. There are advantages.

As the irradiation conditions, the same conditions as those in the normal sterilization step can be used. For example, in the case of electron beam irradiation, a sample cut out to a thickness of about 1 cm may be irradiated with 1 to 60 kGy. Further, when gamma ray irradiation is performed, the sample cut out to a thickness of about 1 cm may be irradiated under the condition of irradiating 1 to 60 kGy.

In the above description, the mode in which the irradiation is performed as a sterilization step has been described, but the radiation irradiation may not be performed as a sterilization step. For example, the sterilization step may be performed by a method other than irradiation such as dry heat sterilization, and a method such as irradiation may be used separately from the sterilization step. That is, the manufacturing process may include a step of irradiating the xerogel with radiation. The radiation irradiation conditions in this case are the same as in the case of performing radiation irradiation as a sterilization step.

The present invention will be described below with reference to specific embodiments, but the present invention is not limited to the embodiments, and various changes and modifications thereof are within the scope of the appended claims by those skilled in the art. It is understood that it can be practiced without departing from the defined scope or gist of the invention.

Example 1: Changes in physical properties of swollen gelatin sponge before and after electron beam irradiation (1) Sample Japanese Pharmacopoeia "Gelatin" (BP-160, manufactured by Nippi Co., Ltd.) and Japanese Pharmacopoeia "Purified Gelatin" (Medigelatin "HMG-BP" , Nippi Co., Ltd.) was mixed so as to have a jelly strength of 200 g, and 540 g of the mixture was added to 8,460 g of water to prepare a gelatin concentration of 6%. This was heated to 40 ° C. or higher and dissolved to obtain an aqueous gelatin solution. This gelatin aqueous solution was filtered through a 0.2 μm membrane filter and poured into a microblender (manufactured by Izumi Food Machinery) at a flow rate of 30 L / h while heating at 28 ° C. At the same time, air was introduced at a flow velocity of 80 L / h and foamed while stirring at 900 rpm. The foamed gelatin aqueous solution was dispensed into a tray and cooled to −40 ° C. using a freeze dryer. The pressure was reduced to about 0.2 Torr and lyophilized to obtain a gelatin xerogel. The xerogel was cut to an appropriate size and thermally crosslinked at 150 ° C. for 6 hours (referred to as sample A). Sample A was electron beam sterilized using an electron accelerator under the condition of 15 kGy to obtain a gelatin sponge (referred to as sample B). As a control sample, a commercially available gelatin sponge (electron beam non-irradiated, Sponzel (registered trademark), manufactured by LTL Pharma Co., Ltd., referred to as sample C) was used.

(2) Confirmation of Changes in Physical Properties Samples A, B, and C were cut into columns having a diameter of about 14 mm and a height of about 10 mm, immersed in water, and swollen. The ratio of the diameters of each sample before and after the obtained swelling (diameter after swelling / diameter before swelling) was examined and used as an index of the difference in physical properties (mainly softness) of the swelling samples. The measurement was repeated 3 times and the physical properties were evaluated using the average value.

The measurement results of the diameter ratio before and after swelling were 1.05 and 1.06 for Sample A and Sample C, respectively, showing almost equal values. On the other hand, in sample B, the ratio of diameters before and after swelling was 1.09, indicating that the physical properties changed slightly and became softer. Photographs of each swollen sample are shown in FIGS. The swollen samples of Sample A and Sample C showed almost no distortion due to their own weight, and their shapes were stable (FIGS. 1 and 2). From this result, it was confirmed that the sample A and the sample C (sample not irradiated with an electron beam) had the same softness in appearance. On the other hand, the swollen sample of sample B (electron beam irradiated product) had a distorted shape as compared with the swollen sample of samples A and C. From this result, it was clarified that the swollen sample of sample B (electron beam irradiation product) had softer physical characteristics than the swollen samples of samples A and C, and the electron beam irradiation step was performed during the manufacturing process. It was confirmed that the inclusion changed the physical characteristics of the gelatin sponge to soft physical characteristics.

Example 2: Difference in clinical results depending on the presence or absence of electron beam irradiation of gelatin sponge (1) Method For patients with perforated eardrum, basic fibroblast growth factor (Fiblast®, registered trademark), according to the method described in Patent Document 1. Kaken Pharmaceutical Co., Ltd.) Performed eardrum perforation recovery by placing a supported gelatin sponge. Patients were divided into group A (62.4 ± 16 years old, 20 cases) and group B (60.9 ± 18 years old, 25 cases), and group A was sample C (commercially available product without electron beam irradiation) as a gelatin sponge. ) Was used, and sample B (electron beam irradiation product) was used as a gelatin sponge in group B. The progress after placement of the gelatin sponge carrying a basic fibroblast growth factor was observed, and the presence or absence of obstruction of the affected area was confirmed. If no obstruction was observed 4 weeks after the operation, the eardrum perforation recovery operation was repeated (number of operations: maximum 4 times). A 4-week follow-up was performed for each eardrum perforation recovery operation, the number of patients with obstructed perforation site was confirmed, and the success rate of eardrum regeneration was calculated. In addition, the patients in each group were divided into three stages of eardrum perforation grades as shown in Table 1, and the success rate of eardrum regeneration in each grade was also compared.

Table 1 Breakdown of eardrum perforation grade

(2) Results The results are shown in Table 2. As shown in this table, the case using sample B (group B) showed a significantly higher success rate of eardrum regeneration as compared with the case using sample C (group A).

Table 2 Success rate of eardrum regeneration in each group

Tables 3 and 4 show the success rate of eardrum regeneration for each eardrum perforation grade. As is clear from these tables, the eardrum in group B (cases using a gelatin sponge that has been irradiated with an electron beam) is higher than that in group A (cases that use a commercially available gelatin sponge that has not been irradiated with an electron beam). The regeneration success rate was high, and this difference was more pronounced as the eardrum perforation grade increased.

Table 3 Success rate of eardrum regeneration for each eardrum perforation grade in group A

Table 4 Success rate of eardrum regeneration for each eardrum perforation grade in Group B

From the above results, it is possible to dramatically improve the results of tympanic membrane regeneration by using a gelatin sponge that has been irradiated with an electron beam, and gelatin suitable for tympanic membrane regeneration by irradiating with an electron beam. It was confirmed that a sponge could be obtained.

The gelatin sponge according to the present invention and a commercially available product (Sponzel (registered trademark)) that has not been irradiated with an electron beam are swollen even though there is no difference in basic performance such as density, water absorption rate, and digestion time. There is a difference in the softness of the case. It is considered that such changes in physical properties are reflected in the difference in clinical results.

The gelatin sponge, which is an embodiment of the present invention, can be used as a therapeutic agent for perforating the eardrum.

Claims

A method for producing a gelatin sponge, which comprises a step of irradiating gelatin xerogel during the production process.
(1) Step of foaming gelatin solution, (2) Step of freeze-drying the foamed gelatin solution in step (1) to obtain xerogel, (3) Sterilization of xerogel obtained in step (2) The method for producing a gelatin sponge according to claim 1, wherein the sterilization step in the step (3) is performed by a irradiation step.
The production method according to claim 1 or 2, wherein the sterilization step in the step (3) is electron beam sterilization.
A gelatin sponge produced by the production method according to any one of claims 1 to 3.