JPH04285561A - Sterilization method for medical material and manufacture of medical instrument - Google Patents

Abstract

PURPOSE:To obtain a medical material sterilization method having the capability of sterilizing a medical material mainly composed of protein having physiologi cal activity without denaturing the protein contained in the material. CONSTITUTION:A sterilization method in the title is used to sterilize a medical material mainly composed of water insoluble protein having physiological activity. The concerned medical material is impregnated with protective solution, and a gamma-ray is irradiated thereto while maintaining an impregnated condition, thereby obtaining the intended sterilization method.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for sterilizing medical materials and a method for manufacturing medical instruments. More specifically, the present invention provides a method for sterilizing medical materials containing physiologically active water-insoluble proteins as a main component, and a method for sterilizing medical materials containing physiologically active water-insoluble proteins as a main component on the surface of a base material. The present invention relates to a method of manufacturing a medical device that is coated with materials and is sterilized.

0002

2. Description of the Related Art Conventionally, various medical devices have been disclosed in which a physiologically active protein is provided on the surface of a base material.

For example, Japanese Patent Application Laid-open No. 62-258666 discloses an artificial blood vessel made of a porous base material coated with gelatin cross-linked with isocyanate. Further, Japanese Patent Application Laid-Open No. 54-63588 discloses an artificial blood vessel in which proteins such as albumin and gelatin are copolymerized into Dacron using glutaraldehyde.

[0004] In these techniques, most of the bacteria contained in the protein are killed by the crosslinking action of isocyanate or glutaraldehyde, but the parts to which the protein is not added are not sterilized. Alternatively, perform γ-ray sterilization.

[0005] However, these sterilization operations cause the protein to denature and lose most of its physiological activity.

[0006]

SUMMARY OF THE INVENTION Therefore, the present invention provides a medical treatment method that enables medical materials containing physiologically active proteins as a main component to be sterilized without denaturing the proteins in the medical materials. The purpose is to provide a method for sterilizing materials for use.

[0007]

[Means for Solving the Problems] What solves the above problems is a method for sterilizing medical materials whose main component is physiologically active water-insoluble proteins, in which the medical materials are impregnated with a protective solution. This method of sterilizing medical materials is characterized by irradiating them with gamma rays while maintaining the impregnated state.

Preferably, the protective solution is selected from the group consisting of distilled water, physiological saline, and aqueous polysaccharide solution.

[0009] The protein is preferably fibrin or a partially hydrolyzed product thereof. The present invention also includes a step of applying a medical material whose main component is a physiologically active water-insoluble protein to the surface of a base material, and impregnating the medical material with a protective solution. The method of manufacturing a medical device is characterized by comprising a step of irradiating a base material and a medical material with gamma rays while maintaining an impregnated state.

0010

[Operation] In the present invention, when sterilizing medical materials whose main component is physiologically active water-insoluble proteins, the medical materials are impregnated with a protective solution and irradiated with gamma rays while maintaining the impregnated state. Therefore, it is possible to reliably sterilize the medical materials without denaturing the proteins in them. Specifically, it is preferable that the medical material be impregnated with a protective solution selected from the group consisting of distilled water, physiological saline, and a polysaccharide aqueous solution, and then irradiated with gamma rays while maintaining the impregnated state.

The present invention will be explained in detail below.

[0012] In the present invention, a "physiologically active protein" refers not only to cases in which the protein itself has physiological activity, but also to cases in which the protein itself serves as a carrier and supports a physiologically active substance (drug). It also includes.

[0013] The protein employed in the present invention is not particularly limited as long as it expresses some physiological activity in vivo and is water-insoluble, but specifically, gelatin, which has cell-inducing properties, or Preferred examples include fibrin or its partial hydrolyzate, which has antithrombotic properties (fibrinolytic activity enhancement effect). Among these proteins, partially hydrolyzed fibrin is preferred because it has a particularly excellent action of enhancing fibrinolytic activity.

[0014] The medical material of the present invention contains the above-mentioned protein as a main component, but may also support or contain other physiologically active drugs. These drugs are not particularly limited, but include acidic mucopolysaccharides with anticoagulant activity such as heparin, chondroitin sulfate, and hyaluronic acid, and enzymes with fibrinolytic activity such as urokinase and elastase. . In addition, when carrying or blending an enzyme with a protein, it is necessary to select an enzyme that does not degrade the protein itself. In addition, when a protein is used to support a drug as described above, the protein itself does not need to have physiological activity. Not done.

[0015] As the fibrin, one obtained by contacting at least one material selected from purified fibrinogen, plasma, and cryoprecipitate with a thrombin solution can be used. In addition, as a hydrolyzate of fibrin, fibrin obtained by the same method as above is treated with a hydrolase such as a plasmin solution to undergo partial hydrolysis, resulting in partial hydrolysis that exposes the amino acid lysine residue at the C-terminus. Degradants (peptides) can be used.

[0016] However, the protein in the present invention is
It is not limited to the one formed by reacting a water-soluble protein precursor (fibrinogen) with an active substance (thrombin) as described above, but it is also possible to use one formed in advance in a solid form.

According to the present invention, the above-mentioned medical material is impregnated with a protective solution and sterilized by irradiation with gamma rays while maintaining the impregnated state.

Specifically, this sterilization operation involves impregnating the medical material with a protective solution selected from the group consisting of distilled water, physiological saline, and polysaccharide aqueous solution, and irradiating it with gamma rays while maintaining the impregnated state. It is implemented by doing the following.

[0019] The polysaccharide mentioned here is not particularly limited as long as it is chemically stable without being activated by γ-ray irradiation and is water-soluble.
Specifically, it is selected from sorbitol, mannitol, xylitol, trehalose, etc. The concentration of these polysaccharide aqueous solutions is preferably 2 to 20 (W/V)%, more preferably 5 to 10 (W/V)%.

The protective solution with which the medical material is impregnated is preferably a solution in which dissolved oxygen has been replaced with an inert gas such as nitrogen, helium, or argon by a method such as bubbling. By replacing oxygen with an inert gas in this manner, no radicals are generated during γ-ray irradiation, thereby preventing denaturation of proteins in medical materials.

[0021] In order to impregnate medical materials with the above-mentioned protective solution and sterilize them while maintaining the impregnated state, the following method can be used, for example.

■Immerse a medical material whose main component is protein in a protective solution to impregnate the medical material with the protective solution, then take out the medical material from the protective solution and remove excess water if desired. This is carried out by sealing this in a container having water vapor barrier properties and impermeability to bacteria, and irradiating it with gamma rays in this state. The temperature of the protective solution that comes into contact with the medical material is preferably about 0 to 65° C., taking into consideration the influence on the proteins in the medical material.

■ A medical material containing protein as a main component is immersed in a protective solution contained in a container made of a material that has water vapor barrier properties and is impermeable to bacteria, and the protective solution is used as a medical material. This is carried out by impregnating the container, then sealing the container with a sealing member having water vapor barrier properties and impermeability to bacteria while maintaining the immersed state, and irradiating the container with gamma rays. The temperature of the protective solution that comes into contact with medical materials is 0 to 65, taking into consideration the effect on the proteins in the medical materials.
It is desired that the temperature is around ℃. In addition, the capacity of the protective solution in which the medical material is immersed is 1 to 100% of the medical material.
It is desired that the capacity be approximately double (capacity).

The intensity of γ-rays irradiated to medical materials varies depending on the type of protein, but is usually 3.8 Mr.
It is desired that it be less than ad, more preferably 3.5 Mrad or less.

[0025] If desired, unreacted protein precursors or drugs such as enzymes may be removed by washing the medical material with distilled water or the like before sterilization. .

Next, a method for manufacturing a medical device according to the present invention will be explained.

[0027] The method for manufacturing a medical device of the present invention includes the steps of applying a medical material whose main component is a physiologically active water-insoluble protein to the surface of a base material, and applying a protective solution to the medical material. The method is characterized by comprising a step of impregnating the base material and the medical material, and irradiating the base material and the medical material with gamma rays while maintaining the impregnated state of the medical material.

[0028] Various materials can be used for the base material in the present invention depending on the intended use of the medical device. For example, if the medical device is an artificial blood vessel, nylon,
Synthetic polymer materials with excellent mechanical properties such as polyester, polypropylene, polyethylene, polyurethane, silicone, and polytetrafluoroethylene, or biologically derived tissues such as natural blood vessels and ureters may also be used. In addition, depending on the purpose and type of medical equipment, polyvinyl chloride,
Polyethylene, polypropylene, nylon, ethylene
Vinyl acetate copolymer, polyvinyl chloride-polyurethane copolymer, polyvinyl chloride-(ethylene-vinyl acetate copolymer) copolymer, silicone rubber, polypropylene, polycarbonate, high-density polyethylene, acrylic resin, etc. can also be used. can.

[0029] Such a base material is preferably porous from the viewpoint of biocompatibility (easiness of cell invasion) and adhesion of proteins. Furthermore, in order to improve the adhesion of the medical material to the surface of the substrate, it is preferable to use a substrate whose surface has been made hydrophilic by treatment with acid, plasma, ozone, or the like.

[0030] As the physiologically active water-insoluble protein, the same ones as mentioned above can be used. The medical material can be applied to the surface of the base material by generally known methods, such as coating, immersion drying, etc., but it is also possible to apply the medical material to the surface of the base material by reacting the protein precursor with the active substance. By doing so, it is also possible to form a film on the surface of the base material. For example, as a method for applying fibrin as described above to the surface of a base material, the base material is immersed in a fibrinogen solution and pulled up, then the base material is immersed in a thrombin solution, and the fibrinogen and thrombin are brought into contact and reacted, so that the fibrin is applied to the surface of the base material. Methods that form fibrin gels are preferred. However, the method is not limited to this method; for example, a fibrin gel may be formed separately and applied to the base material.

[0031] Furthermore, as a method for applying a partially hydrolyzed fibrin product to the surface of a base material, after applying fibrin gel to the surface of the base material, a hydrolase such as a plasmin solution is brought into contact with the fibrin gel, and a predetermined amount of This is carried out by performing partial hydrolysis and then adding a trypsin inhibitor or antifibrinolytic agent to stop the enzymatic reaction. The contact time between the plasmin solution and fibrin varies depending on the plasmin concentration in the plasmin solution, but is usually desirably about 5 to 15 minutes.

After the medical material has been applied to the surface of the substrate in this manner, unreacted protein precursors or drugs such as enzymes may be removed by washing with distilled water or the like, if desired. It is something.

[0033] Subsequently, the medical instrument having the medical material applied to the surface of the base material is irradiated with gamma rays to perform a sterilization operation. This sterilization operation may be performed in the same manner as the above-mentioned sterilization operation for medical materials.

Specific examples of the medical devices of the present invention include various artificial organs such as artificial blood vessels, artificial hearts, artificial kidneys, artificial lungs, etc., but are not limited thereto. For example, gas exchange membranes, dialysis membranes, etc. that are the main bodies of these devices or parts of these devices, as well as various tubes that constitute extracorporeal blood circulation circuits, connectors that connect tubes, sedative/arterial insertion catheters, Blood transfusion or blood collection devices such as bubble traps, blood bags, chambers, centrifugal pumps, intravascular catheters, syringes, blood collection tubes, blood bags and their accessories can also be included.

[0035] The present invention will be explained in more detail below by showing examples.

[0036]

【Example】

(Example 1) Human fibrinogen preparation (Kabi
Vitrum (Grade L) was dissolved in distilled water and applied to a gelatin-Cellulofine column (Seikagaku Corporation) and a lysine-Sepharose 4B column (Pharmacia Corporation) to remove fibronectin and plasminogen. to remove fibronectin and plasminogen. The fibrinogen eluted from the column was precipitated with ethanol and then added to a 10mM Hepes buffer (pH 7.00) containing 150mM sodium chloride.
4) and filtered through a 0.8 μm filter. The concentration of fibrinogen solution was determined by the clotting time measurement method (D
ata-Fi, manufactured by Dade), 30
It was adjusted to mg/ml.

[0037] This fibrinogen solution was diluted to 150mM.
10mM Hepes buffer containing NaCl (pH
7.4) and readjusted to 5 mg/ml, and dispensed 200 μl into 6 wells of a 24-hole polystyrene petri dish.

Next, 50uni containing calcium ions
A fibrin gel was prepared by adding 30 μl of t/ml thrombin solution (Mochida Pharmaceutical Co., Ltd.). After standing overnight at 37°C, 3 of the 6 holes were filled with 0.05 unit/ml plasmin (Boehringer Mannheim Yamanouchi Co., Ltd.) 0.5 m.
After processing at 37℃ for 15 minutes, 1000 units/
The reaction was stopped by adding 10 μl of trasylol (manufactured by Bayer). Then, soak the fibrin gel in distilled water for 2~
After washing three times, 1 ml of 10% sorbitol aqueous solution was dispensed and left to stand at room temperature overnight to make the inside of the fibrin gel 10%
% sorbitol aqueous solution. Note that the sorbitol aqueous solution was replaced three times in total. Next, after discarding the sorbitol aqueous solution and lightly draining the water, add 2Mr without drying.
Sample 1 was prepared by irradiating ad gamma rays. Furthermore, as a control, a fibrin gel (Sample 2) was prepared in the same manner without irradiation with γ-rays.

The activation effects of the plasminogen activators of Samples 1 and 2 were compared as follows. That is, samples 1 and 2 were treated with 50mM Tris
-0.08 adjusted with HCl buffer (pH.8.8)
mg/ml Glu-Plasminogen (Bi
opol) 200μl, 1.74mM H-D-
Valyl-L-leucyl-L-lysine-P
-nitroanilide dihydrochlor
ide (chromogenic substrate) (manufactured by Daiichi Chemical Co., Ltd., product name S-
2251) 200 μl, 400 μl of 80 unit/ml plasminogen activator (manufactured by Biopool) were added, and after incubating at 37°C for about 20 minutes, 2%
Add 1 ml of citric acid solution to stop the reaction, and
The absorbance was measured at In addition, fibrin not treated with plasmin (sample 3: 2.5 Mrad γ-ray irradiation,
The absorbance of sample 4 (not irradiated with γ-rays) was measured in the same manner. The results are shown in Figure 1. As is clear from the figure, according to the sterilization method according to the present invention, γ-ray sterilization is possible without almost changing the activation effect of the plasminogen activator contained in the fibrin partial hydrolyzate. confirmed.

(Comparative Example 1) 400 μl of 5 mg/ml fibrinogen solution prepared in the same manner as in Example was added to 3
After dispensing into 24 5mm glass petri dishes, 50un
It/ml thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) was added in 60 μl portions to prepare a fibrin gel. After standing overnight at 37°C, 12 of these sheets were processed at 0.05 unit/
Treated with 0.5 ml of plasmin (manufactured by Boehringer Mannheim Yamanouchi) at 37°C for 30 minutes, and 1000 u
20 μl of nit/ml Trasylol (manufactured by Bayer) was added to stop the reaction.

Next, three sheets of plasmin-treated fibrin were sterilized with ethylene oxide gas (dry at 50°C,
3 hours), γ-ray sterilization, autoclave sterilization (121℃,
20 minutes) and samples 5, 6 and 7 were prepared. Sample 8 was prepared without further sterilization.

The activation effect of the plasminogen activator of these samples 5 to 8 was measured in the same manner as in Example 1. The results are shown in FIG. As is clear from the figure,
The plasminogen activation effect was significantly changed by autoclave sterilization and ethylene oxide sterilization, and denaturation of the fibrin hydrolyzate was confirmed. In addition, denaturation of the fibrin hydrolyzate was also confirmed in cases where γ-rays were simply irradiated without impregnation with a protective solution.

(Comparative Example 2) 400 μl of a 5 mg/ml fibrinogen solution prepared in the same manner as in Example was added to
After dispensing into a 2-hole polystyrene petri dish, 50 units
A fibrin gel was prepared by adding 60 μl of thrombin solution (manufactured by Mochida Pharmaceutical Co., Ltd.) at a volume of 60 μl/ml each. After standing overnight at 37°C, 0.5ml of 0.05 unit/ml plasmin (manufactured by Boehringer Mannheim Yamanouchi) was dispensed into a total of 9 wells, and timed at 37°C for 15, 30, and 60 minutes, 3 wells each. Processed accordingly. The reaction was stopped by adding 20 μl of 1000 unit/ml Trasylol (manufactured by Bayer).

[0044] Then, plasmin-treated fibrin and untreated fibrin (0 minute treatment) were each treated with 0.1%
Add 1 ml of glutaraldehyde (2 holes each),
Treated overnight at °C. Thereafter, it was thoroughly washed with distilled water to prepare a fibrin gel. A sample that was not treated with glutaraldehyde in the same manner was used as a control.

The activation effect of the plasminogen activator of these fibrin gels was measured in the same manner as in Example 1. The results are shown in FIG. As is clear from the figure,
Glutaraldehyde treatment significantly changed the plasminogen activation effect, and denaturation of untreated fibrin and fibrin hydrolyzate was confirmed.

(Example 2) A polyester tubular body with an inner diameter of 4 mmφ was immersed in 13 ml of a fibrinogen solution obtained in the same manner as in Example 1, and then a 50 unit/ml thrombin solution containing calcium ions (manufactured by Mochida Pharmaceutical Co., Ltd.) was immersed. ) to coat the surface of the artificial blood vessel with fibrin gel. Furthermore, a 3.8 mmφ stainless steel rod was inserted into the lumen of the artificial blood vessel, and the fibrin gel was pressed onto the lumen surface of the artificial blood vessel. Furthermore, after allowing this fibrin-coated artificial blood vessel to stand for a day and night, it was added to 0.05 unit/ml plasmin solution (manufactured by Boehringer Mannheim Yamanouchi) for 37 hours.
℃ for 15 minutes, and immersed in a 50 unit/ml tradiol solution (manufactured by Bayer) to stop the reaction, thereby producing a plasmin-treated fibrin-coated artificial blood vessel 1.

[0047] Gently wash the artificial blood vessel 1 in distilled water, and
I cut out 15 lengths of m. Three of them were immersed in 3 ml of a 10% sorbitol aqueous solution at room temperature for several hours to impregnate the fibrin gel with the 10% sorbitol aqueous solution. Next, these were taken out from the 10% sorbitol aqueous solution, drained on filter paper, and then sealed in a 15 ml polystyrene tube (manufactured by Falcon) without drying. The opening of the polystyrene tube was sealed, and in this state, γ-rays of 2.5 Mrad were irradiated to obtain Sample 9.

(Example 3) Three of the cut artificial blood vessels 1 were immersed for several hours at room temperature in 15 ml of a 10% sorbitol aqueous solution filled in a polystyrene tube, and the polystyrene tube was then sealed. 2.5 Mra
Sample 10 was obtained by irradiating with gamma rays. (Example 4) Sample 11 was obtained in the same manner as in Example 2 except that the immersion solution was changed to 3 ml of distilled water.

(Example 5) The solution to be immersed in 15 m of distilled water.
Sample 12 was obtained in the same manner as in Example 3 except for changing to 1.

[Experimental Example] Using Samples 9 to 12 and using samples without γ-ray irradiation as a control, the activation effect of plasminogen activator of plasmin-treated fibrin was compared in the same manner as in Example 1. The results are shown in FIG. As is clear from the figure, all of Samples 9 to 12 obtained by the production method of the present invention showed an activation effect on the plasminogen activator that was almost the same as that of samples that had not been irradiated with γ-rays.

[0051]

Effects of the Invention As described above in detail, the method for sterilizing medical materials according to the present invention is a method for sterilizing medical materials whose main component is a physiologically active, water-insoluble protein. The material is impregnated with a protective solution and irradiated with gamma rays while maintaining the impregnated state, so it has the effect of making it possible to sterilize physiologically active proteins without denaturing them. It is something.

[Brief explanation of the drawing]

FIG. 1 shows the results of comparing the activation effect of plasminogen activator of fibrin or its partial hydrolyzate obtained by the sterilization method according to the present invention with that of unsterilized fibrin or its partial hydrolyzate. This is a graph showing.

FIG. 2 is a graph showing the degree of denaturation when a fibrin partial hydrolyzate is sterilized with ethylene oxide, γ-ray sterilization, and autoclave without being impregnated with a protective solution.

FIG. 3 is a graph showing the degree of denaturation when partially hydrated fibrin is treated with glutaraldehyde.

FIG. 4: The activation effect of the plasminogen activator of the fibrin partial hydrolyzate applied to the inner surface of the artificial blood vessel obtained by the production method according to the present invention is imparted to the inner surface of the unsterilized artificial blood vessel. 2 is a graph showing the results of comparison with a partially hydrolyzed fibrin product.

Claims (5)

[Claims] (1) A method for sterilizing medical materials whose main component is physiologically active water-insoluble proteins, which involves impregnating the medical materials with a protective solution and irradiating them with gamma rays while maintaining the impregnated state. Characteristic method of sterilizing medical materials. (2) The method for sterilizing medical materials according to claim 1, wherein the protective solution is selected from the group consisting of distilled water, physiological saline, and an aqueous polysaccharide solution. (3) The method for sterilizing medical materials according to claim 1 or 2, wherein the protein is fibrin or a partially hydrolyzed product thereof. (4) A step of applying a medical material whose main component is a physiologically active water-insoluble protein to the surface of the base material, impregnating the medical material with a protective solution, and checking the impregnated state of the medical material. 1. A method for manufacturing a medical device, comprising the step of irradiating a base material and a medical material with gamma rays while maintaining the same. (5) The method for manufacturing a medical device according to claim 4, wherein the medical device is an artificial blood vessel.