JP2008206914A - Sterilization method for compound and manufacturing method of medical material using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sterilization method for a compound being labile in relation to radiation, which suppresses functional decline due to the degeneration or the like of the compound by radiation sterilization, does not need processing such as freezing because of few generation of deleterious by-products, and causes little degeneration of the compound by drying. <P>SOLUTION: In the sterilization method for the compound, the radiation is emitted in the state of dissolving and/or dispersing the compound labile in relation to the radiation in an organic solvent aqueous solution satisfying conditions A and B. A: Moisture content is 25 to 90 vol% and B: at least one secondary or tertiary hydroxyl group is contained. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a compound sterilization technique suitably used in fields such as medicine, medicine, proteome analysis and food production.

  When pharmaceuticals and medical devices are used in the medical setting, they must be sterilized. That is, it is necessary to finally sterilize when producing pharmaceuticals and medical instruments. In the Japanese Pharmacopoeia, a heating method, a gas method, and an irradiation method are shown as final sterilization methods for pharmaceuticals and medical devices.

  Irradiation methods are broadly classified into radiation methods and high-frequency methods. Among them, the radiation method is a sterilization method that has been widely used in recent years because it hardly causes denaturation of an object to be sterilized by heat and there are few problems of toxicity due to residual gas. High-energy radiation is used for irradiation, and γ-ray is typically used. When γ-ray energy is absorbed by a substance, the molecule of the substance is ionized or excited. Then, a dissociation reaction occurs, and as a result, highly reactive radicals are generated and react one after another with ordinary molecules. When this series of reactions occurs with the most important nucleic acid to maintain the life of a microorganism, the nucleic acid molecule is cleaved and directly kills the microorganism.

  However, radicals generated by γ-ray irradiation may react not only with microorganisms but also with molecules constituting pharmaceuticals and medical materials, thereby deteriorating their functions and physical properties. In recent years, the surface of substrates has been chemically or biochemically modified to give specific adsorption, reactivity, or biocompatibility. When chemical or biochemical modification is performed, the surface characteristics obtained by these methods have a problem that their functions are lowered by γ-ray irradiation.

  With respect to such problems, a method of radiation sterilization with a base material impregnated with an antioxidant such as sodium pyrosulfite or ascorbic acid (see Patent Document 1), or a method of radiation sterilization in a frozen state (Patent Document 2). And a method of radiation sterilization in a state in which the amount of residual solvent is reduced (see Patent Document 3). In recent years, a method of radiation sterilization in a state where the organic solvent content is increased and the moisture content is decreased (see Patent Document 4) has also been proposed.

  In the method of impregnating the base material with the above-mentioned antioxidant, since the antioxidant is a compound unstable to radiation, there is a problem that it becomes a harmful substance by being modified by irradiation. For example, when sodium pyrosulfite is used, harmful substances such as sulfurous acid and sulfur dioxide are generated. Therefore, it is not preferable to use it for final sterilization of pharmaceuticals and medical devices. In addition to the cost of raw materials for antioxidants, it is also disadvantageous in terms of cost because the number of processes for adding antioxidants increases and the associated equipment is required.

  In addition, the method of irradiating with radiation in a frozen state has a big problem that the physiological activity is lost when a compound having physiological activity is frozen. Further, there is a problem of cost increase such as equipment necessary for freezing, electric power necessary for the freezing, and transportation in a frozen state.

  In addition, the method of radiation sterilization in a state where the residual solvent is lowered is that the moisture content is 2% or less, and it cannot be used for a compound that needs to be kept in a wet state. For example, hemodialysis membranes and the like depend on the microporous structure of the material, and there is a problem that the originally planned performance cannot be obtained because these structures change due to drying.

Further, in the method of radiation sterilization in a state where the organic solvent content is increased and the moisture content is lowered, a method of reducing the moisture content to 20% by weight or less is preferable. However, in this method, for example, when this method is used for the production of medical materials, there is a description or suggestion that the organic solvent residue after washing affects biocompatibility or the performance of the material is deteriorated. For example, when used for radiation grafting, there has been no consideration for a decrease in graft efficiency, which may not be suitable for practical use.
Japanese Patent No. 3432240 U.S. Pat. No. 4,620,908 Special table 2003-527210 gazette JP 2006-289071 A

  Therefore, an object of the present invention is to suppress a functional deterioration due to denaturation of a compound by radiation sterilization in a compound unstable to radiation, and generates no harmful by-products and does not require a treatment such as freezing. Another object of the present invention is to provide a method for sterilizing a compound that causes little modification of the compound by drying and a method for producing a medical material using the method.

In order to achieve the above object, the method for sterilizing a compound of the present invention has the following constitution.
1. A method for sterilizing a compound, comprising irradiating a compound unstable to radiation in a state of being dissolved and / or dispersed in an organic solvent aqueous solution satisfying the following conditions A and B.
A: Water content is 25 vol% or more and 90 vol% or less B: At least one contains a hydroxyl group that is secondary or tertiary 2. 2. The method for sterilizing a compound according to 1 above, wherein the compound unstable to radiation comprises an amino acid as a constituent element.
3. 3. The method for sterilizing a compound according to 1 or 2 above, wherein the compound unstable to radiation has blood anticoagulant activity.
4). 4. The method for sterilizing a compound as described in 3 above, wherein the compound having blood anticoagulant activity comprises a portion having blood anticoagulant activity and a polymer chain portion.
5. The polymer chain portion contains at least one selected from polyethylene glycol residue, polyvinyl pyrrolidone residue, polypropylene glycol residue, polyvinyl alcohol residue and any copolymer residue thereof, 5. A method for sterilizing the compound according to 4.
6). 6. The method for sterilizing a compound according to any one of 3 to 5, wherein the compound unstable to radiation has antithrombin activity.
7). A method for producing a medical material, wherein the compound unstable to radiation is immobilized on a base material using the method for sterilizing a compound according to any one of 1 to 6 above.
8). 8. A method for producing a medical device, wherein the medical material according to 7 is incorporated in a container.
9. 9. The method for producing a medical device according to 8 above, wherein the medical device is an artificial kidney.

  According to the present invention, it is possible to prevent functional degradation due to denaturation of such a compound when radiation sterilizing a compound unstable to radiation, and there is little generation of harmful by-products, and processing such as freezing is not required, In addition, sterilization can be performed with little denaturation of the compound by drying. Furthermore, when a medical material is produced by immobilizing the compound on a substrate by, for example, radiation grafting, it is expected that the graft efficiency is improved by increasing the moisture content.

  The radiation used in the present invention refers to high-energy particle beams and electromagnetic waves, and examples include α rays, β rays, γ rays, X rays, ultraviolet rays, electron beams, and neutron rays. Of these, γ-rays, electron beams and X-rays are suitable for sterilization because they can kill microorganisms efficiently. The radiation dose is preferably 5 kGy or more, more preferably 10 kGy or more, and further preferably 15 kGy or more. However, irradiation with excessive radiation not only denatures the compound, but also increases the time required for irradiation and decreases productivity, so 5000 kGy or less is preferable, 1000 kGy or less is more preferable, and 100 kGy or less is more preferable.

  In addition, the compound unstable to radiation used in the present invention means that when irradiated with radiation, at least a part of the compound causes a change in molecular structure due to chemical bond breaking or generation, or oxidation reaction. Mechanical properties such as strength, electrical properties such as conductivity, optical properties such as refractive index, physical functions such as surface tension and / or chemical functions such as catalytic activity and adsorption properties and / or biocompatibility It refers to a compound that changes biological functions such as sex, enzyme activity and antibody activity. Details of the compound unstable to radiation used in the present invention will be described later.

  A compound that is unstable to radiation may be sterilized alone or together with a base material such as a medical material. It is also possible to modify the substrate by immobilizing the compound unstable to the radiation on the substrate by irradiation. Thus, in the method for producing a medical material according to the present invention, since the compound can be immobilized and the medical material can be sterilized at the same time, the number of production steps can be reduced. In this case, it is also possible to chemically graft a compound unstable to radiation onto the substrate surface.

  The base material used in the present invention refers to a material to which a compound unstable to radiation is to be imparted, and a polymer compound material is preferably used. Examples of the polymer compound constituting the polymer compound material include, for example, polymethyl methacrylate, polypropylene, polyethylene, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyvinyl acetate, polycarbonate, cellulose, cellulose acetate, and cellulose triacetate. , Polyacrylonitrile, polysulfone, polyethersulfone, polystyrene, polyurethane and the like, but are not limited thereto.

  In the present invention, a medical base material can be used as the base material. Examples of medical base materials include artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, and surgical aids. Biological component separation modules and blood purification modules, preferably medical devices such as artificial kidneys In addition, a separation membrane that is used by being incorporated in a device is also included. Here, the separation membrane for separating biological components refers to a membrane for separating a biological material by filtration or dialysis and collecting a part thereof.

  Examples of the organic solvent suitably used in the present invention include a solvent containing a carbon atom and / or a silicon atom in the molecule. Hydroxyl groups have a high effect of stabilizing radicals generated by radiation irradiation, are nonionic functional groups, have little interaction with compounds having strong surface charges, have low redox power, and are not modified. Few. In particular, secondary and tertiary hydroxyl groups are more effective in stabilizing radicals. Therefore, in the present invention, an organic solvent having at least one secondary or tertiary hydroxyl group, such as glycerin, propylene glycol, or isopropanol. 2-butanol, 2,3-butanediol, 1,3-butanediol and the like are used.

In addition, when the method for sterilizing a compound of the present invention is used for the production of a medical material or a medical device in which the compound is incorporated, it is necessary to consider its safety. Therefore, a non-aqueous solvent having low toxicity is preferably used. It is done.
Moreover, the upper limit of the moisture content in organic solvent aqueous solution is 90 vol%, and when it exceeds 90 vol%, the radical stabilization effect of an organic solvent is not fully acquired. In the range of 90 vol% or less, it is preferable that the moisture content is larger. On the other hand, when the moisture content is low, for example, when used for medical devices, there is a concern that the residual organic solvent after washing may affect biocompatibility, and for example, when used for radiation grafting, the grafting efficiency decreases. Can be considered. Therefore, the water content is preferably 25 vol% or more, more preferably 50 vol% or more of the total solvent amount.

  In the present invention, dissolution of a compound unstable to radiation means that the compound is dissolved in an aqueous solution of an organic solvent to form a uniform mixture, that is, a solution. Further, dispersion of a compound unstable to radiation means that the compound is scattered in an organic solvent aqueous solution. The concentration of the compound in the organic solvent is not particularly limited. However, depending on the compound, if the concentration is too high, a cross-linking reaction proceeds between the compounds, and the original physical properties of the compound may be lost due to gelation or the like. The concentration of the compound in the aqueous solution of the organic solvent is preferably 50% by weight or less, more preferably 30% by weight or less, and still more preferably 20% by weight or less.

The moisture content in the present invention is defined by the following equation.
(Volume of water contained in an organic solvent aqueous solution in which a compound unstable to radiation and the compound is dissolved and / or dispersed) / (A compound unstable to radiation and the compound dissolved in and / or dispersed therein) Volume of organic solvent aqueous solution) × 100 (%)
The radiation-labile compound in the present invention preferably contains an amino acid as a constituent element. A compound containing an amino acid as a constituent element has high biocompatibility, and therefore has high safety when used for medical purposes. Furthermore, a compound containing an amino acid as a constituent element has physiological activity, and is effective when used on its own or immobilized on the surface of a medical material, and its use is not limited. A compound having an amino acid as a constituent element is a compound containing a naturally occurring amino acid, such as a compound composed only of an amino acid such as a protein or a peptide, a glycoprotein, an amino acid complex, an aminoacyl adenylic acid, or the like. Although what is comprised from a thing other than these amino acids and amino acids is mentioned, It is not limited to these.

The compound having blood anticoagulant activity refers to a compound in which the prothrombin time is prolonged by 30% or more when the compound is added to blood so as to have a concentration of 10 μg / ml as compared with unadded blood. The prothrombin time can be measured by the method described in the following document.
Masamitsu Kanai et al., “Proposal for Clinical Examination Revised 30th Edition”, Kanehara Publishing, 1993, pp. 416-418
That is, mix 1 volume of 3.2 vol% sodium citrate and 9 volumes of blood, and take 0.1 ml of citrated plasma collected in a small test tube (inner diameter 8 mm, length 7.5 cm) in a 37 ° C. constant temperature water bath. Heat for about 3 minutes. At the same time as adding 0.2 ml of tissue thromboplastin / calcium reagent kept at the same temperature, start the second clock, shake gently, and measure the time until fibrin precipitates while tilting.

In addition, the radiation labile compound in the present invention is preferably a compound having blood anticoagulant activity. In general, when blood comes into contact with a material, a coagulation reaction proceeds. Therefore, when a material that comes into contact with blood, particularly a material that comes into contact with blood for a long time, is used for medical purposes, it is necessary to suppress blood coagulation. Since the present invention considers the production of a material to be used in contact with blood, it is preferably a compound having blood anticoagulant activity. Examples of the compound having anticoagulant activity, heparin, nafamostat mesylate, sodium citrate, sodium oxalate, alpha ₁ antitrypsin, alpha ₂ macroglobulin, C1 inhibitor, but thrombomodulin and protein C, etc., in these It is not limited. Examples of the compound having blood anticoagulant activity include a compound having a portion having blood anticoagulant activity and a polymer chain portion. Such a polymer chain portion preferably includes a polyethylene glycol residue, a polyvinylpyrrolidone residue, Examples thereof include a polymer chain selected from the group consisting of a polypropylene glycol residue, a polyvinyl alcohol residue, and a copolymer residue containing them.

  In addition, among compounds having blood anticoagulant activity, there are compounds that exhibit strong blood anticoagulant activity by suppressing thrombin activity. Accordingly, the compound is more preferably a compound having antithrombin activity. The compound having antithrombin activity in the present invention is a compound that suppresses the activity of thrombin, which is a coagulation-related substance in the blood. A compound whose measured value in “kit” is increased by 50% or more compared to that of plasma without compound. The measuring device is not limited to this as long as it can measure with the same accuracy. An example of a compound having antithrombin activity is represented by the following chemical formula.

  4-methoxy-benzenesulfonyl-Asn (PEG2000-Ome) -Pro-4amidinobenzylamide (hereinafter abbreviated as Compound A), antithrombin III, hirudin and the like.

(In the formula, PEG represents a polyethylene glycol residue, and Me represents a methyl group.)
Moreover, the form with which a buffer solution is contained in the organic-solvent aqueous solution is mentioned as a preferable aspect of this invention. Due to the presence of such a buffer solution, it is possible to suppress the pH fluctuation of the organic solvent aqueous solution, to prevent functional deterioration due to denaturation of the compound, etc., generation of harmful by-products is small, and treatment such as freezing is not required, and drying Sterilization can be performed with little denaturation of the compound. The buffer here refers to a solution in which the pH does not change even if a small amount of acid or base is added or the concentration slightly changes. For example, phosphate buffer, trishydroxymethylamino Examples include methane buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer, acetate buffer, citrate buffer, and borate buffer. Among these, phosphate buffer, trishydroxymethylaminomethane buffer and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer have a buffering action in neutral and / or acidic regions, and have physiological activity. This is preferable because it is often added as a solvent for a compound having the above. However, under strongly acidic conditions such as less than pH 3 or strongly basic conditions such as pH 10 or more, some molecular structures in the compound having physiological activity change, resulting in physical functions and / or chemical properties. Since it is considered that the function and / or biological function is changed or the performance of the material itself is lowered when, for example, a graft-immobilized material is created, the buffering range is pH 3 or more, preferably 5 or more. Moreover, as an upper limit, it is less than pH10 and 8 or less is preferable. The glass electrode method is used for measuring the pH, but it is not limited to this as long as it can be measured with the same accuracy.

  Hereinafter, the method for sterilizing the compound of the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

(Measurement of antithrombin activity value)
Since compound A was used as a radiation labile compound, the residual ratio after irradiation of the antithrombin activity value was used as an index indicating the activity of antithrombin. For the measurement of the antithrombin activity value, an ECA-T kit manufactured by HaemoSys was used as a reagent, and COATRON M1 (code 80 800 000) manufactured by TECO Medical Instruments Production was used as an apparatus. First, 20 μl of the solution to be measured was added to 80 μl of human plasma (Cosmo Bio release Human Plasma 12271210, lot. 16878) and stirred. This solution is used as a sample solution. The sample solution is cooled on an ice bath until immediately before the measurement. 100 μl of ECA prothrombin buffer, 30 μl of sample solution and 25 μl of ECA-T substrate were mixed, incubated at a temperature of 37 ° C. for 60 seconds, and set in the apparatus. To this, 50 μl of ECA ecarin reagent was added for measurement. Measurement was performed on a sample prepared using ultrapure water as a blank measurement target solution. The residual rate was calculated by the following formula (1).
(Example 1)
An aqueous solution of isopropanol was prepared by dissolving 0.25 ml of ultrapure water and 0.75 ml of isopropanol (manufactured by Sigma-Aldrich). Compound A was dissolved in the aqueous isopropanol solution to prepare an aqueous Compound A isopropanol solution having a concentration of 5000 ppm by weight. The aqueous solution of Compound A isopropanol was irradiated with γ rays. At this time, the absorbed dose of γ rays was 25 kGy. The antithrombin activity value of the compound A isopropanol aqueous solution before irradiation and the compound A isopropanol aqueous solution after irradiation was measured, and the residual ratio of the antithrombin activity value was calculated from the following formula (1). As a result, the antithrombin residual rate was 79.4%.
A = 100 × (BD) / (CD) Formula (1)
The definitions of the symbols in the above formula (1) are as follows.
A: Antithrombin activity remaining rate (%)
B: Sample measurement value after irradiation (sec.)
C: Sample measurement value before irradiation (sec.)
D: Blank measurement value (sec.)
(Example 2)
An aqueous solution of isopropanol was prepared by dissolving 0.5 ml of ultrapure water and 0.5 ml of isopropanol (manufactured by Sigma-Aldrich). Compound A used in Example 1 was dissolved in the aqueous isopropanol solution to prepare an aqueous solution of Compound A isopropanol having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 61.4%.
(Example 3)
An aqueous solution of isopropanol was prepared by dissolving 0.9 ml of ultrapure water and 0.1 ml of isopropanol (manufactured by Sigma Aldrich). Compound A used in Example 1 was dissolved in the aqueous isopropanol solution to prepare an aqueous solution of Compound A isopropanol having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 33.0%.
Example 4
An aqueous glycerin solution was prepared by dissolving 0.5 ml of ultrapure water and 0.5 ml of glycerin (manufactured by Sigma-Aldrich). Compound A used in Example 1 was dissolved in the glycerin aqueous solution to prepare a Compound A glycerin aqueous solution having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 53.8%.
(Example 5)
An aqueous propylene glycol solution was prepared by dissolving 0.5 ml of ultrapure water and 2 ml of propylene glycol (Wako Pure Chemical Industries, Ltd.). Compound A used in Example 1 was dissolved in the propylene glycol aqueous solution to prepare a compound A propylene glycol aqueous solution having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 64.0%.
(Comparative Example 1)
Compound A used in Example 1 was dissolved in ultrapure water to prepare a Compound A aqueous solution having a concentration of 5000 ppm by weight. The aqueous solution was irradiated with γ rays. At this time, the absorbed dose of γ rays was 25 kGy. The antithrombin activity values of the solution before irradiation and the solution after irradiation were measured, and the residual ratio of the antithrombin activity value was calculated from the above equation (1). As a result, the antithrombin residual rate was 0%.
(Comparative Example 2)
An ethylene glycol aqueous solution was prepared by dissolving 0.5 ml of ultrapure water and 0.5 ml of ethylene glycol (manufactured by Sigma-Aldrich). Compound A used in Example 1 was dissolved in the ethylene glycol aqueous solution to prepare a compound A ethylene glycol aqueous solution having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Comparative Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 8.3%.
(Comparative Example 3)
A polyethylene glycol aqueous solution was prepared by dissolving 0.5 ml of ultrapure water and 0.5 ml of polyethylene glycol (manufactured by Nacalai Tesque). Compound A was dissolved in the polyethylene glycol aqueous solution to prepare a compound A polyethylene glycol aqueous solution having a concentration of 5000 ppm by weight. Thereafter, the same treatment as in Comparative Example 1 was performed, and the residual ratio of the antithrombin activity value was calculated. As a result, the antithrombin residual rate was 6.9%.

  The present invention relates to a method and / or radiation graft immobilization material for radiation sterilization of a compound having blood anticoagulant activity which is unstable to radiation, while maintaining the activity, and can be sterilized without using an antioxidant. It can be expected to reduce the risk of possible and harmful antioxidant degradation products. Moreover, since the amount of the organic solvent in the solvent to be used can be suppressed, biocompatibility is maintained. Furthermore, a radiation graft fixing material can be created without degrading the performance of the material to be fixed. As described above, according to the present invention, it is possible to sterilize physiologically active substances unstable to various radiations while suppressing denaturation, and it is possible to create a radiation graft immobilization material.

Claims (9)

A method for sterilizing a compound, comprising irradiating a compound unstable to radiation in a state of being dissolved and / or dispersed in an organic solvent aqueous solution satisfying the following conditions A and B.
A: Water content is 25 vol% or more and 90 vol% or less B: At least one contains a hydroxyl group that is secondary or tertiary   The method for sterilizing a compound according to claim 1, wherein the radiation labile compound comprises an amino acid as a constituent element.   The method for sterilizing a compound according to claim 1 or 2, wherein the compound unstable to radiation has blood anticoagulant activity.   4. The method for sterilizing a compound according to claim 3, wherein the compound having blood anticoagulant activity comprises a part having blood anticoagulant activity and a polymer chain part.   The polymer chain portion contains at least one selected from a polyethylene glycol residue, a polyvinyl pyrrolidone residue, a polypropylene glycol residue, a polyvinyl alcohol residue, and a residue of any copolymer thereof. Item 5. A method for sterilizing a compound according to Item 4.   The method for sterilizing a compound according to any one of claims 3 to 5, wherein the compound unstable to radiation has antithrombin activity.   A method for producing a medical material, wherein the compound unstable to radiation is immobilized on a substrate using the method for sterilizing a compound according to any one of claims 1 to 6.   A method for producing a medical device, wherein the medical material according to claim 7 is contained in a container.   The method for producing a medical device according to claim 8, wherein the medical device is an artificial kidney.