JP2009077877A - Manufacturing method for medical material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medical material which shows less interference by an aqueous solution in which a base material is immersed with a limulus test. <P>SOLUTION: The medical material is obtained by immersing the base material in the aqueous solution which contains a cationic water-soluble polymer and a chemical compound, having destabilizing energy accompanying a radical production of 99.5 kcal/mol or less, at a concentration of 0.0008 mol/L or more and 0.008 mol/L or less and by performing radiation irradiation. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a medical material with less interference with the Limulus reaction of an aqueous solution in which a substrate is immersed.

  Conventionally, as a method for modifying the base material of medical materials, the method of immersing in a polymer aqueous solution and irradiating with radiation to chemically bond the polymer to the base material has a strong binding force between the base material and the polymer and is difficult to elute. It is excellent in that it can be performed simultaneously with radiation sterilization and is widely used.

  It is generally known that a cationic polymer exhibits an antibacterial action, and it is effective to chemically bond a cationic water-soluble polymer to a substrate in order to impart an antibacterial action to medical materials and the like. However, there are concerns such as modification of the cationic water-soluble polymer by radiation, an increase in the amount of eluate, and damage to the substrate. Therefore, a technique has been disclosed in which an antioxidant is added to an aqueous polymer solution during irradiation to reduce the influence of radiation on the polymer and the substrate (Patent Document 1).

  It is necessary to measure the endotoxin activity contained in filling liquids for the purpose of preventing endotoxin, which is a component that causes fever and shock, from medical materials. As a method for measuring endotoxin activity, the Limulus test is widely used because a large amount of sample can be easily measured.

  However, the Limulus test is susceptible to interference such as inhibition and activity due to the components contained in the test solution. In particular, when an aqueous solution in which a substrate is immersed is irradiated with radiation, the interference with the Limulus test changes due to denaturation or deterioration due to irradiation, and endotoxin activity cannot be accurately measured in many cases.

  The technique disclosed in Patent Document 1 does not consider such interference with the Limulus test.

  There is a rabbit fever test as a method for measuring endotoxin activity other than the Limulus test, but it takes a long time to obtain a result and it is difficult to maintain a reliable result. Moreover, it is not preferable also from the aspect of animal welfare.

In other words, there has been no medical material in which a cationic water-soluble polymer is chemically bonded to a base material by irradiation so that the endotoxin activity of an aqueous solution in which the base material is immersed can be easily measured. It was.
JP2005-239761

  An object of the present invention is to provide a medical material having an antibacterial action with less interference with the limulus test of an aqueous solution in which a substrate is immersed.

In order to achieve the above object, the present invention has the following configuration.
1. Immerse the substrate in an aqueous solution containing a cationic water-soluble polymer aqueous solution and a compound having a destabilization energy accompanying radical generation of 99.5 kcal / mol or less at a concentration of 0.0008 mol / L or more and 0.008 mol / L or less. And the manufacturing method of the medical material characterized by irradiating with radiation.
2. 2. The method for producing a medical material according to 1 above, wherein the compound contains a secondary hydroxyl group.
3. 3. The method for producing a medical material according to 1 or 2, wherein the compound is any one of isopropyl alcohol, 2-butanol, and glycerin or a combination thereof.
4). The cationic water-soluble polymer is a primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine. Any one or more of a group, a pyrrolidine group, a thiazole group, and a purine group is contained, The manufacturing method of the medical material in any one of said 1-3 characterized by the above-mentioned.
5). 5. The method for producing a medical material according to any one of 1 to 4 above, wherein the substrate contains a polysulfone-based polymer.
6). The endotoxin addition recovery rate in the limulus test of an aqueous solution after irradiation produced by the method for producing a medical material according to any one of 1 to 5 above is 50% or more and 200% or less after 48 hours. A medical material characterized by

  According to the present invention, as described below, even when a medical material is immersed in an aqueous solution containing a cationic water-soluble polymer and irradiated with radiation, the endotoxin activity is measured by a limulus test on the immersed aqueous solution. It can be performed.

  1. When the substrate is immersed in an aqueous solution containing a cationic water-soluble polymer and irradiated with radiation, the cationic water-soluble polymer is chemically bonded to the surface of the substrate. The cationic water-soluble polymer has an antibacterial action and can impart an antibacterial action to the substrate by chemically bonding to the surface of the substrate. By providing an antibacterial action, it can be utilized as a medical material.

  2. Examples of the medical material referred to in the present invention include materials used for artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, surgical aids, and the like. Biological component separation modules and blood purification modules Also included are separation membranes built in and used. A separation membrane for separating biological components refers to a membrane that separates a biological substance by filtration or dialysis and collects a part thereof. A blood purification module is a module that has the function of removing waste and harmful substances in the blood by adsorption, filtration, and diffusion when circulating blood outside the body. There are toxin adsorption columns.

  3. For the purpose of preventing endotoxin contamination, it is necessary to measure the presence or absence of endotoxin activity in the aqueous solution in which the base material is immersed. However, when the aqueous solution is irradiated with radiation, components in the aqueous solution interfere with the Limulus test, and the endotoxin activity of the material cannot be measured. Examples of interference with the Limulus test include disruption of the endotoxin micelle structure and direct action on the reagent to suppress or enhance gelation. When interference occurs in the Limulus test, detection of endotoxin activity often decreases over time.

  4). Therefore, in the present invention, as a result of intensive studies, in a medical material obtained by immersing a substrate in an aqueous cationic water-soluble polymer and irradiating with radiation, the substrate has a destabilization energy of 99. By immersing an aqueous solution containing a compound of 5 kcal / mol or less (hereinafter referred to as Compound A) and having a concentration before irradiation of the compound of 0.0008 mol / L or more and 0.008 mol / L or less. It was found that the interference with the Limulus test of the cationic water-soluble polymer aqueous solution in which the material is immersed can be suppressed.

  5). The compound A used here is added for the purpose of suppressing interference with the Limulus test due to modification of the cationic water-soluble polymer due to its protective effect against radiation. However, the compound A itself has a conflicting effect of interfering with the Limulus reagent. Therefore, it is effective to contain the compound A having a high protection effect against radiation at a low concentration to suppress interference with the Limulus test.

  6). In the present invention, the lower the radical destabilization energy of Compound A, the less the interference with the Limulus test due to the modification of the cationic water-soluble polymer, 99.5 kcal / mol or less is necessary. The lower the destabilization energy, the higher the protection effect against radiation, and therefore 99.0 kcal / mol or less is more preferable.

  7. When the concentration of the compound A is low, the radiation protection effect is lowered. On the other hand, when the concentration is high, interference of the compound A with the Limulus test becomes a problem. Therefore, the concentration of Compound A needs to be in the range of 0.0008 mol / L or more and 0.008 mol / L or less, more preferably 0.001 mol / L or more, and more preferably 0.005 mol / L or less. .

  8). Further, due to the radiation protection effect of the compound A, the influence of radiation irradiation can be weakened, and modification of the cationic water-soluble polymer and damage to the substrate can be suppressed.

  The destabilization energy accompanying radical generation in the present invention is a value obtained by the equation (1).

_{E I = E H + E M} -E N (1)
In formula (1), E _I is the destabilization energy associated with radical generation, E _H is the total energy of the hydrogen radical, E _M is the total energy of the molecular radical, and E _N is the quantum chemical calculation value of the total energy of the neutral molecule. These are values specific to each compound. The smaller the destabilizing energy associated with radical generation, the more stable the radicals when irradiated with radiation, and thus the higher the protection effect against radiation.

  For quantum chemistry calculations, various quantum chemistry calculation program packages available for a fee or free of charge can be used. In the present invention, Gaussian 03 (Gaussian 03 (B.05 revised edition) (Gaussian 03 ( Revision B.05)), MJ Frisch et al., Gaussian, Inc., Pittsburgh PA, 2003.].

  As a calculation method, it is preferable to use a hybrid method of the density functional method and the ab initio molecular orbital method from the viewpoint of the calculation accuracy of the molecular structure and total energy. In the present invention, the B3LYP method [AD Becke (AD Becke), Journal of Chemical Physics, 1993, 98, p.5648 / C. Lee, W. Yang, Earl G. Pearl ( RG Parr), Physical Review (Phys. Rev.) B, 1988, 37, p.785 / B. Miehlich, A. Savin, Etch Stoll, Etch -Pruss (H. Preuss), Chemical Physics Letters (Chem. Phys. Lett.), 1989, 157, p.200.

  In addition, as a basis function for expanding one electron orbital, in order to obtain sufficient calculation accuracy for the molecular structure and total energy, the double valence orbital basis function by Pople et al. [WJ Hehre, R. R. Ditchfield, JA Pople, Journal of Chemical Physics (J. Chem. Phys. 1972, 56, p.2257.) And polarization function [PC Haliharan (PC) Hariharan), JA Pople, Theoret. Chimica Acta 1973, 28, p.213.] 6-31G (d, p) basis functions were used.

  9. In the present invention, in particular, a compound having a secondary hydroxyl group is unstable or low in energy accompanying radical generation, has a small interaction with a compound having a strong surface charge, which is a nonionic functional group, and redox Since the force is also small, the cationic water-soluble polymer is less modified and therefore preferably used. Examples of the compound having a secondary hydroxyl group include glycerin, 2-butanol, and isopropanol. These compounds may be used alone or in combination of two or more. Moreover, since it is necessary to consider safety | security for a medical material, the thing with low toxicity is suitable.

  10. 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. Among these, γ rays and electron beams are more preferably used because they have particularly high energy and can be modified efficiently. Further, γ-rays, X-rays, and electron beams are preferable because sterilization can be performed simultaneously by controlling the dose.

  In the case of irradiating the substrate with radiation, the dose of radiation is preferably 1 kGy or more because it is difficult to control the dose because the absorbed dose in the substrate varies when the dose of radiation is small. More preferably, it is 5 kGy or more. Moreover, when sterilizing a medical material simultaneously, it is preferable that the radiation dose is 15 kGy or more, more preferably 25 kGy or more. However, since irradiation with excessive radiation degrades the substrate itself, the radiation dose is preferably 5000 kGy or less, more preferably 1000 kGy or less, and even more preferably 100 kGy or less.

  11. The cationic water-soluble polymer used in the present invention is a substance having a charge of 1 meq / g or more at pH 4.5, a solubility in water of 10 g / L or more, and a molecular weight of 50,000 or more. Point to. Among them, primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine group, pyrrolidine group, A substance having a functional group such as a thiazole group or a purine group is preferably used. From the viewpoint of availability, polyethyleneimine, polyvinylamine, polyallylamine, diethylaminoethyldextran, polylysine, polydiallyldimethylammonium chloride, a copolymer of vinylimidazolium methochloride and vinylpyrrolidone, and the like are preferably used. Two or more cationic water-soluble polymers may be used in combination.

  The range of the concentration of the cationic water-soluble polymer in which the substrate is immersed in the aqueous solution is generally preferably 1 ppm or more, more preferably 10 ppm or more, although it depends on the type of the cationic water-soluble polymer. Moreover, 50 wt% or less is preferable and 10 wt% or less is more preferable. When the concentration of the cationic water-soluble polymer is high, the amount immobilized on the surface corresponding to the cost cannot be obtained. In addition, residual surplus material becomes a problem, and it is necessary to enhance cleaning. On the other hand, when the concentration of the cationic water-soluble polymer is low, there are few cationic water-soluble polymers chemically bonded to the surface of the substrate, and a sufficient antibacterial action cannot be obtained.

  12 As the base material used in the present invention, 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. Of these, polysulfone polymers having high resistance to radiation are preferably used.

  13. In order to accurately measure the endotoxin activity by the Limulus test, the endotoxin addition recovery rate is 50% or more, preferably 70% or more after 48 hours from the addition of endotoxin, while 200% or less, preferably 150%. It is necessary that: Outside this range, endotoxin activity cannot be accurately measured, and there is a risk of endotoxin contamination due to measurement errors.

  The endotoxin addition recovery rate is the percentage of the endotoxin activity obtained by adding a known amount of endotoxin to an aqueous solution and conducting a limulus test, and the activity of the added endotoxin. .

P = (U ₁ / U ₀ ) × 100 (2)
In the formula (2), P = endotoxin addition recovery rate (%), U ₁ = endotoxin activity (EU) of the sample, U ₀ = activity of added endotoxin (EU).

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
1. Measurement of endotoxin addition recovery rate by Limulus test Endotoxin standard product (Japanese Pharmacopoeia standard product 10000 EU / vial) was dissolved in pyrogen-free water for injection (Japan Pharmacopoeia water for injection: Otsuka Pharmaceutical Co., Ltd.) Diluted with pyrogen-free water for injection to prepare an endotoxin aqueous solution having a concentration of 0.12 EU / ml. The endotoxin aqueous solution and the test solution were mixed at a 1: 1 (volume ratio) in a polystyrene test tube 2054 (Falcon) to prepare a sample solution having an endotoxin activity of 0.06 EU / ml.

  After preparing the above sample solution, immediately add 0.2 ml to the Limulus test tube (for Limulus reagent for dialysate 0.2 ml: Wako Pure Chemical Industries), and stir for 5 seconds with a desktop lab mixer to dissolve the reagent and immediately It was set in a toxinometer (ET-301: Wako Pure Chemical Industries, Ltd.) (within 10 seconds), and endotoxin activity was measured by a turbidimetric time analysis method. In order to measure the activity of endotoxin added to the sample, a standard solution of endotoxin dissolved in pyrogen-free water for injection was diluted with pyrogen-free water for injection in turn, and the endotoxin activity was 0.06 EU / ml. A sample solution was prepared.

  The above sample solution and control sample solution were allowed to stand at room temperature for 48 hours after preparation, and endotoxin activity was similarly measured using a toxinometer.

From the above formula (2), the endotoxin addition recovery rate immediately after the endotoxin addition and the endotoxin addition recovery rate after 48 hours from the endotoxin addition were determined. In the formula (2), U ₁ is the endotoxin activity of the measurement sample, and U ₀ is the endotoxin activity of the control.
Example 1
Polyethyleneimine (Sigma MW 750,000) 1000ppm aqueous solution was introduced from the blood outlet of "Toresulfone BS-1.6L" (registered trademark) module manufactured by Toray Industries, Inc., which is a hollow fiber type artificial kidney, and filtered through the hollow fiber membrane The whole amount of polyethyleneimine was introduced and fixed to the hollow fiber membrane. At this time, the blood inlet and the dialysate inlet were plugged. Moreover, the flow rate of the aqueous solution was 500 mL / min, and the flow rate was 1 L. Subsequently, an isopropanol 0.0016 mol / L aqueous solution was introduced from the blood outlet, and the hollow fiber membrane was filtered and discharged from the dialysate outlet. At this time, the blood inlet and the dialysate inlet were plugged. The flow rate was 500 mL / min and the flow rate was 2 L. Subsequently, an isopropanol 0.0016 mol / L aqueous solution was introduced from the blood outlet and discharged from the blood outlet. At this time, the dialysate inlet and the dialysate outlet were plugged. The flow rate was 500 mL / min and the flow rate was 0.5 L. The filling was performed at room temperature, and the polyethyleneimine aqueous solution and isopropanol aqueous solution were set to a liquid temperature of 20 ± 5 ° C. Thereafter, the module was irradiated with γ rays. The amount of γ-ray irradiation was 27 kGy. The blood side filling solution was extracted and an endotoxin addition recovery rate test was conducted. As a result, it was 107.8% immediately after endotoxin addition and 70.1% after 48 hours, and there was little interference with the Limulus test.

  The radical destabilization energy of isopropanol was determined according to the following procedure using quantum chemical calculation. All quantum chemical calculations were performed on a Linux server with an Itanium2 (registered trademark) processor.

(1) Total energy of hydrogen radicals The total energy of hydrogen atoms was calculated using the Gaussian 03 program. The charge was set to zero, the spin multiplicity was set to 2, and the calculation keyword was B3LYP / 6-31G **. The total energy obtained was -0.500272784191 Hartree (1 Hartree = 627.5095 kcal / mol).

(2) Total energy of molecular radicals A molecular model was created by extracting secondary hydrogen atoms of isopropanol, and the structure optimization and total energy calculation of isopropanol secondary radicals were performed using Gaussian 03. The charge was set to zero, the spin multiplicity was set to 2, and the calculation keyword was B3LYP / 6-31G ** FOpt. The total energy gained for the optimized structure was -193.712006644 Hartree.

(3) Total energy of neutral molecules A molecular model of isopropanol was created, and structural optimization and total energy calculation were performed using Gaussian 03. The charge was set to zero, the spin multiplicity was set to 1, and B3LYP / 6-31G ** FOpt was used as the calculation keyword. The total energy gained for the optimized structure was -194.368606865 Hartree.

(4) Radical destabilization energy The radical destabilization energy calculated from the above total energy according to equation (1) was 0.156327437 Hartree = 98.10 kcal / mol.

(Comparative Example 1)
The experiment was performed under the same conditions as in Example 1 except that an ethanol 0.0016 mol / L aqueous solution was introduced into the hollow fiber membrane instead of the isopropanol 0.0016 mol / L aqueous solution. As a result of the endotoxin addition recovery rate test, it was 136.8% immediately after endotoxin addition, and 16.0% after 48 hours, and it became impossible to accurately measure endotoxin activity by the limulus test over time.

  The radical destabilization energy of ethanol was calculated according to the following procedure.

(1) Total energy of hydrogen radical By the same method as in Example 1, -0.500272784191 Hartree was obtained.

(2) Total energy of molecular radicals The structure optimization and total energy calculation of ethanol radicals were carried out in the same way as in Example 1, except that a molecular model was created by extracting one hydrogen atom adjacent to the ethanol hydroxy group. did. The total energy gained for the optimized structure was -154.386809202 Hartree.

(3) Total energy of neutral molecule A molecular model of ethanol was created, and structural optimization and total energy calculation were performed in the same manner as in Example 1. The total energy gained for the optimized structure was -155.046209287 Hartree.

(4) Radical destabilization energy The radical destabilization energy calculated from the above total energy according to equation (1) was 0.159127301 Hartree = 99.85 kcal / mol.
(Comparative Example 2)
The experiment was performed under the same conditions as in Example 1 except that the concentration of the aqueous isopropanol solution was 0.016 mol / L. As a result of the endotoxin addition recovery rate test, it was 96.3% immediately after the endotoxin addition and 33.1% at the end of 48 hours, and the endotoxin activity by the limulus test could not be measured accurately over time. When the radical destabilization energy of isopropanol was calculated in the same manner as in Example 1, it was 98.10 kcal / mol.

Claims (6)

  Immerse the substrate in an aqueous solution containing a cationic water-soluble polymer aqueous solution and a compound having a destabilization energy accompanying radical generation of 99.5 kcal / mol or less at a concentration of 0.0008 mol / L or more and 0.008 mol / L or less. And the manufacturing method of the medical material characterized by irradiating with radiation.   The method for producing a medical material according to claim 1, wherein the compound contains a secondary hydroxyl group.   The method for producing a medical material according to claim 1 or 2, wherein the compound is one or a combination of isopropyl alcohol, 2-butanol, and glycerin.   The cationic water-soluble polymer is a primary amino group, secondary amino group, tertiary amino group, quaternary amino group, pyrrole group, pyrazole group, imidazole group, indole group, pyridine group, pyridazine group, quinoline group, piperidine. The method for producing a medical material according to any one of claims 1 to 3, comprising any one or more of a group, a pyrrolidine group, a thiazole group, and a purine group.   The method for producing a medical material according to any one of claims 1 to 4, wherein the base material contains a polysulfone-based polymer.   The endotoxin addition recovery rate in the limulus test of the aqueous solution after irradiation produced by the method for producing a medical material according to any one of claims 1 to 5 is 50% or more and 200% or less after 48 hours. A medical material characterized by that.