JPH0335974B2 - - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to endotoxin detoxification adsorbents. (Prior art and its problems) Endotoxin is a substance that exhibits pyrogenicity and toxicity when it enters the blood of mammals. It is a lipopolysaccharide derived from the outer cell wall membrane of Gram-negative bacteria. Escherichia coli, a typical gram-negative bacterium, always resides in the intestines, but when the body is healthy, its endotoxins are detoxified by the liver even if absorbed, so they do not appear in the blood. However, if your liver function is impaired, if you are receiving anticancer drugs or immunosuppressive drugs, if you have a large burn injury, or if you have undergone major surgery, you may be susceptible to severe bacterial infections. This can result in endotoxemia and shock. In addition, endotoxemia may occur in cases of liver abscess or cholecystitis, which may lead to shock. In addition, if an indwelling catheter, dialysis equipment, or circulatory fluid is contaminated with endotoxin, endotoxemia may suddenly occur, which may cause a shock. Gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, are generally weakly virulent bacteria, but bacterial infections are a major problem because antibiotics are becoming increasingly resistant to them. Endotoxins are heat-stable substances and cannot be detoxified by autoclaving. Ion-exchange resins are said to non-specifically adsorb endotoxins, but their capacity is small, so they are not effective in treating endotoxemia. Polymyxin is known as a drug that detoxifies endotoxins. This is a heptide antibiotic that is not absorbed in the intestinal tract, but it is highly toxic to the central nervous system and kidneys, so it can only be administered orally or locally.
Therefore, it cannot be used directly to treat endotoxemia. Polymyxin immobilized on sepharose is known as an endotoxin adsorbent, but it cannot be used to treat endotoxemia because it has high swelling properties and low mechanical strength. Although it has been confirmed in the present invention that the action of immobilized polymyxin is the decomposition of endotoxin, in this case, it is possible that this decomposition product exhibits other toxic effects. (Objective of the invention) To create a material that can detoxify and adsorb and remove endotoxins. (Structure of the Invention) A detoxification adsorbent formed by bonding a functional group having a basic nitrogen atom and a polymixing molecule to an insoluble vinyl polymer molded article. (Explanation of structure) The insoluble vinyl polymer molded article in the present invention is
A vinyl polymer that is substantially insoluble under the conditions of use, into which a functional group having a basic nitrogen atom can be introduced, and to which polymyxin molecules can be covalently bonded, and which is suitable for fibers and hollow fibers. , membranes and their highly processed products. In order for the insoluble vinyl polymer molded article of the present invention to covalently bond to a polymyxin molecule, the polymer must include an α-halogenated acyl group, a halogenated alkyl group, an isocyanate group, a carboxyl group, or a carboxylic acid ester group. It is necessary that the functional group is bonded to the polymer directly or via a spacer. The length of this spacer is preferably 4 or more methylene groups. Examples of such insoluble vinyl polymers include homopolymers or copolymers of monovinyl compounds represented by styrene, vinyl chloride, acrylic amide, acrylonitrile, maleic anhydride, methacrylic esters, or divinylbenzene or methylene. In addition to copolymers with polyvinyl compounds such as bisacrylamide, examples include those obtained by crosslinking the monovinyl compound polymer molded products with formaldehyde, chlorosulfonic acid, and the like. Crosslinked polymers have no fluidity and are difficult to mold, so when the polymer molded product is a fiber, membrane, etc., a method of crosslinking after molding is preferably employed. The greater the surface area of the molded article of the present invention, the greater its ability to detoxify and adsorb endotoxins; however, the liquid permeability of a column filled with the molded article becomes worse, so the surface area should be 0.01 or more and 100 m ² /g. More preferably, it is 0.05 or more and 10 m ² /g or less. In the present invention, the functional group having a basic nitrogen atom is a secondary amino group represented by a primary amino group, a monomethylaminoalkyl group, a monoethylaminoalkyl group, a monobutylaminoalkyl group, etc.
dimethylaminoalkyl group, diethylaminoalkyl group, di-n-propylaminoalkyl group, di-n
- Tertiary amino groups represented by butylaminoalkyl groups, etc.; and tertiary amino groups represented by trimethylammonium alkyl groups, triethylammonium alkyl groups, tri-n-propylammonium alkyl groups, tri-n-butylammonium alkyl groups, etc. It means a quaternary ammonium group. The density of the functional group having a basic nitrogen atom is 0.1 meq/g or more, more preferably 0.5 meq/g.
The above is good. The group that connects the functional group having a basic nitrogen atom and the insoluble vinyl polymer may be any group that is stable under the conditions of use. Polymyxin as used in the present invention refers to Bacillus
It is an antibiotic produced by Polymyxa, including polymyxin A, polymyxin B ₁ , polymyxin
B ₂ , polymyxin D, polymyxin E ₁ , and
Refers to substances that have antibacterial activity against Gram-negative bacteria such as polymyxin E ₂ . The bonding state between the polymyxin molecule and the insoluble vinyl polymer is such that when all the amino groups in the polymyxin molecule are amidated, the detoxifying activity against endotoxins is lost.
At least one amino group must be present as a basic group. If the density of polymyxin molecules is too low, there will be no detoxifying activity against endotoxins, so it is preferably 1 mg or more, more preferably 20 mg or more per gram of molded product. If the chain length of the group that binds the polymyxin molecule and the insoluble vinyl polymer is too short, the detoxifying activity of polymyxin against endotoxins will not be exhibited, so the chain length is preferably 4 or more carbon atoms. A typical example of the method for preparing the detoxifying adsorbent of the present invention is when the molded product is a polystyrene fiber that has been insolubilized and α-chloroacetamide methylated by formaldehyde/N-methylol-α-chloroacetamide/sulfuric acid treatment. (1) A method in which the molded product is reacted with polymyxin at a pH of 7 or higher and then reacted with an amine; (2) The molded product is treated with a mixture of a primary diamine and a secondary amine to perform mixed amination; Furthermore, there is a method of succinylating with succinic anhydride and then reacting with polymyxin in the presence of a peptide condensing agent. The detoxification adsorbent of the present invention can be used to remove endotoxins from transfusions and dialysis fluids, as well as to prevent infection when using an indwelling catheter, to prevent infection during severe burns, and to prevent endotoxin blood loss after surgery for the digestive tract, heart, brain, etc. Typical examples of its use include circulating infusion fluid, dialysate, or blood through a column filled with the detoxification adsorbent, and placing the detoxification agent of the present invention on the outside of an indwelling catheter. There are methods such as placing an adsorbent in parallel, and coating the surface of the burn area with the detoxifying adsorbent of the present invention. (Mechanism of action of the invention) The action mechanism of the detoxification adsorbent of the present invention is considered to be that the polymyxin molecular group decomposes endotoxin, and the functional group having a basic nitrogen atom adsorbs the decomposition products of endotoxin. (Effects of the Invention) The detoxification adsorbent of the present invention shares a polymyxin molecular group with a functional group having a basic nitrogen atom, thereby decomposing endotoxins and adsorbing and removing the decomposition products. It has features that are highly effective. Furthermore, even if the immobilization density of polymyxin is low, a large effect can be obtained, so that expensive polymyxin can be saved. Examples are shown below. Example 1 Polypropylene (Mitsui “Noblen” J3HG)
50 parts as an island component, polystyrene (“Styron”)
666) 46 parts, polypropylene (Sumitomo “Noblen”)
Sea-island composite fiber containing 4 parts of WF-727-F) as sea component (number of islands: 16, single yarn fineness: 2.6 denier,
Tensile strength 2.9g/d, elongation 50%, number of filaments
42) 50g, N-methylol-α-chloroacetamide 60g, nitrobenzene 400g, 98% sulfuric acid 400g
g and 0.85 g of paraformaldehyde, and reacted at 20° C. for 1 hour. The fibers were taken out from the reaction solution and poured into ice water 5 at 0° C. to stop the reaction, washed with water, and then the nitrobenzene adhering to the fibers was extracted and removed with methanol. This fiber (fiber A) was vacuum dried at 50°C, and 71g of chloroacetamide methylated fiber was obtained.
I got it. Polymyxin B sulfate (Sigma; 8000 units/mg)
Dissolve 4.8g in 600ml of water and add 22g of fiber A in this.
After shaking for 5 hours, 1.5 g of magnesium oxide was added and shaking at room temperature for 12 hours. Next, this fiber was taken out from the reaction mother liquor, packed into a chromatography column, and washed with 4800 ml of 0.1N hydrochloric acid to obtain a polymyxin-immobilized fiber (fiber B).
The amount of polymyxin in this washing solution and the reaction mother liquor was measured by the microbiuret method, and the difference between the measured amount and the amount charged was regarded as the amount of polymyxin immobilized on the fibers. The immobilized amount was 39 mg/g. 20 g of fiber B was immersed in a 50% dimethylamine aqueous solution and heated at 55° C. for 4 hours. This fiber is sequentially
Water, 1N-hydrochloric acid, water, 0.17M-phosphate buffer (PH
7.4) to obtain the detoxifying adsorbent 1 of the present invention. The amount of amino groups in the detoxification adsorbent of the present invention is 2.1 milliequivalent/
It was hot at g. Example 2 23.7 g of 1,12-diaminododecane was added to 1050 ml of 50
34 g of the chloroacetamidomethylated fiber (fiber A) obtained in Example 1 was added to a mixed solution obtained by dissolving the fiber in a % dimethylamine aqueous solution, and the mixture was reacted for 72 hours. The fibers were taken out and thoroughly washed with dilute hydrochloric acid and water to obtain mixed aminated fibers (fiber C). From the exchange capacity of fiber C and the amount acetylated with acetic anhydride/pyridine, the amount of dimethylamino groups in the fiber is
The amount of 1,12-diaminododecane was 0.16 mmol/g. 1N-30g of fiber C
After being treated with a caustic soda aqueous solution, the fibers were thoroughly washed with water and then dried to obtain aminated fibers (fibers D). 23.4 in DMSO400ml containing 6.0g of succinic anhydride
After soaking g of fiber D and heating it at 50℃ for 5 hours,
A succinylated fiber (fiber E) was obtained by washing with water. 20 g (dry weight) of fiber E was immersed in 250 ml of sulfuric acid polymyxin B (manufactured by Sigma) aqueous solution (15 mg/ml), and while adjusting the pH of the solution to 4.5 to 6.0 with 1N caustic soda aqueous solution and 1N hydrochloric acid aqueous solution, 1- 500 mg of ethyl-3-(3-dimethylaminopropyl)carbodiimide was added over 3 hours. after that,
After shaking the reaction mixture at room temperature for 5 days, the fibers were packed into a chromatography column and washed with 4 portions of water and 1 portion of phosphate buffer to obtain detoxifying adsorbent 2 of the present invention. The amount of polymyxin in the reaction mother liquor and washing solution was measured by the microbiuret method, and the difference between that amount and the amount charged was regarded as the amount of polymyxin immobilized on the fibers. The immobilization density was 119 mg/g. Example 3 Escherichia Coli 055: A solution of 100 mg of B5 lipopolysaccharide (manufactured by Difco Laboratories, trichloroacetic acid extraction method) dissolved in 200 ml of physiological saline 20
Add 2g of adsorbent 1 or 2 of the present invention to ml and heat at 37°C.
After shaking for 60 minutes, 1 ml of the supernatant was administered to ICR mice via the tail vein, and the mortality rate was examined 48 hours later. The amount of sugar components contained in the same supernatant was determined by the phenol/sulfuric acid method (1 ml of sample solution + 1 ml of 5% phenol water + 5 ml of concentrated sulfuric acid: 485 mμ). In addition, the amount of endotoxin contained in the same supernatant was examined using a Toxicolor Test (manufactured by Seikagaku Corporation). For comparison, fiber A, instead of the adsorbent of the present invention,
Fiber B, Fiber C and ion exchange resin IRA-938
A similar experiment was conducted for The results are shown in Table 1. Table 1 shows that polymyxin-immobilized fibers (fiber B) degrade endotoxins but do not adsorb them, and that anion exchange fibers (fiber C) and resins adsorb sugar components but do not reduce mouse mortality. It can be seen that the adsorbent of the present invention in which functional groups coexist has both detoxification and adsorption effects. 【table】

Claims (1)

[Claims] 1. A detoxification adsorbent made by bonding a functional group having a basic nitrogen atom and a polymyxin molecule to an insoluble vinyl polymer molded article.