JPH0364153B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel blood treatment agent and a method for producing the same.

Lipopolysaccharide derived from the cell wall of Gram-negative bacteria is a substance also called exotoxin, endotoxin, or pyrodiene, and is known to exhibit harmful effects such as fever, Schwarzmann activity, and lethal toxicity. On the other hand, it is also known as a substance that exhibits antitumor effects against malignant tumors.

Currently, there is no specific drug for malignant tumors, and substances with antitumor effects are worthy of attention. However, lipopolysaccharide cannot be used safely because its lethal dose is close to the minimum effective antitumor effect dose.

Therefore, the present inventors have conducted intensive studies on a method for making use of the antitumor action of the lipopolysaccharide and attenuating its lethal toxicity. As a result, the present inventors have determined that the lipopolysaccharide can be treated with a substance obtained by solidifying the lipopolysaccharide into an insoluble carrier. The inventors have discovered that by circulating the collected plasma into the blood vessels of a tumor-bearing animal, it is possible to eliminate the tumor in the animal, and have arrived at the present invention.

That is, the present invention provides a blood treatment agent in which a lipopolysaccharide derived from the cell wall of a gram-negative bacterium is immobilized on an insoluble carrier containing a primary or secondary amino group or a carboxyl group, and a blood treatment agent comprising the lipopolysaccharide and the insoluble carrier. The present invention provides a method for producing the blood treatment agent, which comprises reacting the mixture with a condensing agent.

A feature of the blood treatment agent of the present invention is that when it comes into contact with plasma, it generates tumor necrosis factor or a factor that activates the body's defense mechanism that causes tumor necrosis in the plasma, and suddenly causes necrosis of tumors in distant areas. In addition to being able to reduce the risk of death, it also has no lethal effects.

In the present invention, the lipopolysaccharide derived from the cell wall of Gram-negative bacteria refers to Gram-negative cocci represented by Neisseria gonorrhoeae, Pseudomonas aeruginosa,
Gram-negative aerobic bacilli such as Brucella abortus and Bordetella pertussis, Escherichia
coli, Salmonella typhi, Shigella dysenteriae,
Klebsiella pneumoniae, Serratia marcescens,
Proteus vulgaris, Yersinia enterocolitica,
Lipid/polysaccharide complexes localized in the outer layer of the cell wall of Gram-negative bacteria such as Gram-negative facultative anaerobic bacilli such as Vibrio cholerae;
Refers to a complex of polysaccharides and a small amount of protein.
The lipopolysaccharide can be extracted from Gram-negative bacteria by known methods. A typical example of this method is extraction with phenol water [Van Otto
Westphal et al., Z.Naturforsch., 7B: 148~
155 (1952)] and the method of extraction with trichloroacetic acid [A. Boivin and L. Meserobeanu., Comp.
Rend.Soc.Biol., 128 5 (1938)]. Usually, in the former method, proteins are
A lipopolysaccharide containing 0.1-2% protein is obtained, and the latter method yields a lipopolysaccharide containing 0.1-10% protein, but the latter method is easier to immobilize. Since the lipopolysaccharide has an associative property, its molecular weight varies from 10,000 to 10,000 depending on the measurement method and conditions.
It is observed that it becomes 1 million.

The insoluble carrier as used in the present invention has a primary or secondary amino group or carboxyl group, and
It means a carrier that is free of eluates and substantially insoluble under the conditions of use.

An example of the insoluble carrier used in the present invention is:
(1) polystyrene or styrene/divinylbenzene copolymer into which a substituent having a primary amino group, secondary amino group, or carbonyl group is introduced as an aromatic nucleus substituent; (2) polystyrene of (1); (3) Acrylic acid/acrylonitrile copolymer, (4) Acrylic acid/styrene copolymer, (5) Nylon 6, (6) Nylon 6/6, (7) polyethylene terephthalate, (8) insolubilized cellulose having a carboxyl group, and (9) aminoethylated cellulose. This carrier is particularly preferably used because it has high acid resistance and alkali resistance.

The shape of the insoluble carrier is not particularly limited as long as it has a suitable surface area and sufficient mechanical strength to withstand external forces during use, and is usually in the form of particles, fibers, etc. Used in hollow fiber and membrane shapes.

The method of solidifying the lipopolysaccharide used in the present invention on the insoluble carrier of the present invention may be any method that utilizes the condensation reactivity of the amino group or carboxyl group of the insoluble carrier, and is not particularly limited. However, typical examples include (1) a method of adding a condensing agent for peptide synthesis to a mixture of the insoluble carrier and an aqueous lipopolysaccharide solution; and (2) a method of adding a condensing agent for peptide synthesis to a mixture of the insoluble carrier and an aqueous lipopolysaccharide solution; After treatment with imide and a condensing agent for peptide synthesis,
There is a method of mixing with the lipopolysaccharide solution. The condensing agent for peptide synthesis used here is 1-
Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 1-cyclohexyl-3-(2-
Examples include morpholinoethyl)carbodiimidemeth-p-toluenesulfonate, N,N'-dicyclohexylcarbodiimide, and Woodward's reagent K.

If the immobilization density of lipopolysaccharide on an insoluble carrier in the present invention is too low, a large amount of carrier will be required, so it is usually desirable to immobilize 10 μg or more of lipopolysaccharide per square meter of carrier surface area. Therefore, the insoluble carrier has an area of 0.01 to 100 m ² /
g, the primary amino group, secondary amino group or carboxyl group is
It is preferred to have a density of 0.001 or more, especially 0.01 meq/g.

Methods for using the blood treatment agent of the present invention include filling a column for extracorporeal circulation and bringing it into continuous or intermittent contact with whole blood or plasma;
There is a method of filling a syringe, sucking whole blood or plasma into the syringe, and then returning the whole blood or plasma to the body.

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 50g, 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 placed in ice water 5 at 0° C. to stop the reaction, washed with water, and then nitrobenzene adhering to the fibers was extracted and removed with methanol. This fiber was vacuum dried at 50° C. to obtain 71 g of chloroacetamidomethylated fiber. Next, 40 g of this fiber was immersed in ethylene diamine at 15°C and reacted at a temperature of 15 to 20°C for 24 hours to obtain ethylene diaminoacetamide methylated fiber.
This fiber has 1.8 mmol/g of ethylene diamino groups, has primary and secondary amino groups, but has no tertiary or quaternary amino groups. Also,
The moisture content at PH7.4 is 1.0g per 1.0g of dry fiber, and the diameter of the single yarn in this state is 40 to 44μm.
It was hot.

300 g of the above amino group-containing polystyrene fiber
After soaking in ml of water for a day and night to swell,
Escherichia Coli O55: Mixed with 500 ml of a 0.4 mg/ml aqueous solution of B5 lipopolysaccharide (trichloroacetic acid extraction method, manufactured by Difco Laboratories), and then
Adjust the pH with 1N hydrochloric acid and 1N sodium hydroxide.
1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide 5.0g
was added little by little and dissolved. After shaking the mixture at room temperature for 2 days, the fibers were taken out and washed three times with 1 part of water. Furthermore, after performing the operation of immersing this fiber in boiling water for 30 minutes in step 1 three times,
The pH of the washing solution is 7.4 with 0.07M phosphate buffer (PH7.4)
The fibers with lipopolysaccharide immobilized thereon were obtained by washing until the fibers were washed. The concentration of lipopolysaccharide in the above immobilization mother liquor and washing solution was determined using the phenol/sulfuric acid method (adding 5 ml of agricultural sulfuric acid to a mixture of 1 ml of sample and 1 ml of 5% phenol water,
Measure the absorbance in mμ. ), and the amount of immobilization was determined using the residual method. In this case, the second and subsequent washes were concentrated 50 times and then subjected to quantitative analysis.

The amount of lipopolysaccharide immobilized was 5.0 mg/g. After washing 0.6 g of this lipopolysaccharide-fixed fiber with 200 ml of physiological saline, immersing it in 50 ml of physiological saline and warming it at 38°C for 3 hours, the lipopolysaccharide in the physiological saline was washed. The amount present was determined by the Limulus-Pregel method and was less than 1 nanogram/ml.

Example 2 Fibrosarcoma MC-B1 induced by inoculating BALB/C mice with methylcholanthrene
Tumor-bearing mice were prepared by taking 5 x 10 ⁵ tumor cells and inoculating them subcutaneously on the back of BALB/C mice. The tumor size was 2.1 mm 5 days after engraftment, and 2.1 mm after 10 days.
6.3 mm, which became 8.9 mm in diameter after 12 days.

0.2 g of lipopolysaccharide-immobilized fiber (1.0 mg of lipopolysaccharide) prepared in Example 1 was added to 20 ml of human plasma at 37°C.
After soaking for 180 minutes, 0.5 ml of this plasma was applied to the above tumor-bearing mice on the 11th day after cancer cell inoculation (tumor diameter: 7
mm) was administered intravenously on the 13th and 15th day, the tumor in 1 out of 10 animals became completely necrotic and disappeared. Significant softening of the tumors in the remaining nine animals was observed. As a control, the same experiment as above was performed using 10 ml of human plasma treated at 56°C for 30 minutes, but no tumor necrosis, softening, or bleeding was observed.

Example 3 40ml of plasma collected from 100 BALB/C mice
The lipopolysaccharide-immobilized fiber prepared in Example 1 was 0.4
g (2.0 mg of lipopolysaccharide) at 37°C for 180 minutes, and 1.0 ml of this plasma was added to tumor-bearing mice prepared in the same manner as in Example 2 on the 11th and 13th day after cancer cell inoculation. When administered intravenously on the 15th day, 1 out of 10 animals
The tumor completely disappeared in one animal, and significant softening of the tumor was also observed in the other nine animals.

Example 4 0.2 g of the lipopolysaccharide-immobilized fiber prepared in Example 1 was immersed in 20 ml of human plasma at 37° C. for 180 minutes. Take 0.5 ml of this plasma, mix it with 2.5 ml of human plasma, and then intraperitoneally inject it into the peritoneal cavity of a tumor-bearing mouse prepared in the same manner as in Example 2 on the 11th, 13th, and 15th day after cancer cell inoculation. When administered on the 17th and 19th day, complete disappearance of tumors was observed in 2 out of 7 animals.

Claims (1)

[Scope of Claims] 1. A blood treatment agent comprising a lipopolysaccharide derived from the cell wall of a Gram-negative bacterium immobilized on an insoluble carrier containing a primary or secondary amino group or carboxyl group. 2 Lipopolysaccharide derived from Gram-negative bacterial cell wall, 1
1. A method for producing a blood treatment agent, which comprises adding a condensing agent to an insoluble carrier containing a primary or secondary amino group or a carboxyl group.