JPH043986B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field of the Invention) The present invention relates to a blood treatment agent having antitumor activity.

(Prior art and its problems) Lipopolysaccharide derived from the cell wall of Gram-negative bacteria is a substance also called intracellular toxin, endotoxin, or pyrodiene, and is known to exhibit harmful effects such as fever and lethal toxicity. However, it is also known as a substance that exhibits antitumor effects against malignant tumors.

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

(Objective of the invention) To attenuate the lethal toxicity of the lipopolysaccharide while taking advantage of its antitumor action, and to store the lipopolysaccharide in an aseptic manner and use it.

(Structure of the Invention) The present invention relates to a blood treatment agent obtained by irradiating a lipopolysaccharide derived from Gram-negative bacterial cell walls immobilized on an insoluble carrier with gamma rays.

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
A complex of lipids and polysaccharides localized in the outer layer of the cell wall of Gram-negative bacteria such as Gram-negative facultative anaerobic bacilli such as enterocolitica Vibrio cholerae.
It also refers to a complex of lipids, 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., 1285 (1938)]. Usually, the former method yields lipopolysaccharide containing 0.1-2% protein, and the latter method yields lipopolysaccharide containing 0.1-10% protein. is easy. Since the lipopolysaccharide is associative, its molecular weight depends on the measurement method and conditions.
They are observed in numbers ranging from several thousand to one million.

The insoluble carrier referred to in the present invention means that there is no eluate under the conditions of use and is substantially insoluble, and
It means a carrier that can immobilize lipopolysaccharide without elution under the conditions of use.

An example of the insoluble carrier used in the present invention is:
(1) Polystyrene, crosslinked polystyrene, or styrene/divinylbenzene copolymer with amino groups,
A substituent having a carboxyl group, a quaternary ammonium group, an active halogenated alkyl group, or an epoxide group is introduced as an aromatic nuclear substituent, (2)
Insoluble copolymer of acrylic acid, (3) insoluble acrylamide copolymer having an amino group, (4) insolubilized cellulose or agarose having a carboxyl group, (5) agarose gel having an epoxide group,
(6) Bromocyanated agarose gel, (8) Nylon 6, (9) Nylon 6.6, (10) Polyethylene terephthalate, etc. Among these, carriers with vinyl polymer as the backbone polymer, especially styrene polymer as the backbone polymer, are used. A polymer carrier is particularly preferably used because it has excellent acid and alkali resistance, durability, and liquid permeability.

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. Although they are used in the form of hollow fibers and membranes, fibrous and hollow fiber forms are particularly preferred since they have both a large surface area and good liquid permeability.

In the present invention, the binding state between the lipopolysaccharide derived from the cell wall of Gram-negative bacteria and the insoluble carrier is not particularly limited as long as the lipopolysaccharide does not substantially flow out under the conditions of use; bond, ether bond, ester bond, ureido bond, urea bond, or alkyl group, or (2) ionic bond, but (1) is more stable. , more preferred. In particular, a state in which they are bonded through an amide bond is preferred because of its high stability.

If the immobilization density of the lipopolysaccharide to the insoluble carrier in the present invention is too low, a large amount of carrier will be required, so it is usually preferably 10 μg or more, especially 1 mg or more per square meter of carrier surface area.

In the present invention, if the dose of γ-rays to be irradiated to the lipopolysaccharide derived from Gram-negative bacterial cell walls immobilized on the insoluble carrier is too small, sterilization will be insufficient, the blood treatment agent of the present invention will not be able to be stored, and Too much will cause decomposition of the carrier and lipopolysaccharide,
0.1 to 100 megaroentgen, especially 1 to 10 megaroentgen is preferred.

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.

(Effect of the invention) The effect of the present invention is that the lipopolysaccharide derived from the cell wall of Gram-negative bacteria is immobilized on an insoluble carrier.
The present invention is capable of exhibiting only an antitumor effect while attenuating lethal toxicity and pyrogenic toxicity, and aseptic storage is possible by irradiating the blood treatment agent with gamma rays.

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 poured into ice water 5 at 0° C. to stop the reaction, washed with water, and then nitrobenzene adhering to the fibers was extracted and removed.
This fiber was vacuum dried at 50° C. to obtain 71 g of chloroacetamidomethylated fiber. Then this fiber 40
Immerse g in ethylenediamine at 15℃ and heat it at 15-20℃.
The mixture was reacted at a temperature of 24 hours to obtain ethylene diaminoacetamide methylated fibers. This fiber is 1.8
It has an ethylene diamino group of mmol/g, and has a primary amino group and a secondary amino group, but has tertiary and quaternary amino groups.
It does not have a grade amino group. Further, the water content at pH 7.4 was 1.0 g per 1.0 g of dry fiber, and the diameter of the single yarn in this state was 40 to 44 μm.

300 g of the above amino group-containing polystyrene fiber
After soaking in ml of water for a day and night to swell,
Escherichia Coli055: 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 concentrated sulfuric acid to a mixture of 1 ml of sample and 1 ml of 5% phenol water,
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 above is 5.0 mg/g (45 mg/
^m2 ).

After sealing 2.0 g of this lipopolysaccharide-fixed fiber in a hard glass tube with 200 ml of pharmacopoeia physiological saline,
Irradiated with 2.5 megaroentgen of 60Co gamma rays and heated at room temperature.
After being left for 90 days, the bags were opened and the toxicity of the filling liquid and the activity of the fibers were examined. When the amount of lipopolysaccharide in this filling liquid was determined using the Limulus Pregel method,
The concentration was less than 0.1 ng/ml, and the results of a fever test using three domestic rabbits were negative (based on the Japanese Pharmacopoeia, Pyrogen Test Method). Also, fiber
Soak 0.5g in 20ml of non-mobilized rabbit serum and incubate at 37℃ for 1 hour.
After incubation for an hour, 5 ml of this serum was added to three rabbits (each weighing 2.9 kg, 2.9 kg, and 2.8 kg).
Kg) was administered intravenously, but no fever was observed in all three animals.

0.5g of fiber left for 90 days after γ-ray irradiation
Immerse in 50 ml of fresh BALB/C mouse serum for 37
After incubation for 1 hour at °C, this serum
Take 0.3 ml of 5 x 10 ⁵ tumor cells of fibrosarcoma MC-B1 induced by inoculating a tumor-bearing mouse (BALB/C mouse with methylcholanthrene) and inoculate subcutaneously into the back of the BALB/C mouse. When administered intravenously on the 10th day (tumor diameter: 6.3 mm), 12th day, and 14th day after tumor cell inoculation, a reduction in tumor size was observed in 13 animals. Six animals had completely disappeared tumors.
Two out of 13 were found. On the other hand, in the untreated group
No reduction in tumor diameter or disappearance was observed in 10 out of 10 animals.

Claims (1)

[Claims] 1. A blood treatment agent obtained by irradiating lipopolysaccharide derived from Gram-negative bacteria cell walls with gamma rays and immobilized on an insoluble carrier.