JPH0472808B2 - - Google Patents

Description

[Detailed description of the invention] (Technical field of invention) The present invention relates to a serum with strong antitumor activity.

(Prior Art and its Problems) Cancer is the leading cause of death in developed countries, but at present no anticancer drugs as strong as antibiotics for infectious diseases have been found.

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, Schwartzmann activity, and lethal toxicity. On the other hand, it is also known as a substance that exhibits antitumor cancer 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.

(Object of the Invention) The present invention utilizes the antitumor action of such lipopolysaccharide to produce a substance that has a strong antitumor action and is not toxic.

(Structure of the Invention) (1) Antitumor serum or plasma obtained by contacting fresh serum or plasma with an insoluble carrier that binds lipopolysaccharide derived from the cell wall of Gram-negative bacteria and then concentrating it.

(Explanation of composition) In the present invention, the lipopolysaccharide derived from the cell wall of Gram-negative bacteria (hereinafter referred to as lipopolysaccharide) refers to the Neisser
Gram-negative cocci, represented by Ia gonorrhoeae,
Pseudomonas aeruginosa, Burcella abortus,
Gram-negative aerobic bacilli such as Bordetella Pertussis, Escherichia coli, and Salmonella
typhi, Shigella dysenteriae, Klebsiella
pneumoniae, Serratia marcescens, Proteus
vulgaris, Yersinia enterocolitica, Vibrio
Refers to 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 cholerae, and a complex of lipids and polysaccharides with 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., Comp.Rend.
Soc.Biol., 128 5 (1938)], butanol extraction method [DCMorrison and L.Leive., J.Biol.
Chem. 250 2911 (1975)] and a method of extraction with ethylenediamine-N,N,N,N-tetraacetic acid water [L. Leive et al. J.Biol.Chem., 243 6384
(1968)]. The protein content of the lipopolysaccharide is determined from the viewpoint of ease of immobilization.
It is preferably 0.1 to 50%, especially 1 to 20%. Since the lipopolysaccharide is associative, its molecular weight varies depending on the measurement method and conditions, and usually
Observed in thousands to millions.

The insoluble carrier as used in the present invention means a carrier that is substantially insoluble and capable of immobilizing lipopolysaccharide. The shape of the insoluble carrier may be spherical, fibrous, or membrane-like, but a shape with a large surface area is preferred in order to immobilize the lipopolysaccharide at high density. A fiber shape is best from the viewpoint of good capture properties and large surface area during separation from serum.

Examples of insoluble carriers used in the present invention include (1) polystyrene or styrene/divinylbenzene copolymer containing amino groups, carboxyl groups,
A functional group capable of bonding to lipopolysaccharide such as an active halogen group, an oxirane group, an active ester group or an isocyanate group has been introduced as an aromatic substituent, (2) A methylene bridge or a sulfone instead of the polystyrene in (1) (3) N-(N'-dimethylaminopropyl)acrylamide/methylenebisacrylamide copolymer, (4) diethylaminoethylated cellulose, etc. Among these, vinyl polymer is used as the backbone polymer. This carrier is particularly preferably used because it has high acid resistance and alkali resistance.

The insoluble carrier to which lipopolysaccharide is bound (hereinafter referred to as immobilized lipopolysaccharide) used in the present invention is one in which lipopolysaccharide is chemically bound to the surface of the insoluble carrier. If the density of lipopolysaccharide in the serum is too low, the activation of the serum will be low, resulting in only antitumor serum with low activity. Therefore, the lipopolysaccharide density of the immobilized lipopolysaccharide is 0.1
It is desirable that the amount is at least mg/g, more preferably at least 1.0 mg/g. Preparation of immobilized lipopolysaccharide is achieved by mixing an insoluble carrier with a solution of lipopolysaccharide, or by further adding a condensing agent for peptide synthesis to this mixture.

The fresh serum or plasma used in the present invention refers to mammalian serum or plasma having a protein concentration of 6 to 9 g/dl and a hemolytic complement value (CH50) of 10 U/ml or more. Here, hemolytic complement value (CH50) 1U/ml means the amount of complement required to hemolyze 50% of 5 x ¹⁰⁸ sheep red blood cells present in 7.5ml of reaction solution, and 30~ for serum
It is 45 U/ml, and around 200 U/ml in guinea pig serum.

The contact between fresh serum and immobilized lipopolysaccharide in the present invention is preferably carried out at a temperature of 25 to 45°C, where complement inactivation and cold activation do not occur, and the contact time is not particularly limited, but Usually 10~
It will be held for 180 minutes. Concentration of serum is carried out using a semipermeable membrane having a molecular weight cut-off of 540,000 to 20,000 under pressure or normal pressure. The mixing ratio of fresh serum and immobilized lipopolysaccharide is usually 1 to 50 ml per 1 mg of lipopolysaccharide.
serum is used. If there is too little serum, the percentage of serum adsorbed to the carrier will be too high, and if there is too much serum, the percentage of serum activation will be too low. Of course, if the serum is highly activated, it will have an antitumor effect even if it is used as it is without being concentrated, but it is possible to concentrate it without any loss of activity, and the effect is more reliable.

The degree of concentration of serum varies depending on the ratio of serum to immobilized lipopolysaccharide used and the immobilization density of lipopolysaccharide, but it is usually concentrated until the protein concentration is 10 g/dl or more, more preferably 20 g/dl or more. be done.
Plasma is heparinized and then processed in the same way as serum. It is also possible to use whole blood.

The antitumor serum or plasma of the present invention can be used as a drug for the treatment of cancer in humans as well as in mammals. Its usage includes intravenous, intraperitoneal, intramuscular,
It can be administered subcutaneously, intradermally, or directly into the tumor.

(Mechanism of action of the invention) In the antitumor serum or plasma of the present invention, complement or a heat-labile protein similar to complement is activated by the immobilized lipopolysaccharide, and is converted into a factor that causes hemorrhagic necrosis of the tumor. Alternatively, it is considered that other components are used to change the factor.

(Effects of the Invention) The antitumor serum of the present invention exhibits antitumor properties similar to lipopolysaccharide, does not exhibit lethal effects, and is concentrated, allowing for a reduction in the amount and frequency of administration, and a greater Antitumor effects can be obtained,
Furthermore, it is effective in small doses and can be administered intratumorally.

Example 1 Polypropylene (Mitsui “Noprene” J3HG)
50 parts as an island component, polystyrene (“Styron”)
666) 46 parts, polypropylene (Sumitomo “Noprene”)
WF-727-F) Sea-island type composite fiber (16 islands, single fiber fineness 2,6-denier, tensile strength 2.9 g/d, elongation 50%, number of filaments 42) 50 g , N-methylol-α-chloroacetamide 50g, nitrobenzene 400g, 98%
It was immersed in a mixed solution consisting of 400 g of sulfuric acid and 0.85 g of paraformaldehyde, and reacted at 20° C. for 1 hour. Take out the fibers from the reaction solution and cool to 0°C with ice water for 5 minutes.
After terminating the reaction, the fibers were washed with water, and the 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.
In addition, the moisture content at PH7.4 is per 1.0g of dry fiber. 0g, and the diameter of the single yarn in this state is
It was 40-44 μm.

300 g of the above amino group-containing polystyrene fiber
After swelling in ml of water for a day and night, Escherichia
Coli055: 0.4 of B5 lipopolysaccharide (trichloroacetic acid extraction method, manufactured by Difco Laboratories)
Mix with 500 ml of mg/ml aqueous solution, then add 5.0 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide little by little while keeping the pH between 4.5 and 6.0 with 1N hydrochloric acid and 1N sodium hydroxide. In addition,
Dissolved. After shaking this mixture at room temperature for 2 days, the fibers were taken out and immersed in boiling water for 30 minutes three times, and then the pH of the washing solution was adjusted with 0.07M phosphate buffer (PH7.4). The fibers were washed to obtain a lipopolysaccharide-immobilized fiber. The concentration of lipopolysaccharide in the above immobilization mother liquor and washing solution was determined using the phenol/sulfuric acid method (1 ml of test material and 1 ml of 5% phenol water).
Add 5 ml of concentrated sulfuric acid to 1 ml of the mixed solution and measure the absorbance at 485 mμ. ). In this case, the second and subsequent washes were concentrated 50 times and then subjected to quantitative analysis.

The amount of lipopolysaccharide fixed above 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.

2.0 g of lipopolysaccharide-immobilized fiber (10 mg of lipopolysaccharide) prepared in Example 1 in 200 ml of fresh human serum at 37°C
After soaking for 180 minutes in
20/32, molecular weight cutoff 10,000 to 20,000, membrane thickness 0.020 mm), and polyethylene glycol (molecular weight 20,000) powder was sprinkled on the outside of the tube to concentrate the serum volume to one quarter. This concentrated serum 0.6ml
When the drug was administered to the above tumor-bearing mice through the tail vein on the 11th day (tumor diameter: 7 mm) and 13th day after cancer cell inoculation, intratumor bleeding was observed in 6 out of 14 mice.
Complete disappearance of the tumor was observed in 5 of them.

As a control, we performed the same experiment as above using 100 ml of human serum treated at 56°C for 30 minutes, but no bleeding or necrosis within the tumor was observed.
There were no cases of complete recovery.

Example 3 Instead of the unreacted conjugate fiber of Example 1, 25 g of polystyrene powder (diameter 0.05 to 1.0 mm) was reacted in the same manner as in Example 1 to obtain 52 g of chloroacetamidomethylated polystyrene powder. 40g of this powder at 15℃
Soaked in ethylenediamine for 24 hours at a temperature of 15-20 °C.
After a period of reaction, ethylenediaminoacetamidomethylated polystyrene beads (3.6 mmol/g of ethylenediamino groups) were obtained. 40g of this powder for 300
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), then 1N
- Adjust pH to 4.5 with hydrochloric acid and 1N sodium hydroxide
While maintaining the temperature at 6.0, 5.0 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide was added little by little and dissolved. This mixture was shaken at room temperature for 2 days and then centrifuged (3000 rpm, 30 minutes).
Take out the powder and wash it 3 times with 1 water.
The centrifugation procedure was repeated. Furthermore, the procedure of immersing the sample in boiling water for 30 minutes and then centrifuging it was performed three times. 0.07M phosphate buffer (PH
7.4) until the pH of the washing solution reached 7.4 to obtain lipopolysaccharide-immobilized powder. The concentration of lipopolysaccharide in the immobilization mother liquor and washing solution is determined using the phenol/sulfuric acid method (add 5 ml of concentrated sulfuric acid to a mixture of 1 ml of the sample and 1 ml of 5% phenol water, and measure the absorbance at 485 mμ).
I asked for it.

The amount of lipopolysaccharide immobilized was 0.5 mg/g.
After washing 0.6 g of this lipopolysaccharide-immobilized powder 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 4 20g of lipopolysaccharide immobilized powder prepared in Example 3
was mixed with 200 ml of fresh human serum, shaken at 37°C for 180 minutes, and then centrifuged (300 rpm, 30 minutes).
The supernatant was taken out and concentrated 4 times as in Example 1. When 0.6 ml of this serum was administered to the tumor-bearing mice obtained in Example 1 through the tail vein on the 11th and 13th day after cancer cell inoculation, intratumor bleeding was observed in 5 out of 14 mice. Complete disappearance of the tumor was observed in 3 of them.

Claims (1)

[Claims] 1. Antitumor serum or plasma obtained by contacting fresh serum or plasma with an insoluble carrier that binds lipopolysaccharide derived from the cell wall of Gram-negative bacteria and then concentrating it.