JPS6072819A - Neutralizing agent for cycotoxin - Google Patents

Abstract

PURPOSE:A neutralizing agent for cytotoxin, consisting of a heat-treated fat globule membranes of animal milk, mass producible more easily than the conventional ganglioside pharmaceutical, effective for preventing and treating infection of cholera and coliform bacilli, etc. and using ganglioside as a receptor. CONSTITUTION:A neutralizing agent for cytotoxin, e.g. cholera toxin, coliform bacilli thermolabile toxin, tetanus toxin, etc. containing heat-treated fat globule membranes of animal milk, and using ganglioside as a receptor. The agent is characterized in that ganglioside GD3, GM2 and GM3, etc. present in the fat globule membranes is used without separation from the membranes. The conventional pharmaceutical contains isolated and purified ganglioside adsorbed on a carrier, and has economical problems. The above-mentioned neutralizing agent is a highly safe agent which can be obtained only by simple separation and heat treatment of the fat globule membranes available in large quantities and at a low cost from a butter manufacturing process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a neutralizing agent for various bacterial toxins (hereinafter sometimes simply referred to as toxins) that uses gangliosides as receptors.

Urella toxin, E. coli heat-labile toxin, tetanus toxin, botulinum toxin, Vibrio parahaemolyticus heat-stable toxin, staphylococcal toxin, etc. all contain acidic glycolipids and gangliosides on the surface of intestinal cells (including several molecules as described below). It has been confirmed that some species (seeds) act as receptors and are taken into the human body, exerting their toxicity. For example, cholera toxin binds to Glyondo GMI, activates adenyl cyclase in the intestinal wall, and activates intracellular azo/syn-3'-
It specifically increases 5'-cyclic monophos-7ate, resulting in large amounts of salt and water loss and causing diarrheal symptoms. In the case of Escherichia coli toxin, it is thought that a similar mechanism is used to cause diarrheal symptoms. Therefore, one possible method for preventing diarrhea and other infectious diseases caused by these toxins is to use substances that can directly act on the toxins and inhibit their toxicity. Methods for preventing or treating inflammation that have been studied from this perspective include gangliosides, which are receptors for toxins, especially ganglioside GM1, which has a strong affinity for many toxins.
There is an antagonistic method in which the toxin is isolated from bovine brain, bound to a carrier such as activated charcoal, and then administered orally to bind to the toxin, thereby preventing the toxin from binding to gangliosides on the intestinal wall (gangliosides can be administered as is). Then, it is adsorbed to the intestinal tissue, increasing the number of receptors and reinforcing the action of the toxin, so it is necessary to fix it by adsorbing it to antibodies.) However, this method has the problem that the ganglioside GMI collected from bovine brain is bulky and expensive, and no method has been found to economically collect ganglioside from biological tissues or biological products other than bovine brain. Because there is no such thing, it has not yet been put into practical use.

The present invention is directed to a novel toxin neutralizing agent that can be used in the above-mentioned antagonistic method, that is, a neutralizing agent for bacterial toxins having ganglioside receptors, which is made of a heat-treated product of the fat globule membrane of animal milk (including its crushed product). It provides:

The animal milk fat globule film is a film that covers the fat globules in animal milk, and in the case of milk, the fat globule diameter is 1 to 10/J,
The film thickness is about 10 nm. This membrane is formed at the same time as animal milk fat is secreted in the mammary gland, and its composition is similar to that of the plasma membrane of mammary gland cells. The main components that make up the fat globule membrane are phospholipids, enzymes, proteins, glycoproteins, triglycerides, cholesterol, etc., but proteins and lipids alone account for more than 90%, of which about 55% are lipids and about 45% is protein. Six types of gangliosides have been confirmed so far, and the total amount contains about 6n+nol (calculated as sialic acid) per 1 mg of protein. Ganglioside GD3 is the most abundant ganglioside, but other gangliosides such as GM2 and GM3 are also found.

The most distinctive feature of the present invention is that whereas conventionally studied ganglioside preparations were made by adsorbing isolated and purified gangliosides onto a carrier, "1. gangliosides GD3, GM2, GM3, etc. By using the fat globule membrane as it is without separating it from the globular membrane, the fat globule membrane, a type of biopolymer, is not only a ganglioside-containing substance but also a carrier of gangliosides. When viewed as a ganglioside' preparation supported on a carrier, the fat globule film exhibits an unexpectedly high stability considering its composition. That is, D'1-13.5
~8.4.Even if heat treatment is performed at 100°C for 160 minutes or treatment with proteolytic enzymes such as pronase or trypsin, the toxin neutralizing ability hardly decreases.
There is no risk that the carrier portion will be digested in the stomach or intestines and liberate gangliosides.

The fat globule film is concentrated in the so-called buttermilk that remains after the cream obtained by centrifuging animal milk is processed in a churn and the resulting butter granules are separated in the conventional butter manufacturing process. Although it is most advantageous to use this animal milk fraction as a raw material for the toxin neutralizer of the invention, it is not limited to this, for example, a treatment in which cream is mixed with water and then washed by centrifugation. A buttermilk equivalent obtained by applying and then churning may also be used.

Buttermilk or an animal milk fraction with a similar composition is not suitable for use as a toxin neutralizer as it contains a large amount of milk components such as milk protein and lactose, so it is usually treated by dialysis or ammonium sulfate. It is desirable to purify by methods such as fractionation, gel filtration, and isoelectric precipitation. Furthermore, since the fat globule membrane contains many enzymes, it is necessary to heat-treat to inactivate them. Heat treatment conditions 3- include, for example, heating at 62°C for 30 minutes or more;
Alternatively, heating equivalent to so-called UHT sterilization treatment, which is heat treatment at a high temperature of 100° C. or more for a short time, is suitable.

The fat globule membrane obtained through the above two treatments has some parts that are the same size as the outside surrounding the fat globule, so even if it is dispersed in water, a part of it tends to precipitate. Therefore, it is usually desirable to subject this to ultrasonic treatment or the like to form fine film fragments so that a stable suspension in water can be formed.

The fat globule membrane that has undergone all the treatments can be lyophilized and stored. The method of formulating the toxin neutralizer is arbitrary, and the lyophilized powder may be taken as it is. The standard dose varies depending on the type of toxin, but for example, in the case of cholera toxin, it is approximately 300 ml per day for an adult.
~5000IT1g.

Since the toxin neutralizer according to the present invention is made from animal milk by only separation treatment and heat treatment without chemical treatment, there is no problem in terms of safety.

As mentioned above, the toxin neutralizer of the present invention advantageously utilizes inexpensive buttermilk, which is produced in large quantities at butter manufacturing factories, as a concentrated raw material, and then subjects it to simple purification and heat treatment. During production, there is no need to perform complicated treatments such as isolating and purifying ganglioside, which is a trace component, and then adsorbing it onto a carrier. Therefore, the toxin neutralizer of the present invention is superior to the conventional ganglioside preparations in that it is easy to mass produce and is extremely inexpensive. Therefore, according to the present invention, highly safe drugs or their raw materials effective for preventing or treating diarrhea and other infectious diseases caused by various bacterial toxins that use gangliosides as receptors, such as cholera toxin and Escherichia coli toxin, can be obtained at low cost. It becomes possible to provide large quantities.

The present invention will be explained below with reference to Examples, and the "toxin neutralizing ability" shown in the Examples was measured by the following method.

First, commercially available purified cholera toxin (concentration 400 ng/ml) or Escherichia coli toxin (concentration as protein 1.08 mg/ml)
l) O, 1m! Add 0.2 ml of the sample (dissolved in phosphate buffer, pH 7.2) to the solution and leave it at 37°C for 2 hours. Then, 1.7 ml of HamF12 culture solution supplemented with 5% calf serum was added, and 0.5 ml of the resulting mixture was added to CH
1 cell (previously 2 to 2 cells in a chamber slide)
(which had been attached to the slide surface by incubating at 37°C under 5% carbon dioxide gas for 3 hours). After culturing the cells for an additional 20 hours under the same conditions as above, the cells are stained with Giemsa solution 1 = and the degree of change in cell morphology caused by the toxin is examined using a microscope.

Using a phosphate buffer instead of the sample, calculate the morphological change inhibition rate based on the cell morphological change rate in this case (the morphological change rate is 400 cells). Count what is happening and display it as a percentage. ).

Example 1 1C of milk with a fat content of 3.3% was centrifuged at 3000 rpm for 15 minutes to obtain cream, which was then washed with water to make a total volume of 44.0 ml by centrifugation. After repeating the same washing process three more times (9 times), the washed cream was stored overnight at 4°C and then processed in a churn to separate the buttermilk and butter granules. was added to achieve 50% saturation, stored overnight, and centrifuged at 300 rpm for 30 minutes. After this, the fat globule membrane was suspended.
After dialyzing 1.9% of the supernatant against 1 liter of distilled water at 4°C, 1.0
Centrifugation was performed at +000 rpm for 30 minutes. The precipitated fat globule membrane was removed and freeze-dried to obtain 650 mg of dried fat globule membrane.

Next, this was suspended in water, subjected to ultrasonication to crush the film, and further heated at 100'C for 30 minutes to deactivate the enzyme, thereby obtaining a toxin neutralizer.

When the toxin neutralizing ability of this neutralizing agent was measured, 500/J g of the toxin neutralizing agent No. 1 inhibited 100% of the cell morphological changes caused by CHO- produced by 10 ng of cholera toxin. Also, CHO-K produced by 2771 g of E. coli toxin (amount as protein).

500 pg of the above-mentioned toxin neutralizer caused almost 1 change in cell morphology.
00% suppressed.

Example 2 Dissolve 10g of powdered buttermilk in water and make a total volume of 100ml
After thorough dialysis against distilled water at 4 to 5°C, the dialyzed fluid was freeze-dried. 1 g of the obtained solid was added to 0.1 M) lithium-hydrochloride buffer (p
H8,3) Dissolve in 20 ml of Blo-Ge1 A-
It was supplied to a 5 m column (3.2 cMφ x 52 cm).

Next, an elution process was performed with the above buffer solution, and a void volume rich in fat globule membranes was collected, and both volumes were dialyzed and freeze-dried. The yield of the fat globule film was 350+nH per 10 g of the raw material powdered buttermilk. Next, this was suspended in water, subjected to ultrasonic treatment, and then heated at 65°C for 30 minutes to obtain a toxin-neutralizing substance.

1) When the toxin neutralizing agent consisting of a fat globule membrane was examined for its ability to neutralize cholera toxin, CHO-K was produced by 10 ng of cholera toxin.

7- The morphological changes of cells are 650) Jg of the above toxin neutralizer is 1
00% suppressed.

Example 3 Goat hole 1C with a fat content of 4.25% was centrifuged at 3000 rp+n, and water was added to the resulting cream to bring the total volume to 57%.
The fat globules in the cream were washed by adjusting the volume to 0 ml and centrifuging again. This washing cycle was repeated three more times, stored overnight at 4°C, and then processed in a churn to separate buttermilk and butter granules. The buttermilk was then heated at 100°C for 10 minutes and then dialyzed against distilled water at 4°C. By freeze-drying the obtained dialyzed content, 380 cm of dried fat globule membrane was obtained.

Next, this was suspended in water, subjected to ultrasonication to crush the film, and further heated at 100'C for 30 minutes to deactivate the enzyme, thereby obtaining a toxin neutralizer.

When the toxin neutralizing ability of this neutralizing agent was measured, it was found that 35 tJg of the above toxin neutralizing agent had almost 100% change in cell morphology due to Cl-10- generated by 10 nH of cholera toxin.
suppressed. In addition, E. coli toxin 277zg (protein equivalent)
CHO- caused by 138p causes cell morphological changes.
, g of the above-mentioned toxin neutralizer inhibited almost 100%.

8-

Claims (1)

[Claims] A neutralizing agent for bacterial toxins that uses ganglioside receptors and is made from heat-treated animal milk fat globule membranes.