Use of compounds containing or binding sialic acid to neutralize bacterial toxins

Abstract: Sialic acid binding proteins, sialic acid-­ binding peptides and sialic acid containing oligo­ saccharides that are effective components of a baterial enterotoxin neutralizer according to the present invention all interfere with the binding of enterotoxins, including the cholera toxin, to receptors so as to exert an effect of neutralziing the toxicity, thus being utilized as an effective neutralizer. Furthermore, all the components according to the present invention are confirmed to be safe and, moreover, available cheaply and abundantly from waste matter produced during processes of cow's milk or the like, which makes the present invention economically advantageous for practical use.

Description: The present invention relates to a neutralizer which is effective for neutralization of bacterial enterotoxins produced by bacteria such as Vibrio cholerae, pathogenic Escherichia coli and Salmonella . Food poisoning due to Vibrio cholerae causes severe diarrhea accompanied by abdominal pain. Consequently, patients infected with Vibrio cholerae are suffering from extreme dehydration due to the diarrhea and, in the worst case, die due to the dehydration. It is well known that the severe diarrhea observed in the base of food poisoning due to vibrio cholerae is caused by an enterotoxin called cholera toxin which Vibrio cholerae produces extracellularly. The cholera toxin is a protein with a molecular weight of 84,000 consisting of one A-subunit and five B-subunits. It is said that, among them, B-subunits bind to receptors located on a mucous cell of the small intestine, while the A component invades into the cell and increases the amount of cyclic-AMP within the cell, causing change in membrane permeability, which induces exudation of intracellular fluid and salts outside the cell and thus causes diarrhea. This kind of toxins are isolated also from pathogenic E . coli and Salmonella , and are believed to be largely responsible for causing food poisoning. The receptors which are present on the mucous cells of the small intestine has been confirmed to be liposaccharides; gangliosides such as G M1 are identified as receptors for the cholera toxin. The utilization of this G M1 as a cholera toxin neutralizer has already been attempted. For example, Japanese Patent Publication No. 500138/1981 disclosed a technique in which gangliosides such as G M1 possessing a potent ability of neutralizing the cholera toxin were immobilized on latex or the like and used as neutralizing agents. Furthermore, Japanese Patent Laid-Open No. 72819/1985 disclosed a technique in which fat globule membranes in cow's milk were treated by heat and used as a toxin neutralizer without extracting gangliosides, G D3 , G M2 and G M3 , containing in the fat globular membranes. However, at the present time G M1 is known to be available only in a minute amount, for example, in the bovine brain. Therefore, it was difficult to supply such G M1 at a low cost in a large amount. Furthermore, the methods of using milk fat globular membranes in cow's milk or the like have disadvantages such that concentrations of the resulting gangliosides, which are effective components in the milk globular membranes are not consistent, and their compositions are not stable. The present invention is a result of the intensive investigations on enterotoxins such as the cholera toxin, and thus an object of the present invention is to provide a bacterial enterotoxin neutralizer which has a potential ability to neutralize bacterial toxins produced by Vibrio cholerae , pathogenic E . coli and Salmonella and which is safe and can be supplied at a low cost and in abundance. Further, the enterotoxins as used herein include cholera toxin and enterotoxins derived from pathogenic E . coli . According to the present invention, sialyllactose which is the effective component of the neutralizer of the present invention, interferes with the binding of bacterial enterotoxins including the cholera toxin to receptors and thus exerts a toxin neutralizing effect; hence the present invention can be effectively applicable to provide a neutralizer for the above-mentioned bacterial enterotoxins. Furthermore, the above-mentioned components of the neutralizer according to the present invention are confirmed to be safe and obtainable at a low cost and in abundance from waste products produced in processes of cow's milk or the like, hence being practical from a viewpoint of cost price. According to the present invention, a non-toxic neutralizer for bacteria enterotoxins such as the cholera toxin can be supplied consistently and at a low cost. Figs. 1 to 3 demonstrate the interfering effect of individual effective components of the bacterial toxin neutralizer according to the present invention on morphological alteration of CHO-K1 cell caused by different toxins. Figs. 4 to 8 demonstrate the interfering effect of the neutralizer according to the present invention on the binding of the cholera toxin to ganglioside G M1 . The present invention is characterized in that sialyllactose is used as a bacterial enterotoxin neutralizer. Sialyllactose is obtained in the manner disclosed in Japanese Patent Laid-Open 181497/1984, sialic acid derivatives disclosed in Japanese Patent Laid-Open 12695/1986 or further saccharide chains which are specific to gangliosides, such as G D3 , G M1 and G M3 , can also be used for this purpose. Sialyllactose is contained in abundance in cow's milk. An enterotoxin neutralizer can be optionally prepared by using known excipients or diluting agents. Amounts of the standard doses of the above-mentioned sialyllactose vary depending on the kind of toxins involved. It may, however, be mentioned that an appropriate dose of the neutralizer is in a range between 6 mg and 300 mg per day for an adult. Furthermore, if desired, it can be mixed in food or administered to animals such as live stock by mixing in feeds for the prevention of diseases. The sialyllactose according to the present invention is derived from cow's milk and and is hence absolutely safe. Sialyllactose can be recovered from cheese whey according to the method disclosed in Japanese Patent Laid-Open No. 181497/1984. The following examples will demonstrate the enterotoxin neutralizing activity of the enterotoxin neutralizer of the present invention. Experimental Example 1 Test for inhibitory effect on morphological alteration of Chinese hamster ovary cells due to the cholera toxin and pathogenic E . coli enterotoxins (1) Determination of concentrations of the cholera toxin and enterotoxin used for measurement of activity for morphologically altering Chinese hamster ovary cells Two kinds of toxin producing E . coli strains (from Tokyo Municipal Institute; serotype H-10407-P strain and Pb-176 strain) which produce two kinds of antigenically different enterotoxins, enterotoxin CFA/I(LT-1) and enterotoxin CFA/II(LT-II), were cultivated with shaking in an ordinary CAYE medium. The culture was centrifuged, and the resultant supernatant was diluted stepwise from 1/10 to 1/1000 and then each diluted fluid was dispensed in a Lab-Tek 8 chamber plate (Flora Laboratories) placed on ice. The cholera toxin CT (List Biological Laboratories) was diluted in the same manner and then placed in a Lab-Tek 8-chamber plate (hereinafter referred to simply as 8-chamber plate). Subsequently, 400 microliters each of Dulbecco's medium containing 1% fetal calf serum (FCS/DMEM) was dispensed in chambers of the 8-chamber plate, then 50 microliters of a cell suspension in which Chinese hamster ovary K1 cells (CHO-K1 cell (ATCC CCL6)) cultured in 10% FCS/DMEM were suspended in 10% FCS/DMEM at a concentration of 5000 cells/ml was dispensed in the chambers, and the chamber plate was incubated at 37°C overnight in an incubator with CO₂ at a concentration of 5%. Thereafter, the medium was discarded and the inside of the chambers were washed with phosphate-buffered saline (PBS(-)). Then the cells inside the chambers were soaked in methanol for 10 minutes, fixed, stained with a Giemsa stain solution and then dried in air. Cells amounting 200 to 400 per sample were observed under microscope and the ratios of morphologically altered cell numbers were counted so as to calculate the morphological alteration rates of CHO-K1 cells at individual concentrations of each toxin. It was revealed that at least 70% of CHO-K1 cells were morphologically altered by CT at a concentration of 10 micrograms/ml and by undiluted supernatants of LT-I and LT-II. In Experimental Examples hereinafter, individual toxins were used at these concentrations. (2) Activity of interfering with CHO-K1 cell morphological alteration Activity of interfering with morphological alteration of cell caused by enterotoxins was confirmed using sialyllactose (SL). Furthermore, the activity was confirmed also with ganglioside G D3 . In each chamber of the 8-chamber plate placed on ice, 10 microliters of 10 micrograms/ml of CT or the culture supernatants of LT-I or LT was dispensed and then 50 microliters of individual samples at a desired concentration in a PBS(-) solution was added, and then the plate was shaken for reaction. Subsequently, 400 microliters of 1% FCS/DMEM and 50 microliters of CHO-K1 cell suspension were added to each chamber, and the plate was incubated overnight with shaking in a 5% CO₂ incubator. Thereafter, the methanol fixation and Giemsa staining were carried out, and then the rates of morphologically altered cells were determined. The rates of morphological alteration by individual toxins, CT, LT-I and LT-II, with the addition of the samples at the amount of 1 micrograms/chamber or 5 micrograms/chamber are shown in Figs. 1, 2 and 3. It was confirmed that SL neutralized the toxicity of CT, LT-I and LT-II against CHO-K1 cells. (3) Inhibitory effect on the binding of CT to ganglioside G M1 As described above, CT exerts the toxicity when the B-subunits bind to ganglioside G M1 receptor on the surface of a cell and then A subunit invades into the cell. The activity of interfering with the binding of G M1 to the cholera toxin B subunits was examined. In each well of a 96-flat-bottom well plate, 50 microliters of a G M1 ethanol solution (1 microgram/ml) was dispensed and then ethanol was dried by evaporation so that G M1 was adsorbed on the plate. Each well thus treated was filled with a 1% bovine serum albumin PBS solution (BSA/PBS), allowed to stand for 1 hour and then washed three times with PBS containing 0.05% Tween® 20 (PBS Tween® 20). On the other hand, each sample was made up to a 1% BSA/PBS solution, 25 microliters of each sample was previously mixed with 25 microliters of a 1% solution of peroxidase-labeled cholera toxin B-subunits (PO-CT-B) in BSA/PBS (1 microgram/ml), and the mixture was incubated at room temperature for 30 minutes. These mixtures were added to the wells of the above-mentioned G M1 -adsorbing plate. After standing for 30 minutes, each well was washed six times with PBS Tween® 20 and added with 100 microliters of 0.2 M citric acid buffer (pH 4.0) containing 2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid-2-ammonium (ABTS) at a concentration of 0.3 mg/ml dissolved in 0.006% H₂O₂. After 10 minutes, the rates of binding of G M1 to the cholera toxin B-subunits were estimated by measuring the absorbance at 405 nm. As demonstrated in Figs. 4 to 8, all the samples interfered with the binding of the cholera toxin B-subunits to G M1 depending on the concentration. From the results described above, it was confirmed that SL neutralized the toxicity of bacterial enterotoxins and interfered with the binding of the enterotoxins to the cell receptors. The following Examples are to explain the present invention in more detail. Example 1 Effect of neutralizing the toxicity of cholera toxin Eighty Balb/c young mice, weighing about 20 grams, were divided into 16 groups, 5 mice in each group. One group was used as a control and the remaining 15 groups were further divided into 5 groups; the animals in each group were administered with samples,i.e. of SL and G D3 . The administration was carried out orally for 7 days at doses of 0.2, 0.5 and 1.0 mg/day/mouse. On day 8, a mixture of 0.25 mg of CT with water or each sample was orally administered, and thereafter, incidence of diarrhea in the mice was observed. As shown in Table 1, the group administered with the samples all demonstrated drastic decreases in the rate of the incidence of diarrhea as compared with the control group. In vivo activity of neutralizing the toxicity of cholera toxin Sample Dose (mg/day) Rate of diarrhea (%) Control - 100 SL 0.2 20 0.5 20 1.0 0 G D3 0.2 20 0.5 20 1.0 0 Example 2 Effect of neutralizing the toxicity of enterotoxin LT-1 derived from phathogenic E . coli Effects of neutralizing the toxicity of LT-I of individual samples were confirmed by the same test method as described in Example 1. LT-I used for administration was 100 microliters of the culture supernatant as described in Experimental Example 1. As shown in Table 2, the group administered with individual samples all demonstrated drastic decreases in the rates of the incidence of diarrhea as compared with the control group. In vivo activity of neutralizing the toxicity of enterotoxin LT-I Sample Dose (mg/day) Rate of diarrhea (%) Control - 100 SL 0.2 40 0.5 20 1.0 20 G D3 0.2 60 0.5 20 1.0 20 Example 3 Effects of neutralizing the toxicity of enterotoxin LT-II derived from phathogenic E . coli Effects of neutralizing toxicity of LT-II of individual samples were confirmed by the same test method as described in Example 1. 100 microliters of the culture supernatant of LT-II was used for administration as described in Experimental Example 1. As shown in Table 3, the group administered with individual samples all demonstrated drastic decreases in the rate of the incidence of diarrhea as compared with the control group. In vivo activity of neutralizing the toxicity of enterotoxin LT-II Sample Dose (mg/day) Rate of diarrhea (%) Control - 100 SL 0.2 40 0.5 20 1.0 0 G D3 0.2 40 0.5 40 1.0 20 Further, the following Examples illustrate the preparation of the bacterial enterotoxin neutralizer according to the present invention. Example 4 Ten grams of G D3 , 50 grams of lactoferrin, 30 grams of GMP and 10 grams of sialyllactose (SL) were mixed, degraded and then granualted. The resultant granular mixture, 100 mg each, was filled in gelatin capsules to prepare 1000 pharmaceutical capsules.

Claims: A bacterial enterotoxin neutralizer which contains as an effective component sialyllactose. A bacterial enterotoxin neutralizer as set forth in claim 1, which is a capsula preparation prepared by mixing ganglioside G D3 , lactoferrin, kappacasein glycomacropeptide and sialyllactose, at rates of 10%, 50%, 30% and 10% respectively, and granulating the resultant mixture. A bacterial enterotoxin neutraliser as set forth in claim 1, which is a tablet containing 6 to 300 mg of siayllactose per tablet.

Inventors: DOSAKO SHUNICHI, IDOTA TADASHI, ISODA HIROKO DE LE SION KYOWA, KAWASAKI YOSHIHIRO, TANIMOTO MORIMASA, DOSAKO, SHUNICHI, IDOTA, TADASHI, ISODA, HIROKO, 503, DE LE SION KYOWA, KAWASAKI, YOSHIHIRO, TANIMOTO, MORIMASA
Applicants: SNOW BRAND MILK PROD CO LTD, SNOW BRAND MILK PRODUCTS CO., LTD.
Application year: 1990
Main classification: A61K31
IPC codes: A61K38, A61P39, A61P31, A61K31, C07K14, C07H7