JPH01153627A - Preventing and treating agent for enterotoxigenic escherichia coli disease - Google Patents

Abstract

PURPOSE:To obtain the title preventing and treating agent containing an aliphatic carboxylic acid and/or alkali (earth) metal salt thereof as an active ingredient and having high safety as well as exhibiting excellent effect. CONSTITUTION:A saturated straight-chain or branched carboxylic acid expressed by the formula R-COOH (R is 1-7C alkyl) and/or alkali metal thereof or alka line earth metal salt thereof is contained as the active ingredient. Since the above-mentioned active ingredient is relatively rapidly absorbed after ad ministering, it is hardly expected to stay in enteric canal cavity being acting point for a long time and express sufficient drug action. Therefore, the agent is preferably administered as a sustained release pharmaceutical used in normal medicine formulation or as prodrug (e.g. ester derivative or amide derivative) biologically synthesizing active ingredient by in vivo metabolism.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a prophylactic and therapeutic agent for diarrhea caused by toxigenic Escherichia coli.

(Prior art and problems to be solved) The first person to discover toxigenic Escherichia coli was De et al.
Op Pathology and Bacteriology (Jou
rnal of Pathology and Bac
teriology), 71.201-209 (19
It was May 1956, 1956. When Escherichia coli was isolated from a patient exhibiting cholera-like symptoms and administered to a rabbit's ligated intestinal tract, remarkable fluid retention was observed, similar to when Vibrio cholerae was administered. Later, Sack et al. [Journal of Infectious Di
sease).

123, 378-385 (1971)].
It was named hia C01i (toxigenic Escherichia coli) and became known as the causative agent of traveler's diarrhea, which plagues travelers to tropical and subtropical regions of the country. Furthermore, it is now assumed that infant diarrhea and the so-called ``food-related'' diarrhea that occurs on a daily basis are related to Japan.

On the other hand, in animals, toxigenic E. coli is found in pigs, cows,
It is known to cause diarrhea in livestock such as sheep. For example, 1- to 3-week-old suckling pigs regularly suffer from diarrhea caused by their home country, and pigs with diarrhea caused by 10-day feeding, which also occurs sporadically in piglets immediately after weaning, excrete watery feces and develop hair loss. growth becomes impaired, and nutritional status deteriorates. At some point, he weakened and died.

Sepsis may develop due to secondary infection. Even if they escape death, their subsequent growth is delayed and they often become so-called pigs, losing their domestic value.

Antibiotics are generally used to treat diarrhea caused by toxigenic E. coli, but many strains of this type of E. coli have acquired resistance to antibiotics, and some are resistant to 4 or 5 antibiotics. Cases of multidrug-resistant bacteria have also been reported. Furthermore, there are problems with the use of antibiotics, such as the fact that antibiotics are generally not used prophylactically under conditions where the onset of symptoms cannot be predicted.

The main reason behind the induction of diarrhea by toxigenic E. coli is that even if only the toxin is administered to the rabbit's ligated intestinal tract, water retention occurs, so the E. coli itself does not induce diarrhea, but rather the diarrhea-causing bacteria produced in the home country. It is considered an enterotoxin. Therefore, even if E. coli is not killed, if only the production of toxins is suppressed, diarrhea will not occur.

From this point of view, the present inventor has conducted intensive research on compounds that affect toxin production by toxigenic E. coli, and has found that aliphatic carboxylic acids can be used to produce heat-labile enterotoxins in amounts that have little effect on the growth of E. coli. The present invention was completed based on the discovery that the production of

(Means for solving the problem) Therefore, the above object is achieved by a preventive and therapeutic agent for toxigenic coliosis containing an aliphatic carboxylic acid and/or an alkali or alkaline earth metal salt of an aliphatic carboxylic acid as an active ingredient. be done.

The aliphatic carboxylic acid used in the present invention is a saturated straight-chain carboxylic acid or a branched carboxylic acid represented by the general formula R-COOH (R represents an alkyl group having 1 to 7 carbon atoms), for example, Saturated linear carboxylic acids include acetic acid and propionic acid; n-carboxylic acids include isobutyric acid,
Examples include isovaleric acid, ethylmethylacetic acid, trimethylacetic acid and isocaproic acid.

The alkali or alkaline earth metal salt of an aliphatic carboxylic acid used in the present invention is a salt of the carboxylic acid with an alkali metal such as sodium or potassium or an alkaline earth metal such as calcium. Further, these aliphatic carboxylic acids and their salts may be used alone or as a mixture of two or more.

The use of these drugs includes certain aliphatic carboxylic acids,
or a salt thereof can be administered orally as a single agent or a mixture of multiple agents. In addition, since aliphatic carboxylic acids are generally absorbed relatively quickly after administration, it is difficult to expect them to remain in the intestinal lumen for a long time and exhibit sufficient medicinal efficacy. It is also necessary to consider flavoring and odor correction. Therefore, this drug can be administered, for example, as a sustained-release preparation used in general pharmaceutical preparations, or as a prodrug (in which the active ingredient is biosynthesized through metabolism in the body).
When used as esters of aliphatic carboxylic acids and alcohols, glycerin, sugars, etc., or amides of amino acids, ion exchange resins having amino groups, etc.), their bioavailability (Bioavailability 1abi)
I1ty) can be increased.

Their dosage forms are generally powders, granules, fine granules, tablets,
Sugar-coated tablets, capsules, water-soluble or enteric film coatings, dry syrups, etc. may also be used as liquid solutions such as suspensions and syrups. Diluents include excipients, binders, disintegrants, etc. used in general pharmaceutical formulations, and in addition to these, colorants, flavoring agents, stabilizers, preservatives, lubricants, emulsifiers, pH You may also add a drug-adjusted diarrhoea or the like.

In addition, for animals, it may be used without dilution, or may be used as a powder, dust, granule, tablet, capsule, liquid, etc. with a diluent. As diluents, various excipients or additives used by humans can be used, such as corn flour, soybean meal, barley flour, bare wheat flour, soybean flour, rice bran, defatted rice bran, rice husk, Livestock feed such as potato powder, kansho powder, tofu dregs, starch, yeast, and fish meal are also used.Liquids include water, physiological saline, breast milk,
It is diluted with artificial milk or the like, and an emulsifying agent, suspending agent, gelling agent, etc. may be added thereto as an auxiliary agent.

Further, any of these preparations may also contain an antidiarrheal agent, an astringent, a mucosal protectant, a bactericide, an antibiotic, an enzyme agent, or a viable bacterial preparation.

The aliphatic carboxylic acids that are the preventive and therapeutic agents for toxigenic coliosis of the present invention are natural products and some are ingested by humans as food, and as shown in Example 5, their toxicity is low. Since it is considered to be extremely low, the dosage can be appropriately determined depending on the subject, body weight, age (age in days), method of administration, purpose of administration, etc. Usually, the dosage ranges from 1 to 100 mg/J of body weight.

toxicity test No acute toxicity was observed in mice.

(Example) The following examples demonstrate that the present drug has an excellent effect as a prophylactic and therapeutic agent for toxigenic coliosis, but the scope of the claims of the present invention is not limited to these examples.

Example 1 Toxigenic Escherichia coli is a heat-labile enterotoxin-producing bacterium (Escherichia coli).
i) ■ Group 3043 (Hereinafter, [Colly [Shun] was used.

Aliphatic carboxylic acids include acetic acid, propionic acid, n-butyric acid,
Isobutyric acid, n-valeric acid, isovaleric acid, n-caproic acid,
Isocaproic acid, n-heptylic acid, n-pyrylic acid with N
It was neutralized with an aOH solution and used in the following experiment.

Add various aliphatic carboxylic acids to CAYE medium at a final concentration of 2 g/
D, F, Collie [■] at 37℃, 2
After shaking culture for 4 hours, the number of viable bacteria and the amount of heat-labile enterotoxin produced were measured. Note that a group without addition of aliphatic carboxylic acids was used as a control.

The amount of heat-labile enterotoxin produced was measured using the reverse passive latex agglutination method, and the sample was serially diluted 2 times.
The highest dilution factor indicating positivity was displayed. In addition, the pH of the medium at the start and end of the culture was also measured. The number of viable bacteria was measured using a normal agar medium supplemented with glucose according to a conventional method.

The results are shown in Table 1.

Table 1 10 - As shown in Table 1, even when various aliphatic carboxylic acids are added to CAYE medium, the pH of the medium before and after cultivation is 8.
The conditions were perfect for toxin production. Under this condition, the growth of F. coli [T.

Example 2 Sodium acetate and sodium heptylate were added as aliphatic carboxylates to CAYE medium at the required concentrations, and one cory was cultured with shaking at 37°C for 24 hours, and the production amount of heat-labile enterotoxin was measured. did. The amount of heat-labile enterotoxin produced was measured according to Example 1.

The results are shown in Table 2.

Table 2 As shown in Table 2, the amount of sodium butyrate added was 0.5
．． When increasing to 1.0, 2.09/fl, E, Collie L
Production of heat-labile enterotoxin from clover
/2.1/32.1/64 and was suppressed in a dose-dependent manner.

In addition, in the case of sodium heptylate, 0.1.0.2.
0.5.1.0.2゜0! ? /! As Q increases, heat-labile enterotoxin production is 1/4.1/16.1/1
It was suppressed in a dose-dependent manner to 6.1/16.1/128 or less.

Example 3 Sodium butyrate or sodium heptylate was added to CAYE medium as an aliphatic carboxylate at a final concentration of 2 g/!
F. and coli [■] were cultured with shaking at 37° C., and the number of viable bacteria, heat-labile enterotoxin production, and pH were measured over time. Note that a system without addition of sodium acetate or sodium heptylate was used as a control. The number of viable bacteria and the amount of heat-labile enterotoxin produced were measured in accordance with Example 1.

The results using sodium acetate are shown in Table 3, and the results using sodium heptylate are shown in Table 4.

Table 3 Table 4 The experimental results are as shown in Tables 3 and 4.
All groups added with sodium heptylate grew well.
The pH change of the culture solution also showed a similar pattern. However, although the amount of toxin produced increased over time in the control group, no increase over time was observed in the sodium butyrate supplemented group, and even after 24 hours, the amount produced was 1/32 of the control group, and the heptyl In the sodium acid addition group, no toxin production was observed during the 24-hour culture period.

Example 4 After culturing E. coli [■] for 6 hours with shaking in CAYE medium, the supernatant was removed by centrifugation and centrifugation, the bacterial cells were washed once with physiological saline, and cultured with fresh CAM medium for 2 hours. A suspension of /108 cells/d was prepared and used in the rabbit intestinal loop experiment. Sodium heptylate and hydroxypropyl cellulose were dissolved in water at a ratio of 1:1 (W:W), lyophilized, and tableted at a compression pressure of 300 KI/cut. I made a pill. Hereinafter, this tablet was used as a test drug in a rabbit intestinal loop experiment. As a control, tablets containing only hydroxypropyl cellulose were also prepared and used in the experiment.

A Japanese white male rabbit (body weight 1.5-2.0 Kfl) was fasted for 2 days (water intake was not restricted) and then general anesthesia (<20 m17 K'J) was performed with bendoparbital.

The following is a routine method for testing Vibrio cholerae and Escherichia coli, written by Miwatani et al.
The abdomen was opened according to Nane Publishing), and approximately 'l0 per head was inserted into the small intestine.
Several cm loops were made with approximately 1 cm between each loop. During this loop, the E and Colli LT suspensions prepared as described above were administered for 2 days each, and the two test tablets were alternately administered. These administrations were performed by making an approximately 1 cm incision between each loop. After abdominal surgery, the rabbit is 2
The animals were left indoors at 4°C, and after 20 hours, they were sacrificed under anesthesia, the abdomen was opened, and the loops were removed. The weight (g>) and length (cm2) of the contents of each loop were then measured, and the liquid storage activity (-/[ratio) was calculated according to the following formula.

Liquid storage activity (W/L) ratio =Weight of the object inside the loop (g> Loop length cm> The results are shown in Table 5.

Table 5 As shown in Table 5, in the F. coli LT infection loop, the W/L ratio in the control group to which only hydroxypropyl cellulose was applied was 1.302 ± 0.228. , test group treated with sodium heptylate-containing hydroxypropylcellulose glue/[ratio is 0.851
±0.242, and the inhibition rate was 34.6%. That is, it was shown that the intestinal water retention induced by E. coli LT infection was successfully suppressed by sodium heptylate.

Example 5 Sodium butyrate or sodium heptylate was dissolved in physiological saline to the required concentration, and the following acute toxicity test was conducted. Group of 5 ICR mice (male and female, weight 30-50
g) The above test solution was administered orally at 101 Wj/Kl, 10
0m! J/ni 9.1, OOOm! j/ni! j, 5°000ml/Ky were administered respectively. As a control, physiological saline was orally administered. The general condition was observed for 7 days after administration of the test solution, and autopsy was performed 8 days later.

As a result, no noteworthy changes were observed in either the control group or the test solution administration group, nor in the membrane condition or autopsy findings. However, only in the sodium heptylate 5000 mg/'j administration group,
After administration, a decrease in locomotor activity and wheezing symptoms were observed in some patients, but they returned to normal after 4 days. From the above, it was concluded that the toxicity of the main principle is extremely low (effects of the invention). It is a prophylactic and therapeutic agent for coliform coliosis, and is highly safe, as well as extremely effective as a prophylactic and therapeutic agent for toxigenic coliosis.

Claims (1)

[Claims] A prophylactic and therapeutic agent for toxigenic coliosis containing an aliphatic carboxylic acid and/or an alkali or alkaline earth metal salt of an aliphatic carboxylic acid as an active ingredient.