JPS61275217A - Prophylactic agent for infection with gram-negative bacillus - Google Patents

Abstract

PURPOSE:To provide a prophylactic agent for infectious disease caused by Gram-negative bacillus, containing lipoteichoic acid and/or teichoic acid as active component, having low toxicity and effective for the prevention and remedy of infectious diseases of Escherichia coil, Pseudomonas aeruginosa, etc. CONSTITUTION:The objective agent contains a lipoteichoic acid of e.g. formula I (R1 is H, Gal or Glc; R2 is alkyl) and/or teichoic acid of e.g. formula II as active components. Especially excellent effect can be attained by the use of a lipoteichoic acid and teichoic acid obtained from Lactobacillus casei YIT-9018 (FERM-BP 665) or Lactobacillus fermentam YIT-0159 (FERM-P 8244). The agent has low toxicity compared with conventional antibiotic substance and can be administered continuously with little side effects. The activity of the agent is caused by the activation of polymorphopuclear leukocyte by the active component and the prevention of the proliferation of Gram-negative bacillus by the activated leukocyte.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a drug effective for the prevention and treatment of Gram-negative bacillus infections such as Escherichia coli infections and Pseudomonas aeruginosa infections.

Gram-negative bacilli such as E. coli and Pseudomonas aeruginosa easily infect hosts with weakened immune systems, and the infection causes serious damage.

Traditionally, as a means to protect against infection with Gram-negative bacilli,
There is a method using antibiotics, but antibiotics have the problem of being highly toxic and having side effects. On the other hand, there have been attempts to protect against the above-mentioned infectious diseases using factors that enhance the host's defense function, but nothing effective has been found.

Problems to be Solved by the Invention An object of the present invention is to provide an agent for protecting against Gram-negative bacillus infections, which does not have the drawbacks of the above-mentioned antibiotics.

The agent for protecting against gram-negative bacillus infections provided by the present invention contains lipoteichoic acid, teichoic acid, or both of these, and these active ingredients activate polymorphonuclear leukocytes and activate the activated leukocytes. It protects against Gram-negative bacillus infections by preventing the proliferation of Gram-negative bacilli.

Lipoteichoic acid teichoic acid is a substance that constitutes the cell membrane and cell wall of Gram-positive bacteria, respectively, and is a high molecular weight compound having a polyglycerol phosphate skeleton as described below.

Teikoic acid However, in both formulas, R, = H, Ic or Ga1R,
=Alkyl group It is known that teichoic acid includes polyribitol phosphate type in addition to the polyglycerol phosphate type mentioned above, but in the present invention, teichoic acid refers to polyglycerol phosphate type. mean something

Lipoteichoic acid and teichoic acid are produced by Staphylococcus,
Although it can be collected from various Gram-positive bacterial cells such as Streptococcus and Lactobacillus, the one obtained from Lactobacillus is particularly preferred for the agent for protecting against Durham-negative bacillus infection of the present invention. That is,
Within the range of the above general formula, lipoteichoic acid and teichoic acid have different molecular weights and sugar compositions depending on their origin, and there are also slight differences in their protective effects against Durham-negative bacillus infections. is preferred because it exhibits particularly excellent effects. Lactobacillus casei YIT-90 is recommended as showing particularly excellent performance.
18 (Feikoken Jokyo No. 665; hereinafter referred to as LC9018) or Lactobacillus fermentum YIT-01
59 (Feikoken Bibori No. 8244; hereinafter referred to as LFO159), botaific acid, teichoic acid, and teichoic acid.

The lipoteichoic acid-derived teichoic acid used in the agent for protecting against Durham-negative bacillus infections of the present invention can be obtained from lactic acid bacilli using any method, but a typical method is the method of Knox et al.
acteriologicalReviews Vol.
, 37.221.1972). This method involves treating freeze-dried bacterial cells with a water-phenol mixture, centrifuging the treated solution, obtaining an extract containing botaific acid and teichoic acid from the resulting aqueous layer, and purifying this. It is.

The lipoteichoic acid and teichoic acid for the Durham-negative bacillus infection protective agent of the present invention are the crude or purified mixture of lipoteichoic acid and teichoic acid obtained as described above. Alternatively, it may be fractionated into each component by means such as gel filtration.

Lipoteichoic acid and teichoic acid are chemically relatively stable substances with a polyglycerol phosphate skeleton.
There is no particular difficulty in formulating it, and the aseptically purified powder may be made into an injection solution or a liquid preparation such as an injection solution by a conventional method.

The appropriate dosage is about 50 to 500 mg per day for adults.

x1 male Lactobacillus LC901B was cultured in Rogosa medium,
The bacterial cells isolated from the culture were heat-treated at 100°C for 30 minutes and then freeze-dried. 5 g of the obtained dry bacterial cell powder is 1
The suspension was suspended in 25 ml of purified water, the same amount of 95% phenol was added thereto, and the mixture was stirred at 4°C for 2 hours.Then, the mixture was separated into an aqueous layer, a phenol layer, and a bacterial cell residue by centrifugation, and the aqueous layer was separated. 125 m1 for the 7-layer layer and bacterial cell residue.
of purified water was added and stirring and centrifugation were performed again. The resulting aqueous layer and the previously obtained aqueous layer were combined, dialyzed against deionized water, the nucleic acids were decomposed with RNase T4, cold phenol extraction and dialysis were repeated, and then lyophilized.

75 mg of the obtained lyophilized product was dissolved in 3 rings of 0.2 M ammonium acetate solution, and 5 ephacryl S-30
Gel filtration was performed at 0, and fraction A (where sugar and phosphoric acid react
Fraction B (molecular weight approximately 300,000) and Fraction B (molecular weight approximately 20,000) were collected. Both fractions were each dialyzed with purified water, freeze-dried, and added to 0.02M Tris-HCl buffer ( DEA E Sep dissolved in 5 ml of pH 7,6) and equilibrated in advance with the same buffer 7-
Place it on haroseCL-4B and add the same buffer to 150
After flowing 1 m1 and collecting the flow-through fraction, elute the column adsorbed substance with a linear gradient of NaCl solution from O to IM, collect the eluted solution in S + al, and collect both fractions A where the reaction between sugar and phosphoric acid occurs. Both fractions A-1 and B-1 were collected and dialyzed with purified water, and then freeze-dried.
About 7+IIg of fraction 1 and about 511Ig of fraction B-1 were obtained.

The same sample preparation as above was carried out for Lactobacillus LFOI S 9
and obtained similar results.

Both A-1 and B-1 obtained from two Lactobacilli were quantified for phosphoric acid, glycerol, fatty acids, and neutral sugars, and their compositions were determined as shown in the following table (molar ratio of phosphorus to 1). ratio) Noribotaifu acid (LTA> and teichoic acid (T
It was confirmed that A).

Phosphorus 1.001.001.001.00 Glycerol
1.13 1.11 1.09 1.10D-glucose 0.25 0.09 0.08 0.04
D-galactose 0.13 0.04 0°O50
,02 fatty acid ester 0.05 - 0.05
- Molecular weight: Approx. 300,000 Approx. 20,000 Approx. 300,000 Approx. 20,000 Next, the protective effect of each lipoteichoic acid and teichoic acid against Gram-negative bacillus infections was tested using the following method. First, sample IH was intraperitoneally administered in the form of physiological saline solution to 7-9 week old C5713L/6jl mice, and after 5 days, 1.4xlO'
Durham-negative bacilli (Pseudomonas aeruginosa strain KC-2 or Escherichia coli strain E77156) are administered intraperitoneally. Thereafter, the survival of the mice is observed for two weeks, and the infection resistance enhancing effect is determined by comparing the final survival rate of the bacteria-administered group with that of the control group (physiological saline administered).

The results are shown in the table below, and all mice treated with lipoteichoic acid or teichoic acid showed strong resistance to infection with Durham-negative bacilli.

LC901B lipoteichoic acid 4/6
3/6 Taif acid S/6 5/6LF
O159 lipoteichoic acid 3/6 3/6 teichoic acid 5/6 4/6 control group) 0/7 0/7 Next, for the above teichoic acid and lipoteichoic acid, Litch-fi
We conducted an acute toxicity test using the eld-Wilcoxon method (20 mice weighing 20-25 gf>I CR strain per group), and found that both teichoic acid and lipoteichoic acid
L.D. is 400mg/Kg or more for intravenous administration, 800B/Kg or more for intraperitoneal administration, and 1000a+g for subcutaneous administration.
/Kg or more, and it was confirmed that the toxicity was extremely low.

Effects of the Invention As shown by the test results in the above examples, the agent for protecting against Durham-negative bacillus infections according to the present invention has lower toxicity than conventionally commonly used antibiotics, and has fewer side effects even when administered continuously. It is extremely effective for the prevention and treatment of gram-negative bacillus infections.

Claims (3)

[Claims] (1) A gram-negative bacillus infection protective agent characterized by containing lipoteichoic acid, teichoic acid, or both of these as active ingredients. (2) The agent for protecting against infectious diseases according to claim 1, wherein lipoteichoic acid and teichoic acid are obtained from the cells of Lactobacillus. (3) Lactobacillus casei YIT-90
18 or Lactobacillus fermentum YIT-0
159. The infectious disease protective agent according to claim 2, which is No. 159.