CS212234B2 - Method of making the immunotherapeutical means against the infection decease uf urine and digesting ways - Google Patents

Abstract

Immunobiotherapeutic medicament for infectious diseases of the urinary and digestive tracts, containing as active principle a bacterial lysate deriving from at least one of the following strains of Escherichia coli: -NCTC: 8603; 8621, 8622, 8623, 9026, 9111, 9119, 9707, 9708. -I-081, I-082, I-083, I-084, I-085, I-086, I-087, I-088, I-089. To obtain the medicament the strains are incubated in a culture medium so that they undergo lysis and/or autolysis and the lysates thereafter harvested.

Description

The invention relates to a method for the production of an immunobiotherapeutic agent against infectious diseases of the urinary and digestive tract.

SUMMARY OF THE INVENTION The present invention provides a method for the manufacture of an immunobiotherapeutic agent against infectious diseases of the urinary and radiant tract comprising as active ingredient a bacterial lysate from at least one of the following strains of Escherichia coli:

NCTC 8603, NCTC 8621, NCTC 8623, NCTC 9026, NCTC 9111, NCTC 9119, NCTC 9707, NCTC 9708, 1-081, 1-082, 1-083, 1-084, 1-085, 1-086 , 1-087, 1-088 and 1-089, characterized in that said Escherichia coli strains are incubated in a solid or liquid medium, subjected to lysis and / or autolysis, and the lysates thus obtained are isolated.

In the process according to the invention, said active ingredient may consist of a mixture of lysates from all said Escherichia coli strains.

The invention also relates to a medium for the production of which it contains * per liter of water.

of the method according to the invention,

22.5 8 meat extract 7.5 β yeast extract, 2.5 β sodium chloride, 0.5 β sodium acetate, 2 β secondary sodium phosphate 2 ml 70% sodium lactate, 2 ml 50% ammonium lactate, 3 mg aneurine, 3 mg nicotinic acid and / 3 β glucose,

said contents of each component being within ± 5%.

The medium according to the invention preferably contains, for one liter of water, 2 g of gelatin and 24 g of agar-agar for solidification.

The liquid medium for carrying out the process according to the invention by autolysis 8 preferably contains per liter of water:

g g

8 meat extract of sodium chloride and secondary sodium phosphate, the contents of which may be in the range of í 5% ·

Eschericia coli NCTC strains are cataloged in the National Collection of Type Cultures, London and are open to the public, whereas bacterial strains designated I were deposited by the inventors on March 7, 1979 in the Collection of Cultures of Micro-organisms, Institut Pasteur, Paris. .

Bacterial cultures are prepared by conventional bacterological techniques under the conditions most favorable to germ propagation.

The cultivation of the cultures to obtain the lysates is preferably carried out in an environment as claimed in points 4 or 5 of the invention, in a fermenter, for example at 35 to 37 ° C for 8 to 14 hours, or in Roux bottles, for example at 33 to 37 ° C for 24 to 48 hours. If it is desired to use a solid culture medium, then the culture medium claimed in clause 5 of the invention may be advantageously used.

Growth cultures are isolated by conventional methods. In each of the thus obtained bacterial suspensions, growth is evaluated, then the suspension is subjected to a progressive alkaline lysis (pH 9-10) at 20 to 40 ° C with sodium hydroxide, potassium hydroxide, a primary amine, a secondary amine or a tertiary amine. The lysis is performed for one to five days under microscopic control.

The volume of lysate from each bacterial strain entering the final concentrate is calculated based on the observed growth results.

The treatment of the cultures to obtain lysates by autolysis is preferably carried out in the environment claimed in clause 6 of the present invention. The inoculated cultures are incubated for 3 months at 37 ° C. The filtered supernatant forms a bacterial lysate of the culture.

The lysates of the individual bacterial strains obtained in the solid and / or liquid nutrient medium are then mixed in a suitable ratio to obtain the immunobiotherapeutic agent of item 1 of the invention. The total number of cells of the bacterial strains used ranges from 1 to 5 billion per day for an adult patient.

The lysate mixture forms a native concentrate which is clarified by centrifugation and filtration. The clarified concentrate is then sterilized by membrane filtration and thus serves as a concentrate of bacterial lysates for the preparation of various galenic formulations.

The immunobiotherapeutic agent of the invention is preferably administered by the oral route in the form of compresses, capsules, gelatin capsules or granules containing said lysate in lyophilized form, or in the form of drinking solutions in single-dose ampoules, syrups or drops containing the lysate of the invention. The composition may also be administered parenterally or also rectally by suppository.

The daily dosage for an adult, preferably taken at once, comprises lysate in an amount of 1 to 50 billion cell equivalents. The children's dose is half the adult dose.

Pharmacological tests

An experimental immunization study was performed in Belb / c mice following oral lysate stimulation according to items 1 and 2 of the subject invention. This study includes both humoral and cell mediated immunization, both specific and non-specific.

Determination of protective effect

Mice were treated orally with 150 mg / kg of lysate for 5 days. It was found that

2122 * 34 that said lysate stimulates a statistically significant protective effect against Escherichia coli infection infected intraperitoneally 10 to 30 days after the start of the lysate application or against infection by Salmonella typhimurium, infected parenterally 30 days after the start of the lysate application.

Plaque forming Cells - test

This technique makes it possible to demonstrate an increase in spleen cell counts over time in mice administered orally with 150 mg / kg lysate for 5 days. The test was performed 14 days after the start of lysate application.

In vivo assay for colloidal carbon phagocytosis

Colloid carbon injected into mice is eliminated from the vascular system by Kupffer cells. Thus, the kinetics of colloidal carbon elimination is an indicator of the effectiveness of the reticuloendothelial system. Mice were treated orally with 150 mg / kg of lysate for 5 days. The test was performed 10 to 30 days after the start of lysate application. It was found that the effect of the lysate resulted in a statistically significant increase in the degree of phagocytosis.

Study of macrophage efficacy

Mice were treated orally with 150 mg / kg of lysate for 5 days. Intraperitoneal macrophages were isolated 10-30 days after the start of lysate administration. A study of the adhesion capacity and the degree of phagocytosis of these macrophages showed that the lysate used had an immunostimulatory effect. A characteristic decrease in the enzymatic activity of these phophrophages, which is probably caused by depletion of these overexposed cells, has also been demonstrated. This hypothesis was also confirmed by transmission electron microscopy; activated macrophages showed strong vacuolization.

Study of chronic toxicity

The lysate of the invention was administered for 13 weeks at doses which were 100 and 1000 times the human therapeutic dose in rats and 20 to 200 times the human therapeutic dose in dogs. During this application, no deterioration in organ function or condition was observed in the test animals.

Clinical trials of patients suffering from chronic urinary tract inflammation were administered a single dose of lysate daily for 6 to 12 months 10 to 15 days per month. In 14 cases total relapse elimination was achieved and in 3 cases less frequent relapses occurred.

Claims (5)

SUBJECT OF THE INVENTION A method for producing an immunobiotherapeutic agent against infectious diseases of the urinary and gastrointestinal tract, characterized in that at least one strain of Escherichia coli is selected from the group comprising NGTC 8603, NCTC 8621, NCTC 8623, NCTC 9026, NCTC 9111, NCTC 9119, NCTC 9707, NCTC 9708, 1-081, 1-082, 1-083, 1-084, 1-085, 1-086, 1-087, 1-088 a 1-089, incubated in a solid or liquid culture medium, subjected to alkaline lysis at pH 9-10 and 20-40 ° C and / or autolysis by fermentation, and the lysate thus obtained is isolated. 2. A method according to claim 1, wherein all said Escherichia coli strains are used. 3. Method according to claim 1, characterized in that a medium containing per liter of water is used: 22.5 & meat extract, 7.5 G yeast extract, 2.5 ε Sodium chloride, 0.5 ε sodium vinegar, 2 ε secondary sodium phosphate 2 m. 70% sodium lactate, 2 ml 50% ammonium methanate, 3 mg aneurine, 3 mg nicotinic acid and 3 ε glucose, said contents of the individual components being within ± 5%. 4. Method according to point 3, except for black, by applying a nutrient medium to one liter of water for the purpose of solidifying:. 2 g gelatin and 24 g agar-agar. 5. Method according to claims 1 and 2, characterized in that a liquid nutrient medium containing per liter of water is used in the autolysis process. 25 g meat extract, 5 g of sodium chloride and 1 g of secondary sodium phosphate, said contents of the individual components being in the range of even 5%.