GB2054374A - Bacterial lysates - 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

SPECIFICATION Medicament for infectious diseases of the urinary tract and digestive tracts The present invention concerns a medicament for infectious diseases of the urinary and digestive tracts.

More particularly, the present invention provides an 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, 9707,9708.1-081: 1-082,1-083,1-084,1-085,1-086, 1-087, 1-088, 1-089.

The lysates are preferably derived from all the strains of E. coli listed above.

The NCTC strains are listed by the National Collection of Type Cultures, London and are accessible to the public, while the strains I were deposited by the holder of the present patent on 7th March, 1979, at the Collection Nationale de Cultures de Microorganismes, I nstitut Pasteur, Paris.

The bacterial cultures are made according to current bacteriological techniques, in the conditions most suitable for the multiplication of microorganisms.

The treatment of the cultures in order to obtain lysates is preferably carried out in a fermentor at 35"-37"C for 8-14 hours, or in a Roux bottle, for example, at 33"-37"C for 24-28 hours in a culture medium containing, per litre of water; meat extract 22.59, yeast extract 7.59, sodium chloride 2.59, sodium acetate 0.5g, sodium monohydrogen phosphate 2.09, sodium lactate 70% p/p 2.0 ml, ammonium lactate 50% p/p 2.0 ml, thiamine 3.0mg, nicotinic acid 3.0mg, glucose 3.09, these amounts being variable by + 5%.

If a solid medium is preferred, then the medium may also contain gelatine 2.09 and agar-agar 24.09.

The cultures are harvested in accordance with the usual methods. The bacterial suspensions thus obtained are counted strain by strain and subjected to progressive alkaiine lysis (pH 9 to 10) atatemper- ature which can vary between 20 and 40"C, with among other things; caustic soda, caustic potash, and primary, secondary and tertiary amines. Lysis is carried out for one to five days under microscopic control.

The volume of lysates from each strain of bacteria in the final concentrate is calculated as a function of the results of the counts.

The treatment of cultures to obtain lysates by autolysis is preferably carried out in a medium containing per litre of water: meat extract 25.09, sodium chloride 5.09, sodium monohydrogen phosphate 1.0g, these amounts being variable by t 5%. The seeded cultures are incubated for 3 months at 370C.

The filtered supernatant of these cultures constitutes the lysate.

Preparation of the bacterial lysate concentrate.

The lysates of each strain of bacteria obtained in solid and/or liquid medium are preferably mixed in suitable proportions to obtain the medicaments of the invention. The quantities of micro-organisms from the strains used in association can vary between certain limits for an adult human daily dose: 1 to 50 milliards of micro-organisms.

The mixture of lysates constitutes a natural concentrate which is clarified by centrifugation and filtration in depth. The clarified concentrate is sterilised by filtration by membrane and constitutes the bacterial lysate concentrate serving as the base for the preparation of various galenical forms.

Galenical forms.

Administration is preferably carried out orally, in the form of tablets, capsules, gelules, or pellets, containing the lysate in freeze-dried form or in aqueous solution to be taken orally in the form of single dose ampoules, linctus, or drops containing the lysate.

Administration can also be carried out parenterally or rectally in the form of suppositories. The daily dose for an adult, preferably taken all at once, can contain in all a lysate varying between 1 and 50 milliards of micro-organism equivalents. The child's dose is half the adult dose.

PHARMACOLOGICAL WORK An experimental study of the immunity state was carried out with mice Baltic after oral stimulation by the lusate defined according to claims 1 and 2. This study covers immunity to humoral mediation and cellular mediation, specific and non-specific.

Protection test The mice were treated orally with 150 mg/kg for 5 days. It was shown that the lysate has a statistically significant protective power with regard to an infec tion by Escherichia coli injected intraperitoneally 10 and 30 days after the beginning of treatment or by Salmonella typhimurium introduced by the enteral route 30 days after the beginning of treatment.

"Plaque forming Cells" test.

This technique allowed demonstration of an increase as a function of time of the number of splenic cells producing lysis plaques in mice treated with 150 mg/kg of lysate, for 5 days orally. The test was carried out 14 days after the beginning of treatment.

Phagocytosis test in vivo with colloidal carbon Colloidal carbon injected into a mouse is eliminated from the vascular system by Küpffer cells. The kinetics of the elimination of the colloid therefore indicates the activity of the reticulo-endothelial system. Mice were treated orally for 5 days with 150 mg/kg of lysate. The test is carried out 10 and 30 days after the beginning of treatment. The phagocytic index is increased in a statistically significant way.

Study ofmacrophagic activity Mice were treated with 150 mg/kg of lysate orally for 5 days. The intraperitoneal macrophages were removed 10 and 30 days after the beginning of treatment. Study of the capacities for adherence and phagocytosis in vitro (Candida albicans) of the macrophages demonstrated that the lysate used has an immuno-stimulant effect at the cellular level. A characteristic fall in the enzymatic activities of these macrophages was also demonstrated, probably due to a depletion of these over-activated cells. Tests made by transmission electron microscopy con firmed this hypothesis; the activated machorphages present greatvacuolization.

STUDY OF CHRONIC TOXICITY The lysate was administered in doses of 100 and 1,000 times the human therapeutic dose to rats and in doses of 20 and 2,000 times to dogs orally for 13 weeks. No functional or organic change was detected in the course of treatment.

CLINICAL TESTS 17 patients suffering from chronic urinary infection were treated at the rate of 1 dose per day of lysate for 8-12 months at the rate of 10 to 15 days of treatment per month. Recurrences were totally eliminated in 14 cases and were at wide intervals in 3 cases.

Claims (6)

1. Immunobiotherapeutic medicamentforinfectious 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.

-- 1-08'1, 1-082, 1-083, 1-084, 1-085, 1-086, 1-087, 1-088, 1-089.

2. Medicament according to claim 1, characterised by the factthatthe said active principle is constituted by a mixture of lysates of all the strains cited.

3. Method of manufacturing a medicament according to claim 1 or 2, characterised by the fact that the said strains are incubated in a solid or liquid culture medium, that they undergo a lysis and/or an autolysis, and that the lysatesthus obtained are harvested.

4. Culture medium for carrying outthe method according to claim 3, containing per litre of water: meat extract 22.5 g, yeast extract 7.5 g, sodium chloride 2.5 g, sodium acetate 0.5 g, sodium monohydrogenphosphate 2.0 g, sodium lactate 70 /O p/p 2.0 ml, ammonium lactate 50% p/p 2.0 ml, thiamine 3.0 mg, nicotinic acid 3.0 mg, glucose 3.0 g, these amounts being variable by t 5%.

5. Medium according to claim 4, containing in addition per litre of water: gelatine 2.0 g and agaragar 24.0 g to make it solid.

6. Liquid culture medium for carrying out the method according to claim 3, by autolysis, containing per litre of water: meat extract 25.0 g, sodium chloride 5.0 g, sodium monohydrogen-phosphate 1.0 g, these amounts being variable by + 5%.