HU181725B - Process for producing immunotherapeutic pharmaceutical composition - Google Patents

Abstract

The invention relates to a process for the preparation of an immunobiotherapeutic preparation for use as a medicament. The aim of the invention is to provide such a preparation for the treatment of urinary and intestinal infections. According to the invention, the preparation contains as active ingredient a bacterial lysate of at least one of the following Escherichia coli strains: NCTC-8603, NCTC-8621, NCTC-8623, NCTC-9026, NCTC-9111, NCTC-9119, NCTC-9707, NCTC-9708, I-081, I-082, I-083, I-084, I-085, I-086, I-087, I-088 and I-089. It is prepared by adding each of the indicated strains in a cultured solid or liquid basal medium, which lyses and / or autolyzed bacteria and isolated the lysate. According to the invention, it is also possible to isolate and mix the lysate of all the stated strains.

Description

The present invention relates to a process for the preparation of an immunotherapeutic drug. The pharmaceutical composition of the present invention is effective against infectious diseases of the urinary tract and gut.

The composition is characterized in that it contains a bacterial lysate as active ingredient which is prepared from at least one of the following strains: Escherichia coli NCTC-8603, NCTC-8621, NCTC-8622, NCTC-8623, NCTC-9026, NCTC-9111, NCTC-9119 , NCTC-9707, NCTC-9708, 1-081, I-0S2, 1-083,1-084,1-085,1-086,1-087,1-088 and 1-089.

Preferably, the composition comprises lysates of all the strains listed.

The NCTC strains are deposited in the National Collection of Type Cultures (London); the tribes are publicly accessible. The I-strains were deposited on March 7, 1979 in the Collection Nationale de Cultures de Microorganisms (Pasteur Institute, Paris).

According to the present invention, the above composition is prepared by culturing the listed strains on solid or liquid media, lysing and / or autolysing the bacteria and isolating the lysate.

Preferably, the microorganisms are cultured in a liquid medium containing 22.5 g of meat extract, 7.5 g of yeast extract, 2.5 g of sodium chloride, 0.5 g of sodium acetate, 2.0 g of sodium monohydrogen phosphate per liter of water. 0 ml of 70% sodium lactate solution, 2.0 ml of 50% ammonium lactate solution, 3.0 mg of aneurine, 3.0 mg of nicotinic acid and 3.0 g of glucose; values may fluctuate by ± 5%.

If a solid medium is used, 2.0 g of gelatin and 24.0 g of agar per liter are added for consolidation.

For example, the bacteria can be fermented at 35-37 ° C for 8-14 hours in a fermentor or, for example, at 33-37 ° C for 24-48 hours in a Roux flask in a manner known per se, under conditions that best promote the growth of microorganisms. .

The cultures are recovered in a manner known per se. Cell counts were determined per strain and the suspensions were subjected to alkaline (pH 9-10) lysis at 20 ° C to 40 ° C. Suitable lysing agents are, for example, sodium hydroxide, potassium hydroxide, and primary, secondary and tertiary amines. Lysis is continued for 1-5 days under microscopic control.

The volume of lysate to be used for each formulation is determined by cell number for each strain.

-1181725

When the lysate of the present invention is prepared by autolysis of the culture, a liquid medium containing 25.0 g of meat extract, 5.0 g of sodium chloride and 1.0 g of sodium monohydrogen phosphate 5 (± 5%) is preferably used. The inoculated medium is incubated for three months at 37 ° C. The filtered supernatant is the lysate.

The lysates obtained using the liquid or solid media are mixed in the appropriate volume ratio. The number of strains employed varies within certain limits, the daily dose for an adult being about 1-50 billion.

The mixture of lysates (first concentrate) was purified by centrifugation and filtration. The resulting concentrate was germinated by membrane filtration and used to prepare various types of formulations.

The pharmaceutical composition of the present invention is preferably administered orally in the form of tablets, capsules, gelatin capsules or granules containing the lysate in lyophilized form, or in the form of syrups or drops or ampoules. Parenteral administration or rectal administration in the form of a suppository is also possible. For adults, the daily dose is 1-50 billion gigabytes of lysate, which should be taken at the same time. For children, the daily dose is halved.

Pharmacological experiments

Balb / c mice were stimulated with the composition according to claim 1 or 2, and subsequently tested for specific and non-specific humoral and cellular immunity.

Examination of protective effect

Mice were dosed orally with 150 mg / kg ⁴⁰ lysate for 5 days. At 10 and 30 days after the start of treatment, the animals were infected with Escherichia coli interperitoneally, and 30 days after the start of treatment, they were inoculated with Salmonella typhimurium enterally. The protective effect of lysate ⁴⁵ in both cases was a statistically significant determination.

Examination of "plaque-forming" cells

Mice were given 150 mg / kg lysate for 5 days. 14 days after treatment, a slowly increasing number of spleen cells around which lytic spots (plaques) develop.

In vivo testing of phagocytosis with colloidal carbon

Mice were given 150 mg / kg lysate for 5 days. Colloidal charcoal is injected 10 and 30 days after treatment. Colloidal carbon is secreted from the system containing Küpffer cells. The kinetics of colloid secretion reflect the activity of the reticulo-endothelial system. The phagocytosis index increased statistically significantly.

Investigation of macrophage activity

Mice were administered orally 150 mg / kg lysate for 5 days. 10 and 30 days after the start of treatment, animals are harvested by intraperitoneal macrophages. Macrophage adhesion and in vitro (Candida albicans) phagocytosis were determined. The results show that the lysate used has an immunomodulatory effect on the cells. Otherwise, the enzyme activity of macrophages has been markedly reduced, which is probably due to the exhaustion of over-utilized cells. Electron microscope images confirmed the latter assumption; Activated macrophages have developed characteristic vacuoles.

toxicity

Rats were dosed 100 times or 1000 times the therapeutic adult dose, and dogs 20 and 200 times the said dose orally for 13 weeks. During this time, no functional or organic lesions were observed.

Clinical trials

17 patients with chronic urinary tract infection were administered lysate for 8-12 months (10-15 days per month). In 14 cases, there was no complaints without relapse, while in 3 cases, the interval between doses was prolonged.

Claims (5)

Patent claims: A process for the manufacture of a medicament for the treatment of infections of the urinary tract and of the gastrointestinal tract comprising as active ingredient Escherichia coli NCTC-8603, NCTC-8621, NCTC-8622, NCTC-8623, NCTC-9026, NCTC-9111, NCTC-9119, NCTC-9707. , NCTC-9708, Ι-08Γ, 1-082,1-083,1-084, 1-085, ΙΌ86, ΙΌ87, 1-088, and ΙΌ89, characterized in that the above strains cultured in solid or liquid media, the bacteria are lysed and / or autolysed, and the lysate is isolated and converted into a pharmaceutical formulation in a manner known per se. 2. The method of claim 1, wherein the lysates of each of the listed strains are isolated and mixed. 3. The method of claim 1, wherein the strains are cultured in a liquid medium that is 22.5 g of meat extract, 7.5 g of yeast extract, 2.5 g of sodium chloride, 0.5 g of sodium acetate, 2.0 g of sodium monohydrogen phosphate, 2.0 ml of 70% w / w sodium lactate solution, 2.0 ml 50 containing 3.0% by weight of ammonium lactate solution, 3.0 mg of aneurine, 3.0 mg of nicotinic acid and 3.0 g of glucose, which values can fluctuate by ± 5%. 4. The method of claim 3, wherein the strains are cultured in a medium containing 2.0 g of gelatin and 24.0 g / l of water. 5. The method of claim 1, wherein said strains are cultured and autolysed in a liquid medium containing 25.0 g of meat extract, 5.0 g of sodium chloride, and 1.0 g of sodium monohydrogen per liter of water. contains phosphate, which can fluctuate by ± 5%.