CS222673B2 - Method of making the immunobiotherapeutic means against infection disease of the breathing ways - Google Patents

Abstract

A medicament for treating infections of the respiratory passages contains as the principal active substance a bacterial lysate obtained from a mixture of:- (i) at least one of the following cultures:- <IMAGE> and (ii) at least one of the following cultures:- <IMAGE> Culture media for the bacteria are also specified.e

Description

The invention relates to a method for the manufacture of an immobiotherapeutic agent against respiratory tract illness.

SUMMARY OF THE INVENTION The present invention provides a method for the manufacture of an immunotherapeutic agent against an infectious airway infection, which is characterized by the fact that it is found in the culture medium of bacteria of at least one bacterial strain from the group that is silent

Staplnylococcus aureus

Streets (:) cbccus virddíns

Neesseria catarrhia.is and at least one stem from the stalk, stunting

1-049,

1-050,

1-051,

1-052, 1-053 and

1-054,

1-046,

1-047 a

1-048,

1-045,

infiu ^ i ^ r ^ z ^ ae, serotype b NCTC 8467, Diplococcus ρneшnobOae, serotype 1 NCTC 7465, Diplocbccrs · pneumaticOL0ae, serotype 2 NCTC 746 Diplococcus pneшnob0ae, Serotype 3 NCTC 7978, Diplococcus> · tire, serotype 47 NCTC 10319, K.ebbseeia pneumoniae NCTC · 204 · NCTC 5056, KLebbsella ozeenae NCTC 5050, Streptc) cbccrs pyogenes sérbstopioa NCTC 8191

and

Neesseria catar * hal.is

NCTC. 3622,

NCTC 3625 under the conventional conditions for the tactivation of said beads, a lysate of the above-described bales is prepared, and the lysate is mixed with a carrier.

In the process according to the invention, preferably all said bacterial strains are brought together. Advantageously, the medium is fed in one liter of water:

22-, 5 g Maasbé. eetradtu,

7.5 g yeast extract,

2.5 g sodium chloride,

0,5 g sodium acetate,

2.0 g of secondary sodium phosphate-phenol,

2.0 ml 70% sodium lactate,

2,0 ml 50% - ammonium lactate,

3.0 mg aneurin,

3.0 mg of nicotinic acid, ^3.0 g of glucose, said contents of the individual components varying within ± 5%

NCTC-labeled bacterial strains are cataloged in the National Collection of 'Type Cultures, London and are available to the professional community, while bacterial strains labeled I were deposited by the authors of this invention on March 14, 1978 in the Collection of National Cultures of Microorganisms, Institut Pasteur , Paris ·

The media are sterilized either by autoclaving or filtration.

The preparation of the bacterial lysate according to the invention is preferably carried out according to the following scheme:

(a) seeding the bacterial strain

(b) incubation

c) isolating the grown colonies from the nutrient medium, concentrating and suspending the bacterial strain,

d) evaluation of the number of cells of the bacterial strain,

(e) skis

f) mixing the lysates and forming a native concentrate

g) removing the solid from the native concentrate by centrifugation or microfiltration

h) sterilizing filtration of the clarified native concentrate to obtain an applicable bacterial lysate concentrate.

Bacterial cultures are produced by known bacteriological techniques under conditions that are optimal for bacterial cell proliferation.

Incubation of the Hemophilus strain

The incubation of the bacterial hemophilus strain is preferably carried out in a fermenter, with 2% of baker's yeast extract and 0.5% of Fildes extract added to the basic nutrient medium.

Incubation is performed at pH 7 and 37 ° C with air bubbling and stirring for 8-14 hours.

Incubation of the pneumoniae strain

The incubation of the bacterial strain pneumoniae is preferably carried out in a fermenter with 0.3% glucose and 0.5% horse serum added to the basic nutrient medium. Otherwise, the same conditions as described above for the incubation of the Hemophilus strain were carried out.

Incubation of Streptococcus strain

The incubation of the bacterial strain Streptococcus is preferably carried out in a fermenter under the same conditions as described for the incubation of the bacterial strain Hemophilus, with 0.3% glucose added to the basic culture medium.

Incubation of K.ebsiella strain

Incubation of bacterial strain Klebsiella is also preferably carried on a solid nutrient medium ⁱⁿ Roux ^{bank b s} t ^ep ture at ³⁷ ° ^C for ⁴⁸ hours, ^e em ^p s ^f with ^{the AK} ladnímu nutrient ^<m $ u. Surroundings added 0.2 % gelatin and 2.4% ago-agar.

Incubation of Staphilococcus strain

The incubation of the Staphilococcus strain is preferably carried out on a solid culture medium under the same conditions as described for the incubation of the K.ebsiella strain.

Incubation of cultures. on liquid culture medium

After the incubation period, the growth of each strain is evaluated either by measuring the opacity or by counting under the microscope. The equilibrium baacteial culture is centrifuged, the centrifuged solids are suspended in a physiological solution, and bacterial cell counts are again evaluated. cultures and baltic cultures are subjected to alkaline lysis (pH 9-10) at 20-40 ° C with sodium hydroxide, potassium carbonate or a primary, secondary or tertiary amine. Skiing is performed for one to five days under microscopic inspection.

Incubation on solid culture medium.

The emerging bacterial strain is isolated from the culture medium by conventional techniques such as flushing. The culture suspensions thus obtained are preferably evaluated for bacterial strain cell numbers and then subjected to alkaline lysis as described above.

Preparation of concentrate in the form of lysates

The lysate volume of each fermentation strain is calculated based on the cell counts of that strain.

The lysates of each of the bacterial strains are then mixed in a suitable proportion to obtain the immobilized preparation of the invention. Total quantity! The cells used in the stem ballasts range from 1 to 50 billion in a daily dose for an adult patient.

The galenic forms of the immunoobo therapeutic composition of. invention *

The immunotherapeutic composition of the invention is preferably administered orally. in the form of a comonomer, capsule, capsule, or capsule, which comprises said lysate in a lyophilized form, or in drinking solutions in barley, syrups, or drops, to form a lysate. according to the invention. The composition may also be administered by aerosol or droplet form, or parenterally.

A Dernti adult dose, preferably taken at once, contains a lysate in a quantity of 1 to 50 billion cell equivalents.

Baby dose. Reading half dose of adult.

Example 1

Method for the production of an immunobiotherapeutic agent against respiratory tract diseases from the iaakertel strain Hemoohhlus

The strain of HemooPilus is cultured by alternating inoculation (every 14 days) at 37 ° C to a solid culture medium (chocolate agar) and a liquid culture medium (operational culture medium).

The inoculum is inoculated into the same nutrient liquid medium used for the culture. The inoculum volume is 1/10 of the total volume of the operating culture. Incubation is carried out for 14-18 hours. at 37 ° C.

Operating kuuturu environment:

It is mixed in one liter of water:

meat extract 22,5 g yeast extract 7,5 g sodium choride 2,0 g sodium acetate 0.5 g sodium phosphate secreted 2,0 g sodium lactate (70%) 2.0 ml ammonium lactate (60%) 2.0 ml aneurin 3.0 mg nicotinic acid 3.0 mg glucose 3,0 - g

The mixture was filtered and 6 g Eugonnroth / BBL powder was added to the filtrate. The reconstituting mixture is placed in an autoclave for 20 minutes, where it is maintained at a temperature of 120 ° C, after which:

yeast extract 20.0 - ml and rUdes / Dirco extract 5.0 ml

The final pH of the culture medium is 7.2.

the operating culture is cultivated under the following conditions:

- the culturing is carried out in a batch fermenter with a feed volume of 8 ИЫ;

- pH control to 7.0 is performed automatically;

- ferrientbr is equipped with an antifoam system;

the cultivation is carried out at 37 ° C, with aeration of 5 l of air per minute, with stirring at 300 to 400 rpm, and for 8 to 14 hours.

The number of cells of the bacterial strain is preferably measured by microbiology and microscopy.

The bacterial strain is thermally inactivated 2-3 times per hour at 56 ° C for 24 hours. The mixture is then centrifuged.

The resulting precipitate is suspended in sterile, promised water. Homibber sediment is performed by meapintide agitation. A second evaluation of the cell number and characterization of the strain per ml of culture is performed.

Progressive alkaline lysis is then carried out at -37 ° C with sodium hydroxide solution to a pH of 9-10. Microscopic examination of the ski is performed. The ski time is equal to 3 days.

The lysate is then stored at 4 ° C up to the preparation point. Bacttrial Lysate Concentrate. The concentrate is then mixed with a pharmaceutically acceptable carrier.

Example 2

Pharmacological tests

A lysate was recovered, which contained 6 billion cell equivalents of each of the following bacceeial strains per dose:

Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus (1-049) , virdd & ns 1-048

Neisseria catarrhalis 1-045

HCT is serotype b NCTC 846 * 7,

Diplococcus pneumoniae, serotype 1 NCTC 7465, Diplococcus pneumoniae, serotype 2 NCTC 7466, Diplococcus ene & clone, serotype 3 NCTC 7978, Diplococcus pneumoniae, serotype 47 NCTC pnPnPnPnPnPnPnPnnPnPnPnPnPnPnPnPnPnPnPnPnPnPnPnPnPnPnPnnnPnPnPnPnnnnPnnnnnn oiamae NCTC 5050

Streptococcus pyogepes, aero group .A NCTC 8191 and Ne ^ Berla. οζΙ ^^ οΙ! NCTC 3622 and cis-rhO-is NCTC 3625.

In this test, the efficacy of said mixed lysate for oral administration in Balb / c carrots on various infectious stimuli was crunched. The daily dose, which was equal to 1/5 of the dernal dose for humans, was administered orally for 10 days to groups of 24 flies.

The animals were infected 5 and 20 days later when the drug was stopped. The results obtained show that in this way a statistically significant protection against infection of the bacterial strain Ώ, Χθ11ζ applied intrispritonally is achieved. 5 days pp cessation of drug administration. The protective effect is also observed when the animals are infected for up to 20 days after cessation of drug administration.

The non-biotherapeutic lysate also induces a non-specific stimulation of the defense mechanism, resulting in reduced orbit of test animals infected with influenza virus in an aerosol.

For samples derived from BsUb / c mice, which were &quot; a bacterial lysate was administered as described above, a biochemical and morphological study of the peritoral β-phophages was performed.

The results obtained show a characteristic increase in the number and volume of cells for 5 and 25 days prior to the end of drug administration. When phosphatase and beta-triazine triase are administered, increased activity is observed over the group of animals.

Severography, n. P., MOST

Price 2,40 Kčs

Oral administration of said lysate results in an increase in IgA in the airway wash fluid. This increase results in a positive clotting reaction of the corresponding antibody at a 1/16 dilution. Given the normally low IgA secretion and the marked increase in the presence of this type of immunoglobulin, it is believed that the increase in total Ig is due to an increase in the immunoglobulin A content.

Example 2

Clinical tests

Twelve healthy volunteers received one capsule containing 6 billion cell equivalents of each of the bacterial strains listed in the preceding Example 1 for ten days each day. serum. This response of the organism is in conformity with the current concept of the development of immunity to infection.

The above lysate-containing capsules of the invention have been used on a clinical scale in the treatment of chronic bronchitis and ENT-spherical diseases such as sinusitis. The therapeutic and preventive efficacy of the immunobiotherapeutic agent of the invention was evaluated. The treatment period was generally ten days per month for several months (during the winter months). The results are summarized in the following table.

Table

- / --------------------------------------------

Number of cases E s s 1 / e d к у good average none Curative value 242 146.0 72.0 24.0 (%) 100 ALIGN! 60.3 29.8 9.9 Preventive value 131 94.0 12.0 25.0 (%) 100 ALIGN! 71, 8 9.1 19.1

The immunobiotherapeutic agent of the invention is particularly recommended for the treatment of:

acute and chronic bronchitis, angina, amygdalitis, laryngitis and faringitis, rhinitis, sinusitis and otitis, infections that are incurable by common antibiotics and diseases of viral origin of the respiratory tract, especially in children and the elderly.

Claims (3)

1-045, and at least one microbial strain from the stalk, comprising Hemoohílus inftueuzjt, serotype b NCTC 8467, Diplococcus Diplococcus Diplococcus Diplococcus hUiWQooLUae, huuuoccine..au, puuiuo ouo, serotype 1 serotype 2 serotype 3 serotype 47 K-ebsiella huuumoniau Kcbsiella ozeenae Streptococcus piens, serostopine A and Neesseria catarrhalis NCTC 7465, NCTC 7466, NCTC 7978, NCTC 10319, NCTC 204 NCTC 5056, NCTC 5050, NCTC 8191 NCTC 3622, NCTC 3625 under the tolerance conditions customary for cultivation of said baakers, followed by preparing a lysate of the so-called Saaterri and mixing the lysate with a pharmaceutically acceptable carrier. 1-048, 1-047 a 1-046, 1-054, 1-053 a 1-052, 1-051, 1-050, 1-049, What is claimed is: 1. A method for the production of an imimobiotsrapeutic agent against an infectious airway disease, characterized in that bacteria in at least one bacterial strain from the stalk, which have been killed and It collapses / lo ^ ccus aureus Streptococcus viridans Neisseria catarríalis 2.0 ml of 50% ammonium lactate, 3.0 mg 3.0 mg ni ceric acid a. 2.0 ml 70% sodium lactate, 2.0 g of secondary sodium phosphate, 2.5 g sodium chloride, 0,5 g sodium acetate, 2. Method according to claim 1, characterized in that all said strains are separated. 3. A method according to claim 1, characterized in that a medium containing 1 liter of water is used. 22,5 g meat extract, 7.5 g yeast extract, 3.0 g of glucose, said contents of the individual components may be up to 5%.