HU180005B - Process for producing pharmaceutical composition utilizable for treating illnesses of respiratory organs - Google Patents

Abstract

According to the invention, at least one of the following bacterial strains is cultivated in a culture medium

Description

Fariné Jean-Claude, chemist, Eysins, Switzerland

Rivkine Anne doctor, Geneva, Switzerland

Pharmacist Bulto Isidro, Barcelona, Spain

patentee:

Laboratories OM Société Anonyme,

Meyrin, Geneva,

Switzerland

A process for the preparation of a medicament for the treatment of respiratory diseases

The present invention relates to a process for the preparation of an immunotherapeutic pharmaceutical composition for the treatment of respiratory diseases.

According to the invention, at least one of Staphylococcus aureus I-049, I-050, I-051, I-052, I-053, I-054; Streptococcus viridans I-046,1-047,1-048; or Neisseria catarhalis I-045, a bacterial strain and at least one Hemophilus influenzae serotype NCTC 8467; Diplococcus pneumoniae serotypes 1, 2, 3 and 47 NCTC 7465, 7466, 7978, 10319; Klebsiella pneumoniae NCTC 204, 5056; Klebsiella ozaenae NCTC 5050; Streptococcus pyogenes Serogroup A NCTC 8191; or bacterial strains of Neisseria catarhalis NCTC 3622, 3625, and the lysate of the bacteria so grown is mixed with a pharmaceutically acceptable carrier and, if desired, other excipients, to form a medicament.

Preferably, all of the bacterial strains listed are co-cultured and their lysates processed into a drug.

In the above list, the abbreviation NCTC in the bacterial deposit numbers refers to the catalog of the "National Collection of Type Cultures" (London), while the numbers beginning with the I are the deposit numbers of the bacterial strains in the Pasteur Institute cultures de microorganismes) was deposited on March 14, 1978.

Preferably, the bacterial strains are cultured in a medium containing 21 to 24 g of meat extract, 7.1 to 7.9 g of yeast extract, 2.3 to 2.7 g of sodium chloride, 0.47 to 0.53 g of sodium acetate, 1.8 to 2 g per liter. , 2 g disodium hydrogen phosphate, 1.8-2.2 ml 70% sodium lactate, 1.8-2.2 ml 50% ammonium lactate,

2.8 to 3.2 mg of aneurine, 2.8 to 3.2 mg of nicotinic acid and

Contains 2.8-3.2 g glucose.

The medium may be sterilized in an autoclave or sterilized by filtration.

The bacterial lysate is conveniently prepared as follows.

The medium is inoculated with the selected bacterial strains and incubated. The bacterial mass is then removed from the medium, concentrated, and suspended. The bacteria in the suspension were then counted and lysis was performed. The Jizates are mixed to give a natural concentrate. This is thoroughly purified by filtration and centrifugation. The product thus obtained is subjected to sterile filtration. The concentrate of the bacterial lysate is then obtained.

Bacteria are cultured by conventional bacteriological methods, always choosing the most appropriate conditions for cell proliferation.

Preferably, the Hemophilus strains are cultured in a fermentor by adding 2% bakery yeast extract and 0.5% "Fildes" extract to the basic medium. The bacteria were grown at pH 7.0 and 37 ° C with agitation and aeration for 8-14 hours.

Pneumococcal strains were also cultured in a fermentor by adding 0.3% glucose and 0.5% horse serum to the basic medium. The culture is carried out under the conditions described in the preceding paragraph.

Strains of Streptococcus are also cultured in a fermentor under similar conditions as Hemophilus strains. 0.3% glucose was added to the medium.

The Klebsiella strains are preferably cultured in solid medium in Roux flasks. 0.2% gelatin and 2.4% gelose were added to the basic medium. The culture was carried out at 37 ° C for 48 hours.

The Staphylococcus strains were also cultured in solid medium under the conditions described in the previous paragraph.

The same procedure was followed for the Neisseria strains, which were also cultured in solid medium under the conditions described above.

In the case of bacterial growth in liquid medium, the cultures are counted per strain at the end of the incubation period. Counting is performed by opacimetry or microscopy. The cultures were then centrifuged, resuspended in physiological saline, counted again, and subjected to vigorous alkaline lysis at pH 9-10 at a temperature between 20 ° C and 40 ° C. For lysis, for example, sodium or potassium hydroxide, primary, secondary or tertiary amur can be used. The lysis is performed for 1-5 days while observing microscopically.

Cultures cultured in solid medium are harvested according to conventional methods. The bacterial supernatants thus obtained are counted every tpt, then the? the living are subjected to serpentine alkaline lysis of the bacterium.

Each bacterial strain lizátutnának .térfogatát calculated as a function of counting eceÍip4 ⁿ Xépek.

Individual strains of bacteria; lysate is mixed in the desired ratio to obtain the medicaments as defined above. The germination rate of bacterial germs for use in goblet bowling can vary within ten limits. For example, for a adult pniber, the germ value for a single daily intake of germinates may be m50 billion.

THE? Lysate prepared according to elqbbiejc can be used to prepare various pharmaceutical compositions. Thus, for the purpose of exemplary oral administration, tablets, sulfur ss soft capsules, granules may be prepared which contain the lipid in an expanded form. However, liquid oral formulations, such as a volatile ampoule formulation, syrup or drops containing the lysate, may also be prepared. You can also prepare medications for inhalation, such as aerosols or drops, and parenteral formulations.

The daily dose of an adult human should preferably contain as many simple lysates as 1 to 50 billion units of germ. The dose for children is half the dose for adults.

The experimental results of the present invention are described below. For our assays, a lysate was prepared in lyophilized form from the bacterial strains listed above containing 6 billion germs per dose.

The effect of the lysate on oral administration was investigated in Balb / c mice, which were exposed to various infections. To establish active protection experiment, a dose corresponding to 1/5 of the human dose was administered orally to groups of 24 mice for 10 days. The animals were infected 5 and 20 days after the drug treatment was completed. We found that the treatment provided statistically significant protection against Klebsiella pneumoniae strain.spl when the strain was infected intraperitopically with the strain 5 days after treatment. When the infection was performed 20 days after the end of the drug treatment, the rate of death was still reduced. It has also been found that the immunotherapeutic lysate exerts a non-specific stimulatory effect on the protective mechanism against the rhinitis virus, which has been shown to reduce the death rate of animals aerosolized with the rhinitis virus.

The lysate was subjected to biochemical and morphological examination of peritoneal macrophages in samples from mice of the Balb / c species as described above. We found that the number and volume of these cells increased significantly 5 and 25 days after the treatment was completed. The addition of phosphatase and beta-galactosidase also showed that the enzymatic activity was increased compared to the controls.

Following oral administration of lysates to mice, an increase in IgA in the respiratory fluid was observed. The sense was that a positive precipitation reaction was observed against the corresponding antisubstituted dilution 1/16. Given the low ratio of section source and the significant increase in lysate activity associated with this type of itrimmunoglobulin, it is believed that the total increase in lg is almost entirely due to an increase in IgA.

The formulation prepared according to the method of the present invention has also been clinically tested. Twelve healthy volunteers were treated with one capsule daily for 10 days, containing a variety of the above bacterial units and _a titer of 6 billion germs. Samples taken for 3 months and beyond showed a statistically significant increase in IgA section in saliva and a statistically significant increase in JgA in serum. This result is consistent with our ideas on the immune response mechanism.

The capsules defined in the previous paragraph were used to investigate the effect on chronic bronchitis and the effect on the area of the larynx, such as inflammation of the sinuses. The curative and preventive effects of the drug were evaluated. The treatment was continued for several months, usually for 10 days per month, especially during the winter months. The results are summarized in the table below.

z Number of cases GOOD Medium Nothing Curative effect,% 242 146 72 24 100 60.3 29.8 9.9 Preventive effect,% 131 94 12 25 100 71.8 9.1 19.1

Medicament manufactured according to the invention is especially efficacious for acute and chronic bronchitis, pharyngitis, tonsillitis, laryngitis, rhinitis, sinusitis and treatment of ear infections and overcoming resistance to treatment with ^one of the usual infections with antibiotics. I may be used according to the invention the produced medicament _v respiratory tract caused by bacteria and viruses ^{1 v} diseases and abuse of good results gyógyí- pouch or alleviation in particular children and the elderly.

Claims (2)

Patent claims A process for the preparation of an immunotherapeutic pharmaceutical composition for use in the treatment of respiratory diseases, wherein at least one of Staphylococcus aureus is 1-049, 1-050,1-051,1-052, 1-053, 1-054; Streptococcus viridans I-046, 1-047, I-048; or a bacterial strain of Neisseria catarrhalis I-045 and at least one Hemophilus influenzae serotype b NCTC 5,8467; Diplococcus pneumoniae serotypes 1, 2, 3 and 47 NCTC 7465, 7466, 7978, 10319; Kelbsiella pneumoniae NCTC 204, 5056; Klebsiella ozaenae NCTC 5050; Streptococcus pyogenes Serogroup A NCTC 8191; or Neisseria catarrhalis NCTC 3622.3625 bacterial strain 10 is cultured in culture medium and the lysate of the bacteria so grown is mixed with a pharmaceutically acceptable carrier and, if desired, other excipients, into a medicament. 2. The method of claim 1, wherein all of the bacterial strains listed are co-cultured. 3. The method of claim 1 or 2, wherein the bacteria are cultured in a medium comprising 21-24 g of meat extract, 7.1-7.9 g of yeast extract, 2,3- 2.7 g sodium chloride, 0.47-0.53 g sodium acetate, 1.8-2.2 g disodium hydrogen phosphate, 1.8-2.2 ml 25 70% sodium lactate, 1.8-2.2 ml 50% ammonium lactate 2.8 It contains -3.2 mg aneurine, 2.8-3.2 mg nicotinic acid, and 2.8-3.2 g glucose.