PT96789B - PREPARATION PROCESS OF A MEDICATION BASED ON BACTERIAL SILENCERS - Google Patents

Abstract

A bacterial lysate derived from at least one of the following strains: Haemophilus influenzae, serotype B NCTC 8467 Diplococcus pneumoniae, serotypes 1, 2, 3 and 47 NCTC 7465, 7466, 7978 and 10319 Klebsiella pneumoniae NCTC 204 and 5056 Klebsiella ozaenae NCTC 5050 Staphylococcus aureus I-049, I-050, I-051, I-052, I-053, and I-054 Streptococcus viridans I-046, I-047 and I-048 Treptococcus pyogenes, group A NCTC 8191 Branhamella catarrhalis (formerly Neisseria catarrhalis) I-045, NCTC 3622 and 3625 is used to manufacture a drug for treating atopic dermatitis.

Description

Process of preparing a medicine based on bacterial lysates so that

LABORATOIRES OM SA, intends to obtain a privilege of invention in Portugal.

RESUME

The present invention relates to the preparation of a medicament from a bacterial lysate in concentrated and lyophilized form, from one of the following strains: Haemophilus influenzae (serotype b), Diplococcus pneumoniae (serotypes 1,2,3, 47), Klebsiella pneumoniae, Klebsiella ozaenae, Staphylococcus aureus, Streptococcus viridans, Streptococcus pyogenes (serogroup A), Branhamella catarrhalis (formerly Neisseria catarrhalis), which, after culture, are lysed, then mixed with a pharmaceutically acceptable lysate. The drug is intended for the treatment of atopic dermatitis.

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The present invention relates to a process of preparing a medicament for the treatment of atopic dermatitis and more particularly to the new use of a known bacterial lysate, for the manufacture of a medicament for the treatment of the aforementioned disease.

Swiss Patent CH 633,188, owned by the same holder, is now known to be an immunobiotic medicine against infectious diseases of the respiratory tract, containing as an active ingredient a bacterial lysate from at least one of the following strains:

- Staphylococcus aureus 1-049, 1-050, 1-051, 1-052,

1-053, 1-054

- Streptococcus viridans 1-046, 1-047, 1-048

- Neisseria catarrhalis 1-045 and at least one of the following strains:

- Hemophilus influenzae serotype b 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 group A NCTC 8191

- Neisseria catarrhalis NCTC 3622, 3625

NCTC strains are cataloged by the National Collection of Type Cultures, London, Colindale Avenue 175, London NW9 5HT (GB) and are accessible to the public, while strains I were deposited by the aforementioned patent holder on March 14, 1978 , next to the Collection nationale de cultures de microorganismo, Pasteur Institute, 28 Rue du Dr. Roux, 75724 Paris (FR).

All of these strains come from germs responsible for infectious diseases of the respiratory tract.

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-30 bacterial lysate which is the subject of the aforementioned Swiss patent has shown a remarkable effect, and this unexpectedly, on atopic dermatitis, which is a hereditary chronic disease.

Recent studies have shown that atopic dermatitis is linked to a complex immune deficiency, characterized especially by weak reactions of delayed hypersensitivity to cell mediation and an increase in the synthesis of immunoglobulins E (IgE). In addition, the gastrointestinal tract can serve as a gateway for allergens responsible for the disease.

Consequently, the aim of the present invention is a process for the preparation of a medicament for the treatment of atopic dermatitis, as defined in the claim.

1.

Preferably, the bacterial lysate comes from all the strains mentioned in that claim 1 and comes in a concentrated and lyophilized form. The drug is preferably made in the form of capsules for oral administration, each containing 7 mg of lyophilized bacterial lysates.

We now describe, by way of example, the preparation of bacterial lysates, as well as a galenical form of the drug and the result of clinical tests.

Preparation of bacterial lysates

A standard, sterile culture medium, based on all strains, can be prepared; contain the following substances, in the quantities indicated for the final volume of 1 1.

Sodium chloride 2.5 g

Sodium monohydrogen phosphate 2 g

Sodium acetate 0.5 g

Aneurine 0.003 g (continued)

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-4 (continued)

Nicotinic acid 0.003 g Sodium lactate (70% solution) 2 ml Ammonium lactate (50% solution) 2 ml Meat extract 22.5 g Yeast extract 7.5 g Glucose 3 g

The different strains are grown separately under optimized conditions, in liquid medium.

The base culture medium is preferably enriched at 2% with yeast extract and at 0.5% with Fildes extract, for the culture of the strain Hemophilus influenza.

Preferably, the base medium is enriched with 0.5% horse serum and 0.3% with glucose for the cultivation of Diplococcus pneumoniae strains.

Finally, the base medium is preferably enriched with 0.3% glucose for the culture of the other strains, respectively Steptococcus viridans and pyogenes, Klebsiella ozaenae and pneumoniae, Staphylococcus aureus and Branhamella catarrhalis.

These separate cultures are carried out in fermentor, at 37 ^and C and at a pH of 7.0.

After harvesting and counting, the germs are centrifuged and suspended in a saline solution, always separately for each of the strains.

The various bacterial suspensions are then subjected to alkaline lysis, in the standard conditions of pH (about 9-10) and temperature (about 20-40 ^and C), the evolution being monitored under the microscope.

Then, the volume of lysate from each bacterial strain is adjusted according to the number of germs and according to the

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-5 final volume of concentrate. The various lysates are then mixed in the appropriate proportions and then centrifuged. The supernatant is diluted in saline to a final volume calculated for each batch of manufacture. The concentrate is then purified by in-depth filtration and finally sterilized on a 0.2 μ membrane. The product is then tested chemically and microbiologically. Can be stored at 4 ° C.

Preparation of the capsules

Before preparing the chosen galenic form, the bacterial lysate concentrate is neutralized in a known way, then lyophilized from that concentrate to obtain a lyophilisate, which is collected aseptically, sieved and stored under vacuum in a container appropriate. The product obtained, called standard lyophilisate, preferably contains, for 40 mg, 7 mg of lyophilized bacterial lysates, which will constitute the active ingredient of the future medicine. This standard lyophilisate is finally subjected to chemical and microbiological quality control.

Then, capsules of the drug for oral administration were also prepared in a known manner, each containing 40 mg of standard lyophilisate, filling the capsules, for example of the Capsugel type, with a mixture of the said lyophilisate and the appropriate excipients chosen. freely by the expert in the art.

Toxicity tests

Acute toxicity studies in mice, to determine the lethal dose 50 (LD50), with doses up to 5000 mg / kg in weight showed that the drug has no toxic effect. Macro and microscopic exams did not reveal any further changes at the organ level.

Clinical tests

In clinic, tests were performed with the drug prepa72 216

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-6 to study its effects on atopic dermatitis. 26 adult patients with long-term atopic dermatitis (AD) were treated with the drug for 5 months. The drug was administered at the rate of one capsule (7 mg of bacterial lysate) per day, for 1 month and for 10 consecutive days for each of the third, fourth and fifth months of the study. Of these patients, 15 had already received the drug, at the same dosage, for 5 months, but a year earlier.

Particular attention was paid to the degree of severity of pruritus and dermatitis with lichenification, which was evaluated monthly, according to the following scale: 0 ~ absent; 1 = slight; 2 = moderate; 3 = severe and 4 = very severe.

It was considered a clinical improvement since the severity of symptoms (itching and dermatitis) and the use of conventional therapies dropped by more than 50%.

The serum rate of immunoglobulins A, G and M (measured by radial immunodiffusion) and the overall serum rate of IgE (measured by radioimmunological techniques) were determined before the study and after 1 and 5 months of treatment, respectively.

Cell mediation skin tests (Multitest CMI) were also used. Clinical improvement was observed in 57.7% of the 26 patients treated during the 5 months of the study. If you consider the 15 patients who were treated for 2 periods of 5 months, an improvement is noted in 73.3% of cases.

The rates of immunoglobulins A, G, Me E in the 26 patients show a statistically significant reduction after 1 and 5 months of treatment (see table I).

In the 15 patients treated with the drug for two periods, there was a statistically significant drop in the IgE rate at the end of the first period, which was accentuated in the second period (see Table II).

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-7 Skin tests (Muititest CMI) showed a weak response before treatment in 26 patients (reflecting a deficiency of cellular immunity), either 7.21 + 1.5, which increases, after 5 months of treatment, to reach normal values, or 22.25 ± 2.7 mm (with normal mean skin tightening values between about 18 and 28 mm).

Thus, it is clear from the results of the tests carried out that the medicine according to the invention has a notable effect on atopic dermatitis.

Table I: Average rate of immunoglobulins A, G, M and E in 26 AD patients treated for only a period of 5 months.

1 1 j Parameter r Values 1 After 1 month | P - 1 After 5 months | p = J Initials 1 1 I 1 1 1 1 Jig (UI / ml) 332.62 ± 17.29 1 313.23 ± 18.42 | 0.077 1 1 306.15 ± 16.36) 0.012 | | igG (UI / ml) 1844.23 ± 68.18 1687.31 ± 59.62 | 0.001 1672.69 ± 60.72 | 0.004 | JigM (UI / ml) 228.84 ± 12.74 182.31 ± 8.22 | 0.0001 195.73 ± 11.86jO, 002 | | igE (UI / ml) 1 1935.54 ± 468, Ó5 1 1553.08 ± 368.49 | 0.007 l 1286.46 ± 281.38) 0.012) II

(See Table II)

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-8Table II: Average IgE rate in the 15 AD patients treated during 2 periods of 5 months.

i- 1 Period Average IgE values (UI / ml) 1 | of of 1 1 1 study Values initials 1 After 1 month I 1 After 5 months | j First 4593.6 ± 1089.6 1 1 | 4315.4 ± 1014.8 * I J 3304 ± 819.5 ** | | Second 1_ 2961.7 ± 681.6 1 1 12329.7 ± 530.9 *** I | 1888.6 ± 384.33 **** | J_1

* p = 0.038 p = 0.007 p = 0.008 p = 0.014

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Claims (4)

1 - Process for the preparation of a medicine for the treatment of atopic dermatitis, characterized by the cultivation, in a culture medium, of bacteria from at least one of the following strains: Haemophilus influenzae, serotype b NCTC 8467 Diplococcus pneumoniae, serotypes 1,2,3,47 NCTC 7465,7466,7978,10319 Klebsiella pneumoniae NCTC 204, 5056 Klebsiella ozaenae NCTC 5050 Staphylococcus aureus 1-049,1-050,1-051,1-052, 1-053,1-054 Streptococcus viridans 1-046,1-047,1-048 Streptococcus pyogenes group A NCTC 8191 Branhamella catarrhalis 1-045, NCTC 3622, 3625 (formerly Neisseria catarrhalis) then prepare a lysate, with the bacteria thus grown, and mix that lysate with a pharmaceutically acceptable support. Process according to claim 1, characterized in that the bacterial lysate is prepared from all the styles mentioned in claim 1. Process according to claim 1 or claim 2, characterized in that the bacterial lysate is in concentrated and lyophilized form. Process according to one of Claims 1 to 3, characterized in that the medicament is prepared in the form of capsules for oral administration, each capsule containing, as a daily dose, 7 mg of the bacterial lysate.