BG67525B1 - Method of obtaining an agent with antitumour activity, an agent with antitumour activity, and use of the agent - Google Patents

Abstract

The present invention relates to a method for obtaining an agent with antitumor activity from a BCG vaccine, to an agent with antitumor activity based on a BCG vaccine, and to its use as a means for the prevention and treatment of cancer diseases. The method according to this invention provides for the production of medications with different potencies of impact which enables their adequate dosing. The agent obtained according to the method exhibits reduced toxicity due to the low content of live bacteria, and a higher curative effect than the known agents. In the present invention the problem is resolved by a method for obtaining the agent by mixing three fractions: fraction 1 containing live Mycobacterium bovis bacteria from BCG vaccine; fraction 2 representing a suspension of inactivated Mycobacterium bovis bacteria from BCG vaccine, and fraction 3 representing the filtrate of a suspension of ultrasonically destroyed Mycobacterium bovis bacteria from BCG vaccine in quantities such that the ratio between the three fractions is 1:10:100 units by volume. The method results in the production of an antitumor agent consisting of fraction 1 which comprises live Mycobacterium bovis bacteria, fraction 2 which comprises a suspension of inactivated Mycobacterium bovis bacteria, and fraction 3 which comprises the filtrate of a suspension of ultrasonically destroyed Mycobacterium bovis bacteria cells in ratio 1:10:100 by volume. In addition to live bacteria, the agent contains killed bacteria and bacterial parts, bacteria cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, wax, proteins, carbohydrates, lipids and nucleic acids. The agent obtained by this method is intended for the treatment of oncological diseases affecting various organs and systems in the human body. It is particularly suitable for preventing the onset of oncological diseases in individuals at risks as well as for the prevention of recurrences.

Description

(54) METHOD FOR OBTAINING AGENT WITH ANTI-TUMOR ACTIVITY, AGENT WITH ANTI-TUMOR ACTIVITY AND ITS USE

Field of technique

The invention relates to a method for obtaining an agent with antitumor activity from a BCG vaccine, to an agent with antitumor activity based on a BCG vaccine and its use as an agent for the prevention and treatment of oncological diseases.

Prior art

From EP 1 881 838 B1 is known a method for obtaining an agent with antitumor activity by mixing two fractions obtained from a standard BCG vaccine containing Mycobacterium bovis, wherein fraction 1 containing live bacteria is obtained as a standard BCG vaccine containing 500 pg dry substance, is suspended in 1 ml of distilled water by first diluting the suspension with saline or distilled water to a concentration of 100 to 10 pg/ml BCG, and second diluting the suspension with saline to a concentration of 10 to 0.001 pg/ml BCG and fraction 2 was obtained by adding BCG vaccine standard solution to distilled water and further diluting with saline, then the suspension was sonicated for 8-12 min, left at 4-8°C for 2 to 60 days, preferably 2 to 20 days, then filtered. The two fractions are mixed in a ratio of 0.01:1 to 1:0.01. The resulting agent is used to obtain a medicament for the treatment of solid tumors, cancer of the throat, breast, stomach, liver, bladder, colon, skin, brain tumors, malignant melanoma and metastases originating from of them in all systems of the human organism.

Technical essence of the invention

The problem to be solved is to develop a method of obtaining an agent with antitumor activity for the treatment of cancer diseases based on BCG vaccine, which has lower toxicity for better patient tolerance and at least equal antitumor activity with the live vaccine and at the same time ensure obtaining preparations with a controlled strength of effect, giving the possibility of choosing an appropriate preparation for the individual patient.

This problem, according to the invention, is solved by a production method that consists in mixing three fractions - fraction 1 containing live bacteria Mycobacterium bovis from BCG vaccine, and fraction 2, representing a suspension of killed bacteria Mycobacterium bovis from BCG vaccine and fraction 3, representing a filtrate from a suspension of sonicated Mycobacterium bovis bacteria from a BCG vaccine taken in quantities providing a ratio between them of 1:10:100 volume units, the first step being the preparation of fraction 1 by dissolving a standard ampoule of BCG vaccine in a standard solvent , diluting the resulting suspension further with saline or distilled water to a concentration of 100 to 10 pg/ml and re-dilution to a concentration of 10 to 0.001 pg/ml, obtaining fraction 2 by heat treatment of a standard BCG vaccine diluted with saline or distilled water to a concentration of 100 to 0.01 μg/ml and obtaining fraction 3 by diluting standard BCG vaccine with 3 to 5 ml saline or 4 to 6 ml distilled water and sonicating the suspension for 8 to 12 min, then the suspension is left for 2 to 60 days, preferably 2 to 20 days at a temperature of 4-8°C, then filtered.

Another object of the invention is an antitumor preparation obtained by the method consisting of fraction 1 containing live Mycobacterium bovis bacteria, fraction 2 representing a suspension of killed Mycobacterium bovis bacteria, and fraction 3 representing a filtrate of a suspension of sonicated bacteria cells Mycobacterium bovis in a volume ratio of 1:10:100 and contains, in addition to live bacteria and killed bacteria, parts of bacteria, bacterial cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, wax D, proteins, carbohydrates, lipids and nucleic acids .

Another object of the invention is the use of the agent obtained by the method to obtain a drug for the treatment of oncological diseases affecting various organs and systems in the human body. The agent is particularly suitable for use in the prevention of the occurrence of oncological diseases in individuals at risk and for the prevention of the occurrence of recurrences.

The advantages of the method according to the invention consist in the fact that it ensures the preparation of medicines with different potency, allowing adequate dosing. The agent obtained by the method exhibits reduced toxicity due to the low content of living bacteria and an unexpectedly higher healing effect than the known agent.

The invention is illustrated by the following exemplary embodiments, without being limited to them.

Exemplary embodiments of the invention Example 1.

Preparation of fraction 1 - live BCG bacteria

One standard ampoule of BCG vaccine containing 500 μg of dry substance is suspended in 1 ml of distilled water, the resulting suspension is diluted with saline to a concentration of 100 μg/ml, then further diluted with saline to a concentration of 10 μg/ml. Live cell content is controlled. Fraction 1 contains, in addition to living cells, products of their vital activity, metabolites.

Obtaining fraction 2 - killed bacteria. A standard ampoule of BCG vaccine is dissolved in saline or distilled water to a concentration of 100 μg/ml and subjected to heat treatment at 125°C and 1.5 atm for 20 min. Fraction 2 contains, in addition to killed cells, products of their vital activity, parts of cells, metabolites.

Preparation of fraction 3 - filtrate. A standard ampoule of BCG vaccine is suspended in a standard solvent. The suspension is diluted with 3 ml of physiological solution and treated with ultrasound for 8 min and left in a refrigerator at a temperature of 4-8°C for 2 days. On the 1st and 2nd day, control is carried out for the presence of live bacteria. The suspension was then filtered through a sterile Falcon filter with a pore size of <0.1 mm. The resulting filtrate contains substances released from the cell when the cell membrane breaks down, such as parts of bacteria, bacterial cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, carbohydrates, lipids and nucleic acids.

The three fractions are mixed in a volume ratio of 5:50:500.

The agent with antitumor activity according to the invention is administered to patients strictly intradermally on the volar part of the hand as a Mantoux in an amount of 0.1 ml to 0.2 ml.

Example 2.

Preparation of fraction 1 - proceed as in example 1, in which the suspension is first diluted to a concentration of 50 pg/ml and then - to 0.1 pg/ml.

Preparation of fraction 2 - proceed as in example 1, at a concentration of 1 pg/ml.

Preparation of fraction 3 - proceed as in example 1, in which the standard ampoule of BCG vaccine is dissolved with a standard solvent and the suspension is diluted with 4 ml of distilled water. It is treated with ultrasound for 9 min and left in a refrigerator at a temperature of 4-8°C for 10 days, during which time it is periodically checked for the presence of live bacteria. Filter as in example 1.

The three fractions are mixed in a volume ratio of 75:750:7500.

Example 3.

Preparation of fraction 1 - proceed as in example 1, where the suspension is diluted with distilled water to a concentration of 10 pg/ml and further diluted with physiological solution to a concentration of 0.001 pg/ml.

Preparation of fraction 2 - proceed as in example 1, at a concentration of 0.01 pg/ml.

Preparation of fraction 3 - proceed as in example 1, in which the suspension is diluted with 5 ml of physiological solution, then treated with ultrasound for 10 min and left in a refrigerator at a temperature of 4-8°C for 20 days, then filtered as in Example 1.

The resulting fractions are mixed in a volume ratio of 5:50:500.

Example 4.

Preparation of fraction 1 - proceed as in example 1.

Preparation of fraction 2 - proceed as in example 1.

Preparation of fraction 3 - proceed as in example 1, where the suspension is treated with ultrasound for 12 min and left at a temperature of 4-8°C for 40 days, after which it is filtered as in example 1.

The resulting fractions are mixed in ratios of 1:10:100.

Example 5.

Preparation of fraction 1 - proceed as in example 1, where the suspension is first diluted with distilled water to a concentration of 20 pg/ml and further diluted with physiological solution to a concentration of 0.01 pg/ml.

Preparation of fraction 2 at a concentration of 0.1 pg/ml.

Preparation of fraction 3 - proceed as in example 1, where the suspension, diluted with 4 ml of physiological solution, is treated with ultrasound for 10 min and left to stand at 4-8°C for 60 days.

The resulting fractions 1, 2 and 3 were mixed in ratios of 1:10:100.

Examples of the use of the product and data on the effect

The data are presented for patients with different localizations of cancer, men and women, without selection by age and stage of the disease for six months in all offices applying the remedy in the country. A total of 582 people passed.

Figure 1 shows all patients who passed, grouped into 5 groups according to their survival time after starting treatment with the agent according to the invention in years. As can be seen from the graph, the most patients visited the offices for 5 years and more, 34.3%, followed by new patients - up to 1 year, which are 29.5%. Over three years, overall survival with treatment with the agent alone is 55.4%, i.e. more than half of the patients, regardless of the stage of the disease, survive for 5 years or more, treated only with the drug. Taking into account the fact that almost no patients come in the 1st and 2nd stages of the disease, this is a pretty good treatment indicator.

The data for breast cancer patients are presented in the following figure 2. It is clear that 95% of these patients are women. While men are a negligible percentage. According to global data, the most common localization of cancer in women is breast cancer. 284 patients treated with the agent according to the invention, after completion of radio- and chemotherapy and regardless of taking hormonal treatment depending on the hormonal status of the patients, are presented.

The following are the data for colon cancer (sigma, rectum, anus, etc.), presented in Figure 3. The results obtained are very similar to the previous ones, with 5 years and more surviving over 34% of the patients. It is the second most frequent location of cancer in men and women in the world.

Prostate cancer is in first or second place in the world in terms of localization in men, immediately before or after lung cancer. The obtained results, according to figure 4, are very promising, where almost 50% of patients survive for more than 5 years and are considered cured in medicine.

Figure 5 includes patients with ovarian cancer, endometrial cancer, and uterine cancer. They are united due to the small number of patients with separate localizations in cancer of the female genital organs. Regardless of the various courses and prognoses of these diseases, the main conclusion is that the five-year survival of this group of patients is significant - over 45%.

For bladder cancer patients, the five-year survival rates presented in Figure 6 (57.9%) are impressive, nearly 60%, which is a serious achievement considering the diverse stages of the disease with which patients were observed after multiple relapses and TURs and often even before total bladder extirpation.

The following localizations (cancer of the throat, tongue, larynx and tonsils) also deserve attention according to Figure 7 and Figure 8, because of their high disability and poor prognosis, as well as melanoma patients.

It is important to note that the treatment with the agent according to the invention is effective, easy to carry out, without side effects (hair loss, radiation complications, severe malaise, etc.), sparing the body, cheap and ensuring a long remission without relapse and dissemination of the disease , in a normal, even comfortable way of life for cancer patients.

Claims (3)

A method of obtaining an agent with antitumor activity, which includes obtaining a first fraction by dissolving a standard ampoule of BCG vaccine in a standard solvent, further diluting the resulting suspension with saline or distilled water to a concentration of 100 to 10 microg/ml, and diluting again to concentration of 10 to 0.001 microg/ml and obtaining another fraction by diluting standard BCG vaccine with 3 to 5 ml of saline or 4 to 6 ml of distilled water and sonicating the suspension for 8 to 12 min, then allowing the suspension to stand for 2 to 60 days, preferably 2 to 20 days at a temperature of 4-8°C, then filtered, characterized in that fraction 1 is obtained containing live Mycobacterium bovis bacteria from a standard BCG vaccine diluted to concentration from 100 microg/ml to 0.001 microg/ml, fraction 2 is obtained by heat treatment carried out at 125°C for 20 min on Mycobacterium bovis bacteria from a standard BCG vaccine diluted to a concentration of 100 to 0.01 microg/ml, containing killed bacteria, as well as products of their vital activity, parts of cells and metabolites, and fraction 3 is obtained, which is a filtrate of a suspension of Mycobacterium bovis bacteria from standard BCG vaccine sonicated bacteria, where all three fractions are mixed in ratios 1: 10:100 volume units. An agent with antitumor activity obtained according to the method of claim 1, characterized in that it consists of fraction 1 containing live bacteria Mycobacterium bovis from BCG vaccine, fraction 2 containing killed bacteria Mycobacterium bovis from BCG vaccine and fraction 3 representing filtrate from suspension bacteria Mycobacterium bovis from BCG vaccine of bacteria destroyed by ultrasound and is in liquid form. Use of an agent with antitumor activity according to claims 1 and 2, for obtaining a medicament for intradermal administration in an amount between 0.1 ml and 0.2 ml in the prevention and treatment of oncological diseases, in particular throat, breast, stomach cancer , of the liver, of the lungs, of the bladder, of the large intestine, of the skin, brain tumors, cancer of the genital system, prostate cancer, as well as metastases from them in all organs and systems of the human body.