WO2021168522A1

WO2021168522A1 - Method for obtaining of an antitumor composition, an antitumor composition and its use

Info

Publication number: WO2021168522A1
Application number: PCT/BG2021/000008
Authority: WO
Inventors: Nasya Spartakova HADJIEVA; Milhail Mihailov PETROV; Dora Spartakova HADJIEVA; Zoya Vangelis RACHKOVSKA
Original assignee: Hadjieva Nasya Spartakova; Petrov Milhail Mihailov; Hadjieva Dora Spartakova; Rachkovska Zoya Vangelis
Priority date: 2020-02-25
Filing date: 2021-02-11
Publication date: 2021-09-02
Also published as: EP4110379A1; BG113091A; BG67525B1

Abstract

The present invention relates to a method for obtaining of an anti-tumor composition with antitumor activity, as well as to a composition with antitumor activity, both of them based on the BCG vaccine and its use for prophylaxis and treatment of oncologic diseases. The method according to the present invention provides the obtaining of medications with differentiated impact of effect and allowing adequate dosing. The composition obtained by this method demonstrates reduced toxicity because of the low content of live bacteria and unexpectedly higher therapeutic effect than the already known one. This problem, according to the present invention is solved by method for preparation that includes the mixing of three fractions - fraction 1 containing live bacteria Mycobacterium bovis from BCG vaccine, fraction 2 consisting of suspension of killed bacteria Mycobacterium bovis from BCG vaccine, and fraction 3 that is a filtrate of a suspension of Mycobacterium bovis bacteria from BCG vaccine, destroyed by ultrasound, the fraction quantities being in a proportion of 1 : 10 : 100 volumetric units. The antitumour composition obtained by the method consisting the fraction 1 with live bacteria Mycobacterium bovis, fraction 2 consisting of suspension of killed bacteria Mycobacterium bovis and fraction 3 containing a filtrate of suspension of destroyed by ultrasound cells of Mycobacterium bovis bacteria in a volumetric proportion of 1 : 10 : 100, which contains not only live and killed bacteria but also parts of bacteria, bacterial cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, wax D, proteins, carbohydrates, lipids and nucleic acids. The composition obtained by the present method is intended for the treatment of oncologic diseases that affect different organs and systems in the human body. The composition is especially suitable for prophylaxis of the occurrence of oncologic diseases in predisposed individuals and for prophylaxis to prevent recurrence.

Description

METHOD FOR OBTAINING OF AN ANTITUMOR COMPOSITION,

AN ANTITUMOR COMPOSITION

FIELD OF THE INVENTION

: _'

The present invention relates to a method for obtaining of an antitumor composition from BCG vaccine, as well as to a composition with antitumor activity on the basis of BCG vaccine and use for prophylaxis and treatment of oncologic diseases thereof.

BACKGROUND OF THE INVENTION

EP 1 881 838 B1 discloses a method for preparing an antitumor composition by mixing two fractions obtained from a standard BCG vaccine containing Mycobacterium bovis, where fraction ^ containing live bacteria and obtained as a standard BCG vaccine containing 500 pg dried substance, is suspended in 1 mi of distilled water by first dilution of the suspension with saline solution or distilled water to a concentration from 100 to 10pkg/ml BCG, and second dilution of the suspension with saline solution to a concentration from 10 to 0,001 pkg/ml BCG, and fraction 2 is obtained by addition of standard solution of BCG vaccine to distilled water and additional dilution with saline solution, then the obtained suspension is treated with ultrasound for 8 - 12 minutes, allowed to stay at temperature of 4 - 8°C for 2 to 60 days, preferably 2 to 20 days, and then it is filtered. Both fractions are mixed in a

; i proportion from 0,01:1 to 1:0,01. The obtained composition is used for preparation of a medication for treatment of massive tumors, cancer of: throat, breast, stomach; liver, urinary bladder, colon, skin, as well as brain tumors, malignant melanoma and metastases of these cancers in all systems of the human body

TECHNICAL FIELD OF DISCLOSURE

The problem to be solved is the development of a method for preparation of an antitumor composition on the basis of BCG vaccine intended for treatment of oncologic diseases, with lower toxicity for better tolerance by the patients where the prepared composition possessing at least a similar antitumor activity with the live vaccine, and capable to ensure the obtaining

of preparations with controlled impact of effect, which provide the possibility for selection of a suitable agent for the individual need of the patient.

According to the present invention, this problem is solved by a method for preparation that includes the mixing of three fractions - fraction 1 containing live bacteria Mycobacterium bovis from BCG vaccine, fraction 2 consisting of suspension of killed bacteria Mycobacterium bovis from BCG vaccine, and fraction 3 that is a filtrate of a suspension of Mycobacterium bovis bacteria from BCG vaccine, destroyed by ultrasound, the fraction quantities being in a proportion of 1 : 10 : 100 volumetric units. The first stage of the present method is the obtaining of fraction 1 by dissolution of a standard ampoule of BCG vaccine in a standard solvent, additional dilution of the obtained suspension with saline solution or distilled water to a concentration of 100 to 10 pg/ml and a second dilution to a concentration of 10 to 0,001 pg/ml, following by preparation of fraction 2 through the heat treatment of a standard BCG vaccine, which is diluted with saline solution or distilled water to a concentration of 100 to 0,01 pg/ml and preparation of fraction 3 by dilution of a standard BCG vaccine with 3 to 5ml of saline solution or 4 to 6 ml of distilled water and treatment-of the suspension by ultrasound for 8 to 12 min, then the resulting suspension is remained for 2 to 60 days, preferably for 2 to 20 days at a temperature of 4 - 8°C, and then it is filtered.

Another subject of the present invention is an antitumor composition obtained by the method that comprises fraction 1 with live bacteria Mycobacterium bovis, fraction 2 consisting of a suspension of killed bacteria Mycobacterium bovis and fraction 3 containing a filtrate of a suspension of destroyed by ultrasound cells of Mycobacterium bovis bacteria in a volumetric proportion of 1 : 10 : 100, which contains not only live and killed bacteria but also parts of bacteria, bacterial cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, wax D, proteins, carbohydrates, lipids and nucleic acids.

Another subject of the present invention is the composition use, obtained by the method for preparation of a medicinal composition, intended for treatment of oncologic diseases that affect different organs and systems in the human body. The composition is especially suitable for prophylaxis of the occurrence of oncologic diseases in predisposed individuals and for prophylaxis to prevent recurrence.

The advantage of the method according to the present invention is that it ensures the obtaining of medications with differentiated impact of effect and allowing adequate dosing, The composition obtained by this method demonstrates lower toxicity because of the low content of live bacteria and unexpectedly higher therapeutic effect than the already known composition .

The invention is illustrated by the following examples, which clarify it, without prejudice to the scope of its protection.

EXAMPLES I

Example 1

Preparation of fraction 1 - live BCG bacteria

One standard ampoule of BCG vaccine containing 500 pg of dried substance is suspended in 1ml of distilled water, the resulting suspension is diluted with saline solution to a concentration of 100 pkg/ml, then additionally is diluted by saline solution to reach a concentration of 10 pkg/ml. The content of living cells is controlled. Resulting fraction 1 contains in ddition to living cells, products of their vital activity and metabolites.

Preparation of fraction 2 - killed bacteria.

A standard ampoule of BCG vaccine is dissolved in saline solution or distilled water to a concentration of 100 pg/ml and subjected to heat treatment at 125° C and 1 ,5 atm for 20min. Fraction 2 contains in addition to killed bacteria, products of their vital activity, parts of cells, and metabolites.

Preparation of fraction 3 - filtrate

Standard ampoule of BCG vaccine is suspended in a standard solvent the resulting suspension is diluted with 3 ml of saline solution and treated with ultrasound for 8 min, then left to stay in a refrigerator at a temperature of 4 - 8° C for 48 hours. Control for the existence of live bacteria during the 1^st and the 2^nd day is made. The resulting suspension is further filtered through a sterile filter Falcon with <0,1 mm pore size. The obtained filtrate contains substances released by the cell during cell membrane destruction such as bacterial parts,

bacterial cell walls, inner cytoplasmic membrane, enzymes, ribosomes, bacterial nuclei, carbohydrates, lipids and nucleic acids.

The resulting three fractions are mixed in a volumetric ratio, respectively 5 : 50 : 500

I i

Example 2

Preparation of fraction 1 -proceed in the manner described in example 1 ,; where the resulting suspension is diluted in advance to a concentration of 50 pkg/ml and then to 0,1 pkg/ml.

Preparation of fraction 2 - proceed as in example 1, the working concentration is 11 m g/ml.

Preparation of fraction 3 - proceed in the manner described in example 1 , where the standard ampoule of BCG vaccine is dissolved with a standard solvent and the resulting suspension is diluted with 4 ml of distilled water. The resulting suspension is further treated by ultrasound for 9 min and left to stay in a refrigerator at a temperature of 4 ^ 8 °C for 10 days. The periodic control for presence of live bacteria is made. Followed by filtration as in example !

The resulting three fractions are mixed in a volumetric ratio respectively 75 : 750 : 7500.

Example 3

Preparation of fraction 1 - proceed in the manner described in example 1, where the suspension is diluted with distilled water to a concentration of 10 pkg/ml and then additionally diluted it with saline solution to a concentration of 0,001 pkg/ml.

Preparation of fraction 2 - proceed in the manner described in example 1 , the working concentration is 0,01 pkg/ml.

Preparation of fraction 3 - proceed in the manner described in example 1, where the suspension is diluted with 5ml of saline solution, then the resulting suspension is treated by ultrasound for 10min and left it to stay in a refrigerator at a temperature of 4 - 8°C for 20 days, then is filtered as in example 1.

The resulting fractions are mixed in a volumetric ratio respectively 5 : 50 : 500.

Example 4

Preparation of fraction 1 - proceed in the manner described in example 1.

it !

Preparation of fraction 2 - proceed in the manner described in example 1 Preparation of fraction 3 - proceed in the manner described in example 1, where the suspension is treated with ultrasound for 12 min, remained to stay at a temperature of 4 - 8°C for 40 days, then is filtered as in example 1.

The obtained fractions are mixed in a ratio, respectively 1 : 10 : 100.

Example 5

Preparation of fraction 1 - proceed in the manner described in example 1 , the suspension firstly is diluted with distilled water to a concentration of 20 pkg/ml and then diluted it additionally with saline solution to a concentration of 0,01 pkg/ml.

Preparation of fraction 2, with concentration of 0,1 pkg/ml.

Preparation of fraction 3 - proceed in the manner described in example 1, where processing the resulting suspension is diluted with 4 ml of saline solution and treated with Ultrasound for 10 min and remained to stay at 4 - 8°C for 60 days.

The obtained fractions 1, 2 and 3 are mixed in ratio, respectively 1 : 10 : 100.

Examples for application of the compositin and data of its effect

The submitted data concern the patients with different localisations of cancer diseases, male and female, without selection in age and stage of disease, who passed in a period of six months through all medical nationwide offices which use the composition in the country. The total of treated patients were 582.

Figure 1 shows all past patients, 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, most patients visited the offices for 5 and more years are 34.3%, followed by new patients - up to 1 year, which are 29.5%.

On Fig.1 is indicated all treated patients divided in 5 groups according to their survival time, after beginning the therapy with the composition subject to the invention, in years. As shown on the graph, most are the patients who visited the consulting medical offices for 5 and more years, who are 34.3%, followed by the new patients - up to 1 year, who are 29.5%.

The general survival rate over 3 years in monotherapy with the composition is 55.4%, i. e. more than half of the patients, irrespective the stage of their disease, treated only with the composition according to this invention, survive five and more than five years.

Considering the fact that patients in T^st and 2 ^nd stage of the disease were almost missing, this is a quite good indicator of healing.

Fig.1

Data of breast cancer patients are given in the next figure 2. It is understood that 95% of these patients are women, while men represent an insignificant percentage. According to world published data the most frequent localisation of cancer in women is breast cancer. The data of 284 patients are presented. The patients are treated with the composition subject to this invention, after completion of radio- and chemotherapy and irrespective of the intake of hormonal treatment depending on the hormonal status of the patients.

Fig.2

Data presented below concern colon cancer (sigma, rectum, anus etc.), given in Fig. 3. The obtained results are very similar to the previous ones, the survival of 5 and more years representing 34% of the patients. This is the second in frequency localisation of male and female cancer in the world.

Fig.3

Prostate cancer is ranked first and second in the world in localisation in men, immediately before or after lung cancer. The obtained results according to Fig. 4 are very promising, where almost 50% of the treated patients survive over 5 years and are considered healed according to medicine. Fig.4

Fig.5 includes the data of patients with ovarian cancer, endometrial cancer and uterine cancer. The patients were united because of the low number of patients with specific cancer localisations in female genitals. Apart from the varying course and forecasts for these diseases, the basic conclusion is that more than 5 years survival rate in this population is significant - more than 45%.

Fig.5

For the patients with bladder cancer, the results presented in Fig. 6 about the 5-year survival rate are impressive (57,9%), almost 60%, which is a serious achievement taking into account the various stages of the disease of the monitored patients after multiple relapses and TURs and often with forthcoming total bladder extirpation. Fig.6

The following human cancer localisations of the (cancer of throat, tongue, larynx and tonsils) also deserve attention according to Fig.7 and Fig.8 because of the high level of disabling conditions and bad prognostication, including the melanoma patients.

Fig.7 Fig.8

It is important to mention that the treatment with the composition subject to this invention is effective, easy to perform, without any adverse effect (hair loss, radiation complications, severe malaise etc.), sparing the body, inexpensive and providing long-lasting remission without recurrence and dissemination of the disease and providing normal and even comfortable way of life for the cancer patients.

Claims

PATENT CLAIMS

1. Method for obtaining of an antitumor composition, comprising preparation of first fraction by dissolution of a standard ampoule of BCG vaccine in a standard solvent, additional dilution of the obtained suspension with saline solution or distilled water to a concentration of 100 to 10 pkg/ml, and second dilution to a concentration of 10 to 0,001 pkg/ml BCG, and preparation of another fraction by dilution of a standard BCG vaccine with 3 to 5 ml of saline solution or 4 to 6 ml of distilled water and treating of the suspension with ultrasound for 8 - 12 minutes, then the suspension is remained to stay for 2 to 60 days, preferably 2 to 20 days at a temperature of 4-8°C, and then it is filtered, characterising in that a fraction 1 containing live bacteria is prepared, a fraction 2 is prepared by heat processing of a standard BCG vaccine diluted to a concentration of 100 to 0,01 pkg/ml, and a fraction 3 is prepared, which is a filtrate of a suspension of bacteria destroyed by ultrasound, and all obtained three fractions are mixed in proportion of ratio respectively 1 : 10 : 100 volumetric units.

2. Antitumor composition obtained according to the method according to claim 1 , characterising in that the composition is consisting of the fraction 1 containing the live Mycobacterium bovis bacteria from BCG vaccine, fraction 2 containing the killed Mycobacterium bovis bacteria and fraction 3, which is a filtrate of a suspension of bacteria destroyed by ultrasound.

3. Use of an antitumor composition according to claims 1 and 2 for the manufacture of a medication for prophylaxis and treatment of oncologic diseases, especially Cancer of the throat, breast, stomach, liver, lungs, urinary bladder, colon, skin, brain tumors, genital cancer as well as metastases of such cancers in all organs and systems of the human body.