RU2555327C2 - Immunobiological agent for treating urinary bladder cancer and method of application thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with an immunobiological agent for treating urinary bladder cancer, based on BCG vaccine, including a peptidoglycan fragment, which contains diaminopimelic acid as a part of its structure. The group of inventions also deals with a method of applying the claimed immunobiological agent for the treatment of urinary bladder cancer.

EFFECT: group of inventions provides an increased anti-tumour activity.

5 cl, 5 ex, 5 dwg, 4 tbl

Description

Technical field

The invention relates to immunology, namely to immunobiological compositions, and can be used as an immunostimulating agent for the treatment of bladder cancer, as well as for the prevention of relapse of bladder cancer after surgical treatment.

State of the art

Bladder cancer (RMP) is one of the urgent problems of modern health care. According to WHO, RMP accounts for 3% of all detected malignant diseases and 70% of all neoplasms of the urinary system in the world. The main treatment for RMP is the surgical method. However, the results of independent surgical treatment of patients with RMP often remain unsatisfactory. After removal of the tumor, relapse of the disease is observed in 60 - 70% of patients.

The search for methods to improve the results of surgical treatment is an important task of modern oncourology.

A known method of treating superficial cancer of the bladder (Matveev B.P., Figurin K.M. and Karyakin O. B. "Bladder cancer", Moscow, 2001), in which the authors propose the introduction of antitumor drugs into the bladder after surgery tumor removal. However, the disadvantage of this method is the high toxicity of chemotherapy drugs that act on the entire wall of the bladder.

A solution is known (RF patent No. RU 2290096), in which a method for administering chemotherapeutic agents using microirrigators that are sutured into the submucosal layer of the bladder of patients after surgery is proposed. This method allows to reduce the overall toxicity of chemotherapeutic agents due to local administration, however, it has the disadvantage associated with the high invasiveness of the method, since it requires additional surgical intervention to remove microirrigators.

Also known is the solution according to patent US 5194257, which claims the use of Calmett-Guerin bacilli (BCG), which are introduced into the bladder after surgical removal of the tumor. This decision is based on the fact that BCG is a powerful nonspecific immunostimulant that causes a complex of local immune reactions, in which T and B lymphocytes, macrophages, and a number of cytotoxic cytokines are involved, which ensures its antitumor activity.

This technical solution as the closest to the claimed by the composition of the active substance and the method of its use is selected by the authors of the claimed invention for the prototype.

The disadvantages of the prototype include the following:

1) a relatively low immunostimulating activity, which requires the introduction of high doses of BCG and leads to an increase in toxicity;

2) a short duration of the effect, which requires multiple instillation of BCG.

Thus, in the prior art there is an urgent need to develop an effective drug for the treatment of bladder cancer after surgical removal of tumors, which would be devoid of these disadvantages.

Disclosure of the present invention.

The objective of the present invention is to provide an immunobiological agent based on BCG vaccine with high immunostimulating activity and prolonged action, with the possibility of its use in the treatment of bladder cancer, with the provision of induction of a wide range of immune reactions generated by additional stimulation of the innate immune system by activating NOD receptors one.

The above objective of the present invention is solved due to the fact that an immunobiological agent for the treatment of bladder cancer based on BCG vaccine has been created, and it further includes a peptidoglycan fragment containing diaminopimelic acid in its structure taken in an effective amount. Peptidoglycan fragments are peptidoglycan fragments obtained from the bacterial cell wall. Peptidoglycan fragments obtained by chemical synthesis can also be used as peptidoglycan fragments. One dose contains:

BCG - from 10 ³ CFU / dose to 10 ¹⁰ CFU / dose,

low molecular weight fragment of peptidoglycan - from 10 pg / dose to 10 mg / dose.

The claimed immunobiological agent is used in an effective amount for the treatment of bladder cancer.

The antitumor effect of BCG is associated with its immunostimulating properties. It is known that these bacteria include structures that are ligands of the receptors of the innate immune system (TLR4, TLR5, etc.). The interaction of BCG components with these receptors leads to the activation of various transcription factors (for example, transcription factor NF-kB) that control the expression of a number of pro-inflammatory molecules.

So, it was shown that the introduction of BCG enhances the expression of adhesion and co-stimulatory molecules, including intracellular adhesion molecules ICAM-1, which affect the binding of T-lymphocytes and neutrophils to tumor cells. There is also literature data indicating that the introduction of these bacteria increases the expression of HLAII, CD80, CD1b molecules, induces the expression of a number of cytokines, such as interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), granulocyte macrophage growth factor (GM-CSF).

A peptidoglycan fragment containing diaminopimelic acid is an agonist of NOD receptor 1. Diaminopimelic acid is the smallest fragment that this receptor recognizes. Moreover, a change in the composition of other amino acids and monosaccharides that make up the peptidoglycan allows you to modulate the activity of this compound.

According to published data, ligands of NOD receptor 1 are capable of enhancing caspase 9 mediated apoptosis. Activation of the NOD1 receptor enhances a number of immune responses, for example, enhanced phagocytosis, activation of mature T cells, proliferation and maturation of B cells, processing and presentation of antigen by dendritic cells, direct activation of memory B cells and subsequent production of antibodies, including IgG, and others (Palm NW Medzhitov R., Pattern recognition receptors and control of adaptive immunity. Immunol Rev. 2009 Jan; 227 (1): 221-33).

The resulting immunobiological agent containing BCG vaccine and low molecular weight fragments of peptidoglycan provides a significantly higher level of induction of immune responses compared to the intensity of those effects that are triggered by the use of individual components that make up its composition.

The minimum number of studies that is necessary to prove the effectiveness of an immunobiological agent developed by the authors includes a comparison of the ability to induce immune responses, the study of antitumor activity and toxicity.

Brief Description of the Figures

Figure 1 presents the scheme of the reporter design, which is under the control of the NF-kB-dependent promoter.

Figure 2 presents a histogram showing the change in the activity of the transcription factor NF-kB in THP-1 cells when immunobiological agents containing various amounts of BCG and diaminopimelic acid are added to them.

Y-axis - optical density OD415

The abscissa axis is the concentration of diaminopimelic acid (mg / dose)

Axis of applicate - BCG concentration (CFU / dose)

Figure 3 presents a histogram showing the change in the concentration of the cytokines IL-1a, IL-6, MCP-1, CXCL-1, MIP-1a in THP-1 cells with the addition of immunobiological agents based on BCG vaccine. The ordinate axis - the concentration of cytokines, PG / ml

Abscissa axis: 1-IL-1α, 2-IL-6, 3-MCP-1, 4-CXCL-1, 5-MIP-1α

Figure 4 presents a diagram of an experiment to test the antitumor activity of an immunobiological agent based on BCG. The abscissa indicates the time from the beginning of the experiment (week), A - the introduction of N-methyl-M-nitrosourea, B - the introduction of physiological saline, C - the introduction of BCG vaccine, D - the introduction of an immunobiological agent based on BCG.

Figure 5 presents a histogram showing the change in the toxicity coefficient in THP-1 cells when immunobiological agents containing different amounts of BCG and diaminopimelic acid are added to them.

The ordinate axis is the toxicity coefficient,%.

The abscissa axis is the concentration of diaminopimelic acid (mg / dose).

Axis of applicate is the concentration of BCG (CFU / dose).

Examples of the implementation of the present invention

Example 1

Determination of doses of BCG and a fragment of peptidoglycan containing diaminopimelic acid in the composition of the developed immunobiological agent Based on published data, the main mechanism of BCG antitumor activity is the stimulation of immune responses. The purpose of this experiment was to determine the dose of the active substances of an immunobiological agent based on BCG by analyzing their immunostimulating properties. In the first stage of invasion, BCG activates various receptors of the innate immune system. Another component of an immunobiological agent, a peptidoglycan fragment containing diaminopimelic acid in its structure, is also an agonist of receptors of the innate immune system — NOD receptors 1. Activation of the corresponding BCG receptors and peptidoglycan fragments leads to an increase in the activity of transcription factors (including the transcription factor NF-kB) that regulate the expression of various pro-inflammatory proteins and, as a consequence, the development of certain immune responses. Thus, the evaluation of the activity of the transcription factor NF-kB will indirectly assess the effectiveness of stimulation of immune responses. To determine the activity of NF-kB, a model based on the THP-SEAP human monocytic leukemia cell line containing the alkaline phosphatase gene under the control of the NF-kB-dependent promoter was selected (see Fig. 1).

Cells were passaged on 25 cm ² culture vials in RPMI growth medium with 10% fetal serum. For analysis, cells were scattered on a 96-well plate in an amount of 10 ⁵ cells per well. After sieving the cells, the plate was incubated at + 37 ° C and 5% CO ₂ for 24 hours.

Further there was obtained a number of immunological agents based on BCG vaccine (in the following concentrations: 0, ¹⁰ February CFU / dose, March ¹⁰ CFU / dose, April ¹⁰ CFU / dose, May ¹⁰ CFU / dose, June ¹⁰ CFU / dose of 10 ⁷ cfu / dose, 10 ⁸ CFU / dose, 10 ⁹ CFU / dose, 10 ¹⁰ CFU / dose, 10 ¹¹ CFU / dose), and a fragment of the peptidoglycan: L-alanine-γ-D-glutamylmesodiaminopimelic acid (in the following concentrations: 1 pg / dose, 10 pg / dose, 100 pg / dose, 1 μg / dose, 10 μg / dose, 100 μg / dose, 1 mg / dose, 10 mg / dose, 100 mg / dose). Dose refers to the amount of the drug intended for a single (per 1 instillation) injection into the bladder. The volume of the drug recommended for one instillation into the bladder is 50 ml (instructions for the preparations Immuron-vak, Uro-BCG medak). Since the preparation contains live bacterial cells, it is recommended to use any isotonic solvent of appropriate pharmaceutical quality for dilution. In this case, we used physiological saline.

After reconstitution, 10 μl from each sample was added to the corresponding wells of the plate.

Then the cells were incubated for 18 hours. Then, growth medium was removed from the plate wells and 100 μl of p-nitrophenyl phosphate solution (4 mg / ml) was added. Next, the activity of the alkaline phosphatase enzyme was determined by measuring the optical density of the solution at a wavelength of 405 nm 3 times with an interval of 10 minutes. In this case, the change in the optical density of the solution is proportional to the amount of alkaline phosphatase enzyme, and therefore, proportional to the activity of the transcription factor NF-kB. The results obtained are presented in Table 1.

Table 1 BCG concentration (CFU / dose) The concentration of L-alanine-γ-D-glutamyl-meso-diaminopimelic acid 0 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ 10 ⁸ 10 ⁹ 10 ¹⁰ 10 ¹¹ 0 pg / dose 0 0.18 0.2 0.25 0.34 0.55 0.62 0.95 1.01 1.05 1.40 1 pg / dose 0.1 0.09 0.21 0.30 0.33 0.57 0.65 0.92 1.05 1.10 1.41 10 pg / dose 0.05 0.08 0.47 0.50 0.70 1,08 0.90 1.00 1.21 1.35 1,58 100 pg / dose 0.25 0.23 0.53 0.74 1,04 1.10 1,11 1.10 1.23 1.42 1.71 1 ng / dose 0.27 0.27 0.55 0.80 1,11 1.41 1.40 1.41 1.41 1,58 1,61 10 ng / dose 0.30 0.28 0.61 0.89 1.19 1.57 1.40 1.42 1.43 1,63 1,63 100 ng / dose 0.41 0.49 0.78 0.91 1.26 1,59 1,56 1,63 1,62 1.71 1.78 1 mcg / dose 0.46 0.51 0.83 1,11 1.32 1,60 1.75 1.75 1.81 1.84 1.90 10 mcg / dose 0.49 0.58 0.92 1.17 1.51 1.72 1.78 1.79 1.87 1.87 1.99 100 mcg / dose 0.57 0.61 1,02 1.23 1.76 1.80 1.89 1.84 1.90 1.98 2.03 1 mg / dose 0.63 0.67 1.30 1.43 1.79 1.91 2.08 1.80 1.95 2.01 1.98 10 mg / dose 0.81 0.75 1,54 1.78 1.93 2.05 2.11 2.15 2.00 2.12 1.85 100 mg / dose 0.94 one 1.80 2.20 2.15 2,30 2.25 2.22 2,35 2.40 1.83

For clarity, the data obtained are also presented in the form of a three-dimensional histogram (see Fig. 2), where the ordinate axis is the optical density OD415, the abscissa axis is the concentration of L-alanine-γ-D-glutamylmesodiaminopimelic acid (mg / dose), the applicate axis is the concentration BCG (CFU / dose)

The L-alanine-γ-D-glutamylmezodiaminopimelic acid used in the experiment was obtained from the bacterial cell wall (E. coli) according to the standard procedure (J. Heijenoort, L. Elbaz, P.MI & Je, A. Petit, E. Bricas, J. Ghuysen, Structure of the meso-Diaminopimelic Acid Containing Peptidoglycans in Escherichia coli B and Bacillus megaterium KM, Biochemistry, 1969, No. 8 (1), 207-213): γ-D-glutamyl mesodiaminopimelic acid was isolated by chromatography using two connected Sephadex G-50-G25 columns sequentially, followed by final cleaning by reverse-phase high-performance liquid chromatography graphs on column C18. An additional experiment was also conducted with L-alanine-γ-D-glutamylmezodiaminopimelic acid obtained by chemical synthesis according to the standard method (A. Roychowdhury, M. Wolfert, G. Boons Synthesis and Proinflammatory Properties of Muramyl Tripeptides Containing Lysine and Diaminopimelic Acid Moieties, ChemBioChem 2005, 6, 2088-2097). L-alanine-γ-D-glutamylmezodiaminopimelic acid was synthesized using standard methods of peptide synthesis from derivatives of mesodiaminopimelic acid, glutamine and alanine.

The results of experiments performed using peptidoglycan fragments obtained by various methods (chemical synthesis and isolation of bacteria from the cell wall) did not differ.

As can be seen from the data presented, an immunobiological agent based on BCG and low molecular weight fragments of peptidoglycan effectively activates the main pro-inflammatory transcription factor NF-kB in the concentration range of BCG (10 ³ CFU / dose - 10 ¹¹ CFU / dose), L-alanyl-γ-D- glutamylmezodiaminopimelic acid (10 pg / dose - 100 mg / dose). In this case, a significant increase in the intensity (potentiation) of the effects occurs, since the activation of the transcription factor NF-kB under the action of an immunobiological agent is much higher than the sum of the levels of NFkB activation by its individual components.

Example 2

Estimation of the duration of the effect of an immunobiological agent based on BCG and peptidoglycan fragments containing diaminopimelic acid in their structure

The purpose of this experiment was to compare the duration of the effect of the developed immunobiological agent compared with the BCG vaccine (by the ability to activate the transcription factor NFkB).

To determine the activity of NF-kB, a model based on the THP-SEAP human monocytic leukemia cell line containing the alkaline phosphatase gene under the control of the NF-kB-dependent promoter was selected (see Fig. 1).

Cells were passaged on 25 cm ² culture vials in RPMI growth medium with 10% fetal serum. For analysis, cells were scattered on a 96-well plate in an amount of 10 ⁵ cells per well. After sieving the cells, the plate was incubated at + 37 ° C and 5% CO ₂ for 24 hours.

After a day, the following samples were added to the cells:

1) an immunobiological agent based on BCG vaccine (10 ⁵ CFU / dose), a fragment of a peptidoglycan containing diaminopimelic acid in its structure (in this case L-alanine-γ-D-glutamylmezodiaminopimelic acid, 100 ng / dose),

2) BCG (10 ⁴ CFU / dose),

3) a fragment of a peptidoglycan containing diaminopimelic acid (L-alanine-γ-D-glutamylmezodiaminopimelic acid, 100 ng / dose).

The activity of the transcription factor NF-kB was determined after 6, 12, 18, 24 hours.

Then, growth medium was removed from the plate wells and 100 μl of p-nitrophenyl phosphate solution (4 mg / ml) was added. Next, the activity of the alkaline phosphatase enzyme was determined by measuring the optical density of the solution at a wavelength of 405 nm 3 times with an interval of 10 minutes.In this case, the change in the optical density of the solution is proportional to the amount of alkaline phosphatase enzyme, and therefore proportional to the activity of the transcription factor NF-kB. The results obtained are presented in Table 2.

table 2 Time after addition of ligands, h 6 12 eighteen 24 Immunobiological
means
based on BCG 0.15 0.94 0.97 0.68 BCG 0.8 0.23 0.11 0.05 Phosphate buffer 0 0.05 0.01 0,03

The results of experiments performed using peptidoglycan fragments obtained by various methods (chemical synthesis and isolation of bacteria from the cell wall) did not differ.

As can be seen from the data presented, an immunobiological agent based on BCG and low molecular weight fragments of peptidoglycan has a prolonged effect compared to BCG.

Example 3

Evaluation of the immunostimulating properties of an immunobiological agent based on BCG vaccine and a fragment of a peptidoglycan containing diaminopimelic acid in its structure

The purpose of this experiment was to compare the immunostimulating properties of the developed immunobiological agent compared to BCG vaccine (in its ability to induce cytokine release).

For this, a THP-1 human monocytic leukemia cell culture model was selected. From literature data it is known that monocytes are one of the main cell populations participating in anti-cancer immunity. In addition, cells of this line express a fairly wide range of cytokines.

To assess the level of expression of cytokines, eBioscience test systems (BMS810FF, BMS821FF) were selected for multiplex analysis, which allow measuring the expression level of 17 cytokines and chemokines.

For the study, we selected such concentrations of the active substances of the immunobiological agent: BCG and a fragment of the peptidoglycan, which lie in the center of the concentration range studied in Example 1.

Cells were passaged on 25 cm2 culture mattresses in RPMI growth medium with 10% fetal serum. For analysis, the cells were scattered on a 96-well plate in an amount of 105 cells per well. After sieving the cells, the plate was incubated at + 37 ° C and 5% CO ₂ for 24 hours. After a day, the following samples were added to the cells:

1) an immunobiological agent based on BCG vaccine (105 CFU / dose), a fragment of a peptidoglycan containing diaminopimelic acid in its structure (in this case L-alanine-γ-D-glutamylmezodiaminopimelic acid, 100 ng / dose),

2) BCG (104 CFU / dose),

3) a fragment of a peptidoglycan containing diaminopimelic acid (L-alanine-γ-D-glutamylmezodiaminopimelic acid, 100 ng / dose).

In the experiment used fragments of peptidoglycan obtained by chemical synthesis (according to the method described in example 1).

Then the cells were incubated for 18 hours. Then determined the level of expression of cytokines using a commercial kit. The results are presented in figure 2, where the ordinate axis is the concentration of cytokines, pg / ml, the abscissa axis: 1-1L-1α, 2-IL-6, 3-MCP-1, 4-CXCL-1, 5-M1P- 1α,

It was shown that the developed immunobiological agent based on BCG vaccine induces the expression of cytokines: IL-1α, IL-6, MCP-1, CXCL-1, MIP-1α, which play an important role in the antitumor immune response. In this case, potentiation of effects occurs: since the level of cytokine expression under the action of an immunobiological agent is much higher than the sum of the levels of cytokine activation by its individual components.

Similar results were obtained in an experiment using peptidoglycan fragments obtained by isolating bacteria from the cell wall (according to the procedure described in Example 1).

Example 4

Evaluation of the antitumor activity of an immunobiological agent based on BCG vaccine and peptidoglycan fragments containing diaminopimelic acid in their structure

The purpose of this example is to evaluate the antitumor efficacy of an BCG-based immunobiological agent in a mouse bladder cancer model induced by N-methyl-N-nitrosourea.

Figure 4 presents a diagram of the experiment. The abscissa indicates the time from the beginning of the experiment (week), A - the introduction of N-methyl-N-nitrosourea, B - the introduction of physiological saline, C - the introduction of BCG, D - the introduction of an immunobiological agent based on BCG.

For the experiment, BALB / c mice were used, females weighing 18-20 g. All animals were catheterized according to the standard method (C. Hung, C. Dodson, S. Hultgren, A murine model of urinary tract infection, Nature protocols, 4, 1230-1243, 2009). Next, all animals were injected with N-methyl-N-nitrosourea (0.2 mg / mouse) in a volume of 50 μl. This procedure was repeated weekly for 10 weeks. Thus, the total dose of N-methyl-N-nitrosourea was 2 mg / mouse. After the course of N-methyl-N-nitrosourea, all animals were divided into three groups, which were introduced

1) an immunobiological agent based on BCG vaccine (10 ⁴ CFU / dose), characterized in that it includes a peptidoglycan fragment containing diaminopimelic acid in its structure (in this case L-alanyl-γ-D-glutamylmesodiaminopimelic acid, 100 ng / dose ),

2) BCG (10 ⁴ CFU / dose),

3) a fragment of a peptidoglycan containing diaminopimelic acid (L-alanyl-γ-D-glutamylmezodiaminopimelic acid, 100 ng / dose.)

For the study, we selected such concentrations of the active substances of the immunobiological agent: BCG and the peptidoglycan fragment, which lie in the middle of the concentration range studied in Example 1.

The drugs were injected into the bladder using a catheter at a therapeutic dose, 3 times at weekly intervals. 12 weeks after the last instillation, the animals were euthanized.

Next, a histological preparation of the bladder was prepared. All organs were placed in casting molds filled with sample freezing medium (Cat. No. 05-9891, Bio-Optica) and frozen in alcohol cooled with liquid nitrogen. Then, using a Shandon cryotome (UK), sections were prepared with a thickness of 10 μm, which were stained with hematoxylin and eosin. Morphological study of sections was carried out at magnifications of a microscope × 100 and × 400. The results are presented in table 3.

Table 3 Histology BCG vaccine BCG vaccine-based immunobiological agent, characterized in that it comprises a peptidoglycan fragment containing in its structure
diaminopimelic acid Physiologists
chesky
solution Number of plots Number of plots Number of plots Transition glue
accurate cancer 43 34 60 Precancer 26 29th 25 Lack of
pathology 31 37 fifteen

Analysis of the histological changes of the obtained samples showed that in the group that received BCG, transitional cell carcinoma occurred in 43% of cases, while in the group that received an immunobiological agent based on BCG vaccine, characterized in that it includes a peptidoglycan fragment containing diaminopimelic acid structure, transitional cell carcinoma was observed in 34% of cases. A precancerous condition was diagnosed in 26% of cases after the introduction of the BCG vaccine and in 29% of cases after the introduction of an immunobiological agent based on BCG.

In the experiment used fragments of peptidoglycan obtained by chemical synthesis (according to the method described in example 1).

Similar results were obtained in an experiment using peptidoglycan fragments obtained by isolating bacteria from the cell wall (according to the procedure described in Example 1).

Based on the results of the work done, we can conclude that the BCG-based immunobiological agent has a pronounced antitumor activity and has a greater therapeutic effect than the prototype BCG.

Example 5

Toxicity assessment of an immunobiological agent based on BCG vaccine and peptidoglycan fragments containing diaminopimelic acid in their structure

The purpose of this study was to determine the safety and potential toxic effects of an immunobiological agent based on BCG vaccine.

At the first stage, the toxicity of the in vitro immunobiological agent was determined. For this, cells of human monocytic leukemia THP-1 were used.

Cells were passaged on 25 cm ² culture vials in DMEM growth medium with 10% fetal serum. For analysis, cells were scattered on a 96-well plate in an amount of 10 ⁵ cells per well. After sieving the cells, the plate was incubated at + 37 ° C and 5% CO ₂ for 24 hours.

Next, BCG-based immunobiological agents were added to the cells with the same concentrations of active substances as in Example 1:

BCG - 0 CFU / dose, February ¹⁰ CFU / dose, March ¹⁰ CFU / dose of 10 ⁴ cfu / dose, May ¹⁰ CFU / dose, June ¹⁰ CFU / dose of 10 ⁷ cfu / dose, 10 ⁸ cfu / dose, 10 ⁹ cfu / dose, 10 ¹⁰ cfu / dose, 10 ¹¹ cfu / dose

L-alanine-γ-D-glutamylmezodiaminopimelic acid - 0 pg / dose, 1 pg / dose, 10 pg / dose, 100 pg / dose, 1 μg / dose, 10 μg / dose, 100 μg / dose, 1 mg / dose 10 mg / dose, 100 mg / dose

Dose refers to the amount of the drug intended for a single (per 1 instillation) injection into the bladder. The volume of the drug recommended for one instillation into the bladder is 50 ml. Since the preparation contains live bacterial cells, it is recommended to use any isotonic solvent of appropriate pharmaceutical quality for dilution. In this case, saline was used.

Cells were incubated for 18 hours. Then, growth medium was removed from the plate wells and 50 μl of chilled alcohol was added to fix the cells. Next, 100 μl of a 0.1% methylene blue solution was added to the fixed cells. After 20 minutes, the wells were washed with distillate from an unbound dye, dried and then extracted with methylene blue with 1% SDS. The optical density was measured at a wavelength of: 540 nm and 620 nm. The result had to be presented as the difference of the values (Z) measured at a wavelength of 620 and 540 nm. The toxicity coefficient of sample T was determined:

, $$T=\frac{\left(Z\mathrm{to}\mathrm{about}ntR\mathrm{about}lb-Z\mathrm{uh}\mathrm{to}\mathrm{from}PeR\mathrm{and}ment\right)*\mathrm{one\; hundred}\%}{Z\mathrm{to}\mathrm{about}ntR\mathrm{about}lb}$$

,

where Z control is the difference in optical density values measured at a wavelength of 620 and 540 nm in the negative control,

Z experiment - the difference in optical density values measured at a wavelength of 620 and 540 nm in the experimental well.

Moreover, T = 0 means that the drug is not toxic and does not affect the survival of this cell culture, and T = 100% means that the drug is toxic and there are no living cells in the well of the tablet.

The results are presented in table 4.

Table 4 BCG concentration (CFU / dose) The concentration of L-alanine-γ-D-glutamyl-meso-diaminopimelic acid 0 10 ² 10 ³ 10 ⁴ 10 ⁵ 10 ⁶ 10 ⁷ 10 ⁸ 10 ⁹ 10 ¹⁰ 10 ¹¹ 0 pg / dose 0 0 0 0 0 0 0 0 0 0 8 1 pg / dose 0 0 0 0 0 0 0 0 0 0 eleven 10 pg / dose 0 0 0 0 0 0 0 0 0 0 fourteen 100 pg / dose 0 0 0 0 0 0 0 0 0 0 fifteen 1 ng / dose 0 0 0 0 0 0 0 0 0 0 17 10 ng / dose 0 0 0 0 0 0 0 0 0 0 21 100 ng / dose 0 0 0 0 0 0 0 0 0 0 29th 1 mcg / dose 0 0 0 0 0 0 0 0 0 0 thirty 10 mcg / dose 0 0 0 0 0 0 0 0 0 0 36 100 mcg / dose 0 0 0 0 0 0 0 0 0 0 38 1 mg / dose 0 0 0 0 0 0 0 one 2 four 41 10 mg / dose 0 0 0 0 0 2 one four 6 9 43 100 mg / dose 9 9 10 fourteen 19 21 24 27 33 38 46

For clarity, the data obtained are also presented in the form of a three-dimensional histogram (see Fig. 5), where the ordinate axis is the toxicity coefficient,%, the abscissa axis is the concentration of L-alanine-γ-D-glutamylmesodiaminopimelic acid (mg / dose), the applicate axis is BCG concentration (CFU / dose)

In the experiment used fragments of peptidoglycan obtained by chemical synthesis (according to the method described in example 1).

Similar results were obtained in an experiment using peptidoglycan fragments obtained by isolating bacteria from the cell wall (according to the procedure described in Example 1).

As can be seen from the presented results, immunobiological agents containing BCG at a concentration of 10 ¹¹ CFU / dose and γ-D-glutamylmesodiaminopimelic acid (concentration 100 mg / dose) had the highest toxicity. The remaining immunobiological agents containing BCG (10 ³ -10 ¹⁰ CFU / dose) and L-alanine-γ-D-glutamylmesodiaminopimelic acid (10 pg / dose -10 mg / dose) either did not have toxicity or had little toxicity. Therefore, these concentrations of BCG and the peptidoglycan fragment can be used to create an immunobiological agent.

The use of BCG and peptidoglycan fragments together in the composition leads to activation of the corresponding BCG receptors and peptidoglycan fragments and to an increase in the activity of transcription factors (including the transcription factor NF-kB), which regulate the expression of various pro-inflammatory proteins and, as a consequence, the development of certain immune reactions.

In the above examples, it was shown that the developed BCG vaccine-based immunobiological agent induces the expression of cytokines: IL-1α, IL-6, MCP-1, CXCL-1, MIP-1α, which play an important role in the antitumor immune response. In this case, potentiation of effects occurs: since the level of cytokine expression under the action of an immunobiological agent is much higher than the sum of the levels of cytokine activation by its individual components. The prolonged effect is achieved due to the potentiation of the induction of immune responses in comparison with the intensity of those effects that are triggered by the application of the individual components included in its composition (example 2).

Similar results were obtained in an experiment using peptidoglycan fragments obtained by isolating bacteria from the cell wall (according to the procedure described in Example 1).

Thus, the inventive step of the present invention is confirmed.

Industrial applicability. The results given in the examples confirm the industrial applicability of the claimed invention and the implementation of the task, namely the creation of an immunobiological agent based on BCG vaccine and a fragment of peptidoglycan containing diaminopimelic acid with high immunostimulating activity and prolonged action, with the possibility of its use in therapy bladder cancer, with the provision of the induction of a wide range of immune reactions, formed due to additional stimulation of the innate immune system through activation of NOD receptors 1.

Claims (5)

1. An immunobiological agent for the treatment of bladder cancer based on BCG vaccine, characterized in that it further includes a peptidoglycan fragment containing in its structure diaminopimelic acid taken in an effective amount. 2. The immunobiological agent according to claim 1, characterized in that peptidoglycan fragments obtained from the bacterial cell wall are used as peptidoglycan fragments. 3. The immunobiological agent according to claim 1, characterized in that peptidoglycan fragments obtained by chemical synthesis are used as peptidoglycan fragments. 4. The immunobiological agent according to claim 1, characterized in that in one dose contains:
BCG - from 10 ³ CFU / dose to 10 ¹⁰ CFU / dose,
low molecular weight fragment of peptidoglycan - from 10 pg / dose to 10 mg / dose. 5. The method of using the immunobiological agent according to claim 1 for the treatment of bladder cancer.

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