RU2396967C2 - Method of treating oncological diseases in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, namely to experimental oncology, can be used for treating oncological diseases. A method involves cytostatic chemotherapy of tumours with cyclophosphan and introduction of a drug of herbal raw material. Said drug of herbal raw material is represented with an agent containing peat, humic acids and fulvic acids, amino acids, vitamins, macro-and microelements and prepared by peat processing with ammonia and hydrogen peroxide added at 140°C. The peat agent is introduced into food or potable water at a daily dose 100-200 mg/kg.

EFFECT: use of the invention allows intensifying anticancer and antimetastatic effects of cyclophosphan, lowering its haematotoxicity.

2 cl, 6 tbl, 6 ex

Description

The invention relates to experimental medicine, namely to experimental oncology, in particular, to methods for increasing the efficiency of cytostatic cancer therapy, and can be used to enhance the direct antitumor and antimetastatic activity of cytostatic drugs, reduce their toxic effects on the hematopoiesis system and restore its impaired functions .

It is known that oncological diseases are one of the main causes of mortality, occupying 18-20% in its structure. One of the leading treatments for malignant tumors is chemotherapy with cytostatics. The most commonly used drugs in this group include cyclophosphamide. The main disadvantage of cytostatic chemotherapy is the development of side effects, including toxic effects on the hematopoietic system: for cyclophosphamide, this side effect is the main / 1 /. In connection with the above, the current direction of modern experimental medicine is the development of means to increase the effectiveness of cytostatic chemotherapy by reducing its toxic side effects on the body and enhancing specific activity, primarily antimetastatic. The search for such drugs, called biological response modifiers (biological reaction modifiers), is actively conducted both in Russia and abroad / 20, 15 /. Most of these drugs are based on plant materials. For the development of antitumor drugs, including biological reaction modifiers, an effective research system has been developed, involving the first stage of experiments on small laboratory animals (mice, rats) in order to work out the optimal doses and modes of application of the studied drugs / 5, 17, 20 /.

The urgent problem of the treatment of cancer in agriculture. Birds occupy one of the first places in the frequency of detection of tumors in different animal species. In birds, tumors are described whose origin is associated with all types of tissues (connective, muscle, nervous, epithelial). The first place in the frequency of detection of tumor diseases is occupied by chickens. Tumor diseases of birds are infectious and non-infectious in nature. The most common chickens are non-plastic ones, for example, Marek’s disease, in which dense or focal diffuse tumors, leukemia characterized by damage to the blood-forming organs, and reticuloendotheliosis are found in various organs of the bird. In Russian farms, only Marek’s disease accounts for more than 20% of infectious pathologies, the remaining / 6, 13 / occupy a much smaller proportion (2-5%). In addition, tumor diseases can be the cause of diseases of the organs of egg formation, the incidence of which is from 9 to 30% of all diseases of laying hens, which causes significant economic damage as a result of mortality, culling, reduction and termination of egg production.

A known method of combined cytostatic therapy of malignant tumors / 10 /, which consists in the use of p-tyrosol as a means for the prevention and treatment of toxic enteropathies caused by a cytostatic agent.

The disadvantage of this method is the lack of a wide range of biological activity in the synthetic compound p-tyrosol.

A known method for the treatment of cancer / 3 /, including radiation therapy, chemotherapy and herbal medicine, i.e. treatment with collections of various plants used before surgery during courses of radiation therapy and chemotherapy.

The disadvantage of this method is the extreme complexity of the composition and the complexity of its standardization for biologically active substances.

A known method of treating cancer / 11 /, taken as a prototype, including chemotherapy with cytostatics and / or radiation therapy and the introduction of drugs, and as drugs use tinctures of common viburnum, Japanese Sophora, garlic and Chernobyl in combination with befungin in a therapeutic dose, and all components are mixed 5 minutes before consumption and taken 20-30 minutes before meals three times a day.

The disadvantage of the prototype is the ability of the alkaloids of one of the components of the proposed composition - Sophora - to cause pronounced side effects on the body and the complexity of preparing a necessarily fresh solution of a mixture of components immediately before use for one patient and for the hospital.

The inventive method of treating cancer in the experiment is aimed at eliminating these drawbacks and includes cytotoxic chemotherapy of tumors with cyclophosphamide and the use of a medicinal product based on plant materials, and a peat product containing humic acids, fulvic acids, amino acids, vitamins, is used as a medicine based on plant materials macro- and microelements obtained by treating peat with ammonia in the presence of hydrogen peroxide at 140 ° C. The peat product is introduced into the composition of the feed or drinking water in an amount of 100-200 mg / kg once a day.

The inventive method differs from the prototype in that as part of the therapy of malignant tumors, a peat product is used as a herbal medicine, which contains a wide range of biologically active substances that promote recovery: humic acids, fulvic acids, vitamins, amino acids, macro- and microelements.

It is shown that humic substances have the ability to enhance the activity of metabolic processes in the body, increase its resistance to adverse environmental factors, including the toxic effects of chemical compounds / 19 /. In the 80s of the last century, the Peat Institute of the Academy of Sciences of Belarus carried out studies on the antitumor activity of various preparations from peat - polysaccharides, humic substances and their total fraction, as well as compounds with antiviral and interferonogenic activity / 9 /. It was shown that a preparation based on humic compounds of peat HCT (ammonium nitrogumate) activates the hematopoietic function of the body, increasing the content of red blood cells, hemoglobin, and also stimulates the body's natural resistance of farm animals / 8 /. Humic substances have a pronounced antioxidant activity, which is extremely important, since the activation of peroxidation is the main mechanism for the development of toxic effects during chemotherapy with cytostatics / 4, 16 /.

The presence of vitamins, macro-, microelements in the peat product enriches the diet and helps to prevent deficient conditions for these elements in the body of animals and birds.

Amino acids, including essential amino acids, normalize metabolic processes in the body. The predominant amino acids in quantitative content are aspartic and glutamic acids, proline, histidine, alanine. Aspartic acid plays an important role in carbohydrate metabolism and neutralization of ammonia. Glutamic acid is a neurotransmitter, and can also be used by brain cells as an energy source. Proline is extremely important for the proper functioning of ligaments and joints and strengthen the heart muscle. Arginine slows the growth of tumors, including cancer, by stimulating the body’s immune system, and promotes detoxification processes in the liver (primarily neutralization of ammonia). Valine is an essential amino acid involved in the repair of damaged tissues and to maintain normal nitrogen metabolism in the body / 12 /.

Of the vitamins that make up the drug from peat, the most important for its use in the complex of cytostatic chemotherapy of tumors are vitamin E and beta-carotene, which have a pronounced antioxidant effect and are widely used in clinical oncology / 14 /. Zinc has the ability to enhance this effect. This microelement is one of the main factors in the formation of immunity 111. Iron, which is also a component of the drug, is an integral element of the hematopoiesis process, which is disturbed during chemotherapy.

Thus, the use of a peat agent containing biologically active substances in the cancer treatment method that plays a vital role for the body and is the regulator of many biochemical and physiological processes, improves the effectiveness of cancer treatment.

Another difference of the proposed method is that the use of peat products does not require preliminary operations for the preparation of tinctures, dosing and mixing of the components that oxidize each other, as in the prototype, which simplifies the treatment method.

An important advantage of the proposed method for the treatment of cancer compared to the prototype is that the peat agent used in the method belongs to the class of low-hazard substances, does not have a cumulative effect, does not exhibit embryotoxicity and is standardized in component composition.

The dosage of the peat product was selected experimentally. The optimal dose for mice is 200 mg / kg, for rats - 100 mg / kg.

Due to the revealed properties of the agent, it is possible to increase the antitumor and antimetastatic effects of the cyclophosphamide cytostatic and also reduce the damaging effect of the cytostatic on the hematopoietic system due to the revealed properties of the agent, including the use of peat along with chemotherapy.

Experiments to evaluate the antitumor and antimetastatic effects of peat and its combinations with cyclophosphamide were performed on C57B1 / 6 mice with Ehrlich adenocarcinoma or B16 melanoma and Wistar rats with Pliss lymphosarcoma in accordance with the classical methods of experimental tumor chemotherapy / 5 /. The peat product was introduced using a probe into the stomach. To recalculate the doses, the Freirech formula (1966) / 18 /, widely used in oncopharmacology, was used. The data obtained were processed using non-parametric statistics methods / 2 /. The research results are shown in tables.

The following is an example of obtaining a concentrated stock solution from peat.

Example 1. Peat is mixed with 1.0% aqueous ammonia and 1.75% aqueous hydrogen peroxide in a ratio of 1: 5: 5, heated to 140 ° C with constant stirring and the liquid phase is separated, which is concentrated to the content of 80 mg of dry matter in 1 ml of the product.

Example 2. The effect of funds from peat on the antitumor effect of cyclophosphamide in mice with Ehrlich adenocarcinoma (table 1-2)

Two series of experiments were carried out. The peat agent was started to be administered to the animals 24 hours after the tumor was inoculated once a day at a dose of 200 mg / kg using a probe into the stomach and continued daily for 8 days, cyclophosphamide was administered intramuscularly at a dose of 100 mg / kg on the 4th and 7th day after inoculation. Evaluation of the effectiveness of therapeutic effects was carried out on the 9th day after transplantation of Ehrlich carcinoma.

When combining drugs, the antitumor effect increases by 1.5-2.6 times compared with the group of animals that received one cytostatic. In the second series of experiments, a peat agent also significantly reduces the toxicity of cyclophosphamide in relation to the hematopoietic system, preventing the decrease in the number of leukocytes.

Thus, in these two series of experiments it was shown that in the method of treating cancer, the use of peat agent increases its effectiveness, significantly enhancing the antitumor activity of cyclophosphamide and also significantly increasing the number of peripheral blood leukocytes, reduced as a result of the use of cytostatic.

Table 1 The effect of peat products on the effectiveness of treatment with cyclophosphamide (CF) in mice with Ehrlich adenocarcinoma (series 1) No. p / p Observation group The number of animals in the group The volume of sediment of tumor cells, ml Inhibition of tumor growth,% one The control 10 1.6 ± 0.1 - 2 CF 10 1.1 ± 0.3 * 31 3 Peat Remedy 10 1.0 ± 0.2 * 38 four CF + peat product 10 0.3 ± 0.08 * ** 81 Note: in this and the following tables: *) the differences are significant in comparison with the Control group at Р≤0.05, **) the differences are significant compared with the Cyclophosphamide group at P≤0.05.

table 2 The effect of peat agents on the effectiveness of treatment with cyclophosphamide (CF) mice with Ehrlich adenocarcinoma (series 2) Group Number of mice The volume of sediment of tumor cells, ml Inhibition of tumor growth,% The number of peripheral blood leukocytes, 10 ⁹ / l The number of red blood cells in peripheral blood, 10 ¹² / l The control 10 1.20 ± 0.09 - 12.81 ± 1.40 6.1 ± 0.52 CF 10 0.62 ± 0.07 * 48.3 3.14 ± 0.25 * 3.6 ± 0.40 Peat Remedy 10 0.70 ± 0.09 * 41.7 13.62 ± 1.29 3.3 ± 0.38 CF + peat product 10 0.33 ± 0.04 * ** 72.5 5.40 ± 0.60 ** 2.73 ± 0.31

Example 3. The effect of funds from peat on the antitumor and antimetastatic effects of cyclophosphamide in mice with metastatic tumor of melanoma B-16 (table 3).

To assess the effect of peat products on the antimetastatic activity of cytostatic cyclophosphamide therapy, an experiment was conducted on a metastatic tumor of mouse B-16 melanoma. The peat agent was administered once a day at a dose of 200 mg / kg from 9 to 19 days after tumor inoculation, cyclophosphamide - twice at a dose of 100 mg / kg on days 13 and 17. The antitumor effect was evaluated on the 22nd day after transplantation by the change in the mass of the tumors, the antimetastatic effect was determined by the number of metastases per animal and the frequency of metastasis.

In the method of treating cancer, the use of a peat preparation during treatment with a cytostatic agent can enhance its antitumor effect and reduce the frequency of tumor metastasis by three times compared with cyclophosphamide monotherapy. The inhibition of the activity of the metastasis process was also the most significant in the group of combined cytostatic therapy.

In addition, treatment with cyclophosphamide alone resulted in the death of 33% of the mice, while in the group with combination therapy using peat agents, no deaths were observed (P _φ = 0.02).

Table 3 The effect of peat products on the effectiveness of cyclophosphamide (CF) treatment in mice with B-16 melanoma Group Number of mice Tumor mass Inhibition of tumor growth,% The number of metastases / mouse, pcs The frequency of metastasis,% Inhibition of the activity of the metastasis process,% The control 10 5.50 ± 0.63 - 13.6 ± 2.40 one hundred - Peat Remedy 10 4.35 ± 0.52 20.90 4.20 ± 0.12 * 80 69.2 CF 10 2.76 ± 0.34 * 49.82 7.14 ± 0.95 90 47.8 CF + peat product 10 2.34 ± 0.27 * 57.50 2.25 ± 1.12 * ** thirty** 83.0

The data obtained indicate that the peat agent not only increases the severity of the antitumor and antimetastatic effects of the cyclophosphamide cytostatic, but also significantly reduces its toxic effect on animals in the method of treating cancer.

Example 4. The effect of funds from peat on the antitumor and antimetastatic effects of cyclophosphamide in rats with metastatic tumor of Pliss lymphosarcoma (Table 4).

The experiment was conducted on another type of animal - Wistar rats with a metastatic tumor of Pliss lymphosarcoma. The peat agent was started to be administered once a day at a dose of 100 mg / kg on the 7th day after the inoculation of the tumor and continued daily for 14 days. Cyclophosphamide was administered twice at a dose of 100 mg / kg with an interval of 96 hours on days 3 and 7 after the start of the use of the drug from peat. The antitumor effect was evaluated on the 22nd day after transplantation.

In the method of treating cancer, the use of a peat preparation during treatment with cyclophosphamide does not affect its antitumor effect, but reduces the frequency of tumor metastasis by a factor of three compared with monotherapy with a cytostatic agent. In this group of rats, the lymph node mass indices are two times less than in animals treated with cytostatic.

When cyclophosphamide was used, the number of peripheral blood leukocytes significantly decreased, indicating a pronounced hematotoxic effect of cytostatic. When combining cyclophosphamide with a peat agent, the number of leukocytes significantly exceeds their level in the group of rats treated with one cytostatic.

Thus, in the method of treating cancer using a combination of cytostatic therapy with cyclophosphamide and a peat agent, the use of peat agents increased the antimetastatic activity of cyclophosphamide and decreased its hematotoxicity, judging by the change in the number of leukocytes in the peripheral blood of animals.

Table 4 The effect of peat products on the effectiveness of cyclophosphamide (CF) treatment of Wistar rats with Pliss lymphosarcoma Groups The number of rats in the group Tumor mass, g Inhibition of tumor growth,% The mass of lymph nodes per rat, mg The number of metastases per rat The frequency of metastasis,% The number of leukocytes, 10 ⁹ / l Intact 7 - - 94.6 ± 8.8 - - 9.4 ± 1.2 The control 12 41.2 ± 6.4 - 345.4 ± 28.6 * 8 one hundred 14.6 ± 1.8 CF 10 23.8 ± 5.4 42.3 252.6 ± 34.8 four 60 4.4 ± 0.6 ** Peat Remedy 10 32.6 ± 3.8 20.9 230.4 ± 26.8 ** four fifty* 10.2 ± 1.2 CF + peat product 10 24.8 ± 3.6 ** 40,0 132.0 ± 15.4 one twenty** 8.6 ± 1.0 *** Note: *) the differences are significant compared with intact animals at P≤0.05, **) the differences are significant compared with the Control group at P≤0.05, ***) the differences are significant compared with the Cyclophosphamide group at P≤0.05.

Example 5. The protective effect of peat agents on the peripheral blood counts of healthy (tumor-free) mice with cyclophosphamide

The experiment was carried out in accordance with the requirements approved by the Pharmaceutical Committee of the Ministry of Health of Russia (Moscow, May 1991), recommendations that came into force in 1992. Healthy mice were chosen insofar as the tumor has a pronounced effect on the hematopoiesis system, which could affect the objectivity of the data obtained (table .5).

The peripheral blood indices of C57B1 / 6 mice were evaluated against the background of the use of cyclophosphamide cytostatic and peat products.

The peat product was used once a day at a dose of 200 mg / kg for 10 days. Cyclophosphamide at a dose of 100 mg / kg was administered on the 4th and 7th day from the start of the administration of peat. Evaluation of the effectiveness of therapeutic effects was carried out on the 11th day of the experiment.

After the use of cytostatic in mice, peripheral blood develops leukopenia, erythropenia and reticulocytopenia. With the introduction of peat products to animals treated with cyclophosphamide, there are significant improvements in the blood picture: the recovery rate of white and red blood counts increases significantly, as evidenced by less pronounced anemia and leukopenia. The number of stab and segmented neutrophils is increasing, the content of reticulocytes in the peripheral blood increases to 70% of the level of intact animals (versus 38% in the Cyclophosphamide group).

The results obtained indicate a significant increase in the effectiveness of cytostatic therapy of cancer by enhancing the direct antitumor and antimetastatic activity of cytostatic drugs, reducing their toxic effects on the hematopoiesis system and restoring its impaired functions

Table 5 The effect of peat agents on the peripheral blood count of mice with the use of cyclophosphamide (CF) cytostatic Groups Red blood cells, 10 ¹² / L Reticulocytes, ‰ White blood cells 10 ⁹ / l Leukocyte formula (%) Stab neutrophils Segmented neutrophils lymphocytes monocytes Intact 7.83 ± 0.48 32.17 ± 2.26 8.3 ± 0.7 1,0 42.0 47.6 9,4 CF 3,50 ± 0,22 * 12.17 ± 1.13 * 3.3 ± 0.2 * 2.1 * 47.1 * 40.0 * 11.8 Peat Remedy 8.50 ± 0.50 33.30 ± 2.0 9.0 ± 0.6 2.0 51.3 37.3 10,4 Peat product + CF 5.61 ± 0.34 ** 22.71 ± 2.14 ** 4.2 ± 0.3 ** 4.0 ** 54.0 ** 29.5 * 12.5 Note to this table and table 6: *) the differences are significant compared with intact animals at P≤0.05 **) the differences are significant compared with the Cyclophosphamide group at P≤0.05

Example 6. The protective effect of peat on the indicators of bone marrow circulation in healthy (without tumors) CBA mice against the background of cyclophosphamide.

The bone marrow hematopoiesis of mice of the C57B1 / 6 line was evaluated against the background of the use of cyclophosphamide cytostatic and peat products.

The peat product was used once a day at a dose of 200 mg / kg for 10 days. Cyclophosphamide at a dose of 100 mg / kg was administered on the 4th and 7th day from the beginning of the administration of peat. Evaluation of the effectiveness of therapeutic effects was carried out on the 11th day of the experiment.

After the use of cytostatics, inhibition of bone marrow hematopoiesis is observed in mice: the total number of myelokaryocytes decreases due to mature and immature granulocyte and erythroid forms, while the number of erythroid and granulocyte-macrophage progenitor cells, elements due to which the restoration of suppressed hematopoiesis is reduced, significantly decreases. 6).

With the introduction of peat products to animals treated with cyclophosphamide, there are significant improvements in bone marrow hematopoiesis: the total number of myelokaryocytes increases, almost to the level of the background. Of particular interest is the fact that under the action of the additive, the number of both erythroid and granulocyte-macrophage progenitor cells increases.

Thus, a peat remedy has a protective effect on the blood-forming tissue during cyclophosphamide hemodepression - it stimulates the erythroid and myeloid hematopoiesis growth both at the level of morphologically differentiated (red blood cells and white blood cells) and the parent blood cells - the precursors of these elements.

The experimental results indicate that in the method of treating cancer, the use of peat not only enhances the specific antitumor and, to a greater extent, the antimetastatic effects of cyclophosphamide cytostatic, but also reduces the severity of its hematotoxicity, enhances the antitumor and antimetastatic activity of cytostatic drugs, reduces them toxic effects on the hematopoietic system and the restoration of its impaired functions.

Table 6 The effect of peat agents on bone marrow hematopoiesis in mice with the use of cyclophosphamide (CF) cytostatic Type of exposure Total bone marrow cellularity (OCC) cell number (× 10 ⁶ ) Myelogram (× 10 ⁶ ) Colony forming units, erythrocyte (× 10 ⁵ ) Colony forming units, granulomacrophagic (× 10 ⁵ ) Granulocytes are immature Mature granulocytes Erythroid cells Intact 19.7 ± 0.8 3.10 ± 0.35 5.17 ± 0.38 9.20 ± 1.05 1.12 ± 0.08 1.34 ± 0.09 CF 12.0 ± 0.9 * 1.17 ± 0.07 * 4.14 ± 0.16 * 6.33 ± 0.64 * 0.25 ± 0.04 * 0.50 ± 0.04 * Peat Remedy 20.0 ± 1.1 2.00 ± 0.40 6.00 ± 0.30 9.17 ± 0.98 1.25 ± 0.05 1,50 ± 0,06 Peat product + CF 18.2 ± 1.3 ** 1.83 ± 0.17 ** 5.17 ± 0.36 ** 8.67 ± 0.83 ** 3.63 ± 0.19 ** 4.10 ± 0.21 **

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Claims (2)

1. A method of treating cancer in an experiment, including cytostatic chemotherapy of tumors with cyclophosphamide and the introduction of a medicinal product based on plant materials, characterized in that as a medicinal product based on plant materials used peat product containing humic acids and fulvic acids, amino acids, vitamins, macro - and trace elements obtained by treating peat with ammonia in the presence of hydrogen peroxide at 140 ° C. 2. The method according to claim 1, characterized in that the dose of peat is 100-200 mg / kg, which is administered once a day.