RU2353623C1 - Corrector of cytostatic polychemotherapy - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to correctors of cytostatic polychemotherapy. As corrector claimed is use of betulonic acid β-alanylamide of formula

(I): which potentiates anti-tumor effect and antimetastatic activity of polychemotherapy (PCT), demonstrates antioxidative and cytoprotecting activity at background of tumor PCT, and also produces anti-toxic impact when complex of cytostatic medications is introduced into organism. Compound (I) is synthesised from plant betulin truterpenoid.

EFFECT: obtaining corrector of cytostatic polychemotherapy.

1 cl, 9 ex, 12 tbl

Description

The invention relates to medicine, specifically to the development of proofreaders of cytostatic polychemotherapy, with the ability to comprehensively protect against the hepato-, nephro- and cardiotoxic effects of cytostatic drugs.

Modern methods of treating tumors, increasingly involving the use of "hard" chemotherapy, run up against limitations due to frequently occurring toxic complications. Represented by such phenomena as hemodepression, structural lesions of the liver and kidneys, necrotic enteropathy, impaired cardiac activity, these toxic complications forced to increase the interval between chemotherapy courses, reduce the dose of cytostatics or completely cancel them. This leads to a decrease in the effectiveness of chemotherapy, to a return to treatment regimens that are easily tolerated, but have less chance of success. “Rigid” polychemotherapy takes place against an unfavorable background of nonspecific reactions arising due to the natural course of the tumor process [1].

The development of drugs that reduce or eliminate toxic complications has been actively carried out in recent decades. But preparations have not yet been developed that provide comprehensive protection against toxic complications that affect vital organs. Selectively acting protectors are known. So, to reduce hemodepression, drugs of recombinant colony-stimulating factors (stimulators of metabolic processes) - ά-, β-, ω-erythropoietins, recombinant myelocytokines - filgrastim, lenograstim, etc. were proposed [2, p. 716]. These drugs are obtained by microbiological means, are not produced in Russia and are expensive.

Damage to the liver can be weakened by the introduction of antioxidants, such as melatonin [2, p.281]. However, the ability to enhance the effect of drugs that depress the central nervous system, and a number of side effects (contraindications for leukemia, lymphomas) limit the use of melatonin. Clinically significant protection of heart tissue from the toxic effects of cytostatics is provided by the drug dexrazoxone [2, p.754]. A side effect of this drug is manifested in dyspepsia, alopecia, leukopenia and thrombocytopenia.

Recently, amifostine has been increasingly used, which protects cells not involved in tumor growth from the toxic effects of alkylating cytostatics and platinum preparations. However, this drug does not eliminate the effects of Dossorubicin therapy and may cause complications. Like other cytoprotectors, amifostine is not produced in Russia and has a high cost [2].

Thus, we can state the absence in the clinic of malignant diseases of correctors of complex action cytostatics. The situation in Russia is complicated by the fact that domestic drugs of this drug group are not produced in the country.

The objective of the invention is to develop an effective domestic drug - corrector of cytostatic polychemotherapy, which can protect vital organs from the toxic effects of cytostatics and potentiate the effect of polychemotherapy.

The problem is solved by a chemical compound of the formula (I),

namely, betulonic acid β-alanylamide (IUPAC name: N- [3-oxo-20 (29) lupen-28-oyl] -3-aminopropionic acid), which potentiates the antitumor effect and antimetastatic activity of polychemotherapy (PCT), which exhibits antioxidant and cytoprotective effect against the background of PCT tumors, as well as having an antitoxic effect when a complex of cytostatic drugs is introduced into the body.

(с 3.04, хлф); брутто-формула С₃₃Н₅₁NO₄; Масс-спектр: Найдено [М]⁺ 525.38371. Вычислено М 525.38178.Compound (I) was synthesized from accessible, extracted from birch bark, betulin (II) triterpenoid according to Scheme 1. The synthesis conditions and physicochemical characteristics of agent (I) are given in [4]. According to the scheme below, betulin II, obtained by hot extraction of birch bark with trichlorethylene, is oxidized with Jones reagent in acetone to betulonic acid III (temperature 0-5 ° С, 7 h, 85% yield, 82% purity by HPLC). The resulting product is treated with oxalyl chloride in methylene chloride (20 ° C, 6 hours) to obtain acid chloride (IV). Isolation of product IV is carried out by three to four times distillation of methylene with temperature control, the formation of a precipitate in diethyl ether and its filtering. The condensation of acid chloride IV with β-alanine methyl ester (anhydrous methylene chloride, argon, 20 ° С, 20 h, 96% yield) yields the corresponding amide containing a fragment of β-alanine methyl ester at atom C (28). Alkaline hydrolysis of the latter by the action of 4 N. a solution of sodium hydroxide in a mixture of methanol and tetrahydrofuran (2: 1, 20 ° C, 24 h) leads to betulonic acid β-alanilamide (N- [3-oxo-20 (29) lupen-28-oyl] -3-aminopropionic acid ) (I) (isolated by pouring the reaction mass onto a mixture of ice and dilute HCl, the precipitate formed is filtered off, washed with water, ethanol and dried in vacuo at 60 ° С, yield 97%. Analytical characteristics of the sample: mp 214-216 ° C (from acetonitrile)

(since 3.04, hlf); gross formula C ₃₃ H ₅₁ NO ₄ ; Mass spectrum: Found [M] ⁺ 525.38371. Calculated: M 525.38178.

Scheme 1

Other amides and peptides of betulonic acid having antitumor activity are known. Thus, in [5], the amide of betulonic acid and N-Boc-lysine was described, which has specific activity in prostate cancer in vitro and in vivo. Betulonic acid amides containing fragments of caprylic, pelargonic and undecanoic acids inhibit the growth of tumor cells MT-4, MOLT-4, CEM and Hep G2 [6]. The betulonic acid dipeptide (N '{N- [3-Oxolup-20 (29) -en-28-oyl] -9-aminononanoyl} -3-amino-3-phenylpropionic acid) is patented as an antiviral agent (anti-HIV, anti-herpes) and immunostimulating activity [7]. It was shown in [8] that this compound inhibits the growth of tumor cells 3 MT-4, MOLT-4, CEM and Hep G2. A dipeptide is an active inducer of apoptosis in leukemia cells and in vitro hepatocarcinoma cells [8].

The use of betulonic acid derivatives as correctors of cytostatics has not been described.

The study of the activity of betulonic acid β-alanilamide (I) as a corrector of cytotoxic drugs included the determination of its effect on the antitumor and antimetastatic effects of polychemotherapy of mice with transplantable tumors of various degrees of malignancy, as well as the study of the antioxidant effect of the agent under polychemotherapy and the possibility of correcting its cytotoxic effect on normal cells of internal organs not affected by malignant degeneration. The data obtained were processed statistically using the standard STATISTICA software package.

Previously, the acute toxicity of beta-alanilamide of betulonic acid was determined on white outbred mice weighing 22-25 g with a single intragastric route of administration using the Kerber method. It was shown that beta-alanilamide of betulonic acid (I) belongs to the IV class of toxicity, LD ₅₀ over 5000 mg / kg.

The effect of betulonic acid β-alanilamide (I) on the antitumor effect of polychemotherapy was studied in mice with inoculated tumors (Lewis lung carcinoma and RLS lymphoma). Polychemotherapy was carried out according to the standard ASOR scheme (single parenteral administration of cyclophosphamide, doxorubicin, vincristine and prednisolone in doses of 1/5 of LD ₅₀ ). The effect of the agent was evaluated in relation to a group of mice that were given the same chemotherapy regimen. Agent (I) was administered intragastrically after 6 or 11 days after inoculation (depending on the tumor strain) in the form of an aqueous twin solution of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg. The introduction of the agent lasted for 8 days. ACOP polychemotherapy was administered once parenterally one day before the start of the agent administration. The control was a group of mice with a tumor without therapy. The antitumor effect was evaluated by the index of inhibition of tumor growth (the ratio of the difference between the average volumes of tumors in the control and experimental groups to its average volume in the control), which was determined in dynamics during the period of administration of the agent.

It was found that the administration of compound (I) enhances the antitumor effect of ACOP polychemotherapy 1.3–1.5 times (Table 1, 1a).

The effect of betulonic acid β-alanilamide (I) on the antimetastatic effect of polychemotherapy was studied in mice with transplanted Lewis lung carcinoma and RLS lymphoma. Polychemotherapy was carried out according to the above scheme. Agent (I) was administered in a regimen of 8 days at a dose of 50 mg / kg in the form of an aqueous twin solution, starting a day after the administration of cytostatics. The effect of the agent was evaluated relative to the group of mice that received only polychemotherapy. A criterion for the antimetastatic effect was a decrease in the bulk density of metastases (relative number of metastatic lesions, Vv%), which were determined by morphometric analysis of histological preparations of animal tissues [9]. In addition, the intensity of the metastasis process was evaluated by the frequency of metastasis (the ratio of the number of animals with metastases to the total number of animals in the group) and the metastasis inhibition index (IIM) [10]. It was found that β-alanilamide of betulonic acid (I) increases the inhibition index of metastasis of ACOP polychemotherapy by 1.3-2.5 times (Table 4).

In the same experiments, the antioxidant properties of compound (I) were studied under the conditions of polychemotherapy of mice with transplanted Lewis lung carcinoma and RLS lymphoma. The mode and methods of administration of cytostatic drugs and the studied agent were similar to the above. The criterion of antioxidant action was a decrease in the concentration of a lipid peroxidation marker - malondialdehyde - in the blood serum of experimental animals compared to the group that was administered only cytostatic drugs. It was found that β-alanilamide of betulonic (I) acid reduces the level of MDA by 2-4 times relative to the comparison group.

A study of the ability of betulonic acid β-alanilamide (I) to stop the toxic side effects of cytotoxic drugs on the tissues of internal organs was also carried out on healthy rats of Wistar females, which were modeled by polychemotherapy. The cytostatic administration schedule corresponded to ASOR: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg). The agent was administered intragastrically in a regimen of: 14 days at a dose of 50 mg / kg in the form of a water-twin solution, starting a day after the administration of cytostatics. The volume and surface density of zones with necrotic and dystrophic lesions were determined by morphometry of histological preparations of animal tissues. The criterion of the cytoprotective effect of the agent was a decrease in morphometric indicators relative to the group with the introduction of certain cytostatic drugs. It was found that compound (I) in the post-cytostatic period has pronounced hepato- and nephroprotective effects, several times reducing the degree of necrobiotic tissue damage.

In animals with transplanted tumors - Lewis lung carcinoma and RLS lymphoma - the ability of betulonic acid β-alanilamide (I) to reduce the cytotoxic effect of polychemotherapy on healthy cells of animal tumor carriers was determined. It was found that an 8-day administration of the agent at a dose of 50 mg / kg in the post-cytostatic period leads to a significant relief of signs of toxic damage to the liver and kidneys of animals. The degree of degenerative damage of hepatocytes and nephrocytes decreases, the area of necrosis and the severity of microcirculatory disorders in the internal organs decreases. In the liver of animals with lymphoma, partial restoration of the plastic processes of the kidneys was noted.

The invention is illustrated by the following examples.

Example 1. The study of the effect of β-alanilamide betulonic acid (I) on the antitumor effect of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma.

The effect of betulonic acid β-alanilamide (I) on the efficacy of antitumor polychemotherapy was studied in C57BL / 6 female mice weighing 20-25 g with transplanted Lewis lung carcinoma. The tumor is solid, metastasizes to the lungs.

The animals were divided into 3 groups of 10-12 animals each. All animals were intramuscularly inoculated with a cell suspension of the tumor in a volume of 5 × 10 ⁶ cells in 0.1 ml of physiological saline. On the 10th day after inoculation, the two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One of these groups, a day after polychemotherapy, was administered intragastrically β-alanilamide of betulonic acid (I) in the form of a water-twin suspension (tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with PCT ASOR was the reference. The control was animals with a tumor. The administration of the agent (I) solution to the experimental mice was continued for 8 days. The control and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. The magnitude of the antitumor effect was determined by the tumor growth inhibition index (SRW), which was calculated on the basis of the volumes of the primary nodes of the tumor during the administration of the agent, and at the end of the experiment, based on the masses of these tumors (the ratio of the difference between the average volumes / masses of the tumors in the control and experimental groups to its average volume / mass in the control).

The results of the experiment are presented in tables 1 and 1A. It was found that β-alanilamide of betulonic acid (I), administered at a dose of 50 mg / kg after polychemotherapy, enhances its antitumor effect by 1.3-1.5 times (Table 1).

Table 1.
Lewis lung carcinoma growth inhibition index (TPO) during the administration of betulonic acid β-alanilamide (I) against the background of ACOP polychemotherapy Group SRW,% (by tumor volume) SRW,% (by tumor mass) 2 days 3 days 4 days 5 days 6 days 9 days PCT ASOR 6 36 ** 37 ** 38 * 37 ** 31 * PCT ASOR + agent (I), (dose 50 mg / kg) 31 * ^# 56 *** ^# 56 *** ^# 53 *** fifty** 41 * * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant
"P <0.05 differences with PCT ASOR reliable

Table 1a.
Changes in the dynamics of growth of Lewis lung carcinoma under the influence of the introduction of agent (I) against the background of cytostatic polychemotherapy ASOR Group SRW,% (by tumor volume) SRW,% (by tumor mass) 2 days 3 days 4 days 5 days 6 days 9 days The control 1.78 ± 0.14 3.06 ± 0.13 3.19 ± 0.20 3.55 ± 0.18 3.74 ± 0.16 5.50 ± 0.21 PCT ASOR 1.67 ± 0.18 1.96 ± 0.19 ** 2.01 ± 0.20 ** 2.20 ± 0.21 * 2.36 ± 0.24 ** 3.81 ± 0.54 * PCT ASOR + agent (I), (dose 50 mg / kg) 1.22 ± 0.12 * ^# 1.33 ± 0.15 *** ^# 1.41 ± 0.16 *** ^# 1.68 ± 0.22 *** 1.89 ± 0.26 ** 3.28 ± 0.59 * * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant
^# P <0.05 differences with PCT ACOP significant

Thus, it was shown that β-alanilamide of betulonic acid (I) potentiates the antitumor effect of the cytostatic polychemotherapy of Lewis lung carcinoma.

Example 2. The study of the effect of β-alanilamide betulonic acid (I) on the antitumor properties of the cytostatic polychemotherapy of mice with transplantable RLS lymphoma.

The effect of betulonic acid β-alanilamide (I) on the effectiveness of antitumor polychemotherapy of RLS lymphoma was studied in mice of male CBA line weighing 15-30 g. This lymphoma strain grows in the form of a solid tumor, differs in malignancy and relative resistance to cyclophosphamide.

The animals were divided into 3 groups of 10-12 animals each. All animals were intramuscularly inoculated with a tumor cell suspension in a volume of 5 × 10 ⁶ tumor cells in 0.1 ml of physiological saline. Five days after inoculation, two groups of animals were given a single parenteral complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One of these groups, a day after polychemotherapy, was injected intragastrically with β-alanilamide of betulonic acid in the form of a water-twin suspension (tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with AST PCT was the reference . The control was animals with a tumor. The introduction of a solution of β-alanilamide betulonic acid (I) to experimental mice lasted 8 days. The control group and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. The magnitude of the antitumor effect was determined by the tumor growth inhibition index (SRW), which was calculated on the basis of the volumes of the primary nodes of the tumor during the period of administration of the agent, and at the end of the experiment, based on the masses of these tumors (the ratio of the difference between the average volumes / masses of the tumors in the control and experimental groups to its average volume / mass in the control).

The experimental results are presented in tables 2 and 2a. It was found that β-alanilamide of betulonic acid (I), administered at a dose of 50 mg / kg for 8 days after chemotherapy, enhances its antitumor effect by 1.3 times (Table 2).

table 2
RLS lymphoma growth inhibition index (TPO) during the administration of betulonic acid β-alanilamide (I) against the background of cytostatic chemotherapy ACOP Group SRW,% (by tumor volume) SRW,% (by tumor mass) 2 days 4 days 7 days 8 days PCT ASOR 49 * 36 * 49 * 44 ** PCT ASOR + agent (I), 50 mg / kg 62 * 80 ** 57 * 54 ** * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant
^# P <0.05 differences with PCT ACOP significant Table 2a
Changes in the dynamics of growth of RLS lymphoma under the influence of the introduction of β-alanilamide betulonic acid (I) against the background of cytostatic chemotherapy ACOP Group SRW,% (by tumor volume) SRW,% (by tumor mass) 2 days 4 days 7 days 8 days The control 0.37 ± 0.03 1.87 ± 0.19 2.47 ± 0.28 2.88 ± 0.25 PCT ASOR 0.19 ± 0.02 * 1.19 ± 0.09 * 1.36 ± 0.14 * 1.61 ± 0.16 ** PCT ASOR + agent (I), 50 mg / kg 0.16 ± 0.02 * 0.67 ± 0.07 ** 1.26 ± 0.16 * 1.34 ± 0.19 ** * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant
^# P <0.05 differences with PCT ACOP significant

Thus, it was shown that β-alanilamide of betulonic acid (I) potentiates the antitumor effect of the cytostatic polychemotherapy of RLS lymphoma.

Example 3. The study of the effect of β-alanilamide betulonic acid (I) on the antimetastatic effect of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma

The antimetastatic properties of betulonic acid β-alanilamide (I) were studied in mice of C57BL / 6 female mice weighing 20-25 g with solid Lewis lung carcinoma. A Lewis lung carcinoma cell suspension was inoculated intramuscularly in a volume of 5 × 10 ⁶ in 0.1 ml of physiological saline. On the 10th day after inoculation, two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One of these groups, a day after polychemotherapy, was injected intragastrically with β-alanilamide of betulonic acid in the form of a water-twin suspension (tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with AST PCT was the reference . The control was animals with a tumor. The introduction of a solution of β-alanilamide betulonic acid (I) to experimental mice lasted 8 days. The control group and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. After 8 days of administration of the agent, the animals were sacrificed, the lungs were removed and, after standard histological treatment, examined by light microscopy. The antimetastatic effect was determined by morphometric analysis of sections of both lung lobes. Bulk density (Vv%) of metastases was calculated according to the Avtandilov method [9] using an ocular network of 289 points. The intensity of the metastasis process was evaluated by the frequency of metastasis (FM) (the ratio of the number of animals with metastases to the total number of animals in the group) and the metastasis inhibition index (IIM) [10]:

, где

where

And _{to the} frequency of metastasis in the control group, A is the frequency of metastasis in the experimental group, In _{to the} density of metastases in animals of the control group,

In - the density of metastases in animals of the experimental group.

Data on changes in the bulk density of metastatic lesions are given in Table 3. It was found that betulonic acid β-alanilamide (I), administered at a dose of 50 mg / kg for 8 days after chemotherapy, dramatically enhances the antimetastatic effect of ACOP polychemotherapy, reducing the bulk density of lung metastases by 56 times. In this case, the inhibition index of PCT metastasis under the influence of the agent increases by 1.3 times (Table 3).

Table 3
Changes in the metastasis of Lewis lung carcinoma in the lungs of mice under the influence of the introduction of agent (I) against the background of cytostatic chemotherapy ACOP Group Vv% World Cup% IMI% The control 0.650 ± 0.200 one hundred 0 PCT ASOR 0.170 ± 0.024 * one hundred 73.8 PCT ASOR + agent (I), 50 mg / kg 0.003 ± 0.003 ** ^## 60 99.5 * P <0.05; ** P <0.01 differences with control are significant;
^## P <0.01 differences with PCT ACOP significant
Vv% is the bulk density of metastasis; FM - frequency of metastasis; IIM - metastasis inhibition index

Thus, it was shown that β-alanilamide of betulonic acid (I) significantly potentiates the antimetastatic effect of cytostatic polychemotherapy of Lewis lung carcinoma.

Example 4. The study of the effect of β-alanilamide betulonic acid (I) on the antimetastatic effect of cytostatic polychemotherapy of mice with transplantable lymphoma RLS

The antimetastatic properties of betulonic acid β-alanilamide (I) were studied in mice of CBA male mice weighing 15-30 g with solid RLS lymphoma. A cell suspension of RLS lymphoma was inoculated intramuscularly in a volume of 5 × 10 ⁶ in 0.1 ml of physiological saline. 5 days after inoculation, the two groups of animals were injected once parenterally with a complex of chemotherapeutic drugs according to the ASOR scheme. After a day, one of these groups began the introduction of agent (I) according to the scheme indicated above. Another group with AST PCT was a reference. The control was animals with a tumor. After 8 days of administration of the agent, the animals were sacrificed, the liver was removed, and after standard histological treatment, examined by light microscopy. The antimetastatic effect was determined by morphometric analysis of the slices according to the method [9] using an ocular network of 289 points. The intensity of the metastasis process was evaluated by the bulk density (Vv%) of metastases, metastasis frequency (FM) and metastasis inhibition index (IIM).

The results are shown in table 4. It was found that betulonic acid β-alanylamide (I), administered at a dose of 50 mg / kg for 8 days after polychemotherapy, enhances the antimetastatic effect of ACOP polychemotherapy, reducing the density of liver metastases by 1.2 times. At the same time, the inhibition index of PCT metastasis under the influence of the agent rises 2.5 times (Table 4).

Table 4
Changes in the metastasis of RLS lymphoma in the liver of mice under the influence of the introduction of agent (I) against the background of cytostatic chemotherapy ACOP Group Vv% World Cup% IMI% The control 11.49 ± 5.17 one hundred 0 PCT ASOR 10.43 ± 8.64 one hundred 9.2 PCT ASOR + agent (I), 50 mg / kg 8.88 ± 9.30 one hundred 22.7 Vv% is the bulk density of metastasis; FM - frequency of metastasis; IIM - metastasis inhibition index

Thus, it was shown that β-alanilamide of betulonic acid (I) significantly potentiates the antimetastatic effect of cytostatic polychemotherapy of RLS lymphoma.

Example 5. The study of the antioxidant effect of β-alanilamide betulonic acid (I) in the conditions of cytostatic polychemotherapy of mice with transplantable Lewis lung carcinoma

C57B L / 6 female mice weighing 20-25 g were divided into 3 groups of 10-12 animals each. A Lewis lung carcinoma cell suspension was inoculated intramuscularly in a volume of 5 × 10 ⁶ in 0.1 ml of physiological saline. On the 10th day after inoculation, the two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) Agent (I) was intragastrically administered to one of these groups a day after polychemotherapy in the form of a water-twin suspension (Tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with ACP PCT was a reference. The control was animals with a tumor. The administration of the agent (I) solution to the experimental mice was continued for 8 days. Control animals and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. Animals were removed from the experiment by decapitation, blood serum was obtained and the concentration of malondialdehyde in the reaction with thiobarbituric acid [11], as well as transaminase activity (using standard Biocon reagent kits) were determined. The results are presented in table 5. It was found that betulonic acid β-alanilamide, administered at a dose of 50 mg / kg for eight days after chemotherapy, reduces the concentration of the MDA lipid peroxidation marker in blood by 4 times. The activity of transaminases decreases by 1.2-1.3 times compared with animals that received only polychemotherapy (Table 5).

Table 5
Changes in biochemical parameters in the blood of mice with Lewis lung carcinoma under the influence of β-alanilamide betulonic acid (I) administration against the background of ASOR cytostatic chemotherapy Group Biochemical parameters of blood serum MDA, mmol / l ACT, mkkat / l ALT, mkkat / l The control 4.83 ± 1.42 17.23 ± 1.33 1.10 ± 0.09 PCT ASOR 4.46 ± 1.27 18.12 ± 0.93 1.49 ± 0.17 PCT ASOR + agent (I), 50 mg / kg 1.12 ± 0.08 * ^# 15.57 ± 0.66 * ^# 1.13 ± 0.07 * ^# * P <0.05 differences with control are significant;
^# P <0.05 differences with PCT ACOP significant

Thus, it was shown that betulonic acid β-alanilamide (I) has an antioxidant effect under the conditions of cytostatic polychemotherapy of Lewis lung carcinoma, reducing the level of lipid peroxidation and cytolysis.

Example 6. The study of the antioxidant effect of β-alanilamide betulonic acid (I) in the conditions of cytostatic polychemotherapy of mice with transplantable lymphoma RLS

Mice of CBA males weighing 15-30 g were divided into 3 groups of 10-12 animals each. All animals were intramuscularly inoculated with a tumor cell suspension in a volume of 5 × 10 ⁶ tumor cells in 0.1 ml of physiological saline. Five days after inoculation, the two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) Compound (I) was intragastrically administered to one of these groups a day after polychemotherapy in the form of a water-twin suspension (Tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with ACP PCT was a reference. The control was animals with a tumor. The administration of the agent (I) solution to the experimental mice was continued for 8 days. The control group and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. Animals were taken out of the experiment by decapitation, blood serum was obtained, and the concentration of malondialdehyde in the reaction with thiobarbituric acid was determined in it [11]. The results are presented in table 6. It was found that compound (I) at a dose of 50 mg / kg for eight days after chemotherapy, reduces the concentration of MDA in the blood by 2 times compared with animals that received only chemotherapy (table 6).

Table 6
Change in the concentration of MDA in the blood of mice with RLS lymphoma under the influence of β-alanilamide betulonic acid (I) administration against the background of cytostatic chemotherapy ACOP Indicator Groups of animals The control PCT ASOR PCT ASOR + agent (I), 50 mg / kg MDA, mmol / l 3.47 ± 0.97 3,55 ± 0,66 1.69 ± 0.28 ** ^## ** P <0.01 differences with control are significant;
^## P <0.01 differences with PCT ACOP significant

Thus, it was shown that β-alanilamide of betulonic acid (I) has an antioxidant effect under the conditions of cytostatic polychemotherapy of RLS lymphoma, reducing the intensity of lipid peroxidation.

Example 7. The study of the effect of β-alanilamide betulonic acid (I) on the organotoxic effect of a combination of cytostatic drugs

Female Wistar rats weighing 180-200 g were divided into 3 groups of 10 animals each. All groups received a single intraperitoneal combination of antitumor drugs: cyclophosphamide - 21 mg / kg, doxorubicin - 2.1 mg / kg, vincristine - 0.04 mg / kg, prednisone - 2.1 mg / kg body weight.

Within 14 days after the administration of cytostatics, rats were injected intragastrically with water (group 1), betulonic acid β-alanilamide (group 2) and the comparison preparation was diquertin (dihydroquercetin, Bianon, Russia), which is an antioxidant with PP-vitamin activity (group 3 ) The studied triterpenoids and reference preparation were administered in an aqueous twin solution at a dose of 50 mg / kg. On the 15th day after the administration of cytostatics, the animals were killed by instant decapitation under light ether anesthesia, the liver, kidneys and thymus were removed, fixed with a 10% solution of neutral formalin and then subjected to standard histological processing on an MICROM automated system (Karl Zeiss). Sections 3-5 microns thick were stained with hematoxylin-eosin and according to Van Gieson. Morphometric study of the areas of sections and structural components of tissues was carried out using an ocular network (289 points) according to the method [9]. In the liver and kidneys, the bulk density (Vv) of the zones with dystrophic and necrotic changes in the cells, as well as the bulk density of the sinusoids of the liver, and the numerical density (Nai) of binuclear hepatocytes were calculated. In the thymus, the bulk density of the cerebral and cortical matter, the number of Gassal bodies was determined. Statistical processing of data was carried out by parametric statistics using t student criterion. The results were considered reliable at P <0.05.

It was found that under the action of compound (I) there was a significant improvement in the structure of the liver and kidneys. The volumetric density of hepatocytes with dystrophic and necrotic lesions decreased by 4 and 1.5 times, respectively, compared with the first group. The introduction of the agent contributed to the restoration of hepatic microcirculation. The bulk density of nephrocytes of the proximal tubules in a state of dystrophy decreased by the action of agent (I) 2.9 times, the bulk density of the necrosis zones - 2.4 times. Under the action of the agent, the cortical-brain index of the thymus increased and the number of Gassal's bodies in its brain part decreased. It was found that the organoprotective effect of β-alanilamide of betulonic acid (I) under conditions of cytotoxic damage is not inferior to the similar effect of the drug of comparison of dikvertin.

Table 7
Changes in the bulk density of structural elements of tissues of the internal organs of rats under the action of β-alanilamide betulonic acid (I) against the background of the introduction of a combination of cytostatic drugs Organ The structural elements of the fabric Bulk density (Vv) of zones,% 1 group 2 group 3 group Liver Hepatocyte dystrophy 64.82 ± 1.15 16.39 ± 0.77 * 7.12 ± 0.97 * Hepatocyte necrosis 11.31 ± 0.60 7.36 ± 1.18 4.78 ± 0.96 * Sine waves 13.74 ± 1.40 23.22 ± 0.99 * 23.50 ± 0.94 * Kidney Nephrocyte Dystrophy 13.14 ± 0.38 4.53 ± 0.20 * 4.88 ± 0.38 * Necrosis of nephrocytes 0.58 ± 0.06 0.24 ± 0.05 * 0.26 ± 0.04 * Thymus Cortical substance 55.62 ± 1.03 68.58 ± 0.68 * 66.87 ± 0.64 * Brain substance 43.41 ± 1.00 31.42 ± 0.68 * 32.42 ± 0.62 * Number of Gassal Taurus 7.16 ± 0.26 3.28 ± 0.12 * 3.98 ± 0.11 * * P <0.05 differences with control are significant

Thus, it was shown that, under the combined effects of cytostatics, the administration of betulonic acid β-alanilamide (I) in the post-cytostatic period has pronounced hepato- and nephroprotective effects, several times reducing the degree of necrobiotic tissue damage.

Example 8. The study of the hepatoprotective properties of β-alanilamide betulonic acid (I) under the conditions of cytostatic polychemotherapy of ASOR mice with transplantable Lewis lung carcinoma

C57B L / 6 female mice weighing 20-25 g were divided into 3 groups of 10-12 animals each. A Lewis lung carcinoma cell suspension was inoculated intramuscularly in a volume of 5 × 10 ⁶ in 0.1 ml of physiological saline. On the 10th day after inoculation, two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One day after polychemotherapy, one of these groups was administered intragastrically compound (I) in the form of a water-twin suspension (Tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with ACP PCT was the reference. The control was animals with a tumor. The introduction of a solution of compound (I) to experimental animals was continued for 8 days. The control and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. After eight days of administration of the agent, the animals were withdrawn from the experience by decapitation. For morphological examination, a liver was taken and fixed in 0.1 M phosphate buffer with a pH of 7.2-7.8 for 4 days, then subjected to standard histological processing on a histological complex MIKROM (Karl Zeiss). Sections with a thickness of 4-5 μm were stained with hematoxylin and eosin, and hematoxylin - orange G, according to the SIC method. The preparations were examined by transmission light microscopy. Morphometric analysis of the slices was carried out according to the method [9] using an ocular network of 289 points. In the liver, the bulk density (Vv) of the zones with dystrophic and necrotic changes in the cells was calculated. The results of the analysis are presented in table.8. It was found that the administration of compound (I) at a dose of 50 mg / kg for eight days after chemotherapy reduces the bulk density of zones with hepatocyte necrosis by 3 times and increases the bulk density of sinusoids by 1.4 times in comparison with the ASOR PCT group. The observed changes indicate the arrest of the processes of cytolysis and cholestasis in the liver caused by the introduction of cytostatic drugs. A morphological study also revealed a decrease in the severity of dystrophic lesions of hepatocytes under the influence of an agent, despite the fact that the bulk density of zones with dystrophy did not change compared to the PCT group. In the group with the introduction of the agent, there was a decrease in cells with balloon and an increase in cells with hydropic and hyaline droplet dystrophy.

Table 8
The change in the bulk density of the elements of the hepatic parenchyma in mice with Lewis lung carcinoma under the influence of the introduction of agent (I) against the background of cytostatic chemotherapy ACOP Group Bulk density (Vv) of zones,% Dystrophy Sine waves Necrosis The control 64.89 ± 1.31 19.36 ± 0.73 8.30 ± 0.11 PCT ASOR 62.50 ± 0.25 15.43 ± 0.51 * 14.59 ± 0.63 * PCT ASOR + agent (I), 50 mg / kg 65.34 ± 1.24 20.99 ± 0.93 ^# 4.78 ± 0.18 * ^# * P <0.05 differences with control are significant;
* P <0.05 differences with PCT ACOP significant

Thus, betulonic acid β-alanylamide has a hepatoprotective effect under the conditions of cytostatic polychemotherapy of Lewis lung carcinoma, reducing the intensity of cytolysis and cholestasis in the hepatic parenchyma.

Example 9. The study of the hepato- and nephroprotective properties of β-alanilamide betulonic acid (I) under the conditions of cytostatic polychemotherapy of ASOR mice with transplantable lymphoma RLS

Mice of CBA males weighing 15-30 g were divided into 3 groups of 10-12 animals each. All animals were intramuscularly inoculated with a tumor cell suspension in a volume of 5 × 10 ⁶ tumor cells in 0.1 ml of physiological saline. Five days after inoculation, the two groups of animals were administered once parenteral a complex of chemotherapeutic drugs according to the ASOR regimen: doxorubicin (4 mg / kg), cyclophosphamide (50 mg / kg), vincristine (0.1 mg / kg), prednisone (5 mg / kg ) One day after polychemotherapy, one of these groups was administered intragastrically compound (I) in the form of a water-twin suspension (Tween-80) of 0.2 ml per 10 g of body weight at a dose of 50 mg / kg, the second group with ACP PCT was the reference. The control was animals with a tumor. The administration of the agent (I) solution to the experimental mice was continued for 8 days. The control and the ASOR polychemotherapy group received at the same time an equivalent amount of water with tween-80. For morphological studies, the liver was fixed in 0.1 M phosphate buffer with a pH of 7.2-7.8 for 4 days, then subjected to standard histological treatment, followed by filling in paraffin blocks on a histological complex MIKROM (Karl Zeiss). Sections with a thickness of 4-5 μm were stained with hematoxylin and eosin, and hematoxylin - orange G, according to the SIC method. The preparations were examined by transmission light microscopy. Morphometric analysis of sections was performed using an ocular network of 289 points. In the liver, the bulk density (Vv) of the zones with dystrophic and necrotic changes in the cells was calculated.

After eight days of administration of the agent, the animals were withdrawn from the experience by decapitation. For morphological examination, a liver was taken and fixed in 0.1 M phosphate buffer with a pH of 7.2–7.8 for 4 days, then it was subjected to standard histological processing on a MIKROM histological complex (Karl Zeiss). Sections with a thickness of 4-5 μm were stained with hematoxylin and eosin, and hematoxylin - orange G, according to the SIC method. The preparations were examined by transmission light microscopy. Morphometric analysis of sections was carried out according to the Avtandilov method [9] using an ocular network of 289 points. In the liver, the bulk density (Vv) of the zones with dystrophic and necrotic changes in the cells was calculated.

The results of the analysis of liver tissue are presented in table 9. It was found that β-alanilamide of betulonic acid (I), administered at a dose of 50 mg / kg for eight days after chemotherapy, reduces the bulk density of zones with hepatocyte necrosis by 2 times and increases the bulk density of sinusoids 1.4 times in comparison with the PCT group ASOR (Table 9). The observed changes indicate the arrest of the processes of cytolysis and cholestasis in the liver caused by the introduction of cytostatic drugs. In the same group of animals, a relative increase in the number of hepatocytes with dystrophy was observed. In this case, the severity of dystrophy is significantly reduced, passing from balloon to slightly pronounced hydropic and hyaline droplet. In hepatocytes throughout the section of the liver, the appearance of glycogen in the form of fine granularity was observed, indicating an increase in the synthetic function of the liver.

Table 9
The change in the bulk density of the elements of the hepatic parenchyma in mice with RLS lymphoma under the influence of the introduction of agent (I) on the background of cytostatic chemotherapy ACOP Group Bulk density (Vv) of zones,% Dystrophy Sine waves Necrosis The control 0.580 ± 0.017 0.110 ± 0.010 0.290 ± 0.036 PCT ASOR 0.42 ± 0.04 0.095 ± 0.020 0.370 ± 0.017 PCT ASOR + agent (I), 50 mg / kg 0.53 ± 0.01 * 0.130 ± 0.011 0.190 ± 0.014 ^# * P <0.05 differences with control are significant;
^# P <0.05 differences with PCT ACOP significant

The results of the analysis of kidney tissue are presented in table 10. It was found that under the influence of agent (I) the degree of structural damage to the kidneys decreases due to a 5.8-fold decrease in the severity of necrotic nephrosis, as well as a 1.8-fold decrease in the lumen of the tubules and swelling of the interstitial tissue in Comparison with the PCT group ASOR. In the group of animals that were injected with agent (I), a statistically significant increase (1.5 times) in the number of cells with weakly expressed hyaline droplet dystrophy was observed compared with the group of animals that received only cytostatic therapy.

Table 10
The change in the bulk density of the elements of the renal parenchyma in mice with RLS lymphoma under the influence of the introduction of agent (III) against the background of cytostatic polychemotherapy ASOR Group Bulk density (Vv) of zones,% Dystrophy Clearance of tubules Necrosis The control 49.0 ± 1.1 14.0 ± 0.7 17.0 ± 3.5 PCT ASOR 49.0 ± 3.2 14.0 ± 0.4 23.0 ± 6.5 PCT ASOR + agent, 50 mg / kg 75.0 ± 1.5 ** ^## 8.0 ± 0.4 ** 4.0 ± 0.4 ** ^# * P <0.05; ** P <0.01 differences with control are significant;
^# P <0.05;^## P <0.01 differences with PCT ACOP significant

It can be seen from the experimental results that betulonic acid β-alanilamide (I) has a hepato- and nephroprotective effect under the conditions of cytostatic chemotherapy for RLS lymphoma, reducing the volume and severity of necrobiotic lesions of the liver and kidneys.

Thus, the proposed compound β-alanilamide betulonic acid (I) as a corrector of cytostatic polychemotherapy has the following advantages:

- high antioxidant, hepato- and nephroprotective activity against the background of cytostatic polychemotherapy;

- potentiation of antitumor and antimetastatic effects of polychemotherapy;

- obtaining it from available raw materials - betulin from birch bark.

INFORMATION SOURCES

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5. B. B. Saxena, L. Zhu, M. Hao, E. Kisilis, M. Katdare, O. Oktem, A. Bomshteyn, P. Rathnam, Bioorg. Med. Chem. Lett., 2006, V.14. 6349-6358.

6. Pokrovsky A.G., Shintyapina A.B., N.V. Pronkina, B.C. Kozhevnikov, Plyasunova O.A., Shults E.E., Tolstikov G.A. Reports of the Academy of Sciences. 2006.V. 407. No. 5. S.698-701.

7. RF patent 2211843 of January 25, 2002 according to the application 2002102338/04. BIPM. 2003. No. 25. Part 3, pp. 498-499.

8. Shintyapina A.B., Shults E.E., Petrenko N.I., Uzenkova N.V., Tolstikov G.A., Pronkina N.V., Kozhevnikov B.C., Pokrovsky A.G. Bioorganic chemistry. 2007.V. 33. No. 6. 620-626.

9. Avtandilov G.G. Medical morphometry. Leadership. M .: Medicine, 1990.384 s.

10. Arkhipov S.A., Junker V.M. The change in the intensity of metastasis to the lungs of transplanted tumors of mice depending on the size of the transplant dose of tumor cells. - Research on the induction and metastasis of tumors in experimental animals. - Novosibirsk, 1984.P.14-32.

11. Kamyshnikov B.C. Handbook of clinical and chemical laboratory diagnostics, Minsk: Belarus, 2000, T.2, p.207.

Claims (1)

The use of β-alanilamide betulonic acid of the formula (I):

as a corrector of cytostatic polychemotherapy.