RU2825825C1 - Method for enhancing antimetastatic action of cisplatin and reducing its side effect on haemostasis system - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, more specifically to oncology and haemostaseology, and can be used to enhance the antimetastatic action of cisplatin and reduce its side effect on the haemostasis system. C57BL/6 female mice with transplanted Lewis lung carcinoma with a single intraperitoneal administration of cisplatin in dose of 9 mg/kg on 10th day after tumour transplantation, daily from 16th to 19th day extract is administered intragastrically, obtained from cell culture Conium maculatum L., in dose of 30 mg/kg. Main indicators of metastasis and characteristics of haemostasis are assessed on 20th day after tumour grafting.

EFFECT: invention provides reducing the number and area of lung metastases, as well as significant changes in the parameters of the haemostasis system.

1 cl, 4 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of medicine, specifically to oncology and hemostasis, and can be used to inhibit metastatic lung damage and pharmacologically correct the hypercoagulable state of the hemostasis system that develops under the influence of cisplatin against the background of a tumor process.

Metastasis is one of the main causes of death from cancer. The most common primary tumors that are characterized by metastasis to the lungs are breast cancer, stomach cancer, intestinal cancer, kidney cancer, liver cancer, etc. [1]. Chemotherapy is widely used to destroy both the tumor itself and possible metastases. Frequent adverse effects that occur in cancer patients are thrombotic complications, including venous thromboembolism and arterial thrombosis. A significant part of the risk of thrombotic complications is associated not only with the course of the disease, but also with its therapy [2].

Cisplatin is a chemotherapeutic drug licensed in 1978 and currently included in the World Health Organization's list of essential medicines. It is widely used both alone and in combination with other cytostatics to treat cancer of the testicles, ovaries, cervix, bladder, head and neck, lungs, esophagus, and stomach. One of the side effects of cisplatin is the development of thrombotic complications, including both venous and arterial thrombosis. Thus, it was shown that among 932 patients receiving cisplatin-based regimens for the treatment of various types of cancer, the incidence of thrombosis was 18.1% [2].

Thus, it is relevant to develop new schemes of additional therapy that lead to an increase in the antimetastatic effect of cytostatics and at the same time do not cause an increase in the incidence of thrombotic complications, but on the contrary, are capable of correcting these disorders. In this aspect, substances of plant origin are of interest, in particular, it is known that furocoumarins of hemlock (Conium maculatum L.) are capable of enhancing the effect of some cytostatics. It is promising to evaluate the effect of an extract obtained from the cell culture of Conium maculatum L. and containing furocoumarins on the state of the hemostasis system in the tumor process.

An antimetastatic agent is known - a liquid extract of the common rowan (Sorbus aucuparia L.), obtained by extracting rowan leaves, crushed to a particle size of 0.3-0.5 cm in diameter, with 40% ethyl alcohol at a temperature of 75°C for 60 minutes, as an antimetastatic agent and enhancing the antimetastatic effect of cyclophosphamide [4].

The product is a sum of phenolic acids in a 40% extract of rowan leaves, 17.86±0.12% in terms of chlorogenic acid; the sum of flavonoids in terms of rutin - 3.70±0.11%, the sum of anthocyanins in terms of cyanide-3-o-glucoside - 5.94±0.04%. This sum of compounds exhibits antimetastatic properties and is able to increase the antimetastatic effect of cyclophosphamide. The extract we use is obtained from biotechnological raw materials, purified and contains furocoumarins, and its use against the background of the tumor process causes not only an increase in the antimetastatic effect of cisplatin, but also has a corrective effect on the hemostasis system.

A method of using 2-(3,4-dihydroxyphenyl)-9-diethylaminoethylimidazole[1,2-a]benzimidazole dihydrobromide (enoxifol) as a means of inhibiting metastases is known. The pharmacological substance 2-(3,4-dihydroxyphenyl)-9-diethylaminoethylimidazole[1,2-a]benzimidazole dihydrobromide (enoxifol) reduces the incidence and number of metastases in the lungs of experimental melanoma B16 mice when administered intragastrically in a single dose of 50 mg/kg once a day [5].

Enoxifol, when administered intragastric once at a dose of 50 mg/kg, causes a decrease in the incidence and number of metastases in the lungs of experimental melanoma B16 mice, while there are no effects on the hemostatic system. While in the method described by us, the extract obtained from the culture of spotted hemlock cells and containing furocoumarins not only causes a decrease in the number of metastases, but also has a corrective effect on the disturbances in the hemostatic potential of female mice caused by the use of cisplatin against the background of the tumor process.

No adequate prototype was found in the analyzed patent and scientific-medical literature.

The problem solved by the present invention is to find ways to enhance the effect of cytostatic therapy while simultaneously reducing its negative effects on the hemostasis system.

The set task is solved by daily intragastric administration to female C57BL/6 mice with transplantable Lewis carcinoma of an extract obtained from a culture of hemlock cells, purified and representing a mixture of 3 furocoumarins (bergapten, isopimpinellin and xanthotoxin) with a content of at least 90%, at a dose of 30 mg/kg from the 16th to the 19th day after tumor transplantation against the background of a single intraperitoneal administration of cisplatin at a dose of 9 mg/kg on the 10th day. On the 20th day after tumor transplantation, biological material is collected to study the effectiveness of chemotherapy and assess the characteristics of hemostasis.

What is new in the proposed invention is that the extract obtained from the culture of hemlock cells (Conium maculatum L.), when administered at a dose of 30 mg/kg from the 16th to the 19th day after tumor transplantation, is capable of increasing the antimetastatic effect of cisplatin with concomitant correction of the hypercoagulable state in experimental animals with transplanted Lewis lung carcinoma.

The new features have demonstrated new properties in the claimed set, which do not clearly follow from the level of technology in this area and are not obvious to a specialist. An identical set of features has not been found in the patent and scientific and medical literature.

The method proposed as an invention can be used to create new treatment regimens that enhance the effect of cytostatics with additional correction of the hypercoagulable state caused by the use of chemotherapeutic drugs, in particular, cisplatin, in oncological practice.

Based on the above, the proposed invention should be considered as meeting the patentability conditions of “Novelty”, “Inventive step”, “Industrial applicability”.

The invention will be clear from the following description and the accompanying figures.

Fig. 1 shows the change in the thrombin activity constant (TAC) of female mice with transplanted Lewis lung carcinoma tumors against the background of the use of cisplatin after correction using an extract obtained from a culture of hemlock cells, where from here on, column a is healthy animals, b is tumor control, c is cisplatin, g is extract, d is warfarin.

Fig. 2 shows the change in the coagulation drive intensity (CDI) of female mice with transplanted Lewis lung carcinoma tumors against the background of cisplatin use after correction using an extract obtained from a culture of hemlock cells: a - healthy animals, b - tumor control, c - cisplatin, d - extract, d - warfarin.

Fig. 3 shows the change in the blood gelling time (t3) of female mice with transplanted Lewis lung carcinoma tumors against the background of the use of cisplatin after correction using an extract obtained from a culture of hemlock cells: a - healthy animals, b - tumor control, c - cisplatin, d - extract, d - warfarin.

Fig. 4 shows the change in the time of clot formation (t5) in female mice with transplanted Lewis lung carcinoma tumors against the background of cisplatin use after correction using an extract obtained from a culture of hemlock cells: a - healthy animals, b - tumor control, c - cisplatin, d - extract, d - warfarin.

Lewis lung carcinoma is transplanted into female C57BL/6 mice, then on the 10th day cisplatin is administered intraperitoneally at a dose of 9 mg/kg, then from the 16th to the 19th day the animals are administered intragastrically daily an extract obtained from a culture of spotted hemlock cells at a dose of 30 mg/kg, and on the 20th day after tumor transplantation biological material (blood, lungs) is collected to assess the effectiveness of the drugs on the development of metastases and hemostasis parameters using the NPTEG method.

The method is carried out as follows. Female mice of the C57BL/6 line are intramuscularly transplanted with tumor cells in the amount of 5×10 ⁶ cells in 0.1 ml of physiological solution, then on the 10th day, cisplatin is administered intraperitoneally once at a dose of 9 mg/kg, then from the 16th to the 19th day, the animals are daily intragastrically administered an extract obtained from a culture of spotted hemlock cells at a dose of 30 mg/kg. Then, on the 20th day, blood is collected by cardiac puncture from anesthetized animals, the blood is placed for 15-20 seconds in a 0.45 ml cuvette located in the thermostat of the hardware and software complex for clinical and diagnostic studies of the rheological properties of blood (thromboelastograph) NPTEG "Mednord" (LLC "Mednord-T", Russia) of the device, while the following parameters are assessed: thrombin activity constant (TAC, o.u.), t3 (gelation time, min), coagulation drive intensity (CDI, o.u.), t5 (clot formation time, min).

To assess the development of the process of hematogenous metastasis, the following criteria are used: tumor growth inhibition (TGI, %) [6], frequency of metastasis (FM), average number of metastases in one animal in a group, and metastasis inhibition index (MII, %) [7].

The technical result achieved is that the use of an extract obtained from a culture of hemlock cells and containing furocoumarins to increase the effectiveness of the antimetastatic action of cisplatin causes a 12.8-fold decrease in the number of metastases and a multiple reduction in the area of metastases in the lungs compared to the group receiving only cisplatin. At the same time, the metastasis inhibition index in animals receiving furocoumarins against the background of cisplatin is 99% versus 74% in the group of mice receiving cisplatin. The extract corrects the hypercoagulable state of the hemostasis system of experimental animals caused by tumor transplantation and cisplatin chemotherapy and causes significant changes in the parameters of the hemostasis system (a decrease in CTA and ICD by 20.9% and 12.5% from the tumor control group and by 18.7% and 19.8% from the group receiving cisplatin) with the achievement of values of healthy animals.

Example

The extract was obtained as follows: dried cellular biomass of hemlock (Conium maculatum L.) cultures was defatted with petroleum ether and infused with the addition of 500 ml of a 70% aqueous acetone solution. Then the resulting solution was processed step by step with 30 ml of a 10% alcoholic solution of potassium hydroxide, 100 ml of a 10% aqueous solution of potassium hydroxide and 100 ml of a 10% hydrochloric acid solution. After that, 150 ml of chloroform were added to the extract and the chloroform layer was separated using a separatory funnel and dried in a fume hood at room temperature. To isolate the sum of furocoumarins, the extract was purified by liquid column chromatography on a Toyopearl HW-50 sorbent (Tosoh Bioscience GmbH, Germany). As a result, a light yellow, odorless crystalline powder was obtained [9].

During the experiments, female C57BL/6 mice were divided into groups:

1. Healthy animals - female mice, which were given a single intraperitoneal injection of 0.5 ml of physiological solution on the 10th day and from the 16th to the 19th day inclusive, 0.5 ml of starch gel intragastrically.

2. Tumor control - female mice that were injected intramuscularly with Lewis lung carcinoma tumor cells, which were given a single intraperitoneal injection of 0.5 ml of saline on the 10th day and from the 16th to the 19th day inclusive, and were given 0.5 ml of starch gel intragastrically.

3. Cisplatin - female mice were injected intramuscularly with Lewis lung carcinoma tumor cells, then given a single intraperitoneal injection of cisplatin at a dose of 9 mg/kg, and from the 16th day through the 19th day, starch gel was administered intragastrically at 0.5 ml.

4. Extract - female mice that were injected intramuscularly with Lewis lung carcinoma tumor cells, then on the 10th day they were injected intraperitoneally with cisplatin at a dose of 9 mg/kg, and from the 16th day to the 19th day inclusive, the extract was injected intragastrically at a dose of 30 mg/kg.

5. Warfarin - female mice were injected intramuscularly with Lewis lung carcinoma tumor cells, then on the 10th day they were given a single intraperitoneal injection of cisplatin at a dose of 9 mg/kg, and from the 16th day to the 19th day inclusive, warfarin was administered intragastrically at a dose of 4 mg/kg.

Blood for the study was collected using an insulin syringe by cardiac puncture. An aliquot of blood was transferred within 15-20 sec into a 0.45 ml cuvette located in the thermostat of the hardware and software complex for clinical and diagnostic studies of blood rheological properties (thromboelastograph) NPTEG "Mednord" and the study was started using the ICS-GEMO 3 program. The following parameters were assessed: thrombin activity constant (TAC, o.u.), t3 (gelation time, min), coagulation drive intensity (CDI, o.u.), t5 (clot formation time, min). Then the animals were killed by euthanasia in a CO ₂ chamber, observing the "Rules for conducting work using experimental animals" [7], an autopsy was performed, internal organs were revised, and the tumor and metastases were isolated.

The following criteria were used to assess the development of the hematogenous metastasis process:

1) Tumor growth inhibition (TGI, %) was calculated using formula (1).

A - average tumor mass in the control group;

B - average tumor mass in the experimental group;

2) the frequency of tumor metastasis (FM) is calculated as a percentage (based on the ratio of the number of animals with metastases to the total number of animals in the group);

2) the average number of metastases in one animal in the group. The average area of metastatic lesions is calculated using the formula S=πr2, determining the diameter of metastases, taking into account that when using Lewis lung carcinoma, metastases were located predominantly subpleurally;

3) the metastasis inhibition index (MII, %) is an integral indicator that allows one to evaluate the magnitude of the difference in tumor metastasis between the control and the experiment, calculated using formula (2).

A1 - frequency of metastasis in the control group;

A2 - frequency of metastasis in the experimental group;

B1 - average number of metastases in animals of the control group;

B2 - the average number of metastases in animals of the experimental group [7];

The criteria for assessing the antimetastatic effect of the studied plant extract is their ability to reliably increase the effectiveness of chemotherapeutic treatment when tested on models of spontaneous tumor metastasis [8].

Statistical processing of the obtained results was performed using the Statistica 10 software package. Intergroup differences were assessed using the nonparametric Mann-Whitney criterion and Fisher's angular transformation. Differences were determined at a 0.05% significance level.

When assessing the effect of the extract obtained from the culture of hemlock cells on the effectiveness of chemotherapy, it was shown that its addition to the cisplatin treatment regimen leads to an increase in the antimetastatic effect of the antineoplastic drug: the number of metastases decreased by 12.8 times (P < 0.05), and the area of metastatic lesions in the lungs also decreased many times. In addition, the combined use of the cytostatic with the extract led to a reliable decrease in the number of animals with metastases relative to the indicator in the monochemotherapy group, while the IMI was 99% versus 74% in the group of animals receiving only cisplatin (Table 1). No significant difference in the mass of the primary tumor node was found between the groups.

Figure 1 shows the CTA changes in female mice with transplanted Lewis lung carcinoma tumors against the background of cisplatin administration after correction using the extract obtained from the culture of spotted hemlock cells. Here and below in the figures the group of healthy animals is taken as 100%, the marker * denotes the detected differences (p<0.05) when compared with the group of healthy animals; the marker # denotes the detected differences (p<0.05) when compared with the tumor control group; the marker ^ denotes the detected differences (p<0.05) when compared with the cisplatin group; the marker & denotes the detected differences (p<0.05) when compared with the warfarin group, column a - healthy animals, b - tumor control, c - cisplatin, g - extract, d - warfarin. As a result of the use of an extract containing furocoumarins in female mice with transplanted Lewis lung carcinoma against the background of cisplatin action, a decrease in CTA was observed in comparison with the group receiving cisplatin and the tumor control group by 18.7% (P < 0.05) and 20.9% (P < 0.05) (Fig. 1).

Figure 2 shows a decrease in ADC when using the extract in female mice by 19.8% (P<0.05) and 12.5% (P<0.05) compared to the groups receiving cisplatin and tumor control (Fig. 2). At the same time, the values of CTA and ADC in animals receiving the extract obtained from the culture of spotted hemlock cells did not differ from the group of healthy animals.

Figure 3 shows an increase in the t3 index in animals receiving the extract by 56.9% (P < 0.05) compared to the cisplatin group and by 56.9% (P < 0.05) compared to the tumor control group. It was noted that in the extract group, the t3 index was higher than the healthy animals by 39.9% (P < 0.05) but lower by 35.0% when compared to the group of animals receiving warfarin (Fig. 3).

Figure 4 shows the growth of the t5 index values both relative to the values of the cisplatin group by 27.0% (P < 0.05) and the tumor control by 25.3% (P < 0.05) and from healthy animals by 29.7% (P < 0.05) (Fig. 4). The introduction of the extract obtained from hemlock and containing furocoumarins leads to a decrease in hypercoagulation caused by the use of cisplatin against the background of the tumor process. At the same time, the corrective effect of the extract is comparable in effectiveness with the comparison drug - warfarin, with the exception of the t3 index values, high values of which in the warfarin group may indicate hypocoagulation changes in the GP.

Thus, the intragastric administration of an extract containing furocoumarins, against the background of cisplatin in animals with transplanted Lewis lung carcinoma, causes a decrease in the number and area of metastases and has a corrective effect on the hemostasis system, similar to the use of warfarin.

References

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Claims (1)

A method for enhancing the antimetastatic effect of cisplatin and reducing its side effects on the hemostasis system, characterized in that female C57BL/6 mice with transplanted Lewis lung carcinoma, against the background of a single intraperitoneal administration of cisplatin at a dose of 9 mg/kg on the 10th day after tumor transplantation, are administered an extract obtained from a Conium maculatum L. cell culture intragastrically daily from the 16th to the 19th day, at a dose of 30 mg/kg, while the main indicators of metastasis and hemostasis characteristics are assessed on the 20th day after tumor transplantation.