RU2270682C2 - Method for enhancing of cyclophosphan antitumor and antimetastatic activity in experiment - Google Patents

Abstract

FIELD: experimental medicine and oncology.

SUBSTANCE: claimed method includes intraperitoneum cyclophosphan administration in dose of 120 mg/kg and intravenous transplantation of fetal liver steam cells to experimental animal. Fetal liver steam cells are transplanted for 3 days before cyclophosphan administration in dose of 25x10⁶ cells/kg of mass.

EFFECT: decreased tumor growth, reduced frequency and area of metastasis due to optimization of cyclophosphan and steam cells administration.

2 ex

Description

The invention relates to oncology and can be used in chemotherapy of malignant tumors.

A known method of increasing the antitumor activity of low doses of cyclophosphamide (60 mg / kg) using sanazole, a synthetic compound from the group of triazoles [1]. Using a Lewis transplantable lung carcinoma model, it was shown that the use of cyclophosphamide at a dose of 60 mg / kg administered on days 3 and 7 and sanazole at a dose of 1 mg / kg administered intraperitoneally daily for 10 days is as effective as therapy high doses of cyclophosphamide (120 mg / kg), as judged by the inhibition of the growth of pulmonary metastases.

The disadvantages of this method is the absence of a pronounced antitumor effect on the primary tumor node and side effects associated with the synthetic nature of sanazole.

One of the currently widely used cytotoxic drugs is the cyclophosphamide alkylating agent. Many organic compounds undergo alkylation, but DNA damage is crucial. However, cyclophosphamide in high doses (120 mg / kg) does not have a significant antitumor effect on the primary tumor node. The main side effects of cyclophosphamide are hematopoiesis, immunosuppression. depletion of the number of hematopoietic cells of the bone marrow [2, 3, 5].

The principles of modern tumor chemotherapy suggest the administration of the most tolerated doses of cytostatics in order to achieve the most complete damage to the tumor cells. The high hematotoxicity of anticancer drugs is a serious limitation in achieving the maximum therapeutic effect. Thus, it is relevant to create optimal conditions for realizing the action of high doses of cytostatics while reducing their side effects.

The technical task of the invention is to increase the severity of the antitumor effect on the primary tumor node while enhancing the antimetastatic action of the cytostatic agent, reducing side effects.

The problem is solved by transplanting an LLC tumor (Lewis lung carcinoma) to an experimental animal and intraperitoneally on days 3 and 7 of cyclophosphamide at a dose of 120 mg / kg and intravenous transplantation of fetal liver stem cells of fetal liver embryos of early stages of gestation at a dose of 25 × 10 ⁶ cells / kg suspension of 0.05 ml a day after inoculation of a tumor LLC (Lewis lung carcinoma).

What is new is that cyclophosphamide is administered at a dose of 120 mg / kg, stem cells of the fetal liver of embryos of the early stages of gestation are transplanted intravenously one day after inoculation of an LLC tumor (Lewis lung carcinoma) at a dose of 25 × 10 ⁶ / kg of 0.05 ml.

Fetal liver cells of embryos of the early stages of gestation are a suspension of individual cells in RPMI-1640 medium with 90-95% viability. At the same time, their immunomodulatory activity was revealed both against T- and B-lymphocytes, and effector cells of natural resistance [4]. The properties of fetal liver cells in early stages of gestation to increase the antitumor and antimetastatic activity of cyclophosphamide are unknown.

These significant features are not identified from the medical, scientific and patent literature. They do not explicitly follow from the prior art for specialists. This method was an experimental study in the laboratory of immunology Research Institute of Oncology TNC SB RAMS, Tomsk.

Thus, this technical solution meets the criteria of the invention of "novelty", "inventive step", "industrially applicable".

The method is as follows.

An LLC tumor (Lewis lung carcinoma) is transplanted to experimental animals and a day after the transplantation, the stem cells of the fetal liver embryos of the early stages of gestation are transplanted at a dose of 25 × 10 ⁶ cells / kg in a suspension of 0.05 ml, then 3 and 7 day cyclophosphamide is administered intraperitoneally.

A specific example of the method.

As experimental animals, C57BL / 6 mice weighing 20-22 grams were used. Lewis hematogenously metastatic lung carcinoma (LLC), transplanted intramuscularly at 10 ⁶ cells / mouse, was used as a model of experimental malignant growth.

For the experiment, all mice with the transplanted LLC were divided into 4 groups:

1) “LLC”: mice injected with nat. 0.05 ml solution intravenously;

2) "LLC + CK" - mice that received an injection of stem cells of the fetal liver at a dose of 25 × 10 ⁶ cells / kg in physical. 0.05 ml solution intravenously;

3) “LLC + cyclophosphamide”: mice treated with cyclophosphamide at a dose of 120 mg / kg ip on the 3rd and 7th day after tumor inoculation and received an injection of phys. 0.05 ml solution intravenously;

4) "LLC + cyclophosphamide + SC": mice treated with cyclophosphamide according to a similar pattern and intravenous stem cell transplantation of fetal liver embryos 10-15 days of gestation at a dose of 25 × 10 ⁶ cells / kg in physical. 0.05 ml solution.

Mice were scored on the 21st day after LLC inoculation.

The effectiveness of the impact was evaluated by inhibition of tumor growth, a decrease in the frequency and intensity of metastasis, inhibition of the growth of metastases.

Statistical processing of results. The research results were processed by methods of variation statistics. The significance of differences between the groups was evaluated using the nonparametric Wilcoxon – Mann – Whitney test at p≤0.05. The calculations were performed using the Statistica 6.0 software package for Windows.

The results of a study on the combined use of cyclophosphamide and early fetal liver stem cell transplantation of embryos in the treatment of Lewis lung carcinoma showed that in mice, combined treatment of stem cell transplantation and cyclophosphamide at a dose of 120 mg / kg showed the most pronounced inhibition of growth of the primary tumor node (growth inhibition the tumor by mass was 84% compared with 63% in the group treated with cyclophosphamide alone at a dose of 120 mg / kg, the inhibition of tumor growth by volume was Responsibly: 71% with combined treatment with cyclophosphamide and with fetal liver stem cell transplantation and 58% with cyclophosphamide alone), while the inhibition of metastasis is observed, for example, the metastasis rate in the group with cyclophosphamide administration was 56%, while in the group with a combination of cyclophosphamide and fetal liver stem cells, it is 22%, the total area of metastatic colonies in the group with a combination of cyclophosphamide and fetal liver stem cells is 3 times less than in the group where cyclophos the fan was introduced without stem cells. The metastasis inhibition index in the experimental LLC + cyclophosphamide + SC group was 98%, while in the LLC + cyclophosphamide group it was 82% (Table 1). Inhibition of growth of already formed metastases was also noted.

Thus, the following positive effect was obtained as a result of the study: the combined use of cyclophosphamide at a dose of 120 mg / kg and intravenous transplantation of stem cells of fetal liver embryos of early gestational doses at a dose of 25 × 10 ⁶ cells / kg in a suspension of 0.05 ml leads to increased inhibition of the primary tumor and a more pronounced antimetastatic effect than when compared with the prototype (table 2).

Literature

1. Konovalova N.P., Dyachkovskaya R.F., Volkova L.M., Kagiya V.T. Potentiation of antimetastatic activity of cyclophosphamide by AK-2123 radiosensitizer // Experimental Oncology. - 1994. - T. 16. - S. 419-422.

2. Goldberg E.D., Dygay A.M., Sherstoboev E.Yu. Mechanisms of local regulation of blood formation. - Tomsk: STT, 2000 .-- 148 p.

3. Experimental evaluation of antitumor drugs in the USSR and the USA // Ed. Sof'ina Z.P. - M.: Medicine. - 1979. - 296 p.

4. Kokorev OV The antitumor effect of fetal tissue transplants on a porous titanium nickelide carrier. Abstract, dis ... Cand. honey. Sciences - M. - 2000.

5. Stem cells and tumor growth: Sat. scientific tr / Ans. ed. Pinchuk V.G., Butenko Z.A. Kiev: Science. Dumka, 1985 .-- 256 s.

Table 1 LLC LLC + CK LLC + CF (120 mg / kg) LLC + CF (120 mg / kg) + SC mass of the primary tumor, g 3,486 ± 0,271 3.626 ± 0.324 1.486 ± 0.114 ¹ 1,026 ± 0,110 ² The volume of the primary tumor, cm ³ 2.6 ± 0.3 2.85 ± 0.23 0.976 ± 0.13 ¹ 0.43 ± 0.14 ² tumor growth inhibition (mass),% 0 12 58 71 tumor growth inhibition (volume),% 0 10 63 84 number of metastases / mouse 8.63 ± 1.38 8.18 ± 1.1 1.56 ± 0.53 ¹ 0.44 ± 0.3 ² frequency of metastasis,% one hundred one hundred 56 ¹ 22 ² area of metastases, mm ² 31.38 ± 4.73 26.25 ± 2.76 2.56 ± 0.78 ¹ 0.56 ± 0.12 ² growth inhibition of metastases,% - 16 87 98 Metastasis Inhibition Index,% 0 2.9 82 98 Note: ¹ - the differences are significant at p <0.05 for LLC + CF in comparison with LLC and LLC + CCK; ² - the differences are significant at p <0.05 for LLC + CF + SC - in comparison with LLC + CF. Table 2. LLC + CF (60 mg / kg) LLC + CF (60 mg / kg) + sanazole LLC + CF (120 mg / kg) LLC + CF (120 mg / kg) + SC Inhibition of tumor growth (volume),% 55 63 63 84 * The frequency of metastasis,% one hundred 25 * one hundred 22 * Metastasis Inhibition Index,% thirty 98 * 82 98 * • - significant differences with "LLC + CF", p <0.05

Claims (1)

A method of increasing the antitumor and antimetastatic effect in an experiment, which consists in the intraperitoneal administration of cyclophosphamide at a dose of 120 mg / kg and the intravenous transplantation of fetal liver stem cells to an experimental animal, characterized in that the fetal liver stem cells are transplanted 3 days before cyclophosphamide administration, at a dose of 25 10 ⁶ cells per kilogram of mass.