WO2019240632A1

WO2019240632A1 - Agent having anti-tumor effect for treating oncological diseases

Info

Publication number: WO2019240632A1
Application number: PCT/RU2019/050085
Authority: WO
Inventors: Рахимджан Ахметджанович РОЗИЕВ; Анна Яковлевна ГОНЧАРОВА; Кенес Тагаевич ЕРИМБЕТОВ; Екатерина Валерьевна БОНДАРЕНКО
Original assignee: Рахимджан Ахметджанович РОЗИЕВ
Priority date: 2018-06-13
Filing date: 2019-06-13
Publication date: 2019-12-19
Also published as: RU2018121545A3; RU2018121545A; RU2761429C2

Abstract

Claimed is an agent having anti-proliferative and anti-metastatic properties, said agent being a 3-(2-phenylethyl)-2-thioxo-1,3 thiazolidin-4-one complex having β-cyclodextrins. Said composition can be used in medicine and pharmacology for treating tumoral diseases occurring at different sites.

Description

An anti-tumor agent for the treatment of cancer.

The invention relates to a new tool, which is a clathrate complex of 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one with b-cyclo-dextrin. The specified complex can find application in medicine and pharmacology for the treatment of tumor diseases of various localization.

At present, cancer chemotherapy is one of the main methods of their treatment. The modern medicines used to treat oncological diseases are not effective enough and have a number of serious undesirable effects caused by a damaging effect on healthy organs and tissues. In this regard, the urgent problem remains the search for low-toxic drugs with high therapeutic activity. Moreover, successes in the development of new antitumor drugs are associated not only with the synthesis of new chemical compounds, but also, to a large extent, with improved properties.

It is known that the compound 3- (2-phenylethyl) -2-thioxo-4-thiazolidinone has an antiproliferative effect on the cell culture of neuroblastoma N2A / US 2003/0195238, C07D207 / 44, publ. 10.16.2003 /.

It is also known that 3- (2-phenylethyl) -2-thioxo-1,3-thiazolidin-4-one has antiproliferative and antimetastatic activity against tumor cells of Lewis epidermoid carcinoma implanted into mice / Patent for invention RF2522449, C07D277 / 36, publ. . July 10, 2014 /.

The available data on 3- (2-phenylethyl) -2-thioxo-1,3-thiazolidin-4-one and b-cyclodextrin as a carrier allow us to create an effective and safe pharmaceutical clathrate complex and expand the arsenal of funds for the treatment of cancer. One of the components of the pharmaceutical composition 3- (2-phenylethyl) -2-thioxo- 1,3 thiazolidin-4-one, being a multikinase inhibitor [Ana Martinez SAR and 3D-QSAR Studies on Thiadiazolidinone Derivatives: Exploration of Structural Requirements for Glycogen Synthase Kinase 3 Inhibitors / Ana Martinez et al. // J. Med. Chem. 2005, 48, 7103-7112], is aimed both at suppressing the proliferation of tumor cells and their metastasis, as well as at inhibiting the growth of tumor vessels. Tumor cell proliferation and vascularization (vascular growth) of the tumor are two important processes that ensure the growth of a malignant tumor. At the same time, the development of new clathrate complexes of active pharmaceutical substances with b-cyclodextrin will increase the solubility in water, improve bioavailability and, therefore, reduce their doses. The absence of low-dose, highly effective drugs is an urgent technical problem in modern medical chemistry. One of the promising areas in the development of such dosage forms is mechanochemistry. The creation of new dosage forms by mechanochemical activation is based on the creation of a complex by combining b-cyclodextrin carriers with the pharmaceutically active substance 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one.

The technical result achieved by the present invention is to increase the efficiency, reduce the dose used.

The technical problem posed is solved by using a clathrate complex consisting of 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one with b-cyclodextrin, with the following content:

3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one 10 wt.% And b-cyclodextrin the rest, as an agent with antiproliferative and antimetastatic properties.

There is no information in the literature on the use of a clathrate complex consisting of 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one with b-cyclodextrin containing an active pharmaceutical substance 10 wt.% And b-cycle o dextrin rest, as an antiproliferative and antimetastatic agent for the treatment of cancer.

New in the present invention is that, as a means of inhibiting the proliferation and metastasis of various tumor cells, a clathrate complex consisting of 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one with b-cyclodextrin c can be used the total content of active pharmaceutical substance 10 wt.% and b-cyclodextrin - the rest. For a specialist, these properties are not explicitly derived from the prior art.

The resulting clathrate complex is storage stable and tested for antiproliferative and antimetastatic efficacy.

Statistical processing of the research results was carried out using parametric and nonparametric methods. Differences between groups will be considered significant at p <0.05.

The complex is 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one in complex with b-cyclodextrin.

The pharmaceutical substance (PS) is 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one.

The invention is illustrated by examples of specific performance.

Examples. The antitumor efficacy of 3- (2-phenylethyl) -2-thioxo-1,3-thiazolidin-4-one PS and its clathrate complex with b-cyclodextrin was evaluated in experiments on mice with implanted tumors of melanoma B 16 and Lewis epidermoid carcinoma and included the following studies :

the antiproliferative and antimetastatic effects of PS and the clathrate complex and the dacarbazine comparison drug were carried out in an experiment in mice with implanted tumor cells with B 16 melanoma; antiproliferative and antimetastatic action of PS and clathrate complex was carried out in an experiment in mice with implanted tumor cells of Lewis epidermoid lung carcinoma;

assessment of antimetastatic activity of PS and clathrate complex in mice with implanted tumor cells of Lewis epidermoid carcinoma of the lung with removal of the primary tumor node.

Criteria for evaluating the effectiveness of the studied compounds

Antiproliferative efficacy was assessed by changes in the volume of the neoplasm. The volume of the neoplasm (V mm3) was calculated according to the following formula (1):

V = axb> <c (l),

where a, b and c are the length, width and height of the tumor node.

Measurement time: 10-11, 15-16 and 20-21 days after implantation. As additional criteria, we used the change in animal body weight, the degree of inhibition of tumor growth, the tumor growth inhibiting effect on the logarithm of dead cells, and the effect of increasing the average life expectancy.

The degree of inhibition of tumor growth was determined by the inhibition of tumor growth (SRW), calculated by the following formula (2):

TPO% = (Control - Experience) / Control ^c 100, (2), where V is the average tumor volume (mm3) in the receiving drug and the control group, respectively, for a specific period.

To evaluate the tumor growth inhibitory effect, the logarithm of dead cells was calculated in the number of subcutaneously inoculated melanoma B 16 and epidermoid lung carcinoma Lewis. For this, in the control (t control) and treated (t experiment) groups, the average time for doubling the tumor volume or reaching a certain value was determined. The difference between the indicators "t control" and "t experience" (time growth retardation in the groups receiving the drugs) was directly related to the pool of dead cells by this time. The lg number of dead cells (lg and) was calculated by the following formula (3):

Lg and = tk - to / 3,32Td, (3),

where tk - to is the time delay of tumor growth in the experiment;

Td — time for doubling the size of the tumor, calculated from the exponential curve of tumor growth in the control;

3.32 is the number of doublings necessary for increasing log and by one order of magnitude.

At the end of the experiments and the death of all animals, an effect was evaluated to increase their average life expectancy. For this, the average life expectancy (LSS, days) in the groups was determined and the indicator of increasing life expectancy (LLL%) was calculated by the following formula (4):

VL% = (LN test - LN control) / LN control 100 (4)

Antimetastatic efficacy was evaluated by the number of metastases in the lungs, which was determined 9-10 and 21 days after the start of administration of the studied drugs. For this, lungs were removed from animals decapitated under anesthesia and transferred to Buen's solution. Metastases were counted macroscopically. The intensity of the metastatic process was assessed by the frequency of tumor metastasis (the percentage of animals with metastases in relation to their total number in the group), the degree of metastatic lung damage according to the criteria developed by Tarin and Price, and the metastasis inhibition index (IIM), which was calculated by the formula (5):

k) - (A B

IIM C A B) ^• 100%

And _to - To _to

(5),

Where

Ak and A - the frequency of metastasis to the lungs in mice of the control group and experimental; Bk and B - the average number of metastases in the lungs in the control and experimental groups.

The antiproliferative and antimetastatic properties of PS 3- (2-phenylethyl) -2-thioxo-1,3-thiazolidin-4-one and its clathrate complex with b-cyclodextrin, as well as a dacarbazine comparison drug, were studied in an experiment in mice with implanted tumor cells melanoma B 16.

The experiments were carried out on 210 male mice and female F1 CBAxC57B1 / 6 (body weight 18-22 g, age 2-2.5 months). Mytti were divided into 7 groups of 30 animals of both sexes. The experimental groups, doses of the studied compounds and the number of animals are shown in table 1.

Table 1 - Experimental groups, doses of the studied compounds and the number of animals

A suspension of B-16 melanoma cells was implanted in the hind paw under the skin of animals in an amount of ~ 106 cells (~ 0.2 cm3). Cells of murine melanoma B-16, adapted for monolayer cultivation (cell bank of the National Research Center for Surgery named after N.N. Blokhin) were cultured in RPMI-1640 medium supplemented with antibiotics (penicillin-streptomycin at 100 IU / ml),

10% fetal calf (10% by volume) serum at temperature +37 ° C in an atmosphere containing 5% CO2. As culture dishes, sterile mattresses with an area of 75 cm3 were used (Coming, Nunc). Reseeding of culture was carried out 3 times a week.

As a comparison drug was used dacarbazine. The comparison drug was administered every two days (a total of 8 doses) at a dose of 5.0 mg / kg. FS and Complex in a volume of 0.4 ml in a 1% starch gel was injected into the stomach with a metal probe for 21 days. Dacarbazine was administered intraperitoneally with a syringe in the form of a solution. Mice of the control group with an implanted tumor were intragastrically injected with 1% starch gel in an amount of 0.4 ml per animal.

The results of the studies indicate that the administration to mice with implanted tumor cells of melanoma B 16 FS, the Complex and the dacarbazine comparison drug statistically significantly reduces their tumor volume compared to the control group (table 2) already 10 days after tumor implantation. Moreover, the inhibition of tumor growth in the treated groups was 32-47%. In the next day after tumor implantation, statistically significant differences from the control were obtained with the introduction of PS, Complex and dacarbazine in all studied doses. Moreover, the best results were achieved by reducing the tumor volume and tumor inhibition efficiency during the observed period of its development with the introduction of the Complex in doses of 10 and 20 mg / kg of mouse body weight. Compared with the reference drug and PS, administration of the Complex mice at doses of 10 and 20 mg / kg provided statistically significant inhibition of tumor growth. Table 2 - Change in tumor volume (mm) and TPO,% in mice depending on the dose of administration of the studied drugs

* P <0.00001-0.05 values in the experimental groups compared with the control;

** P <0.00001-0.05 values in group 7 compared with the control;

# P <0, 00001-0, 05 values in groups 4 and 5 compared with group 7;

& P <0.00001-0.05 values in groups 4 and 5 compared with group 2.

The average life expectancy of animals to which the Complex was administered in all studied doses increased compared with the mice of the control group, dacarbazine and FS. The most significant data for an increase in the average life expectancy of mice was obtained with the introduction of the Complex in doses of 10-40 mg / kg compared with animals that were administered dacarbazine and FS (table 3).

Table 3 - Inhibitory tumor growth effect on the logarithm of dead cells and average life expectancy of mice depending on the dose of administration of the studied drugs

^* P <0, 00001-0, 05 values in the experimental groups compared with the control;

P <0.00001-0.05 values in group 7 compared with the control;

^# P <0, 00001-0, 05 values in groups 4 and 5 compared with group 7.

^& P <0.00001-0.05 values in groups 4 and 5 compared with group 2. The administration of dacarbazine, PS and Complex to tumor-bearing mice in all studied doses leads to an increase in lg and the number of dead cells, and these values are higher than 0.7, which may indicate their high activity. It should be noted that the greatest inhibitory tumor growth effect on the logarithm of dead cells was achieved when the Complex was administered to mice at doses of 10-40 mg / kg (table 3).

When analyzing the number of metastases in the lungs, a statistically significant decrease was found in comparison with the control, dacarbazine and PS for tumor-bearing mice of the studied groups, which were administered Complex in all doses administered (table 4). It should also be noted that the best results were achieved, as in the case of a decrease in tumor volume with the introduction of the Complex at doses of 10 and 20 mg / kg.

Table 4 - The number of metastases in the lungs depending on the dose of administration of the studied drugs

P <0.00001-0.05 values in the 7th group compared with the control;

^# P <0, 00001-0, 05 values in groups 4 and 5 compared with group 7;

^& P <0, 00001-0, 05 values in groups 4 and 5 compared with group 2.

A study of the antitumor activity of FS, the Complex, and the cisplatin comparison drug was carried out in an experiment in mice with implanted tumor cells of Lewis epidermoid carcinoma of the lung.

The experiments were carried out on 210 male and male mice ¥ СВАхС57В1 / 6 (body weight 19-21 g, age 2-2.5 months). A suspension of Lewis epidermoid carcinoma cells in the amount of ~ 106 cells (~ 0.2 cm) was implanted in the hind paw under the skin of mice. Lewis mouse epidermoid carcinoma cells were obtained from the cell bank of the State Pediatric Oncology Center named after N.N. Flea.

Mytti were divided into 7 groups of 30 animals of both sexes. The experimental groups, the doses of the test compounds and the number of animals are shown in table 5. As a comparison drug, cisplatin was used. FS and Complex in a volume of 0.4 ml in a 1% starch gel was injected into the stomach with a metal probe for 21 days. The comparison drug cisplatin at a dose of 3.5 mg / kg (per administration) was administered 3 times for 21 days with a syringe in the form of an injection for injection in a volume of 0.2 ml intraperitoneally. Mice of the control group with an implanted tumor were intragastrically injected with 1% starch gel in an amount of 0.4 ml per animal.

Table 5 - Experimental groups, doses of the studied compounds and the number of animals

The results of the studies show that the administration of cisplatin, PS, and the Complex to mice implanted with tumor cells with epidermoid carcinoma Lewis statistically significantly reduces their tumor volume compared to animals in the control group (table 6) already 11 days after tumor inoculation. Moreover, the inhibition of tumor growth in the treated groups was 26-47%. In the next day after tumor implantation, statistically significant differences persisted with the administration of cisplatin, PS and Complex in all studied doses. Moreover, the best results were achieved in reducing the tumor volume and inhibition of tumor growth during the observed period of its development with the introduction of the Complex in doses of 10 and 20 mg / kg of body weight of mice. Compared with cisplatin and FS, administration of Complex mice at doses of 10 and 20 mg / kg provided statistically significant inhibition of tumor growth.

Table 6 - Change in tumor volume (mm) and TPO,% in mice depending on the dose of administration of the studied drugs

^** P <0.00001-0.05 values in group 7 compared with the control;

^# P <0, 00001-0, 05 values in groups 4 and 5 compared with group 7;

& P <0, 00001-0, 05 values in groups 4 and 5 compared with group 2.

The average life expectancy of animals that were injected with cisplatin, FS and Complex in all studied doses increased by 37-60% compared with mice of the control group. The most statistically significant data on the increase in the average life expectancy of mice compared to animals that were injected with cisplatin and PS were obtained with the introduction of the Complex in doses of 10-40 mg / kg (table 7).

Table 7 - Inhibitory tumor growth effect on the logarithm of dead cells and average life expectancy of mice depending on the dose of administration of the studied drugs

* P <0, 00001-0, 05 values in the experimental groups compared with the control; ^** P <0.00001-0.05 values in group 7 compared with the control;

^# P <0, 00001-0, 05 values in groups 4 and 5 compared with group 7;

^& P <0.00001-0.05 values in groups 4 and 5 compared with group 2.

When analyzing the number of metastases in the lungs, a statistically significant decrease was found in comparison with the control for tumor bearing mice of the studied groups, which were administered cisplatin, FS and Complex in all doses administered (table 8). It should be noted that the best results were achieved, as in the case of a decrease in tumor volume, with the introduction of the Complex at doses of 10 and 20 mg / kg compared with FS and cisplatin.

Table 8 - the Number of metastases in the lungs depending on the dose of the study drugs

P <0.00001-0.05 values in the 7th group compared with the control;

# P <0.00001-0.05 values in groups 4 and 5 compared with group 7;

^& P <0, 00001-0, 05 values in groups 4 and 5 compared with group 2.

Evaluation of the antimetastatic activity of FS and the Complex was carried out in mice with implanted Lewis lung epidermoid carcinoma with removal of the primary tumor node.

The experiments were carried out on 180 male mice and female F1

СВАхС57В1 / 6 (body weight 18-21 g, age 2-2.5 months). A suspension of Lewis epidermoid carcinoma cells was implanted into the pad of the hind paw of mice, in an amount of ~ 106 cells (~ 0.2 cm3). Lewis mouse epidermoid carcinoma cells were obtained from the cell bank of the State Pediatric Oncology Center named after N.N. Flea. On the 15th day after the transplantation of Lewis epidermoid carcinoma cells, amputation of the legs with the tumor along the knee joint was performed under ether anesthesia with the application of a pulling tourniquet in mice. The surgical wound was treated with medical glue.

Mytti were divided into 6 groups of 30 animals of both sexes.

The experimental groups, doses of the studied compounds and the number of animals are shown in table 9. PS and Complex mice were injected into the stomach with a metal probe in 1% starch gel in a volume of 0.4 ml per mouse. The introduction of the FS and the Complex began 1 hour before the operation and continued to be administered daily for 9 days. Half of the animals from each group were left to assess the effectiveness of therapy to increase their average life expectancy. Mice in the control groups with and without tumor removal were administered intragastrically 1% starch gel in an amount of 0.4 ml per animal.

Table 9 - Experimental groups, doses of the studied compounds and the number of animals

The results of the studies indicate that the introduction of the Complex to mice with implanted epidermoid lung carcinoma Lewis with removal of the primary tumor node provided an increase of 56-57% compared with mice of the control group (with the removed primary tumor node) and FS of the average life span of animals (table 10) .

Table 10 - Average life expectancy of mice, depending on the dose of administration of the studied drugs

t

P <0.00001-0.05 values in groups 5 and 6 compared with group 2;

^# P <0, 00001-0, 05 values in the 2nd group compared to the 1st group;

n P <0, 00001-0, 05 values in 5 and 6 groups compared with 1 group.

^& P <0, 00001-0, 05 values in groups 5 and 6 compared with groups 3 and 4. When analyzing the number of metastases in the lungs, a statistically significant decrease was found in comparison with animals from the control groups (with and without removal of the tumor) and PS for mice of the studied groups with the removed primary tumor node, which were injected with Complex at doses of 10 and 20 mg / kg. It should also be noted that a favorable picture according to the degree of least metastatic lesion of the lungs of mice was revealed in groups with a primary tumor node removed with the introduction of the Complex at doses of 10 and 20 mg / kg (table

P).

Table 11 - The number of metastases in the lungs and their degree of damage depending on the dose of administration of the studied drugs

^* P <0.00001-0.05 values in groups 5 and 6 compared with group 2;

^# P <0, 00001-0, 05 values in the 2nd group compared to the 1st group; P <0, 00001-0, 05 values in groups 5 and 6 compared with group 1.

^& P <0, 00001-0, 05 values in groups 5 and 6 compared with groups 3 and 4. The introduction of the Complex at doses of 10 and 20 mg / kg to mice with removed primary tumor nodes compared with the control groups and FS provided effective inhibition of the metastatic process, as shown by the data presented in table 12.

Table 12 - Effect of PS and Complex on the intensity of the metastatic process in mice

As a result of studies on the antitumor effect of the clathrate complex - 3- (2-phenylethyl) -2-thioxo-1,3-thiazolidin-4-one with b-cyclodextrin, it was established that the Complex under study has a pronounced antiproliferative and antimestatic effect against tumors as melanoma In 16, both epidermoid carcinoma Lewis. The use of the Complex in tumor-bearing mice in comparison with FS provides effective inhibition of tumor growth, an increase in the average duration of animals, and a decrease in the number of lung metastases. It should be noted that the Complex revealed a rather high antimetastatic effect. The use of the Complex in mice with a remote primary tumor node provided effective inhibition of the metastatic process, an increase in the average duration of animals, and a decrease in the number of metastases in the lungs.

The best results were achieved with the introduction of the Complex compared with FS in doses of 10-20 mg / kg. In the experiments performed, the Antitumor Action Complex was superior to the comparison drugs dacarbazine and cisplatin.

The present invention provides an increase in efficiency, a reduction in the doses used and an expansion of the arsenal of agents for treating various types of tumor diseases.

Claims

Claim

An agent with antiproliferative and antimetastatic properties, consisting of 3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one with b-cyclodextrin in the following contents:

3- (2-phenylethyl) -2-thioxo-1,3 thiazolidin-4-one - 10 wt.%

b-cyclo dextrin - the rest.