METHOD OF OBTAINING A PHARMACEUTICAL AGENT USED FOR INHIBITING THE PROLIFERATION OF TUMOR CELLS

Abstract

The given invention is related to the field of medicine, namely to experimental research in oncology, and can be used to obtain a pharmaceutical agent for inhibiting the proliferation of HeLa cervical cancer cells. It entails the use of a metal powder obtained in a gaseous medium by means of an electric explosion of a wire made of low-carbon steel carried out with a specific energy of 7-18 kJ/g and a pulse duration of 1.2-2 s. The explosion is carried out inside a reactor that is pre-evacuated to a residual pressure of 10.sup.2 Pa, and then filled with carbon monoxide to a pressure of 10.sup.5 Pa at a gas circulation rate flow of 10 m/s. The products of the explosion deposited in a hopper are passivated in air for at least 48 hours. The resulting powder is extracted and mixed with a solution of the RPMI-1640 nutrient medium with L-glutamine, a pH of 7.2, in the proportion of the mass of the powder to the volume of the specified solution from 1:10 to 2.7:10. The solution is then centrifuged until the phases are separated. The liquid phase is decanted and used as a pharmaceutical agent. The purpose of the given invention is to expand the arsenal of tools for inhibiting the proliferation of tumor cells.

Claims

1. A method for obtaining a pharmaceutical agent for inhibiting proliferation of cervical cancer cells, comprising: use of a metal powder obtained in a gaseous medium by means of an electric explosion of a metal wire, wherein the metal wire is made of a low-carbon steel and the electric explosion is carried out at a specific energy of 7-18 kJ/g and a pulse duration of 1.2-2 s in a reactor that is pre-evacuated to a residual pressure of 10.sup.2 Pa and then filled with carbon monoxide to a pressure of 105 Pa at a circulation rate of a gas flow in the reactor of 10 m/s, products of the electric explosion deposited in a hopper are passivated in air for at least 48 hours, a resulting powder is extracted and mixed with a solution of a nutrient medium RPMI-1640 with L-glutamine, the solution having pH of 7.2, in a ratio of a mass of the resulting powder to a volume of said solution ranging from 1:10 to 2.7:10, the solution is then centrifuged until phases are separated, and a liquid phase is decanted and used as the pharmaceutical agent.

2. A method comprising inhibiting proliferation of cervical cancer cells using the pharmaceutical agent obtained according to claim 1.

3. A method comprising treating cervical cancer using the pharmaceutical agent obtained according to claim 1.

4. A method for obtaining a pharmaceutical agent for inhibiting proliferation of cervical cancer cells, comprising: providing a metal wire made of a low-carbon steel; providing a reactor that is pre-evacuated to a residual pressure of 10.sup.2 Pa; filling the reactor with carbon monoxide to a pressure of 105 Pa at a circulation rate of a gas flow in the reactor of 10 m/s; generating a metal powder obtained in a gaseous medium through an electric explosion of the metal wire, wherein the electric explosion is carried out at an energy of 7-18 kJ/g and a pulse duration of 1.2-2 s in the reactor; depositing products of the electric explosion in a hopper and passivating the products in air for at least 48 hours, thereby creating a resulting powder; extracting the resulting powder and mixing the resulting powder with a solution of a nutrient medium RPMI-1640 with L-glutamine, the solution having pH of approximately 7.2, in a ratio of a mass of the resulting powder to a volume of the solution ranging from 1:10 to 2.7:10; centrifuging the solution as mixed with the resulting powder until phases are separated; and decanting a liquid phase and obtaining the pharmaceutical agent therefrom.

5. A method comprising inhibiting proliferation of cervical cancer cells using the pharmaceutical agent obtained according to claim 4.

6. A method comprising treating cervical cancer using the pharmaceutical agent obtained according to claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, with emphasis instead being placed upon clearly illustrating the principles of the disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0016] FIG. 1 is a simplified circuit diagram of a Doherty power amplifier in the related art.

[0017] FIG. 1 shows a diagram of the rig for producing the metal powder.

[0018] FIG. 2 shows photographs of the particles of the powder obtained in Example 1.

[0019] FIGS. 3 and 4 show the particle size distribution of the powder obtained in Example 1.

[0020] FIG. 5 shows an X-ray diffraction pattern obtained in Example 1 of the powder.

[0021] FIG. 6 illustrates the change in the cell index in HeLa cells after the addition of a pharmaceutical agent.

[0022] FIGS. 7 and 8 show photographs of the particles of the powder obtained in Example 2.

[0023] FIG. 9 shows the particle size distribution of the powder obtained in Example 2.

[0024] FIGS. 10 and 11 show photographs of the particles of the powder obtained in Example 3.

[0025] FIG. 12 shows the particle size distribution of the powder obtained in Example 3.

DETAILED DESCRIPTION

[0026] The rig for producing the metal powder consists of a horizontally installed reactor 1, which contains high-voltage 2 and grounded electrodes 3, as well as a feed mechanism 4 of the wire. The electrode 2 is connected to power supply 5 (PS). The bottom of the reactor 1 is connected by a pipeline to the inlet to the cyclone 6 of a cylindrical type, the lower part of which is equipped with a hopper 7 for collecting the powder. The outlet of the cyclone 6 is connected to the upper part of the reactor 1 by a pipeline, which contains the fan 8. The cyclone 6 is connected through pipelines equipped with valves to the gas cylinder 9 (GC) containing carbon monoxide, to the foreline pump 10 (FP) and to the valve 25 for discharging the gas.

Example 1

[0027] A coil of low-carbon steel wire of SV-08 grade alloy was placed in the wire feed mechanism 4 in the reactor 1. The diameter of the wire was 0.3 mm, and the length of the interelectrode gap was 80 mm.

[0028] Using a foreline pump 10 (FP), the internal volume of the rig was pre-evacuated to a residual pressure of 10.sup.2. Then, from the gas cylinder 9 (GS), the internal volume of the rig was filled with carbon monoxide to a pressure of 105 Pa. By turning on the fan 8, carbon monoxide was continuously circulated through the pipeline connecting it to the reactor 1 at a speed of 10 m/s. Upon turning on of the feed mechanism 4, a continuous wire feed was injected in the direction from the grounded electrode 3 to the high-voltage electrode 2. The distance of the interelectrode gap was 80 mm. A high voltage with a duration of 1.5 s was applied to the high-voltage electrode 2 from the power source 5 (PS). When the wire injected into the reactor 1 came into contact with the high-voltage electrode 2, it exploded. The specific energy expended was 14 kJ/g. The products of the explosion of the wire were extracted by gas flow out of the reactor 1 into the cyclone 6, where they were separated from the carbon monoxide and deposited in the hopper 7. The purified gas from the cyclone 6 returned to the inlet of the fan 8 and entered the reactor 1 again. After the hopper 7 was filled with the accumulated products of the wire's explosion, the power source 5 (PS) was turned off, and the wire feeder 4, the fan 8 and the hopper 7 were disconnected from the cyclone 6. The hopper 7 was covered with a lid with an opening of a diameter of 1 mm and kept in this state for 48 hours to bring the resulting product into equilibrium. Afterwards, the resulting metal powder was removed from the hopper 7 and placed in a storage container.

[0029] The resulting metal powder is a mixture of nanoparticles ranging in size from 20 to 300 nm (FIG. 2, 3) with a maximum distribution of 80 nm and microparticles with a size of up to 2 m and a maximum distribution of about 0.8 In this case, the number of particles larger than 500 nm is no greater than 1% (FIG. 4).

[0030] X-ray phase analysis showed that the obtained metal powder consists of pure iron particles in the form of a-Fe phase and austenite compound in the form of FeC (FIG. 5). Its specific surface area was 9.3 m.sup.2/g.

[0031] Under sterile conditions, a 270 g sample was taken from the obtained metal powder, and 1,000 l of a nutrient solution RPMI-1640 with L-glutamine with a pH of 7.2 was added to it. The resulting mixture was stirred by ultrasound for 10 minutes in an ultrasonic bathWiseCleanwith an operating frequency of 300 kHz. Then the resulting suspension was centrifuged for 10 minutes in an Allegra 64R centrifuge (USA) at 3,000 rpm to separate the liquid and solid phases. The liquid phase, which is the end pharmaceutical agent, was poured into a container.

[0032] The resulting pharmaceutical agent was used for in vitro testing on HeLa cells.

[0033] A HeLa cervical cancer cell line obtained from a Chinese hamster ovary CHO-K1 was used for the experiment. The cells were kept in a wholesome nutrient mediumRPMI-1640containing 10% inactivated fetal calf serum and antibiotics (50 U/ml penicillin and 50 g/ml streptomycin), at 37 C., at 5% CO.sub.2 content, in a humid environment (in a CO.sub.2 incubator).

[0034] The effect of the obtained pharmaceutical agent on epithelioid carcinoma of human cervical cancer HeLa cancer cells was carried out using a plate. The cells were seeded into the wells of an 8-well plate from the iCelligence system (ACEA Bioscience, USA) at the rate of 70 thousand cells per well.

[0035] 4 hours after seeding of the cells, 200 l of the obtained pharmaceutical agent was poured into some of the wells, and 200 l of the RPMI-1640 nutrient solution were added to others.

[0036] The iCelligence real-time assessment system (ACEA Bioscience, USA) was used to determine the level of proliferation. The readings were taken in automated mode with an interval of 1 hour. The iCelligence system performed an automatic recalculation of the readings into the cell index, indicating the level of cell proliferation, as well as the calculation of the standard deviation. The data was statistically processed using the Statistica software.

[0037] The crimson curve (270 g/ml) in FIG. 6 shows the inhibition of the proliferation of HeLa tumor cells relative to the control group (red curve). The inhibition of the proliferation of the tumor cells begins almost immediately after the addition of the pharmaceutical agent. The cell index was 0.5 rel. units, and 3.25 rel. units in the control group over 120 hours of observation.

Example 2

[0038] Under conditions similar to Example 1, an electric explosion of a workpiece with a diameter of 0.3 mm and a length of 80 mm was carried out on a low-carbon steel wire of SV08 grade alloy, at an energy supply of 7 kJ/g for 2 s.

[0039] The corresponding images of the resulting powder particles are shown in FIG. 7 and FIG. 8.

[0040] The powder is a mixture of particles with a size of up to 6 microns and a maximum distribution of 2 microns and nanometer particles with a size of 20 to 300 nm with a maximum distribution of 80 nm.

[0041] The number of particles larger than 500 nm is more than 2% (FIG. 9). The phase composition of the resulting powder is the same as in Example 1 (FIG. 5). The specific surface area of this powder was 4 m.sup.2/g.

[0042] Under sterile conditions, a sample weighing 100 g was taken from the obtained metal powder, and 1,000 l of a nutrient solution RPMI-1640 with L-glutamine with a pH of 7.2 was added to it. The pharmaceutical agent was prepared under the same conditions as in Example 1.

[0043] The resulting pharmaceutical agent was added to one of the wells at a rate of 100 g/ml, and a nutrient solution was added to the control wells. The observation results are represented by the blue curve in FIG. 6 (100 g/ml). The inhibition of the proliferation of the HeLa tumor cells relative to the red control curve begins almost immediately after the addition of the pharmaceutical agent. The cell index was 0.75 rel. units, and 3.25 rel. units in the control group over 120 hours of observation.

Example 3

[0044] Under conditions similar to Example 1, an electric explosion of a workpiece with a diameter of 0.3 mm and a length of 80 mm was carried out on a low-carbon steel wire of the SV08 grade alloy, at an energy supply of 18 kJ/g for 1.2 s.

[0045] The corresponding images of the resulting powder particles are shown in FIG. 10, 11.

[0046] The resulting powder is a mixture of micron particles with a size of up to 2 m and a maximum distribution of 500 nm and partially sintered nanoscale particles with a size of 20 to 300 nm with a maximum distribution of 80 nm. The number of particles larger than 500 nm is not more than 0.5% (FIG. 12). The phase composition of the powder is the same as in Example 1 (FIG. 5). The specific surface area of this powder was 11 m.sup.2/g.

[0047] From the obtained powder, a weighed portion of 170 g was taken and 1,000 l of a nutrient solution RPMI-1640 with L-glutamine with a pH of 7.2 was added to it. Under the same conditions as in Example 1, a pharmaceutical agent was prepared. The resulting pharmaceutical agent was added to some of the wells at a rate of 170 g/ml, and a nutrient solution was added to the control wells. The observation results are represented by a green curve in FIG. 6 (170 g/ml). The inhibition of the proliferation of the HeLa tumor cells relative to the red control curve begins almost immediately after the addition of the pharmaceutical agent. The cell index is 0.75 rel. units, and 3.25 rel. units in the control group for 120 hours of observation.

[0048] The above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.