JP6736004B2 - Anticancer agent and infusion solution, method for producing them, and anticancer substance - Google Patents

Description

The technical field of the present specification relates to anticancer agents and infusion solutions, methods for producing them, and anticancer substances. More specifically, the present invention relates to an anticancer agent and an infusion solution produced by using plasma, a method for producing them, and an anticancer substance.

Plasma technology has been applied in the fields of electricity, chemistry and materials. In recent years, application to medicine has been actively studied. Inside the plasma, ultraviolet rays and radicals are generated in addition to charged particles such as electrons and ions. It has been found that these have various effects on living tissues, including sterilization of living tissues.

For example, in Patent Document 1, blood coagulation (see Example 4 of Patent Document 1, paragraphs [0063] to [0068]) and tissue sterilization (Example 5 of Patent Document 1, paragraph [] are performed by direct plasma irradiation. 0069]-[0074]) and leishmaniasis (see Example 6 of Patent Document 1, paragraphs [0075]-[0077]). Then, it is described that plasma has an effect of killing melanoma cells (malignant melanoma cells) (see Example 7, paragraph [0078] of Patent Document 1).

Further, Patent Document 2 discloses a technique of sterilizing bacteria in the liquid by irradiating plasma to a liquid adjusted to have a pH of 4.8 or less (paragraph [Patent Document 2 paragraph [ [0020] etc.). Further, it is described that superoxide anion radicals, hydroperoxy radicals and the like may have a bactericidal effect (see paragraphs [0090]-[0099] of Patent Document 2).

Japanese Patent Publication No. 2008-533007 International Publication No. 2009/041049 International Publication No. 2013/128905

By the way, in the treatment of such cancer, it is generally preferable to 1) kill the cancer cells and 2) selectively kill the cancer cells so as not to affect the normal cells. This is because even if cancer cells can be killed, killing a large number of normal cells for that purpose imposes a great physical burden on the patient. Therefore, a therapeutic technique for selectively killing cancer cells in this way is desired. However, it is not easy to selectively kill cancer cells. Further, in Patent Document 1, the degree of influence on normal cells is not clear.

Therefore, as described in Patent Document 3, the present inventors have researched and developed a technique relating to an antitumor aqueous solution that selectively kills cancer cells (paragraphs [0085] to [0087] of Patent Document 3 and FIG. 16 etc.). This anti-tumor aqueous solution can selectively kill cancer cells with almost no effect on normal cells. Further, this antitumor aqueous solution exerted an antitumor effect not only on cultured cells but also on mice (see paragraphs [0145]-[0152] of Patent Document 3 and FIGS. 45, 46, etc.).

However, it is difficult to administer an antitumor aqueous solution containing culture components into the body of a patient. This is because the effect of the culture components on the patient's body cannot be ignored.

The technique of the present specification has been made to solve the problems of the above-described conventional technique. That is, the problem is that anti-cancer agents and infusions capable of selectively killing cancer cells with little effect on normal cells and administrable into the body of a patient, and methods for producing them and anti-cancer agents. To provide a substance.

The method for producing an anticancer agent according to the first aspect includes an aqueous solution preparation step of preparing a first aqueous solution and a plasma irradiation step of irradiating the first aqueous solution with plasma to obtain a second aqueous solution. The first aqueous solution is lactic acid, sodium lactate, potassium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, calcium acetate, citric acid, sodium citrate, and potassium citrate. calcium citrate and contains at least one of.

This method for producing an anticancer agent is a method for producing an anticancer agent having an antitumor effect. Moreover, this anticancer agent may be Ringer's solution. Therefore, it can be used as an infusion solution. That is, this anticancer agent is an intravenous drip for intravenous injection into a patient. It is also possible to wash the patient's organs and the like.

In the method for producing an anticancer agent according to the second aspect, the first aqueous solution is Ringer's solution.

In the method for producing an anticancer agent according to the third aspect, the first aqueous solution is lactated Ringer's solution.

In the method for producing an anticancer agent according to the fourth aspect, the first aqueous solution is Ringer's acetate solution.

In the method for producing an anticancer agent according to the fifth aspect, the first aqueous solution is a bicarbonate Ringer's solution.

In the method for producing an anticancer agent according to the sixth aspect, the first aqueous solution contains at least one of sodium chloride, potassium chloride, and calcium chloride.

The method for producing an anticancer agent according to the seventh aspect has a component addition step of adding at least one of sodium chloride, potassium chloride, and calcium chloride to the second aqueous solution to form a third aqueous solution.

The method for producing an anticancer agent according to the eighth aspect has a freezing step of freezing the second aqueous solution or the third aqueous solution. The second aqueous solution or the third aqueous solution contains sodium chloride, potassium chloride, and calcium chloride.

In the method for producing an anticancer agent according to the ninth aspect, in the freezing step, the second aqueous solution or the third aqueous solution is frozen in the range of -196°C or higher and 0°C or lower.

In the method for producing an anticancer agent according to the tenth aspect, in the plasma irradiation step, the first electrode having the tubular portion is arranged outside the first aqueous solution and the second electrode is arranged in the first aqueous solution. Then, gas is irradiated from the tubular portion of the first electrode toward the first aqueous solution, and in that state, a voltage is applied between the first electrode and the second electrode.

The method for producing an infusion solution according to the eleventh aspect includes an aqueous solution preparation step of preparing a first aqueous solution, and a plasma irradiation step of irradiating the first aqueous solution with plasma to form a second aqueous solution. The first aqueous solution is lactic acid, sodium lactate, potassium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, calcium acetate, citric acid, sodium citrate, and potassium citrate. calcium citrate and contains at least one of.

Provided herein are anticancer agents and infusions capable of selectively killing cancer cells with little effect on normal cells and administrable into the body of a patient, a method for producing them, and an anticancer substance. Has been done.

It is a conceptual diagram for demonstrating the structure of the robot arm which scans the gas ejection port of the plasma generator of embodiment. Figure 2. 2A is a cross-sectional view showing the configuration of the first plasma generator, and FIG. B is a figure which shows the shape of an electrode. Figure 3. FIG. 3A is a sectional view showing the configuration of the second plasma generator, and FIG. B is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma region. It is a figure which shows schematic structure of the 3rd plasma generator in embodiment. It is a schematic block diagram which shows the upper structure of the 3rd plasma generator in embodiment. It is a schematic block diagram which shows the lower structure of the 3rd plasma generator in embodiment. It is a figure for demonstrating the case where the 3rd plasma generation apparatus is irradiating plasma in embodiment. It is a graph which shows the ratio of the number of viable cells at the time of supplying an anticancer agent to 5000 U251SP cells using the 2nd plasma generator in experiment A. It is a graph which shows the ratio of the number of viable cells at the time of supplying an anticancer agent to 10,000 U251SP cells using the 2nd plasma generator in experiment A. It is a graph which shows the ratio of the number of viable cells at the time of supplying an anticancer agent to 5000 U251SP cells using the 3rd plasma generator in experiment A. It is a graph which shows the ratio of the number of viable cells at the time of supplying an anticancer agent with respect to 10,000 U251SP cells using the 3rd plasma generator in experiment A. It is a graph which shows the ratio of the viable cell number at the time of supplying various sample aqueous solution with respect to 10000 U251SP cells in experiment B. 7 is a graph showing the ratio of the number of viable cells in the case of supplying an anticancer agent that has undergone a freezing step and a thawing step to U251SP cells in Experiment C. 7 is a graph showing the selectivity of anticancer agents in Experiment D. It is a figure for demonstrating the experimental method in the experiment E. It is a photograph which shows the subcutaneous tumor extracted in experiment E. 7 is a graph showing the change over time in the volume of subcutaneous tumors in Experiment E. 7 is a graph showing changes in body weight of nude mice in Experiment E. 7 is a graph showing the volume and weight of subcutaneous tumors removed in Experiment E. It is a figure which shows the experiment procedure of experiment F. 7 is a graph showing the survival rate of SKOV3 in the case of treating SKOV3 with an aqueous solution in which a lactated Ringer's solution was irradiated with plasma in Experiment F. 7 is a graph showing the survival rate of SKOV3 in the case of treating SKOV3 with an aqueous solution in which the Ringer's acetate solution was irradiated with plasma in Experiment F. FIG. 7 is a graph showing the survival rate of SKOV3 in the case of treating SKOV3 with an aqueous solution obtained by irradiating a bicarbonate Ringer's solution with plasma in Experiment F. FIG. It is a graph which shows the result of having observed ¹ H by NMR in Experiment G. It is the graph which expanded FIG. ¹³ is a graph showing the results of observing ¹³ C by NMR in Experiment G. It is the graph which expanded FIG. It is a graph (the 1) which shows the experimental result which investigated the antitumor effect of the sample aqueous solution in Experiment H. It is a graph (the 2) which shows the experimental result which investigated the antitumor effect of the sample aqueous solution in Experiment H.

Hereinafter, specific embodiments will be described with reference to the drawings by taking an anticancer agent, an infusion solution, a method for producing them, and an anticancer substance as examples.

(First embodiment)
The first embodiment will be described.

1. Anticancer agent manufacturing apparatus 1-1. Configuration of Anticancer Agent Manufacturing Apparatus The anticancer agent manufacturing apparatus PM of the present embodiment has a plasma irradiation unit P1 and an arm robot M1 as shown in FIG. The plasma irradiation device P1 is for generating plasma and irradiating the plasma toward the solution.

As shown in FIG. 1, the arm robot M1 can move the position of the plasma irradiation device P1 in each of the x-axis, y-axis, and z-axis directions. For convenience of description, the direction of plasma irradiation is the −z axis direction. Thereby, the distance between the liquid surface of the solution and the plasma irradiation part P1 can be adjusted. Further, this anticancer agent manufacturing apparatus PM is capable of irradiating plasma only for the time by setting the plasma irradiation time in advance.

The plasma irradiation device P1 has three types (first plasma generation device P10, second plasma generation device P20, and third plasma generation device P30) as described later. And any method may be used. The third plasma generation device P30 does not have the robot arm M1 and the like as shown in FIG.

1-2. First plasma generator FIG. A is a sectional view showing a schematic configuration of a plasma generator P10. Here, the plasma generation device P10 is a first plasma generation device that ejects plasma in spots. Figure 2. B is shown in FIG. It is a figure which shows the detail of the shape of the electrodes 2a and 2b of the plasma generator P10 of A.

The plasma generator P10 includes a housing 10, electrodes 2a and 2b, and a voltage applying unit 3. The casing 10 is made of a sintered body made of alumina (Al ₂ O ₃ ) as a raw material. The housing 10 has a tubular shape. The inner diameter of the housing unit 10 is 2 mm or more and 3 mm or less. The thickness of the housing 10 is 0.2 mm or more and 0.3 mm or less. The length of the housing unit 10 is 10 cm or more and 30 cm or less. A gas introduction port 10i and a gas ejection port 10o are formed at both ends of the housing 10. The gas introduction port 10i is for introducing a gas for generating plasma. The gas ejection port 10o is an irradiation unit for irradiating plasma to the outside of the housing unit 10. The direction in which the gas moves is the direction of the arrow in the figure.

The electrodes 2a and 2b are a pair of counter electrodes arranged so as to face each other. The lengths of the electrodes 2 a and 2 b in the facing surface direction are smaller than the inner diameter of the housing 10. For example, it is about 1 mm. The electrodes 2a and 2b have a structure shown in FIG. As shown in B, a large number of recesses (hollows) H are formed on each of the facing surfaces. Therefore, the opposing surfaces of the electrodes 2a and 2b have fine irregularities. The depth of the recess H is about 0.5 mm.

The electrode 2a is arranged inside the housing 10 and near the gas inlet 10i. The electrode 2b is arranged inside the housing 10 and near the gas ejection port 10o. Therefore, in the plasma generator P10, the gas is introduced from the opposite side of the facing surface of the electrode 2a, and the gas is jetted to the opposite side of the facing surface of the electrode 2b. The distance between the electrodes 2a and 2b is 24 cm. The distance between the electrodes 2a and 2b may be smaller than this.

The voltage application unit 3 is for applying an AC voltage between the electrodes 2a and 2b. The voltage application unit 3 boosts the voltage to 9 kV using a commercial AC voltage of 60 Hz and 100 V, and applies a voltage between the electrodes 2a and 2b.

When argon is introduced from the gas inlet 10i and a voltage is applied between the electrodes 2a and 2b by the voltage application unit 3, plasma is generated inside the housing unit 10. Figure 2. As indicated by the hatched line A, the region where plasma is generated is referred to as plasma generation region P. The plasma generation region P is covered by the housing 10.

1-3. Second plasma generator Fig. 3. A is a sectional view showing a schematic configuration of a plasma generator P20. Here, the plasma generator P20 is a second plasma generator that ejects plasma in a linear shape. Figure 3. B is shown in FIG. It is a fragmentary sectional view in a section perpendicular to the longitudinal direction of plasma field P of plasma generator P20 of A.

The plasma generator P20 has a housing 11, electrodes 2a and 2b, and a voltage application unit 3. The casing 11 is made of a sintered body made of alumina (Al ₂ O ₃ ) as a raw material. A gas introduction port 11i and a large number of gas ejection ports 11o are formed at both ends of the casing 11. The gas inlet 11i is shown in FIG. It has a slit shape with the left-right direction of A as the longitudinal direction. The slit width from the gas inlet 11i to just above the plasma region P (width in the left-right direction in FIG. 3.B) is 1 mm.

The gas ejection port 11o is an irradiation unit for irradiating plasma to the outside of the housing unit 11. The gas ejection port 11o has a cylindrical shape or a slit shape. The gas ejection port 11o in the case of a cylindrical shape is formed in a straight line along the longitudinal direction of the plasma region. The inner diameter of the gas ejection port 11o is in the range of 1 mm or more and 2 mm or less. Further, in the case of the slit shape, it is preferable that the slit width of the gas ejection port 11o is 1 mm or less. As a result, stable plasma is formed. Further, the gas introduction port 11i is adapted to introduce gas in a direction intersecting a line connecting the electrodes 2a and 2b.

The electrodes 2a and 2b and the voltage application unit 3 are the same as those of the plasma generator P10 shown in FIG. Then, similarly, a voltage is applied between the electrodes 2a and 2b using a commercial AC voltage. As a result, plasma can be ejected in a straight line.

Further, a plasma generator P20 for ejecting plasma in a straight line is shown in FIG. By arranging them side by side in the left-right direction of B, plasma can be ejected in a plane over a rectangular region.

1-4. Third Plasma Generating Device FIG. 4 is a conceptual diagram showing a schematic configuration of the third plasma generating device P30. The plasma generator P30 is for irradiating the contained solution with plasma.

As shown in FIG. 4, the plasma generator P30 includes a first electrode 110, a second electrode 210, a first potential applying section 120, a second potential applying section 220, and a first lead wire 130. A second lead wire 230, a gas supply unit 140, a gas pipe coupling connector 150, a gas pipe 160, a first electrode protection member 170, a second electrode protection member 240, and a first electrode support member 180. It has a sealing member 191, a coupling member 192, a container 250, a sealing member 260, and a pedestal 270.

1-4-1. Schematic Configuration of Electrode The first electrode 110 has a tubular portion 110a. Then, the plasma gas can be supplied to the inside of the tubular portion 110a. That is, the inside of the first electrode 110 communicates with the gas supply unit 140. The first electrode 110 blows gas from the tubular portion 110a toward the second electrode 210. The tip of the first electrode 110 has an injection needle shape. That is, the tip of the first electrode 110 has an inclined surface that is inclined with respect to the direction perpendicular to the axial direction of the first electrode 110. A micro hollow is formed at the tip of the first electrode 110.

The second electrode 210 is an electrode facing the first electrode 110. The second electrode 210 is a rod-shaped electrode. The second electrode 210 has a cylindrical shape. Alternatively, it may have a polygonal prism shape. Alternatively, it may have a needle shape with a sharp tip. Here, the second electrode 210 has a tip portion 211. The tip portion 211 of the second electrode 210 is made of an iridium alloy containing iridium. For example, it is an alloy of iridium and platinum. Alternatively, it is an alloy of iridium, platinum, and osmium. Iridium alloy has high hardness and excellent heat resistance. Therefore, the iridium alloy is suitable for the second electrode 210. Further, platinum may be used instead of iridium. Alternatively, it may be palladium. Alternatively, a metal or an alloy containing at least one kind of iridium, platinum, and palladium is preferable. Further, at the time of discharge, the second electrode 210 is immersed in the solution contained in the container 250.

The first potential applying section 120 is for applying a periodically changing potential to the first electrode 110. The second potential applying section 220 is for applying a potential that changes periodically to the second electrode 210. Here, either one of the first potential applying unit 120 and the second potential applying unit 220 may be grounded. The first lead wire 130 is for electrically connecting the first electrode 110 and the first potential applying section 120. The first lead wire 130 is preferably nickel alloy or stainless steel. The second lead wire 230 is for electrically connecting the second electrode 210 and the second potential applying section 220. The second lead wire 230 is preferably nickel alloy or stainless steel. As a result, a high frequency voltage is applied between the first electrode 110 and the second electrode 210. That is, the first potential applying section 120 and the second potential applying section 220 are voltage applying sections for applying a voltage between the first electrode 110 and the second electrode 210.

1-4-2. Gas Supply Path The plasma generation device P30 includes the gas supply unit 140, the gas pipe coupling connector 150, and the gas pipe 160, as described above. Therefore, the gas supply unit 140 supplies the plasma gas to the inside of the tubular portion of the first electrode 110 via the gas pipe 160 and the gas pipe coupling connector 150. Here, the gas supply unit 140 supplies, for example, Ar gas. Alternatively, other rare gas may be supplied. Alternatively, a small amount of other gas such as oxygen gas may be contained. Therefore, the plasma gas is blown from the first electrode 110 toward the solution contained in the container 250.

1-4-3. Configuration of Upper Structure FIG. 5 is a diagram showing an upper structure of the plasma generator P30. As shown in FIG. 5, the first electrode 110 has a tip portion 111. As shown in FIG. 4, the tip portion 111 is arranged at a position facing the second electrode 210. The tip portion 111 of the first electrode 110 has an inclined surface 111a. The inclined surface 111a is a surface that is inclined with respect to a surface perpendicular to the axial direction of the first electrode 110. A micro hollow 111b is formed on the tip portion 111. The micro hollow 111b is a minute recess having a length of 0.5 mm or more and 1 mm or less and a width of 0.3 mm or more and 0.5 mm or less.

Further, as described above, the plasma generation device P30 has the sealing member 191 and the coupling member 192. The sealing member 191 is attached to the container 250 shown in FIG. 4 and seals the inside of the container 250. The coupling member 192 is a member for coupling the first electrode 110 and the gas pipe coupling connector 150 via the sealing member 191 or the like.

1-4-4. Configuration of Lower Structure FIG. 6 is a diagram showing a lower structure of the plasma generator P30. As described above, the plasma generator P30 includes the container 250, the sealing member 260, and the pedestal 270. The container 250 can store a solution therein. Here, the solution includes an aqueous solution such as a culture solution, other aqueous solution, and an organic solvent. Moreover, the container 250 accommodates the first electrode 110 and the second electrode 210 therein. Further, the container 250 may have a scale. This is for measuring the amount of the solution contained in the container 250.

The sealing member 260 is for closing the gap between the second electrode protection member 240 and the container 250. An example of the sealing member 260 is an O-ring. Other members may be applied as long as they ensure the hermeticity of the container 250 and prevent the solution from leaking to the bottom of the container 250. The gantry 270 is for supporting the container 250 and other members.

2. Plasma generated by plasma generator 2-1. First Plasma Generator and Second Plasma Generator Plasmas generated by the plasma generators P10 and P20 are non-equilibrium atmospheric pressure plasmas. Here, the atmospheric pressure plasma refers to plasma having a pressure within the range of 0.5 atm to 2.0 atm.

In the present embodiment, Ar gas is mainly used as the plasma generating gas. Inside the plasma generated by the plasma generators P10 and P20, of course, electrons and Ar ions are generated. Then, the Ar ions generate ultraviolet rays. Further, since this plasma is released into the atmosphere, it generates oxygen radicals, nitrogen radicals, and the like.

The plasma density of this plasma is in the range of 1×10 ¹⁴ cm ⁻³ or more and 1×10 ¹⁷ cm ⁻³ or less. The plasma density of the plasma generated by the dielectric barrier discharge is about 1×10 ¹¹ cm ⁻³ to 1×10 ¹³ cm ⁻³ . Therefore, the plasma density of the plasma generated by the plasma generators P10 and P20 is about three orders of magnitude higher than the plasma density of the plasma generated by the dielectric barrier discharge. Therefore, more Ar ions are generated inside this plasma. Therefore, a large amount of radicals and ultraviolet rays are generated. The plasma density is almost equal to the electron density inside the plasma.

The plasma temperature at the time of plasma generation is in the range of approximately 1000K to 2500K. The electron temperature in this plasma is higher than the gas temperature. Moreover, although the electron density is in the range of 1×10 ¹⁴ cm ⁻³ or more and 1×10 ¹⁷ cm ⁻³ or less, the gas temperature is approximately 1000 K or more and 2500 K or less. The temperature of the plasma is the temperature in the plasma region P where the plasma is generated. Therefore, the plasma temperature at the position of the liquid surface can be set to about room temperature by changing the plasma condition and the distance from the gas ejection port to the liquid surface.

The density (radical density) of triplet oxygen atoms is in the range of 2×10 ¹⁴ cm ⁻³ or more and 1.6×10 ¹⁵ cm ⁻³ or less. The density of this triplet oxygen atom can be adjusted by adjusting the amount of oxygen gas mixed with the argon gas.

2-2. Third Plasma Generator FIG. 7 is a diagram schematically showing how the plasma generator P30 is generating plasma. The plasma generated by the plasma generator P30 is non-equilibrium atmospheric pressure plasma.

As shown in FIG. 7, the plasma gas supplied from the gas supply unit 140 is discharged from the first electrode 110 in the direction of arrow K1. Then, when a high frequency voltage is applied between the first electrode 110 and the second electrode 210, a plasma generation region PG1 is formed between the first electrode 110 and the second electrode 210. The plasma generation region PG1 in FIG. 7 is conceptually drawn.

When the first potential applying section 120 and the second potential applying section 220 apply a voltage between the first electrode 110 and the second electrode 210, the second electrode 210 is arranged inside the liquid. ing. Thus, between the first electrode 110 and the second electrode 210, there is the liquid contained in the container 250 and the atmosphere. The line connecting the first electrode and the second electrode intersects with the liquid surface LL1 of the liquid.

Therefore, plasma is generated between the liquid surface LL1 of the liquid and the first electrode 110. At this time, the liquid surface LL1 of the liquid receives the wind pressure of the plasma gas emitted from the first electrode 110 in the direction of the arrow K1 and is recessed toward the liquid side. Then, inside the liquid, the solution is partially electrolyzed and vaporized. Plasma is also generated inside the vaporized gas. The plasma generation region PG1 is in contact with the liquid surface LL1 of the liquid.

As described above, radicals derived from the air or water are generated. Then, the solution is irradiated with radicals. This causes the radicals to react with water molecules or solutes in the solution.

3. Effect of anti-cancer agent (anti-tumor aqueous solution) The anti-tumor aqueous solution of this embodiment is obtained by irradiating an aqueous solution containing L-sodium lactate with plasma. This antitumor aqueous solution has an antitumor effect, as described later. That is, the cancer cells immersed in this antitumor aqueous solution die, but the normal cells hardly die. Therefore, the antitumor aqueous solution of this embodiment can be used as an anticancer agent.

4. Treatment method using anti-cancer agent (anti-tumor aqueous solution) 4-1. Assumed Treatment Method The following method is assumed as a treatment method using the anticancer agent (antitumor aqueous solution) of the present embodiment. An anticancer agent is directly or indirectly administered to tumorous lesions (whether benign or malignant) and disease states (metastasis, dissemination, etc.) related to tumorous lesions. The term "administration" as used herein refers to all actions in which an anticancer agent is brought into direct or indirect contact with or affects an organ, tissue, or cell. That is, the administration is, for example, spraying or exposure. Examples of the case of indirect administration include a case of being contained in a cloth, absorbent cotton or the like and brought into contact with a neoplastic lesion.

4-2. Specific Example For example, an anticancer agent is directly or indirectly administered to a tumorous lesion that has arisen from the digestive organs, hepatobiliary tract, blood vessels, or organs or tissues or cells related thereto. Alternatively, an anticancer agent is intrathecally, thoracically or intraperitoneally administered to a disseminated brain tumor or cancer (intrathecal, thoracic or intraperitoneal dissemination).

5. Method for producing anticancer agent (antitumor aqueous solution) 5-1. Aqueous Solution Preparation Step First, a first aqueous solution is prepared. The first aqueous solution means an aqueous solution before being irradiated with plasma. The first aqueous solution is lactic acid, sodium lactate, potassium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, calcium acetate, citric acid, sodium citrate, and potassium citrate. It contains at least one of calcium citrate, potassium hydrogen carbonate, and calcium hydrogen carbonate. Further, the first aqueous solution may contain at least one of sodium chloride, potassium chloride, and calcium chloride. Then, the first aqueous solution is preferably Ringer's solution. Ringer's solution includes lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution.

5-2. Plasma irradiation step 5-2-1. First Plasma Generation Device and Second Plasma Generation Device Next, the first aqueous solution is irradiated with the non-equilibrium atmospheric pressure plasma generated in the plasma generation region by the anticancer drug manufacturing device PM. The distance between the liquid surface and the plasma ejection port when irradiating the plasma is, for example, 1 cm. Further, this distance may be changed within the range of 1 mm or more and 3 cm or less. The plasma density of this plasma is in the range of 1×10 ¹⁴ cm ⁻³ or more and 1×10 ¹⁷ cm ⁻³ or less. The plasma temperature in this plasma is within the range of approximately 1000 K or more and 2500 K or less. However, the plasma temperature can be lowered to about room temperature (about 300 K) on the liquid surface. The oxygen radical density is in the range of 2×10 ¹⁴ cm ⁻³ or more and 1.6×10 ¹⁵ cm ⁻³ or less. Table 1 shows these plasma conditions. These conditions are just examples.

[Table 1]
Conditions Numerical range Liquid level-Spouting distance 1 mm or more and 3 cm or less Plasma density 1×10 ¹⁴ cm ⁻³ or more 1×10 ¹⁷ cm ⁻³ or less Plasma temperature 1000 K or more and 2500 K or less

In order to produce an anticancer drug having an antitumor effect, the plasma density time product should satisfy the following conditions.
1.2×10 ¹⁸ sec·cm ⁻³ or more Here, the plasma density time product is the product of the plasma density in the plasma generation region and the time (irradiation time) at which this aqueous solution is irradiated with atmospheric pressure plasma.

5-2-2. Third Plasma Generator Instead of the plasma generators P10 and P20, the atmospheric pressure plasma generated in the plasma generation region by the plasma generator P30 may be applied to the first aqueous solution. The first electrode 110 is placed outside the first aqueous solution and the second electrode 210 is placed in the first aqueous solution. Then, gas is irradiated from the tubular portion 110a of the first electrode 110 toward the first aqueous solution. Then, in that state, a voltage is applied between the first electrode 110 and the second electrode 210.

In this way, by irradiating the first aqueous solution with plasma, the first aqueous solution becomes the second aqueous solution. This second aqueous solution is an anticancer agent having an antitumor effect.

5-3. Component Addition Step Next, at least one of sodium chloride, potassium chloride, and calcium chloride is added to the second aqueous solution to form a third aqueous solution.

6. Modification 6-1. Component addition step The component addition step may not necessarily be performed.

6-2. First Electrode In the plasma generator P30 of the present embodiment, the tubular portion 110a of the first electrode 110 has a cylindrical shape. However, the shape is not limited to the cylindrical shape. A polygonal shape may be used as long as it has a cylindrical shape.

7. Summary of the present embodiment As described in detail above, the anticancer agent of the present embodiment, lactic acid, sodium lactate, potassium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, calcium acetate, A first aqueous solution containing at least one of citric acid, sodium citrate, potassium citrate, calcium citrate, potassium hydrogen carbonate, and calcium hydrogen carbonate is irradiated with plasma. .. This anticancer agent preferably kills cancer cells. The first aqueous solution is Ringer's solution containing sodium chloride, potassium chloride, and calcium chloride. Then, the second aqueous solution after plasma irradiation has almost no effect on normal cells. Therefore, even if it is administered into the patient's body, the patient's body is hardly loaded. Therefore, there are various administration methods.

(Second embodiment)
The second embodiment will be described. The second embodiment differs from the first embodiment in the method of producing an anticancer agent. Therefore, only the method for producing an anticancer agent will be described.

1. Method for producing anticancer agent (antitumor aqueous solution) The method for producing an anticancer agent of the present embodiment has an aqueous solution preparation step, a plasma irradiation step, and a freezing step. The aqueous solution preparation process and the plasma process are the same as those in the first embodiment. Therefore, the freezing process will be described.

1-1. Freezing Step This freezing step is performed on the second aqueous solution or the third aqueous solution after the plasma irradiation. The second aqueous solution or the third aqueous solution is an anticancer agent. In this step, the second aqueous solution or the third aqueous solution is frozen. Therefore, the 2nd aqueous solution or the 3rd aqueous solution is frozen above -196 °C and below 0 °C. Specifically, it is stored in a freezer. As the freezer, for example, a biological experiment refrigerator (for example, Biofreezer GS-5203KHC manufactured by Japan Freezer Co., Ltd.) can be used.

The temperature of the second aqueous solution or the temperature of the third aqueous solution frozen in this freezer is within the range of -28°C or higher and -14°C or lower. Further, the temperature of the second aqueous solution or the temperature of the third aqueous solution is not limited to this range. It may be a normal freezing temperature. For example, it is in the range of -196°C or higher and 0°C or lower. It is preferably −196° C. or higher and −10° or lower. More preferably, it is −150° C. or higher and −20° C. or lower. More preferably, it is −80° C. or higher and −30° C. or lower. As described above, in the freezing step, the second aqueous solution or the third aqueous solution is frozen to produce the fourth aqueous solution in a frozen state.

2. Duration of antitumor effect in frozen anticancer agent (antitumor aqueous solution) 2-1. Antitumor effect of second aqueous solution (before freezing) The antitumor effect of the second aqueous solution (before freezing) will be described. The second aqueous solution before freezing exhibits an antitumor effect. The duration of the antitumor effect of the anticancer agent described in Patent Document 3 was 8 hours or more and less than 18 hours (see Patent Document 3). It can be estimated that the duration of the antitumor effect of the anticancer agent of the first embodiment is similar.

2-2. Antitumor Effect of Fourth Aqueous Solution (After Frozen) On the other hand, the anticancer agent produced by the production method of the present embodiment, that is, the third aqueous solution in a frozen state, continues to retain the antitumor effect. In fact, an anticancer agent having a freezing/cooling period of 28 days or longer exerts an antitumor effect after thawing. That is, the anticancer agent can be stored frozen for a long period of time. The antitumor effect is hardly lost by freezing and thawing the anticancer drug. This will be described later.

2-3. Consideration on Antitumor Effect of Second Aqueous Solution (Before Freezing) The anticancer drug before freezing is considered to contain some antitumor substance. It is considered that this antitumor substance is not a radical such as a hydroxy radical, a superoxide anion radical, or a hydroperoxy radical as described in Patent Document 2. The reason is that (1) the effect itself is different between the bactericidal effect and the antitumor effect, (2) the duration of the effect is different, (3) the relationship between the effect and pH dependence is different, There are three of them.

First, it goes without saying that the first bactericidal effect is different from the antitumor effect. Further, the anticancer agent of the present embodiment has selectivity. This anti-cancer agent has little effect on normal cells, but selectively kills cancer cells. This means that instead of creating a harsh survival environment for all cells, the cancer cells are targeted and selectively killed.

Next, the duration will be described. Patent Document 2 describes that, for example, a superoxide anion radical can exist in water for several seconds (see paragraphs [0090] to [0093] of Patent Document 2). On the other hand, it is considered that the second aqueous solution (before freezing) has a sustained antitumor effect for at least 8 hours or longer.

3. Modified Example The raw material of the second aqueous solution (before freezing) and the third aqueous solution (before freezing) may be Ringer's solution. That is, the second aqueous solution (before freezing) and the third aqueous solution (before freezing) may contain sodium chloride, potassium chloride, and calcium chloride.

1. Experiment A (antitumor effect of anticancer drug)
This experiment is an experiment conducted on the anticancer agent produced using the second plasma generator P20 and the third plasma generator P30.

1-1. Cancer cells used In this experiment, glioma was used as the cancer cells. Gliomas are gliomas that develop in glial cells. That is, it is a type of brain tumor. Specifically, U251SP was used as the glioma.

1-2. Experimental method 1-2-1. Culturing of cancer cells The above cancer cells were cultured in a 96-well plate to prepare a cancer cell culture medium. The culture medium used was a solution in which DMEM, serum (FBS), and an antibiotic (penicillin/streptomycin) were mixed. The number of cells seeded per well was 5000 and 10000. The volume of the culture solution supplied per well was 0.2 mL. The culture period for culturing the cancer cells was 24 hours.

Here, the culture components contained in DMEM include calcium chloride, ferric nitrate 9H ₂ O, magnesium sulfate (anhydrous), potassium chloride, sodium hydrogen carbonate, sodium chloride, monosodium phosphate (anhydrous), and L-arginine. · HCl, L- cystine · 2HCl, L- glutamine, glycine, L- histidine · HCl · H ₂ O, L- isoleucine, L- leucine, L- lysine · HCl, L- methionine, L- phenylalanine, L- serine , L-threonine, L-tryptophan, L-tyrosine·2Na·2H ₂ O, L-valine, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid, pyridoxine·HCl, riboflavin, thiamine·HCl, D-glucose and phenol red/Na.

1-2-2. Preparation of anticancer agent (antitumor aqueous solution) Separately from preparing a cancer cell culture medium, an anticancer agent (antitumor aqueous solution) was prepared. The anticancer agent is a solution (PAL: Plasma Activated Lactec (Lactec is a registered trademark)) obtained by irradiating an aqueous solution of the same component as Lactec (registered trademark) with plasma. Lactec (registered trademark) contains sodium chloride, potassium chloride, calcium chloride, and sodium L-lactate. The concentration of sodium chloride is 6.0 g/L. The concentration of potassium chloride is 0.3 g/L. The concentration of calcium chloride hydrate is 0.2 g/L. The concentration of L-sodium lactate is 3.1 g/L.

The plasma irradiation time was 5 minutes. Argon gas was used as the type of gas. In the plasma generator P20, the distance between the plasma generation region and the solution 1 was 2 mm. In the plasma generator P30, the distance between the first electrode 110 and the liquid surface of the solution 1 was 6 mm.

1-2-3. Supply of anti-cancer agent (anti-tumor aqueous solution) to the cancer cell culture medium Next, the culture solution of the 96-well plate in which the cancer cells were cultured was replaced with the sample aqueous solution. The time for which the cancer cells were immersed in the sample aqueous solution was 24 hours. Then, after that, the sample aqueous solution was exchanged with a normal culture solution. Then, the ratio of the number of surviving cells was examined by MTS assay.

1-3. Experimental Results FIG. 8 shows a case where an anticancer agent is supplied to 5000 cells using the second plasma generator P20. The vertical axis of FIG. 8 is the ratio of the number of viable cells. Here, “Unmanaged” indicates a case where plasma is not irradiated. Then, the case where the plasma was not irradiated was set as 100% and used as a reference.

As shown in FIG. 8, the anticancer agent showed a strong antitumor effect. The antitumor effect was shown for each of the anticancer drug diluted 4 times, 16 times, and 64 times. However, the antitumor agent diluted 256 times did not show an antitumor effect.

FIG. 9 shows a case where an anticancer agent is supplied to 10,000 cells using the second plasma generator P20. The vertical axis of FIG. 9 is the ratio of the number of viable cells. Here, “Unmanaged” indicates a case where plasma is not irradiated. Then, the case where the plasma was not irradiated was set as 100% and used as a reference.

As shown in FIG. 9, the anticancer drug showed a strong antitumor effect. The antitumor effect was shown for each of the anticancer drug diluted 4 times and 16 times. However, the anticancer drug was not shown to have the anti-tumor agent diluted 64 times or 256 times.

FIG. 10 shows a case where an anticancer agent is supplied to 5000 cells using the third plasma generator P30. The vertical axis of FIG. 10 is the ratio of the number of viable cells. Here, “Unmanaged” indicates a case where plasma is not irradiated. Then, the case where the plasma was not irradiated was set as 100% and used as a reference.

As shown in FIG. 10, the anticancer agent showed a strong antitumor effect. The antitumor effect was shown for each of the anticancer drug diluted 4 times, 16 times, and 64 times. However, the antitumor agent diluted 256 times did not show an antitumor effect.

FIG. 11 shows a case where an anticancer agent is supplied to 10,000 cells using the third plasma generator P30. The vertical axis of FIG. 11 is the ratio of the number of viable cells. Here, “Unmanaged” indicates a case where plasma is not irradiated. Then, the case where the plasma was not irradiated was set as 100% and used as a reference.

As shown in FIG. 11, the anticancer agent showed a strong antitumor effect. The antitumor effect was shown for each of the anticancer drug diluted 4 times and 16 times. However, the anticancer drug was not shown to have the anti-tumor agent diluted 64 times or 256 times.

2. Experiment B (raw material for anti-cancer drug)
This experiment is an experiment conducted on an anticancer agent produced using the plasma generator P20.

2-1. Cancer cells used In this experiment, glioma was used as the cancer cells. Gliomas are gliomas that develop in glial cells. That is, it is a type of brain tumor. Specifically, U251SP was used as the glioma.

2-2. Experimental method 2-2-1. Culturing of cancer cells The above cancer cells were cultured in a 96-well plate to prepare a cancer cell culture medium. The culture medium used was a solution in which DMEM, serum (FBS), and an antibiotic (penicillin/streptomycin) were mixed. The number of cells seeded per well was 10,000. The volume of the culture solution supplied per well was 0.2 mL. The culture period for culturing the cancer cells was 24 hours.

2-2-2. Preparation of Sample Aqueous Solution Separately from preparing the cancer cell culture medium, a sample aqueous solution was prepared. The sample aqueous solution was prepared by irradiating the following aqueous solution with plasma. Lactec (registered trademark) was used as a standard as the drip component. Lactec (registered trademark) contains sodium chloride, potassium chloride, calcium chloride, and sodium L-lactate. The concentration of sodium chloride is 6.0 g/L. The concentration of potassium chloride is 0.3 g/L. The concentration of calcium chloride hydrate is 0.2 g/L. The concentration of L-sodium lactate is 3.1 g/L.

Then, as shown in Table 2, 11 types of sample aqueous solutions were prepared. The sample aqueous solutions of Examples 1-11 shown in Table 2 all have almost the same components as Lactec (registered trademark). As shown in Table 2, the sample aqueous solution is an aqueous solution obtained by mixing Solution 1 and Solution 2. Solution 1 is a solution irradiated with plasma. Solution 2 is a solution that has not been irradiated with plasma. When the solution 1 and the solution 2 are mixed, they have almost the same components as Lactec (registered trademark) described above. That is, sodium chloride, potassium chloride, calcium chloride, and sodium L-lactate were mixed with either Solution 1 or Solution 2.

For example, in Example 2 of Table 2, as the solution 1, a sodium chloride aqueous solution having a concentration twice that of Lactec (registered trademark) was prepared. Further, as the solution 2, potassium chloride, calcium chloride, and L-sodium lactate were mixed, and the concentration was double that of Lactec (registered trademark). If these solutions 1 and 2 are mixed without irradiating plasma, the same thing as Lactec (registered trademark) is manufactured.

The sample aqueous solution of Example 1 is the same as ordinary Lactec (registered trademark). Example 2 is NaCl-GOF (Gain of Function). That is, the sodium chloride aqueous solution was irradiated with plasma and other components were added. Example 3 is KCl-GOF. That is, the potassium chloride aqueous solution is irradiated with plasma and other components are added. Example 4 is a CaCl ₂ -GOF. That is, the calcium chloride aqueous solution is irradiated with plasma and other components are added. Example 5 is L-sodilumlacate-GOF. That is, the L-sodium lactate aqueous solution was irradiated with plasma and other components were added.

Example 6 is NaCl-LOF (Loss of Function). That is, an aqueous solution containing components other than sodium chloride was irradiated with plasma and sodium chloride was added. Example 7 is KCl-LOF. That is, an aqueous solution containing components other than potassium chloride was irradiated with plasma and potassium chloride was added. Example 8 is a CaCl ₂ -LOF. That is, an aqueous solution containing components other than calcium chloride is irradiated with plasma and calcium chloride is added. Example 9 is L-sodium lacte-LOF. That is, an aqueous solution containing components other than L-sodium lactate was irradiated with plasma and L-sodium lactate was added.

Example 10 is a plasma irradiated lactec. That is, plasma was irradiated to twice the aqueous solution of Lactec (registered trademark) and diluted twice with Milli-Q water. Example 11 is plasma-irradiated water. That is, Milli-Q water was irradiated with plasma and mixed with twice the aqueous solution of Lactec (registered trademark).

The plasma irradiation time was 2 minutes. The gas flow rate was 0.4 slm. Argon gas was used as the type of gas. The distance between the plasma generation region and the solution 1 was 13 mm.

2-2-3. Supply of anti-cancer agent (anti-tumor aqueous solution) to the cancer cell culture medium Next, the culture solution of the 96-well plate in which the cancer cells were cultured was replaced with the sample aqueous solution. The time for which the cancer cells were immersed in the sample aqueous solution was 24 hours. Then, after that, the sample aqueous solution was exchanged with a normal culture solution. Then, the ratio of the number of surviving cells was examined by MTS assay.

2-3. Experimental Results Experimental results are shown in FIG. The vertical axis of FIG. 12 is the ratio of the number of viable cells. Here, the example 1 was set as the reference 1.

As shown in FIG. 12, the sample aqueous solutions of Examples 5-8 and 10 exhibited antitumor effect. The sample aqueous solution of Example 5 showed the highest antitumor effect. The ratio of the number of viable cells in Example 5 was about 0.1. The ratio of the number of viable cells in Example 10 was about 0.2 to 0.3. The ratio of the number of viable cells in Examples 6-8 was about 0.4 to 0.5.

A matter common to Examples 5-8 and 10 is that Solution 1 contains sodium L-lactate. That is, it is shown that the anticancer agent is produced by irradiating the first aqueous solution containing L-sodium lactate with plasma. Moreover, even if sodium chloride, potassium chloride, or calcium chloride is contained, the antitumor effect is hardly lost. Therefore, for example, it is possible to administer the anticancer agent of Example 5 into a patient's body.

In this experiment, L-sodium lactate was used. However, it is considered that even D-sodium lactate has the same effect. In addition, lactic acid, potassium lactate, and calcium lactate are considered to have the same effect.

3. Experiment C (frozen anticancer drug)
This experiment is an experiment conducted on an anticancer agent (antitumor aqueous solution) produced using the plasma generator P30.

3-1. Cancer cells used In this experiment, glioma was used as the cancer cells. Gliomas are gliomas that develop in glial cells. That is, it is a type of brain tumor. Specifically, U251SP was used as the glioma.

3-2. Experimental method 3-2-1. Culturing of cancer cells The above cancer cells were cultured in a 96-well plate to prepare a cancer cell culture medium. The culture medium used was a solution in which DMEM, serum (FBS), and an antibiotic (penicillin/streptomycin) were mixed. The number of cells seeded per well was 5000. The volume of the culture solution supplied per well was 0.2 mL. The culture period for culturing the cancer cells was 24 hours.

3-2-2. Preparation of anti-cancer agent (anti-tumor solution) Separately from preparing a cancer cell culture medium, an anti-cancer agent was prepared. The anticancer agent is obtained by irradiating plasma with an aqueous solution containing the same components as Lactec (registered trademark). Lactec (registered trademark) contains sodium chloride, potassium chloride, calcium chloride, and sodium L-lactate. The concentration of sodium chloride is 6.0 g/L. The concentration of potassium chloride is 0.3 g/L. The concentration of calcium chloride hydrate is 0.2 g/L. The concentration of L-sodium lactate is 3.1 g/L.

The plasma irradiation time was 2 minutes. Argon gas was used as the type of gas. In the plasma generator P30, the distance between the first electrode 110 and the liquid surface of the solution 1 was 6 mm.

Then, the anticancer agent was frozen inside the Biofreezer GS-5203KHC (manufactured by Japan Freezer Co., Ltd.). The freezing temperature was -150°C. The freezing time was 12 hours. Then, an anti-cancer agent that was not frozen, an anti-cancer agent that was frozen and thawed only once, and an anti-cancer agent that was frozen and thawed twice were manufactured.

3-2-3. Supply of anti-cancer agent (anti-tumor aqueous solution) to the cancer cell culture medium Next, the culture solution of the 96-well plate in which the cancer cells were cultured was replaced with the anti-cancer agent. The time during which the cancer cells were immersed in the anticancer drug was 24 hours. Then, after that, the anti-cancer agent was replaced with a normal culture solution. Then, the ratio of the number of surviving cells was examined by MTS assay.

3-3. Experimental Results FIG. 13 is a graph showing changes in the ratio of the number of viable cells due to the execution of freezing and thawing. The vertical axis of FIG. 13 is the ratio of the number of living cells.

As shown in FIG. 13, when it was not frozen, the antitumor agent of 1-fold and the anticancer agent diluted 4-fold showed the antitumor effect. Further, the anticancer agent diluted 16 times showed some antitumor effect. The anticancer drug diluted 64 times did not show an antitumor effect.

As shown in FIG. 13, when frozen and thawed only once, a 1-fold anticancer agent showed an antitumor effect. The anticancer drug diluted 4 times showed some antitumor effect. The 16-fold diluted anti-cancer drug and the 64-fold diluted anti-cancer drug showed no antitumor effect.

As shown in FIG. 13, when freeze-thawing and thawing were repeated twice, 1-fold anticancer drug showed an antitumor effect. However, the 4-fold diluted anti-cancer drug and the 16-fold diluted anti-cancer drug did not show an antitumor effect.

Thus, the antitumor effect can be maintained to some extent even if the anticancer drug is stored frozen. However, its antitumor effect was lost to some extent by repeated freezing and thawing.

4. Experiment D (selectivity of anticancer drug)
FIG. 14 is a graph showing the selectivity of anticancer agents. FIG. 14(a) is a graph showing the results for U251 cells. FIG. 14(b) is a graph showing the results for MCF10A cells. FIG. 14(c) is a graph showing the results for neonatal keratinocyte cells. As mentioned above, U251 cells are cancer cells. MCF10A cells and neonatal keratinocyte cells are normal cells.

In this experiment, Lactec (registered trademark), which was obtained by irradiating these cells with plasma under equivalent conditions, was used. In FIG. 14(a), U251 cells are killed by exposure to an anticancer drug irradiated with plasma for 40 seconds. On the other hand, as shown in FIGS. 14(b) and 14(c), MCF10A cells and neonatal keratinocyte cells were not killed when exposed to an anticancer drug irradiated with plasma for 40 seconds. That is, the anticancer agent of the embodiment kills cancer cells and hardly kills normal cells. Thus, the anti-cancer agent can selectively kill cancer cells.

5. Experiment E (Biological experiment)
5-1. Mouse FIG. 15 is a diagram schematically showing the experimental method of this experiment. As experimental mice, 8-week-old female BALB/c-nu/nu nude mice (manufactured by Japan SLC, Inc.) were used.

5-2. Cancer cells Human cervical cancer cell lines (SiHa, ATCC) were used as cancer cells.

5-3. Experimental Method 1500 cells of human cervical cancer cell line (SiHa) suspended in 100 μL of PBS were mixed with 100 μL of Matrigel (manufactured by BD Biosciences) to prepare a total of 200 μL of cell suspension. Then, the cell suspension was subcutaneously injected into the flank of a nude mouse. At that time, one mouse was seeded on each flank, and a total of two mice were seeded. Then, 10 nude mice seeded with cells were divided into two groups. The first group of mice was injected with normal Lactec® on both flanks. A second group of mice was injected with 5.5 mL of Lactec (registered trademark) irradiated with plasma for 10 minutes (PAL: Plasma Activated Lactec) on both flanks. This PAL is the anticancer agent described in the embodiment. These Lactec and the like were administered three times a week. The dose administered to one site per administration was 200 μL. Then, 42 days later, subcutaneous tumors were taken out from these two groups of mice.

5-4. Experimental Results FIG. 16 is a photograph showing a subcutaneous tumor taken out from a mouse 42 days after the start of the experiment. As shown in FIG. 16, a subcutaneous tumor excised from a mouse (Control) to which normal Lactec (registered trademark) was administered was obtained from a subcutaneous tumor excised from a mouse (PAL) to which plasma was irradiated with Lactec (registered trademark) Also tends to be large. Also, there are some individual differences among mice. However, in the same mouse, there was almost no difference in tumor size between left and right.

FIG. 17 is a graph showing changes over time in subcutaneous tumor volume of mice. The horizontal axis of FIG. 17 is the number of days. The vertical axis of FIG. 17 is the volume of subcutaneous tumor. The subcutaneous tumor volume V1 was calculated by approximation using the following equation. That is, the shape of the subcutaneous tumor was approximated by a spheroid.
V1=(π/6)×a1×b1 ²
V1: Subcutaneous tumor volume
a1: major axis of subcutaneous tumor
b1: Short diameter of subcutaneous tumor Incidentally, about the long diameter a1 and the short diameter b1, approximate values were measured using a digital caliper.

As shown in FIG. 17, a subcutaneous tumor excised from a mouse (Control) to which normal Lactec (registered trademark) was administered was obtained from a subcutaneous tumor excised from a mouse (PAL) to which Lactec (registered trademark) was irradiated with plasma. Was also great. Further, in a mouse to which usual Lactec (registered trademark) was administered, a subcutaneous tumor grew rapidly after 30 days.

FIG. 18 is a graph showing changes over time in body weight of mice. The horizontal axis of FIG. 18 is the number of days. The vertical axis of FIG. 18 is the weight of the mouse. The body weight of the nude mice was almost the same in the first group of mice administered with normal Lactec (registered trademark) and the second group of mice administered with plasma-irradiated Lactec (registered trademark). From the start of the experiment, the weight of nude mice has tended to increase, but has not changed so much.

FIG. 19 is a graph showing the volume and weight of subcutaneous tumors extracted on day 42. The subcutaneous tumor volume V2 was calculated by approximation using the following equation.
V2=(π/6)×a2×b2×h2
V2: Subcutaneous tumor volume
a2: length of subcutaneous tumor
b2: short diameter of subcutaneous tumor
h2; height (thickness) of subcutaneous tumor
The major axis a2, the minor axis b2, and the height h2 were measured using a digital caliper.

As shown in FIG. 19( a ), the volume of subcutaneous tumor of mice irradiated with plasma-irradiated Lactec (registered trademark) (PAL) was administered with plasma-unirradiated Lactec (registered trademark) (Control). It was about 30% of the volume of the subcutaneous tumor of the mouse. Further, as shown in FIG. 19( b ), the weight of the subcutaneous tumor of the mouse irradiated with Lactec (registered trademark) (PAL) irradiated with plasma was the same as that of Lactec (registered trademark) (Control) not irradiated with plasma. It was about 30% of the weight of the subcutaneous tumor of the administered mouse.

As described above, ordinary Lactec (registered trademark) that was not irradiated with plasma had no anticancer effect. On the other hand, plasma-irradiated Lactec (registered trademark) suppressed the volume and weight of subcutaneous tumors by about 70% as compared with ordinary Lactec (registered trademark). As described above, Lactec (registered trademark) irradiated with plasma exhibited an anticancer effect.

6. Experiment F (other Ringer's solution)
This experiment is an experiment conducted on an anticancer agent (antitumor aqueous solution) produced using the plasma generator P30.

6-1. Cancer cells used In this experiment, ovarian cancer cells were used as the cancer cells. Specifically, SKOV3 was used.

6-2. Experimental method 6-2-1. Culturing of cancer cells The above cancer cells were cultured in a 96-well plate to prepare a cancer cell culture medium. The culture solution used is a solution in which RPMI, serum (FBS), and an antibiotic (penicillin/streptomycin) are mixed. The number of cells seeded per well was 5000. Further, the volume of the culture solution supplied per well was 0.1 mL. The culture period for culturing the cancer cells was 24 hours.

6-2-2. Preparation of anti-cancer agent (anti-tumor solution) Separately from preparing a cancer cell culture medium, an anti-cancer agent was prepared. Lactate Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution were used as materials for the anticancer agent. The components of the lactated Ringer's solution are the same as those of Lactec (registered trademark) used in Experiment A and the like.

Ringer's acetate solution contains sodium chloride, potassium chloride, calcium chloride, and sodium acetate. The concentration of sodium chloride is 6.0 g/L. The concentration of potassium chloride is 0.3 g/L. The concentration of calcium chloride hydrate is 0.2 g/L. The concentration of sodium acetate hydrate is 3.8 g/L.

The Ringer's bicarbonate solution contains sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium hydrogen carbonate, and sodium citrate. The concentration of sodium chloride is 5.84 g/L. The concentration of potassium chloride is 0.3 g/L. The concentration of calcium chloride is 0.22 g/L. The concentration of magnesium chloride is 0.2 g/L. The concentration of sodium hydrogen carbonate is 2.35 g/L. The concentration of sodium citrate is 0.2 g/L.

The three types of Ringer's solutions were irradiated with plasma. The plasma irradiation time was 10 minutes. Argon gas was used as the type of gas. In the plasma generator P30, the distance between the first electrode 110 and the liquid surface of the solution 1 was 3 mm.

6-2-3. Supply of anti-cancer agent (anti-tumor aqueous solution) to cancer cell culture medium Then, as shown in FIG. 20, the culture solution of the 96-well plate in which the cancer cells were cultured was replaced with the anti-cancer agent. The time during which the cancer cells were immersed in the anticancer drug was 24 hours. Then, after that, the anti-cancer agent was replaced with a normal culture solution. Then, the ratio of the number of surviving cells was examined by MTS assay.

6-3. Experimental result 6-3-1. Lactated Ringer's Solution FIG. 21 is a graph showing the survival rate of SKOV3 when SKOV3 was treated with an aqueous solution in which lactated Ringer's solution was irradiated with plasma. As shown in FIG. 21, the aqueous solution obtained by irradiating the lactated Ringer's solution with plasma showed an antitumor effect on ovarian cancer cells (SKOV3). The strength was a 78-fold dilution that could kill 50% of all SKOV3 cells.

6-3-2. Ringer's Acetate Solution FIG. 22 is a graph showing the survival rate of SKOV3 when SKOV3 was treated with an aqueous solution in which the Ringer's acetate solution was irradiated with plasma. As shown in FIG. 22, the aqueous solution obtained by irradiating the Ringer's acetate solution with plasma showed an antitumor effect on ovarian cancer cells (SKOV3). Its strength was a 53-fold dilution that could kill 50% of all SKOV3 cells.

6-3-3. Bicarbonate Ringer's Solution FIG. 23 is a graph showing the survival rate of SKOV3 when SKOV3 was treated with an aqueous solution in which bicarbonate Ringer's solution was irradiated with plasma. As shown in FIG. 23, the aqueous solution obtained by irradiating the bicarbonate Ringer's solution with plasma exhibited an antitumor effect on ovarian cancer cells (SKOV3). The strength was such that the dilution ratio capable of killing 50% of all SKOV3 cells was 1/3-fold.

As described above, the aqueous solutions obtained by irradiating the lactate Ringer's solution, the acetate Ringer's solution, and the bicarbonate Ringer's solution with plasma each showed an antitumor effect on ovarian cancer cells (SKOV3). The strength of the antitumor effect was in the order of the aqueous solution in which the lactated Ringer's solution was irradiated with the plasma, the aqueous solution in which the acetic acid Ringer's solution was irradiated with the plasma, and the aqueous solution in which the bicarbonate Ringer's solution was irradiated with the plasma. As described above, not only the lactated Ringer's solution but also these various Ringer's solutions exhibited an antitumor effect upon irradiation with plasma. In addition, it showed an antitumor effect on ovarian cancer cells (SKOV3).

Further, the raw material of the anticancer agent is not limited to sodium acetate, but may be acetic acid, potassium acetate, or calcium acetate. Similarly, the raw material of the anticancer agent is not limited to sodium hydrogen carbonate or sodium citrate, but may be citric acid, potassium citrate, calcium citrate, potassium hydrogen carbonate, or calcium hydrogen carbonate.

7. Experiment G (NMR)
7-1. Sodium lactate aqueous solution In this experiment, NMR was performed on the sodium lactate aqueous solution not irradiated with plasma and the sodium lactate aqueous solution irradiated with plasma. Therefore, a commercially available sodium lactate aqueous solution was used. The concentration of the aqueous sodium lactate solution is 50%. The plasma-irradiated aqueous solution of sodium lactate was prepared by irradiating the aqueous solution of sodium lactate with plasma for 5 minutes. At that time, the plasma generator P20 was used. Then, ¹ H and ¹³ C were observed using NMR.

7-2. Experimental Results FIG. 24 is a graph showing the results of ¹ H observation. The result of the sodium lactate aqueous solution which is not irradiated with plasma is shown on the upper side of FIG. The same applies to the figures after this figure. The results of the sodium lactate aqueous solution irradiated with plasma are shown on the lower side of FIG. As shown in FIG. 24, a peak derived from OH, a peak derived from CH, and a peak derived from CH ₃ were observed regardless of the presence or absence of plasma irradiation. And, no significant difference was observed between the peak of the aqueous sodium lactate solution irradiated with plasma and the peak of the aqueous sodium lactate solution not irradiated with plasma.

FIG. 25 is an enlarged view of FIG. As shown in FIG. 25, peaks showing structures such as CH ₃ COCOOH and CH ₃ CO are large after plasma irradiation. In other respects, the appearance has not changed significantly before and after plasma irradiation.

FIG. 26 is a graph showing the results of observation of ¹³ C. As shown in FIG. 26, a COOH-derived peak, a CH-derived peak, and a CH ₃ -derived peak were observed. As a result of observing ¹³ C, the appearance did not change significantly before and after plasma irradiation.

FIG. 27 is an enlarged view of FIG. 26. In FIG. 27, similar to FIG. 26, the appearance is not largely changed before and after the plasma irradiation.

As described above, the basic structure of sodium lactate is not significantly changed by irradiating with plasma. Then, by irradiating with plasma, CH ₃ COCOOH and CH ₃ CO increased. Therefore, it is considered that CH ₃ COCOOH and CH ₃ CO contribute to the antitumor effect in some way. That is, the anticancer substance is considered to have the functional group CH ₃ COCOO or the functional group CH ₃ CO.

8. Experiment H (component of Ringer's acetate solution)
This experiment is an experiment conducted on an anticancer agent (antitumor aqueous solution) produced using the plasma generator P30.

8-1. Cancer cells used In this experiment, ovarian cancer cells were used as the cancer cells. Specifically, SKOV3 was used.

8-2. Experimental method 8-2-1. Culture of cancer cells Cancer cells were cultured in the same manner as in Experiment F above.

8-2-2. Preparation of sample aqueous solution In this experiment, seven types of sample aqueous solutions were used. These sample aqueous solutions are aqueous solutions manufactured according to the same concept as GOF of Experiment B. That is, the aqueous solution A1 and the aqueous solution A2 are prepared. Here, when the aqueous solution A1 and the aqueous solution A2 are mixed, the acetate Ringer's solution used in the experiment F is obtained. In this experiment, only the aqueous solution A1 is irradiated with plasma, and then the aqueous solution A1 and the aqueous solution A2 are mixed.

The first sample aqueous solution is Ringer's acetate solution which is not irradiated with plasma. The second sample aqueous solution is obtained by irradiating pure water with plasma and then mixing a high concentration Ringer's acetate solution. The third sample aqueous solution was obtained by irradiating an aqueous solution of sodium acetate with plasma and then mixing the other components of the Ringer's acetate solution. The fourth sample aqueous solution was obtained by irradiating the Ringer's acetate solution with plasma and then mixing the Ringer's acetate solution. The fifth sample aqueous solution was obtained by irradiating the sodium chloride aqueous solution with plasma and then mixing the other components of the Ringer's acetate solution. The sixth sample aqueous solution was obtained by irradiating a potassium chloride aqueous solution with plasma and then mixing the other components of the Ringer's acetate solution. The seventh sample aqueous solution was obtained by irradiating an aqueous calcium chloride solution with plasma and then mixing the other components of the Ringer's acetate solution.

In the above, the components of the Ringer's acetate solution are the same as those used in Experiment F. The plasma irradiation time was 5 minutes. Argon gas was used as the type of gas. In the plasma generator P30, the distance between the first electrode 110 and the liquid surface of the solution 1 was 10 mm.

8-2-3. Supply of Sample Aqueous Solution to Cancer Cell Culture Site Then, in the same manner as in Experiment F, cancer cells were supplied with sample aqueous solution 1 to sample aqueous solution 7. Then, the ratio of the number of surviving cells was examined by MTS assay.

8-3. Experimental Results FIG. 28 and FIG. 29 are graphs showing the experimental results. As shown in FIGS. 28 and 29, the third sample aqueous solution and the fourth sample aqueous solution showed antitumor effect. Other sample aqueous solutions did not show antitumor effect. This indicates that, among the components contained in Ringer's acetate solution, sodium acetate is a raw material for the antitumor substance. This result is consistent with Experiment G.

P1... Plasma irradiation device M1... Robot arm PM... Anticancer agent manufacturing device P10, P20, P30... Plasma generation device 10, 11... Housing parts 10i, 11i... Gas inlets 10o, 11o... Gas ejection ports 2a, 2b... Electrode P ... Plasma area H... Recess (hollow)
110... 1st electrode 120... 1st electric potential provision part 130... 1st lead wire 140... Gas supply part 150... Gas pipe coupling connector 160... Gas pipe 170... 1st electrode protection member 210... 2nd electrode 220... Second potential applying section 230... Second lead wire 240... Second electrode protection member 250... Container 260... Sealing member 270... Stand

Claims (11)

An aqueous solution preparation step of preparing a first aqueous solution;
A plasma irradiation step of irradiating the first aqueous solution with plasma to form a second aqueous solution;
Have
The first aqueous solution is
Lactate, and sodium lactate, and potassium lactate, and the calcium lactate, acetate, and sodium acetate, and potassium acetate, and calcium acetate, and citric acid, sodium citrate and, potassium citrate, calcium citrate, the A method for producing an anticancer agent, which comprises at least one of them. The method for producing an anticancer agent according to claim 1,
The first aqueous solution is
A method for producing an anticancer agent, which is a Ringer's solution. The method for producing an anticancer agent according to claim 2,
The first aqueous solution is
A method for producing an anticancer agent, which is a lactated Ringer's solution. The method for producing an anticancer agent according to claim 2,
The first aqueous solution is
A method for producing an anticancer agent, which is a Ringer's acetate solution. The method for producing an anticancer agent according to claim 2,
The first aqueous solution is
A method for producing an anticancer agent, which is a bicarbonate Ringer's solution. The method for producing an anticancer agent according to any one of claims 1 to 5,
The first aqueous solution is
A method for producing an anticancer agent, which comprises at least one of sodium chloride, potassium chloride, and calcium chloride. The method for producing an anticancer agent according to any one of claims 1 to 6,
A method for producing an anticancer agent, comprising a component addition step of adding at least one of sodium chloride, potassium chloride and calcium chloride to the second aqueous solution to form a third aqueous solution. The method for producing an anticancer agent according to any one of claims 1 to 7,
A freezing step of freezing the second aqueous solution or the third aqueous solution,
The second aqueous solution or the third aqueous solution is
A method for producing an anticancer agent, which comprises sodium chloride, potassium chloride, and calcium chloride. The method for producing an anticancer agent according to claim 8,
In the freezing step,
A method for producing an anticancer agent, which comprises freezing the second aqueous solution or the third aqueous solution within a range of -196°C to 0°C. The method for producing an anticancer agent according to any one of claims 1 to 9,
In the plasma irradiation step,
A first electrode having a tubular portion is arranged outside the first aqueous solution, and a second electrode is arranged in the first aqueous solution,
Irradiating gas from the tubular portion of the first electrode toward the first aqueous solution,
A method for producing an anticancer agent, comprising applying a voltage between the first electrode and the second electrode in that state. An aqueous solution preparation step of preparing a first aqueous solution;
A plasma irradiation step of irradiating the first aqueous solution with plasma to form a second aqueous solution;
Have
The first aqueous solution is
Lactate, and sodium lactate, and potassium lactate, and the calcium lactate, acetate, and sodium acetate, and potassium acetate, and calcium acetate, and citric acid, sodium citrate and, potassium citrate, calcium citrate, the A method for producing an infusion solution, which comprises at least one of the above.