JP6381111B2 - Anti-tumor aqueous solution, anti-cancer agent and method for producing them - Google Patents

Description

  The technical field of the present specification relates to an antitumor aqueous solution and an anticancer agent and methods for producing them. More specifically, the present invention relates to an antitumor aqueous solution and an anticancer agent capable of killing cancer cells, and methods for producing them.

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

  For example, Patent Document 1 discloses blood coagulation (see Example 4 of Patent Document 1, paragraphs [0063] to [0068]) and tissue sterilization (Example 5 of Patent Document 1, paragraph [0069] by plasma irradiation. And Leishmaniasis (see Example 6 of Patent Document 1, paragraphs [0075]-[0077]). And it is described that there exists an effect which kills a melanoma cell (malignant melanoma cell) (refer Example 7 of patent document 1, paragraph [0078]).

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

Special table 2008-539007 gazette 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 cancer cells and 2) selectively kill cancer cells so as not to affect normal cells. This is because even if cancer cells can be killed, killing a large number of normal cells for that purpose causes a great physical burden on the patient. Therefore, a treatment technique that selectively kills cancer cells in this way is desired. However, it is not easy to selectively kill cancer cells. 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 related to an antitumor aqueous solution that selectively kills cancer cells (Patent Document 3, paragraphs [0085]-[0087] and FIG. 16 etc.). This anti-tumor aqueous solution can selectively kill cancer cells. In addition, this anti-tumor aqueous solution exhibited an anti-tumor effect not only on cultured cells but also on mice (see paragraphs [0145]-[0152] and FIGS. 45 and 46 of Patent Document 3). However, the antitumor effect of the antitumor aqueous solution was less than 18 hours (see paragraphs [0088]-[0091] and FIG. 17 of Patent Document 3). In order to use the anti-tumor aqueous solution for cancer treatment, it is preferable that the anti-tumor effect of the anti-tumor aqueous solution lasts longer so that the anti-tumor aqueous solution can be used at a place away from the place where the plasma generator is installed.

  The technique of this specification has been made to solve the problems of the conventional techniques described above. That is, the subject is to provide an antitumor aqueous solution and an anticancer agent capable of killing cancer cells with little effect on normal cells and maintaining an antitumor effect over a long period of time, and a method for producing them. .

The method for producing an anti-tumor aqueous solution in the first aspect is a method for producing an anti-tumor aqueous solution that selectively kills cancer cells. The method for producing this anti-tumor aqueous solution includes disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-histidine. And an aqueous solution preparation step of preparing a culture solution containing at least one of L-tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) as a first aqueous solution, and plasma generated by a plasma generator A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in the generation region to form a second aqueous solution; and a freezing step of freezing the second aqueous solution.

  The antitumor aqueous solution produced by this production method kills cancer cells and hardly kills normal cells. That is, cancer cells can be selectively killed. Therefore, this anti-tumor aqueous solution is directly contacted with cancer cells, the patient is given an anti-tumor aqueous solution, or the patient's abdominal cavity is filled or washed with the anti-tumor aqueous solution around the organ where the cancer has occurred. By using the method, human cancer can be treated. The antitumor aqueous solution produced by this production method maintains the antitumor effect for a long time. The antitumor effect is considered to be that some antitumor substance is generated inside the antitumor aqueous solution, not the ions or radicals irradiated from the plasma generator. Moreover, it shows that the antitumor substance does not react with other chemical species in the antitumor aqueous solution without destroying the chemical structure for a long time by freezing.

The method for producing an anti-tumor aqueous solution in the second aspect is a method for producing an anti-tumor aqueous solution that selectively kills cancer cells. The method for producing this anti-tumor aqueous solution includes disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-histidine. An aqueous solution preparation step of preparing a first aqueous solution in which a solute containing at least one of L-tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) is added to water, and plasma generation A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in the plasma generation region by the apparatus to form a second aqueous solution; a culture solution addition step of adding a culture solution to the second aqueous solution; and a culture solution And a freezing step of freezing the second aqueous solution to which is added .

In the method for producing an antitumor aqueous solution according to the third aspect, in the freezing step, the second aqueous solution is frozen within a range of −196 ° C. or higher and 0 ° C. or lower.

The method for producing an anticancer agent in the fourth aspect is a method for producing an anticancer agent that selectively kills cancer cells. The method for producing this anticancer agent includes disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, L-histidine, L-histidine, and L-histidine. -Aqueous solution preparation step for preparing a culture solution containing at least one of tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) as a first aqueous solution , and a plasma generation region by the plasma generator A plasma irradiation step of irradiating the first aqueous solution with the atmospheric pressure plasma generated in step 1 to form a second aqueous solution, and a freezing step of freezing the second aqueous solution.

The method for producing an anticancer agent in the fifth aspect is a method for producing an anticancer agent that selectively kills cancer cells. The method for producing this anticancer agent includes disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, L-histidine, L-histidine, and L-histidine. An aqueous solution preparing step of preparing a first aqueous solution in which a solute containing at least one of tyrosine disodium dihydrate (L-Tyrosine · 2Na · 2H ₂ O) is added to water, and a plasma generator A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in the plasma generation region to form a second aqueous solution, a culture solution adding step of adding a culture solution to the second aqueous solution, and adding the culture solution And a freezing step of freezing the second aqueous solution.

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

  In the present specification, an antitumor aqueous solution and an anticancer agent capable of killing cancer cells with little effect on normal cells and an antitumor effect that lasts for a long time and methods for producing them are provided.

It is a conceptual diagram for demonstrating the structure of the robot arm which scans the gas jet nozzle of a plasma irradiation apparatus. FIG. FIG. 2A is a cross-sectional view illustrating a configuration of a first plasma irradiation apparatus, and FIG. B is a figure which shows the shape of an electrode. FIG. FIG. 3A is a cross-sectional view showing a configuration of a second plasma irradiation apparatus, and FIG. B is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma region. In Experiment A, it is a graph which shows the relationship between the time from the time of manufacture in an anti-tumor aqueous solution, and an antitumor effect. It is the graph which compared the effect of the anti-tumor aqueous solution with the cancer cell and the normal cell in Experiment B. It is a microscope picture which shows the result at the time of immersing a cancer cell culture place in the anti-tumor aqueous solution in Experiment B. It is a graph which shows the duration of the anti-tumor effect of the anti-tumor aqueous solution in Experiment C. It is a graph which shows the result of having investigated the antitumor effect in the antitumor aqueous solution which irradiated the plasma to the culture solution in experiment D. FIG. It is a graph which shows the result of having investigated the antitumor effect in the antitumor aqueous solution which added the culture solution after irradiating plasma to the disodium hydrogenphosphate aqueous solution in Experiment D. It is a graph which shows the result of having investigated the anti-tumor effect in the anti-tumor aqueous solution which added the culture solution after irradiating plasma to the sodium hydrogencarbonate aqueous solution in Experiment D. It is a graph which shows the result of having investigated the anti-tumor effect in the anti-tumor aqueous solution which added the culture solution after irradiating plasma to the L-glutamine aqueous solution in Experiment D. It is a graph which shows the result of having investigated the anti-tumor effect in the anti-tumor aqueous solution which added the culture solution after irradiating plasma to L-histidine aqueous solution in Experiment D. It is a graph which shows the result of having investigated the anti-tumor effect in the anti-tumor aqueous solution which added the culture solution after irradiating plasma to the L-tyrosine disodium dihydrate aqueous solution in Experiment D. It is a graph which shows the result of having investigated the anti-tumor effect in the anti-tumor aqueous solution which added the culture solution after irradiating plasma to various single-component aqueous solution in Experiment D. It is a photograph which compares the case where the anti-tumor aqueous solution or a normal culture solution is administered to the nude mouse which administered the ovarian cancer cell in Experiment E.

  Hereinafter, specific embodiments will be described with reference to the drawings, taking an anti-tumor aqueous solution and an anti-cancer agent and their production methods as examples.

1. Anti-tumor aqueous solution production apparatus 1-1. Configuration of Antitumor Aqueous Solution Manufacturing Device As shown in FIG. 1, the antitumor aqueous solution manufacturing device PM of this embodiment includes a plasma irradiation unit P1 and an arm robot M1. The plasma irradiation apparatus P1 is for generating plasma and irradiating the plasma toward the solution. As will be described later, there are two types of plasma irradiation apparatus P1 (first plasma irradiation apparatus 100 and second plasma irradiation apparatus 200). Any method may be used.

  As shown in FIG. 1, the arm robot M1 can move the position of the plasma irradiation apparatus P1 in each of the x-axis, y-axis, and z-axis directions. For convenience of explanation, the direction of plasma irradiation is the −z axis direction. Thereby, the distance between the liquid level of a solution and the plasma irradiation part P1 can be adjusted. Moreover, this anti-tumor aqueous solution manufacturing apparatus PM can irradiate plasma only for the time by setting plasma irradiation time beforehand.

1-2. First plasma irradiation apparatus FIG. A is a cross-sectional view showing a schematic configuration of the plasma irradiation apparatus 100. Here, the plasma irradiation apparatus 100 is a first plasma irradiation apparatus that ejects plasma in a dot shape. FIG. 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 irradiation apparatus 100 of A. FIG.

The plasma irradiation apparatus 100 includes a housing unit 10, electrodes 2a and 2b, and a voltage application unit 3. The casing 10 is made of a sintered body made of alumina (Al ₂ O ₃ ) as a raw material. And the shape of the housing | casing part 10 is a cylinder shape. The internal diameter of the housing | casing part 10 is 2-3 mm. The thickness of the housing | casing part 10 is 0.2-0.3 mm. The length of the casing 10 is 25 cm. A gas inlet 10 i and a gas outlet 10 o are formed at both ends of the housing 10. The gas inlet 10i is for introducing a gas for generating plasma. The gas outlet 10 o is an irradiation unit for irradiating the outside of the housing unit 10 with plasma. The direction in which the gas moves is the direction of the arrow in the figure.

  The electrodes 2a and 2b are counter electrode pairs arranged 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 portion 10. For example, it is about 1 mm. For the electrodes 2a and 2b, FIG. As shown to B, many recessed parts (hollow) H are formed in each of an opposing surface. Therefore, the opposing surfaces of the electrodes 2a and 2b have a fine uneven shape. In addition, the depth of this recessed part H is about 0.5 mm.

  The electrode 2a is disposed inside the housing 10 and in the vicinity of the gas inlet 10i. The electrode 2b is disposed inside the housing portion 10 and in the vicinity of the gas ejection port 10o. Therefore, in the plasma irradiation apparatus 100, the gas is introduced from the opposite side of the facing surface of the electrode 2a, and the gas is ejected 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 a smaller distance.

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

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

1-3. Second plasma irradiation apparatus FIG. A is a cross-sectional view showing a schematic configuration of the plasma irradiation apparatus 110. Here, the plasma irradiation apparatus 110 is a second plasma irradiation apparatus that ejects plasma linearly. FIG. B is shown in FIG. It is a fragmentary sectional view in the cross section perpendicular | vertical to the longitudinal direction of the plasma area | region P of the plasma irradiation apparatus 110 of A. FIG.

The plasma irradiation apparatus 110 includes a casing unit 11, electrodes 2 a and 2 b, and a voltage application unit 3. The casing 11 is made of a sintered body using alumina (Al ₂ O ₃ ) as a raw material. At both ends of the housing portion 11, a gas introduction port 11 i and a large number of gas ejection ports 11 o are formed. 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 (the width in the left-right direction in FIG. 3.B) from the gas inlet 11i to just above the plasma region P is 1 mm.

  The gas ejection port 11o is an irradiation unit for irradiating the outside of the casing unit 11 with plasma. The gas ejection port 11o has a cylindrical shape or a slit shape. The gas outlet 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 to 2 mm. In the case of a slit shape, the slit width of the gas ejection port 11o is preferably 1 mm or less. Thereby, a stable plasma is formed. The gas inlet 11i introduces gas in a direction intersecting with a line connecting the electrode 2a and the electrode 2b.

  The electrodes 2a and 2b and the voltage application unit 3 are the same as those in the plasma irradiation apparatus 100 shown in FIG. Similarly, a voltage is applied between the electrodes 2a and 2b using a commercial AC voltage. Thereby, plasma can be ejected in a straight line.

  Further, a plasma irradiation apparatus 110 for ejecting plasma in a straight line is shown in FIG. If arranged in a line in the left-right direction of B, the plasma can be ejected in a plane over a rectangular region.

2. Plasma generated by the plasma irradiation apparatus The plasma generated by the plasma irradiation apparatuses 100 and 110 is non-equilibrium atmospheric pressure plasma. Here, atmospheric pressure plasma refers to plasma having a pressure in the range of 0.5 to 2.0 atmospheres.

  In this embodiment, Ar gas is mainly used as the plasma generating gas. Of course, electrons and Ar ions are generated in the plasma generated by the plasma irradiation apparatuses 100 and 110. 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 to} 1 × 10 ¹⁷ cm ⁻³ . The plasma density in 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 plasma generated by the plasma irradiation apparatuses 100 and 110 is about three orders of magnitude higher than the plasma density of plasma generated by dielectric barrier discharge. Therefore, more Ar ions are generated inside the plasma. Therefore, the amount of radicals and ultraviolet rays is also large. This plasma density is approximately equal to the electron density inside the plasma.

And the plasma temperature at the time of this plasma generation exists in the range of about 1000K-2500K. Moreover, the electron temperature in this plasma is larger than the gas temperature. Moreover, although the electron density is in the range of 1 × 10 ¹⁴ cm ^{−3 to} 1 × 10 ¹⁷ cm ⁻³ , the gas temperature is about 1000K to 2500K. The temperature of this plasma is the temperature in the plasma region P where plasma is generated. Therefore, the plasma temperature at the position of the cancer cells can be set to about room temperature by setting the plasma conditions and the distances from the gas ejection port to the cancer cells to be different. Therefore, when cancer cells and normal cells are irradiated with this plasma, there is almost no possibility that these cells will be damaged by heat.

The oxygen radical density is in the range of 2 × 10 ¹⁴ cm ^{−3 to} 1.6 × 10 ¹⁵ cm ⁻³ . This oxygen radical density can be adjusted by adjusting the amount of oxygen gas mixed into the argon gas.

3. Antitumor aqueous solution The antitumor aqueous solution of the present embodiment is obtained by irradiating a first aqueous solution, which is a raw material, with atmospheric pressure plasma to form a second aqueous solution, and then freezing the second aqueous solution. The solvent of the first aqueous solution is an aqueous solvent. As the solute of the first aqueous solution, disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-histidine. A solute containing at least one of L-tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) may be used. Alternatively, a culture solution can be used as the first aqueous solution. An example of the culture solution is DMEM. DMEM contains sugars such as glucose. Here, the culture component is a component contained in a culture solution for culturing cells and the like. For example, it is described in Table 3 (DMEM component) described later.

4). 2. Manufacturing method of anti-tumor aqueous solution There are two types of manufacturing methods of the anti-tumor aqueous solution of this embodiment. Therefore, each method will be described.

4-1. Method for producing antitumor aqueous solution (first method)
4-1-1. Aqueous solution preparation step (first method)
The first method will be described. First, a first aqueous solution is prepared. The first aqueous solution refers to an aqueous solution before being irradiated with plasma. In this first method, a culture solution is prepared as a first aqueous solution. That is, a culture solution in which these culture components are added to water is prepared. The pH of the culture solution at this stage is approximately 7. For example, it is in the range of 5.5 or more and 8.5 or less. The culture components will be described later (see Table 3).

4-1-2. Plasma irradiation process (first method)
Next, the first aqueous solution (culture solution) is irradiated with atmospheric pressure plasma generated in the plasma generation region by the antitumor aqueous solution manufacturing apparatus PM. The distance between the liquid level and the plasma jet outlet when the plasma is irradiated is, for example, 1 cm. Further, this distance may be changed within a range of 0.5 cm to 3 cm. The plasma density of this plasma is in the range of 1 × 10 ¹⁴ cm ^{−3 to} 1 × 10 ¹⁷ cm ⁻³ . And the plasma temperature in this plasma exists in the range of about 1000K-2500K. However, the plasma temperature can be lowered to about room temperature (about 300 K) at the liquid level. The oxygen radical density is in the range of 2 × 10 ¹⁴ cm ^{−3 to} 1.6 × 10 ¹⁵ cm ⁻³ . These plasma conditions are shown in Table 1. These conditions are merely examples.

[Table 1]
Conditions Numerical range Liquid surface-jet distance 0.5 cm or more 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 2500 K or less Oxygen radical density 2 × 10 ¹⁴ cm ⁻³ or more 1.6 × 10 ¹⁵ cm ^-3 or less

Note that, as will be described later in the experiment, in order to produce an antitumor aqueous solution having an antitumor effect, the plasma density time product is set to satisfy the following condition.
1.2 × 10 ¹⁸ sec · cm ⁻³ or more Here, the plasma density time product is a product of the plasma density in the plasma generation region and the time (irradiation time) of irradiating this aqueous solution with atmospheric pressure plasma.

  In this way, the first aqueous solution (culture solution) is changed to the second aqueous solution (culture solution) by irradiating the first aqueous solution (culture solution) with plasma. The pH of the first aqueous solution (culture solution) before plasma irradiation is close to 7. For example, it is in the range of 5.5 or more and 8.5 or less. In general, the culture solution contains a substance that attempts to maintain the pH in the vicinity of 7. For example, hydrogen carbonate ions. Therefore, the pH of this aqueous solution hardly changes before and after plasma irradiation. The pH of the second aqueous solution (culture solution) after plasma irradiation is close to 7. For example, it is in the range of 5.5 or more and 8.5 or less.

4-1-3. Freezing process (first method)
Next, the second aqueous solution (culture solution) is frozen. For this purpose, the second aqueous solution (culture solution) is frozen at −196 ° C. or higher and 0 ° C. or lower. Specifically, it stores in a freezer. As the freezer, a biological experiment refrigerator (for example, BioFreezer GS-5203KHC manufactured by Nippon Freezer Co., Ltd.) can be used. The temperature of the second aqueous solution (culture solution) frozen in this freezer is in the range of −28 ° C. or higher and −14 ° C. or lower. Further, the temperature of the second aqueous solution (culture solution) is not limited to this range. Any ordinary freezing temperature may be used. For example, it is in the range of −196 ° C. or more and 0 ° C. or less. Preferably, it is -196 degreeC or more and -10 degrees or less. More preferably, it is -150 degreeC or more and -20 degrees C or less. More preferably, it is −80 ° C. or higher and −30 ° C. or lower. Thus, in the freezing step, the third aqueous solution (culture solution) in a frozen state is produced by freezing the second aqueous solution (culture solution).

4-2. Method for producing antitumor aqueous solution (second method)
4-2-1. Aqueous solution preparation process (second method)
The second method will be described. First, a first aqueous solution is prepared. The first aqueous solution refers to an aqueous solution before being irradiated with plasma. In the second method, the following aqueous solution is prepared as the first aqueous solution. Here, disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-tyrosine disodium A first aqueous solution in which a solute containing at least one of dihydrate (L-Tyrosine · 2Na · 2H ₂ O) is added to water is prepared.

4-2-2. Plasma irradiation process (second method)
Next, the first aqueous solution is irradiated with atmospheric pressure plasma generated in the plasma generation region by the antitumor aqueous solution manufacturing apparatus PM. Various conditions for plasma irradiation are the same as in the first method. Thus, the second aqueous solution is produced by irradiating the first aqueous solution with plasma.

4-2-3. Culture component addition step (second method)
Next, a culture component (for example, a DMEM component in Table 3 described later) is added to the second aqueous solution. For convenience, the aqueous solution obtained by adding the culture components to the second aqueous solution is also referred to as the second aqueous solution.

4-2-4. Freezing process (second method)
Next, the second aqueous solution is frozen. For this purpose, the second aqueous solution is frozen at −196 ° C. or higher and 0 ° C. or lower. Preferably, it is -196 degreeC or more and -10 degrees or less. More preferably, it is -150 degreeC or more and -20 degrees C or less. More preferably, it is −80 ° C. or higher and −30 ° C. or lower. Thus, freezing is the same as in the case of the first method. Thereby, the 3rd aqueous solution of a frozen state is obtained.

5. 5. Duration of anti-tumor effect in frozen anti-tumor aqueous solution 5-1. 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 described in this item refers to the second aqueous solution in the first method and the second aqueous solution in the second method after adding culture components. The antitumor aqueous solution (second aqueous solution) before freezing exhibits an antitumor effect as described later. The duration of the antitumor effect in the second aqueous solution was 8 hours or more and less than 18 hours.

5-2. Antitumor Effect of Third Aqueous Solution (After Freezing) On the other hand, the antitumor aqueous solution produced by the production method of the present embodiment, that is, the frozen third aqueous solution, keeps the antitumor effect. In fact, as will be described later, the antitumor aqueous solution having a frozen and kept period of 28 days or longer exhibited an antitumor effect after thawing. That is, the antitumor aqueous solution can be stored frozen for a long period of time. The antitumor effect is hardly lost by freezing and thawing the antitumor aqueous solution. Details will be described later.

5-3. Consideration of antitumor effect of second aqueous solution (before freezing) The antitumor aqueous solution before freezing is considered to contain some antitumor substance. This antitumor substance is not considered to be a radical such as a hydroxy radical, a superoxide anion radical, or a hydroperoxy radical, as described in Patent Document 2. The reasons are as follows: (1) The bactericidal effect and the antitumor effect are different, (2) the duration of the effect is different, (3) the relationship between the effect and pH dependence is different, There are three.

  First, it goes without saying that the difference between the first bactericidal effect and the antitumor effect. Moreover, the anti-tumor aqueous solution of this embodiment has selectivity. This anti-tumor aqueous solution has little effect on normal cells, but selectively kills cancer cells. This means that it does not bring about a harsh living environment for all cells, but rather selectively kills cancer cells as a target.

  Next, the duration will be described. Patent Document 2 describes that, for example, a superoxide anion radical can exist in water for several seconds. There is also a description that the lifetime is relatively long (see paragraphs [0090]-[0093] etc. of Patent Document 2). In contrast, the second aqueous solution (before freezing) maintains the antitumor effect for at least 8 hours.

  Next, effects and pH dependency will be described. The radical described in Patent Document 2 provides a sufficient bactericidal effect under acidic conditions at a low pH, but hardly provides a bactericidal effect under conditions close to neutrality (paragraphs [0084]-[0089] of Patent Document 2). And FIG. 8 to FIG. 11). In addition, it is described that hydroperoxy radicals increase as pH decreases (paragraph [0093] of Patent Document 2). On the other hand, the anti-tumor aqueous solution of this embodiment has an anti-tumor effect under neutral conditions.

  From the above, it is considered that this antitumor aqueous solution contains an antitumor substance.

5-4. Consideration of Antitumor Effect of Third Aqueous Solution (After Freezing) In a frozen antitumor aqueous solution (third aqueous solution), it is considered that this antitumor substance exists without destroying the chemical structure. Further, it is suggested that the antitumor substance in a frozen state does not react with other chemical species in the antitumor aqueous solution and the antitumor substance does not disappear. In the frozen state, the chemical reaction is considered to hardly progress. Furthermore, this antitumor substance is not broken by the phase transition (solidification) of water by freezing.

  Therefore, this anti-tumor aqueous solution can be stored while retaining its anti-tumor effect. Of course, it can also be transported in a frozen state. The antitumor aqueous solution stored in a frozen state can be used whenever necessary if it is thawed immediately before use.

6). 6. Treatment of cancer using antitumor aqueous solution 6-1. Properties and uses of antitumor aqueous solution (anticancer agent)
This anti-tumor aqueous solution has an anti-tumor effect that selectively kills cancer cells. That is, this antitumor aqueous solution can be used as an anticancer agent having an anticancer effect. This anti-cancer action lasts for a long time by performing frozen storage. And the anti-tumor aqueous solution of this embodiment hardly damages a normal cell so that it may mention later.

7). Experiment A (Duration of antitumor effect in antitumor aqueous solution)
This experiment was an experiment conducted on the relationship between the elapsed time from the production in the frozen antitumor aqueous solution (third aqueous solution) and the antitumor effect.

7-1. Cancer cell used In this experiment, glioma was used as a cancer cell. Glioma is a glioma that develops in glial cells (glial cells). That is, it is a kind of brain tumor. Specifically, the gliomas shown in Table 2 were used. That is, U251SP. Table 2 also describes the cells used in the experiments described below.

[Table 2]
Cell name Status Type Resistance U251SP Cancer cell Glioma-
RI-371 Normal cells Astrocytes −
SKOV3 cancer cell ovarian cancer cell −
NOS2TR cancer cell ovarian cancer cell paclitaxel resistance

7-2. Experimental method 7-2-1. Culture of cancer cells The above cancer cells were cultured on a plate to prepare a cancer cell culture medium. The plate is a plastic container. And the culture solution was put into the inside of the plate. The culture solution is a mixed solution of DMEM, serum (FBS) and antibiotics (penicillin / streptomycin). The components of DMEM are shown in Table 3.

[Table 3]
Calcium chloride Ferric nitrate 9H ₂ O
Magnesium sulfate (anhydrous)
Potassium chloride Sodium bicarbonate Sodium chloride Phosphate-sodium (anhydrous)
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 phenol red Na

7-2-2. Preparation of anti-tumor aqueous solution Separately from preparing a cancer cell culture medium, an anti-tumor aqueous solution was prepared. In this experiment, the first method was used. In this experiment, a plate provided with six holes was used. This hole is a non-through hole. Therefore, the solution can be put inside the hole. First, 3 mL of culture solution is put into the hole of this plate. The culture solution used here is a solution obtained by mixing the aforementioned DMEM, serum (FBS) and antibiotics (penicillin / streptomycin). The components of DMEM are as shown in Table 3.

  Next, plasma is irradiated to a culture solution using antitumor aqueous solution manufacturing apparatus PM. At that time, the aqueous solution was irradiated with atmospheric pressure plasma in a state in which the liquid level of the culture solution was arranged at a position where the plasma generation region was not brought into contact with the culture solution. And the counter electrode of antitumor aqueous solution manufacturing apparatus PM was arrange | positioned facing the position which does not pinch | interpose the liquid level of a culture solution outside the culture solution. In this state, the aqueous solution was irradiated with atmospheric pressure plasma. Thus, although the plasma generation region is not in contact with the culture solution, the culture solution is irradiated with various radicals generated in the plasma. The plasma is forced out of the atmosphere above the culture in the plate. Therefore, during the plasma irradiation time, the culture solution is hardly exposed to the atmosphere. Thereby, the 2nd aqueous solution was produced.

Table 4 shows the plasma conditions. Only argon gas was used as a gas for generating plasma. The gas flow rate was 2.0 slm. Moreover, the distance between the plasma jet nozzle and the liquid level was 13 mm. The plasma irradiation time was 5 minutes. The plasma density in the plasma generation region was 2 × 10 ¹⁶ cm ⁻³ .

[Table 4]
Gas flow rate 2.0 slm
Distance between plasma jet and liquid level 13mm
Plasma irradiation time 5 minutes Plasma density (when generated) 2 × 10 ¹⁶ cm ^-3

  Next, the anti-tumor aqueous solution (second aqueous solution) was frozen in a freezer. The freezer used was BioFreezer GS-5203KHC manufactured by Nippon Freezer Co., Ltd. About the temperature of this freezer, it can adjust within the range of -28 degreeC or more and -14 degrees C or less. And the anti-tumor aqueous solution (2nd aqueous solution) was frozen at the temperature of -20 degreeC inside this freezer. And it kept the state frozen for a certain time. And the culture solution (3rd aqueous solution) from which freezing time differs was produced. It should be noted that the time from when the culture solution (first aqueous solution) is irradiated with plasma until the culture solution (second aqueous solution) is put into the freezer is about 2 to 3 minutes. Then, the culture solution (third aqueous solution) was thawed by being left at room temperature. The volume of the culture solution (third aqueous solution) is 0.2 mL. Therefore, the time required for this thawing is very short.

7-2-3. Supply of anti-tumor aqueous solution to cancer cell culture medium Next, the thawed anti-tumor aqueous solution (third aqueous solution) is supplied to the cancer cell culture medium. Specifically, the culture solution is removed from the cancer cell culture medium, and an antitumor aqueous solution (third aqueous solution) is put into the cancer cell culture medium. The supply amount of the anti-tumor aqueous solution (third aqueous solution) at that time is 0.2 mL. Then, after a predetermined time has elapsed since the culture solution was replaced with the antitumor aqueous solution (third aqueous solution), the culture solution is replaced again. Here, the culture solution supplied again to the cancer cell culture medium is a normal culture solution.

  Thus, the survival rate of cancer cells was examined. And the survival rate of the cancer cell was investigated 16 hours after supplying the anti-tumor aqueous solution (third aqueous solution) to the cancer cell culture medium. At that time, the number of surviving cancer cells was counted by microscopic observation.

7-3. Experimental results The experimental results are shown in FIG. The horizontal axis of FIG. 4 is the elapsed time from the time when the culture solution is irradiated with plasma. The vertical axis | shaft of FIG. 4 is a cell survival rate at the time of supplying anti-tumor aqueous solution (3rd aqueous solution) etc. FIG. The numerical value of the vertical axis | shaft of each cancer cell (U251SP) in FIG. 4 has shown the survival rate of the cancer cell. When this value is “1”, it indicates that the cancer cell is alive. “0” indicates that all cancer cells are dead. For example, in the case of 0.6, it indicates that about 60% of the number of cancer cells are alive compared to before the anti-tumor aqueous solution (third aqueous solution) is supplied.

  As shown in FIG. 4, immediately after the plasma irradiation, 1 day after plasma irradiation, 3 days, 7 days, 14 days, 21 days, and 28 days, the survival rate of cancer cells is Is almost zero. That is, the antitumor aqueous solution (third aqueous solution) exhibits an antitumor effect within at least 28 days after the plasma irradiation.

  In FIG. 4, “unirradiated” indicates the survival rate of cancer cells in a culture solution not irradiated with plasma. This “non-irradiation” is a comparative example.

  Thus, the anti-tumor aqueous solution (third aqueous solution) has an anti-tumor effect. That is, the antitumor aqueous solution (third aqueous solution) is an anticancer agent having an antitumor effect.

8). Experiment B (Comparison of effects on cancer cells and normal cells)
8-1. Cancer cells and normal cells used In this experiment, the selectivity of the antitumor effect was examined for an anti-tumor aqueous solution (second aqueous solution) that was not frozen. U251SP (glioma) shown in Table 2 was used as a cancer cell. On the other hand, RI-371 (astrocytes) shown in Table 2 was used as normal cells. This is to compare the effect of the anti-tumor aqueous solution (second aqueous solution) on cancer cells with the effect on normal cells.

8-2. Experimental Method An antitumor aqueous solution (second aqueous solution) was supplied to a cancer cell culture medium and a normal cell culture medium. The method is the same as in Experiment A described above. In this experiment, the number of cells was 10,000 in any culture place. These culture sites were immersed in an antitumor aqueous solution (second aqueous solution).

8-3. Experimental Results The experimental results are shown in FIG. As shown in FIG. 5, cancer cells (glioma: U251SP) immersed in an antitumor aqueous solution (second aqueous solution) are dead. On the other hand, normal cells (astrocytes: RI-371) immersed in an antitumor aqueous solution (second aqueous solution) are hardly killed. The number of normal cells immersed in the antitumor aqueous solution (second aqueous solution) is almost the same as the number of normal cells immersed in a normal culture solution. This indicates that cancer cells are killed and normal cells are rarely killed. Thus, by using the anti-tumor aqueous solution (second aqueous solution), it is possible to selectively kill only cancer cells. That is, brain tumor cancer can be treated.

8-4. Induction of apoptosis FIG. 6 is a photomicrograph showing cancer cells after dropping the second aqueous solution. The cancer cell is U251SP. FIG. 6 is a photomicrograph when immersed in an antitumor aqueous solution (second aqueous solution). Here, the bar in each figure has a length of 100 μm. The killed cancer cells are shown at the positions of the arrows in FIG. As shown in FIG. 6, apoptotic vesicles contracted into a spherical shape are seen. That is, these cells are cells that have been killed by the induction of apoptosis.

9. Experiment C (Duration of anti-tumor effect)
Here, an experiment conducted on the duration of the antitumor effect of the antitumor aqueous solution (second aqueous solution) will be described.

9-1. Used cancer cell In this experiment, U251SP (glioma) shown in Table 2 was used as a cancer cell.

9-2. Experimental Method As described in Experiment A, the cancer cell culture medium was immersed in an antitumor aqueous solution (second aqueous solution). In that case, an antitumor aqueous solution (second aqueous solution) in which the elapsed time from the plasma irradiation was changed was prepared, and the antitumor effect was examined in each case. The prepared anti-tumor aqueous solution (second aqueous solution) is one in which 0 hour, 1 hour, 8 hours, and 18 hours have elapsed after irradiation with plasma for 1 minute, respectively.

9-3. Experimental Results The experimental results are shown in FIG. As shown in FIG. 7, the antitumor effect of the antitumor aqueous solution (second aqueous solution) lasts for at least 8 hours immediately after plasma irradiation. Then, before 18 hours elapses, the antitumor effect of the antitumor aqueous solution (second aqueous solution) disappears. That is, the anti-tumor effect of the anti-tumor aqueous solution (second aqueous solution) lasts for an elapsed time of less than 18 hours from the start of plasma irradiation after the start of plasma irradiation. That is, when not stored in a frozen state, the antitumor effect is lost after an elapsed time of less than 18 hours.

10. Experiment D (second method)
In this experiment, a single component aqueous solution containing any single component among the many culture components shown in Table 3 was used as the first aqueous solution.

10-1. Cells used In this experiment, SKOV3 (ovarian cancer cells) was used as a cancer cell as shown in Table 2.

10-2. Preparation of anti-tumor aqueous solution (second aqueous solution) Disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine, and L-histidine (L-histidine) ), L-tyrosine disodium dihydrate (L-Tyrosine · 2Na · 2H ₂ O), and other culture components, single-component aqueous solutions were prepared and allowed to stand for 1 hour. After irradiating the first aqueous solution thus produced with plasma, a culture solution is added to obtain a second aqueous solution.

10-3. Experimental Results Experimental results are shown in FIGS. As shown in these figures, disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-histidine. When a single component aqueous solution containing L-tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) is used as the first aqueous solution, the second aqueous solution has an antitumor effect. Demonstrated.

10-4. Experimental Consideration As described above, the antitumor effect was confirmed with a solution obtained by irradiating plasma to five types of single-component aqueous solutions and then adding a culture solution. That is, the antitumor substance is not necessarily generated from one kind of component. In addition, both amino acids and inorganic salts can be raw materials for antitumor substances. That is, it is considered that these five kinds of substances react with some radicals supplied from plasma and the like, and an antitumor substance is generated after a multi-step reaction.

11. Experiment E (Animal experiment: anticancer drug resistance)
11-1. Mouse used In this experiment, an animal experiment using a female nude mouse was performed. Nude mice were seeded with ovarian cancer cells subcutaneously on both sides. The ovarian cancer cell used is NOS2TR in Table 2. Then, 2000 ovarian cancer cells were administered per site and the same amount of Matrigel was administered.

11-2. Experimental Method From the day after the ovarian cancer cells were seeded on the mice, an anti-tumor aqueous solution (second aqueous solution) was locally administered three times per week. The plasma irradiation time was 10 minutes. Then, 0.2 ml per site where ovarian cancer cells were seeded was locally injected. In addition, a simple culture solution not irradiated with plasma was injected into a mouse to be compared.

11-3. Experimental Results FIG. 15 is a photograph showing the state of the 4th week of a mouse seeded with NOS2TR. The left side of FIG. 15 shows a mouse administered with a normal culture solution. The right side of FIG. 15 shows a mouse administered with an anti-tumor aqueous solution (second aqueous solution). In mice administered with a normal culture solution, swelling due to tumor is observed, but in mice administered with an anti-tumor aqueous solution (second aqueous solution), swelling due to tumor is hardly observed.

  As described above, for Experiment A, a frozen antitumor aqueous solution (third aqueous solution) was used. For Experiment BE, a non-frozen antitumor aqueous solution (second aqueous solution) was used. However, the frozen anti-tumor aqueous solution (third aqueous solution) is also considered to exhibit the effects investigated in Experiment BE.

12 PH of antitumor aqueous solution
Further, the pH of the antitumor aqueous solution was examined. Here, the antitumor aqueous solution (culture solution) of the first method was used. The culture component of the culture solution is DMEM in Table 3. The pH of the first aqueous solution (culture solution) was 7.8. The pH of the second aqueous solution (culture solution) after irradiation with plasma for 5 minutes was 8.0. As a comparison, the pH of distilled water was examined. The pH of distilled water was 7.6. The pH of distilled water after irradiation with plasma for 5 minutes was 3.9. The antitumor aqueous solution is almost neutral before and after plasma irradiation. Distilled water becomes acidic when irradiated with plasma.

13. Summary of this embodiment As described in detail above, the antitumor aqueous solution according to this embodiment is obtained by irradiating a culture solution with plasma. Alternatively, an aqueous solution containing a specific culture component as a solute is irradiated with plasma, and then the culture component is added. These aqueous solutions are frozen. This anti-tumor aqueous solution has an anti-tumor effect. By freezing, the sustained effect of the antitumor effect is prolonged. Furthermore, it has the effect of killing cancer cells and rarely killing normal cells. That is, cancer cells can be selectively killed.

  The anti-tumor aqueous solution of this embodiment is an anti-cancer agent that can induce apoptosis only for cancer cells and shrink the tumor. Moreover, since this anticancer agent has selectivity, it is expected to cause almost no side effects.

DESCRIPTION OF SYMBOLS 100,110 ... Plasma irradiation apparatus 10, 11 ... Housing | casing part 10i, 11i ... Gas inlet 10o, 11o ... Gas jet 2a, 2b ... Electrode P ... Plasma area | region H ... Recessed part (hollow)
P1 ... Plasma irradiation device M1 ... Robot arm PM ... Antitumor aqueous solution manufacturing device

Claims (6)

In a method for producing an antitumor aqueous solution for selectively killing cancer cells,
Disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-tyrosine disodium dihydrate An aqueous solution preparation step of preparing, as a first aqueous solution, a culture solution containing at least one of a product (L-Tyrosine · 2Na · 2H ₂ O);
A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in a plasma generation region by a plasma generator to form a second aqueous solution;
A freezing step of freezing the second aqueous solution;
A method for producing an antitumor aqueous solution, comprising: In a method for producing an antitumor aqueous solution for selectively killing cancer cells,
Disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-tyrosine disodium dihydrate An aqueous solution preparation step of preparing a first aqueous solution in which a solute containing at least one of a product (L-Tyrosine · 2Na · 2H ₂ O) is added to water;
A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in a plasma generation region by a plasma generator to form a second aqueous solution;
A culture solution addition step of adding a culture solution to the second aqueous solution;
A freezing step of freezing the second aqueous solution to which the culture solution has been added ;
A method for producing an antitumor aqueous solution, comprising: In the manufacturing method of the anti-tumor aqueous solution of Claim 1 or Claim 2 ,
In the freezing step,
The method for producing an antitumor aqueous solution, wherein the second aqueous solution is frozen within a range of -196 ° C or higher and 0 ° C or lower. In a method for producing an anticancer agent that selectively kills cancer cells,
Disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-tyrosine disodium dihydrate An aqueous solution preparation step of preparing, as a first aqueous solution, a culture solution containing at least one of a product (L-Tyrosine · 2Na · 2H ₂ O);
A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in a plasma generation region by a plasma generator to form a second aqueous solution;
A freezing step of freezing the second aqueous solution;
The manufacturing method of the anticancer agent characterized by having. In a method for producing an anticancer agent that selectively kills cancer cells,
Disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium bicarbonate (NaHCO ₃ ), L-glutamine, L-histidine, and L-tyrosine disodium dihydrate An aqueous solution preparation step of preparing a first aqueous solution in which a solute containing at least one of a product (L-Tyrosine · 2Na · 2H ₂ O) is added to water;
A plasma irradiation step of irradiating the first aqueous solution with atmospheric pressure plasma generated in a plasma generation region by a plasma generator to form a second aqueous solution;
A culture solution addition step of adding a culture solution to the second aqueous solution;
A freezing step of freezing the second aqueous solution to which the culture solution has been added ;
The manufacturing method of the anticancer agent characterized by having. In the manufacturing method of the anticancer agent of Claim 4 or Claim 5 ,
In the freezing step,
The method for producing an anticancer agent, wherein the second aqueous solution is frozen within a range of -196 ° C or higher and 0 ° C or lower.