ANTICANCER AGENT, PRODRUG OF ANTICANCER AGENT, METHOD FOR KILLING CANCER CELL IN VITRO, CANCER TREATEMENT METHOD, AND CANCER TREATMENT DEVICE

Abstract

Provided is an anticancer agent containing a substance that contains nitrogen-15 and that specifically accumulates in a cancer cell. Also provided is a method for killing a cancer cell in vitro, including accumulating nitrogen-15 in a cancer cell in vitro and irradiating the cancer cell with a proton beam in vitro. Additionally provided is a cancer treatment method including causing an accumulation of the nitrogen-15 in a cancer cell in a human or a nonhuman animal and irradiating the human or the nonhuman animal with a proton beam.

Claims

1. An anticancer agent comprising a substance that causes a specific accumulation of nitrogen-15 in a cancer cell.

2. The anticancer agent according to claim 1, wherein the anticancer agent is to be administered into a body of a patient and the patient is to be irradiated with a proton beam after the administration.

3. The anticancer agent according to claim 1, wherein the substance is 5-aminolevulinic acid in which nitrogen is nitrogen-15.

4. The anticancer agent according to claim 1, wherein the substance is 5-fluorouracil in which nitrogen is nitrogen-15.

5. The anticancer agent according to claim 1, wherein the anticancer agent is a molecular-targeted therapeutic drug in which nitrogen is nitrogen-15.

6. The anticancer agent according to claim 5, comprising a portion that binds to the cancer cell.

7. The anticancer agent according to claim 6, wherein the portion that binds to the cancer cell is an antibody or a part of an antibody.

8. The anticancer agent according to claim 7, wherein a drug is bound to the antibody or the part of the antibody.

9. The anticancer agent according to claim 7, wherein the antibody or the part of the antibody is trastuzumab in which nitrogen is nitrogen-15.

10. A prodrug of an anticancer agent comprising a substance that causes a specific accumulation of nitrogen-15 in a cancer cell.

11. The prodrug according to claim 10, wherein the prodrug is to be administered into a body of a patient and the patient is to be irradiated with a proton beam after the administration.

12. The prodrug according to claim 10, wherein the anticancer agent is 5-fluorouracil in which nitrogen is nitrogen-15.

13. The prodrug according to claim 10, wherein the prodrug is selected from the group consisting of tegafur, tegafur/uracil, tegafur/gimeracil/oteracil potassium, doxifluridine, and capecitabine, in which nitrogen is nitrogen-15.

14. A method for killing a cancer cell in vitro, comprising: causing in vitro an accumulation of nitrogen-15 in a cancer cell; and irradiating the cancer cell with a proton beam in vitro.

15. The method for killing a cancer cell in vitro according to claim 14, wherein causing the accumulation of the nitrogen-15 in the cancer cell comprises administering the anticancer agent according to claim 1 to the cancer cell.

16. The method for killing a cancer cell in vitro according to claim 14, wherein causing the accumulation of the nitrogen-15 in the cancer cell comprises administering, to the cancer cell, a prodrug of the anticancer agent according to claim 5.

17. A cancer treatment method comprising: causing an accumulation of nitrogen-15 in a cancer cell of a human or nonhuman animal; and irradiating the human or nonhuman animal with a proton beam.

18. The cancer treatment method according to claim 17, wherein causing the accumulation of the nitrogen-15 in the cancer cell of the human or the nonhuman animal comprises administering the anticancer agent according to claim 1 to the human or the nonhuman animal.

19. The cancer treatment method according to claim 17, wherein causing the accumulation of the nitrogen-15 in the cancer cell of the human or the nonhuman animal comprises administering a prodrug of the anticancer agent according to claim 5 to the human or the nonhuman animal.

20. A cancer treatment device comprising an irradiator that irradiates a human or a nonhuman animal in which nitrogen-15 is accumulated in a cancer cell with a proton beam.

21. The cancer treatment device according to claim 20, further comprising an accelerator that accelerates the proton beam.

22. The cancer treatment device according to claim 21, wherein the accelerator comprises a laser plasma.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a schematic diagram that shows the pathway by which protoporphyrin IX, derived from 5-aminolevulinic acid, is converted to heme.

[0065] FIG. 2 is a schematic diagram that shows the kinetic energy positions due to the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction.

[0066] FIG. 3 is a diagram that shows the relationship between the laboratory system and the center-of mass system for the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction.

[0067] FIG. 4 shows a comparison of the LETs of the proton and product ions.

[0068] FIG. 5 shows the dependence on proton energy of the reaction cross section of the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction.

[0069] FIG. 6 is a diagram that describes the relationship between the resonance energy of the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction and the reaction position (residual range) of the proton in the body, and describes the range of the reaction product ions on the scale of this residual range.

[0070] FIG. 7 shows the resonance energy of the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction, the reaction position (residual range) of the proton in the body, the range of reaction product ions (.sup.4He.sup.2+, .sup.12C.sup.6+), and the biological ionization effect (Bragg's peak).

[0071] FIG. 8 is a schematic diagram that shows the occurrence of the .sup.15N resonance nuclear reaction in cancer cells.

[0072] FIG. 9 shows the resistance to thermal hydrolysis of Torelina (registered trademark) (refer to Non-Patent Document 8).

[0073] FIG. 10 is a table that shows the gas permeabilities of Torelina (registered trademark) film (refer to Non-Patent Document 9).

[0074] FIG. 11 is a schematic diagram that shows a cancer treatment device according to an embodiment.

[0075] FIG. 12 is a diagram that shows a method, according to Example 1, for quantitating the .sup.15N that has accumulated in normal cells and in cancer cells.

[0076] FIG. 13 is a graph that shows the gamma-ray spectrum obtained in the quantitation according to Example 1 of the .sup.15N that has accumulated in normal cells and in cancer cells.

[0077] FIG. 14 is a graph that shows the amount of intake of .sup.15N_5-ALA, according to Example 1, into normal cells and into cancer cells.

[0078] FIG. 15 is a gene structural diagram of the pUC4-KIXX plasmid according to Example 2.

[0079] FIG. 16 contains photographs that show E. coli colonies when the .sup.15N isotopic ratio in pUC4-KIXX was changed according to Example 2.

[0080] FIG. 17 is a graph that shows, when the .sup.15N isotopic ratio in pUC4-KIXX was changed according to Example 2, the E. coli count (left vertical axis) and the yield of 4.43 MeV gamma rays (right vertical axis) emitted by the .sup.15N(.sup.1H, .sub.1).sup.2C resonance nuclear reaction (E.sub.r=0.987 MeV).

[0081] FIG. 18 contains photographs of RGM-GFP cells cultured on .sup.15N_5-ALA-free culture medium, immediately after the execution of proton beam irradiation according to Example 3.

[0082] FIG. 19 is a graph of the calculated results for the surviving fraction versus the proton beam irradiation charge amount, for RGM-GFP cells cultured on .sup.15N_5-ALA-free culture medium, according to Example 3.

[0083] FIG. 20 contains photographs of RGK-KO cells immediately after the execution of proton beam irradiation according to Example 3. These are photographs of cells cultured on .sup.15N_5-ALA-containing culture medium and .sup.15N_5-ALA-free culture medium.

[0084] FIG. 21 contains photographs of RGK-KO cells after the elapse of 24 hours after the execution of proton beam irradiation according to Example 3, and contains photographs of cells cultured on .sup.15N_5-ALA-containing culture medium.

[0085] FIG. 22 contains a photograph of a cell holder, according to Example 4, that enables cells to be held in a live state in a vacuum.

[0086] FIG. 23 is a graph according to Example 4 that shows timewise changes in the vacuum when cells were held in a live state in a vacuum.

DESCRIPTION OF EMBODIMENTS

[0087] Modes for executing the present invention (referred to as embodiments in the following) are described below. The present invention is not limited to the following embodiments, and the present invention can be executed in various modifications within the scope of its essential features. The embodiments given in the following are examples of, e.g., methods and so forth, for realizing the technical concepts of this invention and are not limited to these examples.

[0088] An anticancer agent according to an embodiment contains a substance that contains nitrogen-15 and that specifically accumulates in a cancer cell.

[0089] Nitrogen (N) is one of the six essential abundant elements (O, C, H, N, Ca, P), takes up 2.6% of the weight of a human, and can be present in all regions of the body. Nitrogen-15 is also referred to as .sup.15N. .sup.15N is one of the stable isotopes of naturally occurring nitrogen and is composed of 7 protons and 8 neutrons. .sup.15N accounts for 0.364% of the total nitrogen on the earth. .sup.15N is also present in the body at the same ratio.

[0090] The substance that contains nitrogen-15 and that specifically accumulates in the cancer cell may be 5-Aminolevulinic acid in which the nitrogen is nitrogen-15. 5-Aminolevulinic acid (5-ALA) is synthesized in all cells and is known as a starting material for the porphyrin synthesis pathway. In normal cells, heme, which is necessary for energy metabolism, is synthesized from 5-aminolevulinic acid through seven steps of enzymatic reactions. When released from protein, heme promotes the generation of active oxygen and thus causes oxidative stress that damages DNA and lipids, and it is therefore rapidly degraded and excreted after energy is produced. The chemical formula of 5-aminolevulinic acid is as follows.

##STR00001##

[0091] In the anticancer agent according to the embodiment, N in 5-aminolevulinic acid is replaced by .sup.15N. 5-Aminolevulinic acid in which the nitrogen is nitrogen-15 is also represented by .sup.15N_5-ALA. The chemical formula of 5-aminolevulinic acid in which the nitrogen is nitrogen-15 is given below.

##STR00002##

[0092] The anticancer agent according to the embodiment is administered to a human or a nonhuman animal. The route of administration can be exemplified by topical administration, enteral administration including oral administration, and parenteral administration, but is not particularly limited. The administered anticancer agent is taken up by cells.

[0093] As shown in FIG. 1, protoporphyrin IX derived from 5-aminolevulinic acid in which the nitrogen is nitrogen-15 is converted to heme by ferrochelatase (FECH) in normal cells. In the normal cells, which can normally synthesize the heme, the nitrogen-15 is excreted and does not accumulate.

[0094] In cancer cells, however, induced nitric oxide synthase (iNOS) is active and the intracellular nitric oxide (NO) concentration is maintained at high levels. As a consequence, in the cancer cells, the iron-sulfur complex rapidly reacts with nitric oxide (NO) to irreversibly form a dinitrosyldithiolate iron complex (DNIC), and as a result the heme cannot be synthesized. As a consequence, 5-aminolevulinic acid in which the nitrogen is nitrogen-15 and derivatives of 5-aminolevulinic acid in which the nitrogen is nitrogen-15, such as protoporphyrin IX in which the nitrogen is nitrogen-15, accumulate in the cancer cells for a long time.

[0095] Thus, when the anticancer agent according to the embodiment is administered to a human or a nonhuman animal, nitrogen-15 specifically accumulates in cancer cells but does not accumulate in normal cells.

[0096] FIG. 2 shows the center-of-mass system energy diagram (unit=MeV) for the proton capture resonance nuclear reaction of the nitrogen-15 atomic nucleus. When the proton and nitrogen-15 collide, in the case where the sum of the binding energy and collision energy for the both atomic nuclei accords (resonates) with the excitation level of the oxygen-16 atomic nucleus, the proton is captured by the nitrogen-15 atomic nucleus to form the oxygen-16 compound atomic nucleus .sup.16O*. The .sup.16O*, which is in an excited state, emits the .sup.4He.sup.2+ atomic nucleus ( ray), which is the most stable of the atomic nucleus species, and immediately assumes the first excitation level of the carbon-12 atomic nucleus; a 4.43 MeV gamma ray is emitted from the .sup.12C* excitation level to yield the .sup.12C atomic nucleus in the ground state. The formula for the resonance nuclear reaction with this sequence is given by the following formula (1).

.sup.15N(.sup.1H,.sub.1).sup.12C(1)

[0097] Here, the 1 in the .sub.1 indicates the first excitation level of .sup.12C*, and the energy difference E.sub.0 from the .sup.16O* excitation level is distributed according to the law of the conservation of momentum into the kinetic energies of the .sup.4He and .sup.12C*. .sup.4He and .sup.12C* are emitted as product ions. When this sequence of reaction processes is viewed as a center-of-mass system, i.e., as a coordinate system that moves at the velocity V.sub.G at which the proton/nitrogen-15 centroid moves, the .sup.16O* compound atomic nucleus is resting. When the mass of the proton and .sup.15N and the velocity in the laboratory system are made, respectively, m.sub.H, m.sub.N, u.sub.H, and u.sub.N=0, the center-of-mass velocity is given by the following formula (2).

[00001]$\left[\mathrm{Math}.1\right]$$\begin{array}{cc}{V}_G=\frac{m_H}{m_H+m_N}{u}_H+\frac{m_N}{m_H+m_N}{u}_N=\frac{m_H}{m_H+m_N}{u}_H.& \left(2\right)\end{array}$

[0098] FIG. 3 shows the relationship between the laboratory system and the center-of-mass system. The velocities V.sub.H and V.sub.N of the proton and .sup.15N in the center-of-mass system are given by u.sub.H-V.sub.G and -V.sub.G, respectively, and the resonance energy .sub.0 is equal to the kinetic energy of the proton in the laboratory system, as shown in the following formula (3).

[00002]$\left[\mathrm{Math}.2\right]$$\begin{array}{cc}{}_0=\frac{m_H{V}_H^2}{2}+\frac{m_N{V}_N^2}{2}=\frac{m_H{u}_H^2}{2}.& \left(3\right)\end{array}$

[0099] The energy difference between the resonance energy level of the .sup.16O* compound atomic nucleus and the first excitation level of .sup.12C* conserves the momenta of the product ions, that is, is distributed into the kinetic energies of the product ions such that the vector sum of the momenta of the respective product ions becomes equal to the zero momentum of the at-rest .sup.16O* compound atomic nucleus (m.sub.He V.sub.He+m.sub.CV.sub.C=0).

[00003]$\left[\mathrm{Math}.3\right]$$\begin{array}{cc}{E}_0=\frac{m_{\mathrm{He}}{V}_{\mathrm{He}}^2}{2}+\frac{m_C{V}_C^2}{2}={}_{\mathrm{He}}+{}_C.& \left(4\right)\end{array}$

[0100] The maximum values of the kinetic energies .sub.He and .sub.C of .sup.4He and .sup.12C* in the laboratory system are given by the following formula (5) and formula (6).

[00004]$\left[\mathrm{Math}.4\right]$$\begin{array}{cc}{}_{\mathrm{He}}=\frac{1}{2}{m_{\mathrm{He}}\left(V_{\mathrm{He}}{V}_G\right)}^2=\frac{m_{\mathrm{He}}}{m_{\mathrm{He}}+m_C}{E}_0+\frac{m_H{m}_C}{{\left(m_H+m_N\right)}^2}{}_0\sqrt{\frac{m_C}{m_{\mathrm{He}}+m_C}\frac{m_H{m}_{\mathrm{He}}}{{\left(m_H+m_N\right)}^2}{E}_0{}_0}.& \left(5\right)\end{array}$$\left[\mathrm{Math}.5\right]$$\begin{array}{cc}{}_C=\frac{1}{2}{m_C\left(V_C{V}_G\right)}^2=\frac{m_{\mathrm{He}}}{m_{\mathrm{He}}+m_C}{E}_0+\frac{m_H{m}_C}{{\left(m_H+m_N\right)}^2}{}_0\sqrt{\frac{m_{\mathrm{He}}}{m_{\mathrm{He}}+m_C}\frac{m_H{m}_C}{{\left(m_H+m_N\right)}^2}{E}_0{}_0}.& \left(6\right)\end{array}$

[0101] The kinetic energies &He and &c given by the preceding formulae (5) and (6) determine the energy transferred from the ions to the surroundings along the track of the product ions (energy-transferring linear energy transfer (LET) per unit range) and define the track of the product ions. Since LET is proportional to the square of the charge on the ion, Z.sup.2, when a .sup.4He.sup.2+ atomic nucleus having Z=2 and a .sup.12C.sup.6+ atomic nucleus having Z=6 are emitted within the cell as product ions, the .sup.4He.sup.2+ atomic nucleus and .sup.12C.sup.6+ atomic nucleus travel within the cell at high kinetic energies and impart high energies to the surroundings and thus can have a cell-killing capability. FIG. 4 is a graph showing a comparison of the LET of the proton and product ions (.sup.4He.sup.2+ atomic nucleus and .sup.12C.sup.6+ atomic nucleus). The product ions exhibit a higher LET than the proton, and, with cancer cells in which .sup.15N has accumulated, it becomes possible to specifically kill the cancer cells with the product ions from the .sup.15N resonance nuclear reaction.

[0102] In addition to the reaction channel of formula (1), the following can also proceed from the excited nucleus level of the .sup.16O* compound atomic nucleus: a direct de-excitation reaction to the ground state of carbon, and a reaction in which only radiation is emitted, without a emission, to achieve the ground state of oxygen-16. When these are expressed with formulae, the reaction channels of the following formula (7) and formula (8) are simultaneously open, respectively.

.sup.15N(.sup.1H,.sub.0).sup.12C(7)

.sup.15N(.sup.1H,.sub.0).sup.16O(8)

[0103] In the resonance nuclear reaction given by formula (8), only highly penetrating gamma rays are emitted and the cancer cell-specific radiation action that is the goal of the present embodiment is not brought about. However, due to the extremely high stability of the .sup.4He.sup.2+ atomic nucleus and the .sup.12C* atomic nucleus, the resonance nuclear reaction channels represented by formula (1) and formula (7) are produced at overwhelmingly higher probabilities than the resonance nuclear reaction channel represented by formula (8). The order of nuclear reaction probabilities is given by the following formula (9).

(reaction probability of formula 1)>(reaction probability of formula 7)>>(reaction probability of formula 8)(9)

[0104] As, in the cancer cells that have accumulated a large amount of nitrogen-15, a resonance nuclear reaction in which a proton collides with the nitrogen-15 atomic nucleus and product ions are emitted occurs with high probability, it is possible to specifically kill the cancer cells.

[0105] FIG. 5 shows the reaction cross section for each proton resonance energy, specified by formula (3), of the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction, and corresponding to the excitation level of the .sup.16O* compound nucleus (refer to Non-Patent Documents 3 and 4). FIG. 6 shows the residual range (residual range from the p-stopping point), which shows the reaction position of the proton in the body for the resonance energy of the proton beam, and shows the range of the reaction product ions on the scale of this residual range. 12 resonance energy positions exist in the range where the proton enters the body and is attenuated to an energy of 10 MeV or less, in a region of about 800 m as the residual range. At the residual range positions corresponding to each resonance energy, the .sup.4He.sup.2+ atomic nucleus and .sup.12C.sup.6+ atomic nucleus product ions are emitted at the kinetic energies given by formula (5) and formula (6), and the range of each product ion is indicated by the radius of the small circle at each resonance energy. High energy is applied to the surroundings over this product ion range and the surroundings are excited at high densities. Damage is thus produced in the cancer cells at these 12 resonance energy positions and the cancer cells die. FIG. 7 gives the resonance energy of the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction, the reaction position (residual range) of protons in the body, the range of reaction product ions (.sup.4He.sup.2+, .sup.12C.sup.6+), and the biological ionization effect (Bragg's peak).

[0106] In the cancer cells to which the anticancer agent according to the embodiment has been provided, particularly at the mitochondria protoporphyrin IX in which the nitrogen is nitrogen-15 is synthesized in large amounts. Due to this, and while not wishing to be bound by theory, it is believed that, in the cancer cells and as shown in FIG. 8, the .sup.15N resonance nuclear reaction occurs at high levels upon proton beam irradiation, the mitochondrial are then damaged, and the cancer cells are killed.

[0107] The cancer cells in the body of a human or nonhuman animal can thus be specifically killed by the proton beam irradiation of the human or nonhuman animal to whom the anticancer agent according to the embodiment has been administered.

[0108] The method for manufacturing 5-aminolevulinic acid in which the nitrogen is nitrogen-15 is not particularly limited. 5-Aminolevulinic acid is ordinarily biosynthesized by supplying glycine and succinic acid, the precursors for 5-aminolevulinic acid, to a 5th mutant strain (CR-520), 6th mutant strain (CR-606), or 7th mutant strain (CR-720) capable of producing 5-aminolevulinic acid under aerobic and dark conditions and created on the basis of the photosynthetic bacterium Rhodobacter sphaeroides IFO12203.

[0109] Therefore, it is possible to manufacture 5-Aminolevulinic acid in which the nitrogen is nitrogen-15 by supplying glycine in which the nitrogen is nitrogen-15 and succinic acid in which the nitrogen is nitrogen-15 to bacteria that produce 5-aminolevulinic acid.

[0110] When 5-aminolevulinic acid in which the nitrogen is nitrogen-15 is manufactured by biosynthesis, a large amount of nitrogen is consumed in the processes of synthesizing, e.g., proteins, nucleic acids, and so forth, in addition to the metabolic pathway for 5-ALA, and it is desirable to efficiently utilize the nitrogen-15, which is rare and exists at only 0.364% in nature. Due to this, it is desirable to recover the .sup.15N that is contained in large amounts in the residue after the biosynthetic production of 5-aminolevulinic acid in which the nitrogen is nitrogen-15, and to return this to the 5-aminolevulinic acid production process. An example of a method for recovering the nitrogen from the residue is the Kjeldahl method, in which organic nitrogen is heated in the presence of sulfuric acid and recovered as the (NH.sub.4).sup.+ ion. Nitrogen utilization in, e.g., microalgae, Escherichia coli, etc., supports the direct uptake of the (NH.sub.4).sup.+ ion and is suitable as a recovery route for nitrogen-15. The intaken (NH.sub.4).sup.+ ion is taken into the glutamate synthesis cycle by glutamine synthetase with the synthesis of glutamic acid. The C5 pathwayin which 5-ALA is synthesized by three enzymes (GltX, HemA, and HemL) from glutamic acid synthesized via -ketoglutarate (-KG) in the glucose-utilizing tricarboxylic acid (TCA) cyclecan be used as a means for producing 5-aminolevulinic acid in which the nitrogen is nitrogen-15 as a biopharmaceutical.

[0111] The nitrogen-15-containing substance that specifically accumulates in the cancer cells is not limited to 5-aminolevulinic acid in which the nitrogen is nitrogen-15. For example, the nitrogen-15-containing substance that specifically accumulates in the cancer cells may be 5-fluorouracil in which a nitrogen is nitrogen-15. Or, the nitrogen-15-containing substance that specifically accumulates in the cancer cells may be a prodrug of 5-fluorouracil in which a nitrogen is nitrogen-15. The prodrug of 5-fluorouracil in which a nitrogen is nitrogen-15 can be exemplified by tegafur in which a nitrogen is nitrogen-15, tegafur/uracil in which a nitrogen is nitrogen-15, tegafur/gimeracil/oteracil potassium in which a nitrogen is nitrogen-15, doxifluridine in which a nitrogen is nitrogen-15, and capecitabine in which a nitrogen is nitrogen-15.

[0112] In addition, the nitrogen-15-containing substance that specifically accumulates in cancer cells according to the embodiment may be a molecular-targeted therapeutic drug in which a nitrogen is nitrogen-15. Molecular-targeted therapeutic drugs, for example, have a portion that binds to cancer cells. Molecular-targeted therapeutic drugs, for example, have a portion that binds to a biomolecule that is specifically expressed by cancer cells. For example, a portion that binds to a biomolecule that is specifically expressed by cancer cells may contain nitrogen-15. The portion that binds to a biomolecule that is specifically expressed by cancer cells may be an antibody or a part of an antibody. The molecular-targeted drug may be an antibody-drug conjugate (ADC) that has nitrogen-15 for its nitrogen and that is targeted to and kills tumor cells while sparing healthy cells. The molecular-targeted drug may be an antibody or part of an antibody that has nitrogen-15 for its nitrogen and that targets the HER2 (human epidermal growth factor receptor 2) glycoprotein present at the surface of breast cancer cells. A HER2-targeting antibody or part of an antibody in which a nitrogen is nitrogen-15 may be obtained, for example, by substituting nitrogen-15 for the nitrogen in trastuzumab (trade name: Herceptin).

[0113] The .sup.15N used in the cancer treatment drug according to the embodiment is one of the six essential abundant elements and can be supplied to all regions of the body.

[0114] The .sup.15N used in the cancer treatment drug according to the embodiment accounts for only 0.364% of the total nitrogen in nature, and it is possible to specifically kill the cancer cells by the accumulating the .sup.15N anticancer agent in high proportions in the cancer cells.

[0115] The .sup.15N used in the cancer treatment drug according to the embodiment is a stable isotopic element. When the N in an already approved cancer treatment drug is replaced with .sup.15N and the cancer treatment drug having N replaced with .sup.15N is administered to a cancer patient, the burden on the patient will be equivalent to that for the already approved cancer treatment drug.

[0116] The chemical action of the .sup.15N used in the cancer treatment drug according to the embodiment is entirely the same as that of nitrogen-14 (.sup.14N), which exists in nature at 99.636%. In the case where an already approved cancer treatment drug contains nitrogen, 99.636% of the nitrogen is .sup.14N. Even if the .sup.14N is replaced with .sup.15N at high concentrations, for example, at a ratio of 98% or more, the chemical action of the cancer treatment drug in the body is entirely the same as the already approved cancer treatment drug and the toxicity does not change.

[0117] A method for killing a cancer cell in vitro according to the embodiment shows the therapeutic effect of the nitrogen-15-containing cancer treatment drug. Since the method for killing a cancer cell in vitro according to the embodiment makes it possible to irradiate the cancer cell with the proton beam while controlling the irradiation energy of the proton beam to the resonance energy at which the .sup.15N resonance nuclear reaction occurs, it is possible to accurately evaluate the therapeutic effects of the nitrogen-15-containing cancer treatment drug.

[0118] This method for killing a cancer cell in vitro includes sealing a cancer cell and optional normal cell, along with the culture solution, in a vacuum using a polymer film. By doing this, the cells in the vacuum chamber through which the proton beam passes can be maintained in a living state.

[0119] The polymer film that seals the cancer cell and optional normal cell along with the culture solution in a vacuum in the method for killing a cancer cell in vitro, may be a polyethylene sulfide material, and a film commercially available as Torelina (Toray Industries, Inc.) may be used. As shown in FIG. 9, polyethylene sulfide film is very stable to thermal hydrolysis (refer to Non-Patent Document 5). In addition, a property of Torelina, as shown in FIG. 10, is a high permeability for oxygen, nitrogen, and carbon dioxide, but a very low permeability for water vapor (refer to Non-Patent Document 5).

[0120] Even in an environment of exposure to a high-density excitation action due to the proton beam while in contact with an aqueous solution for the culture, a stable sealing of the cancer cells and optional normal cells along with the culture solution in a vacuum is made possible utilizing the extremely stable properties of Torelina film with respect to thermal hydrolysis. Due to the property of Torelina film of a very low water vapor permeability and due to the very high hydrophobicity of Torelina fiber at the interface where the aqueous solution and Torelina film are in contact, even when the vacuum side surface of the film assumes a negative pressure, it is difficult for the water molecules to become water vapor and the culture solution can be present in a liquid state. In the interval in which the culture solution maintains the liquid state, the highly permeable oxygen and carbon dioxide are maintained in a dissolved state in the liquid and an environment is obtained in which the oxygen and carbon dioxide required to maintain cells in a living state are maintained in the culture solution.

[0121] In order to confine the cancer cells and optional normal cells along with the culture solution in a vacuum, a device may be used that has the capability of sealing with a polymer film such as Torelina film. This sealing-capable device may be configured such that the vacuum required for the irradiation of a high energy proton beam is obtained, desirably a vacuum of 110.sup.3 Pa or below. This sealing-capable device may have CLEANSTAR B (product name, Daido Steel Co., Ltd.), a soft stainless steel with an ultralow carbon content (equal to or less than 0.007%), for a material. This sealing-capable device may have a sealing surface having a semicircular cross section. The configuration may be such that a high hardness layer of iron nitride is formed in a surface layer thickness of 100 nm by N.sub.2 ion implantation at this sealing surface, to have close contact by elastic deformation of the sealing surface upon tightening and have an easy separation behavior when opening (refer to Patent Documents 2 and 3).

[0122] The nitrogen-15-containing anticancer agent is administered to the cancer cells, and optionally to the normal cells, prior to the introduction of the cancer cells and optional normal cells into the vacuum chamber. Sealing with the polymer film, e.g., Torelina film, at the sealing surface is carried out and the cancer cells and optional normal cells are maintained along with the culture solution in a live state in the vacuum chamber. As a consequence of this, the cancer cells and optional normal cells, which have taken up .sup.15N into the cell along with the anticancer agent, are maintained in the vacuum chamber. Accordingly, the cancer cells can be killed in vitro by producing the resonance nuclear reaction through the collision of protons with the .sup.15N within the cancer cells and optional normal cells.

[0123] The cancer treatment device according to the embodiment comprises, as shown in FIG. 11, an irradiator 20 that irradiates a proton beam onto a human 10 or a nonhuman animal in whom nitrogen-15 has accumulated in cancer cells. The above-described anticancer agent or anticancer agent prodrug has been administered to the human 10 or nonhuman animal. The cancer treatment device according to the embodiment may additionally comprise an accelerator that accelerates the proton beam. The accelerator may comprise a laser plasma.

Example 1

[0124] Rat gastric mucosal-derived cells (RGM-GFP) were prepared as normal cells and rat gastric mucosal-derived cancer cells (RGK-KO) were prepared as cancer cells, and the difference in cellular intake of .sup.15N_5-ALA between the RGM-GFP cells and RGK-KO cells was confirmed as follows. The rat gastric mucosal-derived cells and rat gastric mucosal-derived cancer cells are both derived from the rat and are widely used in research on anticancer agents because they have the same gene sequence (refer to Non-Patent Documents 6 and 7).

[0125] The RGM-GFP cells and RGK-KO cells were cultured for 3 or 4 days on a culture solution adapted to each and were grown to 80% confluence; this was followed by the addition of .sup.15N_5-ALA and the amount of .sup.15N intake per cell with elapsed time was measured. A schematic diagram of measurement of the amount of .sup.15N intake is given in FIG. 12. .sup.15N_5-ALA was added to the RGM-GFP cells and RGK-KO cells that had grown to 80% confluence, and cell samples were prepared after the passage of each of 0 hours, 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after the addition. Washing with fresh culture solution was performed three times in order to remove the .sup.15N_5-ALA that remained on the cell surface. The cells, which had grown with attachment to the bottom of the Petri dish, were detached from the Petri dish by the addition of trypsin, and the concentration of the liberated cells was measured.

[0126] The concentration was adjusted, and 10.sup.5 cells/2 L was dripped onto a crystalline silicon substrate and drying was carried out. The sample size was adjusted to a diameter of approximately 2.5 mm. The cell sample, set into a sample holder, was inserted into a vacuum chamber and was exposed to a proton beam. Considering the thickness of the sample and considering that the energy of the proton beam attenuates in the interior of the sample, the energy of the proton beam was set at 22 points between 0.894 MeV and 0.994 MeV so the .sup.15N in the sample interior could be quantitated by the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction (E.sub.r=0.897 MeV). The 4.43 MeV gamma radiation emitted by this resonance nuclear reaction with .sup.15N was measured using a Bi.sub.4Ge.sub.3O.sub.12 (BGO) detector.

[0127] The measured gamma-ray spectra are given in FIG. 13. Since the 4.43 MeV main peak and the two annihilation gamma ray peaks (3.92 MeV S.E. and 3.41 MeV D.E.) formed a broad peak, the gamma ray yield in the 2.98 MeV to 4.85 MeV energy range was integrated and the natural background (white dots in the figure) was subtracted to provide the effective gamma-ray yield. The cellular intake of .sup.15N_5-ALA was determined as the absolute value based on the gamma ray yield from samples provided by dripping an aqueous solution of .sup.15N_5-ALA of stipulated concentration. The results are given in FIG. 14. At 24 hours after the addition of the .sup.15N_5-ALA, the intake of .sup.15N_5-ALA by cancer cells was at least 5-times the intake of .sup.15N_5-ALA by the normal cells.

[0128] On the basis of these results, it was shown that the nitrogen-15 taken in as .sup.15N_5-ALA by the cancer cells accumulated within the cells and the cancer cells could be specifically killed.

[0129] The specific procedure for quantitating the .sup.15N_5-ALA intake by the RGM-GFP cells and RGK-KO cells was as follows.

[0130] RGM-GFP and RGK-KO were cultured in 35-mm dishes in an incubator (37 C., 5% CO.sub.2) over 3 to 4 days until 80% confluence.

[0131] 1000 L of a culture medium solution containing 1 mmol/L .sup.15N_5-ALA was introduced into each dish. Treatment with trypsin was performed on each of the cells at each of the stipulated elapsed times.

[0132] Washing of the cells with phosphate-buffered saline (PBS) or 5% glucose solution and centrifugal separation was carried out three times.

[0133] The cell count was measured using an automatic counter (Invitrogen Countess (registered trademark)) and was adjusted to 1.010.sup.5 cell/2 L. The cells were dripped onto a silicon substrate and after drying the silicon substrate was then set in a vacuum chamber. The cells on the silicon substrate were irradiated with a proton beam from a 1 MV tandem accelerator (Applied Accelerator Department, Tsukuba University), and the 4.43 MeV gamma rays emitted by the resonance nuclear reaction with .sup.15N were measured using a BGO detector located outside of the vacuum chamber.

[0134] The proton beam was irradiated onto a sapphire plate and the beam area was measured from the fluorescence image, and the gamma ray yield normalized to the unit proton beam charge amount was determined from the relative ratio with the area of the sample dripped onto the silicon substrate.

[0135] The intracellular nitrogen-15 was quantitated by normalizing the gamma ray yield from the aqueous .sup.15N_5-ALA solution sample of stipulated concentration to the proton beam charge amount and comparing the proton beam charge amount-normalized gamma ray yield.

[0136] The following reagents were used in Example 1. [0137] 5-aminolevulinic acid hydrochloride: 018-13133 (FUJIFILM Wako Pure Chemical Corporation) [0138] .sup.15N-aminolevulinic acid hydrochloride: N1371 (Medical Isotope) [0139] 0.5% Trypsin-EDTA (10): 15400054 [0140] TrypLE Select Enzyme (1), no phenol red 100 mL: 12563011 (Gibco) [0141] 10D-PBS: 048-29805 (FUJIFILM Wako Pure Chemical Corporation) [0142] D (+)-glucose: 049-31165 (FUJIFILM Wako Pure Chemical Corporation)

Example 2

[0143] FIG. 15 shows the gene structure of pUC4-KIXX (3914 bps), which is circular artificial plasmid DNA. Because pUC4-KIXX has an ampicillin-resistance gene and a kanamycin-resistance gene, E. coli transformed with pUC4-KIXX exhibits resistance to ampicillin and kanamycin. Modified pUC4-KIXX's were prepared by changing the isotopic ratio .sup.15N/(.sup.14N+.sup.15N) for the N in pUC4-KIXX in steps to the natural abundance ratio of 0.364% and to 25%, 50%, 75%, and at least 98%.

[0144] E. coli transformed with each of the modified pUC4-KIXX's was cultured on culture medium containing 100 g/mL ampicillin and culture medium containing 20 g/mL kanamycin, and was subjected to proton beam irradiation. According to the results, and as shown in FIG. 16 and FIG. 17, in comparison to E. coli transformed with pUC4-KIXX not containing .sup.15N, E. coli transformed with .sup.15N-containing pUC4-KIXX presented an increase in the number of cells killed by proton beam irradiation as the .sup.15N isotopic ratio increased. In comparison to E. coli transformed with pUC4-KIXX not containing .sup.15N, the colony count for E. coli transformed with pUC4-KIXX having a .sup.15N isotope abundance ratio of 75% or more was decreased by at least 1 over 10.sup.3.

[0145] Proton beam irradiation did not kill large numbers of the E. coli transformed with pUC4-KIXX not containing .sup.15N. It is thus thought that the killing of large numbers of E. coli transformed with a modified pUC4-KIXX was due to a loss of drug resistance by the E. coli due to damage to the ampicillin-resistance gene and kanamycin-resistance gene due to the .sup.15N(.sup.1H, .sub.1).sup.12C resonance nuclear reaction.

Example 3

[0146] Rat gastric mucosal-derived cancer cells (RGK-KO) and normal cells (RGM-GFP) were uniformly cultured for seven days in 35-mm dishes filled with culture medium adapted to each, and the cells were prepared so all the dishes reached 80% confluence. .sup.15N_5-ALA was introduced into the culture medium at 24 hours before proton beam irradiation, and holding was carried out for 24 hours using the same culture conditions for both culture media in order to enable comparison with the culture medium that did not contain .sup.15N_5-ALA. The RGK-KO cells and RGM-GFP cells cultured on a culture medium containing .sup.15N_5-ALA and the RGK-KO cells and RGM-GFP cells cultured on a culture medium not containing .sup.15N_5-ALA were in each instance held sealed in a vacuum along with the culture medium and were irradiated with a proton beam at a prescribed ion charge amount. With regard to the proton beam energy, considering the attenuation of the proton beam energy by the Torelina film and the culture solution sealed in the vacuum, the sample was irradiated at 1.3 MeV to match the 1.21 MeV resonance energy.

[0147] FIG. 18 shows fluorescence microscope photographs taken immediately after (within 3 hours) the proton beam irradiation of RGM-GFP cells cultured on culture medium that did not contain .sup.15N_5-ALA. The upper sections show images of RGM-GFP cells made to metabolize a fluorescent protein by genetic recombination; the lower sections each show photographs in which the dead cells are identified by the DAPI staining method, which stains the DNA in the nucleus of dead cells. The cell mortality, i.e., the number of fluorescent protein-imaged cells (number of surviving cells) versus the sum of the number of fluorescent protein-imaged cells and the number of DAPI staining fluorescent-imaged cells (number of dead cells), increased accompanying the increase in the irradiation charge amount of the proton beam to 2.5 nC, 4.0 nC, 5.0 nC, 10 nC, and 25 nC. FIG. 19 shows the relationship between the irradiated charge amount and the cell surviving fraction for the proton beam irradiation of RGM-GFP cells cultured on culture medium that did not contain .sup.15N_5-ALA. A logarithmic relationship is seen between the two, and the characteristics of the biological radiation damage due to a proton beam were demonstrated.

[0148] FIG. 20 shows photographs for immediately after the proton beam irradiation of RGK-KO cells cultured on culture medium that did not contain .sup.15N_5-ALA and culture medium that contained .sup.15N_5-ALA. The photographs in FIG. 20 show the case of a proton beam irradiation amount of charge of 4.0 nC for both cells. Genes for the metabolism of fluorescent protein are also incorporated in the RGK-KO cells, and the cells present a red color when observed with a fluorescence microscope. For the RGK-KO cells cultured on culture medium that did not contain .sup.15N_5-ALA, the number of cells in the fluorescent protein image was 4671 and the number of cells in the DAPI staining fluorescent image was 3179, and the surviving fraction was 59.5%. On the other hand, for the RGK-KO cells cultured on culture medium that contained .sup.15N_5-ALA, the number of cells in the fluorescent protein image was 4522 and the number of cells in the DAPI staining fluorescent image was 1622, and the surviving fraction was 73.5%.

[0149] FIG. 21 gives photographs of the cells after the passage of 24 hours after proton beam irradiation, at a charge amount of 4.0 nC, for RGK-KO cells that had been cultured on culture medium that contained .sup.15N_5-ALA. For the cells that exhibited a surviving fraction within three hours immediately after irradiation of 73.5% and 85.5%, the surviving fraction after 24 hours had declined to 40.5% and 10.6%, respectively. The trend of the RGK-KO cell surviving fraction declining sharply with elapsed time shows that the .sup.15N_5-ALA taken up by the RGK-KO cells is connected to the metabolic pathway in the cancer cells that leads to protoporphyrin IX.

Example 4

[0150] In a method for killing cancer cells in vitro, the RGM-GFP cells and RGK-KO cells were sealed using a vacuum sealing joint (referred to as a CS flange in the following) having a scaling capability, for which the materials were Torelina film (thickness of 4 m, Toray Industries, Inc.) and CLEANSTAR; and this was set in a vacuum chamber and held in a vacuum. FIG. 22 shows a photograph in which a crystalline silicon substrate with a diameter of 15 mm and a thickness of 0.5 mm has been placed in a 1.0 mm-deep circular dish made of high-purity silicon; the RGM-GFP cells and RGK-KO cells with culture solution have been dripped onto this substrate; and sealing with Torelina film has been carried out. It was possible to avoid discharge damage due to charging from the proton beam by carrying out the vapor deposition (Toray KP Films Inc.) of a thin carbon film on the surface on one side of the Torelina film.

[0151] When RGM-GFP cells and RGK-KO cells that had been kept in a vacuum for 20 to 30 minutes were released from the seal and cultured, regrowth of the cells was confirmed for both cell types, as shown in the cell photograph in FIG. 22, and maintenance in a living condition in a vacuum for a certain period of time was demonstrated.

[0152] According to FIG. 23, when RGM-GFP cells and RGK-KO cells were sealed together with the culture solution and vacuum evacuation was performed using a dry pump (TSU071E, exhaust speed=60 L/s, achieved vacuum=10.sup.5 Pa, PFEIFFER), it was confirmed that the vacuum dropped at a rate that was unchanged from the vacuum exhaust process when a cell sample was not present.