JP6994700B2 - Radiation therapy sensitizer - Google Patents

Description

The present invention is a radiotherapy that selectively damages or kills malignant neoplasms such as cancer cells; cells in lesions such as cancer precursor cells, virus-infected cells, and bacterial-infected cells; by irradiation. Regarding the sensitizer for radiotherapy used in.

In conventional radiotherapy, when high-energy radiation such as X-rays and gamma rays is intensively applied to a tumor, intracellular water is decomposed into hydroxy radicals and hydrogen atoms, which react with intracellular oxygen. Therefore, superoxide and hydroperoxy radicals are generated. These active oxygen and free radicals damage the DNA in the cell, and further extract hydrogen from the lipid of the cell membrane and transform it into the lipid peroxide by the chain of radical peroxides, causing cell membrane damage. However, since radiation cannot be selectively applied only to target cells, DNA damage and cell membrane damage have progressed even in normal cells around the affected area.

Therefore, we focused on the fact that 5-aminolevulinic acid (5-ALA) is selectively taken up by tumors to generate protoporphyrin IX (PpIX), and then active oxygen and free radicals are generated from PpIX by X-ray irradiation. An X-ray therapeutic sensitizer containing 5-ALA as an active ingredient has been investigated (see, for example, Patent Document 1). When 5-aminolevulinic acids (5-ALA) are administered to subjects, the physiological phenomenon in which PpIX is specifically accumulated in lesions such as tumors is applied to other therapeutic methods, and in lesions where PpIX is accumulated. A photodynamic treatment method in which active oxygen is generated from PpIX by irradiating a laser beam having a wavelength of 400 to 700 nm to selectively injure and destroy the lesion tissue has been investigated (see, for example, Patent Document 2).

Ingestion of 5-ALA is known to bring about other physiological effects. It was found that ingestion of 5-ALA and iron compounds could alleviate hematopoietic disorders and weight loss in radiation-exposed organisms, and preventive and / or therapeutic agents for radiation disorders containing 5-ALA and iron compounds were investigated. (See, for example, Patent Document 3). Furthermore, it was found that ingestion of 5-ALA and iron compounds prevents electron leakage from the electron transport chain of mitochondria and suppresses the generation of active oxygen, and contains 5-ALA and iron compounds. A preventive / ameliorating composition for mitochondria was investigated (see, for example, Patent Document 4).

By the way, it is known that administration of 5-ALA and an iron compound suppresses the accumulation of PpIX in normal cells and specifically accumulates PpIX in cancer cells (see, for example, Non-Patent Document 1).

Japanese Patent No. 5182858 Japanese Unexamined Patent Publication No. 2017-79940 Japanese Patent No. 5920902 Japanese Unexamined Patent Publication No. 2011-16753

PLOS ONE, Hayashi M. et al. DOI: 10.1371 / journal.pone.0122351 March 30, 2015

In X-ray therapy, a small dose of X-rays is often repeatedly applied to the subject. When the X-ray therapeutic sensitizer shown in Patent Document 1 was repeatedly administered to a subject for X-ray treatment, there was concern about side effects due to the accumulation of PpIX in normal cells. Therefore, there has been a demand for a radiotherapy sensitizer capable of suppressing the accumulation of PpIX in normal cells even when repeatedly administered to a subject, but such a radiotherapy sensitizer has not been provided. That is, an object of the present invention is to provide a radiotherapy sensitizer capable of suppressing the accumulation of PpIX in normal cells even when repeatedly administered to a subject.

According to the findings shown in Non-Patent Document 1, the amount of PpIX accumulated in cancer cells when 5-ALA and an iron compound are administered is smaller than the amount of PpIX accumulated in cancer cells when only 5-ALA is administered. Therefore, the X-ray sensitizing effect when 5-ALA and the iron compound are administered is lower than the X-ray sensitizing effect when only 5-ALA is ingested, and PpIX and iron are administered by the administration of 5-ALA and the iron compound. Is expected to be specifically accumulated in cancer cells, while the accumulation of PpIX in normal cells is suppressed. The present inventors have stated that administration of 5-ALA and an iron compound suppresses the accumulation of PpIX in normal cells, and that when the accumulated PpIX and iron are irradiated with radiation, active oxygen and free radicals are generated. I found it and came to complete the present invention.

（式中、Ｒ^１は、水素原子又はアシル基を表し、Ｒ^２は、水素原子、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す）
［２］Ｒ^１及びＲ^２が、水素原子であることを特徴とする上記［１］に記載の放射線治療用増感剤。
［３］鉄化合物が、塩化第二鉄、三二酸化鉄、硫酸鉄、ピロリン酸第一鉄、クエン酸第一鉄、クエン酸鉄ナトリウム、クエン酸第一鉄ナトリウム、クエン酸鉄アンモニウム、ピロリン酸第二鉄、乳酸鉄、グルコン酸第一鉄、ジエチレントリアミン五酢酸鉄ナトリウム、ジエチレントリアミン五酢酸鉄アンモニウム、エチレンジアミン四酢酸鉄ナトリウム、エチレンジアミン四酢酸鉄アンモニウム、ジカルボキシメチルグルタミン酸鉄ナトリウム、ジカルボキシメチルグルタミン酸鉄アンモニウム、フマル酸第一鉄、酢酸鉄、シュウ酸鉄、コハク酸第一鉄、コハク酸クエン酸鉄ナトリウム、ヘム鉄、デキストラン鉄、トリエチレンテトラアミン鉄、ラクトフェリン鉄、トランスフェリン鉄、鉄クロロフィリンナトリウム、フェリチン鉄、含糖酸化鉄、及び硫化グリシン鉄から選ばれる１種又は２種以上の化合物であることを特徴とする上記［１］又は［２］に記載の放射線治療用増感剤。
［４］鉄化合物が、クエン酸第一鉄であることを特徴とする上記［１］～［３］のいずれかに記載の放射線治療用増感剤。 That is, the present invention is as follows.
[1] A compound represented by the following formula (I) or a salt thereof and an iron compound are contained.
A radiotherapy sensitizer that is administered to a subject to be treated before irradiation and is used for the subject's radiation therapy.

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group).
[2] The sensitizer for radiotherapy according to the above [1], wherein R ¹ and R ² are hydrogen atoms.
[3] The iron compounds are ferric chloride, iron sesquioxide, iron sulfate, ferrous pyrophosphate, ferrous citrate, sodium iron citrate, sodium ferrous citrate, ammonium iron citrate, and pyrophosphate. Iron ferric, iron lactate, ferrous gluconate, sodium diethylenetriamine pentaacetate, ammonium diethylenetriamine pentaacetate, sodium ethylenediamine tetraacetate, ammonium ethylenediamine tetraacetate, sodium dicarboxymethylglutamate, ammonium iron dicarboxymethylglutamate , Ferrous fumarate, iron acetate, iron oxalate, ferrous succinate, sodium iron citrate succinate, hem iron, dextran iron, triethylenetetraamine iron, lactoferrin iron, transferase iron, iron chlorophyllin sodium, ferritin The sensitizer for radiotherapy according to the above [1] or [2], which is one or more compounds selected from iron, sugar-containing iron oxide, and iron sulfide.
[4] The sensitizer for radiotherapy according to any one of the above [1] to [3], wherein the iron compound is ferrous citrate.

Further, another aspect of the present invention is as follows.
[5] A radiotherapy method comprising administering a radiotherapy sensitizer according to any one of the above [1] to [4] to a subject and then irradiating the subject in need of radiotherapy with radiation. ..
[6] a) The compound represented by the above formula (I) or a salt thereof;
b) Iron compounds;
A radiation therapy method comprising irradiating a subject in need of radiation therapy with radiation after administering to the subject at the same time or before or after.
[7] Administered to subjects in need of radiation therapy prior to irradiation,
a) The compound represented by the above formula (I) or a salt thereof;
b) Iron compounds;
A radiation therapy kit for use in radiation therapy, including.
[8] A compound represented by the above formula (I) or a salt thereof and an iron compound for use as a sensitizer for radiotherapy, which is administered to a subject in need of radiotherapy before irradiation.
[9] Use of the compound represented by the above formula (I) or a salt thereof and an iron compound for preparing a radiotherapy sensitizer to be administered to a subject in need of radiotherapy before irradiation.

The sensitizer for radiotherapy of the present invention exhibits a radiosensitizing effect equal to or higher than that of the sensitizer for X-ray therapy containing only 5-ALA as an active ingredient. Furthermore, even if the sensitizer for radiotherapy of the present invention is repeatedly administered to a subject, the accumulation of PpIX in normal cells can be suppressed.

It is a figure which shows the PpIX concentration which accumulates in a mouse melanoma cell when 5-ALA which has a different mixed molar ratio is used in combination with an iron agent. It is a figure which shows the PpIX concentration accumulated in the human alveolar basal epithelial adenocarcinoma cell when 5-ALA having a different mixed molar ratio is used in combination with an iron preparation. It is a figure which shows the iron ion concentration accumulated in the human alveolar basal epithelial adenocarcinoma cell when 5-ALA alone and 5-ALA and iron are used in combination. The iron ion concentration accumulated in each cell of normal tissue (brain; FIG. 4A) and cancer tissue (melanoma; FIG. 4B) when 5-ALA alone or 5-ALA in combination with iron is administered in vivo. It is a figure which shows. Comparison results of fluorescence intensity of PpIX accumulated in the whole body of mice when 5-ALA alone or in combination with 5-ALA and iron in vivo (Fig. 5-A), results of dynamic observation (Fig. 5-A) B) is shown. It is a figure which shows the relationship between the intensity of X-rays irradiating various iron compounds, and the amount of active oxygen species produced. The results of ferrous citrate are shown in FIG. 6A, the results of iron (II) sulfate are shown in FIG. 6B, and the results of iron (II) acetate are shown in FIG. 6C. It is a figure which shows the verification result of the antitumor effect in vivo when 5-ALA alone and 5-ALA and iron are administered in combination.

The radiotherapy sensitizer of the present invention contains the compound represented by the above formula (I). 5-ALA, in which both R ¹ and R ² of the above formula (I) are hydrogen atoms, is one of the amino acids also called δ-aminolevulinic acid. In the 5-ALAs contained in the sensitizer for radiotherapy of the present invention, R ¹ of the above formula (I) is a hydrogen atom or an acyl group, and R ² of the above formula (I) is a hydrogen atom, a linear or linear group. It is a branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group.

Specific examples of the acyl group in the above formula (I) include linear or branched carbon atoms 1 to 8 such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl and benzylcarbonyl groups. Alkanoyl group; an aloyl group having 7 to 14 carbon atoms such as a benzoyl, 1-naphthoyl, 2-naphthoyl group and the like.

Specific examples of the alkyl group in the above formula (I) include linear or linear groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl groups. It is a branched alkyl group having 1 to 8 carbon atoms.

Specific examples of the cycloalkyl group in the above formula (I) include saturated or partially unsaturated bonds such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, and 1-cyclohexenyl group. It is also a good cycloalkyl group having 3 to 8 carbon atoms.

Specific examples of the aryl group in the above formula (I) are aryl groups having 6 to 14 carbon atoms such as phenyl, naphthyl, anthryl, and phenanthryl groups.

The aryl moiety of the aralkyl group in the above formula (I) is exemplified in the same manner as the above aryl group, and the alkyl moiety of the above aralkyl group is exemplified in the same manner as the above alkyl group. Specific examples of the above-mentioned aralkyl group are aralkyl groups having 7 to 15 carbon atoms such as benzyl, phenethyl, phenylpropyl, phenylbutyl, benzhydryl, trityl, naphthylmethyl and naphthylethyl groups.

The above-mentioned 5-ALAs may exert the effect of the present invention in the state of 5-ALA represented by the above formula (I) or a derivative thereof in vivo, and in order to increase the solubility depending on the form to be administered. It is administered as a prodrug (precursor) such as various 5-ALA salts of 5 or 5-ALA ester derivatives that are degraded by enzymes in vivo. Specific examples of salts of 5-ALA and its derivatives are pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts and the like. Specific examples of the acid addition salt are inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate and sulfate; formate, acetate, propionate and toluenesulfone. Acid, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. It is an organic acid addition salt. Specific examples of the metal salt are alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as magnesium and calcium salt; aluminum salt; zinc salt and the like. Specific examples of the above-mentioned ammonium salt are alkylammonium salts such as ammonium salt and tetramethylammonium salt. Specific examples of the organic amine salt are triethylamine salt, piperidine salt, morpholine salt, toluidine salt and the like. In addition, these salts may be used as a solution at the time of use.

Among the above 5-ALA compounds, particularly preferable compounds are 5-ALA; 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester and the like. ALA ester derivatives; as well as these hydrochlorides, phosphates, sulfates. 5-ALA, 5-ALA hydrochloride and 5-ALA phosphate are most preferred.

The above-mentioned 5-ALAs may be produced by any known method of chemical synthesis, production by microorganisms, and production by enzymes. The above-mentioned 5-ALAs may form a hydrate or a solvate. Any one of the above 5-ALAs can be used alone or in combination of two or more as appropriate.

The radiotherapy sensitizer of the present invention contains an iron compound. The iron compound may be an organic salt or an inorganic salt. Specific examples of the above-mentioned inorganic salt are ferric chloride, iron sesquioxide, iron sulfate, ferrous pyrophosphate and the like. Specific examples of the organic salt include carboxylates such as hydroxycarboxylate, ferrous citrate, sodium iron citrate, sodium ferric citrate, ammonium ferric citrate, ferric pyrophosphate, iron lactate, and the like. Ferric gluconate, diethylenetriamine ferric acetate sodium, diethylenetriamine pentairon ammonium acetate, ethylenediamine sodium ferrictetraacetate, ethylenediamine iron ammonium tetraacetate, sodium dicarboxymethylglutamate, ammonium iron dicarboxymethylglutamate, ferrous fumarate, Iron acetate, iron oxalate, ferric succinate, sodium ferric citrate, hem iron, dextran iron, triethylenetetraamine iron, lactoferrin iron, transferrin iron, iron chlorophyllin sodium, ferritin iron, sugar-containing iron oxide, Glycin sulfide iron and the like. One or more of the above iron compounds can be used.

The dose of the iron compound may be 0.00001 to 100 times, preferably 0.00003 to 10 times, more preferably 0, in terms of molar ratio with respect to the dose of 5-ALA. It is 0.0005 times to 2 times.

The 5-ALA and the iron compound contained in the radiotherapy sensitizer of the present invention can be administered to the subject as a composition containing the 5-ALA and the iron compound, or individually. When 5-ALA and iron compounds are administered independently, they may be administered to the subject at the same time, or these may be administered to the subject at different times, and any of these may be used as a sustained release preparation. It may be administered to the subject. When the peak of PpIX concentration and the peak of iron concentration accumulated in the cells in the lesion overlap, the therapeutic effect of radiation therapy is enhanced. For example, administration of 5-ALA to a subject causes the peak time of PpIX concentration in cancer cells to be 6 hours after administration of 5-ALA, and administration of iron compound to a subject causes the peak time of iron concentration in blood to be iron compound administration. If the time of administration of the iron compound to the subject is delayed by about 2 hours from the time of administration of the iron compound to the subject, the radiation sensitizing effect can be further enhanced, or the dose of these compounds can be increased. Can be reduced.

The administration route of the sensitizer for radiotherapy of the present invention is oral administration including sublingual administration; direct kidney administration by catheter; inhalation administration; intravenous administration including infusion; transdermal administration by propagating agent or the like; suppository, nasal administration. Parenteral administration by forced enteral nutrition using a gastrointestinal tract, nasal intestinal tract, gastric fistula tube, or intestinal fistula tube.

The dosage form of the sensitizer for radiotherapy of the present invention can be appropriately determined according to the above-mentioned route administration. Specific examples of the above dosage forms are injections, drip infusions, tablets, pills, capsules, fine granules, scattered, liquids, liquids dissolved in syrups, spreading agents, suppositories and the like.

In order to prepare the sensitizer for radiotherapy of the present invention, if necessary, pharmacologically acceptable carriers, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, etc. Lubricants, lubricants, flavors, sweeteners, solubilizers, solvents, gelling agents, nutrients and the like can be added. Specific examples of these include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin. And so on. When preparing the sensitizer for radiotherapy of the present invention as an aqueous solution, it is desirable to be careful not to make the aqueous solution alkaline in order to prevent the decomposition of 5-ALA, and when the aqueous solution becomes alkaline, oxygen is used. Removal can prevent the decomposition of 5-ALAs.

After administration of the sensitizer for radiotherapy of the present invention to a subject, radiotherapy is usually started within 24 hours, preferably within 18 hours, and more preferably within 3 to 12 hours. The dose of the sensitizer for radiotherapy of the present invention can be changed according to the caution, weight, age, symptoms and the like of the patient to be treated with radiotherapy. The dose of 5-ALA contained in the radiotherapy sensitizer of the present invention is per 1 kg of the subject in terms of 5-ALA (that is, R ¹ and R ² are hydrogen atoms in the formula (I)). It can be adjusted in the range of 1 mg to 1,500 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg, still more preferably 15 mg to 25 mg.

After administration of the sensitizer for radiotherapy of the present invention to a subject, the radiation radiated to the subject is ionizing radiation such as γ-ray, X-ray, electron beam, α-ray, and neutron beam.

The present invention will be described in detail below with reference to Experimental Examples and Examples, but the present invention is not limited to these Experimental Examples and Examples.

[Experimental Example 1]
Mouse melanoma B16 / BL6 cells cultured in RPMI1640 medium containing 10% FBS under a carbon dioxide atmosphere (adjusted to 5% by volume of carbon dioxide concentration in the air) were seeded on a 96-well microplate to make a confluent. It was cultured until it became. 100 μg / mL 5-ALA so that the molar ratio of 5-ALA to iron is 1: 0.00005, 1: 0.0005, 1: 0.005, 1: 0.05, 1: 0.5. Add ferrous citrate to the culture medium added to the plate in which mouse melanoma B16 / BL6 cells are cultured and incubate for 3 hours. Using a microplate reader, light at 405 nm, which is the excitation wavelength of PpIX. Was irradiated to the cells, and the fluorescence at 636 nm emitted from the cells was observed to measure the PpIX concentration. The results are shown in FIG. The value on the vertical axis (ratio) is shown as a ratio to the case where no iron agent is added (n = 4). As a result, it was found that the concentration of PpIX accumulated in mouse melanoma B16 / BL6 cells decreased as the molar ratio of iron to 5-ALA in the medium increased.

[Experimental Example 2]
Human alveolar basal epithelial adenocarcinoma A549 cells cultured in RPMI1640 medium containing 10% FBS under a carbon dioxide atmosphere (adjusted to 5% by volume of carbon dioxide concentration in the air) were inoculated into 96-well microplates and confined. Incubated until fluent. 100 μg / mL 5-ALA so that the molar ratio of 5-ALA to iron is 1: 0.00005, 1: 0.0005, 1: 0.005, 1: 0.05, 1: 0.5. The culture medium to which ferrous citrate was added was added to a plate in which human alveolar basal epithelial adenocarcinoma A549 cells were cultured and cultured for 3 hours, and the excitation wavelength of PpIX was used using a microplate reader. The cells were irradiated with light of 405 nm, and the fluorescence of 636 nm emitted from the cells was observed to measure the PpIX concentration. The results are shown in FIG. The value (ratio) was shown as a ratio to the case where no iron agent was added (n = 4). As a result, it was found that the concentration of PpIX accumulated in human alveolar basal epithelial adenocarcinoma cells decreased as the molar ratio of iron to 5-ALA in the medium increased.

[Experimental Example 3]
Human alveolar basal epithelial adenocarcinoma A549 cells were seeded in a flask in an area of 25 cm ² and conquered in RPMI 1640 medium containing 10% FBS under a carbon dioxide atmosphere (adjusted to 5% by volume of carbon dioxide in the air). It was cultured until it became a fluent. The culture solution in the above flask was prepared so that 5-ALA was 100 μg / mL and ferrous citrate was 0.30 mM (5-ALA to iron molar ratio = 1: 0.5). After culturing for 2 hours, the cells were replaced with a normal culture medium containing 5-ALA and ferrous citrate. Two hours later and the next morning, the cells were replaced with normal culture medium, and 5-ALA, ferrous citrate and PpIX were completely removed from the cells and the medium. Two hours after the medium was changed the next morning, the cells were replaced with a culture solution containing 5-ALA and ferrous citrate, and the cells were cultured for 2 hours. The cells were cultured for 2 days using this cell culture method. After completion of the second day of culture, the cultured cells were rinsed with phosphate buffered saline (PBS). Then, 10% trypsin-EDTA was added, and the cells were allowed to stand at 37 ° C. for 5 minutes, and the cultured cells were peeled from the flask and collected. 500 μL of PBS was added to the collected cells, and 5 μL of 6M-HCl was further added to prepare a sample. Then, the intracellular iron ion concentration of the sample was measured by the ferrosin method using a metalloassay iron measurement kit (Metallogenics). Further, the same as above, using a culture solution prepared to have 5-ALA at 100 μg / mL instead of the culture solution prepared to contain 5-ALA and ferrous citrate at the above concentrations. Was cultured, and the intracellular iron ion concentration was measured. The intracellular iron ion concentration when human alveolar basal epithelial adenocarcinoma A549 cells were cultured in a normal culture medium for 2 days was also measured. The results are shown in FIG.
In the ferrosin method, iron bound to a transport protein such as transferrin is dissociated with a weak acid and a modifier to form an iron-ferrodine complex, and this complex is detected by light having a wavelength of 560 nm to measure the iron concentration. is doing.

The amount of iron accumulated in human alveolar basal epithelial adenocarcinoma cells cultured in a medium containing 5-ALA and ferrous citrate was cultured in a medium containing 5-ALA and not containing ferrous citrate. The amount of iron accumulated in human alveolar basal epithelial adenocarcinoma cells and the amount of iron accumulated in human alveolar basal epithelial adenocarcinoma cells cultured in a medium free of 5-ALA and ferrous citrate. Turned out to be more than.

[Experimental Example 4]
1 × 10 ⁵ mouse melanoma B16 / BL6 cells were inoculated subcutaneously into C57BL / 6J mice (6 week old females), which are mice for inbred strains, to prepare cancer-bearing mice. A control group in which 5-ALA and PBS containing no iron compound are intraperitoneally administered to the prepared cancer-bearing mice for 7 consecutive days, and a group in which 5-ALA of 200 mg / kg / day is intraperitoneally administered for 7 consecutive days. (ALA), a group (ALA + Fe) in which 5-ALA of 200 mg / kg / day and ferrous citrate (5-ALA to iron molar ratio = 1: 0.5) are intraperitoneally administered for 7 consecutive days. The mice were divided into 3 groups, and each cancer-bearing mouse was euthanized 4 hours after intraperitoneal administration on the 7th day, perfused with PBS, and then tumor tissue (B16 / BL6 cells) and normal tissue (brain) were removed. 500 μL of PBS is added to 50 mg of the excised tissue, 5 μL of 6M-HCl is added, homogenized, and the supernatant obtained by centrifugation at 8000 rpm for 5 minutes is used as a sample by the ferrosin method. The iron concentration in the tissue was measured. The iron concentration accumulated in normal tissue is shown in FIG. 4A, and the iron concentration accumulated in tumor tissue is shown in FIG. 4B.

The iron concentrations accumulated in the normal tissues of the three groups were almost the same. On the other hand, the iron concentration accumulated in the tumor tissue increases in the order of the iron concentration accumulated in the ALA + Fe tumor tissue, the iron concentration accumulated in the ALA tumor tissue, and the iron concentration accumulated in the control tumor tissue. Was there.

[Experimental Example 5]
In BALB / c nu / nu mice (6 weeks old female), 200 mg / kg of 5-ALA and ferrous citrate were added in a molar ratio of 1: 0, 1: 0.05, 1: 0.1. Was administered intraperitoneally. The fluorescence of PpIX in the whole body of the mouse 0, 1, 2, 3, 4, 6 hours after the abdominal administration was dynamically observed with the IVIS Imaging System (Suisho Pharma International Co., Ltd., excitation wavelength 430 nm, fluorescence wavelength 640 nm). Mice not receiving 5-ALA were used as the control group. The amount of PpIX accumulated peaked 3 to 4 hours after intraperitoneal administration. The comparison result of the fluorescence intensity is shown in FIG. 5-A, and the result of the dynamic observation is shown in FIG. 5-B.

It was found that the higher the molar ratio of ferrous citrate to 5-ALA, the weaker the fluorescence and the less PpIX was accumulated.

[Experimental Example 6]
A microplate in which cells are not seeded with a solution of iron preparations (ferrous citrate, iron (II) sulfate, iron (II) acetate) dissolved in PBS to a concentration of 0 μM, 0.1 μM, 1 μM, and 10 μM. Was added to. Next, after adding a solution in which aminophenylfluorescein (APF), which is an active oxygen detection reagent, was added to PBS to a concentration of 5 μM, the prepared plate was irradiated with X-rays, and fluorescence of APF was performed using a microplate reader. (Excitation wavelength 480 nm, fluorescence wavelength 520 nm) was observed with a multi-detection mode microplate reader device (TECAN), and the amount of generated hydroxy radicals was measured. The results are shown in FIG.

It was found that, regardless of the type of iron compound, as the iron concentration and the irradiated X-ray dose increased, the fluorescence intensity became stronger and the amount of generated active oxygen (mainly hydroxyl radical) increased.

[Example 1]
^Four B16-luc cells (1 × 10), which are luciferase-expressing mouse melanoma cells, were inoculated into the brains of BALB / c nu / nu mice (6-week-old females) to prepare cancer-bearing mice. Two days after inoculation of B16-luc cells, 150 mg / kg of luciferin was administered to cancer-bearing mice, and the luciferase fluorescence emitted by B16-luc cells was quantified by the IVIS Imaging System (Sumisho Pharma International Co., Ltd.). A group that only irradiates X-rays so that the average is even, a group that irradiates X-rays 4 hours after administration of 200 mg / kg 5-ALA, 200 mg / kg 5-ALA and ferrous citrate. Four hours after the administration of (5-ALA to iron molar ratio = 1: 0.5), the group was divided into groups to be irradiated with X-rays. X-rays were irradiated using a Faxitron CP-160 type X-ray irradiator (Acrobio Co., Ltd.) at an intensity of 1 Gy / min for 2 minutes per day for 7 consecutive days. Two days, four days, six days, and nine days after the inoculation of B16-luc cells, 150 mg / kg of luciferin was administered to each group of cancer-bearing mice, and the above IVIS Imaging System was used to fluoresce the luciferase emitted by the B16-luc cells. It was measured. The measurement results of luminescence in cancer tissue are shown in FIG. 7-A, and the results of dynamic observation are shown in FIG. 7-B.

Tumors in the cancer-bearing mice in the X-ray-only group continued to grow for 9 days. Tumor division ability of cancer-bearing mice in the group irradiated with X-rays 4 hours after administration of 5-ALA and ferrous citrate was 5 days after inoculation of B16-luc cells and 4 from administration of 5-ALA. Tumor division ability of the cancer-bearing mice in the group irradiated with X-rays after hours was lost 7 days after inoculation with B16-luc cells.

The above results can be summarized as follows.
(1) The amount of PpIX accumulated in cancer cells by administration of 5-ALA and an iron compound is smaller than the amount of PpIX accumulated in cancer cells by administration of 5-ALA, but 5-ALA and iron. The amount of iron accumulated in cancer cells by administration of the compound is higher than the amount of iron accumulated in cancer cells by administration of 5-ALA.
(2) Irradiation generates active oxygen and free radicals from PpIX and iron accumulated in cells.
(3) PpIX and iron are selectively accumulated in lesion cells such as cancer cells rather than normal cells.
(4) The effect of radiation therapy in which 5-ALA and the iron compound are administered and then irradiated is equal to or higher than the effect of radiation therapy in which 5-ALA is administered and then irradiated.

The sensitizer for radiotherapy of the present invention selectively selects cells in lesions such as malignant neoplasms such as cancer cells, cancer precursor cells, virus-infected cells, and bacterial-infected cells by irradiation. Useful for radiation therapy that damages or kills.

Claims (3)

It contains a compound represented by the following formula (I) or a salt thereof and an iron compound.
A sensitizer for X -ray therapy that is administered to a subject to be treated before X -ray irradiation and is used for the subject's X -ray treatment.

(In the equation, R ¹ represents a hydrogen atom and R ² represents a hydrogen atom ). The iron compounds are ferric chloride, iron sesquioxide, iron sulfate, ferrous pyrophosphate, ferrous citrate, sodium iron citrate, sodium ferrous citrate, ammonium iron citrate, ferric pyrophosphate. , Iron Lactate, ferrous gluconate, sodium diethylenetriamine pentaacetate, ammonium diethylenetriamine pentaacetate, sodium ethylenediamine tetraacetate, ammonium iron diamine tetraacetate, sodium dicarboxymethylglutamate, ammonium iron dicarboxymethylglutamate, fumaric acid Contains ferrous iron, iron acetate, iron oxalate, ferrous succinate, sodium iron citrate succinate, hem iron, dextran iron, triethylenetetraamine iron, lactoferrin iron, transferase iron, iron chlorophyllin sodium, ferritin iron, The sensitizer for X -ray therapy according to claim 1, wherein the sensitizer is one or more compounds selected from iron glycooxide and glycine iron sulfide. The sensitizer for X -ray therapy according to claim 1 or 2 , wherein the iron compound is ferrous citrate.

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