Radiation sensitivity enhancer

Abstract

The purpose of the present application is to provide: an enhancer of radiation sensitivity of cancer cells such as prostate cancer cells; and others. The present application discloses, as one embodiment of the invention, an Artemis inhibitor that contains a miconazole compound as an active ingredient.

Description

Radiosensitizers

[0001] As one embodiment, the present invention discloses an Artemis inhibitor containing miconazoles as an active ingredient, which is useful, for example, as a radiosensitizer in cancer radiotherapy, and is useful, for example, in the pharmaceutical field.

[0002] Radiation therapy is one of the standard treatments for localized prostate cancer. However, more than 15% of intermediate-risk patients and approximately half of high-risk or higher patients experience recurrence after radical irradiation (Non-Patent Documents 1 and 2). Furthermore, internal radiotherapy has recently attracted attention, and the effectiveness of radium chloride 223 (Ra223) for castration-resistant prostate cancer (CRPC) with bone metastasis and 177Lu-PSMA for metastatic CRPC (mCRPC) has been reported.

[0003] Aizawa, et al. Int J Clin Oncol. 2021 Narang, et al. Int. J. Radiat. Oncol. Biol. Phys. 2016 Japanese Cancer Society Academic Conference Abstracts, Vol. 82th, 2023, p. 256 (P-3333)

[0004] As described above, internal use of radioisotopes (RIs) has been investigated in radiotherapy for prostate cancer. However, the lack of sufficient prognostic benefits remains a clinical challenge. Therefore, research and development of agents that enhance the radiosensitivity of cancer cells is needed to further improve the therapeutic effects of radiotherapy.

[0005] (1) In light of the fact that the molecular mechanisms controlling the radiosensitivity of prostate cancer have not been elucidated, the present inventors investigated genes that control the radiosensitivity of prostate cancer in order to solve the above-mentioned problem, and identified potential candidate genes that control the radiosensitivity of prostate cancer using CRISPR screening applying the CRISPR-Cas9 system, which was previously reported (Non-Patent Document 3). The outline of this study is as follows. A single guide RNA library was introduced into three prostate cancer cell lines, 22RV1, DU145, and LNCaP, to create a mutant cell library. The cells were divided into an untreated group and an irradiated group, and after 11 days of culture, the amount of single guide RNA in surviving cells was analyzed. The decrease in the amount of single guide RNA in the irradiated group indicated that the gene was a target gene for radiosensitivity enhancement (i.e., knockout of the gene increased radiosensitivity). Based on this, the Artemis gene was identified as the top-ranked candidate gene for suppressing radiosensitivity in all three cell lines. (2) Based on the above findings, the inventors further verified the relationship between the Artemis gene and radiosensitivity and diligently identified compounds that bind to the Artemis gene. They discovered for the first time that miconazoles represented by the following formula (I) enhance the radiosensitivity of prostate cancer, thereby completing the present invention. Because Artemis is expressed in many cancers, the radiosensitizing effect of Artemis inhibition based on this finding is expected to be similar in other cancers, and application to such other cancers is also within the scope of the present invention. The present invention will be described below with reference to specific embodiments. However, the present invention is not limited to these embodiments.

[0006] [1] Formula (I):

[0007]

[0008] (Wherein, m R １ each represents a halogen atom, and n R ２each represent a halogen atom, m and n each independently represent an integer of 0 to 3, and ring A represents a nitrogen-containing aromatic heterocyclic group.) or a salt thereof. [2] The inhibitor according to the above [1], which is a radiosensitizer for cancer radiotherapy. [3] The inhibitor according to the above [2], wherein the enhancement of radiosensitivity is due to enhancement of DNA damage in cancer cells. [4] The inhibitor according to the above [2] or [3], wherein the enhancement of radiosensitivity is for curing cancer or for improving the therapeutic effect of internal radiotherapy for metastatic cancer. [5] The inhibitor according to any of the above [2] to [4], wherein the cancer radiotherapy is performed in combination with another cancer therapy. [6] The inhibitor according to any of the above [2] to [5], wherein the cancer is resistant to radiotherapy. [7] The inhibitor according to any one of the above-mentioned [2] to [6], wherein the cancer is prostate cancer or colon cancer. [8] The inhibitor according to any one of the above-mentioned [1] to [7], wherein compound (I) is a compound represented by the formula (I), wherein m and n are each independently 1 or 2, and ring A is a 5- or 6-membered nitrogen-containing aromatic heterocyclic group. [9] The inhibitor according to any one of the above-mentioned [1] to [8], wherein compound (I) is miconazole.

[0009]

[10] A method for enhancing radiosensitivity in cancer radiotherapy, comprising administering an effective amount of compound (I) or a salt thereof defined in [1] above to a subject in need thereof.

[11] Compound (I) or a salt thereof defined in [1] above for use in enhancing radiosensitivity in cancer radiotherapy.

[12] Use of compound (I) or a salt thereof defined in [1] above for the manufacture of a medicament for enhancing radiosensitivity in cancer radiotherapy.

[0010] In one embodiment, the present invention provides an Artemis inhibitor containing miconazoles as an active ingredient, which is useful as a radiosensitizer in cancer radiotherapy.

[0011] FIG. 1 shows the results of an evaluation of the effect of radiation exposure on the cell viability of cancer cells (wild-type cancer cells and Artemis-deficient cancer cells) performed in Reference Example 2, described below. The upper graph shows the results of the evaluation using Artemis-deficient cancer cell clone #1 (lower graph), and the lower graph shows the results of the evaluation using the same clone #2 (lower graph). FIG. 2 shows the results of an evaluation (evaluation by antibody staining of cells) of the effect of radiation exposure on the formation of γ-H2AX foci, an indicator of DSBs, in cancer cells (wild-type cancer cells and Artemis-deficient cancer cells), performed in Reference Example 3, described below. FIG. 3 shows the results of an evaluation (evaluation by the percentage of cells containing a specified fluorescent foci) of the effect of radiation exposure on the formation of γ-H2AX foci, an indicator of DSBs, in cancer cells (wild-type cancer cells and Artemis-deficient cancer cells), performed in Reference Example 3, described below. FIG. 4 shows the results of an evaluation of the effect of miconazole on cancer cell viability, performed in Example 1, described below. Figure 5 shows the results of an evaluation of the effect of miconazole on luciferase activity in cancer cell supernatants, as performed in Example 2 below. Figure 6 shows the results of an evaluation of the effect of miconazole on the formation of γ-H2AX foci, an indicator of DSBs in cancer cells irradiated with radiation (evaluation by antibody staining of cells), as performed in Example 3 below. Figure 7 shows the results of an evaluation of the effect of miconazole on the formation of γ-H2AX foci, an indicator of DSBs in cancer cells irradiated with radiation (evaluation by the percentage of cells containing a specified fluorescent foci), as performed in Example 3 below. Figure 8 shows the results of an evaluation of the effect of miconazole on cancer cell viability, as performed in Example 4 below. Figure 9 shows the results of an investigation of the inhibitory (or destructive) effect of miconazole on Artemis, as performed in Example 5 below. Figure 10 shows the results of an evaluation of the effect of miconazole on tumor volume in an animal model, as performed in Example 6 below.

[0012] The present invention will now be described with reference to specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0013] One embodiment of the present invention is a compound of formula (I):

[0014]

[0015] (Wherein, m R １ each represents a halogen atom, and n R ２ each represent a halogen atom, m and n each independently represent an integer of 0 to 3, and ring A represents a nitrogen-containing aromatic heterocyclic group.) (Hereinafter, this is also referred to as "compound (I)" or "miconazoles"), or a salt thereof, is an Artemis inhibitor containing as an active ingredient. (Embodiment A) (hereinafter, this inhibitor is also referred to as "the present inhibitor.")

[0016] [Artemis] As mentioned above, the "Artemis gene" (also referred to as "Artemis" herein) has been identified as a gene that suppresses radiation sensitivity. This gene is one of the molecules that constitute the non-homologous end joining (NHEJ) pathway and has been reported to be involved in the repair of DNA double-strand breaks (DSBs) caused by radiation exposure (Morio T, et al. Int J Biochem Cell Biol. 2008; 40(4): 598-603., Carusillo A, et al. Cells. 2020; 10; 9(7): 1665). As described in detail in the Examples section below, to verify the involvement of the Artemis gene in the radiosensitivity of prostate cancer, Artemis-deficient cells were created and cell viability after radiation exposure was examined. It was found that the deficient cells had a significantly reduced cell viability after radiation exposure. Furthermore, it was shown that Artemis gene deficiency increases γH2AX (an indicator of DSBs) after radiation exposure, enhancing DNA damage. These findings demonstrate that Artemis gene deficiency increases the radiosensitivity of prostate cancer cells by delaying DSB repair and enhancing DNA damage.

[0017] [Radiosensitivity Enhancement Effect of Artemis Inhibitory Compounds] (Artemis Inhibitory Compounds) As used herein, "Artemis inhibition" includes the binding of a compound to an Artemis protein and / or the inhibition of the function of the Artemis gene (e.g., inhibition of its expression). The inventors searched for compounds that specifically bind to Artemis using in silico compound screening with LIGHTHOUSE, an artificial intelligence prediction tool that quickly finds candidate compounds based solely on the amino acid sequence of a protein, without using any 3D protein structure. As a result, the aforementioned miconazole was identified.

[0018] (Radiosensitivity Enhancement) Miconazole is a well-known compound used as an antifungal agent for fungal infections. However, as detailed in the Examples section below, miconazole has surprisingly been found to enhance the radiosensitivity of 22RV1 cells, a prostate cancer cell line. Based on these novel findings, the present inhibitor was conceived, in which the miconazoles (compound (I)) or salts thereof, consisting of miconazole and its structural analogs, are used as radiosensitivity enhancers in cancer radiotherapy. Here, "enhanced radiosensitivity" refers to the improvement in the sensitivity of cancer cells to radiation exposure by the co-administration of compound (I) or its salt compared to before administration (i.e., compared to the case without administration). In this inhibitor, the enhanced radiosensitivity is believed to be achieved by enhancing DNA damage in cancer cells. In one embodiment, the enhanced radiosensitivity of this inhibitor can be used to cure cancer or to improve the therapeutic effect of internal radiotherapy for metastatic cancer. In one embodiment, the inhibitor can be used for cancers that exhibit resistance to radiation therapy. For example, the Artemis gene may be responsible for the resistance. The cancer may be prostate cancer or colon cancer.

[0019] (Actions other than radiosensitivity enhancement) The above has focused on the radiosensitivity enhancement effect of this inhibitor and explained its usefulness. However, this inhibitor is also expected to enhance the anticancer effect of anticancer drugs (e.g., bleomycin, etoposide, cisplatin, etc.) used in cancer chemotherapy (enhancing the sensitivity of cancer to anticancer drugs) through its inhibition of Artemis (see, for example, Oncol Res. 2018 Dec 27; 27(1): 29-38).

[0020] [Compound (I) or its salt] (Compound (I)) The compound that inhibits Artemis in this inhibitor is a compound represented by the above formula (I) (compound (I)) or a salt thereof. Each group in compound (I) and its preferred embodiments are described below.

[0021] m R １ Each of the n R ２ and m and n each represent a halogen atom. Here, examples of "halogen atom" include fluorine, chlorine, bromine, and iodine, with chlorine being more preferred. Furthermore, m and n each independently represent an integer of 0 to 3, more preferably 1 or 2, and even more preferably 2.

[0022] Ring A represents a nitrogen-containing aromatic heterocyclic group. In the present specification, examples of the "aromatic heterocyclic group" (including "5- to 14-membered aromatic heterocyclic group") include 5- to 14-membered (preferably 5- to 10-membered) aromatic heterocyclic groups containing, as ring-constituting atoms other than carbon atoms, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Preferable examples of the "aromatic heterocyclic group" include 5- or 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl, etc.; Benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thia Examples of the aromatic heterocyclic group include 8- to 14-membered fused polycyclic (preferably bi- or tricyclic) aromatic heterocyclic groups such as pyrazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl. In this specification, the term "nitrogen-containing aromatic heterocyclic group" refers to the above-mentioned "aromatic heterocyclic group" containing at least one nitrogen atom as a ring-constituting atom. Here, 5- or 6-membered nitrogen-containing aromatic heterocyclic groups are preferred, and among these, groups containing 1 to 3 nitrogen atoms are more preferred, and groups containing 2 nitrogen atoms are even more preferred.

[0023] The compound (I) has the following structural formula:

[0024]

[0025] The most preferred compounds are compounds represented by the following formula (I): or optical isomers thereof, and mixtures thereof (for example, racemates; miconazole).

[0026] Miconazole is a known compound and is available. Its synthesis method has been reported, for example, in Godefroi et al., J. Med. Chem. 12, 784 (1969). Therefore, those skilled in the art can appropriately synthesize and obtain compounds (I) other than miconazole by referring to these reports. (Salts of Compound (I), etc.) In the present inhibitor, compound (I) can be used in either the free form or its salt (preferably, its pharmaceutically acceptable salt). Those skilled in the art can appropriately select from either form based on the properties of the individual compound (I) used to implement this embodiment. Those skilled in the art can appropriately convert compound (I) to its salt form or convert a salt of compound (I) to its free form. Pharmaceutically acceptable salts include, for example, salts with inorganic acids such as hydrochloride, hydrobromide, sulfate, phosphate, and nitrate; salts with organic acids such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, tosylate, and maleate; and salts with amino acids such as glutamate and aspartate. When compound (I) is a solvate (e.g., hydrate), the starting compound can be reacted in an appropriate solvent, and then the solvate of the target compound can be isolated from the reaction mixture by various methods, such as distillation and crystallization. A solvate-free compound can be produced by desolvating the solvate by heating or drying. When compound (I) may have optical isomers, both these individual optical isomers and mixtures thereof are encompassed within the scope of compound (I). If desired, these isomers can be optically resolved or individually produced according to known methods. When compound (I) exists as diastereomers, they can be isolated by separation and purification means commonly used in the pharmaceutical field, if desired. Furthermore, when compound (I) is a racemate, it can be separated into S- and R-isomers by conventional optical resolution means. When compound (I) has stereoisomers, both the isomers alone and mixtures thereof are included in the scope of compound (I).

[0027] [Use forms of compound (I) or its salt] (dosage form, administration target, dosage, etc.) In the present inhibitor, compound (I) or its salt (hereinafter also collectively referred to as "the compound") can be used for enhancing radiosensitivity in cancer radiotherapy either in its alone form or in the form of a pharmaceutical composition containing the compound as an active ingredient together with a pharmaceutically acceptable carrier. In this specification, the above two forms may be collectively referred to as "medicine".

[0028] Examples of such pharmaceutical compositions include tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., immediate-release preparations, sustained-release preparations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosal patch films), injections (e.g., subcutaneous injections, intravenous injections (e.g., bolus), intramuscular injections, intraperitoneal injections), drip infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, etc.

[0029] As the "pharmaceutically acceptable carrier", various carriers commonly used in the field of pharmaceutical formulation technology can be used.

[0030] Specific examples of "pharmaceutically acceptable carriers" that can be used in solid preparations include excipients (e.g., lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc.), lubricants (e.g., magnesium stearate, talc, colloidal silica, etc.), binders (e.g., crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose, etc.), and disintegrants (e.g., starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylstarch, L-hydroxypropylcellulose, etc.).

[0031] Liquid preparations may contain solvents (e.g., water for injection, isotonic saline, alcohol, propylene glycol, macrogol, sesame oil, etc.), solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.), suspending agents (e.g., surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, glycerin monostearate, etc.; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, etc.), buffers (e.g., buffer solutions such as phosphates and citrates, etc.), and soothing agents (e.g., benzyl alcohol, etc.).

[0032] If necessary, formulation additives such as preservatives (e.g., paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, sorbic acid, etc.), antioxidants (e.g., sulfites, ascorbic acid, α-tocopherol, etc.), colorants, sweeteners, etc. may be further added.

[0033] Pharmaceutical compositions of the present inhibitors may be prepared by adding the present compound to the total formulation in an amount of usually 0.01 to 99% (w / w), preferably 0.1 to 85% (w / w), depending on the dosage form, administration method, carrier, etc. Pharmaceutical compositions can be prepared by conventional methods in the field of formulation technology depending on the form. Pharmaceutical compositions may also be formed into sustained-release preparations containing the active ingredient.

[0034] The present compound is expected to have low toxicity and few side effects, and has excellent properties as a pharmaceutical. Therefore, the present inhibitor can be safely administered to mammals (e.g., humans, dogs, or cats, particularly humans).

[0035] (Regarding administration route) The present inhibitor is used in combination with cancer radiotherapy before and / or simultaneously in medical practice, and can be administered orally or parenterally (e.g., intravenously, intramuscularly, subcutaneously, intraorgan, intranasally, intradermally, by instillation, intracerebral, rectally, intravaginally, intraperitoneally, and into a lesion). Among these, intravenous administration can be useful.

[0036] The dose of the present compound varies depending on the subject, route of administration, and age and symptoms of the subject, but is not particularly limited as long as it is an amount that can enhance the radiosensitivity of the target cancer in radiotherapy (hereinafter also referred to as an "effective amount"). For example, the effective amount of the present compound per dose is 1 to 250 mg when administered orally, and 0.1 to 1000 mg when administered parenterally.

[0037] Cancer radiotherapy using this inhibitor may be performed in combination with other cancer therapies. Such other cancer therapies include surgical therapy, drug therapy, immunotherapy, and photoimmunotherapy. These therapies can be used in combination as appropriate in clinical practice, taking into consideration the stage of cancer progression, the patient's condition, etc.

[0038] Another embodiment of the present invention is "[B] A method for enhancing radiosensitivity in cancer radiotherapy, comprising administering to a subject in need thereof an effective amount of compound (I) or a salt thereof defined in [1] above (Embodiment B)" (hereinafter also referred to as "the present enhancement method"). With regard to the significance and specific aspects (preferred aspects) of each invention-specifying feature ("enhancement of radiosensitivity," "compound (I) or a salt thereof," "form of use of compound (I) or a salt thereof," etc.) in "the present enhancement method" of Embodiment B, reference can be made to the explanation of the corresponding invention-specifying feature in "the present therapeutic agent" of Embodiment A.

[0039] Another embodiment of the present invention is "[C] Compound (I) or a salt thereof defined in the above [1], used for enhancing radiosensitivity in cancer radiotherapy (Embodiment C)" (hereinafter also referred to as "the present compound / salt"). With regard to the meaning of each of the invention-specifying matters (such as "radiosensitivity enhancement," "compound (I) or a salt thereof," and "mode of use of compound (I) or a salt thereof") in "the present compound / salt" of Embodiment C and their specific aspects (preferred aspects), reference can be made to the explanation of the corresponding invention-specifying matters in "the present therapeutic agent" of Embodiment A.

[0040] A further embodiment of the present invention is "[D] Use of compound (I) or a salt thereof defined in the above [1] for the manufacture of a medicament for enhancing radiosensitivity in cancer radiotherapy (Embodiment D)" (hereinafter also referred to as "the present use"). With regard to the significance of each invention-specifying feature ("radiosensitivity enhancement," "compound (I) or a salt thereof," "use form of compound (I) or a salt thereof," etc.) in "the present compound / salt" of Embodiment D and its specific aspects (preferred aspects), reference can be made to the explanation of the corresponding invention-specifying feature in "the present therapeutic agent" of Embodiment A.

[0041] The present invention will be described in detail below with reference to the following examples, but the present invention is not limited to these examples. Those skilled in the art can modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included within the scope of the present invention.

[0042] Reference Example 1: Creation of Artemis-deficient cells Two sgRNAs were designed to target a portion of exon 8 of the Artemis gene and the intron between exons 10 and 11. A plasmid vector expressing the two sgRNAs and a blasticidin resistance gene was added to the prostate cancer cell line DU145 (RRID: CVCL_0105; obtained from RIKEN BRC CELL BANK) and electroporated with NEON. The cells were cultured for three days with 20 μg / ml blasticidin for selection, and colonies were harvested to obtain Artemis-deficient cells. The sequences of the two sgRNAs are shown below (Table 1). gRNA5 targets a portion of exon 8, and gRNA3 targets the intron between exons 10 and 11.

[0043]

[0044] Reference Example 2: Effect of Radiation on Cell Viability Control or knockout DU145 cells obtained in Reference Example 1 were plated and irradiated at different doses. After 11-14 days of culture, the cells were fixed and stained with crystal violet. Colonies with 30 or more cells were counted and the viability calculated. The results are shown in Figure 1. To ensure experimental accuracy, two clones of knockout DU145 cells were collected and used (in Figure 1, these are designated DU145_KO_Artemis#1 (lower graph in the upper figure) and #2 (lower graph in the lower figure)). Artemis-deficient cells were shown to have a significantly reduced cell viability after irradiation.

[0045] Reference Example 3: γ-H2AX Assay. Control or Artemis-deficient DU145 cells obtained in Reference Example 1 were seeded onto glass slides and incubated for 48 hours, after which they were either irradiated (4 Gy) or left untreated. Four hours after irradiation, γ-H2AX foci formation was assessed. Cells were fixed with 4% paraformaldehyde for 15 minutes, treated with 0.2% Triton X-100 for 10 minutes, blocked with 1% BSA for 1 hour, and incubated with γ-H2AX antibody (1:500) for 30 minutes. Cells were labeled with Alexa Fluor antibody (Invitrogen) for 30 minutes and counterstained with Prolong Gold with DAPI (Invitrogen). Foci were visualized using a BZ-X710 fluorescence microscope (KEYENCE). Images were taken with the same exposure time for each experiment. The results are shown in Figure 2. In each experiment, we counted more than 200 cells from three or more random fields and calculated the percentage of cells containing more than 10 fluorescent foci. The results are shown in Figure 3. Artemis deficiency was shown to increase γ-H2AX (an indicator of DSBs) after radiation exposure, enhancing DNA damage.

[0046] The results of Reference Examples 2 and 3 revealed that Artemis deficiency increases the radiosensitivity of prostate cancer by delaying DSB repair and enhancing DNA damage.

[0047] Example 1: Radiosensitivity Enhancement Effect of Miconazole (1) 22Rv1 or PC3 cells were cultured for 24 hours in DMSO or 10 μM miconazole, then plated and irradiated at different doses. After 11 days of culture, the cells were fixed and stained with crystal violet. Colonies with 30 or more cells were counted and the survival rate was calculated. The results are shown in Figure 4. The upper panel shows the results for 22Rv1 cells, and the lower panel shows the results for PC3 cells.

[0048] Example 2: Radiosensitivity Enhancement Effect of Miconazole (2) LNCaP cells transfected with Metridia luciferase were used. Luciferase activity in the cell supernatant is proportional to the number of viable cells (PLoS One. 2012;7:e36535.). LNCaP-MLuc cells were cultured for 24 hours in medium containing various concentrations of miconazole, and then irradiated (4 Gy) or left untreated. After 11 days of culture, luciferase activity in the culture supernatant was measured. The results are shown in Figure 5.

[0049] Example 3: Radiosensitivity Enhancement Effect of Miconazole (3) 22Rv1 or PC3 cells were cultured for 24 hours in DMSO or 10 μM miconazole, then irradiated (4 Gy) or left untreated. Four hours after irradiation, the formation of γ-H2AX foci was assessed. Cells were fixed with 4% paraformaldehyde for 15 minutes, treated with 0.2% Triton X-100 for 10 minutes, blocked with 1% BSA for 1 hour, and incubated with γ-H2AX antibody (1:500) for 30 minutes. Cells were labeled with Alexa Fluor antibody (Invitrogen) for 30 minutes and counterstained with Prolong Gold with DAPI (Invitrogen). Foci were visualized using a BZ-X710 fluorescence microscope (KEYENCE). Images were taken with the same exposure time for each experiment. The results are shown in Figure 6. In each experiment, 200 or more cells were counted from three or more random fields, and the percentage of cells containing 10 or more fluorescent foci was calculated. The results are shown in Figure 7.

[0050] Example 4: Radiosensitivity Enhancement Effect of Miconazole (4) DU145 cells were cultured for 24 hours in DMSO or 20 μM miconazole, then plated and irradiated at different doses. After 11 days of culture, the cells were fixed and stained with crystal violet. Colonies with 30 or more cells were counted, and the survival rate was calculated. The results are shown in Figure 8.

[0051] Example 5: Investigation of the mechanism of action of miconazole. DU145 or 22Rv1 cells were exposed to DMSO or various concentrations of miconazole for 2–3 days, and then proteins were collected. Samples were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with Bullet Blocking One for Western Blotting (Nacalai Tesque, Kyoto, Japan) and incubated with anti-Artemis antibody (1:1000). After washing, proteins were detected with anti-rabbit IgG, HRP-conjugated antibody (Cell Signaling Technology) for approximately 1 hour at room temperature and visualized using a Chemi Doc XRS+ system (BIO-RAD). Signals for each protein were normalized using ACTB. The results are shown in Figure 9. The upper panel shows the results in DU145 cells, and the lower panel shows the results in 22Rv1 cells.

[0052] Example 6: Radiosensitivity enhancing effect of miconazole (animal model) 1×10 ７ A mixture of 50 μL of PBS containing 1000 PC3 cells and 50 μL of Matrigel was injected subcutaneously. ３ Treatment began after the tumor volume exceeded 100 μg / day. Miconazole 50 mg / kg or DMSO was administered intraperitoneally for three consecutive days, starting the day before irradiation. Radiation was performed using a Gamma Cell, with the mouse shielded except for the right lower limb, to deliver a dose of 6 Gy. The results are shown in Figure 10. Administration of miconazole reduced tumor volume.

[0053] In one embodiment, the present invention discloses an Artemis inhibitor containing miconazoles as an active ingredient, which is useful, for example, as a radiosensitizer in cancer radiotherapy, and is useful, for example, in the field of medicine. This application is based on Japanese Patent Application No. 2024-070674 (filing date: April 24, 2024), the contents of which are incorporated in full herein.

Claims

1. A compound of the following formula (I): (Wherein, m R １ each represents a halogen atom, and n R ２ each represent a halogen atom; m and n each independently represent an integer of 0 to 3; and ring A represents a nitrogen-containing aromatic heterocyclic group.) An Artemis inhibitor comprising a compound represented by the formula ("Compound (I)") or a salt thereof as an active ingredient.

2. The inhibitor according to claim 1, which is a radiosensitizer in cancer radiotherapy.

3. The inhibitor according to claim 2, wherein the enhanced radiosensitivity is due to enhanced DNA damage in cancer cells.

4. The inhibitor according to claim 2, wherein the enhancement of radiosensitivity is for curing cancer or for enhancing the therapeutic effect of internal radiotherapy for metastatic cancer.

5. The inhibitor according to any one of claims 2 to 4, wherein cancer radiotherapy is performed in combination with other cancer therapies.

6. The inhibitor according to any one of claims 2 to 4, wherein the cancer exhibits resistance to radiotherapy.

7. The inhibitor according to claim 6, wherein the cancer is prostate cancer or colon cancer.

8. The inhibitor according to any one of claims 1 to 4, wherein compound (I) is a compound represented by formula (I), wherein m and n are each independently 1 or 2, and ring A is a 5- or 6-membered nitrogen-containing aromatic heterocyclic group.

9. The inhibitor according to any one of claims 1 to 4, wherein compound (I) is miconazole.

10. A method for enhancing radiosensitivity in cancer radiotherapy, which comprises administering an effective amount of compound (I) or a salt thereof as defined in claim 1 to a subject in need thereof.

11. Compound (I) or a salt thereof as defined in claim 1, used for enhancing radiosensitivity in cancer radiotherapy.

12. Use of compound (I) or a salt thereof as defined in claim 1 for the manufacture of a drug for enhancing radiosensitivity in cancer radiotherapy.

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