Combined medicine for intra-arterial infusion therapy for cancer treatment

Abstract

The present invention addresses the problem of providing a combined medicine for an intra-arterial infusion therapy for cancer treatment.　This combined medicine for an intra-arterial infusion therapy for cancer treatment comprises: a compound represented by general formula (1) [wherein R represents a hydrogen atom, a linear or branched alkyl or alkenyl group having 1 to 12 carbon atoms, an aralkyl or arylalkenyl group having 7 to 12 carbon atoms and optionally having a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms and optionally having a substituent] or a pharmaceutically acceptable salt thereof; and a platinum preparation, which is an active ingredient for cancer treatment.

Description

Combination drugs for intra-arterial infusion therapy in cancer treatment

[0001] The disclosure in this application relates to a combination agent for intra-arterial infusion therapy for cancer treatment.

[0002] Platinum compounds are the most common anti-cancer drugs, and are covered by insurance for a wide range of cancers, including testicular tumors, bladder cancer, renal pelvis and ureter tumors, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer, neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumors (testicular tumors, ovarian tumors, extragonadal tumors), malignant pleural mesothelioma, biliary tract cancer, malignant osteosarcoma, uterine cancer, recurrent / refractory malignant lymphoma, and pediatric solid tumors (rhabdomyosarcoma, neuroblastoma, hepatoblastoma and other primary liver malignant tumors, medulloblastoma, etc.).

[0003] Furthermore, intra-arterial injection therapy is known as a method for administering anti-cancer agents such as platinum preparations, as described in Patent Document 2. Since intra-arterial injection therapy allows the anti-cancer agent to be locally introduced into tumor tissue, it has the advantage of being able to reliably introduce the anti-cancer agent into tumor tissue with a smaller amount than when administered systemically by intravenous drip.

[0004] However, platinum-based drugs are known to cause kidney damage. Platinum-based kidney damage affects whether cancer treatment can be continued, as well as the patient's quality of life and prognosis. Furthermore, some patients may already have impaired kidney function before starting cancer treatment. Therefore, there is a need to develop treatment methods that can prevent kidney function from declining due to anticancer drug administration, or that can safely administer anticancer drug therapy to cancer patients who already have impaired kidney function.

[0005] To solve this problem, Patent Document 1 describes that an agent capable of suppressing interleukin-11 (IL-11)-mediated signal transduction can be used in a method for treating or preventing cisplatin-induced nephropathy.

[0006] Special Publication No. 2022-521591 Publication Patent No. 3296545

[0007] The invention described in Patent Document 1 focuses on the mechanism of action of suppressing interleukin-11 (IL-11)-mediated signal transduction. Cells utilize various survival signals to enable their own survival. Therefore, it is desirable to develop pharmaceutical compositions that are effective against the same disorder (disease) through different mechanisms of action.

[0008] The present application has been made to solve the above-mentioned problems. As a result of intensive research, the present inventors have newly discovered that (a) a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof has a function of treating or preventing nephropathy and a function of inhibiting CCBL1, and (b) by using a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof in combination with a platinum preparation in intra-arterial injection therapy, cancer can be treated while treating or preventing nephropathy induced by administration of the platinum preparation.

[0009] That is, an object of the disclosure in the present application is to provide a combination agent for use in intra-arterial injection therapy for cancer treatment.

[0010] The disclosure of the present application relates to the following combination agents for use in intra-arterial injection therapy for cancer treatment:

[0011] [1] A combination drug for arterial injection therapy for cancer treatment, the combination drug being represented by the following general formula (1): [In formula (1), R represents a hydrogen atom, a linear or branched alkyl group or alkenyl group having 1 to 12 carbon atoms, an aralkyl group or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent.] or a pharmaceutically acceptable salt thereof; and a platinum agent which is an active ingredient for cancer treatment. [2] The combination drug according to the above-mentioned [1], wherein the compound represented by general formula (1) functions as an active ingredient for treating or preventing nephropathy induced by administration of a platinum agent. [3] The combination drug according to the above-mentioned [2], wherein the CCBL1 inhibitory function of the compound represented by general formula (1) enables treatment or prevention of nephropathy induced by administration of a platinum agent. [4] The combination drug according to the above-mentioned [1], wherein the platinum agent is cisplatin. [5] The combination drug according to the above-mentioned [1], wherein the cancer is liver cancer. [6] The combination drug according to the above-mentioned [1], wherein general formula (1) is the following formula (2): [7] The combination drug according to any one of the above [1] to [5]. [7] The combination drug according to the above [1] to [5], wherein the general formula (1) is the following formula (3): [8] The combination drug according to any one of the above [1] to [5]. [8] The combination drug according to the above [1] to [5], wherein the general formula (1) is the following formula (4): [9] A combination drug according to any one of [1] to [5] above. [9] A combination drug for intra-arterial injection therapy for cancer treatment, comprising a CCBL1 inhibitor and a platinum preparation as an active ingredient for cancer treatment, wherein the CCBL1 inhibitor is represented by the following general formula (1): [In formula (1), R represents a hydrogen atom, a linear or branched alkyl group or alkenyl group having 1 to 12 carbon atoms, an aralkyl group or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent] or a pharmaceutically acceptable salt thereof as an active ingredient.

[10] The combination drug according to the above-mentioned [9], wherein the CCBL1 inhibitor functions as an active ingredient for treating or preventing nephropathy induced by administration of a platinum agent.

[11] The combination drug according to the above-mentioned [9], wherein the platinum agent is cisplatin.

[12] The combination drug according to the above-mentioned [9], wherein the cancer is liver cancer.

[13] The combination drug according to the above-mentioned [9], wherein general formula (1) is the following formula (2):

[14] The combination drug according to any one of the above [9] to

[12] .

[14] The combination drug according to the above [9] to

[12] , wherein the general formula (1) is the following formula (3):

[15] The combination drug according to any one of the above [9] to

[12] .

[15] The combination drug according to the above [9] to

[12] , wherein the general formula (1) is the following formula (4): The combination drug according to any one of [9] to

[12] above.

[0012] The combination agent for arterial infusion therapy for cancer treatment disclosed in the present application can treat cancer while treating or preventing nephropathy induced by the administration of platinum compounds.

[0013] Figure 1 is a graph showing the results of an investigation into the inhibitory activity of the compound of formula (2) against CCBL1β elimination activity. Figure 2 is a graph showing the results of an investigation into the effects of the compound of formula (2) on renal tubular cells and cancer cells. Figure 2A shows the results using CCBL1-overexpressing LLC / PK1 cells, Figure 2B shows the results using a human breast cancer cell line (MDA-MB-231 cells), and Figure 2C shows the results using a Lewis lung carcinoma cell line (LLC cells). Figure 3 shows the results of an investigation into the effects of the compound of formula (2) on renal tubular cells and cancer cells. Figure 3A shows the experimental procedure. Figure 3B is a photograph (substituting a drawing) showing the CCBL1 protein expression level in the kidney of each group examined by immunohistochemistry. Figure 4 is a photograph (substituting a drawing) showing the results of immunostaining human kidney tissue. Figure 5 is a graph showing the results of an investigation into the effects of the compound of formula (2) on body weight and kidneys, where Figure 5A shows body weight change and Figure 5B shows relative kidney weight. Figure 6 is a graph showing the results of an investigation into the effects of the compound of formula (2) on renal damage, where Figure 6A shows Cre concentration and Figure 6B shows BUN concentration. Figure 7 shows the results of histopathological observation of mice administered with the compound of formula (2). Figure 7A is a photograph, substituted for a drawing, showing an optical microscope photograph. Figure 7B is a graph showing scores. Figure 8 shows the results of an investigation into the inhibition of apoptosis in the kidney by the compound of formula (2), induced by cisplatin. Figure 8A shows the results for BAX, and Figure 8B shows the results for Cleaved Caspase-3. Figure 9 shows the results of an investigation into whether the compound of formula (2) has no adverse effect on the anticancer activity of cisplatin. Figure 9A is a photograph of tumor tissue, substituted for a drawing, Figure 9B is a graph showing changes in tumor volume, and Figure 9C is a graph showing the tumor inhibition rate. Figure 10 shows the results of an investigation into whether the compound of formula (2) has no adverse effect on the anti-cancer effect of cisplatin. Figure 10A is a graph showing body weight change, and Figure 10B is a graph showing relative kidney weight. Figure 11 shows the results of an investigation into whether the compound of formula (2) has no adverse effect on the anti-cancer effect of cisplatin. Figure 11A is a graph showing Cre concentration, and Figure 11B is a graph showing BUN concentration.Figure 12 shows the results of an investigation into whether the compound of formula (2) has no adverse effect on the anticancer activity of cisplatin. Figure 12A is a photograph, a drawing substitute, that is an optical microscope photograph. Figure 12B is a graph showing scores. Figure 13 is a graph showing the results of an investigation into the inhibitory activity of the compound of formula (3) against CCBL1β elimination activity. Figure 14 is a graph showing the results of an investigation into the effect of the compound of formula (3) on renal damage, with Figure 14A showing Cre concentration and Figure 14B showing BUN concentration.

[0014] The concomitant drug for arterial injection therapy for cancer treatment disclosed in the present application (hereinafter, sometimes simply referred to as "concomitant drug") will be described below.

[0015] (Embodiment of the Concomitant) The concomitant is a compound represented by the following general formula (1): or a pharmaceutically acceptable salt thereof, and a platinum preparation that is an active ingredient in cancer treatment.

[0016] In general formula (1), R represents a hydrogen atom, a linear or branched alkyl or alkenyl group having 1 to 12 carbon atoms, an aralkyl or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent.

[0017] Examples of linear or branched alkyl or alkenyl groups having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpentyl, n-hexyl, isohexyl, vinyl, allyl, butenyl, hexenyl, n-heptyl, 2,4-dimethylpentyl, 1-n-propylbutyl, n-octyl, 2-ethylhexyl, n-nonyl, 1-methylnonyl, n-decyl, 3,7-dimethyloctyl, 2-isopropyl-5-methylhexyl, n-undecyl, n-dodecyl, and decenyl groups.

[0018] Examples of the aralkyl group or arylalkenyl group having 7 to 12 carbon atoms, which may have a substituent, include a benzyl group, a hydroxybenzyl group, a dihydroxybenzyl group, a phenylethyl group, a phenylethenyl group, a hydroxyphenylethyl group, a dihydroxyphenylethyl group, a hydroxyphenylethenyl group, a dihydroxyphenylethenyl group, a phenylpropyl group, a phenylpropenyl group, a phenylbutyl group, a phenylbutenyl group, a phenylpentyl group, a phenylpentenyl group, a phenylhexyl group, and a phenylhexenyl group.

[0019] Examples of the aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent include a phenyl group, a hydroxyphenyl group, a dihydroxyphenyl group, a trihydroxyphenyl group, a naphthyl group, a hydroxynaphthyl group, and a dihydroxynaphthyl group.

[0020] The description of R in general formula (1) describes a more preferred range, and R is not limited to the substituents exemplified above as long as it is within the range in which the effects disclosed in the present application are achieved. Substituents that can be adopted other than the substituents exemplified above are described below (for convenience, the substituents described below are defined as "R'"). Examples of R' include a hydroxy group, a halogen group such as F, -CF ３ Ya-CF ２ CF ３ Examples of such fluoroalkyl groups include fluoroalkyl groups such as fluoroalkyl groups, cyclolower alkyl groups, lower alkoxy groups, lower amino groups, trifluoromethoxy groups, amino lower alkyl groups, mono- or di-substituted amino lower alkyl groups, cyclic amino lower alkyl groups, lower alkenyl groups, and styryl groups. The cyclolower alkyl group means a C3 to C8 cycloalkyl group, such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group, and these rings may be substituted with a C1 to C3 linear or branched alkyl group, a hydroxy group, or a halogen atom.

[0021] Furthermore, general formula (1) may be general formula (1') shown below.

[0022] X in the general formula (1') is CH２ , O, or NH. R in general formula (1′) is the same as R in general formula (1).

[0023] The present inventors have created a screening system for inhibitors of enzymes involved in the onset of cisplatin-induced nephropathy, and have conducted screening tests on various compounds, finding that the compound represented by the above general formula (1) is useful. Specific compounds included in the above general formula (1) include, for example, compounds represented by the following formulas (2), (3), and (4).

[0024]

[0025] The compound represented by formula (2) above is called 2,4,6-trihydroxyacetophenone (THA), also known as phloracetophenone. THA is a natural compound obtained from the rhizomes of Curcuma comosa. THA is known as a matrix used in mass spectrometry using matrix-assisted laser desorption / ionization (MALDI-TOF) spectrometry.

[0026] The compound represented by formula (3) above is a compound known as 1-(2,4,6-trihydroxyphenyl)propan-1-one, also known as flopropione. Flopropion is sometimes used as an antispasmodic. When administered to humans, flopropione is known to have a strong effect on specific areas, such as the biliary system around the duodenum, and may be administered to target spasms of the muscle of Oddi, bile duct, and urinary tract. For example, in patients with hepatobiliary and pancreatic diseases, flopropione is administered to facilitate the flow of bile from the bile duct and pancreatic juice from the pancreatic duct into the duodenum. Similarly, in the presence of urinary stones, flopropione is administered to facilitate the excretion of urinary stones from the body with urination.

[0027] The compound represented by formula (4) is 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one, also known as phloretin. Phloretin is a dihydrochalcone and polyphenol found in apple tree leaves.

[0028] The above compounds may be pharmaceutically acceptable salts. "Pharmaceutically acceptable" generally means safe, non-toxic, and biologically or otherwise useful for preparing desirable pharmaceutical compositions, including being acceptable for veterinary and human pharmaceutical use. Pharmaceutically acceptable salts are not particularly limited as long as they exhibit the desired pharmacological activity, and examples thereof include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, oxalate, and phosphate, as well as acetate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethoxybenzoylbenzoate, benzoic acid salt ... and organic acid salts such as benzoxanthate, benzoxanthate, p-chlorobenzoxanthate, 2-benzoxanthate, p-toluenesulfonate, camphorsulfonate, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylate, glucoheptanoate, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylate), 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, and muconate.

[0029] Furthermore, the pharmaceutically acceptable salts of the above compounds may exist as various solvates, for example with water, methanol, ethanol, dimethylformamide, and the like.

[0030] The combination drug according to the embodiment is administered locally via intra-arterial injection therapy. This is a therapeutic method in which a catheter is inserted into a blood vessel, its tip is brought close to a blood vessel supplying the cancer, and an anticancer drug (the combination drug in this application) is locally introduced into the cancer tissue. The combination drug may be provided in any form suitable for intra-arterial injection therapy. For example, (i) the combination drug may be provided as a pre-mixed combination drug comprising a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof and a platinum preparation, or (ii) the compound represented by general formula (1) or a pharmaceutically acceptable salt thereof and a platinum preparation may be provided separately and mixed together at the time of use (combined injection). The combination drug may contain a sterilized aqueous or non-aqueous solution, suspension, emulsion, etc. Examples of non-aqueous diluents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic ester compositions such as ethyl oleate. The aqueous carrier may include water, an alcoholic aqueous solution, an emulsion, a suspension, saline, a buffered medium, etc. Also included may be sodium chloride solution, Ringer's dextrose, dextrose, sodium chloride, Ringer's lactate and bound oils, fluid replenishers, nutrients and electrolytes (eg, based on Ringer's dextrose), and the like.

[0031] The dosage form of the combination drug is not particularly limited as long as it can be used by intra-arterial injection therapy. For example, it may be provided as a tablet, pill, powder, etc., which may be converted to a liquid at the time of use using the above-mentioned sterilized aqueous or non-aqueous solution, or may be provided in advance as an injection.

[0032] As shown in the Examples below, administration of the compound represented by general formula (1) disclosed in the present application prior to administration of cisplatin improved the values ​​of creatinine (Cre) and urea nitrogen (BUN), indicators of renal function. Because the CCBL1 pathway is also involved in the development of drug- (chemical)-induced nephropathy caused by other halogenated alkene-derived products, the compound represented by general formula (1) disclosed in the present application is useful for treating platinum- (cisplatin)-induced nephropathy caused by the administration of platinum preparations such as cisplatin, but is also useful for treating platinum-independent chemical-induced acute nephropathy, chronic nephropathy, nephrotoxicity, and the like (hereinafter, these may be collectively referred to as "platinum-independent chemical-induced nephropathy"). In this specification, "treatment" refers to treating platinum-induced nephropathy caused by the platinum preparation contained in the concomitant drug, and "prevention" refers to preventing the occurrence of platinum-induced nephropathy. Platinum preparations include cisplatin, carboplatin, and oxaliplatin. Examples of chemical substances that induce nephrotoxicity other than platinum preparations include trichloroethylene, tetrafluoroethylene, and busulfan. Platinum preparations and chemical substances that induce nephrotoxicity other than platinum preparations are sometimes collectively referred to as "nephrotoxicity-inducing chemical substances." Busulfan, which is given as an example of a chemical substance that induces nephrotoxicity other than platinum preparations, is known to be used as an anti-cancer agent. Drugs that have anti-cancer effects but may induce nephrotoxicity, such as platinum preparations and busulfan, are sometimes referred to as nephrotoxicity-inducing anti-cancer agents. Nephrotoxicity induced by nephrotoxicity-inducing anti-cancer agents is sometimes referred to as anti-cancer agent-induced nephrotoxicity.

[0033] The combination drug may be (i) a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof and a platinum drug administered simultaneously, (ii) a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof may be administered to a patient before administering a platinum drug to treat or reduce the severity of platinum drug-induced nephropathy if it occurs (prevention), or (iii) a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof may be administered to a patient if platinum drug-induced nephropathy occurs in a patient after administration of a platinum drug. When the combination drug is provided as a combination drug, only the administration method (i) above can be used, but when provided as a combined injection, the administration methods (i) to (iii) above can be used.

[0034] As described in the Examples below, the present inventors investigated the various activities of the screened compounds and found that all of the compounds represented by formulas (2) to (4) exhibited KAT1 (cysteine ​​conjugate β-lyase 1: CCBL1) inhibitory activity. It can be presumed that the newly discovered compound represented by general formula (1) exerts a therapeutic effect on nephropathy by acting on the CCBL1 pathway. Therefore, it can be said that the compound represented by general formula (1) or a pharmaceutically acceptable salt thereof contained in the combination drug: functions as an active ingredient for treating or preventing nephropathy induced by the administration of a platinum drug; and the CCBL1 inhibitory function of the compound represented by general formula (1) enables the treatment or prevention of nephropathy induced by the administration of a platinum drug.

[0035] Furthermore, the compound represented by general formula (1) can be referred to as a CCBL1 inhibitor. When the compound represented by general formula (1) or a pharmaceutically acceptable salt thereof is contained in a concomitant drug as a CCBL1 inhibitor, the CCBL1 inhibitor functions as an active ingredient for treating or preventing nephropathy induced by the administration of a platinum preparation.

[0036] As mentioned above, platinum preparations are covered by insurance for a wide range of cancers, including testicular tumors, bladder cancer, renal pelvis and ureter tumors, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer, neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumors (testicular tumors, ovarian tumors, extragonadal tumors), malignant pleural mesothelioma, biliary tract cancer, malignant osteosarcoma, uterine cancer, recurrent and refractory malignant lymphoma, and pediatric solid tumors (rhabdomyosarcoma, neuroblastoma, hepatoblastoma and other primary malignant liver tumors, medulloblastoma, etc.).

[0037] Therefore, the combination drug according to the embodiment can be used in intra-arterial injection therapy for the above-mentioned cancers. IA-call (available from Nippon Kayaku Co., Ltd.) is known as an anti-cancer drug for intra-arterial injection therapy using cisplatin, a platinum preparation. IA-call is already being used in intra-arterial injection therapy for liver cancer by administration into the hepatic artery. The combination drug according to the embodiment, like IA-call, can be suitably used, for example, in intra-arterial injection therapy for liver cancer. Patent Document 2 describes that cisplatin, a platinum preparation, can be used in embolization therapy in addition to intra-arterial injection therapy for cancer treatment. In this specification, the term "arterial injection therapy" may be interpreted literally as "arterial injection therapy" or may be interpreted as a concept including embolization therapy.

[0038] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the technical scope of the disclosure in the present application.

[0039] <Materials> (1) Animals All mice were housed in plastic cages and maintained under a 12-hour light-dark cycle (lights on at 8:00 AM). Food and water were provided ad libitum. All experiments were conducted in accordance with the guidelines established by the Fujita Health University Animal Care and Use Committee. The study protocol was approved by the Fujita Health University Animal Care and Use Committee (Permit Number: AP20004).

[0040] (2) Cisplatin-Induced Nephropathy Model Mice To generate cisplatin-induced nephropathy model mice, male C57BL / 6J mice (Japan SLC Co., Ltd.) weighing approximately 20-25 g were intraperitoneally administered cisplatin (20 mg / kg) once. To examine the renal protective (preventive) effect of the compounds described in formulas (2) and (3) (hereinafter simply referred to as "compounds") on cisplatin-induced nephropathy, the compounds were intraperitoneally administered at doses of 0, 25, 50, and 100 mg / kg 30 minutes before cisplatin injection. Mice were sacrificed three days after cisplatin injection. Blood and kidney samples were collected and stored at -80°C for further analysis.

[0041] (3) Tumor allograft model mice: Male C57BL / 6J mice weighing approximately 20-25 g were inoculated with Lewis lung carcinoma cells (1 × 10 ６ The tumors were subcutaneously injected into the dorsum of each mouse. Tumors were measured using a caliper, and tumor volume was calculated as [length x width x width]. After the tumors reached a certain size, the compound (100 mg / kg) was intraperitoneally administered 30 minutes before intraperitoneal administration of 20 mg / kg cisplatin. Mice were sacrificed 3 days after cisplatin injection. Blood, tumor, and kidney samples were collected and stored at -80°C for further analysis.

[0042] (4) Body Weight Change, Kidney / Body Weight Ratio, and Tumor Inhibition Body weight change (%), kidney / body weight ratio, and tumor inhibition were calculated using the following formulas: Body weight change (%) = [(After treatment with cisplatin and compound) / (Before administration of cisplatin and Compound X)] × 100 Relative kidney weight (%) = [Kidney weight (g) / Kidney weight (g) after treatment with cisplatin and compound] × 100 Tumor inhibition (%) = Before administration of cisplatin and compound - [(After treatment with cisplatin and Compound X) / (Before administration of cisplatin and compound)]

[0043] (5) Cell Culture. LLC / PK1 (porcine kidney epithelial), LLC (Lewis lung carcinoma), and MDA-MB-231 (human breast cancer) cells were obtained from the JCRB Cell Bank (Japan). LLC / PK1 cells were transfected with a human CCBL1 expression vector, and CCBL1-stably overexpressing cells were cloned and used as CCBL1-overexpressing LLC / PK1 cells for further experiments. CCBL1-overexpressing LLC / PK1 cells were grown in 199 / EBSS medium (10% FBS, 1% penicillin / streptomycin, and 400 μg / ml G418), while LLC and MDA-MB-231 cells were grown in DMEM medium (10% FBS and 1% penicillin / streptomycin) at 37°C in a humidified atmosphere of 5% CO2.

[0044] (6) Cell viability assay Cell viability was measured using a cell viability assay kit. Cells were cultured in a 96-well plate (2 × 10 ４ Cells were seeded in 1000 μL / well, treated with different concentrations of cisplatin and / or THA (the compound described in formula (2)), and cultured for 24 hours. Then, 20 μL of CellTiter 96 aqueous solution (Promega) was added to each well, and the cells were cultured for an additional hour. The absorbance at 490 nm was measured using a microplate reader (Molecular Devices, USA). Cell viability was calculated as the absorbance of treated cells relative to that of untreated cells.

[0045] (7) Histopathological Observation Pathological changes in the kidneys were examined by periodic acid-Schiff (PAS) staining. Half of the kidney was immersion-fixed in 10% buffered formalin, embedded in paraffin, and thinly sliced ​​to a thickness of 4 μm. After staining, the sections were observed under a light microscope. Tubular damage was scored as follows: Six fields of view were observed for each section, and the average percentage of damaged renal tubules was calculated. The results of renal tubular damage were converted into an index of renal tubular necrosis, with an index of 0 assigned for less than 10% damage, an index of 1 for 10% to 25% damage, an index of 2 for 25% to 50% damage, an index of 3 for 51% to 75% damage, and an index of 4 for 75% or more damage.

[0046] (8) Immunostaining. Human and mouse paraffin sections were first dewaxed in xylene for 5 minutes (3 times), then replaced with fresh xylene. The resulting sections were treated with absolute ethanol for 2 minutes, followed by repeated treatments with 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, and 70% ethanol for 2 minutes. The deparaffinized kidney sections were washed with water for 2 minutes. Then, endogenous peroxidase treatment (3% H) was performed. ２ O ２ ) for 20 minutes. Then, sections were washed twice for 2 minutes with distilled water and three times for 3 minutes with 1x PBS. Then, sections were blocked with 10% goat serum in 1x PBS for 20 minutes (CCBL1) or 30 minutes (B cell / CLL lymphoma 2; Bcl-2). Sections were incubated overnight at 4°C with primary antibodies: anti-CCBL1 (1:500, GTX32492), anti-Bcl-2 (1:500, ab182858), and rabbit IgG isotype control antibody (1:500, ER1621011). The next day, sections were washed three times for 3 minutes with 1x PBS. The slides were then incubated with secondary antibody (Histofine Simple Stain MAX PO; Nichirei Biosciences) for 30 minutes and washed three times with 1x PBS for 3 minutes. The slides were then incubated with chromogen / substrate reagent for 30 seconds. Nuclear staining was then performed with hematoxylin. After staining, the sections were observed under a light microscope.

[0047] (9) Measurement of blood urea nitrogen and creatinine in serum Serum creatinine (Cre) and blood urea nitrogen (BUN) levels were measured using a fully automated blood biochemistry analyzer (Biomajesty JCA-BM9130) according to the manufacturer's method.

[0048] (10) Western Blotting. Immunoblotting analysis of proteins and tissue lysates from human CCBL1-overexpressing LLC / PK1 cells was performed by homogenization in TNE buffer containing 10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% NP40, and protease inhibitors. Lysate supernatants were obtained by centrifugation at 14,000 rpm for 10 minutes at 4°C. Total protein concentrations in the lysates were determined using a Pierce 660 nm Protein Assay Kit (Thermo Scientific). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%-12.5% ​​gel) and blotted onto membranes. After blocking with 5% nonfat milk, the membrane was incubated overnight with primary antibodies: anti-CCBL1 (1:1000, GTX32492, GeneTex), anti-Bcl-2-associated X protein (BAX; 1:5000, 50599-2-Ig, Thermo Fisher Scientific), anti-Cleaved Caspase-3 (1:2000, #9661, Abcam), and anti-β-actin (1:2000 or 1:5000, A5441, Santa Cruz). After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated anti-IgG, and target bands were visualized with enhanced chemiluminescence (ECL) reagents according to the manufacturer's instructions. The intensity of the target bands was analyzed by densitometry and normalized to β-actin.

[0049] (11) Production of Recombinant CCBL1 Protein. Sf9 cells infected with baculovirus containing the human CCBL1 gene were pelleted by centrifugation and lysed in 50 mM phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole. After sonication, the cell lysate was centrifuged at 10,000 × g for 20 minutes at 4°C, and the recombinant CCBL1 in the supernatant was loaded onto a pre-equilibrated Ni-NTA resin (Qiagen). The enzyme / resin complex was transferred to a column and washed with a buffer containing 300 mM NaCl and 20 mM imidazole in 50 mM phosphate buffer (pH 8.0). The recombinant CCBL1 was eluted with a buffer containing 300 mM NaCl, 250 mM imidazole, and 50 mM phosphate (pH 8.0). Enzyme fractions were pooled based on purity as determined using SDS-PAGE and desalted using a PD-10 column (GE Healthcare, UK).

[0050] (12) Inhibition of CCBL1 activity by compounds The beta-elimination activity of CCBL1 was measured according to the method described in "Arun Kumar Selvam et al., "A Novel Assay Method to Determine the Elimination of Se-Methylselenocysteine ​​to Monomethylselenool by Kynurenine Aminotransferase 1" Antioxidants 2020, 9, 139; doi: 10.3390 / antiox9020139. Briefly, 500 ng of recombinant CCBL1 protein in 5 μL was mixed with 1 μL of compounds at different concentrations and incubated at room temperature for 30 minutes. After incubation, 44 μL of the reaction mixture contained 100 mM potassium phosphate buffer, pH 7.4, 5 mM Se-methylselenocysteine, 100 μM dimethyl-2-oxoglutaraate, 100 μM α-keto-γ-methylthiobutyric acid sodium salt, 10 μM 2-amino-2-methyl-1,3-propanediol, and pyridoxal 5'-phosphate hydrate. The reaction mixture was incubated at 37°C, and the reaction was terminated by adding 20 μL of 2 M HCl containing 5 mM 2,4-dinitrophenylhydrazine. Then, 70 μL of the assay mixture was incubated at 37°C for 10 minutes, 130 μL of 1 M NaOH was added, and the increase in absorbance at 520 nm was measured using a spectrophotometer (Nivo Multiplate Microplate Reader, PerkinElmer). The assay mixture without enzyme was used as a blank.

[0051] (13) Data Analysis All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, USA). Significant differences in comparisons between two groups were evaluated using one-way analysis of variance. The significance criteria were ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. All data were expressed as standard error ± mean.

[0052] Example 1 The following experiment was carried out using THA represented by formula (2). The THA used in Example 1 may be referred to as compound X (simply "X" in the drawings).

[0053] [In vitro experiment] (a) Inhibition of CCBL1 activity To examine whether compound X inhibits CCBL1 enzyme activity, the inhibitory activity of compound X against CCBL1 β-elimination activity was examined using recombinant human CCBL1. Figure 1 is a graph showing the results. As is clear from Figure 1, it was confirmed that CCBL1 activity was inhibited by compound X in a concentration-dependent manner.

[0054] (b) Effects of Compound X on Renal Tubular Cells and Cancer Cells To investigate whether Compound X prevents cisplatin-induced renal tubular cell death, CCBL1-overexpressing LLC / PK1 cells were treated with various concentrations of cisplatin and Compound X for 24 hours, and cell viability was assessed by cell proliferation assay. The expression level of human CCBL1 in LLC-PK1 cells was confirmed by immunoblotting. The results are shown in Figure 2. As shown in Figure 2A, Compound X did not affect LLC-PK1 cell death in the cisplatin-free group (0 μM). On the other hand, Compound X prevented cisplatin-induced renal tubular cell death in a dose-dependent manner in the cisplatin-treated group (50 μM). Similar experiments were then performed using a human breast cancer cell line (MDA-MB-231 cells) and a Lewis lung carcinoma cell line (LLC cells). The results are shown in Figures 2B and 2C. As is clear from Figures 2B and 2C, Compound X did not affect cisplatin-induced tumor cell death in human breast cancer cell line (MDA-MB-231 cells) and Lewis lung carcinoma cell line (LLC cells). The results shown in Figures 2A to 2C confirmed that Compound X protects against the cytotoxicity of cisplatin in renal tubular cells in vitro without affecting the anticancer activity of cisplatin in cancer cells.

[0055] [In Vivo Experiment] (c) Effect of Compound X on Cisplatin-Induced Nephropathy in Vivo Figure 3A shows the experimental procedure. As shown in Figure 3A, to investigate whether Compound X prevents renal tubular cell death in vivo, C57BL / 6J mice were injected with Compound X 30 minutes before cisplatin injection. Next, the expression level of CCBL1 protein in the kidneys of each group was examined by immunohistochemistry. The results are shown in Figure 3B. As is clear from Figure 3B, CCBL1 is mainly expressed in renal tubular cells, and administration of cisplatin and Compound X did not affect the CCBL1 protein expression level.

[0056] Figure 4 is a photograph showing the results of immunostaining human kidney tissue. As is clear from Figure 4, it was confirmed that CCBL1 was expressed in renal tubular cells in human kidney sections.

[0057] (d) Effect of Compound X on Body Weight and Kidneys Since cisplatin administration is known to reduce body weight and increase kidney weight, body weight and kidney weight were measured for each group. The results are shown in Figure 5. Figure 5A shows the change in body weight, and Figure 5B shows the relative kidney weight. As is clear from Figures 5A and 5B, administration of cisplatin alone reduced body weight and increased relative kidney weight. However, administration of both cisplatin and Compound X did not increase relative kidney weight.

[0058] (e) Effect of Compound X on Renal Damage Next, the level of renal damage was examined by measuring Cre and BUN in serum. The results are shown in Figure 6. Figure 6A is a graph showing Cre concentration, and Figure 6B is a graph showing BUN concentration. As is clear from Figure 6, administration of cisplatin alone increased Cre and BUN levels. On the other hand, while administration of cisplatin increased Cre and BUN levels, administration of Compound X dose-dependently decreased Cre and BUN. Furthermore, when Compound X was administered alone, no change in Cre and BUN levels was observed.

[0059] (f) Histopathological Observation Furthermore, renal tubular cell damage, such as tubular cell death, cell shedding, interstitial edema, and renal cortical tubular damage, was examined by histopathological observation. The results are shown in Figure 7. Figure 7A shows an optical micrograph, with the arrows indicating damaged cells. Figure 7B is a graph showing the scores. As is clear from Figure 7, Compound X was confirmed to dose-dependently inhibit cisplatin-induced renal tubular cell death. The cisplatin and Compound X groups had lower tubular damage scores than the cisplatin-only group. These results suggest that Compound X prevents cisplatin-induced renal damage. Furthermore, as is clear from the group administered with Compound X alone, Compound X itself was confirmed to not damage renal tubular cells.

[0060] (g) Inhibition of cisplatin-induced apoptosis in the kidney by Compound X. Cisplatin is known to activate the mitochondrial apoptosis pathway (Shaomin Shi et al., "ER stress and autophagy are involved in the apoptosis induced by cisplatin in human lung cancer cells," Oncol Rep, 2016; 2606-14). Therefore, we examined the effects of Compound X and cisplatin on apoptosis markers, such as Bcl-2-associated X protein (BAX) and cleaved caspase-3, by immunoblotting in kidney samples after cisplatin and Compound X administration. Figure 8A shows the results for BAX, and Figure 8B shows the results for Cleavage caspase-3. As can be seen from Figures 8A and 8B, the levels of BAX and Cleavage caspase-3 were reduced by Compound X administration in the Compound X and cisplatin groups compared to the cisplatin alone group. These results confirmed that Compound X reduces cisplatin-induced apoptosis of renal tubular cells.

[0061] (h) Regarding the absence of adverse effects of Compound X on the anticancer activity of cisplatin: Using the tumor allograft mouse model (3) above, we investigated whether Compound X can reduce renal tubular cell damage without adversely affecting the anticancer activity of cisplatin. The results are shown in Figures 9 to 12. Figure 9A is a photograph of tumor tissue, Figure 9B is a graph showing changes in tumor volume, and Figure 9C is a graph showing tumor inhibition rates. Figure 10A is a graph showing changes in body weight, and Figure 10B is a graph showing relative kidney weight. Figure 11A is a graph showing Cre concentration, and Figure 11B is a graph showing BUN concentration. Figure 12A is an optical microscope photograph, with arrows in the photograph indicating damaged cells. Figure 12B is a graph showing scores. As is clear from Figures 9A and 9B, administration of cisplatin reduced tumor size, and co-administration of Compound X with cisplatin did not alter this effect. Similar to the cisplatin-induced nephrotoxicity mouse model, administration of cisplatin alone resulted in a decrease in body weight and an increase in relative kidney weight. However, as shown in Figures 10A and 10B, administration of both cisplatin and Compound X resulted in little change in body weight, while relative kidney weight decreased. Furthermore, as shown in Figures 11A and 11B, administration of Compound X dose-dependently reduced the cisplatin-induced increases in BUN and Cre. Furthermore, as shown in Figures 12A and 12B, in tumor allograft model mice, Compound X reduced the renal tubular injury score compared to administration of cisplatin alone. (3) In tumor allograft model mice, Compound X was intraperitoneally administered 30 minutes before intraperitoneal administration of cisplatin near the tumor (sham arterial infusion therapy). However, given the function of Compound X, simultaneous administration of cisplatin and Compound X would obviously produce similar effects.

[0062] These results demonstrate that Compound X (THA) prevents cisplatin-induced renal tubular cell injury (i.e., treats existing damage) both in vitro and in vivo. Furthermore, Compound X protects the kidney from cisplatin-induced renal damage in vivo without affecting the anticancer effects of cisplatin. Therefore, Compound X may be a potential adjuvant for clinical cisplatin therapy.

[0063] Example 2 Experiments were conducted in the same manner as in "(a) Inhibition of CCBL1 activity" and "(e) Effect of Compound X on renal damage" in Example 1, except that the compound represented by formula (3) (flopropion) was used instead of THA. Figure 13 is a graph showing the results of CCBL1 activity inhibition. For comparison, the results for THA are also shown in Figure 13. Figure 14A is a graph showing Cre concentration, and Figure 14B is a graph showing BUN concentration. As is clear from Figures 13 and 14, CCBL1 activity was confirmed to be inhibited by flopropion in a concentration-dependent manner, similar to THA. Furthermore, while Cre and BUN levels increased with administration of cisplatin, flopropion dose-dependently decreased Cre and BUN. Furthermore, no change in Cre and BUN levels was observed when flopropion was administered alone. These results confirmed that flopropion exerts an effect similar to that of THA.

[0064] Example 3 Screening of CCBL1 Inhibitory Compounds CCBL1 inhibitors were obtained by screening the KAT1 inhibitory activity of compounds contained in the compound library (The Spectrum Collection) provided by the Center for Drug Discovery Science Research Support, Graduate School of Pharmaceutical Sciences, Osaka University. A mixture of 10 ng / μL recombinant CCBL, 1 mM L-kynurenine, 1 mM sodium pyruvate, 100 μM PLP, and 0.005% Tween 20 in 150 mM 2-amino-2-methyl-1-propanol buffer was added to a 384-well plate containing 10 μM compound and incubated at room temperature for 2 hours. Next, 20 μL of 300 mM zinc acetate solution was added, and the fluorescence intensity was measured at an excitation wavelength of 340 nM and an emission wavelength of 460 nM using an Infinite M1000 plate reader (TECAN). The inhibitory activity of the compound (sample) was calculated based on the following formula, where the control containing no compound was defined as 100% reaction and the background containing no enzyme was defined as 0% reaction: Inhibitory activity (%) = 100 x {1 - (sample - background) / (control - background)}

[0065] The results of compound screening are shown in Table 1. The inhibitory activities of fluoroacetophenone (a compound represented by formula (2)) used in Example 1 and flopropione (a compound represented by formula (3)) used in Example 2 are also shown.

[0066] As is clear from the results shown in Table 1, PHLORETIN (the compound described in formula (4)) also has the same inhibitory function against CCBL1 as the compounds of Examples 1 and 2, and therefore exhibits the same effects as those of Examples 1 and 2.

[0067] On the other hand, 4-O-methylfluoroacetophenone (see formula (5) below, which has antifungal activity), in which the 4-hydroxy group in general formula (2) is replaced with 4-O-methyl, exhibited almost no CCBL1 inhibitory activity, even though it was the same as general formula (2) except that the hydroxy group at the 4-position of the benzene ring was replaced with an O-methyl group.

[0068]

[0069] From the above results, the compounds whose effects were confirmed in Examples 1 to 3 share a common skeleton represented by formula (1), and therefore, it is expected that any compound represented by formula (1) will exhibit similar effects. Furthermore, since the compound represented by formula (5) had almost no CCBL1 inhibitory activity, it can be inferred that in order for the compound represented by formula (1) to exhibit CCBL1 inhibitory activity, it is important that positions 2, 4, and 6 of the benzene ring are hydroxy groups.

[0070] The compound represented by formula (1) can alleviate renal damage. It also has CCBL1 inhibitory activity. Therefore, by using it in combination with a platinum agent in intra-arterial infusion therapy, cancer can be treated while suppressing platinum-induced renal damage.

Claims

1. A combination drug for arterial injection therapy for cancer treatment, the combination drug being represented by the following general formula (1): [In formula (1), R represents a hydrogen atom, a linear or branched alkyl or alkenyl group having 1 to 12 carbon atoms, an aralkyl or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent] or a pharma- ceutically acceptable salt thereof, and a platinum preparation which is an active ingredient for cancer treatment.

2. The combination drug according to claim 1, wherein the compound represented by general formula (1) functions as an active ingredient for treating or preventing nephropathy induced by administration of a platinum preparation.

3. The combination drug according to claim 2, which is capable of treating or preventing nephropathy induced by administration of a platinum agent by the CCBL1 inhibitory function of the compound represented by general formula (1).

4. The combination drug according to claim 1, wherein the platinum agent is cisplatin.

5. The combination drug according to claim 1, wherein the cancer is liver cancer.

6. The general formula (1) is the following formula (2): The combination drug according to any one of claims 1 to 5.

7. The general formula (1) is the following formula (3): The combination drug according to any one of claims 1 to 5.

8. The general formula (1) is the following formula (4): The combination drug according to any one of claims 1 to 5.

9. A combination drug for intra-arterial injection therapy for cancer treatment, comprising a CCBL1 inhibitor and a platinum preparation as an active ingredient for cancer treatment, the CCBL1 inhibitor being represented by the following general formula (1): [In formula (1), R represents a hydrogen atom, a linear or branched alkyl or alkenyl group having 1 to 12 carbon atoms, an aralkyl or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent, or an aromatic hydrocarbon group having 6 to 12 carbon atoms which may have a substituent] or a pharma- ceutical acceptable salt thereof, as an active ingredient.

10. The combination drug according to claim 9, wherein the CCBL1 inhibitor functions as an active ingredient for treating or preventing nephropathy induced by administration of a platinum preparation.

11. The combination drug according to claim 9, wherein the platinum agent is cisplatin.

12. The combination drug according to claim 9, wherein the cancer is liver cancer.

13. The general formula (1) is the following formula (2): The combination drug according to any one of claims 9 to 12.

14. The general formula (1) is the following formula (3): The combination drug according to any one of claims 9 to 12.

15. The general formula (1) is the following formula (4): The combination drug according to any one of claims 9 to 12.

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