Pharmaceutical composition for use in a therapeutic method or prevention method of renal injury, combination comprising same, and ccbl1 inhibitor

Abstract

The present invention addresses the problem of providing a pharmaceutical composition for use in a method for treating or preventing renal injury. A pharmaceutical composition for use in a method for treating or preventing renal injury, the pharmaceutical composition comprising, as an active ingredient, a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof, and R represents a hydrogen atom, a linear or branched alkyl group or alkenyl group having 1 to 12 carbon atoms, an aralkyl group or aryl alkenyl 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. # imgabs0 #

Description

Technical Field

[0001] The disclosure in the present application relates to a pharmaceutical composition for use in a method for treating or preventing renal injury, a combination drug comprising the pharmaceutical composition, and a CCBL1 inhibitor. Background Art

[0002] Platinum preparations are the most representative anticancer agents. Moreover, platinum preparations are used in a wide range of cancers, including testicular tumors, bladder cancer, renal pelvis / ureteral 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, embryonal cell tumors (testicular tumors, ovarian tumors, extragonadal tumors), malignant pleural mesothelioma, biliary tract cancer, malignant osteosarcoma, uterine body cancer, relapsed / refractory malignant lymphoma, pediatric solid tumors (rhabdomyosarcoma, neuroblastoma, hepatoblastoma, other primary liver malignant tumors, medulloblastoma, etc.).

[0003] However, it is known that platinum preparations cause renal damage. Renal damage caused by platinum preparations affects whether cancer treatment can be continued, the patient's QOL (Quality of Life), and life prognosis. In addition, depending on the patient, renal function may have decreased before the start of cancer treatment. Therefore, it is desirable to develop a treatment method that can prevent renal function from decreasing due to the administration of anticancer agents, or that can safely implement anticancer treatment even for cancer patients whose renal function has already decreased.

[0004] In order to solve this problem, Patent Document 1 describes that a drug capable of inhibiting interleukin 11 (IL-11)-mediated signal transduction can be used in a method for treating or preventing cisplatin-induced renal injury.

[0005] Prior Art Literature

[0006] Patent Literature

[0007] Patent document 1: Japanese Patent Application Publication No. 2022-521591. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] The invention described in the above-mentioned patent document 1 is an invention focusing on the mechanism of action of inhibiting interleukin 11 (IL-11)-mediated signal transduction. However, cells drive various survival signals to survive themselves. Therefore, it is expected to develop a pharmaceutical composition that exerts effects with different mechanisms of action even for the same injury (disease).

[0010] The present invention is intended to solve the above problems. The present inventors have conducted intensive research and have newly discovered that the compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof can be used in a method for treating or preventing renal injury and has a CCBL1 inhibitory function.

[0011] [Chemical formula 1]

[0012]

[0013] That is, the object disclosed in the present application is to provide a pharmaceutical composition and a combination drug comprising the pharmaceutical composition, and a CCBL1 inhibitor, which are used in a method for treating or preventing renal injury.

[0014] Means of solving the problem

[0015] The disclosure in the present application relates to the pharmaceutical composition and the combination drug containing the pharmaceutical composition, and the CCBL1 inhibitor shown below, for use in the method for treating or preventing renal injury.

[0016] [1] A pharmaceutical composition for use in a method for treating or preventing renal injury, the pharmaceutical composition comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] [Chemical formula 2]

[0018]

[0019] 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 aralkenyl 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.

[0020] [2] The pharmaceutical composition according to [1] above, wherein the compound represented by the general formula (1) has a CCBL1 inhibitory function.

[0021] [3] The pharmaceutical composition according to [1] above, wherein the general formula (1) is the following formula (2).

[0022] [Chemical formula 3]

[0023]

[0024] [4] The pharmaceutical composition according to the above [1], wherein the general formula (1) is the following formula (3).

[0025] [Chemical formula 4]

[0026]

[0027] [5] The pharmaceutical composition according to the above [1], wherein the general formula (1) is the following formula (4).

[0028] [Chemical formula 5]

[0029]

[0030] [6] The pharmaceutical composition according to any one of [1] to [5] above, wherein the renal damage is induced by administration of a platinum preparation.

[0031] [7] The pharmaceutical composition according to [6] above, wherein the renal injury is cisplatin-induced renal injury.

[0032] [8] A combination drug comprising:

[0033] A first medicament comprising the pharmaceutical composition described in any one of [1] to [5] above; and

[0034] A second medicament comprising a kidney damage-inducing anti-malignant tumor agent as an active ingredient.

[0035] [9] The combination drug according to [8] above, wherein the first drug is administered before or simultaneously with the second drug.

[0036]

[10] A CCBL1 inhibitor comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0037] [Chemical formula 6]

[0038]

[0039] 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 aralkenyl 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.

[0040]

[11] The CCBL1 inhibitor according to

[10] above, wherein the general formula (1) is the following formula (2).

[0041] [Chemical formula 7]

[0042]

[0043]

[12] The CCBL1 inhibitor according to

[10] above, wherein the general formula (1) is the following formula (3).

[0044] [Chemical formula 8]

[0045]

[0046]

[13] The CCBL1 inhibitor according to

[10] above, wherein the general formula (1) is the following formula (4).

[0047] [Chemical formula 9]

[0048]

[0049]

[14] The CCBL1 inhibitor according to any one of

[10] to

[13] above, which is used in a method for treating or preventing renal injury.

[0050] Effects of the Invention

[0051] The pharmaceutical composition disclosed in the present application can be used in a method for treating or preventing renal injury. In addition, a combination drug comprising the pharmaceutical composition and a renal injury-inducing anti-malignant tumor agent can prevent the occurrence of anti-malignant tumor agent-induced renal injury, or even if anti-malignant tumor agent-induced renal injury occurs, it can reduce (treat) the degree of injury. The compound represented by the general formula (1) disclosed in the present application can be used as a CCBL1 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graph showing the results of studying the inhibitory activity of the compound represented by formula (2) on CCBL1β-detachment activity.

[0053] Figure 2 This is a graph showing the results of studying the effects of the compound described in formula (2) on renal tubular cells and cancer cells; Figure 2 A represents the results using CCBL1-overexpressing LLC / PK1 cells; Figure 2 B represents the results using human breast cancer cell line (MDA-MB-231 cells); Figure 2 C shows the results using Lewis lung cancer cell line (LLC cells).

[0054] Figure 3 The results show the effects of the compounds described in formula (2) on renal tubular cells and cancer cells; Figure 3 A indicates the experimental steps; Figure 3 B is a photograph substituted for a drawing, which is a photograph showing the expression level of CCBL1 protein in the kidney of each group investigated by immunohistochemistry.

[0055] Figure 4 This is a photograph substituted for the attached drawing, and is a photograph showing the results of immunostaining of human kidney tissue.

[0056] Figure 5This is a graph showing the results of studying the effects of the compound described in formula (2) on body weight and kidney; Figure 5 A represents weight change; Figure 5 B represents relative kidney weight.

[0057] Figure 6 This is a graph showing the results of studying the effects of the compound described in formula (2) on renal damage; Figure 6 A represents the Cre concentration; Figure 6 B represents the concentration of BUN.

[0058] Figure 7 The results of histopathological observation of mice administered with the compound described in formula (2); Figure 7 A is a substitute photo for the attached figure, which is an optical microscope photo; Figure 7 B is a graph showing the scores.

[0059] Figure 8 The present invention shows the results of studying the inhibition of cisplatin-induced apoptosis in kidney by the compound described in formula (2); Figure 8 The A indicates the result of BAX; Figure 8 B shows the result of activated caspase-3.

[0060] Fig. 9 The results of the study indicate that the compound described in formula (2) does not adversely affect the anticancer effect of cisplatin; Fig. 9 A is a photograph of tumor tissue used as a substitute photograph for the attached drawing; Fig. 9 B is a graph showing changes in tumor volume; Fig. 9 C is a graph showing the tumor inhibition rate.

[0061] Fig.10 The results of the study indicate that the compound described in formula (2) does not adversely affect the anticancer effect of cisplatin; Fig.10 A is a graph showing weight changes; Fig.10 B is a graph showing relative kidney weights.

[0062] Fig.11 The results of the study indicate that the compound described in formula (2) does not adversely affect the anticancer effect of cisplatin; Fig.11 A is a graph showing Cre concentration; Fig.11 B is a graph showing the concentration of BUN.

[0063] Fig.12 The results of the study indicate that the compound described in formula (2) does not adversely affect the anticancer effect of cisplatin; Fig.12 A is a substitute photo for the attached figure, which is an optical microscope photo; Fig.12 B is a graph showing the scores.

[0064] Fig.13 This is a graph showing the results of examining the inhibitory activity of the compound represented by formula (3) on CCBL1β elimination activity.

[0065] Fig.14 is a graph showing the results of studying the effects of the compound described in formula (3) on renal damage; Fig.14 A represents the Cre concentration; Fig.14 B represents the concentration of BUN. DETAILED DESCRIPTION

[0066] Hereinafter, the pharmaceutical composition (hereinafter sometimes referred to as "pharmaceutical composition"), the combination drug containing the pharmaceutical composition, and the CCBL1 inhibitor in the treatment method or prevention method for renal injury disclosed in the present application (hereinafter sometimes collectively referred to as "treatment method") are described.

[0067] (Embodiment of the pharmaceutical composition)

[0068] The pharmaceutical composition comprises a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0069] [Chemical formula 10]

[0070]

[0071] In the 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 aralkenyl 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.

[0072] Examples of the linear or branched alkyl or alkenyl group 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.

[0073] Examples of the aralkyl or arylalkenyl group having 7 to 12 carbon atoms which may have a substituent include benzyl, hydroxybenzyl, dihydroxybenzyl, phenylethyl, phenylvinyl, hydroxyphenylethyl, dihydroxyphenylethyl, hydroxyphenylvinyl, dihydroxyphenylvinyl, phenylpropyl, phenylpropenyl, phenylbutyl, phenylbutenyl, phenylpentyl, phenylpentenyl, phenylhexyl, and phenylhexenyl.

[0074] 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.

[0075] In addition, the description of R in the 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 of the effects disclosed in the present application. The substituents that can be used other than the substituents exemplified above are described below (for convenience, the substituents described below are defined as "R'"). As R', for example, hydroxyl, halogen groups such as F, fluoroalkyl groups such as -CF3, -CF2CF3, cyclo-lower 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 can be cited. Cyclo-lower alkyl groups refer to cycloalkyl groups of C3 to C8, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups can be cited, and these rings can be substituted by straight-chain or branched alkyl groups of C1 to C3, hydroxyl groups, or halogen atoms.

[0076] In addition, the general formula (1) may be the general formula (1') shown below.

[0077] [Chemical formula 11]

[0078]

[0079] X in the general formula (1') represents CH2, O or NH. In addition, R in the general formula (1') is the same as R in the general formula (1).

[0080] The present inventors prepared an inhibitor screening system for enzymes involved in the pathogenesis of cisplatin-induced renal injury and conducted screening tests for various compounds. As a result, it was found that the compound represented by the above general formula (1) is useful. As specific compounds contained in the above general formula (1), for example, compounds represented by the following formulas (2), (3), and (4) can be cited.

[0081] [Chemical formula 12]

[0082]

[0083] The above formula (2) is a compound called 2,4,6-trihydroxyacetophenone (THA), also known as phlorothenone. THA is a natural compound obtained from the rhizome of Curcuma comosa. THA is known as a matrix for mass spectrometry analysis by matrix-assisted laser desorption ionization (MALDI-TOF).

[0084] The above formula (3) is a compound called 1-(2,4,6-trihydroxyphenyl)propane-1-one, also known as flopropione (FLO, abbreviated as FLO). Flopropione is used as a spasmodic drug. It is known that flopropione administered to humans strongly acts on a specific place such as the biliary system around the duodenum, and is sometimes administered with the goal of sedating spasms of the sphincter of Oddi, the bile duct, and the urinary tract. For example, hepatobiliary and pancreatic diseases are administered in order to facilitate the bile and pancreatic juice from the bile duct to flow from the pancreatic duct into the duodenum. Similarly, in the presence of urinary stones, it is administered with the purpose of easily excreting urinary stones to the outside of the body with urination.

[0085] The above formula (4) is a compound called 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propane-1-one, also called phloretin. Phloretin is one of dihydrochalcone and polyphenol, and is a compound found in apple leaves.

[0086] The above compounds may be pharmaceutically acceptable salts. "Pharmaceutically acceptable" means generally safe and non-toxic, and can be used to prepare the desired pharmaceutical composition biologically and in other aspects, including those permitted for animal use and human drug use. Pharmaceutically acceptable salts are not particularly limited as long as they are salts that exhibit the desired pharmacological activity, and examples include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, oxalates and phosphates; as well as acetates, propionates, caproates, cyclopentanepropionates, glycolates, pyruvates, lactates, malonates, succinates, malates, fumarates, tartrates, citrates, benzoates, o-(4-hydroxybenzoyl)benzoate, cinnamates, mandelates, methanesulfonates, ethanesulfonates, Organic acid salts such as 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, 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, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate and muconate.

[0087] In addition, the pharmaceutically acceptable salts of the above compounds may exist in the form of various solvates with water, methanol, ethanol, dimethylformamide, and the like.

[0088] The pharmaceutical composition can be administered topically or systemically. The mode of administration is not particularly limited, as long as it is administered orally or parenterally. Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, emulsions, etc. As examples of non-aqueous diluents, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and organic ester compositions, such as ethyl oleate, etc. can be cited. Aqueous carriers may include any one of water, alcoholic aqueous solutions, emulsions, suspensions, saline and buffered media. Parenteral carriers may include any one of sodium chloride solution, Ringer's dextrose, glucose, sodium chloride, Ringer's lactate and combined oils. Intravenous carriers may include, for example, any one of liquid supplements, nutrients and electrolytes (such as substances based on Ringer's dextrose), etc.

[0089] Examples of dosage forms of the pharmaceutical composition include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid or liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, and sterile encapsulated powders.

[0090] As shown in the embodiments described later, the pharmaceutical composition disclosed in the present application is administered before cisplatin administration, and the numerical improvement of creatinine (Cre) and urea nitrogen (BUN) as renal function indicators is observed. CCBL1 pathway is also involved in the occurrence of drug (chemical substance) renal injury caused by products from other halogenated olefins, therefore, the pharmaceutical composition disclosed in the present application is not limited to the treatment of platinum preparation (cisplatin) induced renal injury caused by platinum preparations such as cisplatin administration, and can also be used for chemical substance-induced acute renal injury, chronic renal injury, nephrotoxicity, etc. of platinum preparation non-participation type (hereinafter, sometimes they are summarized and recorded as "platinum preparation non-participation type chemical substance-induced renal injury"). In addition, in this specification, "treatment method" refers to treatment of platinum preparation-induced renal injury or platinum preparation non-participation type chemical substance-induced renal injury (hereinafter, sometimes two kidney injuries are collectively referred to as "chemical substance-induced renal injury"), and "prevention method" refers to the generation of prevention of chemical substance-induced renal injury. As platinum preparations, cisplatin, carboplatin, oxaliplatin, etc. can be cited. As chemical substances other than platinum preparations that induce renal damage, trichloroethylene, tetrafluoroethylene, busulfan, etc. can be cited. In addition, platinum preparations and chemical substances other than platinum preparations that induce renal damage are sometimes summarized and recorded as "renal damage inducing chemical substances". In addition, busulfan, which is known as an example of chemical substances other than platinum preparations that induce renal damage, is used as an anti-malignant tumor agent. Sometimes platinum preparations, busulfan, etc., which have anti-malignant tumor effects but may induce renal damage, are recorded as renal damage inducing anti-malignant tumor agents. In addition, renal damage induced by renal damage inducing anti-malignant tumor agents is sometimes recorded as anti-malignant tumor agent-induced renal damage.

[0091] As described in the examples described below, the present inventors investigated the various activities of the screened compounds, and found that KAT1 (Cysteine ​​Conjugate β-lyase1: CCBL1) inhibitory activity was observed in all formulas (2) to (4). It can be inferred that the newly discovered pharmaceutical composition for renal injury can achieve a therapeutic effect on renal injury by acting on the CCBL1 pathway.

[0092] The pharmaceutical composition disclosed in the present application may be used, for example, as described below.

[0093] (1) To treat or reduce the extent of occurrence (prevention) of anti-cancer agent-induced renal damage, by administering the drug to a patient before administration of an anti-cancer agent that induces renal damage;

[0094] (2) concurrent administration with kidney damage-inducing anti-malignant tumor agents;

[0095] (3) Administration to patients who have developed renal damage induced by anti-tumor drugs after administration of anti-tumor drugs;

[0096] (4) Administration to patients confirmed to have developed chemical-induced renal damage (excluding anti-tumor agent-induced renal damage).

[0097] (Embodiment of Combination Drug)

[0098] Next, the embodiment of the combination drug is described. The combination drug involved in the embodiment includes: a first drug, which includes any of the pharmaceutical compositions described in the embodiment of the pharmaceutical composition; and a second drug, which includes a kidney damage-inducing anti-malignant tumor agent as an active ingredient. The pharmaceutical composition as the first drug and the kidney damage-inducing anti-malignant tumor agent contained in the second drug have been described in the embodiment of the above-mentioned pharmaceutical composition. Therefore, since it becomes a duplicate record, the description of the pharmaceutical composition as the first drug and the kidney damage-inducing anti-malignant tumor agent contained in the second drug is omitted.

[0099] As long as the second drug contains the kidney damage-inducing anti-malignant tumor agent as an active ingredient, the administration method, dosage form, etc. may be the same as the first drug. Therefore, the administration method, dosage form, etc. are redundant and thus omitted.

[0100] The first drug contained in the combination drug is used to prevent the occurrence of anti-malignant tumor agent-induced renal damage induced by the second drug containing a renal damage-inducing anti-malignant tumor agent as an active ingredient, or to reduce (treat) the degree of damage even if anti-malignant tumor agent-induced renal damage occurs. Therefore, it is desirable that the first drug is administered before or simultaneously with the second drug. The dosing schedule and dosage can be determined by the doctor based on the patient's condition, etc.

[0101] (Embodiments of CCBL1 Inhibitors)

[0102] Next, the embodiment of the CCBL1 inhibitor is described. The CCBL1 inhibitor of the embodiment contains the compound represented by the general formula (1) described in the above (embodiment of the pharmaceutical composition) or a pharmaceutically acceptable salt thereof as an active ingredient. As described in the above (embodiment of the pharmaceutical composition), the compound represented by the general formula (1) has KAT1 (Cysteine ​​Conjugate β-lyase1: CCBL1) inhibitory activity. Therefore, the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof can be used as a CCBL1 inhibitor.

[0103] The CCBL1 inhibitor of the embodiment is the same as the pharmaceutical composition used in the method for treating or preventing renal injury, except that the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof is used as the CCBL1 inhibitor. Therefore, the embodiment of the CCBL1 inhibitor can be obtained by replacing the "pharmaceutical composition" in the embodiment of the pharmaceutical composition with "CCBL1 inhibitor", and thus the detailed description of the embodiment of the CCBL1 inhibitor is omitted.

[0104] In addition, it is speculated that CCBL1 inhibitors can achieve a therapeutic effect on renal injury by acting on the CCBL1 pathway. Therefore, CCBL1 inhibitors can be used in methods for treating or preventing renal injury, and CCBL1 inhibitors can also be used as the first drug in an embodiment of a combination drug.

[0105] The following examples are given to specifically illustrate the embodiments disclosed in the present application, but the examples are only used to illustrate the embodiments and do not limit or restrict the technical scope disclosed in the present application.

[0106] Example

[0107] <Materials>

[0108] (1) Animals

[0109] All mice were housed in plastic boxes and maintained on a 12-hour light-dark cycle (lights on at 8:00 AM), and were given food and water (ad libitum). All experiments were performed in accordance with the guidelines established by the Animal Experimentation Committee of Fujita Medical University. The experimental protocol was approved by the Animal Experimentation Committee of Fujita Medical University (Permission No.: AP20004).

[0110] (2) Cisplatin-induced renal injury model mice

[0111] In order to prepare a cisplatin-induced renal injury model mouse, male C57BL / 6J mice weighing about 20 to 25 g (SLC Co., Ltd., Japan) were given cisplatin (20 mg / kg) once intraperitoneally. In order to investigate the renal protective effect (preventive effect) of the compounds described in formulas (2) and (3) (hereinafter, simply described as "compounds") in cisplatin-induced renal injury, the compounds were intraperitoneally administered at doses of 0, 25, 50 and 100 mg / kg 30 minutes before cisplatin injection. Mice were killed on the third day after cisplatin injection. Blood and kidney samples were collected and stored at -80°C for further analysis.

[0112] (3) Tumor homograft model mice

[0113] Male C57BL / 6J mice weighing about 20-25 g were subcutaneously injected with Lewis lung cancer cells (1×10 6The tumor was measured using a vernier caliper, and the tumor volume was calculated as [length × width × width]. After the tumor reached a certain size, the compound (100 mg / kg) was intraperitoneally administered 30 minutes before 20 mg / kg cisplatin was intraperitoneally administered. The mice were killed on the third day after the injection of cisplatin. Blood, tumor, and kidney samples were collected and stored at -80°C for further analysis.

[0114] (4) Body weight changes, kidney / body weight ratio, and tumor suppression

[0115] Body weight change (%), kidney / body weight ratio and tumor inhibition were calculated using the following formula:

[0116] Body weight change (%) = [(after treatment with cisplatin and compound X) / (before administration of cisplatin and compound X)] × 100

[0117] ·Relative kidney weight (%) = [kidney weight (g) / kidney weight after cisplatin and compound treatment (g)] × 100

[0118] Tumor inhibition (%) = before administration of cisplatin and compound - [(after treatment with cisplatin and compound X) / (before administration of cisplatin and compound)]

[0119] (5) Cell culture

[0120] LLC / PK1 (pig kidney epithelium), LLC (Lewis lung cancer) and MDA-MB-231 (human breast cancer) cells were obtained from JCRB cell bank (Japan). LLC / PK1 cells were transfected using human CCBL1 expression vector, and CCBL1 stable overexpression cells were cloned and used for further experiments as CCBL1 overexpression LLC / PK1 cells. LLC / PK1 cells overexpressed by CCBL1 were propagated in 199 / EBSS culture medium (10% FBS, 1% penicillin / streptomycin and 400 μg / ml G418), and LLC and MDA-MB-231 cells were propagated in DMEM culture medium (10% FBS and 1% penicillin / streptomycin) at 37°C in a humid atmosphere of 5% CO2.

[0121] (6) Cell survival rate detection

[0122] The cell viability was determined using a cell viability assay kit. Cells were seeded in 96-well plates (2 × 10 4Cells / well) were treated with different concentrations of cisplatin and / or THA (compounds described in formula (2)) and cultured for 24 hours. Then, 20 μL of CellTiter 96 (registered trademark) aqueous solution (produced by Promega) was added to each well and further cultured for 1 hour. The absorbance at 490 nm was measured using an ELISA reader (Molecular Devices, USA). The cell survival rate was calculated as the absorbance of the treated cells relative to the absorbance of the untreated cells.

[0123] (7) Histopathological observation

[0124] The pathological changes of the kidney were studied by periodic acid Schiff (PAS) staining. Half of the kidney was fixed by immersion in 10% buffered formalin, embedded in paraffin, and sliced ​​thinly into 4 μm thickness. After staining, the slices were observed under an optical microscope. The tubular injury was scored as follows. Each slice was observed in 6 fields of view, and the average proportion of damaged tubules was calculated. The results of tubular injury were converted into an index of tubular necrosis, with an index of 0 assigned to injuries less than 10%, an index of 1 assigned to injuries between 10% and 25%, an index of 2 assigned to injuries between 25% and 50%, an index of 3 assigned to injuries between 51% and 75%, and an index of 4 assigned to injuries above 75%.

[0125] (8) Immunostaining

[0126] Paraffin sections of humans and mice were first filtered in xylene for 5 minutes (3 times) and then replaced with fresh xylene. The obtained sections were treated with anhydrous ethanol for 2 minutes, and then repeatedly treated with 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, and 70% ethanol for 2 minutes. The deparaffinized sections of the kidney were washed with water for 2 minutes. Subsequently, endogenous peroxidase treatment (3% H2O2) was performed for 20 minutes. Then, the sections were washed with distilled water for 2 minutes (2 times) and 1×PBS for 3 minutes (3 times). Then, the sections were blocked with 10% goat serum in 1×PBS for 20 minutes (CCBL1) or 30 minutes (B cell / CLL lymphoma 2; Bcl-2). The sections were incubated overnight at 4°C with primary antibodies against CCBL1 (1:500, GTX32492), anti-Bcl-2 (1:500, ab182858) and rabbit IgG isotype control antibody (1:500, ER1621011). The next day, wash with 1×PBS for 3 minutes (3 times). Then, incubate the slides with secondary antibody for 30 minutes (Histofine Simple Stain MAX PO; manufactured by NICHIREI BIOSCIENCES) and wash with 1×PBS for 3 minutes (3 times). Then, incubate the slides with chromogen / substrate reagent for 30 seconds. Then, perform nuclear staining with hematoxylin. After staining, observe the sections with an optical microscope.

[0127] (9) Determination of blood urea nitrogen and creatinine in serum

[0128] Serum creatinine (Cre) and blood urea nitrogen (BUN) levels were measured using a fully automated blood biochemical analyzer (BioMajesty JCA-BM9130) according to the manufacturer's method.

[0129] (10) Western blotting

[0130] In TNE buffer containing 10mM Tris-HCl, 0.15M 5M NaCl, 1mM 0.5M EDTA, 1% NP40 and protease inhibitors, proteins and tissue lysates in human CCBL1 overexpressing LLC / PK1 cells were homogenized. Centrifugation was performed at 14000rpm, 10 minutes, and 4°C to obtain the supernatant of the lysate. The total protein concentration in the lysate was determined using Pierce (registered trademark) 660nm protein detection kit (manufactured by Thermo Scientific). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% to 12.5% ​​gel) and blotted on a membrane. After blocking with 5% fat-free milk, the membrane was incubated overnight with anti-CCBL1 (1:1000, GTX32492, manufactured by GeneTex), anti-Bcl-2 associated X protein (BAX; 1:5000, 50599-2-Ig, manufactured by Thermo Fisher Scientific), anti-activated caspase-3 (1:2000, #9661, manufactured by Abcam), and anti-β-actin (1:2000 or 1:5000, A5441, manufactured by SantaCruz) as the primary antibody. After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated anti-IgG, and the target band was visualized with enhanced chemiluminescence (ECL) reagent according to the manufacturer's instructions. The intensity of the target band was analyzed by densitometer and normalized to β-actin.

[0131] (11) Production of recombinant CCBL1 protein

[0132] Sf9 cells infected with baculovirus containing human CCBL1 gene were pelleted by centrifugation and dissolved in 50mM phosphate buffer (pH8.0) containing 300mM NaCl and 10mM imidazole. After ultrasonic treatment, the cell lysate was centrifuged at 10000×g for 20 minutes at 4°C, and the recombinant CCBL1 in the supernatant was added to the pre-equilibrated Ni-NTA resin (QIAGEN). The enzyme / resin complex was transferred to the column, 50mM phosphate buffer (pH8.0) was washed with a buffer containing 300mM NaCl and 20mM imidazole, and the recombinant CCBL1 was eluted with a buffer containing 300mM NaCl, 250mM imidazole and 50mM phosphoric acid (pH8.0). The enzyme fraction was extracted based on the purity determined using SDS-PAGE and desalted using a PD-10 column (GE Healthcare, UK).

[0133] (12) Inhibition of CCBL1 activity by compounds

[0134] The beta-elimination activity of CCBL1 was determined according to the method described in "Arun Kumar Selvam et al., "A Novel Assay Method to Determine the-Elimination of Se-Methylselenocysteine ​​to Monomethylselenol by Kynurenine Amino transferase 1", Antioxidants 2020, 9, 139; doi: 10.3390 / antiox9020139". Briefly, 500 ng of recombinant CCBL1 protein in 5 μL was mixed with different concentrations of compounds in 1 μL and incubated at room temperature for 30 minutes. After incubation, 44 μL of the reaction mixture included 100 mM potassium phosphate buffer at pH 7.4, 5 mM seleno-methylselenocysteine, 100 μM dimethyl 2-oxoglutarate, 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 20 μL of 2M HCl containing 5 mM 2,4-dinitrophenylhydrazine was added to terminate the reaction. Next, 70 μL of the detection mixture was incubated at 37°C for 10 minutes, 130 μL of 1M NaOH was added, and the increase in absorbance at 520 nm was measured using a spectrophotometer (Nivo multifunctional microplate reader, PerkinElmer). The detection mixture without enzyme was used as a blank.

[0135] (13) Data Analysis

[0136] All statistical analyses were performed using GraphPad Prism8 software (GraphPad Software, San Diego, USA). One-way ANOVA was used to evaluate significant differences in comparisons between two groups. The significance criteria were ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. All data are expressed as standard error ± mean.

[0137] <Example 1>

[0138] The following experiments were conducted using THA represented by formula (2). In addition, THA used in Example 1 may be referred to as compound X (abbreviated as "X" in the figure).

[0139] [In vitro experiments]

[0140] (a) Inhibition of CCBL1 activity

[0141] In order to investigate whether compound X inhibits CCBL1 enzyme activity, recombinant human CCBL1 was used to investigate the inhibitory activity of compound X on CCBL1β dissociation activity. Figure 1 is a graph showing the results. Figure 1 It was found that CCBL1 activity was inhibited by Compound X in a concentration-dependent manner.

[0142] (b) Effects of Compound X on renal tubular cells and cancer cells

[0143] To investigate whether compound X prevents cisplatin-induced tubular cell death, LLC / PK1 cells overexpressing CCBL1 were treated with different concentrations of cisplatin and compound X for 24 hours, and cell survival was evaluated by cell proliferation assay. The expression level of human CCBL1 in LLC-PK1 cells was confirmed by immunoblotting. Figure 2 Indicates the result. Figure 2 As shown in A, compound X had no effect on LLC-PK1 cell death in the group without cisplatin (0 μM). On the other hand, in the group with cisplatin (50 μM), cisplatin-induced renal tubular cell death was prevented in a dose-dependent manner. Next, the results of the same experiment using human breast cancer cell line (MDA-MB-231 cells) and Lewis lung cancer cell line (LLC cells) are shown in Figure 2 B and Figure 2 C. By Figure 2 is B and Figure 2 From C, it can be seen that compound X does not affect cisplatin-induced tumor cell death in human breast cancer cell line (MDA-MB-231 cells) and Lewis lung cancer cell line (LLC cells). Figure 2 A~ Figure 2 The results shown in C confirm that compound X does not affect the anticancer activity of cisplatin in cancer cells in vitro and protects against the cytotoxicity of cisplatin in renal tubular cells.

[0144] [In vivo experiments]

[0145] (c) Cisplatin-induced renal injury in vivo in relation to compound X

[0146] Figure 3 The A in the figure indicates the experimental step. Figure 3As shown in A, in order to study whether compound X prevents renal tubular cell death in vivo, compound X was injected into C57BL / 6J mice 30 minutes before cisplatin injection. Then, the expression level of CCBL1 protein in the kidney of each group was investigated by immunohistochemistry. The results are shown in Figure 3 B. By Figure 3 As shown in Figure B, CCBL1 is mainly expressed in renal tubular cells, and the administration of cisplatin and compound X does not affect the expression level of CCBL1 protein.

[0147] Figure 4 This is a photograph showing the results of immunostaining of human kidney tissue. Figure 4 It was found that CCBL1 is expressed in renal tubular cells in human kidney sections.

[0148] (d) Effects of Compound X on Body Weight and Kidney

[0149] It is known that cisplatin administration reduces body weight and increases kidney weight, so the body weight and kidney weight of each group were measured. The results are shown in Figure 5 . Figure 5 A represents weight change, Figure 5 B represents the relative kidney weight. Figure 5 A and Figure 5 As shown in Figure B, the administration of cisplatin alone reduced body weight and increased relative kidney weight. However, the administration of both cisplatin and compound X did not increase relative kidney weight.

[0150] (e) Effect of Compound X on Renal Damage

[0151] Next, the level of renal damage was investigated by measuring Cre and BUN in serum. The results are shown in Figure 6 . Figure 6 A is a curve graph showing Cre concentration. Figure 6 B is a graph showing the concentration of BUN. Figure 6 It is known that the administration of cisplatin alone increases the levels of Cre and BUN. On the other hand, the administration of cisplatin increases the levels of Cre and BUN, but the administration of compound X decreases them in a dose-dependent manner, and when compound X is administered alone, no changes in the levels of Cre and BUN are observed.

[0152] (f) Histopathological observation

[0153] Furthermore, tubular cell damage such as tubular cell death, cell shedding, interstitial edema, and renal cortical tubular damage was investigated through histopathological observation. Figure 7 Indicates the result. Figure 7 A is an optical microscope photograph, in which the arrows indicate damaged cells. Figure 7B is a graph showing the ratings. Figure 7 It can be seen that compound X was confirmed to inhibit cisplatin-induced renal tubular cell death in a dose-dependent manner. The renal tubular injury score of the cisplatin and compound X group was lower than that of the group containing only cisplatin. These results teach that compound X prevents cisplatin-induced renal injury. In addition, from the group administered with compound X alone, it can be seen that compound X itself does not damage renal tubular cells.

[0154] (g) Inhibition of cisplatin-induced apoptosis in kidney by compound X

[0155] Cisplatin is known to activate the mitochondrial apoptosis pathway (Shaomin Shi et al., "ER stress and autophagy are involved in the apoptosisinduced by cisplatin in human lung cancer cells", Oncol rep, 2016; 2606-14.). Therefore, the effects of administration of compound X and cisplatin on apoptosis markers such as Bcl-2-associated protein X (BAX) and activated caspase-3 were studied by immunoblotting in kidney samples after administration of cisplatin and compound X. Figure 8 A indicates the result of BAX, Figure 8 B indicates the result of activated cysteine ​​protease 3 (Cleaved Caspase-3). Figure 8 A and Figure 8 B shows that, in the group administered with compound X and cisplatin, the levels of BAX and cleaved caspase-3 were reduced by the administration of compound X, compared with the group administered with cisplatin alone. These results confirmed that compound X reduced the apoptosis of renal tubular cells caused by cisplatin.

[0156] (h) Compound X does not adversely affect the anticancer effect of cisplatin.

[0157] Using the tumor homograft model mice described above (3), it was investigated whether Compound X could alleviate renal tubular cell damage without adversely affecting the anticancer effect of cisplatin. Figures 9 to 12 Indicates the result. Fig. 9 A is a photo of tumor tissue. Fig. 9 B is a graph showing the change in tumor volume. Fig. 9 C is a graph showing the tumor inhibition rate. Fig.10 A is a graph showing weight changes. Fig.10 B is a graph showing relative kidney weights. Fig.11 A is a curve graph showing Cre concentration. Fig.11 B is a graph showing the concentration of BUN. Fig.12 A is an optical microscope photograph, in which the arrows indicate damaged cells. Fig.12 B is a graph showing the ratings. Fig. 9 A and Fig. 9 As shown in B, the tumor size was reduced by the administration of cisplatin, and this effect did not change even when compound X was administered together with cisplatin. As in the cisplatin-induced renal injury model mice, the body weight decreased and the relative kidney weight increased by the administration of cisplatin alone. However, Fig.10 A and Fig.10 As shown in Figure B, when both cisplatin and compound X were administered, body weight was almost unchanged, while relative kidney weight was reduced. Fig.11 A and Fig.11 As shown in Figure B, the administration of compound X dose-dependently reduced the increase of BUN and Cre induced by cisplatin. Fig.12 A and Fig.12 As shown in B, in tumor homograft model mice, it was confirmed that compound X reduced the renal tubular injury score compared with cisplatin alone.

[0158] The above results confirm that compound X (THA) prevents cisplatin-induced renal tubular cell damage (damage caused by treatment) both in vitro and in vivo. In addition, it is confirmed that compound X does not affect the anticancer effect of cisplatin in vivo and protects the kidneys from cisplatin-induced renal damage. Therefore, compound X can become a potential adjuvant for clinical cisplatin therapy.

[0159] <Example 2>

[0160] The experiment was conducted in the same manner as in "(a) Inhibition of CCBL1 activity" and "(e) Effect of compound X on renal damage" of Example 1, except that the compound represented by formula (3) (flopropion) was used instead of THA of Example 1. Fig.13 is a graph showing the results of CCBL1 activity inhibition. Fig.13 The results of THA are also recorded. Fig.14 A is a curve graph showing Cre concentration. Fig.14 B is a graph showing the concentration of BUN. Fig.13 and Fig.14It is known that CCBL1 activity is confirmed to be inhibited by floxacin in a concentration-dependent manner like THA. In addition, Cre and BUN levels increase by administration of cisplatin, but floxacin decreases Cre and BUN in a dose-dependent manner, and no changes in Cre and BUN levels are found when floxacin is administered alone. It is confirmed from the above results that floxacin has the same effect as THA.

[0161] <Example 3>

[0162] [Screening of CCBL1 inhibitory compounds]

[0163] CCBL1 inhibitory compounds were obtained by screening the KAT1 inhibitory activity of compounds contained in the compound library (The Spectrum Collection) provided by the Pharmaceutical Science Research Support Center of Osaka University. A mixture of 10 ng / μl recombinant CCBL, 1 mM L-kynurenine, 1 mM sodium pyruvate, 100 μM PLP, and 0.005% Tween20 contained 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 at an excitation wavelength of 340 nM and a fluorescence wavelength of 460 nM was measured using an Infinite M1000 microplate reader (manufactured by TECAN). The control without the compound was taken as the reaction 100%, and the background without the enzyme was taken as the reaction 0%, and the inhibitory activity of the compound (Sample) was calculated based on the following formula.

[0164] Inhibitory activity (%) = 100 x {1-(sample-background) / (control-background)}

[0165] The results of compound screening are shown in Table 1. In addition, the inhibitory activities of phloracetophenone (the compound described in formula (2)) used in Example 1 and floxacin (the compound described in formula (3)) used in Example 2 are also described.

[0166] [Table 1]

[0167]

[0168] From the results shown in Table 1, it is understood that phloretin (the compound represented by formula (4)) also has the CCBL1 inhibitory function similarly to the compounds of Examples 1 and 2, and therefore exhibits the same effects as those of Examples 1 and 2.

[0169] On the other hand, 4-O-methyl phenacetophenone (refer to the following formula (5), which has antifungal activity) in which the 4-hydroxyl group of the general formula (2) is replaced by a 4-O-methyl group is the same as the general formula (2) except that the hydroxyl group at the 4-position of the benzene ring is replaced by an O-methyl group, but it shows almost no CCBL1 inhibitory activity.

[0170] [Chemical formula 13]

[0171]

[0172] According to the above results, the compounds that have been confirmed to have an effect in Examples 1 to 3 have a common skeleton shown in formula (1), so if it is the compound shown in formula (1), it can be expected to exert the same effect. In addition, the compound shown in formula (5) has almost no CCBL1 inhibitory activity, so it can be inferred that in the compound shown in formula (1), it is important that the 2, 4, and 6 positions of the benzene ring are hydroxyl groups in order to exert CCBL1 inhibitory activity.

[0173] Industrial Applicability

[0174] The compound represented by formula (1) can alleviate renal damage and has CCBL1 inhibitory function. Therefore, it can treat cancer while inhibiting renal damage induced by platinum agents.

Claims

1. A pharmaceutical composition for use in a method for treating or preventing renal injury, the pharmaceutical composition comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, [Chemical 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 aralkenyl 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.

2. The pharmaceutical composition according to claim 1, wherein The compound represented by the general formula (1) has a CCBL1 inhibitory function.

3. The pharmaceutical composition according to claim 1, wherein The general formula (1) is the following formula (2), [Chemical formula 2] 4. The pharmaceutical composition according to claim 1, wherein The general formula (1) is the following formula (3), [Chemical formula 3] 5. The pharmaceutical composition according to claim 1, wherein The general formula (1) is the following formula (4), [Chemical formula 4] 6. The pharmaceutical composition according to any one of claims 1 to 5, wherein Renal injury is an injury induced by the administration of platinum preparations.

7. The pharmaceutical composition according to claim 6, wherein Renal injury was cisplatin-induced renal injury.

8. A combination drug comprising: A first medicament comprising the pharmaceutical composition according to any one of claims 1 to 5; and A second medicament comprising a kidney damage-inducing anti-malignant tumor agent as an active ingredient.

9. The combination drug according to claim 8, wherein: The first drug is administered prior to or simultaneously with the second drug.

10. A CCBL1 inhibitor comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, [Chemical formula 5] 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 aralkenyl 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.

11. The CCBL1 inhibitor according to claim 10, wherein The general formula (1) is the following formula (2), [Chemical formula 6] 12. The CCBL1 inhibitor according to claim 10, wherein The general formula (1) is the following formula (3), [Chemical formula 7] 13. The CCBL1 inhibitor according to claim 10, wherein The general formula (1) is the following formula (4), [Chemical formula 8] 14 . The CCBL1 inhibitor according to claim 10 , for use in a method for treating or preventing renal injury.

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