Methods for the treatment of nephrotoxicity induced by chemotherapy agents - Patents.com

Abstract

Cisplatin is a potent chemotherapeutic drug that is widely used to treat various solid cancers. However, its clinical effectiveness is strongly limited by frequent and severe adverse effects, such as nephrotoxicity. Therefore, there is an urgent medical need to identify novel strategies to limit the toxicity induced by cisplatin. Here, the inventors have identified adenosine A, which is approved by the US Food and Drug Administration. 2A We provide evidence that the receptor antagonist istradefylline (KW-6002) significantly protects against cisplatin-induced nephrotoxicity in acute and subchronic cisplatin intoxication experimental models. In particular, the present invention provides a therapeutically effective dose of selective A 2A Adenosine receptor (A 2A The present invention relates to a method for the treatment of cisplatin-induced nephrotoxicity in a subject in need thereof, comprising the step of administering to said subject a cisplatin (R) antagonist.

Description

[Technical field]

[0001] Field of the invention: The present invention relates to a therapeutically effective amount of selective A 2A Adenosine receptor (A 2A The present invention relates to a method for the treatment of chemotherapeutic drug-induced nephrotoxicity in a subject in need thereof, comprising the step of administering to said subject an anti-nephrotoxic agent (R) antagonist.

[0002] Background of the invention: Cisplatin is a potent antitumor agent that is widely used to treat various solid tumors, such as lung, ovarian, or testicular cancer, as well as certain forms of lymphoma (1). The antitumor effect of cisplatin requires its intracellular bioactivation by replacement of chloride by water molecules, resulting in the formation of highly reactive molecules that bind to DNA and induce cytotoxic lesions in tumors (2). However, the unwanted accumulation of cisplatin in healthy cells can also trigger genotoxicity. Indeed, the clinical use of cisplatin is limited by a variety of serious adverse effects, including nephrotoxicity (3-5). In the kidney, cisplatin mainly promotes the injury and death of proximal tubule cells through several pathways, including apoptosis. Both the antitumor properties as well as the side effects of cisplatin depend on its intracellular accumulation, which is mediated, at least in part, by membrane transporters. In the kidney, organic cation transporter type 2 (OCT2) and copper transporters (Ctr1 and Ctr2) mediate the basolateral uptake of cisplatin in renal proximal tubule cells, whereas other efflux transporters located at the apical plasma membrane, including copper-transporting ATPase α and β (ATP7A, ATP7B) and multidrug and toxin efflux proteins 1 and 2 (MATE1, MATE2), are involved in the secretion of cisplatin into the urine (6-7). Cisplatin nephrotoxicity is cumulative and dose-dependent, resulting in tubular lesions associated with lower glomerular filtration rates (8-9). Cisplatin has subsequently been reported to promote acute renal failure in up to 35% of patients, leading to dose adjustments or even discontinuation of cisplatin, which adversely affects patient outcomes (10-11).

[0003] In clinical practice, prevention of cisplatin-induced nephrotoxicity still relies heavily on nonspecific interventions, such as saline hydration or magnesium infusion (3, 11).

[0004] Therefore, there is an urgent medical need for strategies to reduce cisplatin-induced nephrotoxicity without interfering with the effectiveness of cisplatin in controlling tumor growth.

[0005] Adenosine plays a major role in intracellular and tissue homeostasis (13-15). Its physiological functions rely on four G protein-coupled receptors, namely A1, A2A, A2B, and A3 (16-19). Adenosine is important for several aspects of renal physiology, and adenosine and its receptors have been implicated in various types of renal injury (22-26). In particular, pharmacological blockade of the A1 receptor using several antagonists, such as tonapophilin (27), 8-cyclopentyl-1,3-dipropylxanthine (28), or KW-3902 (29), has been reported to protect against cisplatin nephrotoxicity in rodent models. Adenosine A 2A Receptor (A 2A A also regulates renal pathologies of various etiologies, such as ischemia-reperfusion injury (30-31), fibrosis (25, 32), diabetic nephropathy (33) or glomerulonephritis (34). 2A The role of the receptor remains unclear in the context of chemotherapy drug-induced toxicity.

[0006] Summary of the invention: The present invention relates to a therapeutically effective amount of selective A 2A Adenosine receptor (A 2A The present invention relates to a method for the treatment of chemotherapy-induced nephrotoxicity in a subject in need thereof, comprising the step of administering to said subject a medicament for the treatment of nephrotoxicity induced by chemotherapy drugs. In particular, the present invention is defined by the claims.

[0007] Detailed description of the invention: Cisplatin is a potent chemotherapeutic drug that is widely used to treat various solid cancers. However, its clinical effectiveness is strongly limited by frequent and severe adverse effects, such as nephrotoxicity. Therefore, there is an urgent medical need to identify novel strategies to limit the toxicity induced by chemotherapeutic drugs. Here, the inventors have found that adenosine A 2A We provide evidence that the receptor antagonist istradefylline (KW-6002) significantly protects against chemotherapy-induced nephrotoxicity in acute and subchronic experimental models of renal cisplatin intoxication. Importantly, we also demonstrate that the antitumor properties of chemotherapy drugs were not altered and could even be enhanced at the molecular level by the antagonist in tumor-bearing mice.

[0008] Indeed, the inventors have surprisingly found that some nephrotoxic chemotherapy drugs, particularly platinum-containing chemotherapy drugs, are apoptotic in the kidney. 2A The inventors further discovered that selective A 2A We have found that administration of adenosine receptor antagonists can reduce nephrotoxicity induced by chemotherapy agents such as DNA alkylating agents, particularly platinum-containing chemotherapy agents. 2A Adenosine receptor antagonists were used to 2A By binding to adenosine receptors, A 2A The reduction or prevention of nephrotoxicity through adenosine receptor blockade was unexpected. Together, the results of the present invention support the use of ADC as a novel and valuable preventative approach for the clinical management of patients undergoing treatment with chemotherapeutic drugs (e.g., cisplatin). 2A The use of adenosine receptor antagonists (eg, istradefylline) is advocated.

[0009] The present invention relates to a therapeutically effective amount of selective A 2A Adenosine receptor (A 2A The present invention relates to a method for the treatment of nephrotoxicity induced by a chemotherapy agent in a subject in need thereof, comprising the step of administering to said subject an anti-nephrotoxic agent (R) antagonist.

[0010] The present invention relates to a therapeutically effective amount of selective A 2A Adenosine receptor (A 2A The present invention relates to a method for the treatment of cisplatin-induced nephrotoxicity in a subject in need thereof, comprising the step of administering to said subject a cisplatin (R) antagonist.

[0011] The present invention provides a selective ADP-1 inhibitor for use in a subject in need thereof for the treatment of nephrotoxicity induced by chemotherapy drugs. 2A Adenosine receptor (A 2A R) relating to antagonists.

[0012] The present invention provides a selective ADP-1 inhibitor for use in a subject in need thereof for the treatment of cisplatin-induced nephrotoxicity. 2A Adenosine receptor (A 2A R) relating to antagonists.

[0013] In some embodiments, the present invention relates to istradefylline (KW-6002) or a derivative thereof for use in a subject in need thereof for the treatment of chemotherapy-induced nephrotoxicity.In some embodiments, the present invention relates to istradefylline (KW-6002) or a derivative thereof for use in a subject in need thereof for the treatment of cisplatin-induced nephrotoxicity.

[0014] The term "subject" as used herein refers to mammals, such as rodents, cats, dogs, and primates. In particular, the subject according to the present invention is a human. In particular, the subject according to the present invention is an adult. In particular, the subject according to the present invention is a child. In particular, the subject according to the present invention is a teenager. In particular, the subject according to the present invention is a newborn. The term "subject" as used herein includes "patient".

[0015] The term "nephrotoxic" as used herein refers to drugs that can induce kidney damage and / or kidney inflammation when administered at or below a pharmacologic effective dose, preferably when administered at a pharmacologic effective dose. In particular, a nephrotoxic drug refers to a drug that can increase plasma urea. Drugs that can induce nephrotoxicity are referred to as "chemotherapeutic drugs that induce nephrotoxicity."

[0016] In some embodiments, the chemotherapy agent that induces nephrotoxicity is induced by cisplatin and is referred to as "cisplatin-induced nephrotoxicity."

[0017] As used herein, "A 2A Adenosine receptor receptors (A 2A The term adenosine receptor (ADR), also known as ADORA2A or RDC8, refers to the adenosine receptor and also refers to the human gene that encodes it. The protein is a member of the G protein-coupled receptor (GPCR) family, with seven transmembrane α-helices and an extracellular N-terminus and an intracellular C-terminus. The protein plays an important role in many biological functions, such as regulating heart rate and circulation, cerebral and renal blood flow, immune function, pain, and sleep. It is also involved in pathophysiological conditions, such as inflammatory diseases and disorders.

[0018] As used herein, the term "antagonist" or "inhibitor" refers to an agent (i.e., a molecule) that inhibits or blocks the activity of a receptor. For example, an antagonist refers to a molecule that inhibits or blocks the activity of a receptor.

[0019] As used herein, A 2A The term "biological activity" of adenosine receptors refers to nephrotoxicity associated with inflammatory responses and apoptosis, as exemplified by increased mRNA expression of interleukin 6 (IL6) and tumor necrosis factor (Tnf).

[0020] Option A 2ATests for determining the ability of a compound to be an A R antagonist are well known to those skilled in the art. In a preferred embodiment, the selective antagonist is 2A in a manner sufficient to inhibit the biological activity of R. 2A Binds specifically to R. A 2A Bonds to R and A 2A Inhibition of the biological activity of R can be determined by any competitive assay known in the art. For example, the assay can be performed using a selective A 2A As an R antagonist, the substance to be tested is an A 2A The binding ability can consist of determining the ability of the antibody to bind to R. The binding ability is reflected by measuring Kd. As used herein, the term "KD" is meant to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). KD values ​​for binding to biomolecules can be determined using methods well established in the art. In a specific embodiment, "A 2A A selective antagonist that specifically binds to human Aβ with a KD of 1 μM or less, 100 nM or less, 10 nM or less, or 3 nM or less. 2A A competitive assay is then set up to determine whether the substance is an inhibitor of A 2A The ability to inhibit the biological activity of R may be determined. Functional assays such as a) evaluating the ability to reduce nephrotoxicity associated with an inflammatory response and / or nephrotoxicity associated with apoptosis may be considered. Those skilled in the art will be able to determine the ability of selective A 2A R antagonist, A 2A Neutralizing, blocking, inhibiting, eliminating, reducing or interfering with the biological activity of R can be readily determined.

[0021] In some embodiments, the selective antagonists of the present invention (i.e., selective A 2A R antagonist) is A 2A It acts through direct interaction with adenosine receptors.

[0022] As used herein, "A 2AAdenosine receptor (A 2A The term "R) antagonist" is used in 2A It refers to an adenosine receptor antagonist.

[0023] In some embodiments, the selective A 2A R antagonists can be used to reduce acute nephrotoxicity induced by injection of nephrotoxic chemotherapeutic drugs, especially chemotherapeutic drugs that contain cisplatin as active compound, especially as the only active compound. These selective antagonists can also be used to reduce chronic nephrotoxicity induced by one or several (e.g. acute, subchronic models) administrations of nephrotoxic chemotherapeutic drugs, as described above; the drug is active for several days after administration.

[0024] As used herein, the term "selective antagonist" refers to a selective A 2A The selective antagonist according to the present invention is an abbreviation of "adenosine receptor antagonist". 2A Refers to an antagonist that specifically targets adenosine receptors.

[0025] As used herein, the term "derivative" refers to a derivative that is a derivative of the base compound (i.e., selective A 2A antagonists), meaning that they share a core structural component.

[0026] As used herein, the term "analog" refers to a compound that is structurally and functionally related to another compound; the compound and the analog share a high degree of structural similarity and have similar biological functions.

[0027] In some embodiments, the selective A 2AThe adenosine receptor antagonist is preferably selected from among istradefylline (KW-6002) or a derivative thereof, MSX-3, SCH-58261, NIR178 (also known as PBF-509), ciforadenant, AB928, AZD4635, EOS100850, inupadenant (EOS-850), EXS21546, TT-10 and TT-53, and pharma- ceutically acceptable salts thereof. Preferably, in all embodiments, the selective A 2A The adenosine receptor antagonist is preferably selected from among istradefylline (KW-6002) or a derivative thereof, MSX-3, SCH-58261, tozadenant (SYN115), preladenant (SCH-420814), NIR178 (also known as PBF-509), ciphoradenant, etrumadenant (AB928), imadenant (AZD4635), EOS100850, inupadenant (EOS-850), EXS21546, TT-10 and TT-53. 2A Receptor (A 2A Examples of substituted purine compounds that act as R) antagonists or derivatives thereof also include compounds disclosed in International Patent Application No. WO2021179074. For example, WO2021179074 includes the following compounds: [ka] [ka] [ka]

[0028] These selective antagonists have demonstrated their effectiveness in reducing nephrotoxicity induced by chemotherapy drugs. They can be used alone, sequentially, or in a mixture of at least two antagonists.

[0029] In some embodiments, the present invention also relates to a selective A inhibitor selected from among istradefylline (KW-6002) or a derivative thereof, MSX-3, SCH-58261, NIR178 (also known as PBF-509), ciphoradenant, AB928, AZD4635, EOS100850, inupadenant (EOS-850), EXS21546, TT-10 and TT-53 for use as a nephrotoxicity reducing agent in the treatment of cancer with at least one chemotherapeutic agent. 2A Regarding adenosine receptor antagonists, the chemotherapeutic agents can induce nephrotoxicity when administered to a subject, and in the subject, 2A The chemotherapeutic agent is capable of inducing overexpression of adenosine receptors, said chemotherapeutic agent being in particular selected from among pharmaceutical compositions comprising as active compound a platinum-containing compound, in particular a compound selected from among cisplatin, carboplatin, oxaliplatin and pharma- ceutical acceptable salts thereof, more particularly cisplatin.

[0030] The term "nephrotoxicity reducing agent" as used herein refers to a pharma- ceutically acceptable compound or composition that, when administered to a subject, can reduce at least one parameter selected from plasma urea concentration, renal histological score, creatinemia, proteinuria, neutrophil gelatinase-associated lipocalin (NGAL) relative expression, and kidney injury molecule-1 (KIM-1) relative expression. The increase in the above parameters is induced by the administration of at least one chemotherapeutic agent. Cisplatin refers to cis-diammine-dichloroplatinum(II).

[0031] As used herein, the term "KW-6002," also known as istradefylline, refers to 8-[(E)-2-(3,4-dimethoxyphenyl)vinyl]-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione. KW-6002 has the following CAS number: 155270-99-8 and the following chemical formula: [ka] It has.

[0032] As used herein, the term "MSX-3" refers to 3,7-dihydro-8-[(1E)-2-(3-methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)propyl-1-(2-propynyl)-1H-purine-2,6-dione disodium salt hydrate. MSX-3 has the following CAS number: 261717-23-1 and the following chemical formula: [ka] It has.

[0033] As used herein, the term "SCH-58261" refers to 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine. SCH-58261 has the following CAS number: 160098-96-4 and the following chemical formula: [ka] It has.

[0034] As used herein, the term "tozadenant" refers to 4-hydroxy-N-(4-methoxy-7-morpholin-4-yl-1,3-benzothiazol-2-yl)-4-methylpiperidine-1-carboxamide. Tozadenant has the following CAS number: 870070-55-6 and the following chemical formula: [ka] It has.

[0035] As used herein, the term "preladenant" refers to 2-(2-furanyl)-7-(2-(4-(4-(2-methoxyethoxy)phenyl)-1-piperazinyl)ethyl)-7H-pyrazolo(4,3-e)(1,2,4)triazolo(1,5-c)pyrimidin-5-amine. Preladenant has the following CAS number: 377727-87-2 and the following chemical formula: [ka] It has.

[0036] As used herein, the term "NIR178," also known as "taminadenanth" or "PBF-509," refers to 5-bromo-2,6-di(pyrazol-1-yl)pyrimidin-4-amine. NIR178 has the following CAS number: 1337962-47-6 and the following chemical formula: [ka] It has.

[0037] As used herein, the term "sifoladenant" refers to (S)-7-(5-methylfuran-2-yl)-3-((6-(((tetrahydrofuran-3-yl)oxy)methyl)pyridin-2-yl)methyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. Sifoladenant has the following CAS number: 1202402-40-1 and the following chemical formula: [ka] It has.

[0038] As used herein, the term "etrmadenant," also known as "AB928," refers to 3-(2-amino-6-(1-((6-(2-hydroxypropan-2-yl)pyridin-2-yl)methyl)-1H-1,2,3-triazol-4-yl)pyrimidin-4-yl)-2-methylbenzonitrile. AB928 has the following CAS number: 2239273-34-6 and the following chemical formula: [ka] It has.

[0039] As used herein, the term "imaladennant," also known as "AZD4635," refers to 6-(2-chloro-6-methyl-4-pyridinyl)-5-(4-fluorophenyl)-1,2,4-triazin-3-amine. Imaladenant has the following CAS number: 1321514-06-0 and the following chemical formula: [ka] It has.

[0040] As used herein, the term "EOS100850" refers to (+)-5-amino-3-{2-[4-(2,4-difluoro-5-{2-[(S)-methanesulfinyl]ethoxy}phenyl)piperazin-1-yl]ethyl}-8-(furan-2-yl)[1,3]thiazolo[5,4-e][1,2,4]triazolo[1,5-c]pyrimidin-2(3H)-one hydrochloride. EOS100850 has the following CAS number: 2411004-22-1 and the following chemical formula: [ka] It has.

[0041] As used herein, the term "inupadenant", also known as "ES-850", refers to 7-amino-10-[2-[4-[2,4-difluoro-5-[2-[(S)-methylsulfinyl]ethoxy]phenyl]piperazin-1-yl]ethyl]-4-(furan-2-yl)-12-thia-3,5,6,8,10-pentazatricyclo[7.3.0.02,6]dodeca-1(9),2,4,7-tetraen-11-one. Inupadenant has the following CAS number: 2246607-08-7 and the following chemical formula: [ka] It has.

[0042] As used herein, the term "TT-10" refers to (2-(allylamino)-4-aminothiazol-5-yl)(5-fluorothiophen-2-yl)methanone. TT-10 has the following CAS number: 2230640-94-3 and the following chemical formula: [ka] It has.

[0043] As used herein, the term "EXS21546" refers to an A2AR selective antagonist that does not penetrate the central nervous system, manufactured by Exscientia, Inc. (Clinical Trial Identifier: NCT04727138).

[0044] As used herein, the term "TT-53" refers to a selective adenosine receptor antagonist manufactured by Tarus therapeutics.

[0045] The present invention also relates to a selective A combination with a chemotherapeutic agent. 2A Use of Adenosine Receptor Antagonists In some embodiments, the therapeutic use of chemotherapeutic agents induces the development of nephrotoxicity.

[0046] The term "chemotherapy" as used herein has its general meaning in the art and refers to treatment consisting of administering to a patient a chemotherapeutic agent. Chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (such as the synthetic analog topotecan); bryostatins; kallistatin; CC-1065 (e.g., its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin I and cryptophycin 8); dolastatins; duocarmycins (e.g., synthetic analogues, such as KW-2189 and CBI-TMI; eleutherobin; pancratistatin; sarcodictyin; spongiostatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11; dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g. morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, Esorubicin, Idarubicin, Marcelomycin, Mitomycins such as Mitomycin C, Mycophenolic acid, Nogalamycin, Olivomycin, Peplomycin, Potfilomycin, Puromycin, Querramycin, Lodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin; Antimetabolites such as Methotrexate and 5-Fluorouracil (5-FU); Folic acid analogues such as Denopterin, Methotrexate, Pteropterin, Trimetrexate; Purine analogues such as Fludarabi purines, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues, e.g. ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens, e.g. calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal drugs, e.g. aminoglutethimide, mitotane, trilostane;folic acid supplements, e.g. folinic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluratone Sil; Amsacrine; Bestravsil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Eflornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium acetate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol: Nitracrine; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex); Razoxane; Rhizoxin;Schizofuran;spirogermanium;tenuazonic acid;triaziquone;2,2',2''-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidin);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("Ara-C");cyclophosphamide;thiotepa;taxoids, such as paclitaxel and docetaxel;chlorambucil;gemcitabine;6-thioguanine;mercaptopurine;methotrexate;platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0047] As used herein, the term "chemotherapeutic agent" refers to any pharma- ceutically acceptable composition that is therapeutically or pharma- ceutical effective in treating cancer. A chemotherapeutic agent can be, for example, a DNA alkylating agent.

[0048] As used herein, the term "nephrotoxic chemotherapeutic agent" refers to any pharma- ceutically acceptable composition that is therapeutically or pharma- ceutical effective in the treatment of cancer that can induce renal damage and / or renal inflammation when administered at or below a pharma- ceutical effective dose, preferably when administered at a pharma- ceutical effective dose.

[0049] In some embodiments, the nephrotoxic chemotherapeutic agent is selected from among DNA alkylating agents, particularly from among platinum-containing chemotherapeutic agents, more particularly from among cisplatin, carboplatin, oxaliplatin, and pharma- ceutically acceptable salts thereof.

[0050] As used herein, the term "cisplatin" refers to cis-diamminedichloroplatinum(II), a drug used in chemotherapy primarily because it blocks DNA synthesis and induces apoptosis through a p53-mediated mechanism of action. Cisplatin has the following CAS number: 15663-27-1 and the following chemical formula: [ka] It has.

[0051] As used herein, the term "oxaliplatin" refers to [(1R,2R)-cyclohexane-1,2-diamine](ethanedioate-O,O')platinum(II). Oxaliplatin has the following CAS number: 63121-00-6 and the following chemical formula: [ka] It has.

[0052] As used herein, the term "carboplatin" refers to cis-diammine(cyclobutane-1,1-dicarboxylate-O,O')platinum(II). Carboplatin has the following CAS number: 41575-94-4 and the following chemical formula: [ka] It has.

[0053] As used herein, "A 2AThe term "capable of inducing overexpression of adenosine receptors" refers to a drug that, when administered in a given amount and in a pharmacologic effective amount or dose, e.g., can induce overexpression of adenosine receptors. 2A Relative expression of adenosine receptors means 1-fold or greater, more particularly 1.1-fold or greater.

[0054] The present invention relates to selective A 2A The present invention relates to novel nephrotoxicity-reducing agents which may be used in the treatment of cancer by administration of an adenosine receptor antagonist and / or at least one nephrotoxic chemotherapeutic agent.

[0055] In some embodiments, the present invention also provides a therapeutically effective amount of a selective A 2A The present invention relates to a method for the treatment of cancer in a subject in need thereof comprising a combination of an adenosine receptor antagonist and a chemotherapeutic agent.

[0056] In some embodiments, the present invention also relates to a method for the treatment of cancer in a subject in need thereof comprising a therapeutically effective amount of a combination of KW-6002 or a derivative thereof and cisplatin.

[0057] The present inventors have 2A 1 shows that adenosine receptor antagonists improve the antitumor effect of cisplatin.

[0058] In some embodiments, the present invention also provides a selective Aβ inhibitor for use as an antitumor treatment in a subject in need thereof. 2A Combinations of adenosine receptor antagonists and chemotherapy agents.

[0059] In some embodiments, the present invention also relates to the use of a combination of KW-6002 or a derivative thereof and cisplatin as an anti-tumor treatment in a subject in need thereof.

[0060] The term "cancer" as used herein has its general meaning in the art and refers to a group of diseases involving abnormal cell growth that has the ability to invade or spread to other parts of the body. The term "cancer" further includes both primary and metastatic cancers. Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, digestive tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, cancer may be classified as any of the following histological types, in particular: malignant neoplasm; cell carcinoma; undifferentiated cell carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial cell carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; familial polyposis coli adenocarcinoma; solid cell carcinoma; malignant carcinoma bronchiolar and alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; Endometrioid carcinoma; skin adnexal carcinoma; apocrine carcinoma; sebaceous gland carcinoma; auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammation foetal carcinoma; Paget's disease of the breast; pancreatic acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; malignant glomus tumor; malignant melanoma; amelanotic melanoma; superficial spreading malignant melanoma; malignant melanoma in giant pigmented nevus tumor;epithelioid cell malignant melanoma;malignant blue nevus;sarcoma;fibrosarcoma;malignant fibrous histiocytoma;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian goiter;choriocarcinoma;malignant mesonephroma;angiosarcoma;Malignant hemangioendothelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblastic sarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Medulloblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Anaplastic neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Malignant meningioma;Neurofibrosarcoma;Malignant The tumor may be, but is not limited to, schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; lateral granuloma; small lymphocytic lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0061] As used herein, the term "administering" or "administration" refers to administering a substance (as it exists outside the body) (e.g., selective A 2A Adenosine receptor (A 2A R) antagonist) to a subject, for example, by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or its symptoms are treated, the administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, the administration of the substance is typically performed before the onset of the disease or its symptoms.

[0062] "Therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of a drug may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or portion of the antibody are outweighed by the therapeutically beneficial effects. The effective dose and dosage regimen for a drug depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start a dose of a drug used in a pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, an appropriate dose of the composition of the present invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the progression of a disease. Those skilled in the art will be able to determine such amounts based on such factors as the patient's size, the severity of the subject's symptoms, and the specific composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a drug is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1 mg-10 mg / kg, such as about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and can be administered, for example, near the target site. The dosage regimen in the above methods of treatment and use is adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of treatment is monitored, eg, at predetermined time points, during therapy.As a non-limiting example, the treatment according to the present invention may be carried out in an amount of about 0.1 to 100 mg / kg per day, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 1 A single or divided dose may be provided every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, on at least one day of the 5th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th or 40th day, or alternatively for at least one week of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th week, or any combination thereof.

[0063] In some embodiments, the selective A 2A The therapeutically effective amount of the adenosine receptor antagonist may be equal to or greater than 10 mg and equal to or less than 350 mg, in particular equal to 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg or 120 mg, 160 mg, 180 mg, 210 mg or 240 mg. 2A When the adenosine receptor antagonist is KW-6002 or a derivative thereof, its therapeutically effective amount is preferably below the maximum value, which may be equal to or greater than 60 mg to 230 mg, more preferably equal to or greater than 70 mg, or equal to or less than 210 mg. As explained later in this specification, when the amount of this particular selective antagonist exceeds a threshold value, it no longer has activity as a nephrotoxicity reducing agent, particularly in reducing nephrotoxicity induced by chemotherapeutic drugs (e.g., cisplatin).

[0064] In some embodiments, the therapeutically effective amount of the chemotherapeutic agent can be equal to 80 mg or more and equal to 1400 mg or less, particularly 140 mg, 147 mg, 175 mg, 630 mg, 692 mg, and 700 mg. When the chemotherapeutic agent contains a chemotherapeutic agent (e.g., cisplatin) as an active compound, particularly when the drug contains a chemotherapeutic agent (e.g., cisplatin) as the only active compound, particularly suitable values ​​are 140 mg, 700 mg, and 1400 mg.

[0065] The term "combination" as used herein is intended to refer to all dosage forms that provide a first drug together with other (second, third) drugs. The drugs may be administered simultaneously, separately, or sequentially in any order. According to the present invention, the drug is administered to the subject using any suitable method that allows the drug to reach the chondrocytes of the bone growth plate. In some embodiments, the drug is administered to the subject systemically (i.e., via systemic administration). Thus, in some embodiments, the drug is administered to the subject so that it enters the circulatory system and is distributed throughout the body. In some embodiments, the drug is administered to the subject by local administration, for example, by local administration to growing bone.

[0066] As used herein, the term "combination treatment," "combination therapy," or "combination of therapies" refers to treatment using more than one drug therapy. The combination therapy can be a dual therapy or a two-drug combination therapy.

[0067] As used herein, the term "concurrent administration" refers to administration of two active ingredients by the same route at the same time or substantially the same time. The term "separate administration" refers to administration of two active ingredients by different routes at the same time or substantially the same time. The term "sequential administration" refers to administration of two active ingredients at different times, where the routes of administration are the same or different.

[0068] In some embodiments, the selective A 2AThe adenosine receptor antagonist will be administered prior to administration of the chemotherapeutic agent, preferably at least one hour prior to administration of the chemotherapeutic agent.

[0069] In some embodiments, the chemotherapy drug can be administered in one dose. In this case, the selective antagonist can be administered before, preferably at least 1 hour before, the administration of the chemotherapy drug. It can also be administered before or after the administration of the chemotherapy drug. For example, it can be administered at least once, the day after the administration of the chemotherapy drug, for example, once a day, for 1, 2, 5, or 8 days after the administration of the chemotherapy drug. In particular, when the chemotherapy drug contains cisplatin as an active compound, preferably as the only anticancer active compound and as the only active compound, the chemotherapy drug is administered several times, once a day for several days, for example (for example, once a day for a week), and the selective antagonist is preferably administered before, for example, at least 1 hour before, each administration of the chemotherapy drug. The selective antagonist can also be administered once a day, after the last administration of the chemotherapy drug, in particular for at least 1 or 2 days after the last administration of the chemotherapy drug, for example, for 5 days after the last administration of the chemotherapy drug.

[0070] The present invention also provides a therapeutically effective amount of a selective A 2A Adenosine receptor (A 2A R) The present invention also relates to combinations of antagonists with chemotherapeutic agents.

[0071] The present invention also provides a therapeutically effective amount of a selective A 2A Adenosine receptor (A 2A R) The present invention also relates to combinations of antagonists with chemotherapeutic agents.

[0072] In a particular embodiment, the present invention relates to a therapeutically effective amount of KW-6002 in combination with cisplatin for use in the treatment of nephrotoxicity induced by chemotherapy agents.

[0073] In a particular embodiment, the present invention relates to a therapeutically effective amount of KW-6002 in combination with cisplatin for use in the treatment of cisplatin-induced nephrotoxicity.

[0074] The present invention also provides a method for treating nephrotoxicity induced by chemotherapy drugs, comprising administering to said patient a combination of: i) a selective A 2A Adenosine receptor (A 2A R) antagonists and ii) chemotherapeutic agents.

[0075] The present invention also provides a method for treating cisplatin-induced nephrotoxicity comprising administering to a subject a therapeutically effective amount of a selective cisplatin-containing compound, comprising administering to said subject a therapeutically effective amount of a selective cisplatin-containing compound, 2A Adenosine receptor (A 2A R) antagonists and ii) chemotherapeutic agents.

[0076] In a particular embodiment, the present invention relates to i) KW-6002 and ii) cisplatin for simultaneous, separate or sequential use in the treatment of nephrotoxicity induced by chemotherapy agents.

[0077] In a particular embodiment, the present invention relates to i) KW-6002 and ii) cisplatin for simultaneous, separate or sequential use in the treatment of cisplatin-induced nephrotoxicity.

[0078] The present invention also provides a therapeutically effective amount of selective A 2A Adenosine receptor (A 2A The present invention relates to a pharmaceutical composition comprising a selective A R antagonist and a chemotherapeutic agent. 2A Adenosine receptor (A 2A R) The antagonist is KW-6002 and the chemotherapy drug is cisplatin.

[0079] More particularly, the pharmaceutical compositions according to the present invention are suitable for treating nephrotoxicity induced by chemotherapeutic agents.

[0080] More particularly, the pharmaceutical compositions according to the present invention are suitable for treating cisplatin-induced nephrotoxicity.

[0081] Option A above 2A Adenosine receptor (A 2AR) antagonists and / or chemotherapeutic agents may be formulated with pharma- ceutically acceptable excipients, optionally with sustained release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered appropriately to a mammal, particularly a human. A pharma- ceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any kind. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, parenteral, topical or rectal administration, the active ingredient may be administered to animals and humans, alone or in combination with another active ingredient, in unit dosage form, in admixture with conventional pharmaceutical supports. Suitable unit dosage forms include oral route dosage forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, parenteral, and intranasal dosage forms, and rectal dosage forms. Typically, pharmaceutical compositions contain a vehicle that is pharma- ceutically acceptable for injectable formulations. These may be, in particular, isotonic and sterile saline solutions (such as monosodium or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dried, in particular lyophilized, compositions that allow the reconstitution of an injection solution when sterile water or saline is added, as the case may be. Pharmaceutical dosage forms suitable for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. The polypeptide (or the nucleic acid encoding it) may be formulated into the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier may also be a solvent or dispersion medium, including, for example, water, ethanol, polyol (such as, for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition substances that delay absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptide in the required amount in the appropriate solvent with several other ingredients as enumerated above, as required, followed by sterilization filtration. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients as enumerated above.In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from the solution thereof that has previously been sterile filtered. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like may also be used. For parenteral administration in aqueous solutions, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or dextrose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of liquid for subcutaneous infusion therapy, or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person administering will, in any event, determine the appropriate dose for the individual subject.

[0082] The present invention will be further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0083] [Figure 1]Illustration of various animal procedures. (A) Acute kidney injury induced by cisplatin. C57BL6 / J 8-week-old male mice were administered a single intraperitoneal injection of 10 mg / kg cisplatin (cis) and sacrificed (E) 3 days after injection (D). (B) Subchronic cisplatin-induced kidney injury. C57BL6 / J 8-week-old male mice were injected intraperitoneally with cisplatin (3 mg / kg) once a day for 6 days and sacrificed 72 hours after the last injection. (C) Dosing schedule of KW-6002 in the acute model. C57BL6 / J 8-week-old male mice were randomized into 4 groups: vehicle, KW-6002, cisplatin, or KW-6002 / cisplatin. Cisplatin administration was performed as indicated in A. The first dose of KW-6002 (3 mg / kg) was administered 1 day before cisplatin treatment and once daily until sacrifice. (D) Dosing schedule of KW-6002 in the subchronic model. C57BL6 / J 8-week-old male mice were randomized into 4 groups: vehicle, KW-6002, cisplatin, or KW-6002 / cisplatin. Cisplatin administration was performed as shown in B. The first dose of KW-6002 (3 mg / kg) was administered 5 days before cisplatin treatment and once daily until sacrifice. (E) Effect of KW-6002 in a subchronic cisplatin-treated LLC syngeneic tumor mouse model. KW-6002 and cisplatin administration were as described in D. LLC cells (10 million) in PBS:Matrigel (1:1, total volume 10 μl) were injected subcutaneously into the right flank of all animals. When tumor volumes reached 100 mm3, mice were randomized into four groups: vehicle, KW-6002, cisplatin, or KW-6002 / cisplatin. [Diagram 2]KW-6002 protects against cisplatin-induced renal injury in acute (A-F) and subchronic (G-L) models. (A,G) Quantification of BUN (blood urea nitrogen). (B,H) Histological scores of renal injury. Gene expression of renal injury markers NGAL (C,I) and KIM-1 (D,J). Gene expression of inflammatory markers Tnfα (E,K) and IL6 (F,L). Results are expressed as mean ± SEM. **p<0.01, ***p<0.001 vs. vehicle; **p<0.01, ***p<0.001 vs. cisplatin (n=6-17 animals / group; one-way ANOVA followed by Tukey post-hoc test). [Diagram 3] KW-6002 protects against nephrotoxicity without attenuating the antitumor properties of cisplatin in a syngeneic in vivo mouse model. Absolute tumor sizes in different animal groups. Results are expressed as mean ± SEM. ***p<0.001 vs. vehicle; ***p<0.001 vs. cisplatin (n=13–25 animals / group; one-way ANOVA followed by Tukey post-hoc test). **p<0.01 (n=6–10 animals / group; two-way ANOVA). [Figure 4] KW-6002 limits cisplatin accumulation in the kidney but not in the tumor. (A) Platinum quantification (n=12-20 mice / group) and relative expression of influx transporters (CTR1 and OCT2) and efflux transporters (ATP7B, MATE1, MATE2, and ABCC2: n=10-12 mice / group) in the kidney. (B) Platinum quantification (n=12-20 mice / group) and relative expression of influx transporters (CTR1) and efflux transporters (ATP7B, MATE1, and ABCC2: n=5-6 mice / group) in the tumor. Results are expressed as mean ± SEM. *p<0.05, ***p<0.001 vs. vehicle; *p<0.05, ***p<0.001 vs. cisplatin (one-way ANOVA followed by Tukey post-hoc test). (C) Active efflux in RPTEC / hTERT cells. ***p<0.05 vs. vehicle; *p<0.05 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test. Results are means ± SEM (n=3 independent experiments). [Diagram 5]KW-6002 protects against cisplatin-induced renal injury in a cisplatin toxicity cumulative model. Quantification of BUN. Data are mean ± SEM. *p<0.05, ***p<0.001 vs. vehicle; p<0.05, **p<0.01, ***p<0.001 vs. cisplatin (n=6 animals / group; 2-way ANOVA followed by Tukey post-hoc test). [Figure 6] KW-6002 reduces cisplatin-induced nephrotoxicity associated with apoptosis, lipid metabolism, and oxidative stress. (A) Tail moment assessed by comet assay; 50 cells were analyzed per condition. (B and C) KW-6002 attenuates cisplatin-induced renal lipid accumulation assessed by oil red staining. (B) Representative quantification (n=5 randomly selected fields per staining). ***p<0.001 vs. vehicle; **p<0.01 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test (n=3 independent experiments). (C) Representative quantification (n=8 randomly selected fields per section). Results are mean ± SEM. ***p<0.05 vs. vehicle; *p<0.05 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test (n=3 mice / group). (D and E) KW-6002 attenuated cisplatin-induced oxidative stress in a subchronic cisplatin mouse model. Gene expression of Nrf2 (D) and HO1 (E). **p<0.01 and ***p<0.001 vs. vehicle; *p<0.05 and **p<0.01 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test. Results are mean ± SEM (n=6-9 mice / group). (F-H) KW6002 attenuated cisplatin-induced oxidative stress in RPTEC / hTERT1 cells. (F and G) Gene expression of Nrf2 (F) and HO1 (G). (H) Catalase activity. *p<0.05 and **p<0.01 vs. vehicle; *p<0.05 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test. Results are means ± SEM (n = 3 independent experiments). [Figure 7]KW6002 does not interfere with the antitumor effect of cisplatin. (A and B) LLC1 cells were exposed to 2 μM cisplatin in the presence or absence of 10 nM KW6002 for 24 h (A) or 6 h (B). Caspase 3 / 7 activity (n = 3 independent experiments) (A) and the number of γH2AX nuclear foci (n = 36-63 nuclei / group) (B) were determined. Results are means ± SEM. ***p < 0.001 vs. vehicle; **p < 0.01 and ***p < 0.001 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test. (C, D, G, and H) H1975 cells were exposed to 20 μM cisplatin in the presence or absence of 10 nM KW6002 for 24 h. Quantification of caspase 3 (C), PARP (poly ADP-ribose polymerase) (D), cyclin D1 (G), and PCNA (proliferating cell nuclear antigen) (H). *p<0.05 and **p<0.01 vs vehicle; **p<0.01 vs cisplatin; one-way ANOVA followed by Tukey post-hoc test. Results are expressed as mean ± SEM (n=3 independent experiments). (E) Tail moment was assessed by comet assay. A total of 50 cells per condition were analyzed. ***p<0.001; one-way ANOVA followed by Tukey post-hoc test. (F) H1975 cells were exposed to 50 μM cisplatin in the presence or absence of 10 nM KW6002 for 6 h, and the number of γH2AX nuclear foci was counted. Results are expressed as mean ± SEM. *p<0.05 vs. vehicle; *p<0.01 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test (n=30–42 nuclei per condition). (I) KW6002 increased the expression of γH2AX in tumors derived from the LLC1 syngeneic mouse model. Quantification of γH2AX nuclear expression. The percentage of positive cells was calculated using 5 randomly selected fields per staining. *p<0.05 vs. vehicle; *p<0.01 vs. cisplatin; one-way ANOVA followed by Tukey post-hoc test (n=5 / group). [Figure 8]KW6002 prevents nephrotoxicity and neurotoxicity without attenuating the antitumor properties of cisplatin in a mEERL syngeneic in vivo mouse model. (A-C) KW6002 attenuated cisplatin-induced nephrotoxicity as estimated by mRNA levels of KIM1 (A) and inflammatory markers tumor necrosis factor alpha and Il6 (B and C). *p<0.05 and ***p<0.001 vs. vehicle; *p<0.01 and **p<0.01 vs. cisplatin; one-way ANOVA (n=5-6 / group). (D) Mechanical sensitivity measured by von Frey hairs in mice in response to cisplatin and / or KW6002. Arrows indicate injection of cisplatin and / or PBS. Data are mean ± SEM. ***p<0.001 vs. vehicle; ***p<0.001 vs. cisplatin; two-way ANOVA (n=5 / group). (E) Absolute tumor size of animals in different groups. Results show mean ± SEM. ***p<0.001 vs. vehicle; *p<0.05 vs. cisplatin; two-way ANOVA (n=5-6 animals / group). [Figure 9] MSX-3 protects against cisplatin-induced renal injury in a mouse model. (A) Quantification of BUN. (B) Gene expression of the inflammatory marker TNFα. Results are expressed as mean ± SEM. **p<0.01, ***p<0.001 vs. vehicle; **p<0.01, ***p<0.001 vs. cisplatin (n=6–17 animals / group); one-way ANOVA followed by Tukey post-hoc test).

[0084] Working Example Materials and Methods Animals and treatments Animal experiments were adapted from 63–63. Animal procedures were performed on 8–10 week old male C57Bl6 / J mice (Janvier Institute). Mice were fed standard laboratory diet with water and food ad libitum and kept under constant environmental conditions with a 12-h light / dark cycle. Istradefylline (KW-6002; Tocris) was dissolved in a carrier solution consisting of 15% DMSO, 15% Cremophor (Sigma), and 70% saline solution (vehicle). Cisplatin (Accord Healthcare) was dissolved in saline solution. Acute cisplatin nephrotoxicity was induced after a single intraperitoneal (ip) injection (day 0) of 10 mg / kg cisplatin. Three days after this single injection, animals were sacrificed by cervical dislocation (Figure 1A). When KW-6002 (3 mg / kg) was tested for acute cisplatin toxicity, the drug was administered intraperitoneally once daily from days -1 to 2 (Figure 1C). Subchronic cisplatin toxicity was assessed after six once-daily injections of cisplatin (3 mg / kg) starting on day 0, and mice were sacrificed 72 hours after the last injection of cisplatin (day 8; Figure 1B). When KW-6002 was tested for subchronic cisplatin toxicity, the drug was administered intraperitoneally once daily from days -5 to 7 (Figure 1D).

[0085] LLC1 in vivo tumor model LLC1 cells (Lewis Lung Carcinoma cells, ATCC® CRL-1642™) were cultured in DMEM containing 10% fetal bovine serum and penicillin-streptomycin. LLC1 cells (10 7 ) was injected subcutaneously into the right flank of the animals. Tumors were measured twice a week with a caliper and their volumes were estimated using the following formula: 1 / 2 (length × width squared). 3 Upon reaching , mice were randomly assigned to four experimental groups (vehicle, KW-6002, cisplatin, and cisplatin / KW-6002) as shown in Figure 1E.

[0086] mEERL in vivo tumor model We used a validated human papillomavirus-positive oropharyngeal squamous cell carcinoma mouse model (not for sale) as previously described (77). This model consists of oropharyngeal epithelial cells derived from C57Bl / 6 male mice stably expressing human papillomavirus 16 viral oncogenes E6 and E7, H-Ras, and luciferase (mEERL cells). mEERL cells were grown to confluence in T75 flasks, after which the cells were trypsinized, harvested, washed three times with sterile PBS, and resuspended in 1 mL of sterile PBS to the appropriate concentration. Mice were injected subcutaneously into the right flank with 20 μL of a solution containing either 1,000,000 mEERL cells or PBS (vehicle). The day of injection of mEERL cells is indicated as day -14. Tumor volumes were monitored using Vernier digital calipers. Tumors with a volume of 100 mm 3 Once this was reached, mice were randomly assigned to one of three experimental groups (vehicle; cisplatin; or cisplatin plus KW6002).

[0087] Sample collection Prior to sacrifice by cervical dislocation, retroorbital blood samples were collected into heparinized tubes and centrifuged at 2000 rpm for 10 min at room temperature. Renal function was assessed by blood urea nitrogen (BUN) measurement using an AU480 Chemistry Analyzer (Beckman Coulter). At the time of sacrifice, kidneys or LLC1 tumors were collected and stored in "RNA later" solution (Thermo Fisher), 4% neutral buffered formalin, or snap frozen in liquid nitrogen.

[0088] Renal histological analysis Formalin-fixed, paraffin-embedded sections (3 μm thick) were stained with hematoxylin and eosin (Sigma-Aldrich) or periodic acid-Schiff (Sigma-Aldrich). Sections were scored by a blinded neuropathologist. A kidney injury score grading scale of 0 to 5 was used to assess the severity of injury as follows: 0 = no lesions, 1 = minimal injury characterized by the occurrence of necrosis and debris; 2 = mild injury including single-cell necrosis, pyknotic cells, and apoptosis; 3 = moderate injury characterized by tubular distension, vacuolization, and some cellular debris; 4 = severe injury with occasional hyaline casts, patchy epithelial necrosis in all segments, and loss of the epithelial lining; and 5 = very severe injury characterized by widespread tubular epithelial necrosis in all segments, loss of the epithelial layer from many tubules, widespread intraluminal cellular debris, and frequent hyaline casts that were particularly prominent within the medullary region (67).

[0089] cell culture Renal proximal tubule epithelial cells (RPTECs) immortalized with the pLXSN-hTERT retroviral vector (ATCC® CRL-4031™) are a valid in vitro model to assess the deleterious effects of cisplatin ( 68 – 70 ). Cells were cultured in DMEM / F12 medium (Dulbecco's modified Eagle's medium and Ham's F12 medium, Thermo Fisher) supplemented with 1% penicillin / streptomycin, 5 pmol / L triiodo-L-thyronine, 10 ng / mL recombinant human epidermal growth factor, 3.5 μg / mL ascorbic acid, 5.0 μg / mL human transferrin, 5.0 μg / mL insulin, 25 ng / mL prostaglandin E1, 25 ng / mL hydrocortisone, 8.65 ng / mL sodium selenite, 0.1 mg / mL G418, and 1.2 g / L sodium bicarbonate (Sigma). Lewis lung carcinoma mouse LLC1 cells were cultured in DMEM Glutamax (Thermo Fisher) containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0090] Cell viability assay RPTEC / hTERT cells were cultured in 96-well plates (40,000 cells / well) and exposed to cisplatin (50 μM) in the absence or presence of KW-6002 (0.5-12.8 μM) for 48 h. Viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's recommendations.

[0091] Caspase 3 / 7 activity RPTEC cells were cultured in 96-well plates (40,000 cells / well) and exposed to cisplatin (50 μM) in the absence or presence of KW-6002 for 48 h. LLC1 cells were cultured in 96-well plates (10,000 cells / well) and after 24 h were exposed to 2 μM cisplatin in the absence or presence of 10 nM KW-6002 for 24 h. Apoptosis was assessed in RPTEC / hTERT and LLC1 cell lysates using the Caspase-Glo3 / 7 assay (Promega) according to the manufacturer's recommendations.

[0092] Catalase activity RPTEC / hTERT1 cells were cultured in 6-well plates (250,000 cells / well) and exposed to cisplatin (50 μM) in the presence or absence of KW-6002 (25 μM) for 48 h. Catalase activity was assessed using a catalase colorimetric activity kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0093] cell discharge Basal cellular efflux function was assessed using the EFLUXX-IDGreen multidrug resistance assay kit (ENZO Life Sciences). 5Cells / condition were harvested, washed with PBS, and incubated with EFLUXX-ID Green detection reagent for 30 min at 37°C, after which shedding was immediately measured by flow cytometry (CytoFLEX LX, Beckman Coulter). All experiments were performed in triplicate and 10,000 individual cells were measured. Data were analyzed using Kaluza analysis software (Beckman Coulter).

[0094] Comet assay Treated cells were suspended (60,000 cells / mL) in 0.5% low melting agarose (1613111, Bio-Rad) in PBS at 42°C. The suspension was then immediately spread onto a Comet slide (4250-200-03, R&D Systems). The agarose was cooled at 4°C for 20 min. Cell membranes were then permeabilized with lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1% Triton X-100 [pH 10]) for 1 h at 4°C. Slides were then equilibrated in electrophoresis buffer (pH 12.3: 2 mM EDTA, pH adjusted to 12.3 with NaOH) for 20 min at 4°C. Then, an electrophoretic magnetic field of 2.06 V / cm (98 V and approximately 176 mA in an electrophoretic system with electrodes 47.5 cm apart) was applied to RPTEC / hTERT1 cells for 5 min at 4°C, or to H1975 cells for 3 min 30 s. Electrophoretic migration was stopped by neutralizing the pH in a cold water bath for 10 min. DNA was stained with SYBR Green (S7563, Invitrogen, Thermo Fisher Scientific) for 20 min at room temperature according to the manufacturer's recommendations. Slides were photographed with an Axio Imager Z1 Apotome microscope (Zeiss). Images were analyzed using an ImageJ informative macro, which defined the boundaries of the comet head (nucleus) and tail (translocated DNA) and obtained the head fluorescence intensity, tail fluorescence intensity, and tail length. Calculation of tail moment was performed using the following formula: (comet tail length × tail fluorescence intensity) / total fluorescence intensity (head + tail).

[0095] Oil Red Staining 10 μm frozen kidney mouse sections were fixed with ethanol (60%) and then incubated with Oil Red O solution (Fisher Biotech) in isopropanol for 15 min. After several washes with double distilled water (ddH2O), the samples were incubated with hematoxylin for 3 min. Lipid droplets were stained red and nuclei appeared blue. RPTEC / hTERT cells were grown in 24-well plates (75,000 cells / well) on glass coverslips and exposed to cisplatin (50 μM) in the absence or presence of 25 μM KW-6002 for 48 h. RPTEC / hTERT cells were fixed with ethanol (60%) and then incubated with Oil Red O solution for 15 min. The cells were washed three times with double distilled water and incubated with hematoxylin for 3 min. Coverslips were rinsed with water and then mounted onto microscope slides using glycerol gelatin aqueous slide mounting medium (Sigma). Quantification was performed blindly using ImageJ software. Briefly, pictures were taken with a light microscope at 400x magnification and processed using color deconvolution with RGB vectors. The resulting red images were quantified using custom thresholds (0.173 for RPTEC / hTERT cells and 0.140 for kidney staining).

[0096] Immunostaining (tissue) Paraffin-embedded sections (3 μm thick) were deparaffinized with xylene and rehydrated in serial dilutions of ethanol. Antigen retrieval was then performed by incubation in 10 mM sodium citrate buffer below the boiling point. Tissues were permeabilized in 0.4% Triton X-100 solution and nonspecific binding was blocked with 5% bovine serum albumin in Tris-buffered saline solution for 2 h. Sections were then incubated overnight with anti-γH2AX antibody (1:50; #9718, Cell Signaling Technology). After washing, secondary antibody (A10042) was incubated for 45 min at room temperature. After washing again, nuclei were stained with 300 nM DAPI solution (D1306, Life Technologies, Thermo Fisher Scientific). Slides were analyzed using a Zeiss LSM880 confocal microscope. Quantification was performed using ImageJ. For immunofluorescence studies of free-floating sections, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, i.p.) and then transcardially perfused with cold NaCl (0.9%) and 4% paraformaldehyde in PBS (pH 7.4). Kidneys were removed, post-fixed in 4% paraformaldehyde for 24 h, cryoprotected in 30% sucrose, then frozen in isopentane (methyl-butane) at -40°C and stored at -80°C. Longitudinal kidney sections (40 μm) were obtained using a Leica cryostat. Free-floating sections were stored in PBS azide (0.2%) at 4°C. Longitudinal kidney sections were incubated with 10% donkey serum (D9663, MilliporeSigma) in PBS Triton X-100 (0.2%) for 1 h, followed by incubation with anti-A2AR primary antibody (1:50; GP-Af1000; Frontiers Institute) in Signal Boost (8114, Cell Signaling Technology) for 72 h at 4°C. Secondary antibody conjugated to Alexa Fluor568 (1:500; Life Technologies, Thermo Fisher Scientific) was incubated overnight at room temperature.Staining for lectin was performed by incubating sections for 1 h at room temperature with FITC conjugate of Lotus tetragonolobus (Vector Laboratories, FL-1321-2) diluted to 2 μg / mL in blocking medium. Sections were counterstained with DAPI (1:5,000; #62247, Thermo Fisher Scientific), mounted on superfrost slides, and air-dried. They were then covered with Vectashield Vibrance Antifade mounting medium (H-1700, Vector Laboratories). Images were acquired at 20x magnification using a Zeiss LSM710 confocal laser scanning microscope. Reconstruction of 3D confocal stack images was performed using Imaris software (Bitplane).

[0097] Immunostaining of γH2AX LLC1 cells were cultured in Lab-tek (15,000 cells / well) and after 24 hours were exposed to cisplatin (2 μM) for 6 hours in the absence or presence of 10 nM KW-6002. Cells were fixed with 4% paraformaldehyde, permeabilized with DPBS / 0.1% Triton X-100, and incubated with γH2AX (Ser139) antibody (1 / 400; #9718, Cell Signaling Technology) followed by Alexa Fluor488 secondary antibody (1 / 200; Life Technologies). Samples were examined under an immunofluorescence microscope (Leica™ DMI8) and nuclear foci of γH2AX were counted.

[0098] RNA extraction Total RNA from kidney samples was extracted with phenol / chloroform followed by precipitation in isopropanol as previously described ( 71 ). Total RNA from cultured kidney cells was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's recommendations.

[0099] RNA sequencing and analysis RNA sequencing libraries (n=5-6 / group) were generated from 500 ng of total RNA using the Illumina® TruSeq® Stranded mRNA Library Preparation Kit version 2. Briefly, after purification using magnetic beads attached to poly-T oligos, mRNA was fragmented using divalent cations at 94°C for 2 min. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second-strand cDNA synthesis with DNA polymerase I and RNase H. After addition of a single "A" base and subsequent ligation of adapters onto the double-stranded cDNA fragments, the products were purified and enriched using PCR [98°C for 30 s; (98°C for 10 s, 60°C for 30 s, 72°C for 30 s) x 12 cycles; 72°C for 5 min] to generate cDNA libraries. Excess PCR primers were further removed by purification using AMPure XP beads (Beckman Coulter), and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Sequencing was performed on an Illumina® Genome Hiseq4000 as single-end 50-base reads according to Illumina's instructions. Reads were mapped onto the mm10 assembly of the Mus musculus genome using STAR version 2.5.3a (72). Only uniquely aligned reads were retained for further analysis. Quantification of gene expression was performed using HTSeq-count version 0.6.1p1 (73) and gene annotations from Ensembl releases 90 and 102 and "integrated" mode. Read counts were normalized between libraries using the method proposed by Ander et al. (74). Comparisons of interest were performed using the test for differential expression proposed by Love et al. (75) and implemented in the DESeq2 Bioconductor library (version 1.16.1). The resulting p values ​​were corrected for multiple testing using the Benjamini and Hochberg method (76).RNA sequencing was performed by the platform GenomEast, French National Center for Scientific Research, UMR 7104 CNRS-UdS / INSERM U964 (Illkirch, France). Sequence data supporting the findings of this study have been deposited in the Gene Expression Bank (GEO) under the primary accession code GSE179247.

[0100] GO (Gene Ontology) terms, STRING, and Ingenuity pathway analysis Functional enrichment analysis was performed using DAVID (Database for Annotation, Visualization, and Integrated Discovery; https: / / david-d.ncifcrf.gove / home.jsp), STRING interaction network, or uploaded to the IPA web portal (Qiagen; www.ingenuity.com). Data were analyzed to predict gene networks, molecular and cellular functions, canonical pathways, and upstream regulators for genes regulated by cisplatin / KW.

[0101] GSEA analysis A prerank analysis was performed using GSEA, version 4.1.0 (82, 83), with the following settings: "no genetic code collapse, classical enrichment statistics, gene sets containing more than 500 genes and less than 50 genes were excluded from the analysis" and gene sets from Gene Ontology (GO). Genes were ranked based on a numerical value computed as follows: -10 × log10(P value) × mean fold change.

[0102] Quantitative reverse transcription PCR Reverse transcription was performed on 1 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer's recommendations. Real-time PCR was performed on a StepOne instrument using Taqman Gene Expression Master Mix (Thermo Fisher) according to the manufacturer's recommendations. The expression levels of the following genes were assessed using the comparative CT method (2ΔCT): neutrophil gelatinase-associated lipocalin (NGAL, assay identification numbers Hs00194353_m1 and Mm01324470_m1), kidney injury molecule-1 (KIM-1, assay identification numbers Hs00273334_m1 and Mm00506686_m1), tumor necrosis factor alpha (TNFα, assay identification numbers Hs00174128_m1 and Mm00443258_m1), interleukin-6 (IL6, assay identification numbers Hs00174131_m1 and Mm00446190_m1), Adenovirus 2A receptor (Adenovirus 2B receptor) and Adenovirus 3A receptor (Adenovirus 4A receptor). 2A R, assay identification numbers Hs00169123_m1 and Mm00802075_m1), Bcl2-associated X (Bax, assay identification numbers Hs00180269_m1 and Mm00432051_m1), B cell lymphoma 2 (Bcl2, assay identification numbers Hs00608023_m1 and Mm00477631_m1), copper transporter receptor 1 (CTR1, assay identification numbers Hs00741015_m1 and Mm00612987_m1), organic cation transporter 2 (OCT2, assay identification numbers Assay identification numbers Hs00533907_m1 and Mm00457295_m1), ATPase 7A (ATP7A, assay identification numbers Hs00163707_m1 and Mm00437663_m1), ATPase 7B (ATP7B, assay identification numbers Hs00163739_m1 and Mm00599675_m1), multidrug and toxin extrusion 1 / 2 (MATE1 / 2, assay identification numbers Hs00979028_m1 / Hs00945652_m1 and assay identification numbers Mm00840361_m1 / Mm02601002_m1), and ATP-binding cassette subfamily C member 2 (Abcc2, assay identification numbers Hs00166123_m1 and Mm00496899_m1). Transcript levels of PPIA (human Hs99999904_m1 and mouse sample Mm02342430_m) were used as endogenous controls.

[0103] Platinum concentration in kidney tissue / cells RPTEC / hTERT cells were cultured in 6-well plates (250,000 cells / well) and exposed to cisplatin (50 μM) in the absence or presence of KW-6002 (25 μM) for 48 h. Tissues (35 mg) and cell pellets were first mineralized with hydrochloric acid (30% Suprapur, Merck) or nitric acid (69.5%, Carlo Erba), respectively. Analysis of tissue samples was performed by graphite furnace atomic absorption spectrometry (GF-AAS) using an Aanalyst 800 (PerkinElmer), whereas cell sample analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS) using an ICAP-Qc (Thermo Scientific). Platinum concentrations were finally normalized to the precisely measured kidney mass or to the previously assessed cell number.

[0104] Immunoblot Cells and tissues were homogenized using RIPA buffer (Sigma) supplemented with protease and phosphatase inhibitors (Pierce). 10 μg of total protein was heated at 70° C. for 10 min and loaded onto a NuPAGE Novex gel (Thermo Fisher Scientific). After protein transfer to nitrocellulose membrane, the membrane was blocked with 5% milk in TBS-tween and subsequently incubated with anti-cleaved caspase 3 (1 / 1000; #9661, Cell Signaling) or anti-cleaved PARP1 (1 / 1000; #5625, Cell Signaling) primary antibodies. Protein visualization was achieved using secondary antibodies conjugated to horseradish peroxidase (1 / 2000; #7074, Cell Signaling). Detection of signals was performed by using ECL select chemiluminescence kit (GE Healthcare) and Image Quant LAS 4000 (GE Healthcare). Data was analyzed using ImageJ. Membranes were probed with anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody as a standard (G9545, Sigma).

[0105] Immunohistochemistry For immunohistochemical studies, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg, i.p.) and then transcardially perfused with cold NaCl (0.9%) and 4% paraformaldehyde in PBS (pH 7.4). Kidneys were removed, postfixed in 4% paraformaldehyde for 24 h, cryoprotected in 30% sucrose, then frozen in isopentane (methyl-butane) at -40°C and stored at -80°C. Longitudinal kidney sections (40 μm) were obtained using a Leica cryostat. Free-floating sections were stored at 4°C in PBS-azide (0.2%). For immunofluorescence staining, longitudinal kidney sections were incubated with 10% donkey serum (Sigma-Aldrich, D9663) in PBS Triton X-100 (0.2%) for 1 h and then stained with anti-A. 2A The sections were incubated overnight at 4°C with the receptor primary antibody (1 / 200; GP-Af1000; Frontiers Institute). The secondary antibody conjugated to Alexa Fluor568 (1 / 500, Life Technologies) was incubated for 1 h at room temperature. The sections were counterstained with DAPI (1 / 5,000, #62247, Thermo Scientific), mounted on superfrost slides, and air-dried. They were then incubated with TrueView Autofluorescence Quench Kit (Vector Laboratories, SP-8400) for 5 min at room temperature to remove autofluorescence and covered with Vectashield Vibrance Antifade mounting medium (Vector Laboratories, H-1700). Images were acquired using a Zeiss LSM710 confocal laser scanning microscope at 20x magnification. Reconstruction of 3D confocal stack images was performed using Imaris software (Bitplane).

[0106] statistics All data are presented as mean ± SEM. Differences between groups were assessed using two-tailed Student's t-test, one-way ANOVA followed by multiple comparison Tukey post-hoc test, or repeated measures two-way ANOVA using Prism (GraphPad Software). Differences were considered statistically significant at p<0.05. Numbers of biologically independent experiments, sample sizes, p-values, and statistical tests are all indicated in the text or figure legends.

[0107] result Cisplatin-induced nephrotoxicity in mice was characterized by renal A 2A Associated with receptor upregulation Mice treated with cisplatin either acutely (acute “A” model; single dose of 10 mg / kg; Fig. 1A) or subchronically (subchronic “SC” model; 3 mg / kg for 6 days; Fig. 1B) showed significant renal dysfunction (data not shown) as indicated by elevated blood urea nitrogen (BUN) levels, as well as severe histological lesions (data not shown), including the presence of necrotic cells and intraluminal casts. Accordingly, the mRNA levels of two renal injury markers, NGAL (neutrophil gelatinase-associated lipocalin) and KIM-1 (kidney injury molecule 1), were significantly increased (data not shown). The nephrotoxicity of cisplatin was accompanied by an inflammatory response and apoptosis, as exemplified by increased mRNA expression of IL6 and Tnf (data not shown) and enhanced Bax / Bcl-2 ratio (data not shown), as previously described (35).

[0108] Interestingly, the present inventors found that cisplatin is 2A We observed that the cisplatin-induced upregulation of the A receptor was observed (data not shown). 2A We showed that the receptor is present on renal resident cells, particularly on proximal tubular epithelial cells (data not shown). 2AR levels were significantly correlated with BUN (r2 = 0.63, p < 0.0001) and NGAL (r2 = 0.71, p < 0.0001) and KIM-1 (r2 = 0.69, p < 0.0001) expression (data not shown). 2A We suggest that receptor upregulation may be related to the pathological processes underlying cisplatin-induced nephrotoxicity.

[0109] A 2A Receptor antagonism attenuates cisplatin-induced toxicity in vivo and in vitro A 2A To assess whether receptor function is indeed involved in cisplatin-induced renal injury, we used the U.S. Food and Drug Administration-approved selective A receptor in both cisplatin-induced acute ("A") and subchronic ("SC") nephrotoxicity models. 2A The effect of the receptor antagonist istradefylline (KW-6002) was evaluated (Figures 1C and 1D). KW-6002 significantly attenuated cisplatin-induced renal dysfunction as indicated by a significant decrease in the expression of BUN (A model: -42.9 ± 7.4%; SC model: -70.2 ± 5.1% vs. cisplatin; Figures 2A, 2G and 5), NGAL (A: -55.5 ± 5.9%; SC: -82.9 ± 1.4% vs. cisplatin; Figures 2C, 2I) and KIM-1 (A: -95.2 ± 0.7%; SC: -79.5 ± 3.8% vs. cisplatin; Figures 2D, 2J), as well as less severe histological lesions (Figures 2B, 2H). Treatment with KW-6002 also significantly reduced renal inflammation, as indicated by lower mRNA levels of Tnfα (A: −36.0 ± 4.3%; Figures 2E, 2K) and IL6 (A: −73.8 ± 5.3%; SC: −77.5 ± 5.6%, versus cisplatin; Figures 2F, 2L).

[0110] MSX-3 significantly attenuated cisplatin-induced renal dysfunction, as indicated by a significant decrease in BUN (Figure 9A). Treatment with MSX-3 also significantly reduced renal inflammation, as indicated by lower mRNA levels of Tnfα (Figure 9B).

[0111] Consistent with the association of lipid accumulation and lipotoxicity with renal disease, and consistent with recent studies (36-37), we also observed that cisplatin induced the accumulation of lipid droplets, which was significantly reduced by KW-6002 (data not shown).

[0112] We next used a human proximal tubule epithelial cell line (RPTEC / hTERT1) to detail the effect of KW-6002 on cisplatin nephrotoxicity in vitro. Consistent with our in vivo data, KW-6002 reduced cisplatin-induced cell death in a concentration-dependent manner (data not shown). KW-6002 specifically inhibited cisplatin-induced apoptosis (data not shown). Furthermore, cisplatin-induced lipid accumulation in RPTEC / hTERT1 cells was also significantly reduced by KW-6002 (data not shown). In summary, these in vivo and in vitro data strongly support that KW-6002 can protect against cisplatin-induced renal injury.

[0113] Transcriptomic signature associated with the protective effect of KW-6002 against cisplatin-induced renal injury A in damaged kidneys 2A To gain insight into the in vivo molecular events underlying the beneficial effects of receptor antagonism, we used an RNA-sequencing transcriptomic approach in a subchronic protocol and performed principal component analysis (PCA) of experimental groups (n = 5–6 per group).

[0114] Cisplatin had a significant effect on the kidney transcriptome, affecting the expression of 4649 genes (corrected p-value < 0.01, log2 ± 1 of fold change), 2350 of which were upregulated and 2299 of which were downregulated (data not shown). Interestingly, KW-6002 reduced the transcriptome changes induced by cisplatin by about 50% (data not shown). Among the 2350 genes upregulated by cisplatin, 811 (about 34%) were normalized by coadministration of KW-6002 (data not shown), whereas KW-6002 had almost no effect on control mice (data not shown). Functional enrichment analysis performed using DAVID (https: / / david.ncifcrf.gov) indicated that these 811 genes were related to cell adhesion and proliferation (data not shown). Similarly, among the 2299 genes downregulated by cisplatin, 635 (about 27%) were normalized by cotreatment with KW-6002 (data not shown), which were mainly related to redox reactions and transport processes (data not shown). We then used Ingenuity Pathway Analysis (IPA) to predict the most important molecular and cellular functions, toxicological features, or upstream regulators affected by cisplatin that were normalized by cotreatment with KW-6002. Our analysis of these 635 KW-6002-regulated genes identified networks and classical pathways involved in amino acid metabolism, molecular transport (data not shown), renal injury, and lipid metabolism (including fatty acid metabolism, data not shown), suggesting that the genes in these networks were particularly related to the protective effect of KW-6002 in cisplatin-treated kidneys. Next, we performed upstream analysis of genes regulated by KW-6002 and identified several important upstream regulators known to affect renal function and / or lipid homeostasis (data not shown).In particular, our analysis predicted activation of the hepatocyte nuclear factor 1A isoform (HNF1A, data not shown). Our analysis further predicted activation of Lim homeobox protein 1 (LHX1, data not shown) or synuclein (SNCA, data not shown).

[0115] The above pathways regulated by KW-6002 that were not covered by RNA-seq analysis were confirmed by additional in vitro and in vivo analyses. First, using human proximal tubule epithelial cell line (RPTEC / hTERT1), we found that KW6002 reduced cisplatin-induced cell death (data not shown) in a concentration-dependent manner, particularly by inhibiting cisplatin-induced apoptosis (data not shown) and DNA damage (Figure 6A). Furthermore, cisplatin-induced lipid accumulation was significantly reduced by KW6002 in both RPTEC / hTERT1 cells (Figure 6B) and kidney samples (Figure 6C). Finally, KW6002 normalized the cisplatin-induced increase in the expression of Nrf2 and HO1, two major regulators of oxidative stress, both in vivo (Figures 6D and 6E) and in vitro (Figures 6F and 6G), as well as the decrease in catalase activity (Figure 6H).

[0116] Altogether, these are important observations supporting a link between the effects of KW-6002 and important protective signaling events in cisplatin-treated kidneys.

[0117] Effect of KW-6002 on the antitumor properties of cisplatin From a clinical perspective, it is important to determine whether KW-6002 preserved renal function after exposure to cisplatin, at least without compromising its antitumor efficacy. To address this question, we used a syngeneic LLC1 lung cancer mouse model. Subcutaneous LLC1 tumors were induced in C57BL6 / J mice, which were then treated with cisplatin, KW-6002, or both (Figure 1E). In this model, we could then simultaneously measure whether KW-6002 modulated the effects of cisplatin on renal injury and tumorigenicity. We observed that the ability of KW-6002 to protect against cisplatin-induced renal injury was preserved in tumor-bearing mice (data not shown). Importantly, KW-6002 did not impair the antitumor response to cisplatin (Figure 3). Furthermore, whole transcriptome analysis was performed to decipher possible molecular changes occurring within the tumor in response to cisplatin and / or KW-6002. Principal component analysis (n=5 per group) (data not shown). Compared to vehicle-treated mice, KW-6002 did not modulate gene expression within the tumor (not shown). Compared to vehicle-treated mice, co-treatment of mice with cisplatin and KW-6002 altered the expression of 3923 genes (corrected p-value <0.05, log2±0.32 fold change), whereas cisplatin alone altered the transcription of 1801 genes. Hence, the effect of cisplatin on the tumor cell transcriptome was enhanced in the presence of KW-6002 (data not shown). Among the 2497 (out of 3923) genes selectively regulated by cisplatin / KW-6002 co-treatment vs vehicle, 1016 genes were down-regulated, with annotations specifically referring to chemokine / cytokine response, cell cycle, and DNA repair and replication (data not shown). Using Ingenuity pathway analysis, we further determined the most important diseases, molecular and cellular functions, and biological networks associated with these 1016 genes specifically down-regulated by KW-6002 upon co-treatment with cisplatin.The altered gene expression patterns were associated with disorders including cancer and immune diseases (data not shown). Furthermore, cellular and molecular functions affected by KW-6002 included increased necrosis (p=9.55×10. -24 ) and apoptosis (p=2.78×10 -22 ), which were predicted to be involved in "cell death and survival, cell development, cell growth and proliferation." Furthermore, our analysis of genes specifically downregulated by KW-6002 upon co-treatment with cisplatin identified 25 biological gene networks (data not shown), the five most important of which are related to "protein synthesis, RNA damage and repair, RNA post-transcriptional modification," "cancer, hematological disease, immune disease," and "antimicrobial response, infectious disease, inflammatory response," "cancer, cardiovascular disease, DNA replication, recombination, and repair," and "molecular transport, post-translational modification, RNA transport" (data not shown). These transcriptome data suggest possible molecular synergistic antitumor and / or antiproliferative effects of KW-6002 when administered in combination with cisplatin. In accordance with these transcriptome analyses, our in vitro data suggest that KW-6002 likely increases the sensitivity of LLC1 to cisplatin, including apoptosis and / or DNA damage (data not shown).

[0118] Consistent with this pathway analysis, in vitro studies performed using two cancer cell lines (LLC1 and H1975) confirmed that KW6002 did not interfere with the antitumor effects of cisplatin in terms of apoptosis (Figures 7A, 7C, and 7D), DNA damage (Figures 7B, 7E, and 7F), and cell cycle (Figures 7G and 7H). Notably, KW6002 even enhanced the efficacy of cisplatin (Figures 7A-7B, 7D, 7F, 7H, and 7I) and showed a specific effect on DNA damage induced by cisplatin, as evidenced by the increased number of H2AX-positive cells observed both in LLC1 and H1975 cells in vitro (Figures 7B and 7F) and in the LLC1 syngeneic model in vivo (Figure 7I).

[0119] KW-6002 reduced the accumulation of cisplatin in the kidney To understand how KW-6002 reduces cisplatin-induced nephrotoxicity while preserving the tumor growth control ability of cisplatin, platinum levels were quantified in both kidney and tumor. This analysis revealed that in mice treated with KW-6002, cisplatin accumulation was reduced in the kidney but remained unchanged in the tumor (Figures 4A and 4B). A correlation was observed between the accumulation of platinum in the kidney and the tumor. 2A The effect of receptor antagonism was repeated in an independent experiment (data not shown; from animals described in Fig. 2G-2L). The different effects of KW-6002 on cisplatin accumulation in kidney and tumor led us to evaluate the mRNA levels of several transporters known to be involved in cisplatin uptake (CTR1 and OCT2) and efflux (ATP7B, MATE1 / SLC47A1, MATE2, and ABCC2) in both tissues (6). In kidney, using quantitative PCR analysis (Fig. 4A), but also using data from our RNA-sequencing experiments (data not shown), we observed that the expression of several efflux transporters was significantly increased by KW-6002. In contrast, no expression changes were observed in tumors (Fig. 4B).

[0120] Furthermore, this difference observed between renal and cancer cells prompted us to evaluate the efflux capacity of RPTEC / hTERT1 renal cells and H1975 cancer cells by flow cytometry. Although efflux was significantly reduced by KW6002 in cancer cells in response to cisplatin (data not shown), it was strongly enhanced by KW6002 in RPTEC / hTERT1 cells treated with cisplatin (Figure 4C).

[0121] A2A receptor antagonism protects against cisplatin-induced nephrotoxicity and chemotherapy-induced peripheral neuropathy (CIPN) while enhancing tumor growth control in a syngeneic model of human papillomavirus-positive squamous cell carcinoma We used an additional cancer mouse model to validate the renal and neuroprotective effects of KW6002 in the context of tumors (77). Subcutaneous mEERL cells were injected into C57Bl6 / J mice, which were then treated with cisplatin alone or in combination with KW6002 (data not shown). Administration of KW6002 to tumor-bearing mice limited cisplatin-induced indices of nephrotoxicity (KIM-1, FIG. 8A) and expression of inflammatory cytokines Tnf and IL6 (FIGS. 8B and 8C). KW6002 also reduced cisplatin-induced hypersensitivity in this model (FIG. 8D). Finally, KW6002 significantly enhanced tumor control by cisplatin (FIG. 8E). In summary, using this additional model with a different cancer etiology, the renal protective effects, reduced pain hypersensitivity, and enhanced tumor control were restored, which suggests that A 2A Highlight the promising therapeutic potential of receptor inhibition.

[0122] Consideration Cisplatin-induced nephrotoxicity remains a serious adverse effect, affecting roughly one-third of exposed patients (11-12). Therefore, identifying targets to reduce such toxicity without attenuating cisplatin-induced tumor control is a major clinical challenge. Moreover, an optimal therapeutic solution would ideally synergize with cisplatin to promote cancer regression while preserving renal and sensory function. In this study, we demonstrate that cisplatin-approved A. 2A We provide evidence that administration of the receptor antagonist istradefylline (KW-6002) efficiently and reproducibly prevents cisplatin-induced nephrotoxicity in mice. These beneficial effects were observed while preserving the tumor growth-regulating properties of cisplatin and even enhancing several antitumor molecular pathways.

[0123] Our targeted and non-targeted (RNA-seq) experiments showed that cisplatin profoundly affected renal function by promoting cell death through multiple pathways, including inflammatory responses, oxidative and reductive reactions, intracellular lipid accumulation, transport disorders, and apoptosis induction (40-42). Treatment with KW-6002 alleviated most of them. Whether the mechanisms underlying KW-6002 action in DRG are similar to those in the kidney will be the focus of future studies.

[0124] A 2A Receptor antagonists have previously been shown to reduce injury by decreasing oxidative stress in various cell types and tissues (43-47). The effect of KW-6002 on renal redox mechanisms is particularly important because, once in the cytosol, cisplatin is highly reactive toward nucleophiles such as glutathione (GSH), cysteine, or methionine (which are metabolically activated to form reactive thiols) (2, 48). The subsequent accumulation of cisplatin in the mitochondria of renal tubular epithelial cells increases the levels of reactive oxygen species and decreases the levels of antioxidant components such as GSH and superoxide dismutase (40, 49), leading to oxidative stress-related injury and death of proximal tubular epithelial cells (50-51).

[0125] Our data also suggest that the renal protective effect of KW-6002 relies on its ability to limit platinum accumulation in the kidney. The latter is consistently associated with upregulation of ABCC2, ATP7b, and MATE-1 (multidrug and toxin efflux 1), the main transporters previously reported to be involved in cisplatin efflux (6). In agreement, it has been shown that upregulation of MATE-1 increases the efflux of cisplatin from renal cells (52), and genetic deletion of MATE-1 exacerbates cisplatin nephrotoxicity in mice (53). How KW-6002 regulates the expression of efflux transporters remains unclear; however, A 2AActivation of the receptor was previously reported to decrease the expression and function of P-glycoprotein (ABCB1), a member of the same family as ABCC2, leading to the accumulation of P-glycoprotein substrates in mouse brain (54). 2A The receptor localization and the fact that KW-6002 limits cisplatin accumulation in the RPTEC / hTERT1 proximal tubular epithelial cell line in vitro (data not shown) suggest that A 2A However, we show that KW-6002 downregulates A receptor expression located on inflammatory cells, as supported by the reduction in the expression levels of TNF and IL-6. 2A It cannot be excluded that adenosine exerts its beneficial effects by modulating the A receptor. Extracellular adenosine has indeed been shown to be important in the regulation of immune cell activation in the kidney, especially in the setting of renal ischemia; however, 2A Activation, rather than blockade, of receptor signaling is recognized for its immunosuppressive effects (30-31, 55).

[0126] It is of great clinical relevance that KW-6002 exerts a strong effect on cisplatin-induced nephrotoxicity without affecting its antitumor properties. Indeed, the reduction in tumor growth rate induced by cisplatin was not affected by co-treatment with KW-6002. Adenosine levels are particularly elevated in the tumor microenvironment (56-57Ohta et al., 2006; 57), and A, specifically expressed by immune cells, is a key regulator of tumor growth. 2A It impairs antitumor immunity through receptor activation (13-58). 2AReceptor antagonists are currently being explored in clinical trials as coadjuvants for autoimmune transplant therapy for immunogenic cancers (https: / / www.cancer.gov / about-cancer / treatment / clinical-trials / intervention / adenosine-a2a-receptor-antagonist-cpi-444). Interestingly, platinum-based chemotherapeutic agents have been suggested to promote adenosine upregulation by cancer cells, confer chemoresistance, and even suppress antitumor immunity (58). In this context, A 2A Receptor blockade is now seen as a valuable strategy to improve chemotherapy and exert cancer immunological effects (58-59). In particular, molecular analysis of syngeneic tumors derived from cisplatin-treated animals demonstrated that co-treatment with KW-6002 resulted in a strongly attenuating effect on cancer-related molecular pathways. Our RNA-seq data also highlighted that in the context of co-treatment with cisplatin, KW-6002 also altered the pattern of genes involved in carcinogenesis, particularly those related to cell proliferation, such as DNA replication and cell cycle. Interestingly, Ingenuity pathway analysis derived from in vivo tumors also predicted that necrosis and apoptosis were specifically activated in tumors (P=2.78×10 -22 ), which is consistent with our in vitro experiments supporting the synergistic effect of cisplatin and KW-6002 on LLC1 cells. 2A These results suggest that caffeine enhances the antitumor properties of cisplatin through the regulation of receptor-mediated tumor-immune-inflammatory interactions. These observations are in agreement with previous data highlighting that caffeine, a nonselective adenosine receptor antagonist, enhances the antitumor effects of cisplatin both in vitro and in vivo (60-61).

[0127] In conclusion, our study clearly demonstrates the high efficacy of KW-6002 in attenuating cisplatin-induced nephrotoxicity without compromising its antitumor properties. Given the safety and tolerability of the US Food and Drug Administration-approved KW-6002 demonstrated in a previous clinical trial targeting Parkinson's disease patients (62), our data encourage its clinical repurposing in cancer patients receiving cisplatin chemotherapy.

[0128] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure. [Table 1] TIFF2025509248000020.tif236169TIFF2025509248000021.tif237169TIFF20255092480 00022.tif245169TIFF2025509248000023.tif245169TIFF2025509248000024.tif105169

Claims

1. A therapeutically effective amount of Selective A ２Ａ Adenosine receptor (A ２Ａ R) A method for the treatment of chemotherapy-induced nephrotoxicity in a subject in need thereof comprising administering to said subject an antagonist.

2. Selective A ２Ａ 2. The method of claim 1, wherein the adenosine receptor antagonist is selected from among istradefylline (KW-6002) or a derivative thereof, MSX-3, SCH-58261, tozadenant (SYN115), NIR178 (also known as PBF-509), ciforadenant, AB928, AZD4635, EOS100850 and preladenant (SCH-420814), inupadenant (EOS-850), EXS21546, TT-10 and TT-53, and pharma- ceutically acceptable salts thereof, more particularly from among istradefylline (KW-6002) or a derivative thereof, MSX-3 and SCH-58261.

3. Selective A ２Ａ The method of claim 1, wherein the adenosine receptor antagonist is KW-6002 or a derivative thereof.

4. Selective A ２Ａ 2. The method of claim 1, wherein the adenosine receptor antagonist is administered orally or parenterally.

5. A therapeutically effective amount of Selective A ２Ａ A method for the treatment of cancer in a subject in need thereof comprising a combination of an adenosine receptor antagonist and a chemotherapeutic agent.

6. 6. The method of claim 5, wherein the chemotherapeutic agent is selected from DNA alkylating agents, in particular from platinum-containing chemotherapeutic agents, more particularly from cisplatin, carboplatin, oxaliplatin, and pharma- ceutically acceptable salts thereof.

7. 6. The method of claim 5, wherein the chemotherapeutic agent is cisplatin.

8. The cancer is cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicle, tongue, or uterus, and further, the cancer is particularly of the following histological types (malignant neoplasm; cell carcinoma; undifferentiated cell carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial cell carcinoma; basal cell carcinoma; hair matrix carcinoma; Transitional cell carcinoma; Papillary transitional cell carcinoma; Adenocarcinoma; Malignant gastrinoma; Cholangiocarcinoma; Hepatocellular carcinoma; Mixed hepatocellular and cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatous polypoid form; Familial polyposis coli adenocarcinoma; Solid cell carcinoma; Malignant carcinoid tumor; Bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma; Chromophobe cell carcinoma; Eosinophil cell carcinoma; Eosinophil adenocarcinoma ; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing cell carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma ; sebaceous gland carcinoma; auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; pancreatic acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; malignant glomus tumor; malignant melanoma; amelanotic melanoma; superficial spreading type malignant melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell malignant melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Mullerian tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant ovarian goiter; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic sarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; malignant pinealoma; chordoma; Malignant glioma; ependymoma; Plasmatic astrocytoma; Fibrillar astrocytoma; astroblastoma; medulloblastoma; glioblastoma; Oligodendroglioma; Oligodendroglioblastoma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma; Small lymphocytic lymphoma; Diffuse large cell lymphoma; Follicular lymphoma; Mycosis fungoides; Other specified non-Hodgkin's lymphoma 6. The method of claim 5, wherein the cancer may be, but is not limited to, myeloid leukemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

9. A therapeutically effective amount of Selective A ２Ａ Adenosine receptor (A ２Ａ R) A pharmaceutical composition comprising an antagonist and a chemotherapeutic agent.

10. Selective A ２Ａ Adenosine receptor (A ２Ａ The pharmaceutical composition according to claim 9, wherein the R) antagonist is KW-6002 or a derivative thereof.

11. 10. The pharmaceutical composition of claim 9, wherein the chemotherapeutic agent is cisplatin.

12. A pharmaceutical composition according to claims 9 to 11 for use in the treatment of nephrotoxicity induced by chemotherapy drugs.

13. Selective A for use in the treatment of chemotherapy-induced nephrotoxicity in a subject in need thereof. ２Ａ Adenosine receptor (A ２Ａ R) antagonist.

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