Drug delivery carrier, drug delivery method and composition for treating renal cell carcinoma utilizing the property of D-allose being taken up by cancer cells

Abstract

To provide a composition for the treatment of renal cell carcinoma and a composition or a method for enhancing the uptake of a drug into renal cell carcinoma cells.SOLUTION: Provided are a D-allose-containing composition for the treatment of renal cell carcinoma, and a D-allose-containing composition or method for enhancing the uptake of a drug into renal cell carcinoma cells. The composition is a pharmaceutical composition to be administered in an effective amount to patients requiring the treatment of renal cell carcinoma in humans or animals other than humans.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a selective drug transport carrier for renal cell carcinoma cells, a drug transport method, and a composition for treating renal cell carcinoma, which utilize the property of D-allose being taken up by cancer cells. [Background technology]

[0002] Cancer treatment methods are broadly divided into surgery, radiation therapy, and chemotherapy. Of these, chemotherapy is a therapy in which anticancer drugs are administered to cancer patients, and is used as pre- and post-operative adjuvant chemotherapy to improve healing before and after surgery or radiation therapy, and to treat metastatic cancer that cannot be treated with surgery or radiation therapy. Currently, anticancer drugs such as metabolic antagonists, topoisomerase inhibitors, molecular targeted drugs, and nucleic acid drugs have been clinically put into practical use, and some cancers are expected to be cured, but chemotherapy for renal cell carcinoma has not yet produced satisfactory results.

[0003] Anticancer drugs often do not selectively act on tumor cells, but also on normal cells, resulting in high toxicity and side effects. Tissues with high cell division rates are particularly susceptible to this adverse systemic toxicity, which limits the dose of anticancer drugs that can be administered to cancer patients and limits their effectiveness. In addition, some anticancer drugs have low solubility and hydrophobicity, which not only results in low membrane permeability, but also leads to the formation of aggregates of anticancer drugs that are not dissolved in aqueous media, which may cause capillary embolization before penetrating into tumors when administered intravenously.

[0004] Furthermore, in the past, in order to selectively deliver drugs to tumors, drug delivery systems have been developed in which drugs are encapsulated in carriers such as micelles, liposomes, microparticles, antibodies, and drug-polymer conjugates (Patent Document 1). Despite these attempts to improve the accumulation of anticancer drugs in tumor tissues, it remains difficult to prevent the accumulation of anticancer drugs in normal tissues such as the liver and kidneys, and there is a demand for the development of drug carriers that can selectively deliver anticancer drugs to cancer cells.

[0005] Meanwhile, among the results of applied research into rare sugars in the medical field, there is the invention of an in vivo antioxidant with D-allose as an active ingredient (Patent Document 2). This is a composition in which D-allose is administered to patients with liver cancer or skin cancer, and the in vivo antioxidant effect of D-allose is used to treat liver cancer or skin cancer. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2015-155392 A [Patent Document 2] Patent No. 5330976 [Patent Document 3] JP 2004-298106 A [Non-patent literature]

[0007] [Non-Patent Document 1] J.Ferment.Biоeng.84,319,1997 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a new use of D-allose that utilizes its property of being taken up by cancer cells, that is, to provide a new use of D-allose having the property of being taken up by cancer cells as a drug transport carrier. Also, the present invention aims to provide a drug transport method using said transport carrier. Still further, the present invention aims to provide a composition for treating renal cell carcinoma as an anticancer agent having excellent antitumor effect against renal cell carcinoma. Also, the present invention aims to provide a composition for treating renal cell carcinoma that can increase drug uptake into renal cell carcinoma cells, and a composition or method for increasing drug uptake into renal cell carcinoma cells. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found for the first time that D-allose is specifically incorporated into human renal cell carcinoma cells, and that the incorporated D-allose has antitumor activity by suppressing the proliferation of renal cell carcinoma cells, thereby completing the present invention. In addition, by using D-allose incorporated into human renal cell carcinoma cells as a carrier for anticancer drugs such as chemotherapeutic agents and nucleic acid drugs, it is possible to increase the uptake of anticancer drugs by renal cell carcinoma cells, and to exert the antitumor activity of both the incorporated D-allose and the anticancer drug.

[0010] The gist of the present invention is an agent that utilizes the properties of being selectively taken up by renal cell carcinoma cells as described below in (1) to (4). (1) An agent comprising D-allose, which utilizes the property of being selectively taken up by renal cell carcinoma cells. (2) The agent according to (1) above, in which D-allose taken up into renal cell carcinoma cells exerts an antitumor effect. (3) The agent according to (1) or (2) above, wherein the D-allose is D-allose and / or a derivative thereof and / or a mixture thereof. (4) The agent described in (3) above, wherein the D-allose derivative is a D-allose derivative selected from a sugar alcohol in which the carbonyl group of D-allose is an alcohol group, a uronic acid in which the alcohol group of D-allose is oxidized, and an amino sugar in which the alcohol group of D-allose is replaced with an NH2 group.

[0011] The present invention relates to the following (5) to (11) selective drug transport carriers for renal cell carcinoma cells. (5) A selective drug transporter for renal cell carcinoma cells, comprising D-allose. (6) A selective drug transporter for renal cell carcinoma cells according to (5) above, in which D-allose taken up into renal cell carcinoma cells exerts an antitumor effect. (7) A selective drug transport carrier for renal cell carcinoma cells according to (4) or (5) above, wherein the D-allose is D-allose and / or a derivative thereof and / or a mixture thereof. (8) A selective drug transporter for renal cell carcinoma cells described in (7) above, wherein the D-allose derivative is a D-allose derivative selected from a sugar alcohol in which the carbonyl group of D-allose is an alcohol group, a uronic acid in which the alcohol group of D-allose is oxidized, and an amino sugar in which the alcohol group of D-allose is replaced with an NH2 group. (9) The selective drug transporter for renal cell carcinoma cells according to any one of (5) to (8) above, wherein the drug comprises an anticancer drug. (10) A selective drug transporter for renal cell carcinoma cells according to any one of (5) to (9) above, in which D-allose and the drug are covalently associated with each other directly or via a linker. (11) The selective drug transporter for renal cell carcinoma cells according to any one of (5) to (10) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiosensitizer, an iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an intercalating antibiotic, an antimetabolite, an aromatase inhibitor, a mitosis inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

[0012] The present invention relates to compositions for treating renal cell carcinoma as set forth below in (12) to (19). (12) A composition for treating renal cell carcinoma, comprising D-allose. (13) The composition for treating renal cell carcinoma described in (12) above, wherein D-allose taken up into renal cell carcinoma cells exerts an antitumor effect. (14) A composition for treating renal cell carcinoma according to (12) or (13) above, wherein the D-allose is D-allose and / or a derivative thereof and / or a mixture thereof. (15) The composition for treating renal cell carcinoma described in (14) above, wherein the D-allose derivative is a D-allose derivative selected from a sugar alcohol in which the carbonyl group of D-allose is an alcohol group, a uronic acid in which the alcohol group of D-allose is oxidized, and an amino sugar in which the alcohol group of D-allose is replaced with an NH2 group. (16) A composition for treating renal cell carcinoma according to any one of (12) to (15) above, further comprising a drug associated with D-allose. (17) The composition for treating renal cell carcinoma according to (16) above, wherein the drug comprises an anticancer drug. (18) A composition for treating renal cell carcinoma described in (16) or (17) above, wherein D-allose and the drug are covalently associated with each other directly or via a linker. (19) The composition for treating renal cell carcinoma according to any one of (16) to (18) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiosensitizer, an iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an intercalating antibiotic, an antimetabolite, an aromatase inhibitor, a mitosis inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

[0013] The present invention relates to the following methods for transporting drugs to renal cell carcinoma cells (20) to (26). (20) A drug transport method for renal cell carcinoma cells, comprising the steps of: loading a drug onto a drug transport carrier to form a drug; applying the drug to renal cell carcinoma cells; and selectively transporting the drug to the renal cell carcinoma cells by selectively taking up the drug, wherein the carrier is D-allose and the drug is loaded onto the D-allose via a chemical bond. (21) The drug transport method according to (20) above, which is a method for increasing drug uptake into renal cell carcinoma cells by utilizing the property of D-allose to be taken up by cancer cells. (22) The drug transport method according to (20) or (21) above, characterized in that the carrier is D-allose and the drug is supported by being covalently associated with the D-allose directly or via a linker. (23) The drug delivery method according to any one of (20) to (22) above, wherein the drug comprises an anticancer drug. (24) The drug delivery method according to any one of (20) to (23) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiosensitizer, an iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an intercalating antibiotic, an antimetabolite, an aromatase inhibitor, a mitosis inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor. (25) The drug delivery method according to any one of (20) to (24) above, wherein the D-allose is D-allose and / or a derivative thereof and / or a mixture thereof. (26) The drug delivery method described in (25) above, wherein the D-allose derivative is a D-allose derivative selected from a sugar alcohol in which the carbonyl group of D-allose is an alcohol group, a uronic acid in which the alcohol group of D-allose is oxidized, and an amino sugar in which the alcohol group of D-allose is replaced with an NH2 group. Effect of the Invention

[0014] D-allose is known to have an antitumor effect against liver cancer and skin cancer due to its antioxidant effect in vivo, but its antitumor effect against renal cell carcinoma has not been known. Furthermore, it has not been demonstrated that D-allose is taken up into human cancer cells. The present inventors have found for the first time that D-allose is taken up by human renal cell carcinoma cells and that the taken up D-allose has antitumor activity. D-allose is water-soluble and therefore has high membrane permeability, and is also soluble in aqueous media, so no aggregates are formed. Furthermore, since it is specifically taken up by renal cell carcinoma cells, particularly among human cancer cells, it can be used as a drug carrier (selective drug transport carrier) that can selectively deliver anticancer drugs to renal cell carcinoma cells, thereby increasing the uptake of anticancer drugs by renal cell carcinoma cells and allowing both the taken up D-allose and the anticancer drug to exert their antitumor activities. Since the 5-year survival rate for clinical stage IV renal cell carcinoma in Japan is still low at 18.1%, the composition for treating renal cell carcinoma containing D-allose of the present invention has the potential to become a groundbreaking molecular targeted therapeutic drug. [Brief description of the drawings]

[0015] [Figure 1] 1 shows the results of a survival assay of a renal cell carcinoma cell line (ACHN). [Diagram 2] 1 shows the results of a survival assay of a renal cell carcinoma cell line (Caki-I). [Diagram 3] 1 shows the results of a survival assay of a renal cell carcinoma cell line (Caki-II). [Figure 4] 1 is a graph showing the time course of D-allose concentration in tumors after intraperitoneal injection of D-allose into a mouse model with a xenograft of renal cell carcinoma. [Diagram 5] 1 is a graph showing the change in tumor volume in a mouse model of renal cell carcinoma xenografted by intraperitoneal injection of D-allose. [Figure 6] 1 is a graph showing the effect of D-allose on a renal cell carcinoma xenograft mouse model: changes in body weight. [Figure 7] 1 is a photomicrograph (×100) showing the effect of D-allose on a renal cell carcinoma xenograft mouse model, showing changes in kidney tissue. [Figure 8] 1 is a photomicrograph (×100) showing the effect of D-allose on a renal cell carcinoma xenograft mouse model, showing changes in liver tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] D-allose is used as a drug that utilizes its property of being selectively taken up by renal cell carcinoma cells. D-allose taken up into renal cell carcinoma cells exerts an antitumor effect. D-allose is also used as a selective drug transport carrier for renal cell carcinoma cells, and a method for transporting a drug to renal cell carcinoma cells is provided. The drug includes an anticancer drug, and the drug is associated with D-allose by covalent bond directly or via a linker. The present invention also provides a composition for treating renal cell carcinoma that contains D-allose, and includes a formulation of an effective amount of D-allose or a pharmacologically acceptable salt or / and hydrate. In the above drug transport method, a carrier made of D-allose is used, and various drugs described below can be used as the drug carried by the carrier, and can be appropriately selected according to the purpose, but since the carried drug is selectively taken up by renal cell carcinoma cells, it is preferable to use a drug such as an anticancer drug. That is, it is preferable to use a drug that carries an anticancer drug, with the aim of the carrier made of D-allose being taken up by cancer cells.

[0017] D-allose used in the present invention is a rare sugar that exists in an overwhelmingly smaller amount than D-glucose, which exists in large quantities in nature. There are a total of 34 types of monosaccharides (monosaccharides with six carbon atoms: hexoses), which are the basic units of sugar, including 16 types of aldoses, 8 types of ketoses, and 10 types of sugar alcohols. In contrast to "natural monosaccharides" represented by D-glucose, which exist in large quantities in nature, monosaccharides (aldoses, ketoses) and their derivatives (sugar alcohols) that exist only in trace amounts in nature are defined as "rare sugars." Currently, rare sugars that can be produced in large quantities are D-allulose (D-psicose) and D-allose. D-Allose is the D form of allose, which is classified as a hexose aldose.

[0018] Methods for obtaining D-allose include a method of synthesizing D-allulose using L-rhamnose isomerase isolated from Pseudomonas stutzeri (Non-Patent Document 1), and a method of obtaining D-allulose by acting D-xylose isomerase on a solution containing D-allulose. For the production of high-purity D-allose, there is a fractionation method using the crystallization method of D-allose (Patent Document 3). The D-allose in the present invention is not limited to these, and may be obtained by any method, such as isomerization by chemical treatment. Currently, D-allulose, which is the raw material for D-allose, is generally obtained by treating fructose with an enzyme (epimerase), but is not limited to these, and may be obtained by a method using a microorganism that produces the enzyme, or may be extracted from a natural product or contained in a natural product, or may be used as is, or may be isomerization by a chemical treatment. In addition, a method for purifying D-allulose using an enzyme is known.

[0019] Derivatives of D-allose are explained. A compound whose molecular structure is changed by chemical reaction from a certain starting compound is called a derivative of the starting compound. Derivatives of hexoses including D-allose are generally sugar alcohols (when monosaccharides are reduced, the aldehyde and ketone groups become alcohol groups, resulting in a polyhydric alcohol with the same number of carbon atoms), uronic acids (monosaccharides in which the alcohol groups are oxidized, and naturally occurring D-glucuronic acid, galacturonic acid, and mannuronic acid are known), and amino sugars (sugar molecules in which the OH group is replaced with an NH2 group, such as glucosamine, chondrosamine, and glycosides), but are not limited to these. The derivatives of D-allose are sugar alcohols in which the carbonyl group of D-allose is an alcohol group, uronic acids in which the alcohol group of D-allose is oxidized, and amino sugars in which the alcohol group of D-allose is replaced with an NH2 group.

[0020] In the therapeutic composition of the present invention containing D-allose and / or its derivatives and / or mixtures thereof, the D-allose and / or its derivatives and / or mixtures thereof are contained in the composition in an effective amount. The term "effective amount" refers to any amount sufficient to achieve the intended purpose (e.g., a desired biological or medical response in a tissue or subject). For example, in the present invention, the effective amount is to suppress the proliferation of renal cell carcinoma cells, deliver a drug to the inside of renal cell carcinoma cells, etc.

[0021] The formulation of D-allose or a pharmacologically acceptable salt and / or hydrate thereof of the present invention will be described below. In addition to using D-allose and / or its derivatives and / or mixtures thereof alone, appropriate additives such as general excipients, stabilizers, preservatives, binders, disintegrants, etc. may be added to the composition for treating renal cell carcinoma of the present invention, and appropriate dosage forms such as liquids, capsules, granules, pills, powders, tablets, etc. may be selected and formulated. The composition for treating renal cell carcinoma of the present invention may be prepared by known formulation techniques in various forms, such as liquids, powders, granules, tablets, injections, suppositories, or external preparations, in which the active ingredient is dissolved in water or various infusion preparations containing medically acceptable carriers, excipients, lubricants, binders, and other additives. For parenteral preparations, dosage forms such as injections, drips, external medicines, or suppositories may be selected. For injections, subcutaneous injections, intramuscular injections, intraperitoneal injections, etc. may be used. Formulation techniques for obtaining the above dosage forms are known.

[0022] When the D-allose or its pharmacologically acceptable salt of the present invention is administered by injection, it is preferably an aqueous injection, an aqueous suspension injection, a fat emulsion, or a liposome injection. In the aqueous injection or aqueous suspension injection, the rare sugar D-allose or its pharmacologically acceptable salt of the present invention is mixed with purified water, and if necessary, a water-soluble or water-swellable polymer, a pH adjuster, a surfactant, an osmotic pressure adjuster, a preservative, or the like is added, mixed, dissolved or suspended while heating if necessary, sterilized, and filled and sealed in an injection container to obtain an aqueous injection or aqueous suspension injection. The aqueous injection can be administered intravenously, subcutaneously, intramuscularly, intradermally, or into an articular cavity. The aqueous suspension injection can also be administered subcutaneously, intramuscularly, intradermally, or into an articular cavity. It can also be administered orally.

[0023] As the water-soluble or water-swellable polymer, gelatin, cellulose derivatives, acrylic acid derivatives, povidone, macrogol, polyamino acid derivatives, or polysaccharides are preferred, and as the gelatins, purified gelatin is preferred, as the cellulose derivatives, methylcellulose, hydroxypropyl methylcellulose 2910, hydroxypropyl methylcellulose 2208, hydroxypropyl methylcellulose 2906, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, carmellose sodium, as the acrylic acid derivatives, aminoacryl methacrylate copolymer, methacrylic acid copolymer, as the polyamino acid derivatives, polylysine, polyglutamic acid are preferred. As the polysaccharides, hyaluronic acid, dextran, or dextrin are particularly preferred. The amount of the water-soluble or water-swellable polymer to be added varies depending on the properties and amount of esculetin, its derivatives, or its pharmacologically acceptable salts, as well as the properties, molecular weight, and application site of the water-soluble or water-swellable polymer, but can be used in the range of about 0.01% to 10% of the total amount of the preparation.

[0024] The pH adjuster is an acid or alkali that is harmless to the human body, and the surfactant is a nonionic surfactant, an anionic surfactant, or an amphoteric surfactant. The osmotic pressure adjuster is exemplified by sodium chloride, glucose, etc., the preservative is exemplified by parabens, and the preservative is exemplified by ascorbic acid or sulfites. The amount of each of these used is not particularly limited, but can be used within the range in which each of them can exert its effect. If necessary, a local anesthetic such as procaine hydrochloride, a pain reliever such as benzyl alcohol, a chelating agent, a buffer, or a water-soluble organic solvent may be added.

[0025] Fat emulsions are prepared by mixing an emulsifier and D-allose or a pharmacologically acceptable salt thereof with a suitable oil, adding purified water, and optionally adding water-soluble or water-swellable polymers, pH adjusters, surfactants, osmotic pressure adjusters, preservatives, or the like, emulsifying the mixture in a suitable emulsifying device, sterilizing the mixture, and filling and sealing the mixture in an injection container.

[0026] The "drug" and "anticancer agent" of the present invention are administered to cancer or precancerous tissue for treatment, and examples thereof include radioisotopes (e.g., iodine-131, lutetium-177, rhenium-188, yttrium-90), toxins (e.g., diphtheria, pseudomonas, ricin, gelonin), enzymes, enzymes that activate prodrugs, radiosensitizers, interfering RNA, superantigens, antiangiogenic agents, alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, metabolic antagonists, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, topoisomerase inhibitors, biological response modifiers, antihormones, and antiandrogens.

[0027] The drug is used by associating (e.g., binding, interacting) with D-allose. The association may be covalent or non-covalent. The association between D-allose and the drug must be strong enough not to dissociate before or during delivery to and uptake into renal cell carcinoma cells, and any chemical, biochemical, or enzymatic coupling known to those skilled in the art may be used.

[0028] When the association between D-allose and a drug is non-covalent, it includes hydrophobic interaction, electrostatic interaction, dipole interaction, van der Waals interaction, and hydrogen bond, and when the association between D-allose and a drug is covalent, it is directly or indirectly bonded through a linker. Such covalent bond is achieved through amide, ester, carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, or carbonate bond.

[0029] Regarding the verification of the safety of D-allose, which is necessary for its use as a pharmaceutical ingredient, it was expected to be safe because rare sugars are monosaccharides that exist in nature, albeit in small amounts. Mutagenicity tests, biodegradability tests, and three types of acute toxicity tests (oral acute toxicity test, primary skin irritation test, and primary eye irritation test) are prescribed as the most basic safety tests, and the inventors requested a designated organization to carry out safety tests on the basic parts of D-allose, and as a result, it was confirmed that there are no problems with its safety.

[0030] The therapeutic composition of the present invention targets animals including humans (humans, mammals such as cows, pigs, dogs, and cats, birds such as chickens, etc.). The cancer cells targeted by the therapeutic composition of the present invention are renal cell carcinoma cells, and examples of the cell lines include human renal cell carcinoma cell lines ACHN, Caki-I, and Caki-II.

[0031] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0032] [Rare sugar uptake experiments using human bladder cancer cell lines, human prostate cancer cell lines, and human renal cell carcinoma cell lines] Using cell lines of three types of cancer, the amount of rare sugars added to the medium was analyzed for uptake into the cancer cells. Four types of rare sugars were used: D-allose, D-allulose (D-psicose), L-allulose (L-psicose), and allitol. D-glucose and fructose were used as non-rare sugar monosaccharides. Cells of each cell line were cultured in RPMI-1640 medium, with six types of sugar added to the medium during the culture, and the amount of sugar taken up into the cells was analyzed after the culture. The control was a medium in which no sugar was added during the culture. 1) Cell line used Human bladder cancer cell lines (RT112, 253J, J82), human prostate cancer cell lines (LNCa, Du145, PC-3), and human renal cell carcinoma cell lines (ACHN, Caki-I, Caki-II) were used.

[0033] 2) Culture medium used After culturing the cells in RPMI-1640 (2000 mg D-glucose / L) medium, each monosaccharide and rare sugar was added to each medium. Specifically, there were seven types of medium: RPMI-1640 alone (control), RPMI-1640 + D-glucose, RPMI-1640 + L-allulose, RPMI-1640 + D-allulose, RPMI-1640 + D-fructose, RPMI-1640 + D-allose, and RPMI-1640 + D-allitol. The final concentration of the added sugar in the medium was 10 mM, and no sugar was added to the control.

[0034] 3)Culture method 3.0 × 10 4 5 ml of the cell suspension (1.5 × 10 5 The cells were cultured in RPMI-1640 medium containing 2000 mg / l D-glucose as a nutrient source for 24 hours. After 24 hours, the medium was changed to one containing the monosaccharides and rare sugars described in 2) above, and the cells were cultured for 48 hours.

[0035] 4) Method for analyzing sugars in cultured cells After 48 hours of culture, the attached human cancer cells were mechanically detached and collected in a spindle including the culture medium and floating cells. After removing the supernatant formed by centrifugation (4°C, 1200 rpm, 5 min), the cell pellet was washed and the cell pellet was suspended in 5 ml of phosphate-buffered saline (PBS), and centrifuged again at 4°C, 1200 rpm for 5 min to remove the supernatant. After washing, the cell pellet was suspended in 1 ml of pure water, and ultrasonic disruption (40% intensity, 30 sec) was performed. The monosaccharides in the cell disruption solution were labeled with ABEE and then analyzed by HPLC. The sugar content of the cells was calculated using the calibration curve from the obtained area. As for ketose, two peaks were confirmed after ABEE labeling, so the sugar content in the cells was calculated from the calibration curve of both peaks. The results are shown in the following Table 1 (sugar content in cell lysate of human bladder cancer cell line RT112), Table 2 (sugar content in cell lysate of human bladder cancer cell line 253J), Table 3 (sugar content in cell lysate of human bladder cancer cell line J82), Table 4 (sugar content in cell lysate of human prostate cancer cell line LNCa), Table 5 (sugar content in cell lysate of human prostate cancer cell line Du145), Table 6 (sugar content in cell lysate of human prostate cancer cell line PC-3), Table 7 (sugar content in cell lysate of human renal cell carcinoma cell line ACHN), Table 8 (sugar content in cell lysate of human renal cell carcinoma cell line Caki-I), and Table 9 (sugar content in cell lysate of human renal cell carcinoma cell line Caki-II).

[0036] [Table 1]

[0037] Analysis of cell lysates of the bladder cancer cell line RT112 by HCPL confirmed intracellular D-glucose in all media, with higher levels observed in cells cultured in RPMI-1640 + D-glucose and RPMI-1640 + D-allose media, both of which have a higher D-glucose content. Cells cultured in RPMI-1640 + L-allulose contained 13.8 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 10.0 to 13.0 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 12.7 to 15.7 μg of D-fructose.

[0038] [Table 2]

[0039] Analysis of cell lysates from the bladder cancer cell line 253J by HCPL confirmed intracellular D-glucose in all media, with higher contents observed in cells cultured in media with a high D-glucose content: RPMI-1640 + D-glucose, RPMI-1640 + D-fructose, and RPMI-1640 + D-allulose. Cells cultured in RPMI-1640 + L-allulose contained 15.5 to 17.0 μg of L-allulose, and cells cultured in RPMI-1640 + D-allulose contained 14.1 to 15.8 μg of D-allulose.

[0040] [Table 3]

[0041] Analysis of cell lysates of the bladder cancer cell line J82 by HCPL confirmed intracellular D-glucose in all media, with higher levels observed in cells cultured in RPMI-1640 + D-glucose and RPMI-1640 + D-allose media, which have higher D-glucose contents. Cells cultured in RPMI-1640 + L-allulose contained 13.2 to 14.1 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 11.8 to 14.3 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 11.1 to 15.0 μg of D-fructose.

[0042] [Table 4]

[0043] Analysis of cell lysates from the prostate cancer cell line LNCa by HCPL revealed that D-glucose was detected intracellularly in all media, with higher concentrations in cells cultured in RPMI-1640, RPMI-1640 + L-allulose, and RPMI-1640 + D-allulose media. Cells cultured in RPMI-1640 + L-allulose contained 16.4 to 19.7 μg of L-allulose, and cells cultured in RPMI-1640 + D-allulose contained 14.6 to 17.8 μg of D-allulose.

[0044] [Table 5]

[0045] HCPL analysis of cell lysates from the prostate cancer cell line Du145 confirmed intracellular D-glucose in all media, with a higher content in cells cultured in RPMI-1640 + D-allulose medium. Cells cultured in RPMI-1640 + L-allulose contained 10.8 to 11.7 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 12.6 to 15.5 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 12.9 to 17.0 μg of D-fructose.

[0046] [Table 6]

[0047] Analysis of cell lysates of the prostate cancer cell line PC-3 by HCPL confirmed intracellular D-glucose in all media, with a higher content in cells cultured in RPMI-1640 + D-glucose medium. Cells cultured in RPMI-1640 + L-allulose contained 17.9 to 21.6 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 14.7 to 19.7 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 16.5 to 20.1 μg of D-fructose. Furthermore, cells cultured in RPMI-1640 + D-allose contained 7.5 μg of D-allulose.

[0048] [Table 7]

[0049] Analysis of cell lysates of the renal cell carcinoma cell line ACHN by HCPL confirmed intracellular D-glucose in all media, and showed a high content in cells cultured in RPMI-1640 + D-glucose medium. Cells cultured in RPMI-1640 + L-allulose contained 16.6 to 23.4 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 15.8 to 22.1 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 16.8 to 19.0 μg of D-fructose. Furthermore, cells cultured in RPMI-1640 + D-allose contained 11.2 μg of D-allulose.

[0050] [Table 8]

[0051] Analysis of cell lysates from renal cell carcinoma cell line Caki-I by HCPL confirmed intracellular D-glucose in all media, and showed a high content in cells cultured in RPMI-1640 + D-glucose medium. Cells cultured in RPMI-1640 + L-allulose contained 16.0 to 21.2 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 17.2 to 23.1 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 16.7 to 20.7 μg of D-fructose. Furthermore, cells cultured in RPMI-1640 + D-allose contained 13.2 μg of D-allulose.

[0052] [Table 9]

[0053] Analysis of cell lysates from renal cell carcinoma cell line Caki-II by HCPL confirmed intracellular D-glucose in all media, and showed a high content in cells cultured in RPMI-1640 + D-glucose medium. Cells cultured in RPMI-1640 + L-allulose contained 14.5 to 17.8 μg of L-allulose, cells cultured in RPMI-1640 + D-allulose contained 16.5 to 21.4 μg of D-allulose, and cells cultured in RPMI-1640 + D-fructose contained 20.4 to 21.3 μg of D-fructose. Furthermore, cells cultured in RPMI-1640 + D-allose contained 11.2 μg of D-allulose.

[0054] Summary of Experimental Results All human cancer cell lines showed uptake of D-allulose and L-allulose into the cells, whereas for D-allose, uptake was not observed in human bladder cancer cell lines and was only observed in one of three human prostate cancer cell lines (PC-3), whereas uptake of D-allose was observed in all human renal cell carcinoma cell lines (ACHN, Caki-I, Caki-II). EXAMPLES

[0055] [Analysis of the antitumor effect of rare sugars on human renal cell carcinoma cell lines] The antitumor effects of rare sugars were analyzed using the three types of human renal cell carcinoma cell lines (ACHN, Caki-I, and Caki-II) used in Example 1. Ten types of rare sugars were used: L-allulose, D-allulose, D-allose, L-fructose, D-mannose, L-sorbose, D-tagatose, D-galactose, D-sorbose, and L-tagatose, and D-glucose and D-fructose were used as non-rare sugar monosaccharides.

[0056] [Culture used] The medium used was a minimum essential medium (MEM) containing 1000 mg / l D-glucose, and the antitumor effects of rare sugars were evaluated using MEM adjusted to contain 10 mM, 25 mM, and 50 mM of each monosaccharide or rare sugar (indicated as (10), (25), and (50) in the figure). As a control, no sugar was added to the MEM.

[0057] [Experimental Procedure] MEM, 5.0 × 10 4 A cell suspension of 1000 cells / ml was prepared and distributed in 0.1 ml / well onto a 96-well plate, and then cultured for 24 hours. After that, the culture medium was removed, and 0.1 ml / well of culture medium containing 10 mM, 25 mM, or 50 mM of each monosaccharide or each rare sugar was added to MEM, and cultured for 24 hours (#1 to #13). #1: MEM solution alone (control) #2: D-glucose solution (D-Glucose in the figure) #3: L-Allulose solution (in the figure, L-Allulose) #4: D-Allulose solution (in the figure, D-Allulose) #5: D-Fructose solution (in the figure, D-Fructose) #6: D-Allose solution (in the figure, D-Allose) #7: L-Fructose solution (in the figure, L-Fructose) #8: D-Mannose solution (in the figure, D-Mannose) #9: L-Sorbose solution (in the figure, L-Sorbose) #10: D-Tagatose solution (in the figure, D-Tagatose) #11: D-galactose solution (in the figure, D-Galactose) #12: D-sorbose solution (D-Sorbose in the figure) #13: L-tagatose solution (in the figure, L-Tagatose)

[0058] [Cell viability assay (MTT assay)] The MTT assay is a test method for measuring cell viability, which utilizes the color reaction of an insoluble formazan dye (blue) that occurs with the reduction of the tetrazolium salt MTT. The reduction of MTT occurs due to succinate-tetrazolium reductase, a mitochondrial reductase. In living cells, this enzyme activity is high, and color is observed, but the color disappears when cell death, including apoptosis, occurs. The cell viability is measured by measuring this color with a microplate reader.

[0059] [Cell viability assay results] The results of measuring the viability of human renal cell carcinoma cells are shown in Figures 1 to 3. That is, Figure 1 shows the results of the viability assay of the renal cell carcinoma cell line (ACHN), Figure 2 shows the results of the viability assay of the renal cell carcinoma cell line (Caki-I), and Figure 3 shows the results of the viability assay of the renal cell carcinoma cell line (Caki-II). As shown in Figures 1 to 3, D-allose significantly reduced the viability of these three cell lines at all concentrations of 10 mM, 25 mM, and 50 mM compared to the control (p<0.05), and exhibited the strongest antitumor effect among the sugars analyzed.

[0060] [Consideration] In cancer cells, mitochondrial ATP production is suppressed. In cancer cells, a metabolic pathway called "glycolysis," which produces ATP from glucose without using oxygen, is enhanced. Glycolysis takes place in the cytoplasm. There are several reasons why cancer cells suppress oxygen respiration in mitochondria. One reason is that a large amount of glucose is required as a material to synthesize cell components. In order for cells to divide and increase in number, they need to create new cell components such as nucleic acids, cell membranes, and proteins. Cells can create nucleic acids, lipids, and amino acids from glucose through glycolysis and various intracellular metabolic pathways derived from it. If all the glucose is used to produce ATP using oxygen in mitochondria, there will be no material left to build cells.

[0061] In addition, oxygen respiration in mitochondria increases the production of reactive oxygen species. Reactive oxygen species damage cells, inhibit proliferation and metastasis, and cause cell death. It is believed that cancer cells suppress the use of oxygen in mitochondria to prevent increased production of reactive oxygen species. For cancer cells, suppressing oxygen-using metabolism in mitochondria is favorable for their survival and proliferation. It is known that in the case of cancer cells, increasing the activity of the mitochondria of cancer cells suppresses proliferation and metastasis and induces cell death. This is because when glucose is completely broken down, there is a shortage of materials for cell proliferation, and increased oxygen respiration increases the production of active oxygen, which causes cancer cells to self-destruct. In other words, it is believed that a treatment that activates the mitochondria of cells can kill only cancer cells while enhancing the function of normal cells.

[0062] It was suggested that D-allose is specifically taken up by human renal cell carcinoma cells, thereby enhancing mitochondrial activity and exerting an antitumor effect. EXAMPLES

[0063] [Method of generating renal cell carcinoma xenograft mice] In this experiment, two types of human renal cell carcinoma cell lines (Caki-1, ACHN) were used. Each cell was cultured in RPMI-1640 culture medium (2000 mg D-glucose / L) containing 10% fetal bovine serum, HEPES buffer, and penicillin-streptmycin at 37°C in a humidified environment of 5% CO2. Renal cell carcinoma xenograft mouse models were cultured in MEM medium at 1.0 × 10 5 The cell suspension was adjusted to 0.1 cells / mL and injected into the subcutaneous tissue of the thigh of female athymic nude mice (BALB / c-nu / nu, 6 weeks old). One week later, the tumor volume was 200 mm 3 It was confirmed that this was the case and the result was used in subsequent experiments.

[0064] [Changes in D-allose concentration in tumors after intraperitoneal injection of D-allose into renal cell carcinoma xenograft mouse models] The rare sugar D-allose was dissolved in saline at 400mg / kg / 0.4mL and injected into the peritoneal cavity of a mouse xenograft model of two types of human renal cell carcinoma cell lines (Caki-I, ACHN) using a 27G needle. Tumors were excised from the mice before and 1, 2, and 4 hours after D-allose administration. The tumors were sonicated in 1mL PBS, centrifuged at 3000 rpm for 5 minutes, and monosaccharides in the supernatant were labeled with ABEE (4-aminobenzol acid ethyl ester), and D-allose was quantitatively analyzed by HPLC (high performance liquid chromatography). Figure 4 shows the change in D-allose concentration in the tumor after intraperitoneal injection of D-allose into a mouse xenograft model of renal cell carcinoma. As shown in Figure 4, D-allose was measured in both Caki-1 and ACHN tumors 1 hour after intraperitoneal injection of D-allose into renal cell carcinoma xenograft mouse models. The D-allose concentration in the tumor reached a maximum 1 hour after intraperitoneal injection in Caki-1 and 2 hours after intraperitoneal injection in ACHN, and then decreased.

[0065] [Changes in tumor volume in renal cell carcinoma xenograft mouse models following intraperitoneal injection of D-allose] Renal cell carcinoma xenograft mouse model with tumor size of 200 mm 3The day when the above was reached was designated as day 0, and from day 1, intraperitoneal injection of a solution containing D-allose was started. The injected solution was 0.4 mL of physiological saline, 100 mg / kg, or 400 mg / kg of D-allose dissolved in 0.4 mL of physiological saline. The mice were divided into three groups (Control, D-allose 100 mg / kg, D-allose 400 mg / kg) and injected once a day into the abdominal cavity for a total of 5 weeks, and the body weight and the long and short diameters of the tumor were measured twice a week over time. The tumor volume was calculated as the long diameter x short diameter x short diameter x 0.5 of the tumor. On the 35th day after the start of intraperitoneal injection, the liver and kidneys were removed from the mice along with the tumor and used for the subsequent experiments. FIG. 5 shows the change in tumor volume in a mouse model of renal cell carcinoma xenografted by intraperitoneal injection of D-allose. As shown in Figure 5, in the evaluation of tumor volume in a xenograft mouse model created using ACHN, the D-allose 400 mg / kg group was significantly smaller than the Control group from day 13 onwards. In a xenograft mouse model created using Caki-1, the tumor volume in the D-allose 400 mg / kg group from day 16 onwards and in the D-allose 100 mg / kg group from day 31 onwards was significantly smaller than that in the Control group. In addition, the tumor volume in the D-allose 400 mg / kg group from day 16 onwards was significantly smaller than the tumor volume before administration. In addition to the tumor growth inhibitory effect, a tumor shrinking effect is also expected depending on the dose of D-allose (Mann-Whitney U test).

[0066] [Antitumor effects of D-allose in renal cell carcinoma xenograft mouse models: necrosis rate and mitotic rate] The tumor, kidney and liver tissues were fixed in 4% paraformaldehyde-phosphate buffer, embedded in paraffin and sliced ​​to a thickness of 4 μm. The slices were stained with hematoxylin and eosin and evaluated by a pathologist. The effect of D-allose on tumor tissue was evaluated by the necrosis rate (%) and the number of mitoses (cells / HPF). The necrosis rate was measured using the image recording software Nikon NIS-Elements D and was rounded down to the nearest whole number. The number of mitoses was calculated as the average of three fields of view and was also rounded down to the nearest whole number. The antitumor effect of D-allose on renal cell carcinoma xenograft mouse models: the necrosis rate and the number of mitoses are shown in Table 10.

[0067] [Table 10]

[0068] As a result, in tumor tissue of a mouse xenograft model of renal cell carcinoma generated using Caki-1 and ACHN, the necrotic rate increased and the number of mitoses decreased in a dose-dependent manner. Figure 6 shows the effect of D-allose on renal cell carcinoma xenograft mouse models: a graph of changes in body weight. No significant difference was observed in the body weight of the mice among the three groups (Mann-Whitney U test). Figure 7 shows the effect of D-allose on a renal cell carcinoma xenograft mouse model: a micrograph (x100) showing changes in kidney tissue. No effect of D-allose on kidney tissue was observed (diagnosis by a pathologist). Figure 8 shows the effect of D-allose on a renal cell carcinoma xenograft mouse model: a microscopic photograph (x100) showing changes in liver tissue. No effect of D-allose on liver tissue was observed (diagnosis by a pathologist). [Industrial Applicability]

[0069] The present inventors have found that D-allose is specifically incorporated into human renal cell carcinoma cells, which are a type of human cancer cell, and that the incorporated D-allose has antitumor activity. D-allose is water-soluble and therefore has high membrane permeability, and is dissolved in an aqueous medium, so that no aggregates are formed. Furthermore, since it is specifically taken up by renal cell carcinoma cells, it can be used as a drug carrier that can selectively deliver anticancer drugs to renal cell carcinoma cells, and can increase the uptake of anticancer drugs by renal cell carcinoma cells, allowing both the taken up D-allose and the anticancer drug to exert their antitumor activities, making it a promising candidate for a groundbreaking molecular targeted therapeutic drug for renal cell carcinoma.

Claims

1. An anticancer agent for renal cell carcinoma, which is any one of a liquid, powder, granule, tablet, injection, suppository, or external preparation, contains D-allose, is selectively taken up into renal cell carcinoma cells, and contains an agent that selectively accumulates in renal cell carcinoma cells, and the D-allose selectively taken up into renal cell carcinoma cells exerts an antitumor effect.

2. D-allose is D-allose and / or its derivative and / or its mixture, The derivatives of D-allose include sugar alcohols in which the carbonyl group of D-allose is an alcohol group, uronic acids in which the alcohol group of D-allose is oxidized, and NH ２ 2. The anticancer agent for renal cell carcinoma according to claim 1, which is a D-allose derivative selected from amino sugars substituted with a group.

3. An anticancer agent for renal cell carcinoma described in claim 1 or claim 2, further comprising a drug associated with D-allose.

4. The anti-cancer drug for renal cell carcinoma according to claim 3 , wherein the drug comprises an anti-cancer drug.

5. An anticancer agent for renal cell carcinoma described in claim 3 or claim 4, wherein D-allose and the drug are covalently associated directly or via a linker.

6. An anticancer agent for renal cell carcinoma described in any of claims 3 to 5, wherein the drug is a radioactive isotope, an enzyme, a prodrug activating enzyme, a radiosensitizer, an iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an intercalating antibiotic, an antimetabolite, an aromatase inhibitor, a mitosis inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

7. An anticancer agent for renal cell carcinoma described in any one of claims 1 to 6, further comprising at least one of an excipient, a stabilizer, a preservative, a binder, and a disintegrant.

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