Drug delivery vehicles, methods of drug delivery, and compositions for treating renal cell carcinoma that exploit the uptake of d-allose by cancer cells

Abstract

The technical problem to be solved by the present application is to provide a renal cell carcinoma treatment composition and to provide a composition or method for increasing drug uptake into renal cell carcinoma cells. The present application is constituted as follows: a renal cell carcinoma treatment composition containing D-allose, or a composition or method for increasing drug uptake into renal cell carcinoma cells containing D-allose. The composition is a pharmaceutical composition for administering an effective amount to a patient in need of renal cell carcinoma treatment in a human or an animal other than a human.

Description

Technical Field

[0001] This invention relates to a carrier for selectively delivering drugs to renal cell carcinoma cells by utilizing the property of D-allose being taken up by cancer cells, a drug delivery method, and a composition for treating renal cell carcinoma. Background Technology

[0002] Cancer treatments are broadly categorized into surgery, radiation therapy, and chemotherapy. Chemotherapy involves administering anticancer agents to cancer patients. It is used as adjuvant chemotherapy before or after surgery or radiation therapy to improve the chances of a cure, and for treating metastatic cancer that cannot be treated with surgery or radiation therapy. Currently, anticancer agents such as metabolic antagonists, topoisomerase inhibitors, molecularly targeted drugs, and nucleic acid agents are clinically available and have achieved a level of potential cure for several types of cancer. However, satisfactory results have not yet been achieved in the chemotherapy of renal cell carcinoma.

[0003] Anticancer agents often do not selectively target tumor cells and can also affect normal cells, causing high toxicity and side effects. Tissues with high cell division rates are particularly vulnerable, and this harmful systemic toxicity limits the dosage of anticancer agents that can be administered to cancer patients, thus limiting their effectiveness. Furthermore, because some anticancer agents are hydrophobic with low solubility, they not only have low membrane permeability but can also form aggregates of anticancer agents that are insoluble in aqueous media. Therefore, when administered intravenously, they may cause capillary embolism before penetrating the tumor.

[0004] Currently, in order to selectively deliver drugs to tumors, drug delivery systems have been developed, such as those encapsulating drugs in carriers like micelles, liposomes, microparticles, antibodies, and drug-polymer conjugates (Patent Document 1). Although these attempts aim to improve the accumulation of anticancer agents in tumor tissues, it remains difficult to prevent the accumulation of anticancer agents in normal tissues such as the liver and kidneys. There is a long-awaited prospect of developing drug carriers that can selectively deliver anticancer agents to cancer cells.

[0005] On the other hand, among the research findings on the application of rare sugars in the medical field, there is an invention of an in vivo antioxidant with D-allose as the active ingredient (Patent Document 2). This invention involves administering D-allose to patients with liver cancer or skin cancer, utilizing the in vivo antioxidant effect of D-allose to treat liver cancer or skin cancer in a composition.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2015-155392

[0009] Patent Document 2: Japanese Patent No. 5330976

[0010] Patent Document 3: Japanese Patent Application Publication No. 2004-298106

[0011] Non-patent literature

[0012] Non-patent literature 1: J. Ferment. Bioeng. 84, 319, 1997 Summary of the Invention

[0013] The technical problem that the invention aims to solve

[0014] The object of this invention is to provide a novel use of D-allose, which has the property of being taken up by cancer cells; specifically, to provide a novel use of D-allose, which has the property of being taken up by cancer cells, as a drug delivery carrier. Another object of this invention is to provide a drug delivery method using this delivery carrier. Furthermore, the object of this invention is to provide a renal cell carcinoma therapeutic composition as an anticancer agent with excellent antitumor effects against renal cell carcinoma. In addition, the object of this invention is to provide a renal cell carcinoma therapeutic composition capable of increasing drug uptake by renal cell carcinoma cells, and a composition or method for increasing drug uptake by renal cell carcinoma cells.

[0015] Technical solutions for solving technical problems

[0016] To solve the aforementioned technical problems, the inventors of this invention conducted in-depth research and discovered for the first time that D-allose is specifically taken up by human renal cell carcinoma cells, and that the taken-up D-allose inhibits the proliferation of renal cell carcinoma cells, thus exhibiting antitumor activity. This led to the completion of this invention. Furthermore, by using D-allose, which can be taken up by human renal cell carcinoma cells, as a carrier for chemotherapeutic agents or nucleic acid drugs used as anticancer agents, the uptake of anticancer agents by renal cell carcinoma cells can be increased, thereby enabling the combined antitumor activity of both the taken-up D-allose and the anticancer agent.

[0017] The essence of the present invention lies in the following (1) to (4) preparations that utilize the property of being selectively taken up by renal cell carcinoma cells.

[0018] (1) An preparation that utilizes the property of being selectively taken up by renal cell carcinoma cells, characterized in that it contains D-allose.

[0019] (2) The preparation as described in (1) above, wherein D-aloose taken up by renal cell carcinoma cells exerts an antitumor effect.

[0020] (3) The formulation as described in (1) or (2) above, wherein D-allose is D-allose and / or its derivatives and / or mixtures thereof.

[0021] (4) The formulation as described in (3) above, wherein the D-allose derivative is a D-allose derivative selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acid formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

[0022] The essence of the present invention is the carrier for selectively delivering drugs to renal cell carcinoma cells as described in (5) to (11) below.

[0023] (5) A carrier for selectively delivering drugs to renal cell carcinoma cells, characterized in that it comprises D-allose.

[0024] (6) A carrier for selectively delivering drugs to renal cell carcinoma cells as described in (5) above, wherein D-aloose taken up by renal cell carcinoma cells exerts an antitumor effect.

[0025] (7) A carrier for selectively delivering drugs to renal cell carcinoma cells as described in (4) or (5) above, wherein D-allose is D-allose and / or its derivatives and / or mixtures thereof.

[0026] (8) The carrier for selectively delivering drugs to renal cell carcinoma cells as described in (7) above, wherein the D-allose derivative is a D-allose derivative selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acid formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

[0027] (9) A carrier for selectively delivering a drug to renal cell carcinoma cells as described in any one of (5) to (8) above, wherein the drug comprises an anticancer agent.

[0028] (10) A carrier for selectively delivering a drug to renal cell carcinoma cells as described in any one of (5) to (9) above, wherein D-aloose and the drug are directly associated or covalently associated via a linker arm.

[0029] (11) A carrier for selectively delivering drugs to renal cell carcinoma cells as described in any one of (5) to (10) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiation sensitizer, iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an embedded antibiotic, a metabolic antagonist, an aromatase inhibitor, a mitotic inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

[0030] The essence of the present invention is the following (12) to (19) compositions for treating renal cell carcinoma.

[0031] (12) A composition for treating renal cell carcinoma, characterized in that it contains D-allose.

[0032] (13) The composition for treating renal cell carcinoma as described in (12) above, wherein D-allose taken up by renal cell carcinoma cells exerts an antitumor effect.

[0033] (14) The composition for treating renal cell carcinoma as described in (12) or (13) above, wherein D-allose is D-allose and / or its derivatives and / or mixtures thereof.

[0034] (15) The composition for treating renal cell carcinoma as described in (14) above, wherein the D-allose derivative is a D-allose derivative selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acid formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

[0035] (16) The composition for treating renal cell carcinoma as described in any one of (12) to (15) above, further comprising a drug associated with D-aloose.

[0036] (17) The composition for treating renal cell carcinoma as described in (16) above, wherein the composition comprises an anticancer agent.

[0037] (18) The composition for treating renal cell carcinoma as described in (16) or (17) above, wherein D-aloose and the above-described drug are directly associated or covalently associated via a linker arm.

[0038] (19) The composition for treating renal cell carcinoma as described in any one of (16) to (18) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiation sensitizer, iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an embedded antibiotic, a metabolic antagonist, an aromatase inhibitor, a mitotic inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

[0039] The essence of the present invention is the following (20) to (26) method for delivering drugs to renal cell carcinoma cells.

[0040] (20) A drug delivery method for delivering a drug to renal cell carcinoma cells, characterized in that the drug is prepared by loading the drug onto a drug delivery carrier, the drug is applied to the renal cell carcinoma cells, and the drug is selectively taken up by the renal cell carcinoma cells, thereby selectively delivering the drug to the cells, wherein the carrier is D-allose, and the drug is loaded onto the D-allose via chemical bonds.

[0041] (21) The drug delivery method described in (20) above is a method of increasing drug uptake to renal cell carcinoma cells by utilizing the property of D-aloose being taken up by cancer cells.

[0042] (22) The drug delivery method as described in (20) or (21) above, characterized in that the carrier is D-allose, and the drug is directly associated with the D-allose or covalently associated and carried by the carrier via a connecting arm.

[0043] (23) The drug delivery method as described in any one of (20) to (22) above, wherein the drug comprises an anticancer agent.

[0044] (24) The drug delivery method as described in any one of (20) to (23) above, wherein the drug is a radioisotope, an enzyme, a prodrug activating enzyme, a radiation sensitizer, iRNA, an alkylating agent, a purine antagonist, a pyrimidine antagonist, a plant alkaloid, an embedded antibiotic, a metabolic antagonist, an aromatase inhibitor, a mitotic inhibitor, a growth factor inhibitor, a cell cycle inhibitor, or a topoisomerase inhibitor.

[0045] (25) The drug delivery method as described in any one of (20) to (24) above, wherein D-allose is D-allose and / or its derivatives and / or mixtures thereof.

[0046] (26) The drug delivery method as described in (25) above, wherein the D-allose derivative is a D-allose derivative selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acid formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

[0047] The effects of the invention

[0048] While D-allose is known to have anti-tumor effects against liver and skin cancer through its antioxidant activity in vivo, its anti-tumor effect on renal cell carcinoma remains unknown. Furthermore, it has not yet been proven that D-allose is absorbed into human cancer cells.

[0049] The inventors of this invention have discovered for the first time that D-allose can be taken up by human renal cell carcinoma cells, and that the taken-up D-allose has antitumor activity. Because D-allose is water-soluble, the extracted membrane has high permeability and does not form aggregates due to its dissolution in aqueous media.

[0050] Moreover, it is specifically taken up by human cancer cells, especially renal cell carcinoma cells. Therefore, it can be used as a drug carrier (selective drug delivery carrier) that can selectively deliver anticancer agents to renal cell carcinoma cells, thereby increasing the uptake of anticancer agents by renal cell carcinoma cells, and enabling both the taken-up D-allose and the anticancer agents to exert antitumor activity.

[0051] The 5-year survival rate for stage IV renal cell carcinoma in Japan is still low at 18.1%. Therefore, the renal cell carcinoma therapeutic composition containing D-allose of the present invention has the potential to become a breakthrough molecularly targeted therapy. Attached Figure Description

[0052] Figure 1 This indicates the results of a survival assay for the renal cell carcinoma cell line (ACHN).

[0053] Figure 2 This indicates the results of a survival test of the renal cell carcinoma cell line (Caki-I).

[0054] Figure 3 This indicates the results of a survival test of the renal cell carcinoma cell line (Caki-II).

[0055] Figure 4 This is a graph showing the shift in D-allose concentration in the tumor after intraperitoneal injection of D-allose into a mouse model of renal cell carcinoma xenograft.

[0056] Figure 5 This is a graph showing the tumor volume shift in a mouse model of renal cell carcinoma induced by intraperitoneal injection of D-allose.

[0057] Figure 6 This is a graph showing the effect of D-allose on a mouse model of renal cell carcinoma xenograft: a curve representing weight gain.

[0058] Figure 7 This is a micrograph (×100) showing the effect of D-allose on a mouse model of renal cell carcinoma xenograft: changes in kidney tissue.

[0059] Figure 8 This is a micrograph (×100) showing the effect of D-allose on a mouse model of renal cell carcinoma xenograft: changes in liver tissue. Detailed Implementation

[0060] D-Allose is used as a formulation that utilizes the property of selective uptake by renal cell carcinoma cells. D-Allose taken up by renal cell carcinoma cells exerts an antitumor effect. Furthermore, D-Allose is used as a carrier for selectively delivering drugs to renal cell carcinoma cells, providing a method for delivering drugs to renal cell carcinoma cells. This drug contains an anticancer agent, which is directly or via a linker arm covalently associated with D-Allose. Additionally, the present invention is a renal cell carcinoma therapeutic composition containing D-Allose, comprising a formulation containing an effective amount of D-Allose or a pharmacologically acceptable salt and / or hydrate. In the above-described drug delivery method, a carrier containing D-Allose is used. Various drugs can be used as the drug carried on this carrier, and the appropriate selection can be made according to the purpose. However, since the carried drug is selectively taken up by renal cell carcinoma cells, it is preferable to use a drug such as an anticancer agent. In other words, it is desirable to prepare a drug that carries an anticancer agent with the goal of being taken up by cancer cells via a carrier containing D-Allose.

[0061] The D-allose used in this invention is a rare sugar, present in far less abundance than D-glucose, which is abundant in nature. Among the 34 types of monosaccharides (six-carbon monosaccharides: hexoses), 16 types of aldoses, 8 types of ketoses, and 10 types of sugar alcohols, monosaccharides (aldecans, ketoses) and their derivatives (sugar alcohols) that exist only in trace amounts in nature are defined as "rare sugars," in contrast to the abundant "natural monosaccharides" represented by D-glucose. Currently, the rare sugars that can be mass-produced are D-allulose (D-allose) and D-allose. D-allose is the D-configuration of allose, an aldose classified as a hexacarbon sugar.

[0062] Methods for obtaining D-allose include methods for synthesizing D-allulose using L-rhamnosyl isomerase isolated from *Pseudomonas stutzeri* (Non-Patent Document 1), methods for obtaining D-allulose by acting D-xylose isomerase on a solution containing D-allulose, and methods for producing high-purity D-allose such as a separation method using D-allose crystallization (Patent Document 3). The D-allose of the present invention is not limited to these methods; it can also be obtained by isomerization through chemical treatment, and can be D-allose obtained by any method. Regarding D-allulose as a raw material for D-allose, the most common method currently is to obtain it by enzymatic (epimerase) treatment of fructose, but it is not limited to this. It can be obtained by using microorganisms that produce this enzyme, extracted from natural substances, or directly used from natural substances, or obtained by isomerization through chemical treatment. In addition, the method of purifying D-allulose using enzymes is well known.

[0063] Derivatives of D-allose are described below. A compound whose molecular structure is altered through a chemical reaction is called a derivative of the starting compound. Common derivatives of hexoses containing D-allose include sugar alcohols (which, upon reduction of monosaccharides, convert aldehyde and ketone groups into alcohol groups, becoming polyols with the same number of carbon atoms), uronic acids (substances formed by the oxidation of the alcohol group of monosaccharides; naturally known examples include D-glucuronic acid, galacturonic acid, and mannuronic acid), and amino sugars (substances formed by replacing the OH group of a sugar molecule with an NH2 group, such as glucosamine, chondroitin, and glycosides), but are not limited to these. D-allose derivatives are selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acids formed by the oxidation of the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

[0064] In the therapeutic compositions of the present invention, which incorporate D-allose and / or its derivatives and / or mixtures thereof, the composition contains an effective amount of D-allose and / or its derivatives and / or mixtures thereof. An "effective amount" refers to any amount sufficient to satisfy the intended purpose (e.g., a desired biological or medical response in a tissue or subject of examination). For example, in the present invention, this includes the amount used to inhibit the proliferation of renal cell carcinoma cells, the amount used to deliver the drug into the renal cell carcinoma cells, etc.

[0065] The present invention describes formulations of D-allose or its pharmacologically acceptable salts and / or hydrates thereof.

[0066] In addition to using only D-allose and / or its derivatives and / or mixtures thereof, appropriate additives such as common excipients, stabilizers, preservatives, binders, and disintegrants can be added, and appropriate formulations such as liquids, capsules, granules, pills, powders, and tablets can be selected for formulation. As the renal cell carcinoma treatment composition of the present invention, it is possible to manufacture various forms containing medically acceptable carriers, excipients, lubricants, binders, and other additives using known formulation techniques. These forms include liquids, powders, granules, tablets, injections, suppositories, and topical preparations dissolved in water or various infusion preparations. As non-oral dosage forms, dosage forms such as injections, infusions, topical preparations, or suppositories can be selected. Examples of injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections. Formulation techniques for producing the above-described formulations are known.

[0067] When administering the D-allose or its pharmacologically acceptable salts according to the present invention by injection, aqueous injections, aqueous suspension injections, fat emulsions, or liposome injections are preferred. In aqueous injections or aqueous suspension injections, the rare sugar D-allose or its pharmacologically acceptable salts according to the present invention are mixed with purified water. Water-soluble or water-swellable polymers, pH adjusters, surfactants, osmotic pressure regulators, preservatives, or other suitable additives are added and mixed as needed. The mixture is then dissolved or suspended while heating as needed, sterilized, and filled and sealed in an injection container to prepare an aqueous injection or aqueous suspension injection. Aqueous injections can be administered intravenously, subcutaneously, intramuscularly, intradermally, or intra-articularly. Aqueous suspension injections can also be administered subcutaneously, intramuscularly, intradermally, or intra-articularly. Oral administration is also possible.

[0068] As water-soluble or water-swellable polymers, gelatin, cellulose derivatives, acrylic acid derivatives, povidone, polyethylene glycol, polyamino acid derivatives, or polysaccharides are preferred. Among gelatin derivatives, refined gelatin is preferred. Among cellulose derivatives, methylcellulose, hydroxypropyl methylcellulose 2910, hydroxypropyl methylcellulose 2208, hydroxypropyl methylcellulose 2906, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, and sodium carboxymethyl cellulose are preferred. As acrylic acid derivatives, aminoacrylate-methylacrylate copolymers and methacrylate copolymers are preferred. As polyamino acid derivatives, polylysine and polyglutamic acid are preferred. As polysaccharides, hyaluronic acid, dextran, or cyclodextrin are particularly preferred. The amount of water-soluble or water-swellable polymer added varies depending on the nature and amount of aesculin, its derivatives, or pharmacologically acceptable salts, as well as the nature, molecular weight, and application site of the water-soluble or water-swellable polymer, but it can generally be used in the range of 0.01% to 10% relative to the total amount of the formulation.

[0069] pH adjusters use acids or bases that are harmless to the human body, and surfactants can be nonionic, anionic, or amphoteric surfactants. Additionally, osmotic pressure regulators can include sodium chloride and glucose; preservatives can include parabens; and ascorbic acid or sulfites can be used as preservatives. There are no particular limitations on their usage; they can be used within the range where they can exert their effects. Furthermore, depending on the need, local anesthetics such as procaine hydrochloride, analgesics such as benzyl alcohol, chelating agents, buffers, or water-soluble organic solvents can be added.

[0070] Fat emulsions are prepared by adding emulsifiers and D-allose or its pharmacologically acceptable salts to appropriate oils, adding purified water, and, as needed, adding water-soluble or water-swellable polymers, pH adjusters, surfactants, osmotic pressure regulators, preservatives or other preservatives, emulsifying using appropriate emulsification equipment, sterilizing, and filling and sealing into injection containers.

[0071] The "medicine" or "anticancer agent" of this invention refers to a substance administered to cancerous or precancerous tissues for treatment, such as radioactive isotopes (e.g., iodine-131, lutetium-177, rhenium-188, yttrium-90), toxins (e.g., diphtheria toxin, pseudomonas toxin, ricin toxin, white tree toxin), enzymes, enzymes that activate prodrugs, radiation sensitizers, 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 altering factors, anti-hormones, and anti-androgens, etc.

[0072] The drug is used after association (e.g., bonding, interaction) with D-allose. Association can be covalent or non-covalent. Regarding the association between D-allose and the drug, sufficient strength is required to prevent dissociation before or during delivery to and uptake into the renal cell carcinoma cells; any chemical, biochemical, or enzymatic coupling known to those skilled in the art can be used.

[0073] When the association between D-allose and a drug is non-covalent, interactions such as hydrophobic interactions, electrostatic interactions, dipole interactions, van der Waals interactions, and hydrogen bonds can be involved. When the association between D-allose and a drug is covalent, the bond can be direct or indirect via a linker arm. Such covalent bonds can be achieved using amide, ester, carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, or carbonate bonds.

[0074] Regarding the safety verification of D-allose required for use as a pharmaceutical ingredient, although it is a rare sugar, it is a monosaccharide that exists in nature, and therefore its safety can be anticipated. Mutagenicity, biodegradability, and three types of acute toxicity tests (oral acute toxicity test, single skin irritation test, and single eye irritation test) were defined as the most basic safety tests. The inventor commissioned a designated institution to conduct safety tests on the basic components of D-allose, and the results confirmed that its safety was not an issue.

[0075] The therapeutic composition of the present invention is applicable to animals including humans (humans, cattle, pigs, dogs, cats and other mammals, birds such as chickens, etc.). Furthermore, the therapeutic composition of the present invention targets renal cell carcinoma cells, and human renal cell carcinoma cell lines such as ACHN, Caki-I, and Caki-II can be cited as cell lines.

[0076] The present invention will now be described in detail using examples. The present invention is not limited to these examples.

[0077] Example 1

[0078] [Rare sugar uptake experiment using human bladder cancer cell lines, human prostate cancer cell lines, and human renal cell carcinoma cell lines]

[0079] Using three cancer cell lines, the uptake of rare sugars added to the culture medium into cancer cells was analyzed. Four rare sugars were used: D-allose, D-allulose (D-alusose), L-allulose (L-alusose), or allitol. D-glucose and fructose were used as monosaccharides that were not rare sugars.

[0080] Cell lines of various types were cultured in RPMI-1640 medium. During culture, six different sugars were added to the medium separately, and the cells were cultured again. After culture, the amount of sugar taken up into the cells was analyzed. Cells cultured without added sugar served as a control.

[0081] 1) Use of cell lines

[0082] 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.

[0083] 2) Use culture medium

[0084] After culturing cells in RPMI-1640 (2000 mg D-glucose / L) medium, various monosaccharides and dilute sugars were added to each medium. Specifically, seven sugars were added: 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-alitol, to achieve a final sugar concentration of 10 mM in the medium. No sugars were added in the control.

[0085] 3) Cultivation methods

[0086] 3.0×10 will be created using RPMI-1640. 4 5 ml of cell suspension (1.5 × 10⁶ cells / ml) was seeded into each 6 cm diameter culture dish. 5 Cells (or culture dishes) were cultured for 24 hours in RPMI-1640 medium containing 2000 mg / L D-glucose as a nutrient source. After 24 hours, the culture was changed to the culture medium containing monosaccharides and rare sugars as described in 2) above, and cultured for 48 hours.

[0087] 4) Methods for analyzing sugars in cultured cells

[0088] After 48 hours of culture, adherent human cancer cells were mechanically detached, and the culture medium and suspended cells were collected in microcentrifuge tubes. The supernatant from centrifugation (4°C, 1200 rpm, 5 min) was removed, leaving only the cell pellet. For washing purposes, the cell pellet was resuspended in 5 ml of phosphate-buffered saline (PBS), and centrifuged again at 4°C, 1200 rpm for 5 min to remove the supernatant. 1 ml of purified water was added to the washed cell pellet to resuspend it, and then it was sonicated (40% intensity, 30 seconds). Monosaccharides in the cell lysate were labeled with ABEE and analyzed by HPLC. The cellular sugar content was determined from the area obtained using a standard curve. Regarding ketoses, two peaks were identified after ABEE labeling, so the cellular sugar content was determined from the standard curve of these two peaks.

[0089] The results are shown in Tables 1 (sugar content in lysate of human bladder cancer cell line RT112), 2 (sugar content in lysate of human bladder cancer cell line 253J), 3 (sugar content in lysate of human bladder cancer cell line J82), 4 (sugar content in lysate of human prostate cancer cell line LNCa), 5 (sugar content in lysate of human prostate cancer cell line Du145), 6 (sugar content in lysate of human prostate cancer cell line PC-3), 7 (sugar content in lysate of human renal cell carcinoma cell line ACHN), 8 (sugar content in lysate of human renal cell carcinoma cell line Caki-I), and 9 (sugar content in lysate of human renal cell carcinoma cell line Caki-II).

[0090] [Table 1]

[0091]

[0092] Sugar content in RT112 cell lysate

[0093] HCPL analysis of lysate of RT112 bladder cancer cell line revealed the presence of D-glucose in cells in all culture media. High levels of D-glucose were observed in cells cultured in RPMI-1640+D-glucose and RPMI-1640+D-aloose media, which are rich in D-glucose.

[0094] Cells cultured with RPMI-1640+L-allulose contained 13.8 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contained 10.0–13.0 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contained 12.7–15.7 μg of D-fructose.

[0095] [Table 2]

[0096]

[0097] Sugar content in 253J cell lysate

[0098] HCPL analysis was performed on the cell lysate of bladder cancer cell line 253J. The results showed that D-glucose was detected in cells in all culture media. The cells cultured in RPMI-1640+D-glucose, RPMI-1640+D-fructose and RPMI-1640+D-alulose media, which have high D-glucose content in their respective media, showed high levels of D-glucose.

[0099] Cells cultured with RPMI-1640+L-allulose contain 15.5–17.0 μg of L-allulose, while cells cultured with RPMI-1640+D-allulose contain 14.1–15.8 μg of D-allulose.

[0100] [Table 3]

[0101]

[0102] Sugar content in J82 cell lysate

[0103] HCPL analysis of lysate of bladder cancer cell line J82 revealed the presence of D-glucose in cells across all culture media. High levels were observed in cells cultured with RPMI-1640+D-glucose and RPMI-1640+D-aloose, which are high in D-glucose-rich media.

[0104] Cells cultured with RPMI-1640+L-allulose contained 13.2–14.1 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contained 11.8–14.3 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contained 11.1–15.0 μg of D-fructose.

[0105] [Table 4]

[0106]

[0107] Sugar content in LNCap cell lysate

[0108] HCPL analysis of LNCa prostate cancer cell lysate revealed the presence of D-glucose in cells across all culture media, with high concentrations observed in cells cultured in RPMI-1640, RPMI-1640+L-alulose, and RPMI-1640+D-alulose media.

[0109] Cells cultured with RPMI-1640+L-allulose contained 16.4–19.7 μg of L-allulose, while cells cultured with RPMI-1640+D-allulose contained 14.6–17.8 μg of D-allulose.

[0110] [Table 5]

[0111]

[0112] Sugar content in Du145 cell lysate

[0113] HCPL analysis of the cell lysate of prostate cancer cell line Du145 revealed the presence of D-glucose in cells across all culture media, with high concentrations observed in cells cultured in RPMI-1640+D-aloxose medium.

[0114] Cells cultured with RPMI-1640+L-allulose contain 10.8–11.7 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contain 12.6–15.5 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contain 12.9–17.0 μg of D-fructose.

[0115] [Table 6]

[0116]

[0117] Sugar content in PC-3 cell lysate

[0118] HCPL analysis of PC-3 prostate cancer cell lysate revealed the presence of D-glucose in cells across all culture media, with high concentrations observed in cells cultured in RPMI-1640+D-glucose medium.

[0119] Cells cultured with RPMI-1640+L-allulose contained 17.9–21.6 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contained 14.7–19.7 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contained 16.5–20.1 μg of D-fructose.

[0120] Furthermore, cells cultured using RPMI-1640+D-allose contained 7.5 μg of D-allose.

[0121] [Table 7]

[0122]

[0123] Sugar content in ACHN cell lysate

[0124] HCPL analysis of the lysate of the renal cell carcinoma cell line ACHN revealed the presence of D-glucose in the cells across all culture media, with high concentrations observed in cells cultured in RPMI-1640+D-glucose medium.

[0125] Cells cultured with RPMI-1640+L-allulose contain 16.6–23.4 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contain 15.8–22.1 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contain 16.8–19.0 ​​μg of D-fructose.

[0126] Furthermore, cells cultured using RPMI-1640+D-allulose contained 11.2 μg of D-allulose.

[0127] [Table 8]

[0128]

[0129] Sugar content in Caki-I cell lysate

[0130] HCPL analysis of the lysate of the renal cell carcinoma cell line Caki-I revealed the presence of D-glucose in the cells across all culture media, with high concentrations observed in cells cultured in RPMI-1640+D-glucose medium.

[0131] Cells cultured with RPMI-1640+L-allulose contain 16.0–21.2 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contain 17.2–23.1 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contain 16.7–20.7 μg of D-fructose.

[0132] Furthermore, cells cultured using RPMI-1640+D-allulose contained 13.2 μg of D-allulose.

[0133] [Table 9]

[0134]

[0135] Sugar content in Caki-II cell lysate

[0136] HCPL analysis of the lysate of the renal cell carcinoma cell line Caki-II revealed the presence of D-glucose in the cells in all culture media, with high concentrations observed in cells cultured in RPMI-1640+D-glucose medium.

[0137] Cells cultured with RPMI-1640+L-allulose contain 14.5–17.8 μg of L-allulose, cells cultured with RPMI-1640+D-allulose contain 16.5–21.4 μg of D-allulose, and cells cultured with RPMI-1640+D-fructose contain 20.4–21.3 μg of D-fructose.

[0138] Furthermore, cells cultured using RPMI-1640+D-allulose contained 11.2 μg of D-allulose.

[0139] <Summary of Experimental Results>

[0140] In all human cancer cell lines, D-allulose and L-allulose uptake was observed. In contrast, D-allulose uptake was not observed in human bladder cancer cell lines, and in human prostate cancer cell lines, uptake was observed in only one of the three cell lines (PC-3). On the other hand, in human renal cell carcinoma cell lines, D-allulose uptake was observed in all cell lines (ACHN, Caki-I, Caki-II).

[0141] Example 2

[0142] [Analysis of the antitumor effects of rare sugars on human renal cell carcinoma cell lines]

[0143] The antitumor effects of rare sugars were analyzed using the three human renal cell carcinoma cell lines (ACHN, Caki-I, and Caki-II) used in Example 1. Ten 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. D-glucose and D-fructose were used as monosaccharides that were not rare sugars.

[0144] [Using culture medium]

[0145] The culture medium used was the minimum essential medium (MEM) containing 1000 mg / L D-glucose. MEM was adjusted to contain 10 mM, 25 mM, and 50 mM of each monosaccharide or oligosaccharide [represented by (10), (25), and (50) in the figure]. The antitumor effects of the oligosaccharides were evaluated. In addition, no sugar was added to the MEM in the control group.

[0146] [Experimental Procedure]

[0147] Fabricated using MEM, each 5.0 × 10 4 Cell suspensions of cells / ml were dispensed into 96-well plates at 0.1 ml / well and cultured for 24 hours. Afterward, the culture medium was removed, and MEM medium containing 10 mM, 25 mM, and 50 mM of monosaccharides or rare sugars was added to each well at 0.1 ml / well, and the plates were cultured for 24 hours (#1–#13).

[0148] #1: MEM solution alone (Figure: Control)

[0149] #2: D-glucose solution (D-glucose in the image)

[0150] #3: L-Allulose solution (L-Allulose in the image)

[0151] #4: D-Allulose solution (D-Allulose in the image)

[0152] #5: D-fructose solution (D-fructose in the image)

[0153] #6: D-Allose Solution (D-Allose in the image)

[0154] #7: L-fructose solution (in the image, L-fructose)

[0155] #8: D-Mannose solution (D-Mannose in the image)

[0156] #9: L-sorbose solution (L-sorbose in the image)

[0157] #10: D-Tagatose solution (D-Tagatose in the image)

[0158] #11: D-galactose solution (D-galactose in the image)

[0159] #12: D-sorbose solution (D-sorbose in the image)

[0160] #13: L-Tagatose solution (L-Tagatose in the image)

[0161] [Cell viability assay (MTT method)]

[0162] The MTT assay is one of the methods for determining viability. It utilizes the insoluble form of MTT, which accompanies the reduction of the tetrazolium salt. The colorimetric reaction of the pigment (blue). The reduction of MTT is caused by the mitochondrial reductase succinate-tetrazolium reductase. This enzyme has high activity in living cells, which is evident in the color development, while the color disappears during cell death, including apoptosis. Cell viability is determined by measuring this color development using a microplate reader.

[0163] [Results of cell survival assay]

[0164] The results of the survival rate determination of human renal cell carcinoma cells are shown in... Figures 1-3 .Right now, Figure 1 This indicates the results of a survival assay for the renal cell carcinoma cell line (ACHN). Figure 2 This indicates the results of a survival assay for the renal cell carcinoma cell line (Caki-I). Figure 3 This indicates the results of a survival assay for the renal cell carcinoma cell line (Caki-II). For example... Figures 1-3 As shown, among these three cell lines, D-allose significantly reduced the survival rate compared to the control at all concentrations of 10mM, 25mM, and 50mM (p<0.05), exhibiting the strongest antitumor effect among the elucidated sugars.

[0165] [Inspection]

[0166] In cancer cells, ATP production within the mitochondria is inhibited. In cancer cells, a metabolic system called glycolysis, which produces ATP from glucose without using oxygen, is overactive. Glycolysis takes place in the cytoplasm. There are several reasons why cancer cells inhibit mitochondrial oxygen respiration.

[0167] One reason is that the materials used to synthesize cells require large amounts of glucose. To divide and increase their number, cells need to generate new cellular components such as nucleic acids, cell membranes, and proteins. Cells can generate nucleic acids, lipids, or amino acids from glucose through the glycolysis system or various intracellular metabolic pathways derived from this pathway. When oxygen is used in mitochondria to dedicate all glucose to ATP production, the materials used to build cells disappear.

[0168] Furthermore, mitochondrial respiration increases the production of reactive oxygen species (ROS). ROS damage cells, inhibit proliferation or metastasis, and contribute to cell death. It is believed that cancer cells inhibit oxygen utilization within mitochondria to prevent increased ROS production. For cancer cells, inhibiting oxygen-using metabolism within mitochondria is beneficial for survival and proliferation.

[0169] It is known that in cancer cells, increasing the activity of mitochondria can inhibit proliferation and metastasis, leading to cell death. This is because when glucose is completely broken down, there is insufficient material for cell proliferation, and increased oxygen respiration leads to the production of reactive oxygen species (ROS), which cause damage to cancer cells, causing them to die. In other words, it is believed that mitochondrial activation therapy can improve the function of normal cells while simultaneously killing cancer cells.

[0170] This suggests that D-allose, through specific uptake by human renal cell carcinoma cells, can enhance mitochondrial activity and exert anti-tumor effects.

[0171] Example 3

[0172] [Methods for creating xenograft mice for renal cell carcinoma]

[0173] This experiment used two human renal cell carcinoma cell lines (Caki-1 and ACHN). The cells were cultured in RPMI-1640 medium (2000 mg D-glucose / L) containing 10% fetal bovine serum, HEPES buffer, and penicillin-streptmycin, at 37°C under humidified conditions of 5% CO2.

[0174] Regarding the mouse model of renal cell carcinoma xenograft, the cultured cells were adjusted to 1.0 × 10⁶ cells / mL using MEM medium. 5 A cell suspension of 0.1 mL was injected subcutaneously into the thigh tissue of female athymic nude mice (BALB / c-nu / nu, 6 weeks old). One week later, the tumor volume was confirmed to be 200 mmHg. 3 The above information is used for subsequent experiments. [Shift in D-allose concentration in tumors after intraperitoneal injection of D-allose in a mouse model of renal cell carcinoma xenograft]

[0175] The dilute sugar D-allose was prepared into a solution at 400 mg / kg / 0.4 mL using physiological saline and injected intraperitoneally into xenograft mouse models of two human renal cell carcinoma cell lines (Caki-I and ACHN) using a 27G needle. Tumors were removed from the mice before D-allose administration and at 1, 2, and 4 hours post-administration. The tumors were sonicated in 1 mL PBS, and the monosaccharides in the supernatant obtained by centrifugation at 3000 rpm for 5 minutes were labeled with ABEE (4-aminobenzoic acid ethyl ester). D-allose was quantitatively analyzed using HPLC (high performance liquid chromatography). Figure 4 This indicates the shift in D-allose concentration in the tumor after intraperitoneal injection of D-allose into a mouse model of renal cell carcinoma xenograft. (Example:) Figure 4 As shown, D-allose was detected in both Caki-1 and ACHN tumors within 1 hour after intraperitoneal injection into a mouse model of renal cell carcinoma xenograft. The highest concentration of D-allose was observed in Caki-1 tumors at 1 hour after intraperitoneal injection and in ACHN tumors at 2 hours after injection. The concentration of D-allose in the tumors then decreased.

[0176] [Tumor volume shift in a mouse model of renal cell carcinoma induced by intraperitoneal injection of D-allose]

[0177] The tumor size in the mouse model of renal cell carcinoma xenograft reached 200 mm. 3 The dates above are designated as day 0. Starting from day 1, a solution containing D-allose was injected intraperitoneally. The injected solution consisted of 0.4 mL of physiological saline, prepared by dissolving 100 mg / kg or 400 mg / kg of D-allose in 0.4 mL of physiological saline. This solution was injected intraperitoneally once daily for 5 weeks into mice divided into three groups (control, D-allose 100 mg / kg, and D-allose 400 mg / kg). Mouse weight and tumor length and width were measured twice weekly. Tumor volume was calculated as length × width × width × 0.5. On day 35 after the start of intraperitoneal injection, the liver and kidneys were removed from the mice along with the tumors for subsequent experiments.

[0178] Figure 5 The figure shows the shift in tumor volume in a mouse model of renal cell carcinoma xenograft induced by intraperitoneal injection of D-allose.

[0179] like Figure 5As shown, in the evaluation of tumor volume in the xenograft mouse model established using ACHN, the D-allose 400 mg / kg group showed significantly smaller tumor volume after day 13 compared to the control group. In the xenograft mouse model established using Caki-1, the tumor volume in the D-allose 400 mg / kg group was significantly smaller after day 16, and in the D-allose 100 mg / kg group after day 31 compared to the control group. Furthermore, the tumor volume in the D-allose 400 mg / kg group after day 16 was also significantly smaller than the tumor volume before administration. These results indicate that not only can a tumor proliferation inhibition effect be expected, but a tumor shrinkage effect can also be expected depending on the dosage of D-allose (Mann-Whitney U test).

[0180] [Antitumor effect of D-allose in a mouse model of renal cell carcinoma xenograft: necrosis rate and mitotic count]

[0181] The excised tumors, kidneys, and livers were fixed in 4% paraformaldehyde-phosphate buffer, embedded in paraffin, and thinly sectioned at a thickness of 4 μm. The thin sections were stained with hematoxylin and eosin and evaluated by pathologists. The effect of D-allose on tumor tissue was evaluated by necrosis rate (%) and mitotic count (Cells / HPF). The necrosis rate was measured using Nikon NIS-Elements D image recording software, and the values ​​were rounded down. The mitotic count was calculated using the average of three fields of view, also rounded down. Table 10 shows the antitumor effect of D-allose in a mouse model of renal cell carcinoma xenograft: necrosis rate and mitotic count.

[0182] [Table 10]

[0183]

[0184] As a result, in tumor tissues of a mouse model of renal cell carcinoma xenograft created using Caki-1 and ACHN, the necrosis rate increased and the number of nuclear divisions decreased depending on the dosage of D-allose.

[0185] Figure 6 The figure shows the effect of D-allose on a mouse model of renal cell carcinoma xenograft: a graph of weight gain. No significant difference in mouse weight was observed among the three groups (Mann-Whitney U test).

[0186] Figure 7 The image shows the effect of D-allose in a mouse model of renal cell carcinoma xenograft: a micrograph (×100) showing changes in kidney tissue. No effect of D-allose on kidney tissue was observed (diagnosed by a pathologist).

[0187] Figure 8 The image shows the effects of D-allose in a mouse model of renal cell carcinoma xenograft: a micrograph (×100) showing changes in liver tissue. No effect of D-allose on liver tissue was observed (diagnosed by a pathologist).

[0188] Industrial utilization potential

[0189] The inventors of this invention discovered that D-allose is specifically taken up by human renal cell carcinoma cells, and that the taken-up D-allose exhibits antitumor activity. Because D-allose is water-soluble, it has high membrane permeability and does not form aggregates due to its solubility in aqueous media.

[0190] Moreover, since it is specifically taken up by renal cell carcinoma cells, it can be used as a drug carrier that can selectively deliver anticancer agents to renal cell carcinoma cells, increase the uptake of anticancer agents by renal cell carcinoma cells, and exert the antitumor activity of both the taken-up D-aloose and the anticancer agents. Therefore, it is expected to be a breakthrough molecularly targeted therapy for renal cell carcinoma.

Claims

1. The use of D-allose in the manufacture of a carrier for selectively delivering drugs to human renal cell carcinoma cells, characterized in that: The carrier contains D-allose.

2. The use as described in claim 1, characterized in that: The human renal cell carcinoma cells are selected from at least one of ACHN, Caki-I, and Caki-II.

3. The use as described in claim 1, characterized in that: D-allose, taken up by the human renal cell carcinoma cells, exerts an anti-tumor effect.

4. The use as described in claim 1 or 2, characterized in that: D-Allose is D-Allose and / or its derivatives and / or mixtures thereof. The D-allose derivatives are selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acids formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

5. The use as described in claim 1 or 2, characterized in that: The drug is a radioactive isotope, enzyme, radiation sensitizer, iRNA, alkylating agent, plant alkaloid, embedded antibiotic, metabolic antagonist, aromatase inhibitor, mitosis inhibitor, growth factor inhibitor, cell cycle inhibitor, or topoisomerase inhibitor.

6. The use as described in claim 5, characterized in that: The drug is a prodrug activator of enzymes, a purine antagonist, or a pyrimidine antagonist.

7. The use as described in claim 1 or 2, characterized in that: D-allose and the drug are directly associated or covalently associated via a linker arm.

8. The use of D-allose in the manufacture of compositions for the treatment of renal cell carcinoma, characterized in that: The composition comprises a carrier for selectively delivering drugs to human renal cell carcinoma cells, the carrier containing D-allose.

9. The use as described in claim 8, characterized in that: The human renal cell carcinoma cells are selected from at least one of ACHN, Caki-I, and Caki-II.

10. The use as described in claim 8, characterized in that: D-allose, taken up by the human renal cell carcinoma cells, exerts an anti-tumor effect.

11. The use as described in claim 8 or 9, characterized in that: D-Allose is D-Allose and / or its derivatives and / or mixtures thereof. The D-allose derivatives are selected from sugar alcohols formed by converting the carbonyl group of D-allose to an alcohol group, uronic acids formed by oxidizing the alcohol group of D-allose, and amino sugars formed by replacing the alcohol group of D-allose with an NH2 group.

12. The use as described in claim 8 or 9, characterized in that: The composition also contains a drug associated with D-allose. The drug is a radioactive isotope, enzyme, radiation sensitizer, iRNA, alkylating agent, plant alkaloid, embedded antibiotic, metabolic antagonist, aromatase inhibitor, mitosis inhibitor, growth factor inhibitor, cell cycle inhibitor, or topoisomerase inhibitor.

13. The use as described in claim 12, characterized in that: The drug is a prodrug activator of enzymes, a purine antagonist, or a pyrimidine antagonist.

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