Cancer treatment drugs and cancer treatment methods

Abstract

Provided is a cancer therapeutic agent comprising: a hydrophobic anti-cancer agent in an effective amount; a cationic liposome in which the hydrophobic anti-cancer agent is encapsulated; and γ-polyglutamic acid in which the cationic liposome is encapsulated, or a salt thereof, wherein the cationic liposome contains a phospholipid or a salt thereof and contains a cationic lipid or a salt thereof.

Description

[Technical Field]

[0001] The present invention relates to a cancer therapeutic agent and a cancer treatment method. [Background technology]

[0002] Because the surface of cells is negatively charged, research has been conducted into methods for promoting cellular uptake of pharmaceuticals using positively charged cationic molecules. However, cationic molecules have the problem of being highly cytotoxic and nonspecifically binding to blood and various organs, causing red blood cell agglutination. In response to this problem, a method has been proposed in which a complex consisting of a drug and a cationic molecule is encapsulated in an anionic molecule in a drug delivery complex (see, for example, Patent Document 1).

[0003] On the other hand, numerous compounds are known as anticancer drugs, including doxorubicin, epirubicin, cyclophosphamide, thiotepa, cytoxin, taxoids, docetaxel, vinblastine, bleomycin, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, caminomycin, aminopterin, dactinomycin, and esperamycin (see, for example, Patent Documents 2 and 3). Anticancer drugs also include hydrophilic anticancer drugs. Examples of hydrophilic anticancer drugs include 5-fluorouracil, cytosine arabinoside, busulfan, methotrexate, cisplatin, melphalan, mitomycin C, daunomycin, and melphalan. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5382682 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-525429 [Patent Document 3] Japanese Patent Application Publication No. 2018-529667 Summary of the Invention [Problem to be solved by the invention]

[0005] For the many anticancer drugs available, methods for efficiently delivering them to cancer cells have yet to be elucidated. Therefore, one of the objects of the present invention is to provide an effective cancer therapeutic drug and cancer treatment method that are easily delivered to cancer cells. [Means for solving the problem]

[0006] According to an aspect of the present invention, there is provided a cancer therapeutic agent comprising an effective amount of a hydrophobic anticancer agent, a cationic liposome encapsulating the hydrophobic anticancer agent, and γ-polyglutamic acid or a salt thereof encapsulating the cationic liposome, wherein the cationic liposome comprises a phospholipid or a salt thereof, and a cationic lipid or a salt thereof.

[0007] In the above-mentioned cancer therapeutic agent, the phospholipid may be a neutral phospholipid.

[0008] In the above-mentioned cancer therapeutic agent, the phospholipid may be an unsaturated phospholipid.

[0009] In the above cancer therapeutic agent, the phospholipid may be 1,2-dioleoyl-sn-glycero-3-phosphocholine.

[0010] In the above cancer therapeutic agent, the cationic lipid may be N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium.

[0011] In the cancer therapeutic agent, the molar ratio of the phospholipid or a salt thereof in the cationic liposome may be 10% or more and 90% or less.

[0012] In the cancer therapeutic agent, the molar ratio of the cationic lipid or a salt thereof in the cationic liposome may be 10% or more and 90% or less.

[0013] In the above cancer therapeutic agent, the molar ratio of the phospholipid or a salt thereof to the cationic lipid or a salt thereof may be 10:90 to 90:10.

[0014] In the above cancer therapeutic agent, the molar ratio of the positively charged functional group of the cationic lipid or a salt thereof to the negatively charged functional group of the γ-polyglutamic acid or a salt thereof may be 1:1 to 1:100.

[0015] In the above-mentioned cancer therapeutic agent, the molecular weight of the γ-polyglutamic acid or a salt thereof may be 2 million or less.

[0016] The cancer therapeutic agent may have a substantially uncharged or negative surface charge.

[0017] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be an anthracycline antibiotic.

[0018] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be doxorubicin or a salt thereof.

[0019] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be a taxane anticancer drug.

[0020] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be paclitaxel or a salt thereof.

[0021] Furthermore, according to an aspect of the present invention, there is provided a cancer therapeutic drug comprising an effective amount of a hydrophobic anticancer drug, a cationic liposome encapsulating the hydrophobic anticancer drug, and γ-polyglutamic acid or a salt thereof encapsulating the cationic liposome, wherein the cationic liposome comprises a phospholipid or a salt thereof and a cationic lipid or a salt thereof, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine, and the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium.

[0022] In the above cancer therapeutic agent, the molar ratio of the phospholipid or a salt thereof to the cationic lipid or a salt thereof may be 10:90 to 90:10.

[0023] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be doxorubicin or a salt thereof.

[0024] In the above-mentioned cancer therapeutic drug, the hydrophobic anticancer drug may be paclitaxel or a salt thereof.

[0025] Furthermore, according to an aspect of the present invention, there is provided the above-described cancer therapeutic agent for use in treating cancer.

[0026] Furthermore, according to an aspect of the present invention, there is provided a method for treating cancer, which comprises administering the above-described cancer therapeutic agent to a human or a non-human animal.

[0027] Furthermore, according to an aspect of the present invention, there is provided a method for delivering a cancer therapeutic agent into a cell, the method comprising contacting the cell with the cancer therapeutic agent. [Effects of the Invention]

[0028] According to the present invention, it is possible to provide an effective cancer therapeutic drug and cancer treatment method. [Brief explanation of the drawings]

[0029] [Figure 1] 1 is a table listing the properties of the composite according to Example 1. [Figure 2] 10 is a fluorescence microscope image of cancer cells administered with an anticancer agent according to Example 2. [Figure 3] 1 is a graph showing the amount of doxorubicin taken up into cancer cells according to Example 2. [Figure 4] 10 is a graph showing the survival rate of cancer cells administered with the anticancer agent according to Example 3. [Figure 5] 10 is a graph showing the proliferation of cancer cells administered with the anticancer agent according to Example 4. [Figure 6] 10 is a graph showing the survival rate of mice administered with the anticancer agent of Example 5. [Figure 7] 10 is a graph showing the survival rate of mice administered with the anticancer agent of Example 6. [Figure 8] 1 is a graph showing the amount of doxorubicin released in Example 7. [Figure 9] 10 is a graph showing the amount of paclitaxel taken up into cancer cells according to Example 8. [Figure 10] 10 is a graph showing the survival rate of cancer cells administered with the anticancer agent of Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0030] The following describes in detail the embodiments of the present invention. Note that the embodiments described below are merely examples of methods for embodying the technical ideas of the present invention, and the present invention is not limited to these examples.

[0031] A cancer therapeutic drug according to an embodiment includes an effective amount of a hydrophobic anticancer drug, cationic liposomes encapsulating the hydrophobic anticancer drug, and gamma-polyglutamic acid (γPGA) or a salt thereof encapsulating the cationic liposomes. In the cancer therapeutic drug according to an embodiment, the cationic liposomes include a phospholipid or a salt thereof, and a cationic lipid or a salt thereof. The cancer therapeutic drug according to an embodiment is in a particulate form.

[0032] The hydrophobic anticancer agent is, for example, a low molecular weight compound. The molecular weight of the low molecular weight compound is, for example, 300 or more or 400 or more and 600 or less. Hydrophobicity refers to a partition coefficient P given by the following formula being greater than 0. P=log 10 P ow In the above formula, P ow is C o / C w and C o is the concentration of the anticancer drug in the 1-octanol layer (mol / L), and C wis the concentration of the anticancer drug in the aqueous layer (mol / L). The partition coefficient is measured, for example, by the shake-flask method. The pH of the aqueous layer is, for example, 7.4.

[0033] The hydrophobic anticancer agent is, for example, an anthracycline antibiotic. Examples of the anthracycline antibiotic include doxorubicin or a salt thereof, daunorubicin or a salt thereof, epirubicin or a salt thereof, amrubicin or a salt thereof, idarubicin or a salt thereof, valrubicin or a salt thereof, aclarubicin or a salt thereof, pirarubicin or a salt thereof, and mitoxantrone or a salt thereof.

[0034] The chemical name of doxorubicin is (2S,4S)-4-(3-Amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyloxy)-2,5,12-trihydroxy-2-hydroxyacetyl-7-methoxy-1,2,3,4-tetrahydrotetracene-6,11-dione monohydrochloride. The molecular formula of doxorubicin is C 27 H 29 NO 11 The chemical structure of doxorubicin is shown in chemical formula (1). [ka]

[0035] An example of a salt of doxorubicin is doxorubicin hydrochloride.

[0036] The hydrophobic anticancer agent is, for example, a taxane anticancer agent, such as paclitaxel or a salt thereof, docetaxel or a salt thereof, cabazitaxel or a salt thereof, taxadiene or a salt thereof, baccatin III or a salt thereof, taxinin A or a salt thereof, brevifoliol or a salt thereof, and taxaspine D or a salt thereof.

[0037] Examples of cancers that can be treated with the cancer therapeutic agents of the embodiments include malignant lymphoma, lung cancer, gastrointestinal cancer, bladder cancer, urothelial cancer, osteosarcoma, breast cancer, uterine cancer, bone and soft tissue tumors, bone tumors, multiple myeloma, and pediatric solid tumors. Examples of gastrointestinal cancers include stomach cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, hepatocellular carcinoma, and colorectal cancer. Examples of colorectal cancers include colon cancer and rectal cancer. Examples of pediatric solid tumors include Ewing's sarcoma family of tumors, rhabdomyosarcoma, neuroblastoma, retinoblastoma, hepatoblastoma, and nephroblastoma.

[0038] The number of molecules of the hydrophobic anticancer drug contained in one particle of the cancer therapeutic drug is 500 or more, 5,000 or more, or 50,000 or more, and the number of molecules of the hydrophobic anticancer drug contained in one particle of the cancer therapeutic drug is 10,000,000 or less, 1,000,000 or less, or 100,000 or less.

[0039] The mass proportion of the hydrophobic anticancer drug contained in one particle of the cancer therapeutic drug is 0.1 mass% or more, 0.5 mass% or more, 1 mass% or more, or 2 mass% or more. The mass concentration of the hydrophobic anticancer drug contained in one particle of the cancer therapeutic drug is 90 mass% or less, 50 mass% or less, or 25 mass% or less.

[0040] Examples of phospholipids contained in cationic liposomes include lecithin, lysolecithin, their hydrogenated derivatives, and their hydroxide derivatives. Examples of phospholipids contained in cationic liposomes also include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristylphosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (also known as dioleylphosphatidylcholine, DOPC), and distearoylphosphatidylserine (DSPS).

[0041] The phospholipid contained in the cationic liposome may be derived from an animal or plant, such as soybean or egg yolk, or may be a synthetic compound. The phospholipid may be, for example, a neutral phospholipid. The phospholipid may be, for example, an unsaturated phospholipid. The molecular formula of DOPC, an example of a phospholipid, is C 44 H 84 NO8P. The chemical structure of DOPC is shown in chemical formula (2). [ka]

[0042] The phospholipids inhibit the aggregation of the cationic lipids when forming cationic liposomes.

[0043] Examples of cationic lipids contained in cationic liposomes include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (also known as 1,2-dioleoyloxy-3-(trimethylammonium)propane, DOTAP), N,N-dioctadecylamidoglycylspermine (DOGS), dimethyldioctadecylammonium bromide (DDAB), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE). Other examples of cationic lipids contained in cationic liposomes include esters of dipalmitoylphosphatidic acid (DPPA) and hydroxyethylenediamine, and esters of distearoylphosphatidic acid (DSPA) and hydroxyethylenediamine.

[0044] The molecular formula of DOTAP, an example of a cationic lipid, is C 42 H 80The chemical structure of DOTAP is shown in chemical formula (3). DOTAP has an amino group as a positively charged functional group. [ka]

[0045] The cationic liposome may be a unilamellar liposome or a multilamellar liposome. The cationic liposome may contain one type of phospholipid or a salt thereof and one type of cationic lipid or a salt thereof. Alternatively, the cationic liposome may contain multiple types of phospholipids or salts thereof and multiple types of cationic lipids or salts thereof.

[0046] The mass ratio of the hydrophobic anticancer drug to the cationic liposome in one particle of the cancer therapeutic drug is, for example, 1:1, 1:5, 1:10, 1:50, or 1:100.

[0047] In one particle of the cancer therapeutic drug, the molar ratio of the phospholipid or a salt thereof to the total mass of the cationic liposome is, for example, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more. Also, in one particle of the cancer therapeutic drug, the molar ratio of the phospholipid or a salt thereof to the total mass of the cationic liposome is, for example, 90% or less, 85% or less, or 80% or less.

[0048] In one particle of the cancer therapeutic drug, the molar ratio of the cationic lipid or its salt to the total mass of the cationic liposome is, for example, 10% or more, 15% or more, or 20% or more. Also, in one particle of the cancer therapeutic drug, the molar ratio of the cationic lipid or its salt to the total mass of the cationic liposome is, for example, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less.

[0049] The molar ratio of the phospholipid or a salt thereof to the cationic lipid in the cationic liposome is, for example, 10:90 to 90:10, 15:85 to 85:10, 20:80 to 80:20, 25:75 to 75:25, or 30:70 to 70:30.

[0050] The cationic liposome may contain lipids other than phospholipids and cationic lipids, such as glycolipids and glycols.

[0051] The cationic liposome may further contain a lipid membrane stabilizer. Examples of the lipid membrane stabilizer include sterols. Examples of sterols include cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, and lanosterol. Examples of the lipid membrane stabilizer include sterol derivatives. Examples of the sterol derivatives include 1-O-sterol glucoside, 1-O-sterol maltoside, and 1-O-sterol galactoside.

[0052] γPGA or its salt is an anionic molecule that electrostatically interacts with the cationic lipid contained in the cationic liposome. The chemical structure of γPGA is shown in chemical formula (4). γPGA has a carboxyl group as a negatively charged functional group. [ka]

[0053] In chemical formula (4), R is a hydrogen atom; an alkali metal atom such as sodium, potassium, or lithium; a tertiary amine such as trimethylamine, triethylamine, dimethylamine, diethylamine, triethanolamine, trimethanolamine, diethanolamine, dimethanolamine, or ethanolamine; or a quaternary amine such as tetramethylamine or tetraethylamine. The R groups present in the molecule may be the same or different. n is an integer of 40 or greater.

[0054] The lower limit of the molecular weight of γPGA is, for example, but not limited to, 1,000 or more, 5,000 or more, or 10,000 or more, and the upper limit of the molecular weight of γPGA is, for example, but not limited to, 2 million or less, 1.5 million or less, 1 million or less, 800,000 or less, 600,000 or less, 400,000 or less, 200,000 or less, or 20,000 or less.

[0055] In the cancer therapeutic agent according to the embodiment, the cationic liposome is encapsulated in γPGA. Due to electrostatic interaction between the cationic lipid or a salt thereof contained in the cationic liposome and the anionic molecule γPGA, the surface charge of the cancer therapeutic agent according to the embodiment is substantially uncharged or negative. A substantially uncharged or negative surface charge means that the positive charge has been reduced to such an extent that the cancer therapeutic agent according to the embodiment does not cause red blood cell aggregation when contacted with blood. Furthermore, a substantially uncharged or negative surface charge means that the positive charge has been reduced to such an extent that the cell viability is at least 50% when the cancer therapeutic agent according to the embodiment is contacted with cells. Because the cancer therapeutic agent according to the embodiment has a substantially uncharged or negative surface charge, it is possible to suppress red blood cell aggregation even when administered systemically, such as intravenously. Furthermore, it is possible to suppress nonspecific delivery of hydrophobic anticancer agents to cells other than the target cancer cells.

[0056] The surface charge (ζ potential) of the cancer therapeutic agent according to the embodiment is, for example, but not limited to, -50 mV or more, -40 mV or more, or -30 mV or more. The surface charge (ζ potential) of the cancer therapeutic agent according to the embodiment is, for example, but not limited to, +30 mV or less, +20 mV or less, +10 mV or less, +5 mV or less, 0 mV or less, -10 mV or less, or -15 mV or less.

[0057] In the cancer therapeutic drug according to the embodiment, γPGA may be encapsulated inside the cationic liposome like a liposome. Alternatively, γPGA may be bound to the surface of the cationic liposome by electrostatic interaction, covering the cationic liposome. Note that, as long as the surface charge of the cancer therapeutic drug according to the embodiment is substantially uncharged or negative, the cationic liposome does not need to be completely covered with γPGA.

[0058] The ratio of cationic liposome to γPGA in the cancer therapeutic agent according to the embodiment is not particularly limited as long as the surface charge of the cancer therapeutic agent according to the embodiment is substantially uncharged or negative, but for example, the molar ratio of positively charged functional groups of the cationic liposome to negatively charged functional groups of γPGA is adjusted to be 1:1 to 1:100, 1:1 to 1:80, 1:1 to 1:60, 1:1 to 1:40, 1:1 to 1:20, 1:1 to 1:10, or 1:1 to 1:6. Because the cancer therapeutic agent according to the embodiment contains γPGA, it is possible to achieve lower cytotoxicity and reduced side effects compared to cationic therapeutic agents that do not contain anionic molecules.

[0059] The cancer treatment drug according to the embodiment is particulate, and the average particle size is, for example, 500 nm or less, 300 nm or less, or 100 nm or less. The particle size distribution and average particle size of the cancer treatment drug according to the embodiment can be calculated from the scattering intensity distribution obtained using, for example, a dynamic light scattering measurement device.

[0060] The cancer treatment drug according to the embodiment is prepared by contacting a phospholipid or a salt thereof with a cationic lipid or a salt thereof at an appropriate mixing ratio to form a cationic liposome, encapsulating a hydrophobic anticancer drug in the cationic liposome, and further contacting the cationic liposome encapsulating the hydrophobic anticancer drug with γPGA at an appropriate mixing ratio.

[0061] Hereinafter, as an example, a method for producing a cancer therapeutic drug according to an embodiment will be described in which the phospholipid is DOPC and the cationic lipid is DOTAP.

[0062] DOTAP and DOPC are dissolved in chloroform, and then mixed. The chloroform is then removed to form a lipid thin film composed of DOTAP and DOPC. An ammonium sulfate solution is added to the lipid thin film composed of DOTAP and DOPC to prepare a solution of cationic liposomes composed of DOTAP and DOPC. The ammonium sulfate in the external phase of the cationic liposome solution is replaced with a HEPES-buffered glucose solution at pH 8.0. A hydrophobic anticancer drug is then added to the cationic liposome solution and incubated at 65°C to prepare cationic liposomes encapsulating the hydrophobic anticancer drug in the internal phase. γPGA is then added to the solution of cationic liposomes encapsulating the hydrophobic anticancer drug, and the cationic liposomes encapsulating the hydrophobic anticancer drug are coated with γPGA to prepare a cancer therapeutic agent according to the embodiment.

[0063] The cancer therapeutic agent according to the embodiment can be used alone or formulated with a pharmacologically acceptable carrier according to conventional methods. The cancer therapeutic agent according to the embodiment can be provided as is. Alternatively, the cancer therapeutic agent according to the embodiment can be provided in the form of a suspension. Examples of liquids for suspending the cancer therapeutic agent include water and physiologically acceptable liquids. The physiologically acceptable liquid may be an aqueous solvent, an organic solvent, or a mixture of an aqueous solvent and an organic solvent. Examples of aqueous solvents include saline, phosphate-buffered saline (PBS), and cell culture medium. Examples of cell culture medium include RPMI 1640, DMEM, HAM F-12, and Eagle's medium. Examples of organic solvents include ethanol, methanol, and DMSO. The cancer therapeutic agent according to the embodiment can be provided together with physiologically acceptable excipients, vehicles, preservatives, stabilizers, and binders, as appropriate.

[0064] When the cancer therapeutic agent according to the embodiment is formulated, the cancer therapeutic agent according to the embodiment may be mixed with a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, and the like.

[0065] Examples of aqueous solutions for parenteral preparations, such as injections, include physiological saline and isotonic solutions containing glucose or other adjuvants. Examples of isotonic solutions include D-sorbitol, D-mannitol, and sodium chloride. A suitable solubilizer may be used in combination with aqueous solutions for parenteral preparations. Examples of solubilizers include alcohols, polyalcohols, and nonionic surfactants. Examples of alcohols include ethanol, examples of polyalcohols include propylene glycol and polyethylene glycol, and examples of nonionic surfactants include polysorbate 80™ and HCO-50. Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

[0066] Furthermore, the cancer therapeutic drug according to the embodiment may be used in combination with, for example, a buffer, a soothing agent, a stabilizer, a preservative, an antioxidant, etc. Examples of buffers include phosphate buffer and sodium acetate buffer, examples of soothing agents include benzalkonium chloride and procaine hydrochloride, examples of stabilizers include human serum albumin and polyethylene glycol, examples of preservatives include benzyl alcohol and phenol, and an example of antioxidants includes ascorbic acid.

[0067] Next, a method for delivering a cancer therapeutic agent into a cell is described, which comprises contacting the cancer therapeutic agent with the cell.

[0068] The type of cell is not particularly limited, and may be derived from a human or a non-human animal. Examples of non-human animals include monkeys, mice, rats, hamsters, and cows. The cells may be cultured cell lines containing cancer cells, cells isolated from an individual or tissue, or cells from tissue or tissue fragments. The cells may also be adherent or non-adherent cells.

[0069] For example, cells are suspended in an appropriate medium several days before contact with the cancer therapeutic agent according to the embodiment and cultured under appropriate conditions. At the time of contact with the cancer therapeutic agent according to the embodiment, the cells may or may not be in the proliferation phase. The culture medium at the time of contact may be a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% or less, and more preferably 20% or less. Excessive serum or other proteins in the medium may inhibit contact between the cancer therapeutic agent according to the embodiment and the cells.

[0070] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the type of cell, etc., but for example, 0.1 × 10 5 From 5 x 10 5 cells / mL, 0.1×10 5 From 4×10 5 cells / mL, 0.1×10 5 From 3 x 10 5 cells / mL, 0.2×105 From 3 x 10 5 cells / mL, or 0.2 x 10 5 From 2 x 10 5 cells / mL range.

[0071] The cancer therapeutic agent according to the embodiment is added to the cell-containing culture medium thus prepared. The amount of the solution containing the cancer therapeutic agent according to the embodiment to be added is not particularly limited and can be appropriately determined taking into account the number of cells, etc., but is, for example, in the range of 1 to 1000 μL, 1 to 500 μL, 1 to 300 μL, 1 to 200 μL, or 1 to 100 μL per 1 mL of culture medium.

[0072] After adding the cancer therapeutic agent according to the embodiment to the culture medium, the cells are cultured. The temperature, humidity, CO2 concentration, and other conditions during culture are appropriately set in consideration of the type of cells. When the cells are derived from mammals, examples of culture conditions include a temperature of approximately 37°C, humidity of approximately 95%, and a CO2 concentration of approximately 5%. The cell culture time can be appropriately set depending on the type of cells used, etc. The cell culture time is, for example, 1 to 72 hours, 1 to 60 hours, 1 to 48 hours, 1 to 40 hours, or 1 to 32 hours.

[0073] After the above-mentioned culturing, the medium may be replaced with a fresh medium or fresh medium may be added to the medium to continue culturing the cells. When the cells are derived from a mammal, the fresh medium may contain serum or nutrient factors.

[0074] Next, a method for treating cancer, which includes administering a cancer treatment drug according to an embodiment to a human or non-human animal, will be described.

[0075] By administering the cancer therapeutic agent according to the embodiment to a subject, the cancer therapeutic agent according to the embodiment reaches and contacts target cells within the subject, and the cancer therapeutic agent according to the embodiment is introduced into the target cells in vivo.

[0076] The subjects to which the cancer therapeutic agents according to the embodiments can be administered are not particularly limited, and include, for example, humans and non-human animals. Examples of non-human animals include monkeys, mice, rats, hamsters, and cows.

[0077] The method of administering the cancer therapeutic agent according to the embodiment is not particularly limited as long as the cancer therapeutic agent according to the embodiment can reach and contact the target cells and can be introduced into the cells, and may be oral administration or parenteral administration. Examples of parenteral administration include intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, and intraperitoneal administration.

[0078] The dose of the cancer therapeutic drug according to the embodiment is not particularly limited as long as it can achieve intracellular delivery of the drug, and can be appropriately selected in consideration of the type of subject, the administration method, the type and site of the target cell, etc. For example, when administered parenterally, such as intravenously, the single dose for a human weighing 60 kg is approximately 0.0001 mg to 10,000 mg.

[0079] The cancer treatment drug according to the embodiment is not particularly limited, but can be used as, for example, a treatment drug for peritoneal dissemination of colon cancer or a treatment drug for ascites liver cancer.

[0080] Example 1 DOTAP (NOF Corporation) and DOPC (NOF Corporation) were dissolved in chloroform and mixed in a recovery flask at a molar ratio of 0:100, 25:75, 50:50, 75:25, or 100:0. The chloroform was then removed from the recovery flask using a rotary evaporator to form a lipid thin film. The recovery flask was then kept under negative pressure using a vacuum pump for 3 hours, and the chloroform was completely removed from the recovery flask.

[0081] A 250 mmol / L ammonium sulfate solution was added to the lipid thin film, shaken at 65°C for 30 minutes, and sonicated for 10 minutes using a bath sonicator, followed by an additional 3 minutes using a probe sonicator to prepare a liposome solution. The ammonium sulfate in the external phase of the prepared liposome solution was replaced with a 10 mmol / L Hepes-buffered 5% glucose solution at pH 8.0 using a gel filtration column to prepare a liposome solution with an ammonium sulfate solution in the inner aqueous phase and a Hepes-buffered 5% glucose solution in the external phase. The particle size and surface charge of the resulting nanoball-like liposomes were measured using a dynamic light scattering analyzer (Zetasizer Nano, Malvern Panalytical). The results are shown in Figure 1.

[0082] To this liposome solution, 1 mg of doxorubicin (Carbosynth) was added per 12.5 mg of lipid and incubated at 65°C for 30 minutes to encapsulate doxorubicin in the liposome internal phase. The solution after preparing the doxorubicin-encapsulated liposomes was ultracentrifuged (245,000 × g, 2 hours) to precipitate the liposomes, and the encapsulation rate of doxorubicin in the liposomes was calculated from the concentration of doxorubicin remaining in the supernatant. The results are shown in Figure 1. Furthermore, γ-PGA was added to the doxorubicin-encapsulating liposomes at a mass ratio of 1:1, and the mixture was incubated at room temperature for 30 minutes to prepare a nanoball-shaped liposome / γ-PGA complex.

[0083] When the molar ratio of DOTAP to DOPC was 0:100, the cationic DOTAP was not present, resulting in a weak interaction between the liposomes and the anionic γ-PGA, making it unsuitable for formulation. When the molar ratio of DOTAP to DOPC was 25:75, 50:50, or 75:25, a good complex between the liposomes and γ-PGA was formed. When the molar ratio of DOTAP to DOPC was 100:0, DOTAP aggregated, preventing the encapsulation of doxorubicin.

[0084] Example 2 A complex of liposomes encapsulating doxorubicin and γ-PGA was prepared in the same manner as in Example 1, except that the molar ratio of DOTAP to DOPC was 50:45. This was used as a cancer treatment drug according to a manufacturing example. The particle size of the cancer treatment drug according to the manufacturing example was 153.0 nm, the surface charge was -43.5 mV, and the doxorubicin encapsulation rate was 93.8%. In addition, commercially available doxorubicin (Cayman Chemical) alone and commercially available Doxil (Janssen Pharmaceuticals) were prepared. In the commercially available Doxil, doxorubicin is encapsulated in PEG liposomes. The particle size of the commercially available Doxil was 82.9 nm, and the surface charge was -38.1 mV.

[0085] Colon cancer cell line (Colon26, provided by RIKEN) derived from Balb / c mice was prepared, and Colon26 cells were seeded at 10,000 cells / well onto a 24-well plate and cultured for 24 hours. Doxorubicin alone, Doxil, or a cancer treatment drug according to the manufacturing example was then added to the Colon26 cells so that the doxorubicin concentration was 10 μg / mL, and the cells were cultured for 4 hours.

[0086] After culturing the cells for 4 hours, the cells were washed with PBS, Hoechst 33342 was added to the cells, and the cells were cultured for an additional 30 minutes to stain the cell nuclei. After nuclear staining, the intracellular fluorescence images were observed using a fluorescence microscope. Note that doxorubicin emits red fluorescence at 590 nm when excited by 485 nm light. As a result, as shown in Figure 2, the cancer treatment drug according to the manufacturing example was observed to be incorporated into or near the nucleus. Doxorubicin alone was observed to be incorporated into or near the nucleus to a small extent. Doxil was not observed to be incorporated into the cells.

[0087] After culturing the cells for 4 hours, the cells were washed with PBS and lysed using lysis buffer (0.1 mol / L Tris / HCl buffer, pH 7.8, containing 0.05% Triton X-100 and 2 mmol / L EDTA). The amount of doxorubicin in the cell lysate was quantitatively measured using a fluorometer (Infinite 200 PRO, Tecan). Furthermore, the protein concentration in the cell lysate was measured, and the amount of doxorubicin taken up per protein was calculated. The results are shown in Figure 3. When the cancer treatment drug according to the manufacturing example was used, the amount of doxorubicin taken up per protein was significantly higher. On the other hand, when doxorubicin alone or Doxil was used, the amount of doxorubicin taken up per protein was significantly lower.

[0088] Example 3 Colon26 cells were seeded at 5,000 cells / well in a 96-well plate and cultured for 24 hours. Then, doxorubicin, Doxil, or the cancer treatment drug according to the manufacturing example was added to the Colon26 cells at a doxorubicin concentration of 10 μg / mL, and the cells were cultured for 6 hours. The doxorubicin-containing medium was then removed, the cells were washed with PBS, and the cells were cultured for an additional 18 hours. Cell viability was then measured using a cell counting kit-8 (Dojindo). Cell viability was calculated as a ratio of the viability of cells not treated with doxorubicin, Doxil, or the cancer treatment drug according to the manufacturing example, converted to 100%. The results are shown in Figure 4. The cancer treatment drug according to the manufacturing example effectively reduced the viability of cancer cells. In contrast, Doxil only slightly reduced the viability of cancer cells.

[0089] Example 4 Three hundred thousand Colon26-Luc cells (Colon26 cells constitutively expressing firefly luciferase, provided by the Institute of Physical and Chemical Research) were intraperitoneally administered to Bulb / c mice. The following day, doxorubicin, Doxil, or a cancer treatment drug from the manufacturing example was intraperitoneally administered to the mice at a dose of 5 mg / kg. On day 14 after administration of Colon26-Luc cells, the mouse peritoneum and intestine were removed and homogenized in lysis buffer (0.1 mol / L Tris / HCl buffer, pH 7.8, containing 0.05% Triton X-100 and 2 mmol / L EDTA). The homogenate was centrifuged, and the luciferase activity of the supernatant was measured to assess Colon26-Luc cell proliferation in the mouse peritoneal cavity. The results are shown in Figure 5. The cancer treatment drug according to the manufacturing example significantly inhibited the proliferation of Colon26-Luc cells in the abdominal cavity of mice, whereas Doxil did not significantly inhibit the proliferation of Colon26-Luc cells in the abdominal cavity of mice.

[0090] Example 5 ddY mice were intraperitoneally administered 300,000 Ehrlich ascites cancer cells per mouse, and the following day, doxorubicin, Doxil, or the cancer treatment drug according to the Manufacturing Example were intraperitoneally administered to the mice at a dose of 5 mg / kg of doxorubicin. The survival rate of the mice after administration of the Ehrlich ascites cancer cells was measured up to day 28. The results are shown in Figure 6. The survival rate of the mice administered the cancer treatment drug according to the Manufacturing Example was significantly higher. On the other hand, the survival rate of the mice administered Doxil was significantly lower.

[0091] Example 6 Doxorubicin, Doxil, or the cancer treatment drug according to the manufacturing example was administered to Bulb / c mice at a dose of 5 mg / kg, 10 mg / kg, or 20 mg / kg of doxorubicin. The survival rate of the mice after administration was measured up to day 14. As a result, as shown in Figure 7, the cancer treatment drug according to the manufacturing example resulted in a high survival rate of the mice, even at high doses. On the other hand, doxorubicin alone, which is known to be highly cardiotoxic, significantly reduced the survival rate of the mice at higher doses.

[0092] Example 7 Doxorubicin alone, Doxil, and the cancer treatment drug according to the Manufacturing Example were prepared at 250 μg / mL. 500 μL of doxorubicin alone, Doxil, or the cancer treatment drug according to the Manufacturing Example were added to the dialysis membrane and stirred in 100 mL of PBS. The PBS was sampled 1 hour, 4 hours, and 24 hours after the start of stirring, and the doxorubicin concentration was measured based on its fluorescence intensity. The results are shown in Figure 8. Since Doxil and the cancer treatment drug according to the Manufacturing Example are microparticles that cannot pass through the dialysis membrane, the doxorubicin leaking into the PBS was doxorubicin released from the microparticles. The cancer treatment drug according to the Manufacturing Example retained more than 75% of the doxorubicin even after 24 hours.

[0093] Example 8 DOTAP (NOF Corporation), DOPC (NOF Corporation), and paclitaxel (Carbosynth) were each dissolved in chloroform and mixed in a recovery flask so that the molar ratio of DOTAP, DOPC, and paclitaxel was 50:45:5. The chloroform was then removed from the recovery flask using a rotary evaporator to form a lipid thin film. The recovery flask was then maintained under negative pressure using a vacuum pump for 3 hours, and the chloroform was completely removed from the recovery flask.

[0094] A 5% glucose solution was added to the lipid thin film, which was then shaken at 65°C for 30 minutes, ultrasonicated for 10 minutes using a bath sonicator, and then ultrasonicated for an additional 3 minutes using a probe sonicator to prepare liposomes encapsulating paclitaxel. γ-PGA was then added to the paclitaxel-encapsulating liposomes at a mass ratio of 1:1, and the mixture was incubated at room temperature for 30 minutes to prepare a complex of nanoball-shaped liposomes and γ-PGA, which was used as the cancer treatment drug according to the manufacturing example of Example 8.

[0095] Colon cancer cell line (Colon26, provided by RIKEN) derived from Balb / c mice was prepared, and Colon26 cells were seeded onto a 24-well plate at 10,000 cells / well and cultured for 24 hours. Paclitaxel alone or the cancer therapeutic agent according to the manufacturing example of Example 8 was then added to the Colon26 cells so that the paclitaxel concentration was 10 μg / mL, and the cells were cultured for 24 hours.

[0096] After culturing the cells for 24 hours, the cells were washed with PBS and lysed using a lysis buffer (0.1 mol / L Tris / HCl buffer at pH 7.8 containing 0.05% Triton X-100 and 2 mmol / L EDTA). The amount of paclitaxel contained in the cell lysate was quantitatively measured using HPLC. Furthermore, the protein concentration in the cell lysate was measured, and the amount of paclitaxel taken up per amount of protein was calculated. The results are shown in Figure 9. When the cancer treatment drug according to the manufacturing example of Example 8 was used, the amount of paclitaxel taken up per amount of protein was significantly higher. On the other hand, when paclitaxel alone was used, the amount of paclitaxel taken up per amount of protein was significantly lower.

[0097] Colon26 cells were seeded at 5,000 cells / well in a 96-well plate and cultured for 24 hours. Subsequently, paclitaxel alone or the cancer therapeutic agent according to the manufacturing example of Example 8 was added to the Colon26 cells so that the paclitaxel concentration was 10 μg / mL, and the cells were cultured for 24 hours. The cell viability was then measured using a cell counting kit-8 (Dojindo). The cell viability was calculated as a ratio when the viability of cells not treated with paclitaxel alone or the cancer therapeutic agent according to the manufacturing example of Example 8 was converted to 100%. The results are shown in Figure 10. The cancer therapeutic agent according to the manufacturing example of Example 8 effectively reduced the viability of cancer cells compared to paclitaxel alone.

Claims

1. A cancer treatment drug, an effective amount of a hydrophobic anticancer agent; a cationic liposome encapsulating the hydrophobic anticancer drug; γ-polyglutamic acid or a salt thereof encapsulating the cationic liposome; Including, the hydrophobic anticancer agent is an anthracycline antibiotic or a taxane anticancer agent; The cationic liposome comprises a phospholipid or a salt thereof, a cationic lipid or a salt thereof, Including, the molar ratio of the positively charged functional group of the cationic liposome to the negatively charged functional group of the γ-polyglutamic acid or a salt thereof is 1:1 to 1:100; the molar ratio of the phospholipid or salt thereof to the cationic lipid or salt thereof is 10:90 to 90:10; the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine; The cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium; Cancer treatment drug.

2. The cancer therapeutic drug according to claim 1, wherein the molecular weight of the γ-polyglutamic acid or a salt thereof is 2,000,000 or less.

3. The cancer therapeutic agent of claim 1 or 2, having a substantially uncharged or negative surface charge.

4. The cancer therapeutic drug according to claim 1 , wherein the hydrophobic anticancer drug is doxorubicin or a salt thereof.

5. The cancer therapeutic drug according to any one of claims 1 to 3, wherein the hydrophobic anticancer drug is paclitaxel or a salt thereof.

6. A cancer treatment drug, an effective amount of a hydrophobic anticancer agent; a cationic liposome encapsulating the hydrophobic anticancer drug; γ-polyglutamic acid or a salt thereof encapsulating the cationic liposome; Including, the hydrophobic anticancer drug is doxorubicin or a salt thereof, or paclitaxel or a salt thereof; The cationic liposome comprises a phospholipid or a salt thereof, a cationic lipid or a salt thereof, Including, the molar ratio of the positively charged functional group of the cationic liposome to the negatively charged functional group of the γ-polyglutamic acid or a salt thereof is 1:1 to 1:100; the molar ratio of the phospholipid or salt thereof to the cationic lipid or salt thereof is 10:90 to 90:10; the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine; The cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium; Cancer treatment drug.

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