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WO2018164116A1 - Liposome, agent anticancéreux et kit de thérapie anticancéreuse - Google Patents

Liposome, agent anticancéreux et kit de thérapie anticancéreuse Download PDF

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Publication number
WO2018164116A1
WO2018164116A1 PCT/JP2018/008560 JP2018008560W WO2018164116A1 WO 2018164116 A1 WO2018164116 A1 WO 2018164116A1 JP 2018008560 W JP2018008560 W JP 2018008560W WO 2018164116 A1 WO2018164116 A1 WO 2018164116A1
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liposome
acid
liposomes
result
tdm
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Japanese (ja)
Inventor
淳 宮▲崎▼
博之 西山
喬之 吉野
英之 赤座
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University of Tokyo NUC
University of Tsukuba NUC
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University of Tokyo NUC
University of Tsukuba NUC
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Priority to JP2019504598A priority Critical patent/JP7220472B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a liposome, an anticancer agent, and a cancer treatment kit.
  • Bladder cancer is known as a cancer with frequent recurrence. Therefore, as a method for treating non-muscle invasive bladder cancer that has not invaded the muscle layer, after removing the cancer tissue by endoscopic surgery, Bacillus Calmette Guerlain (BCG) is used in the bladder to suppress recurrence. The standard of treatment is infused. BCG administered into the bladder is thought to invade the patient's cancer cells and activate the patient's immune system to damage the cancer cells. BCG may also be used to treat intraepithelial cancers other than bladder cancer.
  • BCG Bacillus Calmette Guerlain
  • BCG is an attenuated strain derived from Mycobacterium bovis, a kind of acid-fast bacterium, and is also used as a preventive vaccine for tuberculosis.
  • mycolic acid present in the cell wall is considered (see, for example, Non-Patent Document 1).
  • Mycolic acid is a cell wall functional molecule characteristic of acid-fast bacteria, and is a long-chain fatty acid having 60 to 90 carbon atoms.
  • Mycolic acid is a characteristic long-chain molecular fatty acid that forms the cell walls of mycobacteria, including tuberculosis, and its hydrophobicity contributes to the stability of the cell wall and is considered essential for maintaining acid resistance. It has been.
  • mycolic acid and its derivatives including free mycolic acid, sugar esters such as trehalose, glucose, glycerol, etc.
  • mycolic acid and its derivatives are administered to animals as a pathogenic factor of Mycobacterium tuberculosis
  • tuberculosis lesions granulomas
  • mycolic acid and its derivatives are distributed in all mycobacteria and the pathogenicity varies depending on the subclass and carbon chain.
  • mycolic acid has many subclasses such as ⁇ -mycolic acid, methoxymycolic acid, ketomycolic acid, wax ester mycolic acid, epoxymycolic acid, dihydroxymycolic acid and the like having different polarities.
  • Mycolic acid of Mycobacterium tuberculosis and BCG is composed of three subclasses: ⁇ -mycolic acid, methoxymycolic acid, and ketomycolic acid.
  • an object of the present invention is to provide a technique for clinical application of mycolic acid.
  • the present invention includes the following aspects.
  • [1] A liposome containing mycolic acid and a sterol compound.
  • [2] The liposome according to [1], further containing a cationic lipid.
  • [3] The liposome according to [1] or [2], wherein the mycolic acid includes ketomycolic acid.
  • [4] The liposome according to any one of [1] to [3], wherein the zeta potential is positive.
  • [5] The liposome according to any one of [1] to [4], having a diameter of 200 nm or less.
  • [6] The liposome according to any one of [1] to [5], which has a polydispersity index of 0.3 or less.
  • An anticancer agent comprising the liposome according to any one of [1] to [6] as an active ingredient.
  • a cancer treatment kit comprising the anticancer agent according to [7] and an immune checkpoint inhibitor.
  • FIG. 6 is a photograph showing the results of thin-layer chromatography of mycolic acid in Experimental Example 1.
  • (A) to (c) are mass spectra showing the results of mass spectrometry of ⁇ -mycolic acid, methoxymycolic acid, and ketomycolic acid in Experimental Example 2, respectively.
  • (A) to (d) are fluorescence micrographs of T24 cells contacted with control liposomes, ⁇ -mycolic acid-containing liposomes, methoxymycolic acid-containing liposomes, and ketomycolic acid-containing liposomes in Experimental Example 5, respectively.
  • Experimental example 5 it is a graph which shows the result of having analyzed T24 cell which contacted the control liposome by flow cytometry.
  • FIG. 7 it is a graph which shows the result of having measured the tumor volume of the mouse
  • Experimental Example 8 it is a graph which shows the result of having measured the tumor volume of the mouse
  • 14 is a graph showing the results of flow cytometry in Experimental Example 10.
  • Experimental example 11 it is a graph which shows the result of having measured the tumor volume of the mouse
  • (A) And (b) is a typical photograph which shows the result of the immuno-staining of the tumor tissue of a mouse
  • FIG. (C) is a graph showing the results of counting CD4-positive T cells infiltrating the tumor tissue of mice in Experimental Example 12.
  • (A) And (b) is a typical photograph which shows the result of the immuno-staining of the tumor tissue of a mouse
  • FIG. (C) is a graph showing the results of counting CD8 positive T cells infiltrating the tumor tissue of mice in Experimental Example 12. It is a figure explaining the experiment schedule of Experimental example 13.
  • (A) to (d) are graphs showing the results of measuring the concentration of TNF released in the medium of bone marrow-derived dendritic cells in Experimental Example 16.
  • (A) to (c) are graphs showing the results of measuring the concentration of TNF released in the medium of bone marrow-derived macrophages in Experimental Example 16.
  • the present invention provides a liposome containing mycolic acid and a sterol compound.
  • the liposome of the present embodiment can be used as an anticancer agent, an immunoactivator, an adjuvant and the like.
  • Mycolic acid is a hydrophobic molecule that forms the surface layer barrier of mycobacteria. On the other hand, it is considered to be a component that induces strong immune activation on the host organism side, but it can be administered directly to the living body. It is difficult to activate the immune system. This is considered to be because mycolic acid has strong hydrophobicity and low cell affinity.
  • the liposome of the present embodiment has high cell affinity and can efficiently deliver mycolic acid into the host cytoplasm.
  • mycolic acid can be delivered to cancer cells, immunity against cancer cells can be activated, and cancer cells can be damaged.
  • Liposomes are a type of artificial vesicle composed of an amphiphile.
  • amphiphilic substances include phospholipids, glycolipids, and surfactants.
  • phosphatidylcholines for example, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), etc.
  • Phosphatidylglycerols for example, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dilauroylphosphatidylglycerol, etc.
  • phosphatidylethanolamines for example, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine) Etc.
  • phosphatidylserine for example, dioleoylphosphatidylserine, dipa Palmi
  • glycolipids examples include glyceroglycolipids such as sulfoquinovosyl diglyceride, diglycosyl diglyceride and digalactosyl diglyceride; glycosphingolipids such as galactosyl cerebroside, lactosyl cerebroside and ganglioside.
  • the surfactant include Span 80, Tween 80, Tween 20, and the like. One kind of amphiphilic substance may be used alone, or two or more kinds may be mixed and used.
  • Liposomes can be produced by any conventionally known method such as an ultrasonic treatment method, reverse phase evaporation method, freeze-thaw method, lipid dissolution method, spray drying method and the like.
  • mycolic acid In the liposome of the present embodiment, mycolic acid can be used without particular limitation as long as it can activate the immune system. In general, mycolic acid is difficult to chemically synthesize. For this reason, it is preferable to use it purified from acid-fast bacteria.
  • Mycolic acid of Mycobacterium tuberculosis and BCG bacteria There are three subclasses of Mycolic acid of Mycobacterium tuberculosis and BCG bacteria: ⁇ -mycolic acid, methoxymycolic acid, and ketomycolic acid.
  • non-tuberculous mycobacterial mycolic acid includes ⁇ -mycolic acid, ketomycolic acid, and wax ester mycolic acid, all of which are known to exhibit strong immune enhancing activity in the form of trehalose ester.
  • ketomycolic acid is preferred because it tends to have a high activity of damaging cancer in vivo.
  • the mycolic acid may be in the form of free mycolic acid, for example, in the form of a sugar ester of mycolic acid such as trehalose monoester, trehalose diester, glucose monoester, glycerol monoester.
  • a sugar ester of mycolic acid such as trehalose monoester, trehalose diester, glucose monoester, glycerol monoester.
  • any one of these may be used as mycolic acid, or a mixture of two or more may be used.
  • the content of mycolic acid is preferably 5 to 20% by mass, and more preferably 10 to 15% by mass, based on the liposome.
  • the liposome of the present embodiment contains a sterol compound.
  • the inventors have clarified that by incorporating a sterol compound in the mycolic acid-containing liposome, the particle size of the liposome can be made smaller and the stability can be improved.
  • Examples of the sterol compound include cholesterol, 3 ⁇ - [N- (dimethylaminoethane) carbamoyl] cholesterol, N- (trimethylammonioethyl) carbamoylcholesterol and the like. Among these, cholesterol can be preferably used.
  • the content of the sterol compound is preferably 5 to 20% by mass, more preferably 10 to 15% by mass, based on the dry weight of the liposome.
  • the liposome of the present embodiment preferably has a positive zeta potential.
  • a positive zeta potential improves cell affinity and facilitates delivery of mycolic acid into the cell.
  • the zeta potential of the liposome is preferably 1 to 20 mV, more preferably 5 to 20 mV.
  • the zeta potential of the liposome can be measured by, for example, electrophoretic light scattering measurement.
  • a cationic substance to the amphiphilic substance constituting the liposome.
  • a cationic lipid may be added as a liposome material.
  • Examples of the cationic lipid include those having a cholesterol skeleton, those having a glutamic acid skeleton, quaternary ammonium salts, dendron lipids and the like.
  • Examples of the cationic lipid having a cholesterol skeleton include [N- (N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol).
  • Examples of the cationic lipid having a glutamic acid skeleton include N- ( ⁇ -trimethylammonioacetyl) -didodecyl-D-glutamic acid (TMAG).
  • Examples of the quaternary ammonium salt include N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium bromide (DOTMA), dimethyldioctadecylammonium bromide (DDAB), 2,3- Dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), Dioleoyl-D-glutamate-N-2 (sperminecarboxyamido) ethyl (DOGS), 1,2-Dimyristoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE) and the like.
  • DOTMA dimethyldioctadecylammonium bromide
  • DOSPA Dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N
  • dendron lipids examples include dendron lipids (types “D1”, “D2”, “D3”, “D12”, “D22”, “D32”, all manufactured by Hygieia Biosciences). It is not limited. Among these, dendron lipids (model “D22”, manufactured by Hygieia Bioscience) are preferable.
  • the content of the cationic lipid is preferably 1 to 10% by mass, more preferably 2 to 5% by mass, based on the dry weight of the liposome.
  • the liposome of the present embodiment is preferably cationic and preferably has a diameter of 200 nm or less.
  • the diameter of the liposome can be measured, for example, by a dynamic light scattering method. Liposomes having a diameter of 200 nm or less can be easily sterilized by filter sterilization. For this reason, it is easy to administer to a living body.
  • the liposome of the present embodiment preferably has a polydispersity index of 0.3 or less.
  • a small polydispersity index means that the particle size of the liposomes is uniform. When the liposomes have the same particle size, loss can be minimized when filter sterilization is performed.
  • the present invention provides an anticancer agent comprising the above-described liposome as an active ingredient.
  • the immune system can be activated and cancer cells can be damaged. Therefore, the anticancer agent of this embodiment can also be called an immune activator, an adjuvant and the like.
  • the anticancer agent of the present embodiment is unlikely to cause infectious diseases, unlike conventional BCG live bacteria, and is easy to manage, supply and dispose of.
  • Examples of the cancer to be treated by the anticancer agent of the present embodiment include cancer that can be easily administered locally, and specific examples include bladder cancer, breast cancer, melanoma, and the like.
  • the anticancer agent of this embodiment is taken into cancer cells by administering the anticancer agent of this embodiment into the bladder or locally by local injection or the like. As a result, cancer cells incorporating the anticancer agent of this embodiment are damaged by the immune system. Thereby, cancer can be treated effectively.
  • the present invention provides a cancer treatment kit comprising the anticancer agent described above and an immune checkpoint inhibitor.
  • immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and the like.
  • anti-PD-1 antibody examples include nivolumab, pembrolizumab and the like.
  • anti-CTLA-4 antibody examples include ipilimumab, tremelimumab and the like.
  • the present invention provides a liposome for the treatment of cancer, comprising a mycolic acid and a sterol compound.
  • the liposome is preferably cationic.
  • the mycolic acid and the sterol compound the same ones as described above can be used.
  • the present invention provides the use of a liposome for producing an anticancer agent, the liposome containing a mycolic acid and a sterol compound.
  • the liposome is preferably cationic.
  • the mycolic acid and the sterol compound the same ones as described above can be used.
  • the present invention provides a method for treating cancer comprising administering an effective amount of a liposome containing mycolic acid and a sterol compound to a patient in need of treatment.
  • the liposome is preferably cationic.
  • the cancer to be treated by the treatment method of this embodiment is the same as described above.
  • the mycolic acid and sterol compound the same ones as described above can be used.
  • the invention treats cancer comprising administering an effective amount of a liposome containing mycolic acid and a sterol compound and an effective amount of an immune checkpoint inhibitor to a patient in need of treatment.
  • the liposome is preferably cationic.
  • the cancer to be treated by the treatment method of this embodiment is the same as described above.
  • the mycolic acid and sterol compound the same ones as described above can be used.
  • the thing similar to what was mentioned above can be used as an immune checkpoint inhibitor.
  • FIG. 1 is a photograph showing the results of thin-layer chromatography of mycolic acid methyl ester developed four times.
  • lane 1 is the result of mycolic acid methyl ester purified from Mycobacterium tuberculosis, which is a human tuberculosis bacterium.
  • Lane 2 is the result of mycolic acid methyl ester purified from BCG.
  • Lane 3 is the result of ⁇ (Alpha) -mycolic acid methyl ester purified from BCG.
  • Lane 4 is the result of methoxy-mycolic acid methyl ester purified from BCG.
  • Lane 5 is the result of keto-mycolic acid methyl ester purified from BCG.
  • 2 (a) to 2 (c) are mass spectra showing the analysis results of ⁇ -mycolic acid, methoxymycolic acid, and ketomycolic acid, respectively.
  • Table 1 below summarizes the results of MALDI-TOF mass spectrometry of each subclass of mycolic acid.
  • the purified subclasses of mycolic acid are ⁇ -mycolic acid having 78 carbon atoms, methoxymycolic acid having 85 carbon atoms, and ketomycolic acid having 84 and 86 carbon atoms. It was confirmed.
  • Table 3 shows the measurement results.
  • ⁇ represents ⁇ -mycolic acid
  • methoxy represents methoxymycolic acid
  • keto represents ketomycolic acid.
  • time after preparation represents the number of days after the preparation of liposomes.
  • liposomes containing mycolic acid, cholesterol and cationic lipid have a diameter of 200 nm or less, a positive zeta potential, and a polydispersity index of 0.3. It became clear that: A small polydispersity index means that the particle size of the liposomes is uniform.
  • the inventors have also clarified that by changing the composition of mycolic acid, cholesterol and cationic lipid, the zeta potential and diameter of the mycolic acid-containing liposome can be adjusted, and a liposome according to the purpose can be prepared.
  • each liposome was added to the medium of the human bladder cancer cell line T24 at a final concentration of 50 ⁇ g / mL, allowed to stand at 37 ° C. for 2 hours, and observed with a confocal microscope, whereby the liposome was incorporated into the cells. We examined whether or not. The fluorescence of NBD indicates the localization of the liposome. In addition, the cell nucleus was fluorescently stained with 4 ', 6-diamidino-2-phenylindole (DAPI).
  • DAPI 6-diamidino-2-phenylindole
  • FIG. 3 (a) to 3 (d) are fluorescence micrographs of T24 cells in contact with each liposome.
  • FIG. 3 (a) shows the result of the control liposome
  • FIG. 3 (b) shows the result of the ⁇ -mycolic acid-containing liposome
  • FIG. 3 (c) shows the result of the methoxymycolic acid-containing liposome
  • FIG. ) Shows the result of ketomycolic acid-containing liposomes.
  • FIG. 4 is a graph showing the results of flow cytometry analysis of T24 cells (with liposomes) contacted with control liposomes.
  • T24 cells not contacted with liposomes were similarly analyzed.
  • a peak of cells showing fluorescence of the control liposome was observed, and it was confirmed that the control liposome was taken up by T24 cells.
  • PBS control liposome
  • each mycolic acid-containing liposome was added to a serum-free medium, and the number of viable cells was measured after 24 hours and 48 hours, respectively.
  • the number of viable cells was measured using a kit (Dojin Chemical Co., Ltd.) using WST-8 which is a water-soluble tetrazolium salt.
  • 5 (a) to 5 (c) are graphs showing the measurement results of the number of viable cells.
  • FIG. 5 (a) shows the results for T24 cells
  • FIG. 5 (b) shows the results for MBT-2 cells
  • FIG. 5 (c) shows the results for MB49 cells.
  • PBS shows the result of adding PBS
  • Control shows the result of adding control liposomes
  • ⁇ shows the result of adding ⁇ -mycolic acid-containing liposomes.
  • Methodoxy indicates the result of adding methoxymycolic acid-containing liposomes
  • keto indicates the result of adding ketomycolic acid-containing liposomes.
  • FIG. 6 is a diagram for explaining an experiment schedule.
  • each reagent alone ( ⁇ -mycolic acid-containing liposome, methoxymycolic acid-containing liposome, ketomycolic acid-containing liposome prepared in the same manner as in Experimental Example 5, Control liposomes or PBS) were each administered subcutaneously at the same site.
  • FIG. 7 is a graph showing the results of measuring the tumor volume over time.
  • PBS indicates the result of the PBS administration group
  • Control indicates the result of the control liposome administration group
  • ⁇ indicates the result of the ⁇ -mycolic acid-containing liposome administration group
  • Methodoxy indicates The result of the methoxymycolic acid-containing liposome administration group is shown
  • keto shows the result of the ketomycolic acid-containing liposome administration group.
  • Example 8 (Study of antitumor effect of mycolic acid-containing liposomes using mouse subcutaneous tumor model 2) The antitumor effect (antitumor activity) of mycolic acid-containing liposomes was examined using a mouse subcutaneous tumor model in the same manner as in Experimental Example 7 except that Beige mice were used instead of C57BL / 6J mice.
  • the Beige mouse is known as a model animal for the Chediak-Higashi syndrome and lacks NK cells.
  • FIG. 8 is a graph showing the results of measuring the tumor volume over time.
  • PBS indicates the result of the PBS administration group
  • Control indicates the result of the control liposome administration group
  • ⁇ indicates the result of the ⁇ -mycolic acid-containing liposome administration group
  • methoxy indicates The result of the methoxymycolic acid-containing liposome administration group is shown
  • keto shows the result of the ketomycolic acid-containing liposome administration group.
  • NK cells are less involved in the expression of antitumor effects of mycolic acid-containing liposomes.
  • Example 9 (Preparation of mycolic acid-containing liposome 2) Except for using a dendron lipid represented by the following chemical formula (2) (model “D12”, Hygieia Biosciences) as the cationic lipid, the composition of Production Example 12 having the composition shown in Table 5 was used. Liposomes (hereinafter sometimes referred to as “D12 liposomes”) were prepared. In Table 5, DOPC represents dioleoylphosphatidylcholine.
  • the diameter, polydispersity index, and zeta potential of the D12 liposome were measured using a molecular property evaluation apparatus (model “ZEN3600”, Malvern). Table 6 shows the measurement results. The measurement was performed on the 11th day after the preparation of the liposome. As a result, the diameter, polydispersity index, and zeta potential of the D12 liposome are each a production example using a dendron lipid represented by the above chemical formula (1) (model “D22”, Hygieia Bioscience) as the cationic lipid. It was clarified that it was almost the same as each value of 6 liposomes (hereinafter sometimes referred to as “D22 liposome”).
  • Example 10 (Investigation of uptake of mycolic acid-containing liposomes into cells 2)
  • the liposome of Production Example 13 was prepared with the composition shown in Table 7 below in the same manner as in Experimental Example 9 except that cholesterol labeled with a fluorescent dye NBD (Avanti Polar Lipid) was used.
  • NBD Advanti Polar Lipid
  • DOPC represents dioleoylphosphatidylcholine.
  • each liposome was added to the medium of the mouse bladder cancer cell line MB49 at a final concentration of 50 ⁇ g / mL, allowed to stand at 37 ° C. for 2 hours, and analyzed by flow cytometry.
  • FIG. 9 is a graph showing the results of flow cytometry.
  • MB49 cells not contacted with liposomes were similarly analyzed.
  • “None” is the result of MB49 cells not contacted with liposomes
  • “D12” is the result of D12 liposomes
  • “D22” is the result of D22 liposomes.
  • the D22 and D12 liposomes had the same degree of color development of the fluorescent dye, and the D12 liposome was taken up by MB49 cells to the same extent as the D22 liposome.
  • the liposome of Production Example 12 prepared in the same manner as in Experimental Example 9 (hereinafter sometimes referred to as “D12 liposome”), the liposome prepared in the same manner as in Production Example 6 of Experimental Example 3 (hereinafter referred to as “D12 liposome”).
  • D12 liposome the liposome prepared in the same manner as in Production Example 6 of Experimental Example 3
  • D22 liposomes liposomes prepared in the same manner as in Production Example 7 of Experimental Example 3
  • control liposomes liposomes prepared in the same manner as in Production Example 7 of Experimental Example 3
  • PBS phosphate buffer
  • FIG. 6 is a diagram for explaining the experiment schedule.
  • day 0 D12 liposome 0.44 mg, D22 liposome 0.44 mg, control liposome 0.5 mg, or PBS 0.1 mL were mixed with 500,000 mouse bladder cancer cell lines MB49, respectively.
  • each reagent alone D12 liposome, D22 liposome, control liposome or PBS was additionally administered subcutaneously to the same site.
  • FIG. 10 is a graph showing the results of measuring the tumor volume over time.
  • PBS indicates the result of the PBS administration group
  • Control indicates the result of the control liposome administration group
  • D22 indicates the result of the D22 liposome administration group
  • D12 indicates the D12 liposome administration group. The results are shown.
  • D22 liposomes using dendron lipids (type “D12”, Hygieia Biosciences) as cationic lipids are more suitable for D22 liposomes using dendron lipids (type “D22”, Hygiea Biosciences) as cationic lipids. It was revealed that the antitumor effect was higher than that.
  • the immunostained tissue was observed under a microscope, and an area where there was much infiltration of CD4-positive T cells and CD8-positive T cells was photographed.
  • CD4 positive T cells and CD8 positive T cells infiltrating the tumor tissue were counted based on the micrographs taken. T cells were counted three times by one observer for the same region, and the average value was statistically analyzed.
  • FIG. 11 (a) is a representative photograph showing the result of immunostaining the tumor tissue of a mouse in the control liposome-administered group with an anti-CD4 antibody.
  • FIG. 11 (b) is a representative photograph showing the result of immunostaining the tumor tissue of a mouse in the D22 liposome administration group with an anti-CD4 antibody.
  • FIG.11 (c) is a graph which shows the result of having counted the CD4 positive T cell in the tumor tissue of the mouse
  • “control” shows the result of the control liposome-administered group
  • D22 shows the result of the D22 liposome-administered group.
  • FIG. 12 (a) is a representative photograph showing the result of immunostaining the tumor tissue of a mouse in the control liposome administration group with an anti-CD8 antibody.
  • FIG. 12 (b) is a representative photograph showing the result of immunostaining the tumor tissue of a mouse in the D22 liposome administration group with an anti-CD8 antibody.
  • FIG.12 (c) is a graph which shows the result of having counted the CD8 positive T cell in the tumor tissue of the mouse
  • “control” shows the result of the control liposome-administered group
  • D22 shows the result of the D22 liposome-administered group.
  • mice in the D22 liposome administration group had significantly more CD8-positive T cells infiltrating the tumor tissue than the mice in the control liposome administration group. Moreover, this result shows that CD8 positive T cells infiltrate the tumor tissue by administration of D22 liposome, and an antitumor effect is exhibited in a CD8 positive T lymphocyte-dependent manner.
  • FIG. 13 is a diagram for explaining the experiment schedule.
  • mycolic acid-containing liposomes were subcutaneously administered to the same site 9 times every 1-2 days, and the tumor volume was measured over time.
  • mycolic acid-containing liposomes As the mycolic acid-containing liposomes, a group was prepared in which 0.44 mg of ketomycolic acid-containing liposomes prepared in the same manner as in Experimental Example 5 was administered per administration. The dose of mycolic acid per administration per mouse was 40 ⁇ g. For comparison, a group was also prepared in which 0.5 mg of control liposome prepared in the same manner as in Experimental Example 5 or 0.1 mL of PBS was administered per administration.
  • FIG. 14 is a graph showing the results of measuring the tumor volume over time.
  • PBS shows the results of the PBS administration group
  • Control shows the results of the control liposome administration group
  • Keto shows the results of the ketomycolic acid-containing liposome administration group.
  • Example 14 (Preparation of mycolic acid-containing liposome 3) A mycolic acid-containing liposome (hereinafter sometimes referred to as “TDM liposome”) using trehalose dimycolic acid (TDM, trehalose diester of mycolic acid) as mycolic acid was prepared.
  • TDM liposome trehalose dimycolic acid
  • BCG Mycobacterium bovis BCG Tokyo 172 strain
  • Mycobacterium phlei Mycobacterium phlei
  • Rhodococcus terae Rhodococcus terae
  • BCG-TDM Mycobacterium bovis BCG Tokyo 172 strain
  • Mycobacterium phlei Mycobacterium phlei
  • Rhodococcus terae Rhodococcus terae
  • BCG-TDM Mycobacterium bovis BCG Tokyo 172 strain
  • Mycobacterium phlei Mycobacterium phlei
  • Rhodococcus terae Rhodococcus terae
  • the liposome of Production Example 14 (hereinafter sometimes referred to as “BCG-TDM liposome”) and the liposome of Production Example 15 (hereinafter referred to as “M. phlei-TDM liposome ”) and the liposome of Production Example 16 (hereinafter also referred to as“ R. terrae-TDM liposome ”).
  • DOPC represents dioleoylphosphatidylcholine.
  • a dendron lipid represented by the chemical formula (1) (model “D22”, manufactured by Hygieia Bioscience) was used.
  • TDM liposome BCG-TDM liposome prepared in Experimental Example 14, M.M. phlei-TDM liposomes, R.I. 0.44 mg of terae-TDM liposome was used per administration.
  • a group was also prepared in which 0.5 mg of control liposome prepared in the same manner as in Experimental Example 5 or 0.1 mL of PBS was administered per administration.
  • FIG. 15 is a graph showing the results of measuring the tumor volume over time.
  • PBS indicates the result of the PBS administration group
  • Control indicates the result of the control liposome administration group
  • BCG indicates the result of the BCG-TDM liposome administration group
  • phlei indicates the M.P.
  • the result of the phlei-TDM liposome administration group is shown.
  • the result of the terrae-TDM liposome administration group is shown.
  • TDM liposomes also have an antitumor effect.
  • mycolic acid liposomes using trehalose dimycolic acid (TDM) derived from BCG, liposomes using TDM derived from Mycobacterium phlei, and TDM derived from Rhodococcus terae are used.
  • TDM trehalose dimycolic acid
  • TDM Immunoreceptor tyrosine-based activation motif
  • bone marrow-derived dendritic cells BDMC
  • bone marrow-derived macrophages BMM
  • TDM liposomes TDM liposomes
  • Bone marrow-derived dendritic cells (BDMC) prepared from wild type mice or Mincle ⁇ / ⁇ mice were respectively seeded in 96-well plates at a cell density of 1 ⁇ 10 5 cells / well. Subsequently, the BCG-TDM liposome prepared in Experimental Example 14 or M.P. Phlei-TDM liposomes were added to a final concentration of 100 ⁇ g / mL and cultured for 48 hours. For comparison, a group in which control liposomes prepared in the same manner as in Experimental Example 5 were added to a final concentration of 100 ⁇ g / mL and a group in which nothing was added were also prepared. Subsequently, the concentration of TNF released into the medium was measured by ELISA.
  • FIG. 16A is a graph showing the results of measuring the concentration of TNF released into the medium.
  • BCG indicates the result of the BCG-TDM liposome addition group
  • M The results of the phlei-TDM liposome added group are shown
  • Control shows the result of the control liposome added group
  • Ni shows the result of the group to which nothing was added
  • WT is derived from the bone marrow of the wild type mouse.
  • the results of dendritic cells are shown, and “Mincle ⁇ / ⁇ ” shows the results of bone marrow derived dendritic cells of Mincle ⁇ / ⁇ mice.
  • BDMC bone marrow-derived dendritic cells
  • FIGS. 16B to 16D are graphs showing the results of measuring the concentration of TNF released into the medium.
  • BCG indicates the result of the BCG-TDM liposome coat group
  • M The result of the phlei-TDM liposome-coated group
  • control shows the result of the control liposome-coated group
  • no shows the result of the group that did not coat anything.
  • FIG.16 (c) shows the result of the group which coated commercially available TDM.
  • FIG. 16D shows the results of the group to which LPS was added.
  • WT shows the results of wild-type mouse bone marrow-derived dendritic cells
  • Mincle ⁇ / ⁇ shows the results of Mincle ⁇ / ⁇ mouse bone marrow-derived dendritic cells. Results are shown.
  • Bone marrow derived macrophages prepared from wild type mice or Mincle ⁇ / ⁇ mice were seeded in 96-well plates at a cell density of 1 ⁇ 10 5 cells / well, respectively. Subsequently, the BCG-TDM liposome prepared in Experimental Example 14 or M.P. Phlei-TDM liposomes were added to a final concentration of 100 ⁇ g / mL and cultured for 48 hours.
  • FIGS. 17A to 17C are graphs showing the results of measuring the concentration of TNF released into the medium.
  • BCG indicates the result of the BCG-TDM liposome addition group
  • M The result of the phlei-TDM liposome added group
  • control shows the result of the control liposome added group
  • no shows the result of the group to which nothing was added.
  • FIG.17 (b) shows the result of the group which coated commercially available TDM.
  • FIG.17 (c) shows the result of the group which added LPS.
  • “WT” indicates the result of bone marrow-derived macrophages of wild-type mice
  • “Mincle ⁇ / ⁇ ” indicates the results of bone marrow-derived macrophages of Mingle ⁇ / ⁇ mice.
  • Example 17 Large-scale preparation of TDM liposomes
  • TDM BCG-TDM purified in Experimental Example 14 was used. Specifically, 15.0 mg of BCG-TDM, 15.0 mg of cholesterol, and 5.0 mg of cationic lipid (type “D22”, Hygieia Bioscience) were weighed and put into a glass vial.
  • the diameter, polydispersity index, and zeta potential of the TDM liposome of Production Example 17 were measured using a molecular property evaluation apparatus (model “ZETA SIZER Nano-ZS”, Malvern). Table 11 shows the measurement results. As a result, it was revealed that the diameter, polydispersity index, and zeta potential of the TDM liposome of Production Example 17 were comparable to the values of the liposome of Production Example 6 produced in Experimental Example 3, respectively.
  • the TDM liposome of Production Example 17 was used in an amount of 0.44 mg per administration.
  • 0.44 mg of BCG-TDM liposome prepared in the same manner as in Production Example 14 of Experimental Example 14 (hereinafter sometimes referred to as “TDM-D22”) per administration.
  • 0.44 mg of the BCG-TDM liposome of Production Example 18 (hereinafter sometimes referred to as “TDM-D12”) produced in the same manner as in Experimental Example 14 with the composition shown in Table 12 below, as in Experimental Example 5.
  • a group in which 0.5 mg of the control liposome prepared as described above or 0.1 mL of PBS was administered was also prepared. Table 12 below shows the composition of each liposome.
  • “D22” represents dendron lipid (model “D22”, manufactured by Hygieia Bioscience)
  • “D12” represents dendron lipid (model “D12”, manufactured by Hygieia Bioscience).
  • FIG. 18 is a graph showing the results of measuring the tumor volume over time.
  • PBS shows the result of the PBS administration group
  • Control shows the result of the control liposome administration group
  • TDM-D22 shows the result of the TDM-D22 administration group
  • TDM- “D12” shows the results of the TDM-D12 administration group described above
  • Production Example 17 shows the results of the TDM liposome administration group of Production Example 17.
  • TDM liposome of Production Example 17 exhibited an antitumor effect. Also in TDM liposomes, when dendron lipid (type “D22”, Hygieia Bioscience) is used as the cationic lipid, dendron lipid (type “D12”, Hygieia Bioscience) is used as the cationic lipid. It was revealed that the antitumor effect was higher than that.

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Abstract

L'invention concerne un liposome qui contient un acide mycolique et un composé stérol.
PCT/JP2018/008560 2017-03-06 2018-03-06 Liposome, agent anticancéreux et kit de thérapie anticancéreuse Ceased WO2018164116A1 (fr)

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JP2009269855A (ja) * 2008-05-07 2009-11-19 Osaka Prefecture Univ 常磁性金属含有ポリアミドアミンデンドロン脂質
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JPH0278623A (ja) * 1988-09-14 1990-03-19 Sawai Seiyaku Kk インターフェロン誘起剤
JP2002179577A (ja) * 2000-12-12 2002-06-26 Club Cosmetics Co Ltd 抗腫瘍物質及び抗腫瘍剤
WO2007132790A1 (fr) * 2006-05-12 2007-11-22 National University Corporation Hokkaido University Liposome ayant une membrane lipidique contenant un composant cellulaire bactérien
JP2009269855A (ja) * 2008-05-07 2009-11-19 Osaka Prefecture Univ 常磁性金属含有ポリアミドアミンデンドロン脂質
WO2013172358A1 (fr) * 2012-05-14 2013-11-21 公立大学法人大阪府立大学 Composé fonctionnel, assemblage moléculaire contenant un composé fonctionnel, composition contenant un assemblage moléculaire, trousse et utilisation de l'assemblage moléculaire, de la composition ou du trousse
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