WO2015125732A1 - 治療抵抗性がんの予防用又は治療用組成物 - Google Patents
治療抵抗性がんの予防用又は治療用組成物 Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0061—5-aminolevulinic acid-based PDT: 5-ALA-PDT involving porphyrins or precursors of protoporphyrins generated in vivo from 5-ALA
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- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
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- A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
- A61N5/00—Radiation therapy
- A61N5/06—Radiation therapy using light
- A61N5/0613—Apparatus adapted for a specific treatment
- A61N5/062—Photodynamic therapy, i.e. excitation of an agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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 composition for preventing or treating treatment-resistant cancer accompanied by treatment-resistant cancer cells in photodynamic therapy.
- PDT photodynamic therapy
- furin is administered and accumulated in cancer cells, followed by irradiation with a laser beam to destroy the cancer cells from inside.
- the photofrin excretion rate is slow and tends to accumulate in the body, so that light in the room and other light causes the patient to become hypersensitive. Therefore, until the photosensitizer is removed from the patient's body (approximately several weeks to about one month after administration), there is a problem that the patient must spend in the dark room.
- ALA-PDT which is a precursor of porphyrin and is administered to 5-aminolevulinic acids (also referred to herein as “ALA”), which is a biological material, and combined with PDT
- ALA 5-aminolevulinic acids
- PDT 5-aminolevulinic acids
- PpIX is known as a photosensitizer having peaks at 410 nm, 510 nm, 545 nm, 580 nm, 630 nm, and the like. Since ALA is metabolized in vivo and excreted within 48 hours, it has a feature that it hardly affects the photosensitivity of the whole body. Therefore, ALA has higher clinical safety than photofrin.
- cancer stem cell hypothesis has recently been proposed as a cause of recurrence and metastasis. According to the cancer stem cell hypothesis, cancer stem cells exist in the tumor tissue, the cells have the ability to replicate themselves, and only a small number of them have the ability to form a tumor similar to the original tumor tissue. It has been. Furthermore, since cancer stem cells have resistance to anticancer drugs and radiation, they are likely to remain during treatment and are considered to cause recurrence and metastasis.
- cancer cells are re-applied from these treatment-resistant cancer cells (cancer stem cells, resistant cancer cells, etc.) remaining after treatment. As they occur, these treatment-resistant cancer cells can cause cancer metastasis and cancer recurrence. That is, in conventional cancer treatment, even if cancer cells are killed by cancer treatment and the cancer seems to have been cured, in many cases, a very small number of treatment-resistant cancer cells remain. And could not be a fundamental treatment for cancer. Accordingly, it has long been desired by society to establish a treatment method targeting treatment-resistant cancer cells to realize cancer treatment with a low risk of recurrence and metastasis. However, ALA-PDT and ALA-PDD for treatment-resistant cancer cells have not been studied so far and their effectiveness is not known.
- An object of the present invention is to provide a composition for preventing or treating treatment-resistant cancer accompanied by treatment-resistant cancer cells.
- the present inventors have focused on ALA-PDT, which has a different mechanism of action on cells than anticancer drug treatment or radiation therapy. As a result of intensive studies, it was surprisingly found that ALA-PDT is effective against treatment-resistant cancer cells.
- the present inventors have conceived that the treatment-resistant cancer cells are more efficiently killed by performing ALA-PDT multiple times (multistage) on the treatment-resistant cancer cells. did. That is, when ALA-PDT is performed a plurality of times, for example, in ALA-PDT after the second stage, the treatment-resistant cancer cells remaining after the first stage ALA-PDT may be eradicated. I came up with what I can do.
- the present inventors have not only solved a very important problem in medicine, but also worked on solving a more important problem. That is, the present inventors assumed a case where even if ALA-PDT was used, an effect on treatment-resistant cancer cells could not be sufficiently obtained. For example, in the deep part of a solid tumor, attention was focused on the possibility that light does not reach the cancer cells to be treated and the therapeutic effect cannot be obtained sufficiently. Moreover, even if a method in which light reaches the deep part of the tumor is used, it cannot be said to be a fundamental treatment for cancer unless treatment-resistant cancer cells can be killed.
- the present inventors have found that a tyrosine kinase inhibitor inhibits ABCG2 involved in PpIX excretion in treatment-resistant cancer cells.
- the present inventors have conceived that inhibition of ABCG2 improves intracellular PpIX accumulation and enhances the effect of ALA-PDT on treatment-resistant cancer cells. That is, the present inventors have found a compound that enhances the therapeutic effect of ALA-PDT on treatment-resistant cancer cells after paying attention to the mechanism of action of PpIX, thereby further increasing the resistance of treatment-resistant cancer cells.
- the present inventors have used a drug capable of inhibiting ABCG2 (preferably a tyrosine kinase inhibitor) together with ALAs to clarify the boundary between cancer tissue and normal tissue and to accumulate PpIX accumulated in cancer cells.
- a drug capable of inhibiting ABCG2 preferably a tyrosine kinase inhibitor
- ALAs a drug capable of inhibiting ABCG2
- ALAs preferably a tyrosine kinase inhibitor
- the present invention provides a compound represented by the following formula (I): (In the formula, R 1 represents a hydrogen atom or an acyl group, and R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group). Or a salt thereof,
- the present invention relates to a composition for preventing or treating treatment-resistant cancer accompanied by treatment-resistant cancer cells in photodynamic therapy.
- this invention relates to the composition for prevention or treatment in which the said treatment resistant cancer cell contains a cancer stem cell in one embodiment.
- the present invention also relates to a preventive or therapeutic composition, wherein in one embodiment, the cancer stem cell is a brain tumor stem cell.
- this invention is one embodiment WHEREIN: The said photodynamic therapy is implemented with respect to the treatment resistant cancer cell in a subject 2 times or more, The composition for prevention or treatment characterized by the above-mentioned.
- the present invention is a combination of (i) the prophylactic or therapeutic composition described above and (ii) an anticancer agent, which are administered simultaneously or sequentially.
- the present invention relates to a preventive or therapeutic drug.
- the tyrosine kinase inhibitor is a tyrosine kinase inhibitor for a molecule having a tyrosine residue
- the molecule having a tyrosine residue is a stem cell factor receptor (KIT), epidermal growth factor receptor (EGFR), nerve growth factor receptor (NGFR), colony stimulating factor receptor (CSF-1R), hepatocyte growth factor Receptor (HGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), human epidermal growth factor receptor 2 (HER2 / neu) , Src family, JAK, Fak, ZAP, Btk, Fps / Fes, and Bcr-Abl.
- KIT stem cell factor receptor
- EGFR epidermal growth factor receptor
- NGFR nerve growth factor receptor
- CSF-1R colony stimulating factor receptor
- HGFR hepatocyte growth factor Receptor
- FGFR
- the present invention also relates to a preventive or therapeutic drug, wherein in one embodiment, the tyrosine kinase inhibitor is an ABCG2 inhibitor.
- the present invention also relates to a prophylactic or therapeutic drug, characterized in that, in one embodiment, the combination aspect is a combination drug or a kit.
- the present invention relates to the preventive or therapeutic composition described above, which is used in combination with an anticancer agent simultaneously or sequentially.
- the present invention provides, in another embodiment, a combination of (i) the preventive or therapeutic composition described above and (ii) an anticancer agent for the treatment of treatment-resistant cancer.
- the present invention relates to a combination wherein (i) the therapeutic composition and (ii) the anticancer agent are administered simultaneously or sequentially.
- the present invention is a method for preventing or treating treatment-resistant cancer involving treatment-resistant cancer cells in a subject, (I) administering a compound represented by the above formula (I) or a salt thereof to a subject suffering from or likely to suffer from treatment-resistant cancer; and (II) It relates to a preventive or therapeutic method including a step of performing photodynamic therapy on the cancer cells in the subject.
- the step (II) is characterized in that the photodynamic therapy is performed twice or more on the treatment-resistant cancer cells.
- the present invention provides an anticancer agent to the subject simultaneously or sequentially before, during or after the execution of the step (I) or (II). Further, the present invention relates to a prophylactic or therapeutic method, further comprising the step of administering. (15) Furthermore, in another embodiment, the present invention relates to a compound represented by the above formula (I) or a compound thereof for producing a medicament for preventing or treating treatment-resistant cancer in photodynamic therapy. Relates to the use of salt. (16) Furthermore, in another embodiment, the present invention is administered simultaneously or sequentially.
- the present invention relates to a medicine for diagnosis of treatment-resistant cancer accompanied by treatment-resistant cancer cells in photodynamic therapy.
- An invention obtained by arbitrarily combining one or a plurality of features of the present invention described above is naturally included in the scope of the present invention as long as there is no technical contradiction.
- treatment-resistant cancer can be prevented or treated by using the prophylactic or therapeutic composition of the present invention in ALA-PDT.
- the treatment or prevention of treatment-resistant cancer is further improved by performing ALA-PDT multiple times (multistage).
- the prophylactic or therapeutic composition of the present invention is optionally treated with an anticancer agent, preferably in combination with a tyrosine kinase inhibitor simultaneously or sequentially.
- the effect of ALA-PDT on cancer can be further enhanced.
- the present invention also reduces the risk of generating resistant cancer cells that are resistant to treatment in the course of treatment, or allows cancer stem cells that originally have therapeutic resistance to be killed.
- a drug capable of inhibiting ABCG2 preferably a tyrosine kinase inhibitor
- ALA a drug capable of inhibiting ABCG2
- a tyrosine kinase inhibitor is used in combination with ALA to clarify the boundary between cancer tissue and normal tissue. According to the fluorescence of PpIX accumulated in the cancer tissue, it is possible to facilitate excision of the cancer tissue containing the treatment-resistant cancer cells. That is, the present invention is a medically extremely important invention.
- FIG. 1A shows a photograph of cells cultured with fetal calf serum (FCS) (non-sphere type).
- FIG. 1B shows a photograph of cells cultured in a culture solution not containing FCS (sphere type).
- FCS fetal calf serum
- FIG. 1B shows a photograph of cells cultured in a culture solution not containing FCS (sphere type).
- FCS fetal calf serum
- FIG. 2A shows a (dot) plot of cells cultured with FCS (non-sphere type).
- FIG. 2B shows a (dot) plot in which cells were cultured in a culture solution not containing FCS (sphere type).
- the light intensity (IC 50 ) that makes the cell viability 50% is indicated by an arrow. It is a figure which shows the microphotograph of a brain tumor cell (A172 cell), and the photosensitivity with respect to ALA-PDT.
- FIG. 3A shows a diagram of cells cultured with FCS.
- FIG. 3B shows a diagram in which cells were cultured without FCS, and the cells formed a sphere characteristic of stem cells.
- A172 cells were cultured at a density of 1.0 ⁇ 10 6 cells / ml, incubated with 0.3 mM ALA for 4 hours, and then irradiated with 405 nm laser light.
- the intensity of the laser beam was measured and the relationship with the cell viability was plotted.
- the light intensity (IC 50 ) that makes the cell viability 50% is indicated by an arrow. It is a figure which shows the photosensitivity with respect to ALA-PDT of a brain tumor cell (A172 cell) from a viewpoint of cell growth ability.
- A172 cells were cultured at a density of 1.0 ⁇ 10 6 cells / ml, incubated with 0.3 mM ALA for 4 hours, and then irradiated with 405 nm laser light.
- the cell growth ability with respect to the light dose (J / cm 2 ) was examined as the colony forming ability.
- the mRNA level of each gene was normalized by using the mRNA level of the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a control.
- Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase
- Measurements genes peptide transporters 1 and 2 (PEPT1 and PEPT2), 5-aminolevulinate synthases 1 and 2 (ALAS1 and ALAS2), 5-aminolevulinate dehydratase (ALAD), uroporphyrinogen III synthase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen oxidase (CPOX), protoporinogenogen oxidase (PPOX), ferrochelatase (FECH), ABC transporters (ABCB6) and ABCG2) It is a figure which shows the screening result of the transport function inhibition of ABCG2 by a tyrosine kinase inhibitor.
- PEPT1 and PEPT2 5-aminolevulinate synthases 1 and 2
- ALAD 5-aminolevulinate dehydratase
- UROS uroporphyrinogen III synthase
- UROD uroporphyrinogen decarbox
- ABCG2 transport function inhibition screening was performed using Erlotinib, Gefitinib, Lapatinib, Imatinib, Nilotinib, Dasatinib as an ABCG2 inhibitor and Verapamil as a control. Inhibitor concentrations ranged from 0 to 100 ⁇ M, and extracellular transport of Hoechst 33342 by ABCG2 was measured. It is a figure which shows the influence with respect to the intracellular accumulation of PpIX by Lapatinib or Gefitinib. The upper row shows a schematic diagram of the assay, and the lower row shows the results. FIG.
- FIG. 4 is a graph showing that the effect of ALA-PDT was increased by the addition of Lapatinib in an experiment using human glioblastoma / astrocytoma U87MG cells. It is a figure which shows the result of having measured the effect with respect to ALA-PDT by combined administration of Lapatinib from the difference in the weight of the tumor in a nude mouse.
- FIG. 6 is a graph showing that the effect of ALA-PDT was improved by the combined administration of Lapatinib in an experiment using nude mice.
- “ALA + TKI-X” in the upper row shows the results of administering ALA and Lapatinib in combination
- ALA in the middle row shows the results of administering ALA alone.
- FIG. 11a shows a decrease in cell viability dependent on the concentration of ALA
- FIG. 11b is an excerpt of the results when 250 ⁇ M of ALA was administered in the graph of FIG. 11a.
- FIG. 12a shows the decrease in cell viability depending on the concentration of ALA
- FIG. 12b is an excerpt of the results when 250 ⁇ M of ALA was administered in the graph of FIG. 12a.
- FIG. 13a shows the decrease in cell viability depending on the concentration of ALA
- FIG. 13b is an excerpt of the results when 250 ⁇ M of ALA was administered in the graph of FIG. 13a.
- FIG. 14a shows a decrease in cell viability depending on the concentration of ALA
- FIG. 14b is an excerpt of the results obtained when 250 ⁇ M of ALA was administered in the graph of FIG. 14a.
- FIG. 15a shows a decrease in ALA concentration-dependent cell viability
- FIG. 15b is an excerpt of the results when 250 ⁇ M of ALA was administered in the graph of FIG. 15a.
- ALA refers to ALA or a derivative thereof or a salt thereof.
- ALA means 5-aminolevulinic acid.
- ALA is also called ⁇ -aminolevulinic acid and is one of amino acids.
- ALA derivative a compound represented by the following formula (I) can be exemplified.
- R 1 represents a hydrogen atom or an acyl group
- R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group.
- ALA corresponds to the case where R 1 and R 2 are hydrogen atoms.
- ALAs may act as active ingredients in the state of ALA of the formula (I) or derivatives thereof in vivo, and can also be administered as prodrugs (precursors) that are degraded by enzymes in the body.
- Examples of the acyl group in R 1 of the formula (I) include linear or branched C 1-8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, benzylcarbonyl group and the like.
- Examples thereof include alkanoyl groups and aroyl groups having 7 to 14 carbon atoms such as benzoyl, 1-naphthoyl and 2-naphthoyl groups.
- alkyl group in R 2 of the formula (I) examples include a straight chain such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl group, etc.
- cycloalkyl group in R 2 of formula (I) there is a saturated or partially unsaturated bond such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, 1-cyclohexenyl group and the like. Examples thereof may include a cycloalkyl group having 3 to 8 carbon atoms.
- Examples of the aryl group in R 2 of the formula (I) include aryl groups having 6 to 14 carbon atoms such as phenyl, naphthyl, anthryl, and phenanthryl groups.
- the aryl moiety can be exemplified as the above aryl group
- the alkyl moiety can be exemplified as the above alkyl group.
- benzyl, phenethyl, phenylpropyl, phenyl Examples thereof include aralkyl groups having 7 to 15 carbon atoms such as butyl, benzhydryl, trityl, naphthylmethyl, and naphthylethyl groups.
- Preferable ALA derivatives include compounds in which R 1 is formyl, acetyl, propionyl, butyryl group or the like. Further, preferable ALA derivatives include compounds in which R 2 is a methyl, ethyl, propyl, butyl, pentyl group or the like. As preferred ALA derivatives, the combination of R 1 and R 2 is (formyl and methyl), (acetyl and methyl), (propionyl and methyl), (butyryl and methyl), (formyl and ethyl), (acetyl) And ethyl), (propionyl and ethyl), and (butyryl and ethyl).
- examples of the salt of ALA or a derivative thereof include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, and organic amine addition salts.
- acid addition salts include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, and other inorganic acid salts, formate, acetate, propionate, toluenesulfonic acid Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc.
- Organic acid addition salts can be exemplified.
- metal salts include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc.
- ammonium salts include ammonium salts and alkylammonium salts such as tetramethylammonium salts.
- organic amine salt include salts such as triethylamine salt, piperidine salt, morpholine salt, and toluidine salt. These salts can also be used as a solution at the time of use.
- ALA a compound formed from ALA and various esters such as ALA methyl ester, ALA ethyl ester, ALA propyl ester, ALA butyl ester, ALA pentyl ester, and their hydrochlorides and phosphoric acids. Salt, sulfate.
- ALA hydrochloride and ALA phosphate can be exemplified as particularly suitable.
- the ALAs can be produced by known methods such as chemical synthesis, production by microorganisms, production by enzymes, and the like.
- the ALAs may form hydrates or solvates, and ALAs may be used alone or in combination of two or more.
- the prophylactic or therapeutic composition of the present invention may contain a metal-containing compound as long as the object and problem of the present invention can be achieved without causing an unacceptable adverse effect on a living body or the like.
- a metal-containing compound examples include, but are not limited to, iron, magnesium, zinc, nickel, vanadium, cobalt, copper, chromium, molybdenum, and the like.
- a person skilled in the art can appropriately select an appropriate dose of the metal-containing compound and administer it together with ALAs in light of the objects and problems of the present invention.
- the preventive or therapeutic composition of the present invention and the metal-containing compound can be administered as a composition containing ALAs and a metal-containing compound, or can be administered alone.
- each When each is administered alone, it may be administered simultaneously.
- the simultaneous is not only performed at the same time, but the administration of ALAs and the metal-containing compound can give an additive effect to the living body, preferably a synergistic effect, even at the same time. Thus, it means that it is performed without a considerable interval between the two.
- ALA-PDT means photodynamic therapy (PDT) using ALAs, and most typically means PDT using ALA.
- ALA-PDD means photodynamic diagnosis (PDD) using ALAs, and most typically means PDD using ALA.
- the ALA-PDT administers a light-sensitive compound and, when performing PDT for treating a target site by irradiating light, administers ALAs that themselves do not have a photosensitizing action, Porphyrins (mainly PpIX) induced through the biosynthetic pathway are accumulated in cancer cells including treatment-resistant cancer cells, and the accumulated PpIX is excited to photoexcite surrounding oxygen molecules, resulting in generation
- the singlet oxygen to be used has a cell killing effect due to its strong oxidizing power.
- a light beam having a peak at 400 nm to 420 nm or a light beam having a peak at 600 nm to 650 nm is preferably used.
- the ALA-PDD detects the presence of cancer tissue containing treatment-resistant cancer cells by detecting fluorescence by exciting PpIX accumulated in cancer cells containing treatment-resistant cancer cells with light. It can be used to detect or diagnose the presence or absence.
- light having a peak at 400 nm to 420 nm.
- treatment-resistant cancer cells refer to advanced cancer, metastatic cancer, recurrent cancer, etc. that have later acquired treatment resistance against conventional anticancer drug treatment or radiation therapy. Some or all of the characteristics of cancer stem cells and cancer stem cells that are inherently resistant to cancer cells (resistant cancer cells), anticancer drug treatment, radiation therapy, etc. Cell (also referred to as “cancer stem cell-like cell” in this specification), a cancer cell with a low degree of differentiation, a cancer cell with slow division, an intractable cancer cell, and the like. Treatment-resistant cancer cells in the present invention are understood by those skilled in the art as another concept from cancer cells having high sensitivity to conventional anticancer drug treatment and radiation therapy.
- a treatment resistant cancer means the cancer containing the treatment resistant cancer cell mentioned above or accompanying.
- cancer stem cells typically means the self-replicating ability to produce exactly the same cell as itself, the multipotency capable of differentiating into many types of cells, and the original cancer when transplanted.
- Cancer stem cells can typically be observed for self-renewal ability, pluripotency, telomerase activity, activation of anti-apoptotic pathways, and / or activation of cell membrane carrier transport.
- Whether a certain cancer cell is a cancer stem cell or a cancer stem cell-like cell can be determined by a person skilled in the art according to a known method. For example, it can be determined that the cell is a cancer stem cell by confirming that the cell is equivalent to a morphologically known cancer stem cell, embryonic stem cell (ES cell) or somatic stem cell.
- the cells may be transplanted under the skin of an immunodeficient mouse, and a tumor tissue formed after a predetermined period of time may be analyzed to be determined as a cancer stem cell.
- a marker gene that is highly expressed in a known cancer stem cell in the cell may be confirmed to be highly expressed in the same manner, and determined to be a cancer stem cell.
- Such marker genes include, but are not limited to, Dclk1 (Nakanishi et al., Nature Gen. 2012.) and the like.
- CD133 + human brain tumor stem cell
- CD44 + CD24 ⁇ / low ESA + human breast cancer stem cell
- ESA epithelial specific antigen
- CD44 + integrin ⁇ 2 ⁇ 1 hi CD133 + or Sca-1 + human prostate cancer stem cells
- CD133 + human colon cancer stem cells
- CD44 + human head and neck squamous cell carcinoma stem cells
- CD44 + CD24 + ESA + (human pancreatic cancer stem cells, ESA : Epithelial specific antigen) may be used as an index to determine whether it is a cancer stem cell or a cancer stem cell-like cell.
- the stem cell is a cancer stem cell by confirming that an undifferentiated marker such as a marker gene specifically expressed in embryonic stem cells or somatic stem cells is expressed.
- an undifferentiated marker such as a marker gene specifically expressed in embryonic stem cells or somatic stem cells.
- the undifferentiation marker include alkaline phosphatase, and it can be determined that the cell is in an undifferentiated state by confirming that alkaline phosphatase staining is positive.
- a gene-wide gene expression pattern may be detected by a microarray or the like, and a cancer stem cell having a high correlation with a known cancer stem cell, embryonic stem cell or somatic stem cell expression pattern may be determined.
- the expression characteristics of cell surface antigens can be compared with those of known cancer stem cells, embryonic stem cells or somatic stem cells, and those with high correlation can be determined as cancer stem cells. Further, DNA methylation in the cells may be detected and compared with known cancer stem cells, embryonic stem cells or somatic stem cells. Whether or not a cancer stem cell or cancer stem cell-like cell is determined can be determined by at least one of the above methods, but can also be determined by combining two or more. For example, when a target cancer cell is cultured in a low serum or serum-free medium, when the cell is in a sphere type form such as an embryonic stem cell or somatic stem cell, The cell may be determined to be a cancer stem cell or at least a cancer stem cell-like cell. In this case, those skilled in the art can understand that the sphere-type cells later acquired treatment resistance.
- the treatment-resistant cancer is preferably malignant, and is not limited to, but includes, but is not limited to, brain tumor, spinal cord tumor, maxillary sinus cancer, pancreatic fluid adenocarcinoma, gingival cancer, tongue cancer, lip cancer, nasopharyngeal cancer, Oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, thyroid cancer, parathyroid cancer, lung cancer, pleural tumor, cancerous peritonitis, cancerous pleurisy, esophageal cancer, stomach cancer, colon cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, liver cancer, Kidney cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, adrenal cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer, bone tumor, breast cancer, skin cancer, melanoma, basal cell tumor, lymphoma Primary or metastatic and invasive or non-invasive cancer or sar
- Examples of the compound that enhances the effect of ALA-PDT or ALA-PDD include molecules that promote the expression of transporters (PEPT1, PEPT2, etc.) that take up ALA into cells, ALA and porphyrin synthase (ALAS1, ALAS2). , UROS, etc.), and molecules that reduce the expression of transporters (ABCG2, ABCB6, etc.) that excrete porphyrin out of the cell (see FIG. 5).
- transporters PEPT1, PEPT2, etc.
- PLAS1, ALAS2 porphyrin synthase
- UROS UROS
- molecules capable of inhibiting ABCG2 are preferred.
- Such molecules include not only anticancer agents that have a therapeutic effect with the compound alone, but also molecules such as compounds and antibodies that do not have an anticancer effect with the compound alone but can inhibit ABCG2. Yes.
- ABCG2 inhibitors are not limited but include, for example: topoisomerase II inhibitor (Mitoxantrone, Bisantrene, Etoposide, Becatecarin, NB-506, J-107088, etc.); Anthracyclines (Daunorubicin, Doxobucincin, Epirubicin, Pirarubicin, etc.); Camptothecin analogs (topoisomerase I inhibitor) (Topotecan, SN-38, CPT-11, 9-aminocamptothecin, NX211, DX-8951f, Homocamptothecins, BN80915 (diflomotecan), Gimatecan, Belotecan, etc.); Tyrosine kinase inhibitors (as described below, Dasatinib, Vandetanib, Nilotin
- cyclin-dependent kinase inhibitor (Flavopiridol etc.); CDK and aurora kinases inhibitor (e.g. JNJ-7706621); non-steroidal anti-androgen (e.g. Bicalutamide); PEITC (such as Phenethyl isothiocyanate); indazole-based tubulin inhibitors (such as TH-337); Sufate and glucuronide conjugates of xenobiotics (Estrone 3-sulfate (E1S), 17beta-estradiol sulfate, DHEAS, 4 [35S] -methylumbelliferone sulfate, E3040 sulfate, Troglitazone sulfate, 3-O-sulfate conjugate of 17alpha-ethinylestradiol, SN-38 -glucuronide, [3H] 17beta-estradiol-17beta-D-glucuronide, [3
- the anticancer agent in the present invention is not particularly limited as long as it is an anticancer agent known to those skilled in the art.
- alkylating agents nitrogen mustards (cyclophosphamide, etc.); nitrosoureas Platinum compounds (cisplatin, etc.); antimetabolites (5-fluorouracil, etc.); folate antimetabolites (dihydropteroate synthase inhibitors; dihydrofolate reductase inhibitors, etc.); pyrimidine metabolism inhibitors; purine metabolism inhibitors Ribonucleotide reductase inhibitors; nucleotide analogs (purine analogs, pyrimidine analogs, etc.); topoisomerase inhibitors (type I topoisomerase inhibitors; type II topoisomerase inhibitors, etc.); microtubule inhibitors (microtubule polymerization inhibitors; microtubule depolymerization inhibitors; Polymerization inhibitors, etc.); antitumor antibiotics; endocrine therapy
- the anticancer agent is preferably an anticancer agent capable of inhibiting ABCG2, and more preferably a tyrosine kinase inhibitor.
- the tyrosine kinase inhibitor is not limited, but is a tyrosine kinase inhibitor for a molecule having a tyrosine residue, and the molecule having the tyrosine residue is a stem cell factor receptor (KIT), an epidermal growth factor receptor (EGFR), nerve growth factor receptor (NGFR), colony stimulating factor receptor (CSF-1R), hepatocyte growth factor receptor (HGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor Body (VEGFR), platelet derived growth factor receptor (PDGFR), human epidermal growth factor receptor 2 (HER2 / neu), Src family (Src, Yes, Fyn, Fgr, Lyn, Lck, Hck, Blk, Frk), Also selected from the group consisting of JAK, Fak, ZAP, Btk, Fps / Fes, and Bcr-Abl It is preferred.
- KIT stem cell factor receptor
- EGFR epidermal
- the tyrosine kinase inhibitor has the following formula (A): Wherein R is halogen, preferably Cl, C 2-5 alkynyl, preferably ethynyl, or phenyl-C 1-5 alkoxy, preferably phenylmethoxy, which may be the same or different, Optionally substituted with F, n is an integer from 1 to 5, preferably 1 or 2, X and Y are organic groups or hydrogen, preferably 2-methoxyethoxy, or 2-methyl Sulfonylethylamino-methyl-2-furin, which may be the same or different, or a salt thereof.
- the compound is Lapatinib (n- [3-chloro-4-[(3-fluorophenyl) methoxy] phenyl] -6- [5-[(2-methylsulfonylethylamino) methyl] -2-furyl] quinazolin-4-amine) , Erlotinib (N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) -4-quinazolinamine), Gefitinib (N- (3-chloro-4-fluoro-phenyl) -7-methoxy-6- ( 3-morpholin-4-ylpropoxy) quinazolin-4-amine), and Lapatinib is more preferable.
- the subject of application of the preventive or therapeutic composition of the present invention is typically a human, but includes cases where it is a non-human animal such as a pet, laboratory animal, or domestic animal. If not preferred, humans may be excluded from the subject.
- the subject includes not only a subject suffering from treatment-resistant cancer, but also a subject suffering from or likely to suffer from treatment-resistant cancer.
- the administration route of the preventive or therapeutic composition of the present invention to a subject may be systemic administration or local administration.
- oral administration including sublingual administration, or inhalation administration, direct administration to a target tissue or organ by catheter, intravenous administration including infusion, transdermal administration by poultice, suppository, or nasogastric And parenteral administration such as administration by forced enteral nutrition using a tube, nasal intestinal tract, gastric fistula tube or intestinal fistula tube.
- the dosage form of the prophylactic or therapeutic composition of the present invention may be appropriately determined according to the administration route, and is not limited, but is not limited to injections, drops, tablets, capsules, fine granules, powders, A liquid medicine, a poultice, a suppository and the like dissolved in a liquid, syrup and the like can be mentioned.
- a pharmacologically acceptable carrier for example, a pharmacologically acceptable carrier, excipient, diluent, additive, disintegrant, binder, coating agent may be used as necessary.
- Lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers, solvents, gelling agents, nutrients, and the like may affect the absorption and blood concentration of things, resulting in changes in pharmacokinetics.
- water, physiological saline, animal fat and oil, vegetable oil, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, glycerin, etc. can be illustrated.
- the preventive or therapeutic drug or diagnostic drug of the present invention means a drug that is a combination of two or more substances and compositions and does not limit the mode of the combination.
- optional components such as other medicinal ingredients, nutrients, and carriers can be added to the preventive or therapeutic drug and diagnostic drug according to the present invention as needed.
- an optional component for example, crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, fats and oils, gums, polyalkylene glycols and the like, pharmaceutically acceptable ordinary carriers and binders
- Various formulation ingredients such as stabilizers, solvents, dispersion media, extenders, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, etc. it can.
- the dose of the prophylactic or therapeutic composition of the present invention may be determined appropriately by those skilled in the art as long as the amount of PpIX accumulated in the target treatment-resistant cancer cell is an effective amount in ALA-PDT.
- the amount, timing, administration frequency, and administration period for the subject include the age, weight, symptoms and condition of the subject, or the state and administration of cells, tissues or organs in the subject to be prevented or treated. It depends on ease of use.
- the dosage of ALAs to humans is not particularly limited as long as a photosensitizing effect by ALAs is obtained, but per 1 kg body weight in terms of ALA, It may be 1 mg to 1,000 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg, and still more preferably 15 mg to 25 mg.
- WO2013 / 187069 Tile of Invention: PDT effect enhancer
- the dose of ALAs is typically smaller than that for the treatment method for treatment-resistant cancer.
- the dose of ALAs to humans is not particularly limited as long as a photosensitizing effect by ALAs is obtained, but per 1 kg body weight in terms of ALA, It may be 0.5 mg to 500 mg, preferably 2.5 mg to 50 mg, more preferably 5 mg to 15 mg, and still more preferably 7.5 mg to 12.5 mg.
- Local administration will require fewer ALAs than systemic administration.
- the administration frequency of ALAs can be exemplified by administration once to several times a day or continuous administration by infusion or the like.
- the administration period of ALAs is, for example, a method known by pharmacologists and clinicians in the technical field based on various clinical indicators in view of symptoms or conditions to be prevented or treated. Can be determined.
- ALA-PDT is administered once or a plurality of times (for example, 2 times, 3 times, 4 or more times) to prevent or treat treatment-resistant cancer.
- treatment resistance in the deep part of the tumor which is not effective in the first PDT Sexual cancer cells can also be killed.
- ALAs when administered multiple times, PDT may be performed multiple times between each administration.
- the remaining (treatment resistant) cancer cells or (treatment resistant) cancer cells ALA-PDT may be performed once or a plurality of times on a tissue that may remain.
- the effect of the first PDT is not obtained. Examples include killing resistant cancer cells.
- first performing conventional cancer treatment for example, surgical excision of tumor, anticancer drug treatment, radiotherapy, etc.
- killing most cancer cells ALAs are used.
- the remaining PDT can be used to kill the remaining cancer cells or prevent cancer recurrence and metastasis. In this case, the first conventional cancer treatment may be continued.
- an anticancer agent when ALA-PDT is performed, an anticancer agent may be combined with ALAs and administered to a subject simultaneously or sequentially.
- the administration method of ALAs and the administration method of the anticancer agent may be the same or different.
- ALAs may be orally administered to a subject and an anticancer agent may be administered intravenously.
- the amount of administration, timing, administration frequency, administration period, etc. of the anticancer drug to the subject can be appropriately determined by those skilled in the art, similarly to ALAs, taking into account the nature and type of the anticancer drug. Needless to say.
- an ABCG2 inhibitor may be used instead of the anticancer agent.
- the anticancer agent also has an ABCG2 inhibitory action.
- the ALA and anticancer agent contained in the preventive or therapeutic composition of the present invention are a composition comprising an ALA and an anticancer agent as a preventive or therapeutic drug ( It may be used as a compounding agent) or as a separate kit.
- each may be administered alone. When each is administered alone, it may be administered simultaneously or sequentially.
- “simultaneously” is not only performed at the same time, but also at the same time so that administration of ALAs and an anticancer agent can exert an additive effect, preferably a synergistic effect. It may mean that the process is performed without a considerable interval between the two.
- ALA-PDT and an anticancer agent are combined to prevent or treat treatment-resistant cancer, for example, after administering an anticancer agent, ALAs are administered, and PDT is administered once or It may be performed multiple times; after administration of ALAs, an anticancer agent may be administered, and then PDT may be performed one or more times; ALAs may be administered and PDT may be administered 1 You may administer an anticancer agent after implementing once or several times.
- any combination of one or more administrations of ALAs, one or more administrations of an anticancer agent, one or more administrations of PDT, etc. is included within the scope of the present invention. Needless to say.
- conventional cancer treatment for example, surgical excision of a tumor, anticancer drug treatment, radiotherapy, etc.
- ALA-PDT is administered. This may be done to kill the remaining cancer cells.
- the first conventional cancer treatment may be continued thereafter, or an anticancer agent (preferably a tyrosine kinase inhibitor) may be used in combination.
- fluorescence excited from PpIX that selectively accumulates in cancer cells including treatment-resistant cancer cells is detected, It is possible to determine or diagnose the presence or absence of cancer tissue containing treatment resistant cancer cells.
- a tyrosine kinase inhibitor or ABCG2 inhibitor together with ALAs, the outflow of PpIX from cancer cells can be reduced and the boundary between cancer tissue and normal tissue can be sharpened. Cancer tissue can be diagnosed and detected more accurately according to the accumulated fluorescence of PpIX, and surgically excised.
- treatment-resistant cancer cells The types of treatment-resistant cancer cells, treatment-resistant cancers, tyrosine kinase inhibitors, ABCG2 inhibitors, etc. targeted by the diagnostic drug of the present invention are those described above for the preventive or therapeutic drug of the present invention. Similar ones may be contemplated.
- the treatment resistant cancer is particularly preferably a brain tumor.
- the dosage, timing, administration frequency, administration period, etc. of administration to the subject in the same manner as those of the preventive or therapeutic medicament of the present invention.
- it can be appropriately examined and implemented.
- one or more times of administration of ALAs, one or more times of tyrosine kinase inhibitor or ABCG2 inhibitor, and one or more times of PDD may be combined as appropriate.
- the dose of ALAs to humans is the presence of cancer cells by detecting fluorescence by exciting PpIX induced by ALAs with light.
- the present invention may relate to a method for diagnosing treatment-resistant cancer involving treatment-resistant cancer cells in a subject.
- the diagnostic method is to use a tyrosine kinase inhibitor or an ABCG2 inhibitor in combination before, at the same time as, or after administration of ALAs to a subject suffering from or possibly suffering from treatment-resistant cancer.
- PDD is performed by irradiating a site where cancer is present or expected to be present in the subject to selectively accumulate in cancer cells including treatment-resistant cancer cells.
- detecting the fluorescence excited from PpIX it may be determined or diagnosed whether the subject suffers from a treatment resistant cancer.
- the expression of ABCG2 is increased, so that the treatment resistance can be increased by using an ABCG2 inhibitor (preferably an anticancer agent capable of inhibiting ABCG2) together with ALAs.
- an ABCG2 inhibitor preferably an anticancer agent capable of inhibiting ABCG2
- This is advantageous because it can promote the accumulation of PpIX in cancer cells and improve the efficiency of ALA-PDT in various cancers such as malignant brain tumors (lung cancer, stomach cancer, colon cancer, breast cancer, etc.).
- invasive cancerous tissue that is difficult to remove by surgery (eg, glioblastoma multiform, grade IV), after surgical excision, it can be localized using an optical fiber or the like.
- cancer cells including treatment resistant cancer cells
- a single or several administration of a tyrosine kinase inhibitor capable of inhibiting the expression of ABCG2 which may be performed several hours before ALA-PDT or ALA-PDD, causes side effects (bone marrow suppression) seen in repeated administration over a long period of time. Leukocytes, neutrophils, thrombocytopenia, skin and liver disorders, etc.) are less likely to appear. Therefore, single or multiple dose tyrosine kinase inhibitors in combination with ALA-PDT or ALA-PDD are advantageous because of their high safety.
- first, second,... are used to represent various elements, it is understood that these elements should not be limited by those terms. These terms are only used to distinguish one element from another, for example, the first element is referred to as the second element, and similarly, the second element is the first element. It is understood that it can be made without departing from the scope of the present invention unless there is a technical contradiction.
- any numerical value used to indicate the component content or numerical range is interpreted as including the meaning of the term “about” unless otherwise specified.
- Example 1 Cancer stem cell culture Experimental methods Cancer stem cell research is being applied by applying normal tissue stem cell enrichment and isolation methods. In leukemia stem cell research, the search for therapeutic methods targeting cancer stem cells and the treatment stage in mouse models Research is progressing. On the other hand, although the establishment of separation and concentration methods is still insufficient for solid cancer, in recent years it has become possible to isolate and culture brain tumor stem cells from patients with malignant brain tumors. Stem cell lines isolated and established from patients with brain tumors based on these methods were used (Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI. Cancerous stem cells can Proc Natl Acad Sci US A.
- glioma stem cells were cultured in a D-MEM / F12 culture medium containing no FCS, all the cells formed a three-dimensional sphere characteristic of stem cells. As shown in FIG. 1, the diameter of the cell sphere was several hundred ⁇ m. On the other hand, when the cells were cultured in a D-MEM / F12 culture solution containing 10% FCS, spheres were not formed, and the cells grew flat.
- Example 2 Photosensitivity of cancer stem cells to ALA-PDT Experimental method Brain tumor stem cells (MD13, MD30, 157NS cells) grown to sphere type and non-sphere type were trypsinized and incubated with 0.3 mM ALA at a density of 1.0 ⁇ 10 6 cells / ml for 4 hours, respectively. , 405 nm laser light was irradiated. After further culturing for 7 days, the cell viability was measured by a viable cell count method (WST-8 assay) using a water-soluble tetrazolium salt as a coloring reagent.
- WST-8 assay viable cell count method
- FIG. 2 is a plot of the relationship between laser light intensity and cell viability.
- a sphere type cell (FIG. 2B) cultured in a D-MEM / F12 culture solution (containing L-glutamine, heparin, B27, EGF, and bFGF) not containing FCS is a D-MEM / F12 culture containing 10% FCS. It was more sensitive to ALA-PDT than non-sphere type cells cultured in liquid (FIG. 2A).
- Table 1 summarizes the light intensity (IC 50 ) that makes the cell viability 50%. It was found that the sensitivity of the sphere type cell (stem cell or stem cell-like cell) was increased by about 2 times compared to the non-sphere type cell.
- Example 3 Photosensitivity of brain tumor cells to ALA-PDT Experimental Method
- brain tumor cell line A172 cells purchased from American Type Culture Collection
- D-MEM / F12 medium containing L-glutamine, heparin, B27, EGF, bFGF
- the cells become stem cells.
- the cells were cultured in a D-MEM / F12 culture solution containing 10% FCS, the cells did not form a sphere type and proliferated in a plane. Cells grown in sphere and non-sphere types were trypsinized.
- each was incubated with 0.3 mM ALA at a density of 1.0 ⁇ 10 6 cells / ml for 4 hours, and then irradiated with 405 nm laser light. After further culturing for 7 days, the cell viability was measured by WST-8 assay in the same manner as in Example 2.
- 500 cells were taken out from each of A172 cells subjected to ALA-PDT, then seeded on a D-MEM agar medium containing 10% FCS and antibiotics, and cultured for 14 days. Thereafter, treatment with Trypan blue containing 10% formalin fixed and stained the cell colonies. The number of colonies consisting of 50 cells or more was counted using a stereo microscope.
- the colony formation assay can measure the proliferation ability of cells. As shown in FIG. 4, as a result of examining by colony formation assay, it was found that the sphere type was significantly more sensitive than the non-sphere type. That is, it can be said that the sphere type (cancer stem cell) is extremely sensitive in terms of cell proliferation ability.
- RNA levels of enzymes involved in porphyrin biosynthesis and transporter genes were measured by quantitative PCR. Total RNA was extracted from sphere-type and non-sphere-type A172 cells using the Magna Pure LC kit (Roche). Then, cDNA was prepared from the RNA using Transscriptor First-strand cDNA Synthesis Kit (Roche Diagnostics). Quantitative PCR was performed using Light Cycler 3 (Roche Diagnostics).
- Example 5 Inhibition of transport function of ABCG2 by tyrosine kinase inhibitor Experimental Method 48 types of ABC transporter genes are encoded in human genomic DNA. Among these, ABCG2 has the ability to physiologically transport intracellular PpIX out of the cell. Moreover, ABCG2 is remarkably high in expression in stem cells, and ABCG2 actively discharges Hoechst 33342 used for isolating stem cells and SP (side population) cells by flow cytometry. (Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP.
- the ABC transporter Bcrp1 / ABCG2 is expressed in a Wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med. 7 (9): 1028-1034, 2001.). So far, the present inventors have developed a rapid screening system that searches for compounds and drugs that inhibit the transport function of ABCG2, and searched for efficient inhibitor molecules (Saito H, Hirano H, Nakagawa H, Fukami). T, Oosumi K, Murakami K, Kimura H, Kouchi T, Konomi M, Tao E, Tsujikawa N, Tarui S, Nagakura M, Osumi M and Ishikawa T.
- an inhibitor screening experiment for ABCG2 was performed using Hoechst 33342 and a plurality of tyrosine kinase inhibitors. Specifically, Ergotinib, Gefitinib, Lapatinib, Imatinib, Nilotinib, and Dasatinib are used as tyrosine kinase inhibitors, and the transport function inhibition screening of ABCG2 is performed using Verapamil, which is an L-type calcium channel inhibitor, as a control. Carried out.
- Flp-In-293 cells Flp-In-293 / ABCG2 cells expressing human ABCG2 were cultured for a certain period of time with Hoechst 33342, which is a substrate for ABCG2, and the tyrosine kinase inhibitor. Thereafter, the level of Hoechst 33342 accumulated in individual cells was measured by FACS (Beckman Coulter). A similar experiment was also performed on Flp-In-293 / Mock (not expressing ABCG2) cells to confirm that it was specific inhibition on ABCG2. The specific experimental procedure is as follows.
- Flp-In-293 / Mock and Flp-In-293 / ABCG2 were washed twice with PBS (phosphate buffered saline) and then peeled off with Trypsin-EDTA. 10 6 cells were suspended in DMEM medium (2% FBS). Subsequently, 5 ⁇ g / mL Hoechst 33342 (Invitrogen) is added alone, or 5 ⁇ g / mL Hoechst 33342 is added in combination with various inhibitors at a concentration of 1 nM to 100 ⁇ M, followed by incubation at 37 ° C. for 90 minutes. did.
- the cells were washed with cold PBS (2% FBS), suspended again in cold PBS (2% FBS), and then the intracellular fluorescence intensity was measured by FACS (Beckman Coulter). 50,000 cells were measured per measurement. Hoechst 33342 accumulated in the cells was excited by a UV laser, the fluorescence 675 nm was detected, and the average fluorescence intensity (MFI) of the measured cells was calculated. Based on the results, the inhibition rate of ABCG2 was calculated.
- FIG. 6 shows the MFI of each addition group when the MFI of Flp-In-293 / Mock cells and Flp-In-293 / ABCG2 cells at the time of adding Hoechst 33342 alone is 100% and 0%, respectively.
- the vertical axis represents the excretion inhibition rate of Hoechst 33342 by a tyrosine kinase inhibitor (reflecting inhibition of ABCG2 function), and the horizontal axis represents the concentration of the tyrosine kinase inhibitor. From these results, it was found that Lapatinib strongly inhibited ABCG2 among the tested tyrosine kinase inhibitors, and Erlotinib and Gefitinib had almost the same ABCG2 inhibitory ability.
- Example 6 Inhibition of ABCG2 transport by Lapatinib and intracellular accumulation of protoporphyrin IX (PpIX) biosynthesized from ALA
- PpIX protoporphyrin IX
- Human glioblastoma / astrocytoma U87MG cells were cultured with 2 mM ALA and various concentrations of Lapatinib for 4 hours, during which the fluorescence intensity (MFI value) of PpIX biosynthesized from ALA and accumulated in the cells was measured. Measured with FACS (Beckman Coulter). The specific experimental procedure is as follows. U87MG cells were washed twice with PBS and then detached with Trypsin-EDTA. 10 6 cells were suspended in low serum DMEM medium (2% FBS).
- Example 7 Inhibition of transport of ABCG2 by Lapatinib and the effect of ALA-PDT: experiment using cultured cells Experimental Method Human glioblastoma / astrocytoma U87MG cells (10 6 cells) are suspended in DMEM (2% FBS) and then 10 mM ALA is added alone, or 10 mM ALA and Lapatinib at a concentration of 1 nM to 300 ⁇ M are added. Was added together, and then cultured at 37 ° C. for 4 hours. Cells were irradiated with an LED having a peak at 635 nm (12 J / cm 2 ), and the subsequent cell viability was measured by the MTT method.
- the MTT method has the same measurement principle as the WST-8 assay, and is a colorimetric quantitative method for measuring enzyme activity for reducing MTT to a formazan dye (purple).
- Example 8 Inhibition of ABCG2 transport by Lapatinib and the effect of ALA-PDT: experiments using nude mice Experimental Method Human glioblastoma / astrocytoma U87MG cells were cultured in D-MEM medium (Sigma-Aldrich) containing 10% FCS, and the number of viable cells was counted using Trypan blue. Then, U87MG cells (5 ⁇ 10 6 cells / 0.1 ml / mouse) were implanted subcutaneously in the back of BALB / c-nu / nu nude mice.
- nude mice were treated with ALA alone (30 mg / kg body weight, po) or ALA (30 mg / kg body weight, po) and Lapatinib (100 mg / kg body weight, po). Orally administered in combination. Then, 3 hours later, an LED having an emission peak at 635 nm was irradiated to the tumor transplantation part. One week after irradiation, tumors were removed and weighed.
- mice subjected to ALA-PDT by oral administration of ALA (30 mg / kg body weight, po) and Lapatinib (100 mg / kg body weight, po) in combination control mice and ALA (30 mg / kg body weight, p.o.) Tumor growth was significantly suppressed as compared with mice administered alone (FIGS. 9 and 10).
- Example 9 Effects of multiple implementations of PDT Experimental Method HeLa cells (human-derived cervical cancer cell line) 1 ⁇ 10 5 cells were seeded in a 60 mm dish, cultured for 24 hours, and then an anticancer agent was administered to the culture solution.
- Table 2 shows the types of anticancer drugs administered and the concentrations of the anticancer drugs in the culture solution.
- Cells were collected by trypsin-EDTA treatment 72 hours after administration of the anticancer agent, and a part of the collected cells was used to confirm the number of viable cells by trypan blue staining.
- Surviving cells were seeded with 5 ⁇ 10 3 cells in a 96-well plate. In this example, these cells that survived 72 hours after administration of the anticancer drug were used as treatment-resistant cancer.
- ALA 5-aminolevulinic acid hydrochloride
- FIGS. 11a, 12a, 13a, 14a, and 15a indicate the concentration of ALA administered.
- 11b, FIG. 12b, FIG. 13b, FIG. 14b, and FIG. 15b are excerpts of the results when 250 ⁇ M of ALA was administered in the graphs of FIGS. 11a, 12a, 13a, 14a, and 15a, respectively.
- the vertical axis of each figure shows the ratio of the cell viability in the experimental group when the cell viability when the experiment was conducted under the same conditions except that ALA was not administered was 100%.
- the HeLa cells were ALA 125 ⁇ M or more, and the cell viability decreased.
- the cell viability decreased depending on the number of times of light irradiation. From this result, it was found that, when light irradiation with the same irradiation energy was performed, the therapeutic effect of ALA-PDT multiple times was higher than that of a single ALA-PDT.
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Abstract
Description
現在、医療現場で実施されているPDTに関しては、肺、食道、胃、子宮頚部の早期がんに対して、特定の波長のレーザー光に反応する、腫瘍親和性の光増感剤であるフォトフリンを投与して、がん細胞に集積させた後に、レーザー光線を照射してがん細胞内部から破壊する治療法が挙げられる。しかしながら、かかる治療法では、フォトフリンの排泄速度が遅く、体内に蓄積しやすいので、部屋の光やその他の光によって患者が光線過敏症を引き起こす。そのため、患者の体内から光増感剤が排除されるまで(投与後、約数週間~約1ヶ月間)は、暗室内で患者が過ごさなくてはならないという難点がある。
ALA類それ自体には光感受性はないものの、ALA類は、細胞内で、ヘム生合成経路の一連の酵素群により代謝活性化されてポルフィリン(主としてプロトポルフィリンIX(PpIX))となることが知られている。そして、PpIXは、410nm、510nm、545nm、580nm、630nm等にピークを有する光増感剤として知られている。ALAは生体内で代謝され、48時間以内に排泄されるため、全身の光感受性にはほとんど影響しないという特徴がある。そのため、フォトフリンよりもALAの方が臨床上の安全性が高い。
一方、ALAを投与した悪性脳腫瘍のPDTは、未だ、臨床的には行われていない。その理由の1つに、悪性脳腫瘍細胞からのPpIX排出によるPpIX蓄積の低下が挙げられる。また、がん細胞から排出されるPpIXによって、がん組織とその周りにある正常組織との境界が不鮮明になる結果、腫瘍が正常脳組織の中に浸潤した部分を切除できなくなり、悪性脳腫瘍の外科手術による摘出の成功率が低下するという問題があった。
また、再発・転移の原因として近年提唱されているのが、がん幹細胞仮説である。がん幹細胞仮説では、腫瘍組織中にがん幹細胞が存在し、その細胞は自己を複製する能力を持つとともに、少数存在するだけで元の腫瘍組織と同様の腫瘍を形成する能力を有すると考えられている。さらに、がん幹細胞は抗がん剤や放射線への抵抗性を有しているため、治療の際に残存しやすく、再発・転移の原因となっていると考えられている。
したがって、治療抵抗性がん細胞を標的とした治療法を確立することで、再発、転移のリスクの少ないがん治療を実現することが、長い間、社会から切望されている。
しかしながら、治療抵抗性がん細胞に対するALA-PDT、ALA-PDDに関しては、これまで研究されておらず、その有効性は知られていない。
これに関連して、ALAから生合成されたPpIXはがん細胞から排出されるために、光増感剤であるPpIXのがん細胞内の蓄積が抑制されて、がん組織に対するPDTの効率の低下が引き起こされることもわかってきていた。
本発明者らは、したがって、これらの重要な課題の解決のために、細胞内のPpIXの蓄積の促進に着目し、試行錯誤を続けた。その結果、治療抵抗性がん細胞において、ALAの細胞内取り込みを行なうトランスポーターPEPT1およびPEPT2の発現、並びにポルフィリン合成酵素やABCB6遺伝子の発現等が亢進している事を見出した。また、本発明者らは、治療抵抗性がん細胞において、チロシンキナーゼ阻害剤がPpIXの排出に関与するABCG2を阻害することを見出した。これらに鑑み、本発明者らは、ABCG2を阻害すると、細胞内PpIXの蓄積が向上し、治療抵抗性がん細胞に対するALA-PDTの効果が増強できることに想到した。すなわち、本発明者らは、PpIXの作用機序に着目した上で、治療抵抗性がん細胞に対するALA-PDTの治療効果を増強させる化合物を見出し、それにより、治療抵抗性がん細胞を一層さらに、増殖阻害/死滅させることをも実現した。
さらに、本発明者らは、ABCG2を阻害できる薬剤(好ましくはチロシンキナーゼ阻害剤)をALA類と共に併用することによって、がん組織と正常組織の境界を鮮明にして、がん細胞に蓄積したPpIXの蛍光に従って、治療抵抗性がん細胞を含むがん組織を検出できることに、驚くべきことに想到した。
又はその塩
を含むことを特徴とする、
光線力学的療法における、治療抵抗性がん細胞を伴う治療抵抗性がんの予防用又は治療用組成物に関する。
(2)また、本発明は、一実施態様において、前記治療抵抗性がん細胞が、がん幹細胞を含むことを特徴とする、予防用又は治療用組成物に関する。
(3)また、本発明は、一実施態様において、前記がん幹細胞が、脳腫瘍幹細胞であることを特徴とする、予防用又は治療用組成物に関する。
(4)また、本発明は、一実施態様において、前記光線力学的療法が、対象における治療抵抗性がん細胞に対して2回以上実施されることを特徴とする、予防用又は治療用組成物に関する。
(5)さらに、本発明は、別の実施態様において、同時または順次に投与される、(i)上述の予防用又は治療用組成物と(ii)抗がん剤とを組み合わせてなることを特徴とする、予防用又は治療用医薬に関する。
(6)また、本発明は、一実施態様において、前記抗がん剤が、チロシンキナーゼ阻害剤であることを特徴とする、予防用又は治療用医薬に関する。
(7)また、本発明は、一実施態様において、前記チロシンキナーゼ阻害剤が、チロシン残基を有する分子に対するチロシンキナーゼ阻害剤であり、
前記チロシン残基を有する分子が、幹細胞因子受容体(KIT)、上皮成長因子受容体(EGFR)、神経成長因子受容体(NGFR)、コロニー刺激因子受容体(CSF-1R)、肝細胞増殖因子受容体(HGFR)、繊維芽細胞増殖因子受容体(FGFR)、血管内皮細胞増殖因子受容体(VEGFR)、血小板由来増殖因子受容体(PDGFR)、ヒト上皮成長因子受容体2(HER2/neu)、Srcファミリー、JAK、Fak、ZAP、Btk、Fps/Fes、および、Bcr-Ablからなる群から選択されることを特徴とする、予防用又は治療用医薬に関する。
(8)また、本発明は、一実施態様において、前記チロシンキナーゼ阻害剤が、ABCG2阻害剤であることを特徴とする、予防用又は治療用医薬に関する。
(9)また、本発明は、一実施態様において、前記組み合わせの態様が、配合剤であるか、または、キットであることを特徴とする、予防用又は治療用医薬に関する。
(10)さらに、本発明は、別の実施態様において、抗がん剤と同時または順次に併用される、上述の予防用又は治療用組成物に関する。
(11)さらに、本発明は、別の実施態様において、治療抵抗性がんの治療のための、(i)上述の予防用又は治療用組成物と(ii)抗がん剤との組み合わせであって、
前記(i)治療用組成物と前記(ii)抗がん剤が、同時または順次に投与されることを特徴とする、組み合わせに関する。
(12)さらに、本発明は、別の実施態様において、対象における治療抵抗性がん細胞を伴う治療抵抗性がんの予防又は治療方法であって、
(I)治療抵抗性がんを患っているか、患う恐れのある対象に対して、上記式(I)で示される化合物又はその塩
を投与するステップ;および、
(II)前記対象における前記がん細胞に対して、光線力学的療法を実施するステップ
を含む、予防又は治療方法に関する。
(13)また、本発明は、一実施態様において、前記ステップ(II)は、前記治療抵抗性がん細胞に対して、前記光線力学的療法を2回以上実施することを特徴とする、予防又は治療方法に関する。
(14)また、本発明は、一実施態様において、前記ステップ(I)または(II)の実施の前、間、または、後において、前記対象に対して、抗がん剤を、同時または順次に、さらに投与するステップを含む、予防又は治療方法に関する。
(15)さらに、本発明は、別の実施態様において、光線力学的療法における、治療抵抗性がんの予防用又は治療用医薬の製造のための、上記式(I)で示される化合物又はその塩の使用に関する。
(16)さらに、本発明は、別の実施態様において、同時または順次に投与される、
(A)上記式(I)で示される化合物又はその塩と、
(B)チロシンキナーゼ阻害剤又はABCG2阻害剤と
を組み合わせてなることを特徴とする、
光線力学的療法における、治療抵抗性がん細胞を伴う治療抵抗性がんの診断用医薬に関する。
(17)以上述べた本発明の一または複数の特徴を任意に組み合わせた発明も、技術的に矛盾しない限り、本発明の範囲に含まれるのは当然である。
また、別の態様において、本発明の予防用又は治療用組成物は、場合により、抗がん剤、好ましくは、チロシンキナーゼ阻害剤と同時または順次に組み合わせて使用することで、治療抵抗性がんに対するALA-PDTの効果をいっそうさらに増強させることができる。
また、本発明は、別の態様において、治療の過程において治療抵抗性の耐性がん細胞を生じさせるリスクを減らし、あるいは、元々治療抵抗性を有するがん幹細胞をも死滅させることも可能とするので、いわゆる難治性がんに対しても有効であり、また、がんの再発および転移を伴うことがない根本的ながん治療をも可能とし得る。また、がんの再発および転移の原因となる治療抵抗性がん細胞を死滅させることにより、がんの再発および転移を予防することも可能とし得る。
また、別の態様において、本発明により、ALA-PDDにおいて、ABCG2を阻害できる薬剤(好ましくはチロシンキナーゼ阻害剤)をALA類と共に併用することによって、がん組織と正常組織の境界を鮮明にして、がん組織に蓄積したPpIXの蛍光に従って、治療抵抗性がん細胞を含むがん組織を外科的に切除することを容易にし得る。
すなわち、本発明は、医学上極めて重要な発明である。
例えば、本発明の予防用又は治療用組成物と金属含有化合物は、ALA類と金属含有化合物とを含む組成物として、あるいは、それぞれ単独で投与することができる。それぞれ単独で投与する場合、同時に投与してよい。ここで、同時とは、同時刻に行われることのみならず、同時刻でなくともALA類と金属含有化合物との投与が、生体に相加的効果、好ましくは相乗的効果を与えることができるように、両者間で相当の間隔をおかずに行われることを意味する。
一方、上記ALA-PDDは、治療抵抗性がん細胞を含むがん細胞に蓄積したPpIXを光線で励起させて蛍光を検出することで、治療抵抗性がん細胞を含むがん組織の存在の有無を検出もしくは診断することに利用できる。ALA-PDDには400nm~420nmにピークを持つ光線を用いるのが好ましい。
また、本明細書において、治療抵抗性がんとは、上述した治療抵抗性がん細胞を含むか、もしくは、伴うがんを意味する。
本明細書において、がん幹細胞とは、典型的には、自らと全く同じ細胞を作り出す自己複製能と、多種類の細胞に分化しうる多分化能と、移植した場合に元のがんと同じ表現型のがんを形成できる能力を有し、がん組織中で、自己複製により自分と同じ細胞を維持しながら、分化によって周辺の大多数のがん細胞を生み出す元になる細胞を指してよい。がん幹細胞は、典型的には、自己複製能、多分化能、テロメラーゼ活性、抗アポトーシス経路の活性化、及び/又は、細胞膜の担体輸送の活性化が観察できる。
あるいは、当該細胞において、公知のがん幹細胞に高発現しているマーカー遺伝子が、同様に高発現していることを確認して、がん幹細胞であると判定してもよい。そのようなマーカー遺伝子には、限定はされないが、Dclk1(Nakanishi et al., Nature Gen. 2012.)等が挙げられる。また、限定はされないが、具体的には、例えば、CD133+(ヒト脳腫瘍幹細胞)、CD44+CD24-/lowESA+(ヒト乳癌幹細胞、ESA:epithelial specific antigen)、CD44+インテグリンα2β1 hiCD133+、又は、Sca-1+(ヒト前立腺癌幹細胞)、CD133+(ヒト大腸癌幹細胞)、CD44+(ヒト頭頸部扁平上皮癌幹細胞)、CD44+CD24+ESA+(ヒト膵臓癌幹細胞、ESA:epithelial specific antigen)を指標として、がん幹細胞又はがん幹細胞様細胞であるか否かを判別してもよい。
あるいは、胚性幹細胞や体性幹細胞で特異的に発現しているマーカー遺伝子等、未分化マーカーが発現していることを確認して、がん幹細胞であると判定してもよい。未分化マーカーとしては、アルカリフォスファターゼも挙げられ、アルカリフォスファターゼ染色が陽性となることを確認して、細胞が未分化状態であることを判定することもできる。
また、ゲノムワイドな遺伝子の発現パターンをマイクロアレイ等で検出し、公知のがん幹細胞、胚性幹細胞又は体性幹細胞の発現パターンと相関の高いものを、がん幹細胞と判定してもよい。
細胞の表面抗原の発現特性を公知のがん幹細胞、胚性幹細胞又は体性幹細胞のそれと比較し、相関の高いものをがん幹細胞と判定することもできる。
さらに、当該細胞におけるDNAのメチル化を検出して公知のがん幹細胞、胚性幹細胞又は体性幹細胞と比較してもよい。
がん幹細胞またはがん幹細胞様細胞であるか否かの判定は、上記方法の少なくとも一つによって行うことができるが、2つ以上を組み合わせて判定することもできる。
例えば、標的とするがん細胞を低血清もしくは無血清培地で培養した場合に、当該細胞が、胚性幹細胞や体性幹細胞の形態のように、sphere型の形態になった場合には、当該細胞はがん幹細胞又は少なくともがん幹細胞様細胞に変化したと判定してよい。この場合、当該sphere型の細胞は、治療抵抗性を後発的に獲得したことを当業者は理解できる。
そのようなABCG2阻害剤としては、例えば、WO2009/072267やInt J Biochem Mol Biol 2012;3(1):1-27に挙げられたものを用いてよい。ABCG2阻害剤は、限定はされないが、例えば、
topoisomerase II inhibitor (Mitoxantrone、Bisantrene、Etoposide、Becatecarin、NB-506,J-107088など);
Anthracyclines (Daunorubicin、Doxobucincin、Epirubicin、Pirarubicinなど);
Camptothecin analogs(topoisomerase I inhibitor) (Topotecan、SN-38、CPT-11、9-aminocamptothecin、NX211、DX-8951f、Homocamptothecins、BN80915(diflomotecan)、Gimatecan、Belotecanなど);
チロシンキナーゼ阻害剤(後述するものの他、Dasatinib、Vandetanib、Nilotinib、Sorafenib、Tandutinib、CI1033 (Pan-HER TKI)、CP-724,714 (HER2 TKI)、Symadex (fms-like tyrosine kinase 3 inhibitor)など);
Antimetabolites(MTX, MTX diglutamate, MTX triglutamate (antifolate)、GW1843, Tomudex (antifolates)、Trimetrexatte, piritrexim, metoprine, pyrimethamine (lipophilic antifolates)、5-fluorouracil (pyrimidine analog)、CdAMP (nucleotide), cladribine (nucleoside)など);cyclin-dependent kinase inhibitor(Flavopiridolなど);
CDK and aurora kinases inhibitor(JNJ-7706621など);
non-steroidal anti-androgen(Bicalutamideなど);
PEITC(Phenethyl isothiocyanateなど);
indazole-based tubulin inhibitors(TH-337など);
Sufate and glucuronide conjugates of xenobiotics (Estrone 3-sulfate (E1S)、17beta-estradiol sulfate、DHEAS、4[35S]-methylumbelliferone sulfate、E3040 sulfate、Troglitazone sulfate、3-O-sulfate conjugate of 17alpha-ethinylestradiol、SN-38-glucuronide、[3H]17beta-estradiol-17beta-D-glucuronide、[14C]4-methylumbelliferone glucuronide、BP-3-sulfate、BP-3-glucuronide、Phenolic MPA glucuronideなど);
Natural compounds and toxins (Folic acid、Urate、Genistein、Riboflavin (vitamin B2)、plumbagin (Vitamin K3)、Glutathione (GSH)、Sphingosine 1-phosphate、PhIP (carcinogen)など);
その他([(125)I]lodoarylazidoprazosin (IAAP), [(3)H]azidopine; Sulfasalazine (anti-inflammatory); Erythromycin (macrolide antibiotic); Ciprofloxacin、ofloxacin、norfloxacin、enrofloxacin、grepafloxacin、ulifloxacin(fluoroquinolone antibiotics); Nitrofurantoin (urinary tract antibiotic); Moxidectin (parasiticide); Albendazole suloxide and oxfendazole (anthelmintics); Ganciclovir (antiviral drug); Zidovudine、Lamivudine (NRTI); Leflunomide、A771726 (antirheumatic drugs); Diclofenac (analgesic and anti-inflammatory drug); Cimetidine (histamine H2-receptor antagonist); ME3277 (hydrophilic glycoprotein IIb/IIIa antagonist); Pitavastatin、Rosuvastatin (HMG-CoA reductase inhibitor); Dipyridamole (thromboxane synthase inhibitor); Glyburide (hypoglycemic agent); Nicardipine、nifedipine、nitrendipine (Ca2+ channel blocker); Olmesartan medoxomil (angiotensin II AT1-R antagonist); Befloxatone (selective monoamine oxidase inhibitor); Prazosin (alpha-1-adrenergic receptor antagonist); Riluzole (Na+ channels blocker); Amyloid-beta Zoledronic acid (osteotropic compound); Hesperetin conjugates、Kaempferol (flavonoid、WO2004/069233も参照); FTC (Fumitremorgin C)誘導体 (Mol. Cancer Ther.,2002,1:417-425参照); エストロゲンや抗エストロゲン(Mol. Cancer Ther.,2003,2:105-112参照); novobiocin (Int.J.Cancer,2004,108:146-151参照); ジフェニルアクリロニトリル誘導体(WO2004/069243参照); 複素環を有するアクリロニトリル誘導体(WO2006/106778、WO2009/072267参照)等が挙げられる。ABCG2阻害剤としては、化合物単独で抗がん作用があり、かつ、ABCG2を阻害できる分子が好ましい。
本発明者らは、がん幹細胞などの治療抵抗性がん細胞においてPpIXの排出に関与するABCG2をチロシンキナーゼ阻害剤が阻害できること、それにより、ALA-PDTによる治療抵抗性がん細胞の増殖阻害/死滅の効果が増強できることに想到した。
したがって、一実施態様において、抗がん剤としては、ABCG2を阻害できる抗がん剤が好ましく、チロシンキナーゼ阻害剤がさらに好ましい。
チロシンキナーゼ阻害剤としては、限定はされないが、チロシン残基を有する分子に対するチロシンキナーゼ阻害剤であって、前記チロシン残基を有する分子が、幹細胞因子受容体(KIT)、上皮成長因子受容体(EGFR)、神経成長因子受容体(NGFR)、コロニー刺激因子受容体(CSF-1R)、肝細胞増殖因子受容体(HGFR)、繊維芽細胞増殖因子受容体(FGFR)、血管内皮細胞増殖因子受容体(VEGFR)、血小板由来増殖因子受容体(PDGFR)、ヒト上皮成長因子受容体2(HER2/neu)、Srcファミリー(Src、Yes、Fyn、Fgr、Lyn、Lck、Hck、Blk、Frk)、JAK、Fak、ZAP、Btk、Fps/Fes、および、Bcr-Ablからなる群から選択されるものが好ましい。
一態様において、チロシンキナーゼ阻害剤は、以下の式(A):
であってもよい。当該化合物は、Lapatinib(n-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine)、Erlotinib(N-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy)-4-quinazolinamine)、Gefitinib(N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine)であるのが好ましく、Lapatinibがより好ましい。
ALA類のヒトへの投与量は、例えば治療抵抗性がんの治療のための経口投与の場合、ALA類による光増感効果が得られる限り特に限定はされないが、ALA換算で体重1kgあたり、1mg~1,000mg、好ましくは5mg~100mg、より好ましくは10mg~30mg、さらに好ましくは15mg~25mgであってもよい。治療及び投与の態様としては、例えば、WO2013/187069(発明の名称:PDT効果増強剤)を参酌してもよい。また、治療抵抗性がんの予防方法の場合には、治療抵抗性がんの治療方法と比較して、典型的には、より少ないALA類の投与量であることが理解される。ALA類のヒトへの投与量は、例えば治療抵抗性がんの予防のための経口投与の場合、ALA類による光増感効果が得られる限り特に限定はされないが、ALA換算で体重1kgあたり、0.5mg~500mg、好ましくは2.5mg~50mg、より好ましくは5mg~15mg、さらに好ましくは7.5mg~12.5mgであってもよい。局所投与は、全身投与よりも、少ないALA類を要求するであろう。
ALA類の投与頻度としては、一日一回~複数回の投与又は点滴等による連続的投与を例示することができる。
ALA類の投与期間は、例えば、対象の、予防又は治療しようとする症状又は状態等に鑑みて、種々の臨床学的指標等に基づいて、当該技術分野の薬理学者や臨床医が既知の方法により決定することができる。
また、ALA類の投与量、タイミング、投与頻度、投与期間等を考慮しながら、対象の治療抵抗性がんに対して、ALA-PDTを1回又は複数回(例えば、2回、3回、4回、又はそれ以上)実施して、治療抵抗性がんの予防又は治療を行ってもよい。例えば、ALA類を投与後に、特定波長の光線を照射してPDTを施した後で、再度、光線照射によるPDTを実施することにより、初回のPDTでは効果が得られない、腫瘍深部の治療抵抗性がん細胞をも死滅させることが可能となる。また、ALA類を複数回投与する場合にも、各投与の間にPDTを複数回実施してもよい。また、従来の抗がん剤治療等により、大部分のがん細胞を死滅又は除去した後で、残存する(治療抵抗性)がん細胞に対して、又は、(治療抵抗性)がん細胞が残存する恐れがある組織に対して、ALA-PDTを1回又は複数回実施してもよい。
例えば、ALA類を用いて特定波長の光を照射することによりPDTを実施した後で、再度、(同様の)PDTを実施することにより、初回のPDTでは効果が得られない、腫瘍深部の治療抵抗性がん細胞を死滅させることが挙げられる。また、例えば、最初に従来のがん治療(例えば、腫瘍の外科的切除、抗がん剤治療、放射線治療等)を実施し、大部分のがん細胞を死滅させた後に、ALA類を用いたPDTにより、残存したがん細胞を死滅させ、又は、がんの再発や転移を予防することが挙げられる。この場合、最初に施した従来のがん治療を継続させて実施してもよい。
抗がん剤の対象への投与の量、タイミング、投与頻度、投与期間等としては、抗がん剤の性質、種類も考慮しつつ、ALA類と同様に、当業者は適宜決定できるのは言うまでもない。
ALA-PDTと抗がん剤とを組み合わせて、治療抵抗性がんの予防又は治療を行う場合には、例えば、抗がん剤を投与後に、ALA類を投与して、PDTを1回又は複数回実施してもよいし;ALA類を投与後に、抗がん剤を投与して、次いで、PDTを1回又は複数回実施してもよいし;ALA類を投与して、PDTを1回又は複数回実施した後に、抗がん剤を投与してもよい。このように、1回又は複数回のALA類の投与、1回又は複数回の抗がん剤の投与、1回又は複数回のPDTの実施等のあらゆる組み合わせが、本発明の範囲内に含まれることは言うまでもない。
ALA類のヒトへの投与量は、例えば治療抵抗性がんの診断のための経口投与の場合、ALA類により誘導されたPpIXを光線で励起させて蛍光を検出することでがん細胞の存在の有無を識別できる限り特に限定はされないが、ALA換算で体重1kgあたり、1mg~1,000mg、好ましくは5mg~100mg、より好ましくは10mg~30mg、さらに好ましくは15mg~25mgであってもよい。診断及び投与の態様としては、例えば、WO2013/150745(発明の名称:センチネルリンパ節がん転移識別装置)を参酌してもよい。
したがって、本発明は、一態様において、対象における治療抵抗性がん細胞を伴う治療抵抗性がんの診断方法に関してもよい。診断方法は、治療抵抗性がんを患っているか、患っている恐れのある対象に対して、ALA類を投与する前、同時、又は、後において、チロシンキナーゼ阻害剤又はABCG2阻害剤を併用して投与した後に、前記対象においてがんが存在するか、存在すると予想される部位に、光線を照射してPDDを実施し、治療抵抗性がん細胞を含むがん細胞に選択的に蓄積するPpIXから励起した蛍光を検出することで、対象が、治療抵抗性がんを患っているかどうかを決定もしくは診断してもよい。
がん幹細胞を含む治療抵抗性がん細胞では、ABCG2の発現が亢進しているので、ABCG2阻害剤(好ましくはABCG2を阻害できる抗がん剤)をALA類と共に併用することによって、治療抵抗性がん細胞内のPpIX蓄積を促進させ、悪性脳腫瘍をはじめとする多様ながん(肺がん、胃がん、大腸がん、乳がんなど)のALA-PDTの効率を向上させることができるので有利である。
また、外科手術によって切除することが困難な浸潤性がん組織(例えば、多型膠芽腫(Glioblastoma multiforme)、グレードIV)に対しては、外科的切除の後、光ファイバーなどを使って局所的に光線を照射する事により、正常組織に浸潤したがん細胞(治療抵抗性がん細胞を含む)を殺傷することができるので有利である。
また、ALA-PDT又はALA-PDDの数時間前に行なってもよい、ABCG2の発現を阻害できるチロシンキナーゼ阻害剤の単回又は数回投与では、長期間の反復投与に見られる副作用(骨髄抑制、白血球・好中球・血小板減少、皮膚及び肝臓の障害など)は現れにくい。したがって、ALA-PDT又はALA-PDDと組み合わせる、単回又は数回投与のチロシンキナーゼ阻害剤の安全性は高いので有利である。
がん幹細胞の培養
実験方法
正常組織幹細胞の濃縮、分離方法を応用してがん幹細胞研究が進められており、白血病幹細胞の研究においては、がん幹細胞を標的とした治療法の探索・マウスモデルでの治療の段階まで研究が進んでいる。一方、固形がんにおいては、分離、濃縮方法の確立もまだ不十分であるが、近年、悪性脳腫瘍の患者から脳腫瘍幹細胞を単離して培養することが可能になった。これらの方法に基づいて脳腫瘍の患者から単離し樹立された幹細胞株を用いた(Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A. 100(25):15178-5183, 2003.;Nakano I, Kornblum HI. Methods for analysis of brain tumor stem cell and neural stem cell self-renewal. Methods Mol Biol. 568:37-56, 2009.)。
具体的には、脳腫瘍患者から単離して樹立した脳腫瘍glioma幹細胞の株3種類(MD13,MD30,157NS細胞)を用いて、ALA-PDTに対する光感受性を検証する実験を行なった。その実験に備えて、まず、これらの細胞を10%牛胎児血清(FCS)を含むD-MEM/F12培養液と、FCSを含まないD-MEM/F12培養液(L-グルタミン,ヘパリン,B27,EGF,bFGFを含む)でそれぞれ培養して、その形態を検証した。
FCSを含まないD-MEM/F12培養液でglioma幹細胞を培養すると、細胞はいずれも幹細胞に特徴的な3次元的な塊(sphere)を形成した。図1に示すように、細胞の塊(sphere)の直径は数百μmであった。一方、細胞を10%FCSを含むD-MEM/F12培養液で培養すると、sphereを形成せず、細胞は平面的に増殖した。
がん幹細胞のALA-PDTに対する光感受性
実験方法
sphere型およびnon-sphere型に増殖した脳腫瘍幹細胞(MD13,MD30,157NS細胞)をトリプシン処理して、それぞれ1.0x106 cells/mlの密度で0.3mM ALAと共に、4時間インキュベートした後、405nmのレーザー光を照射した。そして、さらに7日間培養した後、細胞の生存率を、水溶性テトラゾリウム塩(Water soluble Tetrazolium salts)を発色試薬として用いた、生細胞数測定法(WST-8 assay)で測定した。
図2は、レーザー光の強度と細胞の生存率との関係をプロットしたものである。FCSを含まないD-MEM/F12培養液(L-グルタミン,ヘパリン,B27,EGF,bFGFを含む)で培養したsphere型の細胞(図2B)は、10%FCSを含むD-MEM/F12培養液で培養したnon-sphere型の細胞(図2A)よりも、ALA-PDTに対して感受性が高かった。表1は、細胞の生存率を50%にする光強度(IC50)をまとめたものである。non-sphere型の細胞に比べて、sphere型の細胞(幹細胞、または、幹細胞様細胞)では感受性が約2倍高まっていたことが判明した。
脳腫瘍細胞のALA-PDTに対する光感受性
実験方法
脳腫瘍細胞株A172細胞(American Type Culture Collectionより購入)を、FCSを含まないD-MEM/F12培養液(L-グルタミン,ヘパリン,B27,EGF,bFGFを含む)で培養すると、細胞は幹細胞に類似したsphereを形成した。一方、細胞を10%FCSを含むD-MEM/F12培養液で培養すると、細胞はsphere型を形成せず、平面的に増殖した。sphere型およびnon-sphere型に増殖した細胞をトリプシン処理した。次いで、それぞれ1.0x106 cells/mlの密度で0.3mM ALAと共に、4時間インキュベートした後、405nmのレーザー光を照射した。そして、さらに7日間培養した後、細胞の生存率を、実施例2と同様に、WST-8 assayで測定した。
また、ALA-PDTを実施したA172細胞から、それぞれ、500細胞を取り出した後、10%FCSと抗生物質とを含むD-MEM寒天培地に播種して、14日間培養した。その後、10%ホルマリンを含むTrypan blueで処理して細胞コロニーを固定・染色した。50細胞以上からなるコロニーの数を、立体顕微鏡を用いてカウントした。
脳腫瘍細胞株A172細胞を、FCSを含まないD-MEM/F12培養液(L-グルタミン,ヘパリン,B27,EGF,bFGFを含む)で培養すると、細胞は幹細胞に類似したsphereを形成した(図3B)。さらに、脳腫瘍幹細胞のマーカーであるCD133とSox-2蛋白質の発現も上昇した(図示せず)。また、脳腫瘍幹細胞MD13,MD30,157NSに対して実施例2で行なったのと同様に、sphere型およびnon-sphere型のA172細胞の、ALA-PDTに対する感受性をそれぞれ調べた結果、non-sphere型と比較して、sphere型は光感受性がより高いことが判明した(図3)。
前述したWST-8 assayでは、ミトコンドリアのNAD(P)Hによる還元反応に基づいて細胞生存率を測定できる。一方、コロニー形成アッセイでは、細胞の増殖能力を測定できる。図4に示すように、コロニー形成アッセイで調べた結果、non-sphere型と比較して、sphere型は光感受性がより顕著に高いことが判明した。すなわち、細胞増殖能力の観点において、sphere型(がん幹細胞)は、光感受性が極めて高いといえる。
脳腫瘍細胞における、ポルフィリン生合成に関与する酵素とトランスポーター
実験方法
脳腫瘍細胞株A172細胞は、FCSを含まないD-MEM/F12培養液(L-グルタミン,ヘパリン,B27,EGF,bFGFを含む)で培養すると、幹細胞に類似したsphereを形成して、且つ、ALA-PDTに対する感受性が高くなった(図3、図4)。その理由を解明するために、ポルフィリン生合成に関与する酵素とトランスポーター遺伝子のmRNAレベルを、定量的PCRで測定した。sphere型およびnon-sphere型のA172細胞から、Magna Pure LC kit(Roche)を用いて、ぞれぞれ、トータルRNAを抽出した。そして、Transcriptor First-strand cDNA Synthesis Kit(Roche Diagnostics)を使って、RNAからcDNAを作製した。定量的PCRは、Light Cycler 3(Roche Diagnostics)を用いて実施した。
non-sphere型の細胞と比較して、がん幹細胞に類似したsphere型の細胞では、ALAの細胞内取り込みを行なうトランスポーターPEPT1及びPEPT2の発現、並びにポルフィリン合成酵素やABCB6遺伝子の発現が亢進していた(図5)。さらに、細胞内PpIXレベルに関しても、non-sphere型の細胞と比較して、sphere型の細胞では2倍以上高くなっていた(図示せず)。
チロシンキナーゼ阻害剤によるABCG2の輸送機能阻害
実験方法
ヒトゲノムDNAの中には48種類のABCトランスポーター遺伝子がコードされている。このうち、ABCG2は、細胞内のPpIXを細胞外へ生理的に輸送する能力を有する。
また、ABCG2は、幹細胞での発現が顕著に高く、フローサイトメトリー法で幹細胞やSP(side population)細胞を単離する際に使うHoechst 33342を、ABCG2が細胞外に能動的に排出していることが報告されている(Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med. 7(9):1028-1034, 2001.)。
これまでに、本発明者らは、ABCG2の輸送機能を阻害する化合物と薬を探索する高速スクリーニング系を開発し、効率的な阻害剤分子を探索した(Saito H, Hirano H, Nakagawa H, Fukami T, Oosumi K, Murakami K, Kimura H, Kouchi T, Konomi M, Tao E, Tsujikawa N, Tarui S, Nagakura M, Osumi M and Ishikawa T. A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions. J. Pharmacol. Exp. Ther. 317(3), 1114-1124, 2006.)。その結果、チロシンキナーゼ阻害剤であるGefitinibがABCG2を効果的に阻害することを実験的に発見した(An R, Hagiya Y, Tamura A, Li S, Saito H, Tokushima D, and Ishikawa T. Cellular phototoxicity evoked through the inhibition of human ABC transporter ABCG2 by cyclin-dependent kinase inhibitors in vitro. Pharm Res. 26(2), 449-458, 2009.)。
本実験においては、Hoechst 33342、及び、複数のチロシンキナーゼ阻害剤を用いて、ABCG2の阻害剤スクリーニング実験を実施した。
具体的には、チロシンキナーゼ阻害剤として、Erlotinib、Gefitinib、Lapatinib、Imatinib、Nilotinib、Dasatinibを用い、また、コントロールとして、L型カルシウムチャネル阻害剤であるVerapamilを用いて、ABCG2の輸送機能阻害スクリーニングを実施した。
ヒトABCG2を発現するFlp-In-293細胞(Flp-In-293/ABCG2細胞)を、ABCG2の基質であるHoechst 33342と前記チロシンキナーゼ阻害剤と共にそれぞれ一定時間培養した。その後、個々の細胞中に蓄積されているHoechst 33342のレベルをFACS(Beckman coulter)で測定した。ABCG2に特異的な阻害であることを裏付けるために、同様の実験をFlp-In-293/Mock(ABCG2を発現していない)細胞でも実施した。具体的な実験手順は以下の通りである。
Flp-In-293/Mock、Flp-In-293/ABCG2をPBS(りん酸緩衝生理食塩水)で2回洗浄後、Trypsin-EDTAにより剥離した。106細胞をDMEM培地(2%FBS)に懸濁した。ついで、5μg/mL Hoechst 33342(Invitrogen)を単独で添加するか、または、5μg/mL Hoechst 33342と1nM~100μMの濃度の各種阻害剤とを併用して添加してから、37℃で90分間培養した。培養後、冷PBS(2%FBS)で洗浄し、再び冷PBS(2%FBS)に懸濁した後、FACS(Beckman coulter)により細胞内蛍光強度を測定した。1回の測定あたり50,000細胞を測定した。細胞内に蓄積したHoechst 33342をUVレーザーにより励起し、その蛍光675nmを検出して、測定した細胞の有する蛍光の平均強度(MFI)を算出した。その結果に基づいて、ABCG2の阻害率を算出した。
Hoechst 33342単独添加時のFlp-In-293/Mock細胞及びFlp-In-293/ABCG2細胞のMFIを、それぞれ100%及び0%とした時の各添加群のMFIを図6に示す。縦軸はチロシンキナーゼ阻害剤によるHoechst 33342の排出阻害率(ABCG2機能の阻害を反映)を示し、横軸はチロシンキナーゼ阻害剤の濃度を示す。この結果から、試験したチロシンキナーゼ阻害剤の中で、Lapatinibが最も強くABCG2を阻害し、ErlotinibとGefitinibは、ほぼ同等のABCG2阻害能力を持つことが判明した。
Lapatinibによる、ABCG2の輸送阻害とALAから生合成されたプロトポルフィリンIX(PpIX)の細胞内蓄積
実験方法
ヒトグリア芽腫・アストロサイトーマU87MG細胞を、2mM ALAと様々な濃度のLapatinibとともに4時間培養して、その間にALAから生合成されて細胞内に蓄積したPpIXの蛍光強度(MFI値)をFACS(Beckman coulter)で測定した。具体的な実験手順は以下の通りである。
U87MG細胞をPBSで2回洗浄後、Trypsin-EDTAにより剥離した。106細胞を低血清のDMEM培地(2%FBS)に懸濁した。ついで、2mM ALAを単独で添加するか、または、2mM ALAと1nM~100μMの濃度のLapatinibもしくはGefitinibとを併用して添加してから、37℃で4時間培養した。培養後、氷冷PBS(2%FBS)で洗浄し、再び冷PBS(2%FBS)に懸濁した後、FACS(Beckman coulter)により細胞内蛍光強度を測定した。1回の測定あたり50,000細胞を測定した。細胞内に蓄積したPpIXをUVレーザー(325nm)により励起し、その蛍光575nMを検出し、測定した細胞の有する蛍光の平均強度(MFI)を算出した。その結果に基づいて、PpIXの細胞内蓄積を相対的に算出し、LapatinibまたはGefitinibによるABCG2のPpIX輸送阻害を推算した。
ヒトグリア芽腫・アストロサイトーマU87MG細胞は、ALAと培養する事によりPpIXを生合成する。2mM ALAと様々な濃度のLapatinibとともに4時間培養すると、細胞内に蓄積したPpIXの蛍光強度が増加した。PpIX蛍光強度の増加はLapatinibの濃度に依存し、1~100μMの濃度で顕著に増加した。10nM以上の濃度領域でLapatinibがABCG2を有意に阻害し、ALAから生合成されたPpIXが細胞内に蓄積したことが判明した。一方、GefinitibでABCG2を阻害してもPpIXの細胞内蓄積は観察されたが、Lapatinibほどは蓄積されなかった(図7)。その理由として、GefitinibはLapatinibと比較してABCG2阻害活性が弱いことも反映したとも考えられる(図6も参照)他、培養条件の相違等も考えられる。
LapatinibによるABCG2の輸送阻害とALA-PDTの効果:培養細胞を用いた実験
実験方法
ヒトグリア芽腫・アストロサイトーマU87MG細胞(106細胞)をDMEM(2%FBS)に懸濁後、10mM ALAを単独で添加するか、または、10mM ALAと1nM~300μMの濃度のLapatinibとを併用して添加してから、37℃で4時間培養した。635nmにピークを持つLEDで細胞を照射(12J/cm2)して、その後の細胞生存率をMTT法で測定した。なお、MTT法は、WST-8 assayと測定原理は同じであり、MTTをホルマザン色素(紫色)へ還元する酵素活性を測定する比色定量法である。
ヒトグリア芽腫・アストロサイトーマU87MG細胞は、ALAを単独で添加して培養した後、LEDで細胞を照射(12J/cm2)する事により、約50%の細胞が死滅した(図8において、Lapatinibの濃度が0の場合に相当)。
一方、10mM ALAと共に様々な濃度のLapatinibでU87MG細胞を培養する事によって、細胞のLED照射(12J/cm2)に対する感受性が濃度依存的に増加した(細胞が死滅しやすくなった)。この事は、LapatinibがABCG2を阻害する事により、ALAから生合成されたPpIXが細胞内に蓄積して、細胞の光照射に対する感受性が増加したことを示す(図8)。
LapatinibによるABCG2の輸送阻害とALA-PDTの効果:ヌードマウスを用いた実験
実験方法
ヒトグリア芽腫・アストロサイトーマU87MG細胞を、10%FCSを含むD-MEM培地(Sigma-Aldrich)で培養したあと、Trypan blueを用いて生存細胞の数を計測した。次いで、U87MG細胞(5x106 cells/0.1ml/mouse)をBALB/c-nu/nuヌードマウスの背部の皮下に注射して移植した。3日後、ヌードマウスに、ALA単独(30mg/kg body weight,p.o.)またはALA(30mg/kg body weight,p.o.)とLapatinib(100mg/kg body weight,p.o.)との併用で経口投与した。次いで、3時間後に635nmに発光ピークを有するLEDを腫瘍移植部分に照射した。照射後一週間後に腫瘍を摘出し、その重さをそれぞれ測った。
ALA(30mg/kg body weight,p.o.)とLapatinib(100mg/kg body weight,p.o.)とを併用で経口投与してALA-PDTを実施したマウスでは、コントロールのマウスおよびALA(30 mg/kg body weight, p.o.)単独投与のマウスと比較して、顕著に腫瘍の成長が抑制された(図9、図10)。
PDTの複数回実施の効果
実験方法
HeLa細胞(ヒト由来子宮頸部がん細胞株)1x105cellsを60mm dishに播種し24時間培養後、培養液に抗がん剤を投与した。投与した抗がん剤の種類、および、培養液中の抗がん剤の濃度を表2に示した。抗がん剤投与72時間後にトリプシン-EDTA処理により細胞を回収し、回収した細胞の一部を用いて、トリパンブルー染色法によって生存細胞数を確認した。生存していた細胞を、96 well plateに5x103cellsを播種した。本実施例においては、抗がん剤投与72時間後に生き残っていたこれらの細胞を、治療抵抗性がんとして用いた。
抗がん剤未投与、治療抵抗性がんにおけるALA-PDT 24時間後の細胞生存率の変化を図11~15に示す。図11a,図12a,図13a,図14a,図15aの横軸は、投与したALAの濃度を示す。図11b,図12b,図13b,図14b,図15bは、それぞれ図11a,図12a,図13a,図14a,図15aのグラフにおいて、ALAを250μM投与した時の結果を抜粋したものである。それぞれの図の縦軸は、ALAを投与しなかったこと以外は同じ条件で実験を行った場合の細胞生存率を100%とした場合の、実験群における細胞生存率の割合を示す。
Claims (16)
- 請求項1に記載の予防用又は治療用組成物であって、
前記治療抵抗性がん細胞が、がん幹細胞を含むことを特徴とする、
予防用又は治療用組成物。 - 請求項1または2に記載の予防用又は治療用組成物であって、
前記がん幹細胞が、脳腫瘍幹細胞であることを特徴とする、
予防用又は治療用組成物。 - 請求項1~3のいずれか1項に記載の予防用又は治療用組成物であって、
前記光線力学的療法が、対象における治療抵抗性がん細胞に対して2回以上実施されることを特徴とする、
予防用又は治療用組成物。 - 同時または順次に投与される、(1)請求項1~4のいずれか1項に記載の予防用又は治療用組成物と(2)抗がん剤とを組み合わせてなることを特徴とする、治療抵抗性がんの予防用又は治療用医薬。
- 請求項5に記載の予防用又は治療用医薬であって、
前記抗がん剤が、チロシンキナーゼ阻害剤であることを特徴とする、
予防用又は治療用医薬。 - 請求項6に記載の予防用又は治療用医薬であって、
前記チロシンキナーゼ阻害剤が、チロシン残基を有する分子に対するチロシンキナーゼ阻害剤であり、
前記チロシン残基を有する分子が、幹細胞因子受容体(KIT)、上皮成長因子受容体(EGFR)、神経成長因子受容体(NGFR)、コロニー刺激因子受容体(CSF-1R)、肝細胞増殖因子受容体(HGFR)、繊維芽細胞増殖因子受容体(FGFR)、血管内皮細胞増殖因子受容体(VEGFR)、血小板由来増殖因子受容体(PDGFR)、ヒト上皮成長因子受容体2(HER2/neu)、Srcファミリー、JAK、Fak、ZAP、Btk、Fps/Fes、および、Bcr-Ablからなる群から選択されることを特徴とする、
予防用又は治療用医薬。 - 請求項6又は7に記載の予防用又は治療用医薬であって、
前記チロシンキナーゼ阻害剤が、ABCG2阻害剤であることを特徴とする、
予防用又は治療用医薬。 - 請求項5~8のいずれか1項に記載の予防用又は治療用医薬であって、
前記組み合わせの態様が、配合剤であるか、または、キットであることを特徴とする、
予防用又は治療用医薬。 - 抗がん剤と同時または順次に併用される、請求項1~4のいずれか1項に記載の予防用又は治療用組成物。
- 治療抵抗性がんの治療のための、(1)請求項1~4のいずれか1項に記載の予防用又は治療用組成物と(2)抗がん剤との組み合わせであって、
前記(1)治療用組成物と前記(2)抗がん剤が、同時または順次に投与されることを特徴とする、
組み合わせ。 - 請求項12に記載の予防又は治療方法であって、
前記ステップ(II)は、前記治療抵抗性がん細胞に対して、前記光線力学的療法を2回以上実施することを特徴とする、
予防又は治療方法。 - 請求項12または13に記載の予防又は治療方法であって、
前記ステップ(I)または(II)の実施の前、間、または、後において、前記対象に対して、抗がん剤を、同時または順次に、さらに投与するステップ
を含む、
予防又は治療方法。
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WO2020170330A1 (ja) | 2019-02-19 | 2020-08-27 | 大塚電子株式会社 | 光線力学的療法条件パラメータの決定方法および光線力学的療法装置 |
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JP2017160171A (ja) * | 2016-03-11 | 2017-09-14 | Sbiファーマ株式会社 | がん細胞におけるPpIX蓄積増強剤 |
WO2020170330A1 (ja) | 2019-02-19 | 2020-08-27 | 大塚電子株式会社 | 光線力学的療法条件パラメータの決定方法および光線力学的療法装置 |
JPWO2020170330A1 (ja) * | 2019-02-19 | 2021-12-16 | 大塚電子株式会社 | 光線力学的療法条件パラメータの決定方法および光線力学的療法装置 |
JP7288951B2 (ja) | 2019-02-19 | 2023-06-08 | 大塚電子株式会社 | 光線力学的療法条件パラメータの決定方法および光線力学的療法装置 |
WO2020217472A1 (ja) * | 2019-04-26 | 2020-10-29 | 株式会社Jimro | 治療抵抗性がんの予防又は治療用の医薬組成物 |
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WO2021192113A1 (ja) * | 2020-03-25 | 2021-09-30 | 大塚メディカルデバイス株式会社 | がんの処置方法およびそのためのシステム |
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