WO2014054601A1 - クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 - Google Patents
クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 Download PDFInfo
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- WO2014054601A1 WO2014054601A1 PCT/JP2013/076629 JP2013076629W WO2014054601A1 WO 2014054601 A1 WO2014054601 A1 WO 2014054601A1 JP 2013076629 W JP2013076629 W JP 2013076629W WO 2014054601 A1 WO2014054601 A1 WO 2014054601A1
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- glucose
- difluoro
- derivative
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- cells
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- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/10—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to heterocyclic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/02—Monosaccharides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Definitions
- the present invention relates to a novel fluorescently labeled sugar derivative to which a specific coumarin derivative is bound, and a cell imaging method and an imaging agent using the same.
- the present invention also relates to a method for detecting and / or imaging cancer cells using an L-glucose derivative (L-glucose derivative to which a specific coumarin derivative is bound) among the fluorescently labeled derivatives, and an imaging agent therefor.
- Visualization imaging of living cells, or visualization imaging targeting molecules in living bodies reveals molecular dynamics, intermolecular interactions, and molecular position information, elucidation of life science mechanisms and drug discovery screening Molecular imaging is being actively conducted to connect to. In particular, research for visualizing abnormal cells such as cancer cells and detecting cancer cells and cancer sites has been actively conducted.
- glucose is known as the most important energy source for maintaining the survival of cells from mammals to Escherichia coli and yeast.
- the brain uses glucose as the only energy source.
- Glucose has enantiomers of D-glucose and L-glucose. Among them, only D-glucose can be used as an energy source by living organisms, and living cells can use D-glucose as a glucose transporter. It has a mechanism for selectively taking in and using transport proteins in cell membranes.
- hexasaccharides that have a large amount of D-form in nature and have no or almost no L-isomer as its optical isomer
- D-galactose is a sugar that is used as an energy source. It is abundant in milk, fruits and vegetables, and about 2 g per day is produced in the human body.
- disaccharide lactose which accounts for 2-8% of milk, is a glycosidic bond between D-galactose and D-glucose, and is separated by lactase when absorbed in the small intestine, and is a kind of glucose transporter.
- L-galactose is an intermediate metabolism of the Smirnoff-Wheeler pathway, one of the pathways when the antioxidant vitamin C (L-ascorbic acid), which cannot be biosynthesized by primates, is biosynthesized from D-glucose in plants.
- D-fructose also called fructose
- fructose is contained in large amounts in berries, melons and other fruits, and certain root vegetables, and is also produced in the body. Ingested D-fructose is taken into the epithelial cells via the glucose transporter GLUT5 in the small intestinal epithelium and then enters the blood mainly via GLUT2.
- fructose that enters liver cells is phosphorylated by fructokinase and used for fatty acid synthesis and energy production, as well as being converted to D-glucose.
- GLUT5 is also expressed in smooth muscle, kidney, adipocyte, brain and testis, so it is considered that GLUT5 has an important function in each of these areas, for example, as an energy source during sperm activity.
- Corn syrup which is widely distributed as a food sweetener, is low in cost, and particularly high in sweetness at low temperatures and with a high sweetness content, is used in large quantities in soft drinks. Overdose is considered dangerous as an adverse effect on brain nerve activity, obesity and cancer.
- L-fructose can be used as energy to some extent when eaten, but there is also a presumption that it is converted by intestinal bacteria.
- D-mannose is contained in fruits and pericarps.
- a polysaccharide composed mainly of mannose is called mannan, which is possessed by plants, yeasts, and bacteria.
- Konjac is mainly composed of glucomannan consisting of mannose and glucose.
- D-mannose is usually almost excreted in urine when ingested orally, but there are many unclear points about the uptake mode in humans.
- D-mannose Once inside the cell, it is phosphorylated and then converted to fructose-6-phosphate, a glycolytic intermediate.
- Mannose receptors to which D-mannose specifically binds help to remove high mannose glycoproteins that increase during inflammation. For example, there is a high mannose sugar chain on the surface of P.
- a method using -yl) amino] -2-deoxy-D-glucose (2-NBDG) was proposed, and its usefulness was demonstrated using various mammalian cells (Non-patent Document 6).
- This method utilizes the property that 2-NBDG is selectively taken up into living cells. By tracking changes in fluorescence intensity due to uptake, the dynamic activity of D-glucose uptake by cells can be monitored. Since it can be quantitatively known, it has been evaluated by researchers all over the world as an innovative method for studying how organisms take D-glucose into cells and uses it. It is positioned as a standard protocol indispensable in this research field (Non-patent Document 7). In order to evaluate the specific uptake of D-glucose, the group of the present inventors further added N- (7-nitrobenz) as a fluorescent chromophore at the 2-position of L-deoxyglucose, an enantiomer of D-deoxyglucose.
- FDG 18- F-2-fluoro-2-deoxy-D-glucose
- a technique for noninvasive imaging diagnosis of cancer from outside the body has been put into practical use by detecting gamma rays emitted by the decay of 18 F in FDG accumulated in cells with a PET (positron tomography) device. .
- This PET test using a radiolabeled D-glucose derivative does not have a spatial resolution that can identify individual cells (the lower limit of the spatial resolution is practically about 5 mm in the PET test), so it may grow rapidly. There is a problem in that fine guns cannot be detected.
- FDG has problems such as short half-life (110 minutes) and large equipment.
- radiolabeled FDG which is a D-glucose derivative, has a major problem of how to avoid the fundamental problem of being taken up not only by tumor cells but also by normal tissues and normal cells.
- adipose tissue and muscle distributed throughout the body, the small intestine epithelium, the liver, and the like take up D-glucose extremely strongly, which is a problem in distinguishing from a tumor.
- other hexoses have been tried to be applied to cancer detection and imaging using the radioactive label.
- D-glucose there are problems that its use is limited because it is D-form, and that differences in individual single cells cannot be detected accurately in real time.
- Non-Patent Document 12 Non-Patent Document 13, Non-Patent Document 14, etc.
- GLUT glucose transporter
- Non-patent Document 15 Cancer cells with active metabolic activity produce a large amount of acid in the form of CO 2 and protons (H + ) due to metabolism. Such so-called waste acid is removed or neutralized in normal cells with the help of the circulatory system such as blood flow to prevent the cells from becoming acidic.
- tissues that are constructed in response to the metabolic activity of normal cells cannot cope with cancer cells that continue to grow unexpectedly.
- removal or neutralization of acid tends to be insufficient in cancer tissues that are distant from blood vessels, and cancer cells are developing various molecular mechanisms to prevent the cells from becoming acidic.
- Non-patent Document 15 a carbonic anhydrase group that is excessively expressed in the cell membrane of cancer cells has attracted attention.
- excess CO 2 which is an acidic waste inevitably generated in cells due to the metabolic activities of cells in the body, is discharged by various biological mechanisms to prevent intracellular acidification.
- the key to these processes is acid removal by the bloodstream.
- cancer cells that are within solid tumors that are a few tens of microns or more away from blood vessels, or that are abnormally proliferating at a position facing the lumen of the digestive tract, and that are away from blood vessels In the case of cells, the supply of oxygen and glucose is insufficient, and at the same time, removal of acids as metabolites tends to be insufficient.
- Non-patent Document 15 transmembrane carbonic anhydrase (CA 9 and CA 12) is overexpressed in the plasma membrane. It has been reported that some of them help to remove CO 2 and neutralize acid generated in cells (Non-patent Document 15).
- Supuran et al. By binding a derivative of coumarin, a fluorescent low-molecular compound, to carbonic anhydrase (eg, CA 9 is assumed) that is strongly expressed in the plasma membrane of some cancer cells in hypoxia They have found that these enzymes inhibit carbonic acid dehydration (Non-patent Document 16, Patent Document 2). These coumarin derivatives are expected as one of the new generation anticancer drug candidates because they damage cancer cells by breaking the pH balance of cancer cells in a hypoxic state (Non-patent Document 21). .
- carbonic anhydrase is an essential enzyme for the survival of all cells, and in mammals, 16 types of isozymes are present not only in the cell membrane surface but also in the cytoplasm and mitochondria. For this reason, it is required that the above-described fluorescent low molecular weight compound inhibit other types of carbonic anhydrase groups in normal cells so as not to cause side effects.
- One effective strategy is to prevent low molecular weight compounds such as coumarin derivatives from selectively entering CA9, which has a reactive site outside the cell membrane of cancer cells, and entering the cells. For this purpose, proposals have been made to introduce a charge into a compound or to give a glycoside a hydrophilic property so that the molecule does not pass through a cell membrane composed of a lipid bilayer membrane.
- Non-patent document 17 Supuran et al. are soluble in molecules by binding various coumarins or derivatives thereof to the first position of natural sugars such as D-glucose, D-mannose, D-galactose, and L-rhamnose.
- To provide cell membrane impermeability Patent Document 2.
- the first place is susceptible to hydrolysis, and if natural type is used, the influence on normal cells is inevitable.
- a fluorescent molecular marker in which a fluorescent molecule is bound to a molecule other than glucose has been actively developed.
- Examples include those using RGD sequences and those using EGF (Non-patent Document 18).
- fluorescent molecules are basically taken up by normal cells even though there are many uptakes. Hold it.
- a molecular marker that targets a specific tumor cell using a specific antibody or the like is difficult to determine other types of tumors, and is difficult to use.
- the present inventors have found that living cells can be imaged using a sugar derivative having a fluorescent molecule group consisting of a specific coumarin skeleton in the molecule, and the present invention is completed.
- the present inventors have also found that an L-glucose derivative bound with a specific coumarin derivative can image cancer cells, and completed the present invention.
- the present invention is as follows. 1. A fluorescently labeled sugar derivative having 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin as a fluorescent molecular group in the molecule; Target cell or target intracellular molecule (target intracellular molecule means a molecule that exists inside the target cell, that is, in the cytoplasm or nucleus, a molecule that exists in the cell membrane of the target cell, or a molecule that exists on the cell membrane of the target cell. A composition for imaging). 2. 2. 2. 2.
- composition according to 1 above wherein the fluorescently labeled sugar derivative is a glucose derivative, a fructose derivative, a galactose derivative or a mannose derivative. 3. 3. The composition according to 2 above, wherein the fluorescent molecular group is bonded to glucose, fructose, galactose or mannose via an —NH— bond. 4).
- the fluorescently labeled sugar derivative is located at the 1st, 2nd, 3rd, 4th or 6th position of glucose (preferably the 2nd, 3rd, 4th or 6th position, more preferably the 2nd, 4th or 6th position).
- 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin as a fluorescent molecular group is bonded via an —NH— bond.
- the fluorescently labeled sugar derivative is 2-deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -D-glucose, 2-deoxy-2- (2- (6,8- Difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -D-glucose, 2-deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -L- 4 above, which is a molecule selected from the group consisting of glucose and 2-deoxy-2- (2- (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -L-glucose
- the fluorescently labeled sugar derivative is in the 1st, 2nd, 3rd, 4th or 6th position of mannose (preferably the 2nd, 3rd, 4th or 6th position, more preferably the 2nd, 4th or 6th position).
- 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin was bound as a fluorescent molecular group through an —NH— bond. 2.
- the fluorescently labeled sugar derivative is 2-deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -D-mannose, 2-deoxy-2-((6,8-difluoro- 7-hydroxycoumarin-3-yl) carboxamide) -L-mannose, 2-deoxy-2- (2- (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -D-
- the above 6 which is a molecule selected from the group consisting of mannose and 2-deoxy-2- (2- (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -L-mannose
- Target cell or target intracellular molecule means a molecule that exists inside the target cell, that is, in the cytoplasm or nucleus, a molecule that exists in the cell membrane of the target cell, or a molecule that exists on the cell membrane of the target cell.
- a. Contacting the target cell (the target cell includes not only the cell itself but also a cell present in the tissue) with the composition according to any one of the above 1 to 7, and b. Detecting the sugar derivative present in the target cell (including inside the target cell, ie, in the cytoplasm or nucleus, in the cell membrane of the target cell, and on the cell membrane of the target cell);
- An imaging method comprising: 9.
- 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy- as a fluorescent molecular group is added to a sugar selected from the group consisting of glucose, fructose, galactose and mannose.
- a method for detecting cancer or cancer cells comprising the following steps: a.
- a target cell (a target cell includes not only a cell itself but also a cell present in a tissue) is added with 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8 as a fluorescent molecular group.
- the fluorescently labeled L-glucose derivative is in the 1-position, 2-position, 3-position, 4-position or 6-position of L-glucose (preferably the 2-position, 3-position, 4-position or 6-position, more preferably 2-position, 4-position, Or 6-position), as a fluorescent molecular group, 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin is —NH-bonded. 13.
- the detection method according to 12 above which is a molecule bound via 14
- the fluorescently labeled L-glucose derivative is 2-deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -L-glucose, or 2-deoxy-2- (2- ( 13.
- 15. The detection method according to any one of 12 to 14 above, wherein the detection in the step a is performed by imaging a target cell. 16.
- the composition in the step a further comprises 2-amino-2-deoxy-L-glucose in which sulforhodamine (preferably sulfodamine 101, sulfodamine B) is sulfonamide bonded at the 2-position, and the step b includes 16.
- sulforhodamine preferably sulfodamine 101, sulfodamine B
- the step b includes 16.
- the detection method according to any one of 12 to 15 which is a step of detecting a fluorescently labeled L-glucose derivative (either or both) present in a target cell.
- the detection method according to any one of 12 to 16 wherein the target cell is a cell in a tumor cell mass.
- target cancer cells include cancer cells existing in the tissue as well as the cells themselves) (for example, in the target cancer cell (inside the target cell, that is, in the cytoplasm or nucleus, the target cell)
- An imaging agent for imaging a cancer cell by incorporating a fluorescently labeled L-glucose derivative into a cell membrane of a target cell), and 3-carboxy-6 An imaging agent for cancer cells comprising a fluorescently labeled L-glucose derivative bound with 8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin.
- the fluorescently labeled L-glucose derivative is in the 1-position, 2-position, 3-position, 4-position or 6-position of L-glucose (preferably the 2-position, 3-position, 4-position or 6-position, more preferably 2-position, 4-position, Or 6-position), as a fluorescent molecular group, 3-carboxy-6,8-difluoro-7-hydroxycoumarin or 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin is —NH-bonded. 19.
- the fluorescently labeled L-glucose derivative is 2-deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -L-glucose, or 2-deoxy-2- (2- ( 19.
- the present invention it is possible to provide a blue imaging agent capable of distinguishing cells or intracellular molecules with high contrast.
- the present invention can also provide a method capable of identifying cancer cells with high contrast and an imaging agent therefor.
- the results of administration of a mixture of a D-glucose derivative (2-PBDG: 100 ⁇ M) emitting blue fluorescence and an L-glucose derivative (2-TRLG: 20 ⁇ M) emitting red fluorescence to normal neurons are shown.
- the results of administering a mixture of an L-glucose derivative (2-PBLG: 100 ⁇ M) emitting blue fluorescence and an L-glucose derivative (2-TRLG: 20 ⁇ M) emitting red fluorescence to normal neurons are shown.
- cancer cell mass (spheroids, MIN6 cells on the 15th day of culture) that showed three-dimensional growth in culture, cells that are apoptotic, necrotic, and DAPI are strongly stained It is the microscope picture which showed the spatial arrangement
- a mixed solution consisting of 100 ⁇ M 2-PBLG, 100 ⁇ M 2-NBDLG and 20 ⁇ M 2-TRLG was obtained by a real-time laser scanning confocal microscope during administration to the tumor cell mass composed of mouse insulinoma cells (MIN6) in Example 7. It is an image. It is the acquired image after 2 minutes from the end of administration.
- FIG. 9 is an image obtained by using a real-time laser scanning confocal microscope before administration of a mixed solution of 100 ⁇ M 2-PBLG and 20 ⁇ M 2-TRLG on the tumor cell mass composed of mouse insulinoma cells (MIN6) in Example 8.
- FIG. It is the acquired image after 2 minutes from the end of administration. It is the acquired image after 8 minutes from the end of administration. It is the acquired image 12 minutes after the end of administration.
- One embodiment of the present invention includes an imaging agent for imaging a cell or an intracellular molecule using a sugar derivative to which a specific coumarin derivative (Pacific Blue or Marina Blue) is bound, and a cell or cell using the imaging agent.
- This is an imaging method for inner molecules.
- One embodiment of the present invention is a fluorescently labeled sugar derivative to which a specific coumarin derivative (Pacific Blue or Marina Blue) that can be used for the imaging agent is bound.
- Another aspect of the present invention relates to an imaging agent for detecting cancer cells using a fluorescently labeled L-glucose derivative obtained by binding a specific coumarin derivative (Pacific Blue or Marina Blue) to L-glucose, and the imaging agent. This is a method for detecting cancer cells.
- Another embodiment of the present invention is a fluorescently labeled L-glucose derivative to which a coumarin derivative (Pacific Blue or Marina Blue) that can be used in the imaging agent is bound.
- 3-carboxy-6,8-difluoro-7-hydroxycoumarin Pacific Blue
- 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin Marina
- the composition of the present invention a composition containing a fluorescent-labeled sugar derivative having blue
- the imaging agent of the present invention a composition containing a fluorescent-labeled sugar derivative having blue
- target cells or target intracellular molecules are molecules inside the target cell, ie, in the cytoplasm or nucleus, molecules present in the cell membrane of the target cell, Including molecules present on the cell membrane.
- it is also possible to image cells in the tissue or molecules in the cells at the level of individual cells by contacting the tissue containing the target cells with the composition of the present invention and performing imaging.
- the sugar in the fluorescently labeled sugar derivative of the present invention may be any sugar as long as it is taken into cells in living cells (normal cells or abnormal cells), preferably glucose, fructose, galactose or mannose. is there.
- D type and L type sugars and any of them can be used in the present invention.
- microorganisms having properties different from that of mammalian cells in the recognition, transport, and metabolism of sugars related to the D-type and L-type configurations are also measured at the cellular level using D-type or L-type fluorescently labeled sugar derivatives. It is possible to elucidate the function by imaging with.
- composition of the present invention is contacted with a target cell.
- the composition of the present invention or “the imaging agent of the present invention”
- the imaging agent of the present invention is contacted with a target cell.
- cancer cells in a tissue can also be detected by performing imaging by bringing the composition of the present invention into contact with a tissue containing target cells.
- cancer cells or tissues containing these cells can be detected by administering the composition of the present invention to a living body and performing imaging, and this method is a method for detecting cancer. Useful.
- the composition of the present invention includes any form of composition that can be applied to cells containing the fluorescently labeled sugar derivative of the present invention, and is particularly limited if it can be applied to a solution, gel, or other cells. There is no. Moreover, the component in a composition can be contained without a restriction
- the fluorescently labeled sugar derivative of the present invention can be dissolved in a buffer solution or a cell culture medium and applied to cells.
- the fluorescently labeled sugar derivative of the present invention that emits blue fluorescence that can be used for imaging of a cell or intracellular molecule includes sugar, Preferably, glucose, fructose, galactose or mannose is mixed with 3-carboxy-6,8-difluoro-7-hydroxycoumarin (pacific blue) or 3-carboxymethyl-6,8-difluoro-7-hydroxy- as a fluorescent molecular group. It is a fluorescently labeled sugar derivative to which 4-methylcoumarin (Marina Blue) is bound.
- the binding site of the fluorescent molecular group in the sugar derivative is not particularly limited as long as it can be synthesized by the method described in this specification or a conventional method, but in the case of glucose, the 1-position, 2-position, 3-position, 4-position or 6-position (preferably 2nd, 3rd, 4th or 6th, more preferably 2nd, 4th or 6th), in the case of fructose, 1st, 3rd, 4th, 5th or 6th (preferably 1st, 5th or 6th, more preferably 1st), in the case of galactose, 1st, 2nd, 3rd, 4th or 6th (preferably 2nd, 3rd, 4th or 6th, More preferably 2nd, 3rd or 6th), in the case of mannose, 1st, 2nd, 3rd, 4th or 6th (preferably 2nd, 3rd, 4th or 6th, more preferably 2nd, 4th or 6th).
- the binding of the fluorescent molecular group to the sugar will be
- the binding position of the fluorescent molecular group to the sugar is not particularly limited, and can be bound to any position according to a conventional method.
- the fluorescent molecular group can be bound at any of the 1-position, 2-position, 3-position, 4-position or 6-position of glucose, but preferably the 2-position, 3-position, Rank, 4th or 6th place.
- the coupling can be performed, for example, via —NH— using glucosamine at the 2-position.
- glucosamine D-glucosamine or L-glucosamine can be used.
- D-glucosamine synthesized D-glucosamine or commercially available D-glucosamine can be used.
- L-glucosamine can be synthesized by the method described in WO2010 / 16587 or the method described in the application specification of PCT / JP2012 / 58439 (the descriptions in these publications or application specifications are cited by reference). Part of this description).
- the method described in the specification of PCT / JP2012 / 58439 application is as follows.
- the fluorescently labeled glucose derivative obtained by binding Pacific Blue (PB) to glucose of the present invention is preferably represented by the following formula (1) or (2).
- Formula (1) D-glucosamine bound to Pacific Blue (PB): referred to as 2-PBDG
- Formula (2) L-glucosamine bound to Pacific Blue (PB): 2-PBLG Is called an enantiomer relationship
- the maximum excitation wavelength (Ex max) and the maximum fluorescence wavelength (Em max) are both 403 nm (Ex max) and 453 nm (Em max).
- the glucose derivative emitting blue fluorescence of the present invention can be used by dissolving in an arbitrary solution, for example, a solvent such as DMSO, and is stable in a solvent or solution used for imaging cells or intracellular molecules. Suitable as an imaging agent.
- the target cells to be imaged using the sugar derivative that emits blue fluorescence according to the present invention are not particularly limited, and are cells derived from mammals, microorganisms such as Escherichia coli and yeast. Cells, plant cells, fertilized eggs, etc., and cells isolated from living organisms, cells existing in tissues isolated from living organisms, cells existing in tissues of living organisms, from living organisms Any form of cells such as primary cultured cells after isolation or established cells may be used. Furthermore, the target cell may be a normal cell or an abnormal cell (for example, a cancer cell).
- the fluorescently labeled sugar derivative of the present invention incorporated into the cells can be detected by a commonly used method for detecting fluorescence.
- a commonly used method for detecting fluorescence For example, it can be performed as follows.
- detection of the fluorescently labeled sugar derivative present in the cell is performed by measuring the fluorescence of the target cell in advance, then contacting the target cell with the fluorescently labeled sugar derivative for a certain period of time, and then washing it off again.
- the fluorescence of the target cell is measured, and the evaluation can be performed with an increase in the fluorescence intensity with respect to the fluorescence intensity of the target cell before contact.
- the cells may be imaged using an appropriate apparatus capable of discriminating the inside, the cell membrane, and the outside of the cell such as a confocal microscope while contacting the fluorescently labeled sugar derivative.
- an appropriate apparatus capable of discriminating the inside, the cell membrane, and the outside of the cell
- a confocal microscope capable of discriminating the inside, the cell membrane, and the outside of the cell
- evaluation may be performed using the sum of the fluorescence intensities exhibited by a large number of cells or the distribution of the fluorescence intensities using a fluorescence plate reader, flow cytometry, or the like.
- the fluorescently labeled sugar derivative of the present invention By using the fluorescently labeled sugar derivative of the present invention, it becomes possible to detect and / or image cells and / or intracellular molecules in blue.
- the fluorescently labeled sugar derivative of the present invention can be used simultaneously with other sugar derivatives having a fluorescent chromophore, such as 2-NBDG and 2-NBDLG that emit green fluorescence, and / or 2-TRLG that emits red fluorescence. it can.
- 2-NBDG, 2-NBDLG, and 2-TRLG are described in WO2010 / 16587 (part of which is incorporated herein by reference). Thereby, evaluation with two colors or three colors becomes possible.
- L-glucose derivative (II-1)
- the L-glucose derivative of the present invention that emits blue fluorescence which can be used for detection or imaging of cancer cells, has 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) or 3- A molecule in which carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin (marina blue) is bound to L-glucose.
- the fluorescent molecular group can be bound at any of the 1-position, 2-position, 3-position, 4-position or 6-position of glucose, preferably 2-position, 3-position, It is 4-position or 6-position, more preferably 2-position, 4-position or 6-position.
- the coupling can be performed, for example, via —NH— using glucosamine at the 2-position.
- the fluorescently labeled L-glucose derivative of the present invention is preferably represented by the following formula (2).
- the fluorescently labeled L-glucose derivative of the present invention is a compound in which a specific coumarin derivative (Pacific Blue or Marina Blue) is used as a key molecule and L-glucose having the property of not being taken up by normal cells is bound thereto.
- Coumarin and its derivatives bind to carbonic anhydrase that is overexpressed in cancer cells that are present in hypoxic and hypotrophic environments and inhibit its function, so when administered to a cell population containing cancer cells,
- the above-mentioned special cancer cells can be selectively visualized with fluorescence, their functions can be inhibited, and the influence on normal cells can be minimized.
- Examples of cells targeted by the method of the present invention include, for example, solid cancer, or in a cancer cell population that shows significant proliferation two-dimensionally or three-dimensionally in the lumen of the digestive tract, etc.
- a cancer cell Non-patent Document 20
- the form of the target cell is not particularly limited, a cell isolated from a living body, a cell present in a tissue isolated from a living body, a cell present in a tissue of a living body, a primary cultured cell after being isolated from a living body, or Any cell form such as established cells may be used.
- Cells that are strongly positive for the fluorescently labeled L-glucose derivative (eg, 2-PBLG) of the present invention are considered to be cancer cells that have acquired excellent traits in their ability to cope with a hypoxic environment. Is a cell that has acquired one of the abilities to survive even in an environment different from the environment in which the cancer cells originally existed at the metastasis destination, and the fluorescently labeled L-glucose of the present invention Derivatives can be used to selectively identify and visualize such cells.
- the fluorescently labeled L-glucose derivative of the present invention (L-glucose derivative having passhook blue or marina blue in the molecule) is used as another fluorescently labeled L-glucose derivative.
- L-glucose derivative having passhook blue or marina blue in the molecule is used as another fluorescently labeled L-glucose derivative.
- the method for detecting cancer according to the present invention and the imaging agent therefor include tissues extracted at the time of surgery, digestive organ tumors using oral tumors and endoscopes, gynecological tumors such as cervical cancer, other lungs, and various It can be used for biopsy specimens obtained at the time of biopsy diagnosis of various organs, etc., for the presence of hypoxia resistant tumor cells, evaluation of the state, and differentiation from normal cells. This makes it possible to quickly perform detailed cell evaluation on a cell-by-cell basis with a simple device equipped with fluorescence, guideline for selection of treatment method, determination of therapeutic effect of drugs, etc., appropriate operation after exposure of affected area This is effective for determining the range.
- the detection of the fluorescently labeled L-glucose derivative present in the cancer cell is performed by, for example, measuring the fluorescence of the target cell in advance and then contacting the fluorescently labeled L-glucose derivative with the target cell for a certain period of time. Then, it is washed away, the fluorescence of the target cell is measured again, and the evaluation can be performed with an increase in the fluorescence intensity with respect to the fluorescence intensity of the target cell before contact.
- the fluorescence intensity as an image, it is possible to detect cells that have fluorescently labeled L-glucose derivatives in the cells and to detect cancer cells or cells that may be.
- the evaluation may be performed using the total of the fluorescence intensities exhibited by a large number of cells or the distribution of the fluorescence intensities using a fluorescence plate reader or flow cytometry.
- a fluorescence plate reader or flow cytometry used to measure the fluorescence intensities.
- cell imaging can be performed by administering to a tissue local to be observed. Is also possible.
- the fluorescently labeled L-glucose derivative of the present invention is useful for detecting cancer cells and also useful as an active ingredient of an imaging agent for visualizing cancer cells, for example. It is.
- the fluorescently labeled L-glucose derivative may be provided in the form of a solution by dissolving it in a solvent for dissolving it (such as physiological saline for injection), or used in combination with a solvent for dissolving it. Sometimes it may be provided in the form of a kit for dissolving and preparing the solution.
- the concentration of the fluorescently labeled L-glucose derivative in the solution may be adjusted in the range of 1 nM to 100 mM, for example.
- the method using the labeled L-glucose derivative of the present invention for detection of cancer cells may be used in combination with a known method related to fluorescence detection or cell detection to further improve the determination accuracy. .
- Example 1 Synthesis of compounds (1) Synthesis of fluorescently labeled sugar derivatives Synthesis of 2-PBDG (2-Deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl) carboxamide) -D-glucose) 2-PBDG represented by the following formula is as follows. Synthesized.
- D-mannosamine hydrochloride (9.5 mg) was dissolved in water (40 ⁇ L), dimethylformamide (100 ⁇ L) and triethylamine (10.3 ⁇ L) were added, and the mixture was stirred at room temperature.
- Pacific Blue TM Succinimidyl Ester (10 mg) / dimethylformamide (800 ⁇ L) was added and stirred at room temperature.
- triethylamine (5.2 ⁇ L) was added and stirred at room temperature.
- the mixture was neutralized with acetic acid and passed through a membrane filter. The filtrate and washings were combined and purified by HPLC. The desired fraction was collected and lyophilized.
- PBDM and PBLM synthesized Pacific blue-labeled D-mannose derivatives in which a fluorescent molecular group is bonded to the 3-position, 4-position or 6-position of D-mannose are respectively 3-amino-3-deoxy-D-mannose, 4 - Pacific 4-deoxy-D-mannose or 6-amino-6-deoxy-D-mannose is used as a raw material and Pacific Blue is introduced into the 3rd, 4th or 6th position of D-mannose according to a conventional method.
- a fluorescent molecular group can be introduced at the 1-position by synthesizing a 1-azido form as an intermediate and immediately fluorescent after reduction.
- the Pacific Blue labeled L-mannose derivative can be synthesized in the same manner by using aminodeoxy-L-mannose as a raw material.
- D-glucosamine hydrochloride (11.7 mg) was dissolved in water (50 ⁇ L), and dimethylformamide (50 ⁇ L) was added and stirred. Triethylamine (11.3 ⁇ L) was added to this, and then a solution of Marina Blue TM Succinimidyl Ester (10 mg) in dimethylformamide was added and stirred at room temperature. The mixture was neutralized with acetic acid, passed through a membrane filter, and the filtrate and washing solution were combined and purified by HPLC. The desired fraction was collected and lyophilized.
- L-glucosamine hydrochloride (7.1 mg) was dissolved in water (56 ⁇ L), and dimethylformamide (400 ⁇ L) was added and stirred.
- Marina Blue TM Succinimidyl Ester (10 mg) / dimethylformamide (1.2 mL) was added, followed by triethylamine (8.3 ⁇ L) and stirring at room temperature.
- L-glucosamine hydrochloride (1.8 mg) and triethylamine (1.1 ⁇ L) were added and stirred at room temperature. Further, after 1 hour, triethylamine (1.9 ⁇ L) was added and stirred at room temperature. Thirty minutes later, acetic acid was added to neutralize the solution and then passed through a membrane filter.
- 2-MBDM (2-Deoxy-2- (2- (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -D-mannose) Similar to the synthesis method of 2-MBDG
- 2-MBDM can be synthesized by using D-mannosamine hydrochloride instead of D-glucosamine hydrochloride used for the synthesis of 2-MBDG.
- 2-MBLM (2-Deoxy-2- (2- (6,8-difluoro-7-hydroxy-4-methylcoumarin-3-yl) acetamide) -L-mannose)
- 2-MBLM can be synthesized using L-mannosamine hydrochloride instead of L-glucosamine hydrochloride used for the synthesis of 2-MBLG.
- Comparative Example 1 Synthesis of comparative compound Synthesis of 2-HCDG (2-Deoxy-2-((7-hydroxycoumarin-3-yl) carboxamide) -D-glucose) 2-HCDG represented by the following formula was synthesized as follows.
- D-glucosamine hydrochloride (11.9 mg) was dissolved in water (2 mL) and cooled on ice. To this was added triethylamine (9.2 ⁇ L), followed by 7-hydroxycoumarin-3-carboxylic acid N-succinimidyl ester (20 mg) / dimethylformamide (2 mL), and the mixture was stirred at room temperature for 3 hours. A 1% aqueous acetic acid solution (4 mL) was added and the mixture was allowed to stand overnight. The membrane was passed through a membrane filter and washed with a 1% aqueous acetic acid solution. The filtrate and washings were combined and purified by HPLC. The desired fraction was collected and lyophilized.
- D-glucosamine hydrochloride (216 mg) was dissolved in water (1 mL), and dimethylformamide (9 mL) was added. To this, MocAc-OH (234 mg) and HOBt (135 mg) were added and ice-cooled. WSCD (187 ⁇ L) was added thereto and stirred at 0 ° C. for 1 hour. After adding WSCD (33.9 ⁇ L) and further stirring for 2 hours, the neutral reaction solution was concentrated under reduced pressure, and water was added to the resulting residue and freeze-dried. The residue was purified by HPLC. The desired fraction was collected and lyophilized.
- Example 2 Application of 2-PBDG to acutely isolated normal nerve cells The method was described in accordance with the method described in WO2010 / 16587. The results are shown in FIG. Viable neurons were isolated from the substantia nigra of the mouse midbrain, and a mixed solution containing 100 ⁇ M 2-PBDG and 20 ⁇ M 2-TRLG was administered at 37 ° C. for 5 minutes. The confocal microscope images immediately before that are shown in FIGS. 1A to 1C. A is a fluorescent image (Blue channel, wavelength range 415-580 nm) in a blue wavelength region. The cell location is known by autofluorescence. The fluorescence signal intensity is displayed in pseudo color.
- A is a fluorescent image (Blue channel, wavelength range 415-580 nm) in a blue wavelength region.
- the cell location is known by autofluorescence.
- the fluorescence signal intensity is displayed in pseudo color.
- B is a fluorescent image in the red wavelength region (Red channel, 580-740 nm). Both A and B are excited simultaneously using 405nm Blue diode laser at 60% intensity, and PMT2 detection sensitivity to detect the presence of 2-TRLG intrusion using photomultiplier (PMT) 1 and 2 respectively. Is acquired higher than PMT1.
- C is a diagram in which a bright field image is superimposed on the fluorescent images of A and B. FIG. Images taken 4 minutes after the administration of the fluorescent liquid mixture is completed and the washing of the administration liquid is started are shown in FIGS. 1D to 1F. The image acquisition conditions are the same as A to C.
- Example 3 Application of 2-PBLG to acutely isolated normal nerve cells
- FIG. 2A-C show confocal microscope images immediately before administration in which mouse midbrain substantia nigra neurons are isolated acutely and a mixed solution containing 100 ⁇ M 2-PBLG and 20 ⁇ M 2-TRLG is administered at 37 ° C. for 5 minutes. Show. Images taken 4 minutes after the administration of the fluorescent liquid mixture is completed and the washing of the administration liquid is started are shown in FIGS. 2D to 2F.
- the image acquisition conditions are the same as A to C. Looking at the Blue channel of D, the intracellular fluorescence intensity was hardly increased compared to that before administration (A).
- FIGS. 2J to L are images 8 minutes and 20 minutes after the start of washing, respectively.
- the slight increase in fluorescence intensity observed in the cells at D returned to the autofluorescence level at 20 minutes after starting washing.
- 2-PBLG which is an L-type glucose derivative
- Comparative Example 1 Application of 2-HCDG to acutely isolated normal neurons An experiment was conducted in the same manner as in Example 2. The results are shown in FIG. FIG. 3 shows confocal microscope images before and after the administration of a mixture containing 100 ⁇ M 2-HCDG and 20 ⁇ M 2-TRLG at 37 ° C. for 3 minutes in neurons isolated acutely from the mouse midbrain substantia nigra. .
- a and B are pre-dose fluorescence images obtained on the Blue channel (415-580 nm) and Red channel (580-740 nm), respectively. The excitation wavelength is 405 nm.
- C is a differential interference (DIC) image.
- D is the above superposition.
- E to H are the same as A to D, but after the 2-HCDG + 2-TRLG fluorescent tracer solution was administered at 37 ° C for 3 minutes, the fluorescent tracer was started to be washed out, and the images obtained 4 minutes after the start of washing. .
- E and F the blue fluorescence intensity increased after administration for cell debris, whereas at a neuron location, no increase in fluorescence intensity was detected before and after administration. .
- 2-TRLG since 2-TRLG has not entered the cell, the cell membrane of the nerve cell is considered to be kept healthy.
- Comparative Example 2 Application of 2-MCDG to acutely isolated normal neurons Comparison of a mixture containing 100 ⁇ M 2-MCDG and 20 ⁇ M 2-TRLG to neurons isolated acutely from the mesencephalic substantia nigra of mice Administration was carried out in the same manner as in Example 1, but no increase in fluorescence intensity in nerve cells was observed before and after administration.
- the optimum excitation wavelength is very low at 320 nm, so using a Nikon Ti-E real-time deconvolution microscope, a xenon lamp with an excitation filter of 320 nm (half-value width 40 nm), a fluorescence filter 435 nm (half-value width 40 nm), and a dichroic mirror Images were acquired with a Q-imaging Retiga-2000R CCD camera through a custom filter with a 409 nm configuration.
- Example 4 Uptake of 2-PBDG (100 ⁇ M) and 2-PBLG (100 ⁇ M) into mouse insulinoma cells (MIN6) and influence of phloretin, a glucose transport inhibitor (experimental method) (1-1) Culture of cells MIN6 cells that had been cryopreserved (cells that were passaged 5-8 times after being provided by Professor Junichi Miyazaki of Osaka University) were transferred to culture according to conventional methods, and passaged 7-9 times. What was done was used for experiment.
- KRB solution A KRB solution having the following composition was used for measurement. NaCl 129.0 mM, KCl 4.75 mM, KH 2 PO 4 1.19 mM MgSO 4 ⁇ 7H 2 O 1.19 mM, CaCl 2 ⁇ 2H 2 O 1.0 mM, NaHCO 3 5.02 mM, D-Glucose 5.6 mM, HEPES 10 mM (in 1M NaOH Adjusted to pH 7.35). In addition, 0.1 mM Carbenoxolone (SIGMA # C4790) was added for the purpose of inhibiting the entry and exit of fluorescently labeled glucose via gap junction / hemichannel. This KRB solution was used as a solution for preparing a 2-PBLG solution.
- 2-NBDLG solution The total amount of one 0.5 mg 2-NBDLG vial was dissolved in 7.3 mL of KRB solution to give a 2-NBDLG solution with a final concentration of 200 ⁇ M.
- Preparation of 2-PBDM solution The entire amount of 0.5 mg 2-PBDM vial was dissolved in 3.1 mL of KRB solution according to the preparation of 2-PBLG solution to give a final concentration of 100 ⁇ M 2-PBDM solution.
- the wells (3C, 3E, 3G) that measure the effect of the glucose transport inhibitor phloretin are pre-administered with phloretin (final 150 ⁇ M) from 5 minutes before 2-PBDG administration, and the other wells (3B, 3D , 3F) added KRB.
- the same operation was performed on the fifth row where 2-PBLG was to be administered.
- 2-PBDG and 2-PBLG were administered at 37 ° C. for 10 minutes.
- the operation of diluting the fluorescent solution in the well with 300 ⁇ L of the KRB solution was repeated for a predetermined number of times every 30 seconds.
- the number of repetitions is determined based on the fact that the fluorescence intensity shown in the wells of rows A and H set as the control group is the same level as the fluorescence intensity of the blank wells without cells, and it is completely washed out Was confirmed in each experiment.
- this washing process took 8 minutes, so fluorescence measurement after administration was performed 9 minutes later.
- this method even if cells that have broken the cell membrane state contact 2-PBDG and 2-PBLG and then take these compounds into the cells, they have already flowed out of the cells at the time of measurement. Since it was washed away, it was judged that the contribution to the increase in fluorescence intensity of the entire observation area was negligible.
- FIG. 4A shows the result of measuring the fluorescence intensity before and after administration with a fluorescence microplate reader. The number in parentheses is the number of observation areas. The fluorescence before administration shows the autofluorescence of the cells.
- FIG. 4B shows the difference in fluorescence intensity before and after administration in A.
- the change in fluorescence intensity when 2-PBDG is administered in the absence of phloretin is shown as 100%. There was no significant difference between the fluorescence intensities of 2-PBDG and 2-PBLG. Further, in the presence of phloretin, a decrease in fluorescence intensity was observed in both cases of 2-PBDG and 2-PBLG compared to the absence, but most of the fluorescence was not inhibited by phloretin. Similar results were obtained in both of the two independent experiments, and the decrease in 2-PBDG and 2-PBLG by phloretin averaged only 22.4% and 20.0%, respectively.
- Example 5 Change in fluorescence intensity by administration of 2-PBDG, 2-PBLG and PB-NH 2 and effect of glucose transport inhibitor D-glucose derivative (2-PBDG), L-glucose on MIN6 cells on day 10 of culture
- PB-NH 2 has the following structure (Ex max. 402 nm, Em max. 451 nm). The results are shown in FIG.
- FIG. 5A shows the GLUT selective inhibitor cytochalasin B (CB, 10 ⁇ M) did not show a significant inhibitory effect on the increase in fluorescence intensity by administration of 2-PBDG (100 ⁇ M).
- the average fluorescence intensity was attenuated in the presence of CB as compared to the absence of CB, but an increase was observed in the results of three independent runs, which were not constant.
- FIG. 5B shows the effect of the glucose transport inhibitor phloretin (PHT, 150 ⁇ M) on the increase in fluorescence intensity by administration of 2-PBLG (100 ⁇ M) or PB-NH 2 (100 ⁇ M).
- the administration experiment to 2-PBLG and PB-NH 2 was carried out on the same culture plate at the same time, and in all three experiments conducted independently, the significant enhancement effect by phloretin on the PB-NH 2 response was confirmed, The increase in fluorescence reached an average of 384.1 ⁇ 24.2% when only PB-NH 2 was administered (n 3).
- PB-NH 2 having no sugar skeleton showed a significantly larger increase in fluorescence intensity than the L-glucose derivative 2-PBLG having a sugar skeleton.
- Example 6 Administration of 2-PBDM (100 ⁇ M) to mouse insulinoma cells (MIN6) and influence of phloretin, a glucose transport inhibitor
- 2-PBDM 100 ⁇ M
- MIN6 mouse insulinoma cells
- FIG. 6 The inhibitory effect of phloretin (150 ⁇ M, PHT) on the increase in fluorescence intensity before and after administration by administering 2-PBDM (100 ⁇ M) to MIN6 cells (20000 cells / well) on the 10th day of culture (10DIV)
- 2-PBDM was confirmed to have a slight but significant inhibitory effect by phloretin.
- the experiment was performed three times independently and all obtained similar results.
- excitation was performed at a maximum excitation light wavelength of 404 nm
- fluorescence was acquired at a maximum fluorescence wavelength of 453 nm.
- Example 7 Imaging of tumor cell mass composed of mouse insulinoma cells (MIN6) using 2-PBDG or 2-PBLG (2-PBDG / 2-TRLG or 2-PBLG / 2-TRLG or 2-PBLG / 2- Use NBDLG / 2-TRLG) (experimental method)
- MIN6 mouse insulinoma cells
- a culture solution in which MIN6 cells are suspended at a rate of 10 ⁇ 10 4 cells / mL is dropped on a glass cover slip
- the culture solution is attached to the glass surface. 3 mL was added and cultured.
- the culture medium was exchanged by half once every 3 days.
- the HEPES solution for image acquisition is a solution for preparing a 2-PBLG solution, and a solution for preparing a 2-PBLG / 2-TRLG solution and a 2-PBLG / 2-NBDLG / 2-TRLG solution. Used as.
- a streamlined hole is drilled in the center (the side that contacts the glass bottom is 10 mm wide x 35 mm long, the radius of curvature is 33 mm, and the side that does not touch the glass surface, that is, the top is slightly wider)
- a 1 mm thick silicon plate (width 20 mm x length 50 mm) was placed and adhered to the cover glass without using silicon grease.
- a 20-gauge catalan needle with a flat tip was set and used as an inlet.
- the stainless steel tube (outlet) for discharging the perfusate is a tube that has been flattened and cut obliquely according to the method described in Non-Patent Document 16, and both air and solution are removed during vacuum suction. The system was stabilized by sucking at the same time.
- Perfusate supply system to the perfusion chamber (a) Warming of the perfusate and supply to the perfusion chamber
- the perfusate supply system consists of one 60 mL syringe for the control solution and 10 mL injection for the drug supply. It is equipped with five tubes and can be perfused by switching at any time with an electromagnetic valve.
- a 5.6 mM glucose-containing HEPES solution was obtained using a 60 mL syringe, and 2-PBLG / 2-NBDLG / 2-TRLG was used using one of five 10 mL syringes.
- a mixed solution, a 2-PBDG / 2-TRLG mixed solution, or a 2-PBLG / 2-TRLG mixed solution was administered.
- both are pre-heated to prevent bubbles in the perfusion chamber, combined with a single tube before being led to the perfusion chamber, and speed adjusted with a flow regulator,
- the mixture was heated again with an in-line heater and supplied to the perfusion chamber on the confocal microscope.
- the HEPES solution for image acquisition is flushed from the 60 mL syringe heated in an aluminum syringe heater (Model SW-61, temperature control unit No. TC-324B, Warner Instruments) in the solution supply line.
- the solution supply speed of the pump was precisely adjusted to be equal to the solution dropping speed. Since the solution supply rate of the peristaltic pump is digitally displayed, if there is a change in the solution supply rate to the perfusion chamber during the experiment, it can be immediately recognized by changing the height of the solution surface. Thus, since this solution is constantly renewed, the syringe heater SW-61 was set to 38.5 ° C. in order to maintain the liquid temperature. On the other hand, 2-PBLG and 2-TRLG mixed solution or 2-PBLG / 2-NBDLG / 2-TRLG mixed solution, etc. are used for syringe heater (Model SW-6, temperature control unit is No. TC-324, Warner Instruments).
- MPP-6 Small manifold
- the outlet of the MPP-6 manifold was connected to a short Pharmed tube, and this tube was inserted into a flow controller that could increase or decrease the opening degree with a screw, and the flow rate was adjusted to 1.2 ⁇ 0.2 mL / min by adjusting the opening degree.
- the Pharmed tube was connected to an in-line heater (Multi-Line In-Line Solution Heater SHM-8, temperature control unit TC-324B, Warner Instruments) at the shortest distance. This is because the temperature of the solution introduced into the perfusion chamber is heated immediately before the introduction.
- SHM-8 Multi-Line In-Line Solution Heater
- the temperature of the SHM-8 in-line heater was adjusted according to the perfusion rate so that the actual temperature of the perfusate in the chamber was 36-37 ° C in the area where the coverslip was present.
- the heated solution was connected to a stainless steel pipe (inlet) arranged upstream of the perfusion chamber through a short Tygon tube (R-3603, inner diameter 1/32 inch) and supplied into the perfusion chamber. Since the solution supply from the syringe determines the supply pressure using hydrostatic pressure, 2-PBLG and 2-TRLG are used so that the difference in height does not cause a change in the water level in the chamber due to the difference in perfusion rate.
- the administration time is short, so liquid supply is not performed during one experiment, and the top surface of the liquid is fluorescent at the end of each experiment.
- the solution was added to approximately the same height as the HEPES solution without glucose.
- the tube was adjusting the length and thickness of the tube connected to the syringe barrel, carefully adjusting the tube so that it is discharged at the same rate as the perfusion rate of the HEPES solution for image acquisition that is the control solution, the liquid level by liquid exchange Fluctuations can be avoided.
- the inside of the tube was sufficiently flushed to ensure a smooth flow.
- An ultrafine thermistor probe (Physitemp IT-23) was used to confirm the temperature of each part in the perfusion solution in the chamber (Non-patent Document 16).
- an operation microscope (POM-50II, KONAN MEDICAL, Nishinomiya) installed on the chamber for each experiment. Checked and cleaned.
- a laser scanning confocal microscope (Leica's TCS-SP5 system, the microscope body was a DMI6000 CS trino electric inverted microscope) was used in conventional mode.
- the laser used is a 405 nm diode laser, excitation of 2-PBLG or 2-PBDG, or excitation of a mixed solution of 2-PBLG (or 2-PBDG) and 2-TRLG with a single light source. Used for live staining.
- the irradiation intensity was appropriately adjusted according to the fluorescent dye used so that sufficient observation intensity could be obtained with an acousto-optic polarizing element (Acoustic Optical Tunable Filter, AOTF).
- 2-NBDLG and 2-TRLG were excited with a 488 nm Argon laser.
- the scan speed was 200Hz or 400Hz.
- Fluorescence detection uses a photomultiplier detector (PMT) 1 for detection of blue fluorescence by 2-PBDG or 2-PBLG with a wavelength detection range of 415-580 nm for 2-PBLG / 2-TRLG two-color detection.
- PMT photomultiplier detector
- the image was acquired with the wavelength detection range set to 415-500 nm.
- PMT2 named green channel, the same shall apply hereinafter
- PMT3 For detection of red fluorescence by 2-TRLG, PMT3 (named red channel, hereinafter the same) was used in the wavelength detection range of 580-740 nm.
- the beam splitter used was 500 nm (RSP500).
- the beam splitter for 405 nm is fixed at 415 nm independently of the above in the SP5 system.
- the detection sensitivity in the red wavelength region was set to be higher than the detection sensitivity in the blue wavelength (blue 617V, red 738V, etc.) at the same time so that intrusion of the light could be detected effectively.
- the acquisition of differential interference (DIC) images used to capture the structural features of the three-dimensional tumor cell mass was detected using the PMT Trans detector for transmitted light at the time of 488 nm (or 405 nm) excitation (typical The detection sensitivity was 145-200 V).
- the DIC polarizer and analyzer should be put in the optical path even when acquiring images by 405 nm excitation. I left it.
- a high resolution x40oil lens (HCX PL APO CS 40.0x1.25 OIL UV, NA1.25) is used as the objective lens to obtain a high resolving power on the xy axis and an angle of view that includes the entire cell mass within the field of view. Used at full aperture.
- the pinhole size was set to 3 airy units. It was confirmed in the acquired image that the nucleus and cytoplasm in the cell could be practically distinguished in the z-axis direction even with this pinhole size.
- Zoom was basically not used (1x), and the image was acquired at a depth of 12 bits with 1024x1024 or 512x512 pixels.
- the administration of the above solutions and acquisition of all images were performed in a dark room maintained at a constant room temperature (24 ° C.) for 24 hours. The results are shown in FIGS.
- FIG. 7 shows that 4 ′, 6-diamidino-2- emitting blue fluorescence appears in the center of a spheroid that has reached a certain diameter (approximately 100 microns or more) and a bulky height (approximately 50 microns or more). There are cells with nuclei that bind phenylindole (DAPI) abnormally strongly. DAPI is applied directly to living cells without being fixed in formalin. In FIG.
- FIG. 7B cells undergoing apoptosis are visualized with green fluorescence by the live apoptosis marker pSIVA-IANBD (IMGENEX, San Diego, USA). Positive cells are scattered around the spheroids.
- red fluorescence indicates cells in which propidium iodide (PI), which is a general necrosis marker, has entered the cells. It can be seen that it is relatively concentrated near the center of the spheroid.
- FIG. 7D shows a differential interference microscope image.
- FIG. 7E shows the above superimposed image. Image acquisition was performed according to the 2-PBLG / 2-NBDLG / 2-TRLG three-color detection method.
- FIG. 8 shows a photomicrograph of a cell mass (a 13th day from the start of culture) in which many MIN6 cells are assembled.
- FIG. 8 shows a state before administration of a fluorescently labeled glucose derivative mixed solution composed of 2-NBDLG, 2-TRLG, and 2-PBLG.
- FIGS. 8A and 8B show the excitation at the 488 nm argon laser simultaneously in the wavelength ranges of 500 to 580 nm (green) and 580 to 740 nm (red), which are optimal for observation of 2-NBDLG and 2-TRLG, respectively. It is the acquired fluorescence image.
- FIG. 8C is a differential interference (Contrast, DIC) microscopic image acquired simultaneously with A and B.
- FIG. 8D is a fluorescence acquisition image in a wavelength range of 415 to 580 nm (blue) obtained by sequential excitation with a 405 nm diode laser following ABC scanning.
- FIG. 8E is the above superimposed image. Compared with C, a slight autofluorescence pattern can be seen.
- FIG. 9 to 12 show the results of imaging using 2-PBLG of a tumor cell mass composed of mouse insulinoma cells (MIN6). Images were acquired with a real-time laser scanning confocal microscope using a mixed solution consisting of 100 ⁇ M 2-PBLG, 100 ⁇ M 2-NBDLG, and 20 ⁇ M 2-TRLG.
- FIG. 9 shows green (A), red (B), and blue (D) fluorescence acquisition of a MIN6 cell mass during administration of a mixed solution consisting of 100 ⁇ M 2-PBDLG, 100 ⁇ M 2-NBDLG, and 20 ⁇ M 2-TRLG.
- FIG. 10 is the same as FIG. 9, but is an image when 2 minutes have elapsed after the administration of the mixed solution of 2-NBDLG, 2-TRLG, and 2-PBLG.
- the fluorescence intensity at the center of the cancer cell mass is stronger than the surrounding area (A, B, D, E).
- the fluorescence image by 2-TRLG that does not pass through the cell membrane is seen, not only the central part of the cell mass but also the peripheral part of the cell mass is dotted with cells that appear to have deteriorated the cell membrane state (B).
- 2-NBDLG and 2-PBLG once enter these cells and have not yet flowed out of the cells. Some cells are emitting. Many of these cells lose strong fluorescence derived from 2-NBDLG (green) and 2-PBLG (blue) within a few minutes after the end of administration of the mixture (see FIG. 11).
- FIG. 11 is an image at the time when 8 minutes have elapsed after the administration of the mixed solution.
- the fluorescence intensity in the central part of the cancer cell cluster does not tend to be particularly strong compared to the peripheral part.
- strong blue fluorescence by 2-PBLG is still maintained for the cells indicated by arrows.
- This cell also shows a stronger tendency for the fluorescence intensity by 2-NBDLG than the surrounding cells, but it is difficult to identify this cell only by the fluorescence image by 2-NBDLG (A).
- 2-TRLG has a property that it does not easily flow out of the cell once it is taken up by a cell that has not completely died but has enhanced cell membrane permeability, and emits strong fluorescence.
- FIG. 12 is an image at the time when 12 minutes have elapsed after the administration of the mixed solution. Only the cells indicated by arrows continue to emit blue fluorescence with 2-PBLG and are distinguished from other cells, suggesting that 2-PBLG is strongly bound to this cell (B, E).
- FIG. 13 is an enlarged image of the vicinity of the central part of the cancer cell cluster shown in FIG. Arrow cells are strongly visualized by 2-PBLG. Arrow indicates 2-PBLG strong positive cells. This cell also has strong green fluorescence due to 2-NBDLG (A), but it is difficult to identify this cell with 2-NBDLG alone.
- Example 8 Imaging of tumor cell mass composed of mouse insulinoma cells (MIN6) using 2-PBLG (use of 2-PBLG / 2-TRLG)
- 2-PBLG use of 2-PBLG / 2-TRLG
- FIG. 14 is an image of the MIN6 cell mass on day 13 of culture before administration of the fluorescently labeled glucose derivative.
- a and B are fluorescence acquisition images in the wavelength range of 580-740 nm (red) and 415-580 nm (blue), respectively.
- C is a differential interference microscope image.
- D is an overlay image of these.
- FIG. 15 is an image at the time when 2 minutes have elapsed after starting to wash out a fluorescent mixture containing 20 ⁇ M 2-TRLG and 100 ⁇ M 2-PBLG to the MIN6 cell mass for 5 minutes.
- 2-TRLG which is impermeable to the cell membrane, invades the interior of the cell, which is mainly in the center of the cell mass and has enhanced cell membrane permeability.
- a part of the debris of the cell tissue outside the cell mass and the cell mass is also stained.
- 2-PBLG once invades into the cell whose permeability to the cell membrane is enhanced.
- the fluorescence intensity distribution of 580-740 nm of 2-TRLG excited at 488 nm is mixed with the increase in fluorescence intensity (long wavelength side) of 2-NBDLG in the region of 580 nm or more.
- the mixed solution of 2-PBLG / 2-TRLG it is possible to avoid both the FRET effect and the influence of the long wavelength side tail, so that the increase in fluorescence intensity can be separated. This is possible and advantageous in quantification.
- FIG. 16 is the same as FIG. 15 but shows an image 8 minutes after the end of administration and the start of washing.
- the distribution pattern of red 2-TRLG and blue 2-PBLG is greatly different (A, B, D), and the distribution of 2-PBLG strongly positive cells is difficult to explain due to the increase in cell membrane permeability.
- Blue 2-PBLG positive cells are scattered on the outer edge of the central part of the cell mass (B, D).
- FIG. 17 is the same as FIG. 16, but is an image 12 minutes after the end of administration and the start of washing. Subsequently, a plurality of cells exhibiting a strong positive signal of 2-PBLG are observed (B, D).
- the present invention provides a novel fluorescently labeled sugar derivative that emits a blue fluorescent color.
- the present invention also provides a method for detecting new tumor cells.
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Abstract
Description
D-ガラクトースは、エネルギー源として利用される糖であり、乳、果実類、野菜類に豊富に含まれているほか、ヒトの体内でも一日2g程度が産生されている。たとえば牛乳の2-8%を占める二糖類ラクトースは、D-ガラクトースとD-グルコースがグリコシド結合したもので、小腸で吸収される際に両者がラクターゼにより分離されて、グルコーストランスポーターの一種であるSGLTを介して体内に吸収されることが知られている。D-ガラクトースが小腸上皮細胞から血管へ運ばれる際にはグルコーストランスポーターGLUT2を通過する。細胞内に取り込まれたガラクトースは、1位にリン酸化を受けた後、解糖系に入りエネルギーとして利用され、あるいは糖脂質や糖タンパクの生合成に利用される。一方、L-ガラクトースは、霊長類が生合成できない抗酸化物質ビタミンC(L-アスコルビン酸)が、植物内においてD-グルコースから生合成される際の経路の一つSmirnoff-Wheeler経路の中間代謝産物としての記載があるが、一般に生物学に登場することがまれな希少糖である。
D-ガラクトースを18Fで標識した2-deoxy-2[18F]fluoro-D-galactoseは、肝臓の代謝解析への適用例がある(非特許文献1)。2-deoxy-2[18F]fluoro-D-galactoseは、がんにおけるガラクトース代謝イメージングへの利用可能性が報告されたが一般化していない(非特許文献2)。
放射性標識体として1-deoxy-1-[18F]fluoro-D-fructoseが合成され、腫瘍への中程度の取り込みが報告されたが、本分子は細胞内で代謝を受けないとみられ、利用されていない。最近では細胞内代謝を受ける6-deoxy-6-[18F]fluoro-D-fructoseが合成され、乳がんにおけるGLUT5を介した取り込みを標的とするPETトレーサー候補として報告されている(非特許文献3)。
D-マンノースが特異的に結合するマンノース受容体は、炎症時に増加する高マンノース糖タンパクの除去に役立つ。例えば、日和見感染症の一種でエイズ患者の死因第一位を占めるカリニ肺炎の原因菌P.cariniの膜表面には高マンノース糖鎖部分があり、肺胞のマクロファージに発現するマンノース受容体がこれを認識して、マクロファージの移動を促進する。D-マンノースのみならず、L-ガラクトースにも、強力なマクロファージ刺激作用があるほか、D-マンノース、L-ガラクトースの両者とも植物におけるビタミンC生合成の前駆体として利用される。
[18F]-2-fluoro-2-deoxy-D-mannoseが、がんのトレーサーとして利用可能であることが報告されているが一般化していない(非特許文献4、非特許文献5)。
従来、生物がD-グルコースをどのようにして細胞内に取り込んで利用するのかについての研究は、例えばラジオアイソトープで標識したD-グルコースやその誘導体(D-デオキシグルコースなど)を用いて細胞内のラジオアイソトープ量を測定することで行われてきた。しかしながら、この方法は定量性に優れるものの、感度が低いといった問題があることに加え、測定手法上、生きた細胞がD-グルコースを取り込む様子をリアルタイムで連続的に観察することができないという欠点を有していた。そこで、本発明者らのグループは、生きた細胞のD-グルコースの動的な取り込みプロセスの研究に使用することができる方法として、D-デオキシグルコースの2位に蛍光発色団としてN-(7-ニトロベンズ-2-オキサ-1,3-ジアゾール-4-イル)アミノ基を結合せしめた、緑色の蛍光を発する2-[N-(7-ニトロベンズ-2-オキサ-1,3-ジアゾール-4-イル)アミノ]-2-デオキシ-D-グルコース(2-NBDG)を用いる方法を提案し、その有用性を哺乳動物の各種の細胞を用いて実証した(非特許文献6)。
また、D-フルクトースの1位にNBDを結合した分子(1-NBDF)を乳がんに応用した報告がある(非特許文献8)。
このようにNBDを分子内に有する、グルコース誘導体やフルクトース誘導体が、生きた細胞を個々の細胞レベルでイメージングできる蛍光標識糖誘導体として知られている。
また、他のヘキソースについても、上記したようにその放射性標識体を用いてがんの検出やイメージングへの応用が試みられてきた。しかし、D-グルコースと同様に、D体であるためにその利用が制限される他、個々の単一細胞における違いをリアルタイムに精度よく検出できないといった問題がある。
1.蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを分子内に有する蛍光標識糖誘導体を含む、標的細胞又は標的細胞内分子(標的細胞内分子とは、標的細胞の内側、すなわち細胞質又は核内に存在する分子、標的細胞の細胞膜中に存在する分子、標的細胞の細胞膜上に存在する分子を含む。)をイメージングするための組成物。
2.蛍光標識糖誘導体が、グルコース誘導体、フルクトース誘導体、ガラクトース誘導体又はマンノース誘導体である、上記1に記載の組成物。
3.前記蛍光分子団が、グルコース、フルクトース、ガラクトース又はマンノースに、-NH-結合を介して結合している、上記2に記載の組成物。
4.蛍光標識糖誘導体が、グルコースの1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを、-NH-結合を介して結合した分子である上記1に記載の組成物。
5.蛍光標識糖誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースからなる群より選ばれる分子である上記4に記載の組成物。
6.蛍光標識糖誘導体が、マンノースの1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した分子である上記1に記載の組成物。
7.蛍光標識糖誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-マンノース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-マンノース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-マンノース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-マンノースからなる群より選ばれる分子である上記6に記載の組成物。
a.標的細胞(標的細胞は、細胞そのもののほか、組織内に存在する細胞も含む)に、上記1~7のいずれか一つに記載の組成物を接触させる工程、及び
b.該標的細胞内(標的細胞の内側、すなわち細胞質又は核内、標的細胞の細胞膜中、及び標的細胞の細胞膜上を含む。)に存在する該糖誘導体を検出する工程、
を含むイメージング方法。
9.グルコース、フルクトース、ガラクトース及びマンノースからなる群から選ばれる糖に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した蛍光標識糖誘導体。
10.2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-マンノース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-マンノース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-マンノース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-マンノースからなる群より選ばれる蛍光標識糖誘導体。
12.がん又はがん細胞を検出するための方法であって、以下の工程、
a.標的細胞(標的細胞は、細胞そのもののほか、組織内に存在する細胞も含む)に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを結合した蛍光標識L-グルコース誘導体を含有する組成物を接触させる工程、及び
b.該標的細胞内(標的細胞の内側、すなわち細胞質又は核内、標的細胞の細胞膜中、及び標的細胞の細胞膜上を含む。)に存在する該L-グルコース誘導体を検出する工程、
を含む検出方法。
13.前記蛍光標識L-グルコース誘導体が、L-グルコースの1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した分子である上記12に記載の検出方法。
14.前記蛍光標識L-グルコース誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである上記12に記載の検出方法。
15.前記工程aにおける検出が標的細胞をイメージングすることにより行う上記12~14のいずれか一つに記載の検出方法。
16.前記工程aにおける組成物が、2位にスルホローダミン(好ましくは、スルホーダミン101、スルホーダミンB)をスルホンアミド結合せしめた2-アミノ-2-デオキシ-L-グルコースをさらに含み、かつ前記工程bが、標的細胞内に存在する(いずれかまたは両方の)蛍光標識L-グルコース誘導体を検出する工程である、上記12~15のいずれか一つに記載の検出方法
17.標的細胞が腫瘍細胞塊中の細胞である、上記12~16のいずれか一つに記載の検出方法。
19.前記蛍光標識L-グルコース誘導体が、L-グルコースの1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した蛍光標識L-グルコース誘導体である上記18に記載のイメージング剤。
20.前記蛍光標識L-グルコース誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである上記18に記載のイメージング剤。
21.前記イメージング剤がさらに、2位にスルホローダミン(好ましくは、スルホーダミン101又はスルホローダミンB)をスルホンアミド結合せしめた2-アミノ-2-デオキシ-L-グルコースを含む上記18~20のいずれか一つに記載のイメージング剤。
22.2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである蛍光標識L-グルコース誘導体。
23.上記17~20のいずれか一つに記載のイメージング剤を含むがん細胞を検出するためのキット。
24.上記11~16のいずれか一つに記載の検出方法を用いてがん細胞を検出することによって、標的細胞ががんであると診断する方法。
本発明の一つの態様は、上記イメージング剤に用いることができる特定のクマリン誘導体(パシフィックブルー又はマリーナブルー)を結合した蛍光標識糖誘導体である。
本発明の他の態様は、L-グルコースに特定のクマリン誘導体(パシフィックブルー又はマリーナブルー)を結合した蛍光標識L-グルコース誘導体を用いてがん細胞を検出するためのイメージング剤及び該イメージング剤を用いてがん細胞を検出する方法である。
本発明の他の一つの態様は、上記イメージング剤に用いることができるクマリン誘導体(パシフィックブルー又はマリーナブルー)を結合した蛍光標識L-グルコース誘導体である。
さらに、D型およびL型の立体配置に関係する糖の認識、輸送、代謝において哺乳動物細胞とは異なった性質をもつ微生物についても、D型又はL型の蛍光標識糖誘導体を用いて細胞レベルでイメージングすることにより、その機能の解明も可能である。
(I-1)蛍光標識糖誘導体
細胞又は細胞内分子のイメージングに用いることができる青色蛍光を発する本発明の蛍光標識糖誘導体は、糖、好ましくは、グルコース、フルクトース、ガラクトース又はマンノースに、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン(パシフィックブルー)又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン(マリーナブルー)を結合した蛍光標識糖誘導体である。
糖誘導体における蛍光分子団の結合部位は、本明細書に記載の方法又は常法により合成できれば特に制限されないが、グルコースの場合は、1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)、フルクトースの場合は、1位、3位、4位、5位又は6位(好ましくは、1位、5位又は6位、より好ましくは1位)、ガラクトース場合は、1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、3位又は6位)、マンノース場合は、1位、2位、3位、4位又は6位(好ましくは、2位、3位、4位又は6位、より好ましくは2位、4位又は6位)をあげることができる。
以下、グルコースを参考に上記蛍光分子団の糖への結合を説明するが、他の糖においても同様である。
グルコサミンは、D-グルコサミン又はL-グルコサミンを用いることができる。D-グルコサミンは、合成したD-グルコサミン又は市販のD-グルコサミンを用いることができる。L―グルコサミンは、WO2010/16587号公報に記載の方法、又はPCT/JP2012/58439号出願明細書に記載の方法により合成することができる(これらの公報又は出願明細書の記載は引用することにより本明細書の一部である)。PCT/JP2012/58439号出願明細書に記載の方法は以下の通りである。
本発明の青色蛍光を発するグルコース誘導体は、任意の溶液、例えば、DMSO等の溶媒に溶解して用いることができ、また、細胞又は細胞内分子のイメージングにおいて用いる溶媒や溶液中でも安定であるので、イメージング剤として適している。
本発明の青色蛍光を発する糖誘導体を用いたイメージングの対象である標的細胞は、特に限定されず、哺乳動物由来の細胞、大腸菌や酵母などの微生物の細胞、植物の細胞、受精卵などを対象とすることができ、また、生体から単離した細胞、生体から単離した組織中に存在する細胞、生体の組織中に存在する細胞、生体から単離後の初代培養細胞、又は株化した細胞など、どのような形態の細胞であってもよい。さらには、対象とする細胞は、正常細胞であっても、異常細胞(例えば、がん細胞)であってもよい。
(II-1)
がん細胞の検出又はイメージングに用いることができる青色蛍光を発する本発明のL-グルコース誘導体は、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン(パシフィックブルー)又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン(マリーナブルー)をL-グルコースに結合した分子である。L-グルコースへの結合は、グルコースの1位、2位、3位、4位又は6位のいずれの位置に上記蛍光分子団を結合することができるが、好ましくは、2位、3位、4位又は6位、より好ましくは、2位、4位又は6位である。また結合は、例えば、2位においては、グルコサミンを用いて-NH-を介して行うことができる。
本発明の蛍光標識L-グルコース誘導体は、好ましくは下記式(2)で表される。
がんは際限なく増殖し続けることで生体にさまざまな不都合を与えるが、特にがんの内部に抗がん剤や放射線治療に対し抵抗性を示すがん細胞の存在することが近年指摘されており、こうした特殊ながん細胞は、正常細胞が生きられない低酸素、低栄養環境に適応するための分子機構を備えている(非特許文献19参照)。
本発明の蛍光標識L-グルコース誘導体は、特定クマリンの誘導体(パシフィックブルー又はマリーナブルー)を鍵分子とし、正常細胞には取り込まれない性質を有するL-グルコースをこれに結合した化合物である。クマリンおよびその誘導体は、低酸素、低栄養環境に存在するがん細胞に過剰に発現する炭酸脱水酵素に結合してその機能を阻害するため、がん細胞を含む細胞集団に投与することにより、上記の特殊ながん細胞を選択的に蛍光可視化するとともに、その機能を阻害し、かつ正常細胞への影響を最小限にとどめることができる。
本発明の蛍光標識L-グルコース誘導体(例えば、2-PBLG)に強陽性の細胞は、低酸素環境への対応力において優れた形質を獲得しているがん細胞と考えられ、こうしたがん細胞は、転移先で本来そのがん細胞が存在していた環境とは異なる環境にあっても生存し得る能力の一つを獲得した細胞である可能性があり、本発明の蛍光標識L-グルコース誘導体を用いて、そのような細胞を選択的に識別・可視化することができる。
(1)蛍光標識糖誘導体の合成
2-PBDG(2-Deoxy-2-((6,8-difluoro-7-hydroxycoumarin-3-yl)carboxamide)-D-glucose)の合成
下記式で表される2-PBDGは以下のようにして合成した。
収量:42.9 mg、収率:72 %
1H-NMR (400 MHz、重メタノール、ppm):
δ9.11 (d, 0.8H, J=9.2 Hz, NH), δ8.98 (d, 0.2H, J=9.2 Hz, NH), δ8.77 (s, 1H, H4’), δ7.43 (dd, 1H, J=10.3 Hz and J=2.1 Hz, H5’), δ5.18 (d, 0.8H, J=3.2 Hz, H-1α), δ4.77 (d, 0.2H, J=8.7 Hz, H-1β), δ3.35-δ4.10 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6).
ESI-MS:calcd for C16H16F2NO9 [M+H]+ 404.07, found 404.0
励起極大波長:403 nm
蛍光極大波長:453 nm
下記式で表される2-PBLGは以下のようにして合成した。
収量:9.2 mg、収率:77 %
1H-NMR (400 MHz、重メタノール、ppm):
δ9.11 (d, 0.8H, J=9.2 Hz, NH), δ8.98 (d, 0.2H, J=9.2 Hz, NH), δ8.77 (s, 1H, H4’), δ7.43 (dd, 1H, J=10.3 Hz and J=2.1 Hz, H5’), δ5.18 (d, 0.8H, J=3.2 Hz, H-1α), δ4.77 (d, 0.2H, J=8.7 Hz, H-1β), δ3.35-δ4.10 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6).
ESI-MS:calcd for C16H16F2NO9 [M+H]+ 404.07, found 404.0
励起極大波長:403 nm
蛍光極大波長:453 nm
D-グルコースの3位、4位又は6位に蛍光分子団が結合したパシフィックブルー標識D-グルコース誘導体は、それぞれ、3-アミノ-3-デオキシ-D-グルコース、4-アミノ-4-デオキシ-D-グルコース、あるいは6-アミノ-6-デオキシ-D-グルコースを原料に用いて常法に基づきパシフィックブルーをD-グルコースの3位、4位又は6位に導入することにより合成できる。また、1位への蛍光分子団の導入は、中間体として1-アジド体を合成し、還元後即座に蛍光化することで可能である。
パシフィックブルー標識L-グルコース誘導体は、原料としてアミノデオキシ-L-グルコースを用いることにより、同様にして合成できる。
下記式で表される2-PBDMは以下のようにして合成した。
収量:10.5 mg、収率:88 %
1H-NMR (400 MHz、重メタノール、ppm):
δ9.14 (m, 0.5H, NH), δ8.74 (m, 1H, Ar), δ7.87 (s, 0.5H, NH), δ7.40 (m, 1H, Ar), δ5.14 (d, 0.5H, J=1.8 Hz, H-1), δ4.93 (d, 0.5H, J=1.4 Hz, H-1), δ3.43-δ4.57 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6).
ESI-MS:calcd for C16H16F2NO9 [M+H]+ 404.07, found 404.0
励起極大波長:404 nm
蛍光極大波長:453 nm
下記式で表される2-PBLMは、その鏡像異性体である上述の2-PBDMと同様の手法により合成することが可能である。
D-マンノースの3位、4位又は6位に蛍光分子団が結合したパシフィックブルー標識D-マンノース誘導体は、それぞれ、3-アミノ-3-デオキシ-D-マンノース、4-アミノ-4-デオキシ-D-マンノース、あるいは6-アミノ-6-デオキシ-D-マンノースを原料に用いて常法に基づきパシフィックブルーをD-マンノースの3位、4位又は6位に導入することにより合成できる。また、1位への蛍光分子団の導入は、中間体として1-アジド体を合成し、還元後即座に蛍光化することで可能である。
パシフィックブルー標識L-マンノース誘導体は、原料としてアミノデオキシ-L-マンノースを用いることにより、同様にして合成できる。
下記式で表される2-MBDGは以下のようにして合成した。
収量:11.4 mg、収率:97 %
1H-NMR (400 MHz、重メタノール、ppm):
δ7.89 (d, 0.4H, J=10.1 Hz, NH), δ7.37 (dd, 1H, J=11.9 Hz and J=2.3 Hz, H5’), δ5.11 (d, 0.7H, J=3.2 Hz, H-1α), δ4.61 (d, 0.3H, J=7.8 Hz, H-1β), δ3.34-δ3.87 (m, 8H, H-2, H-3, H-4, H-5, H-6, H-6, C3’-CH2), δ2.41 (s, 3H, C4’-CH3)
ESI-MS:calcd for C18H20F2NO9 [M+H]+ 432.10, found 432.1
励起極大波長:364 nm
蛍光極大波長:458 nm
下記式で表される2-MBLGは以下のようにして合成した。
収量:10.0 mg、収率:85 %
1H-NMR (400 MHz、重メタノール、ppm):
δ7.86 (d, 0.2H, J=9.2 Hz, NH), δ7.36 (dd, 1H, J=11.9 Hz and J=2.3 Hz, H5’), δ5.10 (d, 0.7H, J=3.2 Hz, H-1α), δ4.61 (d, 0.3H, J=8.2 Hz, H-1β), δ3.35-δ3.86 (m, 8H, H-2, H-3, H-4, H-5, H-6, H-6, C3’-CH2), δ2.40 (s, 3H, C4’-CH3)
ESI-MS:calcd for C18H20F2NO9 [M+H]+ 432.10, found 432.1
励起極大波長:365 nm
蛍光極大波長:458 nm
1位、3位、4位又は6位にマリーナブルーを有する他のMBDG及びMBLGは、PBDG及びPBLGと同様にして合成できる。
2-MBDGの合成方法と同様にして、2-MBDGの合成に用いるD-グルコサミン塩酸塩の代わりにD-マンノサミン塩酸塩を用いて、2-MBDMを合成できる。
2-MBLGの合成方法と同様にして、2-MBLGの合成に用いるL-グルコサミン塩酸塩の代わりにL-マンノサミン塩酸塩を用いて、2-MBLMを合成できる。
2-HCDG(2-Deoxy-2-((7-hydroxycoumarin-3-yl)carboxamide)-D-glucose)の合成
下記式で表される2-HCDGは以下のようにして合成した。
収量:10.6 mg、収率:44 %
1H-NMR (400 MHz、重水、ppm):
δ8.58 (s x 2, 1H, Ar), δ7.53-δ7.56 (m, 1H, Ar), δ6.79 (m, 1H, Ar), δ6.67 (m, 1H, Ar), δ5.24 (d, 0.7H, J=3.7 Hz, H-1α), δ4.84 (d, 0.3H, J=8.2 Hz, H-1β), δ3.41-δ4.06 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6).
ESI-MS:calcd for C16H18NO9 [M+H]+ 368.10, found 368.1
励起極大波長:402 nm
蛍光極大波長:447 nm
下記式で表される2-MCDGは以下のようにして合成した。
収量:69.6 mg、収率:18 %
1H-NMR (400 MHz、重メタノール、ppm):
δ7.66 (m, 1H, Ar), δ6.85 (m, 2H, Ar), δ6.23 (s x 2, 1H, Ar),δ5.03 (d, 0.6H, J=3.2 Hz, H-1α), δ4.54 (d, 0.4H, J=7.3 Hz, H-1β), δ3.26-δ3.81 (m, 9H, H-2, H-3, H-4, H-5, H-6, H-6, OMe).
ESI-MS:calcd for C18H22NO9 [M+H]+ 396.13, found 396.1
励起極大波長:325 nm
蛍光極大波長:392 nm
WO2010/16587に記載の方法に従って行った。結果を図1に示す。
マウス中脳の黒質網様部から生きた神経細胞を急性単離した上、これに 2-PBDG を100μM、 2-TRLGを20μM含む混合液を37度で5分間投与した。その直前の共焦点顕微鏡画像を図1A~Cに示す。Aは、青色波長領域における蛍光像(Blue channel、波長範囲415-580 nm)である。自家蛍光により細胞位置がわかる。蛍光シグナル強度は疑似カラー表示してある。Bは、赤色波長領域における蛍光像(Red channel、580-740 nm)。AとBは共に405nm Blue diode laserを60%の強度で用いて同時に励起し、それぞれphotomultiplier(PMT)1と2を用いて、2-TRLGの侵入の有無を鋭敏に検出できるようPMT2の検出感度をPMT1よりも高めて取得したものである。Cは、明視野像(Bright field image)をA, Bの蛍光像に重ねた図である。
蛍光混合液の投与が終了して投与液の洗い流しを開始してから4分後の映像を図1D~Fに示す。画像取得条件は、A~Cと同様である。DのBlue channelでは、投与前(A)に比較して、核以外の細胞内蛍光強度が増加している様子が確認できた。中央部の暗い部分が細胞の核を示す。これに対してEにみられるようにRed channelの蛍光強度は増加していなかった(緑色の点は、細胞表面に一過性に認められた蛍光信号を疑似カラーで示したものである)。2-TRLGは比較的大型の蛍光基を分子内に有する赤色蛍光L-グルコース誘導体で、2-TRLGが細胞内に侵入していないことは、Blue channelでみられた蛍光強度の増加が2-TRLGの通過を許すような細胞膜破綻により生じたものではないことを示している。
それぞれ洗い流し開始後8分後、20分後の映像を図1G~I、及び図1J~Lに示す。いったん細胞内に取り込まれた2-PBDGが容易に減衰しない様子が確認できた。
実施例2と同様にして実験を行った。結果を図2に示す。
マウス中脳黒質網様部神経細胞を急性単離し、2-PBLG を100μM、 2-TRLGを20μM含む混合液を37度で5分間投与する投与直前の共焦点顕微鏡画像を図2A~Cに示す。
蛍光混合液の投与が終了し、投与液の洗い流しを開始してから4分後の映像を図2D~Fに示す。画像取得条件はA~Cと同様である。DのBlue channelを見ると、投与前(A)に比較して細胞内蛍光強度はほとんど増加していなかった。EのRed channelの蛍光強度もほとんど増加しておらず、2-TRLGの侵入を許すような細胞膜の破綻は見られなかった。図2G~I、及び図2J~Lは、それぞれ洗い流し開始後8分後、20分後の映像である。Dでわずかに細胞内に認められた蛍光強度の増加は洗い流し開始後20分のJでは自家蛍光レベルに戻っていた。このようにL型グルコース誘導体である2-PBLGは、 D型グルコース誘導体である2-PBDGを投与した結果(図1)に比較して、細胞内にほとんどとりこまれないことがわかる。
実施例2と同様にして実験を行った。結果を図3に示す。
マウス中脳黒質網様部から急性単離した神経細胞に2-HCDGを100μM、2-TRLGを20μM含む混合液を37℃にて3分間投与した前後の共焦点顕微鏡画像を図3に示す。A, BはそれぞれBlue channel (415-580 nm)およびRed channel (580-740 nm)で取得した投与前蛍光画像。 励起波長は405 nm。Cは、微分干渉(Differential Interference contrast, DIC)画像。Dは以上の重ね合わせである。E~HはA~Dと同様だが、 2-HCDG+2-TRLG 蛍光トレーサー液を37℃にて3分間投与した後、蛍光トレーサーの洗い流しを開始、洗い流し開始から4分後に取得した画像である。E, Fを見ると、細胞の破片(debris)については投与後、青色の蛍光強度が増加しているのに対して、神経細胞のある位置においては投与前後で蛍光強度の増加は全く検知できない。 また2-TRLGが細胞内に侵入していないことから、神経細胞の細胞膜は健全に保たれていると考えられる。
マウス中脳黒質網様部から急性単離した神経細胞に2-MCDGを100μM、2-TRLGを20μM含む混合液を比較例1と同様に投与したが、投与前後で神経細胞における蛍光強度の増加を認めなかった。
なお本実験においては最適励起波長が320nmと非常に低いため、Nikon Ti-Eリアルタイムデコンボリューション顕微鏡を用い、キセノンランプで励起フィルター320nm (半値幅40nm)、蛍光フィルター435nm(半値幅40nm)、ダイクロイックミラー409nmの構成の特注フィルターを介して画像をQ-imaging社Retiga-2000R CCDカメラで取得した。
(実験方法)
(1-1)細胞の培養
凍結保存していたMIN6細胞(大阪大学の宮崎純一教授より供与を受けて5-8回継代した細胞)を常法に従って培養に移し、7-9回継代したものを実験に供した。
(1-2)MIN6細胞の培養に用いた培養液の組成
高グルコース含有Dulbecco's modified Eagle's Medium(DMEM-HG)(SIGMA #D5648) 13.4 g, NaHCO3(Wako, No.191-01305) 3.4 g, 2-Mercaptoethanol(Wako, No.135-14352) 5 μLを1 Lの超純水(Mili Q)に溶解し、37℃のCO2インキュベーター中でpH 7.3 - 7.35となるようpHを調整した。Hyclone Fetal Bovine Serum(Cat# SH30070.03)を終濃度10 %となるように、またペニシリン-ストレプトマイシン(Gibco #15140)を終濃度0.5 %となるよう添加した。
(1-3)KRB溶液
計測には下記の組成のKRB溶液を用いた。
NaCl 129.0 mM, KCl 4.75 mM, KH2PO4 1.19 mM MgSO4・7H2O 1.19 mM, CaCl2・2H2O 1.0 mM, NaHCO3 5.02 mM, D-Glucose 5.6 mM, HEPES 10 mM (1M NaOHにてpH 7.35に調整)。なおgap junction/hemichannelを経由する蛍光標識グルコースの出入りを阻害する目的で0.1 mM Carbenoxolone(SIGMA #C4790)を加えた。なお本KRB溶液は、2-PBLG溶液を作成するための溶液として使用した。
2-PBLG溶液の調製
0.5 mg 2-PBLGバイアル全量を合計30 μL dimethyl sulfoxide(DMSO) を用いて回収、3.1 mLのKRB溶液にYamada K. et al., Nat. Protoc. 2, 753-762, 2007に準じた方法で加えることで溶解した。
2-PBDG溶液の調製
2-PBDGの代わりに2-PBLGを用いて、同様にして行った。
PB-NH2溶液の調製
0.3 mg PB-NH2バイアル一本全量を、同様にして3.1 mLのKRB溶液に溶解することで、終濃度 200 μMのPB-NH2溶液とした。
2-NBDLG溶液の調製
0.5 mg 2-NBDLGバイアル一本全量をKRB溶液7.3 mLに溶解することで、終濃度 200 μMの2-NBDLG溶液とした。
2-PBDM溶液の調製
0.5 mg 2-PBDMバイアル一本全量を、2-PBLG溶液の調整に準じて3.1 mLのKRB溶液に溶解することで、終濃度 100 μMの2-PBDM溶液とした。
2-PBDGおよび2-PBLGは、8連ピペットを用いて、それぞれ3列目および5列目のウエルに投与した。投与前には、あらかじめ各ウェルの自家蛍光を蛍光マイクロプレートリーダー(Flex Station, Molecular Device社)で計測した。測定条件は、Bottom Readで、Ex 401 nm, Em 453 nm, Cut off 420 nm, Averaging 3 、Photomultiplier感度 highにておこなった。測定方法には、Well Scan Modeを用いた。Well Scan Modeは、一つのウェル中を9つの観察領域(直径1.5 mm)に分割して、それぞれ独立に計測する。
次いで、グルコース輸送阻害剤フロレチンの効果を計測するウェル(3C, 3E, 3G)には、2-PBDG投与の5分前からフロレチン(final 150 μM)を前投与し、その他のウェル(3B, 3D, 3F) にはKRBを加えた。2-PBLGを投与する予定の5列目にも同様の操作を行った。2-PBDGおよび2-PBLGの投与は、37℃で10分間おこなった。
投与終了後は、300 μL のKRB溶液を用いてウェル中の蛍光溶液を希釈する操作を30秒づつ、決められた回数繰り返した。繰り返し回数は、対照群として設定したA行およびH行のウェルの示す蛍光強度が、細胞のないブランクのウェルの蛍光強度と同レベルになることを基準として決定し、完全に洗い流されていることを毎回の実験で確認した。2-PBDGおよび2-PBLGの場合にはこの洗い流し過程に8分を要したため、投与後の蛍光計測は9分後に実施した。
なお、この方法によれば、細胞膜状態の破綻を来たした細胞が2-PBDGおよび2-PBLGに接触後、これらの化合物をいったん細胞内に取り込んだとしても、計測時点では既に細胞外に流出し洗い流されているために、観察エリア全体の蛍光強度の増加に対する寄与は無視しうる程度と判断された。このことは別途薬理学的阻害実験で、阻害剤存在下に蛍光強度の増加がほぼ消失することにより裏付けられた。上記の方法は、他の阻害剤たとえばサイトカラシンB (10μM)を投与する場合にも同様に実施した。
結果を図4に示す。
クマリン誘導体であるパシフィックブルーをD-グルコサミンに結合した2-PBDG 、ならびにL-グルコサミンに結合した2-PBLGを、いずれも100μMの濃度で培養開始後10日目の多数のMIN6マウスインスリノーマ細胞に対して投与した結果を図4に示す。グルコース輸送阻害剤であるフロレチン(PHT) 150 μM による阻害効果も併せて示している。図4Aは、投与前後の蛍光強度を蛍光マイクロプレートリーダーで計測した結果である。括弧内の数字は、観察領域数である。投与前の蛍光は、細胞の自家蛍光を示す。蛍光強度はいずれの場合にも投与前に比較して有意に増加している(ANOVA, Bonferroni-Dunn post hoc test)。励起および蛍光波長はそれぞれ401 nmおよび453 nmとした。図4Bは、Aにおける投与前後の蛍光強度の差を示したものである。フロレチン非存在下で2-PBDGを投与した際の蛍光強度の変化を100%として表示している。2-PBDGと2-PBLGの蛍光強度の間には有意の差が認められなかった。またフロレチン存在下では、非存在下に比較して2-PBDG、2-PBLGいずれの場合にも蛍光強度の低下を認めたが、蛍光の大部分はフロレチンにより阻害されなかった。独立に実施した二回の実験のいずれにおいても同様の結果が得られ、2-PBDG、2-PBLGのフロレチンによる減少分はそれぞれ平均 22.4%および 20.0%にとどまった。
培養10日目のMIN6細胞に対するD-グルコース誘導体(2-PBDG)、L-グルコース誘導体(2-PBLG)、及びパシフィックブルー(PB)発色団をアミド化したPB-NH2投与による蛍光強度の変化とグルコース輸送阻害剤による効果を実施例4と同様にして確認した。PB-NH2は以下の構造である(Ex max.402nm, Em max. 451nm)。結果を図5に示す。
図5Aから判るように、2-PBDG (100μM)投与による蛍光強度の増加に対し、 GLUT選択的阻害剤サイトカラシンB (CB, 10μM)は有意な阻害効果を示さなかった。本例では平均蛍光強度がCB存在下で非存在下に比較して減弱しているが、独立に3回実施した結果では増加したものも認められ、一定しなかった。図5Bは、2-PBLG (100μM)あるいはPB-NH2 (100μM)投与による蛍光強度の増加に対するグルコース輸送阻害剤フロレチン(PHT, 150μM)の効果を示している。フロレチンは、2-PBLGによる蛍光強度の増加を図4と同様わずかに阻害したが、逆にPB-NH2による蛍光強度の増加を著しく促進した。Bの縦軸の単位は、A、Cと異なることに注意されたい。 2-PBLGおよびPB-NH2への投与実験は同一培養プレート上で同時に実施し、独立に実施した3回の実験のいずれにおいてもPB-NH2応答へのフロレチンによる著しい増強効果が確認され、蛍光増加はPB-NH2のみ投与した場合の平均384.1 ± 24.2%に達した(n = 3)。また糖骨格を有しないPB-NH2は、糖骨格を有するL-グルコース誘導体2-PBLGより有意に大きな蛍光強度の増加を示した。
実施例4と同様にして実験を行った。結果を図6に示す。
培養10日目(10DIV)のMIN6細胞(20000 cells/well)に対し、2-PBDM(100μM)を投与して、投与前後の蛍光強度の増加に対するフロレチン (150μM, PHT)による阻害効果をフレックスステーションで計測したところ、2-PBDMはフロレチンによりわずかながら有意な阻害効果が確認された。実験は独立に3回実施し、すべて同様の結果が得られた。2-PBDM投与実験では極大励起光波長404nmで励起し、極大蛍光波長453nmで蛍光取得した。
(実験方法)
(1)マウスインスリノーマ細胞(MIN6)の調製
MIN6細胞を10 x 104 cells/mLの割合で懸濁させた培養液をガラスカバースリップ上に10μL滴下した後、ガラス面に付着させ、培養液を 3 mL加えて培養した。培養液は3日に一回半量を交換した。
(1-1)MIN6細胞の培養
凍結保存していたMIN6細胞(大阪大学の宮崎純一教授より供与を受けて5-8回継代した細胞)を常法に従って培養に移し、7-9回継代したものを実験に供した。培養液は2日に一回半量を交換した。
(1-2)MIN6細胞の培養に用いた培養液の組成
高グルコース含有Dulbecco's modified Eagle's Medium(DMEM-HG)(SIGMA #D5648) 13.4 g, NaHCO3(Wako, No.191-01305) 3.4 g, 2-Mercaptoethanol(Wako, No.135-14352) 5 μLを1 Lの超純水(Mili Q)に溶解し、37℃のCO2インキュベーター中でpH 7.3 - 7.35となるようpHを調整した。Hyclone Fetal Bovine Serum(Cat# SH30070.03)を終濃度10 %となるように、またペニシリン-ストレプトマイシン(Gibco #15140)を終濃度0.5 %となるよう添加した。
(1-3)MIN6細胞を10 x 104 cells/mLの割合で懸濁させた培養液
MIN6細胞を、細胞数が10 x 104 cells/mLになるよう培養液を用いて調製した。
2-PBLG溶液の調製
0.5 mg 2-PBLGバイアル全量を合計30 μL dimethyl sulfoxide(DMSO) を用いて回収、6.25mLの画像取得用HEPES溶液にYamada K. et al., Nat. Protoc. 2, 753-762, 2007に準じた方法で加えることで溶解した。
2-PBDG溶液の調製
2-PBDGの代わりに2-PBLGを用いて、同様にして行った。
2-NBDLG溶液の調製
0.5 mg 2-NBDLGバイアル一本全量を画像取得用HEPES溶液14.6 mLに溶解することで、終濃度 100 μMの2-NBDLG溶液とした。
2-TRLG溶液の調製
0.2 mg 2-TRLGバイアル全量を合計100μLのDMSOを用いて回収。6.5mLのKRB溶液に加えることで溶解した。
2-PBLG + 2-TRLG混合溶液の調製
上記の2-PBLG溶液ならびに2-TRLG溶液を1:1で混合して目的の蛍光誘導体混合液を作成した。
(2-1)画像取得用HEPES溶液
フレックスステーション実験で用いたKRB溶液と同一の下記の組成の溶液を用いた。
NaCl 129.0 mM, KCl 4.75 mM, KH2PO4 1.19 mM MgSO4・7H2O 1.19 mM, CaCl2・2H2O 1.0 mM, NaHCO3 5.02 mM, D-Glucose 5.6 mM, HEPES 10 mM (1M NaOHにてpH 7.35に調整)。なおgap junction/hemichannelを経由する蛍光標識グルコースの出入りを阻害する目的で0.1 mM Carbenoxolone(SIGMA #C4790)を加えた。なお本画像取得用HEPES溶液は、2-PBLG溶液を作成するための溶液として、また、2-PBLG/2-TRLG溶液及び2-PBLG/2-NBDLG/2-TRLG溶液を作成するための溶液として使用した。
MIN6細胞を付着させ10-13日間培養したガラスカバースリップを、35 mmディッシュに満たしたD-グルコース5.6 mMを含有するDAPI溶液中に移し、37℃で加温しながら45分から1時間静置して細胞にDAPIを取り込ませた。別実験で、共焦点顕微鏡上で継時的に観察しながらDAPIを投与する実験を行い、実験時間内にDAPI投与および405 nmのレーザー光照射による細胞の形態変化は認められないことを確認した。
DAPI溶液の調製:4',6-Diamidino-2-phenylindole DAPI(No. 049-18801, Wako Pure Chemical Industries, Osaka)を画像取得用HEPES溶液に終濃度1μg/mL の割合で溶解して用いた。
レーザースキャン共焦点顕微鏡(ライカ社TCS SP5)上のユニバーサルステージ(Leica 11600234)上にセットされた灌流チャンバー内の画像取得用HEPES溶液中に、MIN6細胞を培養したガラスカバースリップを移し、チャンバー底部のガラス面上に軽く密着させた。静置後、カバースリップの両側を左右からカバースリップの長軸に平行に2枚の長方形の金属ガイド(長さ10 mm、幅2 mm、厚み0.7 mm、銀製)を用いて押さえ、慎重に押し当てることで、流れの中でもカバースリップが動かないようにした。またこの2枚の金属ガイドに挟まれた空間内では、灌流液が層流となってスムーズに流れ、すみやかな液交換が可能となるという優れた効果がある。
(4-1)レーザースキャン共焦点顕微鏡ステージ上の蛍光測定用灌流チャンバー
底部に対物レンズ用の丸穴(直径18 mm)のあいたアルミ製加温制御プラットフォーム(PH1、Warner Instruments, USA、温度制御装置TC-324により37℃に加温, Warner Instruments)上に、シリコングリース(HIVAC-G, Shin-Etsu Silicone, Tokyo)を用いて、カバーガラス(幅24 mm x 長さ50 mm、厚さ No. 1, Warner Instruments No. CS-24/50)をプラットフォーム中央の丸穴以外の部分に密着させた。次いでカバーガラス上に、中央に流線型に穴開け加工(ガラス底面に接する側は幅10 mm x 長さ35 mm、曲率半径33 mm、ガラス面に接しない側すなわち上方がわずかに広くなるように加工した)を施した厚さ1 mmのシリコン板(幅20 mm x 長さ50 mm)を載せ、シリコングリースを用いずにカバーガラスと密着させた。
シリコン板上の流線型穴の上流隅に、先端をフラットにした太さ20ゲージのカテラン針をセットして、インレットとして用いた。
灌流液の排出用ステンレス管(アウトレット)は、非特許文献16に記載の方法に準じて先端部を平らにつぶした上で斜めにカットしたものを用い、真空吸引時、空気と溶液の両者を同時に吸引することで安定させる方式とした。
(a)灌流液の加温と灌流チャンバーへの供給
灌流液供給システムは、コントロール溶液用の60 mL注射筒一本と、薬剤供給用の10 mL注射筒5本を備え、電磁バルブで随時切り替えて灌流することができる。本発明に関わる実験では、60 mL注射筒を用いて5.6 mMグルコース含有画像取得用HEPES溶液を、5系統ある10 mL注射筒のうち一本を用いて2-PBLG/2-NBDLG/2-TRLG混合溶液もしくは2-PBDG/2-TRLG混合溶液、もしくは2-PBLG/2-TRLG混合溶液を投与した。以下に述べるように、両者は灌流チャンバー内でバブルを生じないよう、あらかじめ加温された上、灌流チャンバーに導かれる前に一本のチューブに合同し、流量調節器で速度調節されてから、再度インラインヒーターにて加温し共焦点顕微鏡上の灌流チャンバーに供給された。
画像取得用HEPES溶液は、アルミ製シリンジヒーター(Model SW-61, 温度制御ユニットはNo. TC-324B, Warner Instruments)中で暖められた60 mL注射筒から、溶液供給ラインのチューブ内をフラッシュするための3方活栓に接続され、続いて細くガス透過性も低いソフトチューブ(Pharmedチューブ, AY242409, Saint-Gobain Performance Plastics, Ohio)を介して超小型電磁バルブ(EXAK-3, 3 way clean valve, Takasago Electric, Nagoya)のnormally open側に接続した。電磁バルブの開閉はパルス発生装置(Master 8, AMPI社, Israel)で制御した。画像取得用HEPES溶液についてはペリスタルティックポンプ(MCPポンプ12 rollers、Ismatec)を用いてメジウムビンから60 mL注射筒内に持続的に供給し、実験中に注射筒内の溶液上面の高さが変化しないよう、溶液落下速度と等しくなるようにポンプの溶液供給スピードを精密に調整した。ペリスタルティックポンプの溶液供給速度はデジタル表示されるため、もしも灌流チャンバーへの溶液供給速度に実験中に変化があれば溶液面の高さが変わることで直ちにわかる。このように本溶液は常時更新されるため、液温維持のためシリンジヒーターSW-61は38.5℃に設定した。
一方、2-PBLGと2-TRLG混合溶液あるいは2-PBLG/2-NBDLG/2-TRLG混合溶液等は、シリンジヒーター(Model SW-6, 温度制御ユニットはNo. TC-324, Warner Instruments)にセットされ37.5℃に加温された10 mL注射筒から供給した。本注射筒は三方活栓を介して、画像取得用HEPES溶液とは別個の電磁バルブのnormally closed側に接続されており、パルス発生装置の制御によりコントロール溶液と随時切り替えて供給できる。シリンジヒーターSW-6には6本の10 mL注射筒がセットでき、1本には蒸留水を入れて加温ブロックの温度モニター用プローブを挿入した。
コントロール溶液である画像取得用HEPES溶液と、2-PBLGと2-TRLG混合溶液あるいは2-PBLG/2-NBDLG/2-TRLG混合溶液等とは、電磁バルブのアウトレットを出た後、6ポートの小型マニフォールド(MPP-6, Warner Instruments)により1本に集められた。MPP-6マニフォールドのアウトレットは短いPharmedチューブに接続し、このチューブをスクリューにより開度を増減できる流量調節器にはさみこみ、開度の調整により流量を1.2 ± 0.2 mL/分に調整した。本Pharmedチューブは、最短距離でインラインヒーター(Multi-Line In-Line Solution Heater SHM-8、温度制御ユニットはTC-324B、Warner Instruments)に接続した。これは灌流チャンバーに導入する溶液温度を、導入直前に加温するためである。SHM-8インラインヒーターの温度は灌流速度にあわせて、チャンバー中での灌流液の実測温度が、カバースリップの存在する領域で36-37℃になるように調整した。加温された溶液は短いタイゴンチューブ(R-3603, inner diameter 1/32 inch)を介して最短距離で灌流チャンバー上流に配置されたステンレスパイプ(インレット)に接続し、灌流チャンバー内に供給した。
注射筒からの溶液供給は静水圧を用いて供給圧力を決めるため、高さの違いが灌流速度の違いとなってチャンバー内の水面高の変化をもたらさないように、2-PBLGと2-TRLG混合溶液あるいは2-PBLG/2-NBDLG/2-TRLG混合溶液等ついては投与時間が短いことから一回の実験では実験中に液供給は行わず、各実験が終了するごとに液の上面が蛍光グルコースを含有しないHEPES溶液とほぼ同じ高さになるように溶液を追加した。また注射筒に接続されたチューブの長さと太さの調整により、コントロール溶液である画像取得用HEPES溶液の灌流速度と同じ速度で排出されるように慎重に調整することで、液交換による液面の変動を避けることができる。また実験終了後および開始前にはチューブ内部を十分フラッシュしてスムーズな流れを確保した。
灌流液の排出用のステンレス管(アウトレット)をタイゴンチューブで二つの大型ガラストラップに順次導き、真空ポンプ(DAP-15, ULVAC KIKO, Inc)で緩やかに吸引した。吸引圧は二つの大型ガラストラップの中間で吸引ラインから枝分かれさせたラインに設置した圧力計でモニターし、三方活栓の開閉度の制御により35kPaとなるように調整した。
灌流チャンバー内の層流の確保は、まず青色色素(Pontamine sky blue, 1 %以下の濃度に希釈して用いる)溶液をインレット付近に滴下して、流れの左右対称性、均一性と再現性を確保した。
チャンバー内灌流溶液中の各部の温度の確認は極細サーミスタープローブ(Physitemp社IT-23)を用いた(非特許文献16)。またアウトレット先端部には実験中にHEPES溶液由来の塩が付着することで吸引圧が変化するのを防ぐため、チャンバー上に設置されたオペレーション顕微鏡(POM-50II, KONAN MEDICAL, 西宮)で実験ごとに確認し、クリーニングを行った。
レーザースキャン共焦点顕微鏡(Leica製TCS-SP5システム、顕微鏡本体はDMI6000 CS trino電動倒立顕微鏡)をコンベンショナルモードで使用した。使用レーザーは、405 nmダイオードレーザーを、2-PBLGや2-PBDGの励起、2-PBLG(もしくは2-PBDG)と2-TRLGとの混合溶液を単一光源で励起する場合、またDAPIによる核のライブ染色に使用した。照射強度は音響光学偏光素子(Acoustic Optical Tunable Filter, AOTF) により十分な観察強度が得られるように使用蛍光色素に合わせて適切に調節した。また2-NBDLGおよび2-TRLGは488 nm Argon laserで励起した。スキャンスピードは200Hzもしくは400Hzを用いた。
蛍光検出は、2-PBDGもしくは2-PBLGによる青色蛍光の検出用にphotomultiplier検出器(PMT)1を2-PBLG/2-TRLGの二色検出の場合には415-580 nmの波長検出範囲で、2-PBLG/2-NBDLG/2-TRLG三色検出の場合には415-500 nmの波長検出範囲に設定して画像取得した。2-NBDLGによる緑色蛍光の検出用にPMT2(緑チャネルと名付けた、以下同じ)を500-580 nmの波長検出範囲で使用した。また、2-TRLGによる赤色蛍光の検出用にPMT3(赤チャネルと名付けた、以下同じ)を580-740 nmの波長検出範囲で使用した。以上の青、緑、および赤等の蛍光検出波長領域の選別は通常用いられるEmissionフィルター方式によらず、プリズム分光とスリットを組み合わせた方式(Leica,TCS-SP5の標準)により取得した。488nmアルゴンレーザーを使用して際のビームスプリッターは500 nm(RSP500)を使用した。405 nm用のビームスプリッターはSP5システムでは上記と独立に415 nmの固定式となる。2-PBLG/2-NBDLG/2-TRLG三色検出の実験では、蛍光励起に際し、最初に488 nm励起による2-NBDLG(緑色)と2-TRLG(赤色)の画像取得をおこない、Sequential modeにてその後直ちに405 nm励起により2-PBDLG(青色)の画像を取得した。2-PBLG/2-TRLGの二色検出の場合には、405 nmダイオードレーザーの単一励起により、2-PBLG(青色)と2-TRLG(赤色)の画像を、2-TRLGの細胞内への侵入を効果的に検出できるように赤色波長領域における検出感度を青色波長における検出感度より高くした感度設定により(青色617V、赤色738V等)同時取得した。
立体的な腫瘍細胞塊の構造的特徴を捉えるために用いた微分干渉(DIC)画像の取得は、488 nm(もしくは405nm)励起時に透過光用検出器PMT Transを同時に使用して検出(典型的な検出感度は145-200 V)したものを用いた。微分干渉方式(DIC)の画像取得に必要なポラライザーやアナライザーを光路に入れる為の切り替え時間や切り替えショックの問題を回避するため、405 nm励起による画像取得時にもDIC用ポラライザーとアナライザーは光路に入れたままとした。
本法では、xy軸への高い解像力と細胞塊の全体を視野内に含む画角を求めて対物レンズは高解像力のx40oilレンズ(HCX PL APO CS 40.0x1.25 OIL UV, NA1.25)を絞り開放で使用した。取得蛍光強度を稼ぐためPinholeサイズは3 airy unitとした。このpinhole sizeでもz軸方向に細胞内の核と細胞質を実用的に区別し得ることが取得画像で確認された。ズームは基本的に使用せず(1倍)、1024x1024もしくは512x512の画素数で、12 bitの深さで画像取得した。
なお以上の溶液の投与およびすべての画像取得は24時間一定室温(24℃)に保持された暗室内で行った。
結果を図7~図17に示す。
図7において、培養下で3次元的な発達を示したがん細胞塊(スフェロイド、培養15日目のMIN6細胞)において、アポトーシスを起こしている細胞と、壊死を起こしている細胞、ならびにDAPIで強く染色される細胞核を有する細胞の空間的配置が確認できる。図7Aは、一定程度以上の直径(およそ100ミクロン以上)とかさ高さ(おおよそ50ミクロン以上)を呈するに至ったスフェロイドの中心部には、青色蛍光を発する4’,6-diamidino-2-phenylindole (DAPI)を異常に強く結合する核を擁する細胞が存在する。DAPIはホルマリン固定して用いずに、生きた細胞にそのまま適用している。図7Bは、ライブアポトーシスマーカーpSIVA-IANBD (IMGENEX, San Diego, USA) により、アポトーシスを起こしている細胞が緑色の蛍光で可視化されている。陽性細胞はスフェロイド周辺部などに点在している。図7Cは、赤色の蛍光は、壊死(ネクローシス)マーカーとして一般的なpropidium iodide (PI)が細胞内に侵入した細胞を示す。スフェロイド中央部付近に比較的集中していることがわかる。図7Dは、微分干渉顕微鏡イメージを示す。図7Eは、以上の重ね合わせ画像を示す。画像取得は2-PBLG/2-NBDLG/2-TRLG三色検出の方法に準じて実施した。
図9は、100μMの2-PBDLG、100μMの2-NBDLG及び20μMの2-TRLGからなる混合溶液を投与中のMIN6細胞塊の緑(A)、赤(B)、青(D)の蛍光取得画像、微分干渉顕微鏡像(C)、および以上の重ね合わせ像(E)である。細胞塊中心部の多くの細胞は細胞膜透過性が亢進しているため、投与中にいずれの蛍光標識グルコース誘導体も強く取り込む傾向がみられる。なお赤緑青の三色は重ね合わせると白色を呈する。画像は、A, B, Cの画像を数秒間かけて同時取得した後、シークエンシャルにDを画像取得したため、Dの画像取得時点では潅流液がより深く細胞塊内部に侵入している。
実施例7と同様にして、2-PBLG/2-NBDLG/2-TRLGの混合溶液の代わりに、2-PBLG/2-TRLGの混合溶液を用いた。
図14は、培養13日目のMIN6細胞塊の蛍光標識グルコース誘導体投与前のイメージである。A、Bはそれぞれ580-740 nm (赤色)および415-580 nm (青色)の波長域における蛍光取得画像。Cは微分干渉顕微鏡像。Dはこれらの重ね合わせ画像である。
図15は、20μMの2-TRLGと100μMの2-PBLGを含む蛍光混合液をMIN6細胞塊に5分間投与した後、洗い流しを開始して2分経過した時点での映像である。Aを見ると、細胞膜不透過性の2-TRLG は、主として細胞塊中心部にあり細胞膜透過性が亢進している細胞の内部に侵入している。また、この時点では細胞塊外縁や細胞塊外にある細胞組織の破片(Debris)の一部も染色されている。Bを見ると、2-PBLGも細胞膜透過性の亢進している細胞内にいったん侵入する。しかし、この時点で既に2-PBLGはこうした細胞内からの流出が進み始め、細胞塊中心部で蛍光強度が減弱しはじめている様子がわかる。その中で、いくつかの細胞の青色蛍光が異常に強い点に注意されたい。ここで実施例7のように、2-PBLGおよび2-TRLGの投与時に、2-NBDLGも同時に投与したケースでは、488 nmのアルゴンレーザーで2-NBDLGならびに2-TRLGの励起を行った後、405 nmダイオードレーザーで2-PBLGの励起を行った。この場合、2-PBLGの蛍光極大が2-NBDLGの励起波長と重なるため、局所濃度によってはFRET効果により2-PBLGの蛍光シグナルが弱まることも考えられる。また488 nmで励起された2-TRLGの580-740 nmの蛍光強度分布の中に、2-NBDLGの580nm以上の領域での(長波長側のすそ野)蛍光強度の増加によるものが混入する。一方、2-PBLG/2-TRLGの混合溶液を用いた場合には、これらFRET効果および長波長側のすそ野による影響のいずれについても回避することができるため、蛍光強度の増加を分離することが可能になり、定量化において有利である。
図17は、図16と同様だが、投与終了し、洗い流しを開始してから12分後のイメージ。引き続き2-PBLGの強陽性信号を呈する細胞が複数認められる(B, D)。
Claims (22)
- 蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを分子内に有する蛍光標識糖誘導体を含む、標的細胞又は標的細胞内分子をイメージングするための組成物。
- 蛍光標識糖誘導体が、グルコース誘導体、フルクトース誘導体、ガラクトース誘導体又はマンノース誘導体である、請求項1に記載の組成物。
- 前記蛍光分子団が、グルコース、フルクトース、ガラクトース又はマンノースに、-NH-結合を介して結合している、請求項2に記載の組成物。
- 蛍光標識糖誘導体が、グルコースの1位、2位、3位、4位又は6位に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した分子である請求項1に記載の組成物。
- 蛍光標識糖誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースからなる群より選ばれる分子である請求項4に記載の組成物。
- 蛍光標識糖誘導体が、マンノースの1位、2位、3位、4位又は6位に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した分子である請求項1に記載の組成物。
- 蛍光標識糖誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-マンノース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-マンノース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-マンノース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-マンノースからなる群より選ばれる分子である請求項6に記載の組成物。
- 標的細胞又は標的細胞内分子をイメージングする方法であって、以下の工程、
a.標的細胞に、請求項1~7のいずれか一つに記載の組成物を接触させる工程、及び
b.該標的細胞内に存在する該糖誘導体を検出する工程、
を含む細胞のイメージング方法。 - グルコース、フルクトース、ガラクトース及びマンノースからなる群から選ばれる糖に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した蛍光標識糖誘導体。
- 2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-マンノース、2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-D-マンノース、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-マンノース、及び2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-マンノースからなる群より選ばれる蛍光標識糖誘導体。
- 2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-グルコース、又は2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-D-マンノースである蛍光標識糖誘導体。
- がん又はがん細胞を検出するための方法であって、以下の工程、
a.標的細胞に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを結合した蛍光標識L-グルコース誘導体を含有する組成物を接触させる工程、及び
b.該標的細胞内に存在する該L-グルコース誘導体を検出する工程、
を含む検出方法。 - 前記蛍光標識L-グルコース誘導体が、L-グルコースの1位、2位、3位、4位又は6位に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した分子である請求項12に記載の検出方法。
- 前記蛍光標識L-グルコース誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである請求項12に記載の検出方法。
- 前記工程aにおける検出が標的細胞をイメージングすることにより行う請求項12~14のいずれか一つに記載の検出方法。
- 前記工程aにおける組成物が、2位にスルホローダミンをスルホンアミド結合せしめた2-アミノ-2-デオキシ-L-グルコースをさらに含み、かつ前記工程bが、標的細胞内に存在する蛍光標識L-グルコース誘導体を検出する工程である、請求項12~15のいずれか一つに記載の検出方法
- 標的細胞が腫瘍細胞塊中の細胞である、請求項12~16のいずれか一つに記載の検出方法。
- 標的がん細胞をイメージングするためのイメージング剤であって、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを結合した蛍光標識L-グルコース誘導体を含むがん細胞のイメージング剤。
- 前記蛍光標識L-グルコース誘導体が、L-グルコースの1位、2位、3位、4位又は6位に、蛍光分子団として3-カルボキシ-6,8-ジフルオロ-7-ヒドロキシクマリン又は3-カルボキシメチル-6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリンを-NH-結合を介して結合した蛍光標識L-グルコース誘導体である請求項18に記載のイメージング剤。
- 前記蛍光標識L-グルコース誘導体が、2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである請求項18に記載のイメージング剤。
- 前記イメージング剤がさらに、2位にスルホローダミンをスルホンアミド結合せしめた2-アミノ-2-デオキシ-L-グルコースを含む請求項18~20のいずれか一つに記載のイメージング剤。
- 2-デオキシ-2-((6,8-ジフルオロ-7-ヒドロキシクマリン-3-イル)カルボキサミド)-L-グルコース、又は2-デオキシ-2-(2-(6,8-ジフルオロ-7-ヒドロキシ-4-メチルクマリン-3-イル)アセタミド)-L-グルコースである蛍光標識L-グルコース誘導体。
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JP2016111982A (ja) * | 2014-12-17 | 2016-06-23 | 富士フイルム株式会社 | 細胞評価統合方法 |
JP2019034311A (ja) * | 2017-08-10 | 2019-03-07 | ユシロ化学工業株式会社 | ダイカスト用離型剤組成物及びその製造方法 |
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JP2015205870A (ja) * | 2014-04-08 | 2015-11-19 | 国立大学法人弘前大学 | 新規なグルコース誘導体、および該誘導体を用いた細胞イメージング方法およびイメージング剤 |
US10001487B2 (en) | 2014-04-08 | 2018-06-19 | Hirosaki University | Glucose derivative, and cell imaging method and imaging agent using said derivative |
US10509041B2 (en) | 2014-04-08 | 2019-12-17 | Hirosaki University | Glucose derivative, and cell imaging method and imaging agent using said derivative |
JP2016111982A (ja) * | 2014-12-17 | 2016-06-23 | 富士フイルム株式会社 | 細胞評価統合方法 |
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JP2019034311A (ja) * | 2017-08-10 | 2019-03-07 | ユシロ化学工業株式会社 | ダイカスト用離型剤組成物及びその製造方法 |
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EP2905620A4 (en) | 2016-04-27 |
US20150369797A1 (en) | 2015-12-24 |
JP6327565B2 (ja) | 2018-05-23 |
EP2905620A1 (en) | 2015-08-12 |
US10288604B2 (en) | 2019-05-14 |
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