JP2011237192A - Fluorescence staining method of mitochondria - Google Patents

Abstract

An object of the present invention is to provide a mitochondrial fluorescent staining agent, a mitochondrial fluorescent staining kit, and a mitochondrial fluorescent staining method.
SOLUTION: General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A 5-aminolevulinic acid or a salt thereof represented by the above and an ABCG2 transporter inhibitor are used in combination.
[Selection figure] None

Description

  The present invention relates to a mitochondrial fluorescent staining agent, a mitochondrial fluorescent staining kit, and a mitochondrial fluorescent staining method.

  Conventionally, vital dyes have been used to stain intracellular mitochondria. For example, in order to observe the mitochondrial intracellular behavior of early embryogenesis sperm, Rhodamine (registered trademark) 123 (manufactured by invitrogen; cat. R302, R22420) or Mito Tracker (registered trademark) (manufactured by invitrogen; cat. M7512) And observing under a fluorescent microscope (for example, see Non-Patent Documents 1 to 4). In addition, mitochondria are visualized by adding a target sequence to mitochondria to green fluorescent protein (GFP) (for example, see Non-Patent Documents 5 to 6).

  However, the above-mentioned vital dyes are easily discolored by excitation light, resulting in a decrease in luminance, a point where dyes are easily dissociated even after staining, and it is necessary to fix cells depending on the dyes. There were insufficient points such as certain points. In addition, the above-described mitochondrial observation method using GFP has been insufficient because it involves genetic recombination. Furthermore, analysis of GFP labeling of organelles and physiologically active substances that are deeply functionally related to mitochondria has been actively conducted. In such cases, while distinguishing these organelles and mitochondria, For simultaneous observation, it was necessary to label mitochondria with a fluorescent protein having wavelength characteristics other than GFP.

  5-Aminolevulinic acid (5-ALA) is a kind of amino acid and is the only raw material of porphyrins essential for chlorophyll in plants and heme in animal blood. In animals, 5-aminolevulinic acid is metabolized in turn to porphobilinogen, hydroxymethylbilane, uroporphyrinogen III, coproporphyrin III, protoporphyrinogen IX, protoporphyrin IX, and protoporphyrin is an iron ion. It is known that heme binds to globin and becomes hemoglobin.

  ABCG2 (ATP-binding cassette, subfamily G, member 2), also called breast cancer resistant protein (BCRP), is a transmembrane ABC (ATP-binding cassette) trans that exists in the mitochondrial membrane. One of the porters. ABCG2 uses protoporphyrin IX as one of its substrates. When ABCG2 is deleted, protoporphyrin IX accumulates in cells (see Non-Patent Documents 7 to 8), and reaction from protoporphyrin IX to heme. Is carried out in mitochondria, and it is also known that protoporphyrin IX can be excreted into the cytoplasm via ABCG2 (see Non-Patent Document 9). Furthermore, it is known that 5-aminolevulinic acid is metabolized to coporphobilinogen III in the cytoplasm via porphobilinogen and then taken into mitochondria via ABCB6 (see Non-Patent Document 10). ). Further, Non-Patent Document 11 describes that when ES cells are treated with ABCG2 inhibitor fmitremorgin C (FTC), intracellular protoporphyrin IX increases, or ES cells are treated with 5-ALA. It is described that the excretion of protoporphyrin IX after induction of endogenous protoporphyrin IX was reduced by the addition of FTC. Non-patent document 12 describes that when ferrochelatase inhibitor DFX (inhibitor of an enzyme that generates heme from protoporphyrin IX) is allowed to act on bladder cancer cell 253JB-V, protoporphyrin via 5-ALA. Although it has been described that the accumulation of IX in cells is increased, this phenomenon was not seen in normal cells.

  As described above, it has not been known that mitochondria can be stained very simply and clearly by using 5-aminolevulinic acid and an ABCG2 transporter inhibitor in combination.

Kaneda, H et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4542-4546. Cummins, J. M et al., (1997) Zygote 5, 301-308. Sutovsky, P. et al., (1996) Biol. Reprod. 55, 1195-1205. Sutovsky, P. et al., (2000) Biol. Reprod. 63, 582-590. Rizzuto, R et al., (1995) Curr. Biol. 5, 635-642. Rizzuto, R et al., (1996) Curr. Biol. 6, 183-188. J. W. Jonker et al., (2007) Am J Physiol Cell Physiol 292, C2204-C2212. S. Zhou et al., (2005) Blood 105, 2571-2576. Tamura, A et al., (2007) Drug Metabolism and Pharmacokinetics 22, No. 6, 428-440. Iqbal Hamza, (2006) ACS Chem. Biol., 1 (10), pp 627-629. Susanto J. et al., (2008) PLoS ONE, vol. 3, issue 12, p.e4023 Inoue, K et al., (2009) Pathology 76, 303-314

  An object of the present invention is to provide a mitochondrial fluorescent staining agent, a mitochondrial fluorescent staining kit, and a mitochondrial fluorescent staining method.

  As described above, conventional vital dyes are easily discolored by excitation light, resulting in a decrease in brightness, the dye easily dissociates even after staining, and some dyes fix cells. There were insufficient points, such as the need to do. In addition, the conventional method for observing mitochondria using GFP has insufficient points such as being complicated due to genetic recombination operations. Therefore, the present inventors conducted extensive studies to improve the insufficient points of the conventional method. As a result, when 5-aminolevulinic acid and an ABCG2 transporter inhibitor are used in combination, a metabolite of 5-aminolevulinic acid is used. The amount of a certain protoporphyrin IX excreted by the ABCG2 transporter into the cytoplasm outside the mitochondria is significantly reduced, and the protoporphyrin IX accumulates in the mitochondria. As a result, the present invention has been completed.

That is, the present invention provides [1] general formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A mitochondrial fluorescent stain characterized by containing a 5-aminolevulinic acid represented by the formula (I) or a salt thereof and an ABCG2 transporter inhibitor;
[2] ABCG2 transporter inhibitor is reserpine, tryprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide And irinotecan, topotecan, SN-38, flavopiridol, CI1033, tamoxifen, and the mitochondrial fluorescent staining agent according to [1] above.

The present invention also provides [3] general formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A mitochondrial fluorescence staining kit comprising a 5-aminolevulinic acid represented by the formula (I) or a salt thereof and an ABCG2 transporter inhibitor;
[4] ABCG2 transporter inhibitor is reserpine, tryprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, And irinotecan, topotecan, SN-38, flavopiridol, CI1033, and tamoxifen. The mitochondrial fluorescence staining kit according to [3] above.

Furthermore, the present invention provides [5] general formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A method for fluorescent staining of mitochondria, comprising a step of using a 5-aminolevulinic acid represented by the following or a salt thereof in combination with an ABCG2 transporter inhibitor;
[6] The method for fluorescent staining of mitochondria according to [5] above, which comprises the following steps (a) and (b):
(A) General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A step of contacting a target cell with a 5-aminolevulinic acid represented by the formula (1) or a salt thereof and an ABCG2 transporter inhibitor;
(B) irradiating target cells with excitation light for protoporphyrin IX;
[7] (c) The mitochondrial fluorescence staining method according to [6] above, further comprising a step of washing the target cell between step (a) and step (b).

  According to the present invention, mitochondria can be fluorescently stained very easily and clearly. Moreover, since the component used for the fluorescent staining of the present invention has no cytotoxicity and can be used for living cells, it is not necessary to fix the cells for observation. In addition, in the fluorescent staining according to the present invention, it is not necessary to consider the dissociation of the dye in principle, and thus clear fluorescence can be observed over a long period of time. Protoporphyrin IX has a maximum fluorescence wavelength of 635 nm and does not overlap with the maximum fluorescence wavelength of GFP (509 nm). Therefore, even when organelles and physiologically active substances that are deeply functionally related to mitochondria are labeled with GFP If mitochondria are fluorescently stained according to the present invention, these organelles and mitochondria can be observed simultaneously while distinguishing them.

It is a figure which shows the result of having observed the fluorescence microscope, after carrying out the fluorescence dyeing | staining of a normal human umbilical vein endothelial cell (HUVEC). FIG. 1 upper left panel: shows the result of fluorescent microscope observation after staining HUVEC (ALA) with NAO. FIG. 1 upper center panel: shows the result of observation with a fluorescence microscope after adding 5-aminolevulinic acid (ALA) to HUVEC (ALA). FIG. 1 upper right panel: shows the result of overlaying the upper left panel of FIG. 1 and the upper center panel of FIG. FIG. 1 lower left panel: shows the result of fluorescent microscope observation after staining HUVEC (ALA + reserpine) with NAO. FIG. 1 middle lower panel: shows the result of observation with a fluorescence microscope after adding 5-aminolevulinic acid (ALA) and reserpine to HUVEC (ALA + reserpine). FIG. 1 lower right panel: shows the result of overlapping the lower left panel of FIG. 1 and the lower center panel of FIG. It is a figure which shows the result of having observed the fluorescence microscope after carrying out the fluorescence stain of the immortal monkey kidney fibroblast (COS cell). Fig. 2 upper left panel: shows the result of fluorescent microscope observation after staining COS cells (ALA) with NAO. Fig. 2 upper center panel: shows the result of fluorescence microscope observation after adding 5-aminolevulinic acid (ALA) to COS cells (ALA). FIG. 2 upper right panel: shows the result of overlaying the upper left panel of FIG. 2 and the upper center panel of FIG. FIG. 2 lower left panel: shows the result of fluorescent microscope observation after staining COS cells (ALA + reserpine) with NAO. FIG. 2 lower center panel: shows the result of fluorescence microscope observation after adding 5-aminolevulinic acid (ALA) and reserpine to COS cells (ALA + reserpine). FIG. 2 lower right panel: shows the result of overlaying the lower left panel of FIG. 2 and the lower center panel of FIG.

1. Mitochondrial fluorescent stain of the present invention The mitochondrial fluorescent stain of the present invention (hereinafter also simply referred to as “fluorescent stain of the present invention”) is a 5-aminolevulin represented by the above general formula (1). It is not particularly limited as long as it contains an acid or a salt thereof (hereinafter also referred to as “5-aminolevulinic acid or the like in the present invention”) and an ABCG2 transporter inhibitor, and is an object of the fluorescent staining agent of the present invention. The mitochondria is not particularly limited as long as it is a mitochondria in a living cell. When the fluorescent stain of the present invention is used, the amount of protoporphyrin IX, which is a metabolite of 5-aminolevulinic acid, excreted by the ABCG2 transporter into the cytoplasm outside the mitochondria is remarkably reduced. Accumulation makes it possible to fluorescently stain mitochondria very easily and clearly.

In the general formula (1), R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group, or an aralkyl group.

  The alkyl group is preferably a linear or branched alkyl group having 1 to 24 carbon atoms, more preferably an alkyl group having 1 to 18 carbon atoms, and particularly preferably an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group and the like.

  As said acyl group, a C1-C12 linear or branched alkanoyl group, an alkenylcarbonyl group, or an aroyl group is preferable, and a C1-C6 alkanoyl group is especially preferable. Examples of the acyl group include formyl group, acetyl group, propionyl group, butyryl group and the like.

  As said alkoxycarbonyl group, a C2-C13 alkoxycarbonyl group is preferable, and a C2-C7 alkoxycarbonyl group is especially preferable. Examples of the alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, and an isopropoxycarbonyl group.

  As said aryl group, a C6-C16 aryl group is preferable, for example, a phenyl group, a naphthyl group, etc. are mentioned.

  As said aralkyl group, the group which consists of a C6-C16 aryl group and the said C1-C6 alkyl group is preferable, for example, a benzyl group etc. are mentioned.

In the general formula (1), R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group, an aryloxy group, an aralkyloxy group or an amino group.

  The alkoxy group is preferably a linear or branched alkoxy group having 1 to 24 carbon atoms, more preferably an alkoxy group having 1 to 16 carbon atoms, and particularly preferably an alkoxy group having 1 to 12 carbon atoms. Examples of the alkoxy group include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, pentyloxy group, hexyloxy group, octyloxy group, decyloxy group, dodecyloxy group and the like.

  As the acyloxy group, a linear or branched alkanoyloxy group having 1 to 12 carbon atoms is preferable, and an alkanoyloxy group having 1 to 6 carbon atoms is particularly preferable. Examples of the acyloxy group include an acetoxy group, a propionyloxy group, and a butyryloxy group.

  As said alkoxycarbonyloxy group, a C2-C13 alkoxycarbonyloxy group is preferable, and a C2-C7 alkoxycarbonyloxy group is especially preferable. Examples of the alkoxycarbonyloxy group include a methoxycarbonyloxy group, an ethoxycarbonyloxy group, an n-propoxycarbonyloxy group, an isopropoxycarbonyloxy group, and the like.

  As said aryloxy group, a C6-C16 aryloxy group is preferable, for example, a phenoxy group, a naphthyloxy group, etc. are mentioned. As the aralkyloxy group, those having the aralkyl group are preferable, and examples thereof include a benzyloxy group.

In general formula (1), R ¹ and R ² are preferably hydrogen atoms. R ³ is preferably a hydroxy group, an alkoxy group or an aralkyloxy group, more preferably a hydroxy group or an alkoxy group having 1 to 12 carbon atoms, and particularly preferably a methoxy group or a hexyloxy group.

In general formula (1), the compound in which R ¹ and R ² are hydrogen atoms and R ³ is a hydroxy group is 5-aminolevulinic acid, and is particularly preferably exemplified. Suitable examples of 5-aminolevulinic acids other than 5-aminolevulinic acid (ie, 5-aminolevulinic acid derivatives) include 5-aminolevulinic acid methyl ester, 5-aminolevulinic acid ethyl ester, 5-aminolevulinic acid propyl ester, and 5-aminolevulinic acid. Examples include 5-aminolevulinic acid esters such as butyl ester, 5-aminolevulinic acid pentyl ester, and 5-aminolevulinic acid hexyl ester, and 5-aminolevulinic acid methyl ester or 5-aminolevulinic acid hexyl ester is particularly preferable.

  The salt of 5-aminolevulinic acid is not particularly limited, but a pharmaceutically acceptable acid addition salt of an inorganic acid or an organic acid is preferable. Addition salts of inorganic acids include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc., and addition salts of organic acids include acetate, lactate, citrate, tartrate, succinic acid Examples thereof include salts, maleate, fumarate, ascorbate and the like, and 5-aminolevulinic acid hydrochloride or 5-aminolevulinic acid phosphate is particularly preferable. These salts can be produced by any of chemical synthesis and methods using microorganisms or enzymes. Examples thereof include the methods described in JP-A-4-9360, JP-A-11-501914, JP-A-2006-182753, JP-A-2005-314361, and JP-A-2005-314360.

As the ABCG2 transporter inhibitor contained in the fluorescent staining agent of the present invention, the activity of the ABCG2 transporter to excrete protoporphyrin IX from the mitochondria into the cytoplasm (hereinafter simply referred to as “ABCG2 transporter excretion activity”) is also indicated. ) Is not particularly limited as long as it is a substance that inhibits, and may be a substance that competitively inhibits the activity or a substance that inhibits non-competitively. Examples of ABCG2 transporter inhibitors include reserpine ((3β, 20α) -11,17α-dimethoxy-18β-[(3,4,5-trimethoxybenzoyl) oxy] yohimbane-16β-carboxylate methyl), tripro Statins A ((8aα) -hexahydro-3β-[[2- (3-methyl-2-butenyl) -6-methoxy-1H-indol-3-yl] methyl] pyrrolo [1,2-a] pyrazine-1 , 4-dione), fumitremolgin C ((1S) -1α-isobutenyl-2,3α- [carbonyl [(2S) -pyrrolidine-2α, 1-diyl] carbonyl] -7-methoxy-1,2,3,4 -Tetrahydro-β-carboline), dipyridamole (N, N, N ', N'-tetrakis (2-hydroxyethyl) -4,8-dipiperidinopyrimy [5,4-d] pyrimidine-2,6-diamine), novobiocin (N- [7-[(3-O-carbamoyl-4-O, 5-C-dimethyl-6-deoxy-β-L- lyxo-hexopyranosyl) oxy] -4-hydroxy-8-methyl-2-oxo-2H-1-benzopyran-3-yl] -4-hydroxy-3- (3-methyl-2-butenyl) benzamide), estrogen, Antiestrogens, acrylonitrile derivatives (acrylonitrile derivatives represented by the general formula (1) of WO / 2009/072267), GF120918 (N- (4- [2- (1,2,3,4-tetrahydro-6,7- Dimethoxy-2-isoquinolinyl) ethyl] -phenyl) -9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide), Totrexate (N- [4-[[(2,4-diamino-6-pteridinyl) methyl] (methyl) amino] benzoyl] -L-glutamic acid), mitoxantrone (5,8-dihydroxy-1,4-bis [[2-[(2-hydroxyethyl) amino] ethyl] amino] -9,10-anthraquinone), daunorubicin ((8S) -8-acetyl-10α-[(3-amino-2,3,6-trideoxy -Α-L-lyxo-hexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,8α, 11-trihydroxy-1-methoxy-5,12-naphthacenedione), doxorubicin ((8S, 10S ) -10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl) oxy] -8-glycoloyl-7,8,9 10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione), bisanthrene (bis (2-imidazolen-2-ylhydrazone) -9,10-anthracenedicarboxaldehyde), Etoposide ((5R) -9α- [4-O, 6-O-[(R) -ethylidene] -β-D-glucopyranosyloxy] -5,8,8aβ, 9-tetrahydro-5β- (4 -Hydroxy-3,5-dimethoxyphenyl) furo [3 ', 4': 6,7] naphtho [2,3-d] -1,3-dioxol-6 (5aαH) -one), gefitinib (N- ( 3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) -4-quinazolinamine), imatinib (N- [3-[[4- (3-pyridinyl) pyri Gin-2-yl] amino] -4-methylphenyl] -4-[(4-methylpiperazin-1-yl) methyl] benzamido), irinotecan ((4S) -4α, 11-diethyl-4β-hydroxy-9 -[(4-Piperidinopiperidino) carbonyloxy] -1H-pyrano [3 ', 4': 6,7] Indolizino [1,2-b] quinoline-3,14 (4H, 12H) -dione ), Topotecan (4α-ethyl-4β, 9-dihydroxy-10-[(dimethylamino) methyl] -1H-pyrano [3 ′, 4 ′: 6,7] indolizino [1,2-b] quinoline-3 14 (4H, 12H) -dione), SN-38 (7-ethyl-10-hydroxycamptothecin), flavopiridol (rel-5,7-dihydroxy-2- (2-chlorophenyl) -8- ( 1-methyl-3α ^* -hydroxypiperidin-4α ^* -yl) -4H-1-benzopyran-4-one), CI 1033 (6-acrylamido-N- (3-chloro-4-fluorophenyl) -7- (3 -Morpholinopropoxy) quinazoline-4-amine), tamoxifen (2- [p-[(Z) -1,2-diphenyl-1-butenyl] phenyloxy] -N, N-dimethylethanamine), derivatives thereof, Among them, reserpine, triprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, geto Itinib, imatinib, irinotecan, topotecan, SN-38, flavopiridol, CI1033, tamoxifen are preferred, and among them reserpine, triprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, More preferred are methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, gefitinib, imatinib, irinotecan, topotecan, SN-38, flavopiridol, CI1033 and tamoxifen. Among the aforementioned ABCG2 transporter inhibitors, as substances that competitively inhibit the excretion activity of ABCG2 transporter, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, gefitinib, imatinib, irinotecan, topotecan, SN-38 , Flavopiridol, CI1033, tamoxifen and the like, and reserpine can be exemplified, and as non-competitive inhibitory substances, tryprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile A derivative, GF120918 can be exemplified.

  The content of 5-aminolevulinic acid and the like in the present invention contained in the fluorescent stain of the present invention is not particularly limited as long as mitochondria can be fluorescently stained. For example, for example, the total amount of the fluorescent stain of the present invention is 0.0001 to 99.9999% by mass, preferably 0.001 to 80% by mass, more preferably 0.001 to 50% by mass, and still more preferably 0.005 to 20% by mass, The content of the ABCG2 transporter inhibitor contained in the fluorescent stain of the present invention is not particularly limited as long as mitochondria can be fluorescently stained. For example, for example, 0.00001 to 0.00001 to the total amount of the fluorescent stain of the present invention 99.9999 mass%, preferably 0.0001-60 mass%, more preferably 0.0001-30 mass%, even more preferred It can be preferably exemplified 0.0005 mass%. In addition, the molar ratio of 5-aminolevulinic acid or the like and ABCG2 transporter inhibitor in the present invention contained in the fluorescent stain of the present invention is 1000: 1 to 1: 1000, preferably 500: 1 to 1: 100. More preferably, 500: 1 to 1: 1, more preferably 200: 1 to 10: 1 can be suitably exemplified.

  As long as the mitochondria can be fluorescently stained, the fluorescent stain of the present invention can be a substance that promotes the accumulation of protoporphyrin IX in mitochondria in addition to the 5-aminolevulinic acid and the like and the ABCG2 transporter inhibitor in the present invention (for example, An optional component such as ferrochelatase) may be contained.

  The 5-aminolevulinic acid and the like and the ABCG2 transporter inhibitor in the present invention can be made into appropriate preparations by a conventional method. The dosage form of the preparation may be a solid preparation such as a powder or a granule, but from the viewpoint of easy use, it is preferably a liquid such as a solution, an emulsion or a suspension. As a method for producing the aforementioned liquid agent, for example, a method of mixing 5-aminolevulinic acid and the like and ABCG2 transporter inhibitor in the present invention with a solvent, and a method of further mixing a suspending agent and an emulsifier are preferably exemplified. Can do. As described above, when a 5-aminolevulinic acid or the like or an ABCG2 transporter inhibitor in the present invention is used as a preparation, an appropriate carrier, for example, an excipient, a binder, a solvent, Solubilizer, suspending agent, emulsifier, isotonic agent, buffer, stabilizer, soothing agent, preservative, antioxidant, coloring agent, lubricant, disintegrant, wetting agent, adsorbent, Optional components such as sweeteners and diluents can be blended.

  As long as the fluorescent staining agent of the present invention is a mitochondrion in a living cell, the mitochondrion can be fluorescently stained regardless of whether it is an in vitro system or an in vivo system. When the fluorescent stain of the present invention is used in an in vitro system, (a) a step of contacting the target cell with the fluorescent stain of the present invention, and (b) excitation light for protoporphyrin IX, And the step (a) includes (a1) a certain time (preferably 5 minutes to 20 minutes) in a state where the target cell is in contact with the fluorescent stain of the present invention. Time, more preferably 10 minutes to 10 hours, further preferably 2 hours to 4 hours, more preferably 3 hours), and (a2) the fluorescent staining agent of the present invention. And then subjecting to a subject after culturing for a certain time (preferably 5 minutes to 20 hours, more preferably 10 minutes to 10 hours, even more preferably 2 hours to 4 hours, more preferably 3 hours). The step of washing the vesicle can be illustrated more suitably. (A3) The target cell is in contact with the fluorescent stain of the present invention for a certain time (preferably 5 minutes to 20 hours, more preferably 10 minutes). 10 hours, more preferably 2 hours to 4 hours, more preferably 3 hours), the target cells are washed, and then the target cells are washed for a certain period of time (preferably 1 minute to 30 minutes, more preferably 5 minutes) The step of incubating for -20 minutes, more preferably 8 minutes to 15 minutes, more preferably 10 minutes can be illustrated more suitably.

  In addition, when the fluorescent stain of the present invention is used in an in vivo system, (A) a step of administering the original fluorescent stain to a subject, (B) irradiation of the subject with excitation light for protoporphyrin IX The step (A) includes the step (A1), wherein (A1) the fluorescent staining agent of the present invention is administered to the subject and then a certain time (preferably 20 minutes to 20 hours, more preferably 10 minutes to 10 hours, more preferably 2 hours to 4 hours, more preferably 3 hours).

  The administration method to the subject in the above step (A) is not particularly limited as long as the subject's mitochondria can be fluorescently stained. Intravenous administration, intramuscular injection, intraarterial administration, etc .; oral administration; transdermal administration; Mucosal administration; rectal administration; transluminal administration; local administration to the brain, etc. can be exemplified, among which intravenous administration, intramuscular injection, intraarterial administration, etc .; oral administration; can do.

The dose of the fluorescent staining agent of the present invention to the subject varies depending on the administration method and the body weight of the subject, but in the case of oral administration, for example, in terms of 5-aminolevulinic acid and the like in the present invention, 0 per kg body weight at a time. 0.001 mg to 10 g, preferably 0.01 mg to 5 g, more preferably 0.1 to 1000 mg, and still more preferably 1 to 600 mg.
In terms of ABCG2 transporter inhibitor, 0.00002 mg to 200 mg, preferably 0.0002 mg to 100 mg, more preferably 0.002 to 20 mg, and still more preferably 0.02 to 12 mg per kg body weight at a time. be able to.

  The “excitation light for protoporphyrin IX” in the step (b) or the step (B) is not particularly limited as long as the protoporphyrin IX is excited, but an excitation light of 405 nm can be preferably exemplified. Such excitation light can be applied to target cells or targets using a commercially available excitation light source.

  Mammals can be preferably exemplified as the origin of the above target cells and the target species, and humans, monkeys, mice, rats, hamsters, guinea pigs, cattle, pigs, horses, rabbits, sheep, goats, Cats, dogs and the like can be preferably exemplified, and among them, humans can be more suitably exemplified.

2. Mitochondrial fluorescence staining kit of the present invention The mitochondrial fluorescence staining kit of the present invention (hereinafter also simply referred to as “fluorescence staining kit of the present invention”) includes 5-aminolevulinic acid and the like in the present invention, and ABCG2 transporter inhibition As long as it contains an agent, it is not particularly limited, and for example, those containing a 5-aminolevulinic acid or the like in the present invention and an ABCG2 transporter inhibitor separately formulated can be suitably exemplified.

  The 5-aminolevulinic acid and the like and the ABCG2 transporter inhibitor in the fluorescent staining kit of the present invention may be used after mixing, or may be used separately without mixing as long as mitochondria can be fluorescently stained. Also good.

3. Mitochondrial fluorescent staining method of the present invention The mitochondria fluorescent staining method of the present invention (hereinafter also simply referred to as “fluorescent staining method of the present invention”) includes 5-aminolevulinic acid and the like in the present invention, and ABCG2 transporter inhibition. (A) a step of bringing 5-aminolevulinic acid or the like in the present invention and an ABCG2 transporter inhibitor into contact with a target cell; and (b) protoporphyrin IX. A method including the step of irradiating the target cell with excitation light for the target cell can be suitably exemplified, and (c) a method including a step of washing the target cell between step (a) and step (b) Can be illustrated more suitably.

  The fluorescent staining method of the present invention can also be applied in vivo. In that case, (A) a step of administering 5-aminolevulinic acid or the like in the present invention and an ABCG2 transporter inhibitor to the subject; and (B) a step of irradiating the subject with excitation light for protoporphyrin IX. The method of including can be illustrated.

  5-aminolevulinic acid is suitably used for photodynamic therapy (PDT) for diseases (particularly malignant tumors). This is because 5-aminolevulinic acid is metabolized up to heme in normal cells, but aerobic respiration is reduced and anaerobic conditions in malignant tumor cells, so only protoporphyrin IX is metabolized. As a result, protoporphyrin IX accumulates specifically in malignant tumor cells. When a patient receiving 5-aminolevulinic acid is irradiated with laser light, etc., protoporphyrin IX accumulated in malignant tumor cells generates reactive oxygen species and active radical species, specifically inhibiting malignant tumor cell activity and proliferation. Thus, a therapeutic effect can be obtained. Our previous studies have revealed that the therapeutic effect on malignant tumor cells is due to apoptosis and necrosis (see Cell Biochem Funct. 2009 27 (8), 503-515). In addition, in the same document, the present inventors found that when protoporphyrin IX was synthesized from 5-aminolevulinic acid, protoporphyrin IX was distributed in mitochondria, and when PDT was performed in that state, apoptosis was induced. It has also been clarified that protoporphyrin IX moves from the cytoplasm to the cell membrane after a long period of time and induces necrosis when PDT is performed in that state. The inventors of the present invention have shown that a significant amount of protoporphyrin IX can be accumulated in mitochondria by using 5-aminolevulinic acid and the like together with an ABCG2 transporter inhibitor according to the present invention. Based on these findings, the present inventors believe that by using 5-aminolevulinic acid and the like together with an ABCG2 transporter inhibitor, apoptosis can be specifically induced in malignant tumors instead of necrosis. When necrosis is caused, scars after treatment may remain, whereas when apoptosis is caused, scars after treatment are difficult to remain. Therefore, 5-aminolevulinic acid and the like and ABCG2 transporter in the present invention A malignant tumor therapeutic agent (apoptosis inducing agent) containing an inhibitor is considered to be a very advantageous therapeutic agent particularly for a malignant tumor at a site visible from the outside.

[Mitochondrial fluorescence staining experiment 1]
In order to confirm whether or not mitochondria in living cells can be fluorescently stained by the fluorescent staining method of the present invention, the following experiment was performed.

  Normal human umbilical vein endothelial cells (HUVEC) were prepared. This HUVEC was cultured in the presence of 1 mM 5-aminolevulinic acid (ALA) and 20 μM reserpine (ABCG2 transporter inhibitor) for 3 hours. Next, HUVECs were washed with FBS medium and then incubated for 10 minutes. Thereafter, this HUVEC (ALA + reserpine) was observed with a fluorescence microscope under an excitation light of 405 nm. The result is shown in the lower center panel of FIG. In addition, the fluorescence observed with 405 nm excitation light is the fluorescence of protoporphyrin IX.

  As a control, HUVEC (ALA) cultured, washed and incubated in the same manner as described above except that reserpine was not used was observed under a fluorescence microscope under 405 nm excitation light. The result is shown in the upper center panel of FIG.

  As another control, the result of HUVEC (ALA + reserpine) is stained with the mitochondrial staining reagent Nonyl Acridine Orange (NAO) and observed with a fluorescence microscope is shown in the lower left panel of FIG. Similarly, the result of observing the aforementioned HUVEC (ALA) with NAO and observing with a fluorescence microscope is shown in the upper left panel of FIG.

  1 is shown in the upper right panel of FIG. 1, and the lower left panel of FIG. 1 and the lower center panel of FIG. 1 are overlapped. Is shown in the lower right panel.

  As can be seen from the results of the lower left panel, the middle lower panel, and the lower right panel in FIG. 1, the fluorescence of the lower left panel of FIG. 1 and the fluorescence of the lower center panel overlapped, and thus the fluorescence of protoporphyrin IX (red fluorescence) Was found to be observed specifically at the mitochondrial site. In addition, as can be seen from the results of the upper center panel and the lower center panel in FIG. 1, when reserpine (ABCG2 transporter inhibitor) is absent, the fluorescence of protoporphyrin IX is quite unclear and stains mitochondria clearly. I couldn't. Furthermore, as can be seen by comparing the results of the lower left panel and the lower central panel of FIG. 1, when 5-aminolevulinic acid (ALA) and reserpine (ABCG2 transporter inhibitor) are used in combination (lower central panel of FIG. 1), It was shown that mitochondria can be stained significantly more clearly than when using conventional NAO (lower left panel in FIG. 1).

[Mitochondrial fluorescence staining experiment 2]
In order to confirm whether the fluorescent staining method of the present invention can be used with cells other than HUVEC, COS cells (immortal monkey kidney fibroblasts) were used instead of HUVEC, PBS instead of FBS medium was used. A fluorescent staining experiment was performed in the same manner as the fluorescent staining experiment of Example 1 except that the used point and the incubation after washing were not performed. The result is shown in FIG. More specifically, the result of fluorescence microscope observation of COS cells (ALA + reserpine) under excitation light of 405 nm is shown in the lower center panel of FIG. 2, and COS cells (ALA + reserpine) are stained with NAO and observed with a fluorescence microscope. The results are shown in the lower left panel of FIG. 2, and the results of observing the COS cells (ALA) with a fluorescence microscope under the excitation light of 405 nm are shown in the upper center panel of FIG. 2, and the COS cells (ALA) are stained with NAO. The results of observation with a fluorescence microscope are shown in the upper left panel of FIG. 2 is shown in the upper right panel of FIG. 2, and the lower left panel of FIG. 2 and the lower center panel of FIG. 2 are overlapped. Is shown in the lower right panel.

  As can be seen from the results in FIG. 2, the results similar to those in FIG. 1 were also obtained when COS cells were used.

The present invention can be suitably used in the field of mitochondrial fluorescence staining.

Claims (7)

General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A mitochondrial fluorescent stain characterized by comprising a 5-aminolevulinic acid represented by the formula (1) or a salt thereof and an ABCG2 transporter inhibitor. ABCG2 transporter inhibitors are reserpine, tryprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, gefitinib 2. The fluorescent dye for mitochondrion according to claim 1, which is selected from topotecan, SN-38, flavopiridol, CI1033, and tamoxifen. General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A mitochondrial fluorescence staining kit comprising a 5-aminolevulinic acid represented by the formula (1) or a salt thereof and an ABCG2 transporter inhibitor. ABCG2 transporter inhibitors are reserpine, tryprostatin A, fumitremolgin C, dipyridamole, novobiocin, estrogen, antiestrogen, acrylonitrile derivative, GF120918, methotrexate, mitoxantrone, daunorubicin, doxorubicin, bisanthrene, etoposide, gefitinib 4. The kit for fluorescent staining of mitochondria according to claim 3, wherein the kit is selected from topotecan, SN-38, flavopiridol, CI1033, and tamoxifen. General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A method for fluorescent staining of mitochondria, comprising the step of using a 5-aminolevulinic acid compound represented by the above or a salt thereof in combination with an ABCG2 transporter inhibitor. The method for fluorescent staining of mitochondria according to claim 5, comprising the following steps (a) and (b):
(A) General formula (1)
R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ (1)
[Wherein, R ¹ and R ² each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aryl group or an aralkyl group; R ³ represents a hydroxy group, an alkoxy group, an acyloxy group, an alkoxycarbonyloxy group; A group, an aryloxy group, an aralkyloxy group or an amino group; ]
A step of contacting a target cell with a 5-aminolevulinic acid represented by the formula (1) or a salt thereof and an ABCG2 transporter inhibitor;
(B) A step of irradiating target cells with excitation light for protoporphyrin IX. (C) The method for fluorescent staining of mitochondria according to claim 6, further comprising a step of washing the target cells between step (a) and step (b).