WO2014192972A1 - Macrophage identification agent, and identification method, sorting method, evaluation method, screening method and kit using the macrophage identifier agent - Google Patents

Macrophage identification agent, and identification method, sorting method, evaluation method, screening method and kit using the macrophage identifier agent Download PDF

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WO2014192972A1
WO2014192972A1 PCT/JP2014/064900 JP2014064900W WO2014192972A1 WO 2014192972 A1 WO2014192972 A1 WO 2014192972A1 JP 2014064900 W JP2014064900 W JP 2014064900W WO 2014192972 A1 WO2014192972 A1 WO 2014192972A1
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group
general formula
atom
macrophage
compound
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French (fr)
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Fumiyo KOIDE
Tsuyoshi Nomoto
Sachiko YAMAUCHI
Kohei Watanabe
Taichi Shintou
Takeshi Miyazaki
Yasuhiko Tabata
Eri SANDA
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Canon Inc
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Canon Inc
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Priority to US14/766,386 priority Critical patent/US10041937B2/en
Priority to EP14804756.6A priority patent/EP2943584B1/en
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Definitions

  • the present invention relates to a macrophage
  • Ml macrophage one having a function to accelerate inflammation
  • M2 macrophage one having a function to inhibit inflammation
  • subtypes are significantly related to pathologic change in inflammatory diseases such as cancer, type II diabetes mellitus related to fatness, arterial sclerosis, and nephritis.
  • inflammatory diseases such as cancer, type II diabetes mellitus related to fatness, arterial sclerosis, and nephritis.
  • pathologic change such as cancer, type II diabetes mellitus related to fatness, arterial sclerosis, and nephritis.
  • identification of a subtype is expected to be applied to. diagnosis and treatment.
  • NPL 1 which a fluorescent dye-labeled antibody is used for identifying a protein marker specifically expressed on cell surfaces of each subtype (hereinafter referred to as the fluorescent antibody method) is known (NPL 1) .
  • porphyrin compound is used to be specifically
  • PTL 1 incorporated into macrophages infiltrated into an inflammatory site in a pancreatic island is disclosed (PTL 1) .
  • diagnostic method for an inflammatory disease can be expected to be developed based on the identification and evaluation of the subtype. Besides, if the subtype is controlled in a disease site or a specific subtype having been adjusted in vitro is administered based on the identification, evaluation, analysis and screening of the subtype, an early treatment method for the inflammatory disease can be developed.
  • NPL 1 Angelo A Manfredi et al., Journal of Leukocyte Biology, pp. 779-787 (2009)
  • NPL 2 Yakugaku Zasshi, Vol., 69, pp. 237-239, 1949
  • NPL 3 Heterocyclic Communications, 15(4), 2009, pp.
  • NPL 4 Heteroatom Chemistry, Volume 1, Number 5, 1990, pp. 389-399
  • NPL 5 Angew. Chem. , Int. Ed. Engl., 2005, 44, pp.
  • NPL 6 Angew. Chem., Int. Ed. Engl., 1997, 36, pp.
  • NPL 7 J. Neuroinflam. , vol. 8, 2011, 58 (URL:
  • NPL 8 Indian Journal of Chemistry, Vol. 6, pp. 136-139,
  • NPL 9 J. Thorac. Oncol., Vol. 7, pp. 1091-1100
  • NPL 10 Mol. Cancer Res., Vol. 10, p. p.727-738, 2012 Summary of Invention
  • This method further has a problem of, for example, expensiveness because storage stability of the antibody is poor and a difference between lots largely affects.
  • the present invention provides a method for identifying macrophage subtypes Ml and M2, in which the macrophages Ml and M2 are identified based on spectral characteristics obtained with an organic compound added thereto.
  • present invention is a macrophage identification agent for identifying the macrophage subtypes Ml and.M2, and contains one or more organic compounds, and a spectral characteristic obtained when added is different between the macrophage Ml and the macrophage M2.
  • a subtype can be identified by a simple and inexpensive method.
  • a subtype can be efficiently identified or separated, the number, density, balance or the like of a subtype can be evaluated, and a substance affecting a subtype can be evaluated or screened.
  • Fig. 1 illustrates a result of gene expression analysis performed on Ml macrophage and M2 macrophage differentiated from mouse bone marrow cells.
  • Fig. 2 illustrates a histogram observed in Example 1.
  • Fig. 3 illustrates a histogram observed in Comparative Example 1.
  • Fig. 4 illustrates a histogram observed in Comparative Example 2.
  • Figs. 5A, 5B, 5C, 5D and 5E illustrate histograms (Figs. 5A and 5B) and cytograms (Figs. 5C, 5D and 5E) observed in Example 55.
  • the cytogram illustrated in Fig.5C is obtained from Ml macrophages stained with a compound
  • the cytogram illustrated in Fig. 5D is obtained from M2 macrophages stained with the compound (1) and the compound (33).
  • the cytogram illustrated in Fig.5E is obtained by overlapping the cytograms illustrated in Figs. 5C and 5D.
  • Figs. 6A, 6B and 6C illustrate cytograms observed in Example 56.
  • the cytogram illustrated in Fig. 6A is obtained from Ml macrophages stained with the compound
  • the cytogram illustrated in Fig. 6B is obtained from M2 macrophages stained with the compound (1)
  • the cytogram illustrated in Fig.6C is obtained from a sample of a mixture of the Ml macrophages and the M2 macrophages stained with the compound (1) .
  • Figs. 7A and 7B illustrate histograms observed in
  • Example 57 and Comparative Example 3 The histogram illustrated in Fig.7A is obtained from Ml macrophages stained with the compound (1), and the histogram
  • Fig.7B is obtained from M2 macrophages stained with the compound (1).
  • Fig. 8 illustrates a result of the gene expression analysis performed on Ml macrophage and M2 macrophage differentiated from RAW264.7 cells.
  • Fig. 9 illustrates a histogram observed in Example 59.
  • Fig. 10 illustrates a result of the gene expression analysis performed on Ml macrophage and M2 macrophage differentiated from THP-1 cells.
  • Fig. 11 illustrates a histogram observed in Example 65.
  • Fig. 12 illustrates a histogram observed in Comparative Example 6.
  • Figs. 13A, 13B and 13C illustrate cytograms observed in Example 78.
  • the cytogram illustrated in Fig.l3A is obtained from Ml macrophages stained with a compound (86)
  • the cytogram illustrated in Fig.l3B is obtained from M2 macrophages stained with the compound (86)
  • the cytogram illustrated in Fig.l3C is obtained from a sample of a mixture of the Ml macrophages and the M2 macrophages stained with the compound (86).
  • Figs. 14A and 14B illustrate histograms observed in Example 79 and Comparative Example 5.
  • the cytogram illustrated in Fig.l4A is obtained from Ml macrophages stained with a compound (113), and the cytogram
  • Fig.l4B is obtained from M2 macrophages stained with the compound (113).
  • Fig. 15 illustrates a histogram observed in Example 81.
  • Fig. 16 illustrates a histogram observed in Comparative Example 8.
  • Figs. 17A, 17B, 17C, 17D and 17E illustrate histograms (Figs. 17A and 17B) and cytograms (Figs. 17C, 17D and 17E) observed in Example 91.
  • the cytogram illustrated in Fig. 17C is obtained from Ml macrophages stained with a compound (116) and a compound (120).
  • the cytogram illustrated in Fig. 17D is obtained from M2 macrophages stained with the compound (116) and the compound (120) .
  • the cytogram illustrated in Fig. 17E is obtained by overlapping the cytograms illustrated in Figs. 17C and 17D.
  • Figs. 18A, 18B and 18C illustrate cytograms observed in Example 92.
  • the cytogram illustrated in Fig. 18A is obtained from Ml macrophages stained with the compound (120)
  • the cytogram illustrated in Fig. 18B is obtained from M2 macrophages stained with the compound (120)
  • the cytogram illustrated in Fig. 18C is obtained from a sample of a mixture of the Ml macrophages and the M2 macrophages stained with the compound (120) .
  • Figs. 19A and 19B illustrate histograms observed in Example 93 and Comparative Example 9.
  • the histogram illustrated in Fig. 19A is obtained from Ml macrophages stained with the compound (120)
  • the histogram illustrated in Fig. 19B is obtained from M2 macrophages stained with the compound (120).
  • Fig. 20 illustrates a histogram observed in Example 95.
  • Fig. 21 illustrates a histogram observed in Example 96.
  • Fig. 22 illustrates a histogram observed in Example 97.
  • Fig. 23 illustrates a histogram observed in Example 98.
  • Figs. 24A, 24B, 24C, 24D and 24E illustrate histograms (Figs. 24A and 24B) and cytograms (Figs. 24C, 24D and 24E) observed in Example 108.
  • the cytogram illustrated in Fig. 24C is obtained from Ml macrophages stained with a compound (139) and a compound (161) .
  • the cytogram illustrated in Fig. 24D is obtained from M2 macrophages stained with the compound (139) and the compound (161) .
  • the cytogram illustrated in Fig. 24E is obtained by overlapping the cytograms illustrated in Figs.24C and 24D.
  • Figs. 25A, 25B and 25C illustrate cytograms observed in Example 109.
  • the cytogram illustrated in Fig. 25A is obtained from Ml macrophages stained with the compound (139)
  • the cytogram illustrated in Fig. 25B is obtained from M2 macrophages stained with the compound (139)
  • the cytogram illustrated in Fig. 25C is obtained from a sample of a mixture of the Ml macrophages and the M2 macrophages stained with the compound (139).
  • Figs. 26A and 26B illustrate histograms observed in Example 110 and Comparative Example 10.
  • the histogram illustrated in Fig. 26A is obtained from Ml macrophages stained with the compound (139), and the histogram illustrated in Fig. 26B is obtained from M2 macrophages stained with the compound (139).
  • Fig. 27 illustrates a histogram observed in Example 112.
  • Fig. 28 illustrates a histogram observed in Example 113.
  • Fig. 29 illustrates a histogram obserbed in Example 114.
  • Figs. 30A, 30B, 30C, 30D and 30E illustrate histograms (Figs. 30A and 30B) and cytograms (Figs. 30C, 30D and 30E) observed in Example 115.
  • the cytogram illustrated in Fig. 30C is obtained from Ml macrophages stained with the compound (1) and the compound (113).
  • the cytogram illustrated in Fig. 30D is obtained from M2 macrophages stained with the compound (1) and the compound (113) .
  • the cytogram illustrated in Fig. 30E is obtained by overlapping the cytograms illustrated in Figs. 30C and 30D.
  • the present invention provides a macrophage
  • identification agent for identifying macrophage
  • the individual embodiments exemplarily describe a novel identification method for macrophage subtypes, a macrophage identification agent, a sorting method, an evaluation method, an analysis method for correlation between a subtype and a
  • a macrophage identification agent according to
  • Embodiment 1 of the present invention contains one or more compounds represented by the following general formula 1) :
  • Ri represents a hydrogen atom, an alkyl group, an aralkyl group, an alkenyl group, or an aryl group
  • R 2 to R 5 each independently represent a hydrogen atom, an alkyl group, an aryl group, a carboxyl group, a carboxylalkyl group, or an
  • R 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom
  • R 7 and RQ each independently represent a hydrogen atom, an alkyl group, or an aryl group
  • Rg represents a dicyanomethylene group, a
  • Ri 0 represents an alkyl group, a carboxylalkyl group, an alkoxycarbonylalkyl group, or an alkylcarbonyloxyalkyl group
  • Rn represents a sulfur atom, a 2-thioxothiazolidin-4-one group, a thiazoline-2, 4-one group, or the following general
  • Ri 2 represents an alkyl group, a carboxylalkyl group, an alkoxycarbonylalkyl group, or an alkylcarbonyloxyalkyl group; and R13 and R i4 each represent a hydrogen atom and may be bound to each other to form a benzene ring.
  • Qi represents an oxygen atom, a sulfur atom, a N-alkyl nitrogen atom, or
  • R15 and Ri6 each independently represent an alkyl group and may be bound to each other to form a ring; and Xi ⁇ represents an anionic group.
  • a sign "*" represents a binding site.
  • Ri 7 represents an alkyl group, a carboxylalkyl group, an alkoxycarbonylalkyl group, or an alkylcarbonyloxyalkyl group; and Ris and R19 each represent a hydrogen atom and may be bound to each other to form a benzene ring.
  • Q 2 represents an oxygen atom, a sulfur atom, a N-alkyl nitrogen atom, or
  • R 2 o and R 2 i each independently represent an alkyl group and may be bound to each other to form a ring; and X 2 ⁇ represents an anionic group.
  • a sign "*" represents a binding site.
  • R 22 represents an alkyl group . or an aryl group; and R 2 3 represents an alkyl group or a carboxyl group.
  • a sign "*" represents a binding site.
  • the alkyl group used as Ri is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group, and a cyclohexyl group.
  • the aralkyl group used as Ri is not especially limited, and examples include a benzyl group and a phenethyl group.
  • the alkenyl group used as Ri is not especially limited, and examples include alkenyl groups having 2 to 20 carbon atoms, such as a vinyl group, a 2 , 2-diphenylvinyl group, a 3-butenyl group, and a cyclohexenyl group.
  • the aryl group used as Ri is not especially limited, and examples include 6- to 14- membered monocyclic or polycyclic aryl groups, such as a phenyl group, a naphthyl group, a phenanthryl group, and an anthracenyl group.
  • Ri may further have a
  • substituent and the substituent is not especially limited as long as the storage stability of the dye compound is not largely inhibited, and examples include alkyl groups such as a methyl group, an ethyl group, a n-propyi group, an isopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group; aryl groups such as a phenyl group and a
  • naphthyl group alkoxy groups such as a methoxy group, an ethoxy group and a butoxy group; aryloxy groups such as a phenoxy group and a naphthyloxy group;
  • alkylsulfanyl groups such as a thiomethyl group, a thioethyl group, a thiopropyl group, a thiobutyl group and a thiophenyl group; di-substituted amino groups such as a dimethylamino group, a N-ethyl-N-phenylamino group, and a diphenylamino group; acyl groups such as an acetyl group and a benzoyl group; a, sulfonyl group; a carboxyl group; a carboxylalkyl group; a carbamoyl group; a sulfamoyl group; hetero ring groups such as a pyridyl group, a triazinyl group, and a benzothiazolyl group; a nitro group; and halogen atoms such as a fluorine atom, a chlorine atom, a bromine atom and an
  • Ri can be arbitrarily and
  • Ri represents an alkyl group, an aryl group or the like because a subtype can be easily identified in this case.
  • Ri represents preferably a methyl group, an ethyl group, a propyl group, a butyl group, a phenyl group, a bromophenyl group, a benzyl group, a
  • bromobenzyl group a methylthiophenyl group, a
  • methoxyphenyl group a methoxynaphthyl group, a
  • Ri benzylphenyl group, a 2 , 2-diphenylvinyl group or a 2,2- diphenylvinylphenyl group. More preferably, Ri
  • Ri particularly preferably represents a methylthiophenyl group because a subtype can be easily identified based on a large stokes shift in this case.
  • the alkyl group used as R2 to R 5 is not especially limited, and examples include alkyl groups having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group and a butyl group.
  • the aryl group used as R 2 to R 5 is not especially limited, and examples include a phenyl group and a naphthyl group.
  • the carboxylalkyl group used as R 2 to R 5 is not especially limited, and examples include a methyl carboxylate group, an ethyl carboxylate group, a propyl carboxylate group and a butyl carboxylate group.
  • the alkylcarbonyl group used as R2 to R5 is not especially limited, and examples include a methylcarbonyl group, an ethylcarbonyl group, a propylcarbonyl group, a butylcarbonyl group, a
  • phenylcarbonyl group a naphthylcarbonyl group, and a phenanthrylcarbonyl group.
  • the aliphatic ring formed when R 2 and R are bound to each other is not especially limited, and examples include saturated aliphatic rings such as a cyclooctane ring, a cycloheptane ring, a cyclohexane ring, a cyclopentane ring, and a
  • cyclobutane ring and partially saturated aliphatic rings such as a cyclopentene ring and a cyclohexene ring.
  • the ring may further have a substituent and the substituent is not especially limited as long as the identification of a subtype is not largely inhibited.
  • substituents include alkyl groups such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group and a butyl group; aryl groups such as a phenyl group; alkoxy groups such as a methoxy group, an ethoxy group and a butoxy group;
  • aryloxy groups such as a phenoxy group; di-substituted amino groups such as a dimethylamino group, a N-ethyl- N-phenylamino group, and a diphenylamino group; acyl groups such as an acetyl group and a benzoyl group; a sulfonyl group, a carboxyl group; a carboxylalkyl group; a carbamoyl group; a sulfamoyl group; hetero ring groups such as a pyridyl group, a triazinyl group, and a benzothiazolyl group; a nitro group; and halogen atoms such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom; a carboxyl group, and a sulfo group.
  • acyl groups such as an acetyl group and a benzoyl
  • R 2 to R 5 each independently represent a hydrogen atom, an alkyl group or an aryl group and R 2 and R 4 are bound to each other to form an aliphatic ring, and in a more preferred aspect, R2 and R 4 are bound to each other to form an aliphatic ring, because a subtype can be easily identified in such a case.
  • the aliphatic ring can be a cyclooctane ring, a cycloheptane ring, a cyclohexane ring, a cyclopentane ring or a cyclobutane ring. More preferably, the aliphatic ring is a
  • the alkyl group used as R6 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group and a butyl group.
  • the alkoxy group used as R 6 is not especially limited, and examples include a methoxy group, an ethoxy group, a propoxy group and a butoxy group .
  • R 3 ⁇ 4 used as R 3 ⁇ 4 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • R6 preferably represents a
  • the alkyl group used as R 7 and Re is not especially limited, and examples include alkyl groups having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group and a butyl group.
  • the aryl group used as R 7 and Rs is not especially limited, and examples include a phenyl group and a methylphenyl group.
  • Y x is not especially limited, and
  • examples include phenyl-substituted carbon
  • R 9 represents a
  • R 9 preferably represents any one of general formulas (2) to (4), and particularly preferably represents general formula (2) because a subtype can be easily identified in this case.
  • the alkyl group used as Ri 0 is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group, and a cyclohexyl group.
  • the carboxylalkyl group used as Rio is not especially limited, and examples include a carboxymethyl group, a carboxyethyl group, a
  • the alkoxycarbonylalkyl group used as R 10 is not especially limited, and examples include a methoxycarbonylmethyl group, a
  • methoxycarbonylethyl group an ethoxycarbonylethyl group, a butoxycarbonylethyl group, a
  • the alkylcarbonyloxyalkyl group used as Rio is not especially limited, and examples include a methylcarbonyloxymethyl group, an ethylcarbonyloxymethyl group, an ethylcarbonyloxyethyl group, ethylcarbonyloxybutyl, and a
  • the 2-thioxothiazolin-4-one group used as Rn is not especially limited, and examples include the following compounds.
  • the sign "*" represents a binding site.
  • the thiazoline-2 , -dione group used as R u is not especially limited, and examples include the following compounds.
  • the sign "*" represents a binding site.
  • R can be represented by the following general formula (5):
  • the alkyl group used as R 12 is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group and a
  • the carboxylalkyl group used as Ri2 is not especially limited, and examples include a carboxymethyl group, a carboxyethyl group, a
  • the alkoxycarbonylalkyl group used as R12 is not especially limited, and examples include a methoxycarbonylmethyl group, a
  • methoxycarbonylethyl group an ethoxycarbonylethyl group, a butoxycarbonylethyl group, a
  • the alkylcarbonyloxyalkyl group used as R12 is not especially limited, and examples include a methylcarbonyloxymethyl group, an
  • ethylcarbonyloxymethyl group an ethylcarbonyloxyethyl group, ethylcarbonylbutyl, and a proxycarbonyloxymethyl group.
  • R13 and R i4 are bound to each other to form a benzene ring, and the ring may further have a substituent.
  • the N-alkyl nitrogen atom used as Qi is not especially limited, and examples include a N-methyl nitrogen atom, a N-ethyl nitrogen atom, a N- propyl nitrogen atom, and a N-butyl nitrogen atom.
  • the alkyl group used as R15 and Ri6 of Qi is not especially limited, and examples include alkyl groups having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group and a butyl group.
  • the ring formed when R ⁇ $ and Ri 6 are bound to each other is not especially limited, and examples include a cyclopentane ring and a cyclohexane ring .
  • the anionic group represented by Xi " is not especially limited, and examples include a chloride ion, a bromide ion, an iodide ion, a sulfate ion, a nitrate ion, a methanesulfonate ion, a p- toluenesulfonate ion, a tetrafluoroborate ion, and a hexafluorophosphate ion.
  • the alkyl group used as Ri 7 is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group and a
  • the carboxylalkyl group used as Ri7 is not especially limited, and examples include a carboxymethyl group, a carboxyethyl group, and a carboxypropyl group.
  • the alkoxycarbonylalkyl group used as Ri 7 is not especially limited, and examples include a methoxycarbonylmethyl group, a
  • methoxycarbonylethyl group an ethoxycarbonylethyl group, a butoxycarbonylethyl group, and a
  • the alkylcarbonyloxyalkyl group used as Ri 7 is not especially limited, and examples include a methylcarbonyloxymethyl group, an
  • ethylcarbonyloxymethyl group an ethylcarbonyloxyethyl group, ethylcarbonylbutyl, and a proxycarbonyloxymethyl group.
  • Ri 8 and Ri 9 are bound to each other to form a benzene ring, and the ring may further have a substituent.
  • the N-alkyl nitrogen atom used as Q 2 is not especially limited, and examples include a N-methyl nitrogen atom, a N-ethyl nitrogen atom, a N- propyl nitrogen atom, and a N-butyl nitrogen atom.
  • the alkyl group used as R 2 o and R21 of Q 2 is not especially limited, and examples include alkyl groups having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group and a butyl group.
  • the ring formed when R 2 o and R 2X are bound to each other is not especially ' limited, and examples include a cyclopentane ring and a cyclohexane ring.
  • the anionic group represented by X 2 ⁇ is not especially limited, and examples include a chloride ion, a bromide ion, an iodide ion, a sulfate ion, a nitrate ion, a methanesulfonate ion, a p- toluenesulfonate ion, a tetrafluoroborate ion, and a hexafluorophosphate ion.
  • the alkyl group used as R 22 is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group and a
  • the aryl group used as R 22 is not especially limited, and examples include 6- to 14- membered monocyclic or polycyclic aryl groups, such as a phenyl group, a naphthyl group, a phenanthryl group, and an anthracenyl group.
  • the aryl group used as R 22 may further have a substituent, and the substituent is not especially limited as long as the identification of a subtype is not largely inhibited.
  • substituents include alkyl groups such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group; aryl groups such as a phenyl group and a
  • naphthyl group alkoxy groups such as a methoxy group, an ethoxy group and a butoxy group; aryloxy groups such as a phenoxy group and a naphthyloxy group;
  • alkylsulfanyl groups such as a thiomethyl group, a thioethyl group, a thiopropyl group, a thiobutyl group and a thiophenyl group; di-substituted amino groups such as a dimethylamino group, a N-ethyl-N-phenylamino group, and a diphenylamino group; acyl groups such as an acetyl group and a benzoyl group; a sulfonyl group; a carboxyl group; a carboxylalkyl group; a carbamoyl group; a sulfamoyl group; hetero ring groups such as a pyridyl group, a triazinyl group, and a benzothiazolyl group; a nitro group; and halogen atoms such as a fluorine atom, a chlorine atom, a bromine atom and an
  • the alkyl group used as R 23 is not especially limited, and examples include linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group, an ethylhexyl group and a
  • the compounds represented by general formula (1) of the present embodiment can be easily synthesized by a method similar to known methods (PTL 2 and the like) .
  • Suitable specific examples of the compounds represented by general formula (1) of the present invention include, but are not limited to, the following compounds (1) to (85) : ]
  • Alkyl group Aliphatic ring - - 0
  • Aryl group Aliphatic ring - atom atom - 0
  • Aryl group Aliphatic ring - atom atom - 0
  • Aryl group Aliphatic ring - atom atom - 0
  • Aryl group Aliphatic ring - atom atom - 0
  • a macrophage identification agent according to
  • Embodiment 2 of the present invention contains one or more compounds represented by the following general formula ( 6) :
  • R 2 and R25 each independently represent an alkyl group, a carboxylalkyl group, an alkoxycarbonylalkyl group, or an alkylcarbonyloxyalkyl group; R 2 6 to R33 each independently represent a
  • R 2 6 and R 2 7, 28 and R29, R30 and R 3i , and R 32 and R33 may be independently cyclized to form a benzene ring;
  • Xi " represents an anionic group;
  • Y 3 represents an anionic group;
  • Y 2 represents a group containing *1, *2 and *5, and containing an oxygen atom, a sulfur atom, a N-alkyl nitrogen atom or -C(R 34 ) ( R35 ) - , or a group containing *1, *2 and *5 and represented by *l-C*5-CH
  • R 36 to R 38 each independently represent a hydrogen atom, an alkyl group or an aryl group; and n represents a value of 0 to 2.
  • R39 represents a hydrogen atom, a phenyl group, a thiol group, an alkoxy group, an aryloxy group, or a halogen atom; and R 40 and R 4 i each independently represent a hydrogen atom, an alkyl group or an
  • the alkyl group used as R24 and R25 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the carboxylalkyl group used as R24 and R25 is not especially limited, and examples include a carboxylmethyl group, a carboxylethyl group, and a carboxylpropyl group.
  • the alkoxycarbonylalkyl group used as R24 and R25 is not especially limited, and examples include a methoxycarbonylmethyl group, a methoxycarbonylethyl group, an ethoxycarbonylethyl group, a butoxycarbonylethyl group, and a
  • the alkylcarbonyloxyalkyl group used as R 24 and R 2 5 is not especially limited, and examples include a methylcarbonyloxymethyl group, an ethylcarbonyloxymethyl group, an ethylcarbonyloxyethyl group, ethylcarbonylbutyl, and a proxycarbonyloxymethyl ' group.
  • the alkyl group used as R 2 6 to R33 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the aryl group used as R 2 6 to R33 is not especially limited, and examples include a phenyl group, a 2-bromophenyl group, a 3-bromophenyl group, a 4-bromophenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, a 2- thiomethylphenyl group, a 3-thiomethylphenyl group, a 4-thiomethylphenyl group and a naphthyl group.
  • the alkoxy group used as R 2 6 to R 33 is not especially limited, and examples include a methoxy group, an ethoxy group a propoxy group and a butoxy group.
  • examples of the halogen atom used as R 2 6 to R 33 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • the alkoxysulfonyl group used as R 2 6 to R33 is not especially limited, and examples include a methoxysulfonyl group and an ethoxysulfonyl group .
  • N-alkylsulfamoyl used as R 2 6 to R 33 is not especially limited, and examples include a N- methylsulfamoyl group, a N-ethylsulfamoyl group, a N,N- dimethylsulfamoyl group and a N, N-ethylsulfamoyl group.
  • the alkyloxycarbonyl group used as R 26 to R33 is not especially limited, and examples include a methyloxycarbonyl group, an ethyloxycarbonyl group, a propyloxycarbonyl group and a butyloxycarbonyl group.
  • the N-alkylcarbamoyl group used as R 2 6 to R33 is not especially limited, and examples include a N-methylcarbamoyl group, a N-ethylcarbamoyl group, a N, N-dimethylcarbamoyl group and a N,N- diethylcarbamoyl group.
  • R 2 6 to R33 each independently
  • ⁇ represent preferably a hydrogen atom, a halogen atom, a phenyl group or an alkoxy group, and more preferably a hydrogen atom or a phenyl group.
  • the anionic group represented by X 3 ⁇ is not especially limited, and examples include a chloride ion, a bromide ion, an iodide ion, a sulfate ion, a nitrate ion, a methanesulfonate ion, a p- toluenesulfonate ion, a tetrafluoroborate ion, a
  • the alkylene group used as Y 2 and Y3 is not especially limited, and examples include a methylene group, an ethylene group, a propylene group and a butylene group.
  • the alkyl group used as R 34 and R35 of Y 2 and Y 3 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and a 2- ethylhexyl group.
  • R 34 and R 35 are preferably the same but may be different from each other.
  • R 34 and R 35 may be bound to each other to form an aliphatic ring, and examples of the ring include a cyclohexane ring and a cyclopentane ring.
  • the alkyl group used as R 3 6 to R 38 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the aryl group used as R 3 6 to R 38 is not especially limited, and examples include a phenyl group, a 2-bromophenyl group, a 3-bromophenyl group, a 4-bromophenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a -methoxyphenyl group, a 2- thiomethylphenyl group, a 3-thiomethylphenyl group, and a 4-thiomethylphenyl group.
  • examples of the thiol group used as R 3 g include a mercaptomethyl group, a
  • examples of the alkoxy group used as R 3 g include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • examples of the aryloxy group used as R 3 g include a phenoxy group and a phenoxy group that may have a substituent.
  • examples of the halogen atom used as R 3 g include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • the alkyl group used as R 40 and R41 is not especially limited, and examples include alkyl groups, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the alkyloxycarbonyl group used as R 40 and R41 is not especially limited, and examples include a methyloxycarbonyl group, an ethyloxycarbonyl group, a propyloxycarbonyl group and a butyloxycarbonyl group.
  • R 42 and R 43 each independently represent an alkyl group, a carboxylalkyl group, an alkylcarbonyloxyalkyl group, or an alkoxycarbonyl alkyl group;
  • R 44 to R51 each independently represent a
  • R 44 and R 4 5 , R 4 6 and R 7 , R 48 and R 49 , and R 50 and R51 may be independently cyclized to form a benzene ring;
  • R 52 to R 54 each independently represent a hydrogen atom, an alkyl group or an aryl group;
  • m represents a value of 0 to 2;
  • X 2 ⁇ represents an anionic group;
  • Y 4 and Y5 each represent an oxygen atom, a sulfur atom or an alkylene group, wherein the
  • alkylene group may have a substituent, the substituent used in this case is an alkyl group, and the
  • substituents may be bound to each other to form an aliphatic ring.
  • the alkyl group used as R 42 and R43 is not especially limited, and examples include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
  • the carboxylalkyl group used as R 42 and R 43 is not especially limited, and examples include a carboxylmethyl group, a carboxylethyl group and a carboxylpropyl group.
  • the alkoxycarbonylalkyl group used as R 2 and R43 is not especially limited, and examples include a methoxycarbonylmethyl group, a methoxycarbonylethyl group, an ethoxycarbonylethyl group, a butoxycarbonylethyl group, and a
  • the alkylcarbonyloxyalkyl group is not especially limited, and examples include a methylcarbonyloxymethyl group, an
  • ethylcarbonyloxymethyl group an ethylcarbonyloxyethyl group, ethylcarbonylbutyl, and a proxycarbonyloxymethyl group.
  • the alkyl group used as R 44 to R 4 8 s not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the aryl group used as R 44 to R 4 8 is not especially limited, and examples include a phenyl group, a 2-bromophenyl group, a 3-bromophenyl group, a 4-bromophenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, a 2- thiomethylphenyl group, a 3-thiomethylphenyl group, a 4-thiomethylphenyl group and a naphthyl group.
  • the alkoxy group used as R 44 to R 4 8 is not especially limited, and examples include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • examples of the halogen atom used as R to R51 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • the alkoxysulfonyl group used as R 44 to R51 is not especially limited, and examples include a methoxysulfonyl group and an ethoxysulfonyl group .
  • R44 to R51 is not especially limited, and examples include a N-methylsulfamoyl group, a N-ethylsulfamoyl group, a N, -dimethylsulfamoyl group and a N,N- ethylsulfamoyl group.
  • the alkyloxycarbonyl group used as R44 to R51 is not especially limited, and examples include a methyloxycarbonyl group, an ethyloxycarbonyl group, a propyloxycarbonyl group and a butyloxycarbonyl group .
  • the N-alkylcarbamoyl group used as R44 to R51 is not especially limited, and examples include a N-methylcarbamoyl group, a N-ethylcarbamoyl group, a N, N-dimethylcarbamoyl group and a N,N- diethylcarbamoyl group.
  • R 44 to R 51 each independently represent preferably a hydrogen atom, a halogen atom, a phenyl group or an alkoxy group, and more preferably a hydrogen atom or a phenyl group.
  • the alkyl group used as R52 to R51 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group and a hexyl group.
  • the aryl group used as R52 to R 5 i is not especially limited, and examples include a phenyl group, a 2-bromophenyl group, a 3-bromophenyl group, a 4-bromophenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, a 2- thiomethylphenyl group, a 3-thiomethylphenyl group, and a 4-thiomethylphenyl group.
  • the anionic group represented by X4 ⁇ is not especially limited, and examples include a chloride ion, a bromide ion, an iodide ion, a sulfate ion, a nitrate ion, a methanesulfonate ion, a p- toluenesulfonate ion, a tetrafluoroborate ion, a hexafluorophosphate ion, and an ammonium ion.
  • Y 4 and Y 5 each represent an oxygen atom, a sulfur atom or an alkylene group, the alkylene group may have a substituent, the substituent used in this case is an alkyl group, and the
  • substituents may be bound to each other to form an aliphatic ring.
  • the alkylene group is not especially limited, and examples include a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, and a 2-ethylhexylene group.
  • the aliphatic ring formed by the substituents is not
  • examples include a cyclohexane ring and a cyclopentane ring.
  • R 69 and R 7 o each independently represent an alkyl group, a carboxylalkyl group, or an alkoxycarbonylalkyl group; and R 7 i to R 78 each
  • R71 and R 2 , R 73 and R 74 , R 7 5 and R 7 6, and R 77 and R 78 may be independently cyclized to form a benzene ring.
  • R 79 represents a hydrogen atom, a phenyl group, a thiol group, an alkoxy group, an aryloxy group, or a halogen atom; Rso and Rsi each independently represent a
  • X 6 ⁇ represents an anionic group
  • Y 7 and Y g each represent an oxygen atom, a sulfur atom or an alkylene group, wherein the alkylene group may have a
  • substituent the substituent used in this case is an alkyl group, and the substituents may be bound to each other to form an aliphatic ring.
  • R 70 is not especially limited, and examples include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
  • the carboxylalkyl group used as R 69 and R 70 is not especially limited, and examples include an acetic acid group, a propionic acid group and a butanoic acid group.
  • the alkoxycarbonylalkyl group used as R 69 and R 70 is not especially limited, and
  • examples include a methoxycarbonylmethyl group, a methoxycarbonylethyl group, an ethoxycarbonylethyl group, a butoxycarbonylethyl group, and a
  • the alkyl group used as R 7 i to R 7 8 is not especially limited, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
  • the aryl group used as R 7 i to R 7 8 is not especially limited, and examples include a phenyl group, a 2-bromophenyl group, a 3-bromophenyl group, a 4-bromophenyl group, a 2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl group, a 2- thiomethylphenyl group, a 3-thiomethylphenyl group, a 4-thiomethylphenyl group, and a naphthyl group.
  • the alkoxy group used as R 7 i to R 78 is not especially limited, and examples include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • examples of the halogen atom used as R 7 i to R 7 8 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • the alkoxysulfonyl group used as R 7 i to R 7 8 is not especially limited, and examples include a methyl sulfonate group and an ethyl sulfonate group .
  • the alkylsulfamoyl group used as R 7 i to R 78 is not especially limited, and examples include a sulfonic acid monomethyl amide group, a monoethyl amide sulfonate group, a dimethyl amide sulfonate group, and a diethyl amide sulfonate group.
  • R 7i to R 78 is not especially limited, and examples include a carboxylic acid methyl ester group, a carboxylic acid ethyl ester group, a carboxylic acid propyl ester group, and a carboxylic acid butyl ester group.
  • R 7i to R 78 is not especially limited, and examples include a N-methylcarbamoyl group, a N- ethylcarbamoyl group, a N, -dimethylcarbamoyl group and a N, -diethylcarbamoyl group.
  • R 7 i to R 78 represent preferably a hydrogen atom, a halogen atom, a phenyl group or an alkoxy group, and more preferably a hydrogen atom or a phenyl group.
  • examples of the thiol group used as R79 include a mercaptomethyl group, a
  • examples of the alkoxy group used as R 7 g include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • examples of the aryloxy group used as R79 include a phenoxy group, or a phenoxy group that may have a substituent.
  • examples of the halogen atom used as R79 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • RB I is not especially limited, and examples include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group.
  • Rso and Rsi is not especially limited, and examples include a methyloxycarbonyl group, an
  • the anionic group represented by ⁇ ⁇ is not especially limited, and examples include a chloride ion, a bromide ion, an iodide ion, a sulfate ion, a nitrate ion, a methanesulfonate ion, a p- toluenesulfonate ion, a tetrafluoroborate ion, a
  • the alkylene group used as Y 7 and Ys is not especially limited, and examples include a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, and a 2-ethylene hexyl group.
  • the alkyl group used as a substituent of the alkylene group is not especially limited, and examples include a methyl group, an ethyl group, a propyl group and a butyl group.
  • Suitable specific examples of the compounds of the present embodiment include, but are not limited to, the following compounds (86) to (115) :
  • a macrophage identification agent according to
  • Embodiment 3 of the present invention contains one or more compounds represented by the following general formula 10) :
  • R 55 and R 56 each independently represent an alkyl group
  • R 57 and R 58 each independently represent a hydrogen atom or an alkyl group, wherein R 55 and R57, and R56 and R 5 e may be independently bound to each other to form a ring
  • R 59 represents a hydrogen atom, an alkyl group, an aryl group, an alkoxy group or a halogen atom
  • X5 represents a sulfur atom, an oxygen atom or -NR 6 -
  • Y 6 represents a carbon atom or a nitrogen atom.
  • the alkyl group used as R55 and R56 is not especially limited, and examples include linear or branched alkyl groups having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group and an ethylhexyl group.
  • R 55 and R 56 can be a methyl group or an ethyl group .
  • the alkyl group used as R 57 and R 5 8 is not especially limited, and examples include linear or branched alkyl groups having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group and an ethylhexyl group.
  • the alkyl group used as R 59 is not especially limited, and examples include linear or branched alkyl groups having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group and an ethylhexyl group.
  • the aryl group used as R 59 is not especially limited, and examples include a phenyl group, a methylphenyl group, a bromophenyl group and a methoxyphenyl group.
  • the alkoxy group used as R59 is not especially limited, and examples include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • the halogen atom used as R59 is not especially limited, and examples include a fluorine atom, a chloro atom, a bromo atom and an iodine atom.
  • X 5 can be a sulfur atom or an oxygen atom because a subtype can be more easily identified in such a case.
  • Suitable specific examples of the compounds represented by general formula (10) of the present embodiment include, but are not limited to, the following
  • a macrophage identification agent according to
  • Embodiment 4 of the present invention contains one or more compounds represented by the following general formula ( 11 ) :
  • R.60 represents a hydrogen atom, an alkyl group, an aryl group, a thioalkyl group, an amino group, a hetero ring group, an alkenyl group, a hydroxyl group, a halogen atom, or an alkoxy group
  • R 6 i to Re6 each independently represent a hydrogen atom, an alkyl group, an aryl group, a hetero ring group, an aralkyl group, or a sulfonyl group, wherein R 6 2 and R 63 , and R65 and R66 may be bound to each other to form a hetero ring
  • R 67 and R 6 8 each independently
  • the alkyl group used as R60 is not especially limited, and examples include linear or branched alkyl groups having 1 to 12 carbon atoms? such as a methyl group, an ethyl group, a propyl group, a butyl group and an ethylhexyl group, and alkyl halide groups such as trifluoromethyl .
  • the aryl group used as R 6 o is not especially limited, and examples include a phenyl group, a methylphenyl group, a methoxyphenyl group, a thiomethylphenyl group, a chlorophenyl group, a bromophenyl group, a fluorophenyl group, a butylphenyl group, a dimethylphenyl group, a trimethylphenyl group and a naphthyl group.
  • the thioalkyl group used as R6o is not especially limited, and examples include linear or branched thioalkyl groups having 1 to 12 carbon atoms, such as a thiomethyl group, a thioethyl group, a thiopropyl group, a thiobutyl group, and a thioethylylhexyl group.
  • the amino group used as R 6 o is not especially limited, and examples include an amino group, a methylamino group, a dimethylamino group, a butylamino group, a dibutylamino group and a
  • the hetero ring group used as R 6 o is not especially limited, and examples include a pyridine ring, a thiophene ring, and a furan ring.
  • the alkenyl group used as R 6 o is not especially limited, and examples include a vinyl group and a 3-butenyl group.
  • the halogen atom used as R 6 o is not especially limited, and examples include a fluorine atom, a chloro atom, a bromo atom and an iodine atom.
  • the alkoxy group used as R6o is not especially limited, and examples include a methoxy group, an ethoxy group, a propoxy group and a butoxy group.
  • R6o represents preferably an alkyl group or an aryl group, and particularly
  • a methyl group preferably a methyl group, an ethyl group, a propyl group, a butyl group or a phenyl group because a subtype can be more easily identified in such a case.
  • the alkyl group used as R 61 to R 6 6 is not especially limited, and examples include linear or branched alkyl groups having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group and an ethylhexyl group.
  • the aryl group used as R 6 i to R.66 is not especially limited, and examples include a phenyl group, a methylphenyl group, a methoxyphenyl group, a thiomethylphenyl group, a chlorophenyl group, a bromophenyl group, a fluorophenyl group, a
  • butylphenyl group a dimethylphenyl group, a
  • the hetero ring group used as R 6 i to R 6 6 is not especially limited, and examples include a pyridine ring, a thiophene ring, and a furan ring .
  • the aralkyl group used as R 6 i to R66 is not especially limited, and examples include a benzyl group or a phenethyl group.
  • the sulfonyl group used as R 6 i to R 6 6 is not especially limited, and examples include a sulfo group and a salt such as sodium sulfonate.
  • R 6 2 and R63 or R 65 and R 6 6 are bound to each other is not especially limited, and an example includes a furan ring .
  • R6o to R 6 6 each may further
  • alkyl groups such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group; aryl groups such as a phenyl group and a
  • naphthyl group alkoxy groups such as a methoxy group, an ethoxy group, and a butoxy group; aryloxy groups such as a phenoxy group and a naphthyloxy group; di- substituted amino groups such as a dimethylamino group, a N-ethyl-N-phenylamino group and a diphenylamino group; acyl groups such as an acetyl group and a benzoyl group; a sulfonyl group; a carbamoyl group; a sulfamoyl group; hetero ring groups such as a pyridyl group, a triazinyl group, and a benzothiazolyl group; a nitro group; and halogen atoms such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.
  • the alkynyl group used as R 67 and R 6 7 is not especially limited, and examples include a vinyl group and a 3-butenyl group.
  • the compounds represented by general formula (11) of the present embodiment can be easily synthesized by a method similar to known methods (for example, NPL 4, NPL 5 and NPL 6) .
  • Suitable specific examples of the compounds represented by general formula (11) of the present invention include, but are not limited to, the following
  • TMS stands for a trimethyl silyl group (-Si(CH 3 ) 3 ).
  • the organic compounds of the present invention can be organic compounds having a molecular weight less than 10,000, preferably 5,000 or less, and more preferably 2,000 or less.
  • the organic compounds of the present invention are preferably dye compounds, and more preferably fluorescent compounds having
  • a fluorescent compound Since a fluorescent compound has high sensitivity, the compound can be used for staining at a low concentration, and hence, the necessary amount of compound can be relatively reduced.
  • a wavelength of 200 to 1400 nm is generally used because of the limit of a laser wavelength for generating excitation light and a range of a wavelength that can be easily transmitted through a living body.
  • the wavelength can be 340 to 800 nm because such a wavelength falls in to a wavelength range in which fluorescence can be detected by various types of apparatuses.
  • a compound having an excitation wavelength in the vicinity of 455 to 530 nm can be suitably used because an inexpensive versatile apparatus containing a 488 nm laser can be used.
  • identification agent of the present invention when used, macrophage subtypes can be identified to be visualized, and therefore, the identification agent of the present invention can be used as a research reagent for clarifying the
  • the identification agent of the present invention can be used as a labeling agent for a subtype to be used for determining or diagnosing the pathology of an inflammatory disease in the research or clinical field.
  • the identification agent of the present invention can be used as a labeling agent for a subtype to be used for determining or diagnosing the pathology of an inflammatory disease in the research or clinical field.
  • inventions can be used as a labeling agent for making pathological diagnosis in vitro or ex vivo, or a
  • labeling agent for making diagnosis in vivo with an endoscope or the like.
  • subtypes are not especially limited as long as the subtypes are those different in the function, the expression gene, the expression protein, the cell producing substance such as a protein or a low-molecular weight substance, or the like, but herein refer to the Ml macrophage and the M2 macrophage.
  • the Ml macrophage herein is not especially limited as long as the Ml macrophage has functions of immune activation, promotion of inflammation, disinfection, antitumor and the like.
  • Examples include macrophages having a large amount of proteins, expressed on cell surfaces, such as cluster of differentiation
  • CD 16 16
  • CD32 16
  • CD64 16
  • CD80 CD169
  • lymphocyte antigen 6C macrophages producing a large amount of inflammatory proteins such as tumor necrosis factor-a (hereinafter referred to as TNF-a)
  • TNF-a tumor necrosis factor-a
  • IL-6 interleukin-6
  • interleukin-12 interleukin-12
  • interleukin-23 interleukin-23
  • interleukin- ⁇ macrophages producing a small amount of antiinflammatory proteins such as interleukin-10
  • IL-10 macrophages producing a large amount of proteins such as CC
  • chemokine ligand 2 CC chemokine ligand 3, CC chemokine ligand 4, CC chemokine ligand 5, CC chemokine ligand 9, CC chemokine ligand 10, CC chemokine ligand 11, CXC chemokine ligand 9, CXC chemokine ligand 10, and CXC chemokine ligand 11, macrophages producing a large amount of reactive nitrogen species such as nitrogen monoxide or reactive oxygen species, and macrophages expressing, at high expression level, genes of any of the aforementioned proteins, inducible nitric monoxide synthase (hereinafter referred to as the iNOS) or the like .
  • iNOS inducible nitric monoxide synthase
  • he M2 macrophage herein is not especially limited as long as the M2 macrophage has functions of suppression of immune or inflammation, healing of wound, tissue remodeling, vascularization, promotion of tumor growth and the like.
  • Examples include macrophages having a large amount of proteins, expressed on cell surfaces, such as CD163, CD206, CD204, CD209, CD301, Dectin-1 and Galectin-3, macrophages producing a large amount of anti-inflammatory proteins such as IL-10, interleukin- 1RA and a decoy receptor for interleukin-1 , macrophages producing a small amount of inflammatory proteins such as interleukin-12 , macrophages producing a large amount of proteins such as CC chemokine ligand 1, CC chemokine 16, CC chemokine ligand 17, CC chemokine ligand 18, CC chemokine ligand 22, CC chemokine ligand 24, CXC chemokine ligand 3, CXC chemokine ligand
  • TGF- ⁇ transforming growth factor ⁇
  • macrophages producing a large amount of polyamine and macrophages expressing, at high expression level, genes of any of the aforementioned proteins, arginase-1 (hereafter referred to as the Argl), Yml, FIZZ1 and the like.
  • a biological sample used in the present invention is not especially limited, and the biological sample can be, for example, a biont, a biological tissue, a biological tissue section, a biological tissue block, human cells, animal cells, human cultured cells, or animal cultured cells.
  • the biological sample may be precedently fixed with formalin or the like.
  • Examples of species for the biological sample of, for example, vertebrates include a teleost such as tiger puffer, grass puffer, green spotted puffer, killifish, or zebra fish; an amphibian such as a xenopus; a bird such as a chicken or a quail; a small animal such as a rat, a mouse or a hamster; a large animal such as a goat, a pig, a dog, a cat, a rabbit, a bovine or a horse; and a primate such as a monkey, a chimpanzee or a human.
  • a teleost such as tiger puffer, grass puffer, green spotted puffer, killifish, or zebra fish
  • an amphibian such as a xenopus
  • a bird such as a chicken or a quail
  • a small animal such as a rat, a mouse or a hamster
  • a large animal such as
  • invertebrates include a fruit fly and a roundworm.
  • Examples of the biological sample of cultured cells include cultured cells derived from normal tissues of any of the aforementioned species, or derived from various diseased tissues.
  • cells of a specific subtype may be
  • a method for producing cells by culture is not
  • he Ml macrophage can be produced, for example, by
  • M-CSF granulocyte macrophage colony stimulating factor or phorbol myristate acetate
  • the granulocyte macrophage stimulating factor or the PMA either of protein combinations of interferon- ⁇ and lipopolysaccharide, and interferon- ⁇ and a tumor necrosis factor may be added for producing the Ml macrophage .
  • the M2 macrophage can be produced, for example, by adding an M-CSF, a granulocyte macrophage stimulating factor or the PMA to a medium for culturing precursor cells of macrophages collected from a bone marrow, a spleen, a tissue, a blood or the like.
  • an M-CSF M-CSF
  • a granulocyte macrophage stimulating factor or the PMA a medium for culturing precursor cells of macrophages collected from a bone marrow, a spleen, a tissue, a blood or the like.
  • the granulocyte macrophage stimulating factor or the PMA any of proteins and protein combinations out of
  • interleukin-4 hereinafter referred to as the IL-4
  • interleukin-13 hereinafter referred to as the IL-13
  • IL-4 and IL-13 interleukin-13
  • IL-10 interleukin-10
  • lipopolysaccharide an immune complex and a toll-like receptor, and an immune complex and interleukin-1 receptor ligand may be added for producing the M2 macrophage .
  • the subtype in a case where, for example, a subtype present in a biological sample or in vivo is to be changed to another subtype, or macrophage having been changed in the subtype in vitro is intravitally administered to find the effect of the subtype change, the subtype can be changed by a method similar to that described above.
  • Embodiment 5 of the present invention provides a method for identifying the macrophage subtypes Ml and M2, specifically, a method for identifying the macrophage Ml and the macrophage M2 based on spectral
  • the organic compound is not especially limited but can be a dye compound having a molecular weight of 2000 or less .
  • the Ml macrophage or the M2 macrophage contained in a biological sample is identified preferably by utilizing a difference in staining properties between the Ml macrophage and the M2 macrophage.
  • the organic compound can be a macrophage identification agent of the present invention .
  • the identification method of the present embodiment can be performed, after staining a biological sample by exposing the macrophage identification agent of the present invention to the biological sample, by a flow cytometry method or by using an identifying apparatus such as a fluorescence activated cell sorting
  • FACS fluorescence activated cell sorting
  • a stained biological sample may be any suitable biological sample.
  • a plurality of excitation lights can be used for irradiation and a plurality of types of fluorescence can be detected, so that fluorescence derived from the macrophage identification agent can be detected by using a combination of a plurality of excitation light and fluorescence wavelengths.
  • a plurality of excitation light and fluorescence can be used for irradiation and a plurality of types of fluorescence, so that fluorescence derived from the macrophage identification agent can be detected by using a combination of a plurality of excitation light and fluorescence wavelengths.
  • wavelengths are thus combined, useful information for identifying the subtypes can be obtained.
  • optical characteristics such as forward
  • the identification can be performed merely on living cells by staining dead cells with a dead cell detection reagent.
  • a dead cell detection reagent a commercially available reagent can be suitably used.
  • a light emitting portion and a non-light emitting portion can be easily detected. Besides, if a bright field image obtained by
  • irradiation with visible light and a fluorescent image obtained by irradiation with excitation light are combined with each other by using image processing means, distributions of the subtypes in the biological sample can be observed in more details. Furthermore, a confocal microscope can be used because an optical section image can be thus obtained. Besides, a
  • multiphoton excitation fluorescence microscope can be suitably used for observing the inside of a biological sample because this type of microscope has deep tissue penetration and high spatial resolution.
  • the macrophage subtypes can be identified by the
  • the identification method of the present invention can be used for clarifying the relationship between the pathologic change of an inflammatory disease and the subtypes. Besides, the identification method of the present invention can be used for
  • the compound represented by general formula (1) may be directly used, or may be dissolved in a suitable solvent before use.
  • the solvent include water, physiological saline, a buffer such as a
  • phosphate buffered saline hereinafter referred to as PBS
  • Tris buffer a cell culture medium such as a Dulbecco's Modified Eagle Medium (hereinafter referred to as D-MEM) , an Iscove's Modified Dulbecco's Medium (hereinafter referred to as IMDM) , a Hanks' Balanced Salt Solutions (hereinafter referred to as HBSS) ,
  • D-MEM Dulbecco's Modified Eagle Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • HBSS Hanks' Balanced Salt Solutions
  • MEM-NEAA Minimum Essential Medium-Eagle, Earle's Salts Base, with Non-Essential Amino Acid
  • RPMI Roswell Park Memorial Institute Medium
  • a solvent can particularly contain water by . 50% or more. Besides, a mixture of two or more of these solvents can be used.
  • a serum such as a fetal bovine serum (hereinafter referred to as the FBS) or a horse serum, or an antibacterial agent such as sodium azide or penicillin-streptomycin (hereinafter referred to as the P/S) can be added before use.
  • a physiological saline, a buffer such as a PBS or a Tris buffer, a cell culture medium such as a D-MEM, an IMD , or a HBSS, a commercially available buffer for FACS analysis, or an infusion solution such as a lactated Ringer's solution can be suitably used from the viewpoint of controlling a salt concentration, pH and the like to be suitable for cells.
  • the compound represented by general formula (1) can be dissolved, before mixing with the aforementioned solvent, in an organic solvent such as dimethyl
  • D SO sulfoxide
  • ethanol sulfoxide
  • Another additive may be added if necessary.
  • examples of the additive include a humectant, a surface tension
  • a thickener such as sodium chloride
  • a salt such as sodium chloride
  • any of various pH adjusters such as sodium chloride
  • any of various pH adjusters such as sodium chloride
  • a pH buffer solution such as sodium chloride
  • an antiseptic agent such as sodium chloride
  • an antibacterial agent such as sodium chloride
  • a sweetener any of various pH adjusters
  • a pH buffer solution such as sodium chloride
  • an antiseptic agent such as sodium chloride
  • an antibacterial agent such as sodium chloride
  • sweetener any of various pH adjusters
  • One of these additives may be singly used, or more of these additives may be used in
  • general formula (1) in the macrophage identification agent of the present invention is not especially limited as long as the compound is used in an amount sufficient for identifying the subtypes, and can be appropriately decreased/increased according to the state of a sample in which a target subtype is present.
  • the compound is used in a concentration of 0.001 nM or more and 1000 ⁇ or less, and more
  • the amount of the compound can be as small as possible .
  • One of the compounds represented by general formula (1) for the macrophage identification agent of the present invention can be singly used, or two or more of the compounds may be used in combination. Besides, two or more of the compounds represented by general formula (1) for the macrophage identification agent of the present invention can be used in combination with a known fluorescent dye.
  • identification agent of the present invention to the biological sample can be performed, in the case of a use in vitro or ex vivo, by mixing the biological sample with the macrophage identification agent in a suitable vessel, or bringing the macrophage
  • identification agent may be administered to a living body by orally administering the agent, or by injecting, spraying or applying the agent intravenously or
  • identification agent may be used together with a probe of an endoscope such as a catheter.
  • the temperature at which the staining is performed is not especially limited, but the macrophage
  • identification agent is exposed to the biological sample at a temperature of preferably 4 to 42°C, more preferably 4 to 38°C, further more preferably 31 to 38°C and most preferably 37°C.
  • the time for the staining is not especially limited, but the macrophage identification agent is exposed to the biological sample for preferably 1 minute or more and 24 hours or less, more preferably 1 minute or more and 4 hours or less, and further more preferably 1 minute or more and 1 hour or less.
  • the staining can be performed generally in 1 hour.
  • the macrophage identification agent of the present invention can identify a subtype based on a difference in the intensity of fluorescence derived from the macrophage identification agent caused depending on the subtypes, and this difference is caused, for example, for the following reason: Depending on the subtypes, the amount or speed for import of the macrophage identification agent into cells is different, or the amount or speed for extracellularly export of the macrophage identification agent having been imported into cells is different, the interaction of the
  • macrophage identification agent having been imported into cells, with a component present in the cells, such as a high molecular weight substance, a low molecular weight substance, a gaseous molecule, or an ion, is different depending on the subtypes, or the interaction of the macrophage identification agent with cell surfaces is different depending on the subtypes.
  • the invention works as a substrate of at least one or more transporters expressed on cell surfaces of a subtype, and therefore, the amount or speed for import of the macrophage identification agent into cells or the amount or speed for extracellularly export of the macrophage identification agent having been imported into cells is different depending on the subtypes.
  • the agent is selectively transported by an efflux transporter, that the agent is transported by an efflux transporter in the absence of an efflux transporter inhibitor but is not transported in the presence of the inhibitor, or that the migration via an efflux transporter is changed in the presence of an efflux transporter inhibitor.
  • the transporter is not especially limited, and examples include an ABC transporter, an SLC transporter, a glucose transporter and a dopamine transporter.
  • an efflux transporter is preferred, an ABC transporter is more preferred, and a transporter working as a substrate of Pgp ( P-glycoprotein) , BCRP (Breast Cancer Resistance Protein), MRP (Multidrug Resistance-associated Protein) or MDR (Multidrug
  • the invention is different in the amount or speed of being imported into cells by endocytosis depending on the subtypes. If the macrophage identification agent of the present invention is modified with a liposome, the macrophage identification agent can be easily imported into cells by endocytosis.
  • a high molecular weight substance such as an enzyme or a protein
  • a low molecular weight substance such as a lipid
  • gaseous molecules such as reactive oxygen species or reactive nitrogen species, or ion species such as hydrogen ions present in the cells .
  • the macrophage identification agent of the present invention shows, depending on the subtypes, a different interaction with cell surfaces.
  • the interaction between the macrophage identification agent and the cell surfaces is caused with at least one or more proteins expressed on the cell surfaces of a subtype, amino acid or a functional group contained in the protein, a cell membrane, a lipid contained in the cell membrane, a charge present on the cell surfaces, a hydrophilic region present on the cell surfaces, a hydrophobic region present on the cell surfaces, or the like.
  • washing operation may be performed as occasion demands. The washing operation will now be described.
  • a cell sample obtained by extraction or culture has been stained, the cell sample is precipitated in a proper vessel by centrifugation for removing the staining solution.
  • a solution not containing a dye compound such as the macrophage identification agent of the present invention i.e., a washing
  • washing operation is performed by exposing, in a proper vessel or on a slide glass or the like, the resultant sample to a solution not containing a dye compound such as the macrophage identification agent of the present invention (i.e., a washing solution).
  • the washed operation may be repeated more than once if necessary.
  • the biological sample may be allowed to stand in a washing solution for a prescribed period of time.
  • a shaking or heating operation may be performed if necessary.
  • the washed biological sample is a sample of cells
  • a stirring operation or a filtering operation may be performed so that the cells cannot be aggregated.
  • a subtype of interest can be sorted.
  • cells can be sorted by selectively collecting or removing cells of a subtype of interest or a subtype other than the subtype of interest. For selectively collecting or removing, an aspirator or the like can be used.
  • Ml macrophages plays a significant role in healing an inflammatory disease.
  • M2 M2
  • the sorting method of the present invention can be utilized in a treatment for an arbitrary subtype. Since an arbitrary subtype can be sorted by the sorting method for a macrophage subtype of the present invention, the sorting method of the present invention can be utilized in a treatment for an arbitrary subtype.
  • Embodiment 7 of the present invention includes exposing the macrophage identification agent of the present invention to a biological sample.
  • a test substance after or simultaneously with exposing the macrophage identification agent to a biological sample, a test substance may be allowed to work on a part or the whole of the biological sample.
  • he evaluation method of the present invention further includes detecting the staining properties of a subtype with the macrophage identification agent.
  • the type, number, ratio, optical characteristics or the like of a subtype contained in the biological sample can be evaluated.
  • test substance If a test substance is allowed to work, an effect of the test substance on the type, number, ratio, optical characteristics or the like of the subtype can be evaluated. At this point, if a biological sample on which the test substance is allowed work and a
  • test substance on the subtype can be evaluated based on change in the type, number, ratio or the like of the subtype depending on the presence or absence of the test substance can be evaluated.
  • Two or more types of test substances may be used for evaluating a
  • the evaluation method of the present invention can be utilized for clarifying the relationship between the pathologic change of an inflammatory disease and a subtype. Besides, in the research or clinical field, the evaluation method of the present invention can be used for determining or diagnosing the pathology of an inflammatory disease. Furthermore, since there are a large number of reports that the balance between the subtypes is correlated with the prognosis or recurrence of an inflammatory disease, the evaluation method of the present invention can be used for predicting the prognosis or recurrence of an inflammatory disease. In addition, if the change in subtype caused after administering a drug is examined by the evaluation method of the present invention, a therapeutic drug can be developed, an optimal therapeutic drug can be selected, or the effect of a selected therapeutic drug can be evaluated.
  • an analysis method in which a macrophage identification agent is exposed to a biological sample simultaneously with, before or after exposing a test substance to the biological sample so as to analyze staining properties of a subtype is provided.
  • the test substance may be a substance affecting or not affecting the staining properties of the subtype with the macrophage
  • test substances may be used for analyzing a difference in the action between the test substances.
  • the influence of the test substance on the staining properties can be evaluated.
  • the correlation between the test substance and a transporter expressed on surfaces of cells in which the macrophage identification agent works as a substrate, or a protein, a functional group or the like expressed on surfaces of cells with which the macrophage identification agent interacts can be analyzed .
  • a test substance not affecting the staining properties of a subtype with the macrophage identification agent may be a substance affecting or not affecting the subtype.
  • the action of the test substance on the subtype can be evaluated based on change in the type, number, ratio or the like of the subtype depending upon the presence or absence of the test substance.
  • characteristics derived from the test substance may be measured for detecting the interaction between the test substance and the subtype. If a test substance or a material used for labeling the test substance has fluorescence, the interaction between the test
  • the test substance or the material used for labeling the test substance can be selected to have a different excitation wavelength or a different fluorescence wavelength from that of the macrophage identification agent of the present invention. If the excitation wavelength or the fluorescence wavelength is different, the binding, incorporation or the like of the test substance to the subtype can be analyzed by detecting a fluorescent signal derived from the
  • test substance derived from the test substance or the material used for labeling the test substance.
  • substance having fluorescence is not especially limited, and may be a fluorescent surface antigen marker, or an organic or inorganic molecule having fluorescence.
  • a therapeutic drug can be developed, an optimal therapeutic drug can be selected, or the effect of a selected therapeutic drug can be analyzed.
  • Embodiment 9 of the present invention a screening method in which a macrophage identification agent is exposed to a biological sample simultaneously with, before or after exposing a substance to the biological sample so as to screen the substance based on the staining properties of a subtype is provided.
  • the evaluation method or the analysis method of the present invention described above can be used, so as to evaluate or analyze the effect or the
  • a biologically active substance working on the action of influx and efflux transporters of a compound expressed in a subtype can be evaluated.
  • the action of an efflux transporter can be suitably evaluated. More preferably, an ABC
  • an efflux transporter can be evaluated.
  • the action of an efflux transporter is inhibited by the influence of a biologically active substance and hence the subtype is poorly separated or satisfactorily separated, or is not different from other cells, it can be evaluated that the biologically active substance acts on the efflux transporter.
  • a fluorescent labeled anti-surface antigen antibody is used as a test substance in order to examine a surface antigen expressed in a subtype identified by the macrophage identification agent, a type of antibody having a high affinity with the subtype can be screened.
  • test substance a fluorescent substance unknown in the incorporation or interaction into or with a subtype
  • the function of the test substance on a subtype identified by the macrophage identification agent can be evaluated based on
  • a therapeutic drug can be developed, an optimal therapeutic drug can be selected or the effect of a selected therapeutic drug can be screened by examining the correlation between a subtype and a drug (i.e., change in the subtype caused after administering a drug) by the screening method of the present invention.
  • An identification kit according to Embodiment 10 of the present invention contains at least one or more
  • the identification kit can contain a vessel, a reagent and the like necessary for exposing the macrophage identification agent to a biological sample.
  • an inflammatory disease can be simply diagnosed in situ at the home, in a place for medical examination or in an emergency medical scene.
  • the obtained substance was confirmed to be the compound (1) of interest by 1 H nuclear magnetic resonance spectrometric analysis (ECA-400, manufactured by JEOL Ltd.) and LC/TOF MS (LC/MSD TOF, manufactured by
  • the culture of subtypes was performed according to a protocol described in NPL 7. Specifically, bone marrow cells collected from the femur of a Balb/c mouse of 8 to 10 weeks old were dispersed to a concentration of 1 x lOVmL in an IMDM containing 20% FBS and 1% P/S, and 1 x 10 7 cells were seeded in a 100 mm dish. After further adding an M-CSF (manufactured by Pepro Tech) . thereto to a concentration of 50 ng/mL, the cells were incubated for 3 to 4 days in the presence of 5% C0 2 at
  • the M-CSF was added to a concentration of 50 ng/mL, and the incubation was performed for further 3 to 4 days in the presence of 5% C0 2 at 37°C, and then, the resulting cells were differentiated to macrophage.
  • the cells were differentiated to the subtype Ml.
  • the cells were differentiated to the subtype M2.
  • the resultant was rinsed with PBS, and PBS containing 0.25% trypsin and 1 mM EDTA
  • RQ Relative Quantification
  • the expression level of each marker gene obtained from the cells differentiated to the M2 macrophages shown as a relative value obtained by assuming that the expression level of the marker gene obtained from the cells differentiated to the Ml macrophage is 1.
  • the expression levels of the iNOS and the TNFa were higher in the cells differentiated to the Ml macrophages than in the cells differentiated to the M2 macrophages.
  • identifying dead cells aqua fluorescent reactive dye, manufactured by Invitrogen; which is hereinafter sometimes abbreviated as aqua
  • aqua aqua fluorescent reactive dye, manufactured by Invitrogen; which is hereinafter sometimes abbreviated as aqua
  • the tube was centrifuged at 180 G for 10 minutes to remove the supernatant, cells were suspended in a FACS buffer to be analyzed by using FACSCanto (trademark) II flow cytometry apparatus manufactured by BD.
  • the analysis was performed on a cell population showing a low signal for the aqua, so as to analyze a cell population from which dead cells were eliminated.
  • a fluorescent signal derived from the compound (1) was measured in a FITC channel (excited at 488 nm, 530/30 nm: center wavelength/wavelength width) .
  • a histogram was prepared by plotting fluorescence intensity
  • Fluorescent signals derived from the compounds (3), (4), (6), (7), (9), (11) to (13), (15), (17), (19) to (26), (28), (30) to (33), (35), (36), (38), (42) to (45), (48) to (62), (65), (67), (74) to (76), (82), (83) and (85) were analyzed in the same manner as in Example 1 except that the compound (1) used in Example 1 was replaced with the compounds (3), (4), (6), (7), (9), (11) to (13), (15), (17), (19) to (26), (28), (30) to (33), (35), (36), (38), (42) to (45), (48) to (62), (65), (67), (74) to (76), (82), (83) and (85).
  • Example 1 except that the compound (1) used in Example 1 was replaced with the comparative compound (1).
  • comparative compound (1) was measured in a PE-Cy7 channel (excited at 488 nm, 780/60 nm: center
  • a histogram was prepared by plotting fluorescence intensity obtained in the PE- Cy7 channel on the abscissa and plotting the number of cells at each fluorescence intensity on the ordinate, which is shown in Fig. 3.
  • Example 1 except that the compound (1) used in Example 1 was replaced with the comparative compound (2).
  • a histogram was prepared by plotting fluorescence
  • the fluorescent signals derived from the compounds (3) , (4), (6), (7), (9), (11) to (13), (15), (17), (19) to (26), (28), (30) to (33), (35), (36), (38), (42) to (45), (48) to (62), (65), (67), (74) to (76), (82), (83) and (85) and the comparative compounds (1) and (2) were measured in channels suitable to the respective compounds. Histograms were prepared by plotting fluorescence intensities obtained in the respective channels on the abscissa and plotting the numbers of cells at each fluorescence intensity on the ordinate.
  • a difference between a fluorescence intensity (F M i) at a peak top of the histogram of the Ml macrophage and a fluorescence intensity (F M2 ) at a peak top of the histogram of the M2 macrophage is divided by a sum of a peak width (W M i) of the histogram of the Ml macrophage and a peak width (W M2 ) of the histogram of the M2 macrophage, so as to obtain a resolution R, which was used as an index of the identification ability for a subtype (see the following expression (1) ) .
  • the compound is good in the subtype identification (namely, the resolution R is 0.15 or more and less than 0.5);
  • the compound cannot identify the subtypes (namely, the resolution R is less than 0.15).
  • a fluorescent signal derived from the compound (1) was measured in the FITC channel (excitation wavelength: 488 nm, center wavelength: 530 nm, wavelength width: 30 nm) and a fluorescent signal derived from the compound (33) was measured in an AmCyan channel (excitation wavelength: 405 nm, center wavelength: 510 nm,
  • the compound (1) has a characteristic to stain the Ml macrophages more strongly than the M2
  • the Ml macrophages can be identified as a cell population having a high fluorescence intensity derived from the compound (1), and on the other hand, the M2 macrophages can be identified as a cell
  • the identified by using the compound (1) could be each sorted with an FACS apparatus.
  • the sorted subtypes were confirmed by a fluorescent antibody method and gene expression analysis.
  • FTC a substance for inhibiting export of the compound (1) having been imported into cells
  • concentration of 10 ⁇ was added thereto to suspend the cells, and the resultant was further incubated at 37°C for 30 minutes.
  • each tube was centrifuged at 180 G for 10 minutes to remove the supernatant, the cells were suspended by adding the dye aqua for identifying dead cells thereto, and the resultant was incubated at 4°C for 10 minutes. Thereafter, the tube was centrifuged at 180 G for 10 minutes to remove the supernatant, and then, the cells were suspended in a FACS buffer and analyzed by using a FACSCanto (trademark) II flow cytometry apparatus manufactured by BD.
  • the analysis was performed on a cell population showing a low signal for the aqua, so as to analyze a cell population from which dead cells were eliminated.
  • a blocking treatment for cell surfaces was performed by using 2.4G2 (anti-mouse CD16/32 antibody, manufactured by Biolegend) .
  • 2.4G2 anti-mouse CD16/32 antibody, manufactured by Biolegend
  • a FACS buffer containing 0.2 ⁇ g of Pacific Blue-labeled anti-CD40 antibody and 0.1 ⁇ g of Alexa Fluor647-labeled anti-Dectin-l antibody was added for performing immunofluorescent staining.
  • fluorescent labeling with these antibodies was selected so that the fluorescence wavelengths of the antibodies could overlap neither with, each other nor with the fluorescence wavelengths of the compound (1) and the aqua .
  • each tube was centrifuged at 180 G for 10 minutes to remove the supernatant, the cells were suspended by adding the dye aqua for identifying dead cells, and the resultant was incubated at 4°C for 10 minutes. Then, after the tube was centrifuged at 180 G for 10 minutes to remove the supernatant, the cells were suspended in a FACS buffer and analyzed by using the FACSCanto (trademark) II flow cytometry apparatus manufactured by BD. The analysis was performed on a cell population showing a low signal for the aqua, so as to analyze a cell population from which dead cells were eliminated.

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JP7095603B2 (ja) * 2017-02-14 2022-07-05 コニカミノルタ株式会社 蛍光標識法
KR102550713B1 (ko) * 2021-01-18 2023-07-03 (주)바이오액츠 히드록시 치환된 트리아진을 포함하는 형광 화합물 및 이의 제조방법

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