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REAGENT FOR FLUORESCENCE IMAGING OF LIPID DROPLETS IN CELL AND TISSUE meetings

Foreign code F200010246
File No. (S2019-0343-N0)
Posted date Oct 30, 2020
Country WIPO
International application number 2020JP012002
International publication number WO2020189721
Date of international filing Mar 18, 2020
Date of international publication Sep 24, 2020
Priority data
  • P2019-051368 (Mar 19, 2019) JP
Title REAGENT FOR FLUORESCENCE IMAGING OF LIPID DROPLETS IN CELL AND TISSUE meetings
Abstract The present invention addresses the problem of providing a fluorescent reagent with which it is possible to perform high-sensitivity imaging of lipid droplets at a scale from the cultured cell level to the individual level. The present invention provides a reagent that contains a compound represented by general formula (I) and that is for detecting lipid droplets. (In the formula, m is an integer between 0 and 5; n is an integer between 0 and 5; X is selected from the group consisting of a sulfur atom, an oxygen atom, and a group indicated by NR; R is a hydrogen atom or a group indicated by -(CH2)yCH3; and y is an integer between 0 and 5.)
Outline of related art and contending technology BACKGROUND ART
Lipid drop (fat drop) is a spherical intracellular organelle in which neutral lipids such as triacylglycerol and cholesterol ester are surrounded by a monolayer phospholipid membrane. Most of them are found in adipocytes, but they are universally present in any cells. In recent years, lipid droplets are involved in the regulation of lipid metabolism in cells. In addition, studies on lipid droplets and autophagy have been reported. On the other hand, excessive accumulation of fat in tissue (individuals) leads to tissue dysfunction, causing diabetes, arteriosclerosis, and the like. In recent years, the development of non-alcoholic steatohepatitis (NASH), which is 1 species of hepatitis, has rapidly increased. If NASH is left to stand, there is a risk of progressing to liver cirrhosis or liver cancer. Therefore, it is important to elucidate the mechanism of formation, growth and degradation of lipid droplets in cells and tissues not only in cell biology but also in diagnosis and treatment of these diseases. Therefore, it is necessary to develop a molecular probe for real-time imaging of lipid droplets in living cells or tissues with high sensitivity.
Fluorescence imaging is a convenient method for imaging cells and tissues in living state, and is widely used in biological and medical studies. Many fluorescent reagents for imaging lipid droplets have been reported at the scientific level, but the reagent used for practical use is limited to several kinds. Fig.1 shows currently commercially available lipid drop fluorescence imaging reagents. BODIPY493/503, Nile Red are used by many researchers. BODIPY493/503 shows green fluorescence near 500 nm and has high lipid droplet selectivity. However, there are problems such as the leakage of excitation light caused by the fact that the light stability is not high, the retention of lipid droplets is low, and the energy difference) between the Stokes shift (absorption maximum wavelength and the fluorescence maximum wavelength is small. Further, since Nile red is distributed in many subcellular organelles other than lipid droplets, lipid droplet selectivity is low. Furthermore, since absorption and fluorescence spectra greatly change depending on the surrounding microenvironment, it is difficult to perform multiple dyeing with other fluorescent reagents. In addition, Patent Document 1 reports an oil droplet staining agent using a ring-fused thiophene compound, however, since the peak of excitation light is widely present from blue to green, imaging is overlapped and multicolor imaging is difficult. To solve these problems, LipiDye and Lipi series (Lipi-Blue , Lipi-Green , Lipi-Red) have been developed. Although these reagents are capable of selectively imaging intracellular lipid droplets, there is no knowledge about lipid droplet imaging in living tissue.
For lipid droplet imaging in living tissues, a Nile Blue derivative with nitrobenzenes substituted, (MNs-NB , Fig. 2) is reported as Japanese Patent Document 2. In MNs-NB, a photoinduced electron transfer reaction occurs between the nitrobenzene unit and Nile blue in a polar solvent. On the other hand, in a low-polarity solvent, photoinduced electron transfer reaction is less likely to occur, and red fluorescence is exhibited. Although MNs-NB is a reagent capable of imaging lipid droplets in tissue, there are problems such as) in (0.21: chloroform having a small fluorescence quantum yield and a small Stokes shift.
Scope of claims (In Japanese)[請求項1]
 下記一般式(I)で表される化合物を含む、脂質滴の検出用試薬。
[化1]
(省略)
(式中、
 mは、0~5の整数を示し、
 nは、0~5の整数を示し、
 Xは、硫黄原子、酸素原子およびNRで示される基からなる群から選ばれ、
 Rは、水素原子または-(CH 2) yCH 3で示される基であり、
 yは、0~5の整数を示す。)

[請求項2]
 mおよびnが1である、請求項1に記載の検出用試薬。

[請求項3]
 yが0である、請求項1または2に記載の検出用試薬。

[請求項4]
 生体試料における脂質滴を検出するための、請求項1~3のいずれか一項に記載の検出用試薬。

[請求項5]
 生体試料が、細胞または組織である、請求項4に記載の検出用試薬。

[請求項6]
 生体個体における脂質滴を検出するための、請求項1~3のいずれか一項に記載の検出用試薬。

[請求項7]
 請求項1~6のいずれか一項に記載の検出用試薬を、生体試料または生体個体(ヒトを除く)に投与する工程、
 を含む、脂質滴の検出方法。

[請求項8]
 検出用試薬および溶解補助剤を含む溶液を、生体試料または生体個体(ヒトを除く)に投与する、請求項7に記載の脂質滴の検出方法。

[請求項9]
 溶解補助剤が、アルブミンである、請求項8に記載の脂質滴の検出方法。

[請求項10]
 下記一般式(I)’で表される化合物。
[化2]
(省略)
(式中、
 mは、0~5の整数を示し、
 nは、0~5の整数を示し、
 X’は、酸素原子およびNR’で示される基からなる群から選ばれ、
 R’は、-(CH 2) yCH 3で示される基であり、
 yは、0~5の整数を示す。)

[請求項11]
 mおよびnが1である、請求項10に記載の化合物。

[請求項12]
 yが0である、請求項10または11に記載の化合物。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • GUNMA UNIVERSITY
  • Inventor
  • YOSHIHARA, Toshitada
  • MARUYAMA, Ryo
  • TOBITA, Seiji
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN WS ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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