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REAGENT FOR MEASURING OXYGEN CONCENTRATION IN CELL AND TISSUE

外国特許コード F210010394
整理番号 (S2019-0657-N0)
掲載日 2021年5月6日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2020JP032028
国際公開番号 WO 2021039789
国際出願日 令和2年8月25日(2020.8.25)
国際公開日 令和3年3月4日(2021.3.4)
優先権データ
  • 特願2019-153754 (2019.8.26) JP
発明の名称 (英語) REAGENT FOR MEASURING OXYGEN CONCENTRATION IN CELL AND TISSUE
発明の概要(英語) The present invention addresses the problem of developing a reagent and a compound having a long phosphorescence lifetime for imaging of a low-oxygen cell or tissue, or measurement or quantification of the oxygen concentration in the low-oxygen cell or tissue. The present invention provides an oxygen concentration measurement reagent which contains a compound represented by general formula (I) or (II). (In the formulae, each of R1 and R2 independently represents a hydrogen atom or a hydrocarbon group having from 1 to 6 carbon atoms; and X- represents a counter anion.)
従来技術、競合技術の概要(英語) BACKGROUND ART
Hypoxic environments in vivo are commonly observed in cancer, stroke, myocardial infarction, and the like, which are three causes of death in our countries. Therefore, the development of a method for non-invasively measuring oxygen concentration in cells and tissues in real time is an important issue in the fields of cell biology and medicine. As methods for quantifying oxygen concentration in biological tissues, there have been conventionally (1) a method of measuring by inserting microelectrodes into tissues, (2) a method of using the ESR signal of paramagnetic probe molecules, (3) a method of using the reduction reaction of nitroimidazole probe molecules, and (4) a method of using the emission of water-soluble porphyrins or ruthenium complexes. In the method using microelectrodes of (1), the oxygen partial pressure at a single point near the electrode can be measured. It also has the disadvantage that it is invasive. In the method based on the ESR signal of the (2), it is not possible to measure oxygen concentration in real time. The method of using the nitroimidazole-based drug of (3) utilizes the reduction of nitroimidazole in hypoxic cells to bind and trap intracellular proteins. This method has the disadvantage that since it takes time to metabolize the drug, data cannot be obtained unless several hours have passed after administration of the drug. The method of (4) is a method of quantifying oxygen concentration using the fact that the phosphorescent life span of water-soluble porphyrin derivatives and ruthenium complexes changes depending on the blood oxygen concentration (is quenched). This method has the great advantage of being able to visualize oxygen partial pressure in tissues non-invasively, but because of the aqueous solubility of reagents, the obtained data is limited to blood oxygen levels (Non-Patent Document 1).
The present inventors developed a method for measuring oxygen concentration in biological tissues using room temperature phosphorescence (intensity, lifetime) of the iridium complex (BTP , Fig. 11) (Patent Document 1). From the measurement of the phosphorescence intensity and life span of BTP, quantification of oxygen concentration in liposome membrane, phosphorescence imaging using cancer cells, and visualization of tumors in responsible mice were successful (Non-Patent Documents 2 and 3). Furthermore, an iridium complex BTPHSA (Fig. 11) exhibiting phosphorescence in the near-infrared region was developed (Non-Patent Document 2, Patent Document 2), and tumors approximately 6-7mm from the skin were successfully visualized (Non-Patent Document 2). However, BTPHSA has a short phosphorescent life of 2.0 μs and has low oxygen responsiveness, making it difficult to distinguish between normal tissues and hypoxic tissues. To solve this, the present inventors synthesized an iridium complex PPZ4DMMD, PPZ3DMMD (Fig. 11) having mesityl dipyridinate (MD) and phenyl pyrazoles (PPZ) as ligands (Patent Document 3). These compounds exhibit phosphorescence in 679 nm and have a phosphorescence lifetime of 18μs, and thus exhibit oxygen responsiveness higher than that of BTPHSA. However, a commercially available microplate reader has not been used to reach the phosphorescent lifetime required to measure intracellular oxygen levels.
On the other hand, a reagent (MitoXpress Intra) for quantifying intracellular oxygen concentration using a microplate reader by incorporating a platinum porphyrin complex having low cellular uptake capability into nanoparticles is commercially available from Agilent (Non-Patent Document 4). However, in order to obtain the signal from the MitoXpress Intra, incubation for 14 hours or longer is required. In addition, there is a concern that adverse effects on cells are negatively affected because the ultraviolet light of the 380 nm is required for photoexcitation. Furthermore, there are no examples in live animals. Non-Patent Document 5 describes a compound corresponding to C6-MEDA. However, it has not been applied to oxygen concentration measurements.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • GUNMA UNIVERSITY
  • 発明者(英語)
  • YOSHIHARA, Toshitada
  • HIROSE, Tatsuya
  • TOBITA, Seiji
国際特許分類(IPC)
指定国 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 IT 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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