Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Diagnostic or test agent produces in vivo fluorescence
Reexamination Certificate
1998-01-15
2001-03-27
Jones, Dameron (Department: 1616)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Diagnostic or test agent produces in vivo fluorescence
C424S001730, C424S009300
Reexamination Certificate
active
06207136
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention, in the field of biochemistry, particularly relates to a method for imaging the saccharide uptake activity of living cells which are in a state of maintaining biological activity and for obtaining information relating to changes in viability of living cells with external stimulations.
2. Description of the Prior Art
It is considered that the most universal indicator for representing the viability of living cells is an energy source uptake activity (uptake rate or uptake quantity). The typical example what of many of those cells can commonly take up as an energy source is glucose. However, no useful imaging methods for the glucose uptake activity has been developed until now.
Heretofore, as a method for imaging the glucose uptake activity in living bodies or tissues thereof, a 2-deoxyglucose (2-DG) method has been conducted. Namely, it is a method in that after a radioisotope labeled 2-deoxyglucose is incorporated instead of glucose, slices of living bodies or tissues thereof are observed by mean of autoradiography. However, this method does not allow, the bodies or tissues to be imaged as they are alive at real time, and requires complicated operations and is, furthermore, not sufficient in spatial resolution.
On the other hand, as techniques for imaging living bodies as they are intact, an X-ray CT, PET, MRI, etc. have already been developed. However, though these can image three-dimensionally, any of them have only a spatial resolution of about 0.1 mm, so that a resolution at a single cell level cannot be obtained. Alternatively, with an X-ray microscope and an atomic force microscope, a resolution superior to an optical microscope is obtained. However, they can image only appearances and shapes. Therefore, there still are many technical difficulties for obtaining images corresponding to the glucose uptake activity.
In order to image living bodies or tissues thereof having bioactivity (referred to as “living tissues, etc.” hereinafter) as they maintain the bioactivity, namely, when they are alive, and to obtain, in combination with a microscope, spatial resolution at a single cell level, many fluorescent reagents have so far been developed. For example, mention may be made of (1) those having a fluorescence intensity or fluorescence wavelength which change with changes in the calcium ion concentrations or pH, (2) those having fluorescence intensity which change with changes in the surface potentials of cells, (3) those having a different permeability of the cell membrane between living cells and dead ones, (4) those which emitting fluorescence upon decomposition by a specific enzyme only in living cells, and (5) those combined with antibodies (fluorescence labeled antibody). If these fluorescent reagents are used, though information relating to living or dead cells, or information about responsiveness to exterior stimulation is obtainable, the information is not always said to be a general and universal indicator showing the viability of the cells of the living tissues, etc.
SUMMARY OF THE INVENTION
Therefore, the principal object of the present invention is to make it possible to image the saccharide uptake activity in living tissues, etc. by using a fluorescent saccharide reagent containing a fluorescent glucose derivative obtained by chemically modifying glucose, which is a common energy source for many cells such as the living tissues, etc.
Another object of the present invention is to effect observation and analysis of the saccharide metabolism in various living tissues, etc. by using a series of fluorescent saccharide compounds comprising fluorescent derivatives of glucose analogues, such as galactose, mannose, N-acetylglucosamine or the like, in combination with a fluorescent saccharide reagent comprising a fluorescent glucose compound.
Further, the ultimate object of the present invention is to establish a bioassay by obtaining in real time and assessing the changes in viability of the cells in the living tissues, etc. with exterior stimulations, namely, the changes in physiological function, and provide implements and apparatuses for this purpose.
The present invention to attain the above objects comprises mainly the establishment of a method for utilizing a fluorescent saccharide reagent in the bioassay of the living tissues, etc.
Firstly, the method for fluorescent-imaging the saccharide uptake activity in the living tissues, etc. according to the present invention is characterized in that an aqueous solution of a fluorescent saccharide compound which comprises at its specific position a saccharide molecule chemically combined with a fluorescent pigment molecule having a molecular weight of at most 300 is contacted with or administered to the living tissues, etc. and the above fluorescent saccharide compound is incorporated with the living tissues, etc.
Further, the imaging method of the viability of the living tissues, etc. according to the present invention comprises giving a chemical or physical exterior stimulation to the living tissues, etc. and obtaining information on changes in the viability of cells such as the living tissues, etc. with said stimulation, from a fluorescence imaging change of the saccharide uptake activity by the uptake of the above fluorescent saccharide compound.
The above exterior stimulation includes those which can give changes in the viability or physiological function of the living tissues, etc. such as chemical stimulation with medicaments, poisons, physiological active substances, and microorganisms such as virus and bacteria, and others, or physical stimulation with light, heat, pressure changes, sonic waves, electric fields, magnetic fields, electromagnetic fields, radioactivity, etc.
The above fluorescent saccharide reagent to be used in the fluorescence imaging method of the saccharide uptake activity or the viability of the living tissues, etc. includes an aqueous solution of a fluorescent saccharide compound having a specific position chemically bonded with a fluorescent pigment molecule having a molecular weight of at most 300. As the saccharide molecule, most preferred is glucose, and the above specific position is suited to be the C-2 position in glucose.
The typical example of such a fluorescent saccharide compound is 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (referred to as “2-NBDG” hereinafter) as shown in the following chemical structural formula (1).
The above fluorescent saccharide reagent to be used in the imaging method of the saccharide uptake activity of the living tissues, etc. according to the present invention is preferred to contain the above fluorescent saccharide compound in a concentration of 10 &mgr;M to 10 mM.
Further, the implements and apparatuses for bioassay according to the present invention assess physiological functions of the above-mentioned exterior stimulation based on changes of the fluorescence imaging of the saccharide uptake activity of the living tissues, etc. and the result of the assessments is utilized.
The fluorescent saccharide compound which is a principal ingredient of the fluorescent saccharide reagent according to the present invention, comprises a fluorescent pigment molecule having a molecular weight of at most 300 combined with a specific position of the saccharide molecule. As such a saccharide, glucose is the most preferred, but not limited thereto. The present inventor has found that analogous saccharides such as galactose, mannose, N-acetylglucosamine or the like are also applicable.
Further, the above specific position is most preferred to be the C-2 position in glucose. For example, as shown in
FIG. 1
, D-glucosamine (GlcN) having an amino group substituted for hydroxyl group at the C-2 position of D-glucose is reacted with a fluorescent pigment molecule having a molecular weight not excessively greater than that of glucose (molecular weight: 180), particularly at most 300, for example, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminochloride (molecular weight: 200,
Flynn ,Thiel, Boutell & Tanis, P.C.
Jones Dameron
Tokyo University of Agriculture and Technology
LandOfFree
Fluorescent imaging method of saccharide uptake activity of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Fluorescent imaging method of saccharide uptake activity of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Fluorescent imaging method of saccharide uptake activity of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2501768