U.S. patent application number 15/423901 was filed with the patent office on 2017-05-25 for use of fluoroquinolone antibiotics.
The applicant listed for this patent is Postech Academy-Industry Foundation. Invention is credited to Bum Ju Kim, Ki Hean Kim, Myoung Joon Kim, Jun Ho Lee, Seong Hun Lee, Jin Hyoung Park, Tae Jun Wang.
Application Number | 20170146546 15/423901 |
Document ID | / |
Family ID | 57325307 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170146546 |
Kind Code |
A1 |
Kim; Ki Hean ; et
al. |
May 25, 2017 |
USE OF FLUOROQUINOLONE ANTIBIOTICS
Abstract
Provided are a method and an apparatus for coordinating
inter-cell interference. A user equipment receives from a serving
cell information on a downlink timing offset between an interfering
cell and an interfered cell and a limited resource measurement, and
applies the downlink timing offset to perform measurement using a
radio resource indicated for the limited resource measurement,
thereby allowing the user equipment to be provided services through
the radio resource which substantially mitigates interference, and
enhancing connectivity with a network.
Inventors: |
Kim; Ki Hean;
(Gyeongsangbuk-do, KR) ; Kim; Myoung Joon; (Seoul,
KR) ; Lee; Jun Ho; (Seoul, KR) ; Lee; Seong
Hun; (Daegu, KR) ; Park; Jin Hyoung;
(Gangwon-do, KR) ; Kim; Bum Ju; (Gyeongsangbuk-do,
KR) ; Wang; Tae Jun; (Busan, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Postech Academy-Industry Foundation |
Gyeongsangbuk-do |
|
KR |
|
|
Family ID: |
57325307 |
Appl. No.: |
15/423901 |
Filed: |
February 3, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14943136 |
Nov 17, 2015 |
|
|
|
15423901 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 33/5091 20130101; G01N 2021/6439 20130101; A61K 49/0052
20130101; C12Q 1/04 20130101; G01N 33/582 20130101 |
International
Class: |
G01N 33/58 20060101
G01N033/58; A61K 49/00 20060101 A61K049/00; G01N 21/64 20060101
G01N021/64; G01N 33/50 20060101 G01N033/50; C12Q 1/04 20060101
C12Q001/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2015 |
KR |
10 2015 0070682 |
Claims
1.-5. (canceled)
6. A method for diagnosing at least one of an infectious bacteria
or fungi, the method comprising labeling cells in a biological
tissue using fluoroquinolone antibiotics.
7. The method of claim 6, wherein the tissue includes tissue from
at least one of a cornea, a skin, and a bladder.
8. The method of claim 6, wherein the infectious bacteria include
at least one of pseudomonas, staphylococcus, and the infectious
fungi include at least one of the aspergillus and candida.
9. The method of claim 6, wherein the labeling uses multi-photon
fluorescence expression.
10. The method of claim 6, wherein the fluoroquinolone antibiotics
includes at least one of moxifloxacin and gatifloxacin.
11. The method of claim 6, comprising: preparing a tissue to be
fluorescent-labeled by cell unit; and administering the
fluoroquinolone antibiotics to the tissue.
12.-14. (canceled)
15. The method of claim 11, further comprising: providing
information on infectious bacteria and fungi by inspecting the
tissue through a multi-photon fluorescence-based optical image.
16.-18. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of Korean
Patent Application No. 10-2015-0070682 filed on May 20, 2015, all
of which are incorporated by reference in their entirety
herein.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The present invention relates to a use of fluoroquinolone
antibiotics.
[0004] Description of the Related Art
[0005] Labeling, fluorescence imaging, and measurement methods
using fluorescent materials which are frequently used in biological
researches are methods of fluorescently expressing only a
predetermined area of interest in body organs of organisms except
for humans and have an advantage of photographing with a high
contrast ratio. However, currently, as a fluorescent label material
targeted to a human body, only ICG and fluorescein are used for
vascular labeling, but there are no materials used for labeling
cells or microorganisms.
[0006] Fluoroquinolone antibiotics generally have single-photon
fluorescence generating one fluorescent photon by one high-energy
incident photon. However, since excited light expressing the
fluoroquinolone antibiotics is in an ultraviolet-ray area in which
moxifloxacin is 280 nm and gatifloxacin is 292 nm, the fluorescent
characteristic is not used to determine bacteria causing diseases
in cells in the human body and the like.
[0007] The material having the single-photon fluorescence may also
have a multi-photon fluorescent characteristic which generates one
fluorescent photon through cooperation of two or three low-energy
incident photons such as two-photon fluorescence and three-photon
fluorescence which are non-linear fluorescence. However, the
multi-photon fluorescent characteristic may not be known until
being actually experimentally verified, and fluorescence efficiency
may be verified only by measuring.
[0008] Accordingly, currently, there are no fluorescent label
materials used for the cells of the human body and the like, and in
order to determine the bacterial, a method of diagnosing infectious
bacteria is used.
[0009] The method of diagnosing the infectious bacteria is a method
of verifying inflammation due to infection through a slit lamp,
extracting a tissue at an inflammation portion by rubbing a cotton
swab, and observing the extracted tissue through labeling or
observing the extracted tissue after culture for several days. In
the method, there is a disadvantage in that it takes a lot of time
and thus a treatment time is delayed.
SUMMARY OF THE INVENTION
[0010] Therefore, an object of the present invention is to provide
a use and a method of labeling cells in at least one of a
biological tissue, bacteria, and fungi with fluoroquinolone
antibiotics, a use of applying labeled cells in the body tissue to
a method of diagnosing infectious bacteria by labeling the cells in
the body tissue, and information on diagnosis of infectious
bacteria and fungi.
[0011] An object of the present invention is achieved by labeling
cells in at least one of a biological tissue, bacteria, and fungi
in a use of fluoroquinolone antibiotics.
[0012] The tissue may include at least one of a cornea, a skin, and
a bladder.
[0013] The labeling may use multi-photon fluorescence
expression.
[0014] The fluoroquinolone antibiotics may include
moxifloxacin.
[0015] The fluoroquinolone antibiotics may include
gatifloxacin.
[0016] Another object of the present invention is achieved by
labeling cells in a biological tissue to be used for a diagnosis
method of at least of infectious bacteria and fungi in a use of
fluoroquinolone antibiotics.
[0017] The tissue may include at least one of a cornea, a skin, and
a bladder.
[0018] The infectious bacteria may include at least one of
pseudomonas, staphylococcus, and the infectious fungi include at
least one of the aspergillus and candida.
[0019] The labeling may use multi-photon fluorescence
expression.
[0020] The fluoroquinolone antibiotics may include at least one of
moxifloxacin and gatifloxacin.
[0021] Yet another object of the present invention is achieved by
including preparing a tissue to be fluorescent-labeled by cell unit
and administering the fluoroquinolone antibiotics to the tissue, in
a labeling method of fluoroquinolone antibiotics.
[0022] The tissue may include at least one of a cornea, a skin, and
a bladder.
[0023] The labeling may use multi-photon fluorescence
expression.
[0024] The fluoroquinolone antibiotics may include at least one of
moxifloxacin and gatifloxacin.
[0025] Still another object of the present invention is achieved by
a method of providing information on at least one of infectious
bacteria and fungi including: preparing a tissue to be
fluorescent-labeled by cell unit; adding the fluoroquinolone
antibiotics into the tissue and labeling the tissue; and providing
information on infectious bacteria by inspecting the tissue through
a multi-photon fluorescence-based optical image.
[0026] At least one of the infectious bacteria may include at least
one of cornea, skin, and bladder bacteria.
[0027] The infectious bacteria may include at least one of
pseudomonas, staphylococcus, and the infection fungi include at
least one of aspergillus and candida.
[0028] The fluoroquinolone antibiotics may include at least one of
moxifloxacin and gatifloxacin.
[0029] According to the present invention, it is possible to
provide an object of the present invention is to provide a use and
a method of labeling cells in at least one of a biological tissue,
bacteria, and fungi with fluoroquinolone antibiotics, a use of
applying labeled cells in at least one of the bacteria and fungi to
a method of diagnosing infectious bacteria and fungi by labeling
the cells in the biological tissue, and information on diagnosis of
infectious bacteria and fungi.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 illustrates a structural formula and a chemical
formula of hydrochloride moxifloxacin used in the present
invention.
[0031] FIG. 2 illustrates a structural formula and a chemical
formula of gatifloxacin used in the present invention.
[0032] FIG. 3A illustrates an excite spectrum of hydrochloride
moxifloxacin M and gatifloxacin G and FIG. 3B illustrates a
fluorescence spectrum of hydrochloride moxifloxacin M and
gatifloxacin G
[0033] FIGS. 4A to 4D illustrate auto-fluorescence expression
photographs of a corneal superficial epithelium, a corneal basal
epithelium, a corneal stroma, and a corneal endothelium of a mouse,
respectively, and FIG. 4E illustrates an average signal graph of
FIGS. 4A to 4D.
[0034] FIGS. 5A to 5D are photographs fluorescent-expressed by
administering hydrochloride moxifloxacin to the cornea of the
mouse, respectively, and FIGS. 6A to 6D are photographs
fluorescent-expressed by administering gatifloxacin to the cornea
of the mouse, respectively. FIGS. 5A and 6A illustrate the corneal
superficial epithelium, FIGS. 5B and 6B illustrate the corneal
basal epithelium, FIGS. 5C and 6C illustrate the corneal stroma,
FIGS. 5D and 6D illustrate the corneal endothelium. FIGS. 5E and 6E
illustrate tomograms and average signal graphs of the superficial
epithelium, the basal epithelium, the stroma, and the endothelium
administered with hydrochloride moxifloxacin and gatifloxacin,
respectively.
[0035] FIGS. 7A to 7D are auto-fluorescence expressed photographs
photographed by a two-photon microscope while descending from
epithelia cells to dermal cells of a ear skin of a mouse, and FIGS.
8A to 8D are photographs fluorescent-expressed at the same position
of FIGS. 7A to 7D by administering hydrochloride moxifloxacin,
respectively.
[0036] FIG. 9 illustrates a protocol of a fluorescence expression
experiment of bladder cells of the mouse administered with the
hydrochloride moxifloxacin.
[0037] FIG. 10A illustrates a photograph fluorescent-expressed by
administering hydrochloride moxifloxacin to a lumen tissue of the
bladder and FIG. 10B illustrates a photograph fluorescent-expressed
by administering hydrochloride moxifloxacin to a serosa tissue
thereof.
[0038] FIGS. 11A, 11B, and 11C illustrate photographs photographing
umbrella cells, intermediate cells, and a laminar propria of the
bladder lumen tissue which are auto-fluorescently expressed,
respectively, and FIGS. 12A, 12B, and 12C are photographs
photographing umbrella cells, intermediate cells, and a laminar
propria of the bladder lumen tissue which are fluorescently
expressed by administering hydrochloride moxifloxacin,
respectively.
[0039] FIGS. 13A, 13B, and 13C illustrate photographs photographing
a bladder serosa tissue which is auto-fluorescently expressed while
going deep into the inside thereof, and FIGS. 14A, 14B, and 14C
illustrate appearances which are fluorescent-expressed by
administering hydrochloride moxifloxacin to the bladder serosa
tissue at the same position as FIGS. 14A, 14B, and 14C,
respectively.
[0040] FIGS. 15A and 15B illustrate appearances of pseudomonas and
staphylococcus used in the present experiment which are observed
through a microscope, respectively.
[0041] FIGS. 16A to 16B and 17A to 17B illustrate fluorescence
expressed photographs of pseudomonas before administering
hydrochloride moxifloxacin, pseudomonas after administering
hydrochloride moxifloxacin, staphylococcus before administering
hydrochloride moxifloxacin, and staphylococcus after administering
hydrochloride moxifloxacin, respectively.
[0042] FIG. 18 illustrates a graph before and after administering
hydrochloride moxifloxacin to pseudomonas and staphylococcus.
[0043] FIGS. 19A and 20A are auto-fluorescent expressed photographs
of aspergillus and candida albican before administering
hydrochloride moxifloxacin which are photographed by laser power of
7 mW, respectively, and FIGS. 19B and 20B are auto-fluorescent
expressed photographs of aspergillus and candida albican which are
photographed by laser power of 50 mW, respectively.
[0044] FIGS. 19C and 20C are photographs of aspergillus and candida
albican after administering hydrochloride moxifloxacin which are
photographed by laser power of 7 mW, respectively
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0045] The present invention provides a use of fluoroquinolone
antibiotics labeling cells in a at least one of a biological
tissue, bacteria, and fungi. and using the labeled cell in a
diagnosis method of infectious bacteria and fungi.
[0046] In the present invention, the labeling method of cells in
the at least one of a biological tissue, bacteria, and fungi. using
the fluoroquinolone antibiotics includes (a) preparing a at least
one of a biological tissue, bacteria, and fungi to be
fluorescent-labeled by cell unit, and (b) adding fluoroquinolone
antibiotics to at least one of the biological tissue, bacteria, and
fungi.
[0047] Further, the present invention provides information on
diagnosis of at least one of infectious bacteria and fungi.
including (a) preparing a tissue to be fluorescent-labeled by cell
unit, (b) adding and labeling fluoroquinolone antibiotics to the
tissue, and (c) providing information on at least one of infectious
bacteria and fungi by inspecting the tissue through a multi-photon
fluorescent-based optical image.
[0048] A moving appearance of molecules, cells, and tissues of the
organism may be observed through a fluorescence microscope when
being treated by a fluorescent probe (FP). While electrons in the
FP become in the excited state by the incident photon and return to
the original site again, fluorescent photons having special colors
are emitted.
[0049] When the fluorescent photons are emitted in the visible
light area, it may be used to determine the bacteria causing the
diseases in cells in the human body and the like. In the present
invention, it was found that the fluoroquinolone antibiotics may be
fluorescent-expressed in the visible light area as well as the
ultraviolet light area. Further, it was found that the
fluoroquinolone antibiotics may label the cells in the human body
and it was verified that the fluoroquinolone antibiotics may label
the bacteria and fungi. Accordingly, the present invention may
provide the method of labeling the cells of the tissue in the human
body and information on diagnosis of the infectious bacteria and
fungi, by using the fluoroquinolone antibiotics.
[0050] As the fluoroquinolone antibiotics, there are moxifloxacin,
gatifloxacin, pefloxacin, difloxacin, nofloxacin, ciprofloxacin,
ofloxacin, enrofloxacin, and the like, but preferably,
fluoroquinolone antibiotics in which auto-fluorescence is expressed
in the visible light area or multi-photon fluorescence including
the visible light area may be expressed are suitable. Hereinafter,
in Experimental Example, the experiment was performed by using
gatifloxacin and hydrochloride moxifloxacin substituted with
hydrochloride which are frequently used as ocular antibiotics among
the fluoroquinolone antibiotics.
[0051] In the following Experimental Example, it was verified that
the hydrochloride moxifloxacin and the gatifloxacin may express
fluorescent signals even in a light source having a near-infrared
wavelength in a range of 700 nm to 800 nm. Further, it was verified
through the following Experimental Examples that fluorescence
efficiency of the body tissue administered with the antibiotics is
10 to 100 times larger than the auto-fluorescence and images of
antibiotics-based cells in the tissue may be photographed without
removing the tissue.
[0052] The present invention may be applied to various at least one
of the following: a biological tissue, bacteria, and fungi, and
preferably, may be applied to cells in the tissue, for example, eye
(cornea), skin, small intestine, stomach, cecum, colon, rectum,
liver, lung, etc. In Experimental Example of the present invention,
the fluorescence expression was measured by administering
hydrochloride moxifloxacin and gatifloxacin to corneal cells, skin
cells, and bladder cells.
[0053] In order to use the fluoroquinolone antibiotics in the
method of diagnosing at least one of the infectious bacteria and
fungi, only when causative bacteria or fungi existing in each cell
as well as the cells in the tissue are labeled, whether infection
exists or not may be determined. The method of the present
invention may be applied to various at least one of bacteria and
fungi, and preferably, may be applied to at least one of bacteria
and fungi, and preferably which may be infected in the cells in the
biological tissue. Further, the method of the present invention may
be applied even to fungus consisting of eukaryotes such as a
human.
[0054] In Experimental Example of the present invention,
pseudomonas and staphylococcus were cultured, fluoroquinolone
antibiotics were administered, and it was verified through the
multi-photon microscope that the fluorescence was expressed.
[0055] First, moxifloxacin and gatifloxacin which are used in the
experiment and areas and intensities of the fluorescence expression
of the antibiotics will be described with reference to FIGS. 1, 2
and 3A to 3B.
[0056] The moxifloxacin as 0.5% of vigamox eye drops being sold in
the market (Alcon Korea Co., Ltd.) is hydrochloride moxifloxacin
(moxifloxacin HCl, 5.45 mg of hydrochloride moxifloxacin)
synthesized with hydrochloride like a structural formula
illustrated in FIG. 1.
[0057] The gatifloxacin is gatiflo eye drops (Handok
pharmaceuticals Co., Ltd., 3 mg of gatifloxacin) having a
structural formula illustrated in FIG. 2.
[0058] FIG. 3A illustrates an excitation spectrum of hydrochloride
moxifloxacin and gatifloxacin. Here, an X axis represents a
wavelength (nm) of a femtosecond laser light source used in the
experiment, and a degree (a.u.) of two-photon excited fluorescence
(TPF) at 700 nm to 800 nm which is a measuring range of a
femtosecond laser wavelength is illustrated as a Y axis. Through
the graph, it can be seen that hydrochloride moxifloxacin and
gatifloxacin may represent fluorescent signals in the light source
having a range of 700 nm to 800 nm which is a near-infrared
wavelength. Further, in the case where the wavelength of the light
source is 700 nm as shown in the intensity of the excited
fluorescence graph of the Y axis, it can be seen that a fluorescent
signal stronger than other wavelengths is shown.
[0059] FIG. 3B illustrates an emission spectrum representing a
fluorescence expression degree of each antibiotic when the
femtosecond laser is applied to hydrochloride moxifloxacin and
gatifloxacin. The X axis represents a wavelength (nm) and the Y
axis represents the degree (a.u.) of the TPF. In the graph, in the
hydrochloride moxifloxacin and the gatifloxacin, it can be seen
that the fluorescent signals having wavelengths of 450 nm to 550 nm
are frequently expressed and the wavelength of about 520 nm is
expressed most strongly. Further, in the intensity of the graph of
the Y axis, it can be seen that the fluorescence expression of the
hydrochloride moxifloxacin is much better than that of the
gatifloxacin.
[0060] Hereinafter, the present invention will be described in more
detail through Experimental Examples. Experimental Examples are
just to describe the present invention in more detail, and the
scope of the present invention is not limited by Experimental
Examples according to the gist of the present invention. In the
following experiment, as cells of a tissue, a cornea, a skin
(ears), and a bladder are targeted, but the present invention is
not limited thereto, and may be applied to cells of various tissues
in the body such as a prostate and a colon.
[0061] The bacteria target pseudomonas and staphylococcus, but the
present invention may be applied to various bacteria which may be
infected in cells of the tissue in the body, and is not limited to
the following Experimental Examples. And the present invention may
be applied to various fungi such as aspergillus and candida, and is
not limited to the following
[0062] Further, the present invention may be applied even to a
fungus which is a eukaryote such as a human and is not limited even
to a kind of fungus.
EXPERIMENTAL EXAMPLE 1
Measurement of Multi-Photon Fluorescence of Corneal Cells of
Fluoroquinolone Antibiotics (Hydrochloride Moxifloxacin and
Gatifloxacin)
[0063] 1) Preparation of Materials and Samples
[0064] Blab/c female mice after five or six weeks, gatifloxacin,
hydrochloride moxifloxacin, and a multi-photon microscope including
a biaxial scanner (a galvano scanner of x axis and a galvano
scanner of y axis) to be used by a point scanning method were
prepared.
[0065] In this Experimental Example, a two-photon microscope using
a femtosecond laser as a light source was used and the multi-photon
fluorescence was equally measured under the following condition
throughout an experimental process.
[0066] Excitation wavelength: 780 nm for vigamox (moxifloxacin)
[0067] Filter set: Ch01: [430 nm, Ch02:]430 nm
[0068] Manufacturer/Product name of Microscope: Leica/TCS SP5II MP
SMD FLIM
[0069] Filter: 500/25 bandpass filter, chroma
[0070] Light source: chameleon vision II, coherent
[0071] Camera: photon multiplier tube (PMT) 6357, Hamamatsu
Photonics
[0072] Objective lens: 25.times.0.95 NA objective lens, leica
[0073] 2)Measurement of Multi-Photon Fluorescence of Corneal Cells
of Fluoroquinolone Antibiotics (Hydrochloride Moxifloxacin and
Gatifloxacin)
[0074] In order to compare fluorescence expression of hydrochloride
moxifloxacin with auto-fluorescence expression of the corneal cells
of the mouse, the corneal cells of the mouse which are not applied
with hydrochloride moxifloxacin were first photographed.
[0075] The Blab/c mouse to be used in the experiment was
anesthetized and then fixed to an eye holder for photographing the
eye, and photographed by the two-photon microscope. Laser power was
set as 30.8 mW, and in this case, the intensity of
auto-fluorescence of the photographed mouse cornea was
recorded.
[0076] In order to measure the fluorescence expression degree of
the mouse cornea by vigamox (hydrochloride moxifloxacin),
hydrochloride moxifloxacin of 10 .mu.l was dropped in a left eye of
the mouse and an eyelid was closed for 30 seconds. About 20 minutes
waited so that hydrochloride moxifloxacin may penetrate into cells
inside the mouse cornea.
[0077] An incubation time when the antibiotic penetrates varies
according to a tissue, but was verified by photographing with for
example, a time lapse such as after 5 minutes and after 10 minutes.
In the cornea, the incubation time was within about several tens of
minutes. The fluorescence image of the cornea was photographed by
the point scanning method through the two-photon microscope. (In
this case, an excitation wavelength of the spectrum was set as 790
nm, the laser power of the hydrochloride moxifloxacin was about
14.8 mW, and the laser power of the gatifloxacin was set as 30.8 mW
which was the same as the laser power before administering the
antibiotic.
[0078] FIG. 4A to 4E illustrate photographs of a cornea of a left
eye of a mouse which is not treated with hydrochloride moxifloxacin
and gatifloxacin photographed by an X-Y plane point scanning method
using the two-photon microscope and average signal graphs.
[0079] FIGS. 4A to 4D illustrate appearances in which hydrochloride
moxifloxacins administered to a corneal superficial epithelial cell
layer, a corneal basal epithelial cell layer, a corneal stroma
layer, and a corneal endothelium layer are fluorescent-expressed,
respectively. Here, a yellow solid line represents a scale bar and
a length means 100 .mu.m.
[0080] FIG. 4E illustrates an average signal graph at positions of
each cornea which is fluorescent-expressed while descending from a
position of FIG. 4A to a position of FIG. 4D, and an X axis
represents the intensity of a signal in a depth direction from a
surface to the corneal endothelium layer in depth. That is, a depth
of 0 is a corneal surface and means the corneal epithelial layer,
the stroma, and the corneal endothelium layer downwards.
[0081] In the photographs illustrated in FIGS. 4A to 4D, it can be
seen that the auto-fluorescence expression in the body tissue is
very weak in the laser power of 30.8 mW, and in this case, it can
be seen that a size of the fluorescent signal is significantly low
as 20 to 50 a.u. as verified in a graph of FIG. 4E.
[0082] FIGS. 5A and 5D are photographs photographing the
fluorescent-expressed cornea of the mouse by hydrochloride
moxifloxacin through a multi-photon microscope. And FIGS. 6A and 6D
are photographs photographing the fluorescent-expressed cornea of
the mouse by gatifloxacin through a multi-photon microscope. Here,
FIGS. 5A and 6A illustrate appearances of the corneal superficial
epithelial cell layer, FIGS. 5B and 6B illustrate appearances of
the corneal basal epithelial cell layer, FIGS. 5C and 6C illustrate
appearances of the corneal stroma layer, FIGS. 5D and 6D illustrate
appearances of the corneal endothelium layer, which are
fluorescent-expressed on an X-Y plane, respectively. Here, a yellow
solid line represents a scale bar and a length means 100 .mu.m.
[0083] In a concentration difference of fluorescent colors
illustrated in FIGS. 5A to 5D and 6A to 6D, it can be seen that
there is a difference in fluorescence expression due to
fluoroquinolone antibiotics (hydrochloride moxifloxacin and
gatifloxacin). It is verified that the fluorescence expression
degree is strongly shown on the corneal superficial epithelial cell
layers of FIGS. 5A and 6A which are the corneal surfaces because
the fluoroquinolone antibiotics are dropped from the corneal
epithelial layer to be diffused and penetrate to the inside of the
cornea.
[0084] FIGS. 5E and 6E illustrate photographs photographing the
cornea labeled with hydrochloride moxifloxacin through point
scanning on an X-Z plane and an X-axial depth represents the
intensity of a signal in a depth direction from the surface to the
corneal endothelium layer. That is, the top of the X axis
represents the corneal epithelial layer and represents the corneal
endothelium layer downwards. In this photograph, it is verified
that the fluorescence expression is strongly shown on the
superficial epithelial cell layer. And in graphs, the depth of 0
means the corneal surface, and a depth represented by a blue dotted
line of the graph means a thickness of the entire corneal
epithelial layer, and lower portions thereof mean the stroma layer
and the corneal endothelium layer, respectively.
[0085] When comparing FIGS. 4A to 4D and 6A to 6D as the same laser
power condition of 30.8 mW, it can be seen that the intensity of
the fluorescence expression when administering the gatifloxacin is
much larger than that of the auto-fluorescence expression. Further,
in the case of the hydrochloride moxifloxacin, even in the laser
power of 14.8 mW, the intensity of the signal is larger than that
of the auto-fluorescence expression and the fluorescence expression
by the gatifloxacin of the mouse cornea which are measured with
30.8 mW, and as a result, It can be seen that the fluorescence
expression is very excellent during administration to the
cornea.
[0086] Further, when comparing graphs of FIGS. 5E and 6E and a
graph of FIG. 4E, even in the corneal endothelium layer, it can be
seen that the fluorescence intensity when administering the
hydrochloride moxifloxacin and the gatifloxacin is higher than the
fluorescence intensity of the auto-fluorescence expression. As a
result, it can be seen that the innermost cell layer in the tissue
may be verified by the fluorescence expression signal through the
hydrochloride moxifloxacin and the gatifloxacin.
[0087] Hereinafter, in Experimental Examples, an experiment was
performed by using only hydrochloride moxifloxacin of which
fluorescence expression is better than that of gatifloxacin.
EXPERIMENTAL EXAMPLE 2
Measurement of Multi-Photon Fluorescence in Skin Cells of
Hydrochloride Moxifloxacin
[0088] 1) Preparation of Materials and Samples
[0089] Blab/c female mice after five or six weeks, hydrochloride
moxifloxacin, and a two-photon microscope including a biaxial
scanner (a galvano scanner of x axis and a galvano scanner of y
axis) to be photographed by a point scanning method, a scotch tape,
a PBS were prepared.
[0090] In this Experimental Example, a two-photon microscope using
a femtosecond laser as a light source was used and the multi-photon
fluorescence was equally measured under the following condition
throughout an experimental process.
[0091] Excitation wavelength: 780 nm for vigamox (moxifloxacin)
[0092] Filter set: Ch01: [490 nm, Ch02: ]490 nm
[0093] Light source: Chameleon Ultra II, Coherent
[0094] Camera: photomultiplier tube (PMT) H7421-40P, Hamamatsu
Photonics
[0095] Objective lens: 20.times.1.0 NA objective lens, XLUMPlanFL,
Olympus
[0096] Photographing area: 300 .mu.m .times.300 .mu.m
(512.times.512 pixels)
[0097] 2) Measurement of Multi-Photon Fluorescence in Skin Cells of
Hydrochloride Moxifloxacin
[0098] In order to compare fluorescence expression of hydrochloride
moxifloxacin with auto-fluorescence expression of the skin cells of
the mouse, the skin cells of the mouse which are not applied with
hydrochloride moxifloxacin were first photographed by the
two-photon microscope. While the mouse was alive through inhalation
anesthesia, a skin (ear) tissue was photographed.
[0099] In order to photograph the fluorescence expression of the
hydrochloride moxifloxacin in the skin tissue of the mouse, a
general scotch tape was repeatedly attached to and detached from
the ear tissue of the mouse about 15 times to remove a horny layer
and vigamox was applied.
[0100] After the vigamox penetrated for about 20 minutes, the
vigamox was washed by using the PBS, and while the mouse was alive
through inhalation anesthesia like the pre-photographing of the
vigamox, the image of the skin (ear) tissue was photographed by the
point scanning method through the multi-photon microscope.
[0101] FIGS. 7A to 7D illustrate auto-fluorescence expressed
photographs of an ear tissue of a mouse before administering
hydrochloride moxifloxacin photographed while descending from
superficial epithelial cells to dermal cells, respectively, and
FIGS. 8A to 8D are fluorescence expressed photographs of the ear
tissue at the same position after administering hydrochloride
moxifloxacin photographed by the same method, respectively. In this
case, the power of the used laser was 121 mW before administering
the hydrochloride moxifloxacin and 17 mW after administering the
hydrochloride moxifloxacin.
[0102] When describing the fluorescence expression degrees of FIGS.
7A to 7D and 8A to 8D, it can be seen that the fluorescence
expression is excellent around the superficial epithelial cells and
the intensity of the fluorescent signal is decreased toward the
dermal cell.
[0103] Further, when comparing FIGS. 8A to 8D with FIGS. 7A to 7D
at the same position, even though the laser power is significantly
low, it can be seen that the tissue administered with the
hydrochloride moxifloxacin was clearly photographed. Particularly,
when comparing FIGS. 7D and 8D as the innermost dermal cells, it
could be seen that cells which are not shown by the
auto-fluorescence expression, particularly, spatial dendritic cells
(langerhans cells) were clearly observed by the fluorescence
expression having the strong intensity of the hydrochloride
moxifloxacin.
EXPERIMENTAL EXAMPLE 3
Measurement of Multi-Photon Fluorescence in Bladder Cells of
Hydrochloride Moxifloxacin
[0104] 1) Preparation of Materials and Samples
[0105] Hydrochloride moxifloxacin, a rat (normal), a vigamox eye
drop (hydrochloride moxifloxacin), an anesthetics, a warmer, a
slide glass, a forcep, a PBS, and a two-photon microscope including
a biaxial scanner (a galvano scanner of x axis and a galvano
scanner of y axis) to be used by a point scanning method for
measuring the multi-photon fluorescence expression degree were
prepared.
[0106] In this Experimental Example, the used vigamox and the
two-photon microscope using a femtosecond laser as a light source
were measured under the following specification condition
throughout an experimental process.
[0107] Excitation wavelength: 780 nm for vigamox (moxifloxacin)
[0108] Filter set: Ch01: [490 nm, Ch02: ]490 nm
[0109] Light source: Chameleon Ultra II, Coherent
[0110] Camera: photomultiplier tube (PMT) H7421-40P, Hamamatsu
Photonics
[0111] Objective lens: 20.times.1.0 NA objective lens, XLUMPlanFL,
Olympus
[0112] Photographing area: 300 .mu.m.times.300 .mu.m (512.times.512
pixels)-FIG. 10A and 10B
[0113] 150 .mu.m.times.150 .mu.m (512.times.512 pixels)-FIGS. 11A
to 11C, 12A to 12C, 13A to 13C and 14A to 14c
[0114] 2) Measurement of Multi-Photon Fluorescence in Bladder Cells
of Hydrochloride Moxifloxacin
[0115] In order to compare fluorescence expression of hydrochloride
moxifloxacin with auto-fluorescence expression of the bladder cells
of the mouse, the rat (normal) which was not applied with
hydrochloride moxifloxacin was prepared and the bladder cells of
the rat (normal) were first photographed by the two-photon
microscope.
[0116] The rat (normal) was sacrificed and the bladder tissue was
extracted. After the extracted bladder tissue was washed in a PBS
solution about five times, the washed bladder tissue was cut with
scissors for surgery and unfolded so that photographed portions
(the lumen and the serosa) may be exposed outside. After the
unfolded bladder tissue was placed on the slide glass so that the
photographed portions (the lumen and the serosa) faced upward,
covered with a coverslip, and fixed well (using a tape), the
auto-fluorescence intensity was measured.
[0117] In Experimental Example, the auto-fluorescence intensity was
measured while the objective lens descended by 2 .mu.m so as to
verify an appearance of cells photographed for each depth of the
lumen and serosa tissues of the bladder tissue. Particularly, the
lumen tissue was photographed so that the umbrella cell, the
intermediate cell, and the laminar propria were verified,
respectively.
[0118] Even like this Experimental Example, the incubation time
when the antibiotic penetrated was photographed and verified with a
time lapse. The fluorescence image of the bladder was photographed
by the point scanning method through the two-photon microscope. In
this experiment, in the lumen of the bladder tissue, the laser
power of 300 mW or more was used, and in the serosa, the laser
power of 200 mW or more was used.
[0119] Hereinafter, an experimental process of the multi-photon
fluorescence measurement of the bladder cells administered with
hydrochloride moxifloxacin will be described with reference to FIG.
9.
[0120] The rat normal was sacrificed, the bladder tissue was
extracted, and the extracted tissue was immersed in vigamox
(hydrochloride moxifloxacin) for 20 minutes and incubated.
Thereafter, the bladder tissue was held with the forceps and gently
washed while being shaken about 2 to 3 times in the PBS.
[0121] Thereafter, a tissue sample was prepared on the slide glass
so that the lumen tissue which is the inside of the cell and the
serasa tissue which is the outside of the cell are exposed during
photographing and TPM-photographed within 30 minutes after the
vigamox was treated so as to complete the photographing when the
cells are activated.
[0122] Even like this Experimental Example, the incubation time
when the antibiotic penetrated was photographed and verified with a
time lapse. The fluorescence image of the bladder was photographed
by the point scanning method through the two-photon microscope. In
the lumen tissue of the bladder tissue treated with the
hydrochloride moxifloxacin, the laser power of 15 mW was used, and
in the serosa tissue, the laser power of 10 mW was used.
[0123] FIG. 10A is a photograph of a lumen of the bladder tissue of
the mouse and FIG. 10B is a photograph of a serosa of the bladder
tissue of the mouse, which are administered with hydrochloride
moxifloxacin photographed by a two-photon microscope in an area of
300 .mu.m.times.300 .mu.m, respectively.
[0124] In the lumen of the bladder tissue of FIG. 10A, it could be
seen that vascular endothelial cells arranged along a vascular wall
were labeled and observed.
[0125] Even in the serosa of the bladder tissue of FIG. 10B, it can
be seen that a muscle observed to be horizontally thick on the top
of the photograph and cells existing between muscular tissues were
observed. Further, it could be seen that the vascular endothelial
cells and the cells distributed around the vascular endothelial
cells were labeled and observed together.
[0126] More effectively, in order to observe expression of the
hydrochloride moxifloxacin in the cells of the bladder tissue, it
will be described in detail with reference to FIGS. 11A to 11C, 12A
to 12C, 13A to 13C and 14A to 14C.
[0127] FIGS. 11A to 11C and 12A to 12C illustrate S1 of FIG. 10A as
the inside of the bladder cell which are enlarged at 150
.mu.m.times.150 .mu.m.
[0128] FIGS. 11A to 11C are the lumen of the bladder tissue before
administering the hydrochloride moxifloxacin which is photographed
by laser power of 300 mW or more while gradually going deep into
the inside, and FIGS. 12A to 12C are the lumen of the bladder
tissue after administering the hydrochloride moxifloxacin which is
photographed by laser power of 15 mW or more while gradually going
deep into the inside.
[0129] That is, FIGS. 11A and 12A illustrate the umbrella cells,
FIGS. 11B and 12B illustrate the intermediate cells, and FIGS. 11C
and 12C illustrate the laminar proprias.
[0130] When comparing the respective cells, even though the laser
power is strong, it was difficult to verify the structure of the
cells in the photograph of auto-fluorescence expression before
administering the hydrochloride moxifloxacin. Particularly, in the
case of the laminar propria, it is difficult to observe the laminar
propria because blood vessels are mainly distributed, and after
administering the hydrochloride moxifloxacin, even though the
intensity of the laser power is decreased to 1/20, the structure of
the laminar propria may also be efficiently observed together with
peripheral blood vessels.
[0131] FIGS. 13A to 13C and 14A to 14C illustrate S2 of FIG. 10B as
the outside of the bladder cell which is enlarged at 150
.mu.m.times.150 .mu.m. FIGS. 13A to 13C are the serosa of the
bladder tissue before administering the hydrochloride moxifloxacin
which is photographed by laser power of 200 mW or more while
gradually going deep into the inside, and FIGS. 14A to 14C are the
serosa of the bladder tissue after administering the hydrochloride
moxifloxacin which is photographed by laser power of 10 mW or more
while gradually going deep into the inside.
[0132] Referring to FIGS. 13A to 13C, a muscular tissue having a
grain-pattern shape may be efficiently observed, but it was
difficult to obtain an image of cells existing between muscular
coats.
[0133] Referring to FIGS. 14A to 14C at the same position, it can
be seen that the muscular tissue having the grain-pattern shape was
observed well even by small laser power and the cells existing
between the muscular coats were also observed.
EXPERIMENTAL EXAMPLE 4
Measurement of Multi-Photon Fluorescence in Bacteria of
Hydrochloride Moxifloxacin
[0134] 1) Preparation of Materials and Samples
[0135] Bacteria (pseudomonas and staphylococcus) used in the
experiment, hydrochloride moxifloxacin, and a biaxial scanner (a
galvano scanner of x axis and a galvano scanner of y axis) to be
used by a point scanning method were prepared.
[0136] In this Experimental Example, a two-photon microscope using
a femtosecond laser as a light source was used, and the
multi-photon fluorescence was equally measured under the following
condition throughout an experimental process.
[0137] Excitation wavelength: 780 nm for vigamox(moxifloxacin)
[0138] Filter set: Ch01:[430 nm, Ch02:]430 nm
[0139] Manufacturer/Product name of Microscope: Leica/TCS SP5II MP
SMD FLIM
[0140] Filter: 500/25 bandpass filter, chroma
[0141] Light source: chameleon vision II, coherent
[0142] Camera: photon multiplier tube (PMT) 6357, Hamamatsu
Photonics
[0143] Objective lens: 250.95 NA objective lens, leica
[0144] 2) Measurement of Multi-Photon Fluorescence in Bacteria of
Hydrochloride Moxifloxacin
[0145] FIGS. 15A and 15B illustrate pseudomonas and staphylococcus
as bacteria used in the present experiment, respectively.
[0146] The bacteria were incubated under the following
condition.
[0147] A. Used medium
[0148] pseudomonas-nutrient broth (nutrient medium)
[0149] staphylococcus-lysogeny broth (LB medium)
[0150] B. Incubation process
[0151] 1. A bacteria stock was put in a medium of 3 mL and cultured
after overnight incubation (37.degree. C. and 200 rpm).
[0152] 2. Here, 0.03 mL of the cultured bacteria stock was taken
and put in a fresh medium of 3 mL, and then cultured (37.degree. C.
and 200 rpm) up to OD600=1.5.
[0153] 3. The cultured bacteria stock was put in two
high-pressure/sterilized e-tubes by 1.0 mL, supernatants thereof
were removed by using a centrifugation (6,000.times. g, 20 min,
4.degree. C.), and then the bacteria stock was resuspended with
sterile PBS.
[0154] 4. In this case, a colony forming unit (CFU) was resuspended
by PBS of 0.1 mL and then serial diluted, and the bacteria stock
was smeared on an agar plate.
[0155] 5. Then, the concentration was shown and a PBS volume to be
107 CFU/5 .mu.L may be calculated by the concentration.
[0156] C. Experiments of auto-fluorescence expression of bacteria
(pseudomonas and staphylococcus) and fluorescence expression of
hydrochloride moxifloxacin
[0157] In order to compare fluorescence expression of hydrochloride
moxifloxacin with auto-fluorescence expression of bacteria,
fluorescence expression of bacteria (pseudomonas and
staphylococcus) which are not applied with hydrochloride
moxifloxacin was first photographed and intensity of the
auto-fluorescence expression was recorded. Subsequently, the
hydrochloride moxifloxacin was administered to the medium and then
the intensity of the fluorescence expression after the incubation
time was measured.
[0158] FIGS. 16A and 17A are auto-fluorescence expressed
photographs of pseudomonas and staphylococcus before administering
hydrochloride moxifloxacin, respectively, and FIGS. 16B and 17B are
fluorescence expressed photographs of pseudomonas and
staphylococcus after administering hydrochloride moxifloxacin,
respectively.
[0159] First, through FIGS. 16B and 17B, it can be seen that
pseudomonas and staphylococcus may be labeled with the
hydrochloride moxifloxacin.
[0160] Further, when comparing FIGS. 16A and 16B and FIGS. 17A and
17B, respectively, in the case where the hydrochloride moxifloxacin
is administered to each bacterium, a fluorescent signal with a
higher intensity than the auto-fluorescence signal may be
verified.
[0161] Values for the accurate fluorescence expression may be
verified through a grape illustrated in FIG. 18. In the case of the
pseudomonas, it can be seen that there is a difference in signal
intensity of about 10 times because a value before administering
the hydrochloride moxifloxacin is 9.5334 a.u. and a value after
administering the hydrochloride moxifloxacin is 101.2196 a.u. Even
in the case of the staphylococcus, it can be seen that there is a
difference in signal intensity of 10 times because a value before
administering the hydrochloride moxifloxacin is 10.4277 a.u. and a
value after administering the hydrochloride moxifloxacin is
110.3189 a.u.
EXPERIMENTAL EXAMPLE 5
Measurement of Multi-Photon Fluorescence in Fungus of Hydrochloride
Moxifloxacin
[0162] 1) Preparation of Materials and Samples
[0163] Aspergillus and candida albican to be used in the
experiment, hydrochloride moxifloxacin, a petri dish, and a biaxial
scanner (a galvano scanner of x axis and a galvano scanner of y
axis) to be used by a point scanning method were prepared.
[0164] In this Experimental Example, a two-photon microscope using
a femtosecond laser as a light source was used, and the
multi-photon fluorescence was equally measured under the following
condition throughout an experimental process.
[0165] Excitation wavelength: 790 nm for moxifloxacin
[0166] Filter set: Ch01: [490 nm, Ch02: ]490 nm
[0167] Light source: Chameleon Ultra II, Coherent
[0168] Camera: photomultiplier tube (PMT) H7421-40P, Hamamatsu
Photonics
[0169] Objective lens: 20.times.1.0 NA objective lens, XLUMPlanFL,
Olympus
[0170] Photographing area: 150 .mu.m.times.150 .mu.m (512.times.512
pixels)
[0171] 2) Measurement of Multi-Photon Fluorescence in Fungi
(Aspergillus and Candida Albican) of Hydrochloride Moxifloxacin
[0172] The aspergillus and the candida albican as the fungi were
cultured on the petri dish.
[0173] A. Used medium
[0174] aspergillus-nigger agar
[0175] candida-LB agar
[0176] B. Incubation process
[0177] 1. First, a nutrient agar was slowly dissolved while being
boiled in 1 L of distilled water.
[0178] 2. An autoclave was performed at 121.degree. C. for about 15
minutes.
[0179] 3. An agar medium of about 25 to 30 ml was spilled on a
plate to prepare a nutrient agar plate.
[0180] 4. The fungi (aspergillus and candida) were smeared on the
prepared agar plate and incubated at 30.degree. C.
[0181] 5. A single fungus colony was found after about 24
hours.
[0182] C. Sample preparing process
[0183] 1. An appropriate amount of fungi formed on the agar plate
with the colony scooped up with a thing such as a toothpick.
[0184] 2. The toothpick coated with the fungi was put into the
e-tube containing distilled water of about 500 ml to be centrifuged
(10 min, 4000.times. g, 4.degree. C.).
[0185] 3. After the centrifuge, the toothpick was removed and
distilled water was mixed with the fungi sunken in the e-tube by
using a pipet. About 50 .mu.l of the fungi and the distilled water
in the e-tube was extracted and placed on a well-slide glass to
prepare a control sample.
[0186] 4. About 20 .mu.l of a moxifloxacin solution (a vigamox
solution) was dropped into the fungi and the distilled water
remaining in the e-tube and then centrifuged (10 min, 4000.times.
g, and 4.degree. C.).
[0187] 5. Thereafter, the remaining solution except for the fungi
sunken in the e-tube was removed by using the pipet, distilled
water of about 500 ml was added, and then mixed by using the
pipet.
[0188] 6. About 50 .mu.l of a mixed solution of the fungi and the
distilled water in the e-tube was extracted to be placed on the
well-slide glass to prepare a moxifloxacin (vigamox) labelled
fungus sample.
[0189] D. Experiment of auto-fluorescence expression of fungi
(aspergillus and candida albican) and fluorescence expression of
hydrochloride moxifloxacin
[0190] In order to compare auto-fluorescence expression of fungi
(aspergillus and candida albican) and fluorescence expression of
hydrochloride moxifloxacin, first, fluorescence expression of fungi
(aspergillus and candida albican ) which are not applied with the
hydrochloride moxifloxacin was photographed and then intensity of
the fluorescence expression was recorded.
[0191] Subsequently, after the hydrochloride moxifloxacin was
administered to the petri dish and sufficiently smeared for 10
minutes, the fungi (aspergillus and candida albican) were collected
by using a centrifuge and the intensity of the fluorescence
expression was measured.
[0192] An experimental result will be described in detail with
reference to FIGS. 19A to 19C and 20A to 20C.
[0193] FIGS. 19A and 20A are auto-fluorescence expression
photographs of aspergillus and candida albican before administering
hydrochloride moxifloxacin which are photographed by laser power of
7 mW, respectively, and FIGS. 19B and 20B are auto-fluorescence
expression photographs of aspergillus and candida albican which are
photographed by laser power of 50 mW, respectively.
[0194] First, referring to FIGS. 19A and 20A, it can be seen that a
structure of the fungi was verified with the laser power of 7 mW.
Further, as illustrated in FIGS. 19B and 20B, structures of
aspergillus having an elongated branch shape and candida albican
having a spherical particle shape may be verified in the laser
power of about 50 mW.
[0195] FIGS. 19C and 20C are photographs of aspergillus and candida
albican after administering hydrochloride moxifloxacin which are
photographed by laser power of 7 mW, respectively. In comparison
with FIGS. 19A and 20A, respectively, it can be seen that the
structures of aspergillus and candida albican may be efficiently
verified even with the laser power of 7 mW.
[0196] Further, in the aspergillus of FIG. 19C, the structure of
the aspergillus may be more clearly observed by using the laser
power with small intensity while minimizing a signal corresponding
to background noise. Even in the candida albican of FIG. 20C, an
empty nucleus existing in the bacteria may be more clearly observed
with smaller laser power than that of FIGS. 20A and 20B.
[0197] That is, the fluoroquinolone antibiotics may label the
bacteria and the fungi, and in this case, the intensity of the
fluorescence expression is 10 times stronger than the
auto-fluorescence. As illustrated in Experimental Examples 1 to 3,
since the cell tissue in the body tissue may be labeled, it can be
seen that whether the bacteria exist in the body may be easily
determined only by administration of the antibiotic.
[0198] The fluoroquinolone antibiotics may be easily obtained as
antibiotics which are sold to be clinically used and have an
advantage of observing the cells while minimizing the damage. In
this case, the tissue to be observed may be high-speed imaged in
vivo without extraction of the tissue or incubation of the
cell.
[0199] Further, even in addition to the cornea, the skin and
bladder cells may be labeled, and as a result, the fluoroquinolone
antibiotics may be used as an inspection method of cell fluorescent
chromosomes and cells of various biological tissues.
[0200] Since the fluoroquinolone antibiotics may express the
fluorescent signal of a minimum of 10 times more than the
auto-fluorescent signal in the cells, even though the laser power
having a small value is used, the cells in the body tissue may be
observed. Particularly, in addition to labeling of the vascular
endothelia cells and fluorescent labeling of the bacteria, the
fungi consisting of a eukaryote such as a human may be labeled, and
as a result, the fluoroquinolone antibiotics may be used for
inspection of the infectious bacteria of various tissues and
cells.
[0201] Particularly, a time may be more shortened than the existing
diagnosis method of the infectious bacteria in which cells are
extracted and cultured for several days, and as a result, there is
an advantage of advancing a treatment time.
[0202] The above Experimental Examples are just examples for
describing the present invention, and the present invention is not
limited thereto. Since those skilled in the art can implement the
present invention through various modifications therefrom, the
technical protection scope should be determined by the appended
claims.
* * * * *