U.S. patent application number 10/181220 was filed with the patent office on 2003-01-30 for method for inhibiting apoptosis under ischemic condition.
Invention is credited to Kim, Kyu-Won, Lee, Jong-Kyun, Lee, Jongwon, Lee, Sang Jong.
Application Number | 20030022847 10/181220 |
Document ID | / |
Family ID | 19638003 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022847 |
Kind Code |
A1 |
Lee, Jongwon ; et
al. |
January 30, 2003 |
Method for inhibiting apoptosis under ischemic condition
Abstract
The present invention relates to a method for inhibiting
apoptosis under ischemic condition, which comprises a step of
administering antibiotics of quinolones, quinones, aminoglycosides
or chloramphenicol to an individual under ischemic condition which
lacks an adequate supply of oxygen and glucose. In accordance with
the present invention, the antibiotics increase cell viability
under hypoxic and hypoglycemic condition, assuring that they can be
applied as a therapeutic agent for ischemia-associated diseases
such as myocardial infarction and cerebral infarction.
Inventors: |
Lee, Jongwon; (Taegu,
KR) ; Kim, Kyu-Won; (Pusan, KR) ; Lee,
Jong-Kyun; (Seoul, KR) ; Lee, Sang Jong;
(Seoul, KR) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
19638003 |
Appl. No.: |
10/181220 |
Filed: |
July 12, 2002 |
PCT Filed: |
January 12, 2001 |
PCT NO: |
PCT/KR01/00049 |
Current U.S.
Class: |
514/39 ; 435/252;
514/152; 514/230.5; 514/253.08 |
Current CPC
Class: |
A61K 31/00 20130101;
A61P 43/00 20180101; A61K 31/5383 20130101; A61K 31/7036 20130101;
A61K 31/496 20130101; A61P 9/10 20180101; A61K 31/65 20130101 |
Class at
Publication: |
514/39 ; 514/152;
514/230.5; 514/253.08; 435/252 |
International
Class: |
A61K 031/70; A61K
031/7036; A61K 031/65; A61K 031/535; A61K 031/497 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 2000 |
KR |
10-2000-0001309 |
Claims
What is claimed is:
1. A method for inhibiting apoptosis under ischemic condition,
which comprises a step of administering antibiotics to an
individual under ischemic condition in an amount of administering
to the individual infected with pathogenic microorganisms.
2. The method for inhibiting apoptosis under ischemic condition of
claim 1, wherein the antibiotics are quinolones, quinones and
aminoglycosides.
3. The method for inhibiting apoptosis under ischemic condition of
claim 2, wherein the quinolone antibiotics are levofloxacin,
ofloxacin and ciprofloxacin.
4. The method for inhibiting apoptosis under ischemic condition of
claim 2, wherein the quinone antibiotics are tetracycline,
minocycline, doxycycline and oxycycline.
5. The method for inhibiting apoptosis under ischemic condition of
claim 2, wherein the aminoglycoside antibiotics are geneticin,
neomycin and gentamycin.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method for inhibiting
apoptosis, more specifically, to a method for inhibiting apoptosis
under ischemic condition.
[0003] 2. Description of the Prior Art
[0004] As the death rate from cardiovascular diseases is increasing
recently, researches on the cardiovascular diseases are now in
rapid progress. Among them, one of the most noticeable field is
that relating to thrombus, wherein efforts to restore blood vessel
functions by dissolving thrombus which is the major cause of
blockage of blood vessels, and, furthermore, to inhibit thrombus
formation are being made. However, there is little progress in
developing method for preventing ripple effects of blockage of
blood vessels caused by thrombus or other causes. Accordingly, in
case of a patient dying of the blockage of blood vessels, it is
almost impossible to alleviate ischemic injury which lacks an
adequate supply of oxygen and glucose.
[0005] It has been reported that administration of antibiotics to
the patient who has antibody against Chlamydia pneumoniae which is
related to onset of acute myocardial infarction reduces the onset
rate of acute myocardial infarction (see: Meier C. R. et al., JAMA,
281(5):427-431, 1999). Antibiotics such as quinolones or quinones
could reduce onset rate of acute myocardial infarction, on the
other hand, antibiotics of macrolide which are known to be the most
effective agents to kill Chlamydia pneumoniae have no effect on
reducing the onset rate of acute myocardial infarction, suggesting
that the antibiotics are not merely killing pathogenic
microorganisms. Thus, antibiotics have been regarded as thrombosis
inhibitors or thrombolytic agents, however, there is no evidence of
relations between antibiotics and thrombus, hence, antibiotics can
be conjectured to work on acute myocardial infarction via other
mechanism than involvement of thrombus. The fact that antibiotics
exert a certain effect on acute myocardial infarction without
involvement of thrombus implies that antibiotics may protect cells
from destruction caused by inadequate supply of oxygen and glucose
due to the blockage of blood vessels. Accordingly, it could be
expected that the patient who has ischemia due to the blockage of
blood vessel can be recovered by using antibiotics, however, there
is still little progress in researches of this area.
[0006] Under the circumstances, there is a continuing need to
understand the effect of antibiotics on the cells under hypoxic and
hypoglycemic condition to contrive its potential application in the
art.
SUMMARY OF THE INVENTION
[0007] The present inventors have made an effort to elucidate the
effect of antibiotics on the cells under a low oxygen (hypoxic) and
a low glucose (hypoglycemic) condition, and, based on the fact that
the death of cells under hypoxic and hypoglycemic condition is
progressed via apoptosis, they discovered that the addition of
antibiotics of quinolones, quinones, aminoglycosides or
chloramphenicol to the cells under hypoxic and hypoglycemic
condition can dramatically inhibit apoptosis, furthermore, the
apoptosis can be inhibited by administering the antibiotics to an
individual under ischemic condition in an amount of administering
to the individual infected with pathogenic microorganisms.
[0008] A primary object of the present invention is, therefore, to
provide a method for inhibiting apoptosis under ischemic condition
which lacks an adequate supply of oxygen and glucose.
BRIEF DESCRIDTION OF THE DRAWINGS
[0009] The above and the other objects and features of the present
invention will become apparent from the following descriptions
given in conjunction with the accompanying drawings, in which:
[0010] FIG. 1 is a graph showing HepG2 cell viability under various
oxygen conditions.
[0011] FIG. 2a is a graph showing the dependency of HepG2 cell
viability on glucose concentration with culture time under a low
oxygen condition.
[0012] FIG. 2b is a graph showing the change in residual glucose
concentration with time depending on initial glucose concentration
under a low oxygen condition.
[0013] FIG. 2c a graph showing the change in pH with time depending
on glucose concentration under a low oxygen condition.
[0014] FIG. 3 is a graph showing HepG2 cell viability at various
geneticin concentrations.
[0015] FIG. 4a is a graph showing HepG2 cell viability with culture
time under a low oxygen and a low glucose condition.
[0016] FIG. 4b is a graph showing the change in glucose
concentration with time under a low oxygen and a low glucose
condition.
[0017] FIG. 4c is a graph showing the change in pH with time under
a low oxygen and a low glucose condition.
[0018] FIG. 5a is a graph showing the HepG2 cell viability with
time under a low oxygen and a high glucose condition.
[0019] FIG. 5b is a graph showing the change in glucose
concentration with time under a low oxygen and a high glucose
condition.
[0020] FIG. 5c is a graph showing the change in pH with time under
a low oxygen and a high glucose condition.
[0021] FIG. 6a is a graph showing HepG2 cell viability with culture
time under a normal oxygen and a low glucose condition.
[0022] FIG. 6b is a graph showing the change in glucose
concentration with time under a normal oxygen and a low glucose
condition.
[0023] FIG. 6c is a graph showing the change in lactic acid
concentration with time under a normal oxygen and a low glucose
condition.
[0024] FIG. 7 is a photograph showing gel electrophoresis pattern
of DNA from cells treated with various antibiotics.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present inventors, first of all, examined that apoptosis
is induced in cells under ischemic condition which lacks an
adequate flow of blood to supply oxygen and glucose due to blockage
of blood vessels by thrombus or other causes, and acknowledged that
glucose generates energy via TCA cycle and electron transfer system
in the presence of oxygen, however, under hypoxic (low oxygen)
condition, glucose is converted to lactic acid which generates only
a little energy, and resumes energy generation if sufficient oxygen
is supplied.
[0026] In order to simulate ischemic cells which lack the supply of
oxygen and glucose due to the blockage of blood vessels by thrombus
or other causes, the present inventors created ischemic condition
by discontinuing supply of oxygen and glucose to the cultured
cells, and then observed the changes occurred in the cells. When
oxygen was depleted in the cells, glucose also became depleted and
the cells were died without utilization of lactic acid. When oxygen
supply was resumed immediately after depletion of glucose, cells
could survive until lactic acid was used up, that is, cells died
with exhaustion of lactic acid. However, when cells were treated
with antibiotics of quinolones, quinones, aminoglycosides or
chloramphenicol, it was found that the cells were viable for a
certain period of time even after exhaustion of glucose and lactic
acid. Preferably, the antibiotics include, but are not intended to
be limited to, quinolones of 10-100 .mu.g/ml levofloxacin, 10-100
.mu.g/ml ofloxacin or 1-10 .mu.g/ml ciprofloxacin; quinones of
tetracycline, minocycline, doxycycline, or oxytetracycline at a
concentration of 0.1-10 .mu.g/ml each; and, aminoglycosides of
10-100 .mu.g/ml geneticin, 500-1000 .mu.g/ml neomycin or 100-1000
.mu.g/ml gentamycin. In case of chloramphenicol, a concentration of
1-10 .mu.g/ml were preferably employed.
[0027] Analyses of various test groups of cells under a condition
of oxygen and glucose depletion have shown that the groups of cells
without antibiotic treatment underwent typical apoptosis, whereas,
the groups of cells treated with said antibiotics did not undergo
apoptosis for a certain period of time. These results imply that
the said antibiotics inhibit apoptosis occurred in cells with
ischemic injury which lacks an adequate supply of oxygen and
glucose. Additional experiments demonstrated that antibiotics
somehow affect the expression of bcl-2 protein which is known to be
an inhibitor of apoptosis in cells with ischemic injury.
[0028] In order to examine if the results obtained with cultured
cells in vitro can be applied to the tissue with ischemic injury,
the mice under ischemic condition were treated with the said
antibiotics and then hearts from the mice with or without
antibiotic treatment were subject to biopsy, and found that the
preservation rate of cardiac tissues from mice treated with the
antibiotics was higher than that without antibiotic treatment.
[0029] The present invention is further illustrated in the
following examples, which should not be taken to limit the scope of
the invention.
EXAMPLE 1
Cell Viability under Various Oxygen Conditions
[0030] HepG2 cells (human hepatoma cell line, ATCC HB 8065,
1.times.10.sup.6 cells/60 mm culture dish) were grown in a minimal
essential medium supplemented with 100 unit/ml penicillin, 100
.mu.g/ml streptomycin, 1 g/l glucose, 2.2 g/l sodium bicarbonate,
and 10% (w/v) fetal calf serum for 2 days, followed by feeding with
the same medium and incubating under an environment of 1, 2, or 5%
(v/v) oxygen, respectively. Numbers of viable cells with time were
determined by trypan blue exclusion assay using hemocytometer after
10-15 minutes of incubation of 1:1 (v/v) mixture of 0.4% (w/v)
trypan blue and cell suspension. Cell viability with time was
represented in the ratio of viable cell number to cell number just
before the incubation condition was changed to a low oxygen
condition (see: FIG. 1). FIG. 1 is a graph showing cell viability
under various oxygen conditions, where (e) indicates 1% (v/v),
(.tangle-soliddn.) indicates 2% (v/v), (.box-solid.) indicates 5%
(v/v), and (.diamond-solid.) indicates 21% (v/v) oxygen,
respectively. As shown in FIG. 1, it was clearly demonstrated that
HepG2 cells were viable in a minimal medium containing low
concentration of glucose under an environment over 5% (v/v) oxygen,
whereas, the cells died under an environment of less than 2% (v/v)
oxygen. Accordingly, a low oxygen condition was set at 1% (v/v)
oxygen in the following examples.
EXAMPLE 2
Cell Viability Depending on Glucose Concentration
[0031] HepG2 cells were cultured analogously as in Example 1 except
for 1% (v/v) oxygen and varied glucose concentrations from 1 to 4.5
g/L. Then, cell viability, changes in glucose concentration and
changes in pH with time were measured (see: FIGS. 2a, 2b and 2c).
FIG. 2a shows cell viability with culture time, 2b shows changes in
glucose concentration with time, and 2c shows changes in pH with
time, where (.circle-solid.) indicates 1 g/L glucose,
(.largecircle.) indicates 2 g/L glucose, (.tangle-soliddn.)
indicates 3 g/L glucose, (.gradient.) indicates 3.5 g/L glucose,
(.box-solid.) indicates 4 g/L glucose, and (.quadrature.) indicates
4.5 g/L glucose, respectively. As shown in FIGS. 2a-2c, it was
clearly demonstrated that cells were died as a result of depletion
of glucose or lowering of pH under a low oxygen condition.
EXAMPLE 3
Cell Viability under Various Geneticin Concentrations
[0032] HepG2 cells were cultured for 2 days in the same manner as
in Example 1, and then, the maximum concentration of geneticin at
which HepG2 cells can survive was determined by replacing the
culture medium with a fresh medium containing 0-1000 .mu.g/ml
geneticin, an aminoglycoside antibiotic under an environment of 1%
(v/v) oxygen, respectively (see: FIG. 3). FIG. 3 is a graph showing
the cell viability at various geneticin concentrations, where
geneticin was added at a concentration of 0 .mu.g/ml
(.circle-solid.), 1 .mu.g/ml (.largecircle.), 3 .mu.g/ml
(.tangle-soliddn.), 10 .mu.g/ml (.gradient.) , 100 .mu.g/ml
(.box-solid.) , and 1000 .mu.g/ml (.quadrature.), respectively. As
shown in FIG. 3, it was clearly demonstrated that cells treated
with 10-100 .mu.g/ml genticin were viable for a certain period of
time under an environment of 1% (v/v) oxygen.
EXAMPLE 4
Dependency of Cell Viability on Geneicin Concentration
[0033] Concentration dependency of cell viability on geneticin was
determined under a low glucose (1 g/L) or a high glucose (4.5 g/L)
condition, as well as under a low oxygen (1%, v/v) or normal oxygen
condition.
EXAMPLE 4-1
Cell Viability under a Low Oxygen (Hypoxic) and a Low Glucose
(Hypoglycemic) Condition
[0034] HepG2 cells were plated in 60 mm culture dishes at a density
of 5.times.10.sup.5 cells per dish under a condition of 1 g/L
glucose and 1% (v/v) oxygen, and then, cell viability, changes in
pH and changes in glucose concentration were determined with time
after adding 10 .mu.g/ml geneticin or without addition,
respectively (see: FIGS. 4a, 4b and 4c). FIG. 4a shows HepG2 cell
viability with incubation time, 4b shows change in glucose
concentration with incubation time, and 4c shows change in pH with
incubation time, where (.circle-solid.) indicates without addition
and (.largecircle.) indicates addition of 10 .mu.g/ml geneticin. As
shown in FIGS. 4a-4c, it was clearly demonstrated that geneticin
maintained cell viability even after glucose was used up under
hypoxic and hypoglycemic condition.
EXAMPLE 4-2
Cell Viability under a Low Oxygen (Hypoxic) and a High Glucose
Condition
[0035] HepG2 cells were grown analogously as in Example 4-1, except
for 4.5 g/L glucose and 1% (v/v) oxygen, and then, cell viability,
changes in pH and changes in glucose concentration were determined
with time after treatment with 10 .mu.g/ml geneticin or without
treatment, respectively (see: FIGS. 5a, 5b and 5c). FIG. 5a shows
HepG2 cell viability with incubation time, 5b shows changes in
glucose concentration with incubation time, and 5c shows change in
pH with incubation time, where (.circle-solid.) indicates without
treatment and (.largecircle.) indicates treatment with 10 .mu.g/ml
geneticin. As shown in FIGS. 5a-5c, it was clearly demonstrated
that geneticin maintained cell viability under a hypoxic and high
glucose condition in a similar manner like under hypoxic and
hypoglycemic condition.
EXAMPLE 4-3
Cell Viability under a Normal Oxygen (Normoxic) and a Low Glucose
(Hypoglycemic) Condition
[0036] HepG2 cells were grown analogously as in Example 4-1, except
for 1 g/L glucose and 21% (v/v) oxygen, and then, cell viability,
change in glucose concentration and change in lactic acid
concentration were determined with time after adding 10 .mu.g/ml
geneticin or without addition, respectively (see: FIGS. 6a, 6b and
6c). FIG. 6a shows HepG2 cell viability with incubation time, 6b
shows change in glucose concentration with incubation time, and 6c
shows change in lactic acid concentration with incubation time,
where (.circle-solid.) indicates without treatment and
(.box-solid.) indicates treatment with 10 .mu.g/ml geneticin. As
shown in FIGS. 6a-6c, it was clearly demonstrated that under
normoxic condition, cells survived while consuming accumulated
lactic acid even after depletion of glucose and died with
exhaustion of lactic acid, whereas, cells treated with geneticin
were viable without being affected by depletion of lactic acid.
EXAMPLE 5
Screening of Antibiotics Exerting Effects on Cell Viability
[0037] In order to examine whether antibiotics with other
structures than aminoglycoside antibiotic of geneticin, can also
enhance cell viability under a hypoxic condition, analyses were
performed as followings: i.e., after analysis of antibiotics such
as geneticin, neomycin, gentamycin, tetracycline, minocycline,
oxytetracycline, doxycycline, chloramphenicol, levofloxacin,
ofloxacin, ciprofloxacin, ampicillin, amoxicillin, cephalosporin,
erythromycin, sulfadiazine, cyclohexamide, 5-fluorouracil,
puromycin and trimetazidine in accordance with the procedure
described in Examples 4-1 and 4-2, antibiotics which showed
enhancement of cell viability under hypoxic condition were selected
and their effective concentrations were determined, respectively
(see: Table 1).
1TABLE 1 Antibiotics exerting enhancement effects on cell viability
and their effective concentration Antibiotics Concentration
(.mu.g/ml) geneticin 10-100 neomycin 1000 gentamicin 100-1000
tetracycline 0.1-10 minocycline 0.1-10 doxycycline 0.1-10
oxytetracycline 0.1-10 chloramphenicol 1-10 levofloxacin 10-100
ofloxacin 10-100 ciprofloxacin 1-10
[0038] Effective concentration ranges in Table 1 represent the
concentration ranges of antibiotics exerting enhancement effects on
HepG2 cell viability under 1% (v/v) oxygen condition. As shown in
Table 1 above, among the antibiotics known to act on 30S subunit of
ribosome in E. coli, neomycin and gentamycin other than geneticin
were effective among aminoglycoside antibiotics. Also, among the
antibiotics known to act on 30S subunit of ribosome in E. coli, a
quinone antibiotic of tetracycline was effective at very low
concentration range of 0.1-10 .mu.g/ml and tetracycline derivatives
such as minocycline, oxytetracycline and doxycycline were effective
at the same range of low concentration. Meanwhile, among the
antibiotics known to act on 50S subunit of ribosome in E. coli, an
aromatic antibiotic of chloramphenicol was effective, but a
macrolide antibiotic of erythromycin was not effective. Among
quinolone antibiotics known to act on DNA gyrase, all analyzed
compounds, levofloxacin, ofloxacin, and ciprofloxacin were
effective. However, antibiotics known to inhibit synthesis of cell
wall of microorganisms, such as ampicillin, amoxillin, and
cephalosporin did not show enhancement effect on cell viability.
Antibiotics such as a sulfadiazine which is known to inhibit
dihydropteroate synthetase in the folic acid metabolism, a
cyclohexamide inhibiting protein synthesis in eukaryotes, a
5-fluorouracil blocking DNA synthesis by competing with uracil, and
puromycin inhibiting protein synthesis did not show any effect on
cell viability. Based on these results, it has been demonstrated
that there is no significant relations between the ability of
antibiotics to enhance cell viability under hypoxic condition and
the action mechanism of antibiotics or the chemical structure of
antibiotics. Although efficacy of antibiotics to maintain cell
viability under hypoxic condition varies, effective concentration
range of antibiotics on enhancement of viability of human hepatoma
cell line was about 0.1 to 1000 .mu.g/ml. Meanwhile, trimetazidine
which is known to enhance cell viability by increasing utilization
of glucose under a hypoxic condition did not show any positive
result in the experiment.
EXAMPLE 6
Analysis of DNA
[0039] Since it has been demonstrated that antibiotics of
quinolones, quinones, and aminoglycosides enhanced cell viability
under a hypoxic condition in Example 5, DNA samples extracted from
the cells treated with various kind of antibiotics were analyzed
and compared with DNA samples from cells without antibiotic
treatment.
[0040] HepG2(Human hepatoma cell line, ATCC HB 8065) cells were
grown under the same condition described in Example 1 for 2 days,
and then, the medium was replaced with a medium proper for test
conditions described below, followed by incubating for 2 days: test
group A with 21% (v/v) oxygen and 4.5 g/L glucose, test group B
with 21% (v/v) oxygen and 1 g/L glucose, test group C with 1% (v/v)
oxygen and 1 g/L glucose, test group D is an aminoglycoside
antibiotic of geneticin (10 .mu.g/ml) treated test group C, test
group E is a quinolone antibiotic of ofloxacin (10 .mu.g/ml treated
test group C, test group F is a quinone antibiotic of doxycycline
(0.1 .mu.g/ml treated test group C. DNA samples exracted from the
cells of said test groups were subject to electrophoresis,
respectively, to compare DNA patterns (see: FIG. 7). FIG. 7 is a
photograph showing gel electrophoresis pattern of DNA from cells of
various test groups, where lane 1 indicates a size marker, lane 2,
test group A, lane 3, test group B, lane 4, test group C, lane 5,
test group D, lane 6, test group E, and, lane 7, test group F,
respectively. As shown in FIG. 7, laddering was observed in test
group C, on the other hand, almost no laddering was observed in
test groups D, E and F, suggesting that, under hypoxic and
hypoglycemic condition, cell death is progressed via apoptosis,
but, apoptic cell death can be inhibited by treatment with
antibiotics.
[0041] As clearly illustrated and demonstrated above, the present
invention provides a method for inhibiting apoptosis under ischemic
condition. The method for inhibiting apoptosis under ischemic
condition comprises a step of administering antibiotics of
quinolones, quinones, aminoglycosides or chloramphenicol to an
individual under ischemic condition which lacks an adequate supply
of oxygen and glucose. In accordance with the present invention,
the antibiotics increase cell viability under hypoxic and
hypoglycemic condition, assuring that they can be applied as a
therapeutic agent for ischemia-associated diseases such as
myocardial infarction and cerebral infarction.
* * * * *