U.S. patent application number 13/825379 was filed with the patent office on 2013-11-28 for methods for predicting and treating myocardial damage.
The applicant listed for this patent is Edward J. Lesnefsky, Marc S. Penn. Invention is credited to Edward J. Lesnefsky, Marc S. Penn.
Application Number | 20130316377 13/825379 |
Document ID | / |
Family ID | 45874358 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316377 |
Kind Code |
A1 |
Penn; Marc S. ; et
al. |
November 28, 2013 |
METHODS FOR PREDICTING AND TREATING MYOCARDIAL DAMAGE
Abstract
A method for predicting myocardial damage in a subject having or
at risk of cardiac disease includes determining a level of
apolipoprotein AI (ApoAI) and a level of Coenzyme Q.sub.10
(CoQ.sub.10) in the subject and comparing the determined levels of
ApoAI and CoQ.sub.10 to control levels.
Inventors: |
Penn; Marc S.; (Beachwood,
OH) ; Lesnefsky; Edward J.; (Richmond, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Penn; Marc S.
Lesnefsky; Edward J. |
Beachwood
Richmond |
OH
VA |
US
US |
|
|
Family ID: |
45874358 |
Appl. No.: |
13/825379 |
Filed: |
September 21, 2011 |
PCT Filed: |
September 21, 2011 |
PCT NO: |
PCT/US11/52591 |
371 Date: |
March 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61384969 |
Sep 21, 2010 |
|
|
|
Current U.S.
Class: |
435/7.92 ;
435/11 |
Current CPC
Class: |
G01N 33/92 20130101;
A61K 9/0053 20130101; A61K 31/122 20130101; A61K 45/06 20130101;
G01N 33/6887 20130101; G01N 2800/324 20130101; G01N 2800/56
20130101 |
Class at
Publication: |
435/7.92 ;
435/11 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for predicting myocardial damage in a subject having or
at risk of cardiac disease, the method comprising: determining a
level of apolipoprotein AI (ApoAI) in the subject; determining a
level of Coenzyme Q.sub.10 (CoQ.sub.10) in the subject; comparing
the determined levels of ApoA1 and CoQ.sub.10 to control levels,
wherein a decreased level of ApoAI and a decreased level of
CoQ.sub.10 compared to control levels are indicative of the subject
having an increased risk of greater myocardial damage following a
myocardial infarction.
2. The method of claim 1, further comprising the step of obtaining
one or more bodily samples from the subject, the one or more bodily
samples including CoQ.sub.10 and ApoAI.
3. The method of claim 2, the one or more bodily samples comprising
plasma.
4. The method of claim 1, wherein the level of ApoAI in the subject
is determined using an ELISA assay.
5. The method of claim 1, wherein the level of CoQ.sub.10 in the
subject is determined using high-performance liquid
chromatography.
6. The method of claim 1, wherein the myocardial damage comprises a
ratio of an area of ischemic myocardial tissue to total area of
myocardial tissue.
7. The method of claim 1, the control levels include normal levels
of ApoA1 and CoQ10 in a population of apparently healthy
subjects.
8. The method of claim 1, wherein the cardiac disease is selected
from the group consisting of myocardial infarction, coronary artery
disease, angina, atherosclerosis, aneurysm, congestive heart
failure, left ventricular dysfunction, cerebrovascular disease, and
cerebrovascular accident.
9. A method of determining increased risk of greater myocardial
damage in a subject having or at risk of cardiac disease, the
method comprising: determining a level of apolipoprotein AI (ApoAI)
in the subject; determining a level of Coenzyme Q.sub.10
(CoQ.sub.10) in the subject; comparing the determined levels of
ApoA1 and CoQ.sub.10 to control levels, wherein a decreased level
of ApoAI and a decreased level of CoQ.sub.10 compared to control
levels are indicative of the subject having an increased risk of
greater myocardial damage following a myocardial infarction.
10. The method of claim 9, further comprising the step of obtaining
one or more bodily samples from the subject, the one or more bodily
samples including CoQ.sub.10 and ApoAI.
11. The method of claim 10, the one or more bodily samples
comprising plasma.
12. The method of claim 9, wherein the level of ApoAI in the
subject is determined using an ELISA assay.
13. The method of claim 9, wherein the level of CoQ.sub.10 in the
subject is determined using high-performance liquid
chromatography.
14. The method of claim 9, wherein the myocardial damage comprises
a ratio of an area of ischemic myocardial tissue to total area of
myocardial tissue.
15. The method of claim 9, the control levels include normal levels
of ApoA1 and CoQ10 in a population of apparently healthy
subjects.
16. The method of claim 9, wherein the cardiac disease is selected
from the group consisting of myocardial infarction, coronary artery
disease, angina, atherosclerosis, aneurysm, congestive heart
failure, left ventricular dysfunction, cerebrovascular disease, and
cerebrovascular accident.
17. A method of determining increased risk of greater myocardial
damage in a subject having or at risk of cardiac disease, the
method comprising: determining a level of HDL in the subject;
determining a level of Coenzyme Q.sub.10 (CoQ.sub.10) in the
subject; comparing the determined levels of ApoA1 and CoQ.sub.10 to
control levels, wherein a decreased level of ApoAI and a decreased
level of CoQ.sub.10 compared to control levels are indicative of
the subject having an increased risk of greater myocardial damage
following a myocardial infarction.
18. The method of claim 17, further comprising the step of
obtaining one or more bodily samples from the subject, the one or
more bodily samples including CoQ.sub.10 and HDL.
19. The method of claim 18, the one or more bodily samples
comprising plasma.
20. The method of claim 17, wherein the level of HDL in the subject
is determined using a fluorometric assay.
21. The method of claim 17, wherein the level of CoQ.sub.10 in the
subject is determined using high-performance liquid
chromatography.
22. The method of claim 17, wherein the myocardial damage comprises
a ratio of an area of ischemic myocardial tissue to total area of
myocardial tissue.
23. The method of claim 17, the control levels include normal
levels of HDL and CoQ10 in a population of apparently healthy
subjects.
24. The method of claim 17, wherein the cardiac disease is selected
from the group consisting of myocardial infarction, coronary artery
disease, angina, atherosclerosis, aneurysm, congestive heart
failure, left ventricular dysfunction, cerebrovascular disease, and
cerebrovascular accident.
25-33. (canceled)
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 61/384,969, filed, Sep. 21, 2010, the subject
matter of which is incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] This application relates generally to methods and kits for
predicting myocardial damage in a subject having or at risk of
cardiac disease and to methods for mitigating ischemic damage in a
subject having an increased risk of myocardial damage resulting
from cardiac disease.
BACKGROUND OF THE INVENTION
[0003] Plasma levels of high-density lipoproteins (HDL) and
apolipoprotein AI (ApoAI) are inversely associated with
cardiovascular morbidity and mortality. ApoAI is primarily
synthesized in the liver and small intestine, and comprises a
single polypeptide of 243 amino acid residues with a molecular
weight of approximately 28,000 Da. ApoAI, through activation of
lecithin:cholesterol acyltransferase, catalyzes the reaction of
cholesterol and phosphatidylcholine to yield cholesterol esterified
with a long-chain fatty acid and 2-lysophosphatidylcholine, an
important step in reverse cholesterol transport.
[0004] The importance of HDL cholesterol as an independent risk
factor for coronary artery disease (CAD) is well known. Novel
therapeutic approaches for administering HDL protein, ApoAI, or
ApoAI analogues to alter the development of atherosclerosis have
been investigated in animal models and in humans. In fact,
individuals with ApoAI deficiency and ApoAI-deficient mice fail to
form normal HDL particles and, as a result, are predisposed to
premature CAD.
[0005] Recent studies have also shown anti-inflammatory properties
of ApoAI. For example, the inhibitory activity of ApoAI appears to
be specifically directed to contact-mediated monocyte activation by
T-cells through inhibition of TNF-.alpha. and IL1.beta.. In
addition to anti-inflammatory and anti-atherogenic function,
reduced plasma concentrations of HDL and ApoAI have been implicated
in the development of Type 2 diabetes.
SUMMARY OF THE INVENTION
[0006] An aspect of the application relates to a method for
predicting myocardial damage in a subject having or at risk of
cardiac disease. The method includes determining a level of
apolipoprotein AI (ApoAI) and a level of CoQ.sub.10 in the subject.
The method further includes comparing the determined levels of
ApoAI and a CoQ.sub.10 to control levels. A decreased level of
ApoAI and a decreased level of CoQ.sub.10 compared to control
levels are indicative of the subject having an increased risk of
greater myocardial damage following a myocardial infarction.
[0007] Another aspect of the application relates to a method for
determining increased risk of greater myocardial damage in a
subject having or at risk of cardiac disease. The method includes
determining a level of apolipoprotein AI (ApoAI) and a level of
CoQ.sub.10 in the subject. The method further includes comparing
the determined levels of ApoAI and a CoQ.sub.10 to control levels.
A decreased level of ApoAI and a decreased level of CoQ.sub.10
compared to control levels are indicative of the subject having an
increased risk of greater myocardial damage following a myocardial
infarction.
[0008] Another aspect of the application relates to a kit for
predicting myocardial damage in a subject having or at risk of
cardiac disease. The kit includes a first reagent for determining a
level of ApoAI in the subject. The kit also includes a second
reagent for determining a level of CoQ.sub.10 in the subject and
instructions for predicting myocardial damage in a subject having
or at risk of cardiac disease.
[0009] A further aspect of the application relates to a method of
mitigating ischemic damage in a subject having an increased risk of
myocardial damage resulting from cardiac disease. The method
includes administering therapeutically effective amounts of
CoQ.sub.10 and a hypolipidemic agent to a subject, which has
decreased levels of ApoAI and CoQ.sub.10 as compared to a
control.
[0010] Yet another aspect of the application relates to a
pharmaceutical composition for mitigating ischemic damage in a
subject having an increased risk of myocardial damage resulting
from cardiac disease. The pharmaceutical composition includes
therapeutically effective amounts of CoQ.sub.10 and a hypolipidemic
agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The foregoing and other features of the present invention
will become apparent to those skilled in the art to which the
present invention relates upon reading the following description
with reference to the accompanying drawings, in which:
[0012] FIG. 1 is a plot comparing infarct size as a percent area at
risk (AAR) in wild-type (WT), ApoAI-heterozygote (+/-), and
ApoAI-null (-/-) mice after 30 minutes of ischemia and 3 hours of
reperfusion (data represent mean+/-SD);
[0013] FIGS. 2A-B are a series of images following hydroethidine
staining for reactive oxygen species (ROS) production in myocardial
tissue from WT (FIG. 2A) and ApoAI-null (FIG. 2B) mice 1 hour after
reperfusion. FIG. 2C illustrates the mean intensity quantified over
4 animals per group. Twelve random images from within the infarct
zone from each animal were quantified (data represent
mean.+-.SD);
[0014] FIGS. 3A-B are a series charts showing electron transport
chain activity. FIG. 3A is a plot comparing the relative activities
of complex I, complex II, and complex III. FIG. 3B is a plot
comparing the relative activity of NADH cytochrome c reductase
(NCR) and succinate cytochrome c reductase (SCR); and
[0015] FIG. 4 is a plot comparing infarct size as a percentage of
AAR in WT and ApoAI-null mice given saline or Coenzyme Q10 daily
for 3 days prior to ischemia induced by 30 minutes of LAD ligation
and 3 hours of reperfusion (data represent mean.+-.SD; n=4-6 per
group; *p<0.0001 compared to strain-matched NT control).
DETAILED DESCRIPTION
[0016] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises, such as
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates). Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the present invention pertains. Commonly
understood definitions of molecular biology terms can be found in,
for example, Rieger et al., Glossary of Genetics: Classical and
Molecular, 5th Ed., Springer-Verlag: New York, 1991, and Lewin,
Genes V, Oxford University Press: New York, 1994. The definitions
provided herein are to facilitate understanding of certain terms
used frequently herein and are not meant to limit the scope of the
present invention.
[0017] As used herein, the term "polypeptide" can refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or to a
fragment, portion, or subunit of any of these, and to naturally
occurring or synthetic molecules. The term "polypeptide" can also
include amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres, and may contain
any type of modified amino acids. The term "polypeptide" can also
include peptides and polypeptide fragments, motifs and the like,
glycosylated polypeptides, and all "mimetic" and "peptidomimetic"
polypeptide forms.
[0018] As used herein, the term "amino acid" can refer to naturally
occurring and synthetic amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally occurring amino acids
are those encoded by the genetic code, as well as those amino acids
that are later modified, e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine. "Amino acid analogs"
can refer to compounds that have the same basic chemical structure
as a naturally occurring amino acid, i.e., a carbon that is bound
to a hydrogen, a carboxyl group, an amino group, and an R group,
e.g., homoserine, norleucine, methionine sulfoxide, methionine
methyl sulfonium. Such analogs have modified R groups (e.g.,
norleucine) or modified peptide backbones, but retain the same
basic chemical structure as a naturally occurring amino acid.
"Amino acid mimetics" can refer to chemical compounds that have a
structure that is different from the general chemical structure of
an amino acid, but function in a manner similar to a naturally
occurring amino acid.
[0019] As used herein, the term "polynucleotide" can refer to
oligonucleotides, nucleotides, or to a fragment of any of these, to
DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin
which may be single-stranded or double-stranded and may represent a
sense or antisense strand, to peptide nucleic acids, or to any
DNA-like or RNA-like material, natural or synthetic in origin,
including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The term
can also encompass nucleic acids, i.e., oligonucleotides,
containing known analogues of natural nucleotides. Additionally,
the term can encompass nucleic acid-like structures with synthetic
backbones.
[0020] As used herein, the term "pharmaceutically acceptable
carrier" can include any material, which when combined with a
conjugate retains the conjugate's activity and is non-reactive with
a subject's immune system. Examples include, but are not limited
to, any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water, emulsions such as
oil/water emulsion, and various types of wetting agents. Other
carriers may also include sterile solutions, tablets including
coated tablets, and capsules. Typically, such carriers contain
excipients, such as starch, milk, sugar, certain types of clay,
gelatin, stearic acid or salts thereof, magnesium or calcium
stearate, talc, vegetable fats or oils, gums, glycols, or other
known excipients. Such carriers may also include flavor and color
additives or other ingredients. Compositions comprising such
carriers are formulated by well known conventional methods.
[0021] As used herein, the term "subject" can refer to any animal,
including, but not limited to, humans and non-human animals (e.g.,
rodents, arthropods, insects, fish (e.g., zebrafish)), non-human
primates, ovines, bovines, ruminants, lagomorphs, porcines,
caprines, equines, canines, felines, ayes, etc.), which is to be
the recipient of a particular treatment. Typically, the terms
"patient" and "subject" are used interchangeably herein in
reference to a human subject.
[0022] As used herein, the terms "administer" or "administering"
can refer to oral administration, administration as a suppository,
topical contact, intravenous, intraperitoneal, intramuscular,
intralesional, intranasal or subcutaneous administration, or the
implantation of a slow-release device e.g., a mini-osmotic pump to
a subject. Administration can be by any route, including parenteral
and transmucosal (e.g., oral, nasal, vaginal, rectal or
transdermal). Parenteral administration can include, e.g.,
intravenous, intramuscular, intra-arteriole, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial.
Other modes of delivery can include, but are not limited to, the
use of liposomal formulations, intravenous infusion, transdermal
patches, etc.
[0023] As used herein, the terms "cardiac disease," "cardiac
disorder," "cardiovascular disease", "cardiovascular disorder," or
"cardiovascular condition" can refer to any disease or disorder
that negatively affects the cardiovascular system. The terms can
also refer to cardiovascular events. "Cardiovascular events", as
used herein, can include acute coronary syndrome, myocardial
infarction, myocardial ischemia, chronic stable angina pectoris,
unstable angina pectoris, angioplasty, stroke, transient ischemic
attack, claudication(s) and vascular occlusion(s). Cardiac diseases
and disorders, therefore, can include acute coronary syndrome,
myocardial infarction, myocardial ischemia, chronic stable angina
pectoris, unstable angina pectoris, angioplasty, stroke, transient
ischemic attack, claudication(s), vascular occlusion(s),
arteriosclerosis, left ventricular dysfunction, heart failure, and
cardiac hypertrophy.
[0024] This application relates to methods for diagnosing,
predicting, and/or determining an increased risk of myocardial
damage in a subject and to methods for mitigating myocardial damage
in a subject having an increased risk of myocardial damage
resulting from cardiac disease. It was found that: (1) myocardial
infarct (MI) size in apolipoprotein AI (ApoAI)-deficient mice is
substantially greater than MI size in wild-type (WT) mice; (2)
prophylactic treatment of ApoAI-deficient mice with Coenzyme
Q.sub.10 (CoQ.sub.10) prior to MI leads to a 100% decrease in MI
size; and (3) analyses of mitochondrial function in WT and
ApoAI-deficient mice suggests that there is a defect in the
mitochondria of ApoAI-deficient mice that leads to decreased flux
in the succinate cytochrome c reductase (SCR) pathway secondary to
a deficiency in the Q pool.
[0025] Based on these discoveries, it was determined that subjects,
which have decreased ApoA1 and CoQ.sub.10 levels compared to
control subjects have increased myocardial damage (e.g., ischemic
damage) following myocardial infarction and that the ApoA1 and
CoQ.sub.10 levels of a subject can be measured to determine or
predict if the subject has increased risk of greater myocardial
damage (e.g., ischemic damage) following myocardial infarction. The
ability to determine a subject as having an increased risk of
greater myocardial damage, therefore, provides a useful diagnostic
tool to predict the amount of myocardial damage in a subject
resulting from cardiac disease and to help mitigate or prevent
myocardial damage in subjects at risk of cardiovascular disease
(e.g., MI).
[0026] Accordingly, an aspect of the application relates a method
for predicting myocardial damage in a subject having or at risk of
cardiac disease. The method includes determining the levels of the
cardiac markers, apolipoprotein and CoQ.sub.10 in a subject.
[0027] As used herein, the term "apolipoprotein" can refer to
apolipoproteins known to those of skill in the art and variants and
fragments thereof. Apolipoproteins are proteins that bind to lipids
and transport dietary fats through the bloodstream. Apolipoproteins
that may be used as cardiac markers to predict the amount of
myocardial damage in a subject include, but are not limited to,
Apolipoprotein (Apo) A (e.g., ApoAI, ApoAII, ApoIV and ApoV), ApoB
(e.g., ApoB48 and ApoB100), ApoC (e.g., ApoCI, ApoCII, ApoCIII and
ApoCIV), ApoD, ApoE and Apo H. In one example, the level of ApoAI
(e.g., an ApoAI polypeptide) in a subject can be used to determine
an increased risk of greater myocardial damage in a subject.
[0028] ApoAI is the major protein component of high-density
lipoprotein complex (HDL) and chylomicrons secreted from the
intestinal enterocyte also contain ApoAI, however it is quickly
transferred to HDL in a subject's bloodstream. Accordingly, the
application also contemplates that a level of HDL in a subject can
be indicative of a level of ApoAI in the subject. Therefore, in
another example, the levels of HDL and CoQ.sub.10 in a subject can
be used to determine an increased risk of greater myocardial damage
in a subject.
[0029] The levels of the cardiac markers (e.g., ApoAI and
CoQ.sub.10) can be determined by first obtaining one or more
biological samples from a subject. In one example, the levels of
the cardiac markers ApoAI and CoQ.sub.10 can both be determined by
first obtaining a single biological sample from a subject. In
another example, the levels of the cardiac markers, ApoAI and
CoQ.sub.10, can each be determined separately by obtaining two or
more biological samples from a subject.
[0030] The subject can be an apparently healthy subject, a subject
at risk for a cardiovascular disease, or a subject known to have
cardiovascular disease. "Apparently healthy", as used herein, can
refer to subjects who have not previously been diagnosed as having
any signs or symptoms indicating the presence of a cardiac disease,
a history of a cardiac disease, or evidence of a cardiac disease.
Apparently healthy subjects may not otherwise exhibit symptoms of a
cardiac disease. In other words, such subjects, if examined by a
medical professional, would be characterized as healthy and free of
symptoms of a cardiac disease.
[0031] Subjects at risk for a cardiac disease can exhibit any one
or combination of risk factors for cardiovascular disease
including, but not limited to, elevated blood pressure, an abnormal
response to a stress test, elevated levels of myeloperoxidase,
C-reactive protein, low density lipoprotein, cholesterol, or
atherosclerotic plaque burden. Techniques for assessing
cardiovascular disease risk factors are known in the art and can
include coronary angiography, coronary intravascular ultrasound,
stress testing (with and without imaging), assessment of carotid
intimal medial thickening, carotid ultrasound studies with or
without implementation of techniques of virtual histology, coronary
artery electron beam computer tomography, cardiac computerized
tomography (CT) scan, CT angiography, cardiac magnetic resonance
imaging, and magnetic resonance angiography.
[0032] The biological sample can include whole blood samples and
samples of blood fractions, such as serum and plasma. The
biological sample may be fresh blood, stored blood (e.g., in a
blood bank), or a blood fraction. The biological sample may be a
blood sample expressly obtained for the assay(s) described herein
or, alternatively, a blood sample obtained for another purpose
which can be sub-sampled. In one example, the biological sample can
comprise whole blood. Whole blood may be obtained from the subject
using standard clinical procedures. In another example, the
biological sample can comprise plasma. Plasma may be obtained from
whole blood samples by centrifugation of anti-coagulated blood.
Such process provides a buffy coat of white cell components and a
supernatant of the plasma. In yet another example, the biological
sample can comprise serum. Serum may be obtained by centrifugation
of whole blood samples that have been collected in tubes that are
free of anti-coagulant. The blood may then be permitted to clot
prior to centrifugation. The yellowish-reddish fluid obtained by
centrifugation is the serum.
[0033] Biological samples can be pretreated as necessary by
dilution in an appropriate buffer solution, heparinized,
concentrated if desired, or fractionated by any number of methods
including, but not limited to, ultracentrifugation, fractionation
by fast performance liquid chromatography, precipitation with
dextran sulfate, or other known methods. Any number of standard
aqueous buffer solutions employing one or a combination of buffers,
such as phosphate, Tris, or the like, at physiological pH can also
be used.
[0034] In one example, a biological sample including CoQ.sub.10 can
include serum. In another example, a biological sample can be
obtained from a subject where the level of CoQ.sub.10 included in
the sample reflects the CoQ.sub.10 tissue level instead of the
dietary intake of a subject. For example a biological sample can
include cultured skin fibroblasts, muscle biopsies and blood
mononuclear cells.
[0035] After obtaining the biological sample from the subject, the
levels of the cardiac markers (e.g., ApoAI and CoQ10) are
determined using any one or combination of known biochemical assays
or techniques. Examples of biochemical assays or techniques that
can be used to determine the level of an ApoAI polypeptide, HDL,
and/or CoQ.sub.10 can include, for example, antibody based assays,
such as ELISA and Western blots, mass spectroscopy (MS) (e.g.,
LC/ESI/MS/MS), fluorometric assays and chromatography (e.g., HPLC,
affinity column, etc.). It will be appreciated that biochemical
assays or techniques may also be used to determine the level of a
cardiac marker comprising a polynucleotide. For example, the level
of an mRNA encoding an ApoAI polypeptide can be determined using
Northern blot analysis. Alternatively, the presence or absence of
the gene encoding an ApoAI polypeptide can be determined using PCR,
for example.
[0036] In an embodiment of the application, the level of CoQ.sub.10
can be determined using high-performance liquid chromatography
(HPLC) with electrochemical detection as described by Tang et al.,
Clinical Chemistry 2001; 47(2)256-265), which is incorporated
herein by reference.
[0037] Once the levels of the cardiac markers have been determined,
the levels of the cardiac markers are compared to control levels in
order to determine an increased risk of greater myocardial damage
in a subject following a myocardial infarction. For example, the
level of an ApoAI polypeptide in a biological sample can be
determined using ELISA and the level of CoQ.sub.10 in a biological
sample can be determined using HPLC and then both levels can be
compared to control levels or values of ApoAI and CoQ.sub.10,
respectively. The control levels can be based upon the level of an
ApoAI polypeptide or CoQ.sub.10 in a comparable biological sample
(or samples) obtained from a control population (e.g., the general
population) or a select population of subjects. For example, the
select population may be comprised of apparently healthy subjects,
subjects determined to have myocardial damage resulting from
cardiac disease, subjects determined to have little or no
myocardial damage resulting from cardiac disease or from subjects
at risk for a cardiac disease.
[0038] The control levels can be related to the levels used to
characterize the levels of the ApoAI polypeptide and CoQ.sub.10
obtained from the subject. For example, if the level of the ApoAI
polypeptide is an absolute value, such as the units of ApoAI
polypeptides per ml of blood, the control level can also based upon
the units of ApoAI polypeptides per ml of blood in subjects of the
general population or a select population. Similarly, if the level
of the ApoAI polypeptide and CoQ.sub.10 is a representative value,
such as an arbitrary unit obtained from an ELISA, the control level
can also be based on the representative value.
[0039] The control levels can also take a variety of forms. For
example, the control levels can be a single cut-off value, such as
a median or mean. The control levels can be established based upon
comparative groups, such as where the risk in one defined group is
double the risk of another defined group. The control levels can
also be divided equally (or unequally) into groups, such as a
low-risk group, a medium-risk group, and a high-risk group, or into
quadrants, the lowest quadrant being subjects with the lowest risk
the highest quadrant being subjects with the highest risk.
[0040] Control levels of ApoAI polypeptides and CoQ.sub.10 in
biological samples, for example, can be obtained (e.g., mean
levels, median levels, or "cut-off" levels) by assaying a large
sample of subjects in the general population or a select population
and then using a statistical model, such as the predictive value
method for selecting a positivity criterion or receiver operator
characteristic curve that defines optimum specificity (highest true
negative rate) and sensitivity (highest true positive rate), as
described in Knapp, R. G. and Miller, M. C. (1992): Clinical
Epidemiology and Biostatistics, William and Wilkins, Harual
Publishing Co. (Malvern, Pa.), which is incorporated herein by
reference.
[0041] Depending upon the levels or values of the cardiac markers
when compared to the control levels, a determination can be made as
to the risk of greater myocardial damage in the subject following a
myocardial infarction. In some embodiments, the myocardial damage
can be defined as the ratio of ischemic or infracted myocardial
area to total myocardial area and, as described below, can be
expressed as a percentage. In an example of the method, a reduced
or decreased level of an ApoAI polypeptide in combination with a
reduced level of CoQ.sub.10 as compared to control value levels may
indicate an increased risk of developing a greater amount of
myocardial damage. Thus, a subject with a reduced level of an ApoAI
polypeptide and a reduced level of CoQ.sub.10 may have an increased
risk of developing a larger infarct area in the left ventricle
(e.g., as a result of MI) as compared to a control subject.
Alternatively, a normal or increased level of an ApoAI polypeptide
and CoQ.sub.10 as compared to a control value level may indicate
little or no risk of a subject developing greater or increased
myocardial damage following MI.
[0042] In another aspect of the application, a kit is provided for
diagnosing an increased risk of myocardial damage resulting from
cardiac disease in a subject. The kit includes at least one first
reagent that specifically detects and/or determines the level of
ApoAI, such as an ApoAI polypeptide, an ApoAI polypeptide fragment,
a polynucleotide encoding an ApoAI polypeptide, or a polynucleotide
encoding a fragment of an ApoAI polypeptide in a subject, at least
one second reagent that specifically detects and/or determines the
level of CoQ.sub.10, in a subject and instructions for using the
kit to determine an increased risk of greater myocardial damage in
a subject following a myocardial infarction.
[0043] In an example of the application, a first reagent can detect
expression levels of an ApoAI polypeptide or fragment thereof via
an antibody that specifically binds to the ApoAI polypeptide or
fragment thereof. In other example, the first reagent can comprises
a nucleic acid probe complementary to a polynucleotide sequence
coding for an ApoAI polypeptide or fragment thereof. For example,
the nucleic acid probe may be a cDNA or an oligonucleotide
immobilized on a substrate surface.
[0044] The instructions of the kit can include instructions
required by a regulatory agency (e.g., the U.S. Food and Drug
Administration) for use in in vitro diagnostic products. For
example, the instructions can be applicable to one or more of an
extraction buffer/reagent(s) and a related protocol, an
amplification buffer/reagent(s) and a related protocol, a
hybridization buffer/reagent(s) and a related protocol, an
immunodetection buffer/reagent(s) and a related protocol, a
labeling buffer/reagent(s) and a related protocol, and/or a control
value or values (as described above).
[0045] This application also relates to a method for mitigating
ischemic damage in a subject having an increased risk of a
myocardial damage resulting from cardiac disease. The method
includes administering therapeutically effective amounts of a
ubiquinone in combination with a hypolipidemic agent to the
subject. In one example, the subject is determined to have an
increased risk of a myocardial damage resulting from cardiac
disease as described herein.
[0046] Administration of a ubiquinone "in combination with" or "in
conjunction with" a hypolipidemic agent includes parallel
administration (administration of both the agents to the patient
over a period-of time, such as administration on alternate days for
one month) co-administration (in which the agents are administered
at approximately the same time, e.g., within about a few minutes to
a few hours of one another), and co-formulation (in which the
agents are combined or compounded into a single dosage form
suitable for oral or parenteral administration).
[0047] As used herein, the term "therapeutically effective amounts"
can refer to the amount of a ubiquinone administered to a subject
in combination with an amount of a hypolipidemic agent that results
in lowering or eliminating the risk of ischemic damage in a subject
found to have an increased risk of myocardial damage. A
therapeutically effective amount can also refer to a
prophylactically effective amount. As used herein, a
"prophylactically effective amount" is an amount of a ubiquinone
and a hypolipidemic agent that, when administered to a subject,
will have the intended prophylactic effect, e.g., preventing or
delaying the onset (or reoccurrence) of cardiac disease or
symptoms, or reducing the likelihood of the onset (or reoccurrence)
of cardiac disease or symptoms. The full prophylactic effect does
not necessarily occur by administration of one dose and may occur
only after administration of a series of doses. Thus, a
prophylactically effective amount may be administered in one or
more administrations.
[0048] The ubiquinone co-administered with a hypolipidemic agent to
the subject can include one or series of quinones, which are widely
distributed in animals, plants and microorganisms, a ubiquinone
mimetic, a ubiquinone variant, or a ubiquinone fragment. In one
example of the present invention the ubiquinone co-administered to
a subject with a hypolipidemic agent is CoQ.sub.10.
[0049] CoQ functions as an agent for carrying out oxidation and
reduction within cells. Its primary site of function is in the
terminal electron transport system where it acts as an electron or
hydrogen carrier between the flavoproteins (which catalyze the
oxidation of succinate and reduced pyridine nucleotides) and the
cytochromes. This process is carried out in the mitochondria of
cells of higher organisms. CoQ plays an important role as an
antioxidant to neutralize potentially damaging free radicals
created in part by the energy-generating process. For example,
CoQ.sub.10 has antioxidant and membrane stabilizing properties that
serve to prevent cellular damage resulting from normal metabolic
processes.
[0050] The term "hypolipidemic agents" as used herein refers to
several classes of pharmaceuticals that are well known to increase
ApoA1 levels and/or HDL levels in vivo. In some embodiments, a
hypolipidemic agent administered to the subject in combination with
ubiquinone in accordance with the applicaion can include ApoAI
polypeptides, ApoAI mimetics, ApoAI analogs, cholesteryl ester
transfer protein (CETP) inhibitors and statins.
[0051] "ApoAI polypeptides" as used herein refers to ApoAI peptide
fragments and full length proteins. By "ApoAI mimetics" or
"mimetics of ApoAI" or "known mimetics of ApoAI" as used in the
specification and in the claims, it is meant mimetics of ApoA1 that
can be identified or derived from any reference and that have ApoA1
behavior. These include mimetics of ApoAI identified in U.S. and
foreign patents and publications. For example, an ApoA1 mimetic
described herein can include any number of peptidomimetics of ApoA1
designed to beneficially influence the lipid parameters and/or
cholesterol levels in the blood. Accordingly, an ApoA1 polypeptide
mimetics contemplated herein may include modified polypeptides from
the ApoA1 forms and variants including, for example, apolipoprotein
A-1 (Brewer et al., (1978)), apolipoprotein A-1 Milano (Weisgraber
(1983) J. Biol. Chem. 258: 2508-2513), apolipoprotein A-1 Marburg,
(Utermann et al., (1982) J. Biol. Chem. 257: 501-507),
apolipoprotein A-1 Paris (Bielicki and Oda (2002) Biochemistry 41,
2089-2096), proapolipoprotein A-1, or any other mutant form of
ApoA1 known in the art whether synthetically formed or naturally
occurring.
[0052] An ApoAI mimetic can also include an ApoAI agonist which
mimics the function of ApoAI in a subject. An example of an ApoAI
agonist includes the recombinant ApoAI mutant protein referred to
as the `milano` mutant. The Milano mutant ApoA1 has an Arginine to
Cysteine mutation at amino acid position 197 (R197c) in the
pre-pro-ApoA1 protein amino acid sequence (corresponding to R173c
in the mature ApoA1 amino acid sequence). The cysteine in the
milano mutant leads to the formation of an ApoA1 dimer, held
together by a disulfide bond, due to the additional cysteine
residue.
[0053] Additional ApoA1 mimetics include ApoA1 oxidant resistant
mimetics, such as those describe in U.S. Pat. Appl. No.
20090149390A1, which is incorporated herein by reference. For
example, an ApoA1 mimetic for use in a method described herein can
include, but is not limited to, an ApoA1 mimetic having an amino
acid sequence that includes at least a portion of the amino acid
sequence of ApoA1 or a mimetic of the ApoA1 where at least one
tryptophan has been substituted with an oxidation resistant amino
acid, (e.g., phenylalanine) in the amino acid sequence of the ApoA1
mimetic. An ApoA1 mimetic for use in the present invention can also
include stabilized Apo A1 protein variants such as those described
in U.S. Pat. Appl. No.: 20100222276A1, which is incorporated herein
by reference.
[0054] As used herein, the term "statins" or "statin drug" can
refer to any compound or agent capable of substantially inhibiting
HMG Co-A reductase. Statins are a family of molecules sharing the
capacity to competitively inhibit the hepatic enzyme
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This
enzyme catalyses the rate-limiting step in the L-mevalonate pathway
for cholesterol synthesis. Consequently, statins block cholesterol
synthesis. Statins that can be administered, or co-administered to
a subject according to the invention include, Compactin,
Atorvastatin, Pravastatin, Lovastatin, Mevinolin, Pravastatin,
Fluvastatin, Mevastatin, Visastatin/RosuvastatinVelostatin,
Cerivastatin, Simvastatin, Synvinolin, Rivastatin (sodium
7-(4-fluorophenyl)-2,6-diisoprop-yl-5-methoxymethylpyridin-3-yl)-3,5-dihy-
-droxy-6-heptanoate), and Itavastatin/Pitavastatin. In one example,
statins are administered orally from about 1 mg/day to about 40
mg/day.
[0055] Additional hypolipidemic agents for use in the compositions
and methods described herein include but are not limited to bile
acid sequestrants (resins), ezetimibe, phytosterols, olistat,
acipimox CETP inhibitors, squalene synthase inhibitors, AGI-1067
and mipomersin. Clinically, the choice of an agent will depend on
the patient's cholesterol profile, cardiovascular risk, and the
liver and kidney functions of the patient evaluated against the
balancing of the risks and benefits of the hypolipdemic agent.
[0056] A hypolipidemic agent administered to a subject in
combination with an ubiquinone (e.g., CoQ.sub.10) in accordance
with the application can also include an agent that increases
HDL-cholesterol in a subject. For example, an agent that increases
HDL-cholesterol in a subject can include niacin and/or fibrates. In
one example, pharmacologic niacin (about 1- to about 3-gram/day)
can be administered to a subject in combination with
CoQ.sub.10.
[0057] The therapeutically effective amounts of ubiquinone (e.g.,
CoQ.sub.10) and/or a hypolipidemic agent can be administered in an
isolated or concentrated form, or as a part of one or more
pharmaceutical compositions and/or formulations. In one embodiment,
a pharmaceutical composition can include ubiquinone and a
hypolipidemic agent as the active ingredient and a pharmaceutically
acceptable carrier or aqueous medium excipient suitable for
administration and delivery in vivo. Combined therapeutics are also
contemplated, and the same type of underlying pharmaceutical
compositions may be employed for both single and combined
medicaments. For example, a pharmaceutical composition described
herein can include ubiquinone and a hypolipidemic agent described
above as the active ingredients and a pharmaceutically acceptable
excipient suitable for administration and delivery in vivo.
[0058] A pharmaceutical composition described herein can be
administered by any appropriate route, such as percutaneous,
parenteral, subcutaneous, intravenous, intraarticular, intrathecal,
intramuscular, intraperitoneal, or intradermal injections, or by
transdermal, buccal, oromucosal, ocular routes, or via inhalation.
The dosage administered will be dependent upon the age, health, and
weight of the subject, kind of concurrent treatment, if any,
frequency of treatment, and the nature of the effect desired. In a
subject with both a decreased level of an ApoAI polypeptide and a
decreased level of CoQ.sub.10, for example, a therapeutically
effective amount of a pharmaceutical composition comprising
CoQ.sub.10 can be prophylactically administered to prevent or
mitigate ischemic damage (e.g., as a result of MI).
[0059] In addition to one or more active ingredients (e.g.,
CoQ.sub.10), pharmaceutical compositions can include
pharmaceutically acceptable carriers comprising excipients and
auxiliaries that facilitate processing of an active ingredients
into pharmaceutical preparations. The pharmaceutical preparations
of the present invention can be manufactured in a known manner by,
for example, means of conventional mixing, granulating,
dragee-making, dissolving, or lyophilizing processes. Thus,
pharmaceutical preparations for oral use can be obtained by
combining the active agents with solid excipients, optionally
grinding the resulting mixture, and processing the mixture of
granules after adding suitable auxiliaries, if desired or
necessary, to obtain tablets or dragee cores.
[0060] Suitable excipients can include fillers, such as saccharides
(e.g., lactose or sucrose, mannitol or sorbitol), cellulose
preparations and/or calcium phosphates (e.g., tricalcium phosphate
or calcium hydrogen phosphate), as well as binders, such as starch
paste using, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, tragacanth, methyl cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or
polyvinyl pyrrolidone. If desired, disintegrating agents can be
added, such as the above-mentioned starches, as well as
carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof, such as sodium alginate.
Auxiliaries are flow-regulating agents and lubricants, for example,
silica, talc, stearic acid or salts thereof, such as magnesium
stearate, or calcium stearate, and/or polyethylene glycol. Dragee
cores can be provided with suitable coatings that, if desired, are
resistant to gastric juices. For this purpose, concentrated
saccharide solutions can be used, which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures.
[0061] To produce coatings resistant to gastric juices, solutions
of suitable cellulose preparations, such as acetylcellulose
phthalate or hydroxypropylmethylcellulose phthalate can be used.
Slow-release and prolonged-release formulations may be used with
particular excipients, such as methacrylic acid-ethylacrylate
copolymers, methacrylic acid-ethyl acrylate copolymers, methacrylic
acid-methyl methacrylate copolymers, and methacrylic acid-methyl
methylacrylate copolymers. Dye stuffs or pigments can be added to
the tablets or dragee coatings, for example, for identification to
characterize combinations of active compound doses.
[0062] Other pharmaceutical preparations that can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules can contain the active compounds in
the form of granules that may be mixed with fillers, such as
lactose, binders, such as starches, and/or lubricants, such as talc
or magnesium stearate and, optionally, stabilizers. In soft
capsules, one or more active ingredients (e.g., CoQ.sub.10) can be
dissolved or suspended in suitable liquids, such as fatty oils or
liquid paraffin.
[0063] Examples of formulations for parenteral administration can
include aqueous solutions of one or more active ingredients in
water-soluble form, for example, water-soluble salts, and alkaline
solutions. Examples of salts can include maleate, fumarate,
succinate, S,S tartrate, or R,R tartrate. In addition, suspensions
of one or more of the active ingredients as oily injection
suspensions can be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, for example, ethyl oleate or triglycerides or
polyethylene glycol-400. Aqueous injection suspensions can contain
substances that increase the viscosity of the suspension, sodium
carboxymethyl cellulose, sorbitol, and/or dextran.
[0064] The therapeutically effective amounts of the ubiquinone
(e.g., CoQ.sub.10) and a hypolipidemic agent can be administered to
a subject on a desired dosing schedule. For example, a
therapeutically effective amount of a pharmaceutical composition
comprising CoQ.sub.10 can be administered about four times daily,
about three times daily, about twice daily, about daily, about
every other day, about three times weekly, about twice weekly,
about weekly, about every two weeks, or less often (as desired). In
one example, a therapeutically effective amount of a pharmaceutical
composition includes 30-1,200 milligrams of CoQ.sub.10 taken orally
in divided doses. In another example CoQ.sub.10 can be administered
intravenously at a dose of around 5 mg/kg of body weight.
[0065] A therapeutically effective amount of the ubiquinone (e.g.,
CoQ.sub.10) and/or a hypolipidemic agent can also be administered
for a duration sufficient to provide a prophylactic effect. For
example, a therapeutically effective amount of CoQ.sub.10 can be
administered daily for one year, for about six months, about a
year, about two years, about five years, about 10 years, or
indefinitely. It will be apparent to those of skill in the art that
the dose, dosing schedule, and duration can be adjusted for the
needs of a particular subject, taking into consideration the
subject's age, weight, severity of disease, and other co-morbid
conditions.
[0066] Toxicity and therapeutic efficacy of compositions comprising
ubiquinone and/or a hypolipidemic agent for use in the invention
can be determined using standard pharmaceutical procedures in cell
culture or experimental animals for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50.
[0067] The following example is for the purpose of illustration
only and is not intended to limit the scope of the claims, which
are appended hereto.
Example 1
Materials and Methods
LAD Ligation/Reperfusion and Quantification of Area at Risk and
Infarct Size
[0068] All animal protocols were approved by the Animal Research
Committee, and all animals were housed in the Association for
Assessment and Accreditation of Laboratory Animal Care
International-approved animal facility of the Cleveland Clinic.
Anterior wall MI (AMI) was performed as recently described (Askari,
A. T. et al., J. Exp. Med. 197:615-624, 2003). Briefly, AMI was
induced in eight 20- to 25-g male littermate wild-type (C57BL/6J),
ApoA1 heterozygote (ApoA1.sup.+/-) or knockout (ApoA1.sup.-/-) mice
by ligation of the LAD 7-0 Prolene. Blanching and dysfunction of
the anterior wall verified LAD ligation. After 30 minutes of LAD
ligation, microsurgical scissors were used to cut the knot in the
ligature at the level of the myocardium, and animals subsequently
underwent reperfusion for 3 hours. Successful reperfusion was
verified by return of red color to the tissue that was initially
blanched at the time of LAD ligation, as well as gross evidence of
some recovery of anterior wall motion Animals were kept on
ventilator for the entire 3 hours of reperfusion and subsequently
analyzed for area at risk and infarct size using 1% solution of
2,3,5-triphenyltetrazolium chloride (TTC) at 37.degree. C. Briefly,
once the LAD was ligated again, Evan's blue dye (1 mg/mL) was
infused to define the volume of myocardium not at risk. After
Evan's blue dye infusion, the heart was harvested and sectioned
into 3 pieces defined as the base, mid, and apex. The sections were
incubated in TTC solution for 15 minutes, rinsed, and then placed
in formalin overnight.
Determination of Reactive Oxygen Species (ROS) Production In
Vivo
[0069] ROS production was assessed using in vivo hydroethidine dye
(Kondo, T. et al., J. Neurosci. 17:4180-4189, 1997; Murakami, K. et
al., J. Neurosci. 18:205-213, 1998), as previously described
(Manabe, Y. et al., Ann. Neurol. 55:668-675, 2004). Hydroethidine,
a cell-permeable oxidative fluorescent dye, is oxidized to ethidium
by superoxide (Carter, W. O. et al., J. Leukoc. Biol. 55:253-258,
1994; Bindokas, V. P. et al., J. Neurosci. 16:1324-1336, 1996).
Ethidium, which exhibits peak absorbance at 520 nm and an emission
maximum at 600 nm, is trapped intracellularly by intercalating with
DNA (Rothe, G. et al., Methods Enzymol. 233:539-548, 1994). The
fluorescence signal attributable to ethidium reflects cumulative
ROS production during the period between administration of
hydroethidine and killing of the animal. Hydroethidine (10 mg/kg)
was injected into the jugular vein of anesthetized and previously
infracted animal as described above and allowed to circulate for 4
h. Mice were killed, and hearts were removed and paraffin-embedded.
Serial sections (n=5) were cut and collected at 600 .mu.m
intervals, and viewed with a confocal microscope. The analysis of
ROS production was performed in a blinded manner by a different
investigator. Five randomly selected areas within the infarct zone
were selected and analyzed. Fluorescence intensity was measured in
five serial sections per animal. The sum of the fluorescence
intensity for each region was divided by the total number of pixels
analyzed and expressed as relative fluorescence units.
TUNEL Assay
[0070] Heart sections were used to perform TUNEL staining with the
In Situ Cell Death Detection kit (Roche Applied Science) per the
manufacturer's instructions. Hearts were collected after 30 minutes
ischemia/3 h reperfusion for assessment of TUNEL. Heart sections
were incubated with TUNEL staining (Roche) for cell death and
co-staining was performed using DAPI and cells were visualized with
a confocal microscope. TUNEL-positive-staining cells were counted
at 40.times. magnification in 5 randomly selected areas within the
infarct zone and expressed as positive cells per mm.sup.2 and then
compared between WT and ApoAI KO mice. At least 10 sections were
analyzed throughout the entire longitudinal axis of the hearts (n=5
hearts per group).
Mitochondrial Techniques
[0071] Three mouse hearts were pooled for isolation of cardiac
mitochondria. Hearts were finely minced, placed in Chappell-Perry
(CP 1) buffer (in mM: 100 KCl, 50 Mops, 5MgSO.sub.4, 1 EGTA, 1
ATP), trypsin added (1 mg/g wet weight), and homogenized with a
polytron tissue processor (Brinkmann Instruments, Westbury, N.Y.)
for 2.5 s at a rheostat setting of 3.5. The polytron homogenate was
incubated in homogenization tube for 10 minutes with stirring at
4.degree. C. CP 2 buffer (CP 1 with 2% fatty-acid free BSA), was
added in the homogenate right after the incubation to stop the
trypsin digestion. Additional mixing and homogenization was
performed using 2 strokes with the loose pestle and 2 strokes with
the tight pestle. Then, the homogenate was centrifuges at
500.times.g for 10 minutes. The supernatant was saved for isolation
of mitochondria and the pallet was washed twice (centrifuge at
3000.times.g) and then resuspended in KME (in mM: 100 KCl, 50 MOPS,
and 0.5 EGTA). Mitochondrial protein concentration was measured by
the Lowry method, using bovine serum albumin as a standard.
[0072] Oxygen consumption in mitochondria was measured using a
Clark-type oxygen electrode at 30.degree. C. Mitochondria were
incubated in a solution including 80 mM KCl, 50 mM MOPS, 1 mM EGTA,
5 mM KH.sub.2PO.sub.4 and 1 mg defatted, dialyzed BSA/ml at pH 7.4.
Glutamate (complex I substrate, 20 mM) plus malate (2 mM),
succinate (complex II substrate, 20 mM) plus rotenone (7.5 mM), and
N,N,N',N'-tetramethyl p-phenylenediamine (TMPD)-ascorbate (complex
IV substrate, 10 mM) plus rotenone (7.5 mM), were used. State 3
(ADP-stimulated), state 4 (ADP-limited) respiration, respiratory
control ratios, the ADP/O ratio, and dinitrophenol-uncoupled
respiration were determined Endogenous substrates were depleted by
addition of 0.1 mM ADP before glutamate stimulated respiration.
[0073] The following enzyme activities were measured in
detergent-solubilized mitochondria using previously described
methods (Hoppel, C. L. et al., J. Clin. Invest. 80:71-77, 1987;
Lesnefsky, E. J. et al., Am. J. Physiol. 273:H1544-H1554, 1997):
NADH-cytochrome c reductase (NCR, rotenone sensitive); succinate
cytochrome c reductase (SCR)-antimycin A sensitive; complex II,
thermoyltrifluoroacetone (TTFA), sensitive; complex III, antimycin
A sensitive, and citrate synthase (CS).
[0074] Net H.sub.2O.sub.2 production from mitochondria was measured
using the oxidation of fluorogenic indicator amplex red in the
presence of horseradish peroxidase. Amplex red assay was obtained
from Molecular Probes (Eugene, Oreg.). Glutamate and succinate were
used as complex I and complex II substrates, and the concentration
of substrates is the same as that used to measure oxidative
phosphorylation (Chen, Q. et al., J. Biol. Chem. 278:36027-36031,
2003.
Statistical Analyses
[0075] All data are expressed as mean.+-.SD. Statistical analysis
was performed with use of SPSS software (version 10.0 for Windows,
SPSS Inc). Comparisons between two groups were statistically
evaluated by Student's t-test. A value of P.ltoreq.0.05 was
considered statistically significant.
Results
Effect of Genetic Background on Infarct Size in Mice
[0076] Due to increased mortality rate in ApoAI KO mice after
chronic ligation of the proximal left anterior descending artery
(LAD) (.about.80-90% within the first 24 hrs), acute myocardial
infarction was achieved by inducing 30 min of LAD ischemia and
subsequent reperfusion for 3 hours. Mice were kept on ventilator
for the entire experiment. There were no differences in the area at
risk following LAD ligation between WT, ApoA-I+/- and ApoA-I-/-
mice following LAD ligation, 49.9.+-.11.2%, 49.7.+-.3.2% and
51.7.+-.4.4%, respectively. Conversely the infarct size as a
percent of the AAR (IS/% AAR) correlated with the level of ApoA-1
with the largest infarcts seen in the ApoA-I null mice compared to
ApoA-I het and WT mice (25.3.+-.7.8%, n=4 vs. 17.8.+-.3.0%, n=6 and
13.1.+-.2.8%, n=4, respectively, FIG. 1), WT vs. ApoA-I het
p=0.042; WT vs. ApoA-I null, p=0.002).
In Situ Detection of ROS
[0077] We postulated that the increase in infarct size in the ApoAI
KO mice could be due to the increased production of ROS since ROS
plays a major pathogenic role in ischemic injury. We used
hydroethidine technique to quantify ROS release in WT and APOAI
null mice after reperfusion (Representative images are shown in
FIGS. 2A-B). There was a trend for an increase in mean fluorescence
intensity in ApoAI null mice compared to WT mice (87.9.+-.47 and
54.6.+-.17.9, respectively, p=0.23, FIG. 2C).
In Situ DNA Fragmentation by TUNEL Staining
[0078] To determine if there was increased apoptosis is responsible
for the observed injury in ApoAI KO mice, the TUNEL method was
employed to detect apoptotic nuclei in myocardial cells.
Quantitatively, the number of TUNEL positive cells/mm.sup.2 trended
higher in the ApoAI null mice compared to WT mice (19.7.+-.13.5 and
12.1.+-.11.1, respectively, p=0.17); however, this increase was not
statistically significant.
Mitochondrial Oxidative Phosphorylation
[0079] Mitochondrial oxidative metabolism was measured with
glutamate, succinate and TMPD-ascorbate as substrates. Oxygen
consumption under ADP-stimulated (state 3), ADP-limited (state 4)
conditions are shown in Table 1.
TABLE-US-00001 TABLE 1 Oxidative phosphorylation ApoA1KO (n = 4) WT
(n = 5) 2 mM 2 mM Substrate State 3 State 4 RCR ADP/O ADP State 3
State 4 RCR ADP/O ADP Glutamate 288 .+-. 1.9 37 .+-. 3.4 8.3 .+-.
0.9 3.2 .+-. 0.1 345 .+-. 40 298 .+-. 17.9 38 .+-. 4 8 .+-. 0.4 3.3
.+-. 0.1 341 .+-. 44.7 Pyruvate + 437 .+-. 16.2 59.6 .+-. 5.6 7.4
.+-. 0.5 3.6 .+-. 0.1 489.3 .+-. 32 416 .+-. 35.5 60 .+-. 14.7 7
.+-. 1 3.7 .+-. 0.3 454 .+-. Malate 40.7 Succinate 575 .+-. 10* 193
.+-. 10 3 .+-. 0*.sctn. 1.6 .+-. 0.1 555 .+-. 40 674 .+-. 29 193
.+-. 12 3.5 .+-. 0.1*.sctn. 1.6 .+-. 0.1 656 .+-. 28 DHQ 663 .+-.
30 217 .+-. 50 3.1 .+-. 0.5 1.6 .+-. 0.1 791 .+-. 48 570 .+-. 125
184 .+-. 19 3 .+-. 0.3 1.5 .+-. 0.1 648 .+-. 72 TMPD 1191 .+-. 65
1918 .+-. 134 206 .+-. 57 1712 .+-. 99 1222 .+-. 52 1818 .+-. 70
246 .+-. 24 15.72 .+-. 55 Values are means .+-. SE. Oxidative
phosphorylation of glutamate in nanoatoms O min.sup.-1 mg
protein.sup.-1. RCR, respiratory control rate; ADP/O, ADP-to-O
ratio. *P < 0.05 vs. corresponding WT. .sctn.P < 0.005.
[0080] State 3 respiration, state 4 respiration, respiration
control ratio (RCR), and the ADP/O ratio were similar in WT and KO
mice when glutamate was used as the substrate (Table 1). With
succinate (plus rotenone), as a complex II substrate, a decrease in
both state 3 respiration, and uncoupled respiration occurred in KO
mice. The decreased coupling of respiration observed in KO mice
indicated by the decrease in RCR was mainly due to a decreased
state 3 respiration, rather than an increased state 4 respiration
or phosphorylation apparatus defect since state 4 respiration was
not altered in KO mice. Furthermore, dinitrophenol-uncoupled
respiration was decreased in KO mice, localizing the respiratory
defect to the electron transport chain (ETC).
[0081] Substrates that donate electrons to specific sites in the
ETC were used under conditions of maximal ADP-stimulated
respiration to identify sites of damage to the ETC. The maximal
ADP-stimulated respiration measured using 2 mM ADP decreased in KO
mice with succinate as substrate when respiration supported by
electron flow from complex II, ubiquinone, complex III, cytochrome
c and complex IV. In contrast, the maximal ADP-stimulated
respiration was not affected in both WT and KO mice with glutamate,
DHQ and TMPD-ascorbate as substrates when electron flow
respectively from complex I, ubiquinone, complex III to final
cytochrome c and complex IV.
ETC Enzyme Activities
[0082] NADH cytochrome c reductase, measures of the activity of
complex I and III was similar in both KO and WT mice (FIG. 3A). The
activity of succinate cytochrome c reductase (SCR, antimycin A
sensitive) was markedly decreased in KO mice, localizing a defect
to complex II, ubiquinone and complex III of the ETC (Table 2).
TABLE-US-00002 TABLE 2 Enzyme activities Enzyme ApoAI KO (n = 4) WT
(n = 5) Complex I 865 .+-. 71 810 .+-. 59 NCR 5120 .+-. 506 5612
.+-. 488 Complex III 5654 .+-. 580 6413 .+-. 449 SCR 334 .+-. 28*
764 .+-. 50 Complex II 759 .+-. 75 789 .+-. 57 Citrate Synthase
3809 .+-. 166 4036 .+-. 78 Values are mean .+-. SE. *P < 0.0001
vs. corresponding WT.
[0083] However, the activity of complex III was not changed
compared to WT mice and the activity of complex II was surprisingly
normal (FIG. 3A). Thus, the defect in ETC of KO mice was likely at
the Q pool, which altered the electron transfer from complex II to
complex III. The activity of citrate synthase, a mitochondrial
matrix marker enzyme, was unaltered in both WT and KO mice (FIG.
3B).
H.sub.2O Production in Mitochondria
[0084] Since horseradish peroxidase and amplex red do not enter
intact mitochondria, only H.sub.2O.sub.2 that is released from
mitochondria (net release of H.sub.2O.sub.2, pmol/mg/30 min) is
detected by this assay. The net production of H.sub.2O.sub.2 in WT
and KO mice was similar with glutamate as complex I substrate, as
well as with succinate plus rotenone as a complex II substrate
(Table 3).
TABLE-US-00003 TABLE 3 Effect of genetic background of mice on
mitochondrial H.sub.2O.sub.2 production ApoAI KO WT (pmol/mg/30
min.) (pmol/mg/30 min.) Complex I substrate Glutamate/malate 570
.+-. 73 872 .+-. 141 Glutamate/malate plus 271 .+-. 58 425 .+-. 66
rotenone Complex II substrate Succinate 686 .+-. 117 923 .+-. 119
Succinate plus rotenone 207 .+-. 79 371 .+-. 8 Means .+-. SEM.
Concentrations used: glutamate 10 mM, malate 2.5 mM, succinate 5
mM. When succinate was used as a substrate, rotenone (2.4 .mu.m)
was added.
Effect of CoQ10 on Infarct Size in ApoAI Null Mice
[0085] The analyses of mitochondrial function in the WT and ApoAI
null mice suggested that there is a defect in the mitochondria of
ApoAI null mice that leads to decreased flux in the succinate
cytochrome c reductase pathway secondary to a deficiency in the Q
pool. To determine if the Q pool was then a target for reversing
the adverse effects of ApoAI deficiency, we inject CoQ10 ip (1
mg/kg/day) into WT and ApoAI null mice for 3 days prior to inducing
ischemia reperfusion. The data in FIGS. 3A-B demonstrate that the
administration of CoQ10 to ApoAI null mice completely corrected the
defect seen in ApoAI null mice leading to >100% decrease in the
infarct size as a percent area at risk (FIG. 4). There was a
non-significant trend (p=0.15) towards a decrease in IS/% AAR in
the WT mice treated with CoQ10 compared to WT controls.
[0086] From the above description of the invention, those skilled
in the art will perceive improvements, changes and modifications.
Such improvements, changes, and modifications are within the skill
of the art and are intended to be covered by the appended
claims.
* * * * *