U.S. patent application number 13/400392 was filed with the patent office on 2012-06-14 for methods for assessing atherogenesis by determining oxidized phospholipid to apolipoprotein b ratios.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Sotirios Tsimikas, Joseph L. Witztum.
Application Number | 20120149596 13/400392 |
Document ID | / |
Family ID | 36780193 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120149596 |
Kind Code |
A1 |
Tsimikas; Sotirios ; et
al. |
June 14, 2012 |
METHODS FOR ASSESSING ATHEROGENESIS BY DETERMINING OXIDIZED
PHOSPHOLIPID TO APOLIPOPROTEIN B RATIOS
Abstract
The present invention relates to the analysis of oxidized
phospholipids (OxPL) on apolipoprotein B-100 in patients at high
risk or with documented coronary artery disease (CAD) or acute
coronary syndromes (ACS) such as unstable angina and acute
myocardial infarction or suspected of being at risk for ACS. Such
methods are useful for diagnostic purposes and for monitoring the
effects of dietary interventions or with drugs such as statins.
More particularly, the present invention relates to methods for
determining OxPL/apoB ratios as indices of atherosclerosis
regression and plaque stability.
Inventors: |
Tsimikas; Sotirios; (San
Diego, CA) ; Witztum; Joseph L.; (San Diego,
CA) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
36780193 |
Appl. No.: |
13/400392 |
Filed: |
February 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11244300 |
Oct 5, 2005 |
8129123 |
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13400392 |
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60615993 |
Oct 5, 2004 |
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Current U.S.
Class: |
506/9 ; 435/7.92;
436/501; 506/18 |
Current CPC
Class: |
G01N 33/5308 20130101;
G01N 2800/32 20130101; G01N 33/92 20130101; G01N 2333/775 20130101;
G01N 2800/323 20130101 |
Class at
Publication: |
506/9 ; 435/7.92;
436/501; 506/18 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10; G01N 33/566 20060101
G01N033/566 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
number 56989 awarded by the National Heart, Lung, and Blood
Institute (NHLBI). The government has certain rights in the
invention.
Claims
1. A method for analyzing atherogenesis in a subject, the method
comprising: a) obtaining a sample comprising plasma from a subject;
b) determining the level of oxidized phospholipid (OxPL) and the
level of all detectable apoB-100 (total apoB-100) in the sample; c)
calculating an atherogenesis index (AI) for the subject comprising
the ratio of the level of OxPL to the level of total apoB-100; d)
comparing the AI for the subject with AIs from subjects at high
risk or with documented coronary artery disease (CAD), acute
coronary syndromes (ACS), or at risk for ACS, wherein if the AI for
the subject falls within the AI range from subjects at high risk or
with documented CAD, ACS, or at risk for ACS, is predictive for the
risk of atherogenesis in the subject.
2. The method of claim 1, wherein the level of OxPL and the level
of total apoB-100 in the sample are measured with two or more
different biomolecules, wherein a first biomolecule specifically
interacts with OxPL and a second biomolecule specifically interacts
with apoB-100.
3. The method of claim 2, wherein the biomolecules are
antibodies.
4. The method of claim 3, wherein the antibodies are monoclonal
antibodies.
5. The method of claim 4, wherein the antibody that interacts with
OxPL is E06 or DLH3.
6. The method of claim 1, wherein the subject is human.
7. The method of claim 1, wherein the oxidized phospholipid is
selected from the group consisting of oxidized forms of
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phos-phorylcholine
(Ox-PAPC),
1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphoryl-choline (POVPC),
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),
1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine
(PEIPC), oxidized
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholin-e
(Ox-SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine
(SOVPC, 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine
(SGPC),
1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine
(SEIPC),
1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(Ox-SAPE),
1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine
(SOVPE),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine
(SGPE), and
1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine
(SEIPE).
8. The method of claim 1, further comprising correlating the AI for
the subject with: a) the age of the subject at the time the sample
is obtained; b) the subject's gender; and/or c) the subject's
race.
9. The method of claim 1, further comprising correlating the AI for
the subject with other atherogenesis risk factors selected from the
group consisting of current smoking, hypertension, LDL cholesterol
levels, and triglyceride levels.
10. The method of claim 1, further comprising determining the level
of Lp(a) liproprotein from the sample and comparing the Lp(a)
liproprotein level for the subject with Lp(a) liproprotein levels
from subjects at high risk or with documented CAD, ACS, or at risk
for ACS, wherein if the Lp(a) liproprotein level for the subject
falls within the Lp(a) liproprotein level range from subjects at
high risk or with documented CAD, ACS, or at risk for ACS, is
predictive for the risk of atherogenesis in the subject.
11. The method of claim 10, further comprising correlating the
Lp(a) lipoprotein levels from the subject with: a) the age of the
subject at the time the sample is obtained; b) the subject's
gender; and/or c) the subject's race.
12. The method of claim 10, further comprising correlating the
Lp(a) lipoprotein levels from the subject with other atherogenesis
risk factors selected from the group consisting of current smoking,
hypertension, LDL cholesterol levels, and triglyceride levels.
13. The method of claim 1, wherein the CAD or ACS is unstable
angina or mycocardial infarction.
14. An article of manufacture comprising packaging material and,
contained within the packaging material, biomolecules that
preferentially interact with OxPL, apoB, and Lp(a) lipoprotein,
wherein the packaging material comprises a label or package insert
indicating that the biomolecules can be used for calculating an AI
and for measuring Lp(a) lipoprotein levels.
15. The article of claim 14, wherein the biomolecules are
antibodies.
16. The article of claim 15, wherein the antibodies are monoclonal
antibodies.
17. The article of claim 16, wherein the antibody that interacts
with OxPL is E06 or DLH3.
18. An array comprising a substrate having a plurality of
addresses, each address having disposed thereon a set of one or
more biomolecules, that specifically interact with OxPL, apoB, and
Lp(a) lipoprotein.
19. A pre-packaged diagnostic kit for analyzing a subject's risk
for atherogenesis, the kit comprising an array of claim 18,
instructions for using the array, instructions for calculating an
AI by determining the ratio of the OxPL level to the apoB level,
and providing information with respect to AI and Lp(a) lipoprotein
levels and the risk for atherogenesis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/244,300, filed Oct. 5, 2005, which claims priority from U.S.
Provisional Application Ser. No. 60/615,993, filed Oct. 5, 2004,
which is incorporated herein by reference.
TECHNICAL FIELD
[0003] The present invention relates to the analysis of oxidized
phospholipids (OxPL) on apolipoprotein B-100 in patients at high
risk or with documented coronary artery disease (CAD) or acute
coronary syndromes (ACS) such as unstable angina and acute
myocardial infarction or suspected of being at risk for ACS. Such
methods are useful for diagnostic purposes and for monitoring the
effects of dietary interventions or with drugs such as statins.
More particularly, the present invention relates to methods for
determining OxPL/apoB ratios as indices of atherosclerosis
regression and plaque stability.
BACKGROUND
[0004] Atherosclerosis is a chronic inflammatory disease that
results from hyperlipidemia and a complex interplay of a variety of
environmental, metabolic and genetic risk factors. The oxidation of
low density lipoprotein (OxLDL) plays a central, if not obligatory
role, in the atherogenic process. Early studies showed that
acetylation of LDL greatly enhanced its uptake by macrophages and
that the uptake occurred via "scavenger receptors" which were
distinct from the classical LDL receptor. Unlike most receptors,
these scavenger receptors were not downregulated following uptake
of OxLDL. Due to the excessive uptake of OxLDL and its associated
lipid by the macrophages, the cells obtained a characteristic
foam-like appearance. The appearance of such cells is one of the
first hallmarks of atherosclerotic disease. Foam cells accumulate
within the intima (under the endothelial lining) of the vessel
walls where they lead to plaque formation, the hallmark of more
advanced disease. Inflammatory conditions develop leading to the
development of complicated lesions.
[0005] There is much evidence that OxLDL contributes to
atherogenesis by a number of mechanisms. The oxidation of
polyunsaturated fatty acids in phospholipids of lipoproteins
generates many breakdown products such as malondialdehyde (MDA),
4-hydroxynonenal (4-HNE), and other reactive moieties attached to
oxidized phospholipids. Many of these intermediate products are
highly reactive and can interact with lysine residues of associated
proteins and phospholipids to generate various adducts. These
adducts are known to occur in vivo and are immunogenic. In murine
models of atherosclerosis, such as apo-E deficient mice
(ApoE.sup.-/-) mice, atherosclerosis is correlated with the
development of high titers of autoantibodies to various oxidation
specific epitopes of OxLDL. The consequences of such cellular and
humoral responses are still poorly understood, but under certain
conditions they can clearly modify the natural history of the
disease.
[0006] It is generally accepted that it is the composition of
atherosclerotic lesions, in particular the content of lipids,
OxLDL, foam cells, and smooth muscle cells that determines their
properties. Foam cells are often found in the sites of lesion that
are susceptible to rupture. Activated macrophages recruited to
clear the apoptotic and necrotic foam cells, as well as OxLDL,
secrete factors that weaken the plaque. Human pathology studies
have shown that atheromas containing a large necrotic core, thin
fibrous cap and large numbers of macrophage/foam cells in the
shoulder are more predisposed to plaque rupture and thrombosis.
These lesions, which frequently appear as mild or moderate coronary
stenoses in angiographic studies, are characterized pathologically
as large atheroma with extensive lipid pools exceeding 40% of
plaque areas. Angiography only provides a measure of arterial
lumen, but fails to detect vessel wall pathology. Diagnostic
methods that provide a measure of the overall extent of the
atherosclerotic lesion, with an emphasis on OxLDL and lipid
content, would therefore be desirable. Moreover, the lipid core of
atheromas can be assumed to contain extensive oxidized lipids that
accumulated within foam cells and set free when cells undergo
necrosis and apoptosis.
[0007] Non-invasive detection of atherosclerotic lesions can now be
performed in animal models using imaging techniques that rely on
antibodies that are specific for OxLDL. (See, for example, U.S.
Pat. No. 6,716,410). Human studies have suggested that plaque
rupture frequently occurs in non-angiographically significant
lesions that contain abundant lipid-laden macrophages and large
lipid pools within atheromas. Therefore imaging of atherosclerosis
directed at lipid rich areas is of value, not only in detecting the
extent of lesion burden, but also in the detecting clinically
silent but "active" lesions.
[0008] In addition to such imaging techniques, there exists a need
to develop simple noninvasive ways of studying atherogenesis that
relate to the complexities of plaque biology rather than on plaque
architecture or lipid profiles as a whole. Accordingly, the present
invention relates to a plasma biomarker that specifically reflects
plaque biology associated with atherogenesis.
SUMMARY
[0009] The present invention relates to methods for analyzing
atherogenesis in a subject comprising the steps of determining the
OxPL level in plasma, determining the apoB level in plasma, and
then calculating the ratio of OxPL/apoB. This ratio provides a
useful "atherogenesis index" (AI) for assessing patients at high
risk or with documented coronary artery disease (CAD) or acute
coronary syndromes (ACS) such as unstable angina and acute
myocardial infarction or suspected of being at risk for ACS.
[0010] In one embodiment, a method of determining whether a therapy
is effective for treating coronary artery disease, is provided. The
method includes obtaining a first sample comprising plasma from a
subject; administering a therapy to the subject; obtaining a second
sample from the subject following administration of the therapy;
determining the level of oxidized phospholipid (OxPL) in the first
sample and second sample; determining the level of apoB in the
first sample and the second sample; calculating an atherogenesis
index (AI) by determining the ratio of the OxPL level to the apoB
level for both samples. An increase in the ratio determined from
the second sample in comparison to the ratio determined for the
first sample, is indicative of an effective therapy for coronary
artery disease. The information may be provided to a caregiver. In
some embodiments, the therapy includes administering to the subject
a composition comprising a compound that modulates the activity of
HMG-CoA reductase, such as, for example, a statin.
[0011] In some embodiments, the level of OxPL and the level of apoB
in the samples obtained from the subject are measured with two or
more different biomolecules. The first biomolecule specifically
interacts with OxPL and the second biomolecule specifically
interacts with apoB. In some aspects, the biomolecules are
antibodies, such as, for example, monoclonal antibodies. The
antibody that interacts with OxPL may be, for example, E06 or
DLH3.
[0012] In other aspects, the biomolecules are antigens. In some
embodiments, the biomolecules are immobilized to form an array
comprising a first set of a plurality of the first biomolecule and
a second set of a plurality of the second biomolecule.
[0013] Exemplary oxidized phospholipid include oxidized forms of
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phos-phorylcholine
(Ox-PAPC),
1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphoryl-choline (POVPC),
1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),
1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine
(PEIPC), oxidized
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholin-e
(Ox-SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine
(SOVPC, 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine
(SGPC),
1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine
(SEIPC),
1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(Ox-SAPE),
1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine
(SOVPE), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine
(SGPE), and
1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine
(SEIPE).
[0014] In another embodiment, the AI index is further correlated
with, the age of the subject at the time the ratios are measured,
the subject's gender, and/or the subject's race.
[0015] In another embodiment, an article of manufacture is
provided. The article may include packaging material containing
biomolecules that preferentially interact with oxidized
phospholipid (OxPL) and apoB. The packaging material may include a
label or package insert indicating that the biomolecules can be
used for calculating an atherogenesis index (AI) by determining the
ratio of the OxPL level to the apoB level.
[0016] In yet another embodiment, an array is provided. The array
may include a substrate having a plurality of addresses, each
address having disposed thereon a set of one or more biomolecules
that specifically interact with oxidized phospholipid (OxPL) and
apoB.
[0017] In another embodiment, a pre-packaged diagnostic kit for
determining whether a therapy is effective for treating coronary
artery disease, is provided. The kit may include an array as
described above, instructions for using the array, and instructions
calculating an atherogenesis index (AI) by determining the ratio of
the OxPL level to the apoB level.
[0018] In other embodiments, a method for identifying plaque
regression or stabilization in a blood vessel in a subject, is
provided. The method includes obtaining a first sample comprising
plasma from a subject; administering a therapy to the subject;
obtaining a second sample from the subject following administration
of the therapy; determining the level of oxidized phospholipid
(OxPL) in the first sample and second sample; determining the level
of apoB in the first sample and the second sample; calculating an
atherogenesis index (AI) by determining the ratio of the OxPL level
to the apoB level for both samples. An increase in the ratio
determined from the second sample in comparison to the ration
determined for the first sample, is indicative of an effective
therapy for coronary artery disease. The information may be
provided to a caregiver. In some embodiments, the therapy includes
administering to the subject a composition comprising a compound
that modulates the activity of HMG-CoA reductase, such as, for
example, a statin.
[0019] In yet another embodiment, a method for determining the
phospholipid content of an apoB-100 particle, is provided. The
method includes obtaining a sample comprising apoB-100; determining
the level of oxidized phospholipid (OxPL) in the sample;
determining the level of apoB in the sample; and calculating an
atherogenesis index (AI) by determining the ratio of the OxPL level
to the apoB level.
[0020] As used herein, the terms "biological molecules" and
"biomolecules" may be used interchangeably. These terms are meant
to be interpreted broadly, and generally encompass polypeptides,
peptides, oligosaccharides, polysaccharides, oligopeptides,
proteins, oligonucleotides, and polynucleotides. Oligonucleotides
and polynucleotides include, for example, DNA and RNA, e.g., in the
form of aptamers. Biomolecules also include organic compounds,
organometallic compounds, salts of organic and organometallic
compounds, saccharides, amino acids, and nucleotides, lipids,
carbohydrates, drugs, steroids, lectins, vitamins, minerals,
metabolites, cofactors, and coenzymes. Biomolecules further include
derivatives of the molecules described. For example, derivatives of
biomolecules include lipid and glycosylation derivatives of
oligopeptides, polypeptides, peptides, and proteins, such as
antibodies. Further examples of derivatives of biomolecules include
lipid derivatives of oligosaccharides and polysaccharides, e.g.,
lipopolysaccharides.
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0022] Other aspects of the invention are discussed throughout the
specification.
DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1A provides a schematic representation of the OxLDL
assay for OxLDL-E06 (OxPL/apoB). The antibody E06 detects oxidized
phospholipids (OxPL) present on apolipoprotein B-100 (apoB) and/or
apolipoprotein (a) [apo(a)].
[0024] FIG. 1B provides a schematic representation of the OxLDL
assay for apoB-Immune complexes (IC/apoB).
[0025] FIG. 1C provides a schematic representation of the OxLDL
assay for autoantibodies to MDA-LDL.
[0026] FIG. 2 depicts the geometric mean (95% CI) percent change
from baseline to 16 weeks in apoB-100, total apoB-OxPL and total
apoB-IC IgG and IgM in the atorvastatin and placebo groups.
[0027] FIG. 3 depicts the geometric mean (95% CI) percent change
from baseline to 16 weeks in OxLDL markers and Lp(a) in the
atorvastatin and placebo groups.
[0028] FIG. 4 depicts the correlation between OxLDL-E06 (OxPL/apoB)
and Lp(a) in all participants at baseline and 16 weeks.
[0029] FIG. 5 depicts the change in total apoB-OxPL and OxPL/apoB
ratio pre-regression and regression.
[0030] FIG. 6, panel A, depicts the frequency distribution of the
oxidized phospholipid:Apo B-100 ratio.
[0031] FIG. 6, panel B, depicts the frequency distribution of the
Lp(a) lipoprotein levels. Oxidized phospholipid:apo B-100 ratio
denotes the oxidized phospholipid content per particle of
apolipoprotein B-100.
[0032] FIG. 7 depicts the association of the oxidized
phospholipid:Apo B-100 ratio and Lp(a) lipoprotein levels to the
extent of coronary artery disease (CAD) in 239 patients 60 Years of
age or younger and 265 patients older than 60 years. The extent of
coronary artery disease is categorized as no disease (stenosis of
less than 10 percent of the luminal diameter), mild disease
(stenosis of 10 to 50 percent of the luminal diameter), or
one-vessel, two-vessel, or three-vessel disease (all patients with
a stenosis of more than 50 percent of the luminal diameter). Each
box represents the median and interquartile range of values, with
the bars (whiskers) extended to the minimum and maximum. Oxidized
phospholipid:apo B-100 ratio denotes the oxidized phospholipid
content per particle of apolipoprotein B-100. Values for the
oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein are
shown on a logarithmic scale.
[0033] FIG. 8 depicts odds ratios for obstructive coronary artery
disease (CAD) associated with selected risk factors among patients
60 years of age or younger and those older than 60 years, from the
multivariable analysis. CI denotes confidence interval, LDL
low-density lipoprotein, CRP C-reactive protein, oxidized
phospholipid:apo B-100 ratio the ratio of oxidized phospholipid
content per particle of apolipoprotein B-100, and HDL high-density
lipoprotein. Risk factors are shown in descending order of
significance. In this analysis, Lp(a) lipoprotein was forced into
the model with the oxidized phospholipid:apo B-100 ratio.
DETAILED DESCRIPTION
[0034] The present invention relates to the analysis of OxPL of
patients at high risk or with documented CAD or acute coronary
syndromes (ACS) or suspected of being at risk for ACS. Such methods
are useful for diagnostic purposes and for monitoring the effects
of dietary interventions, as well as for monitoring treatment with
anti-ACS drugs such as statins. More particularly, the present
invention relates to methods for determining OxPL/apoB ratios as
indices of atherosclerosis regression and plaque stability (i.e.
"atherogenesis").
[0035] In one embodiment, a method of determining whether a therapy
is effective for treating coronary artery disease, is provided. The
method includes obtaining a first sample comprising plasma from a
subject; administering a therapy to the subject; obtaining a second
sample from the subject following administration of the therapy;
determining the level of oxidized phospholipid (OxPL) in the first
sample and second sample; determining the level of apoB in the
first sample and the second sample; calculating an atherogenesis
index (AI) by determining the ratio of the OxPL level to the apoB
level for both samples. An increase in the ratio determined from
the second sample in comparison to the ratio determined for the
first sample, is indicative of an effective therapy for coronary
artery disease. The information may be provided to a caregiver. In
some embodiments, the therapy includes administering to the subject
a composition comprising a compound that modulates the activity of
HMG-CoA reductase, such as, for example, a statin.
[0036] In some embodiments, the level of OxPL and the level of apoB
in the samples obtained from the subject are measured with two or
more different biomolecules. The first biomolecule specifically
interacts with OxPL and the second biomolecule specifically
interacts with apoB. In some aspects, the biomolecules are
antibodies, such as, for example, monoclonal antibodies. The
antibody that interacts with OxPL may be, for example, E06 or
DLH3.
[0037] In other aspects, the biomolecules are antigens. In some
embodiments, the biomolecules are immobilized to form an array
comprising a first set of a plurality of the first biomolecule and
a second set of a plurality of the second biomolecule.
[0038] In one embodiment, the present invention relates to a method
for measuring the plasma content of oxidized phospholipids on
apolipoprotein B-100 particles (OxPL/apoB). For example, the
content of OxPL and apoB may be measured with monoclonal antibodies
that are specific for each of these OxPL constituents.
[0039] An exemplary biochemical test for identifying specific
proteins, such as OxPL and apoB, employs a standardized test
format, such as the Enzyme Linked Immunosorbent Assay or ELISA
test, although the information provided herein may apply to the
development of other biochemical or diagnostic tests and is not
limited to the development of an ELISA test (see, e.g., Molecular
Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc.,
New York and Basel 1984, for a description of ELISA tests). It is
understood that commercial assay enzyme-linked immunosorbant assay
(ELISA) kits for various plasma constituents are available.
[0040] In another embodiment, the AI index is further correlated
with, the age of the subject at the time the ratios are measured,
the subject's gender, and/or the subject's race.
[0041] In another embodiment, an article of manufacture is
provided. The article may include packaging material containing
biomolecules that preferentially interact with oxidized
phospholipid (OxPL) and apoB. The packaging material may include a
label or package insert indicating that the biomolecules can be
used for calculating an atherogenesis index (AI) by determining the
ratio of the OxPL level to the apoB level.
[0042] In other embodiments, the invention provides methods for
predicting the efficacy of a treatment for CAD through the use of
proteomics. Proteomics is an evolving technology capable of testing
for the presence of minute amounts of a vast array of proteins
using small samples of human tissue. Using proteomic tools,
increased or decreased levels of certain proteins in a biological
sample such as serum can be ascertained. The invention encompasses
plasma proteomic analysis as a non-invasive approach to determining
whether a subject is responding to a treatment.
[0043] In yet another embodiment, an array is provided. The array
may include a substrate having a plurality of addresses, each
address having disposed thereon a set of one or more biomolecules
that specifically interact with oxidized phospholipid (OxPL) and
apoB.
[0044] The invention provides an array (i.e., "biochip" or
"microarray") that includes immobilized biomolecules that
facilitate the detection of a particular molecule or molecules in a
biological sample. Biomolecules that identify the biomarkers
described above can be included in a custom array for detecting
OxPL or apoB. The array can also include biomolecules that identify
additional factors indicative of the efficacy of a treatment for
CAD. Additional biomolecules can be included in a custom array of
the invention.
[0045] The term "array," as used herein, generally refers to a
predetermined spatial arrangement of binding islands, biomolecules,
or spatial arrangements of binding islands or biomolecules. Arrays
according to the present invention that include biomolecules
immobilized on a surface may also be referred to as "biomolecule
arrays." Arrays according to the present invention that comprise
surfaces activated, adapted, prepared, or modified to facilitate
the binding of biomolecules to the surface may also be referred to
as "binding arrays." Further, the term "array" may be used herein
to refer to multiple arrays arranged on a surface, such as would be
the case where a surface bore multiple copies of an array. Such
surfaces bearing multiple arrays may also be referred to as
"multiple arrays" or "repeating arrays." The use of the term
"array" herein may encompass biomolecule arrays, binding arrays,
multiple arrays, and any combination thereof; the appropriate
meaning will be apparent from context. The biological sample can
include fluid or solid samples from any tissue of the body
including plasma.
[0046] An array of the invention comprises a substrate. By
"substrate" or "solid support" or other grammatical equivalents,
herein is meant any material appropriate for the attachment of
biomolecules and is amenable to at least one detection method. As
will be appreciated by those in the art, the number of possible
substrates is very large. Possible substrates include, but are not
limited to, glass and modified or functionalized glass, plastics
(including acrylics, polystyrene and copolymers of styrene and
other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TEFLON.RTM., etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, ceramics, and a variety of other polymers. In addition,
as is known the art, the substrate may be coated with any number of
materials, including polymers, such as dextrans, acrylamides,
gelatins or agarose. Such coatings can facilitate the use of the
array with a biological sample derived from serum.
[0047] A planar array of the invention will generally contain
addressable locations (e.g., "pads", "addresses," or
"micro-locations") of biomolecules in an array format. The size of
the array will depend on the composition and end use of the array.
Arrays containing from about 2 different biomolecules to many
thousands can be made. In some embodiments, the compositions of the
invention may not be in an array format; that is, for some
embodiments, compositions comprising a single biomolecule may be
made as well. In addition, in some arrays, multiple substrates may
be used, either of different or identical compositions. Thus, for
example, large planar arrays may comprise a plurality of smaller
substrates.
[0048] As an alternative to planar arrays, bead based assays in
combination with flow cytometry have been developed to perform
multiparametric inimunoassays. In bead based assay systems the
biomolecules can be immobilized on addressable microspheres. Each
biomolecule for each individual immunoassay is coupled to a
distinct type of microsphere (i.e., "microbead") and the
immunoassay reaction takes place on the surface of the
microspheres. Dyed microspheres with discrete fluorescence
intensities are loaded separately with their appropriate
biomolecules. The different bead sets carrying different capture
probes can be pooled as necessary to generate custom bead arrays.
Bead arrays are then incubated with the sample in a single reaction
vessel to perform the immunoassay.
[0049] Product formation of the biomarker with their immobilized
capture biomolecules can be detected with a fluorescence based
reporter system. Biomarkers can either be labeled directly by a
fluorogen or detected by a second fluorescently labeled capture
biomolecule. The signal intensities derived from captured
biomarkers are measured in a flow cytometer. The flow cytometer
first identifies each microsphere by its individual color code.
Second the amount of captured biomarkers on each individual bead is
measured by the second color fluorescence specific for the bound
target. This allows multiplexed quantitation of multiple targets
from a single sample within the same experiment. Sensitivity,
reliability and accuracy are compared to standard microtiter ELISA
procedures. With bead based immunoassay systems serum components
can be simultaneously quantified from biological samples. An
advantage of bead-based systems is the individual coupling of the
capture biomolecule to distinct microspheres.
[0050] An array of the present invention encompasses any means for
detecting a biomarker molecule such as, for example, apoB. For
example, microarrays can be biochips that provide high-density
immobilized arrays of recognition molecules (e.g., antibodies),
where biomarker binding is monitored indirectly (e.g., via
fluorescence). In addition, an array can be of a format that
involves the capture of proteins by biochemical or intermolecular
interaction, coupled with direct detection by mass spectrometry
(MS).
[0051] Arrays and microarrays that can be used with the new methods
to detect the biomarkers described herein can be made according to
the methods described in U.S. Pat. Nos. 6,329,209; 6,365,418;
6,406,921; 6,475,808; and 6,475,809, and U.S. patent application
Ser. No. 10/884,269, which are incorporated herein in their
entirety. New arrays, to detect specific selections of sets of
biomarkers described herein can also be made using the methods
described in these patents.
[0052] Surfaces useful according to the present invention may be of
any desired shape (form) and size. Non-limiting examples of
surfaces include chips, continuous surfaces, curved surfaces,
flexible surfaces, films, plates, sheets, tubes, and the like.
Surfaces preferably have areas ranging from approximately a square
micron to approximately 500 cm.sup.2. The area, length, and width
of surfaces according to the present invention may be varied
according to the requirements of the assay to be performed.
Considerations may include, for example, ease of handling,
limitations of the material(s) of which the surface is formed,
requirements of detection systems, requirements of deposition
systems (e.g., arrayers), and the like.
[0053] In certain embodiments, it is desirable to employ a physical
means for separating groups or arrays of binding islands or
immobilized biomolecules: such physical separation facilitates
exposure of different groups or arrays to different solutions of
interest. Therefore, in certain embodiments, arrays are situated
within wells of 96, 384, 1536, or 3456 microwell plates. In such
embodiments, the bottoms of the wells may serve as surfaces for the
formation of arrays, or arrays may be formed on other surfaces and
then placed into wells. In certain embodiments, such as where a
surface without wells is used, binding islands may be formed or
biomolecules may be immobilized on a surface and a gasket having
holes spatially arranged so that they correspond to the islands or
biomolecules may be placed on the surface. Such a gasket is
preferably liquid tight. A gasket may be placed on a surface at any
time during the process of making the array and may be removed if
separation of groups or arrays is no longer necessary.
[0054] Modifications or binding of biomolecules in solution or
immobilized on an array may be detected using detection techniques
known in the art. Examples of such techniques include immunological
techniques such as competitive binding assays and sandwich assays;
fluorescence detection using instruments such as confocal scanners,
confocal microscopes, or CCD-based systems and techniques such as
fluorescence, fluorescence polarization (FP), fluorescence resonant
energy transfer (FRET), total internal reflection fluorescence
(TIRF), fluorescence correlation spectroscopy (FCS);
colorimetric/spectrometric techniques; surface plasmon resonance,
by which changes in mass of materials adsorbed at surfaces may be
measured; techniques using radioisotopes, including conventional
radioisotope binding and scintillation proximity assays so (SPA);
mass spectroscopy, such as matrix-assisted laser
desorption/ionization mass spectroscopy (MALDI) and MALDI-time of
flight (TOF) mass spectroscopy; ellipsometry, which is an optical
method of measuring thickness of protein films; quartz crystal
microbalance (QCM), a very sensitive method for measuring mass of
materials adsorbing to surfaces; scanning probe microscopies, such
as AFM and SEM; and techniques such as electrochemical, impedance,
acoustic, microwave, and IR/Raman detection. See, e.g., Mere L, et
al., "Miniaturized FRET assays and microfluidics: key components
for ultra-high-throughput screening," Drug Discovery Today
4(8):363-369 (1999), and references cited therein; Lakowicz J R,
Principles of Fluorescence Spectroscopy, 2nd Edition, Plenum Press
(1999).
[0055] In another embodiment, a pre-packaged diagnostic kit for
determining whether a therapy is effective for treating coronary
artery disease, is provided. The kit may include an array as
described above, instructions for using the array, and instructions
calculating an atherogenesis index (AI) by determining the ratio of
the OxPL level to the apoB level.
[0056] Arrays of the invention suitable for identifying coronary
artery disease, and the efficacy of a treatment therefore, may be
included in kits. Such kits may also include, as non-limiting
examples, reagents useful for preparing biomolecules for
immobilization onto binding islands or areas of an array, reagents
useful for detecting modifications to immobilized biomolecules, or
reagents useful for detecting binding of biomolecules from
solutions of interest to immobilized biomolecules, and instructions
for use. Likewise, arrays comprising immobilized biomolecules may
be included in kits. Such kits may also include, as non-limiting
examples, reagents useful for detecting modifications to
immobilized biomolecules or for detecting binding of biomolecules
from solutions of interest to immobilized biomolecules.
[0057] In other embodiments, a method for identifying plaque
regression or stabilization in a blood vessel in a subject, is
provided. The method includes obtaining a first sample comprising
plasma from a subject; administering a therapy to the subject;
obtaining a second sample from the subject following administration
of the therapy; determining the level of oxidized phospholipid
(OxPL) in the first sample and second sample; determining the level
of apoB in the first sample and the second sample; calculating an
atherogenesis index (AI) by determining the ratio of the OxPL level
to the apoB level for both samples. An increase in the ratio
determined from the second sample in comparison to the ration
determined for the first sample, is indicative of an effective
therapy for coronary artery disease. The information may be
provided to a caregiver. In some embodiments, the therapy includes
administering to the subject a composition comprising a compound
that modulates the activity of HMG-CoA reductase, such as, for
example, a statin.
[0058] In yet another embodiment, a method for determining the
phospholipid content of an apoB-100 particle, is provided. The
method includes obtaining a sample comprising apoB-100; determining
the level of oxidized phospholipid (OxPL) in the sample;
determining the level of apoB in the sample; and calculating an
atherogenesis index (AI) by determining the ratio of the OxPL level
to the apoB level.
[0059] The invention provides compositions and methods for the
identification of a subject that may or may not be responding to a
treatment for CAD or ACS such that a theranostic approach can be
taken to test such individuals to determine the effectiveness of a
particular therapeutic intervention (pharmaceutical or
non-pharmaceutical) and to alter the intervention to 1) reduce the
risk of developing adverse outcomes and 2) enhance the
effectiveness of the intervention. Thus, the methods and
compositions of the invention also provide a means of optimizing
the treatment of a subject having such a disorder. The invention
provides a theranostic approach to treating such a disorder by
integrating diagnostics and therapeutics to improve the real-time
treatment of a subject having, for example, CAD and/or ACS.
Practically, this means creating tests that can identify which
patients are most suited to a particular therapy, and providing
feedback on how well a drug is working to optimize treatment
regimens. In the area of diseases associated with atherosclerosis,
theranostics can flexibly monitor changes in important parameters
over time. For example, theranostic multiparameter immunoassays
specific for a series of diagnostically relevant molecules such as
OxPL or apoB can be used to follow the progress of a subject
undergoing treatment. The markers provided herein are particularly
adaptable for use in diagnosis and treatment because they are
available in easily obtained body fluids such as blood or
serum.
[0060] Within the clinical trial setting, a theranostic method or
composition of the invention can provide key information to
optimize trial design, monitor efficacy, and enhance drug safety.
For instance, "trial design" theranostics can be used for patient
stratification, determination of patient eligibility
(inclusion/exclusion), creation of homogeneous treatment groups,
and selection of patient samples that are representative of the
general population. Such theranostic tests can therefore provide
the means for patient efficacy enrichment, thereby minimizing the
number of individuals needed for trial recruitment. "Efficacy"
theranostics are useful for monitoring therapy and assessing
efficacy criteria. Finally, "safety" theranostics can be used to
prevent adverse drug reactions or avoid medication error.
[0061] In other embodiments, the invention provides databases and
computerized methods of analyzing and storing data associated with
treatment regimens for athersclerosis related diseases. A database
generated by the methods and analyses described herein can be
included in, or associated with, a computer system for determining
whether a treatment is successful. The database can include a
plurality of digitally encoded "reference" (or "control") profiles.
Each reference profile of the plurality can have a plurality of
values, each value representing a level of, for example, OxPL or
apoB detected in blood or serum of an individual having, or
predisposed to having, an athersclerosis related disorder.
Alternatively, a reference profile can be derived from an
individual who is normal. Both types of profiles can be included in
the database for consecutive or simultaneous comparison to a
subject profile. The computer system can include a server
containing a computer-executable code for receiving a profile and
identifying from the database a matching reference profile that is
diagnostically relevant to the subject profile. The identified
profile can be supplied to a caregiver for diagnosis or further
analysis.
[0062] Using standard programs, electronic medical records (EMR)
can be accumulated to provide a database that combines, for
example, AI index data with additional information such as the age
of a patient or any other parameter useful for predicting whether
or not a subject will or is responding to a treatment. Patient
information can be randomly assigned a numerical identifier to
maintain anonymity with testing laboratories and for security
purposes. All data can be stored on a network that provides access
to multiple users from various geographic locations.
[0063] Thus, the various techniques, methods, and aspects of the
invention described herein can be implemented in part or in whole
using computer-based systems and methods. Additionally,
computer-based systems and methods can be used to augment or
enhance the functionality described herein, increase the speed at
which the functions can be performed, and provide additional
features and aspects as a part of, or in addition to, those of the
invention described herein.
[0064] A processor-based system can include a main memory,
preferably random access memory (RAM), and can also include a
secondary memory. The secondary memory can include, for example, a
hard disk drive and/or a removable storage drive, e.g., a floppy
disk drive, a magnetic tape drive, or an optical disk drive. The
removable storage drive reads from and/or writes to a removable
storage medium. The removable storage medium can be a floppy disk,
magnetic tape, optical disk, or the like, which is read by and
written to by a removable storage drive. As will be appreciated,
the removable storage medium can comprise computer software and/or
data.
[0065] The computer system can also include a communications
interface. Communications interfaces allow software and data to be
transferred between the computer system and external devices.
Examples of communications interfaces include a modem, a network
interface (such as, for example, an Ethernet card), a
communications port, a PCMCIA slot and card, and the like. Software
and data transferred via a communications interface are in the form
of signals, which can be electronic, electromagnetic, optical, or
other signals capable of being received by a communications
interface. These signals are provided to a communications interface
via a channel capable of carrying signals and can be implemented
using a wireless medium, wire or cable, fiber optics or other
communications medium. Some examples of a channel include a phone
line, a cellular phone link, an RF link, a network interface, and
other communications channels. In this document, the terms
"computer program medium" and "computer usable medium" are used to
refer generally to media such as a removable storage device, a disk
capable of installation in a disk drive, and signals on a channel.
These computer program products are means for providing software or
program instructions to a computer system.
[0066] Computer programs (also called computer control logic) are
stored in main memory and/or secondary memory. Computer programs
can also be received via a communications interface. Such computer
programs, when executed, enable the computer system to perform the
features of the methods discussed herein. In particular, the
computer programs, when executed, enable the processor to perform
the features of the invention. Accordingly, such computer programs
represent controllers of the computer system.
[0067] In an embodiment where the elements are implemented using
software, the software may be stored in, or transmitted via, a
computer program product and loaded into a computer system using a
removable storage drive, hard drive, or communications interface.
The control logic (software), when executed by the processor,
causes the processor to perform the functions of the methods
described herein.
[0068] In another embodiment, the computer-based methods can be
accessed or implemented over the World Wide Web by providing access
via a Web Page to the methods of the invention. Accordingly, the
Web Page is identified by a Universal Resource Locator (URL). The
URL denotes both the server machine and the particular file or page
on that machine. In this embodiment, it is envisioned that a
consumer or client computer system interacts with a browser to
select a particular URL, which in turn causes the browser to send a
request for that URL or page to the server identified in the URL.
Typically the server responds to the request by retrieving the
requested page and transmitting the data for that page back to the
requesting client computer system (the client/server interaction is
typically performed in accordance with the hypertext transport
protocol ("HTTP")). The selected page is then displayed to the user
on the client's display screen. The client may then cause the
server containing a computer program of the invention to launch an
application to, for example, perform an analysis according to the
invention.
LDL Architecture
[0069] Early atherosclerotic lesions are characterized by the
appearance of lipid-rich "foam cells", which are
monocyte/macrophages that have taken up lipoproteins and are
localized in the subendothelial space. It is not native LDL that is
taken up by these cells, since native LDL cannot induce cholesterol
accumulation. In contrast, these cells take up modified forms of
LDL, and in particular oxidized LDL. Oxidized LDL (OxLDL) contains
OxPL in two distinctly different forms: it is present in the lipid
phase of the OxLDL, or it is covalently attached to apolipoprotein
B-100 (apoB-100) during LDL oxidation.
OxLDL Metabolism
[0070] OxPL are present in the vessel wall and are pro-inflammatory
and pro-atherogenic. During cholesterol lowering by diet or drugs,
such as HMG-CoA-reductase inhibitors (statins), the plasma levels
of OxPL/apoB actually increase compared to placebo. Animal
experiments show that there is depletion of OxPL from the vessel
wall into the circulation. This implies that a measurement of
OxPL/apoB reflects both plaque regression and plaque
stabilization.
[0071] This is documented in two studies described below. In
studies with cynomolgous monkeys that were first placed on
atherogenic diets ("Pre-regression") for six months and then
subjected to regression diets ("regression"), there was a depletion
of OxPL epitopes within atherosclerotic lesions by immunostaining
with antibody E06, while at the same time, there was an increase in
plasma OxPL/apoB levels (see FIG. 5). These experimental data
support an interpretation that the increase in OxPL/apoB in plasma
is a reflection of net efflux of OxPL from the vessel wall.
LDL: Individual Components
[0072] OxPL:
[0073] A monoclonal antibody, designated E06 has been reported that
binds specifically to the phosphorylcholine head group of oxidized
but not native phospholipids.sup.13, 19. Accordingly, this antibody
can be used to determine the level of oxidized phospholipids in the
OxLDL complex. This antibody can be adapted for use in any
immunoassay. For example, chemiluminsecent ELISA assays are
described elsewhere herein.
[0074] Additional antibodies have been described in the literature
that can also bind OxPL, such as DLH3 (Itabe et al., J Lipid Res.
1996; 37:45-53).
[0075] ApoB:
[0076] The total apoB-100 level in plasma can be determined using
known immunoassay techniques.
[0077] Lp(a): Lp(a) consists of a particle of low-density
lipoprotein cholesterol (LDL-C) linked by a disulfide bond to a
large hepatically derived glycoprotein, apolipoprotein(a), which is
structurally similar to plasminogen. In theory, then, Lp(a) could
promote cardiovascular disease in two ways: its apolipoprotein(a)
moiety could promote thrombogenesis and its LDL-C moiety could
promote atherogenesis. The apolipoprotein portion of Lp(a)
competitively displaces plasminogen from binding sites on both
fibrin and endothelial cells. Lp(a) is associated with increased
levels of plasminogen activator-inhibitor (PAI-1) and decreased
activity of tissue plasminogen activator (t-PA). These effects all
promote thrombosis and inhibit fibrinolysis. Lp(a) can, like LDL-C
alone, be oxidized, taken up by macrophages, and recovered from
atherosclerotic plaque. Lp(a) appears to facilitate the oxidation
of LDL-C, and can impair endothelial function.
LDL: Relative Component Levels
[0078] As discussed herein, "OxLDL-E06" or "OxPL/apoB" is a measure
of the content of oxidized phospholipids (OxPL) per apoB-100
particle. Also as discussed herein, "apoB-IC", or "IC/apoB", refers
to the total amount of apoB in circulating LDL immune complexes.
Collectively, the OxPL/apoB and IC/ApoB measurements are used to
specifically quantify the content of OxPL and IC, respectfully, on
each captured apoB particle.
[0079] Alternatively, the Total apoB-OxPL and Total apoB-IC
measurements reflect the OxPL and IC content on all apoB-100
containing particles in plasma, and are determined by multiplying
the plasma "OxPL/apoB" and "IC/apoB" values by the plasma apoB-100
levels.
[0080] Accordingly, the present invention relates to the
measurement of OxPL/apoB with or without simultaneous measurement
of IC/apoB levels and/or total OxPL/apoB and total IC/apoB as
indices of atherogenesis.
Statins
[0081] Statins inhibit the enzyme, HMG-CoA reductase that controls
the rate of cholesterol production in the body. These drugs lower
cholesterol by slowing down the production of cholesterol and by
increasing the liver's ability to remove the LDL-cholesterol
already in the blood. The large reductions in total and
LDL-cholesterol produced by these drugs results in large reductions
in heart attacks and heart disease deaths.
[0082] Examples of commercially available statins include, for
example, Lipitor from Pfizer (atorvastatin), Zocor from Merck
(simvastatin), Pravachol from Bristol-Myers Squibb (pravastatin),
Lescol from Novartis (fluvastatin) and Mevacor from Merck
(lovastatin). These statins are routinely administered using
well-known clinical protocols.
[0083] Using the methods described herein, statin treatment can be
readily monitored for efficacy.
Statin Studies
[0084] Randomized trials have clearly shown that
hydroxymethylglutaryl (HMG)-CoA reductase inhibitors (statins)
reduce all-cause mortality and cardiovascular events in patients
with stable coronary artery disease (CAD) when given over 5
years..sup.1;2 Retrospective.sup.3 and observational.sup.4 studies
have also suggested that statins given to patients with acute
coronary syndromes (ACS) improve event-free survival over one year.
The MIRACL study demonstrated that in-hospital initiation of 80 mg
atorvastatin reduced recurrent ischemic events over a 16 weeks
period..sup.5 The PROVE-IT study recently demonstrated superior
outcomes in patients with ACS following two years' treatment with
80 mg atorvastatin versus 40 mg pravastatin, resulting in median
LDL cholesterol levels of 62 and 95 mg/dl, respectively..sup.6
[0085] The mechanisms underlying the early benefits of statins are
not well delineated, but have been loosely attributed to plaque
stabilization..sup.7;8 However, it is not established whether
statins exert their benefits primarily through reduction of
LDL-cholesterol alone and/or through additional pleiotropic
effects, such as direct anti-inflammatory or anti-oxidant
actions..sup.9 Although statins have been shown to reduce in vitro
measures of oxidative stress (reviewed in.sup.10), their effects on
plasma OxLDL levels in patients, particularly those with ACS, are
not well known.
[0086] Increased levels of OxLDL in the vessel wall and circulation
are present in patients with unstable or "vulnerable"
plaques,.sup.11;12 in ACS.sup.11-14 and are associated with
endothelial dysfunction..sup.15-17 In mouse and rabbit aortic
atherosclerotic lesions, OxLDL becomes depleted following
regression diets, out of proportion to LDL depletion or other
measures of plaque regression..sup.8;18 These reductions in OxLDL
are associated with increased collagen and smooth muscle cell
content, increased eNOS production and reduced inflammatory
markers,.sup.7 suggesting that removal of OxLDL from the vessel
wall may serve as an early marker of plaque stabilization. In this
analysis of the MIRACL trial, we determined whether favorable
changes in plasma OxLDL levels may provide insights into the early
clinical benefits of intensive statin treatment following ACS.
Example 1
[0087] a. Study Design and Patient Sample:
[0088] The MIRACL study design was previously published..sup.5
Briefly, the study recruited 3086 patients with unstable angina or
non-Q-wave acute myocardial infarction between 24-96 hours after
hospital admission at 122 centers in 19 countries. Patients were
randomly assigned to double-blind treatment with atorvastatin 80
mg/day or placebo for 16 weeks. The primary efficacy measure was
the time to first occurrence of death, nonfatal acute myocardial
infarction, cardiac arrest with resuscitation, or worsening angina
with new objective evidence of ischemia and requiring emergency
re-hospitalization. 2739 patients completed the entire 16 week
follow-up period, of whom 2442 had baseline blood samples and 2341
had baseline and Week 16 blood samples available for analysis.
Blood was collected in EDTA and stored at -70.degree. C. until
analysis. Patients were analyzed on an intention-to-treat
basis.
[0089] b. Determination of OxLDL-E06, Apolipoprotein B-100 Immune
Complexes and OxLDL (MDA-LDL) Autoantibody Titers:
[0090] Chemiluminescent ELISA was used to measure OxLDL markers
(FIG. 1A -1C). All samples for a given assay were run in a single
assay and internal controls consisting of high and low standard
plasma samples were included to detect potential variations between
microtitration plates. Each sample was assayed in triplicate and
data are expressed as relative light units (RLU) in 100
milliseconds. The intra-assay coefficients of variation for all
assays were 6-10%.
[0091] OxLDL-E06 is a measure of the content of oxidized
phospholipids (OxPL) per apoB-100 particle using the murine
monoclonal antibody E06, which specifically binds to the
phosphorylcholine head group of oxidized but not native
phospholipids (reviewed in Tsimikas.sup.13;39 and references
therein). A 1:50 dilution of plasma in phosphate-buffered saline
(PBS) is added to microtiter wells coated with the monoclonal
antibody MB47 (5 .mu.g/ml), which specifically binds apoB-100
particles. Under these conditions, a saturating amount of apoB-100
is added to each well and consequently an equal number of apoB-100
particles are captured in each well for all assays. The content of
OxPL per apoB-100 is then determined with biotinylated E06 as
previously described..sup.13 In a previous study we directly
measured the amount of apoB-100 bound in each well by the degree of
binding of a biotinylated anti-apoB-100 antibody, and compared the
OxPL RLU values alone to the ratio of OxPL RLU/apoB-100 RLU and
found a correlation of r.sup.2=0.99 (n=1500 samples, data not
shown). Therefore, we arbitrarily assigned the apoB-100 RLU value
in the denominator as 1 and report the OxPL/apoB values as OxPL RLU
counts only. Note that by design, this assay normalizes the OxPL
content per apoB-100 particle and is therefore independent of
plasma apoB-100 (and thus LDL-cholesterol) levels.
[0092] The data for OxLDL-E06 and apoB-IC are presented in two
ways: 1) as OxPL/apoB and IC/apoB, which specifically quantifies
the content of OxPL and IC, respectively, on each captured apoB-100
particle, and 2) as Total apoB-OxPL and Total apoB-IC, which
reflects the OxPL and IC content on all apoB-100 containing
particles in plasma, by multiplying the plasma "OxPL/apoB" and
"IC/apoB" value by the plasma apoB-100 levels measured
independently as noted below.
[0093] Plasma titers of IgG and IgM apoB-IC and malondialdehyde
(MDA-LDL) (1:200 plasma dilution) autoantibodies and apoB-IC were
measured as previously described..sup.13
[0094] c. Lipoprotein (a) and ApoB-100 Levels:
[0095] Lp(a) is a modified LDL particle to which apo(a) is
covalently linked. We have recently shown that a strong correlation
exists between plasma OxLDL-E06 and Lp(a) levels and that 75-90% of
the E06 immunoreactivity on apoB-100 particles is associated
specifically with Lp(a) at steady state..sup.13;19 E06
immunoreactivity is found in both the lipid and protein moieties of
Lp(a), and kringle V of apolipoprotein (a) appears to contain up to
2 moles of covalently bound OxPL..sup.20 However, in some settings,
such as immediately following percutaneous coronary intervention
(PCI), OxPL are found equally on apoB-100 particles without apo(a)
and Lp(a)..sup.19
[0096] Plasma Lp(a) levels were measured by a chemiluminescent
ELISA with monoclonal antibody LPA4, as previously
described..sup.19 LPA4 does not cross-react with plasminogen.
ApoB-100 levels were measured by a commercially available kit
(Behring) and CRP levels as previously described..sup.5
[0097] d. Statistical Analysis:
[0098] Since the baseline and Week 16 distributions of the OxLDL
markers were positively skewed, log-transformed values were used in
the statistical models and analyses and antilog-transformed for
descriptive statistics yielding geometric means and 95% confidence
intervals (CI) for baseline, Week 16 and percent change from
baseline to Week 16. Inferential analyses included paired-sample
t-tests for within treatment group differences in markers at
baseline versus Week 16, and independent-sample t-tests for between
treatment group differences in markers at Week 16 and between
treatment group differences in absolute change in log-transformed
markers from baseline to Week 16. Logistic regression models were
constructed to summarize the relationships between baseline OxLDL
values and the primary efficacy measure of the trial. For each
marker, the logistic regression model included terms for
log-transformed marker and randomized treatment assignment; odds
ratios were expressed as the relative odds for each one unit
increase in the log-transformed marker. Pearson correlations were
calculated to summarize the relationship between OxPL/apoB and
Lp(a) at baseline and at Week 16. Statistical significance was
defined as P<0.05.
[0099] a. Patient Characteristics:
[0100] Baseline characteristics did not differ significantly
between groups (as shown below in Table 1) or compared to the
entire MIRACL population and there were no significant differences
between patients with and without available blood samples for
analysis..sup.5
TABLE-US-00001 TABLE 1 Baseline characteristics of the 2341 study
subjects Placebo Atorvastatin Characteristic (n = 1190) (n = 1151)
Age, mean (SD), years 64 (11) 65 (11) Men, n (%) 796 (67) 752 (65)
Presenting Syndrome, n (%) Unstable angina 534 (45) 527 (46) Non-Q
wave myocardial infarct 656 (55) 624 (54) Past myocardial infarct,
n (%) 288 (24) 269 (23) Prior coronary revascularization, n (%) 131
(11) 109 (9) Hyperlipidemia, n (%) 408 (34) 421 (35) Hypertension,
n (%) 636 (53) 629 (55) Diabetes mellitus type II, n (%) 280 (24)
251 (22) Total cholesterol, mean (SD), mg/dl 207 (37) 206 (38) LDL
cholesterol, mean (SD), mg/dl 125 (33) 124 (34) HDL cholesterol,
mean (SD), mg/dl 46 (12) 47 (12) Triglycerides, mean (SD), mg/dl
186 (93) 182 (86)
[0101] For the 2442 patients with baseline data, 10.7% had an
endpoint in the atorvastatin group and 12.8% in the placebo group
during the 16-week follow-up period. Among the entire MIRACL
population, 14.6% suffered a primary endpoint event in the
atorvastatin group, compared with 17.2% in the placebo group. Thus,
the incidence of recurrent events was lower in the present analysis
cohort than the entire MIRACL population, but the risk reduction
associated with atorvastatin treatment was similar.
[0102] b. Effect of Atorvastatin on OxLDL Markers and Lp(a):
[0103] Baseline levels of apoB-100, Total apoB-OxPL, Total apoB-IC,
MDA-LDL autoantibodies and Lp(a) did not differ between groups. The
atorvastatin-treated group had a 42% reduction in LDL cholesterol
(124.+-.34 to 72.+-.35 mg/dL, P<0.0001) while the
placebo-treated group had modest increases (124.+-.34 to 135.+-.37
mg/dL, P<0.0001). From baseline to Week 16, significant
reductions in absolute and relative levels of apoB-100, Total
apoB-OxPL, and Total apoB-IC (both IgG and IgM) were noted in the
atorvastatin group compared to the placebo group: apoB-100, -33.0%
vs. 5.8%; Total apoB-OxPL, -29.7% vs.-0.2%; Total apoB-IC IgG,
-29.5% vs. 2.1% and Total apoB-IC IgM, -25.7% vs. 13.2%,
P<0.0001 for all comparisons (see Table 2 below and FIG. 2).
TABLE-US-00002 TABLE 2 Baseline and 16 Week Mean Levels (95%
confidence intervals) Of ApoB, OxLDL markers and Lp(a) Atorvastatin
P- Placebo P- P- Variable Baseline Week 16 Value** Baseline Week 16
Value** Value*** ApoB-100* 132 87 <0.0001 133 138 <0.0001
<0.0001 (130, 134) (85, 89) (131, 135) (136, 140) Total apoB-
1297 912 <0.0001 1300 1299 0.8290 <0.0001 OxPL (1252, 1342)
(876, 949) (1257, 1344) (1254, 1345) Total apoB- 1067 749
<0.0001 1090 1103 0.1963 <0.0001 IC IgG (1030, 1106) (723,
778) (1051, 1131) (1064, 1143) Total apoB- 439 325 <0.0001 456
517 <0.0001 <0.0001 IC IgM (421, 458) (311, 339) (437, 477)
(492, 542) OxPL/apoB 10085 11048 <0.0001 10062 9690 <0.0001
<0.0001 (9770, 10409) (10667, 11442) (9756, 10378) (9356, 10036)
Lp(a)* 11.9 13.0 <0.0001 11.9 11.9 0.6164 0.0651 (11.2, 12.6)
(12.2, 14.0) (11.2, 12.7) (11.1, 12.7) IC/apoB IgG 8323 9048
<0.0001 8406 8204 0.2212 <0.0001 (8069, 8585) (8763, 9342)
(8137, 8685) (7944, 8472) IgM 3426 3933 <0.0001 3525 3852
<0.0001 <0.0001 (3293, 3565) (3785, 4087) (3387, 3669) (3695,
4016) Autoantibodies to MDA-LDL IgG 4317 4922 <0.0001 4587 5084
<0.0001 0.1403 (4176, 4464) (4772, 5076) (4451, 4728) (4933,
5239) IgM 10025 1088 <0.0001 10198 11256 <0.0001 0.1608
(9696, 10365) (10531, 11256) (9879, 10528) (10895, 11627) For Total
apoB-OxPL and Total apoB-IC IgG and IgM units are relative light
units (RLU mg/dl) .times. 10.sup.3 For OxPL/apoB, IgG and IgM IgG
IC/apoB and autoantibodies to MDA-LDL units are in RLU *units are
mg/dl **Within treatment group t-test p-values for changes Baseline
to Week 16 ***Atorvastatin vs. placebo t-test p-values for Week
16
[0104] In contrast, absolute OxPL/apoB levels increased
significantly in the atorvastatin group, but actually decreased
significantly in the placebo group (Table 2), suggesting that
atorvastatin resulted in OxPL enrichment of apoB-100 particles,
despite a reduction in Total apoB-OxPL levels. In parallel to the
rise in OxPL/apoB, Lp(a) levels also increased to a similar extent
in response to atorvastatin (Table 2). There was a significant
relative increase in OxPL/apoB (9.5% vs.-3.9%) and Lp(a) levels
(8.8% vs.-0.7%, P<0.0001 for both, FIG. 3) in the atorvastatin
group compared to the placebo group. Indeed, strong correlations
were noted between OxPL/apoB and Lp(a) at baseline (r=0.82) and
Week 16 (r=0.85), P<0.0001 for both, FIG. 4. Similar
correlations were noted for Total apoB-OxPL (data not shown). These
findings are consistent with our previous observation that the OxPL
recognized by E06 are predominantly associated with
Lp(a)..sup.13;39
[0105] The relative increases in IgG IC/apoB (9.6% vs.-1.8%,
P<0.0001) and IgM IC/apoB (15.4% vs. 9.3%, P=0.0053) were also
higher in the atorvastatin group compared to placebo (FIG. 3). The
IgG and IgM MDA-LDL autoantibody levels increased equally in both
groups (range 9.1-14.4%, P<0.0001 for both, FIG. 3), as has been
shown previously in ACS..sup.13 The treatment group difference was
significant for IgG (P=0.035) but not IgM (P=0.16). Pearson
correlations between log-transformed CRP levels and OxLDL markers
and Lp(a) at baseline and Week 16 were not statistically
significant.
[0106] c. Relationship of Baseline OxLDL Markers and Lp(a) to
Clinical Outcomes:
[0107] With increasing baseline levels of either Total apoB-IC IgM
or IC/apoB IgM, there was reduced risk (odds ratio 0.81 for both)
of recurrent events (P=0.032 and 0.013, respectively, FIG. 5). In
this analysis, a one unit increase on the log scale was slightly
greater than the interquartile range (the difference between the
25th and 75th percentiles). Baseline levels of other OxLDL markers,
LDL-C, apoB-100 or Lp(a) were not predictive of risk at 16
weeks.
[0108] This study reveals that high-dose atorvastatin significantly
reduced the total content of OxPL present on all circulating
apoB-100 particles and suggests that the early clinical benefit of
atorvastatin in ACS may be mediated in part through a reduction of
vasoactive and pro-inflammatory OxPL in plasma..sup.21
Interestingly, on average, individual apoB-100 particles at the new
steady state were actually enriched in OxPL (i.e. an increased
OxPL/apoB ratio), in conjunction with a strikingly similar increase
in Lp(a), which we have previously shown binds OxPL..sup.13;19;20
Although our observations do not establish a causal mechanism, we
hypothesize that with the reduction of LDL cholesterol levels and
inflammation,.sup.22 there ensues a mobilization of OxPL from the
vessel wall, transient binding by apoB-100 particles [chiefly
Lp(a)] and clearance from the circulation. In addition, these data
provide further evidence for a novel physiological and/or
pathophysiological role of Lp(a), which we.sup.13;19 and
others.sup.23 have proposed binds and transports inflammatory
OxPL.
[0109] It should be appreciated that different antibodies are used
to detect oxidation epitopes on OxLDL and, depending on the epitope
measured, different information may be obtained. We have therefore
suggested that authors utilize the antibody used in their assay in
their designation of OxLDL to call attention to this possibility at
this early stage of such measurements..sup.13 Thus, we designate
our measure of OxLDL as OxLDL-E06 (OxPL/apoB). Because our assay
was designed to provide a physical estimate of the OxPL epitope
detected by E06 already normalized for apoB-100 levels, e.g. to
yield OxPL/apoB, our methodology allows for two complementary but
unique sets of measurements. First, it quantitates the number of
E06 epitopes per apoB-100 particle [OxPL/apoB], which by design is
independent of plasma LDL-cholesterol levels..sup.13 Secondly, when
the OxPL/apoB is multiplied by (independently measured) plasma
apoB-100 levels, one derives Total apoB-OxPL levels present on all
apoB-100 particles.
[0110] The role of statins in reducing plasma OxLDL in ACS has not
been previously described. Based on the present study, it can be
hypothesized that statin-mediated reduction in total plasma levels
of OxPL and apoB-IC may be through both a reduction in the
substrate for oxidation (i.e. reducing LDL levels and its
associated lipids) and possibly through direct anti-inflammatory
effects of atorvastatin metabolites, which have potent antioxidant
effects..sup.24 In vitro studies using several different statins,
and/or their metabolites, demonstrate both a reduction in markers
of generalized oxidative stress and LDL susceptibility to
oxidation..sup.10 For example, in patients with
hypercholesterolemia, simvastatin has been shown to reduce the
formation of F.sub.2-isoprostanes and plasma OxLDL levels, although
the epitope of OxLDL measured in this study was not defined..sup.25
Moreover, statins also have other reported pleiotropic effects, at
least in animal models, such as nitric-oxide sparing
properties..sup.9
[0111] OxPL are known to be highly inflammatory and to induce
vasoconstriction.sup.21 and it is possible that removal of such
OxPL contributes to rapid improvement in endothelial function. This
is supported by several studies showing improvement in
coronary.sup.17 and brachial endothelial function.sup.16 with LDL
apheresis or lovastatin treatment..sup.15 More specifically, Tamai
et al.sup.16 have shown that acetylcholine-induced brachial artery
vasodilatation rapidly improves within 4 hours following LDL
apheresis [Lp(a) is also removed] and the best correlate of
improvement was reduced plasma OxLDL levels, measured using
monoclonal antibody DLH3, which binds to an OxPL epitope nearly
identical to that bound by E06. Penny et al.sup.15 have shown that
OxPL-E06 was the best correlate of acetylcholine-induced coronary
vasodilatation following lovastatin therapy in patients with CAD.
LDL apheresis also decreases plasma concentrations of another model
OxLDL, MDA-LDL, by 61%..sup.26 A reduction in total OxPL may also
produce anti-inflammatory effects which, in turn, may be related to
clinical benefit of statin treatment..sup.22However, in this and
previous studies,.sup.13 we have not found an association between
any plasma OxLDL markers and CRP.
[0112] Our analyses also reveal the complementary observation that
the plasma apoB particles of atorvastatin-treated patients were
enriched in OxPL (an absolute 13.4% difference compared to placebo)
despite the fact that there was an overall reduction in the content
of total OxPL on all apoB-100 particles. What are the potential
mechanisms of the increase in OxPL/apoB plasma levels following
treatment with atorvastatin? In human studies, Crisby et al.sup.27
have shown that 3-months' treatment with pravastatin prior to
carotid endarterectomy markedly reduced OxLDL immunostaining in
carotid plaques, using the oxidation-specific antibody NA59, which
recognizes 4-hydroxynonenal oxidation-specific epitopes. Tsimikas
et al.sup.18 in an LDLR.sup.-/- mouse model and Aikawa et al.sup.8
in a New Zealand White rabbit model, both using the
oxidation-specific antibody MDA2, have shown decreased OxLDL
content in aortic plaques following aggressive dietary lipid
lowering. Additional unpublished data from both the murine and
rabbit experiments, [which do not have Lp(a)], as well as similar
studies with cynomolgous monkeys, show that following regression of
established atherosclerosis by dietary induced lipid lowering,
there are similar increases in OxPL/apoB plasma levels, but
markedly diminished Total apoB-OxPL levels, similar to the MIRACL
study. In all three animal studies, a direct immunochemical
analysis of the arterial tissue with antibody E06 demonstrated a
marked depletion of OxPL epitopes from the vessel wall, even as the
plasma OxPL/apoB ratios were increased from the baseline
measurements. Thus, in association with lesion regression, there
was a clear net efflux of OxPL from the vessel wall at a time when
the OxPL/apoB ratio in plasma was increased. (Tsimikas/Witztum,
unpublished observations). In addition, unpublished in vitro data
in our laboratory show that even in a PBS buffer there is
preferential physical transfer of OxPL (derived from OxLDL) to
Lp(a), compared to LDL. These data strongly support the hypothesis
that the increase in OxPL/apoB associated with atorvastatin
treatment is a surrogate marker of net OxPL efflux from the vessel
wall. This hypothesis deserves further study.
[0113] Lp(a) levels were also modestly increased in response to
atorvastatin in this study, which has previously been observed
during the treatment of hypercholesterolemia with other
statins,.sup.28-32 but has been underappreciated. One might
speculate that increased Lp(a) levels occur in response to the
enhanced efflux of OxPL from the vessel wall in order to facilitate
their transport and elimination, though the mechanisms mediating
such processes are unknown. In addition, it is also possible that
anti-inflammatory and anti-atherogenic functions of HDL may have
been improved by atorvastatin, leading to increased OxPL
efflux..sup.33 HDL, and in particular, a pre-beta fraction of HDL,
may be the preferred initial acceptor of cholesterol from cellular
sources. Recently, Navab et al.sup.34 have observed that an apoA-I
mimetic effects efflux of OxPL from cells to such a pre-beta HDL
fraction and we speculate that in turn, Lp(a) would then
preferentially accept such OxPL from the pre-beta HDL. This
potential mechanism of efflux of OxPL from the vessel wall may be
analogous to the rapid effects of ApoA-1/phospholipid complexes in
reducing coronary atheroma volume, which presumptively also
mobilized lipids out of the vessel wall..sup.35 In support of this
hypothesis is the recent observation that the OxPL/apoB ratio
increased, as did Lp(a), in subjects consuming a low-fat diet,
another condition in which one might speculate there was
mobilization of OxPL from the artery wall..sup.36
[0114] In support of a potential transport function of OxPL by
Lp(a), we have recently documented an .about.50% increase in plasma
OxLDL-E06 (i.e. OxPL/apoB) levels immediately following PCI,
presumably released from disrupted plaques, with a simultaneous and
similar increase in Lp(a) levels..sup.19 Furthermore, the released
OxPL epitopes were initially equally present on both apoB-100 and
Lp(a) particles, but appeared to transfer to Lp(a) nearly
exclusively by six hours..sup.19 In patients presenting with ACS or
undergoing PCI,.sup.13;19 we have also shown a strong association
between plasma levels of OxLDL-E06 (OxPL/apoB) and Lp(a), further
defining a novel pathophysiological association between OxPL and
Lp(a).
[0115] It is also possible that Lp(a) directly contributes to the
degradation of such OxPL, as Lp(a) was reported to be greatly
enriched in platelet activating factor acetyl hydrolase, an enzyme
that can degrade such OxPL..sup.21;37 We have previously suggested
that this potential physiological function of Lp(a) may be
beneficial acutely, particularly in patients with normal Lp(a)
levels. However, in patients with chronically elevated levels,
Lp(a) with its predilection for enhanced binding to the
extracellular matrix of atherosclerotic lesions (reviewed
in.sup.13), may be proinflammatory and proatherogenic because of
the enhanced OxPL content.
[0116] The highest baseline levels of IgM IC/apoB and Total apoB-IC
were associated with reduced risk of recurrent events (OR 0.81 and
0.84, respectively) and there was a similar trend with IgM MDA-LDL
autoantibodies (OR 0.90). Although the underlying mechanisms are
unclear, this suggests a potential protective effect of IgM OxLDL
autoantibodies, as has been shown in animal models immunized with
OxLDL or pneumococcal vaccine (which contains the same OxPL
epitopes as OxLDL), which induce high circulating levels of
OxLDL-specific IgM autoantibodies and decreased
atherosclerosis..sup.38 This is also consistent with previous
studies showing an inverse correlation between IgM OxLDL
autoantibody titers and CAD,.sup.13 hypertension,.sup.39 and
carotid and femoral atherosclerosis..sup.40;41
[0117] Limitations of this study include the absence of blood
samples at an intermediate timepoint during randomized treatment.
Had samples been available for such measurements, the change in
OxPL markers from baseline to the intermediate time point could
have been related to the risk of an event following the
intermediate time point. The high correlation of OxLDL-E06 with
Lp(a) raises the question of whether measurements of OxLDL-E06 will
provide incremental information above and beyond measurement of
Lp(a). Additional experimental and appropriately powered clinical
studies will be needed to establish whether OxPL markers are useful
in predicting clinical outcomes. Nonetheless, our data do suggest
novel physiological and/or pathophysiological functions of Lp(a)
which warrant further investigation in future studies.
[0118] This study shows that atorvastatin therapy, compared to
placebo, results in marked reduction in total plasma OxPL
associated with apoB-100, while at the same time enlarging a pool
of Lp(a) particles enriched in OxPL. These observations support the
hypothesis that early atorvastatin treatment after ACS enhances
mobilization and subsequent clearance of OxPL from the arterial
wall, a mechanism that may contribute to the clinical benefit of
statin therapy.
Example 2
[0119] Lp(a) lipoprotein binds proinflammatory oxidized
phospholipids. We investigated whether levels of oxidized
low-density lipoprotein (LDL) measured with use of monoclonal
antibody E06 reflect the presence and extent of obstructive
coronary artery disease, defined as a stenosis of more than 50
percent of the luminal diameter.
[0120] Levels of oxidized LDL and Lp(a) lipoprotein were measured
in a total of 504 patients immediately before coronary angiography.
Levels of oxidized LDL are reported as the oxidized phospholipid
content per particle of apolipoprotein B-100 (oxidized
phospholipid:apo B-100 ratio).
[0121] Measurements of the oxidized phospholipid:apo B-100 ratio
and Lp(a) lipoprotein levelswere skewed toward lower values, and
the values for the oxidized phospholipid:apo B-100 ratio correlated
strongly with those for Lp(a) lipoprotein (r=0.83, P<0.001). In
the entire cohort, the oxidized phospholipid:apo B-100 ratio and
Lp(a) lipoprotein levels showed a strong and graded association
with the presence and extent of coronary artery disease (i.e., the
number of vessels with a stenosis of more than 50 percent of the
luminal diameter) (P<0.001). Among patients 60 years of age or
younger, those in the highest quartiles for the oxidized
phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels had odds
ratios for coronary artery disease of 3.12 (P<0.001) and 3.64
(P<0.001), respectively, as compared with patients in the lowest
quartile. The combined effect of hypercholesterolemia and being in
the highest quartiles of the oxidized phospholipid:apo B-100 ratio
(odds ratio, 16.8; P<0.001) and Lp(a) lipoprotein levels (odds
ratio, 14.2; P<0.001) significantly increased the probability of
coronary artery disease among patients 60 years of age or younger.
In the entire study group, the association of the oxidized
phospholipid:apo B-100 ratio with obstructive coronary artery
disease was independent of all clinical and lipid measures except
one, Lp(a) lipoprotein. However, among patients 60 years of age or
younger, the oxidized phospholipid:apo B-100 ratio remained an
independent predictor of coronary artery disease.
[0122] Circulating levels of oxidized LDL are strongly associated
with angiographically documented coronary artery disease,
particularly in patients 60 years of age or younger. These data
suggest that the atherogenicity of Lp(a) lipoprotein may be
mediated in part by associated proinflammatory oxidized
phospholipids.
[0123] Human coronary atherosclerosis is a chronic inflammatory
disease that is superimposed on a background of lipid
abnormalities. Proinflammatory oxidized low-density lipoprotein
(LDL) may be a unifying link between lipid accumulation and
inflammation in the vessel wall. In humans, oxidized LDL in plasma
and within atherosclerotic lesions is strongly associated with
coronary artery disease, acute coronary syndromes, and vulnerable
plaques.
[0124] Lp(a) lipoprotein is a lipoprotein of unknown physiologic
function that is composed of apolipoprotein B-100 (apo B-100) to
which apolipoprotein(a) is covalently bound. Increased plasma
levels of Lp(a) lipoprotein are independent predictors of the
presence of angiographically documented and clinical coronary
artery disease, particularly in patients with hypercholesterolemia.
However, the underlying mechanisms by which Lp(a) lipoprotein
contributes to the pathogenesis of atherosclerosis are not well
understood. We recently showed that proinflammatory oxidized
phospholipids are strongly associated with Lp(a) lipoprotein in
human plasma. Therefore, we hypothesized that the presence of
oxidized phospholipids on apo B-100--containing lipoproteins may
explain some of the atherogenic properties of Lp(a) lipoprotein,
and we designed this study to evaluate the relationship between
circulating oxidized LDL, Lp(a) lipoprotein, and angiographically
documented coronary artery disease.
[0125] We designed the current study on the basis of a previous
study in which we had enrolled a total of 504 consecutive patients
(97.2 percent of whom were white), 18 to 75 years of age, who were
undergoing clinically indicated coronary angiography at the Mayo
Clinic between June 1998 and December 1998.10 Race was
self-reported. The exclusion criteria, which have been described
previously, included prior coronary revascularization and the
presence of diabetes mellitus. 10 Arterial plasma samples were
obtained from the femoral sheath before angiography and were placed
in tubes containing EDTA and frozen at 70.degree. C. until the
analyses were performed. Hypercholesterolemia was defined as a
total cholesterol level of at least 250 mg per deciliter (6.5 mmol
per liter), an LDL level of at least 150 mg per deciliter (3.9 mmol
per liter), or ongoing treatment with lipid-lowering agents. The
study was approved by the Mayo Clinic institutional review board,
and all patients gave written informed consent. See Sotirios et
al., "Oxidized Phospholipids, Lp(a) Lipoprotein, and Coronary
Artery Disease," N Engl J Med 2005; 353:46-57, which is
incorporated by herein by reference in its entirety.
[0126] The maximal stenosis in each of 27 coronary-artery segments
was assessed by a cardiologist, who was unaware of risk factors,
with the use of handheld calipers or in visual analysis according
to the segmental classification system of the Coronary Artery
Surgery Study. The extent of angiographically documented coronary
artery disease was quantified as follows: normal coronary arteries
(smooth, with either no stenosis or a stenosis of <10 percent of
the luminal diameter), mild disease (a stenosis of 10 to 50 percent
of the luminal diameter in one or more coronary arteries or their
major branches), or onevessel, two-vessel, or three-vessel disease,
defined as a stenosis of more than 50 percent of the luminal
diameter in one, two, or three coronary arteries or their major
branches.
[0127] Analyses of apo B-100, Lp(a) lipoprotein, total cholesterol,
high-density lipoprotein (HDL) cholesterol, and triglycerides were
performed with the use of commercially available kits. LDL
cholesterol was estimated with the use of the Friedewald formula.
High-sensitivity C-reactive protein (CRP) (lower limit of
detection, 0.15 mg per liter) was measured as described
elsewhere.
[0128] Our assay of oxidized LDL determines the content of oxidized
phospholipids per particle of apo B-100 (oxidized phospholipid:apo
B-100 ratio) and is performed with the use of the murine monoclonal
antibody E06, which specifically binds to the phosphorylcholine
moiety of oxidized but not native phospholipids. We have previously
used the term OxLDL-E06 to describe the name of this assay. In
brief, a dilution of plasma at 1:50 in phosphate-buffered saline
was added to microtiter wells coated with monoclonal antibody MB47,
which specifically binds apo B-100 particles. Under these
conditions, a saturating amount of apo B-100 was added to each
well, and consequently, equal numbers of apo B-100 particles were
captured in each well for all assays. The oxidized phospholipid:apo
B-100 ratio was measured by chemiluminescent enzyme-linked
immunosorbent assay with the use of biotinylated E06, as described
elsewhere.
[0129] Discrete data are presented as frequencies and percentages,
and continuous variables as means and standard deviations or as
medians and interquartile ranges if the distributions were skewed.
Spearman's correlation coefficient was used to measure the linear
associations between the rank values of the oxidized
phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels as well
as lipid levels and other clinical risk factors. The association of
the oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein
levels with the extent of coronary artery disease was tested by
one-way analysis of variance of the log-transformed values followed
by a one-degree-of-freedom test for trend. The percentages of
patients with obstructive coronary artery disease and the odds
ratios were calculated for quartiles of the oxidized
phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels for all
patients, according to age (.ltoreq.60 years or >60 years), and
according to the presence or absence of hypercholesterolemia.
[0130] Logistic-regression models were used to estimate the
associations between patients' characteristics and lipid
measurements and obstructive coronary artery disease. Multiple
logistic-regression analysis was used to estimate the partial
associations between the oxidized phospholipid:apo B-100 ratio and
Lp(a) lipoprotein levels and obstructive coronary artery disease,
with adjustment for age, sex, smoking status, the presence or
absence of hypertension, and levels of LDL cholesterol, HDL
cholesterol, triglycerides, and CRP. The base-2 logarithms (log 2)
of the oxidized phospholipid:apo B-100 ratio and the levels of
Lp(a) lipoprotein, triglycerides, and CRP were used in all the
logistic regression models to account for skewness in the
distributions. Thus, odds ratios for these variables reflect the
change in odds for an increase of 1 log 2 (the equivalent of a
doubling of the value) in the measure.
[0131] The baseline clinical characteristics of the patients,
indications for coronary angiography, lipid measurements, and CRP
levels are shown in Table 3. The distributions of both the oxidized
phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels were
skewed toward lower values, with 85 percent of the patients having
levels lower than 0.4 and 45 mg per deciliter, respectively (FIG.
6). In the entire population, a strong correlation (r=0.83,
P<0.001) was noted between the oxidized phospholipid:apo B-100
ratio and Lp(a) lipoprotein levels.
[0132] Association with the extent of angiographically documented
disease: In the entire study group, the oxidized phospholipid:apo
B-100 ratio and Lp(a) lipoprotein levels were strongly associated
with a graded increase in the extent of coronary artery disease
(P<0.001 for both analyses) (data not shown). These
relationships were markedly stronger for patients 60 years of age
or younger than for patients older than 60 years (FIG. 7).
[0133] Association with obstructive coronary artery disease: The
proportion of patients with obstructive coronary artery disease
increased consistently with increases in the oxidized
phospholipid:apo B-100 ratio and in Lp(a) lipoprotein levels (Table
4). This association was particularly evident among patients 60
years of age or younger, among whom the highest quartiles of the
oxidized phospholipid:apoB-100 ratio (odds ratio, 3.12; P<0.001)
and Lp(a) lipoprotein levels (odds ratio, 3.64, P<0.001) were
associated with a significantly higher risk, as compared with the
lowest quartiles. This association was not present among patients
older than 60 years.
[0134] The combined effects of hypercholesterolemia plus either the
oxidized phospholipid:apo B-100 ratio or Lp(a) lipoprotein levels
greatly increased the probability of obstructive coronary artery
disease. When compared with patients in the lowest quartile who did
not have hypercholesterolemia, patients in the highest quartile of
the oxidized phospholipid:apo B-100 ratio or Lp(a) lipoprotein
levels who had hypercholesterolemia were significantly more likely
to have obstructive coronary artery disease (Table 5). These
relationships were markedly accentuated among patients 60 years of
age or younger (for the oxidized phospholipid:apo B-100 ratio, odds
ratio, 16.8 [P<0.001]; for Lp(a) lipoprotein levels, odds ratio,
14.2 [P<0.001]), as compared with those older than 60 years (for
the oxidized phospholipid:apo B-100 ratio, odds ratio, 4.95
[P=0.003]; for Lp(a) lipoprotein levels, odds ratio, 4.92
[P=0.007]).
[0135] The relationship of the oxidized phospholipid:apo B-100
ratio and Lp(a) lipoprotein levels to coronary artery disease
remained fundamentally similar after the exclusion from analysis of
41 patient with acute myocardial infarction within six weeks before
enrollment. Also, there was a stronger association between the
oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels
and coronary artery disease in patients with hypercholesterolemia
who were taking statins than among such patients who were not
taking statins, but differences in the odds ratios were not
statistically significant.
[0136] Predictors of obstructive coronary artery disease: Without
adjustment for other risk factors, the oxidized phospholipid:apo
B-100 ratio was predictive of obstructive coronary artery disease
(odds ratio per doubling, 1.19; 95 percent confidence interval,
1.05 to 1.34; P=0.005) as was the Lp(a) lipoprotein level (odds
ratio per doubling, 1.22; 95 percent confidence interval, 1.07 to
1.40; P=0.003). Similarly, male sex (odds ratio, 4.33; 95 percent
confidence interval, 2.95 to 6.35; P<0.001), age (odds ratio per
decade, 1.48; 95 percent confidence interval, 1.25 to 1.75;
P<0.001), current smoking (odds ratio, 1.65; 95 percent
confidence interval, 1.16 to 2.35; P=0.006), hypertension (odds
ratio, 1.81; 95 percent confidence interval, 1.27 to 2.58;
P=0.001), LDL cholesterol (odds ratio per increase of 25 mg per
deciliter [0.65 mmol per liter], odds ratio, 1.28; 95 percent
confidence interval, 1.12 to 1.45; P=0.003), and triglyceride
levels (odds ratio per doubling, 1.27; 95 percent confidence
interval, 1.00 to 1.61; P=0.05) were also predictive, whereas HDL
cholesterol (odds ratio per increase of 10 mg per deciliter [2.3
mmol per liter], 0.64; 95 percent confidence interval, 0.56 to
0.74; P<0.001) was a negative predictor. CRP (odds ratio per
doubling, 1.08; 95 percent confidence interval, 0.98 to 1.19;
P=0.12) was not a predictor of obstructive coronary artery
disease.
[0137] Among patients 60 years of age or younger, the odds ratios
per doubling for the oxidized phospholipid:apo B-100 ratio (1.43;
95 percent confidence interval, 1.20 to 1.71; P<0.001) and Lp(a)
lipoprotein level (1.41; 95 percent confidence interval, 1.16 to
1.73; P<0.001) were significant, whereas among those older than
60 years they were no longer significant (for the oxidized
phospholipid:apo B-100 ratio: odds ratio per doubling, 1.05; 95
percent confidence interval, 0.89 to 1.25; P=0.58; and for Lp[a]
lipoprotein levels: odds ratio per doubling, 1.09; 95 percent
confidence interval, 0.90 to 1.32; P=0.37).
[0138] Multivariable analysis with the use of logistic regression
models to derive adjusted odds ratios for coronary artery disease
showed that an increase in the oxidized phospholipid:apo B-100
ratio (odds ratio per doubling, 1.21; 95 percent confidence
interval, 1.05 to 1.39; P=0.007) was an independent predictor of
obstructive coronary artery disease, as were male sex (odds ratio,
4.27; 95 percent confidence interval, 2.59 to 7.03; P<0.001),
age (odds ratio per decade, 1.72; 95 percent confidence interval,
1.41 to 2.10; P<0.001), an increase in LDL cholesterol (odds
ratio per 25 mg per deciliter, 1.28; 95 percent confidence
interval, 1.11 to 1.48; P<0.001), and hypertension (odds ratio,
1.67; 95 percent confidence interval, 1.10 to 2.52; P=0.016),
whereas an increase in HDL cholesterol levels (odds ratio per 10 mg
per deciliter, 0.75; 95 percent confidence interval, 0.63 to 0.90;
P=0.002) was a negative predictor. An increase in CRP (odds ratio
per doubling, 1.09; 95 percent confidence interval, 0.97 to 1.22;
P=0.16) was not a predictor of obstructive coronary artery disease.
When Lp(a) lipoprotein was added to the model and the oxidized
phospholipid:apo B-100 ratio was removed, Lp(a) lipoprotein was
also an independent predictor (odds ratio per doubling, 1.20; 95
percent confidence interval, 1.02 to 1.40; P=0.02). As in the
unadjusted data, the odds ratios per doubling for the oxidized
phospholipid:apo B-100 ratio (1.49; 95 percent confidence interval,
1.20 to 1.84; P<0.001) and for Lp(a) lipoprotein (1.42; 95
percent confidence interval, 1.12 to 1.81; P=0.004) among patients
60 years of age or younger were significantly accentuated, whereas
among those older than 60 years they were no longer significant
(for the oxidized phospholipid:apo B-100 ratio: odds ratio per
doubling, 1.00; 95 percent confidence interval, 0.82 to 1.22;
P=0.96; for Lp(a) lipoprotein: odds ratio per doubling, 1.05; 95
percent confidence interval, 0.84 to 1.31; P=0.69).
[0139] Interestingly, in the entire study group, when Lp(a)
lipoprotein was forced into the model with the oxidized
phospholipid:apo B-100 ratio, there was a trend toward significance
of the oxidized phospholipid:apo B-100 ratio (odds ratio per
doubling, 1.21; 95 percent confidence interval, 0.95 to 1.54;
P=0.12), whereas Lp(a) lipoprotein levels no longer remained an
independent predictor of coronary artery disease (odds ratio per
doubling, 1.00; 95 percent confidence interval, 0.76 to 1.32;
P=0.99). However, when patients were analyzed according to age, the
oxidized phospholipid:apo B-100 ratio, but not Lp(a) lipoprotein
levels, was an independent predictor of obstructive coronary artery
disease among those 60 years of age or younger, but not among those
older than 60 years (FIG. 8). CRP was also a predictor of
obstructive coronary artery disease among patients 60 years of age
or younger, but not among those older than 60 years. When the 41
patients with acute myocardial infarction, who also had the highest
levels of CRP, were removed from the analysis, CRP was no longer a
predictor of obstructive coronary artery disease (odds ratio per
doubling, 1.06; 95 percent confidence interval, 0.85 to 1.33;
P=0.58), but the oxidized phospholipid:apo B-100 ratio (odds ratio
per doubling, 1.55; 95 percent confidence interval, 1.05 to 2.27;
P=0.03) remained a significant predictor. When the data were
evaluated according to the absence of coronary artery disease, as
compared with the presence of any coronary artery disease, the odds
ratios were slightly smaller, but in general, the trends described
were maintained, so that younger patients had higher odds ratios
than older patients.
[0140] Correlations between oxidized LDL levels and other
biomarkers: Levels of LDL cholesterol were weakly associated with
levels of Lp(a) lipoprotein (r=0.17, P<0.001), and with the
oxidized phospholipid:apo B-100 ratio (r=0.09, P=0.05). CRP levels
correlated weakly with LDL cholesterol levels (r=0.10, P=0.02) and
triglyceride levels (r=0.11, P=0.01). There were no significant
correlations between the oxidized phospholipid: apo B-100 ratio or
Lp(a) lipoprotein levels and CRP levels, age, body-mass index,
blood pressure, and serum creatinine level.
[0141] This study shows an association between the oxidized
phospholipid:apo B-100 ratio in plasma and the presence and extent
of angiographically documented coronary artery disease. The
association is independent of all clinical and lipid-related risk
factors, except one, Lp(a) lipoprotein, which also has a strong
association with angiographically documented coronary artery
disease. The odds ratios for angiographically documented coronary
artery disease associated with the Lp(a) lipoprotein level were
nearly identical with those associated with the oxidized
phospholipid:apo B-100 ratio. However, among patients younger than
60 years of age, the oxidized phospholipid:apo B-100 ratio remained
an independent predictor of obstructive coronary artery disease.
There was a strong correlation between levels of Lp(a) lipoprotein
and the oxidized phospholipid:apo B-100 ratio. These observations,
in conjunction with previous studies from our laboratory showing
that in plasma such oxidized phospholipids are predominantly
physically present on Lp(a) lipoprotein, 5-7,9 as opposed to other
lipoproteins, lend strong support to the hypothesis that, in the
setting of enhanced oxidative stress, proinflammatory.
[0142] Oxidized phospholipids may, in part, mediate the
atherogenicity of Lp(a) lipoprotein. The natural murine monoclonal
IgM autoantibody E06, cloned from apolipoprotein E
receptor--deficient mice is functionally identical with classic
natural T15 murine antibodies that bind phosphorylcholine on the
cell-wall polysaccharide of pathogens such as pneumococcus and
provide optimal protection from pneumococcal infections.13 In
vitro, E06 binds to and prevents the uptake of oxidized LDL and
apoptotic cells by scavenger receptors of macrophages. Binder et
al. have also shown that the immunization of mice with
Streptococcus pneumoniae results in increased titers of IgM
oxidized LDL autoantibodies and reduction in the progression of
atherosclerosis. These observations suggest that seemingly
unrelated proatherogenic processes, such as oxidation, apoptosis,
and infection, share molecular mimicry of the phosphorylcholine
epitopes found on proinflammatory oxidized phospholipids.
[0143] Although previous studies have shown that plasma oxidized
LDL levels are elevated in patients with clinically manifest stable
coronary artery disease and acute coronary syndromes our study
shows that oxidized phospholipids present on particles of apo B-100
and primarily on Lp(a) lipoprotein correlate with both the presence
and extent of angiographically documented coronary artery disease.
Although most of the oxidized LDL is present within the vessel
wall, this study suggests that the small amounts of minimally
modified LDL (e.g., particles of apo B-100 that contain oxidized
phospholipids) are present in the circulation. This finding is also
consistent with previous studies from our laboratory showing that
the oxidized phospholipid:apo B-100 ratio (with oxidized LDL
measured with use of antibody E06) rises abruptly after acute
coronary events and immediately after percutaneous coronary
intervention--situations in which the release of oxidized
phospholipids (or oxidized LDL, or both) from the vessel wall might
be postulated.
[0144] A potential pathophysiological relationship between levels
of oxidized phospholipids and Lp(a) lipoprotein is strongly
supported by this study and by data from earlier studies from our
laboratory showing that oxidized phospholipids are physically
associated with Lp(a) lipoprotein bound to lysine residues on
isolated fragments of kringle V of apolipoprotein(a) 9 and also in
the lipid phase of Lp(a) lipoprotein (unpublished data). In
addition, the kringle V fragments containing such oxidized
phospholipids induce inflammatory responses by up-regulating
secretion of interleukin-8 by cultured human macrophages.
[0145] In this study, we have shown that the predictive abilities
of levels of oxidized LDL and Lp(a) lipoprotein for obstructive
coronary artery disease are highly interdependent. In the entire
study group, when Lp(a) lipoprotein was excluded from the
multivariable analysis, the odds ratios for the oxidized
phospholipid:apo B-100 ratio were similar to those for traditional
risk factors such as age, hypertension, and LDL cholesterol.
Similarly, without the oxidized phospholipid:apo B-100 ratio in the
analysis, Lp(a) lipoprotein levels stood as an independent
predictor, as has been shown in a recent meta-analysis.8 In the
entire study group, with the oxidized phospholipid:apo B-100 ratio
in the model, there was no added ability of Lp(a) lipoprotein
levels to explain the risk of obstructive coronary artery disease,
suggesting that measures of oxidized LDL and Lp(a) lipoprotein
represent a common path of biologic influence on the risk for
coronary artery disease. However, in patients 60 years of age or
younger, the oxidized phospholipid:apo B-100 ratio maintained its
independent predictive power even with Lp(a) lipoprotein in the
model. This observation supports the hypothesis that much of the
risk attributable to Lp(a) lipoprotein levels can be explained by
the binding of oxidized phospholipids by Lp(a) lipoprotein, but
that in younger patients, an additional risk associated with
oxidized phospholipids may be present, perhaps through
proinflammatory pathways independent of Lp(a) lipoprotein.
[0146] The physiologic role of Lp(a) lipoprotein is unknown. We and
others have suggested that a potential physiologic role of Lp(a)
lipoprotein may be to bind and detoxify proinflammatory oxidized
phospholipids. Lp(a) lipoprotein, which is present only in humans
and Old World primates (although a partially related gene arose
separately in hedgehogs), may have evolved to provide protection
against various oxidative stressors. For example, Lp(a) lipoprotein
has been shown to be involved in wound healing and possibly in
preventing angiogenesis in tumor models, and elevated levels have
been noted in centenarians in a manner consistent with human
longevity.
[0147] Similarly, oxidized phospholipids are generated not only
during atherogenesis but also in inflammation and apoptosis, which
suggests that housekeeping functions involving the clearance of
such oxidized phospholipids may have evolved for maintaining
general health as well as vascular health. In this regard, Lp(a)
lipoprotein may act in a way similar to CRP, which Chang et al.
have shown also binds specifically to the phosphorylcholine moiety
of oxidized phospholipids and apoptotic cells. Indeed, we and
others have shown that Lp(a) lipoprotein acts as an acute-phase
reactant in patients with acute coronary syndromes. It has also
been reported to be highly enriched (higher by a factor of 7 than
LDL) in platelet-activating factor acetyl hydrolase an enzyme that
potentially could detoxify such oxidized phospholipids by removing
the oxidized fatty acid.
[0148] Thus, when present at low levels, Lp(a) lipoprotein may
serve a protective function by binding and participating in the
transfer and possible degradation of oxidized phospholipids formed
during normal homeostasis or in acutely stressful situations.
However, when Lp(a) lipoprotein levels are chronically elevated (as
determined genetically), especially in a milieu of chronically
increased oxidative stress, Lp(a)\ lipoprotein, with its content of
oxidized phospholipids, may be proatherogenic, particularly since
it has enhanced binding to the extracellular matrix of the artery
wall.
[0149] The association between the oxidized phospholipid:apo B-100
ratio and angiographically documented coronary artery disease in
our study was much stronger for patients 60 years of age or younger
than for older patients. The reasons for this association are not
entirely clear, but many previous studies have documented a
strikingly similar relationship between Lp(a) lipoprotein levels
and angiographically documented disease among younger patients
only. By excluding patients with diabetes and previous coronary
revascularization from our study, we may have preferentially
enriched the study group with younger patients with fewer
traditional risk factors. In addition, increasing age, which is a
surrogate for known and unknown risk factors, is itself one of the
strongest risk factors for coronary artery disease. Thus, the
independent effects of oxidized LDL and Lp(a) lipoprotein levels
appear to diminish with age, presumably because of the cumulative
contributions of additional risk factors that affect the clinical
expression of atherosclerosis.
[0150] The limitations of this study include the fact that
angiography is not a precise method for quantifying
atherosclerosis. In addition, we have not yet defined the exact
oxidized phospholipids, their physical location within Lp(a)
lipoprotein, or the rates of flux, binding, and removal of oxidized
phospholipids that are on Lp(a) lipoprotein. We have documented
that plasma levels of oxidized phospholipids present on apo
B-100--containing lipoproteins and predominantly on Lp(a)
lipoprotein reflect the presence and extent of angiographically
documented coronary artery disease. We propose that in settings of
enhanced oxidative stress and elevated Lp(a) lipoprotein levels, a
proinflammatory milieu may predominate that contributes to the
clinical expression of cardiovascular disease.
[0151] The examples set forth above are provided to give those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the preferred embodiments of the
compositions, and are not intended to limit the scope of what the
inventors regard as their invention. Modifications of the
above-described modes for carrying out the invention that are
obvious to persons of skill in the art are intended to be within
the scope of the following claims. All publications, patents, and
patent applications cited in this specification are incorporated
herein by reference as if each such publication, patent or patent
application were specifically and individually indicated to be
incorporated herein by reference.
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