U.S. patent application number 11/716996 was filed with the patent office on 2008-03-06 for soluble cd40l (cd 154) as a prognostic marker of atherosclerotic diseases.
This patent application is currently assigned to The Brigham and Women's Hospital, Inc.. Invention is credited to Peter Libby, Paul Ridker, Uwe Schonbeck.
Application Number | 20080058360 11/716996 |
Document ID | / |
Family ID | 23326383 |
Filed Date | 2008-03-06 |
United States Patent
Application |
20080058360 |
Kind Code |
A1 |
Schonbeck; Uwe ; et
al. |
March 6, 2008 |
Soluble CD40L (CD 154) as a prognostic marker of atherosclerotic
diseases
Abstract
This invention involves the new use of a diagnostic test to
determine the risk of atherosclerotic diseases such as myocardial
infarction and stroke, particularly among individuals with no signs
or symptoms of current disease and among nonsmokers. Further, this
invention involves the new use of a diagnostic test to assist
physicians in determining which individuals at risk will
preferentially benefit from certain treatments designed either to
prevent first or recurrent myocardial infarctions and strokes, or
to treat acute and chronic cardiovascular disorders. Methods for
treatment are also described.
Inventors: |
Schonbeck; Uwe; (Randolph,
MA) ; Ridker; Paul; (Chestnut Hill, MA) ;
Libby; Peter; (Boston, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
The Brigham and Women's Hospital,
Inc.
Boston
MA
02115
|
Family ID: |
23326383 |
Appl. No.: |
11/716996 |
Filed: |
March 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10288253 |
Nov 5, 2002 |
7189518 |
|
|
11716996 |
Mar 12, 2007 |
|
|
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60338841 |
Nov 5, 2001 |
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Current U.S.
Class: |
514/277 ; 436/86;
514/356; 514/415; 514/460; 514/568 |
Current CPC
Class: |
G01N 2800/32 20130101;
A61P 9/10 20180101; G01N 33/6893 20130101; G01N 2800/324 20130101;
A61P 3/06 20180101; G01N 2800/323 20130101; A61K 38/1709
20130101 |
Class at
Publication: |
514/277 ;
436/086; 514/356; 514/415; 514/460; 514/568 |
International
Class: |
A61K 31/19 20060101
A61K031/19; A61K 31/40 20060101 A61K031/40; A61K 31/435 20060101
A61K031/435; A61K 31/44 20060101 A61K031/44; A61P 9/10 20060101
A61P009/10; G01N 33/00 20060101 G01N033/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The work leading to the present invention was funded in part
by grant numbers HL-34636, HL-56985, HL-58755, HL-63293 from the
National Heart, Lung and Blood Institute. Accordingly, the United
States Government may have certain rights to this invention.
Claims
1.-11. (canceled)
12. A method for characterizing an individual's risk profile of
developing a future cardiovascular disorder associated with
atherosclerotic disease, comprising: obtaining a level of sCD40L in
the individual, comparing the sCD40L level to a first predetermined
value to establish a first risk value, obtaining a level of a
cholesterol or C-Reactive Protein (CRP) in the individual,
comparing the level of the cholesterol or C-Reactive Protein (CRP)
to a second predetermined value to establish a second risk value,
and characterizing the individual's risk profile of developing the
cardiovascular disorder based upon the combination of the first
risk value and the second risk value, wherein the combination of
the first risk value and second risk value establishes a third risk
value different from said first and second risk values.
13. The method of claim 12, wherein said individual is an
apparently healthy, non-smoking individual.
14.-17. (canceled)
18. The method of claim 12, wherein the cardiovascular disorder is
stroke.
19. The method of claim 12, wherein the cardiovascular disorder is
nonfatal myocardial infarction.
20. (canceled)
21. A method for evaluating the likelihood that an individual will
benefit from treatment with an agent for reducing the risk of a
cardiovascular disorder associated with atherosclerotic disease,
the agent selected from the group consisting of anti-inflammatory
agents, anti-thrombotic agents, anti-platelet agents, fibrinolytic
agents, lipid reducing agents, direct thrombin inhibitors, and
glycoprotein II b/IIIa receptor inhibitors comprising: obtaining a
level of sCD40L in the individual, and comparing the level of
sCD40L to a predetermined value, wherein the level of sCD40L in
comparison to the predetermined value is indicative of whether the
individual will benefit from treatment with said agents, and
characterizing whether the individual is likely to benefit from
said treatment based upon said comparison.
22. (canceled)
23. The method of claim 21, wherein said individual is an
apparently healthy; non-smoking individual.
24.-27. (canceled)
28. The method of claim 21, wherein the cardiovascular disorder is
stroke.
29. The method of claim 21, wherein the cardiovascular disorder is
myocardial infarction.
30. A method for treating a subject to reduce the risk of a
cardiovascular disorder, comprising: selecting and administering to
a subject who is known to have an above-normal level of sCD40L an
agent for reducing the risk of the cardiovascular disorder in an
amount effective to lower the risk of the subject developing a
future cardiovascular disorder, wherein the agent is an
anti-inflammatory agent, an antithrombotic agent, an anti-platelet
agent, a fibrinolytic agent, a lipid reducing agent, a direct
thrombin inhibitor, a glycoprotein IIb/IIIa receptor inhibitor, an
agent that binds to cellular adhesion molecules and inhibits the
ability of white blood cells to attach to such molecules, a calcium
channel blocker, a beta-adrenergic receptor blocker, a
cyclooxygenase-2 inhibitor, or an angiotensin system inhibitor.
31. The method of claim 30, wherein the subject is otherwise free
of symptoms calling for treatment with the agent.
32. The method of claim 30, wherein the subject is apparently
healthy and the subject does not have an elevated risk of an
adverse cardiovascular event.
33. The method of claim 30, wherein the subject is
nonhyperlipidemic.
34. The method of claim 30, wherein the agent is a non-aspirin,
anti-inflammatory agent.
35.-38. (canceled)
39. The method of claim 30, wherein the agent is a lipid reducing
agent.
40. The method of claim 39, wherein the lipid reducing agent is
gemfibrozil, cholystyramine, colestipol, nicotinic acid, probucol
lovastatin, fluvastatin, simvastatin, atorvastatin, pravastatin, or
cerivastatin.
41.-70. (canceled)
71. A method for reducing sCD40L levels in a subject to lower the
risk of an adverse cardiovascular disorder, comprising: selecting
and administering to a subject having elevated levels of sCD40L an
agent that reduces sCD40L levels in an amount effective to reduce
the sCD40L levels in the subject.
72. The method of claim 71, wherein the agent is a lipid lowering
agent.
73. The method of claim 71, wherein the subject is apparently
healthy.
74. The method of claim 71, wherein the subject is not otherwise at
an elevated risk of having an adverse cardiovascular event.
75. The method of claim 71, wherein the subject has elevated
C-Reactive Protein (CRP) levels.
76. The method of claim 71, wherein the subject is otherwise free
of indications calling for treatment with a lipid reducing agent.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) from U.S. Provisional Application Ser. No. 60/338,841,
filed Nov. 5, 2001, and entitled: SOLUBLE CD40L(CD154) AS A
PROGNOSTIC MARKER OF ATHEROSCLEROTIC DISEASES incorporated herein
in its entirety by reference.
FIELD OF THE INVENTION
[0003] This invention describes the new use of a diagnostic test to
determine the risk of cardiovascular disorders, such as myocardial
infarction and stroke, particularly among individuals with no signs
or symptoms of current disease and among nonsmokers. Further, this
invention describes the new use of a diagnostic test to assist
physicians in determining which individuals at risk will
preferentially benefit from certain treatments designed to prevent
or treat cardiovascular disorders. Methods for treatment also are
described.
BACKGROUND OF THE INVENTION
[0004] Despite significant advances in therapy, cardiovascular
disease remains the single most common cause of morbidity and
mortality in the developed world. Thus, prevention of
cardiovascular disorders such as myocardial infarction and stroke
is an area of major public health importance. Currently, several
risk factors for future cardiovascular disorders have been
described and are in wide clinical use in the detection of
individuals at high risk. Such screening tests include evaluations
of total and HDL cholesterol levels. However, a large number of
cardiovascular disorders occur in individuals with apparently low
to moderate risk profiles, and our ability to identify such
patients is limited. Moreover, accumulating data suggests that the
beneficial effects of certain preventive and therapeutic treatments
for patients at risk for or known to have cardiovascular disorders
differs in magnitude among different patient groups. At this time,
however, data describing diagnostic tests to determine whether
certain therapies can be expected to be more or less effective are
lacking.
[0005] Certain cardiovascular disorders, such as myocardial
infarction and ischemic stroke, are associated with
atherosclerosis. The mechanism of atherosclerosis is not well
understood. CD40 ligand (also known as CD40L, CD154, and/or gp39)
is a 261 amino acid, type II transmembrane protein. One or more
biologically active soluble forms of the molecule, collectively
designated sCD40L, are produced by proteolytic cleavage of the
full-length form, which may occur intracellularly or on the cell
surface.
[0006] CD40L is a multipotent immunomodulator that together with
its receptor, CD40, are expressed on a broad variety of cells
including vascular endothelial (EC) and smooth muscle cells (SMC),
mononuclear phagocytes (MO), and platelets.
[0007] Engagement of the CD40 receptor on any of the foregoing cell
types, reportedly triggers the expression of various
pro-inflammatory mediators, such as the cytokines IL1, IL-6, IL-12,
TNF.alpha., or IFN.gamma., the chemokines IL-8, MCP-1, or RANTES,
the adhesion molecules ICAM-1 or VCAM-1, the matrix
metalloproteinases MMP-1/-2/-3/-7/-8/-9/-10/-11/-12/-13, as well as
the procoagulant tissue factor. Expression of these
pro-inflammatory mediators has been, reportedly, linked to the
promotion of a wide array of pro-atherogenic functions in vitro.
These observations have implicated CD40L in the various stages of
atherogenesis.
[0008] Elevated levels of sCD40L have been described among patients
with unstable angina. Further, concentrations of sCD40L in serum or
other body fluids have been used to assess the immune,
inflammatory, or malignant status of human patients. Such patients
include patients suffering from systemic autoimmunity or
inflammation, vascular diseases, viral diseases, or malignancies,
or patients undergoing immunosuppressive therapy. These patients
are not healthy individuals. Since levels of sCD40L increase during
inflammation, it has been uncertain whether statistical
associations observed in these prior studies of acutely ill or
high-risk populations are causal, are due to short-term
inflammatory changes or are due to interrelations with other risk
factors, in particular, smoking and hyperlipidemia.
[0009] Elevated levels of markers of inflammation have been shown
previously to be predictive of future adverse cardiovascular
disorders. This has not previously been demonstrated for sCD40L, a
mediator of certain aspects of inflammation although not
conventionally regarded previously as a systemic marker of
inflammation.
SUMMARY OF THE INVENTION
[0010] This invention describes in one aspect new diagnostic tests
which broadly include (1) the prediction of risk of future
cardiovascular disorders such as myocardial infarction and stroke
and peripheral arterial disease; and (2) the determination of the
likelihood that certain individuals will benefit to a greater or
lesser extent from the use of certain treatments designed to
prevent and/or treat cardiovascular disorders. These new tests are
based in part upon the following discoveries.
[0011] It has been discovered that elevated levels of sCD40L are
predictive of future cardiovascular disorders. For example,
elevated levels of sCD40L in apparently healthy, nonsmokers are
predictive of an increased risk of myocardial infarction. As
another example, elevated levels of sCD40L are predictive of an
increased likelihood of a future stroke.
[0012] It has been discovered also that the likelihood that certain
individuals will benefit to a greater or a lesser extent from the
use of certain therapeutic agents for reducing the risk of a future
cardiovascular disorder can be determined from the base-line level
sCD40L in an individual. The invention is based in part on the
surprising discovery that that sCD40L has a predictive value
independent of other predictors of future cardiovascular disorders.
In particular, sCD40L predicts future adverse cardiovascular
disorders independent of the systemic inflammatory marker
C-Reactive Protein (CRP). Thus, sCD40L may be used alone as a
predictor future adverse cardiovascular disorders or in combination
with prior art predictors such as cholesterol and CRP. Thus, the
present invention does not involve simply duplicating a measurement
that previously could be made using other predictors. Instead,
levels of sCD40L are additive to prior art predictors.
[0013] As mentioned above, these discoveries have led to new
diagnostic tests.
[0014] According to one aspect of the invention, a method is
provided for characterizing an individual's risk profile of
developing a future cardiovascular disorder. The method involves
obtaining a level of sCD40L in the individual. The level of sCD40L
then is compared to a predetermined value, and the individual's
risk profile of developing a future cardiovascular disorder then is
characterized based upon the level of sCD40L in comparison to the
predetermined value.
[0015] The predetermined value can be a single value, multiple
values, a single range or multiple ranges. Thus, in one embodiment,
the predetermined value is a plurality of predetermined marker
level ranges, and the comparing step comprises determining in which
of the predetermined marker level ranges the individual's level
falls. In preferred embodiments, a preferred predetermined sCD40L
value is about or above 2.9 ng/mL of blood. Another preferred
predetermined sCD40L value is about or above 3.2 ng/mL of blood. A
further preferred predetermined sCD40L value is about or above 5.5
ng/mL of blood. When ranges are employed, it is preferred that one
of the plurality of ranges be below about 2.9 ng/mL of blood and
that another of the ranges be above about 2.9 ng/mL of blood.
[0016] In certain embodiments the individual is an apparently
healthy, non-smoking individual. In some embodiments, the
individual is not otherwise at an elevated risk of a myocardial
infarction or stroke.
[0017] In some embodiments of this aspect of the invention, the
cardiovascular disorder is associated with atherosclerotic disease.
In some embodiments, the cardiovascular disorder is other than
fatal myocardial infarction. In some embodiments, the
cardiovascular disorder is a stroke.
[0018] According to still another aspect of the invention, a method
is provided for characterizing an individual's risk profile of
developing a future cardiovascular disorder associated with
atherosclerotic disease. A level of sCD40L in the individual is
obtained. The level of sCD40L is compared to a predetermined value.
The individual's risk profile of developing the future
cardiovascular disorder associated with atherosclerotic disease,
then is characterized based upon the level of sCD40L in comparison
to the predetermined value. The predetermined value can be as
described above. The individual characterized may be any
individual, but preferably is an apparently healthy individual. The
apparently healthy individual can be a smoker or a nonsmoker. In
certain embodiments the subject does not otherwise have an elevated
risk of an adverse cardiovascular disorder. In certain embodiments,
the future cardiovascular disorder associated with atherosclerotic
disease does not include a fatal myocardial infarction. The
preferred markers and predetermined values are as described above.
In one important embodiment, the cardiovascular disorder is stroke.
In another important embodiment, the cardiovascular disorder is
myocardial infarction. In another important embodiment, the
cardiovascular disorder is peripheral artery disease. In a further
important embodiment, the cardiovascular disorder is non-fatal
myocardial infarction.
[0019] According to another aspect of the invention, a method is
provided for characterizing an apparently healthy, non-smoking
individual's risk profile of developing a future myocardial
infarction. The method involves obtaining a level of sCD40L in the
individual. The level of sCD40L then is compared to a predetermined
value, and the individual's risk profile of developing a future
myocardial infarction then is characterized based upon the level of
sCD40L in comparison to the predetermined value. In certain
embodiments, the individual does not otherwise have an elevated
risk of an adverse cardiovascular event.
[0020] As in the previous aspect of the invention, the
predetermined value may be a single value, a plurality of values, a
single range or a plurality of ranges. In one embodiment, the
predetermined value is a plurality of predetermined marker level
ranges and the comparing step involves determining in which of the
predetermined marker level ranges the individual's level falls.
Preferred predetermined values and the like for sCD40L are as
described above.
[0021] According to another aspect of the invention, a method is
provided for characterizing an individual's risk profile of
developing a future cardiovascular disorder associated with
atherosclerotic disease. A level of sCD40L in the individual is
obtained. The level of sCD40L is compared to a predetermined value.
The individual's risk profile of developing the future
cardiovascular disorder associated with atherosclerotic disease,
then is characterized based upon the level of sCD40L in comparison
to the predetermined value. As in the previous aspect of the
invention, the predetermined value may be a single value, a
plurality of values, a single range or a plurality of ranges. In
one embodiment, the predetermined value is a plurality of
predetermined marker level ranges and the comparing step involves
determining in which of the predetermined marker level ranges the
individual's level falls. Preferred predetermined values and the
like for sCD40L are as described above.
[0022] The individual characterized may be any individual, but
preferably is an apparently healthy individual. The apparently
healthy individual can be a smoker or a nonsmoker. In certain
embodiments the subject does not otherwise have an elevated risk of
an adverse cardiovascular event. In certain embodiments, the future
cardiovascular disorder associated with atherosclerotic disease
does not include a fatal myocardial infarction. The preferred
markers and predetermined values are as described above. In one
important embodiment, the cardiovascular disorder is stroke. In
another important embodiment, the cardiovascular disorder is
myocardial infarction. In another important embodiment, the
cardiovascular disorder is peripheral artery disease. In a further
important embodiment, the cardiovascular disorder is non-fatal
myocardial infarction.
[0023] According to still another aspect of the invention, a method
is provided in which one uses a blood sCD40L level together with a
cholesterol fraction or C-Reactive Protein (CRP) for characterizing
an individual's risk profile of developing a future cardiovascular
disorder associated with atherosclerotic disease. A level of sCD40L
in the individual is obtained. The level of the sCD40L is compared
to a first predetermined value to establish a first risk value. A
level of a cholesterol or CRP in the individual also is obtained.
The level of the cholesterol or CRP in the individual is compared
to a second predetermined value to establish a second risk value.
The individual's risk profile of developing the cardiovascular
disorder then is characterized based upon the combination of the
first risk value and the second risk value, wherein the combination
of the first risk value and second risk value establishes a third
risk value different from the first and second risk values. In
particularly important embodiments, the third risk value is greater
than either of the first and second risk values. The preferred
individuals for testing, markers and predetermined values are as
described above. The cardiovascular disorder can be any
cardiovascular disorder associated with atherosclerotic disease,
although in certain important embodiments the cardiovascular
disorder is nonfatal myocardial infarction or ischemic stroke
[0024] According to yet another aspect of the invention, a method
is provided for evaluating the likelihood that an individual will
benefit from treatment with an agent for reducing the risk of a
cardiovascular disorder, and particularly cardiovascular disorders
associated with atherosclerotic disease. The agent can be selected
from the group consisting of anti-inflammatory agents,
anti-thrombotic agents, anti-platelet agents, fibrinolytic agents,
lipid reducing agents, direct thrombin inhibitors, and glycoprotein
II b/IIIa receptor inhibitors and agents that bind to cellular
adhesion molecules and inhibit the ability of white blood cells to
attach to such molecules (e.g. anti-cellular adhesion molecule
antibodies). To practice the method, a level of sCD40L in an
individual is obtained. This level then is compared to a
predetermined value, wherein the level of sCD40L in comparison to
the predetermined value is indicative of the likelihood that the
individual will benefit from treatment with the agent. The
individual then can be characterized in terms of the net benefit
likely to be obtained by treatment with the agent.
[0025] As mentioned above, the invention is particularly adapted to
determining which individuals will preferentially benefit from
treatment with an agent for reducing the risk in the individuals of
a cardiovascular disorder such as a future stroke or a future
myocardial infarction, including nonfatal myocardial infarctions.
It also permits selection of candidate populations for clinical
trials and for treatment with candidate drugs, by identifying, for
example, the individuals most likely to benefit from a new
treatment or from a known treatment with a high risk profile of
adverse side effects. Thus, the invention provides information for
evaluating the likely net benefit of certain treatments for
candidate patients.
[0026] The invention also contemplates kits comprising a package
including an assay for sCD40L and instructions, and optionally
related materials such as number or color charts, for correlating
the level of sCD40L as determined by the assay with a risk of
developing a future cardiovascular disorder or with other patient
criteria as described above. In important embodiments, the kits
also include an assay for a cholesterol.
[0027] In another aspect of the invention, a method for treating a
subject to reduce the risk of a cardiovascular disorder, is
provided. The method involves selecting and administering to a
subject who is known to have an above-normal level of sCD40L an
agent for reducing the risk of the cardiovascular disorder. The
agent can be an anti-inflammatory agent (including aspirin and
nonaspirin anti-inflammatory agents), an antithrombotic agent, an
anti-platelet agent, a fibrinolytic agent, a lipid reducing agent,
a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptor
inhibitor, an agent that binds to cellular adhesion molecules and
inhibits the ability of white blood cells to attach to such
molecules, a calcium channel blocker, a beta-adrenergic receptor
blocker, a cyclooxygenase-2 inhibitor, an angiotensin system
inhibitor, and/or combinations thereof. The agent is administered
in an amount effective to lower the risk of the subject developing
a future cardiovascular disorder. The preferred subjects are
apparently healthy subjects otherwise free of current need for
treatment with any one or combination of the foregoing agents. In
further important embodiments, the subject treated is a
nonhyperlipidemic subject. In another embodiment, the subjects are
not at an elevated risk of an adverse cardiovascular event (e.g.,
subjects with no family history of such events, subjects who are
nonsmokers, subjects who are nonhyperlipidemic subjects with normal
levels of systemic inflammatory markers), other than having an
elevated level of sCD40L.
[0028] In certain embodiments, the agent is an anti-inflammatory
agent selected from the group consisting of Alclofenac;
Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase;
Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride;
Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium;
Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;
Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;
Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;
Cloticasone Propionate; Cormethasone Acetate; Cortodoxone;
Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate;
Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate;
Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl
Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;
Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;
Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;
Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide
Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl;
Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;
Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide;
Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen;
Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;
Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone
Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride;
Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium;
Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid;
Mesalamine; Meseclazone; Methylprednisolone Suleptanate;
Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol;
Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin;
Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate
Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam;
Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate;
Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate;
Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;
Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;
Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;
Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;
Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;
Triclonide; Triflumidate; Zidometacin; Glucocorticoids or Zomepirac
Sodium.
[0029] The invention also involves a method for treating subjects
with a lipid reducing agent, to prevent cardiovascular disorders.
Such an agent is administered to a subject selected on the basis of
having an above-normal level of sCD40L. The agent is administered
in an amount effective to lower the risk of the subject developing
a future cardiovascular disorder. In one embodiment, the subject
already has had a cardiovascular event, such as a heart attack or
an angioplasty. In this embodiment, the lipid reducing agent can
limit further injury or help prevent restenosis, post-myocardial
infarction or post-angioplasty injury. In another important
embodiment, the subjects are apparently healthy subjects otherwise
free of current need for lipid reducing agent treatment. In
important embodiments, the subjects are not an elevated risk of an
adverse cardiovascular event, other than having elevated levels of
sCD40L. In further important embodiments, the subject treated is a
nonhyperlipidemic subject. In any of the foregoing embodiments, the
lipid reducing agent may be, but is not limited to, gemfibrozil,
cholystyramine, colestipol, nicotinic acid, probucol lovastatin,
fluvastatin, simvastatin, atorvastatin, pravastatin, or
cerivastatin. In preferred embodiments, the lipid reducing agent is
pravastatin.
[0030] The invention also involves a method for treating subjects
with an agent that binds to a cellular adhesion molecule and that
inhibits the ability of white blood cells to attach to such
molecules, to prevent cardiovascular disorders. Such an agent is
administered to a subject selected on the basis of having an
above-normal level of sCD40L. The agent is administered in an
amount effective to lower the risk of the subject developing a
future cardiovascular disorder. In one embodiment, the subject
already has had a cardiovascular event, such as a heart attack or
an angioplasty. In this embodiment, the agent that binds to a
cellular adhesion molecule and that inhibits the ability of white
blood cells to attach to such molecules, may limit further injury
or help prevent restenosis, post-myocardial infarction or
post-angioplasty injury. In another important embodiment, the
subjects are apparently healthy subjects otherwise free of current
need for treatment with an agent that binds to a cellular adhesion
molecule and that inhibits the ability of white blood cells to
attach to such molecules. In important embodiments the subjects are
not an elevated risk of an adverse cardiovascular event, other than
having elevated levels of sCD40L. In further important embodiments,
the subject treated is a nonhyperlipidemic subject.
[0031] The invention also involves a method for treating subjects
with a calcium channel blocker, to prevent cardiovascular
disorders. Such an agent is administered to a subject selected on
the basis of having an above-normal level of sCD40L. The agent is
administered in an amount effective to lower the risk of the
subject developing a future cardiovascular disorder. In one
embodiment, the subject already has had a cardiovascular event,
such as a heart attack or an angioplasty. In another important
embodiment, the subjects are apparently healthy subjects otherwise
free of current need for calcium channel blocker treatment. In
important embodiments the subjects are not an elevated risk of an
adverse cardiovascular event, other than having elevated levels of
sCD40L. In further important embodiments, the subject treated is a
nonhyperlipidemic subject. In any of the foregoing embodiments, the
calcium channel blocker may be but is not limited to,
dihydropyridines, phenyl alkyl amines, and/or benzothiazepines. In
preferred embodiments, calcium channel blockers useful according to
the invention, include, but are not limited to, amrinone,
amlodipine, bencyclane, diltiazem, felodipine, fendiline,
flunarizine, isradipine, nicardipine, nifedipine, nimodipine,
perhexilene, gallopamil, tiapamil and tiapamil analogues (such as
1993RO-11-2933), verapamil, phenytoin, barbiturates, and the
peptides dynorphin, omega-conotoxin, and omega-agatoxin, and the
like and/or pharmaceutically acceptable salts thereof.
[0032] The invention also involves a method for treating subjects
with a beta-adrenergic receptor blocker, to prevent cardiovascular
disorders. Such an agent is administered to a subject selected on
the basis of having an above-normal level of sCD40L. The agent is
administered in an amount effective to lower the risk of the
subject developing a future cardiovascular disorder. In one
embodiment, the subject already has had a cardiovascular event,
such as a heart attack or an angioplasty. In another important
embodiment, the subjects are apparently healthy subjects otherwise
free of current need for beta-adrenergic receptor blocker
treatment. In important embodiments the subjects are not an
elevated risk of an adverse cardiovascular event, other than having
elevated levels of sCD40L. In further important embodiments, the
subject treated is a nonhyperlipidemic subject. In any of the
foregoing embodiments, the beta-adrenergic receptor blocker may be
but is not limited to, atenolol, acebutolol, alprenolol, befunolol,
betaxolol, bunitrolol, carteolol, celiprolol, hedroxalol,
indenolol, labetalol, levobunolol, mepindolol, methypranol,
metindol, metoprolol, metrizoranolol, oxprenolol, pindolol,
propranolol, practolol, practolol, sotalolnadolol, tiprenolol,
tomalolol, timolol, bupranolol, penbutolol, trimepranol,
2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitril
HCl, 1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,
1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,
3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,
2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,
7-(2-hydroxy-3-t-butylaminpropoxy) phthalide.
[0033] The invention also involves a method for treating subjects
with a cyclooxygenase-2 inhibitor, to prevent cardiovascular
disorders. Such an agent is administered to a subject selected on
the basis of having an above-normal level of sCD40L. The agent is
administered in an amount effective to lower the risk of the
subject developing a future cardiovascular disorder. In one
embodiment, the subject already has had a cardiovascular event,
such as a heart attack or an angioplasty. In another important
embodiment, the subjects are apparently healthy subjects otherwise
free of current need for cyclooxygenase-2 inhibitor treatment. In
important embodiments the subjects are not an elevated risk of an
adverse cardiovascular event, other than having elevated levels of
sCD40L. In further important embodiments, the subject treated is a
nonhyperlipidemic subject. In any of the foregoing embodiments, the
cyclooxygenase-2 inhibitor may be, but is not limited to, a phenyl
heterocycle, a diaryl bicyclic heterocycle, an aryl substituted 5,5
fused aromatic nitrogen compound, a N-benzylindol-3-yl propanoic
acid and/or its derivatives, a 5-methanesulfonamido-1-indanone, a
N-benzyl indol-3-yl butanoic acid and/or its derivatives, a
diphenyl-1,2-3-thiadiazole, a diaryl-5-oxygenated-2-(5H) -furanone,
a 3,4-diaryl-2-hydroxy-2,5-dihydrofuran, a stilbene and/or its
derivatives, a diphenyl stilbene, an alkylated styrene, a bisaryl
cyclobutene and/or its derivatives, a substituted pyridine, a
pyridinyl-2-cyclopenten-1-one, and/or a substituted
sulfonylphenylheterocycle.
[0034] The invention also involves a method for treating subjects
with an angiotensin system inhibitor, to prevent cardiovascular
disorders. Such an agent is administered to a subject selected on
the basis of having an above-normal level of sCD40L. The agent is
administered in an amount effective to lower the risk of the
subject developing a future cardiovascular disorder. In one
embodiment, the subject already has had a cardiovascular event,
such as a heart attack or an angioplasty. In another important
embodiment, the subjects are apparently healthy subjects otherwise
free of current need for angiotensin system inhibitor treatment. In
important embodiments the subjects are not an elevated risk of an
adverse cardiovascular event, other than having elevated levels of
sCD40L. In further important embodiments, the subject treated is a
nonhyperlipidemic subject. In any of the foregoing embodiments, the
angiotensin system inhibitor may be, but is not limited to, an
angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II
antagonist, an angiotensin II receptor antagonist, agents that
activate the catabolism of angiotensin II, and/or agents that
prevent the synthesis of angiotensin I.
[0035] According to another aspect of the invention, a method is
provided for evaluating the likelihood that an individual will
benefit from treatment with an agent for reducing the risk of a
cardiovascular disorder associated with atherosclerotic disease.
The agent can be selected from the group consisting of
anti-inflammatory agents, anti-thrombotic agents, anti-platelet
agents, fibrinolytic agents, lipid reducing agents, direct thrombin
inhibitors, glycoprotein II b/IIIa receptor inhibitors, agents that
bind to cellular adhesion molecules and inhibit the ability of
white blood cells to attach to such molecules (e.g. anti-cellular
adhesion molecule antibodies), calcium channel blockers,
beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors,
angiotensin system inhibitors, and/or combinations of the foregoing
agents thereof. To practice the method, a level of sCD40L in an
individual is obtained. This level then is compared to a
predetermined value, wherein the level of sCD40L in comparison to
the predetermined value is indicative of the likelihood that the
individual will benefit from treatment with the agent. The
individual then can be characterized in terms of the net benefit
likely to be obtained by treatment with the agent.
[0036] The predetermined value can be as described above.
[0037] As mentioned above, the invention is particularly adapted to
determining which individuals will preferentially benefit from
treatment with an agent for reducing the risk in the individuals of
a cardiovascular disorder such as a future stroke or a future
myocardial infarction, including nonfatal myocardial infarctions.
It also permits selection of candidate populations for clinical
trials and for treatment with candidate drugs, by identifying, for
example, the individuals most likely to benefit from a new
treatment or from a known treatment with a high risk profile of
adverse side effects. Thus, the invention provides information for
evaluating the likely net benefit of certain treatments for
candidate patients.
[0038] According to another aspect of the invention, a method for
reducing sCD40L levels in a subject to lower the risk of an adverse
cardiovascular disorder is provided. The method involves selecting
and administering to a subject having elevated levels of sCD40L an
agent that reduces sCD40L levels in an amount effective to reduce
the sCD40L levels in the subject. In one embodiment, the agent is a
lipid reducing agent. The preferred subject is an apparently
healthy subject. In some embodiments, the subject is not otherwise
at an elevated risk of having an adverse cardiovascular event. In
certain embodiments, the subject has elevated C-Reactive Protein
(CRP) levels. In some embodiments of this aspect of the invention,
the subject is otherwise free of indications calling for treatment
with a lipid reducing agent.
[0039] According to a further aspect of the invention, a method for
evaluating the likelihood for vascular intra-plaque lipid
accumulation in an individual at risk of developing a
cardiovascular disorder, is provided. The method involves obtaining
a level of sCD40L in the individual, comparing the level of sCD40L
to a predetermined value, and characterizing the individual's risk
profile for vascular intra-plaque lipid accumulation, based upon
the level of sCD40L in comparison to the predetermined level. The
predetermined value can be a plurality of predetermined sCD40L
level ranges and said comparing step comprises determining in which
of said predetermined sCD40L level ranges said individual's sCD40L
level falls. In certain embodiments, the predetermined value is
about 2.5 ng/mL of blood or higher. In some embodiments, the
predetermined value is about 3.0 ng/mL of blood or higher. In
important embodiments, the predetermined value is a plurality of
predetermined sCD40L level ranges, one of said plurality being
below about 2.5 ng/mL blood and another of said ranges being about
2.5 ng/mL blood, and said comparing step comprises determining in
which of said plurality of predetermined sCD40L level ranges said
individual's sCD40L level falls. In further important embodiments,
the vascular intra-plaque lipid accumulation occurs in the carotid
artery. Other preferred ranges and important embodiments are as
described above and below.
[0040] According to a further aspect of the invention, methods for
preparing medicaments useful in the treatment of cardiovascular
conditions, are provided.
[0041] These and other aspects of the invention will be described
in more detail below in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0042] FIG. 1 is a graph demonstrating the relative risk of
cardiovascular events in the study population according to sCD40L
blood levels. The dotted line depicts the 99.sup.th percentile
cutpoint for the control distribution.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The primary basis for this invention is evidence from a
prospective, nested case control analysis among participants in the
Women's Health Study (WHS), an ongoing primary prevention trial
evaluating the efficiency of vitamin E and low dose aspirin in
28,263 middle aged American women with no history of cardiovascular
disease or cancer. In this trial, baseline level of sCD40L, a
mediator of inflammation, was found to determine the future risk of
myocardial infarction and stroke, independent of a large series of
lipid and non-lipid risk factors, and independent of other
predictors, including markers of systemic inflammation (see, e.g.,
U.S. Pat. No. 6,040,147). Specifically, individuals with the
highest baseline levels of sCD40L were found to have at least 3
fold increases in risk of developing future cardiovascular events
(FIG. 1).
[0044] Moreover, in data from the Women's Health Study, the risk of
future myocardial infarction and stroke associated with sCD40L,
appear to be additive to that which could otherwise be determined
by usual assessment of total cholesterol and HDL cholesterol. In
this trial, the predictive value of sCD40L was present for
non-fatal as well as fatal events, was stable over long periods of
time, and was present for non-smokers as well as smokers. Further,
data from this trial indicate the magnitude of benefit that
apparently healthy individuals can expect from therapeutic agents
used in the prevention and treatment of atherosclerotic
disorders.
[0045] The current invention in one aspect describes the use of
sCD40L to predict risk of cardiovascular disorders associated with
atherosclerosis such as myocardial infarction and stroke among
individuals without current evidence of disease. Thus, these data
greatly extend prior observations regarding the use of sCD40L, to
predict risk among already identified high-risk populations or
among symptomatic ischemia patients such as those with unstable
angina pectoris. Indeed, since levels of sCD40L increase following
acute ischemia, it has been uncertain whether statistical
associations observed in prior studies of acutely ill or high-risk
populations are casual or due to short-term inflammatory changes,
or to interrelations with other risk factors, in particular smoking
and hyperlipidemia.
[0046] In marked contrast, data from the Women's Health Study
indicate for the first time the utility of sCD40L to predict risk
among currently healthy and otherwise low-risk individuals, to
predict non-fatal as well as fatal events, to predict risk among
non-smokers, and to predict risk above and beyond that associated
with screening for total and HDL cholesterol. Data from the Women's
Health Study also indicate for the first time that the likelihood
of efficacy of interventions designed to reduce risk of
atherosclerotic events such as myocardial infarction and stroke
differs in magnitude based upon a measure of the sCD40L
plasma/blood levels. The invention will be better understood with
reference to the following brief explanation of terms.
[0047] "Cardiovascular myocardial ischemia, disorders" includes
myocardial infarction, stroke, myocardial ischemia, angina pectoris
and peripheral arteriovascular disease. Cardiovascular disorders do
not include venous thrombosis.
[0048] "Apparently healthy", as used herein, means individuals who
have not previously had an acute adverse cardiovascular event such
as a myocardial infarction (i.e., individuals who are not at an
elevated risk of a second adverse cardiovascular event due to a
primary adverse cardiovascular event). Apparently healthy
individuals also do not otherwise exhibit symptoms of disease. In
other words, such individuals, if examined by a medical
professional, would be characterized as healthy and free of
symptoms of disease.
[0049] In important embodiments, the subject does not otherwise
have an elevated risk of an adverse cardiovascular event. Subjects
having an elevated such risk include those with a family history of
cardiovascular disease, elevated lipids, smokers, prior acute
cardiovascular event, etc. (See, e.g., Harrison's Principles of
Experimental Medicine, 15th Edition, McGraw-Hill, Inc.,
N.Y.--hereinafter "Harrison's"). According to one important aspect
of the invention, a method for treating a subject to reduce the
risk of a cardiovascular disorder, is provided. The method involves
selecting and administering to a subject who is known to have an
above-normal level of sCD40L an agent for reducing the risk of the
cardiovascular disorder. The agent can be an anti-inflammatory
agent, an antithrombotic agent, an anti-platelet agent, a
fibrinolytic agent, a lipid reducing agent, a direct thrombin
inhibitor, a glycoprotein IIb/IIIa receptor inhibitor, an agent
that binds to cellular adhesion molecules and inhibits the ability
of white blood cells to attach to such molecules, a calcium channel
blocker, a beta-adrenergic receptor blocker, a cyclooxygenase-2
inhibitor, an angiotensin system inhibitor, and/or combinations
thereof. The agent is administered in an amount effective to lower
the risk of the subject developing a future cardiovascular
disorder.
[0050] The preferred subjects are apparently healthy subjects
otherwise free of current need for treatment with the agent
prescribed according to the present invention. For example, if
treatment with a particular agent occurs based on elevated levels
of sCD40L, then the patient preferably is free of symptoms calling
for treatment with that agent (or the category of agent into which
the agent falls), other than the symptom of having elevated levels
of sCD40L. In some embodiments, the subject is otherwise free of
symptoms calling for treatment with any one of any combination of
or all of the foregoing categories of agents. Such as, for example,
with respect to anti-inflammatory agents, free of symptoms of
rheumatoid arthritis, chronic back pain, autoimmune diseases,
vascular diseases, viral diseases, malignancies, and the like. In
further important embodiments, the subject treated is a
nonhyperlipidemic subject. In another embodiment, the subjects are
not at an elevated risk of an adverse cardiovascular event (e.g.,
subjects with no family history of such events, subjects who are
nonsmokers, subjects who are nonhyperlipidemic, subjects who do not
have elevated levels of a systemic inflammatory marker), other than
having an elevated level of sCD40L. In some embodiments, the
subject is otherwise free of symptoms calling for treatment with
any one of, any combination of or all of the foregoing categories
of agents.
[0051] In some embodiments, the subject is otherwise free of
symptoms calling for treatment with any one of, any combination of
or all of the foregoing categories of agents. In further important
embodiments, the subject treated is a nonhyperlipidemic subject. A
"nonhyperlipidemic" is a subject that is a nonhypercholesterolemic
and/or a nonhypertriglyceridemic subject. A
"nonhypercholesterolemic" subject is one that does not fit the
current criteria established for a hypercholesterolemic subject. A
nonhypertriglyceridemic subject is one that does not fit the
current criteria established for a hypertriglyceridemic subject
(See, e.g., Harrison's Principles of Experimental Medicine, 15th
Edition, McGraw-Hill, Inc., N.Y.--hereinafter "Harrison's").
Hypercholesterolemic subjects and hypertriglyceridemic subjects are
associated with increased incidence of premature coronary heart
disease. A hypercholesterolemic subject has an LDL level of >160
mg/dL, or >130 mg/dL and at least two risk factors selected from
the group consisting of male gender, family history of premature
coronary heart disease, cigarette smoking (more than 10 per day),
hypertension, low HDL (<35 mg/dL), diabetes mellitus,
hyperinsulinemia, abdominal obesity, high lipoprotein (a), and
personal history of cerebrovascular disease or occlusive peripheral
vascular disease. A hypertriglyceridemic subject has a triglyceride
(TG) level of >250 mg/dL. Thus, a nonhyperlipidemic subject is
defined as one whose cholesterol and triglyceride levels are below
the limits set as described above for both the hypercholesterolemic
and hypertriglyceridemic subjects.
[0052] In some embodiments, the subject has normal levels of
systemic inflammatory markers. For the purposes of this
application, normal such levels means the same things as the
absence of elevated levels. Normal levels will depend on the
particular systemic inflammatory marker. (See U.S. Pat. No.
6,040,147, incorporated herein in its entirety by reference.)
[0053] "Nonsmoking", as used herein, means an individual who, at
the time of the evaluation, is not a smoker. This includes
individuals who have never smoked as well as individuals who in the
past have smoked but presently no longer smoke.
[0054] Agents for reducing the risk of a cardiovascular disorder
include, but are not limited to, those selected from the group
consisting of anti-inflammatory agents, anti-thrombotic agents,
anti-platelet agents, fibrinolytic agents, lipid reducing agents,
direct thrombin inhibitors, glycoprotein II b/IIIa receptor
inhibitors, agents that bind to cellular adhesion molecules and
inhibit the ability of white blood cells to attach to such
molecules (e.g. anti-cellular adhesion molecule antibodies),
calcium channel blockers, beta-adrenergic receptor blockers,
cyclooxygenase-2 inhibitors, angiotensin system inhibitors, and/or
any combinations thereof.
[0055] "Anti-inflammatory" agents include but are not limited to,
Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha
Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose
Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;
Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine
Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen;
Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;
Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;
Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;
Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;
Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide;
Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole;
Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac;
Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort;
Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin
Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone;
Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen;
Furobufen; Halcinonide; Halobetasol Propionate; Halopredone
Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen
Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen;
Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam;
Ketoprofen; Lofemizole Hydrochloride; Lornoxicam; Loteprednol
Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone
Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;
Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;
Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;
Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;
Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate;
Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine;
Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;
Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;
Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride;
Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin;
Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium;
Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol
Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate;
Zidometacin; Glucocorticoids; Zomepirac Sodium.
[0056] "Anti-thrombotic" and/or "fibrinolytic" agents include but
are not limited to, Plasminogen (to plasmin via interactions of
prekallikrein, kininogens, Factors XII, XIIIa, plasminogen
proactivator, and tissue plasminogen activator[TPA]) Streptokinase;
Urokinase: Anisoylated Plasminogen-Streptokinase Activator Complex;
Pro-Urokinase; (Pro-UK); rTPA (alteplase or activase; r denotes
recombinant); rPro-UK; Abbokinase; Eminase; Sreptase Anagrelide
Hydrochloride; Bivalirudin; Dalteparin Sodium; Danaparoid Sodium;
Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;
Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase;
Trifenagrel; Warfarin; Dextrans.
[0057] "Anti-platelet" agents include but are not limited to,
Clopridogrel; Sulfinpyrazone; Aspirin; Dipyridamole; Clofibrate;
Pyridinol Carbamate; PGE; Glucagon; Antiserotonin drugs; Caffeine;
Theophyllin Pentoxifyllin; Ticlopidine; Anagrelide.
[0058] "Lipid reducing" agents include but are not limited to,
gemfibrozil, cholystyramine, colestipol, nicotinic acid, probucol
lovastatin, fluvastatin, simvastatin, atorvastatin, pravastatin,
cerivastatin, and other HMG-CoA reductase inhibitors.
[0059] HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is
the microsomal enzyme that catalyzes the rate limiting reaction in
cholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductase
inhibitor inhibits HMG-CoA reductase, and as a result inhibits the
synthesis of cholesterol. A number of HMG-CoA reductase inhibitors
has been used to treat individuals with hypercholesterolemia. More
recently, HMG-CoA reductase inhibitors have been shown to be
beneficial in the treatment of stroke (Endres M, et al., Proc Natl
Acad Sci USA, 1998, 95:8880-5).
[0060] HMG-CoA reductase inhibitors useful according to the
invention include, but are not limited to, simvastatin (U.S. Pat.
No. 4,444,784), lovastatin (U.S. Pat. No. 4,231,938), pravastatin
sodium (U.S. Pat. No. 4,346,227), fluvastatin (U.S. Pat. No.
4,739,073), atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin,
and numerous others described in U.S. Pat. No. 5,622,985, U.S. Pat.
No. 5,135,935, U.S. Pat. No. 5,356,896, U.S. Pat. No. 4,920,109,
U.S. Pat. No. 5,286,895, U.S. Pat. No. 5,262,435, U.S. Pat. No.
5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat. No. 5,283,256, U.S.
Pat. No. 5,256,689, U.S. Pat. No. 5,182,298, U.S. Pat. No.
5,369,125, U.S. Pat. No. 5,302,604, U.S. Pat. No. 5,166,171, U.S.
Pat. No. 5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No.
5,196,440, U.S. Pat. No. 5,091,386, U.S. Pat. No. 5,091,378, U.S.
Pat. No. 4,904,646, U.S. Pat. No. 5,385,932, U.S. Pat. No.
5,250,435, U.S. Pat. No. 5,132,312, U.S. Pat. No. 5,130,306, U.S.
Pat. No. 5,116,870, U.S. Pat. No. 5,112,857, U.S. Pat. No.
5,102,911, U.S. Pat. No. 5,098,931, U.S. Pat. No. 5,081,136, U.S.
Pat. No. 5,025,000, U.S. Pat. No. 5,021,453, U.S. Pat. No.
5,017,716, U.S. Pat. No. 5,001,144, U.S. Pat. No. 5,001,128, U.S.
Pat. No. 4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat. No.
4,994,494, U.S. Pat. No. 4,992,429, U.S. Pat. No. 4,970,231, U.S.
Pat. No. 4,968,693, U.S. Pat. No. 4,963,538, U.S. Pat. No.
4,957,940, U.S. Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S.
Pat. No. 4,946,860, U.S. Pat. No. 4,940,800, U.S. Pat. No.
4,940,727, U.S. Pat. No. 4,939,143, U.S. Pat. No. 4,929,620, U.S.
Pat. No. 4,923,861, U.S. Pat. No. 4,906,657, U.S. Pat. No.
4,906,624 and U.S. Pat. No. 4,897,402, the disclosures of which
patents are incorporated herein by reference.
[0061] "Direct thrombin inhibitors" include but are not limited to,
hirudin, hirugen, hirulog, agatroban, PPACK, thrombin aptamers.
[0062] "Glycoprotein IIb/IIIa receptor inhibitors" are both
antibodies and non-antibodies, and include but are not limited to
ReoPro (abcixamab), lamifiban, tirofiban.
[0063] "Calcium channel blockers" are a chemically diverse class of
compounds having important therapeutic value in the control of a
variety of diseases including several cardiovascular disorders,
such as hypertension, angina, and cardiac arrhythmias
(Fleckenstein, Cir. Res. v. 52, (suppl. 1), p.13-16 (1983);
Fleckenstein, Experimental Facts and Therapeutic Prospects, John
Wiley, New York (1983); McCall, D., Curr Pract Cardiol, v. 10, p.
1-11 (1985)). Calcium channel blockers are a heterogenous group of
drugs that prevent or slow the entry of calcium into cells by
regulating cellular calcium channels. (Remington, The Science and
Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company,
Eaton, Pa., p. 963 (1995)). Most of the currently available calcium
channel blockers, and useful according to the present invention,
belong to one of three major chemical groups of drugs, the
dihydropyridines, such as nifedipine, the phenyl alkyl amines, such
as verapamil, and the benzothiazepines, such as diltiazem. Other
calcium channel blockers useful according to the invention,
include, but are not limited to, amrinone, amlodipine, bencyclane,
felodipine, fendiline, flunarizine, isradipine, nicardipine,
nimodipine, perhexilene, gallopamil, tiapamil and tiapamil
analogues (such as 1993RO-11-2933), phenytoin, barbiturates, and
the peptides dynorphin, omega-conotoxin, and omega-agatoxin, and
the like and/or pharmaceutically acceptable salts thereof.
[0064] "Beta-adrenergic receptor blocking agents" are a class of
drugs that antagonize the cardiovascular effects of catecholamines
in angina pectoris, hypertension, and cardiac arrhythmias.
Beta-adrenergic receptor blockers include, but are not limited to,
atenolol, acebutolol, alprenolol, befunolol, betaxolol, bunitrolol,
carteolol, celiprolol, hedroxalol, indenolol, labetalol,
levobunolol, mepindolol, methypranol, metindol, metoprolol,
metrizoranolol, oxprenolol, pindolol, propranolol, practolol,
practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,
bupranolol, penbutolol, trimepranol,
2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl-
, 1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,
1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,
3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,
2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,-
7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identified
compounds can be used as isomeric mixtures, or in their respective
levorotating or dextrorotating form.
[0065] Cyclooxygenase-2 (COX-2) is a recently identified new form
of a cyclooxygenase. "Cyclooxygenase" is an enzyme complex present
in most tissues that produces various prostaglandins and
thromboxanes from arachidonic acid. Non-steroidal, antiinflammatory
drugs exert most of their antiinflammatory, analgesic and
antipyretic activity and inhibit hormone-induced uterine
contractions and certain types of cancer growth through inhibition
of the cyclooxygenase (also known as prostaglandin G/H synthase
and/or prostaglandin-endoperoxide synthase). Initially, only one
form of cyclooxygenase was known, the "constitutive enzyme" or
cyclooxygenase-1 (COX-1). It and was originally identified in
bovine seminal vesicles.
[0066] Cyclooxygenase-2 (COX-2) has been cloned, sequenced and
characterized initially from chicken, murine and human sources
(See, e.g., U.S. Pat. No. 5,543,297, issued Aug. 6, 1996 to
Cromlish, et al., and assigned to Merck Frosst Canada, Inc.,
Kirkland, Calif., entitled: "Human cyclooxygenase-2 cDNA and assays
for evaluating cyclooxygenase-2 activity"). This enzyme is distinct
from the COX-1. COX-2, is rapidly and readily inducible by a number
of agents including mitogens, endotoxin, hormones, cytokines and
growth factors. As prostaglandins have both physiological and
pathological roles, it is believed that the constitutive enzyme,
COX-1, is responsible, in large part, for endogenous basal release
of prostaglandins and hence is important in their physiological
functions such as the maintenance of gastrointestinal integrity and
renal blood flow. By contrast, it is believed that the inducible
form, COX-2, is mainly responsible for the pathological effects of
prostaglandins where rapid induction of the enzyme would occur in
response to such agents as inflammatory agents, hormones, growth
factors, and cytokines. Therefore, it is believed that a selective
inhibitor of COX-2 has similar antiinflammatory, antipyretic and
analgesic properties to a conventional non-steroidal
antiinflammatory drug, and in addition inhibits hormone-induced
uterine contractions and also has potential anti-cancer effects,
but with reduced side effects. In particular, such COX-2 inhibitors
are believed to have a reduced potential for gastrointestinal
toxicity, a reduced potential for renal side effects, a reduced
effect on bleeding times and possibly a decreased potential to
induce asthma attacks in aspirin-sensitive asthmatic subjects, and
are therefore useful according to the present invention.
[0067] A number of selective "COX-2 inhibitors" are known in the
art. These include, but are not limited to, COX-2 inhibitors
described in U.S. Pat. No. 5,474,995 "Phenyl heterocycles as cox-2
inhibitors"; U.S. Pat. No. 5,521,213 "Diaryl bicyclic heterocycles
as inhibitors of cyclooxygenase-2''; U.S. Pat. No. 5,536,752
"Phenyl heterocycles as COX-2 inhibitors"; U.S. Pat. No. 5,550,142
"Phenyl heterocycles as COX-2 inhibitors"; U.S. Pat. No. 5,552,422
"Aryl substituted 5,5 fused aromatic nitrogen compounds as
anti-inflammatory agents"; U.S. Pat. No. 5,604,253
"N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase
inhibitors"; U.S. Pat. No. 5,604,260
"5-methanesulfonamido-1-indanones as an inhibitor of
cyclooxygenase-2"; U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl
butanoic acid derivatives as cyclooxygenase inhibitors"; U.S. Pat.
No. 5,677,318 Diphenyl-1,2-3-thiadiazoles as anti-inflammatory
agents"; U.S. Pat. No. 5,691,374 "Diaryl-5-oxygenated-2-(5H)
-furanones as COX-2 inhibitors"; U.S. Pat. No. 5,698,584
"3,4-diaryl-2-hydroxy-2,5-dihydrofurans as prodrugs to COX-2
inhibitors"; U.S. Pat. No. 5,710,140 "Phenyl heterocycles as COX-2
inhibitors"; U.S. Pat. No. 5,733,909 "Diphenyl stilbenes as
prodrugs to COX-2 inhibitors"; U.S. Pat. No. 5,789,413 "Alkylated
styrenes as prodrugs to COX-2 inhibitors"; U.S. Pat. No. 5,817,700
"Bisaryl cyclobutenes derivatives as cyclooxygenase inhibitors";
U.S. Pat. No. 5,849,943 "Stilbene derivatives useful as
cyclooxygenase-2 inhibitors"; U.S. Pat. No. 5,861,419 "Substituted
pyridines as selective cyclooxygenase-2 inhibitors"; U.S. Pat. No.
5,922,742 "Pyridinyl-2-cyclopenten-1-ones as selective
cyclooxygenase-2 inhibitors"; U.S. Pat. No. 5,925,631 "Alkylated
styrenes as prodrugs to COX-2 inhibitors"; all of which are
commonly assigned to Merck Frosst Canada, Inc. (Kirkland, Calif.).
Additional COX-2 inhibitors are also described in U.S. Pat. No.
5,643,933, assigned to G. D. Searle & Co. (Skokie, Ill.),
entitled: "Substituted sulfonylphenylheterocycles as
cyclooxygenase-2 and 5-lipoxygenase inhibitors."
[0068] A number of the above-identified COX-2 inhibitors are
prodrugs of selective COX-2 inhibitors, and exert their action by
conversion in vivo to the active and selective COX-2 inhibitors.
The active and selective COX-2 inhibitors formed from the
above-identified COX-2 inhibitor prodrugs are described in detail
in WO 95/00501, published Jan. 5, 1995, WO 95/18799, published Jul.
13, 1995 and U.S. Pat. No. 5,474,995, issued Dec. 12, 1995. Given
the teachings of U.S. Pat. No. 5,543,297, entitled: "Human
cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2
activity," a person of ordinary skill in the art would be able to
determine whether an agent is a selective COX-2 inhibitor or a
precursor of a COX-2 inhibitor, and therefore part of the present
invention.
[0069] An "angiotensin system inhibitor" is an agent that
interferes with the function, synthesis or catabolism of
angiotensin II. These agents include, but are not limited to,
angiotensin-converting enzyme (ACE) inhibitors, angiotensin II
antagonists, angiotensin II receptor antagonists, agents that
activate the catabolism of angiotensin II, and agents that prevent
the synthesis of angiotensin I from which angiotensin II is
ultimately derived. The renin-angiotensin system is involved in the
regulation of hemodynamics and water and electrolyte balance.
Factors that lower blood volume, renal perfusion pressure, or the
concentration of Na.sup.+ in plasma tend to activate the system,
while factors that increase these parameters tend to suppress its
function.
[0070] Angiotensin I and angiotensin II are synthesized by the
enzymatic renin-angiotensin pathway. The synthetic process is
initiated when the enzyme renin acts on angiotensinogen, a
pseudoglobulin in blood plasma, to produce the decapeptide
angiotensin I. Angiotensin I is converted by angiotensin converting
enzyme (ACE) to angiotensin II (angiotensin-[1-8] octapeptide). The
latter is an active pressor substance which has been implicated as
a causative agent in several forms of hypertension in various
mammalian species, e.g., humans.
[0071] Angiotensin (renin-angiotensin) system inhibitors are
compounds that act to interfere with the production of angiotensin
II from angiotensinogen or angiotensin I or interfere with the
activity of angiotensin II. Such inhibitors are well known to those
of ordinary skill in the art and include compounds that act to
inhibit the enzymes involved in the ultimate production of
angiotensin II, including renin and ACE. They also include
compounds that interfere with the activity of angiotensin II, once
produced. Examples of classes of such compounds include antibodies
(e.g., to renin), amino acids and analogs thereof (including those
conjugated to larger molecules), peptides (including peptide
analogs of angiotensin and angiotensin I), pro-renin related
analogs, etc. Among the most potent and useful renin-angiotensin
system inhibitors are renin inhibitors, ACE inhibitors, and
angiotensin II antagonists. In a preferred embodiment of the
invention, the renin-angiotensin system inhibitors are renin
inhibitors, ACE inhibitors, and angiotensin II antagonists.
[0072] "Angiotensin II antagonists" are compounds which interfere
with the activity of angiotensin II by binding to angiotensin II
receptors and interfering with its activity. Angiotensin II
antagonists are well known and include peptide compounds and
non-peptide compounds. Most angiotensin II antagonists are slightly
modified congeners in which agonist activity is attenuated by
replacement of phenylalanine in position 8 with some other amino
acid; stability can be enhanced by other replacements that slow
degeneration in vivo. Examples of angiotensin II antagonists
include: peptidic compounds (e.g., saralasin,
[(San.sup.1)(Val.sup.5)(Ala.sup.8)] angiotensin -(1-8) octapeptide
and related analogs); N-substituted imidazole-2-one (U.S. Pat. No.
5,087,634); imidazole acetate derivatives including
2-N-butyl-4-chloro-1-(2-chlorobenzile) imidazole-5-acetic acid (see
Long et al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988));
4,5,6,7-tetrahydro-1H-imidazo [4,5-c] pyridine-6-carboxylic acid
and analog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazole
beta-glucuronide analogs (U.S. Pat. No. 5,085,992); substituted
pyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127);
phenol and heterocyclic derivatives such as 1, 3-imidazoles (U.S.
Pat. No. 5,073,566); imidazo-fused 7-member ring heterocycles (U.S.
Pat. No. 5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684);
antibodies to angiotensin II (e.g., U.S. Pat. No. 4,302,386); and
aralkyl imidazole compounds such as biphenyl-methyl substituted
imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891
(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl
(35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide,
Sankyo Company, Ltd., Tokyo, Japan); SKF108566
(E-alpha-2-[2-butyl-1-(carboxy phenyl) methyl]
1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid, Smith Kline
Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPont Merck
Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRoche
AG); A.sub.2 agonists (Marion Merrill Dow) and certain non-peptide
heterocycles (G.D. Searle and Company).
[0073] "Angiotensin converting enzyme (ACE), is an enzyme which
catalyzes the conversion of angiotensin I to angiotensin II. ACE
inhibitors include amino acids and derivatives thereof, peptides,
including di and tri peptides and antibodies to ACE which intervene
in the renin-angiotensin system by inhibiting the activity of ACE
thereby reducing or eliminating the formation of pressor substance
angiotensin II. ACE inhibitors have been used medically to treat
hypertension, congestive heart failure, myocardial infarction and
renal disease. Classes of compounds known to be useful as ACE
inhibitors include acylmercapto and mercaptoalkanoyl prolines such
as captopril (U.S. Pat. No. 4,105,776) and zofenopril (U.S. Pat.
No. 4,316,906), carboxyalkyl dipeptides such as enalapril (U.S.
Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829),
quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No.
4,587,258), and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyl
dipeptide mimics such as cilazapril (U.S. Pat. No. 4,512,924) and
benazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoyl prolines
such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.
[0074] "Renin inhibitors" are compounds which interfere with the
activity of renin. Renin inhibitors include amino acids and
derivatives thereof, peptides and derivatives thereof, and
antibodies to renin. Examples of renin inhibitors that are the
subject of United States patents are as follows: urea derivatives
of peptides (U.S. Pat. No. 5,116,835); amino acids connected by
nonpeptide bonds (U.S. Pat. No. 5,114,937); di and tri peptide
derivatives (U.S. Pat. No. 5,106,835); amino acids and derivatives
thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diol sulfonamides
and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides (U.S.
Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates
(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No.
5,075,451); fluorine and chlorine statine or statone containing
peptides (U.S. Pat. No. 5,066,643); peptidyl amino diols (U.S. Pat.
Nos. 5,063,208 and 4,845,079); N-morpholino derivatives (U.S. Pat.
No. 5,055,466); pepstatin derivatives (U.S. Pat. No. 4,980,283);
N-heterocyclic alcohols (U.S. Pat. No. 4,885,292); monoclonal
antibodies to renin (U.S. Pat. No. 4,780,401); and a variety of
other peptides and analogs thereof (U.S. Pat. Nos. 5,071,837,
5,064,965, 5,063,207, 5,036,054, 5,036,053, 5,034,512, and
4,894,437).
[0075] Agents that bind to cellular adhesion molecules and inhibit
the ability of white blood cells to attach to such molecules
include polypeptide agents. Such polypeptides include polyclonal
and monoclonal antibodies, prepared according to conventional
methodology. Such antibodies already are known in the art and
include anti-ICAM 1 antibodies as well as other such antibodies.
Significantly, as is well-known in the art, only a small portion of
an antibody molecule, the paratrope, is involved in the binding of
the antibody to its epitope (see, in general, Clark, W. R. (1986)
The Experimental Foundations of Modern Immunology, Wiley &
Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th
Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc
regions, for example, are effectors of the complement cascade but
are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd Fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0076] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (Frs), which maintain the tertiary
structure of the paratope (see, in general, Clar, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FR1 through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDR3). The CDRs, and in particular the CDR3
regions, and more particularly the heavy chain CDR3, are largely
responsible for antibody specificity.
[0077] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of nonspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. Thus,
for example, PCT International Publication Number WO 92/04381
teaches the production and use of humanized murine RSV antibodies
in which at least a portion of the murine FR regions have been
replaced by FR regions of human origin. Such antibodies, including
fragments of intact antibodies with antigen-binding ability, are
often referred to as "chimeric" antibodies.
[0078] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab').sub.2, Fab, Fv
and Fd fragments; chimeric antibodies in which the Fc and/or Fr
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric
F(ab').sub.2 fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or nonhuman sequences. The present invention also
includes so-called single chain antibodies.
[0079] Thus, the invention involves polypeptides of numerous size
and type that bind specifically to cellular adhesion molecules.
These polypeptides may be derived also from sources other than
antibody technology. For example, such polypeptide binding agents
can be provided by degenerate peptide libraries which can be
readily prepared in solution, in immobilized form or as phage
display libraries. Combinatorial libraries also can be synthesized
of peptides containing one or more amino acids. Libraries further
can be synthesized of peptoids and non-peptide synthetic
moieties.
[0080] Phage display can be particularly effective in identifying
binding peptides useful according to the invention. Briefly, one
prepares a phage library (using e.g. m13, fd, or lambda phage),
displaying inserts from 4 to about 80 amino acid residues using
conventional procedures. The inserts may represent, for example, a
completely degenerate or biased array. One then can select
phage-bearing inserts which bind to the cellular adhesion molecule.
This process can be repeated through several cycles of reselection
of phage that bind to the cellular adhesion molecule. Repeated
rounds lead to enrichment of phage bearing particular sequences.
DNA sequences analysis can be conducted to identify the sequences
of the expressed polypeptides. The minimal linear portion of the
sequence that binds to the cellular adhesion molecule can be
determined. One can repeat the procedure using a biased library
containing inserts containing part of all of the minimal linear
portion plus one or more additional degenerate residues upstream or
downstream thereof. Yeast two-hybrid screening methods also may be
used to identify polypeptides that bind to the cellular adhesion
molecules. Thus, cellular adhesion molecules, or a fragment
thereof, can be used to screen peptide libraries, including phage
display libraries, to identify and select peptide binding partners
of the cellular adhesion molecules.
[0081] In practicing the methods of the present invention, it is
required to obtain a level of sCD40L in an individual. Soluble
CD40L is well-known to those of ordinary skill in the art. The
level of sCD40L for the individual can be obtained by any art
recognized method. Typically, the level is determined by measuring
the level of the marker in a body fluid, for example, blood, lymph,
saliva, urine and the like. The level can be determined by ELISA,
or immunoassays or other conventional techniques for determining
the presence of the marker. Conventional methods include sending
samples of a patient's body fluid to a commercial laboratory for
measurement.
[0082] The invention also involves comparing the level of sCD40L
for the individual with a predetermined value. The predetermined
value can take a variety of forms. It can be single cut-off value,
such as a median or mean. It can be established based upon
comparative groups, such as where the risk in one defined group is
double the risk in another defined group. It can be a range, for
example, where the tested population is divided equally (or
unequally) into groups, such as a low-risk group, a medium-risk
group and a high-risk group, or into quadrants, the lowest quadrant
being individuals with the lowest risk and the highest quadrant
being individuals with the highest risk.
[0083] The predetermined value can depend upon the particular
population selected. For example, an apparently healthy, nonsmoker
population with no detectable disease and no prior history of a
cardiovascular disorder will have a different `normal` range of
sCD40L than will a smoking population or a population the members
of which have had a prior cardiovascular disorder. Accordingly, the
predetermined values selected may take into account the category in
which an individual falls. Appropriate ranges and categories can be
selected with no more than routine experimentation by those of
ordinary skill in the art.
[0084] The preferred body fluid is blood. For sCD40L, one important
cut-off for a population of apparently healthy, nonsmokers is 2.9
ng/mL or below. Another important cut-off for sCD40L is 3.0, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,
5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 or
11.0 ng/mL. In characterizing risk, numerous predetermined values
can be established.
[0085] There presently are commercial sources which produce
reagents for assays for sCD40L. These include, but are not limited
to, BenderMedSystems (Vienna, Austria), and Abbott Pharmaceuticals
(Abbott Park, Ill.).
[0086] In preferred embodiments the invention provides novel kits
or assays which are specific for, and have appropriate sensitivity
with respect to, predetermined values selected on the basis of the
present invention. The preferred kits, therefore, would differ from
those presently commercially available, by including, for example,
different cut-offs, different sensitivities at particular cut-offs
as well as instructions or other printed material for
characterizing risk based upon the outcome of the assay.
[0087] As discussed above the invention provides methods for
evaluating the likelihood that an individual will benefit from
treatment with an agent for reducing risk of a future
cardiovascular disorder. This method has important implications for
patient treatment and also for clinical development of new
therapeutics. Physicians select therapeutic regimens for patient
treatment based upon the expected net benefit to the patient. The
net benefit is derived from the risk to benefit ratio. The present
invention permits selection of individuals who are more likely to
benefit by intervention, thereby aiding the physician in selecting
a therapeutic regimen. This might include using drugs with a higher
risk profile where the likelihood of expected benefit has
increased. Likewise, clinical investigators desire to select for
clinical trials a population with a high likelihood of obtaining a
net benefit. The present invention can help clinical investigators
select such individuals. It is expected that clinical investigators
now will use the present invention for determining entry criteria
for clinical trials.
[0088] In another surprising aspect of the invention, it has been
discovered that sCD40L has predictive value independent of other
known predictors of future adverse cardiovascular disorders. Thus,
the present invention does not involve simply duplicating a
measurement that previously could be made using other predictors.
Instead, levels of sCD40L are additive to prior art predictors.
Prior art predictors include markers of systemic inflammation, such
as C-Reactive Protein, cytokines, and cellular adhesion molecules.
Cytokines are well-known to those of ordinary skill in the art and
include human interleukins 1-17. Cellular adhesion molecules are
well-known to those of ordinary skill in the art and include
integrins, ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM.
Prior art predictors also include cholesterol.
[0089] In a further surprising aspect of the invention, it has been
discovered that sCD40L has predictive value on the presence of
vascular intra-plaque lipid accumulation in individuals at an
elevated risk of developing a cardiovascular disorder. As used
herein, individuals "at risk of developing a cardiovascular
disorder" are a category of subjects determined according to
conventional medical practice. (See, e.g., Harrison's Principles of
Experimental Medicine, 15th Edition, McGraw-Hill, Inc., New
York).
[0090] Typically, an individual at risk of developing a
cardiovascular disorder has one or more risk factors associated
with cardiovascular disease. Such risk factors include family
history of a cardiovascular disorder, hypertension,
hypercholesterolemia, diabetes, smoking, atherosclerosis, etc. In
addition, atrial fibrillation, or recent stroke and/or myocardial
infarction are important risk factors. Previously, determination of
vascular intra-plaque lipid accumulation in individuals at risk of
developing a cardiovascular disorder could only be accomplished
using expensive, and in certain cases limiting, technology (e.g.,
MRI).
[0091] It is known in the art that a major factor invoking coronary
thrombosis is disruption of an atherosclerotic plaque. As explained
elsewhere herein, determining intra-plaque lipid accumulation in
individuals at risk of developing a cardiovascular disorder is an
important step in determining plaque vulnerability (i.e. plaques at
risk of disruption), and thus assessing risk for the future
occurrence of a thrombotic event. Studies comparing intact and
disrupted plaques have been used to define the characteristics of
vulnerable plaques. The characteristics are a lipid core occupying
over 50% of overall plaque volume, a thin plaque cap, a large
absolute number and density of macrophages, and a reduction in the
smooth muscle content of the plaque. Thus, after determining
intra-plaque lipid accumulation in individuals at risk of
developing a cardiovascular disorder according to the methods of
the present invention, one of skill in the art could evaluate
whether the plaque is vulnerable and devise appropriate
theraputic/interventional regimens to prevent the occurrence of a
subsequent thrombotic event in the individual.
[0092] The invention also involves a method for treating subjects,
with therapies, to prevent cardiovascular disorders. An agent
selected from the group consisting of an anti-inflammatory agent,
an antithrombotic agent, an anti-platelet agent, a fibrinolytic
agent, a lipid reducing agent, a direct thrombin inhibitor, a
glycoprotein IIb/IIIa receptor inhibitor, or an agent that binds to
cellular adhesion molecules and inhibits the ability of white blood
cells to attach to such molecules, and/or any combinations thereof,
is administered to a subject who has an above-normal level of a
marker of systemic inflammation. The agent is administered in an
amount effective to lower the risk of the subject developing a
future cardiovascular disorder. In some embodiments the agent is a
non-aspirin anti-inflammatory agent. Agents are described elsewhere
herein.
[0093] An effective amount is a dosage of the agent sufficient to
provide a medically desirable result e.g., reduction in risk. The
effective amount will vary with the particular condition being
treated, the age and physical condition of the subject being
treated, the severity of the condition, the duration of the
treatment, the nature of the concurrent therapy (if any), the
specific route of administration and the like factors within the
knowledge and expertise of the health practitioner. For example, an
effective amount can depend upon the degree to which an individual
has abnormally elevated levels of sCD40L. It should be understood
that the agents used according to the invention are intended to
lower the risk of a cardiovascular disorder, that is, they are used
prophylactically. Thus, an effective amount is that amount which
can lower the risk of, slow or perhaps prevent altogether the
development of a cardiovascular disorder.
[0094] Generally, doses of active compounds would be from about
0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses
ranging from 50-500 mg/kg will be suitable, preferably orally and
in one or several administrations per day. Lower doses will result
from other forms of administration, such as intravenous
administration. In the event that a response in a subject is
insufficient at the initial doses applied, higher doses (or
effectively higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Multiple doses per day are contemplated to achieve
appropriate systemic levels of compounds.
[0095] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptably compositions. Such preparations may
routinely contain salt, buffering agents, preservatives, compatible
carriers, and optionally other therapeutic agents. When used in
medicine, the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0096] The anti-inflammatory agents may be combined, optionally,
with a pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or
more compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy.
[0097] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt.
[0098] The pharmaceutical compositions also may contain,
optionally, suitable preservatives, such as: benzalkonium chloride;
chlorobutanol; parabens and thimerosal.
[0099] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous preparation of the
anti-inflammatory agent, which is preferably isotonic with the
blood of the recipient. This aqueous preparation may be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
also may be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables. Carrier
formulation suitable for oral, subcutaneous, intravenous,
intramuscular, etc. administrations can be found in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
[0100] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular drug selected, the severity of the condition being
treated and the dosage required for therapeutic efficacy. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Such modes of administration include oral, rectal, topical, nasal,
intradermal, or parenteral routes. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, or infusion. Intravenous
or intramuscular routes are not particularly suitable for long-term
therapy and prophylaxis. They could, however, be preferred in
emergency situations. Oral administration will be preferred for
prophylactic treatment because of the convenience to the patient as
well as the dosing schedule.
[0101] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the anti-inflammatory agent into association with
a carrier which constitutes one or more accessory ingredients. In
general, the compositions are prepared by uniformly and intimately
bringing the anti-inflammatory agent into association with a liquid
carrier, a finely divided solid carrier, or both, and then, if
necessary, shaping the product.
[0102] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the anti-inflammatory
agent. Other compositions include suspensions in aqueous liquids or
non-aqueous liquids such as a syrup, elixir or an emulsion.
[0103] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of an agent of the present
invention, increasing convenience to the subject and the physician.
Many types of release delivery systems are available and known to
those of ordinary skill in the art. They include polymer base
systems such as poly(lactide-glycolide), copolyoxalates,
polycaprolactones, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the
foregoing polymers containing drugs are described in, for example,
U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer
systems that are: lipids including sterols such as cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-
di- and tri-glycerides; hydrogel release systems; sylastic systems;
peptide based systems; wax coatings; compressed tablets using
conventional binders and excipients; partially fused implants; and
the like. Specific examples include, but are not limited to: (a)
erosional systems in which an agent of the invention is contained
in a form within a matrix such as those described in U.S. Pat. Nos.
4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974
and 5,407,686. In addition, pump-based hardware delivery systems
can be used, some of which are adapted for implantation.
[0104] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic conditions.
Long-term release, are used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
[0105] The invention will be more fully understood by reference to
the following example. This example, however, is merely intended to
illustrate the embodiments of the invention and is not to be
construed to limit the scope of the invention.
EXAMPLES
Example 1
High Plasma Concentrations of sCD40L are Associated with Increased
Vascular Risk in Apparently Healthy Women
[0106] We performed a prospective, nested case control analysis
among participants in the Women's Health Study (WHS), an ongoing
primary prevention trial evaluating the efficiency of vitamin E and
low dose aspirin in 28,263 middle aged American women with no
history of cardiovascular disease or cancer..sup.11 Blood samples
were collected in EDTA and stored in liquid nitrogen until
analysis. For this study, 130 women who subsequently developed
either non-fatal myocardial infarction or stroke, or died from
acute cardiovascular events during the initial four-year follow-up
period were selected as `case` subjects. A committee of physicians
using standardized procedures classified endpoints. For each
confirmed case, a `control` participant of same age (.+-.2 years),
similar smoking status (former, current, never) and who remained
free of reported cardiovascular disease was selected.
[0107] Measurement of baseline plasma sCD40L concentrations used an
ELISA (BenderMedSystems; Vienna, Austria). Briefly, diluted (1:5)
plasma samples were applied in triplicate to 96-well plates
precoated with mouse-anti-human CD40L antibody and mixed (1:2) with
a horseradish-peroxidase-labeled secondary mouse-anti-human CD40L
antibody (2 h). Subsequently, plates were washed and antibody
binding determined by colorimetry employing TMB substrate.
Absorbance was read at 650 nm and plasma concentrations of sCD40L
determined by comparison with serial dilutions of recombinant human
CD40L. The analysis was performed in a blinded fashion. The
intra-assay variation among the triplicates for all samples was
less than 15 percent. Lipid levels were measured in a laboratory
which participates in the Centers for Disease Control
Standardization.
[0108] Means and proportions for baseline clinical characteristics
of the study participants were computed and compared using either
Student's T-test or the chi-square statistic. Relative risk of
developing future cardiovascular events associated with increasing
levels of sCD40L at baseline were then computed in a series of
logistic regression analysis which divided the study sample
according to the 50.sup.th, 75.sup.th, 90.sup.th, 95.sup.th, and
99.sup.th percentile cutpoints of the control distribution for
sCD40L. All p values are two-tailed and all confidence intervals
computed at the 95% level.
Results
[0109] Table I shows the baseline clinical characteristics of the
study participants. As expected, women who developed cardiovascular
disease during follow-up were more likely at study entry to be
obese, hypertensive, diabetic, or have a family history of
premature atherosclerosis compared to women who remained free of
disease. LDL cholesterol and triglyceride levels were higher at
baseline among cases, whereas HDL cholesterol levels were lower
(all p.ltoreq.0.01). Use of hormone replacement therapy did not
differ significantly between the two groups.
[0110] Overall, plasma levels of sCD40L at baseline among cases
exceeded that in controls (2.86.+-.0.35 vs. 2.09.+-.0.19 ng/mL;
p.ltoreq.0.02). This difference resulted almost completely from an
excess of particularly high values among the case subjects. The
great majority of cases and control subjects had similar levels of
sCD40L at study entry (FIG. 1). However, 11 cases had baseline
levels of sCD40L in excess of the 99.sup.th percentile cutpoint for
the control distribution as compared to only 1 study subject in the
control group (p.gtoreq.0.01).
[0111] Relative risks of developing future cardiovascular events,
according to the pre-specified cutpoints defined by the
distribution of the study controls, rose with increasing
concentrations of sCD40L and became statistically significant with
levels of sCD40L in excess of the 95.sup.th and 99.sup.th
percentile cutpoints (RR: 3.29 (p.ltoreq.0.02) and 11.83
(p.ltoreq.0.01), respectively) (Table II).
[0112] An additional post-hoc analysis was performed comparing
clinical characteristics among the 12 participants with levels of
sCD40L in excess of the 99.sup.th percentile cutpoint to the 248
participants with lower levels. Age, smoking, body mass index, LDL,
and HDL cholesterol levels and hormone replacement therapy were
similar between these two study groups (Table III). Study
participants with particularly elevated levels of sCD40L had
somewhat higher rates of hypertension and a family history of
premature coronary artery disease, but neither of these differences
achieved statistical significance. None of the 12 women with
markedly elevated baseline levels of sCD40L had diabetes. Moreover,
there were no significant differences in the time from
randomization to the time of the cardiovascular event between the
11 cases with extreme sCD40L levels and the remaining cases with
lower sCD40L (15.9 vs. 19.5 months, p=0.3). Assignment to aspirin
vs. placebo presumably did not affect our observation, since within
the group of 12 subjects showing the highest sCD40L concentrations
six were randomly assigned to aspirin and six to placebo. In
addition, the bloods assayed for sCD40L were drawn before
randomization.
[0113] Previous reports on this cohort have documented association
between plasma levels of C-reactive protein, IL-6, serum amyloid A
and ICAM-1 with increased cardiovascular risk..sup.12 However, we
observed no significant correlation between these parameters and
sCD40L.
Discussion
[0114] In this prospective, nested case control study of apparently
healthy middle aged women, markedly elevated plasma concentrations
of sCD40L at baseline (.gtoreq.5.5 ng/mL) foretold a significantly
increased risk of future cardiovascular events. Previous studies
demonstrated that patients with unstable angina had significantly
raised serum levels of sCD40L when compared with patients with
stable angina and controls..sup.10 In this circumstance activated
platelets and/or T lymphocytes may release sCD40L secondarily. The
present study, however, demonstrates elevation of sCD40L
concentrations in some women before events that may result from
acute thrombosis.
[0115] Little is known regarding the mechanisms yielding release of
soluble forms of CD40L. Potential source(s) for sCD40L in plasma
include platelets and T lymphocytes as well as mononuclear
phagocytes and endothelial cells..sup.1-5 The tendency of family
history for cardiovascular disease to correlate with enhanced
sCD40L plasma levels suggests that genetic factors, might
contribute to our observation. We found no association between
sCD40L and C-reactive protein, IL-6, and ICAM-1 levels. It is
therefore implied that CD40/CD40L-independent mechanisms may well
pertain to women developing cardiovascular complications despite
low sCD40L levels. Our discoveries suggest that high plasma
concentrations of sCD40L reflect aspects of risk distinct from
those gauged by other inflammatory markers.
REFERENCES INCORPORATED IN EXAMPLE 1
[0116] 1. Alderson M R, Armitage R J, Tough T W, et al. J Exp Med.
1993; 178:669-74.
[0117] 2. Reul R M, Fang J C, Denton M D, et al. Transplantation.
1997; 64:1765-74.
[0118] 3. Mach F, Schonbeck U, Sukhova G K, et al. Proc Natl Acad
Sci USA. 1997; 94:1931-6.
[0119] 4. Henn V, Slupsky J R, Grafe M, et al. Nature.
1998;391:591-4.
[0120] 5. Schonbeck U, Libby P. Cell Mol Life Sci. 2001;
58:4-43.
[0121] 6. Mach F, Schonbeck U, Sukhova G K, et al. Nature. 1998;
394:200-3.
[0122] 7. Schonbeck U, Sukhova G K, Shimizu K, et al. Proc Natl
Acad Sci U S A. 2000; 97:7458-63.
[0123] 8. Lutgens E, Cleutjens K B, Heeneman S, et al. Proc Natl
Acad Sci U S A. 2000; 97:7464-9.
[0124] 9. Graf D, Muller S, Korthauer U, et al. Eur J Immunol.
1995; 25:1749-54.
[0125] 10. Aukrust P, Muller F, Ueland T, et al. Circulation. 1999;
100:614-20.
[0126] 11. Buring J E, Hennekens C H. J. Myocard. Ischemia. 1992;
4:19-27.
[0127] 12. Ridker P M, Hennekens C H, Buring J E, et al. N Engl J
Med. 2000; 342:836-43.
DETAILED DESCRIPTION OF THE DRAWING
[0128] FIG. 1. Baseline serum concentrations of sCD40L among middle
aged healthy women which either stayed free of (controls, n=130) or
developed cardiovascular events (cases, n=130). Samples were
analyzed in triplicates, mean values are shown. The dotted line
depicts the 99.sup.th percentile cutpoint for the control
distribution. TABLE-US-00001 TABLE I Baseline characteristics of
study participants. Controls Cases (N = 130) (N = 130) P-value Age,
years 60.3 60.3 Matching criteria Smoking Status (%) Matching
criteria Current 26.9 26.9 Former 31.6 31.6 Never 41.5 41.5 Body
Mass Index (kg/m.sup.2) 25.7 27.6 0.004 Hypertension (%) 34.9 56.9
0.001 Family history of CAD (%)* 10.8 22.7 0.01 Diabetes (%) 3.1
10.8 0.02 Current HRT** (%) 40.0 44.6 0.1 LDL (mg/dL) 118.4 128.5
0.02 HDL (mg/dL) 48.4 42.6 0.01 Triglycerides (mg/dL) 136.5 161.0
0.01 sCD40L (ng/mL) 2.09 2.86 0.02 *Before age 60; **HRT, hormone
replacement therapy
[0129] TABLE-US-00002 TABLE II Baseline characteristics of study
participants. Cutpoint sCD40L Controls Cases P- (percentile)
(ng/mL) % (N) % (N) RR 95% CI value 50.sup.th >1.76 50.4 (65)
50.8 (66) 1.02 0.62-1.65 0.95 75.sup.th >2.15 24.9 (32) 30.8
(40) 1.35 0.78-2.33 0.29 90.sup.th >2.92 10.1 (13) 17.7 (23)
1.92 0.93-3.98 0.08 95.sup.th >3.71 5.0 (6) 13.9 (18) 3.29
1.26-6.59 0.02 99.sup.th >5.54 0.8 (1) 8.5 (11) 11.83 1.50-93.0
0.01
[0130] TABLE-US-00003 TABLE III Clinical characteristics of study
participants with sCD40L concentrations above 99.sup.th percentile
of the control distribution (.gtoreq.5.54 ng/mL sCD40L) sCD40L,
>99.sup.th sCD40L, .ltoreq.99.sup.th (N = 12) (N = 248) P-value
Age, years 63.1 60.2 0.3 Smoking Status (%) 0.7 Current 25.0 27.1
Former 33.3 47.7 Never 41.7 31.2 Body Mass Index (kg/m.sup.2) 27.3
26.7 0.7 Hypertension (%) 58.3 45.1 0.4 Family history of CAD (%)*
30.0 16.0 0.2 Diabetes (%) 0 7.3 0.9 Current HRT** (%) 50.0 41.7
0.8 LDL (mg/dL) 126.9 127.7 0.9 HDL (mg/dL) 49.8 48.4 0.7
Triglycerides (mg/dL) 165.5 174.3 0.8 *Before age 60; **HRT,
hormone replacement therapy
Example 2
HMG-CoA Reductase Inhibitors Limit CD40 and CD40L Expression in
Human Vascular Cells
[0131] This study tested the hypothesis that HMG-CoA reductase
inhibitors (statins) can diminish the expression of the
receptor/ligand dyad on these cells in vitro as well as of sCD40L
plasma levels in vivo, and that oxidatively modified LDL induces
the expression of CD40/CD40L on cells involved in atherosclerosis,
namely human vascular EC and SMC, as well as MO.
Materials and Methods
Materials
[0132] Native and oxidized (5 .mu.M CuSO.sub.4, 37.degree. C., 24
h) LDL (TBARS: 0.4 and 8.3 nM MDA/mg protein, respectively) were
obtained from Biomedical Technologies, Inc. (Stoughton, Mass.).
Human recombinant IL-1.beta., TNF.alpha., and IFN.gamma. were
obtained from Endogen (Woburn, Mass.).
Cell Isolation and Culture
[0133] Human vascular EC and SMC were isolated from saphenous veins
and cultured as described previously..sup.4,26 Mononuclear
phagocytes (MOO) were isolated from leukocyte concentrates by
density gradient centrifugation employing Lymphocyte Separation
Medium (Organon-Teknika, Durham, N.C.) and were cultured (10 days)
in RPMI 1640 (BioWhittaker, Walkersville, Md.) containing 2% human
serum (Sigma; St. Louis, Mo.)..sup.26 All cell types were cultured
24 h before and during the experiment in media lacking serum.
Viability of the cultures was determined by trypan blue (Sigma, St.
Louis, Mo.) exclusion count as well as an oligonucleosome formation
assay (Cell Death Detection ELISA, Boehringer Mannheim,
Germany).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
[0134] Total RNA isolated from cultured EC, SMC, or MO employing
RNAzol (Tel-Test; Friendswood, Tex.) was assessed for purity and
yield spectrophotometrically (2100 Bioanalyzer, Agilent
Technologies, Wilmington, Del) and was reverse-transcribed (2 .mu.g
total RNA; 50min, 42.degree. C.) employing Superscript II Reverse
Transcriptase (LifeTechnologies, Carlsbad, Calif.). PCR was
performed for 35 cycles at 95.degree. C. (120 sec), 62.degree. C.
(120 sec), and 72.degree. C. (180 sec, 2 sec prolongation per
cycle) after hot start, employing primers for CD40 or CD40L
previously described..sup.4 Semiquantitative PCR studies employing
20, 25, 30, 35, and 40 cycles verified that the conditions used
yielded PCR products within the exponential range of amplification
and were optimized for signal:background ratios . PCR products were
analyzed on ethidium bromide-containing 1.3% agarose gels and
visualized by UV transillumination. Loading of equal template
amounts was verified by RT-PCR for GAPDH. Mock RT reactions, either
lacking reverse transcriptase or employing H.sub.2O as template,
demonstrated specificity of the signals obtained.
Western Blot
[0135] Culture lysates (50 .mu.g total protein/lane) and
supernatants were separated by SDS-PAGE and blotted to
polyvinylidene difluoride membranes (Bio-Rad, Hercules, Calif.)
using a semi-dry blotting apparatus (3 mA/cm.sup.2, 30 min;
Bio-Rad, Hercules, Calif.). Blots were blocked and primary
(mouse-anti-human CD40 or CD40L; both 1:1,000; PharMingen, San
Diego, Calif.) antibodies were added in 5% defatted dry
milk/PBS/0.1% Tween 20. After 1 h, blots were washed three times
(PBS/0.1% Tween 20) and secondary, peroxidase-conjugated,
goat-anti-mouse antibody (Jackson Immunoresearch, West Grove, Pa.)
was added (1 h). Finally, blots were washed and immunoreactive
proteins were visualized using the Western blot chemiluminescence
system (NEN.TM., Boston, Mass.). Data were verified by employing
anti-CD40/-CD40L antibodies from Santa Cruz (Santa Cruz,
Calif.).
Flow Cytometry
[0136] Human vascular EC, SMC, or MO were washed with ice-cold PBS,
harvested by trypsinization, and fixed (PBS/4% paraformaldehyde,
15min). Subsequently, the cells were washed once with PBS/2% BSA
before being incubated (1 h, 4.degree. C.) with either buffer alone
or FITC-conjugated control IgG, mouse-anti-human CD40, or
mouse-anti-human CD40L antibody (1 .mu.g/ml; PharMingen, San Diego,
Calif.). Finally, cells were washed with PBS/2% BSA and analyzed in
a Becton Dickinson FACSCAN.RTM. flow cytometer employing
CELLQUEST.RTM. software (Becton Dickinson; San Jose, Calif.). At
least 20,000 viable cells per condition were analyzed.
Patient Studies
[0137] Whole blood (10 mL) was collected in EDTA from 27 patients
presenting for coronary arteriography who had at least a 30%
stenosis in one coronary artery. The cohort was divided into
patients who were or were not treated with any statin at the time
of catheterization. Blood was drawn at baseline (0 month) and final
follow-up visit (6 months), and plasma was stored at -70.degree. C.
All subjects were studied in the fasting state. Written informed
consent was obtained from all subjects and the study was approved
by the Human Research Committee of Brigham and Women's Hospital.
Plasma lipids as well as IL-1.beta., IL-6, TNF.alpha., IFN.gamma.,
sVCAM, C-reactive protein (CRP), and sCD40L were measured by ELISA
(Sigma, St. Louis, Mo.; Endogen, Woburn, Mass.; BenderMedSystems,
Vienna, Austria). The two groups did not differ significantly in
age, gender, diabetes mellitus, smoking, triglycerides, or HDL
(Table I). The statin-treated group had significantly lower total
cholesterol and LDL, as expected.
Ex vivo Fibrin Clot Formation
[0138] Blood was collected from mice by retro-orbital bleeding into
0.1 volume of 0.13M trisodium citrate using non-coated capillary
tubes. Platelet-rich plasma (PRP) was prepared by centrifugation
(500.times.g, 5 min, 20.degree. C.) and fibrin clot formation was
examined using a modification of a microtiter-plate clot lysis
assay described previously..sup.27 Clots were prepared with 2.94
.mu.M fibrinogen, 0.24 .mu.M plasminogen, 36 .mu.M t-PA, 3.8 nM
thrombin, and 5.3 mM CaCl.sub.2 (all final concentrations). PRP
(final concentration of 10% (v/v)) was incorporated into clots.
Clot formation was monitored at 405nm for up to 15 min.
Statistical Analysis
[0139] Data are presented as mean+SD and groups were compared using
the Student's t-test. A value of p.ltoreq.0.05 was considered
significant.
Results
HMG-CoA Reductase Inhibitors Diminish the Expression of CD40 and
CD40L on Human Vascular Endothelial and Smooth Muscle Cells, as
Well as Macrophages in vitro
[0140] Exposure to HMG-CoA reductase inhibitors
concentration-dependently diminished the expression of both CD40
and CD40L in human vascular EC, SMC, and MO. At concentrations
.gtoreq.2 nM cerivastatin diminished the constitutive as well as
IL-1.beta./TNF.alpha./IFN.gamma.-induced expression of the receptor
in EC. Maximal inhibition was achieved at 10-50 nM cerivastatin.
Mevalonate reversed the diminished expression of CD40 and CD40L by
statins. Parallel studies employing oxLDL as a stimulus or SMC or
MO as the cell type yielded similar results. To determine whether
the modulation of the CD40/CD40L expression extends to other
statins, parallel experiments employed atorvastatin or simvastatin.
These HMG-CoA reductase inhibitors similarly reduced CD40/CD40L
expression, but required higher concentrations (100 nM).
Re-development of the Western blots with a mouse-anti-human GAPDH
antibody, providing similar band-intensities across the blots,
verified equal loading among the lanes, and also suggested that
statin treatment per se did not affect Western blot analysis. Of
note, neither statin affected cell number or viability at the
concentrations analyzed (up to 250 nM), as determined by trypan
blue exclusion cell count, as well as mono/oligonucleosome
formation.
[0141] HMG-CoA reductase inhibitors moreover diminished CD40 and
CD40L mRNA expression in human vascular EC and MO stimulated either
with pro-inflammatory cytokines (IL-1.beta./TNF.alpha./IFN.gamma.)
or oxLDL. Atorvastatin or simvastatin yielded similar results.
Mevalonate reversed the diminished expression of CD40 and CD40L
transcripts. Parallel studies analyzing the expression of GAPDH
transcripts demonstrated application of equal amounts of reverse
transcribed mRNA to each reaction and furthermore suggested that
treatment with statins per se did not affect the RT-PCR.
[0142] In accord with the findings for whole cell lysates,
cerivastatin concentration-dependently diminished the cell surface
expression of both CD40 and CD40L on human vascular EC, as well as
MO. Notably, the cerivastatin concentrations required for minimal
and maximal reduction in CD40 and CD40L surface expression
resembled those observed for whole cell lysates in the Western blot
as well as the RT-PCR studies. All three statins acted similarly,
but required different concentrations to achieve equal reduction in
CD40/CD40L expression
(cerivastatin>simvastatin=atorvastatin).
[0143] Since CD40L can also be shed as a biologically active
soluble form, sCD40L, we tested whether treatment of EC, SMC, and
MO with HMG-CoA reductase inhibitors affected the secretion of
sCD40L. Indeed, cerivastatin treatment markedly lowered sCD40L
concentrations in culture.
[0144] Combined, RT-PCR, Western blot, and FACS studies suggest
that oxLDL induces and statins diminish the cell surface expression
and/or release of CD40 or CD40L by regulating gene activity rather
than intracellular translocation.
HMG-CoA Reductase Inhibitors Diminish sCD40L Plasma Levels in
Humans
[0145] To assess the potential clinical relevance of these in vitro
findings, we performed a pilot study to determine whether treatment
of patients with HMG-CoA reductase inhibitors diminished sCD40L
plasma levels. Plasma of statin-treated atherosclerotic subjects
had significantly lower levels of sCD40L compared to non-treated
patients (8.3.+-.3.1 ng/ml (n=11) vs. 13.1.+-.2.5 ng/ml (n=16);
p<0.05). The individual sCD40L levels did not vary significantly
if the treatment status of the patient at baseline and 6 month
follow-up did not change. However, sCD40L plasma levels at 6 month
follow-up decreased markedly in patients changed to statin
treatment (13.1.+-.5.74 ng/ml vs. 5.21.+-.2.36 ng/ml (n=4);
p<0.05). Plasma levels of IL-1.beta., IL-6, TNF.alpha.,
IFN.gamma., and sVCAM-1 did not change with statin treatment.
Plasma concentrations of C-reactive protein (CRP) were lower in the
statin-treated group, although the difference did not achieve
statistical significance.
Oxidized LDL Induces Expression of CD40 and CD40L in Human Vascular
Cells
[0146] Oxidatively modified LDL (oxLDL) concentration-dependently
enhanced the faint constitutive expression of CD40 and CD40L
protein in human vascular EC and MO. Augmentation of basal
CD40/CD40L expression on either cell type required 1-3 .mu.g
oxLDL/ml. Maximal expression of CD40 (5.1.+-.1.1 fold above
non-stimulated control; n=4) and CD40L (4.2.+-.2.2 fold; n=3)
immunoreactive protein was achieved with 10-30 .mu.g oxLDL/ml.
Native LDL also induced expression of the receptor (2.8.+-.1.1
fold; n=2) and ligand (2.2.+-.0.8 fold; n=2), although to a lesser
extent. Furthermore, oxLDL concentration-dependently augmented the
expression of CD40 and CD40L mRNA in human vascular EC or MO.
Parallel studies analyzing the expression of GAPDH transcripts
demonstrated application of equal amounts of reverse transcribed
mRNA amounts to each reaction and furthermore suggested that oxLDL
stimulation per se did not affect the RT-PCR.
Deficiency of CD40L Prolongs Time of Coagulation of Murine Blood ex
vivo
[0147] In light of recent data implicating CD40L in thrombosis and
the statin-mediated lowering of plasma sCD40L levels shown above,
we further tested the hypothesis that diminished expression of
CD40L affects blood coagulation. Indeed, platelet-rich plasma from
CD40L-deficient mice showed delayed coagulation in a fibrin clot
formation assay when compared to wildtype mice preparations,
supporting a role for CD40L in blood coagulation. These
observations on the role of CD40L in blood coagulability provide a
pathway by which reduction in CD40 signaling via HMG-CoA reductase
inhibitors might reduce thrombotic complications of
atherosclerosis.
Discussion
[0148] Clinical benefits in patients with average or below average
LDL levels and reduced cardiovascular risk independent of the
degree of LDL-lowering in a consistent series of previous clinical
trials have highlighted the potential clinical relevance of the
putative "pleiotropic" effects of statins..sup.21-24,28 In addition
to their lipid-lowering effects, numerous clinical and experimental
studies have suggested anti-inflammatory pathways of statins, such
as diminished expression of chemokines, major histocompatibility
complex II molecules, matrix-degrading enzymes, and the
procoagulant tissue factor, as well as the augmented expression of
nitric oxide..sup.22,23,25,28 Moreover, treatment of Watanabe
heritable hyperlipidemic rabbits with HMG-CoA reductase inhibitors
diminishes the expression of numerous pro-atherogenic inflammatory
mediators in vivo..sup.29,30 Although statins lowered lipids only
modestly in these rabbits lacking LDL-receptors, these in vivo
observations could not conclusively distinguish the degree to which
effects on lipoproteins account for the anti-inflammatory effects
observed. The present report provides evidence for a novel
anti-inflammatory pathway by which statins may act both dependently
and independently of lipid-lowering. Three members of this drug
class (cerivastatin, atorvastatin, or simvastatin) significantly
diminished the constitutive as well as cytokine-induced expression
of CD40 and CD40L protein and transcript in cell types implicated
in atherosclerosis, namely human vascular EC, SMC, and MO, arguing
for a lipid-lowering independent function of statins. However, the
identification of oxLDL as an inducer of CD40 and CD40L in these
cell types further suggests that statins might affect CD40/CD40L
expression, at least in part, also via their lipid-lowering
properties. Of note, previous studies have colocalized oxLDL with
CD40 and CD40L within early human atherosclerotic lesions,.sup.31 a
finding in accord with our hypothesis that oxLDL provides an
initial signal for the expression of the CD40 receptor/ligand dyad
in atherosclerotic plaques.
[0149] The pilot observation that patients treated with statins
have diminished levels of sCD40L supports the clinical relevance of
the present in vitro observations. Several cell types might give
rise to sCD40L. Platelets release sCD40L upon ligation of the
thrombin receptor in vitro as well as upon thrombus formation in
vivo..sup.32,33 However, as suggested by our own and other previous
studies, other cell types, including EC, MO, and T lymphocytes,
might also generate sCD40L..sup.11,12,17,18
[0150] The present observation that CD40L-deficient platelet-rich
plasma clots more slowly than preparations from wild type mice
suggests that CD40L, in its membrane-bound and/or soluble form,
modulates thrombosis, a crucial determinant of cardiovascular risk.
The finding that CD40L can activate platelets by functioning as an
.alpha..sub.IIb.beta..sub.3 ligand further supports this
hypothesis..sup.35
REFERENCES INCORPORATED IN EXAMPLE 2
[0151] 1. Libby P, Hansson G K. Lab. Invest. 1991; 64:5-15.
[0152] 2. Ross R. N Engl J Med 1999; 340:115-126.
[0153] 3. Reul R M, Fang J C, Denton M D, et al. Transplantation.
1997; 64:1765-1774.
[0154] 4. Mach F, Schonbeck U, Sukhova G K, et al. Proc Natl Acad
Sci U S A. 1997; 94:1931-1936.
[0155] 5. Gaweco A S, Wiesner R H, Yong S, et al. Liver Transpl.
Surg. 1999; 5:1-7.
[0156] 6. Afford S C, Randhawa S, Eliopoulos A G, et al. J Exp Med.
1999; 189:441-446.
[0157] 7. Malik N, Greenfield B W, Wahl A F, et al. J Immunol.
1996; 156:3952-3960.
[0158] 8. Zhou L, Stordeur P, de Lavareille A, et al. Thromb.
Haemost. 1998; 79:1025-1028.
[0159] 9. Miller D L, Yaron R, Yellin M J. J Leukoc Biol. 1998;
63:373-379.
[0160] 10. Slupsky J R, Kalbas M, Willuweit A, et al. Thromb
Haemost. 1998; 80:1008-1014.
[0161] 11. Schonbeck U, Libby P. Cell Mol Life Sci. 2001;
58:4-43.
[0162] 12. Schonbeck U, Libby P. Circ Res. 2001; 89:1092-1103.
[0163] 13. Mach F, Schonbeck U, Sukhova G K, et al. Nature. 1998;
394:200-203.
[0164] 14. Lutgens E, Gorelik L, Daemen M J, et al. Nat Med. 1999;
5:1313-1316.
[0165] 15. Schonbeck U, Sukhova G K, Shimizu K, et al. Proc Natl
Acad Sci U S A. 2000; 97:7458-7463.
[0166] 16. Lutgens E, Cleutjens K B, Heeneman S, et al. Proc Natl
Acad Sci U S A. 2000; 97:7464-7469.
[0167] 17. Graf D, Muller S, Korthauer U, et al. Eur J Immunol.
1995; 25:1749-1754.
[0168] 18. Ludewig B, Henn V, Schroder J M, et al. Eur J Immunol.
1996; 26:3137-3143.
[0169] 19. Aukrust P, Muller F, Ueland T, et al. Circulation. 1999;
100:614-620.
[0170] 20. Schonbeck U, Varo N, Libby P, et al. Circulation. 2001;
104:2266-2268.
[0171] 21. Vaughan C J, Murphy M B, Buckley B M. Lancet. 1996;
348:1079-1082.
[0172] 22. Dangas G, Smith D A, Unger A H, et al. Thromb Haemost.
2000; 83:688-692.
[0173] 23. Ni W, Egashira K, Kataoka C, et al. Circ Res. 2001;
89:415-421.
[0174] 24. Heeschen C, Hamm C W, Laufs U, et al. Circulation. 2002;
105:1446-1452.
[0175] 25. Kwak B, Mulhaupt F, Myit S, et al. Nat Med. 2000;
6:1399-1402.
[0176] 26. Schonbeck U, Mach F, Sukhova G K, et al. J Exp Med.
1999; 189:843-853.
[0177] 27. Robbie L A, Booth N A, Croll A M, et al. Thromb Haemost.
1993; 70:301-306.
[0178] 28. Libby P, Aikawa M, Schonbeck U. Biochim Biophys Acta.
2000; 1529:299-309.
[0179] 29. Bustos C, Hernandez-Presa M A, Ortego M, et al. J Am
Coll Cardiol. 1998; 32:2057-2064.
[0180] 30. Aikawa M, Rabkin E, Okada Y, et al. Circulation. 1998;
97:2433-2444.
[0181] 31. Hakkinen T, Karkola K, Yla-Herttuala S. Virchows Arch
2000; 437:396-405.
[0182] 32. Henn V, Slupsky J R, Grafe M, et al. Nature. 1998;
391:591-594.
[0183] 33. Viallard J F, Solanilla A, Gauthier B, et al. Blood.
2002; 99:2612-2614.
[0184] 34. Garlichs C D, John S, Schmeisser A, et al. Circulation.
2001; 104:2395-2400.
[0185] 35. Andre P, Prasad K S, Denis C V, et al. Nat Med. 2002;
8:247-252.
Example 3
Soluble CD40 Ligand Levels Indicate Lipid Accumulation in Carotid
Atheroma
[0186] A major factor invoking coronary thrombosis is disruption of
an atherosclerotic plaque. Studies comparing intact and disrupted
plaques have been used to define the characteristics of vulnerable
plaques i.e. those at risk of disruption. The characteristics are a
lipid core occupying over 50% of overall plaque volume, a thin
plaque cap, a large absolute number and density of macrophages, and
a reduction in the smooth muscle content of the plaque. Such
vulnerable plaques make up a small proportion of all the plaques
present in most individuals. Angiographic stenosis, however, does
not predict vulnerability because there is no relation between core
size or plaque size with stenosis. A large proportion of disruption
episodes go unnoticed clinically because the thrombus does not
sufficiently encroach on the lumen to cause ischaemia. These
subclinical episodes, however, will invoke plaque growth. Plaque
disruption is followed by a smooth muscle proliferative repair
response analogous to that occurring after angioplasty. In both
situations, exuberant repair leads to post event stenosis.
Reconstruction of coronary lesions at autopsy shows that 70% of
high grade stenosis (angiographic >50% diameter) have had an
episode of healed disruption. Such data highlight the role of
plaque disruption in the generation of advanced stenotic lesions
irrespective of whether an acute clinical event occurred.
[0187] In summary, the structure and the dynamic biology of the
atheroma, rather than the severity of stenosis, largely determine
cardiovascular events. Large lipid pools and thin fibrous caps
characterize vulnerable plaque, and inflammatory mechanisms play a
pivotal role in determining plaque stability..sup.1,2 Much of our
knowledge of the unstable atheroma derives from post-mortem
examination, and less is known about the relationships of
inflammatory mechanisms and lesion structure in vivo.
[0188] Evidence from animal studies supports the importance of CD40
ligand as inhibition of CD40 signaling in atherosclerosis-prone
mice reduced the size and lipid content of aortic lesions, and
yielded a relative increase in smooth muscle content and fibrillar
collagen..sup.9 Moreover, as discussed elsewhere herein (see
Example 1), elevated plasma levels of soluble CD40 ligand at
baseline predict prospectively cardiovascular events among
apparently healthy women.
[0189] Recent advances in magnetic resonance imaging (MRI) have
permitted non-invasive assessment of carotid plaque
composition..sup.11-13 Specifically, use of a custom-made phased
array carotid coil has demonstrated high levels of agreement
between carotid MRI findings and results at histology among 22
patients undergoing carotid endarterectomy (89% agreement;
kappa=0.83; 95% confidence interval 0.67-1.0)..sup.14 Furthermore,
high-resolution carotid MRI accurately detects intra-plaque
lipid-rich cores..sup.12,13,15,16
[0190] This study tested the hypothesis that elevated plasma levels
of soluble CD40 ligand correlated with features suggestive of
lipid-rich cores on high-resolution carotid MRI.
Methods
[0191] We invited men and women with stenoses greater than or equal
to 30 percent in either internal or common carotid artery by
carotid ultrasonography to participate in the study, from January
2001 to January 2002. Any patient with a pacemaker or implantable
cardioverter defibrillator was excluded, as well as patients who
had received surgical clips or coronary stents in the previous two
months. Patients requiring systemic corticosteroids for a systemic
inflammatory condition were also excluded. The study population
comprised the 49 consecutive patients who gave informed written
consent to participate in the study. The study was approved by the
Human Research Committee of Brigham and Women's Hospital.
[0192] A detailed medical history including prior cardiovascular
history, risk factors, and medication use was recorded by a study
physician for each participant. A blood sample was drawn by
non-traumatic venipuncture, centrifuged, and the plasma stored in
EDTA at -80.degree. C.
[0193] MRI protocol: The patients underwent high resolution MRI of
the carotid arteries using a dedicated phased array carotid coil
(IGC, Inc.) on a 1.5T Signa CV/i MRI scanner (GE Medical Systems,
Milwaukee, Wis.). 3D time-of-flight images, moderately proton
density weighted images, and fat-suppressed moderately T2-weighted
images were obtained. For the 3D time-of-flight sequences,
parameters were as follows: echo time (TE) 3.5 ms, repetition time
(TR) 33-40 ms, flip angle 25 degrees, bandwidth 15.63 kHz, field of
view 12-14 cm, slice thickness 2 mm interpolated to 1 mm, 32
slices, acquired matrix 256.times.256, reconstructed matrix
512.times.512, with one excitation. For the moderately proton
density weighted sequence a TE of 21-22 ms was used, and for the
moderately T2-weighted acquisition a TE of 53-58 ms was used with
chemical-selective fat suppression. Parameters for these scans
were: TR 2 R-R intervals, echo train length 16, bandwidth 62.5 kHz,
field of view 14 cm, slice thickness 3 mm, acquired matrix
256.times.256, reconstructed matrix 512.times.512, with one
excitation. Slice levels were centered at the carotid bifurcation
in each patient. Two board-certified radiologists blinded to all
other information determined the presence or absence of
intra-plaque lipid, based on the loss of signal between the proton
density weighted images and the fat-suppressed moderately-T2
weighted fast spin echo images with iso-intense signal on 3D
time-of-flight imaging..sup.15 The percent diameter stenosis was
calculated as the difference between reference and stenotic
diameters on 3D time-of-flight axial images, divided by the
reference diameter and multiplied by 100. The images were read
independently off-line. Inter-observer variability was <10%.
[0194] Baseline plasma soluble CD40 ligand concentrations were
measured by ELISA as previously described..sup.10 In brief, 1:5
diluted plasma samples were applied in triplicate to 96-well plates
precoated with mouse anti-human CD40 ligand antibody and mixed
(1:2) with a horseradish-peroxidase-labeled secondary mouse
anti-human CD40 ligand antibody for two hours. Plates were then
washed and antibody binding determined by colorimetry using
3,3'-5,5'-tetramethyl benzidine substrate. Absorbance was read at
650 nm and plasma concentrations of soluble CD40 ligand were
determined by comparison with serial dilutions of recombinant human
CD40 ligand. Intra-assay variation among the triplicates for all
samples was less than 10% and inter assay variability was 7.4%.
[0195] We divided the study participants into two groups, those
with evidence of intra-plaque lipid on carotid MRI, and those
without evidence of intra-plaque lipid. Median levels of soluble
CD40 ligand were computed and compared between the two groups using
Wilcoxon's ranked sum test. Relative risks of having intra-plaque
lipid associated with elevated soluble CD40 ligand levels were
computed by use of logistic regression models that divided the
study sample according to the median level of soluble CD40 ligand
among those without evidence of intra-plaque lipid.
Results
[0196] Of the 49 patients enrolled, carotid MRI images could not be
obtained for 3 patients due to claustrophobia. We utilized the
observations for analyses of the remaining 46 patients. The
baseline clinical characteristics of the patients (Table IV)
revealed that there was a high prevalence of a history of
hypertension, diabetes, and hypercholesterolemia in the overall
study cohort. Thirteen of the 46 patients (28.3%) had a prior
history of transient ischemic attack (TIA) (n=8) or stroke (n=6);
one patient had suffered both TIA and stroke. The remaining 33
patients (71.7%) were asymptomatic.
[0197] Fourteen patients had evidence of intra-plaque lipid and 32
did not. Patients with evidence of intra-plaque lipid more likely
had diabetes (p=0.02) than those patients without evidence of
intra-plaque lipid. There was also a trend towards an increased
proportion of women (p=0.1), patients with a history of
hypertension (p=0.16), and current smokers (p=0.13) in the group
with intra-plaque lipid. Mean percent carotid diameter stenosis
(58.+-.20 vs 56.+-.24) did not differ between those with and
without evidence of intra-plaque lipid.
[0198] Subjects with intra-plaque lipid had higher baseline levels
of soluble CD40 ligand than among those without lipid accumulations
(median 2.54 ng/ml [interquartile range (IQR) 1.85-3.52] vs median
1.58 ng/ml [IQR 1.21-2.39]; p=0.02). In contrast, soluble CD40
ligand levels did not correlate with percent diameter stenosis
(r=-0.19; p=0.21). The relative risk for intra-plaque lipid
associated with soluble CD40 ligand levels above the median was 6.0
(95% confidence interval 1.15-31.23; p=0.03). The magnitude of this
predictive effect did not substantially change when analyzed by a
multivariable model controlling for the effects of gender,
diabetes, hypertension, current smoking, percent stenosis, and
ratio of total cholesterol to high density lipoprotein cholesterol
(relative risk 5.12, 95% confidence interval 0.78-33.73; p=0.09).
TABLE-US-00004 TABLE IV Baseline Clinical Characteristics of the
Study Population. Intra-plaque P value Total No Intra-plaque lipid
(Group 1 Cohort lipid Group 1 Group 2 vs Group (n = 46) (n = 32) (n
= 14) 2) Age (years) 70.5 .+-. 8.1 71.0 .+-. 7.9 69.4 .+-. 8.8 0.5
mean .+-. SD Male Gender 31/46 24/32 (75%) 7/14 (50%) 0.10 (67.4%)
History of 15/46 7/32 (21.9%) 8/14 (57.1%) 0.02 Diabetes (32.6%)
History of 37/46 24/32 (75%) 13/14 (92.9%) 0.16 Hypertension
(80.4%) Current 5/46 2/32 (6.3%) 3/14 (21.4%) 0.13 Smoker (10.9%)
History of High 34/46 23/32 (71.9%) 11/14 (78.6%) 0.6 Cholesterol
(73.9%) Prior TIA or 13/46 8/32 (25%) 5/14 (35.7%) 0.46 Stroke
(28.3%) Statin use 31/46 20/32 (62.5%) 11/14 (78.6%) 0.28 (67.4%)
Percent 57% .+-. 23 56% .+-. 24 58% .+-. 20 0.6 diameter stenosis
Soluble CD40 1.89 1.58 2.54 0.02 ligand (ng/ml) [1.35- [1.21-2.39]
[1.85-3.52] median [inter- 2.64] quartile range] TIA = transient
ischemic attack
Discussion
[0199] As described elsewhere herein (Example 1), baseline plasma
levels of soluble CD40 ligand prospectively predict cardiovascular
events among apparently healthy women. The current data provide
novel insight into the mechanism through which elevated levels of
soluble CD40 ligand may reflect future cardiovascular risk in
humans. We found an association between elevated plasma levels of
soluble CD40 ligand and carotid plaques with features of high risk
without relation to the severity of stenosis. These data agree with
evidence from studies showing that interruption of CD40 signaling
reduced the size and lipid content of aortic lesions in
atherosclerosis-prone mice..sup.9
[0200] Previous work has found that high-resolution carotid MRI,
using a similar phased array carotid coil, can accurately predict
histological findings of lipid pool following carotid
endarterectomy .sup.15; our MRI protocol relies more on T2-weighted
protocols, which other studies have shown can accurately
distinguish lipid pool..sup.12,13,16 The predictive effect of
soluble CD40 ligand persisted after adjustment for traditional
cardiovascular risk factors, although the confidence intervals did
widen in the adjusted analysis, as might be expected given that
CD40 ligation appears to represent a common causal pathway in lipid
pool formation.
[0201] In conclusion, we believe that this study establishes a link
between plasma levels of CD40 ligand and intra-plaque lipid, which
represents one potentially important marker of plaque
vulnerability.
REFERENCES INCORPORATED IN EXAMPLE 3
[0202] 1. Davies M J. Circulation 1996; 94:2013-20.
[0203] 2. Libby P Circulation 1995; 91:2844-50.
[0204] 3. Karmann K, et al. Proc Natl Acad Sci USA 1995;
92:4342-6.
[0205] 4. Kornbluth R S, et al. Proc Natl Acad Sci USA 1998;
95:5205-10.
[0206] 5. Mach F, et al. J Clin Invest 1999; 104:1041-50.
[0207] 6. Denger S, et al. Atherosclerosis 1999; 144:15-23.
[0208] 7. Mach F, et al. Circulation 1997; 96:396-9.
[0209] 8. Schonbeck U, Am J Pathol 2000; 156:7-14.
[0210] 9. Schonbeck U, J Exp Med 1999; 189:843-53.
[0211] 10. Schonbeck U, Circulation2001; 104:2266-8.
[0212] 11. Fayad Z A, Fuster V. Circ Res 2001; 89:305-16.
[0213] 12. Shinnar M, et al. Arterioscler Thromb Vasc Biol 1999;
19:2756-61.
[0214] 13. Serfaty J M, et al. Radiology 2001; 219:403-10.
[0215] 14. Hatsukami T S, et al. Circulation 2000; 102:959-64.
[0216] 15. Yuan C, et al. Circulation 2001; 104:2051-6.
[0217] 16. Toussaint J F, et al. Arterioscler Thromb Vasc Biol
1995; 15:1533-42.
Equivalents
[0218] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0219] All references disclosed herein are incorporated by
reference in their entirety.
* * * * *