Note: Descriptions are shown in the official language in which they were submitted.
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ALDOSTERONE ANTAGONIST AND NON-STEROIDAL ANTI
INFLAMMATORY AGENT COMBINATION THERAPY TO PREVENT OR
TREAT CARDIOVASCULAR DISORDERS
Field of the Invention
This invention is in the field of preventing or
treating cardiovascular disorders. More specifically,
this invention relates to the use of aldosterone
antagonist and non-steroidal anti-inflammatory drug
(NSAID) combination therapy in preventing or treating
cardiovascular disease including atherosclerosis.
Background of the Iaventioa
Prostaglandins play a major role in the
inflammation process and the inhibition of prostaglandin
production, especially production of PGG~, PGH2 and
PGE2~ has been a common target of anti-inflammatory drug
discovery. However, common non-steroidal anti-
inflammatory drugs (NSAIDs) that are active in reducing
the prostaglandin-induced pain and swelling associated
with the inflammation process are also active in
affecting other prostaglandin-regulated processes not
associated with the inflammation process. Thus, use of
high doses of most common NSAID's can produce severe
side effects, including life threatening ulcers, that
limit their therapeutic potential. An alternative to
NSAID's is the use of corticosteroids, which also
produce severe adverse effects, especially when long
term therapy is involved.
NSAIDs have been found to prevent the production of
prostaglandins by inhibiting enzymes in the human
arachidonic acid/prostaglandin pathway, including the
enzyme cyclooxygenase (COX). Recently an inducible
enzyme associated with inflammation (named
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"cyClooxygenase-2 (COX-2)" or "prostaglandin G/H
synthase II") was discovered.
Several studies have suggested that inflammation
plays a role in cardiovascular diseases. For example,
Ridker et al. (New Eng. J. Med., 336, 973-9 (1997))
describes a possible role of inflammation in
cardiovascular disease. J. Boyle (J. Path., 181, 93-9
(1997)) describes the association of plaque rupture and
atherosclerotiC inflammation.
In the treatment or prevention of cardiovascular
disorders, present drug therapies are not always
effective or well tolerated by the subjects undergoing
therapy. Accordingly new drug therapies are necessary
to fill this need. The present invention is therefore
directed to a novel drug therapy employing a combination
of an aldosterone antagonist and NSAID to treat or
prevent cardiovascular disorders. More specifically,
this invention relates to the use of aldosterone
antagonist and NSAID combination therapy in preventing
or treating cardiovascular disorders.
Brief Description of the Drawings
Fig. 1 shows changes in systolic blood pressure in
angiotensin II infused rat study.
Fig. 2 shows prevention by eplerenone
(epoxymexrenone) of vascular inflammation in the heart
of angiotensin II infused rats.
Fig. 3 shows lack of cyclooxygenase-2 (COX-2)
expression in the heart of a vehicle infused rat.
Fig. 4 shows induction of COX-2 expression in heart
of Ang II infused rat.
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Fig. 5 shows prevention by eplerenone of induction
of COX-2 expression in heart of Ang II infused rat.
Fig. 6 shows lack of osteopontin expression in the
heart of a vehicle infused rat.
Fig. 7 shows prevention by eplerenone of induction
of osteopontin expression in heart of aldosterone
infused rat.
Fig. 8 shows prevention by eplerenone of
osteopontin upregulation in myocardium of aldosterone
infused rats.
Fig. 9 shows prevention by eplerenone of COX-2
upregulation in myocardium of aldosterone infused rats.
Fig. 10 shows prevention by eplerenone of
myocardial injury in aldosterone infused rats.
Fig. 11 shows upregulated co-expression of C0X-2
and osteopontin in coronary artery media of aldosterone
infused rat.
Fig. 12 shows.some of the mechanisms for
aldosterone-induced vascular inflammation and injury.
Fig. 13 shows inhibition of increased urinary
protein excretion by eplerenone treatment in angiotensin
II infused, captopril treated stroke prone spontaneously
hypertensive rats.
Fig. 14 shows reduction in histopathological scores
for renal injury with eplerenone treatment in
angiotensin II infused, captopril treated stroke prone
spontaneously hypertensive rats.
Fig. 15 shows increased survival and reduced
cerebral injury with eplerenone~ treatment in stroke-
prone spontaneously hypertensive rats.
Fig. 16 shows decrease in cerebral injury with
eplerenone treatment in stroke-prone spontaneously
hypertensive rats.
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Fig. 17 shows inhibition of early time-course
expression of myocardial COX-2 in aldosterone-infused,
hypertensive rats treated with eplerenone.
Fig. 18 shows inhibition of early time-course
expression of myocardial osteopontin in aldosterone-
infused, hypertensive rats treated with eplerenone.
Fig. 19 shows inhibition of early time-course
expression of myocardial MCP-1 in aldosterone-infused,
hypertensive rats treated with eplerenone.
Fig. 20 shows inhibition of early time-course
expression of myocardial ICAM-1 and VCAM-1 in
aldosterone-infused, hypertensive rats treated with
eplerenone.
Fig. 2l shows systolic blood pressure elevation
with aldosterone infusion, and depression of this
elevation with aldosterone infusion and eplerenone
treatment. °
Fig. 22 shows myocardial histopathology scores at
28 days for control rats, for rats infused with
aldosterone, and for rats infused with aldosterone and
treated with eplerenone, and the ratio of heart weight
to body weight for rats infused with aldosterone, and
for rats infused with aldosterone and treated with
eplerenone.
Fig. 23 shows 28 day circulating osteopontin levels
for control rats, for rats infused with aldosterone, and
for rats infused with aldosterone and treated with
eplerenone.
Fig. 24 shows the relative mRNA expression at 28
days for inflammatory cytokines in control rats, in rats
infused with aldosterone, and in rats infused with
aldosterone and treated with eplerenone.
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Detailed Description of the Invention
The present invention provides a method for
5 preventing or treating cardiovascular disorders in a
subject in need thereof. The method comprises treating
the subject with a therapeutically effective amount of
an aldosterone receptor antagonist (including, but not
limited to, derivatives or pharmaceutically-acceptable
salts thereof) in combination with a NSAID (including,
but not limited to, derivatives or pharmaceutically-
acceptable salts thereof).
The method above would be useful for, but not
limited to, preventing or treating inflammation-related
disorders in a subject, including but not limited to
inflammation-related disorders of the heart, kidney and
brain, particularly vascular inflammation-related
disorders. The method would be useful for prevention or
treatment of hypertension, heart failure, heart failure
folloing myocardial infarction, congestive heart
failure, coronary artery disease, aneurysm,
arteriosclerosis, atherosclerosis including cardiac
transplant atherosclerosis, myocardial infarction,
embolism, stroke, thrombosis, including venous
thrombosis, angina including unstable angina,
calcification (such as vascular calcification and valvar
calcification), Kawasaki disease and inflammation (such
as coronary plaque inflammation, bacterial-induced
inflammation including Chlamydia-induced inflammation
and viral induced inflammation).
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The method is useful for, but not limited to,
treating or preventing inflammation-related disorders by
altering the expression of one or more expression
products that directly or indirectly regulate
inflammation. Inflammation-related disorders,
particularly inflammation-related cardiovascular
disorders, may be mediated, in whole or in part, by one
or more expression products, which may undergo increased
or decreased expression. Said expression products may
include but are not limited to organic molecules,
proteins, DNA-based or RNA-based molecules, and networks
or aggregates of such products, acting together or
alone, to directly or indirectly produce an effect.
Changes in patterns of expression of said expression
products may occur sequentially or simultaneously,
involving two or more expression products. These
expression products may have direct or indirect affects
on the tissues or organs of the subject, inducing or
amplifying a pathological effect induced by other
molecules, or expression products. These expression
products may produce pro-inflammatory effects by
increased expression or decreased expression, depending
on their function as pro-inflammatory or anti-
inflammatory expression products, respectively.
The method is particularly useful for treating or
preventing conditions by moderating 'the upregulation of
pro-inflammatory components found in affected tissues,
including cyclooxygenase and osteopontin, while also
inhibiting the activity of cyclooxygenase in the kidney,
particularly the macula densa where aldosterone
antagonism can induce expression of cyclooxygenase.
While the use of an aldosterone antagonist leads to a
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reduction in cyclooxygenase expression induced by an
inflammation-related disorder, it may not completely
prevent cyclooxygenase activity. The co-action of
adding an NSAID that inhibits cyclooxygenase activity
will also lead to a reduction in inflammation of the
affected tissue or organ. The use of an aldosterone
antagonist can induce upregulation of cyclooxygenase in
the macula densa and cortical thick ascending limb
(CTAL) of Henle's loop in the kidney. In the kidney,
prostaglandins, the product of cyclooxygenase, are
involved in the regulation of renal hemodynamics and
saltwater homeostasis. As a result the non-
inflammatory aldosterone antagonist induction of
cyclooxygenase in the macula densa and CTRL region of
the kidney can lead to pathological effects such as
increased blood pressure and retention of salt and
water. Accordingly, co-administration of a NSAID that
inhibits cyclooxygenase, with an aldosterone antagonist,
will slow, stop, or reverse the progression of the
pathological renal response to the aldosterone
antagonist induction of cyclooxygenase in the kidney.
In the method above, cardiovascular disorder
includes, but is not limited to, those disorders which
are known to have an inflammation component and those
that may be mediated by aldosterone or cyclooxygenase or
both. The method above also includes treatment of
patients with an aldosterone antagonist and NSAID
combination requiring moderation of the upregulated
expression of cyclooxygenase or osteopontin. In
tissues, including but not limited to the kidney, heart,
and brain, cyclooxygenase may be induced resulting~in
upregulated expression of this pro-inflammatory enzyme,
which can cause mild to severe tissue and organ damage.
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In the method above, administration of an aldosterone
antagonist and NSAID combination is used to moderate the
upregulated expression of cyclooxygenase. The method
above would also be useful for preventing or treating
conditions which may arise in tissues, including but not
limited to the kidney, heart, and brain, wherein the
upregulated expression of the pro-inflammatory protein
osteopontin, may be induced, resulting in mild to severe
tissue and organ damage. In the method above,
administration of an aldosterone antagonist and NSAID
combination is used to moderate the upregulated
expression of osteopontin.
In another embodiment, the present invention would
be useful in preventing or treating conditions in
tissues and organs, including but not limited to the
kidney, heart and brain, wherein the upregulated
expression of any one of the pro-inflammatory expression
products MCP-1, IL-1, IL-6, VCAM-1 and ICAM-1 may occur,
resulting in mild to severe tissue and organ damage. In
the method above, administration of an aldosterone
antagonist and NSAID combination is used to moderate the
upregulated expression of any one of MCP-1, IL-1, IL-6,
VCAM-1 and ICAM-1.
Non-limiting examples of expression products, whose
expression can be moderated to reduce inflammation-
related cardiovascular disease by treatment with an
aldosterone antagonist and NSAID combination, are shown
in Figure 24. Non-limiting examples of pro-inflammatory
expression products that may be upregulated include one
or more of the following:
(a) receptors for angiotensin II and~endothelin,
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(b) monocyte activating molecules avf~3 (adhesion,
proliferation, migration) and CD44 (migration),
(c) mediators of vascular inflammation interferon-'y
(Inf-'y) , interleukin-1 (IL-1) and tumor necrosis
factor-a (TNF-a),
(d) NADH/NADPH oxidase to produce tissue damaging
superoxide radicals, and
(e) prothrombotic plasminogen activator inhibitor-1
(PAI-1) causing a decrease in active tissue
plasminogen activator (t-PA).
In another embodiment of the present invention,
non-limiting examples of expression products, whose
expression can be moderated to reduce inflammation-
related cardiovascular disease by treatment with an
aldosterone antagonist and NSAID combination, include
one or more of the following:
acute phase reactants like C-reactive protein
( CRP ) ,
pleiotropic cytokines like interleukin-6 (IL-6),
IL-10, IL-12, soluble intracellular adhesion
molecule-1 (sICAM-1),
troponin T or I, heat shock protein 65 (HSP65),
amyloid, phospholipase A2, fibrinogen, CD40/CD40L
signaling pathway
and adhesion mediators like collagen-binding
integrins alf~1 (mesenchymal cells) and a2f31
(epithelial cells).
In another embodiment of the present invention, one
or more of the inflammation-related expression products
can be moderated or altered by combination therapy of an
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aldosterone receptor antagonist and a NSAID, through an
increase or decrease in expression of at least 10%. In
another embodiment, said expression products can be
moderated or altered by combination therapy of an
5 aldosterone receptor antagonist and a NSAID, through an
increase or decrease in expression of at least 25%. In
another embodiment, said expression products can be
moderated or altered by combination therapy of an
aldosterone receptor antagonist and a NSAID, through an
10 increase or decrease in expression of at least 50%. In
another embodiment, said expression products can be
moderated or altered by combination therapy of an
aldosterone receptor antagonist and a NSAID, through an
increase or decrease in expression of at least 100%.
Inhibitors of the cyclooxygenase pathway in the
metabolism of arachidoniC acid used in the prevention of
cardiovascular disorder may inhibit enzyme activity
through a variety of mechanisms. By the way of example,
the inhibitors used in the methods described herein may
inhibit expression of the enzyme activity. Blocking
expression of Cyclooxygenase-2, at the site of
inflammatory damage, using an aldosterone antagonist, is
highly advantageous in that it minimizes the gastric
side effects that can occur with non-selective NSAID's,
especially where prolonged prophylactic treatment at a
high dose of NSAID is expected.
Dosages and Treatment Regimen
The amount of aldosterone receptor antagonist
blocker that is administered and the dosage regimen for
the methods of this invention depend on a variety of
factors, including the age, weight, sex and medical
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condition of the subject, the severity of the pathogenic
effect, the route and frequency of administration, and
the particular aldosterone blocker employed, and thus
may vary widely. A daily dose administered to a subject
of about 0.001 to 30 mg/kg body weight, preferably
between about 0.005 and about 20 mg/kg body weight, more
preferably between about 0.01 and about 15 mg/kg body
weight, still more preferably between about 0.05 and
about 10 mg/kg body weight, and most preferably between
about 0.01 to 5 mg/kg body weight, may be appropriate.
The daily dose of aldosterone antagonist
administered to a human subject typically will range
from about 0.1 mg to about 2000 mg. In one embodiment
of the present invention, the daily dose range is from
about 0.1 mg to about 400 mg. In another embodiment of
the present invention, the daily dose range is from
about 1 mg to about 200 mg. In a further embodiment of
the present invention, the daily dose range is from
about 1 mg to about 100 mg. In another embodiment of
the present invention, the daily dose range is from
about 10 mg to about 100 mg. In a further embodiment of
the present invention, the daily dose range is from
about 25 mg to about 100 mg. In another embodiment of
the present invention, the daily dose is selected from
the group consisting of 5 mg, 10 mg, 12.5 mg, 25 mg, 50
mg, 75 mg, and 100 mg. In a further embodiment of the
present invention, the daily dose is selected from the
group consisting of 25 mg, 50 mg, and 100 mg. A daily
dose of aldosterone blocker that produces no substantial
diuretic and/or anti-hypertensive effect in a subject is
specifically embraced by the present method. The daily
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dose can be administered in one to four doses per day.
Dosing of the aldosterone blocker can be determined
and adjusted based on measurement of blood pressure or
appropriate surrogate markers (such as natriuretic
peptides, endothelins, and other surrogate markers
discussed below). Blood pressure and/or surrogate
marker levels after administration of the aldosterone
blocker can be compared against the corresponding
baseline levels prior to administration of the
aldosterone blocker to determine efficacy of the present
method and titrated as needed. Non-limiting examples of
surrogate markers useful in the method are surrogate
markers for renal and cardiovascular disease.
Prophylatic Dosing
It is beneficial to administer the aldosterone
blocker prophylatically, prior to a diagnosis of said
inflammation-related cardiovascular disorders, and to
continue administration of the aldosterone blocker
during the period of time the subject is susceptible to
the inflammation-related cardiovascular disorders.
Individuals with no remarkable clinical presentation but
that are nonetheless susceptible to pathologic effects
therefore can be placed upon a prophylatic dose of an
aldosterone blocking compound. Such prophylactic doses
of the aldosterone blocker may, but need not, be lower
than the doses used to treat the specific pathogenic
effect of interest.
Cardiovascular Pathology Dosing
Dosing to treat pathologies of cardiovascular
function can be determined and adjusted based on
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measurement of blood concentrations of natriuretic
peptides. Natriuretic peptides are a group of
structurally similar but genetically distinct peptides
that have diverse actions in cardiovascular, renal, and
endocrine homeostasis. Atrial natriuretic peptide
("ANP") and brain natriuretic peptide ("BNP") are of
myocardial cell origin and C-type natriuretic peptide
("CNP") is of endothelial origin. ANP and BNP bind to
the natriuretic peptide-A receptor ("NPR-A"), which, via
3', 5'-cyclic guanosine monophosphate (cGMP), mediates
natriuresis, vasodilation, renin inhibition,
antimitogenesis, and lusitropic properties. Elevated
natriuretic peptide levels in the blood, particularly
blood BNP levels, generally are observed in subjects
under conditions of blood volume expansion and after
vascular injury such as acute myocardial infarction and
remain elevated for an extended period of time after the
infarction. (Uusimaa et al.: Int. J. Cardiol 1999; 69:
5-14) .
A decrease in natriuretic peptide level relative to
the baseline level measured prior to administration of
the aldosterone blocker indicates a decrease in the
pathologic effect of aldosterone and therefore provides
a correlation with inhibition of the pathologic effect.
Blood levels of the desired natriuretic peptide
level therefore can be compared against the
corresponding baseline level prior to administration of
the aldosterone blocker to determine efficacy of the
present method in treating the patologic effect. Based
upon such natriuretic peptide level measurements, dosing
of the aldosterone blocker can be adjusted to reduce the
cardiovascular pathologic effect.
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Similarly, cardiac pathologies can also be
identified, and the appropriate dosing determined, based
on circulating and urinary cGMP Levels. An increased
plasma level of cGMP parallels a fall in mean arterial
pressure. Increased urinary excretion of cGMP is
correlated with the natriuresis.
Cardiac pathologies also can be identified by a
reduced ejection fraction or the presence of myocardial
infarction or heart failure or left ventricular
hypertrophy. Left ventricular hypertrophy can be
identified by echo-cardiogram or magnetic resonance
imaging and used to monitor the progress of the
treatment and appropriateness of the dosing.
In another embodiment of the invention, therefore,
the methods of the present invention can be used to
reduce natriuretic peptide levels, particularly BNP
levels, thereby also treating related cardiovascular
pathologies.
Renal Pathology Dosing
Dosing to treat pathologies of renal function can
be determined and adjusted based on measurement of
proteinuria, microalbuminuria, decreased glomerular
filtration rate (GFR), or decreased creatinine
clearance. Proteinuria is identified by the presence of
greater than 0.3 g of urinary protein in a 24 hour urine
collection. Microalbuminuria is identified by an
increase in immunoassayable urinary albumin. Based upon
such measurements, dosing of the aldosterone blocker can
be adjusted to reduce the renal pathologic effect.
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Neuropathy Pathology Dosing
Neuropathy, especially peripheral neuropathy, can
be identified by and dosing adjustments based on,
neurologic exam of sensory deficit or sensory motor
5 ability.
Retinopathy Pathology Dosing
Retinopathy can be identified by, and dosing
adjustments based on, ophthalmologic exam.
Inflammation Markers
Certain markers may be indicative of or responsible
for inflammation, or pre-inflammatory conditions.
Measurement of these markers may be useful in
determination of an appropriate dosage of aldosterone
blocker to be administered, or determination of an
efficatious dose of an aldosterone blocker after
administration. Non-limiting examples of such markers
are: osteopontin; acute phase reactants such as C
reactive protein (CRP), fibrinogen, Factor VIII, serum
copper (carrier protein ceruloplasmin), serum iron
(carrier protein ferritin), Plasminogen activator
Inhibitor-1 (PAI-1) and lipoprotein(a); natriuretic
peptides; endothelins; VCAM-l; ICAM-1; IL-1,Q; TNF-cx; IL-
6; COX-2; fractalkine; MCP-1; and triglyceride.
NSAIDs useful in the present invention include
compounds listed in Table 1 (including derivatives of
these compounds). Each published document listed in
Table 1 describes selected aspects of the NSAID, such as
the chemical preparation or the biological properties of
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such compound. The content of each of these documents
is incorporated herein by reference.
Table 1: NSAIDs
REFERENCE
COMPOUND NAME CHEMICAL
NAME/CAS
acetaminophen US 3125598
benoxaprofen US 3912748 a
carprofen US 3896145
diclofenac 15307-86-5 US 3778470
diflunisal FR 1522570
Etodolac 1,8-diethyl-1,3,4,9-GB 1391005 fenoprofen
tetrahydropyrano[3,4-
b]indole-1-acetic
acid /41340-
25-4
fenoprofen 31879-05-7 US 3649679
fh~rbiprofen (alphaR)-2-fluoro-alpha-,EP 103265
methyl-[1,1'-biphenyl]-4-acetic
acid/ 5104-49-4
--- ibuprofen - alpha-methyl-4-(2- GB 1538636
methylpropyl)benzeneacetic
acid 2-methoxyphenyl
ester/
66332-77-2
indomethacin 53-86-1 US 3161654
ketoprofen (R)-3-benzoyl-alpha-GB 1164585
methylbenzeneacetic
acid/
22071-15-4
I~etorolac (+,-)-5-benzoyl-2,3-dihydro-GB 1554057
1 H-pyrrolizine-1-carboxylic
acid/ 74103-06-3
meclofenamate
mefenamic acid US 3294813
nabumetone 4-(6-methoxy-2-naphthyl)-2-GB 1476721
butanone/ 42924-53-8
Naproxen 22204-53-1 US 3637767
oxaprozin 4,5-diphenyl-2- GB 1206403
oxazolepropanoic
acid /
21256-18-8
oxyphenbutazone US 3482021
henylbutazone US 3265577
piroxicam 3-phenyl-propenoic EP 79639
acid, 2-
methyl-3-[(2-pyridinylamino)-
carbonyl]-2 H-1,2-
benzothiazin-4-yl
ester, S,S-
dioxide/ 36322-90-4
Sulindac 38194-50-2 US 3692651
Suprofen US 4035376
Tenidap (Z)-5-chloro-2,3-dihydro-3-EP 156603
(hydroxy-2-thienylmethylene)-
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2-oxo-1 H-indole-1-
carboxamide 1120210-48-2
Tolmetin 26171-23-3 US 3752826
zomepirac US 3752826
Aspirin
In one embodiment, the NSAID is selected from the
group consisting of acetaminophen, benoxaprofen,
carprofen, diclofenac, diflunisal, etodolac, fenoprofen,
flurbiprofen, ibuprofen, indomethacin, ketoprofen,
ketorolac, meclofenamate, mefenamic acid, nabumetone,
naproxen, oxaprozin, oxyphenbutazone, phenylbutazone,
piroxicam, sulindac, suprofen, tenidap, tolmetin,
zomepirac, and aspirin.
In another embodiment, the NSAID is selected from
the group consisting of acetaminophen, benoxaprofen,
carprofen, diclofenac, diflunisal, etodolac, fenoprofen,
and flurbiprofen.
In another embodiment, the NSAID is selected from
the group consisting of ibuprofen, indomethacin,
ketoprofen, ketorolac, meclofenamate, mefenamic acid,
nabumetone, naproxen, and oxaprozin.
In another embodiment, the NSAID is selected from
the group consisting of oxyphenbutazone, phenylbutazone,
piroxicam, sulindac, suprofen, tenidap, tolmetin,
zomepirac, and aspirin.
The term "NSAID" includes any compounds (such as
derivatives and pharmaceutically acceptable salts),
which are structurally related to a NSAID and which
possess the substantially equivalent biologic activity.
By way of example, such compounds may include, but are
not limited to, prodrugs thereof.
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The term "aldosterone receptor antagonist" or
"aldosterone antagonist" denotes a compound capable of
binding to an aldosterone receptor, as a competitive
5' inhibitor of the action of aldosterone itself at the
receptor site, so as to modulate the receptor-mediated
activity of aldosterone.
Aldosterone Antagonists
The aldosterone antagonists used in the methods
of the present invention generally are spirolactone-
type steroidal compounds. The term "spirolactone-
type" is intended to characterize a structure
comprising a lactone moiety attached to a steroid
nucleus, typically at the steroid "D" ring, through a
spiro bond configuration. A subclass of spirolactone-
type aldosterone antagonist compounds consists of
epoxy-steroidal aldosterone antagonist compounds such
as eplerenone. Another subclass of spirolactone-type
antagonist compounds consists of non-epoxy-steroidal
aldosterone antagonist compounds such as
spironolactone.
The epoxy-steroidal aldosterone antagonist
compounds used in the method of the present invention
generally have a steroidal nucleus substituted with an
epoxy-type moiety. The term "epoxy-type" moiety is
intended to embrace any moiety characterized in having
an oxygen atom as a bridge between two carbon atoms,
examples of which include the following moieties:
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O O O
~CH~ CH2
epoxyethyl 1,3-epoxypropyl 1,2-epoxypropyl
The team "steroidal", as used in the phrase
"epoxy-steroidal", denotes a nucleus provided by a
cyclopenteno-phenanthrene moiety, having the
conventional "A", "B", "C" and "D" rings. The epoxy-
type moiety may be attached to the
cyclopentenophenanthrene nucleus at any attachable or
substitutable positions, that is, fused to one of the
rings of the steroidal nucleus or the moiety may be
l0 substituted on a ring member of the ring system. The
phrase "epoxy-steroidal" is intended to embrace a
steroidal nucleus having one or a plurality of epoxy-
type moieties attached thereto.
Epoxy-steroidal aldosterone antagonists suitable
for use in the present methods include a family of
compounds having an epoxy moiety fused to the °C" ring
of the steroidal nucleus. Especially preferred are 20-
spiroxane compounds characterized by the presence of a 9
a,lloc-substituted epoxy moiety. Compounds 1 through
11, Table 1 below, are illustrative 9oc,110c-epoxy-
steroidal compounds that may be used in the present
methods. These epoxy steroids may be prepared by
procedures described in Grob et al., U.S. Patent No.
4,559,332. Additional processes for the preparation of
9,11-epoxy steroidal compounds and their salts are
disclosed in Ng et al., W097/21720 and Ng et al.,
W098/25948.
TABLE 2: Aldosterone Receptor Antagonist
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Compound # Structure and Name
O
O
1 s ~R
0:~~ R i c~Me ,
R
M R H OMe
O
O
Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-
17-hydroxy-3-oxo-,Y-lactone, methyl ester,
(7a, 11a, 17(3) -
OMe
Me ~ OOH
R H S R
S
R
~MR RH
,,~ OMe
O
O
Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-
17-hydroxy-3-oxo-,dimethyl ester,(7a,11a,17(3)-
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21
T~TA
3
0
0
3'H-Cyclopropa[6,7]pregna-4,6-dime-21-carboxylic
acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-,
'y-lactone, (6(3, 7(3, 11a, 17(3) -
C02H
4
~+
R
R~
~MR H
~,~ OPr-i
O
O
Pregn-4-ene-7,21-dicarboxyliC acid,9,11-epoxy-17-
hydroxy-3-oxo-,7-(1-methylethyl) ester,
monopotassium salt, (7a, 11a, 17(3) -
Me OH C02H
R S R
MR RH .K+
S'R
OMe
O
O
Pregn-4-ene-7,21-dicarboxyliC acid,9,11-epoxy-17-
hydroxy-3-oxo-,7-methylethyl) ester,monopotassium
salt, (7a, 11a, 17(3) -
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6
22
Me
R-. /~
Me ~a S p- ' O
S
S ~~ ~~.
S. H H
O
3'H-cyclopropa[6,7]pregna-1,4,6-triene-21-carboxylic
acid,9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-,
'y-lactone (6(3, 7[3, 11a) -
7
O
3'H-cyclopropa[6,7]pregna-4,6-dime-2l-carboxylic
acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-,
methyl ester, (6(3, 7(3, lla,, 17(3) -
Me HO
COZH
Me R ~~a '~~
' S
S
S ~~
~t/I H H ~ I~
O
3'H-cyclopropa[6,7]pregna-4,6-dime-21-carboxylic
acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-,
monopotassium salt, (6~i, 7(3, 11a, 17(3) -
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Me
R.~ ~~~.~0~ O
Me
S
S
S. H H
O
3'H-cyclopropa[6,7]pregna-1,4,6-triene-21-carboxylic
acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-,y-
lactone (6~i,7~3,11a,17(3)-
O
O
S 'R
R H Me
S' '
Me K H OE t
R
O
O
Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-
17-hydroxy-3-oxo-,y-lactone, ethyl ester,
(7a,11a,17(3)-
0
O
11 S 'R
R H ~Me
S ~ R
K
~M R H
O P r-i
O
O
Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-
17-hydroxy-3-oxo-,y-lactone, 1-methylethyl
ester (7a,11a, 17(3)-
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24
Of particular interest is the compound eplerenone
(also known as: epoxymexrenone and CGP 30 083) which is
compound 1 as shown above. The chemical name for
eplerenone is pregn-4-ene-7,21-dicarboxylic acid, 9,11-
epoxy-17-hydroxy-3-oxo, 'y-lactone, methyl ester, (7cx,
llcx, l7cx) -. This chemical name corresponds to the CAS
registry name for eplerenone (the CAS registry number
for eplerenone is 107724-20-9). U.S. Patent No.
4,559,332 identifies eplerenone by the alternative name
of 9cx, llcx-epoxy-7cx-methoxycarbonyl-20-spirox-4-ene-3 , 21-
dione. Such "spiroxane" nomenclature is further
described, for example, at column 2, line 16 through
column 4, line 48 of U.S. Patent No. 4,559,332.
Eplerenone is an aldosterone receptor antagonist and has
a higher specificity for aldosterone receptors than
does, for example, spironolactone. Selection of
eplerenone as the aldosterone antagonist in the present
method would be beneficial to reduce certain side-
effects such as gynecomastia that occur with use of
aldosterone antagonists having less specificity.
Non-epoxy-steroidal aldosterone antagonists
suitable for use in the present methods include a family
of spirolactone-type compounds defined by Formula I:
0
--n (I)
wherein ~ ~ is
C6 '~ C7
C o ~~ SCOR,
H2 H
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wherein R is lower alkyl of up to 5 carbon atoms,
and
wherein ~ C15" C1 G is
\~ ~ ~. ~ 0r ~~
C ' C
HZ H2
5
Lower alkyl residues include branched and
unbranched groups, preferably methyl, ethyl and n-
propyl.
10 Specific compounds of interest within Formula I are
the following:
7a-acetylthio-3-oxo-4,15-androstadiene-[17((3-1')-
spiro-5']perhydrofuran-2'-one;
3-oxo-7a-propionylthio-4,15-androstadiene-[17(((3-
15 1')-spiro-5']perhydrofuran-2'-one;
6(3, 7(3-methylene-3-oxo4, 15-androstadiene- [17 ( ((3-1' ) -
spiro-5']perhydrofuran-2'-one;
15a,16a-methylene-3-oxo-4,7a-propionylthio-4-
androstene [17 ((3-1' ) -spiro-5' ] perhydrofuran-2' -one;
20 6(3, 7(3, 15a, 16a-dimethylene-3-oxo-4-androstene [17 ((3-
1')-spiro-5']-perhydrofuran-2'-one;
7a-acetylthio-15(3,16(3-Methylene-3-oxo-4-androstene-
[17 ((3-1' ) -spiro-5' ] perhydrofuran-2' -one;
15(3, 16(3-methylene-3-oxo-7(3-propionylthio-4-
25 androstene-[17((3-1')-spiro-5']perhydrofuran-2'-one; and
6(3, 7(3, 15(3, 163-dimethylene-3-oxo-4-androstene- [17 ((3-
1')-spiro-5']perhydrofuran-2'-one.
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26
Methods to make compounds of Formula I are
described in U.S. Patent No. 4,129,564 to Wiechart et
al. issued on 12 December 1978.
Another family of non-epoxy-steroidai compounds of
interest is defined by Formula II:
wherein R1 is Cl_3-alkyl or Cl_3 aryl and R2 is H or Cl_3-
alkyl .
Specific compounds of interest within Formula II
are the following:
la,-acetylthio-15(3, 16(3-methylene-7a-methylthio-3-
oxo-17a-pregn-4-ene-21,17-carbolactone; and
15(3, 16(3-methylene-la, 7a-dimethylthio-3-oxo-17a-
pregn-4-ene-21,17-Carbolactone.
Methods to make the compounds of Formula II are
described in U.S. Patent No. 4,789,668 to Nickisch et
al. which issued 6 December 1988.
Yet another family of non-epoxy-steroidal compounds
of interest is defined by a structure of Formula III:
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27
.R
wherein R is lower alkyl, with preferred lower alkyl
groups being methyl, ethyl, propyl and butyl. Specific
compounds of interest include:
3(3,21-dihydroxy-170c-pregna-5,15-dime-17-carboxylic
acid 'y-lactone;
3(3,21-dihydroxy-170(-pregna-5,15-diene-17-carboxylic
acid ~r-lactone 3-acetate;
3~3,21-dihydroxy-17a-pregn-5-ene-17-carboxylic acid
'y-lactone;
3(3,21-dihydroxy-l7oc-pregn-5-ene-17-carboxylic acid
'y-lactone 3-acetate;
21-hydroxy-3-oxo-l7oc-pregn-4-ene-17-carboxylic acid
'y-lactone ;
21-hydroxy-3-oxo-l7oc-pregna-4,6-dime-17-carboxylic
acid y-lactone;
21-hydroxy-3-oxo-l7oc-pregna-1,4-dime-17-carboxylic
acid y-lactone;
70(-acylthio-21-hydroxy-3-oxo-l7oc-pregn-4-ene-17-
carboxylic acid ~lactone; and
70c-acetylthio-21-hydroxy-3-oxo-l7oc-pregn-4-ene-17-
carboxylic acid 'y-lactone.
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28
Methods to make the compounds of Formula III are
described in U.S. Patent No. 3,257,390 to Patchett which
issued 21 June 1966.
Still another family of non-epoxy-steroidal
compounds of interest is represented by Formula IV:
0
0
wherein E' is selected from the group consisting of
ethylene, vinylene and (lower alkanoyl)thioethylene
radicals, E" is selected from the group consisting of
ethylene, vinylene, (lower alkanoyl)thioethylene and
(lower alkanoyl)thiopropylene radicals; R is a methyl
radical except when E' and E" are ethylene and (lower
alkanoyl) thioethylene radicals, respectively, in which
case R is selected from the group consisting of hydrogen
and methyl radicals; and the selection of E' and E" is
such that at least one (lower alkanoyl)thio radical is
present.
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A preferred family of non-epoxy-steroidal compounds
within Formula IV is represented by Formula V:
~o
I0
lower (V)
A more preferred'compound of Formula V is
1-acetylthio-17a-(2-carboxyethyl)-17(3-hydroxy-androst-4-
en-3-one lactone.
Another preferred family of non-epoxy-steroidal
compounds within Formula IV is represented by Formula
VI:
~o
0
(VI)
lower alkyl
More preferred compounds within Formula VI include
the following:
7oc-acetylthio-17a- (2-carboxyethyl) -17(3-hydroxy-
androst-4-en-3-one lactone;
7(3-acetylthio-l7oc- (2-carboxyethyl) -17(3-hydroxy-
androst-4-en-3-one lactone;
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loc, 7oc-diacetylthio-170c- (2-carboxyethyl) -17(3-
hydroxy-androsta-4,6-dien-3-one lactone;~
7oc-acetylthio-17a- (2-carboxyethyl) -17(3-hydroxy-
androsta-1,4-dien-3-one lactone;
5 7a-acetylthio-l7oc- (2-carboxyethyl) -17(3-hydroxy-19-
norandrost-4-en-3-one lactone; and
7a-acetylthio-l7oc- (2-carboxyethyl) -17(3-hydroxy-6o~-
methylandrost-4-en-3-one lactone;
6'
10 In Formulae IV-VI, the term "alkyl" is intended to
embrace linear and branched alkyl radicals containing
one to about eight carbons. The term "(lower
alkanoyl)thio" embraces radicals of the formula lower
0
II
alkyl -~-s .
Of particular interest is the compound
spironolactone having the following structure and formal
name:
"spironolactone": 17-hydroxy-70~-mercapto-3-oxo-170~-
pregn-4-ene-21-carboxylic acid y-lactone acetate.
Methods to make compounds of Formulae IV-VI are
described in U.S. Patent No. 3,013,012 to Cella et al.
which issued 12 December 1961. Spironolactone is sold
by G.D. Searle & Co., Skokie, Illinois, under the
Jl: V l:rig
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31
trademark "ALDACTONE", in tablet dosage form at doses of
25 mg, 50 mg and 100 mg per tablet.
Another family of steroidal aldosterone antagonists
is exemplified by drospirenone, [6R-
(6alpha,7alpha,8beta,9alpha,lObeta,l3beta,l4alpha,l5alph
a,l6alpha, l7beta)]-
1,3',4',6,7,8,9,10,11,12,13,14,15,16,20,21-hex
adecahydro-10,13-dimethylspiro[17H-
dicyclopropa[6,7:15,16]cyclopenta[a]phenanthrene-
17, 2' (5'H) -furan] -3, 5' (2H) -dione, CAS registration
number 67392-87-4. Methods to make and use drospirenone
are described in patent GB 1550568 1979, priority DE
2652761 1976.
Definitions
The term "treatment" or "treating" includes the
administration, to a person in need, of an amount of an
aldosterone antagonist and NSAID combination that will
inhibit or reverse development of a pathological
cardiovascular condition.
The term "prevention" or "preventing" includes
either preventing the onset of clinically evident
cardiovascular disorders altogether or preventing the
onset of a preclinically evident stage of cardiovascular
disorder in individuals. This includes prophylactic
treatment of those at risk of developing a
cardiovascular disorder.
The phrase "therapeutically-effective" is intended
to qualify the amount of the two agents given in
combination which will achieve the goal of improvement
in disorder severity and the frequency of incidence,
while avoiding adverse side effects.
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The term "subject" for purposes of treatment
includes any human or animal subject (preferably
mammalian and including, but not limited to,
domesticated animals such as those from the bovine,
porcine, ovine or equine families, and companion animals
such as those from the canine and feline family),
susceptible to or suffering from a cardiovascular
disorders, and preferably is a human subject. The
subject, for example, may be at risk due to diet,
exposure to bacterial or viral infection, having common
markers present, being genetically predisposed to the
cardiovascular disorders, and the like.
The terms "aldosterone antagonist" and "aldosterone
receptor antagonist" include a compound that inhibits
the binding of aldosterone to mineralocorticoid
receptors thereby blocking the biological effects of
aldosterone.
The terms "non-steroidal anti-inflammatory drug'' or
"NSAID" include a compound whose structure lacks a
steroid ring and prevents, reduces or inhibits an
inflammatory response in a tissue or organ.
The term "pro-inflammmatory" characterizes
molecules produced in the body to induce, activate or
enhance an inflammatory response in a tissue or organ.
The term "hydrido" denotes a single hydrogen atom
(H). This hydrido radical may be attached, for example,
to an oxygen atom to form a hydroxyl radical or two
hydrido radicals may be attached to a carbon atom to
form a methylene (-CH2-) radical. Where used, either
alone or within other terms such as "haloalkyl",
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"alkylsulfonyl", "alkoxyalkyl" and "hydroxyalkyl", the
term "alkyl" embraces linear or branched radicals having
one to about twenty carbon atoms or, preferably, one to
about twelve carbon atoms. More~preferred alkyl radicals
are "lower alkyl" radicals having one to about ten
carbon atoms. Most preferred are lower alkyl radicals
having one to about six carbon atoms. Examples of such
radicals include methyl, ethyl, n-propyl, isopropyl, n-
butyl, isobutyl, sec-butyl, tart-butyl, pentyl, iso-
amyl, hexyl and the like. The term "alkenyl" embraces
linear or branched radicals having at least one carbon-
carbon double bond of two to about twenty carbon atoms
or, preferably, two to about twelve carbon atoms. More
preferred alkyl radicals are "lower alkenyl" radicals
having two to about six carbon atoms. Examples of
alkenyl radicals include ethenyl, propenyl, allyl,
propenyl, butenyl and 4-methylbutenyl. The term
"alkynyl" denotes linear or branched radicals having two
to about twenty carbon atoms or, preferably, two to
about twelve carbon atoms. More preferred alkynyl
radicals are "lower alkynyl" radicals having two to
about ten carbon atoms. Most preferred are lower
alkynyl radicals having two to about six carbon atoms.
Examples of such radicals include propargyl, butynyl,
and the like. The terms "alkenyl", "lower alkenyl",
embrace radicals having "cis" and "traps" orientations,
or alternatively, "E" and "Z" orientations. The term
"cycloalkyl" embraces saturated carbocyclic radicals
having three to twelve carbon atoms. More preferred
cycloalkyl radicals are "lower cycloalkyl" radicals
having three to about eight carbon atoms. Examples of
such radicals include cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl. The term "cycloalkenyl"
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34
embraces partially unsaturated carbocyclic radicals
having three to twelve carbon atoms. More preferred
cycloalkenyl radicals are "lower cycloalkenyl" radicals
having four to about eight carbon atoms. Examples of
such radicals include cyclobutenyl, cyclopentenyl,
cyclopentadienyl, and cyclohexenyl. The term "halo"
means halogens such as fluorine, chlorine, bromina or
iodine. The term "haloalkyl" embraces radicals wherein
any one or more of the alkyl carbon atoms is substituted
with halo as defined above. Specifically embraced are
monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals.
A monohaloalkyl radical, for one example, may have
either an iodo, bromo, chloro or fluoro atom within the
radical. Dihalo and polyhaloalkyl radicals may have two
or more of the same halo atoms or a combination of
different halo radicals. "Lower haloalkyl" embraces
radicals having 1-6 carbon atoms. Examples of haloalkyl
radicals include fluoromethyl, difluoromethyl,
trifluoromethyl, chloromethyl, dichloromethyl,
trichloromethyl, trichloromethyl, pentafluoroethyl,
heptafluoropropyl, difluorochloromethyl,
dichlorofluoromethyl, difluoroethyl, difluoropropyl,
dichloroethyl and dichloropropyl. The term
"hydroxyalkyl" embraces linear or branched alkyl
radicals having one to about ten carbon atoms any one of
which may be substituted with one or more hydroxyl
radicals. More preferred hydroxyalkyl radicals are
"lower hydroxyalkyl" radicals having one to six carbon
atoms and one or more hydroxyl radicals. Examples of
such radicals include hydroxymethyl, hydroxyethyl,
hydroxypropyl, hydroxybutyl and hydroxyhexyl. The terms
"alkoxy" and "alkyloxy" embrace linear or branched oxy-
containing radicals each having alkyl portions of one to
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about ten carbon atoms. More preferred alkoxy radicals
are "lower alkoxy" radicals having one to six carbon
atoms. Examples of such radicals include methoxy,
ethoxy, propoxy, butoxy and tert-butoxy. The term
5 "alkoxyalkyl" embraces alkyl radicals having one or more
alkoxy radicals attached to the alkyl radical, that is,
to form monoalkoxyalkyl and dialkoxyalkyl radicals. The
"alkoxy" radicals may be further substituted with one or
more halo atoms, such as fluoro, chloro or bromo, to
10 provide haloalkoxy radicals. More preferred haloalkoxy
radicals are "lower haloalkoxy" radicals having one to
six carbon atoms and one or more halo radicals.
Examples of such radicals include fluoromethoxy,
chloromethoxy, trifluoromethoxy, trifluoroethoxy,
15 fluoroethoxy and fluoropropoxy. The term "aryl", alone
or in combination, means a carbocyclic aromatic system
containing one, two or three rings wherein such rings
may be attached together in a pendent manner or may be
fused. The term "aryl" embraces aromatic radicals such
20 as phenyl, naphthyl, tetrahydronaphthyl, indane and
biphenyl. Aryl moieties may also be substituted at a
substitutable position with one or more substituents
selected independently from alkyl, alkoxyalkyl,
alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl,
25 aminocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino,
halo, nitro, alkylamino, aryl, cyano, carboxy,
aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl. The
term "heterocyclyl" embraces saturated, partially
unsaturated and unsaturated heteroatom-containing ring-
30 shaped radicals, where the heteroatoms may be selected
from nitrogen, sulfur and oxygen. Examples of saturated
heterocyclyl radicals include saturated 3 to 6-membered
heteromonocylic group containing 1 to 4 nitrogen atoms
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36
(e. g. pyrrolidinyl, imidazolidinyl, piperidino,
piperazinyl, etC.); saturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 oxygen atoms
and 1 to 3 nitrogen atoms (e. g. morpholinyl, etC.);
saturated 3 to 6-membered heteromonocycliC group
containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms
(e. g., thiazolidinyl, etc.). Examples of partially
unsaturated heterocyClyl radicals include
dihydrothiophene, dihydropyran, dihydrofuran and
dihydrothiazole. The term "heteroaryl" embraces
unsaturated heterocyclyl radicals. Examples of
unsaturated heterocyclyl radicals, also termed
"heteroaryl" radicals include unsaturated 3 to 6
membered heteromonocycliC group containing 1 to 4
nitrogen atoms, for example, pyrrolyl, pyrrolinyl,
imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl,
pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-
1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.) tetrazolyl
(e. g. 1H-tetrazolyl, 2H-tetrazolyl, etC.), etc.;
unsaturated condensed heterocyclyl group containing 1 to
5 nitrogen atoms, for example, indolyl, isoindolyl,
indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,
indazolyl, benzotriazolyl, tetrazolopyridazinyl (e. g.,
tetrazolo[1,5-b]pyridazinyl, etC.), etc.; unsaturated 3
to 6-membered heteromonocyclic group containing an
oxygen atom, for example, pyranyl, furyl, etc.;
unsaturated 3 to 6-membered heteromonocycliC group
containing a sulfur atom, for example, thienyl, etc.;
unsaturated 3- to 6-membered heteromonocyclic group
containing 1 to 2 oxygen atoms and 1 to 3 nitrogen
atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl
(e. g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-
oxadiazolyl, etc.) etc.; unsaturated condensed
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37
heterocyclyl group containing 1 to 2 oxygen atoms and 1
to 3 nitrogen atoms (e. g. benzoxazolyl, benzoxadiazolyl,
etc.); unsaturated 3 to.6-membered heteromonocyclic
group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen
atoms, for example, thiazolyl, thiadiazolyl (e. g.,
1,2,4- thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-
thiadiazolyl, etc.) etc.; unsaturated condensed
heterocyclyl group containing 1 to 2 sulfur atoms and 1
to 3 nitrogen atoms (e. g., benzothiazolyl,
benzothiadiazolyl, etc.) and the like. The term also
embraces radicals where heterocyclyl radicals are fused
with aryl radicals. Examples of such fused bicyclic
radicals include benzofuran, benzothiophene, and the
like. Said "heterocyclyl group" may have 1 to 3
substituents such as alkyl, hydroxyl, halo, alkoxy, oxo,
amino and alkylamino. The term "alkylthio" embraces
radicals containing a linear or branched alkyl radical,
of one to about ten carbon atoms attached to a divalent
sulfur atom. More preferred alkylthio radicals are
"lower alkylthio" radicals having alkyl radicals of one
to six carbon atoms. Examples of such lower alkylthio
radicals are methylthio, ethylthio, propylthio,
butylthio and hexylthio. The term "alkylthioalkyl"
embraces radicals containing an alkylthio radical
attached through the divalent sulfur atom to an alkyl
radical of one to about ten carbon atoms. More
preferred alkylthioalkyl radicals are "lower
alkylthioalkyl" radicals having alkyl radicals of one to
six carbon atoms. Examples of such lower alkylthioalkyl
radicals include methylthiomethyl. The term
"alkylsulfinyl" embraces radicals containing a linear or
branched alkyl radical, of one to ten carbon atoms,
attached to a divalent -S(=O)- radical. More preferred
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38
alkylsulfinyl radicals are "lower alkylsulfinyl"
radicals having alkyl radicals of one to six carbon
atoms. Examples of such lower alkylsulfinyl radicals
include methylsulfinyl, ethylsulfinyl, butylsulfinyl and
hexylsulfinyl. The term "sulfonyl", whether used alone
or linked to other terms such as alkylsulfonyl, denotes
respectively divalent radicals -S02-. "Alkylsulfonyl"
embraces alkyl radicals attached to a sulfonyl radical,
where alkyl is defined as above. More preferred
alkylsulfonyl radicals are "lower alkylsulfonyl"
radicals having one to six carbon atoms. Examples of
such lower alkylsulfonyl radicals include
methylsulfonyl, ethylsulfonyl and propylsulfonyl. The
"alkylsulfonyl" radicals may be further substituted with
one or more halo atoms, such as fluoro, chloro or bromo,
to provide haloalkylsulfonyl radicals. The terms
"sulfamyl", "aminosulfonyl" and "sulfonamidyl" denote
NH202S-. The term "aryl" denotes a radical provided by
the residue after removal of hydroxyl from an organic
acid. Examples of such acyl radicals include alkanoyl
and aroyl radicals. Examples of such lower alkanoyl
radicals include formyl, acetyl, propionyl, butyryl,
isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl,
trifluoroacetyl. The term "carbonyl", whether used
alone or with other terms, such as "alkoxycarbonyl",
denotes -(C=O)-. The term "aroyl" embraces aryl
radicals with a carbonyl radical as defined above.
Examples of aroyl include benzoyl, naphthoyl, and the
like and the aryl in said aroyl may be additionally
substituted. The terms "carboxy" or "carboxyl", whether
used alone or with other terms, such as "carboxyalkyl",
denotes -C02H. The term "carboxyalkyl" embraces alkyl
radicals substituted with a carboxy radical. More
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39
preferred are "lower carboxyalkyl" which embrace lower
alkyl radicals as defined above, and may be additionally
substituted on the alkyl radical with halo. Examples of
such lower carboxyalkyl radicals include carboxymethyl,
carboxyethyl and carboxypropyl. The term
"alkoxycarbonyl" means a radical containing an alkoxy
radical, as defined above, attached via an oxygen atom
to a carbonyl radical. More preferred are "lower
alkoxycarbonyl" radicals with alkyl porions having 1 to
6 carbons. Examples of such lower alkoxycarbonyl
(ester) radicals include substituted or unsubstituted
methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
butoxycarbonyl and hexyloxycarbonyl. The terms
"alkylcarbonyl", "arylcarbonyl" and "aralkylcarbonyl"
include radicals having alkyl, aryl and aralkyl
radicals, as defined above, attached to a carbonyl
radical. Examples of such radicals include substituted
or unsubstituted methylcarbonyl, ethylcarbonyl,
phenylcarbonyl and benzylcarbonyl. The term "aralkyl"
embraces aryl-substituted alkyl radicals such as benzyl,
diphenylmethyl, triphenylmethyl, phenylethyl, and
diphenylethyl. The aryl in said aralkyl may be
additionally substituted with halo, alkyl, alkoxy,
halkoalkyl and haloalkoxy. The terms benzyl and
phenylmethyl are interchangeable. The term
"heterocyclylalkyl" embraces saturated and partially
unsaturated heterocyclyl-substituted alkyl radicals,
such as pyrrolidinylmethyl, and heteroaryl-substituted
alkyl radicals, such as pyridylmethyl, quinolylmethyl,
thienylmethyl, furylethyl, and quinolylethyl. The
heteroaryl in said heteroaralkyl may be additionally
substituted with halo, alkyl, alkoxy, halkoalkyl and
haloalkoxy. The term "aralkoxy" embraces aralkyl
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radicals attached through an oxygen atom to other
radicals. The term "aralkoxyalkyl" embraces aralkoxy
radicals attached through an oxygen atom to an alkyl
radical. The term "aralkylthio" embraces aralkyl
5 radicals attached to a sulfur atom. The term
"aralkylthioalkyl" embraces aralkylthio radicals
attached through a sulfur atom to an alkyl radical. The
term "aminoalkyl" embraces alkyl radicals substituted
with one or more amino radicals. More preferred are
10 "lower aminoalkyl" radicals. Examples of such radicals
include aminomethyl, aminoethyl, and the like. The term
"alkylamino" denotes amino groups which have been
substituted with one or two alkyl radicals. Preferred
are "lower N-alkylamino" radicals having alkyl portions
15 having 1 to 6 carbon atoms. Suitable lower alkylamino
may be mono or dialkylamino such as N-methylamino, N-
ethylamino, N,N-dimethylamino, N,N-diethylamino or the
like. The term "arylamino" denotes amino groups which
have been substituted with one or two aryl radicals,
20 such as N-phenylamino. The "arylamino" radicals may be
further substituted on the aryl ring portion of the
radical. The term "aralkylamino" embraces aralkyl
radicals attached through an amino nitrogen atom to
other radicals. The terms "N-arylaminoalkyl" and "N-
25 aryl-N-alkyl-aminoalkyl" denote amino groups which have
been substituted with one aryl radical or one aryl and
one alkyl radical, respectively, and having the amino
group attached to an alkyl radical. Examples of such
radicals include N-phenylaminomethyl and N-phenyl-N-
30 methylaminomethyl. The term "aminocarbonyl" denotes an
amide group of the formula -C(=O)NH2. The term
"alkylaminocarbonyl" denotes an aminocarbonyl group
which has been substituted with one or two alkyl
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radicals on the amino nitrogen atom. Preferred are "N-
alkylaminocarbonyl" "N,N-dialkylaminocarbonyl" radicals.
More preferred are "lower N-alkylaminocarbonyl" "lower
N,N-dialkylaminocarbonyl" radicals with lower alkyl
portions as defined above. The term "alkylaminoalkyl"
embraces radicals having one or more alkyl radicals
attached to an aminoalkyl radical. The term
"aryloxyalkyl" embraces radicals having an aryl radical
attached to an alkyl radical through a divalent oxygen
atom. The term "arylthioalkyl" embraces radicals having
an aryl radical attached to an alkyl radical through a
divalent sulfur atom.
The compounds utilized in the methods of the
present invention may be present in the form of free
bases or pharmaceutically acceptable acid addition salts
thereof. The term "pharmaceutically-acceptable salts"
embraces salts commonly used to form alkali metal salts
and to form addition salts of free acids or free bases.
The nature of the salt is not critical, provided that it
is pharmaceutically-acceptable. Suitable
pharmaceutically-acceptable acid addition salts of
compounds of the present invention may be prepared from
an inorganic acid or from an organic acid. Examples of
such inorganic acids are hydrochloric, hydrobromic,
hydroiodic, nitric, carbonic, sulfuric and phosphoric
acid. Appropriate organic acids may be selected from
aliphatic, cycloaliphatic, aromatic, araliphatic,
heterocyclic, carboxylic and sulfonic classes of organic
acids, example of which are formic, acetic, propionic,
succinic, glycolic, gluconic, lactic, malic, tartaric,
citric, ascorbic, glucuronic, malefic, fumaric, pyruvic,
aspartic, glutamic, benzoic, anthranilic, mesylic, 4-
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hydroxybenzoic, phenylacetic, mandelic, embonic
(pamoic), methanesulfonic, ethanesulfonic,
benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic,
toluenesulfonic, sulfanilic, cyclohexylaminosulfonic,
stearic, algenic, b-hydroxybutyric, salicylic,
galactaric and galacturonic acid. Suitable
pharmaceutically-acceptable base addition salts include
metallic salts made from aluminum, calcium, lithium,
magnesium, potassium, sodium and zinc or organic salts
made from N,N'-diben~ylethylenediamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, meglumine (N-
methylglucamine) and procaine. All of these salts may
be prepared by conventional means from the corresponding
compound by reacting, for example, the appropriate acid
or base with the compound.
COMBINATIONS
The present invention is further directed to
combinations comprising an aldosterone antagonist and a
NSAID. In one embodiment, the combination is a
pharmaceutical composition comprising an aldosterone
antagonist and a NSAID. One illustrative, nonlimiting
example is a pharmaceutical composition comprising
eplerenone and diclofenac.
PHARMACEUTICAL COMPOSITIONS
The present invention comprises a pharmaceutical
composition for the prevention or treatment of
cardiovascular disorders, comprising a therapeutically-
effective amount of an aldosterone antagonist and NSAID
combination in association with at least one
pharmaceutically-acceptable carrier, adjuvant or diluent
(collectively referred to herein as "carrier" materials)
and, if desired, other active ingredients. The active
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compounds of the present invention may be administered
by any suitable route known to those skilled in the art,
preferably in the form of a pharmaceutical composition
adapted to such a route, and in a dose effective for the
treatment intended. The active compounds and
composition may, for example, be administered orally,
intravascularly, intraperitoneally, intranasally,
intrabronchially, subcutaneously, intramuscularly or
topically (including aerosol).
Administration of aldosterone antagonist and
NSAID combination may take place sequentially in
separate formulations, or may be accomplished by
simultaneous administration in a single formulation or
separate formulations. Administration may be
accomplished by oral route, or by intravenous,
intramuscular or subcutaneous injections. The
formulation may be in the form of a bolus, or in the
form of aqueous or non-aqueous isotonic sterile
injection solutions or suspensions. These solutions and
suspensions may be prepared from sterile powders or
granules having one or more pharmaceutically-acceptable
carriers or diluents, or a binder such as gelatin or
hydroxypropyl-methyl cellulose, together with one or
more of a lubricant, preservative, surface-active or
dispersing agent.
For oral administration, the pharmaceutical
composition may be in the form of, for example, a
tablet, capsule, suspension or liquid. The
pharmaceutical composition is preferably made in the
form of a dosage unit containing a particular amount of
the active ingredient. Examples of such dosage units are
tablets or capsules. These may contain, for example, an
amount of each active ingredient from about 1 mg to
about 1000 mg, or about 5 mg to about 500 mg, or about
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mg to about 250 mg, or about 25 mg to about 150 mg. A
suitable daily dose for a mammal may vary widely
depending on the condition of the patient and other
factors. However, a dose of from about 0.01 to 30 mg/kg
5 body weight, particularly from about 1 to 15 mg/kg body
weight, may be appropriate.
The active ingredients may also be
administered by injection as a composition wherein, for
10 example, saline, dextrose or water may be used as a
suitable carrier. A suitable daily dose of each active
component is from about 0.01 to 15 mg/kg body weight
injected per day in multiple doses depending on the
disease being treated. A preferred daily dose would be
from about 1 to 10 mg/kg body weight. Compounds
indicated for prophylactic therapy will preferably be
administered in a daily dose generally in a range from
about 0.1 mg to about 15 mg per kilogram of body weight
per day. A more preferred dosage will be a range from
about 1 mg to about 15 mg per kilogram of body weight.
Most preferred is a dosage in a range from about 1 to
about 10 mg per kilogram of body weight per day. A
suitable dose can be administered, in multiple sub-doses
per day. These sub-doses may be administered in unit
dosage forms.
In one embodiment the aldosterone receptor
antagonist may be present in an amount in a range from
about 1 mg to about 200 mg, and the NSAID may be present
in an amount in a range from about 1 mg to about 800 mg,
which represents aldosterone antagonist-to-NSAID ratios
ranging from about 200:1 to about 1:800.
In another embodiment, the aldosterone
receptor antagonist may be present in an amount in a
range from about 5 mg to about 400 mg, and the NSAID may
be present in an amount in a range from about 1 mg to
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about 200 mg, which represents aldosterone antagonist-
to-NSAID ratios ranging from about 400:1 to about 1:40.
In another embodiment, the aldosterone
5 receptor antagonist may be present in an amount in a
range from about 10 mg to about 200 mg, and the NSAID
may be present in an amount in a range from about 5 mg
to about 100 mg, which represents aldosterone
antagonist-to-NSAID ratios ranging from about 40:1 to
10 about 1:10.
In another embodiment, the aldosterone
receptor antagonist may be present in an amount in a
range from about 20 mg to about 100 mg, and NSAID may be
15 present in an amount in a range from about 10 mg to
about 80 mg, which represents aldosterone antagonist-to-
NSAID ratios ranging from about 10:1 to about 1:4.
The NSAID dose administered to the subject or
20 contained in the pharmaceutical composition can vary and
generally wil l depend on the particular NSAID used,
inherent potency, bioavailability and metabolic
lability of the composition and whethter it has been
formulated for immediate release or extended release.
25 Non-limiting examples of dose ranges for specific NSAIDs
are listed below:
Component COMPOUND ILLUSTRATIVE ILLUSTRATIVE
Number DOSAGE FORM DOSE
N-1 acetaminophen ca sule/oral 2.5 mg - 650
m
N-2 benoxaprofen
N-3 ca rofen
N-4 diclofenac tablet/oral; 0.2 mg - 75 mg
gel; 3%
solution 0.1
N-5 diflunisal tabletloral 250 mg - 500
m
N-6 etodolac ca sule/oral 400 mg - 600
m
N-7 feno rofen ca sule/oral 200 m - 400 mg
N-8 flurbiprofen capsule/oral; 50 mg - 100 mg
solution 0.03%
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N-9 ibuprofen tablet/oral; 7.5 mg - 800
sus ension; mg
40 mg/ml - 100
mg/5 ml
N-10 indomethacin capsule/oral; 25 mg - 75 mg
suppository; 25 mg- 50 mg
injectable/injection1 mg
N-11 ketoprofen capsule/oral 25 mg- 200 mg
N-12 ketorolac
N-13 meclofenamate
N-14 mefenamic acidcapsule/oral 250 mg
N-15 nabumetone tablet/oral 500 mg - 750
mg
N-16 naproxen suspension 25 mg
tablet/oral 250 m - 500 mg
N-17 oxa rozin tablet/oral 600 m
N-18 ox henbutazone
N-19 phenylbutazone
N-20 iroxicam ca sule/oral 10 m - 20 m
N-21 sulindac tablet/oral 150 mg-200 mg
N-22 su rofen solution 1%
N-23 tenidap
N-24 tolinetin tablet/oral 200 mg - 600
mg
N-25 zome irac
N-26 aspirin tablet/oral ~ 0.19 mg - 770
mg
One of ordinary skill in the art will be
capable of using these dose ranges as a suitable
starting point to administer this therapy, after which
the dose may be titrated up or down, depending on the
response of the subject being treated.
The dosage regimen for treating a disease
condition with the combination therapy of this invention
is selected in accordance with a variety of factors,
including the type, age, weight, sex and medical
condition of the patient, the severity of the disease,
the route of administration, and the particular compound
employed, and thus may vary widely.
Below, non-limiting examples of combinations
of the present invention are listed wherein the
combination comprises a first amount of an aldosterone
receptor antgonist and a second amount of a NSAID
wherein the first amount and second amount together
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comprise a therapeutically-effective amount of an
aldosterone receptor antagonist and a NSAID:
EXAMPLE COMPONENT 1 COMPONENT 2
1 Eplerenone N 1
2 Eplerenone N-2
3 Eplerenone N-3
4 Eplerenone N-4
Eplerenone N-5
Eplerenone N-6
7 Eplerenone N-7
8 Eplerenone N-8
Eplerenone N-g
Eplerenone N-10
11 Eplerenone N-11
12 Eplerenone N-12
13 Eplerenone N-13
14 Eplerenone N-14
Eplerenone N-15
Eplerenone N-16
Eplerenone N-17
18 Eplerenone N-18
19 Eplerenone N-19
Eplerenone N-20
21 Eplerenone N-21
22 Eplerenone N-22
23 Eplerenone N-23
24 Eplerenone N-24
Eplerenone N-25
26 Eplerenone N-26
27 Spironolactone N-1
28 Spironolactone N-2
29 Spironolactone N-3
Spironolactone N-4
31 Spironolactone N-5
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32 Spironolactone N-6
33 Spironolactone N-7
34 Spironolactone N-8
35 Spironolactone N-9
36 Spironolactone N-10
3~ Spironolactone N-11
38 Spironolactone N-12
39 Spironolactone N-13
40 Spironolactone N-14
41 Spironolactone N-15
42 Spironolactone N-16
43 Spironolactone N-17
44 Spironolactone N-18
45 Spironolactone N-19
46 Spironolactone N-20
4~ Spironolactone N-21
48 Spironolactone N-22
49 Spironolactone N-23
50 Spironolactone N-24
51 Spironolactone N-25
52 Spironolactone N-26
For therapeutic purposes, the active
components of this combination therapy invention are
ordinarily combined with one or more adjuvants
appropriate to the indicated route of administration. If
administered per os, the components may be admixed with
lactose, sucrose, starch powder, cellulose esters of
alkanoic acids, cellulose alkyl esters, talc, stearic
acid, magnesium stearate, magnesium oxide, sodium and
calcium salts of phosphoric and sulfuric acids, gelatin,
acacia gum, sodium alginate, polyvinylpyrrolidone,
and/or polyvinyl alcohol, and then tableted or
encapsulated for convenient administration. Such
capsules or tablets may contain a controlled-release
formulation as may be provided in a dispersion of active
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compound in hydroxypropylmethyl cellulose. Formulations
for parenteral administration may be in the form of
aqueous or non-aqueous isotonic sterile injection
solutions or suspensions. These solutions and
suspensions may be prepared from sterile powders or
granules having one or more of the carriers or diluents
mentioned for use in the formulations for oral
administration. The components may be dissolved in
water, polyethylene glycol, propylene glycol, ethanol,
corn oil, cottonseed oil, peanut oil, sesame oil, ben~yl
alcohol, sodium chloride, and/or various buffers. Other
adjuvants and modes of administration are well and
widely known in the pharmaceutical art.
The present invention further comprises kits
that are suitable for use in performing the methods of
treatment and/or prophylaxis described above. In one
embodiment, the kit contains a first dosage form
comprising one or more of the epoxy-steroidal
aldosterone antagonists previously identified and a
second dosage form comprising a NSAID identified in
Table 1 in quantities sufficient to carry out the
methods of the present invention. Preferably, the first
dosage form and the second dosage form together comprise
a therapeutically effective amount of the compounds. In
another embodiment, the kit further comprises written
instructions stating how the contents of the kit can be
used by the subject. The written instructions will be
useful, for example, for the subject to obtain a
therapeutic effect without inducing unwanted side-
effects. In another embodiment the written instructions
comprise all or a part of the product label approved by
a drug regulatory agency for the kit.
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Crystalline Forms of Active Com ounds
It is particularly useful to select a form of each
active compound that is easily handled, reproducible in
5 form, easily prepared, stable and which is non-
hygroscopic. By way of illustration and not limitation,
several crystalline forms have been identified for the
aldosterone antagonist eplerenone. These include Form H,
Form L, various crystalline solvates and amorphous
10 eplerenone. These forms, methods to make these forms
and use of these forms in preparing compositions and
medicaments, are disclosed in the following
publications, incorporated herein by reference:
WO 01/41535 and WO 01/42272.
Subject Populations
Certain groups are more prone to disease
modulating effects of aldosterone. Members of these
groups that are sensitive to aldosterone are typically
also salt sensitive, wherein individuals blood
pressure generally rises and falls with increased and
decreased sodium consumption, respectively. While the
present invention is not to be construed as limited in
practice to these groups, it is contemplated that
certain subject groups may be particularly suited for
therapy with an anti-inflammatory dose of an
aldosterone blocker of the present invention.
Accordingly, subjects who can benefit from treatment
or prophylaxis in accordance with the method of the
present invention are human subjects generally
exhibiting one or more of the following
characteristics:
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(a) the average daily intake of sodium chloride by
the subject is at least about 4 grams, particularly
where this condition is satisfied over any one month
interval for at least one or more monthly intervals over
a given annual period. The average daily intake of
sodium by the subject preferably is at least about 6
grams, more preferably at least about 8 grams, and still
more preferably at least about 12 grams.
(b) the subject exhibits an increase in systolic
blood pressure and/or diastolic blood pressure of at
least about 5%, preferably at least about 7%, and more
preferably at least about 100, when daily sodium
chloride intake by the subject is increased from less
than about 3 g/day to at least about 10 g/day.
(C) the activities ratio of plasma aldosterone
(ng/dL) to plasma renin (ng/ml/hr) in the subject is
greater than about 30, preferably greater than about 40,
more preferably greater than about 50; and still more
preferably greater than about 60.
(d) the subject has low plasma renin levels; for
example, the morning plasma renin activity in the
subject is less than about 1.0 ng/dL/hr, and/or the
active renin value in the subject is less than about 15
pg/mL .
(e) the subject suffers from or is susceptible to
elevated systolic and/or diastolic blood pressure. In
general, the systolic blood pressure (measured, for
example, by seated cuff mercury sphygmomanometer) of the
subject is at least about 130 mm Hg, preferably at least
about 140 mm Hg, and more preferably at least about
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about 150 mm Hg, and the diastolic blood pressure
(measured, for example, by seated cuff mercury
sphygmomanometer) of the subject is at least about 85 mm
Hg, preferably at least about 90 mm Hg, and more
preferably at least about 100 mm Hg.
(f) the urinary sodium to potassium ratio
(mmol/mmol) of the subject is less than about 6,
preferably less than about 5.5, more preferably less
than about 5, and still more preferably less than about
4.5.
(g) the urinary sodium level of the subject is at
least 60 mmol per day, particularly where this condition
is satisfied over any one month interval for at least
one or more monthly intervals over a given annual
period. The urinary sodium level of the subject
preferably is at least about 100 mmol per day, more
preferably at least about 150 mmol per day, and still
more preferably 200 mmol per day.
(h) the plasma concentration of one or more
endothelins, particularly plasma immunoreactive ET-1, in
the subject is elevated. Plasma concentration of ET-1
preferably is greater than about 2.0 pmol/L, more
preferably greater than about 4.0 pmol/L, and still more
preferably greater than about 8.0 pmol/L.
(I) the subject has blood pressure that is
substantially refractory to treatment with an ACE
inhibitor; particularly a subject whose blood pressure
is lowered less than about 8 mm Hg, preferably less than
5 mm Hg, and more preferably less than 3 mm Hg, in
response to 10 mg/day enalapril compared to the blood
pressure of the subject on no antihypertensive therapy.
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(j) the subject has blood volume-expanded
hypertension or blood volume-expanded borderline
hypertenision, that is, hypertension wherein increased
blood volume as a result of increased sodium retension
contributes to blood pressure.
(k) the subject is a non-modulating individual, that
is, the individual demonstrates a blunted positive
response in renal blood flow rate and/or in adrenal
production of aldosterone to an elevation in sodium
intake or to angiotensin II administration, particularly
when the response is less than the response of
individuals sampled from the general geographical
population (for example, individuals sampled from the
subject's country of origin or from a country of which
the subject is a resident), preferably when the response
is less than 40% of the mean of the population, more
preferably less than 30%, and more preferably still less
than 200.
(I) the subject has or is susceptible to renal
dysfunction, particularly renal dysfunction selected
from one or more members of the group consisting of
reduced glomerular filtration rate, microalbuminuria,
and proteinuria.
(m) the subject has or is susceptible to
cardiovascular disease, particularly cardiovascular
disease selected from one or more members of the group
consisting of heart failure, left ventricular diastolic
dysfunction, hypertrophic cardiomyopathy, and diastolic
heart failure.
(r1) the subject has or is susceptible to liver
disease, particularly liver cirrhosis.
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(o) the subject has or is susceptible to edema,
particularly edema selected from one or more members of
the group consisting of peripheral tissue edema, hepatic
or splenic congestion, liver ascites, and respiratory or
lung congestion.
(p) the subject has or is susceptible to insulin
resistance, particularly Type I or Type II diabetes
mellitus, and/or glucose sensitivity.
(q) the subj ect is at least 55 years of age,
preferably at least about 60 years of age, and more
preferably at least about 65 years of age.
(r) the subject is, in whole or in part, a member of
at least one ethnic group selected from the Asian
(particularly from the Japanese) ethnic group, the
American Indian ethnic group, and the Black ethnic
group.
(S) the subject has one or more genetic markers
associated with salt sensitivity.
(t) the subject is obese, preferably with greater
than 25% body fat, more preferably with greater than 30%
body fat, and even more preferably with greater than 350
body fat.
(U) the subj ect has one or more 1St, 2nd or 3ra
degree relatives who are or were salt sensitive, wherein
1St degree relatives means parents or relatives sharing
one or more of the same parents, 2na degree relatives
means grandparents and relatives sharing one or more of
the same grandparents, and 3ra degree relatives means
great-grandparents and relatives sharing one or more of
the same great-grandparents. Preferably, such
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individuals have four or more salt sensitive 1St, 2nd, or
3rd degree relatives; more preferably, eight or more
such relatives; even more preferably, 16 or more such
relatives; and even more preferably still, 32 or more
5 such relatives.
Unless otherwise indicated to the contrary, the
values listed above preferably represent an average
value, more preferably a daily average value based on at
least two measurements.
10 Preferably, the subject in need of treatment
satisfies at least two or more of the above-
characteristics, or at least three or more of the above-
characteristics, or at least four or more of the above-
characteristics.
Biological Evaluation
Human cardiovascular disorders are complex
conditions, often initiated by vascular hypertension or
a myocardial infarction (MI). In order to determine the
probable effectiveness of a therapy for cardiovascular
disorders, it is important to determine the potency of
components in several assays. Accordingly, in Assay
"A", the efficacy of the aldosterone antagonist
eplerenone (epoxymexrenone) was determined in a
hypertensive rat model with vascular inflammation, using
angiotensin II infusion. In Assay "B" a study is
described evaluating the efficacy of the aldosterone
antagonist eplerenone (epoxymexrenone) in a rat model
using aldosterone infusion to produce hypertension with
vascular inflammation. In Assay "C" a further study is
described evaluating the efficacy of the aldosterone
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antagonist eplerenone (epoxymexrenone) in a rat model
using aldosterone infusion to produce hypertension with
vascular inflammation.
In addition, clinical trials can be used to
evaluate aldosterone antagonist therapy in humans.
Numerous examples of such therapeutic tests have been
published, including those of the RAZES 003 study
described in American Journal of Cardiology 78, 902-907
(1996) or the RAZES 004 study described in New England
Journal of Medicine 341, 709-717 (1999).
Assay A: In Vivo Angiotensin II Infusion Model
Protocol:
Methods:
~ Male Wistar rats (n=50, 10/group; BW=200 g)
~ 1% NaCl to drink
~ Experimental groups
1. Control
2. Angiotensin II (25 ng/min, sc via alzet
minipump)
3. Angiotensin II (25 ng/min, sc) + eplerenone 100
mpk
4. Angiotensin II (25 ng/min, sc) + adrenalectomy +
dexamethasone (12 ~,g/kg/d, sc)
5. Angiotensin II (25 ng/min, sc) + adrenalectomy +
dexamethasone (12 ~.g/kg/d, sc) + aldosterone (40
mg/kg/d, sc via alzet minipump)
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~ SBP measured by tail-cuff every week
~ 24-hours food and fluid intake and urine output
measured every day
~ Urine samples collected every day for determination
S of urinary electrolytes.
~ Sacrifice by exanguination after 4 weeks. Blood was
be collected in dry tubes for determination of serum
electrolytes and in EDTA-containing tubes for
measurement of aldosterone and corticosterone levels
~ Hearts were stained with hematoxylin&eosin and have
been analyzed for determination of morphologic
abnormalities (i.e. necrosis, vascular injury).
Results
Blood pressure. Systolic blood pressure increased in all
animals receiving angiotensin II infusion. Neither
eplerenone nor adrenalectomy reduced blood pressure when
compared to animals receiving vehicle. Aldosterone
infusion increased blood pressure in angiotensin
II/salt, adrenalectomized rats. Fig. 1 demonstrates
this increase in systolic blood pressure.
Electrolyte excretion. The ratio between daily urinary
Na+ excretion and urinary K+ excretion (U Na+/K+ ratio)
was used as an index for natriuresis. Urinary Na+/K+
ratio was similar in all groups before the start of the
treatments, and increased similarly in all animals upon
initiation of the high salt diet. Urinary Na+/K+ ratio
was not unchanged in animals receiving angiotensin II
infusion until day 17 when it was significantly
increased in these animals with respect to the vehicle-
infused rats. A similar effect occurred in angiotensin
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II-infused animals receiving eplerenone, which
demonstrated increases in urinary Na+/K+ ratio from day
14 of infusion. However, at no time-point did
eplerenone-treated rats demonstrate higher urinary
Na+/K+ ratio than angiotensin II-infused rats treated
with vehicle. In fact, a significant difference was
only observed at day 21, when angiotensin II-infused,
vehicle treated rats demonstrated higher urinary Na+/K+
ratio than eplerenone-treated animals indicating that,
under these experimental conditions eplerenone did not
produce a significant diuretic or natriuretic effect.
Adrenalectomized animals with or without aldosterone
infusion always demonstrated higher urinary Na+/K+ ratio
than the adrenal-intact animals.
Myocardial injury. Seven out of the ten angiotensin
II/salt-treated animals developed vascular inflammatory
changes in the coronary arteries. These changes were
characterized by leukocyte infiltration of the
perivascular space, mainly by macrophages. Fibrinoid
necrosis of the media was also observed in some
arteries. In some cases, when the lesions were
extensive there was cardiomyocyte necrosis associated in
the surrounding myocardium. P,arenchymal hemorrhages
were observed in these cases, consistent with findings
of myocardial necrosis. These vascular inflammatory
lesions were observed in only one of the ten angiotensin
II~-infused animals receiving eplerenone, despite the
fact that these animals were as hypertensive as the
vehicle-treated angiotensin II-infused rats. (See Fig.
2). Similarly, adrenalectomy prevented the vascular
inflammatory lesions in the heart. However, aldosterone
replacement restored the severe coronary and myocardial
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inflammation and injury observed in angiotensin-II
infused, adrenal-intact, vehicle-treated rats.
Immunostaining of the hearts from angiotensin II-infused
rats with a cyclooxgenase-2 specific antibody identified
the presence of this enzyme in areas of inflammation
around the arteries, mainly in monocyte/macrophages.
Cycloxygenase-2 staining was also observed in the
vascular smooth muscle cells of the media of coronary
arteries, even when there was no evidence of morphologic
alterations or inflammatory aggregates in the
perivascular space (Fig. 4). Eplerenone treatment, as
well as adrenalectomy markedly reduced and in most cases
completely prevented the expression of cycloxygenase-2
in the hearts from angiotensin II-infused rats (See
Figs. 3 and 5). Replacement of aldosterone in
angiotensin-II, adrenalectomized rats restored the
presence of cycloxygenase-2 in coronary arteries.
Osteopontin (also known as early T-cell activation-
1, Eta-1) is a secreted glycoprotein with pro-
inflammatory characteristics that mediates
chemoattraction, activation and migration of monocytes.
Immunostaining of the hearts from angiotensin II-
infused, saline-drinking rats with an osteopontin-
specific antibody identified the presence of osteopontin
in the media of coronary arteries. Both eplerenone
treatment and adrenalectomy prevented osteopontin
expression in the hearts of angiotensin II-infused,
saline-drinking rats (Figs. 6 and 7). Aldosterone
replacement restored osteopontin expression in
adrenalectomized animals.
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Assay B: In Vivo Aldosterone Infusion Model
Protocol 2:
5 Methods:
~ Male Sprague Dawley rats (n=39; BW=250 g)
~ 1% NaCl to drink
~ Uni-nephrectomy performed during implantation of
mini-pumps
10 ~ Experimental groups
1. Con trol
2. Aldosterone (0.75 mg/hr, sc via alzet minipump)
2. Aldosterone (0.75 mg/hr, sc tria alzet minipump)
15 + eplerenone 100 mpk, p.o
1. Aldosterone (0.75 mg/hr, sc via alzet minipump)
+ 0.6o KCl in the drinking fluid
~ Groups l, 2 and 3 received only 0.3% KCl in the
20 drinking solution
~ SBP measured by radio-telemetry probes inserted in
the abdominal aorta
~ Sacrifice after 4 weeks.
-~ Hearts were harvested and divided by half through a
25 transverse section at the mid-ventricles: The upper
half was stored into formalin. The bottom part was
snap-frozen in liquid nitrogen for biochemical
analysis.
~ Hearts were stained with hematoxylin & eosin and the
30 collagen specific dye picro-sirius red and were
analyzed for determination of interstitial collagen
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volume fraction and morphologic abnormalities (i.e.
necrosis, vascular injury).
~ Hydroxyproline concentration was measured in the
frozen hearts.
~ Determination of osteopontin and COX-2 was performed
by quantitative RT-PCR (Taqman). Osteopontin was
also identified in the heart by immunohistochemistry.
Results
Blood pressure. Systolic blood pressure increased in all
animals receiving aldosterone infusion. Eplerenone
treatment significantly reduced, but did not normalize
blood pressure. Fig. 21 shows these results
graphically.
Myocardial injury. Saline-drinking, uni-nephrectomized
rats did not have myocardial injury. Determination of
interstitial collagen by histologic determination of
interstitial collagen volume fraction or by biochemical
determination of hydroxyproline concentration evidenced
the absence of myocardial fibrosis in animals receiving
aldosterone/salt treatment. However, examination of the
hematoxilin-eosin-stained hearts from aldosterone/salt-
treated rats evidenced severe vascular inflammatory
lesions. These lesions were identical to those
described in protocol 1. Administration of eplerenone
completely prevented the vascular inflammatory changes
in aldosterone-infused, saline-drinking, uni-
nephrectomized rats (Fig. 10), even though it did not
normalize blood pressure. Elevations of dietary
potassium did not have significant effects in the
development of aldosterone-induced injury, as these
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animals demonstrated similar levels of injury as the
aldosterone/salt treated rats receiving vehicle.
Serum osteopontin levels were determined at 28
days, and measured for each group (NaCl 1o drinking
rats, NaCl 1o drinking rats with aldosterone, and NaCl
1% drinking rats with aldosterone and eplerenone). Fig.
23 shows the marked decrease in circulating osteopontin
levels in the eplerenone treated rats.
Osteopontin immunostaining was also performed in
the hearts from these animals. Osteopontin was not
detected in saline-drinking, uninephrectomized animals
receiving no aldosterone. However, osteopontin was
clearly identified in the media of coronary arteries in
animals receiving aldosterone infusion. Eplerenone
treatment, prevented the expression of osteopontin in
the hearts from aldosterone-infused rats (Figs. 8 and
18). Increases in dietary potassium did not reduce
osteopontin expression. Determination of osteopontin
mRNA by quantitative RT-PCR, demonstrated a marked (7-
fold) upregulatoin of this Cytokine in the hearts of
aldosterone/salt-treated rats receiving vehicle
(relative mRNA expression: 1.7~.2 vs 12.25~1.7,
P<.0001). This effect was prevented by eplerenone
(relative mRNA expression: 2.5~.6, P<.0001 vs
aldosterone/salt+vehicle group). Consistent with a role
for cycloxygenase-2 in the development aldosterone-
induced vascular inflammation in the heart, COX-2 mRNA
expression was 3-fold increased in rats with
aldosterone/salt+vehicle treatment (relative mRNA
expression: 1.2~.12 vs 3.7~.46, P<.0001). Similar to
the effects on osteopontin expression, eplerenone
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prevented the increase in COX-2 expression in
aldosterone/salt-treated rats (relative mRNA expression:
1.8~.36, P<.01 vs aldosterone/salt+vehicle group, see
Figs. 9 and 17). In like fashion, MCP-1 expression and
IL-6 expression was attenuated by eplerenone treatment
(Fig. 24).
The above data suggest that aldosterone mediates a
vascular inflammatory phenotype in the heart of
hypertensive rats. This phenotype is associated with
up-regulation of the cytokine osteopontin and the enzyme
cycloxygenase-2 in vascular smooth muscle cells in the
arterial media, which may mediate the perivascular
inflammation observed and the consequent
ischemic/necrotic injury of coronary arteries and
myocardium. Without wishing to be bound by any theory,
it is believed that this is the mechanism that mediates
the vascular alterations observed in diseases such as
heart failure, coronary artery disease, auto-immune or
viral myocarditis, periateritis nodosa, stroke, and
nephrosclerosis. Fig. 11 reveals that osteopontin and
cyclooxygensase-2 are expressed in similar regions of
the coronary arterial wall. While~theory plays no part
in the instant invention, Fig. 12 shows a proposed
mechanism for this model. In these examples, eplerenone
treatment prevented the vascular inflammation in the
heart to an extent similar to that of adrenalectomy, as
demonstrated in protocol #1. The effects of eplerenone
were largely independent of major reductions in systolic
blood pressure as demonstrated in protocol #1. The lack
of a diuretic or natriuretic effect of eplerenone in
angiotensin II/salt hypertensive rats suggests that the
protective effects of the selective aldosterone
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antagonist were also independent of its potential
effects on epithelial tissues. In addition, the fact
that an elevated dietary potassium failed to mimic the
effects of eplerenone, argue against the possibility
that eplerenone provides benefit through its potassium-
sparing properties. Thus, we propose that aldosterone
may have direct deleterious effects in the coronary
vasculature unrelated to the effects of this hormone in
electrolyte homeostasis in epithelial tissues or its
effects on blood pressure. Administration of eplerenone
to humans could provide benefit by its anti-inflammatory
effects in vascularized organs, including but not
limited to heart, kidney, and brain, as suggested by the
present experiment.
Assay C: Further In Vivo Aldosterone Infusion Study
The procedure of Assay B was expanded upon in a
further study. Uninephrectomized, Sprague-Dawley rats
were given 1%NaCl-0.3%KCl to drink and one of the
following treatments: vehicle; aldosterone infusion; or
aldosterone infusion in combination with eplerenone (100
mg/kg/day). Aldosterone/salt treatment induced severe
hypertension in rats after 30 days, which was
significantly reduced by eplerenone. Myocardial tissue
from animals in each treatment group was examined after
7, 14, or 30 days of treatment. Histopathologic
analysis revealed vascular inflammatory lesions starting
at 14 days that extended to surrounding myocardium and
resulted in focal ischemic/necrotic changes. Lesions
were preceded by the expression and progressive
upregulation of proinflammatory molecules. Upregulation
of proinflammatory molecules and associated vascular and
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myocardial damage were markedly attenuated by eplerenone
treatment. These data demonstrate that eplerenone is
effective in reducing blood pressure and providing end-
organ protection against aldosterone-induced vascular
5 inflammatory damage in the heart.
Animals
Male Sprague-Dawley rats, weighing 230 to 250 g, (Harlan
Sprague-Dawley Industries, Indianapolis, IN) were housed
10 in a room 12-hours light/12-hours dark daily cycle at an
ambient temperature of 22~1°C (n=96). Animals were
allowed one week to adjust after arrival and had free
access to TEKLAD 22/5 rodent diet (Harlan TEKLAD,
Madison, WI) and tap water until the initiation of the
15 experiment.
Experimental Protocol
Prior to surgery the animals were individually weighed
and placed in one of the following groups: (I) high salt
20 control (vehicle/normal chow/1% NaCl & 0.3o KCl drinking
water, n=31 for 3 time point groups), (II) aldosterone
control (aldosterone/normal chow/1o NaCl & 0.3% KCl
drinking water, n=28 for 3 time point groups), (III) 100
mg/kg/day eplerenone (aldosterone/eplerenone chow/1%NaCl
25 & 0.3o KCl drinking water, n=30 for 3 time points).
Potassium chloride supplementation was added to the
saline solution in order to prevent the potential
hypokalemia associated with aldosterone excess.
Treatment
30 At the time of the surgery, an Alzet 2002 osmotic
minipump (Alza Corp., Palo Alto, CA) containing either
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vehicle (9% ethanol/87% propylene glycol/4% dH20) or 1.0
mg/mL d-aldosterone (Sigma Chemical, St. Louis, MO) was
inserted subcutaneously at the nape of the neck.
Aldosterone was administered at a dose of 0.75 Og/hour.
Eplerenone was incorporated into TEKLAD 22/5 rodent diet
(Harlan TEKLAD, Madison, WI) at a concentration of lmg/g
of chow (calculated to deliver 100 mg/kg/day). Previous
analytical work has demonstrated the stability of
eplerenone in this diet, as well as the homogeneity
obtained after preparation. Animals were sacrificed
from each group (n=8-13) after 7, 14, or 30 days of
treatment.
Surgical procedure
Animals to be sacrificed after 7 or 14 days of treatment
were uninephrectomized and implanted with an Alzet
minipump. Animals treated for 30 days were
uninephrectomized, fitted for Alzet minipumps, and
implanted with radio telemetry units (model# TA11PA-C40,
Data Sciences Inc., St. Paul, MN) according to the
following procedure. Animals were anesthetized with 50
isoflurane (SOLVAY Animal Health Inc., Mendota Heights,
MN), which was delivered in 02 using a VMS anesthesia
instrument (Matrix Medical, Inc., Orchard Park, NY).
Anesthesia was maintained with 1-2% isoflurane
throughout the surgical procedure. The surgery site was
clipped, scrubbed with nolvasan, and sprayed with
betadine. A rostral-caudal incision was made through
the skin from the base of the rib cage to the pubic
region using a #11 scalpel blade. A second incision was
made through the muscles of the abdominal wall to expose
the peritoneal cavity. The urethra, renal artery and
vein of the left kidney were isolated, tied off with 4-0
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silk, and the kidney excised and discarded. Organs were
carefully displaced with tissue retractors in order to
expose the abdominal aorta. A 1.5 cm segment just
rostral to the bifurcation of the abdominal aorta into
the iliac arteries was cleared of excessive connective
tissue and 4-0 silk was used to make an anchor adjacent
to the aorta. A microvascular clip was then placed at
both ends of the cleaned region to stop excessive blood
flow. Using a bent, 21 gauge needle, the abdominal
aorta was penetrated. The cannula of the radio
telemetry unit was inserted and stabilized in the aorta
using the 4-0 silk anchor. Organs were repositioned and
the telemetry unit was placed over the organs. Using a
non-interrupted suture pattern with 4-0 silk, the
abdominal wall was closed, and the skin was subsequently
closed using a 4-0 silk in an interrupted suture
pattern. Animals were injected around the sutures with
100 ~,L of the anesthetic Marcaine HCl (Sanofi Winthrop
Pharmaceuticals, New York, NY) and given an injection
(i.m.) of the antibiotic Mandol (Eli Lilly & Co.,
Indianapolis, IN). Post-operative care included
monitoring the animals on a heating pad during recovery
from anesthesia until sternal recumbency was
reestablished. Animals were monitored daily for signs
of distress and infection at the surgical site. Animals
displaying continued discomfort after surgery were
treated with 0.1-0.5 mg/kg, s.c. Buphrenorphine
(Rickett & Colman Pharmaceuticals, Inc. Richmond, VA).
Animals were then placed on tap water and TEKLAD 22/5
rodent diet (Harlan TEKLAD, Madison, WI).
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Blood Pressure Analysis
Radiotelemetrized arterial blood pressure was calculated
with the DATAQUEST A.R.T Version 1.1-Gold software (Data
Sciences International, St. Paul, MN). Data points were
collected over a 24 hour period with the collection rate
set for a 10 second reading every 5 min for each animal.
The 24 hour period used was from 6:00 a.m. to 6:00 a.m.
Sacri fi ce
At the cessation of each experimental time point, the
animals were anesthetized with pentobarbital (65 mg/kg
i.p., Sigma Chemical, St. Louis MO) and weighed with a
Mettler PM6000 balance (Mettler-Toledo, Inc.,
Hightstown, NJ). The abdominal cavity was opened to
expose the abdominal aorta. A 16-gauge needle was
inserted into the abdominal aorta and the animal was
exsanguinated into a l2cc syringe. The blood sample was
transferred immediately into glass serum collection
tubes (Terumo Medical Corp., Elkton, MD) for drug level
analysis. The samples were placed on wet ice until
sample collection was complete and centrifuged for 15
min at 3000 rev/min at 4°C.
Following exsanguination, hearts and kidneys were
isolated, removed, rinsed in cold phosphate-buffered
saline, and blotted dry. Kidneys were immediately
bifurcated through the long axis with a razor blade and
placed in loo neutral buffered formalin (NBF, Richard-
Allen Scientific, Kalamazoo, MI). For the hearts, the
right ventricle (RV) was cut away from the left
ventricle (LV), both ventricles were weighed using a
Mettler AE163 balance (Mettler-Toledo, Inc., Hightstown,
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NJ), and the RV was placed in 10% NBF. A 2 mm coronal
slab of the L~V apex was removed and frozen with dry
ice/isopentane for analysis of gene expression and the
remaining portion of the LV was placed in 10o NBF for
fixation. Final wet trimming was completed after 3-4
days fixation where a second 2 mm coronal slab was
removed for hydroxyproline analysis and a third 2mm slab
was removed from the equatorial region for histology.
Tissue Processing & Staining
The equatorial regions of the heart were routinely
processed into paraffin with an automated tissue
processor (Hypercenter XP, Shandon/Lipshaw Inc.,
Pittsburgh, PA) and embedded into fresh paraffin apical
side down (Shandon Embedding Center, Shandon/Lipshaw
Inc.). Five and 10 um sections were cut from each block
of tissue using a Leica RM2035 rotary microtome (Leica
Inc., Houston, Texas) and mounted on Superfrost/Plus
microscope slides (Fisher Scientific, Pittsburgh, PA).
Ten ,um sections were stained with the collagen specific
stain, Picrosirius Red F3BA (Saturated Picric Acid
(Sigma Chemical, St. Louis, MO) with 0.10 (w/v) Sirius
Red F3BA (C. I. #35780, Pfaltz & Bauer, Inc. VJaterbury,
CN) (6). Mounted tissues were hydrated with water.
Slides were subsequently incubated in distilled water
with 0.20 (w/v) Phosphomolybdic Acid (Sigma Chemical,
St. Louis MO) for 15 min, transferred to 0.10
Picrosirius Red F3BA stain for 110 min, placed in 950
ethanol w/ 1% acetic acid (v/v) for 1 min followed by
two, 1-min incubations in 1000 ethanol, and cleared in
xylene for 1 min. Slides were coverslipped with #1
cover glass using Permount Histological Mounting Media
(Fisher Scientific). Two slides mounted with 5 ~m
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sections were cut for each animal. One slide was
processed for H&E staining and one for Periodic Acid
Schiff (PAS) staining. The H&E and PAS were used for
pathological scoring of the hearts.
5
Histopathologic Analysis
Semi-quantification of myocardial injury was performed
as described previously with minor modifications (7).
Briefly, a scale from 0 to 4 was used to score the level
10 of myocardial injury. A score of 0 represented no
damage. A score of 1 represented the presence of
vascular and perivascular inflammatory lesions without
cardiomyocyte injury. A score of 2 was given when one
clear area of myocardial necrosis was observed.
15 Myocardial necrosis was defined as the presence of
necrotic changes in cardiomyocytes such as nuclear
' pyknosis or karyolysis, non-contracting marginal wavy
fibers and hypereosinophilia of the cytoplasm, or clear
evidence of destruction of the cardiomyocyte membrane.
20 When two or more separate areas of necrosis were found
(implicating the presence of two different infracted
regions), hearts received a score of 3. A score of 4
was assigned to hearts that demonstrated extensive areas
of necrosis compromising more than 500 of the left
25 ventricle.
Image Analysis
Picrosirius Red F3BA stained slides were used to
quantify interstitial collagen with a Videometric 150
30 Image Analysis System (Oncor Inc., Gaitherburg, MD).
Briefly, images were captured using a Nikon E Plan
10/0.25; 160/- Objective (Nikon Inc. Garden City, NY)
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attached to a Nikon Optiphot microscope (Nikon Inc.). A
Toshiba 3 CCD Color Video Camera (Model#IK-T30T, Toshiba
Corp. Japan) relayed the images in RGB format from the
microscope to a 386 computer with a V150 video board.
The V150 video board/V150 software application (Oncor
Inc.) converted RGB images to HIS (Hue, Intensity,
Saturation) format for display and analysis on a Sony
Trinitron Color Video Monitor (Model#PVM-1342Q, Sony
Corp, Tokyo, Japan) at a magnification of 305x. Once
the image was displayed on the image monitor; hue,
intensity, and saturation of pixels to be measured were
defined by a process called thresholding. The V150
application then measured only pixels which fell into
thresholding limits. The system was calibrated with a
micrometer scale (EM Sciences, FT. Washington, PA
19034), which allowed data to be expressed in mm2 or
Omz. After each measurement, data was automatically
saved in ASCII file format and transferred to Microsoft
Excel version 7.0 for final summation.
Immunohistochemistry
Five ~,m sections were deparaffinized in xylene (two, 5-
10 min incubations) and rehydrated by 3 min incubations
in ethanol as follows: two incubations in 100% ethanol
followed by two incubations in 95% alcohol and one
incubation in 70% alcohol. Once hydrated, sections were
rinsed in tap water for 1 min and distilled water for 1
min. Endogenous peroxide activity was blocked by
placing slides in 3.0% H~02 for 15 min followed by a 5
min rinse in distilled water. Slides were processed for
antigen retrieval using citric acid, pH6Ø Slides were
heated to boiling, cooled for 20 min at 25°C, and rinsed
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in distilled water. Slides were stained using a DAKO
autostainer (DAKO Corporation, Carpinteria, CA). Prior
to staining, slides were rinsed and incubated in
blocking buffer for 20 min. Blocking buffer is
described in the Vectastain ABC kit (Vector Labs,
Burlingame, CA) and contains 10 mL TNB (NEN TSA Biotin
System kit, Cat#NEL700A, NEN Life Science Products,
Boston, MA) and 3 drops of normal (corresponding to the
secondary antibody) serum.
Primary antibodies used for staining include:
Osteopontin, diluted at 1:100 (Mouse monoclonal,
Cat#MPIIIbIO, Developmental Studies Hybridoma Bank, The
University of Iowa, Iowa City, IA); ED-1 diluted at
1:500 (anti-macrophage glycoprotein, mouse monoclonal,
MAB1435, Chemicon International Inc., Temecula, CA); CD-
3 diluted at 1:300 (anti-T-cell, rabbit polyclonal-
affinity purified antibody, A0452, DAKO Corporation,
Carpineria, CA); ICAM-1 diluted at 1:100 (goat
polyclonal-affinity purified, M-l9:sc-1511, Santa Cruz
Biotechnology, Santa Cruz, CA); VCAM-1 diluted at 1:100
(goat polyclonal-affinity purified, C-l9:sc-1504, Santa
Cruz Biotechnology). Slides were incubated with primary
antibodies for 60 min, followed by biotinylated
antibodies at a final concentration of 5 ~,L/mL for 30
min at 25°C. Staining was visualized with the
Vectastain ABC-AP kit (Vector Laboratories) and
diaminobenzidine staining (DAKO Corporation,e
Carpinteria, CA). Slides were rinsed in water and
counter-stained with hematoxylin for approximately 30
sec. Isotype-matched IgG (Sigma Chemical, St. Louis MO)
was used as a negative control for the primary
antibodies.
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In situ Hybridization for Osteopontin mRNA
RNA probes were generated based on a sequence for rat
osteopontin (GenBank accession# NM 008608-1). Briefly,
a cDNA fragment of rat osteopontin was generated by RT-
PCR using the following primers: forward primer, 5'-TGG
CAC ATT TGT CTT; reverse primer 3'AGC CCA TCC AGTC. The
cDNA fragment was inserted into the PCR II plasmid using
the TA cloning kit (Invitrogen Corporation, Carlsbad,
CA) . Probes were labeled in 100 ~,L in vitro
transcription reaction containing: rRNasin (2 U), DNase
(0.5 U) , TE Buffer (1X) , rGTP (10 mM) , rCTP (10 mM) ,
rATP ( 10 mM) , rUTP ( 10 mM) , ( PROMEGA, Madi son, WI ) , 5/~,L
(50~Ci) 33P-UTP (Elkin Pelmer, Boston, MA) and
appropriate RNA polymerases (Sp6 RNA Polymerase (20
U/~.L) ; T7 RNA Polymerase (15 U-~.L) , PROMEGA) for 60 min
at 37°C. Free label was removed from the reaction using
Microcon YM-50 Microconcentrators (Amicon, Bedford, MA).
Sections were deparaffinized in xylene, rehydrated in
graded ethanol solutions as described above, and fixed
in 4% paraformaldehyde (EMS, Ft. Washington, PA) for 10
min at 4°C. Tissues were then digested with Proteinase
K (5 mg/mL; 10 min, 37°C, Roche, Indianapolis, IN) and
washed in 0.5 X SSC buffer (Saline-Sodium Citrate
buffer) (10 min) . Prehybridization was performed after
sequential dehydration in graded series of ethanol, the
reverse process as described above for rehydration,
followed by incubation in hybridization buffer (50%
formamide, 2 X SSC, 10% dextran sulfate, v/v) for 2
hours at 42°C. Hybridization was performed overnight
using hybridization buffer containing tRNA (50 ~,g/mL,
Sigma, St. Louis, MO) and the appropriate labeled probe
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at 55°C. Hybridized tissues were then washed
successively in ZX SSC buffer, O.1X SSC-EDTA buffer
(O.1X SSC, 1mM EDTA), and 2X SSC buffer for 1 hour 40
min. Slides were finally dehydrated in graded series of
ethanol as described above containing NH40Ac (2 min
each) and dried in a vacuum desiccator for 1.5 hours at
room temperature. Tissues were exposed overnight to a
phosphorus screen. Slides were coated with photographic
emulsion (Kodak, Rochester, NY) and exposed at 4°C for
3-5 weeks prior to development. Developed slides were
counterstained with hematoxylin and eosin.
Principles of TaqMan Analysis
The fluorogenic 5'-nuclease assay (TaqMan PCR) using
Applied Biosystems' 7700 Sequence Detection System
(Applied Biosystems, Foster City, CA) allowed for real
time detection/quantitation of a specific gene by
monitoring the increase in fluorescence of a gene-
specific, dye-labeled oligonucleotide probe. Probes for
target and reference genes were labeled at the 5'-end
with a 6-carboxyfluorescein (6FAM) reporter dye and at
the 3'-end with a 6-carboxy-N,N,N',N'-
tetramethylrhodamine (TAMRA) quencher dye. When the
probe was annealed to the target gene, fluorescence of
6FAM was prevented by the close proximity of TAMRA. The
exonuclease activity of Taq polymerase released the dyes
from the oligonucleotide probe by displacing the probe
from the target sequence resulting in fluorescence
excitation in direct proportion to the amount of target
message present. Data analysis was performed using the
Sequence Detection System software from Applied
Biosystems.
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T5
TaqMan Primers and Probes: TGF~i 1, ANP, Collagen I,
Collagen III
Primers and probes were designed using Oligo Primer
Analysis Software, Version 5.0 (National Biosciences
Inc. (NBI)-Wojciech Rychlik, Cascade, CO). Primers were
synthesized by Life Technologies (Grand Island, NY) and
probes were synthesized by Applied Biosystems.
Primer/probe sets were designed from known sequences of
rat genes to be analyzed. All target gene values were
normalized to a reference gene, constitutively expressed
cyclophilin. Primer/probe sets sequences can be found
in Table 8
Table 8 TaqMan RT-PCR Gene Marker Primer/Probe Sets
Gene Forward Primer Reverse Probe
Primer
Transform CACCATCCATGACA ACCTTGCTGTACT TCAGCTCCACAG
ing TGAACC ' GTGTGTCC AGAAGAACTGC
growth
factor
beta
1 ( TGF(31
)
Atrial TGGGCTCCTTCTCC AGCAGAGCCCTCA CCATATTGGAGC
natriuret ATCAC GTTTG AAATCCCGTATA
is factor C
(ANP )
Collagen ACCAAGGCTGCAAC GCAGGAAGGTCAG CCATACTCGAAC
I CTGGA CTGGAT TGGAATCCATCG
Collagen GGCTTTCAGTTCAG GACTGTCTTGCTC CCTGATCTTCCT
III CTATGG CATTCAC GAAGATGTCCTT
G
Cyclophil CTTGTCCATGGCAA GTGATCTTCTTGC CCACAATGCTCA
in ATGCTG TGGTCTTGC TGCCTTCTTTCA
CC
Cyclooxeg TCAAAGACACTCAG CGGCACCAGACCA CACGTCCCTGAG
enase-2 GTAGA AAGACTT CACCTGCGG
(COX-2) CATGATCT
Osteopont CCAGCACACAAGCA TCAGTCCATAAGC CAGTCGATGTCC
in GACGTT CAAGCTATCAC CTGACGGCCG
Monocyte GCAGGTCTCTGTCA GGCTGAGACAGCA CCTGTTGTTCAC
Chemoattr CGCTTCT CGTGGAT AGTTGCTGCCTG
actant TAGC
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76
Protein-1
(MCP-1)
Intercell ACCTGCAGCCGGAA CCCGTTTGACAGA CCGATAGGCAGC
ular AGC CTTCACCAT GGGACACCA
Adhesion
Molecule-
1 ( I CAM-
1)
Vascular GAAGCCGGTCATGG GGTCACCCTTGAA TGGCTCCTGATG
Cell TCAAGT CAGTTCTATCTC TTTACCCAATTG
Adhesion ACAGA
Molecule
-1 (VCAM-
1)
Cyclophil AGAGAAATTTGAGG TTGTGTTTGGTCC AAGCATACAGGT
in ATGAGAACTTCAT AGCATTTG CCTGGCATCTTG
TCCAT
All oligonucleotides are written 5' - 3'. Primers are
unlabeled and all probes are labeled at the 5' end with
6-carboxyfluorescein (6FAM) reporter dye and at the 3'
end with 6-carboxy-N,N,N',N'-tetramethylrhodamine
(TAMRA) quencher dye
RNA isolation: TGF(31, ANP, Collagen I, Collagen III
RNA was extracted from frozen (-70°C) left ventricle
(LV) tissue (approximately 10-20 mg) using 1.5 mL RNA-
STAT 60 according to manufacturer's instructions (Leedo
Medical Laboratories, Inc., Houston, Texas). Briefly,
tissues were homogenized using a tissue homogenizer
equipped with a 5 mm probe (Ultra-Turrax T8 Homogenizer,
IKA Works, Inc. Wilmington, NC). Following
homogenization, an equal volume of molecular grade
chloroform (Sigma Chemical Co., St. Louis, Mo.) was
incubated with periodic mixing for 10 min at room
temperature. Samples were centrifuged at 12,OOOg for 10
min and RNA was precipitated from the top layer by
adding an equal volume of molecular grade isopropanol
(Sigma Chemical Co.) followed by an overnight incubation
at -80°C. RNA was pelleted by centrifugation at
12,OOOg, washed with 75% ethanol, and solubilized in
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nuclease-free water (Promega, Madison, WI). RNA was
diluted and analyzed spectrophotometrically for
concentration and purity (A260/A280 = 1.9 - 2.0, with an
average yield of 2-5 ~,g RNA).
Reverse Transcription: TGF/31, ANP, Collagen I, Collagen
III
Double-stranded CDNA was synthesized by adding 400 ng
RNA (4uL) to a final volume of 20 uL containing 15%
nuclease-free water (Promega, Madison, WI), 1X RT Buffer
(Life Technologies, Grand Island, NY), 10 mM DTT (Life
Technologies), 0.5 mM each of dATP, dTTP, dGTP, dCTP (PE
Biosystems, Foster City, CA), 2.5~,M Oligo d(T)15 (Oligo
Therapeutics, InC., Wilsonville, OR), 40 units RNAsin
(Promega), and 200 units Superscript II Reverse
Transcriptase (Life Technologies). The reactions were
performed in thin-walled reaction tubes with caps
(Applied Biosystems) to ensure accurate reaction
temperatures. Reactions were performed using a GeneAmp
9600 thermal Cycler (Applied Biosystems) according to
the following protocol: 1 hour at 37°C, 5 min at 95°C,
and 10 min at 4°C.
TaqMan Analysis: TGF~31, ANP, Collagen I, Collagen III
Each PCR reaction contained the following: 2.5 ~,L (50
ng) of each CDNA added to 22.5 ~,L of a PCR mix
containing: 38.50 nuclease-free water (Promega), 1X PCR
Buffer II, 2 mM MgCl~, 0.05 U/~,L AmpliTaq Gold (PCR Core
Reagent Kit, N808-0228, Applied Biosystems), 300 nM each
of a forward and a reverse primer (Life Technologies),
200 nM probe (Applied Biosystems) and 200 ~M each of
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78
dATP, dTTP, dGTP, and dCTP (Applied Biosystems). Single
reactions were set up in MicroAmp optical tubes with
MicroAmp optical caps (Applied Biosystems) and loaded
into the 7700 Sequence Detector. The following protocol
was applied to all reactions: 10 min at 95°C (polymerase
activation), 40 cycles of 10 seconds at 95°C
(denaturation) and 1 min at 57°C (annealing).
TaqMan Primers and Probes: COX-2, Osteopontin, MCP-1,
ICAM-1, VCAM-1
All primers and probes were designed using Primer
Express software supplied with the 7700 Sequence
Detection System and synthesized by Applied Biosystems.
Standard curves using 5-fold dilutions of total RNA
(from 200 ng to 320 pg) were performed to determine the
efficiency of each primer/probe set in the TaqMan
reaction prior to the analysis of the experimental
samples. Primer/probe sets were designed from known
sequences of rat genes to be analyzed. All target gene
values were normalized to a reference gene,
constitutively expressed cyclophilin. Primer/probe set
sequences can be found in Table 8.
RNA isolation: COX-2, Osteopontin, MCP-1, ICAM-1, VCAM-1
RNA was extracted from frozen (-80°C) rat heart tissue
using the Totally RNA Isolation Kit (Ambion, Inc.,
Austin, TX). Tissue was crushed using a stainless steel
mortar and pestle, which had been chilled to -80°C and
transferred to a Bounce homogenizer (Kontes, Vineland,
NJ) containing 3-10 mL cold denaturation buffer. Tissue
was homogenized and transferred to a sterile, 15 mL
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polypropylene centrifuge tube. An equal volume of
phenol: chloroform:isoamyl alcohol (25:24:1) was added,
samples were shaken vigorously for 1 min, and incubated
on ice for at least 15 min. Samples were centrifuged
for 30 min at 10,OOOg. The aqueous phase was removed,
1/10 volume of a sodium acetate solution (3.0 M NaOAc pH
4.5) was added, samples were shaken or inverted for 10
seconds, and acid-phenol (premixed with isoamyl
alcohol):chloroform (5:1, Ambion, Inc.) was added at an
volume equivalent to the starting sample volume.
Samples were shaken vigorously for 1 min, followed by a
15-min incubation on ice, and centrifuged for 30 min at
10,OOOg. The aqueous phase removed and placed in a
clean polypropylene tube,. An equal volume of
isopropanol (Sigma, St. Louis, MO) was added and the
samples were mixed and incubated overnight at -20°C.
The samples were centrifuged for 30 min at 10,000g, the
supernatant was removed and the RNA pellet was
resuspended in DNAse/RNAse-free water. Samples were
frozen at -80°C for at least 2 hours, thawed on wet ice,
and diluted for quantitation.
All RNA was further purified by DNase digestion to
remove genomic DNA and LiCl precipitation to remove
carbohydrates. Each RNA (100 ~,g) was incubated for 45
min at 37°C with 1 unit of DNAse (Roche Diagnostics,
Indianapolis, IN) and 10 units RNAse inhibitor (Applied
Biosystems, Foster City, CA) in a buffer containing 40
mM Tris pH 7.8, 6 mM MgCl2, 10 mM CaCl2. The DNAse and
buffer were removed using the RNeasy Mini protocol for
RNA cleanup (Qiagen, Valencia, CA). The RNA was then
precipitated with 7.5M LiCl/50 mM EDTA (Ambion, Inc.,
Austin, TX) in a volume equal to half the sample volume,
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incubated overnight at -20°C, and centrifuged for 30 min
at 13-16,OOOg at 4°C. All RNA was frozen for at least 2
hours at -80°C, thawed, diluted, and analyzed
spectrophotometrically for concentration and purity.
5
TaqMan Analysis: COX-2o Osteopontin, MCP-1, ICAM-1,
VCAM-1
TaqMan reactions were performed as follows. Ten ~,L (200
ng) of total RNA (DNAsed and LiCl precipitated) was
10 added to 15 ~,L of a RT-PCR reaction mix containing: 12.5
~,L of 2X One-Step PCR Master Mix without uracil-N-
glycosylase (contains AmpliTaq Gold DNA Polymerase,
dNTPs with dUTP, passive reference, and optimized buffer
components), 0.625 ~,L of a 40X MultiScribe and RNAse
15 Inhibitor Mix, 0.625 ~,L of 20 ~M forward primer, 0.625
~,L of 20 ~,M reverse primer, 0.5 ~L of 5 ~,M TaqMan probe,
and 0.125 ~.L of DNAse/RNAase-free water. Reactions were
set up in duplicate in MicroAmp optical 96-well reaction
plates with MicroAmp optical caps or adhesive covers
20 (Applied Biosystems) and loaded into the 7700 Sequence
Detector. The following protocol was applied to all
reactions: 30 min at 48°C (reverse transcription), 10
min at 95°C (inactivation of reverse transcriptase and
polymerase activation), 40 cycles of 15 seconds at 95°C
25 (denaturation), and 1 min at 60°C (annealing).
Hydroxyproline Assay
Myocardial hydroxyproline concentration was measured by
a Colorimetric assay that quantifies the reaction
30 between oxidized hydroxyproline, and p-
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dimethylaminobenzaldehyde as described previously (4).
Briefly, tissues (180-250 mg) were dried for 18 hours at
60°C using a Reacti-Therm heating block (Pierce,
Rockford, IL) and weighed. Dried tissues and a positive
collagen control (Bovine Collagen Type I, Sigma, St.
Louis, MO) were hydrolyzed with 2 mL 6N HCl for 3 hours
at 150°C in the Reacti-Therm heating block. Acid was
evaporated under nitrogen gas, samples were rehydrated
in 1 mL of citrate-acetate buffer (0.7 M NaOAc, 0.2 M
citrate, 45 mM citric acid, pH 6.0) in the presence of 4
mL isopropanol, and filtered through a 0.45 ~m Millex
LCR filter (Gelman Sciences, Ann Arbor, MI).
Hydroxyproline content was measured by incubating 60 ~,L
of hydrolyzed sample or collagen standard with 350 ,~.L
citrate-acetate-isopropanol buffer (citrate-acetate
buffer with 40o isopropanol, v/v) and 100 ~,L of 300 mM
Chloramine T (J.T. Baker, Phillipsburg, NJ) for 5 min at
25°C. Erlich's Reagent (1.25 mL, 3.5 M p-
dimethylaminobenzaldehyde in 70% perchloric acid with
80% isopropanol, v/v) was added for visualization and
quantitation of hydroxyproline. Samples were incubated
at 60°C for 30 min, cooled to room temperature, and
absorbance was monitored at 558 nm. Hydroxyproline
content was quantitated from a freshly prepared standard
curve of trans-4-hydroxy-L-proline (Sigma, St. Louis,
MO). All samples and standards were performed in
duplicate.
.Statistical Analysis
Data were analyzed using one-way analysis of variance
(ANOVA). Because the assumptions of normality within
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82
groups and equality of variance across groups could not
be consistently met, the analysis was performed on the
rank transformed values of the raw data (nonparametric
analysis). The alpha=0.05 level of significance was
used for the planned comparisons between the means. The
Least Significant Differences (LSD) method was used for
planned comparisons between groups. Data were analyzed
using PROC TTEST in the SAS statistical software package
(SAS PC, version 6.12,,SAS Institute, Cary, NC). All
data are reported as mean ~ standard error of the mean
(SEM) .
Animal Exclusioxi
Three animals died during the experiment: rat #17
(aldosterone + salt group, found dead after 24 days of
infusion), rat #64 (aldosterone + salt group, died
following surgery), and rat 5 (vehicle group, died
following surgery). Additional animals were excluded if
multiple parameters were found not to represent the
treatment group to which they were assigned (e. g. more
than 3 standard deviations from the mean for that
treatment group). Three such animals were excluded from
the study: rat #57 (from 7-day protocol, aldosterone +
salt group), rat #97 (from 14-day protocol, aldosterone+
salt group), and rat 24 (from 30-day protocol, 100
mg/kg/day eplerenone group). These three animals
demonstrated expression of inflammatory marker genes
(COX-2, Osteopontin, MCP-1, ICAM-l, and VCAM-1} that
were greater than 3 standard deviations from the mean
for the treatment group. Rat #24 was also excluded as a
result of telemetry unit dysfunction. Values generated
for these animals are shown in Table 9.10-Table 9.19,
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separated from the data for the other animals in the
data tables.
Table 9.10 Ix~,dividual data used for Table 10
Control: vehicle + salt
Rat 1 2 4 6 7 8 9 10
#
Day Systolic
Blood
Pressure
(mmHg)
3 118 130 121 -- -- -- -- 118
4 120 122 125 -- -- -- -- 123
5 126 123 125 -- -- -- -- 127
6 132 129 130 -- -- -- -- 131
7 133 132 134 -- -- -- -- 131
8 135 133 133 -- -- -- -- 129
9 131 131 133 -- -- -- -- 128
130 132 128 124 -- 116 135 127
11 130 130 129 125 -- 118 138 128
12 130 128 126 124 -- 124 143 128
13 131 127 128 121 -- 123 143 126
14 142 122 126 125 -- 128 148 128
144 128 127 128 -- 125 134 127
16 132 133 127 128 -- 125 134 123
17 133 133 127 123 -- 124 140 128
18 134 133 129 121 -- 126 143 128
19 125 129 120 125 -- 124 140 128
119 131 121 125 -- 122 139 126
21 123 131 125 126 -- 120 136 128
22 127 128 128 126 -- 125 133 129
23 129 133 131 125 -- 128 138 131
24 132 134 130 125 -- 132 140 130
133 131 125 125 -- 128 136 129
26 132 131 127 126 -- 132 141 130
-- - No data were collected due to technical
difficulties.
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Table 9.10 (continued)
Aldosterone + salt
Rat 11 12 13 14 15 18
16 19
20
Day Systolic
Blood
Pressure
(mmIIg)
3 116 152 115 127 143 122 -- 124 159
4 120 149 122 134 129 135 -- 125 152
126 158 124 142 129 137 -- 128 151
6 132 170 136 157 144 149 -- 135 158
7 140 179 139 165 153 154 -- 145 165
8 145 182 143 160 158 154 -- 146 163
9 150 191 148 172 172 159 -- 151 169
156 196 149 175 175 165 -- 151 172
11 154 201 155 178 181 163 -- 155 175
12 159 207 161 190 186 170 -- 163 190
13 161 210 166 196 191 172 -- 166 194
14 164 208 170 204 192 181 159 172 192
171 200 164 205 183 173 160 175 194
16 179 218 165 200 194 176 166 187 198
17 174 222 178 209 220 185 170 192 202
18 181 226 174 212 213 186 175 198 203
19 189 219 185 208 231 188 177 201 203
192 225 190 220 212 198 180 207 204
21 197 227 197 218 220 201 186 213 211
22 198 227 204 213 223 204 190 221 204
23 200 221 203 223 214 204 187 220 199
24 204 218 199 222 219 207 194 212 212
215 209 205 231 219 210 198 196 210
26 219 211 215 224 207 202 192 212 205
-- - No data were collected due to technical
difficulties.
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Table 9.10 (continued)
Eplerenone + aldosterone + salt
Rat 21 23 25 26 27 28 29 30 24*
22
Day Systolic
Blood
Pressure
(mmHg)
3 123 126 130 128 119 125 126 125 130 --
4 130 128 131 139 122 126 128 130 134 --
5 132 134 132 143 123 127 127 133 142 --
6 133 142 136 152 126 133 137 140 150 --
7 140 142 143 156 132 140 140 141 156 --
8 142 146 141 156 131 138 138 139 152 --
9 142 146 139 154 130 133 137 141 151 --
10 143 143 138 158 134 136 139 142 149 --
11 145 139 138 160 136 137 140 145 152 --
12 147 140 139 165 137 139 140 148 154 --
13 148 144 137 170 140 140 140 149 153 --
14 146 142 138 178 143 144 143 152 161 --
15 145 143 137 173 143 144 141 149 156 --
16 148 137 137 179 145 145 143 150 164 --
17~ 148 141 143 182 149 148 143 160 174 --
18 151 146 144 187 152 149 148 162 177 --
19 156 147 145 192 153 154 150 166 177 --
20 159 147 146 192 155 151 151 168 176 --
21 162 148 152 200 159 154 155 175 182 --
22 162 149 153 203 160 158 155 176 185 --
23 169 157 157 209 163 160 159 180 191 --
24 168 164 159 211 163 162 161 180 195 --
25 174 165 161 215 165 161 161 182 198 --
26 178 168 163 223 167 166 162 192 202 --
-- - No data were collected due to technical
5 difficulties.
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
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Table 9.11 Individual data used for Table 11
Control: vehicle + salt
Left
Right
Final Left Right Ventricle
R B t l Tibia W Ventricle ~P
t d i V ht / '
l t i
i
a o en r t e ht /
y r en L g Wei
c c
eng g
# Weight a Weight a Weight Tibia (p,U)
(cm) Tibia Lengt
(g) (mg) (mg) Length (mg/cm)
(mg/cm)
0.
47
291 771 194 3.9 198 50 90
0.
48
283 699 155 3.8 184 41 70
3.
49
2g4 696 166 3.8 183 44 59
3.
50
267 562 175 3.8 148 46 96
1.
51
268 636 178 3.8 167 47 11
0.
52
273 709 185 3.7 192 50 94
0.
53
269 699 197 3.8 184 52 64
1.
54
245 612 189 3.8 161 50 06
0.
55
2g6 667 190 3.8 176 50 93
1.
56
245 616 149 3.8 162 39 10
1~
Mea
271 667 178 3.8 175 47 49
0
SEM
19 5 0.01 5 1 38
5 Table 9.11 (continued)
Aldosterone + salt
Tibi Left Right
Final Left Right a Ventricle Ventricle
Rat Body VentriclVentricl ANP
L W W
i i
ht / ht /
eng e e
g g
# Weight a Weighta Weight ('~U)
(g) (mg) (mg) th ibia Lengt ibis Lengt
(cm) (mg/cm) (mg/cm)
11.
58 266 784 183 3.8 206 48 92
3.9
59 271 719 178 3.6 200 49 9
13.
60 299 719 223 3.9 184 57 41
61 286 779 185 3.9 200 47 3.6
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4
9.0
62
274 746 168 3.8 196 44 9
13
.
63
27& 620 154 3.8 163 41 13
6.1
65
-- g49 197 3.9 218 51 3
3.8
66
266 674 174 3.7 182 47 8
8.1
Mea
277 736 183 3.8 194 . 48 5
0.0 1.5
SEM
25 7 3 6 2 1
-- - No data were collected due to technical
difficulties.
57* 267 778 208 3 8 205 55 I13 32I
* Data from this animal were not considered for
5 statistical analysis and not included in the final
results.
Table 9.11 (continued)
Eplerenone + aldosterone + salt
Tibi Left Right
Final Left Right
a Ventricle Ventricle
Rat Body entricl Ventricle ANP
L W W
ht / i
i ht /
eng e e
g g
# weight a Weight Weight (AU)
th ibia Lengt ibis Lengt
(g) (mg) (mg)
(cm) (mg/cm) (mg/cm)
1.
67
306 859 216 3.9 220 55 26
1.
68
295 712 181 3.8 187 48 81
0
69
286 618 154 3.7 167 42 59
2.
70
277 658 174 3.8 173 46 58
4.
71
295 754 192 3.8 198 51 48
4.
72
281 733 171 3.8 193 45 98
3.
73
273 726 181 3.8 191 48 82
3.
74 286 696 190 3.8 183 50 59
0.
75 -- 700 170 3.8 184 45 95
3.
76 276 688 187 3.8 181 49 67
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Mea 2~
286 714 182 3.8 188 48 77
SEM 0'~ 0.
4 20 5 1 5 1 49
-- - Lvo data were collected due to technical
difficulties.
Table 9.12 Individual data used for Table 12
Control: vehicle + salt
Left Right
a Final Left Right Tibia entricle entricleANP
Body Ventricle Ventricl Weight Wei ht
# Length/ g / (AU)
WeightWeight Weight (cm) Tibia
(g) (mg) (mg) . Tibia Length
Length (mg/cm)
(mg/cm)
87 319 760 188 3.9 195 48 0.16
88 337 782 238 3.9 201 61 0.92
89 322 665 179 3.9 171 46 0.36
90 322 802 208 3.8 211 55 0.89
91 -- 742 174 3.8 195 46 7.04
92 327 790 200 3.8 208 53 1.89
93 324 747 303 3.8 197 80 3.33
94 301 826 184 3.80 217 48 1.80
95 303 745 178 3.8 196 47 1.08
96 295 756 206 3.9 194 53 0.17
127 313 777 174 3.9 199 45 nd
128 295 677 178 3.8 178 47 nd
129 278 657 165 3.8 173 43 nd
ea 311 748 198 3.8 195 52 1.76
'SE 5 15 10 0.01 ~ 4 3 0.66
-- - m~ uaza were collected due to technical
difficulties.
nd = No data were reported due to insufficient mRNA
sample .
Table 9.12 (continued)
Aldosterone + salt
Final Left Right Tibi Left Right
a Ventricle Ventricle
Rat Body VentriclVentricl '~P
# Weight a weighta Weight Leng Weight / weight / '
(AU)
(g) (mg) (mg) th ibia Lengt ibia Lengt
(cm) (mg/em) (mg/cm)
98 4.5
298 846 194 3.8 223 51 8
99 261 784 ~ 189 ~ 3.8 206 I 50 7
~ ~ ~ 7
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89
5
100 7'3
307 912 208 3.9 234 53 4
101 4.1
242 720 174 3.8 189 46 8
102 1.5
307 923 217 3.9 237 56 9
103 3~8 17.
279 854 186 0 225 49 81
104 6.4
308 894 216 3.9 229 55 8
105 8.0
290 859 171 3.9 220 44 8
106 2.5
264 750 153 3.8 197 40 1
130 275 818 202 3.8 215 53 nd
131 193 746 195 3.7 202 53 nd
132 215 700 172 3.6 194 48 nd
Mea 6.7
270 817 189 3.8 214 50 0
SEM 0'0 1.5
11 22 5 2 5 1 9
na = lvo aata were reported. d.ue to insufficient mRNA
sample.
97* 235 809 178 3.9 207 46 5.96
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.12 (continued)
Eplerenone + aldosterone + salt
Final Left Right Tibi Left Right
Rat Body Ventricl Ventricla Ventricle Ventricle ~
# weight a Weight a weightLeng Weight / Weight / (p,,U)
(g) (mg) (mg) th ibia Lengt ibia Lengt
(cm) (mg/cm) (mg/cm)
133 281 804 182 3.8 212 48 nd
134 2.8
304 898 188 3.8 236 49 4
135 3.2
293 789 176 3.8 208 46 2
136 6.3
268 851 189 3.9 221 49 9
137 4.0
267 668 139 3.8 176 37 4
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138 25.9
247 833 371 3.7 225 100 0
139 5.5
296 886 193 3.8 233 51 2
140 3.5
291 756 188 3.8 199 49 7
2.2
141
297 751 158 3.8 198 42 9
142 8.3
264 795 155 3.7 215 42 7
143 4.2
302 915 225 3.9 235 58 4
6.6
Mea
283 813 197 3.8 214 52 4
0.0 2.2
SEM
6 22 19 2 6 5 2
nd = No data were reported due to insufficient mRNA
sample.
Table 9.13 Individual data used for Table 13
5
Control: vehicle + salt
Final Left Right Tibi Left Right
a Ventricle Ventricle
Rat Body Ventricl 'entricl ANP
# Weight a Weight e WeightLeng Weight / Weight / (gU)
(g) (mg) (mg) th ibia Lengt ibia Lengt
(cm) (mg/cm) (mg/cm)
1 0.9
308 686 160 4.0 172 40 5
2 0.3
337 763 194 4.1 186 47 0
4 0.1
316 728 162 4.0 182 41 2
1.0
343 721 162 4.1 176 40 6
7 1.9
291 664 153 4.0 166 38 3
8 0.2
294 612 180 4.1 149 44 4
1.1
291 613 141 4.0 153 35 7
10 0.1
332 812 184 4.2 193 44 1
Mea 0.7
314 700 167 4.1 172 41 4
SEM 0.0 0.2
8 25 6 3 5 1 3
Aldosterone + salt
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Tibi Left Right
Final Left Right Ventricle
Rat Body VentriclVentricla Ventricle Weight / ANP
# Weight a Weighta WeightLeng Weight / Tibia (AU)
(g) (mg) (mg) th ibia Lengt Length
(cm) (mg/cm)
(mg/cm)
11 23
.
289 934 196 4.0 234 49 59
12 43
.
219 726 148 3.8 191 39 11
13 14
.
289 963 215 3.9 247 55 83
14 18.
282 942 176 3.9 242 45 90
15 14
.
290 1030 224 3.9 264 57 83
16 23.
267 837 173 3.9 215 44 43
18 15.
319 962 220 3.9 247 56 14
19 6.7
263 873 187 4.0 218 47 7
20 20.
234 919 185 3.8 242 49 97
Mea 20.
272 910 192 3.9 233 49 17
SEM 0-0 3.3
10 29 8 2 7 2 6
Table 9.13 (continued)
Eplerenone + aldosterone + salt
Final Left Right Tibi Left Right
Rat Body Ventricl Ventricla Ventricle Ventricle ~P
# Weight a Weight a WeightLeng Weight / Weight / (AU)
(g) (mg) (mg) th ibia Lengt ibia Lengt
(cm) (mg/cm) (mg/cm)
21 1.9
310 873 177 3.9 224 45 3
22 1.1
343 908 202 4.1 233 52 5
23 4.8
334 899 200 3.9 231 51 9
25 21.
299 1063 209 3.9 273 54 26
26 10.
361 958 187 3.9 246 48 63
27 20.
351 1129 242 3.9 289 62 25
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10.
28
316 929 189 3.9 238 48 20
4.8
29
352 805 181 4.0 206 46 2
7.6
30
317 861 195 3.9 221 50 7
9.2
Mea
331 936 198 3.9 240 51 0
0.0 2.4
SEM
7 34 6 0 9 2 4
24* 273 822 178 3.9 211 46 13.45
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.14 Individual data used for Table 14
Control: vehicle + salt
Myocardial Interstitial ollagen-ollagen-
Rat Collagen ydroxyproline I III
Necrosis Volume
( 0 - 4 ) ( N~g /mg ) mRNA mRNA
Fraction (%) (AU) (AU)
47 0.0 2.9 5.11 1.72 1.39
48 0.0 7.1 5.72 0.63 0.80
49 0.0 3.1 3.15 1.97 2.00
50 0.0 4.1 2.37 1.08 1.19
51 0.0 3.4 2.23 1.40 1.09
52 0.0 4.5 2.48 0.73 0.92
53 0.0 2.3 2.35 1.22 1.27
54 0.0 6.6 2.42 0.78 0.91
55 0.0 4.1 4.68 0.54 0.70
56 0.0 6.3 5.21 0.93 0.61
ea 0.0 4.4 3.57 1.10 1.09
SEM 0.0 0.5 0.45 0.15 0.13
Table 9.14 (continued)
Aldosterone + salt
Interstitial ollagen-ollagen-
Rat Myocardial Collagen IydroxyprolineI III
Necrosis
Volume (~,g/mg) mRNA mRNA
(0-4)
Fraction (%) (AU) (AU)
58 0.0 nd 4.48 0.84 0.65
59 0.0 3.2 4.06 1.40 1.29
60 0.0 6.5 2.32 1.97 1.67
61 0.0 nd 2.14 1.89 1.67
62 0.0 6.1 2.18 1.36 1.59
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63 0.0 6.9 2.31 1.05 1.59
65 0.0 6.5 2.10 1.33 1.58
66 0.0 4.4 2.22 1.07 1.30
Mean 0.0 5.6 2.73 1.36 1.42
SEM 0.0 0.6 0.34 I 0.14 ( 0.12
nd = No data were reported due to insufficient mRNA
sample.
57* 0 . 0 3 . 1 3 . 86 - ~1-.71 I 1 . 15
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.14 (continued)
E~lerenone + aldosterone + salt
Myocardial Interstitial ollagen- ollagen-
Rat Collagen ydroxyproline I III
# Necrosis Volume m mRNA mRNA
(8-4) (N~9'/ g)
Fraction (AU) (AU)
(%)
67 0.0 4.3 2.02 0.62 0.93
68 0.0 7.2 4.18 0.92 0.95
69 0.0 2.9 4.08 0.29 0.43
70 0.0 3.3 3.96 1.79 1.25
71 0.0 4.2 4.26 0.78 1.03
72 0.0 6.6 4.17 0.85 1.14
73 0.0 4.4 1.90 0.29 0.45
74 0.0 4.9 1.53 0.42 0.64
75 0.0 8.8 2.08 1.28 1.33
76 0.0 6.9 2.41 1.21 2.71
Mean 0.0 5.4 3.06 0.85 1.09
SEM 0.0 0.6 0.36 0.15 0.21
Table 9.15 Individual data used for Table 15
Control: vehicle + salt
Collagen ollagen- ollagen-
Rat Myocardial Volume IydroxyprolineI III
Necrosis
# Fraction (~,g/mg) mRNA mRNA
(0-4)
(%) (AU) (AU)
g7 0.0 4.6 . 2.03 0.90 0.96
88 0.0 3.9 2.20 1.60 1.60
89 0.0 6.5 4.51 0.92 0.80
90 0.0 4.4 4.07 0.58 0.65
91 0.0 6.3 4.93 1.28 1.42
92 0.0 3.1 4.00 0.94 1.05
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93 0.0 4.9 2.89 1.14 1.00
94 0.0 3.9 3.24 1.07 1.02
95 0.0 3.2 3.21 1.56 1.00
96 0.0 3.7 3.16 0.80 0.56
127 0.0 4.9 2.66 nd nd
128 0.0 6.0 2.70 nd nd
129 0.0 6.1 2.84 nd nd
Mean 0 . 0 4-. 7 3 . 2~ 1 . 0$-- 1 . 01
SEM 0.0 ~ 0=-4 ~ __-0.24 -- I O--10 0.10
I
nd = No data were reported due to insufficient mRNA
sample.
Table 9.15 (continued)
Aldosterone + salt
Myocardial Collagen
Rat Ydroxyproline ollagen- ollagen-
ecrosis (0- Volume I III
4) Fraction ( g~mg)
(%) mRNA mRNA
(AU) (AU)
9g 0.0 4_4 2.89 1.15 0,76
99 1.0 5.4 2.91 2.31 1.80
100 0.0 3.2 6.28 0.25 0.44
101 0.0 5.9 5.63 1.89 1.39
102 0.0 4.6 4.83 2.03 1.17
103 1.0 3.9 5.64 1.00 1.24
104 0.0 4.8 5.29 1.20 1.06
105 0.0 4.6 2.76 1.70 1.31
106 1.0 5.9 2.68 0.43 0.59
130 0.0 3.4 2.60 nd nd
131 3.0 6.4 3.00 nd nd
132 3.0 9.0 3.99 nd nd
Mean 0.8 5.1 4.04 1.33 1.08
SEM 0.3 0.5 0.40 0.24 0.14
nd = No data were reported due to insufficient mRNA
sample.
97* 3.0 3.2 2.73 2.69 1.22
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.15 (continued)
Eplerenone + aldosterone + salt
Myocardial Collagen
ollagen- ollagen-
Rat Ydroxyproline I III
ecrosis (0- Volume (~'g~mg) ~.NA mRNA
4) Fraction (AU) (AU)
(%)
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134 0.0 6.2 5.97 0.86 1.19
135 1.0 3.9 6.52 0.90 1.16
136 0.0 3.7 5.35 1.65 1.24
137 0.0 4.2 6.80 1.14 1.70
138 0.0 3.5 5.32 1.44 1.81
139 1.0 3.3 2.72 0.50 0.60
140 0.0 3.7 3.13 1.24 1.61
141 0.0 5.2 2.41 1.69 2.21
142 2.0 5.6 2.81 2.03 1:80
143 0.0 6.0 5.03 3.02 3.77
Mean0.5 4.5 4.46 1.39 1.61
SEM 0.2 0.3 0.50 0.21 0.26
Table 9.16 Individual data used for Table 16
Control: vehicle + salt
Myocardial Collagen ollagen-ollagen-
Rat ecrosis (0- Volume Ydroxyproline I III
4) Fraction (~g~mg) ANA mRNA
(o) (AU) (AU)
1 0.0 4.3 2.00 1.69 1.43
2 0.0 4.1 2.71 0.90 0.98
4 0.0 6.4 2.95 1.65 1.02
~
6 0.0 7.9 3.02 0.90 1.28
7 0.0 5.8 2.81 0.97 0.62
8 0.0 7.7 5.84 1.03 0.54
9 0.0 6.0 5.45 0.69 0.94
10 0.0 7.1 7.03 0.92 0.48
Mean0.0 6.2 3.98 1.09 0.91
SEM 0.0 0.5 0.65 0.13 0.12
Aldosterone + -salt
Myocardial Collagen ollagen-ollagen-
Rat ecrosis (0- Volume ~YdroxyprolineI III
4) Fraction (~'g~mg) ANA mRNA
(%) (AU) (AU)
11 1.5 6.6 7.24 2.20 0.75
12 2.5 8.8 8.01 2.02 0.58
13 3.0 7.2 3.62 5.88 1.99
14 2.0 7.1 3.69 1.05 0.72
15 3.0 9.3 4.00 1.32 2.04
16 0.5 6.8 3.54 2.02 1.43
18 2.0 4.0 3.07 1.98 1.82
19 0.3 7.2 3.25 1.63 1.89
20 3.5 14.5 3.09 2.54 1.28
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Mean 2.0 7.9 4.39 2.29 1.39
SEM 0.4 1.0 0.62 0.47 0.20
Table 9.16 (continued)
Eplerenone + aldosterone + salt
Myocardial Collagen ollagen-ollagen-
Rat ecrosis (0- Volume Ydroxyproline I III
# 4) Fraction (~'g~mg) ANA mRNA
(%) (AU) (AU)
21 0.0 3.4 5.18 1.89 0.95
22 0.0 5.0 6.11 1.54 0.72
23 0.0 6.5 5.17 2.65 1.37
25 0.0 7.9 6.40 1.97 0.89
26 0.0 7.1 2.73 2.98 1.26
27 0.0 6.3 2.84 2.65 1.87
28 0.0 6.1 2.97 2.90 1.66
29 0.0 5.4 2.82 2.88 2.89
30 0.0 7.8 2.72 3.35 2.16
Mean 0.0 6.2 4.10 2.53 1.53
SEM 0.0 0.5 0.53 0.20 0.23
24* 0.0 4.4 5.75 2.01 0.73
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.17 Individual data used for Table 17
Control: vehicle + salt
Rat COX-2 Osteoponti MCP1 ~TGF-~CiICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
47 nd nd nd 1.32 nd nd
48 nd nd nd 0.66 nd nd
49 nd nd nd 1.46 nd nd
50 0.57 1.28 1.13 0.72 1.15 1.19
51 1.04 0.94 1.00 1.17 0.94 nd
52 0.99 0.73 0.71 0.80 1.17 1.17
53 0.87 1.00 0.84 1.11 0.82 0.60
54 1.88 nd nd 0.90 nd nd
55 1.01 nd nd 0.52 nd nd
56 nd 1.66 1.67 1.50 1.00 0.86
Mean 1.06 1.12 1.07 0.98 1.02 0.96
SEM 0.18 0.16 0.17 0.12 0.07 0.14
na = lvo aaza was reported due to insufficient mRNA
sample .
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Table 9.17 (continued)
Aldosterone + salt
Rat COX-2 Osteoponti MCP1 TGF-~3 ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
58 2.10 1.84 2.05 1.23 1.39 3.49
59 0.70 0.84 1.78 0.98 0.80 0.85
60 2.01 0.95 3.06 1.31 1.09 2.06
61 2.95 1.05 2.36 1.89 1.61 2.51
62 2.05 1.08 1.95 1.22 1.11 1.65
63 1.94 4.92 2.33 1.45 1.15 0.61
65 3.54 3.29 3.14 1.47 1.56 0.94
66 2.45 1.32 2.40 1.21 1.06 0.27
Mean 2.22 1.91 2.38 1.35 1.22 1.55
SEM 0.29 0.51 0.17 0.09 0.10 0.39
57* 0.82 28.64 5.17 1.35 1.68 5.23
* Data from this animal were not considered for
statistical analysis and not included in the final .
results.
Table 9.17 (continued)
Eplerenone + aldosterone + salt
Rat COX-2 Osteoponti MCP1 TGF-~3 ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
67 1.19 0.54 2.35 0.80 0.91 0.67
68 2.85 1.24 1.60 0.81 0.89 0.58
69 0.60 0.52 0.85 0.51 0.89 0.22
70 nd nd nd 1.31 nd nd
71 1.16 0.27 0.83 0.80 0.40 0.57
72 0.82 0.60 1.74 1.02 1.23 nd
73 1.86 1.13 2.38 0.61 nd nd
74 nd nd nd 0.84 nd nd
75 0.60 0.96 0.67 1.51 0.58 0.53
76 0.91 0.75 2.03 1.64 1.00 1.00
Mean 1.25 0.75 1.56 0.99 0.83 0.56
SEM 0.29 0.12 0.25 0.12 0.10 0.08
nd = No data were reported due to insufficient mRNA
sample.
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Table 9.18 Individual data used for Table 18
Control: vehicle + salt
Rat COX-2 Osteoponti MCP1 TGF-~(i ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
87 1.69 1.28 1.28 1.21 1.45 0.92
88 0.74 1.13 0.94 1.19 1.11 0.64
89 nd nd nd 1.00 nd nd
90 1.00 0.94 0.73 0.84 1.14 nd
91 1.43 1.00 1.38 1.32 1.23 0.93
92 0.61 1.28 0.91 1.26 0.98 1.00
93 0.84 1.40 1.00 0.86 0.94 1.35
94 1.18 0.87 1.05 0.82 1.00 nd
95 nd nd nd 1.00 nd nd
96 nd nd nd 0.74 nd nd
127 nd nd nd nd nd nd
128 nd nd nd nd nd nd
129 nd nd nd nd nd nd
Mean 1.07 1.13 1.04 1.02 1.12 0.97
SEM 0.15 0.08 0.08 0.07 0.07 0.11
na = loo exaLa were reported due to insufficient mRNA
sample.
Table 9.18 (continued)
Aldosterone + salt
Rat COX-2 Osteoponti MCP1 TGF-~3 ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
98 nd nd nd 1.26 nd nd
99 7.39 8.14 2.42 1.85 1.16 0.89
100 1.83 1.02 1.87 0.55 1.18 0.69
101 5.80 6.19 4.59 1.91 1.75 0.84
102 2.59 4.06 3.19 1.49 1.15 0.72
103 6.63 12.04 3.34 1.18 1.91 2.23
104 4.18 2.35 1.91 1.32 1.19 1.03
105 3.71 8.25 2.50 1.27 1.82 1.65
106 2.62 10.41 2.22 0.56 1.57 1.24
130 nd nd nd nd nd nd
131 nd nd nd nd nd nd
132 nd nd nd nd nd nd
Mean 4.34 6.56 2.76 1.27 1.47 1.16
SEMI 0.72 ~ 1.37 ~ 0.32 ~ 0.16 0.12 I 0.19
I
nd
=
No
data
were
reported
due
to
insufficient
mRNA
sample.
97*~ 23.34 ~ 81.29 ~ 5.88 I 1.29 I 1.84 I 1.75
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10
* Data from this animal were not considered for
statistical analysis and not included in the final
results.
Table 9.18 (continued)
Eplerenone + aldosterone + eali-.
Rat COX-2 Osteoponti MCP1 TGF-~i ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
133 1.56 4.03 1.78 0.58 1.20 0.54
134 1.04 1.00 1.37 0.62 1.36 0.66
135 0.70 0.77 1.27 1.04 0.95 0.61
136 1.41 8.43 1.75 1.42 1.26 0.61
137 3.78 1.59 1.60 1.29 1.56 0.67
138 1.86 3.97 1.24 1.49 0.98 0.86
139 6.19 3.93 1.92 0.71 1.51 1.21
140 1.87 2.13 1.24 1.21 0.79 1.00
141 0.99 0.72 1.89 1.44 0.98 0.68
142 1.92 4.76 2.21 1.69 1.72 1.60
143 0.86 0.99 1.20 2.41 0.83 0.68
Mean 2.02 2.94 1.59 1.26 1.19 0.83
SEM 0.49 0.72 0.10 0.16 0.09 0.10
Table 2.19 Individual data used for Table 19
Control: vehicle + ~alt-
Rat COX-2 Osteoponti MCP1 TGF-~i ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
1 1.15 0.81 2.39 0.53 1.01 0.96
2 1.75 1.46 1.79 0.52 2.29 1.93
4 0.96 0.57 1.00 1.00 0.99 nd
6 0.95 0.82 0.81 1.19 1.60 1.38
7 0.86 1.13 0.52 1.00 nd nd
8 1.07 1.16 0.53 1.68 0.55 0.45
9 1.00 1.00 1.52 0.90 0.96 1.00
nd nd nd 1.24 nd nd
Mean 1.11 0.99 1.22 1.01 1.23 1.14
SEM 0.11 0.11 0.27 0.13 0.25 0.25
~~~ ua~a. wire reporzea aue to lnsutticient mRNA
sample .
Aldosterone + salt
Rat COX-2 Osteoponti MCP1 TGF-~i ICAM VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
11 nd nd nd 1.41 nd nd
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12 4.26 13.13 3.94 1.27 nd nd
13 4.81 11.43 7.19 2.11 2.67 3.48
14 nd nd nd 1.20 nd nd
1.54 13.78 1.61 1.95 1.63 1.87
16 nd nd nd 1.49 nd nd
18 3.10 7.97 9.35 0.83 1.69 2.99
19 5.28 18.44 2.30 0.54 1.50 1.64
8.20 14.88 2.86 1.21 1.54 0.72
Mean4.53 X3.27 4.54 1.33 1.81 2.14
SEM 0.92 1.43 1.25 0.16 0.22 0.49
na = 1vo aata were reported due to insufficient mRNA
sample.
Table 9.19 (continued)
Eplerenone + aldosterone + salt
Rat COX-2 Osteoponti MCP1 TGF-~i ICAM 'VCAM
# (AU) (AU) (AU) (AU) (AU) (AU)
21 2.44 1.53 2.11 1.00 1.54 1.42
22 0.55 3.28 1.70 1.49 2.06 1.29
23 1.97 1.98 2.21 1.40 1.01 1.49
3.41 8.91 1.38 1.31 1.21 1.27
26 3.71 1.88 2.10 0.96 1.26 0.79
27 3.04 1.97 2.02 1.93 1.06 0.52
28 2.11 1.28 1.43 1.54 0.60 0.57
x
29 1.34 1.43 5.58 1.32 0.99 0.61
1.92 1.01 2.11 0.89 nd 1.42
Mean 2.28 2.59 2.29 1.32 1.22 1.04
SEM 0.33 0.82 0.42 0.11 0.15 0.14
nd = No data were reported due to insufficient mRNA
sample.
24* 12.21 54.57 8.14 1.35 2.92 4.01
10 * Data from this animal were not considered for
statistical analysis and not included in the final
results
15 Results
Blood pressure
Blood pressure remained normal in vehicle + salt
controls throughout the experiment (Table 10).
Aldosterone + salt induced a progressive increase in
20 blood pressure with time. In animals receiving
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eplerenone + aldosterone + salt, systolic blood pressure
was significantly reduced at days 8-30. However, blood
pressure remained elevated compared to vehicle + salt
controls.
Table 10. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
blood pressure over time
Systolic Pressure (mmHg)
Blood
Day Vehicle ldosterone Eplerenone
+ n + n + n
salt salt aldosterone
+
salt
3 122 3 4 132 6 8 126 1
4 123 1 4 133 4* 8 130 2*
5 125 1 4 137 4* 8 132 2*
6 130 1 4 148 5* 8 139 3*
7 132 1 4 155 5* 8 143 3*
8 132 1 4 156 4* 8 142 3*# 9
131 1 4 164 5* 8 142 3*# 9
127 2 7 168 6* 8 142 2*# 9
11 128 2 7 171 6* 8 143 3*#
12 129 2 7 178 6* 8 145 3*# 9
13 128 3 7 182 6* 8 147 3*# 9
14 131 4 7 182 6* 9 150 4*# 9
130 2 7 181 5* ~ 148 4*# 9
16 129 2 7 187 6* 9 150 4*#
17 130 2 7 195 7* 9 154 5*# 9
18 131 3 7 196 6* 9 157 5*# 9
19 127 2 7 200 6* 9 160 5*# 9
126 3 7 203 5* 9 160 5*# 9
21 127 2 7 208 4* 9 165 6*# 9
22 128 1 7 209 4* 9 167 6*# 9
23 131 2 7 208 4* 9 172 6*# 9
24 132 2 7 210 3* 9 174 6*# 9
130 2 7 210 4* 9 176 6*# 9
26 131 2 7 210 3* 9 180 7*# 9
10 These data are expressed graphically in Figure 1.
Values are mean ~ SEM of values obtained every 5 min
over 24-hour period.
*Significantly different from vehicle + salt, p<0.05.
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# Significantly different from aldosterone + salt,
p<0.05.
Body weight, Myocardial Hypertrophy and ANP
Body weights were significantly lower in animals
receiving aldosterone + salt treatment at days 7, 14,
and 30 compared to vehicle + salt normotensive controls
(Tables 11-13). The decrease in body weight induced by
aldosterone + salt treatment was significantly
attenuated by administration of eplerenone at day 30
(Table 11). Significant left and right ventricular
hypertrophy occurred in response to aldosterone + salt
treatment. Left ventricular hypertrophy was evident
after 7 days of aldosterone + salt treatment (Table 11)
whereas right ventricular hypertrophy was only evident
after 30 days of aldosterone + salt treatment (Table
13). Eplerenone did not impact absolute ventricular
weights or ventricular weight to tibia length ratios
induced by aldosterone + salt treatment (Tables 11-13).
Significant elevations in atrial natiuretic peptide
(ANP) mRNA levels were also observed in animals treated
with aldosterone + salt (Tables 11-13). The ANP mRNA
upregulation was significantly reduced by eplerenone
after 30 days of treatment but not after 14 days (Table
13 ) .
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Table 11. Effects of aldosterone + salt treatment
alone or in combination with eplerenone in
rats after 7 days of treatment
Fina Left Right
Left Right
1 Tibia entricle Ventricle .
VentriVentri
ANp
Grou Body cle cle Lengt Weight Weight
p / /
Weig h Tibia Tibia U A
~
ht WeightWeight (cm) Length Length ,
(g) (mg) (mg) (mg/mm) (mg/mm)
2715 3.80
490
1
Vehicle 66719 1785 .01 1755 471 .
-
+ salt (n_1 (n=10)(n=10) (n=10 (n=10) (n=10) 38
0) (n=10)
Aldoster 2775 73625 3.80 8
721
.
one + (n=7 * 1837 -03 1946* 482 .
51*
salt ) (n=g) (n=8) (n=g) (n=8) (n=8) (n=g)
Eplereno
2874 3.80
ne + 2.770.
* 71420 1825 01 1885 481
aldoster . 49
(n=9 (n=10)(n=10) (n=10 (n=10) (n=10)
one +
) (n=10)
salt )
Values are mean ~ SEM measured after 7 days of
treatment.
*Significantly different from vehicle + salt control,
p<0.05.
#Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
ANP = atrial natiuretic peptide.
AU = arbitrary units, measured relative to cyclophilin
expression.
Table 12. Effects of aldosterone + salt treatment
alone or in combination with eplerenone in
rats after l4ays of treatment
Left Right Left Right
Final VentriVentr Tibia VentricleVentricle ,
A
p Body cle icle Lengt Weight Weight ~p
Grou / / ,
~gN
Weigh Weigh h Tibia Tibia A
t (g) Weightt (cm) Length Length (AU)
(mg) (mg) (mg/mm) (mg/mm)
3115 1981 3.80
76~'0
1
Vehicle 74825 0 .01 1954 523 .
(n=12 ~
+ salt (n=13)(n=13 (n=13 (n=13) (n=13) 66
(n=10)
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2 7 - -3
Aldoster 01 81722 1895 = 6.701.
8+0
1* .02 2145* 501
(n=12 (n~12 (n=12 (n=12) (n=12) n9
salt (n (
12) 9)
Eplereno
2836 1971 3.80
ne + 81322 6.642.
aldoster * * 9 .02 2146* 525
(n 11 (n (n=11) (n=11)
11 (n 11
one +
~ (n=11)~ ~ _
(n 10)
salt
Values are mean ~ SEM measured after 14 days of
treatment.
* Significantly different from vehicle + salt, p<0.05.
Eplerenone dose was 100 mg/kg/day.
ANP = atrial natiuretic peptide.
AU = arbitrary units, measured relative to cyclophilin
expression.
Table 13. Effects of aldosterone + salt treatment
alone or in combination with eplerenone in
rats after 30 days of treatment
Left Right Left Right
Final
Ventr Ventr entricle Ventricle
Bod icle icle Tibia Weight Weight
y / /
Group Weigh weigh Weigh LengthTibia Tibia ~'NA
(em) (AU)
fig) t t Length Length
(mg) (mg) (mg/nun) (mg/~)
Vehicle
+ salt 3148 7052 1676 4 1725 411 0-73+_0.2
0
(n=8) 03
Aldoste
rove 2721 9102 1928 3.90. 20.173.
+
salt p* g* * 02* 2337* 492* 36*
(n=9)
Epleren
one +
aldoste # 9363 1986 3.90. 9.202.4
3317 2409* 512* #
rone 4* * 00* 4*
+
salt
(n=9)
Values are mean ~ SEM measured after 30 days of
treatment.
*Significantly different from vehicle + salt, p<0.05.
# Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
ANP = atrial natiuretic peptide.
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AU = arbitrary units, measured relative to cyclophilin
expression.
Myocardial Fibrosis
Interstitial collagen volume fraction and hydroxyproline
levels were not statistically different at any time
point among the experimental groups (Tables 14-16). A
modest increase in collagen type-I message was detected
in aldosterone + salt and aldosterone + eplerenone +
salt treatment at 30 days, compared to vehicle + salt
controls (Table 16). Collagen type III mRNA levels were
not significantly increased at any time point (Tables
14-16).
Table 14. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
myocardial injury and fibrosis in rats after
7 days of treatment
yocardia ydroxypro ollagen-
1 ICVF line I Collagen-
Group
Necrosis (o) III (AU)
(p_4) (N~g/m9') (AU)
4.40
3.570.4 1.100.
0'00.0 -5 090
13
1
Vehicle + salt (n=10) 5 15 .
.
(n=10)
(n=10 (n=10) (n=10)
Aldosterone 0.00.0 5-60 2.730.3 1.360. 1.420.12
+
n-8) .6 4 14 _
salt ( (n 8)
(n=6) (n=8) (n=8)
Eplerenone + 5-40
3.060.3 0
850
0.00.0 .6 . 1,090.21
aldosterone 6 .
+ 15
(n=10) (n=10 (n=10)
salt )
(n=10) (n=10)
Values are mean ~ SEM measured after 7 days of
treatment.
Eplerenone dose was 100 mg/kg/day.
ICVF = interstitial collagen volume fraction.
Collagen-I = Collagen type I mRNA.
Collagen-III = Collagen type III mRNA.
AU=arbitrary units, measured relative to cyclophilin
expression.
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Table 15. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
myocardial injury and fibrosis in rats after
14 days of treatment
yocardia ydroxypr
1 ICVF line ollagen- Collagen-
Group Necrosis (%) I (AU) III (AU)
( !~g/m9' '
)
4.70
Vehicle + 0.00.0 .4 3.260.2 1.080. 1,010.10
salt (n=13) 4 10
(n=13 (n=13) (n=10) (n=10)
5.10
Aldosterone 0 . 80 , 5 4 . 040 1 . 240 1 _ ~B...E.O.,
+ . 3 0 . 4 . 14
salt (n=12) (n=12 (n=9)
(n=12) (n=9)
Eplerenone 4.50
+ 4.460.5 1
390
0.50.2 ,3 . 1,610.26
aldosterone 0 .
+ 21
(n=11) (n=11 (n=11)
salt )
(n=11) (n=11)
Values are mean ~ SEM measured after 14 days of
treatment.
Eplerenone dose was 100 mg/kg/day.
ICVF = interstitial collagen volume fraction.
Collagen-I = collagen type I mRNA.
Collagen-III - collagen type III mRNA.
AU = arbitrary units, measured relative to cyclophilin
expression.
Table 16. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
myocardial injury and fibrosis in rats after
30 days of treatment
yocardia ydroxypro ollagen-
1 ICVF line Collagen-
I
Group Necrosis (%) III (AU)
(N~g/mg) (AU)
Vehicle + salt 6.20 3.980.6 1
090
.
(n=8) 0.00.0 5 . 0-910.12
.5 13
Aldosterone 2.00.4 7.91 4.390.6 2.290.
+
salt (n=9) * .0 2 47* 1.390.20
Eplerenone +
Aldosterone 0.00.0# 6.20 4.100.5 2.530. 1,530.23
+
salt (n=9)
.5 3 20*
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Data are mean ~ SEM measured after 30 days of treatment.
* Significantly different from vehicle, p<0.05.
# Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
ICVF = interstitial collagen volume fraction
Collagen-I = collagen type I mRNA.
Collagen-III = collagen type III mRNA.
AU = arbitrary units, measured relative to cyclophilin
expression.
Myocardial Histopathology
Myocardial tissue damage was evaluated after 7, 14, and
30 days of treatment using a semi-quantitative scoring
system. Hearts from vehicle + salt controls were
histologically normal at all timepoints. No vascular or
myocardial lesions were identified in hearts from rats
receiving aldosterone + salt after 7 days of treatment
(Table 14). In contrast, focal arterial and myocardial
alterations were observed starting at 14 days of
treatment (Tables 15 and 16). Qualitative changes in
the arteries and myocardium were similar after 14 days
and 30 days of aldosterone + salt treatment, but the
frequency and severity increased with time.
Administration of eplerenone markedly attenuated
myocardial injury at all time points (Tables 14-16; Fig.
44) .
Gene Expression of Inflammatory Mediators
The expression levels of multiple proinflammatory
molecules were assessed using quantitative Taqman PCR
analysis (Tables 17-19). Expression levels of
cyclooxygenase-2 (COX-2) and monocyte chemoattractant
protein-1 (MCP-1) were similarly and significantly
increased by aldosterone + salt treatment at all time
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points. Osteopontin expression was also markedly
upregulated after 14 days (~6-fold) and 30 days (~13-
fold) of aldosterone + salt treatment (Tables 18-19).
Transforming growth factor beta one (TGF-~o, mRNA levels
were not upregulated at any of the time points examined.
Intracellular adhesion molecule-1 (ICAM-1) mRNA
expression was upregulated at day 14 and 30 of
aldosterone + salt treatment, although increases were
modest (Tables 9-10). Gene expression for vascular cell
adhesion molecule-1 (VCAM-1) was increased two-fold at
day 30 of aldosterone + salt treatment, however this
increase did not reach statistical significance (Table
19). Expression of all marker genes was significantly
reduced by eplerenone compared to gene expression in
animals treated with aldosterone + salt.
Table 17. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
the relative mRNA expression of the
inflammatory markers in rats after 7 days of
treatment
COX-2 Osteopo MCP-1 TGF-~i1 ICAM VCAM
ntin
Group
mRNA mgNA mRNA mRNA mRNA
(AU) ~ (AU) (AU) (AU) (AU)
U p'
,
1.060 1.120 1.07'0. 0.980 1.020 0:960
Vehicle + .18 17 .12 .12 .14
salt (n=6) ~16 (n=5) (n=10 (n=5) (n=5)
(n=5) )
Aldosterone 2-220 1.910 2.380.1 1.350 1.220 1.550
.29* .51 7* .09 .10 .39
+ salt (n=8) (n=8) (n=8) (n=8) (n=8) (n=8)
Eplerenone 1.250 0.750 1.560.2 0.990 0.830 0.560
+
.27 .12 5 # .12 .10 .08
#
aldosterone (n=8) (n=8) (n=8) (n=10 (n=7) (n=6)
+ salt )
Values are mRNA expression means in arbitrary units ~
SEM after 7 days of treatment (relative to cyclophilin
expression).
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* Significantly different from vehicle + salt, p<0.05.
# Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
COX-2=cyclooxygenase-2.
MCP-1=monocyte chemoattractant protein-1.
TGF-(31= transforming growth factor beta 1.
ICAM=intracellular adhesion molecule-1.
VCAM=vascular cell adhesion molecule-1.
Table 18. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
the relative mRNA expression of the
inflammatory markers in rats after 14 days
of treatment
COX-2 OsteopontMCP-1 TGF-ail ICAM VCAM
Group ANA , mRNA mRNA mRNA mRNA
(AU) ~N (AU) (AU) (AU) (AU)
A
(AU)
1.070 1.130. 1.040 1.020 1.120 0.970
Vehicle .15 .08 .07 .07 .11
+
salt (n=7) 08 (n=7) (n=10 (n=7) (n=5)
(n=7) )
Aldosteron 4'340. 6.561.3 2.760. 1.270 1.470 1.160
72* 7* 32* .16 .12* .19
a + salt (n=g) (n=8) (n=8) (n=9) (n=8) (n=8)
Eplerenone
1.260 0.830
2.020. 2.940.7 1.590. 1
190
+ 49* # 2 # * 10* # .16 . .10
.09#
aldosteron (n=11) (n=11) (n=11) (n~11 (n=11) (n~ll
a + salt
Values are mRNA expression means in arbitrary units ~
SEM after 14 days of treatment (relative to cyclophilin
expression).
* Significantly different from vehicle + salt, p<0.05.
# Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
COX-2=cyclooxygenase-2.
MCP-1=monocyte chemoattractant protein-1.
TGF-(31= transforming growth factor beta 1.
ICAM=intracellular adhesion molecule-1.
VCAM=vascular cell adhesion molecule-1.
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Table 19. Effects of aldosterone + salt treatment
alone or in combination with eplerenone on
the relative mRNA expression of the
inflammatory markers in rats after 30 days
of treatment
COX-2 Ostt I,qCP-1 TGF-ailICAM VCAM
opon
Group mRNA , mRNA mRNA mRNA mRNA
(AU) ~N (AU) (AU) (AU) (AU)
A
(AU)
Vehicle + 1-110.1 0.990.1 1,220.2 1.010 1.230. 1.140.
1 1 7 .13 25 25
salt (n=7) (n=7) (n=8) (n=6) (n=5)
(n=7)
Aldosterone 4.530.9 13.271. 4.541.2 1.330 1.810. 2.140.
2* 43* 5* .16 22* 49
+ salt (n=6) (n=6) (n=6) (n=9) (n=5) (n=5)
Eplerenone 2-2g0,3 2.590.8 2.290.4 1.320 1.220. 1.040.
+
aldosterone 3* # 2* # 2* # .11 15 # 14#
+ (n=9) (n=9) (n=9) (n=9) (n=8) (n=9)
lt
sa
Values are mRNA expression means ~ SEM after 30 days of
treatment (relative to Cyclophilin expression).
* Significantly different from vehicle + salt, p<0.05.
Significantly different from aldosterone + salt,
p<0.05.
Eplerenone dose was 100 mg/kg/day.
COX-2=cyclooxygenase-2.
MCP-1=monocyte chemoattractant protein-1.
TGF-(31= transforming growth factor beta 1.
ICAM=intracellular adhesion molecule-1.
VCAM=vascular cell adhesion molecule-1.
Immunohistochemistry
The molecular analysis of the aldosterone + salt-induced
proinflammatory response was further characterized using
immunohistochemical analysis. The majority of cells
adhering to the endothelium and infiltrating the
perivascular space stained positive for a
monocyte/macrophage antibody (ED-1) and negative for a
T-cell antibody (CD-3). Significant expression of
osteopontin was evident in hearts from aldosterone +
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salt treated rats, compared with the absence of
osteopontin staining in hearts from vehicle + salt
controls. Osteopontin expression was primarily
localized to medial cells of affected and some
unaffected coronary arteries, but was also present in
some macrophages in the perivascular space and areas of
myocardial necrosis. No evidence of significant
osteopontin expression was found in cardiomyocytes.
ICAM-1 staining was identified in endothelial cells and
in the perivascular space; however, VCAM-1 was primarily
expressed in endothelial cells. Administration of
eplerenone markedly blunted the aldosterone + salt
treatment induced staining in myocardial tissue for all
marker proteins evaluated.
In-situ Hybridization for Osteopontin mRNA
In-situ hybridization was performed to localize
osteopontin expression in myocardial tissue. The
majority of osteopontin mRNA was found in the medial
cells of coronary arteries (Figure 3); however,
osteopontin message was also identified in perivascular
cells and cells infiltrating ischemic and necrotic
areas. Osteopontin mRNA was not evident in
cardiomyocytes_or in unaffected interstitial areas.
CONCLUSION
Treatment of rats with aldosterone in the presence of
salt induced vascular inflammation and cardiac tissue
damage. This damage induced by aldosterone + salt
treatment was preceded by an inflammatory response that
was characterised by the upregulation of proinflammatory
molecules. Eplerenone markedly attenuated this initial
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vascular inflammatory response and subsequent myocardial
ink ury .
Renal Hypertensive Rat Model
A combination therapy of an aldosterone inhibitor
and a cyclooxygenase-2 selective inhibitor may be
evaluated for blood pressure lowering activity in the
renal-artery ligated hypertensive rat, a model of high
renin hypertension. In this model, six days after
litigation of the left renal artery, both plasma renin
activity and blood pressure are elevated significantly
(J. L. Cangiano et al, J. Pharmacol. Exp. Ther., 206,
310-313 (1979)). Male Sprague-Dawley rats are
instrumented with a radiotelemetry blood pressure
transmitter for continuous monitoring of blood pressure.
The rats are anesthetized with a mixture of ketamine-HCl
(100 mg/kg) and acepromazine maleate (2.2 mg/kg). The
abdominal aorta is exposed via a midline incision.
Microvascular clamps are placed on the aorta distal to
the renal arteries and the iliac bifurcation. The aorta
is punctured with a 22-gauge needle and the tip of a
catheter is introduced. The catheter, which is held in
place by a ligature in the psoas muscle, is connected to
a radiotelemetry blood pressure transmitter (Mini-Mitter
Co., Inc., Sunriver, OR). The transmitter is placed in
the peritoneal cavity and sutured to abdominal muscle
upon closing of the incision. Rats are housed singly
above a radiotelemetry receiver and are allowed standard
rat cho and water ad libitum. At least five days are
allowed for recovery from surgery. Mean arterial
pressure and heart rate are measured on a data recorder
as is appropriate, such as a mini-computer. Data Data
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are sampled for 10 seconds at 200-500 Hz at 2.5 to 10
min intervals 24 hours per day. After collecting
control data for 24 hours, the rats are anesthetized
with methohexital (30 mg/kg, i.p.) and supplemented as
needed. A midline abdominal incision is made,
approximately 2 cm in length to expose the left kidney.
The renal artery is separated from the vein near the
aorta, with care taken not to tramatize the vein. The
artery is completely ligated with sterile 4-O silk. The
incision is closed by careful suturing of the muscle
layer and skin. Six days later, when MAP is typically
elevated by 50-70 mmHg, an aldosterone antagonist or a
combination with one or more Cyclooxygenase-2 selective
inhibitors are administerd by gavage each day for about
8 weeks. Single drug dosing is carried out using 20 and
200 mg/kg/day of the aldosterone inhibitor (for example,
eplerenone) and 1, 3, 10, 30, and 100 mg/kg/day of the
cycloogenase-2 selective inhibitor. Drug mixtures are
obtained by administering a combination of a dose of 1,
3, 10, 30, or 100 mg/kg/day of the cycloogenase-2
selective inhibitor with a dose of either 20 or 200
mg/kg/day of the aldosterone inhibitor. Blood pressure
lowering is monitored by the radiotelemetry system and
responses with the compounds are compared to a response
obtained in vehicle-treated animals. Plasma and urinary
sodium and potassium levels are monitored as a measure
of the effectiveness of the aldosterone blockade. Urine
samples are collected overnight using metabolic cages to
isolate the samples. Plasma samples are obtained by
venous catheterization. Sodium and potassium are
measured by flame photometry. Cardiac fibrosis is
determined by histological and chemical measurements of
the excised hearts following perfusion fixation. Left
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and right ventricles are weighed, embedded, and
sectioned. Subsequently, sections are stained with
picrosirius red and the red staining collagen areas are
quantitated by computerized image analysis. The apex of
the heart is acid digested and the free hydroxyproline
measured colorimetrically. It is expected that MAP will
be significantly lowered toward normal pressures in the
test animals, treated with the combination therapy and
that the condition of myocardial fibrosis will be
arrested or avoided.
Several other animal models are available which are
appropriate for evaluation of prevention of
cardiovascular conditions including the prevention of
atherosclerosis. See Stehbens, Prog. Card. Dis., XXIX,
1007-28 (1986) and Zhang et al., Science, 258, 468-71
(1992) .
An APOe mouse model for atherosclerosis has been
described by Roselear et al. (Arterioscle. Thromb. Vasc.
Biol., 16, 1013-18 (1996)). The aldosterone blocker
should be active in preventing atherosclerotic lesions.
The biological evaluations described herein are
useful in demonstrating the efficacy of combination
therapies comprising an aldosterone receptor antagonist,
and a NSAID, for the treatment or prevention of a
cardiovascular disorder.
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Although this invention has been described with
respect to specific embodiments, the details of these
embodiments are not to be construed as limitations.
All patent documents referenced herein are
incorporated by reference.
