Note: Descriptions are shown in the official language in which they were submitted.
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UPREGULATION OF TYPE III ENDOTHELIAL CELL NITRIC OXIDE
s SYNTHASE BY HMG-CoA RF,DUCTASE INHIBITORS
Field of the Invention
This invention describes the new use of HMG-CoA reductase inhibitors as
upregulators
of Type III endothelial cell Nitric Oxide Synthase. Further, this invention
describes methods that
employ HMG-CoA reductase inhibitors to treat conditions that result from the
abnormally low
expression and/or activity of endothelial cell Nitric Oxide Synthase in a
subject.
Is
Background of the Invention
Nitric oxide (NO) has been recognized as an unusual messenger molecule with
many
physiologic roles, in the cardiovascular, neurologic and immune systems
(Griffith. TM et al., .I
zo Am Coll Caf~diol, 1988, 12:797-806). It mediates blood vessel relaxation,
neurotransmission and
pathogen suppression. NO is produced from the guanidino nitrogen of L-arginine
by NO
Synthase (Moncada, S and Higgs, EA, Eur,l Clin Invest, 1991, 21(4):361-374) .
In mammals,
at least three isoenzymes of NO Synthase have been identified. Two, expressed
in neurons
(nNOS) and endothelial cells (Type III-ecNOS), are calcium-dependent, whereas
the third is
2s calcium-independent and is expressed by macrophages and other cells after
induction with
cytokines (Type II-iNOS) (Bredt, DS and Snyder, SH, Ps°oc Natl Acad Sci
USA, 1990,
87:682-685, Janssens, SP et al., JBiol ChenZ, 1992, 267:22964, Lyons, CR et
al., JBiol Cheuz,
1992, 267:6370-6374). The various physiological and pathological effects of NO
can be
explained by its reactivity and different routes of formation and metabolism.
3o Recent studies suggest that a loss of endothelial-derived NO activity may
contribute to
the atherogenic process (O'Driscoll, G, et al., Cii°culation, 1997,
9:1126-1131). For example,
endothelial-derived NO inhibits several components of the atherogenic process
including
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monocyte adhesion to the endothelial surface (Tsao, PS et al., Circulation,
1994, 89:2176-2182),
platelet aggregation (Radomski, MW, et al., Proc Natl Acad Sci USA, 1990,
87:5193-5197),
vascular smooth muscle cell proliferation (Garg, UC and Hassid, A, J Clin
Invest, 1989,
83:1774-1777), and vasoconstriction (Tanner, FC et al., Circulation, 1991,
83:2012-2020). In
addition, NO can prevent oxidative modification of low-density lipoprotein
(LDL) which is a
major contributor to atherosclerosis, particularly in its oxidized form (Cox,
DA and Cohen, ML,
Pharm Rev, 1996, 48:3-19).
It has been shown in the prior art that hypoxia downregulates ecNOS expression
and/or
activity via decreases in both ecNOS gene transcription and mRNA stability
(Liao, JK et al., J
1o Clin Invest, 1995, 96:2661-2666, Shaul, PW et al., Am JPhysiol, 1997, 272:
LI005-LI012).
Thus, ischemia-induced hypoxia may produce deleterious effects, in part,
through decreases in
ecNOS activity.
HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is the microsomal
enzyme that catalyzes the rate limiting reaction in cholesterol biosynthesis
(HMG
CoA6Mevalonate). An HMG-CoA reductase inhibitor inhibits HMG-CoA reductase,
and as a
result inhibits the synthesis of cholesterol. A number of HMG-CoA reductase
inhibitors has been
used to treat individuals with hypercholesterolemia. Clinical trials with such
compounds have
shown great reductions of cholesterol levels in hypercholesterolemic patients.
Moreover, it has
been shown that a reduction in serum cholesterol levels is correlated with
improved
2o endothelium-dependent relaxations in atherosclerotic vessels (Treasure, CB
et al., N Engl J Med,
1995, 332:481-487). Indeed, one of the earliest recognizable benefits after
treatment with
HMG-CoA reductase inhibitors is the restoration of endothelium-dependent
relaxations (supra,
Anderson, TJ et al., NEngl JMed, 1995, 332:488-493).
Although the mechanism by which HMG-CoA reductase inhibitors restore
endothelial
function is primarily attributed to the inhibition of hepatic HMG-CoA
reductase and the
subsequent lowering of serum cholesterol levels, little is known on whether
inhibition of
endothelial HMG-CoA reductase has additional beneficial effects on endothelial
function.
Pulmonary hypertension is a major cause of morbidity and mortality in
individuals
exposed to hypoxic conditions (Schemer, U et al., N Engl J Med, 1996, 334:624-
629). Recent
3o studies demonstrate that pulmonary arterial vessels from patients with
pulmonary hypertension
have impaired release of NO (Giaid, A and Saleh, D, NEngl JMed, 1995, 333:214-
221, Shaul,
PW, AnZ J Physiol, 1997, 272: L1005-L1012). Additionally, individuals with
pulmonary
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hypertension demonstrate reduced levels of ecNOS expression in their pulmonary
vessels and
benefit clinically from inhalation nitric oxide therapy (Roberts, JD et al., N
Engl J Med, 1997,
336:605-610, Kouyoumdjian, C et al., J Clin Invest, 1994, 94:578-584).
Conversely, mutant
mice lacking ecNOS gene or newborn lambs treated with the ecNOS inhibitor, N-
omega-
s rnonomethyl-L-arginine (LNMA, or N-omega-nitro-L-arginine), develop
progressive elevation
of pulmonary arterial pressures and resistance (Steudel, W et al., Circ Res,
1997, 81:34-41,
Fineman, JR et al., JClin Invest, 1994, 93:2675-2683). It has also been shown
in the prior art
that hypoxia causes pulmonary vasoconstriction via inhibition of endothelial
cell nitric oxide
synthase (ecNOS) expression and activity (Adnot, S et al., J Clin Invest,
1991, 87:155-162,
Liao, JK et al., J Clin Invest, 1995, 96, 2661-2666). Hence, hypoxia-mediated
downregulation
of ecNOS may lead to the vasoconstrietive and structural changes associated
with pulmonary
hypertension.
Often cited as the third most frequent cause of death in the developed
countries, stroke
has been defined as the abrupt impairment of brain function caused by a
variety of pathologic
IS changes involving one or several intracranial or extracranial blood
vessels. Approximately 80%
of all strokes are ischemic strokes, resulting from restricted blood flow.
Mutant mice lacking the
gene for ecNOS are hypertensive (Huang, PL et al., Nature, 1995, 377:239-242,
Steudel, W et
al., Circ Res, 1997, 81:34-41) and develop greater intimal smooth muscle
proliferation in
response to cuff injury. Furthermore, occlusion of the middle cerebral artery
results in 21%
greater infarct size in "ecNOS knockout" mice compared to wildtype mice
(Huang, Z et al., J
Cereb Blood Flow Metab, 1996, 16:981-987). These findings suggest that the
ecNOS production
may play a role in cerebral infarct formation and sizes. Additionally, since
most patients with
ischemic strokes have average or normal cholesterol levels, little is known on
what the potential
benefits of HMG-CoA reductase inhibitor administration would be in
cerebrovascular events.
There exists a need to identify agents that improve endothelial cell function.
There also exists a need to identify agents that can be used acutely or in a
prophylactic
manner to treat conditions that result from low levels of endothelial cell
Nitric Oxide Synthase.
Summary of the Invention
The invention involves the discovery that HMG-CoA reductase inhibitors can
upregulate
endothelial cell Nitric Oxide Synthase (Type III) activity other than through
preventing the
formation of oxidative LDL. It previously was believed that such reductase
inhibitors functioned
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by lowering serum cholesterol levels by blocking hepatic conversion of HMG-CoA
to
L-mevalonate in the cholesterol biosynthetic pathway. It has been discovered,
surprisingly, that
HMG-CoA reductase inhibitors can increase Nitric Oxide Synthase activity by
effects directly
on endothelial rather than hepatic HMG-CoA reductase. This upregulation of
activity does not
depend upon a decrease in cholesterol synthesis and in particular does not
depend upon a decrease
in the formation of ox-LDL. The invention, therefore, is useful whenever it is
desirable to restore
endothelial cell Nitric Oxide Synthase activity or increase such activity in
an affected cell or
tissue. The tissue is defined as to include both the cells in the vasculature
supplying nutrients to
the tissue, as well as cells of the tissue that express endothelial cell
Nitric Oxide Synthase.
to Nitric Oxide Synthase activity is involved in many conditions, including
impotence, heart
failure, gastric and esophageal motility disorders, kidney disorders such as
kidney hypertension
and progressive renal disease, insulin deficiency, etc. Individuals with such
conditions would
benefit from increased endothelial cell Nitric Oxide Synthase activity. It
also was known that
individuals with pulmonary hypertension demonstrate reduced levels of Nitric
Oxide Synthase
/5 expression in their pulmonary vessels and benefit clinically from
inhalation of Nitric Oxide. The
invention therefore is particularly useful for treating pulmonary
hypertension. It also has been
demonstrated that hypoxia causes an inhibition of endothelial cell Nitric
Oxide Synthase activity.
The invention therefore is useful for treating subjects with hypoxia-induced
conditions. It also
has been discovered, surprisingly, that HMG-CoA reductase inhibitors are
useful for reducing
20 brain injury that occurs following a stroke.
According to one aspect of the invention, a method is provided for increasing
endothelial
cell Nitric Oxide Synthase activity in a nonhypercholesterolemic subject who
would benefit from
increased endothelial cell Nitric Oxide Synthase activity in a tissue. The
method involves
administering to a nonhypercholesterolemic subject in need of such treatment a
HMG-CoA
25 reductase inhibitor that increases endothelial cell Nitric Oxide Synthase
activity in an amount
effective to increase endothelial cell Nitric Oxide Synthase activity in the
tissue of the subject.
In certain embodiments, when the subject in need of such treatment has an
abnormally
elevated risk of an ischemic stroke use of HMG CoA reductase inhibitors is
excluded.
In one important embodiment, HMG-CoA reductase inhibitors do not affect
cholesterol
30 levels in a subject. In atherosclerotic patients, reduction in serum
cholesterol is correlated with
improved endothelium-dependent relaxation in atherosclerotic vessels
(Treasure, et al.,
N.EngI.JMed., 1995, 332:481-487). HMG-CoA reductase inhibitors have been
demonstrated to
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reduce serum cholesterol in a matter of weeks, and maximum level of
cholesterol reduction can
be achieved after a few months of chronic administration. In contrast, the
effect of HMG-CoA
reductase inhibitors on up-regulation of ecNOS occurs within a few days. Thus,
treatment
according to the present invention provides significant advantages, e.g., when
administered to
address short term increases in risk of stroke or other embolic events, such
as that due to surgical
intervention, even for hypercholesterolemic patients.
In certain embodiments, the subject is not hypercholesterolemic or not
hypertriglyceridemic or both (i.e., nonhyperlipidemic). In other embodiments,
the amount is
sufficient to increase endothelial cell Nitric Oxide Synthase activity above
normal baseline levels
1o established by age-controlled groups, described in greater detail below. In
certain embodiments,
the HMG-CoA reductase inhibitor is administered in an amount which alters the
blood LDL
cholesterol levels in the subject by less than 10%. The alteration may even be
less than 5%. In
certain embodiments the amount is sufficient to increase endothelial cell
Nitric Oxide Synthase
activity above normal baseline levels established by age-controlled groups,
described in greater
I5 detail below.
The subject can have a condition characterized by an abnormally low level of
endothelial
cell Nitric Oxide Synthase activity which is hypoxia-induced. In other
embodiments the subject
can have a condition comprising an abnormally low level of endothelial cull
Nitric Oxide
Synthase activity which is chemically induced. In still other embodiments the
subject can have
2o a condition comprising an abnormally low level of endothelial cell Nitric
Oxide Synthase activity
which is cytokine induced. In certain important embodiments, the subject has
pulmonary
hypertension or an abnormally elevated risk of pulmonary hypertension. In
other important
embodiments, the subject has experienced an ischemic stroke or has an
abnormally elevated risk
of an ischemic stroke. In still other important embodiments, the subject has
heart failure or
25 progressive renal disease. In yet other important embodiments, the subject
is chronically exposed
to hypoxic conditions.
According to any of the foregoing embodiments, the preferred HMG-CoA reductase
inhibitor is selected from the group consisting of simvastatin and lovastatin.
In further important
embodiments, the subject has experienced a thrombotic event or has an
abnormally elevated risk
30 of thrombosis. In still other embodiments, the subject has an abnormally
elevated risk of
arteriosclerosis or has arteriosclerosis. In other important embodiments, the
subject has an
abnormally elevated risk of developing a myocardial infarction or has
experienced a myocardial
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infarction. In yet further important embodiments, the subject has an
abnormally elevated risk of
reperfusion injury. In preferred embodiments, the subject with an elevated
risk of reperfusion
injury is an organ transplant recipient (e.g., heart, kidney, liver, etc.). In
other important
embodiments, the subject has homocystinuria. In certain other important
embodiments, the
subject has Cerebral autosomal dominant arteriopathy with subcortical infarcts
and
leucoencephalopathy (CADASIL) syndrome. In further important embodiments, the
subject has
a degenerative disorder of the nervous system. In preferred embodiments, the
subject with a
degenerative disorder of the nervous system has Alzheimer's disease.
In certain other embodiments, when the subject in need of a treatment
according to the
1o present invention has an abnormally elevated risk of an ischemic stroke,
HMG-CoA reductase
inhibitors are excluded as treatments for such subjects.
According to any of the foregoing embodiments, the method can further comprise
co-administering an endothelial cell Nitric Oxide Synthase substrate (L-
arginine preferred) and/or
co-administering a nonHMG-CoA reductase inhibitor agent that increases
endothelial cell Nitric
IS Oxide Synthase activity. A preferred such agent is selected from the group
consisting of
estrogens and angiotensin-converting enzyme (ACE) inhibitors. The agents may
be administered
to a subject who has a condition or prophylactically to a subject who has a
risk, and more
preferably, an abnormally elevated risk, of developing a condition. The
inhibitors also may be
administered acutely.
2o According to another aspect of the invention, a method is provided for
increasing
endothelial cell Nitric Oxide Synthase activity in a subject to treat a
nonhyperlipidemic condition
favorably affected by an increase in endothelial cell Nitric Oxide Synthase
activity in a tissue.
Such conditions are exemplified above. The method involves administering to a
subject in need
of such treatment a HMG-CoA reductase inhibitor in an amount effective to
increase endothelial
25 cell Nitric Oxide Synthase activity in the tissue of the subject. Important
conditions are as
described above. Also as described above, the method can involve co-
administration of substrates
of endothelial cell Nitric Oxide Synthase and/or a nonHMG-CoA reductase
inhibitor agent that
increases endothelial cell Nitric Oxide Synthase activity. Preferred compounds
are as described
above. As above, the reductase inhibitor can be administered, inter alia,
acutely or
3o prophylactically.
In certain embodiments, when the subject in need of such treatment has an
abnormally
elevated risk of an ischemic stroke, then HMG CoA reductase inhibitors are
excluded from
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treating such subjects.
According to another aspect of the invention, a method is provided for
reducing brain
injury resulting from stroke. The method involves administering to a subject
having an
abnormally high risk of an ischemic stroke a HMG-CoA reductase inhibitor in an
amount
effective to increase endothelial cell Nitric Oxide Synthase activity in the
brain of the subject.
As above, important embodiments include the inhibitor being selected from the
group consisting
of simvastatin and lovastatin. Also as above, important embodiments include co-
administering
a substrate of endothelial cell Nitric Oxide Synthase (L-arginine preferred)
and/or a
nonHMG-CoA reductase inhibitor agent that increases endothelial cell Nitric
Oxide Synthase
activity. Likewise, important embodiments include prophylactic and acute
administration of the
inhibitor.
In certain embodiments, when the subject in need of such treatment has an
abnormally
elevated risk of an ischemic stroke, HMG CoA reductase inhibitors are excluded
from treating
such subjects.
l5 According to another aspect of the invention, a method is provided for
treating pulmonary
hypertension. The method involves administering to a subject in need of such
treatment a
HMG-CoA reductase inhibitor in an amount effective to increase pulmonary
endothelial cell
Nitric Oxide Synthase activity in the subject. Particularly important
embodiments are as
described above in connection with the methods for treating brain injury.
Another important
embodiment is administering the inhibitor prophylactically to a subject who
has an abnormally
elevated risk of developing pulmonary hypertension, including subjects that
are chronically
exposed to hypoxic conditions.
According to another aspect of the invention, a method for treating heart
failure is
provided. The method involves administering to a subject in need of such
treatment a HMG-CoA
reduetase inhibitor in an amount effective to increase vascular endothelial
cell Nitric Oxide
Synthase activity in the subject. As discussed above, important embodiments
include
prophylactic and acute administration of the inhibitor. Another important
embodiment is treating
a subject that is nonhyperlipidemic. Preferred compounds and co-administration
schemes are as
described above.
3o According to yet another aspect of the invention, a method is provided for
treating
progressive renal disease. The method involves administering to a subject in
need of such
treatment a HMG-CoA reductase inhibitor in an amount effective to increase
renal endothelial
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cell Nitric Oxide Synthase activity in the kidney of the subject. Important
embodiments and
preferred compounds and schemes of co-administration are as described above in
connection with
heart failure.
According to another aspect of the invention, a method for increasing blood
flow in a
tissue of a subject is provided. The method involves administering to a
subject in need of such
treatment a HMG-CoA reductase inhibitor in an amount effective to increase
endothelial cell
Nitric Oxide Synthase activity in the tissue of the subject. In preferred
embodiments, the tissue
in which blood flow is increased includes tissue in the brain. In a
particularly preferred
embodiment, cerebral blood flow is enhanced. As discussed above, important
embodiments
l0 include prophylactic and acute administration of the inhibitor. Preferred
compounds and co-
administration schemes are also as described above. Other important
embodiments include co-
administering a second agent to the subject with a condition treatable by the
second agent in an
amount effective to treat the condition, whereby the delivery of the second
agent to a tissue of the
subject is enhanced as a result of the increased blood flow.
In certain embodiments, the tissue is brain and the second agent comprises an
agent
having a site of action in the brain.
According to another aspect of the invention, a method of screening for
identifying an
inhibitor of HMG-CoA reductase for treatment of subjects who would benefit
from increased
endothelial cell Nitric Oxide Synthase activity in a tissue, is provided. The
method involves
2o identifying an inhibitor of HMG-CoA reductase suspected of increasing
endothelial cell Nitric
Oxide Synthase activity, and determining whether or not the inhibitor of HMG-
CoA reductase
produces an increase in endothelial cell Nitric Oxide Synthase activity in
vivo or in vitro. In
certain embodiments, the subject who would benefit from increased endothelial
cell Nitric Oxide
Synthase activity has an abnormally elevated risk of stroke.
The invention also involves the use of HMG-CoA reductase inhibitors in the
manufacture
of medicaments for treating the above-noted conditions. Important conditions,
compounds, etc.
are as described above. The invention further involves pharmaceutical
preparations including the
HMG-CoA reductase inhibitors for treating the above-noted conditions. The
preparations can
include other agents such as second agents, ecNOS substrates, ecNOS cofactors,
as described
3o above, or can be cocktails of the HMG-CoA reductase inhibitors together
with a nonHMG-CoA
reductase inhibitor agent that increases ecNOS activity in a cell, directly or
indirectly
(synergistically, cooperatively, additively, etc.).
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In certain embodiments, compositions and pharmaceutical preparations that are
cocktails
of a HMG-CoA reductase inhibitor and L-arginine are provided. In other
embodiments, the
HMG-CoA reductase inhibitor and the L-arginine are in amounts effective to
increase blood flow.
In further embodiments, the HMG-CoA reductase inhibitor and the L-arginine are
in amounts
effective to increase blood flow in brain tissue. In preferred embodiments,
administration of the
HMG-CoA reductase inhibitor and the L-arginine results in increased blood
flow. In particularly
preferred embodiments, administration of the HMG-CoA reductase inhibitor and
the L-arginine
results in increased blood flow to the brain. Any of the above cocktail
compositions may also
include other cofactors that enhance ecNOS substrate connversion by ecNOS to
nitric oxide, the
to preferred cofactors being NADPH and tetrahydrobiopterin.
The invention also involves methods for increasing ecNOS activity in a cell by
contacting
the cell with an effective amount of a HMG-CoA reductase inhibitor, alone, or
together with any
of the agents co-administered as described above, or as a cocktail as
described above. Any of the
above cocktails may also include a substrate for endothelial cell Nitric Oxide
Synthase, the
I5 preferred substrate being L-arginine, and/or other cofactors that enhance
ecNOS substrate
connversion by ecNOS to nitric oxide, the preferred cofactors being NADPH and
tetrahydrobiopterin.
These and other aspects of the invention are described in greater detail
below.
2o Brief Description of the Drawings
Figure 1. Western blots showing the effects of oxidized (ox)-LDL on ecNOS
protein
levels in the presence and absence of simvastatin. Figure 1 A depicts the
effects of increasing
concentrations of simvastatin on ecNOS protein levels. Figure 1 B depicts the
effects of
increasing concentrations of simvastatin on ecNOS protein levels in a time-
dependent manner.
25 Figure 2. Northern blots showing the effects of ox-LDL on ecNOS mRNA levels
in
the presence and absence of HMG-CoA reductase inhibitors. Figure 2A depicts
the effects of
simvastatin on ecNOS mRNA levels. Figure 2B depicts the effects of lovastatin
on ecNOS
mRNA levels.
Figure 3. Western blots showing the concentration-dependent effects of
simvastatin
30 (Figure 3A) and lovastatin (Figure 3B) on ecNOS protein levels after 48
hours.
Figure 4. Volume of cerebral infarction after 2 h filamentous middle cerebral
artery
occlusion and 22 h reperfusion as % of control, with and without simvastatin
treatment.
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Figure 4A depicts the cerebral infarction in wild-type SV-129 mice. Figure 4B
depicts the
cerebral infarction in ecNOS-deficient mice.
Figure S. Bar graph showing regional CBF changes in wild type and eNOS null
mice
for 40 min after L-arginine or saline infusion.
Figure 6. Bar graph showing regional CBF changes in simvastatin-treated mice
for
40 min after L-arginine or saline infusion at the same dose.
Detailed Description of the Invention
The invention is useful whenever it is desirable to increase endothelial cell
Nitric Oxide
/0 Synthase (Type III isoform) activity in a subject. Nitric Oxide Synthase is
the enzyme that
catalyzes the reaction that produces nitric oxide from the substrate L-
arginine. As the name
implies, endothelial cell nitric oxide Synthase refers to the Type III isoform
of the enzyme found
in the endothelium.
By "ecNOS activity", it is meant the ability of a cell to generate nitric
oxide from the
substrate L-arginine. Increased ecNOS activity can be accomplished in a number
of different
ways. For example, an increase in the amount of ecNOS protein or an increase
in the activity of
the protein (while maintaining a constant level of the protein) can result in
increased "activity".
An increase in the amount of protein available can result from increased
transcription of the
ecNOS gene, increased stability of the ecNOS mRNA or a decrease in ecNOS
protein
2o degradation.
The ecNOS activity in a cell or in a tissue can be measured in a variety of
different ways.
A direct measure would be to measure the amount of ecNOS present. Another
direct measure
would be to measure the amount of conversion of arginine to citrulline by
ecNOS or the amount
of generation of nitric oxide by ecNOS under particular conditions, such as
the physiologic
conditions of the tissue. The ecNOS activity also can be measured more
indirectly, for example
by measuring mRNA half life (an upstream indicator) or by a phenotypic
response to the
presence of nitric oxide (a downstream indicator). One phenotypic measurement
employed in the
art is detecting endothelial dependent relaxation in response to a
acetylcholine, which response
is affected by ecNOS activity. The level of nitric oxide present in a sample
can be measured
3o using a nitric oxide meter. All of the foregoing techniques are well known
to those of ordinary
skill in the art, and some are described in the examples below.
The present invention, by causing an increase in ecNOS activity, permits not
only the
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re-establishment of normal base-line levels of ecNOS activity, but also allows
increasing such
activity above normal base-line levels. Normal base-line levels are the
amounts of activity in a
normal control group, controlled for age and having no symptoms which would
indicate alteration
of endothelial cell Nitric Oxide Synthase activity (such as hypoxic
conditions, hyperlipidemia and
s the like). The actual level then will depend upon the particular age group
selected and the
particular measure employed to assay activity. Specific examples of various
measures are
provided below. In abnormal circumstances, e.g. hypoxic conditions, pulmonary
hypertension,
etc., endothelial cell Nitric Oxide Synthase activity is depressed below
normal levels.
Surprisingly, when using the reductase inhibitors according to the invention,
not only can normal
1o base-line levels be restored in such abnormal conditions, but endothelial
cell Nitric Oxide
Synthase activity can be increased desirably far above normal base-line levels
of endothelial cell
Nitric Oxide Synthase activity. Thus, "increasing activity" means any increase
in endothelial cell
Nitric Oxide Synthase activity in the subject resulting from the treatment
with reductase inhibitors
according to the invention, including, but not limited to, such activity as
would be sufficient to
IS restore normal base-line levels and such activity as would be sufficient to
elevate the activity
above normal base-line levels.
As mentioned above, Nitric Oxide Synthase activity is involved in many
conditions,
including stroke, pulmonary hypertension, thrombosis, arteriosclerosis,
myocardial infarction,
reperfusion injury (e.g., in an organ transplant recipient), impotence, heart
failure, gastric and
2o esophageal motility disorders, kidney disorders such as kidney hypertension
and progressive renal
disease, insulin deficiency, hypoxia-induced conditions, homocystinuria,
neurodegenerative
disorders, CADASIL syndrome, etc. In one embodiment of the invention the
decrease in
endothelial cell Nitric Oxide Synthase activity is cytokine induced. Cytokines
are soluble
polypeptides produced by a wide variety of cells that control gene activation
and cell surface
25 molecule expression. They play an essential role in the development of the
immune system and
thus in the development of an immune response. However, besides their numerous
beneficial
properties, they have also been implicated in the mechanisms for the
development of a variety
of inflammatory diseases. For example, the cytokines TNF-a and IL-1 are
thought to be part of
the disease causing mechanism of non-cholesterol induced atherosclerosis,
transplant arterial
30 sclerosis, rheumatoid arthritis, lupus, scleroderma, emphysema, etc.
Subjects of such disorders
exhibit lower levels of endothelial cell Nitric Oxide Synthase activity (which
is thus "cytokine
induced"), and may benefit from therapy using the agents of the instant
invention.
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One important embodiment of the invention is treatment of ischemic stroke.
Ischemic
stroke (ischemic cerebral infarction) is an acute neurologic injury that
results from a decrease in
the blood flow involving the blood vessels of the brain. Ischemic stroke is
divided into two broad
categories, thrombotic and embolic.
A surprising finding was made in connection with the treatment of ischemic
stroke. In
particular, it was discovered that treatment according to the invention can
reduce the brain injury
that follows an ischemic stroke. Brain injury reduction, as demonstrated in
the examples below,
can be measured by determining a reduction in infarct size in the treated
versus the control
groups. Likewise, functional tests measuring neurological deficits provided
further evidence of
l0 reduction in brain injury in the treated animals versus the controls.
Cerebral blood flow also was
better in the treated animals versus the controls. Thus, in the various
accepted models of brain
injury following stroke, a positive effect was observed in the treated animals
versus the control
animals. It is believed that all of the foregoing positive results are
attributable to the upregulation
of endothelial cell Nitric Oxide Synthase activity, which is believed
demonstrated in the examples
/5 below.
An important embodiment of the invention is treatment of a subject with an
abnormally
elevated risk of an ischemic stroke. As used herein, subjects having an
abnormally elevated risk
of an ischemic stroke are a category determined according to conventional
medical practice; such
subjects may also be identified in conventional medical practice as having
known risk factors for
20 stroke or having increased risk of cerebrovascular events. Typically, the
risk factors associated
with cardiac disease are the same as are associated with stroke. The primary
risk factors include
hypertension, hypercholesterolemia, and smoking. In addition, atrial
fibrillation or recent
myocardial infarction are important risk factors. As used herein, subjects
having an abnormally
elevated risk of an ischemic stroke also include individuals undergoing
surgical or diagnostic
25 procedures which risk release of emboli, lowering of blood pressure or
decrease in blood flow to
the brain, such as carotid endarterectomy, brain angiography, neurosurgical
procedures in which
blood vessels are compressed or occluded, cardiac catheterization,
angioplasty, including balloon
angioplasty, coronary by-pass surgery, or similar procedures. Subjects having
an abnormally
elevated risk of an ischemic stroke also include individuals having any
cardiac condition that may
30 lead to decreased blood flow to the brain, such as atrial fibrillation,
ventrical tachycardia, dilated
cardiomyopathy and other cardiac conditions requiring anticoagulation.
Subjects having an
abnormally elevated risk of an ischemic stroke also include individuals having
conditions
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including arteriopathy or brain vasculitis, such as that caused by lupus,
congenital diseases of
blood vessels, such as cadasil syndrome, or migraine, especially prolonged
episodes. In certain
embodiments, the subject is not hypercholesterolemic or not
hypertriglyceridemic or both (i.e.,
nonhyperlipidemic).
The treatment of stroke can be for patients who have experienced a stroke or
can be a
prophylactic treatment. Short term prophylactic treatment is indicated for
subjects having
surgical or diagnostic procedures which risk release of emboli, lowering of
blood pressure or
decrease in blood flow to the brain, to reduce the injury due to any ischemic
event that occurs as
a consequence of the procedure. Longer term or chronic prophylactic treatment
is indicated for
subjects having cardiac conditions that may lead to decreased blood flow to
the brain, or
conditions directly affecting brain vasculature. If prophylactic, then the
treatment is for subjects
having an abnormally elevated risk of an ischemic stroke, as described above.
If the subjects
have an abnormally elevated risk of an ischemic stroke because of having
experienced a previous
ischemic event, then the prophylactic treatment for these subjects excludes
the use ofHMG CoA
IS reductase inhibitors. If the subject has experienced a stroke, then the
treatment can include acute
treatment. Acute treatment for stroke subjects means administration of the HMG-
CoA reductase
inhibitors at the onset of symptoms of the condition or at the onset of a
substantial change in the
symptoms of an existing condition.
Another important embodiment of the invention is treatment of pulmonary
hypertension.
Pulmonary hypertension is a disease characterized by increased pulmonary
arterial pressure and
pulmonary vascular resistance. Hypoxemia, hypocapnia, and an abnormal
diffusing capacity for
carbon monoxide are almost invariable findings of the disease. Additionally,
according to the
present invention, patients with pulmonary hypertension also have reduced
levels of ecNOS
expression in their pulmonary vessels. Traditionally, the criteria for
subjects with, or at risk for
pulmonary hypertension are defined on the basis of clinical and histological
characteristics
according to Heath and Edwards (Circulation, 1958, 18:533-547).
Subjects may be treated prophylactically to reduce the risk of pulmonary
hypertension or
subjects with pulmonary hypertension may be treated long term and/or acutely.
If the treatment
is prophylactic, then the subjects treated are those with an abnormally
elevated risk of pulmonary
hypertension. A subject with an abnormally elevated risk of pulmonary
hypertension is a subject
with chronic exposure to hypoxic conditions, a subject with sustained
vasoconstriction, a subject
with multiple pulmonary emboli, a subject with cardiomegaly and/or a subject
with a family
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history of pulmonary hypertension.
Another important embodiment of the invention involves treating hypoxia-
induced
conditions. Hypoxia as used herein is defined as the decrease below normal
levels of oxygen in
a tissue. Hypoxia can result from a variety of circumstances, but most
frequently results from
impaired lung function. Impaired lung function can be caused by emphysema,
cigarette smoking,
chronic bronchitis, asthma, infectious agents, pneumonitis (infectious or
chemical), lupus,
rheumatoid arthritis, inherited disorders such as cystic fibrosis, obesity, a,-
antitrypsin deficiency
and the like. It also can result from non-lung impairments such as from living
at very high
altitudes. Hypoxia can result in pulmonary vasoconstriction via inhibition of
ecNOS activity.
Jo Another important embodiment of the invention is the treatment of heart
failure. Heart
failure is a clinical syndrome of diverse etiologies linked by the common
denominator of
impaired heart pumping and is characterized by the failure of the heart to
pump blood
commensurate with the requirements of the metabolizing tissues, or to do so
only from an
elevating filling pressure.
The invention involves treatment of the foregoing conditions using HMG-CoA
reductase
inhibitors. "HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A)" is the
microsomal
enzyme that catalyzes the rate limiting reaction in cholesterol biosynthesis
(HMG-CoA6Mevalonate). An "HMG-CoA reductase inhibitor" inhibits HMG-CoA
reductase,
and therefore inhibits the synthesis of cholesterol. There is a large number
of compounds
2o described in the art that have been obtained naturally or synthetically,
which have been seen to
inhibit HMG-CoA reductase, and which form the category of agents useful for
practicing the
present invention. Traditionally these agents have been used to treat
individuals with
hypercholesterolemia. Examples include some which are commercially available,
such as
simvastatin (U.S. Patent No. 4, 444,784), lovastatin (U.S. Patent No.
4,231,938), pravastatin
sodium (LJ.S. Patent No. 4,346,227), fluvastatin (U.S. Patent No. 4,739,073),
atorvastatin (U.S.
Patent No. 5,273,995), cerivastatin, and numerous others described in U.S.
Patent No. 5,622,985,
U.S. Patent No. 5,135,935, U.S. Patent No. 5,356,896, U.S. Patent No.
4,920,109, U.S. Patent No.
5,286,895, U.S. f atent No. 5,262,435, U.S. Patent No. 5,260,332, U.S. Patent
No. 5,317,031, U.S.
Patent No. 5,283,256, U.S. Patent No. 5,256,689, U.S. Patent No. 5,182,298,
U.S. Patent No.
5,369,125, U.S. Patent No. 5,302,604, U.S. Patent No. 5,166,171, U.S. Patent
No. 5,202,327, U.S.
Patent No. 5,276,021, U.S. Patent No. 5,196,440, U.S. Patent No. 5,091,386,
U.S. Patent No.
5,091,378, U.S. Patent No. 4,904,646, U.S. Patent No. 5,385,932, U.S. Patent
No. 5,250,435, U.S.
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Patent No. 5,132,312, U.S. Patent No. 5,130,306, U.S. Patent No. 5,116,870,
U.S. Patent No.
5,112,857, U.S. Patent No. 5,102,911, U.S. Patent No. 5,098,931, U.S. Patent
No. 5,081,136, U.S.
Patent No. 5,025,000, U.S. Patent No. 5,021,453, U.S. Patent No. 5,017,716,
U.S. Patent No.
5,001,144, U.S. Patent No. 5,001,128, U.S. Patent No. 4,997,837, U.S. Patent
No. 4,996,234, U.S.
Patent No. 4,994,494, U.S. Patent No. 4,992,429, U.S. Patent No. 4,970,231,
U.S. Patent No.
4,968,693, U.S. Patent No. 4,963,538, U.S. Patent No. 4,957,940, U.S. Patent
No. 4,950,675, U.S.
Patent No. 4,946,864, U.S. Patent No. 4,946,860, U.S. Patent No. 4,940,800,
U.S. Patent No.
4,940,727, U.S. Patent No. 4,939,143, U.S. Patent No. 4,929,620, U.S. Patent
No. 4,923,861, U.S.
Patent No. 4,906,657, U.S. Patent No. 4,906,624 and U.S. Patent No. 4,897,402,
the disclosures
of which patents are incorporated herein by reference.
Important embodiments of the invention involve populations never before
treated with
an HMG-CoA reductase inhibitor. Thus, the invention involves in certain
aspects treatments of
individuals who are otherwise free of symptoms calling for treatment with an
HMG-CoA
reductase inhibitor. It is believed that the only clinically accepted such
condition is
hypercholesterolemia, wherein the reductase inhibitor is administered for the
purpose of
preventing the biosynthesis of cholesterol. Thus, in certain embodiments the
treated population
is nonhypercholesterolemic. In other embodiments, the subject is
nonhypertriglyceridemic. In
still other embodiments, the subject is nonhypercholesterolemic and/or
nonhypertriglyceridemic,
i.e., nonhyperlipidemic.
2o A nonhypercholesterolemic subject is one that does not fit the current
criteria established
for a hypercholesterolemic subject. A noWypertriglyceridemic subject is one
that does not fit the
current criteria established for a hypertriglyceridemic subject (See, e.g.,
Hanison's Principles of
Experimental Medicine, 13th Edition, McGraw-Hill, Inc., N.Y., hereinafter
"Harrison's").
Hypercholesterolemic subjects and hypertriglyceridemic subjects are associated
with increased
incidence of premature coronary heart disease. A hypercholesterolemic subject
has an LDL level
of > 160 mg/dL or > 130 mg/dL and at least two risk factors selected from the
group consisting
of male gender, family history of premature coronary heart disease, cigarette
smoking (more than
10 per day), hypertension, low HDL (<35 mg/dL), diabetes mellitus,
hyperinsulinemia,
abdominal obesity, high lipoprotein (a), and personal history of
cerebrovascular disease or
occlusive peripheral vascular disease. A hypertriglyceridemic subject has a
triglyceride (TG)
level of >250 mg/dL. Thus, a hyperlipidemic subject is defined as one whose
cholesterol and
triglyceride levels equal or exceed the limits set as described above for both
the
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hypercholesterolemic and hypertriglyceridemic subjects.
Another important embodiment of the invention is treatment of thrombosis.
Thromboembolism is the collective term used for diseases characterized by the
formation,
development, or presence of a thrombus and the blocking of a vessel by a
thrombus brought
to a thrombotic vascular site by the blood current. Thromboembolism can reduce
blood flow
to almost all organs including the brain and myocardium. Thromboembolism
involving the
brain is otherwise known as an ischemic stroke and is described elsewhere in
this application.
Thromboembolism involving the heart is otherwise known as a myocardial
infarction and is
also described elsewhere in this application. According to Harrison's, certain
patient groups
1o have been identified who are particularly prone to thrombosis and embolism.
These include
patients: (1) immobilized after surgery; (2) with chronic congestive heart
failure; (3) with
atherosclerotic vascular disease; (4) with malignancy; or (5) who are
pregnant.
An important embodiment of the invention is treatment of subjects with an
abnormally
elevated risk of thrombosis (or thromboembolism). As used herein, subjects
having an
abnormally elevated risk of thrombosis are a category determined according to
conventional
medical practice. Typically, prethrombotic patients can be identified by a
careful history.
There are, according to Harrison's, three important clues to this diagnosis:
(1) repeated
episodes of thromboembolism without an obvious predisposing condition; (2) a
family history
of thrombosis; and (3) well-documented thromboembolism in adolescents and
young adults.
2o Subjects may be treated prophylactically to reduce the risk of a thrombotic
episode or
subjects with thrombosis may be treated long-term and/or acutely. If the
treatment is
prophylactic, then the subjects treated are those with an abnormally elevated
risk of
thrombosis.
Another important embodiment of the invention is treatment of myocardial
infarction.
Myocardial infarction is the diseased state which occurs with the abrupt
decrease in coronary
blood flow that follows a thrombotic occlusion of a coronary artery previously
narrowed by
artheosclerosis. Such injury is produced or facilitated by factors such as
cigarette smoking,
hypertension and lipid accumulation.
An important embodiment of the invention is treatment of a subject with an
abnormally
3o elevated risk of myocardial infarction. As used herein, subjects having an
abnormally elevated
risk of myocardial infarction are the category of patients that include those
with unstable
angina, multiple coronary risk factors (similar to those described for stroke
elsewhere herein),
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and Prinzmetal's variant angina. Less common etiologic factors include
hypercoagulability,
coronary emboli, collagen vascular disease, and cocaine abuse.
Subjects may be treated prophylactically to reduce the risk of myocardial
infarction, or
subjects with myocardial infarction, may be treated long-term and/or acutely.
If the treatment
is prophylactic, then the subjects treated are those with an abnormally
elevated risk of
myocardial infarction. A subject with an abnormally elevated risk of
myocardial infarction is
a subject that falls in the above-described categories.
Another important embodiment of the invention, is the treatment of subjects
with an
abnormally elevated risk of reperfusion injury damage. Preferred subjects are
about to receive
or have received a transplant. According to the present invention, increase in
ecNOS
expression and/or activity in the vessels of the transplanted organ is
believed to reduce
reperfusion injury damage. Reperfusion injury is the functional, metabolic, or
structural
change that includes necrosis in ischemic tissues, thought to result from
reperfusion to ischemic
areas of the tissue. The most common example involves myocardial reperfusion
injury. In
myocardial reperfusion injury, changes in ischemic heart muscle are thought to
result from
reperfusion to the ischemic areas of the heart. Changes can be fatal to muscle
cells and may
include oedema with explosive cell swelling and disintegration, sarcolemma
disruption,
fragmentation of mitochondria, contraction and necrosis, enzyme washout and
calcium
overload.
2o Another important embodiment of the invention, is the treatment of subjects
with a
homocystinuria. The homocystinurias are seven biochemically and clinically
distinct disorders,
each characterized by increased concentration of the sulfur-containing amino
acid homocysteine
in blood and urine. This is because the enzyme cystathione synthetase that
converts homocysteine
and serine into cystathione, a precursor of cysteine, is missing. Subjects
with a homocystinuria
are also likely to suffer from thrombosis, and can benefit from increased
ecNOS expression
and/or activity.
Another important embodiment of the invention, is the treatment of subjects
with
Cerebral autosomal dominant arteriopathy with subcortical infarcts and
leucoencephalopathy
(CADASIL) syndrome. The disorder is characterized by relapsing strokes with
neuropsychiatric
3o symptoms and affects relatively young adults of both sexes. CT scans have
demonstrated
occlusive cerebrovascular infarcts in the white matter, which was usually
reduced. Subjects with
CADASIL syndrome can also benefit from increased ecNOS expression and/or
activity.
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Another important embodiment of the invention, is the treatment of subjects
with a
neurodegenerative disease. The term "neurodegenerative disease" is meant to
include any
pathological state involving neuronal degeneration, including Parkinson's
Disease, Huntington's
Disease, Alzheimer's Disease, and amyotrophic lateral sclerosis (ALS). In
preferred
embodiments, the neurodegenerative disease is Alzheimer's Disease. Alzheimer's
Disease is a
progressive, neurodegenerative disease characterised by loss of function and
death of nerve cells
in several areas of the brain leading to loss of cognitive function such as
memory and language.
The cause of nerve cell death is unknown but the cells are recognised by the
appearance of
unusual helical protein filaments in the nerve cells (neurofibrillary tangles)
and by degeneration
1o in cortical regions of brain, especially frontal and temporal lobes.
Increase of cerebral blood flow
mediated by an increase in ecNOS expression and/or activity can also be of
benefit to subjects
suffering from a neurodegenerative disease.
According to another aspect of the invention, a method of screening for
identifying an
inhibitor of HMG-CoA reductase inhibitor for treatment of subjects who would
benefit from
increased endothelial cell Nitric Oxide Synthase activity in a tissue, is
provided. The method
involves identifying an inhibitor of HMG-CoA reductase suspected of increasing
endothelial cell
Nitric Oxide Synthase activity, and determining whether or not the inhibitor
of HMG-CoA
reductase produces an increase in endothelial cell Nitric Oxide Synthase
activity in vivo or in
vitro. HMG-CoA reductase inhibitors according to this invention can be
identified by confirming
2o that the inhibitor produces increased ecNOS activity in a model system
compared to a control,
using any of the model systems described herein, and also inhibits at least
one other HMG-CoA
reductase dependent function as determined in any of the model systems
described herein and/or
other model systems known in the art.
The invention also involves the co-administration of agents that are not HMG-
CoA
reductase inhibitors but that can act cooperatively, additively or
synergistically with such
HMG-CoA reductase inhibitors to increase ecNOS activity. Thus, ecNOS
substrates which are
converted by ecNOS to nitric oxide and cofactors enhancing such conversion,
can be
co-administered with the HMG-CoA reductase inhibitors according to the
invention. Such
ecNOS substrates (e.g. L-arginine) and cofactors (e.g., NADPH,
tetrahydrobiopterin, etc.) may
be natural or synthetic, although the preferred substrate is L-arginine.
Likewise, there are other agents besides HMG-CoA reductase inhibitors that are
not
substrates of ecNOS and that can increase ecNOS activity. Agents belonging to
these categories
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are therefore nonHMG-CoA reductase inhibitors and can be used in co-
administrations with rho
GTPase function inhibitors in cocktails. Examples of categories of such agents
are estrogens and
ACE inhibitors. Estrogens are a well defined category of molecules known by
those of ordinary
skill in the art, and will not be elaborated upon further herein. All share a
high degree of
structural similarity. ACE inhibitors also have been well characterized,
although they do not
always share structural homology.
Angiotensin converting enzyme, or ACE, is an enzyme which catalyzes the
conversion
of angiotensin I to angiotensin II. ACE inhibitors include amino acids and
derivatives thereof,
peptides, including di and tri peptides and antibodies to ACE which intervene
in the
1o renin-angiotensin system by inhibiting the activity of ACE thereby reducing
or eliminating the
formation of presser substance angiotensin II. ACE inhibitors have been used
medically to treat
hypertension, congestive heart failure, myocardial infarction and renal
disease. Classes of
compounds known to be useful as ACE inhibitors include acylmercapto and
mercaptoalkanoyl
prolines such as captopril (US Patent Number 4,105,776) and zofenopril (US
Patent Number
I5 4,316,906), carboxyalkyl dipeptides such as enalapril (US Patent Number
4,374,829), lisinopril
(US Patent Number 4,374,829), quinapril (US Patent Number 4,344,949), ramipril
(US Patent
Number 4,587,258), and perindopril (US Patent Number 4,508,729), carboxyalkyl
dipeptide
mimics such as cilazapril (US Patent Number 4,512,924) and benazapril (US
Patent Number
4,410,520), phosphinylalkanoyl prolines such as fosinopril (US Patent Number
4,337,201 ) and
20 trandolopril.
This invention also contemplates co-administration of agents that increase the
production
of NO by ecNOS without affecting ecNOS expression, as do ACE inhibitors or
administration
of ecNOS substrate and/or ecNOS cofactors. Estrogens upregulate Nitric Oxide
Synthase
expression whereas ACE inhibitors do not affect expression, but instead
influence the efficiency
25 of the action of Nitric Oxide Synthase on L-arginine. Thus, activity can be
increased in a variety
of ways. In general, activity is increased by the reductase inhibitors of the
invention by
increasing the amount of the active enzyme present in a cell versus the amount
present in a cell
absent treatment with the reductase inhibitors according to the invention.
The reductase inhibitors are administered in effective amounts. In general, an
effective
3o amount is any amount that can cause an increase in Nitric Oxide Synthase
activity in a desired
tissue, and preferably in an amount sufficient to cause a favorable phenotypic
change in the
condition such as a lessening, alleviation or elimination of a symptom or of a
condition.
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In general, an effective amount is that amount of a pharmaceutical preparation
that alone,
or together with further doses, produces the desired response. This may
involve only slowing the
progression of the disease temporarily, although more preferably, it involves
halting the
progression of the disease permanently or delaying the onset of or preventing
the disease or
condition from occurring. This can be monitored by routine methods. Generally,
doses of active
compounds would be from about 0.01 mg/kg per day to 1000 mglkg per day. It is
expected that
doses ranging from 50-500 mg/kg will be suitable, preferably orally and in one
or several
administrations per day.
Such amounts will depend, of course, on the particular condition being
treated, the
to severity of the condition, the individual patient parameters including age,
physical condition, size
and weight, the duration of the treatment, the nature of concurrent therapy
(if any), the specific
route of administration and like factors within the knowledge and expertise of
the health
practitioner. Lower doses will result from certain forms of administration,
such as intravenous
administration. In the event that a response in a subject is insufficient at
the initial doses applied,
higher doses (or effectively higher doses by a different, more localized
delivery route) may be
employed to the extent that patient tolerance permits. Multiple doses per day
are contemplated
to achieve appropriate systemic levels of compounds. It is preferred generally
that a maximum
dose be used, that is, the highest safe dose according to sound medical
judgment. It will be
understood by those of ordinary skill in the art, however, that a patient may
insist upon a lower
2o dose or tolerable dose for medical reasons, psychological reasons or for
virtually any other
reasons.
The reductase inhibitors useful according to the invention may be combined,
optionally,
with a pharmaceutically-acceptable carrier. The term "pharmaceutically-
acceptable carrier" as
used herein means one or more compatible solid or liquid fillers, diluents or
encapsulating
substances which are suitable for administration into a human. The term
"carrier" denotes an
organic or inorganic ingredient, natural or synthetic, with which the active
ingredient is combined
to facilitate the application. The components of the pharmaceutical
compositions also are capable
of being co-mingled with the molecules of the present invention, and with each
other, in a manner
such that there is no interaction which would substantially impair the desired
pharmaceutical
3o eff cacy.
The pharmaceutical compositions may contain suitable buffering agents,
including: acetic
acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric
acid in a salt.
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The pharmaceutical compositions also may contain, optionally, suitable
preservatives,
such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
A variety of administration routes are available. The particular mode selected
will
depend, of course, upon the particular drug selected, the severity of the
condition being treated
s and the dosage required for therapeutic efficacy. The methods of the
invention, generally
speaking, may be practiced using any mode of administration that is medically
acceptable,
meaning any mode that produces effective levels of the active compounds
without causing
clinically unacceptable adverse effects. Such modes of administration include
oral, rectal, topical,
nasal, interdermal, or parenteral routes. The term "parenteral" includes
subcutaneous,
1o intravenous, intramuscular, or infusion. Intravenous or intramuscular
routes are not particularly
suitable for long-term therapy and prophylaxis.
The pharmaceutical compositions may conveniently be presented in unit dosage
form and
may be prepared by any of the methods well-known in the art of pharmacy. All
methods include
the step of bringing the active agent into association with a carrier which
constitutes one or more
IS accessory ingredients. In general, the compositions are prepared by
uniformly and intimately
bringing the active compound into association with a liquid carrier, a finely
divided solid carrier,
or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete
units, such as
capsules, tablets, lozenges, each containing a predetermined amount of the
active compound.
20 Other compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a
syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of reductase inhibitors, which is preferably isotonic with
the blood of the
recipient. This aqueous preparation may be formulated according to known
methods using
25 suitable dispersing or wetting agents and suspending agents. The sterile
injectable preparation
also may be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable
diluent or solvent, for example, as a solution in 1,3-butane diol. Among the
acceptable vehicles
and solvents that may be employed are water, Ringer's solution, and isotonic
sodium chloride
solution. In addition, sterile, fixed oils are conventionally employed as a
solvent or suspending
3o medium. For this purpose any bland fixed oil may be employed including
synthetic mono-or di-
glycerides. In addition, fatty acids such as oleic acid may be used in the
preparation of
injectables. Carrier formulation suitable for oral, subcutaneous, intravenous,
intramuscular, etc.
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administrations can be found in Remington's Pharmaceutical Sciences, Mack
Publishing Co.,
Easton, PA.
Other delivery systems can include time-release, delayed release or sustained
release
delivery systems. Such systems can avoid repeated administrations of the
active compound,
increasing convenience to the subject and the physician. Many types of release
delivery systems
are available and known to those of ordinary skill in the art. They include
polymer base systems
such as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of
the foregoing
polymers containing drugs are described in, for example, U.S. Patent
5,075,109. Delivery
Io systems also include non-polymer systems that are: lipids including sterols
such as cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-
glycerides; hydrogel
release systems; sylastic systems; peptide based systems; wax coatings;
compressed tablets using
conventional binders and excipients; partially fused implants; and the like.
Specific examples
include, but are not limited to: (a) erosional systems in which the active
compound is contained
IS in a form within a matrix such as those described in U.S. Patent Nos.
4,452,775, 4,675,189 and
5,736,152, and (b) diffusional systems in which an active component permeates
at a controlled
rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974
and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some of which
are adapted for
implantation.
2o Use of a long-term sustained release implant may be desirable. Long-term
release, are
used herein, means that the implant is constructed and arranged to delivery
therapeutic levels of
the active ingredient for at least 30 days, and preferably 60 days. Long-term
sustained release
implants are well-known to those of ordinary skill in the art and include some
of the release
systems described above.
25 According to another aspect of the invention, a method for increasing blood
flow in a
tissue of a subject is provided. The method involves administering to a
subject in need of such
treatment a HMG-CoA reductase inhibitor in an amount effective to increase
endothelial cell
Nitric Oxide Synthase activity in the tissue of the subject.
In important embodiments a second agent is co-administered to a subject with a
condition
3o treatable by the second agent in an amount effective to treat the
condition, whereby the delivery
of the second agent to a tissue of the subject is enhanced as a result of the
increased blood flow
from administering the first agent of the invention (a HMG CoA reductase
inhibitor). The
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"second agent" may be any pharmacological compound or diagnostic agent, as
desired. Preferred
second agents are agents having a site of action in the brain. Such agents
include analeptic,
analgetic, anesthetic, adrenergic agent, anti-adrenergic agent, amino acids,
antagonists, antidote,
anti-anxiety agent, anticholinergic, anticolvunsant, antidepressant, anti-
emetic, anti-epileptic,
antihypertensive, antifibrinolytic, antihyperlipidemia, antimigraine,
antinauseant, antineoplastic
(brain cancer), antiobessional agent, antiparkinsonian, antipsychotic,
appetite suppressant, blood
glucose regulator, cognition adjuvant, cognition enhancer, dopaminenergic
agent, emetic, free
oxygen radical scavenger, glucocorticoid, hypocholesterolemic, holylipidemic,
histamine H2
receptor antagonists, immunosuppressant, inhibitor, memory adjuvant, mental
performance
JO enhancer, mood regulator, mydriatic, neuromuscular blocking agent,
neuroprotective, NMDA
antagonist, post-stroke and post-head trauma treatment, psychotropic,
sedative,
sedative-hypnotic, serotonin inhibitor, tranquilizer, and treatment of
cerebral ischemia, calcium
channel blockers, free radical scavengers - antioxidants, GABA agonists,
glutamate antagonists,
AMPA antagonists, kainate antagonists, competitive and non-competitive NMDA
antagonists,
/5 growth factors, opioid antagonists, phosphatidylcholine precursors,
serotonin agonists, sodium-
and calcium-channel blockers, and potassium channel openers.
In addition to the foregoing brain-specific categories of agents, examples of
categories of
other pharmaceutical agents that can be used as second agents include:
adrenergic agent;
adrenocortical steroid; adrenocortical suppressant; alcohol deterrent;
aldosterone antagonist;
20 amino acid; ammonia detoxicant; anabolic; analeptic; analgesic; androgen;
anesthesia, adjunct
to; anesthetic; anorectic; antagonist; anterior pituitary suppressant;
anthelmintic; anti-acne
agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen; anti-
anemic; anti-anginal;
anti-anxiety; anti-arthritic; anti-asthmatic; anti-atherosclerotic;
antibacterial; anticholelithic;
anticholelithogenic; anticholinergic; anticoagulant; anticoccidal;
anticonvulsant;
25 antidepressant; antidiabetic; antidiarrheal; antidiuretic; antidote; anti-
emetic; anti-epileptic;
anti-estrogen; antifibrinolytic; antifungal; antiglaucoma agent;
antihemophilic;
antihemorrhagic; antihistamine; antihyperlipidemia; antihyperlipoproteinemic;
antihypertensive; anti-infective; anti-infective, topical; anti-inflammatory;
antikeratinizing agent;
antimalarial; antimicrobial; antimigraine; antimitotic; antimycotic,
antinauseant, antineoplastic,
30 antineutropenic, antiobessional agent; antiparasitic; antiparkinsonian;
antiperistaltic,
antipneumocystic; antiproliferative; antiprostatic hypertrophy; antiprotozoal;
antipruritic;
antipsychotic; antirheumatic; antischistosomal; antiseborrheic; antisecretory;
antispasmodic;
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antithrombotic; antitussive; anti-ulcerative; anti-urolithic; antiviral;
appetite suppressant; benign
prostatic hyperplasia therapy agent; blood glucose regulator; bone resorption
inhibitor;
bronchodilator; carbonic anhydrase inhibitor; cardiac depressant;
cardioprotectant; cardiotonic;
cardiovascular agent; choleretic; cholinergic; cholinergic agonist;
cholinesterase deactivator;
coccidiostat; cognition adjuvant; cognition enhancer; depressant; diagnostic
aid; diuretic;
dopaminergic agent; ectoparasiticide; emetic; enzyme inhibitor; estrogen;
fibrinolytic; fluorescent
agent; free oxygen radical scavenger; gastrointestinal motility effector;
glucocorticoid; gonad-
stimulating principle; hair growth stimulant; hemostatic; histamine H2
receptor antagonists;
hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;
imaging agent;
immunizing agent; immunomodulator; immunoregulator; immunostimulant;
immunosuppressant;
impotence therapy adjunct; inhibitor; keratolytic; LNRH agonist; liver
disorder treatment;
luteolysin; memory adjuvant; mental performance enhancer; mood regulator;
mucolytic; mucosal
protective agent; mydriatic; nasal decongestant; neuromuscular blocking agent;
neuroprotective;
NMDA antagonist; non-hormonal sterol derivative; oxytocic; plasminogen
activator; platelet
activating factor antagonist; platelet aggregation inhibitor; post-stroke and
post-head trauma
treatment; potentiator; progestin; prostaglandin; prostate growth inhibitor;
prothyrotropin;
psychotropic; pulmonary surface; radioactive agent; regulator; relaxant;
repartitioning agent;
scabicide; sclerosing agent; sedative; sedative-hypnotic; selective adenosine
Al antagonist;
serotonin antagonist; serotonin inhibitor; serotonin receptor antagonist;
steroid; stimulant;
2o suppressant; symptomatic multiple sclerosis; synergist; thyroid hormone;
thyroid inhibitor;
thyromimetic; tranquilizer; treatment of amyotrophic lateral sclerosis;
treatment of cerebral
ischemia; treatment of Paget's disease; treatment of unstable angina;
uricosuric; vasoconstrictor;
vasodilator; vulnerary; wound healing agent; xanthine oxidase inhibitor.
In another aspect of the invention, the reductase inhibitor is "co-
administered," which
means administered substantially simultaneously with another agent. By
substantially
simultaneously, it is meant that the reductase inhibitor is administered to
the subject close enough
in time with the administration of the other agent (e.g., a nonHMG-CoA
reductase inhibitor agent,
a "second agent", etc.), whereby the two compounds may exert an additive or
even synergistic
effect, i.e. on increasing ecNOS activity or on delivering a second agent to a
tissue via increased
3o blood flow.
Examples
"Upregulation of endothelial cell Nitric Oxide Synthase by HMG CoA Reductase
Inhibitors "
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Experimental Procedures
All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS).
Unless
indicated otherwise, all reagents were purchased from Sigma Chemical Co. (St.
Louis, MO).
[a-32P]CTP (3000 Ci/mmol) was supplied by New England Nuclear. Purified human
LDL was
s obtained from Calbiochem (San Diego, CA; lot#730793) and Biomedical
Technologies Inc.
(Stoughton, MA; lot#9030197). The level of endotoxin was determined by the
chromogenic
Limulus amebocyte assay (BioWhittaker Ine., Walkersville, MD). The antibody
detection kit
(Enhanced Chemiluminescence) and the nylon nucleic acid (Hybond) and protein
(PVDF)
transfer membranes were purchased from Amersham Corp. (Arlington Heights, IL).
Simvastatin
IO and lovastatin were obtained from Merck, Sharp, and Dohme, Inc. (West
Point, PA). Since
endothelial cells lack lactonases to process simvastatin and lovastatin to
their active forms, these
HMG-CoA reductase inhibitors were chemically activated prior to their use as
previously
described (Laufs, U et al., J Biol Chem, 1997, 272:31725-31729).
/5 Cell Culture:
Human endothelial cells were harvested from saphenous veins and cultured as
described
(15). For transfection studies, bovine aortic endothelial cells of less than 3
passages were cultured
in a growth medium containing DMEM (Dulbecco's Modified Eagle's Medium), 5
mmol/L
L-glutamine (Gibco), and 10% fetal calf serum (Hyclone Lot#1114577). For all
experiments, the
2o endothelial cells were placed in 10% lipoprotein-deficient serum (Sigma,
Lot#26H94031 ) for 48
h prior to treatment conditions. In the indicated experiments, endothelial
cells were pretreated
with actinomycin D (5 mg/ml) for 1 h prior to treatment with ox-LDL and/or
simvastatin. Cellular
viability as determined by cell count, morphology, and Trypan blue exclusion
was maintained
for all treatment conditions.
Preparation of LDL:
The LDL was prepared by discontinuous ultracentrifugation according to the
method of
Chung et al. with some modification (Methods Enzymol, 1984, 128:181-209).
Fresh plasma from
a single donor was anticoagulated with heparin and filtered through a Sephadex
G-25 column
3o equilibrated with PBS. The density was adjusted to 1.21 g/ml by addition of
KBr (0.3265 g/ml
plasma). A discontinuous NaCI/KBr gradient was established in Beckman Quick-
Seal centrifuge
tubes (5.0 nil capacity) by layering 1.5 ml of density-adjusted plasma under
3.5 ml of 0.154 M
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NaCI in Chelex-100-treated water (BioRad, Hercules, CA). After
ultracentrifugation at 443,000
x g and 7°C for 45 min in a Beckman Near Vertical Tube 90 rotor
(Beckman L8-80M
ultracentrifuge), the yellow band in the upper middle of the tube
corresponding to LDL was
removed by puncturing with a needle and withdrawing into a syringe. The KBr
was rcmovcd
from the LDL by dialyzing with three changes of sterile PBS, pH 7.4,
containing 100 :g/ml
polymyxin B.
The purity of the LDL samples was confirmed by SDS/polyacrylamide and
cellulose
acetate gel electrophoresis. Cholesterol and triglyceride content were
determined as previously
described (Liao, JK et al., J Biol Chem, 1995, 270:319-324.). The LDL protein
concentration
1o was determined by the method of Lowry et al., (J Biol Chem, 1951, 193:265-
275.). For
comparison, commercially-available LDL (Biomedical Technologies Inc.,
Stoughton, MA;
Calbiochem, San Diego, CA) were characterized and used in selected
experiments.
Oxidation of LDL:
Oxidized LDL was prepared by exposing freshly-isolated LDL to CuS04 (5-10 mM)
at
37°C for various duration (6-24 h). The reaction was stopped by
dialyzing with three changes of
sterile buffer (150 :mol/L NaCI, 0.01% EDTA and 100 :g/ml polymyxin B, pH 7.4)
at 4°C. The
degree of LDL oxidation was estimated by measuring the amounts of
thiobarbituric acid reactive
substances (TBARS) produced using a fluorescent assay for malondialdehyde as
previously
described (Yagi, KA, Biochem Med, 1976, 15:212-21.). The extent of LDL
modification was
expressed as nanomoles of malondialdehyde per mg of LDL protein. Only mild to
moderate
ox-LDL with TBARS values between 12 and 16 nmol/mg LDL protein (i.e. 3 to 4
nmol/mg LDL
cholesterol) were used in this study. All oxidatively-modified LDL samples
were used within 24
h of preparation.
Northern Blotting:
Equal amounts of total RNA (10-20 mg) were separated by 1.2% formaldehyde-
agarose
gel electrophoresis and transferred overnight onto Hybond nylon membranes.
Radiolabeling of
human full-length ecNOS cDNA (Verbeuren, TJ et al., Circ Res, 1986, 58:552-
564, Liao, JK et
al., J Clin Invest, 1995, 96:2661-2666) was performed using random hexamer
priming,
[a-3zP]CTP, and Klenow (Pharmacia). The membranes were hybridized with the
probes overnight
at 45°C in a solution containing SO% formamide, 5 X SSC, 2.5 X
Denhardt's Solution, 25 mM
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sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA.
All Northern
blots were subjected to stringent washing conditions (0.2 X SSC/0.1% SDS at
65°C) prior to
autoradiography. RNA loading was determined by rehybridization with human
GAPDH probe.
Western Blottine:
Cellular proteins were prepared and separated on SDS/PAGE as described (Liao,
JK et
al., J Biol Chem, 1995, 270:319-324.). Immunoblotting was performed using a
marine
monoclonal antibody to human ecNOS (1:400 dilution, Transduction Laboratories,
Lexington,
KY). Immunodetection was accomplished using a sheep anti-mouse secondary
antibody (1:4000
dilution) and the enhanced chemiluminescence (ECL) kit (Amersham Corp.,
Arlington i-leights,
IL). Autoradiography was performed at 23°C and the appropriate
exposures were quantitated by
densitometry.
Assa~for ecNOS Activity:
IS The ecNOS activity was determined by a modified nitrite assay as previously
described
(Misko, TP et al., Analytical Biochemistry, 1993, 214:11-16, Liao, JK et al.,
J Clin Invest, 1995,
96:2661-2666). Briefly, endothelial cells were treated for 24 h with ox-LDL in
the presence and
absence of simvastatin (0.1 to 1 mM). After treatment, the medium was removed,
and the cells
were washed and incubated for 24 h in phenol red-free medium. After 24 h, 300
:l of conditioned
medium was mixed with 30 :1 of freshly prepared 2,3-diaminonaphthalene (1.5
mmol/L DAN in
1 mol/L HCl). The mixture was protected from light and incubated at
20°C for 10 min. The
reaction was terminated with 15 :1 of 2.8 mol/L NaOH. Fluorescence of 1-(H)-
naphthotriazole
was measured with excitation and emission wavelengths of 365 and 450 nm,
respectively.
Standard curves were constructed with known amounts of sodium nitrite.
Nonspecific
fluorescence was determined in the presence of LNMA (5 mmol/L).
Nuclear Run-on Assav:
Confluent endothelial cells (~5 x 10' cells) grown in LPDS were treated with
simvastatin
(1 mM) or 95%Oz for 24 h. Nuclei were isolated and in vitro transcription was
performed as
previously described ( Liao, JK et al., JClin Invest, 1995, 96:2661-2666).
Equal amounts (1 mg)
of purified, denatured full-length human ecNOS, human ~3-tubulin (ATCC
#37855), and
linearized pGEM-3z cDNA were vacuum-transferred onto nitrocellulose membranes
using a slot
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blot apparatus (Schleicher & Schuell). Hybridization of radiolabeled mRNA
transcripts to the
nitrocellulose membranes was carried out at 45°C for 48 h in a buffer
containing 50% formamide,
X SSC, 2.5 X Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1%
SDS, and
250 mg/ml salmon sperm DNA. The membranes were then washed with 1 x SSC/0.1%
SDS for
5 1 h at 65°C prior to autoradiography for 72 h at -80°C.
Transfection Assavs:
For transient transfections, bovine rather than human endothelial cells were
used because
of their higher transfection efficiency by the calcium-phosphate precipitation
method (12% vs
<4%) (Graham, FL and Van der Erb, AJ, Virology, 1973, 52:456-457). We used the
human
ecNOS promoter construct, FI.LUC, which contains a -1.6 kb 5'-upstream
sequence linked to
the luciferase reporter gene as described by Zhang et al. (JBiol Chem, 1995,
270:15320-15326).
Bovine endothelial cells (60%-70% confluent) were transfected with 30 mg of
the indicated
constructs: p.LUC (no promoter), pSV2.LUC (SV40 early promoter), or F1.LUC. As
an internal
I5 control for transfection efficiency, pCMV.bGal plasmid ( 10 mg) was co-
transfected in all
experiments. Preliminary results using b-galactosidase staining indicate that
cellular transfection
efficiency was approximately 10% to 14%.
Endothelial cells were placed in lipoprotein-deficient serum for 48 h after
transfection and
treated with ox-LDL (50 mg/ml, TBARS 12.4 nmol/mg) in the presence and absence
of
simvastatin (1 mM) for an additional 24 h. The luciferase and b-galactosidase
activities were
determined by a chemiluminescence assay (Dual-Light, Tropix, Bedford, MA)
using a Berthold
L9501 luminometer. The relative promoter activity was calculated as the ratio
of luciferase-to
(3-galactosidase activity. Each experiment was performed three times in
triplicate.
Data Anal,:
Band intensities were analyzed densitometrically by the National Institutes of
Health
Image program (Rasband, W, NIH Image program, v 1.49, National Institutes of
Health,
Bethesda, 1993). All values are expressed as mean ~ SEM compared to controls
and among
separate experiments. Paired and unpaired Student's t tests were employed to
determine any
significant changes in densitometric values, nitrite production, and promoter
activities. A
significant difference was taken for P values less than 0.05.
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Example 1: Cell Culture
Relatively pure (>95%) human endothelial cell cultures were confirmed by their
morphological features (i.e. cuboidal, cobble-stone, contact inhibited) using
phase-contrast
microscopy and by immunofluorescent staining with anti-Factor VIII antibodies
(Gerson, RJ et
al., Am JMed,1989, 87:28-38). For all experimental conditions, there were no
observable adverse
effects of ox-LDL or HMG-CoA reductase inhibitors on cellular morphology, cell
number,
immunofluorescent staining, and Trypan blue exclusion (>95%). Higher
concentrations of
ox-LDL (>100 mg/ml) with greater oxidative modification (i.e. TBARS values of
>30 nmol/mg)
caused vacuolization and some detachment of endothelial cells after 24 h.
Neither simvastatin
(0.01 to 0.1 mmol/L) nor lovastatin (10 mmol/L) produced any noticeable
adverse effects on
human endothelial cell for up to 96 h. However, higher concentrations of
simvastatin (> 15
mmol/L) or lovastatin (>50 mmol/L) caused cytotoxicity after 36 h, and
therefore, were not used.
Example 2: Characterization of LDL
I5 SDS/polyacrylamide gel electrophoresis of native or unmodified LDL revealed
a single
band 0510 kD) corresponding to ApoB-100 (data not shown). Similarly, cellulose
acetate
electrophoresis revealed only one band corresponding to the presence of a
single class of
low-density lipids (density of 1.02 to 1.06 g/ml). The LDL had a protein,
cholesterol, and
triglyceride concentration of 6.3 ~ 0.2, 2.5 ~ 0.1, and 0.5 ~ 0.1 mg/ml,
respectively. In contrast,
2o lipoprotein-deficient serum was devoid of both apoB-100 protein and low-
density lipid bands,
and had non-detectable levels of cholesterol. There was no detectable level of
endotoxin (<0.10
EU/ml) in the lipoprotein-deficient serum or ox-LDL samples by the chromogenic
Limulus
amebocyte assay.
In addition, there was no apparent difference between our own preparation and
25 commercially-obtained LDL samples in terms of electrophoretic mobility.
Native LDL had a
TBARS value of 0.3 ~ 0.2 nmol/mg, but after exposure to human saphenous vein
endothelial cells
in lipoprotein-deficient media for 72 h, this value increased to 3.1 ~ 0.4
nmol/mg.
Copper-oxidized LDL had TBARS values ranging from 4.6 ~ 0.5 to 33.1 ~ 5.2
nmol/mg. The
degree of ox-LDL used in this study was mild to moderate with TBARS value
ranging from 12
3o to 16 nmol/mg LDL protein (i.e. 3 to 4 nmol/mg LDL cholesterol).
Example 3: Effect of ox-I~DI. and 1IMG-CoA Rcductasc Inhilaitors on ccN()S
1'rotcin
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We have previously shown that ox-LDL (50 mg/ml) downregulates ecNOS expression
(Liao, JK et al., JBiol Chem, 1995, 270:319-324). Compared to untreated cells,
treatment with
ox-LDL (50 mg/ml, TBARS 12.2 nmol/mg) caused a 54% ~ 6% decrease in ecNOS
protein after
48 h (p < 0.01, n=4) (Western blots, 40 mg protein/lane, Figure lA). There was
no difference
between our preparation of ox-LDL and commercially-available ox-LDL with
similar TBARS
values in terms of the degree of ecNOS downregulation. Addition of simvastatin
(0.01 mmol/L)
did not significantly affect the downregulation of ecNOS protein by ox-LDL
(57% ~ 8%
decrease, p > 0.05, n=4). However, in the presence of 0.1 mmol/L of
simvastatin, ox-LDL no
longer produce any significant decrease in ecNOS protein levels (4% ~ 7%
decrease, p < 0.01,
1o n=4). Higher concentrations of simvastatin (1 and 10 mmol/L) resulted in
not only a reversal of
ecNOS downregulation by ox-LDL, but also significant increases in ecNOS
protein levels above
baseline (146% ~ 9% and 210% ~ 12%, respectively, p < 0.05, n=4). Simvastatin
or lovastatin
(10 mmol/L) which were not chemically-activated had no effect on ecNOS
expression.
In a time-dependent manner, treatment with ox-LDL (50 mg/ml, TBARS 12.2
nmol/mg)
I5 decreased ecNOS protein expression by 34% ( 5%, 67% ( 8% and 86 ( 5% after
24 h, 72 h, and
96 h, respectively (p < 0.05 for all values, n=4,) (Figure 1B). Compared to ox-
LDL alone,
co-treatment with simvastatin (O.I mmol/L) attenuated the decrease in ecNOS
protein level after
24 h (15% ( 2% vs 34% ( 5%, p < 0.05, n=4). Longer incubation with simvastatin
(0.1 mmol/L)
for 72 h and 96 h not only reversed ox-LDL's inhibitory effects on ecNOS
expression, but also
20 increased ecNOS protein levels by 110% ( 6% and 124% ( 6% above basal
expression (p < 0.05,
n=4). Thus, compared to ox-LDL alone, co-treatment with simvastatin produced a
1.3-, 3.3-and
8.9-fold increase ecNOS protein levels after 24 h, 72 h, and 96 h,
respectively. These blots are
representative of four separate experiments.
25 Example 4: Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOS mRNA
The effect of simvastatin on ecNOS mRNA levels occurred in a time-dependent
manner
and correlated with its effect on ecNOS protein levels. Northern blot analyses
(Northern blots,
20 mg total RNA/lane, Figure 2A) showed that ox-LDL (50 mg/ml, TBARS 15.1
nmol/mg)
produced a time-dependent 65 t 5% and 91 t 4% decrease in ecNOS mRNA levels
after 48 h and
3o 72 h, respectively (p < 0.01, n=3). Compared to ox-LDL at the indicated
time points,
co-treatment with simvastatin 0.1 mmol/L) increased ecNOS mRNA levels by 6.3-
fold after 48
h and 14.5-fold after 72 h (p < 0.01 for all values, n=3).
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To determine whether treatment with another HMG-CoA reductase inhibitor have
similar
effect as simvastatin, we treated endothelial cells with lovastatin. Again, ox-
LDL decreased
steady-state ecNOS mRNA by 52 ~ 5% after 24 h (p < 0.01, n=3) (Figure 2B).
Treatment with
lovastatin (10 mmol/L) not only reversed the inhibitory effects of ox-LDL on
ecNOS mRNA, but
also caused a 40 ~ 9% increase in ecNOS mRNA level compared to that of
untreated cells.
Compared to ox-LDL alone, co-treatment with lovastatin caused a 3.6-fold
increase in ecNOS
mRNA levels after 24 h. Treatment with lovastatin alone, however, produced 36%
increase in
ecNOS mRNA levels compared to untreated cells (p < 0.05, n=3). Each experiment
was
performed three times with comparable results. The corresponding ethidium
bromide-stained 28S
/o band intensities were used to standardize loading conditions.
Example 5: Effect of ox-LDL and Simvastatin on ecNOS Activity
The activity of ecNOS was assessed by measuring the LLAMA-inhibitable nitrite
production from human endothelial cells (Liao, JK et al., J C'lin Invest,
1995, 96:2661-2666).
/s Basal ecNOS activity was 8.8 ~ 1.4 nmol/500,000 cells/24 h. Treatment with
ox-LDL (50
mg/ml, TBARS 16 nmol/mg) for 48 h decreased ecNOS-dependent nitrite production
by 94 ~ 3%
(0.6 t 0.5 nmol/500,000 cells/24 h, p < 0.001). Co-treatment with simvastatin
(0.1 mmol/L)
significantly attenuated this downregulation resulting in a 28 ~ 3% decrease
in ecNOS activity
compared to untreated cells (6.4 ~ 0.3 nmol/500,000 cells/24 h, p < 0.05). Co-
treatment with a
2o higher concentration of simvastatin (1 mmol/L) not only completely reversed
the downregulation
of ecNOS by ox-LDL, but also, resulted in a 45 ~ 6% increase in ecNOS activity
compared to
baseline (12.8 ~ 2.7 nmol/500,000 cells/24 h, p < 0.05). Experiments were
performed three times
in duplicate.
25 Example 6: Effect of Simvastatin on ecNOS mRNA Stability
The post-transcriptional regulation of ecNOS mRNA was determined in the
presence of
the transcriptional inhibitor, actinomycin D (5 mg/ml). Oxidized LDL (50
mg/ml, TBARS 13.1
nmol/mg) shortened the half life of ecNOS mRNA (tl/2 35 ~ 3 h to 14 ~ 2 h, p <
0.05, n=3).
Co-treatment with simvastatin (0.1 mmol/L) prolonged the half life of ecNOS
mRNA by 1.6-fold
30 (tl/2 22 ~ 3 h, p < 0.05, n=3). Treatment with simvastatin alone prolonged
ecNOS mRNA
half life by 1.3-fold over baseline (tl/2 43 ~ 4 h, p < 0.05, n=3). Band
intensities of ecNOS
mRNA (relative intensity) were plotted as a semi-log function of time (h). The
data points
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obtained represented mean t SEM of three separate experiments.
Example 7: Effect of Simvastatin on ecNOS Gene Transcription
To determine whether the effects of simvastatin on ecNOS expression occurs at
the level
of eeNOS gene transcription, we performed nuclear run-on assays using
endothelial cells treated
with simvastatin ( 1 mmol/L) for 24 h. Preliminary studies using different
amounts of
radiolabelled RNA transcripts demonstrate that under our experimental
conditions, hybridization
was linear and nonsaturable. The density of each ecNOS band was standardized
to the density
of its corresponding ~i-tubulin. The specificity of each band was determined
by the lack of
Io hybridization to the nonspecific pGEM cDNA vector. In untreated endothelial
cells (control),
there was constitutive ecNOS transcriptional activity (relative index of 1.0).
Treatment with
simvastatin (1 mmol/L) did not significantly affect ecNOS gene transcription
compared to that
of untreated cells (relative index of 1.2 ~ 0.3, p > 0.05, n=4). However,
treatment of endothelial
cells with hyperoxia (95% OZ) significantly increased ecNOS gene expression
(relative index of
IS 2.5, p < 0.05, n=4). The blots shown are representative of four separate
experiments.
To further confirm the effects of simvastatin on ecNOS gene transcription by a
different
method, we transfected bovine aortic endothelial cells using a -1600 to +22
nucleotide ecNOS
5'-promoter construct linked to a luciferase reporter gene (F 1.LUC) (Zhang, R
et al., JBiol Chem,
1995, 270:15320-15326). This promoter construct contains putative cis-acting
elements for
2o activator protein (AP)-1 and -2, sterol regulatory element-1,
retinoblastoma control element, shear
stress response element (SSRE), nuclear factor-1 (NF-1), and cAMP response
element (CRE).
Treatment with ox-LDL (SO mg/ml, TBARS 14.5 nmol/mg), simvastatin (1 mol/L),
alone or in
combination, did not significantly affect basal F 1 promoter activity.
However, laminar fluid
shear-stress (12 dynes/cm2 for 24 h) was able to induce F1 promoter activity
by 16-fold after 24
25 h (data not shown) indicating that the F 1 promoter construct is
functionally-responsive if
presented with the appropriate stimulus.
Example 8: Effect of Simvastatin and Lovastatin on ecNOS Expression
To further characterize the effects of HMG-CoA reductase inhibitors on the
upregulation
3o ecNOS expression, we treated endothelial cells with simvastatin (0.1
mmol/L) for various
durations (0-84 h). Treatment with simvastatin (0.1 mmol/L) increased ecNOS
protein levels by
4(6%,21(9%,80(8%,90(12%,and95(16%after12h,24h,48h,72 h,and84h,
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respectively (p < 0.05 for all time points after 12 h, n=4). Higher
concentrations of simvastatin
similarly increased ecNOS protein levels, but in significantly less time
compared to lower
concentrations of simvastatin. (Western blots, 40 mg protein/lane).
In a concentration-dependent manner, treatment with simvastatin (0.01 to 10
mmol/L, 48
h) increased ecNOS expression by 1 ( 6%, 80 ( 8%, 190 ( 10% and 310 ( 20%,
respectively (p <
0.05 for concentrations ( 0.1 mmol/L, n=4) (Figure 3A). The upregulation of
ecNOS expression
by simvastatin, therefore, is dependent upon both the concentration and
duration of simvastatin
treatment. For comparison, treatment with lovastatin (0.1 to 10 mmol/L, 48 h)
also increased
ecNOS expression in a concentration-dependant manner (10 ( 6%, 105 ( 8% and
180 ( 11%,
respectively, p < 0.05 for concentrations > 0.1 mmol/L, n=3) (Figure 3B) but
significantly less
effectively than simvastatin at comparable concentrations. Therefore, at the
same concentration,
simvastatin had greater effects on ecNOS expression compared to lovastatin.
These results are
consistent with reported IC50 values for simvastatin and lovastatin (4 nmol/L
and 19 nmol/L,
respectively) (Van Vliet, AK et al., l3iochem Pharmacol, 1996, 52:1387-1392).
Example 9: Effect of L-Mevalonate on ecNOS Expression
To confirm that the effects of simvastatin on ecNOS expression were due to the
inhibition
of endothelial HMG CoA reductase, endothelial cells were treated with ox-LDL
(50 mg/ml,
TBARS 15.1 nmol/mg), simvastatin (1 mmol/L), alone or in combination, in the
presence of
L-mevalonate (100 mmol/L). Treatment with ox-LDL decreased ecNOS expression by
55% ( 6%
after 48 h which was completely reversed and slightly upregulated in the
presence of simvastatin
(1 mmol/L) (150% ( 8% above basal expression) (p < 0.05 for both, n=3).
Compared to endothelial cells treated with ox-LDL and simvastatin, addition of
L-mevalonate reduced ecNOS protein by 50% ~ 5% (p < 0.05, n=3). Furthermore,
the
upregulation of ecNOS expression by simvastatin alone (2.9-fold increase, p <
0.05, n=3) was
completely reversed by co-treatment with L-mevalonate. Treatment with L-
mevalonate alone did
not have any appreciable effects on basal ecNOS expression (p > 0.05, n=3).
Similar findings
were also observed with L-mevalonate and lovastatin.
"HMG-CoA Reductase Inhibitors Reduce Cerebral Infarct Size by Upregulating
endothelial cell
Nitric Oxide Synlhasc "
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Experimental Procedures
Cell Culture:
Human endothelial cells were harvested from saphenous veins using Type II
collagenase
(Worthington Biochemical Corp., Freehold, NJ) as previously described. Cells
of less than three
s passages were grown to confluence in a culture medium containing Medium 199,
20 mM HEPES,
50 mg/ml ECGS (Collaborative Research Ine., Bedford, MA), 100 mg/ml heparin
sulfate, 5 mM
L-glutamine (Gibco), 5% fetal calf serum (Hyclone, Logan, UT), and antibiotic
mixture of
penicillin ( 100 U/ml)/ streptomycin ( 100 mg/ml)/Fungizone ( 1.25 mg/ml). For
all experiments,
the endothelial cells were grown to confluence before any treatment
conditions. In some
Io experiments, cells were pretreated with actinomycin D (5 mg/ml) for 1 h
prior to treatment with
HMG-CoA reductase inhibitors.
Exposure of Endothelial Cells to Hypoxia:
Confluent endothelial cells grown in 100 mm culture dishes were treated with
HMG-CoA
I5 reductase inhibitors and then placed without culture dish covers in
humidified airtight incubation
chambers (Billups-Rothenberg, Del Mar, CA). The chambers were gassed with 20%
or 3% O2,
5% CO2, and balanced nitrogen for 10 min prior to sealing the chambers. The
chambers were
maintained in a 37°C incubator for various durations (0-48 h) and found
to have less than 2%
variation in OZ concentration as previously described (Liao, JK et al., J Clin
Invest, 1995,
20 96:2661-2666). Cellular confluence and viability were determined by cell
count, morphology, and
trypan blue exclusion.
In vitro Transcription Assav:
Confluent endothelial cells (S x 10' cells were treated with simvastatin ( 1
mM) in the
25 presence of 20% or 3% OZ for 24 h. Nuclei were isolated and in vitro
transcription was performed
as previously described (Liao, JK et al., JClin Invest, 1995, 96:2661-2666).
Equal amounts (1
mg) of purified, denatured full-length human ecNOS, human ~3-tubulin (ATCC
#37855), and
linearized pGEM-3z cDNA were vacuum-transferred onto nitrocellulose membranes
using a slot
blot apparatus (Schleicher & Schuell). Hybridization of radiolabeled mRNA
transcripts to the
3o nitrocellulose membranes was carried out at 45°C for 48 h in a
buffer containing 50% formamide,
X SSC, 2.5 X Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1%
SDS, and
250 mg/ml salmon sperm DNA. The membranes were then washed with 1 x SSC/0.1%
SDS for
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1 h at 65°C prior to autoradiography for 72 h at -80°C. Band
intensities were subjected to
analyses by laser densitometry.
Asst for Nitrite Accumulation:
The amount of NO produced by ecNOS was determined by nitrite accumulation in
the
conditioned medium. Nitrite accumulation was determined by measuring the
conversion of
2,3-diaminonaphthalene (1.5 mM of DAN in 1 M of HCl) and nitrite to 1-(H)-
naphthotriazole as
previously described ( 13,24). Nonspecific fluorescence was determined in the
presence of LNMA
(5 mM). Previous studies with nitrate reductase indicate that the nitrite to
nitrate concentration
/0 in the medium was approximately 5: l and that this ratio did not vary with
exposure to 20% or 3%
OZ concentration.
Murine Model of Cerebral Vascular Ischemia:
Adult male (18-20 g) wildtype SV-129 mice (Taconic farm, Germantown, NY) and
ecNOS mutant mice (Huang, PL et al., Nature, 1995, 377:239-242.) were
subcutaneously
injected with 0.2, 2, or 20 mg of activated simvastatin per kg body weight or
saline (control) once
daily for 14 days. Ischemia was produced by occluding the left middle cerebral
artery (MCA)
with a coated 8.0 nylon monofilament under anesthesia as described (Huang, Z
et al., J Cereb
Blood Flow Metab, 1996, 16:981-987, Huang, Z et al., Science, 1994, 265:1883-
1885, Hara, H
2o et al., J Cereb Blood Flow Metab, 1997, 1:515-526). Arterial blood
pressure, heart rate, arterial
oxygen pressure, and partial pressure of carbon dioxide were monitored as
described (Huang, Z
et al., J Cereb Blood Flow Metab, 1996, 16:981-987, Huang, Z et al., Science,
1994,
265:1883-1885, Hara, H et al., J Cereb Blood Flow Metab, 1997, 1:515-526). The
filaments
were withdrawn after 2 hours and after 24 h, mice were either sacrificed or
tested for neurological
deficits using a well-established, standardized, observer-blinded protocol as
described (Huang,
Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987, Huang, Z et al.,
Science, 1994,
265:1883-1885, Hara, H et al., JCereb Blood Flow Metab, 1997, 1:515-526). The
motor deficit
score range from 0 (no deficit) to 2 (complete deficit).
Brains were divided into five coronal 2-mm sections using a mouse brain matrix
(RBM-200C, Activated Systems, Ann Arbor, MI, USA). Infarction volume was
quantitated with
an image analysis system (M4, St. Catharines, Ontario, Canada) on 2% 2,3,5-
triphenyltetrazolium
chloride stained 2-mm slices. The levels of serum cholesterol, creatinine and
transaminases were
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determined by the Tufts University Veterinary Diagnostic Laboratory (Grafton,
MA).
Assay for ecNOS Activity from Tissues:
The ecNOS activities in mice aortae and brains were measured by the conversion
of
[3H]arginine to [3H]citrulline in the presence and absence of LNMA (5 mM) as
described earlier.
(quantitative Reverse Transcription-Pol~merase Chain Reaction:
Total RNA from mouse aortae and brains was isolated by the guanidinium
isothiocyanate
method and reverse transcribed using oligo-dT (mRNA Preamplification reagents;
Gibco BRL)
and Taq ploymerase (Perkin-Elmer). One tenth of the sDNA was used as template
for the PCR
reaction. Approximately 0.2 nmol of the following primers amplifying a 254-by
fragment of
marine ecNOS cDNA were used: 5'Primer: 5'-GGGCTCCCTCCTTCCGGCTGCCACC-3' (SEQ
ID NO. 1) and 3'Primer: 5'-GGATCCCTGGAAAAGGCGGTGAGG-3' (SEQ ID NO. 2) (Hara,
H et al., J Cereb Blood Flow Metab, 1997, 1:51 S-526). For amplification of
glyceraldehyde 3-
phosphate dehydrogenase (GAPDH), 0.1 nmol of the following primers amplifying
a 452-by
fragment were used: 5'Primer: 5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO. 3) and 3'
Primer: 5'-TCCACCACCCTGTTGCTGTA-3'(SEQ ID NO. 4). Denaturing was performed at
94°C for 30 s, annealing at 60°C for 30 s, and elongation at
72°C for 60 s. Preliminary results
indicated that the linear exponential phase for ecNOS and GAPDH polymerization
was 30-35
cycles and 20-25 cycles, respectively.
Example I0: Cell Culture
Relatively pure (>98%) human saphenous vein endothelial cell cultures were
confirmed
by their morphological features (ie. cuboidal, cobble-stone, contact
inhibited) using
phase-contrast microscopy and immunofluorescent-staining with antibodies to
Factor VIII. There
were no observable adverse effects of HMG-CoA reductase inhibitors, L-
mevalonic acid, or
hypoxia on cellular morphology. However, higher concentrations of simvastatin
(> 15 mmol/L)
or lovastatin (>50 mmol/L) caused cytotoxicity after 36 h, and therefore, were
not used.
Otherwise, cellular confluency and viability as determined by trypan blue
exclusion were
3o maintained for all treatment conditions described.
Example I 1: Effects of HMG-CoA Reductase Inhibitors on ecNOS Activity
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The activity of ecNOS was assessed by measuring the LNMA-inhibitable nitrite
accumulation from human endothelial cells (Liao, JK et al., JClin Invest,
1995, 96:2661-2666).
The ratio of nitrite to nitrate production under our culture condition was
approximately 5:1 and
was similar for hypoxia and normoxia. Basal ecNOS activity at 20% OZ was 6.0 ~
3.3
nmol/500,000 cells/24 h. Exposure of endothelial cells to 3% Oz for 24 h
decreased nitrite
production by 75 ~ 14% (1.5 ~ 0.9 nmol/500,000 cells/24 h, p < 0.01).
Treatment with
simvastatin (1 mM) not only completely reversed the downregulation of ecNOS by
hypoxia, but
resulted in a 3-fold increase in ecNOS activity over basal activity (18 ~ 5.0
nmol/500,000 cells/24
h, p < 0.05). This upregulation of ecNOS activity was attenuated by the
addition of L-mevalonate
to (400 mM) (9.6 ~ 1.3 nmol/500,000 cells/24 h, p < 0.05). Interestingly,
simvastatin (I mM) alone
upregulated nitrite production 5-fold (30 ~ 6.5 nmol/500,000 cells/24 h, p <
0.0,1), which was
completely blocked by L-mevalonate (400 mM) (8.6 ~ 2.9 nmol/500,000 cells/24
h, p < 0.05).
Similar findings were observed with lovastatin, but at 10-fold higher
concentration compared to
that of simvastatin.
IS
Example 12: Effects of HMG-CoA Reductase Inhibitors on ecNO'~~ Protein and
mRNA Levels
In a concentration-dependent manner, treatment with simvastatin (0.01 to 10
mM, 48 h)
increased ecNOS expression by 1 ( 6%, 80 ( 8%, 190 ( 10% and 310 ( 20%,
respectively (p < 0.05
for concentrations ( 0.1 mM, n=4). Treatment with simvastatin (0.1 mM)
increased ecNOS
2o protein levels in a time-dependent manner by 4 ( 6%, 21 ( 9%, 80 ( 8%, 90 (
12%, and 95 ( 16%
after 12 h, 24 h, 48 h, 72 h, and 84 h, respectively (p < 0.05 for all time
points after 12 h, n=4).
Another HMG-CoA reductase inhibitor, lovastatin, also increased ecNOS protein
levels in a
time-, and concentration-dependent manner. Because lovastatin has a higher
IC50 value for
HMG-CoA reductase compared to that of simvastatin, it was 10-fold less potent
in upregulating
25 ecNOS protein levels than simvastatin at equimolar concentrations.
We have previously shown that hypoxia downregulates ecNOS protein expression
(Liao,
JK et al., J Clin Invest, 1995, 96:2661-2666). Compared to normoxia (20% Oz),
exposure to
hypoxia (3% OZ) resulted in a 46 ~ 4 % and 75 ~ 3 % reduction in ecNOS protein
levels after 24
h and 48 h, respectively (p<0.01, n=3). In a concentration-dependent manner,
treatment with
3o simvastatin produced a progressive reversal of hypoxia-mediated
downregulation of ecNOS
protein levels after 48 h. At higher concentrations of simvastatin (1 and 10
mM), ecNOS protein
levels were upregulated to 159 ~ 13 % and 223 ~ 21 % of basal levels (p< 0.05,
n=3).
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Co-treatment with L-mevalonic acid (400 mM) completely blocked simvastatin-
induced increase
in ecNOS protein levels after 48 h (35 ~ 2.4 %). Treatment with L-mevalonic
acid alone,
however, did not produce any significant effects on basal ecNOS protein levels
in untreated cells
exposed to hypoxia (25 ~ 3.9 %, p>0.05, n=3). In addition, simvastatin which
was not
chemically-activated had no effect on ecNOS expression. These results indicate
that
simvastatin-and lovastatin-mediated increases in ecNOS protein expression are
mediated by
inhibition of endothelial HMG-CoA reductase.
To determine whether changes in ecNOS protein levels are due to changes in
ecNOS
steady-state mRNA levels, we performed Northern blotting on endothelial cells
exposed to
IO normoxia and hypoxia in the presence or absence of simvastatin (1 mM) and
lovastatin (10 :M).
Simvastatin alone increased ecNOS mRNA levels to 340 ~ 24 % (p<0.01, n=3).
Exposure of
endothelial cells to hypoxia reduced ecNOS mRNA levels by 70% ~ 2 % and 88 ~ 4
% after 24
h and 48 h with respect to GAPDH mRNA levels, respectively. Co-treatment with
simvastatin
not only completely reversed hypoxia-mediated decrease in ecNOS mRNA levels,
but increased
ecNOS mRNA levels to 195 ~ 12~ % and 530 ~ 30% of basal levels after 24 h and
48 h,
respectively (p<0.01, n=3). Similarly, lovastatin (10 :M) alone increased
ecNOS message to 350
~ 27 % under hypoxia and 410 ~ 21 % alone (p<0.01, n=3). Neither simvastatin
nor lovastatin
caused any significant change in G-protein as and b-actin mRNA levels under
normoxic or
hypoxic conditions. These results indicate that the effects of HMG-CoA
reductase inhibitors are
relatively selective in terms of their effects on ecNOS mRNA expression.
Example 13: Effects of HMG-Co A Reductase Inhibitors on ecNOS mRNA Half life
The half life of ecNOS mRNA was determined in the presence of actinomycin D (5
mg/ml). Hypoxia shortened the half life of ecNOS mRNA from 28 ~ 4 h to 13 ~ 3
h. Treatment
with simvastatin (1 mM) increased ecNOS half life to 46 ~ 4 h and 38 ~ 4 h
under normoxic and
hypoxic conditions, respectively (p<0.05 for both, n=3). These results suggest
that HMG-CoA
reductase inhibitors prevent hypoxia-mediated decrease in ecNOS expression by
stabilizing
ecNOS mRNA.
Example 14: Effects of HMG-CoA Reductase Inhibitors on ecNOS Gene
Transcription
Nuclear run-on assays showed that hypoxia caused a 85 ~ 8% decrease in ecNOS
gene
transcription (p<0.01, n=3). Treatment with simvastatin (1 mM) did not produce
any significant
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affect on hypoxia-mediated decrease in ecNOS gene transcription (83 ~ 6%
decrease in ecNOS
gene transcription, p>0.05 compared to hypoxia alone). Furthermore,
simvastatin alone produced
minimal increase in ecNOS gene transcription under normoxic condition (20 ~ 5%
increase in
ecNOS gene transcription, p<0.05 compared to normoxia control).
s Preliminary studies using different amounts of radiolabeled RNA transcripts
demonstrate
that under our experimental conditions, hybridization was linear and
nonsaturable. The density
of each ecNOS band was standardized to the density of its corresponding ((3-
tubulin band, relative
intensity). To exclude the possibility that changes in ((3-tubulin gene
transcription are caused by
hypoxia or simvastatin, another gene, GAPDH, was included on each of the
nuclear run-on blots.
Similar relative indices were obtained when ecNOS gene transcription was
standardized to
GAPDH gene transcription. The specificity of each band was determined by the
lack of
hybridization to the nonspecific pGEM cDNA vector.
Example 15: Effect of HMG-CoA Reductase Inhibitors on Mouse Ph. s~logy
IS To determine whether the upregulation of ecNOS by HMG-CoA reductase
inhibitors
occurs in vivo, SV-129 wild-type and ecNOS knockout mice were treated with 2
mg/kg
simvastatin or saline subcutaneously for 14 days. The mean arterial blood
pressures of wild-type
and ecNOS mutant mice were as reported previously (Huang, PL et al., Nature,
1995,
377:239-242). The ecNOS mutants were relatively hypertensive. There was no
significant change
in mean arterial blood pressures of wild-type mice after 14 days of
simvastatin treatment (81 ~
7 mmHg vs. 93 ~ 10 mmHg, p > 0.05, n=8). There was also no significant group
difference in
heart rate, arterial blood gases and temporalis muscle temperature before
ischemia or after
reperfusion. Furthermore, there was no significant difference in the levels of
serum cholesterol
(control: 147 ~ 10 vs. simvastatin 161 ~ 5.2 mg/dl), creatinine and
transaminases after treatment
with simvastatin compared to control values.
Example 16: Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression and
Function in
Mouse Aorta
The activity of ecNOS in the aortae of simvastatin-treated (2 mg/kg, s.c., 14
days) and
saline-injected mice was determined by measuring the LNMA-inhibitable
conversion of arginine
to citrulline (C'4). The ecNOS activity in aortae from simvastatin-treated
mice was significantly
higher than in the control group (0.39 ~ 0.09 vs. 0.18 ~ 0.04 U/mg protein,
n=8, p < 0.05).
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The ecNOS mRNA expression in the aortae of simvastatin-treated and -untreated
mice
was examined by quantitative RT-PCR. There was a significantly dose-dependent
3-fold increase
of ecNOS message compared to that of GAPDH in simvastatin-treated mice (n=3).
These findings
indicate that simvastatin upregulates ecNOS expression and activity in vivo.
Example 17: Effect of HMG-CoA Reductase Inhibitors on Cerebral Ischemia in
Mice
Endothelium-derived NO protects against ischemic cerebral injury (Huang, Z et
al., J
Cereb Blood Flow Metab, 1996, 16:981-987). Therefore we examined, wether the
observed
upregulation of ecNOS by simvastatin in vivo has beneficial effects on
cerebral infarct size.
Following treatment for 14 days with 2 mglkg of simvastatin, cerebral ischemia
was produced
by occluding the left middle cerebral artery for 2 hours. After 22 hours of
reperfusion, mice were
tested for neurological deficits using a well-established, standardized,
observer-blinded protocol.
The neurological motor deficit score improved in simvastatin-treated mice
(n=18) by almost
2-fold compared to that of controls (n=12) (0.8 ~ 0.2 vs. 1.7 ~ 0.2, p <
0.01).
Simvastatin-treated wild-type mice (n=18) had 25% smaller cerebral infarct
sizes
compared to untreated animals (73.8 ~ 8.5 mm3 vs. 100.7 t 7.3 mm3, n=12, p <
0.05) (Figure
4A). This effect was concentration-dependent (0.2, 2, 20 mg/kg simvastatin),
persisted for up to
3 days, and also occurred with lovastatin treatment, albeit at higher relative
concentrations.
Furthermore, simvastatin increase cerebral blood flow by 23% and 35% over
basal values at
concentrations of 2 mg/kg and 20 mg/kg, respectively (n=8, p <0.05 for both).
These findings
suggest, that simvastatin decreases cerebral infarct size and neurological
deficits.
Finally, to demonstrate that the reduction of cerebral infarct sizes by
simvastatin is due
to the upregulation of ecNOS, cerebral ischemia was applied to ecNOS mutant
mice lacking
ecNOS gene in the presence and absence of simvastatin (2 mg/kg, 14 days).
There was no
significant difference between the cerebral infarct sizes of simvastatin-
treated and -untreated
ecNOS mutant mice (n=6, p < 0.05) (Figure 4B). These findings indicate that
the upregulation
of ecNOS mediates the beneficial effects of HMG-CoA reductase inhibitors on
cerebral infarct
size.
3o Example 18: Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression in
Mouse
Brain
The ecNOS mRNA expression in the ischemic and contralateral (non-ischemic)
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hemispheres of mouse brain was examined by quantitative RT-PCR with respect to
GAPDH
mRNA levels. Simvastatin-treated mice (n=3) (2 mg/kg, 14 days) showed a 1.5-to
2-fold increase
in ecNOS expression in the infarcted, ipsilateral (I) hemisphere compared to
the contralateral (C),
non-infarcted side. In contrast, there was no difference in ecNOS expression
in untreated mice
s between their infarcted and non-infacted hemispheres. These findings suggest
that simvastatin
may reduced cerebral infarct size by selectively increasing ecNOS expression
in the ischemic and
hypoxic infarct zone.
Example 19: Effect of L-ar~inine on cerebral blood flow
L-arginine infusion at 300 mg/kg, i.v., caused modest (10%) and variable
elevations in
regional cerebral blood flow (rCBF) after infusion in several preliminary
experiments (n=4, data
not shown). In the present experiments, 450 mg/kg or saline was infused at a
constant rate of 100
microliter/kg/min over 15 minutes into wild type mice, mutant mice deficient
in endothelial nitric
oxide synthase (eNOS null), and mice which had received chronic daily
administration of
IS simvastatin (2 mg/kg). Regional cerebral blood flow (rCBF) was monitored by
laser-Doppler
flowimetry in groups of urethane-anesthetized, ventilated mice. Additional
physiological
variables were also monitored in the mice, including mean arterial blood
pressure (MABP), heart
rate, blood pH, PaOz, and PaC02.
Results
Physiological variables during laser-Doppler flowimetry in urethane-
anesthetized
ventilated wild type, simvastatin-treated and eNOS null mice infused with L-
arginine or saline
are shown in Table 1. Number of mice in each group is shown in parenthesis.
Values are
reported as mean +/- SEM. * denotes statistically significant difference
(P<0.05) compared with
eNOS null mice; # denotes statistically significant difference (P<0.05)
compared with baseline
by one-way ANOVA followed by Scheffe test. MABP indicates mean arterial blood
pressure;
sim indicates mice chronically administered simvastatin.
There were no within-group differences during observation time in mean
arterial blood
pressure and heart rate, although those values were elevated in eNOS null mice
as reported
previously. PaC02 values were not different between two time points in all
groups nor between-
group, although pH values were lower after infusion of L-arginine.
rCBF response to L-argininc:
Figure 5 is a bar graph showing regional CBF changes in wild type and eNOS
null mice
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for 40 min after L-arginine (450 mg/kg) or saline infusion at a constant rate
of 100
microliter/kg/min over 15 min. The number of mice in each group is indicated
in parenthesis.
Error bars denote standard error of the mean (SEM), and an asterisk (*)
denotes statistically
significant difference (P<0.05) compared with baseline control by one-way
ANOVA followed
s by Fisher's protected least-squares difference test.
L-arginine infusion (450 mg/kg, i.v.) increased rCBF in parietal cortex in
wild type mice,
as shown in Figure 1 (Fig. 1). The increase in rCBF began at 5-10 minutes and
achieved
statistical significance at 10-15 minutes after infusion. Maximum values
achieved at 20-25 min
reached 26% above, after which values decreased to control levels. By
contrast, L-arginine did
1o not increase rCBF in eNOS null mice. Values in these mutants ranged from -4
to +5% during the
40 minute recording period. Saline infusion in wild type mice did not increase
rCBP
significantly.
rCBF response to L-arginine plus simvastatin:
Figure 6 is a bar graph showing regional CBF changes in simvastatin-treated
mice for 40
~s min after L-arginine or saline infusion at the same dose. The number of
mice in each group is
indicated in parenthesis; sim indicates simvastatin. Error bars denote SEM and
an asterisk (*)
denotes statistically significant difference (P<0.05) compared with baseline
control by one-way
ANOVA followed by Fisher's protected least-squares difference test.
After chronic daily administration of simvastatin alone, the baseline rCBF was
increased
2o by 25%. L-arginine but not saline infusions increased rCBF significantly
above the simvastatin
baseline. Marked elevation was observed in the 10-15 minute epoch. The maximum
increase
was observed at 15-20 min and was 29-31 % over baseline. These increases
sustained for an
additional 20 minutes which was considerably longer than after L-arginine
treatment alone. The
maximum response to L-arginine in the presence of simvastatin was not
statistically increased.
2s However, the response to L-arginine was more sustained in the simvastatin-
treated mice. In the
30-40 minute epoch, the increase in blood flow was larger in the simvastatin
treated compared
to non treated control (P<0.05).
TABLE 1
MABP heart ratepH Pa02 PaC02
3oGroup (n) mmHg bpm mmHg mmHg
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wild + saline (6)
baseline 94.7+/-3.4543+/-20 7.36+/-0.02154+/-1335.8+/-2.0
0-5 min 94.8+/-3.3549+/-19
10-IS min 96.2+/-3.2548+/-16
S 20-25 min 95.8+/-3.1550+/-16
35-40 min 96.5+/-2.9547+/-15
after infusion 7.35+/-0.02180+/-5 33.4+/-1.8
wild + L-arginine
(7)
baseline 92.9+/-3.5*545+/-11 7.40+/-0.02127+/-9 39.2+/-2.0
100-5 min 92.7+/-3.5548+/-11
10-15 min 94.6+/-3.5561+/-9
20-25 min 93.3+/-3.4554+/-10
35-40 min 89.9+/-3.3533+/-9
after infusion 7.32+/-0.03169+/-5#36.4+/-1.7
15eNOS null+L-arginine
(4)
baseline 116.3+/-9.7618+/-9 7.40+/-0.0315+/-4 3G.4+/-l./
0-5 min I 15.8+/-10.0621+/-8
10-15 min 113.3+/-7.4623+/-6
20-2~ min I 13.8+/-8.1630+/-13
2035-40 min 94.8+/-8.1604+/-10
after infusion 7.28+/-0.04#178+/-7#35.8+/-2.4
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Table 1 continued-----
sim (2mg/kg)+saline
(3)
baseline 88.0+/-3.0*541+/-1 7.46+/-0.03159+/-1932.7+/-2.2
0-5 min 90.7+/-2.8543+/-3
10-15 min 93.3+/-3.3547+/-9
20-25 min 95.0+/-3.5553+/-10
35-40 min 94.0+/-4.0558+/-4
after infusion 7.41+/-0.01177+/-732.5+/-2.8
sim (2mg/kg)+L-arginine(5)
baseline 87.4+/-3.1505+/-9* 7.44+/-0.03144+/-1531.2+/-2.8
*
0-5 min 88.4+/-3.3*503+/-7*
10-15 min 92.2+/-3.2507+/-6*
20-25 min 88.4+/-3.4*502+/-5*
35-40 min 83.8+/-4.2490+/-4*
after infusion 7.30+/-0.02#163+/-1334.2+/-2.1
sim (20mg/kg)+L-arginine
(6)
baseline 91.7+/-2.8*566+/-26 7.44+/-0.01169+/-g32.0+/-1.5
0-5 min 91.5+/-3.7*571+/-26
10-15 min 92.7+/-4.9574+/-26
20-25 min 89.7+/-4.9*571+/-26
35-40 min 84.8+/-5.0551+/-21
after infusion 7.33+/-0.02#178+/-732.4+/-2.7
All references disclosed herein are incorporated by reference in their
entirety.
Presented below are the claims followed by the Sequence Listing follows the
claims.
We claim:
CA 02368187 2001-09-17
WO 00/56403 PCT/US00/07221
SEQUENCE LISTING
<110> The Brigham and Women's Hospital, Inc.
Liao, James K.
Laufs, Ulrich
Endres, Matthias
Moskowitz, Michael A.
<120> UPREGULATION OF TYPE III ENDOTHELIAL
CELL NITRIC OXIDE SYNTHASE BY HMG-COA REDUCTASE INHIBITORS
<130> R0547/7007W0/ERG/KA
<150> US 09/273,445
<151> 1999-03-19
<160> 4
<170> FastSEQ for Windows Version 3.0
<210> 1
<2i1> 25
<212> DNA
<213> Mus Musculus
<400> 1
gggctccctc cttccggctg ccacc 25
<210> 2
<211> 24
<212> DNA
<213> Mus Musculus
<40C> 2
ggatccctgg aaaaggcggt gagg 24
<210> 3
<211> 20
<212> DNA
<213> Mus Musculus
<400> 3
accacagtcc atgccatcac 20
<210> 4
<211> 20
<212> DNA
<213> Mus Musculus
<400> 4
tccaccaccc tgttgctgta 20
