U.S. patent application number 10/618835 was filed with the patent office on 2004-04-29 for enhancement of vascular function by modulation of endogenous nitric oxide production or activity.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Cooke, John P., Dzau, Victor J., Gibbons, Gary H..
Application Number | 20040082659 10/618835 |
Document ID | / |
Family ID | 27372858 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082659 |
Kind Code |
A1 |
Cooke, John P. ; et
al. |
April 29, 2004 |
Enhancement of vascular function by modulation of endogenous nitric
oxide production or activity
Abstract
Vascular function and structure is maintained or improved by
long term administration of physiologically acceptable compounds
which enhance the level of endogenous nitric oxide or other
intermediates in the NO induced relaxation pathway in the host.
Alternatively, or in combination, other compounds may be
administered which provide for short term enhancement of nitric
oxide, either directly or by physiological processes
Inventors: |
Cooke, John P.; (Palo Alto,
CA) ; Dzau, Victor J.; (Newton, MA) ; Gibbons,
Gary H.; (Atlanta, GA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
27372858 |
Appl. No.: |
10/618835 |
Filed: |
July 15, 2003 |
Related U.S. Patent Documents
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Application
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10618835 |
Jul 15, 2003 |
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10060252 |
Feb 1, 2002 |
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6646006 |
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10060252 |
Feb 1, 2002 |
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09930833 |
Aug 15, 2001 |
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6642208 |
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09930833 |
Aug 15, 2001 |
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09075509 |
May 8, 1998 |
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6337321 |
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09075509 |
May 8, 1998 |
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08556035 |
Nov 9, 1995 |
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5891459 |
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08556035 |
Nov 9, 1995 |
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08336159 |
Nov 8, 1994 |
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08336159 |
Nov 8, 1994 |
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08076312 |
Jun 11, 1993 |
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5428070 |
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Current U.S.
Class: |
514/565 ;
514/15.1; 514/19.1; 514/564; 514/8.2 |
Current CPC
Class: |
A23V 2200/326 20130101;
A23V 2250/0606 20130101; A61K 2300/00 20130101; A23V 2200/3262
20130101; A61K 45/06 20130101; A61K 38/063 20130101; A23V 2002/00
20130101; C12N 9/0075 20130101; C12N 2800/107 20130101; A23V
2002/00 20130101; C12N 15/85 20130101; A61K 31/00 20130101; A61K
31/195 20130101; A61K 31/198 20130101; A61K 48/00 20130101; A23L
33/175 20160801; A61K 38/063 20130101; A61K 48/0075 20130101; C12N
9/0044 20130101 |
Class at
Publication: |
514/565 ;
514/564; 514/002 |
International
Class: |
A61K 031/198; A61K
038/00 |
Goverment Interests
[0001] This invention was supported in part by the United States
Government under Grant 1KO7HCO2660 (NHLBI). The U.S. Government may
have an interest in this application.
Claims
What is claimed is:
1. A method of improving function and structure of the vascular
system of a human host, said method comprising: administering
orally to said host in accordance with a predetermined regimen a
prophylactic dose of a source of at least one of L-arginine and
L-lysine as other than a natural food source to enhance the level
of endogenous NO in the vascular system to improve vascular
function.
2. A method according to claim 1, wherein said dose comprises at
least 50% by weight of at least one of amino acid compounds
L-arginine or L-lysine, polypeptides comprising at least about 40
mol % of at least one of said amino acids, or physiologically
acceptable salt thereof.
3. A method according to claim 2, wherein said polypeptide is an
oligopeptide of at least one of L-arginine and L-lysine.
4. A method according to claim 2, wherein said dose comprises
L-arginine.
5. A method according to claim 4, wherein L-arginine is
administered in a daily amount in the range of 1 to 25 g per
day.
6. A method according to claim 4, wherein L-arginine is
administered at a dosage in the range of 0.5 to 5 g per dose.
7. A method according to claim 2, wherein said dose comprises
L-lysine.
8. A method according to claim 7, wherein L-lysine is administered
in a daily amount in the range of 1 to 25 g per day.
9. A method according to claim 7, wherein L-lysine is administered
at a dosage in the range of 0.5 to 5 g per dose.
10. A method according to claim 1, wherein said dose comprises at
least one of calcium, an amino acid absorption enhancing compound,
a cofactor for NO synthase activity, or an antioxidant in an amount
sufficient to enhance the prophylactic effect of said L-arginine
and L-lysine.
11. A method according to any of claims 5, 6, 8 or 9, wherein said
dose is administered as a tablet, capsule, or powder.
12. A method according to claims 5, 6, 8 or 9, wherein said dosage
is administered as a prepared solid food, nutritional supplement or
liquid.
13. A method of preventing a reduction in vascular function of the
vascular system of a human host as evidenced by reduced
vasodilation, said method comprising: administering orally to said
human host in accordance with a predetermined regimen a
prophylactic dosage of at least one of L-arginine, L-lysine or
physiologically acceptable salt thereof as other than a natural
food source in a daily amount to provide a plasma level in the
range of 0.15 to 3 mM to enhance the level of endogenous NO in the
vascular system, whereby reduction in said vasodilation is
inhibited.
14. A method according to claim 14, wherein said L-arginine,
L-lysine or a physiologically acceptable salt thereof is present in
a prepared food, nutritional supplement or liquid at from about
0.5-25 g.
15. A method according to claim 13, wherein said dose of
L-arginine, L-lysine or a physiologically acceptable salt thereof,
is in the range of 0.5-10 g in combination with at least one of
calcium, folate, B.sub.12 or B.sub.6 in sufficient amount to
enhance the effect of said L-arginine, L-lysine or a
physiologically acceptable salt thereof.
16. A method according to claim 13, wherein said L-arginine,
L-lysine or its physiologically acceptable salt is administered as
a tablet, capsule, or powder.
17. A physiologically acceptable formulation comprising at least
one of L-arginine, L-lysine or its physiologically acceptable salt
in from about 0.5 to 5 g and at least one of calcium, folate,
B.sub.6, or B.sub.12, in sufficient amount to enhance the effect of
said L-arginine, L-lysine or its physiologically acceptable salt on
enhancing the amount of NO in a human host.
18. A physiologically acceptable formulation comprising L-arginine
or its physiologically acceptable salt.
19. A physiologically acceptable formulation comprising L-lysine or
its physiologically acceptable salt.
20. A method for inhibiting vascular smooth muscle cell
proliferation at a site of injury in the vascular system, said
method comprising: administering at said site an effective amount
of at least one of L-arginine, L-lysine or its physiologically
acceptable salt to enhance NO production; whereby vascular smooth
muscle cell proliferation is inhibited.
21. A method according to claim 20, wherein said injury is as a
result of angioplasty.
Description
TECHNICAL FIELD
[0002] The field of this invention is the modulation of NO
activity, which finds application in maintaining and improving
vascular function and thereby preventing or improving vascular
degenerative diseases.
BACKGROUND
[0003] Atherosclerosis and vascular thrombosis are a major cause of
morbidity and mortality, leading to coronary artery disease,
myocardial infarction, and stroke. Atherosclerosis begins with an
alteration in the endothelium, which lines the blood vessels. The
endothelial alteration results in adherence of monocytes, which
penetrate the endothelial lining and take up residence in the
subintimal space between the endothelium and the vascular smooth
muscle of the blood vessels. The monocytes absorb increasing
amounts of cholesterol (largely in the form of oxidized or modified
low-density lipoprotein) to form foam cells. Oxidized low-density
lipoprotein (LDL) cholesterol alters the endothelium, and the
underlying foam cells distort and eventually may even rupture
through the endothelium.
[0004] Platelets adhere to the area of endothelial disruption and
release a number of growth factors, including platelet derived
growth factor (PDGF). PDGF, which is also released by foam cells
and altered endothelial cells, stimulates migration and
proliferation of vascular smooth muscle cells into the lesion.
These smooth muscle cells release extracellular matrix (collagen
and elastin) and the lesion continues to expand. Macrophages in the
lesion elaborate proteases, and the resulting cell damage creates a
necrotic core filled with cellular debris and lipid. The lesion is
then referred to as a "complex lesion." Rupture of this lesion can
lead to thrombosis and occlusion of the blood vessel. In the case
of a coronary artery, rupture of a complex lesion may precipitate a
myocardial infarction, whereas in the case of a carotid artery,
stroke may ensue.
[0005] One of the treatments that cardiologists and other
interventionalists employ to reopen a blood vessel which is
narrowed by plaque is balloon angioplasty (approximately 300,000
coronary and 100,000 peripheral angioplasties are performed
annually). Although balloon angioplasty is successful in a high
percentage of the cases in opening the vessel, it unfortunately
denudes the endothelium and injures the vessel in the process. This
damage causes the migration and proliferation of vascular smooth
muscle cells of the blood vessel into the area of injury to form a
lesion, known as myointimal hyperplasia or restenosis. This new
lesion leads to a recurrence of symptoms within three to six months
after the angioplasty in a significant proportion of patients
(30-40%).
[0006] In atherosclerosis, thrombosis and restenosis there is also
a loss of normal vascular function, such that vessels tend to
constrict, rather than dilate. The excessive vasoconstriction of
the vessel causes further narrowing of the vessel lumen, limiting
blood flow. This can cause symptoms such as angina (if a heart
artery is involved), or transient cerebral ischemia (i.e. a "small
stroke", if a brain vessel is involved). This abnormal vascular
function (excessive vasoconstriction or inadequate vasodilation)
occurs in other disease states as well. Hypertension (high blood
pressure) is caused by excessive vasoconstriction, as well as
thickening, of the vessel wall, particularly in the smaller vessels
of the circulation. This process may affect the lung vessels as
well causing pulmonary (lung) hypertension. Other disorders known
to be associated with excessive vasoconstriction, or inadequate
vasodilation include transplant atherosclerosis, congestive heart
failure, toxemia of pregnancy, Raynaud's phenomenon, Prinzmetal's
angina (coronary vasospasm), cerebral vasospasm, hemolytic-uremia
and impotence.
[0007] Because of their great prevalence and serious consequences,
it is critically important to find therapies which can diminish the
incidence of atherosclerosis, vascular thrombosis, restenosis, and
these other disorders characterized by abnormality of vascular
function and structure. Ideally, such therapies would inhibit the
pathological vascular processes associated with these disorders,
thereby providing prophylaxis, retarding the progression of the
degenerative process, and restoring normal vasodilation.
[0008] As briefly summarized above, these pathological processes
are extremely complex, involving a variety of different cells which
undergo changes in their character, composition, and activity, as
well as in the nature of the factors which they secrete and the
receptors that are u- or down-regulated. A substance released by
the endothelium, "endothelium derived relaxing factor" (EDRF), may
play an important role in inhibiting these pathologic processes.
EDRF is now known to be nitric oxide (NO) or a labile nitroso
compound which liberates NO. (For purposes of the subject
invention, unless otherwise indicated, nitric oxide (NO) shall
intend nitric oxide or the labile nitroso compound which liberates
NO.) This substance relaxes vascular smooth muscle, inhibits
platelet aggregation,. inhibits mitogenesis and proliferation of
cultured vascular smooth muscle, and leukocyte adherence. Because
NO is the most potent endogenous vasodilator, and because it is
largely responsible for exercise-induced vasodilation in the
conduit arteries, enhancement of NO synthesis could also improve
exercise capacity in normal individuals and those with vascular
disease. NO may have other effects, either direct or indirect, on
the various cells associated with vascular walls and degenerative
diseases of the vessel.
Relevant Literature
[0009] Girerd et al. (1990) Circulation Research 67:1301-1308
report that intravenous administration of L-arginine potentiates
endothelium-dependent relaxation in the hind limb of
cholesterol-fed rabbits. The authors conclude that synthesis of
EDRF can be increased by L-arginine in hypercholesterolemia.
Rossitch et al. (1991) J. Clin. Invst. 87:1295-1299 report that in
vitro administration of L-arginine to basilar arteries of
hypercholesterolemic rabbits reverses the impairment of
endothelium-dependent vasodilation and reduces vasoconstriction.
They conclude that the abnormal vascular responses in
hypercholesterolemic animals is due to a reversible reduction in
intracellular arginine availability for metabolism to nitric
oxide.
[0010] Creager et al. (1992) J. Clin. Invest. 90:1248-1253, report
that intravenous administration of L-arginine improves
endothelium-derived NO-dependent vasodilation in
hypercholesterolemic patients.
[0011] Cooke et al., "Endothelial Dysfunction in
Hypercholesterolemia is Corrected by L-arginine," Endothelial
Mechanisms of Vasomotor Control, eds. Drexler, Zeiher, Bassenge,
and Just; Steinkopff Verlag Darmstadt, 1991, pp. 173-181, review
the results of the earlier references and suggest, "If the result
of these investigations may be extrapolated, exogenous
administration of L-arginine (i.e., in the form of dietary
supplements) might represent a therapeutic adjunct in the treatment
and/or prevention of atherosclerosis".
[0012] Cooke (1990) Current Opinion in Cardiology 5:637-644
discusses the role of the endothelium in the atherosclerosis and
restenosis, and the effect that these disorders have on endothelial
function.
[0013] Cooke (1992) J. Clin. Invest. 90:1168-1172, describe the
effect of chronic administration of oral L-arginine in
hypercholesterolemic animals on atherosclerosis. This is the first
demonstration that oral L-arginine supplements can improve the
release of NO from the vessel wall. The increase in NO release from
the vessel wall was associated with a striking reduction in
atherosclerosis in hypercholesterolemic animals. This is the first
evidence to support the hypothesis that increasing NO production by
the vessel wall inhibits the development of atherosclerosis.
[0014] Cooke and Tsao (1992) Current Opinion in Cardiology
7:799-804 describe the mechanism of the progression of
atherosclerosis and suggest that inhibition of nitric oxide may
disturb vascular homeostasis and contribute to atherogenesis.
[0015] Cooke and Santosa (1993) In: Steroid Hormones and
Dysfunctional Bleeding, AAAS Press, review the activities of EDRF
in a variety of roles and suggest that reversibility of endothelial
dysfunction may be affected by the stage of atherosclerosis. They
conclude that EDRF is a potent vasodilator, plays a key role in
modulating conduit and resistance vessel tone, has important
effects on cell growth and interactions of circulatory blood cells
with a vessel wall, and that disturbances of EDRF activity may
initiate or contribute to septic shock, hypertension, vasospasm,
toxemia and atherosclerosis.
[0016] Fitzpatrick et al., American Journal of Physiology 265
(Heart Circ. Physiol. 34):H774-H778, 1993 report that wine and
other grape products may have endothelium-dependent vasorelaxing
activity in vitro.
[0017] Wang et al. (1994) J. Am. Cell. Cardiol. 23:452-458, report
that oral administration of arginine prevents atherosclerosis in
the coronary arteries of hypercholesterolemic rabbits.
[0018] Drexler et al. (1994) Circulation 89:1615-1623 describe the
effect of intravenous arginine upon coronary vascular tone. This
was the first evidence that intravenous arginine could restore
normal NO-dependent vasodilation in the coronary arteries of
patients with cardiac transplants, Tsao et al. (1994) Circulation
89:2176-2182 demonstrates that oral administration of arginine to
hypercholesterolemic rabbits enhances the release of nitric oxide
by the vessel wall, and inhibits monocytes from sticking to the
vessel.
[0019] Tsao et al. (1994) J. Arterioscl. Thromb. 14:1529-1533
reveals that oral arginine administration to hypercholesterolemic
rabbits inhibits platelet aggregation (blood clotting). Platelet
aggregation plays an important role in causing vascular thrombosis
in vascular degenerative disorders.
[0020] Von de Leyen et al. (1995) PNAS USA, show that the gene
encoding nitric oxide synthase (the enzyme that produces NO) can be
inserted into the carotid artery of the rat. This causes the rat
carotid artery to make more NO, and thereby enhances vasodilation
and inhibits thickening of the vessel wall after balloon
angioplasty.
[0021] Noruse et al. (1994) Arterioscler. Thromb. 14:746-752,
report that oral administration of an antagonist of NO production
accelerates atherogenesis in hypercholesterolemic rabbits.
[0022] Cayette et al. (1994) Arterioscler. Thromb. 14:753-759, also
report that oral administration of an antagonist of NO production
accelerates plaque development in hypercholesterolemic rabbits.
[0023] Other references which refer to activities attributed to NO
or its precursor include: Pohl and Busse (1989) Circ. Res.
65:1798-1803; Radomsli et al. (1987) Br. J. Pharmacol. 92:181-187;
Stamler et al. (1989) Circ. Res. 65:789-795; anti-platelet
activity); Garg and Hassid (1989) J. Clin. Invest. 83:1774-1777;
Weidinger et al. (1990) Circulation 81:1667-1679; NO activity in
relation to vascular smooth muscle growth); Ross (1986) N. Engl. J.
Med. 314:488-500; Bath et al. (1991) Arterioscler. Thromb.
11:254-260; Kubes et al. (1991) Proc. Natl. Acad. Sci. USA
89:6348-6352; Lefer et al. (1990) In: Endothelium-Derived
Contracting Factors. Basel, S. Karger, pp.190-197; NO activity in
relation to leukocyte adhesion and migration); Heistad et al.
(1984) Circ. Res. 43:711-718; Rossitch et al. (1991) J. Clin.
Invest. 87:1295-1299; Yamamoto et al. (1988) ibid 81:1752-1758;
Andrews et al. (1987) Nature 327:237-239; Tomita et al. (1990)
Circ. Res. 66:18-27; Kugiyama et al. (1990) Nature 344:160-162;
Mitchell et al. (1992) J. Vasc. Res. 29:169 (abst.); Minor et al.
(1990) J. Clin. Invest. 86:2109-2116; NO activity in relation to
hypercholesterolemia); Tanner et al. (1991) Circulation
83:2012-2020; Kuo et al. (1992) Circ. Res. 70:f465-476; Drexler et
al. (1991) Lancet 338:1546-1550; Schuschke et al. (1994) Int. J. of
Microcircu: Clin. and Erper. 14(4):204-211; Yao et al. (1992)
Circulation 86:1302-1309; Higashi et al. (1995) Hypertension 25(4
Pt 2):898-902; Kharitonov et al. (1995) Clin. Sci. 88(2):135-139;
Smulders et al. (1994) Clin. Sci. 87(1):37-43; Bode-boger et al.
(1994) Clin. Sci. 87(3):303-310; Bode-Boger et al. (1994) Clin.
Sci.; Randall et al. (1994) Clin. Sci. 87(1):53-59; Dubois-Rande et
al. (1992) J. Card. Pharm. 20 Suppl. 12:S211-3; Otsuji et al.
(1995) Am. Heart J. 129(6): 1094-1100; Nakanishi et al. (1992) Am.
J. of Physio. 263(6 Pt 2):H1650-8; Kuo et al. (1992) Circ. Research
70(3): 465-476; Tanner et al. (1991) Circulation 83(6):2012-2020;
Meng et al. (1995) J. Am. Col. Card. 25(1):269-275; Lefer and Ma
(1993) Arterioscl. and Thromb. 13(6):771-776; McNamara et al.
(1993) Biochem. and Biophys. Res. Comm. 193(1):291-296; Tarry and
Makhoul (1994) Arter. and Thromb. 14(6):983-943; Davies et al.
(1994) Surgery 116(3):557-568; and Raij (1994) Kidney Institute
45:775-781.
SUMMARY OF THE INVENTION
[0024] Methods are provided for improving vascular function and
structure, particularly modulating vascular relaxation, cellular
adhesion, infiltration and proliferation by modulating the level of
nitric oxide or active precursor at a physiological site. The
methods find use in preventing the degradation of vascular
function, particularly as involved with the occurrence of
atherosclerosis, restenosis, thrombosis, hypertension, impotence,
and other disorders characterized by reduced or inadequate
vasodilation. The enhancement of endogenous nitric oxide or
secondary messenger availability at a physiological site improves
vascular relaxation and thereby relieves symptoms due to inadequate
blood flow (such as angina) and can counteract inappropriate
elevation of blood pressure. The enhancement of endogenous nitric
oxide also inhibits initiation and the progression of
atherosclerosis, restenosis, vascular hypertrophy or hyperplasia
and thrombosis. This is due to the fact that nitric oxide is not
only a potent modulator, but can also inhibit platelets and white
blood cells from adhering to the vessel wall. As a prophylaxis or
treatment for vascular function deterioration, particularly in
atherosclerotic susceptible hosts, the agent is chronically
administered at an effective dosage. For restenosis, the agent may
be administered for a limited period since this pathological
process generally abates 3-6 months after the vascular injury (i.e.
angioplasty or atherectomy). Oral administration of L-arginine,
precursors to L-arginine, e.g. oligopeptides or polypeptides
comprising L-arginine, or proteins comprising high levels of
L-arginine, by itself or in combination with L-lysine, particularly
further supplemented with GRAS substances which enhance the
effectiveness of the active agents, as a dietary supplement will
increase NO elaboration and thereby diminish the effects of
atherogenesis. Other techniques to enhance NO or secondary
messenger availability may be utilized such as increasing the
availability of NO synthase, for example, as a result of enhanced
expression of NO synthase in the vessel wall, particularly at the
lesion site, release of NO from the vessel wall or reduction of
degradation of NO or the secondary messenger, cyclic guanosine
monophosphate ("cGMP").
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a bar diagram of histomorphometric studies of the
effect of L-arginine on atherosclerotic plaque in
hypercholesterolemic animals. (See Ex. 1)
[0026] FIG. 2 is a nephelometric scan of the effect of L-arginine
diet supplement on platelet reactivity as evidenced by platelet
aggregation initiated by adenosine diphosphate. (See Ex. 2) A)
aggregation of platelets from hypercholesterolemic rabbit; B)
reduced aggregation of platelets from hypercholesterolemic rabbit
treated with L-arginine; C) antagonism of NO synthase by LNMMA
reverses the beneficial effect of L-arginine.
[0027] FIG. 3 is a bar diagram comparing the effect of L-arginine
diet supplement on cell binding to aortic endothelium of
hypercholesterolemic animals. (See Ex. 4)
[0028] FIG. 4. Lesion surface area of thoracic aortae from all
arginine treated hypercholesterolemic animals (ARG, weeks 14-23) is
reduced in comparison to that of hypercholesterolemic animals
receiving vehicle (CHOL, weeks 14-23). (See Ex. 5)
[0029] FIG. 5. Macrophage accumulation in iliac arteries 4 weeks
following balloon injury. (Macrophage infiltration into the vessel
wall initiates and accelerates plaque formation). Data is expressed
as a percent of the vessel that contain macrophages. Balloon injury
in hypercholesterolemic rabbits (CHOL) results in a marked increase
in arterial macrophage accumulation compared with injured iliac
arteries from rabbits on normal chow (CONT). Macrophage
accumulation in iliac arteries from hypercholesterolemic rabbits
receiving L-arginine (ARG) is significantly reduced compared to the
CHOL group. (*; p<0.01, ARG v. CHOL). This study revealed that
oral arginine treatment markedly reduced the infiltration of
monocytes/macrophages into the vessel wall, explaining in part the
effect of arginine to inhibit plaque formation. (See Ex. 6)
[0030] FIG. 6. Stimulation of cultured endothelial cells with fluid
flow causes them to secrete nitric oxide. Flow-induced secretion of
nitric oxide decreases endothelial adhesiveness induced by oxidized
LDL cholesterol (oxLDL; 30 .mu.g/ml). Exposure of human aortic
endothelial cells to oxLDL increased the or ex vivo binding of
monocytes when compared to Control. In comparison to cells not
exposed to flow (static), previous exposure to flow inhibited the
monocyte adhesion induced by oxLDL. These effects of flow were
blocked by NO synthase inhibitors and mimicked by NO donors
(PAPA-NO) or cyclic GMP (cGMP). Bars represent mean .+-.SEM.
*p<0.05; **p<0.01. (See Ex. 8)
[0031] FIG. 7 is a bar diagram of morphometric measurements of
intimal lesion thickening two weeks after a balloon angioplasty in
animals treated with a plasmid construct containing the gene for NO
synthase (INJ+NOS) in comparison to control vector (INJ+CV) or
untreated injured vessels (INJ). (See Ex. 11)
[0032] FIG. 8 is a histogram showing the effect of
local-intraluminal administration of arginine on restenosis.
Hypercholesterolemic rabbits had balloon angioplasty of the iliac
artery. Immediately thereafter some animals received an infusion of
arginine directly into the vessel by means of a catheter designed
to apply high local concentrations of arginine to the vessel. Two
to four weeks later, vessels were removed from the animals, and
examined microscopically. Thickening of the vessel wall (internal
thickening or "restenosis") was reduced in the animals treated with
intraluminal infusion of arginine (ARG) in comparison to those
treated with vehicle. (See Ex. 12)
[0033] FIG. 9 is a set of dose-response curves showing the effect
of chronic lysine administration on endothelium dependent
vasodilation in hypercholesterolemic rabbits. Chronic oral
administration of lysine (for ten weeks) improved NO-mediated
vasodilation; this improvement in NO activity was also associated
with a marked reduction in plaque area. Chronic administration of
lysine was just as effective as arginine in restoring vascular
function and structure. (See Ex. 14)
[0034] FIG. 10 is a scatter-diagram illustrating the relationship
between the level of blood LDL-cholesterol and monocyte binding.
Monocytes were isolated from the blood of humans with normal or
elevated cholesterol levels. The binding of these monocytes to
endothelial cells in culture was observed. Monocytes from
individuals with high cholesterol levels have a greater
adhesiveness for endothelial cells. This monocyte-endothelial cell
interaction in vivo is the first step in the development of
atherosclerotic plaque. (See Ex. 15)
[0035] FIG. 11 is a bar diagram showing the adhesiveness of
monocytes obtained from subjects with normal cholesterol levels
(CONT) and those from hypercholesterolemic (HC) humans, before,
during, and after treatment with arginine (the NO precursor). Prior
to initiating arginine (Arg) or placebo (plac) treatment, monocytes
from hypercholesterolemic individuals have a greater tendency to
bind to endothelial cells ex vivo (baseline). After 2 weeks of
arginine treatment monocytes from these hypercholesterolemic
individuals have a significantly reduced adhesiveness and are no
different from those of the normal subjects. At this point arginine
therapy was discontinued and there was a washout (4 weeks). At this
time point, monocytes from the patients previously treated with
arginine now have increased adhesiveness, off of the arginine
treatment. (See Ex. 15)
[0036] FIG. 12 is a bar diagram which shows that monocytes from
individuals with elevated cholesterol (CHOL) have greater
adhesiveness for endothelial cells. However, after treatment with
sodium nitroprusside (CHOL+SNP), the adhesiveness of these
monocytes is normalized. SNP is an NO donor. (See Ex. 15)
[0037] FIG. 13 is a set of histograms showing aggregation of
platelets obtained in hypercholesterolemic humans (hc), and
individuals with normal cholesterol levels (nc). Platelet
aggregation ex vivo in response to adenosine diphosphate (ADP) is
increased in hc individuals in comparison to normal individuals.
After 2 weeks of treatment with oral L-arginine, platelet
aggregation is attenuated in the hypercholesterolemic individuals,
while an even greater effect of the treatment is seen at four
weeks. (See Ex. 16)
[0038] FIG. 14 is a bar graph showing increases in coronary blood
flow in response to intracoronary infusions of acetylcholine (ACH)
before and after intravenous infusion of L-arginine (30 g), in
patients with transplant atherosclerosis. Acetylcholine stimulates
the release of NO from the vessel wall causing vasodilation and
increased blood flow. There is improved NO-dependent vasodilation
after L-arginine administration. (See Ex. 18)
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0039] In accordance with the subject invention, vascular function
is maintained or its deterioration inhibited or retarded by
enhancing the level or activity of endogenous nitric oxide. By
enhancing the level or activity of endogenous nitric oxide, common
vascular degenerative diseases such as atherosclerosis, restenosis,
hypertension, vasospasm, impotence, angina, and vascular
thrombosis, can be treated prophylactically and/or therapeutically.
The enhanced level or activity of nitric oxide (which is intended
to include any precursor of nitric oxide which results in such
enhanced level) can be achieved by modulating the activity,
synthesis or concentration of any of the components associated with
the formation of nitric oxide in the nitric oxide synthetic
pathway, or inhibiting the rate of degradation of nitric oxide, its
precursors, or the secondary messengers associated with the
relaxation signal. In referring to the enhanced level or activity,
the term "effect" will be used to encompass the two situations. The
enhanced effect of nitric oxide may be a result of oral or
intravenous administration to the patient of a precursor in the
metabolic pathway to the production of nitric oxide (such as
L-arginine, L-lysine, polypeptides comprising these amino acids,
and the like), providing an enzyme in the metabolic pathway to NO,
particularly NO synthase, by introduction of the gene for NO
synthase under conditions for integration of the gene into the
endothelial or other cells and expression of the gene, or by
directly adding an enzyme associated with the production of nitric
oxide. The enhanced level of nitric oxide may also result from
administration of an agent to protect the NO from degradation, such
as an oxidant, reductant or superoxide dismutase. Alternatively,
the agent may serve to enhance the bioavailability or effectiveness
of the primary active agent, such as L-arginine or L-lysine. The
agent, individually or in combination, will be administered in a
form of other than a natural food source, such as meat or plants as
natural protein sources, fruits or products derived therefrom.
[0040] One approach is to employ L-arginine and/or L-lysine, as
individual amino acids, in combination, or as a precursor to
L-arginine, e.g. a monomer or a polypeptide, as a dietary
supplement. The amino acid(s) are administered as any
physiologically acceptable salt, such as the hydrochloride salt,
glutamate salt, etc. They can also be administered as a peptide
(e.g., poly-L-arginine, poly-L-lysine, or combinations thereof) so
as to increase plasma levels of the NO precursor. Oligopeptides of
particular interest include oligopeptides of from 2 to 30, usually
2 to 20, preferably 2 to 10 amino acids, having at least 50 mol %
of L-arginine and/or L-lysine, preferably at least about 75 mol %
of L-arginine and/or L-lysine, more preferably having at least
about 75 mol % of L-arginine and/or L-lysine. The oligopeptides can
be modified by being ligated to other compounds, which can enhance
absorption from the gut, provide for enhancement of NO synthesis or
stability, e.g. reducing agents and antioxidants, and the like.
[0041] Naturally occurring sources include protamine or other
naturally occurring L-arginine or -lysine containing protein, which
is high in one or both of the indicated amino acids, e.g. greater
than about 40%, preferably greater than about 50%.
[0042] The administration of L-arginine, other convenient NO
precursor, or other agent which enhances NO availability, would be
in accordance with a predetermined regimen, which would be at least
once weekly and over an extended period of time, generally at least
one month, more usually at least three months, and as a chronic
treatment, could last for one year or more, including the life of
the host. The dosage administered will depend upon the frequency of
the administration, the blood level desired, other concurrent
therapeutic treatments, the severity of the condition, whether the
treatment is for prophylaxis or therapy, the age of the patient,
the natural level of NO in the patient, and the like. Desirably,
the amount of L-arginine and/or L-lysine (R and/or K) or
biologically equivalent compound which is used would generally
provide a plasma level in the range of about 0.15 to 30 mM. The
oral administration of R and/or K can be achieved by providing R
and/or K, other NO precursor, or NO enhancing agent as a pill,
powder, capsule, liquid solution or dispersion, particularly
aqueous, or the like. Various carriers and excipients may find use
in formulating the NO precursor, such as lactose, terra alba,
sucrose, gelatin, aqueous media, physiologically acceptable oils,
e.g. peanut oil, and the like. Usually, if daily, the
administration of L-arginine and/or L-lysine for a human host will
be about 1 to 12 g per day.
[0043] Furthermore, other agents can be added to the oral
formulation of the amino acids or polypeptides to enhance their
absorption, and/or to enhance the activity of NO synthase, e.g.
B.sub.6 (50-250 mg/d), folate (0.4-10 mg per daily dose), B.sub.12
(0.5-1 mg/d) or calcium (250-1000 mg per daily dose). Furthermore,
agents known to protect NO from degradation, such as antioxidants
(e.g. cysteine or N-acetyl cysteine 200-1000 mg/d Vitamin C
(250-2000 mg daily dose), (coenzyme Q 25-90 mg daily dose,
glutathione 50-250 mg daily dose), Vitamin E (200-1000 I.U. daily
dose), or .beta.-carotene (10-25,000 I.U. daily dose) or other
naturally occurring plant antioxidants such as tocopherols,
phenolic compounds, thiols, and ubiquinones can be added to the
oral or intravenous formulations of R and/or K, or R and/or
K-containing peptides. Alternatively, one may include the active
agent in a nutritional supplement, where other additives may
include vitamins, amino acids, or the like, where the subject
active agent will be at least 10 weight %, more usually at least
about 25 weight % of the active ingredients.
[0044] The administration of R and/or K or its physiologic
equivalent in supporting NO can be administered prophylactically to
improve vascular function, serving to enhance vasodilation and to
inhibit atherogenesis or restenosis, or therapeutically after
atherogenesis has been initiated. Thus, for example, a patient who
is to undergo balloon angioplasty can have a regimen of R and/or K
administered substantially prior to the balloon angioplasty,
preferably at least about a week or substantially longer.
Alternatively, in a patient, the administration of R and/or K can
begin at any time. Conveniently, the amino acid composition can be
administered by incorporating the appropriate dose in a prepared
food. Types of foods include gelatins, ice creams, cereals,
candies, sugar substitutes, soft drinks, and the like. Of
particular interest is the incorporation of R and/or K as a
supplement in a food, such as a health bar, e.g. granola, other
grains, fruit bars, such as a date bar, fig bar, apricot bar, or
the like. The amount of R and/or K or the equivalent would be about
1-25 g per dosage or bar, preferably about 2-15 g.
[0045] Instead of oral administration, intravascular administration
can also be employed, particularly where more rapid enhancement of
the nitric oxide level in the vascular system is desired (i.e. as
with acute thrombosis of a critical vessel), so that combinations
of oral and parenteral administrations can be employed in
accordance with the needs of the patient. Furthermore, parenteral
administration can allow for the administration of compounds which
would not readily be transported across the mucosa from the
gastrointestinal tract into the vascular system.
[0046] Another approach is to administer the active ingredient of
grape skin extract, which is known to enhance NO activity. See
Fitzpatrick et al. (1993), supra. The extract can be enriched for
the active component by employing separation techniques and
assaying the activity of each of the fractions obtained. The grape
skin extract can be divided into fractions using a first gel
permeation separation to divide the extract by the size of the
components. The active fraction(s) can be determined by an
appropriate assay, see the experimental section. The active
fraction(s) can be further separated using HPLC and an appropriate
eluent, conveniently either an isocratic eluent of aqueous
acetonitrile or propanol or a linearly varying eluent, using the
same solvents. Fractions which are shown to be active and
substantially pure, e.g. at least 80 weight %, by thin layer
chromatography, mass spectrometry, gas phase chromatography, or the
like can then be characterized using infra-red, nuclear magnetic
resonance, mass or other spectroscopy.
[0047] For oral or intravascular administration, one can provide R
and/or K, by itself or in a polypeptide, or its physiological
equivalent in supporting NO, together with antioxidants or
scavengers of oxygen-derived free radicals (such as sulfhydryl
containing compounds) or compounds that prevent the production of
oxygen-derived free radicals (such as superoxide dismutase), as it
is known that oxygen-derived free radicals (such as superoxide
anion) can inactivate nitric oxide. Alternatively, or in addition,
one can administer cofactors required for NO synthase activity,
such as calcium or folate. The amounts of each of these co-agents
can be determined empirically, using the assays in the experimental
section to determine NO activity.
[0048] The various cofactors that may be used with the NO
precursors will vary in amount in relation to the amount of NO
precursor and the effectiveness of the cofactor, particularly for
oral administration. Generally, the cofactors may be present in
amounts that would provide daily doses of folate (0.4-10 mg),
B.sub.6 (50-250 mg), B.sub.12 (0.5-1 mg) and/or calcium (250-1000
mg). Usually, where the amount of the NO precursor is greater than
about 2 g, it may be administered periodically during the day,
being administered 2 to 4 times daily. For the most part, the
cofactors will be GRAS substances and will be able to be taken at
high dosages without adverse effects on the recipient host.
[0049] The subject compositions will be for the most part
administered orally and the dosage may take a variety of forms. The
dosage may be tablets, pill, capsules, powders, solutions,
dispersions, bars, ice creams, gelatins, and the like, formulated
with physiologically acceptable carriers, and optionally
stabilizers, colorants, flavoring agents, excipients, tabletting
additives, and the like. Depending upon the mode of administration,
the amount of active agent may be up to about 25 g. For
formulations as dietary supplements, individual dosages will
generally range from about 0.5 to 5 g, more usually from about 1 to
3 g of the NO precursor.
[0050] Alternatively, one can enhance, either in conjunction with
the enhancement of precursors to nitric oxides or independently,
components of the nitric oxide metabolic pathway. For example, one
can enhance the amount of nitric oxide synthase present in the
vessel wall, particularly at the site of lesions. This can be done
by local administration to the lesion site or systemically into the
vascular system. The synthase can be administered using liposomes,
slow release particles, or in the form of a depot, e.g. in
collagen, hyaluronic acid, biocompatible gels, vascular stents, or
other means, which will provide the desired concentration of the NO
synthase at the lesion site.
[0051] Instead of providing for the enhancement of NO at the
physiological site of interest, one can choose to extend the
lifetime of the signal transduced as a result of the presence of
nitric oxide. Since cGMP is produced intracellularly as a result of
a nitric oxide induced signal, employing agents which result in the
production of or extending the lifetime of cGMP can be employed.
Illustrative agents include cGMP phosphodiesterase inhibitors or
agents which increase the amount of guanylate cyclase.
[0052] Alternatively, cells can be genetically engineered to
provide for constitutive or inducible expression of one or more
genes, which will provide for the desired relaxation response, by
expressing NO synthase, or other enzyme or protein which is
secreted and acts extracellularly. Thus, expression vectors (viral
or plasmid) can be prepared which contain the appropriate gene(s)
and which can be introduced into host cells which will then produce
high concentrations of nitric oxide or other intermediate in the
relaxation pathway. These cells can be introduced at the lesion
site or at another site in the host, where the expression will
induce the appropriate response as to relaxation, proliferation,
etc. The NO synthase or cells expressing the NO synthase can be
present as depots by encapsulation and positioning at the site of
interest. For example, porous stents can be produced which
encapsulate the enzyme or cells to protect the enzyme from
degradation or being washed away.
[0053] Cultured cells can be transfected with expression vectors
containing the NO synthase or other gene ex-vivo and then
introduced into the vessel wall, using various intra-arterial or
intra-venous catheter delivery systems. Alternatively, techniques
of in vivo gene transfer car be employed to transfect vascular
cells within the intact vessel in vivo. The gene(s) can be
expressed at high constitutive levels or can be linked to an
inducible promoter (which can have tissue specificity) to allow for
regulation of expression.
[0054] DNA constructs are prepared, where the appropriate gene,
e.g. a NO synthase gene, is joined to an appropriate promoter,
either with its native termination region or a different
termination region, which can provide for enhanced stability of the
messenger RNA. Constitutive promoters of particular interest will
come from viruses, such as Simian virus, papilloma virus,
adenovirus, HIV, Rous sarcoma virus, cytomegalovirus or the like,
where the promoters include promoters for early or late genes, or
long terminal repeats. Endogenous promoters can include the
.beta.-actin promoter, or cell-type specific promoters.
[0055] A construct is prepared in accordance with conventional
techniques, the various DNA fragments being introduced into an
appropriate plasmid or viral vector, normally a vector capable of
replication in a bacterial and/or eucaryotic host. Normally, the
vector will include a marker, which allows for selection of cells
carrying the vector, e.g. antibiotic resistance. The vector will
normally also include an origin which is functional in the host for
replication. Other functional elements can also be present in the
vector.
[0056] Once the vector has been prepared and replicated, it can
then be used for introduction into host cells. The plasmid vector
construct can be further modified by being joined to viral elements
which allow for ease of transfection, can provide a marker for
selection, e.g. antibiotic resistance, or other functional
elements. Introduction of the plasmid vector construct into the
host cells can be achieved by calcium phosphate precipitated DNA,
transfection, electroporation, fusion, lipofection, viral
capsid-mediated transfer, or the like. Alternatively, the
expression construct within viral vectors can be introduced by
standard infection techniques. For somatic cell gene therapy,
autologous cells will generally be employed, although in some
instances allogeneic cells or recombinantly modified cells can be
employed. Usually the cells employed for genetic modification will
be mature endothelial or vascular smooth muscle cells.
Occasionally, the cells employed for genetic modification will be
progenitor cells, particularly early progenitor cells. For example,
myoblasts can be employed for muscle cells or hematopoietic stem
cells or high proliferative potential cells can be employed for
lymphoid and/or myelomonocytic cells.
[0057] Depending upon the nature of the cells, they can be injected
into tissue of the same or different cellular nature, they can be
injected into the vascular system, where they may remain as mobile
cells or home to a particular site (i.e. the lesion). For the NO
synthase gene, the number of cells which are administered will
depend upon the nature of the cells, the level of production of the
NO synthase, the desired level of NO synthase in the host vascular
system, at the lesion site, or the like, whether the enhanced level
of NO synthase is the only treatment or is used in conjunction with
other components of the nitric oxide synthetic pathway, and the
like. Therefore, the particular number of cells to be employed will
be determined empirically in accordance with the requirements of
the particular patient.
[0058] These cells can also be introduced into the circulation by
first growing them on the surface of standard vascular graft
material (i.e. Dacron or polytetrafluoroethylene grafts) or other
synthetic vascular conduits or vascular bioprostheses.
[0059] Alternatively, one can use viral vectors, which are capable
of infecting cells in vivo, such as adenovirus or retroviruses. In
this case, the viral vector containing the NO synthase gene or
other gene involved with the relaxation pathway will be
administered directly to the site of interest, where it will enter
into a number of cells and become integrated into the cell genome.
Thus, one can titer the desired level of nitric oxide synthase
which is secreted or other protein which is expressed, by providing
for one or more administrations of the virus, thus incrementally
increasing the amount of synthase which is secreted or other
protein which is produced.
[0060] Alternatively, one can use modified liposomes as a vehicle
for endovascular administration of the vector containing the NO
synthase or other gene. One such modified liposome technique
involves mixing the liposomes with the vector containing NO
synthase. Once the gene expression construct-containing vector is
incorporated into the liposome, the liposomes are coated with a
protein (e.g. the viral coat protein of the Hemagglutinating Virus
of Japan) that increases the affinity of the liposome for the
vessel wall.
[0061] In some situations, the NO synthase or other gene in the
relaxation pathway can be co-transfected with an artificial gene
encoding an arginine and/or lysine rich polypeptide susceptible to
proteolytic cleavage as an intracellular source of L-arginine
and/or L-lysine. In other situations, the NO synthase or other gene
can be co-transfected with the superoxide dismutase gene, so as to
inhibit the degradation of the nitric oxide.
[0062] In some situations, acute treatment may be involved,
requiring one or a few administrations. This will normally be
associated with compounds which can act as nitric oxide precursors
and are other than naturally occurring compounds or are compounds
which can be added with naturally occurring compounds to enhance
the rate of formation of nitric oxide. Thus, one can provide for
acute administration of L-arginine and/or L-lysine and superoxide
dismutase to increase the nitric oxide concentration over a
restricted period of time. These administrations can be independent
of or in conjunction with long term regimens.
[0063] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
Anti-Atherogenic Effects of Oral Arginine
[0064] Study design: (See, Cooke et al., 1992, supra) Male New
Zealand white rabbits (n=49) were assigned to one of three
treatment groups: 10 were fed with normal rabbit chow for ten weeks
(Control); 19 received chow enriched with 1% cholesterol (Chol);
and 20 received a 1% cholesterol diet supplemented with 2.25%
L-arginine hydrochloride in the drinking water (Arg.). Following
ten weeks of the dietary intervention, animals were lightly sedated
and the central ear artery cannulated for measurement of
intra-arterial blood pressure, followed by collection of blood
samples for serum chemistries and plasma arginine. Subsequently the
animals were sacrificed and the left main coronary artery and the
thoracic aorta were harvested for studies of vascular reactivity
and histomorphometry. In some animals, blood was collected for
studies of platelet and monocyte reactivity.
[0065] Results: Biochemical and physiological measurements.
[0066] Hypercholesterolemic animals maintained on oral L-arginine
supplementation (Arg) experienced a twofold elevation in plasma
arginine levels in comparison to animals on a normal (Control) or
1% cholesterol (Chol) diet alone; the elevation in plasma arginine
was maintained throughout the course of the study. Serum
cholesterol measurements were elevated equally in both groups
receiving the 1% cholesterol diet [50.+-.6 vs. 1629.+-.422 vs.
1852.+-.356 mg/dl respectively for Control (=10), Chol (=13), and
Arg (=14)]. There were no significant differences in hemodynamic
measurements between groups.
[0067] Organ chamber studies of isolated vessels: For
NO-independent responses, there were no differences between the
treatment groups in maximal response or sensitivity to
norepinephrine (a vasoconstrictor), or to nitroglycerin (a
nitrovasodilator). By contrast, NO-dependent relaxations were
attenuated in vessels harvested from hypercholesterolemic animals
with a reduction in the maximal response to acetylcholine. In
comparison, vessels harvested from hypercholesterolemic animals
receiving L-arginine supplementation had improved NO-dependent
relaxation to acetylcholine. In a separate study, the effect of
chronic arginine supplementation to improve NO-dependent relaxation
was confirmed in the hypercholesterolemic rabbit abdominal
aorta.
[0068] Histomorphometric studies (planimetry of EVG-stained
sections): A blinded histomorphometric analysis revealed that
medial cross-sectional areas of thoracic aortae were not different
between the groups. By contrast, the intimal cross-sectional area
(i.e. amount of atherosclerotic plaque) of vessels from
hypercholesterolemic animals receiving L-arginine supplementation
was reduced in comparison to those from animals receiving
cholesterol diet alone. In the Arg animals the reduction in the
intimal, lesion was most pronounced in the ascending thoracic aorta
and left main coronary artery. In the left main coronary artery of
hypercholesterolemic animals receiving arginine, essentially no
atherosclerotic plaque developed.
[0069] Changes in lesion surface area: In a second series of
studies, the extent of the thoracic aorta involved by lesions was
examined. In hypercholesterolemic rabbits receiving vehicle (n=6)
or L-arginine supplement (n=6), thoracic aortae (from left
subclavian artery to diaphragm) were harvested after ten weeks of
treatment, bisected longitudinally, and stained with oil-red O.
Vessels were photographed and vessel and lesion surface area
determined by planimetry. Approximately 40% of the total surface
area was covered with plaque in thoracic aortae from
hypercholesterolemic animals receiving vehicle, whereas in thoracic
aortae from arginine-treated hypercholesterolemic animals, less
than 10% of the surface area was covered with plaque (FIG. 1).
[0070] To summarize, dietary arginine supplementation increases
plasma arginine levels, but does not alter serum cholesterol. This
is associated with significant improvement in NO-dependent
vasodilation as judged by bioassay. Finally, the improvement in
NO-dependent vasodilation is associated with reduction in thickness
and area of the lesions in vessels from hypercholesterolemic
animals.
EXAMPLE 2
Inhibition of Platelet Aggregation by Oral L-arginine
[0071] The effect of L-arginine supplementation on platelet
reactivity in rabbits that had normal chow (Control; n=6), a 1%
cholesterol diet (Chol; n=5), or a 1% cholesterol diet supplemented
with oral arginine (Arg; n=6), as detailed above, was examined.
Arterial blood obtained after central ear artery cannulation was
anticoagulated with 13 mM sodium citrate. Platelet-rich suspension
was prepared by washing platelets in calcium-free Krebs-Henseleit
solution and resuspending them in Tyrode's solution with albumin.
Aggregation was initiated by addition of adenosine diphosphate and
monitored by standard nephelometric techniques. In platelets
derived from Chol animals, aggregation was not different in rate or
maximum extent in comparison to platelets from Control animals (A,
in FIG. 2). By contrast, aggregation of platelets from Arg animals
was reduced by 50% (B, in FIG. 2).
[0072] This reduction in platelet aggregation was associated with a
two-fold greater cGMP content in aggregated platelets from
arginine-treated animals. The reduction of platelet reactivity
could be reversed by administration of N-methylarginine (10.sup.-4
M) in vitro (C, in FIG. 2). Therefore, the anti-platelet effect of
chronic oral arginine administration can be credited to an
increased synthesis of endogenous NO. Furthermore, NO synthesis may
be induced in both the platelets and the endothelium.
EXAMPLE 3
Inhibition of Monocyte Adherence
[0073] A. Functional Binding Assay: To determine if oral arginine
supplementation affects monocyte adherence, blood was collected
from rabbits fed normal chow (=6) a 1% cholesterol diet (=6), or a
1% cholesterol diet supplemented with L-arginine (=6), as described
above. Mononuclear cells were purified from blood by Ficoll-paque
density gradient centrifugation. In these preliminary studies,
adhesion was examined of blood leukocytes to a transformed
endothelial cell line, bEnd3 (mouse brain-derived polyoma middle T
antigen transformed endothelial cells). The Bend3 cells display the
morphology of endothelial cells, and like human endothelial cells
are capable of uptake of acetylated low-density lipoprotein and
express adhesion molecules in a cytokine-regulatable fashion.
Cultured cells were grown to confluence in 0.5 cm.sup.2 Lab-Tek
chamber slides (MilesScientific) and treated with control medium or
with LPS (1 mg/ml) or TNF.alpha. (25 U/ml) for 18 hours. Cultures
were washed with fresh assay buffer, and low, medium, or high
concentrations of leukocytes (0.75, 1.5, or 3.times.10.sup.5
cells/ml, respectively) were added per well. Following a 30-minute
incubation on a rocking platform at room temperature to allow
binding, the slides were inverted and immersed in buffer containing
2% (v/v) glutaraldehyde, such that non-adherent cells were lost and
adherent cells were fixed to the monolayer.
[0074] The adherent mononuclear cells were enumerated using
video-light microscopy.
[0075] Monocytes from hypercholesterolemic animals (Chol) exhibited
greater adherence, consistent with observation by others, that
monocytes from hypercholesterolemic cats or humans exhibit greater
adherence to cultured endothelial cells. (deGruijter et al. (1991)
Metabol. Clin. Exp. 40:1119-1121; Fan et al. (1991) Virchows Arch.
B Cell Pathol. 61:19-27).
[0076] In comparison to monocytes derived from vehicle-treated
hypercholesterolemic animals (Chol), those from arginine-treated
hypercholesterolemic animals (Arg) were much less adherent. This
data shows that the arginine treatment inhibits adhesion of
monocytes to the endothelium, which is the first observable event
in atherogenesis.
EXAMPLE 4
Dietary L-Arginine Inhibits the Enhanced Endothelial-Monocyte
Interaction In Hypercholesterolemia
[0077] The earliest observable abnormality of the vessel wall in
hypercholesterolemic animals is enhanced monocyte adherence to the
endothelium, which occurs within one week of a high cholesterol
diet. This event is thought to be mediated by the surface
expression of endothelial adhesion molecules and chemotactic
proteins induced by hypercholesterolemia.
[0078] Another endothelial alteration that occurs in parallel is a
reduced activity of nitric oxide (i.e., NO), derived from
metabolism of L-arginine. As shown above chronic dietary
supplementation with L-arginine restores NO-dependent
vasodilatation in hypercholesterolemic rabbits, and that this
improvement in NO activity is associated with a striking
anti-atherogenic effect. In the following study was tested the
hypothesis that the anti-atherogenic effect of dietary arginine was
mediated by endothelial derived NO which inhibits
monocyte-endothelial cell interaction.
[0079] Methods. Animals. Male New Zealand White rabbits were pair
fed, receiving one of the following dietary interventions for two
weeks: normal rabbit chow (Cont, n=7); rabbit chow enriched with 1%
cholesterol (Chol, n=7); or 1% cholesterol chow supplemented with
2.25% L-arginine HCl in the drinking water (Arg, n=7) ad libitum
throughout the course of the study. In a second series of studies
designed to further explore the role of endogenous NO on
monocyte-endothelial cell interaction, another group of animals
were pair fed, receiving a normal rabbit diet supplemented with
either vehicle control (N=5) or the NO synthase antagonist,
nitro-L-arginine (L-NA, 10 mg/100 ml; n=5), administered in the
drinking water ad libitum throughout the course of the study (for
an average daily dose of 13.5 mg/kg/day). In a third series of
experiments animals received a normal diet and either vehicle
(n=4), L-NA (13.5 mg/kg/day; n=4), or L-NA and hydralazine (n=4)
added to the drinking water for two weeks. At this dose,
hydralazine (5 mg/kg/day) reversed the increase in blood pressure
induced by L-NA One day before sacrifice (after 2 weeks of dietary
intervention), animals were lightly sedated and the central ear
artery was cannulated for collection of blood samples.
[0080] Mononuclear cell culture and isolation. Murine monocytoid
cells, WEHI 78/24 cells were grown in Dulbecco's Modified Eagle's
Medium supplemented 10% fetal calf serum (vol/vol) and were kept in
an atmosphere of 5% CO.sub.2/95% air. Prior to binding studies,
mononuclear cells were fluorescently labeled with TRITC (3
.mu.g/ml). To confirm the results using WEHI cells, in some studies
binding studies were performed in parallel using rabbit mononuclear
cells. Mononuclear cells were isolated from fresh whole blood of
Control rabbits before sacrifice.
[0081] Preparation of aortic endothelium and binding assay. After 2
weeks of the dietary intervention, the thoracic aortae were removed
and placed in cold, oxygenated saline. A 15 mm segment of thoracic
aorta was excised from a point immediately distal to the left
subclavian artery to the mid-thoracic aorta. The segments were then
carefully opened longitudinally and placed into culture dishes
containing HBSS medium. Aortic strips were fixed to the culture
dish using 25 gauge needles so as to expose the endothelial surface
to the medium. Culture dishes were then placed on a rocking
platform at room temperature.
[0082] After 10 minutes the HBSS medium was replaced by binding
medium containing WEHI cells. The aortic strips were incubated with
the mononuclear cells for 30 minutes. The medium was then replaced
by fresh binding medium without cells to remove non-adherent cells.
The aortic segments were then removed and placed on a glass slide,
and adherent cells counted under epifluorescent microscopy from at
least 30 sites on each segment.
[0083] Results. Monocyte adhesion to rabbit aortic endothelium.
Exposure of WEHI 78/24 cells to normal rabbit aortic endothelium
results in a minimal cell binding in this ex vivo adhesion assay.
However, when WEHI cells were incubated with aortic endothelium
from hypercholesterolemic animals (Chol; n=7), cell binding was
enhanced 3-fold in comparison to Cont (n=7). The increased cell
binding manifested by aortic endothelium of hypercholesterolemic
animals was significantly attenuated by L-arginine supplementation
(n=7). (FIG. 3) Similar results were achieved when adhesion assays
were performed in parallel with mononuclear cells that were freshly
isolated from Cont animals (n=2) in each of the three groups.
[0084] Effect of chronic NO synthase inhibition on endothelial
adhesiveness. To further investigate the role of
endothelium-derived NO in modulating endothelial-monocyte
interaction, an addition series of binding studies were performed
using thoracic aorta from animals that received regular chow
supplemented with vehicle (n=5) or the NO synthase inhibitor, L-NA
(n=5). The adhesion of WEHI cells was markedly increased when
incubated with aortic endothelium from L-NA animals compared to
control endothelium. This effect could not be attributed to
hypertension caused by L-NA since concomitant administration of
hydralazine normalized blood pressure but did not reverse the
augmentation of cell binding induced by L-NA.
[0085] In a separate series of studies it was confirmed that
chronic administration of L-NA (the inhibitor of NO synthase)
significantly inhibited generation and release of NO from the
vessel wall (as measured by chemiluminescence), compared to vessels
from animals treated with vehicle or arginine.
[0086] The salient findings of this investigation are: 1) monocyte
binding to the endothelium ex vivo is increased in vessels from
hypercholesterolemic animals; 2) this increase in monocyte binding
is attenuated in hypercholesterolemic animals treated chronically
with the NO precursor L-arginine; 3) monocyte binding to the
endothelium is increased in vessels from normocholesterolemic
animals treated with the NO synthase antagonist L-nitro-arginine;
and 4) this effect of NO synthase antagonism was not reversed by
administration of hydralazine in doses sufficient to normalize
blood pressure. These findings are consistent with the hypothesis
that NO inhibits monocyte-endothelial cell interaction.
[0087] To conclude, an ex vivo model of monocyte binding has been
used to study the increase in endothelial adhesiveness induced by
hypercholesterolemia. Endothelial adhesiveness is attenuated by
oral administration of the NO precursor L-arginine is shown.
Conversely, inhibition of NO synthase activity by oral
administration of nitro-arginine strikingly increases endothelial
affinity for monocytes ex vivo. The data are consistent with NO
being an endogenous anti-atherogenic molecule.
EXAMPLE 5
Oral Arginine Causes Regression of Atherosclerosis in
Hypercholesterolemic Rabbits
[0088] Our previous work demonstrated that oral arginine could
prevent the development of plaque in hypercholesterolemic animals
but it was not known if pre-existing plaque could be affected by
arginine treatment. This is clinically important if arginine is to
be useful in the treatment of pre-existing atherosclerosis in
humans. Accordingly, New Zealand white rabbits (n=85) received
normal chow or 0.5% cholesterol chow for 10 weeks. Subsequently,
half of the hypercholesterolemic rabbits were given 2.25% (W/V)
L-arginine in their drinking water. Thoracic aortae were harvested
at weeks 10, 14, 18, or 23. Rings of aorta were used to assess
NO-dependent vasodilation to acetylcholine (ACh). Maximal
relaxation to ACh in the hypercholesterolemic rabbits receiving
vehicle (CHOL) became progressively attenuated from 53.4% (at week
10) to 17.4% by week 23). Planimetry of the Iuminal surface of the
aortae from CHOL animals revealed a progressive increase in plaque
area from 30.3% (at week 10) to 56.5% (by week 23) of the total
surface of the thoracic aorta. By contrast, hypercholesterolemic
animals receiving arginine (ARG) manifested improved
endothelium-dependent relaxation associated with a reduction of
plaque area at 14 and 18 weeks. Lesion surface area in all arginine
treated hypercholesterolemic animals (weeks 14-23) was
significantly reduced in comparison to vehicle-treated
hypercholesterolemic animals (FIG. 4). The arginine-induced
improvement in endothelium-dependent relaxation was associated with
an increased generation of vascular NO, and a reduced generation of
vascular superoxide anion. By 23 weeks, 3 of 7 ARG animals had
persistent improvement in NO-dependent vasodilation and exhibited a
further reduction of plaque area to 5.4%
[0089] Conclusions: hypercholesterolemia induces a progressive loss
of NO-dependent vasodilation associated with progressive intimal
lesion formation. Administration of L-arginine to animals with
pre-existing intimal lesions augments vascular NO elaboration,
reduces superoxide anion generation, and is associated with a
reduction in plaque area. This is the first demonstration that
restoration of NO activity can induce regression of pre-existing
intimal lesions, and provides evidence that L-arginine therapy may
be of potential clinical benefit.
EXAMPLE 6
Oral Arginine Administration Restores Vascular NO Activity and
Inhibits Myointimal Hyperplasia after Balloon Injury in
Hypercholesterolemic Rabbits
[0090] Purpose. The purpose of this study was to determine if the
alterations in vascular function and structure following balloon
angioplasty in hypercholesterolemic rabbits could be inhibited by
restoration of endogenous nitric oxide (NO) activity.
[0091] Methods. Twenty-eight New Zealand white rabbits were
randomized into one of three dietary groups and received either
normal rabbit chow, 0.5% cholesterol diet, or 0.5% cholesterol diet
plus L-arginine hydrochloride (2.25% W/V) in the drnking water.
After six weeks of dietary intervention, the left iliac artery of
each animal was subjected to a balloon angioplasty. Four weeks
later, the iliac arteries were harvested for vascular reactivity
studies and immunohistochemistry.
[0092] Results. The bioassay studies indicated that
endothelium-derived NO activity was inhibited in
hypercholesterolemic animals in comparison to normocholesterolemic
animals. The administration of arginine partially restored
endothelium-derived NO activity. Balloon angioplasty induced
intimal thickening which was largely composed of vascular smooth
muscle cells and extracellular matrix. In the setting of
hypercholesterolemia, vascular injury induced an exuberant
myointimal lesion that was augmented by the accumulation of
lipid-laden macrophages. Administration of L-arginine induced a
quantitative as well as qualitative change in the lesion. Dietary
arginine reduced intimal thickening in the injured vessels of
hypercholesterolemic animals, and substantially inhibited the
accumulation of macrophages in the lesion (FIG. 5).
[0093] Conclusions. We report that the lesions induced by balloon
angioplasty in hypercholesterolemic animals are markedly reduced by
oral administration of arginine. Moreover, we find that the nature
of the lesion is altered, with a striking reduction in the
percentage of macrophages comprising the lesion.
Hypercholesterolemia induces an endothelial vasodilator dysfunction
in the rabbit iliac artery that is reversible by chronic oral
administration of arginine.
EXAMPLE 7
Nitric Oxide Regulates Monocyte Chemotactic Protein-1
[0094] Our previous studies had established that oral arginine
administration could enhance. vascular NO synthesis. This increase
in vascular NO synthesis was associated with inhibition of monocyte
adherence and accumulation in the vessel wall (thereby reducing the
progression, and even inducing regression, of plaque). The question
remained: "How does vascular nitric oxide inhibit monocyte
adherence and accumulation in the vessel wall?"
[0095] Monocyte chemotactic protein-1 (MCP-1) is a 76-amino acid
chemokine thought to be the major chemotactic factor for monocytes
(chemotactic factors are proteins that attract white blood cells).
We hypothesized that the anti-atherogenic effect of NO may be due
in part to its inhibition of MCP-1 expression.
[0096] Methods and Results. Smooth muscle cells (SMC) were isolated
from normal rabbit aortae by explant method. Cells were then
exposed to oxidized LDL (30 .mu.g/ml) (which is known to induce
vascular cells to synthesize MCP-1). The expression of MCP-1 in SMC
was associated with an increased generation of superoxide anion by
the SMC, and increased activity of the transcriptional protein
NF.kappa.B. All of these effects of oxidized LDL cholesterol were
reduced by previous exposure of the SMC to the NO-donor
DETA-NONOate (100 .mu.M) (p<0.05). To determine if NO exerted
its effect at a transcriptional level, SMC and COS cells were
transfected with a 400 bp fragment of the MCP-1 promoter. Enhanced
promoter activity by oxLDL was inhibited by DETA-NO.
[0097] To investigate the role of endogenous NO in the regulation
of MCP-1 in vivo, NZW rabbits were fed normal chow, normal chow
plus nitro-L-arginine (L-NA) (to inhibit vascular NO synthesis),
high cholesterol diet (Chol), or high cholesterol diet supplemented
with L-arginine (Arg) (to enhance NO synthesis). After two weeks,
thoracic aortae were harvested and total RNA was isolated. Northern
analysis demonstrated increased expression of MCP-1 in Chol and
L-NA aortae; this expression was decreased in aortae from Arg
animals. These studies indicate that the anti-atherogenic effect of
NO may be mediated in part by its inhibition of MCP-1 expression.
NO inhibits the generation of superoxide anion by the vascular
cells and thereby turns off an oxidant-responsive transcriptional
pathway (i.e. NF.kappa.B-mediated transcription) activating MCP-1
expression.
EXAMPLE 8
Nitric Oxide Inhibits the Expression of an Endothelial Adhesion
Molecule known to be Involved in Atherosclerosis
[0098] Vascular cell adhesion molecule (VCAM-1) is an endothelial
adhesion molecule that binds monocytes. This molecule is expressed
by the endothelium of hypercholesterolemic animals, and is
expressed by endothelial cells overlying plaque in animals and
humans. This adhesion molecule is believed to participate in
monocyte adherence and accumulation in the vessel wall during the
development of plaque. Other workers have shown that the expression
of this molecule is regulated by an oxidant-responsive
transcriptional pathway mediated by the transcriptional factor
NF.kappa.B. Endothelial cells exposed to oxidized LDL cholesterol
(or cytokines like TNF-.alpha.) begin to generate superoxide anion.
Superoxide anion turns on oxidant-responsive transcription leading
to the expression of VCAM-1 and MCP-1 (and probably other genes
that participate in atherosclerosis). Our data indicates that NO
inhibits the generation of superoxide anion, thereby turning off
these oxidant-responsive transcriptional pathways.
[0099] Methods and Results: Confluent monolayers of human aortic
endothelial cells (HAEC) were exposed to static or fluid flow
conditions for 4 hours (fluid flow stimulates the production of
endogenous nitric oxide). Medium was then replaced and cells were
then incubated with native LDL (50 .mu.g/ml), oxidized LDL (30
.mu.g/ml), or LPS (10 ng/ml)+TNF-.alpha. (10 U/ml) for an
additional 4 hours. Functional binding assays utilizing THP-1
monocytes were then performed. Superoxide production by HAECs was
monitored by lucigenin chemiluminescence and expression of the
adhesion molecules VCAM-1 and ICAM-1 was quantitated by flow
cytometry. Whereas native LDL had little effect, incubation with
either oxLDL or LPS/TNF significantly increased superoxide
production, NF-.kappa.B activity, VCAM-1 expression and endothelial
adhesiveness for monocytes. Previous exposure to fluid flow
inhibited endothelial adhesiveness for monocytes (FIG. 6) and the
other sequelae of exposure to cytokines or oxidized lipoprotein.
The effect of fluid flow was due to shear-induced release of nitric
oxide since coincubation with L-nitro-arginine completely abolished
these effects of flow. Furthermore, the NO donor PAPA-NONOate
mimicked the effects of flow.
[0100] Conclusions. Previous exposure to fluid flow decreased
cytokine or lipoprotein-stimulated endothelial cell superoxide
production, VCAM-1 expression and monocyte binding; the effects of
flow are due at least in part to nitric Oxide. NO participates in
the regulation of the endothelial generation of superoxide anion
and thereby inhibits oxidant-responsive transcription of genes
(i.e. VCAM-1 and MCP-1) that are involved in atherogenesis.
EXAMPLE 9
Transfection of the Gene Encoding NO Synthase Increases NO
Generation and Inhibits Monocyte Adherence
[0101] The following experiment was done to determine if transfer
of the gene encoding NO synthase (the enzyme that produces NO)
could increase generation of nitric oxide and thereby inhibit
monocyte adherence. Cultured endothelial cells (bEnd-3; a murine
endothelial cell line) were transfected with a plasmid construct
encoding the NO synthase gene, using lipofectamine liposomal
technique. Forty-eight hours later, generation of nitric oxide was
measured using chemiluminescence. Nitric oxide generation was
increased 2-fold in cells transfected with the NO synthase
construct (but not in cells transfected with a control construct).
In parallel, binding assays were performed using a murine
monocytoid cell line. The binding of monocytoid cells to the
endothelial cells was reduced by 30% in those cells transfected
with the NO synthase construct.
[0102] Conclusion: endothelial cells transfected with a plasmid
construct containing the NO synthase gene were able to elaborate
more nitric oxide. The increased elaboration of nitric oxide was
associated with an inhibition of monocyte binding to the
endothelial cells.
EXAMPLE 10
Effect of NO Synthase Expression on Proliferation of Vascular
Smooth Muscle Cells
[0103] Cultured rat aortic vascular smooth muscle cells under
confluent quiescent conditions were studied. An efficient viral
coat protein-mediated DNA transfer method was employed to transfect
the cells with the NO synthase gene driven by the .beta.-actin
promoter and CMV enhancer. This resulted in increased NO synthase
activity (as measured by the arginine-to-citrulline conversion
assay) in comparison to control vector transfected cells.
Transfection of the NO synthase gene completely abolished
serum-stimulated DNA synthesis compared to control vector
transfection. These results indicated that increased expression of
NO synthase (associated with increased production of NO) inhibits
excessive proliferation of vascular smooth muscle cells. This
inhibition can be correlated with treatment of atherosclerosis and
restenosis.
EXAMPLE 11
Gene Therapy Using NO Synthase cDNA Prevents Restenosis
[0104] The study above indicated that NO inhibits proliferation of
vascular smooth muscle cells. In atherogenesis and restenosis,
excessive proliferation of vascular smooth muscle cells contributes
to lesion formation. Injury to the endothelium in atherosclerosis
and after catheter interventions apparently reduces or removes the
salutary influence of NO. The following study shows delivery of the
gene for NO synthase to the vessel wall-inhibits lesion
formation.
[0105] A plasmid construct encoding the cDNA of endothelial-type NO
synthase (EC-NOS) was synthesized. A full length cDNA encoding for
EC-NOS was inserted into the EcoRI site of the pUCcaggs expression
vector. Balloon angioplasties of the carotid artery in
Sprague-Dawley rats were performed and HVJ-liposomes with plasmids
encoding EC-NOS cDNA infused, or plasmids lacking EC-NOS cDNA
(control vector) infused. After 4 days to 2 weeks, the rats were
sacrificed and the carotid arteries harvested for: 1)
histomorphometry; 2) measurement of DNA synthesis; and 3) ex vivo
determination of NO synthesis and release by bioassay and by
chemiluminescence.
[0106] Results. Morphometric measurements 2 weeks after injury
revealed a significant (68%) reduction of intimal lesion thickness
in EC-NOS treated (Inj+NOS) in comparison to control vector treated
(Inj+CV) or untreated (Inj) injured vessels. (FIG. 7) Measurements
of DNA synthesis were performed four days after injury using
bromodeoxyuridine. EC-NOS transfection significantly limited
bromodeoxyuridine incorporation (by 25%) in comparison to control
vector treated or untreated injured vessels. Vessel segments were
studied ex vivo using organ chamber technique to bioassay for NO
release. Calcium ionophore increases intracellular calcium and
activates NO synthase to produce NO. Calcium ionophore induced
relaxations in injured carotid arteries transfected with control
vector that were only 15% of uninjured vessels. Injured arteries
that had been transfected with EC-NOS relaxed to a much greater
degree, approximately 50% of that observed in uninjured vessels.
Direct measurement of NO (by chemiluminescence) released into the
medium revealed that NO released by injured tissues (transfected
with the control vector) was only 20% of that released by normal
uninjured tissues. By contrast, injured tissues transfected with
EC-NOS released more NO (about 75% of normal).
[0107] To conclude, balloon angioplasty of the rat carotid artery
removes the endothelial source of NO, induces excessive vascular
smooth muscle DNA synthesis and proliferation, resulting in an
intimal lesion (restenosis). Transfection of the vessel with EC-NOS
at the time of balloon injury partially restores NO production by
the vessel, and this is associated with reduced DNA synthesis and
vascular smooth muscle proliferation, thereby reducing lesion
formation. These results are consistent with the conclusion that NO
is an endogenous anti-atherogenic molecule.
EXAMPLE 12
Local Application of L-arginine to the Vessel Wall Inhibits
Myointimal Hyperplasia
[0108] The previous studies revealed that oral administration of
arginine could enhance vascular NO activity and inhibit lesion
formation induced by a high cholesterol diet and/or vascular injury
(with balloon angioplasty). To determine if intraluminal
application of arginine to the vessel wall at the time of balloon
angioplasty could inhibit lesion formation, the following study was
performed. Rabbits (n=7) were fed a 1% cholesterol diet. After one
week, angioplasty of the iliac arteries was performed. After
angioplasty of one iliac artery, a local infusion catheter was used
to expose the injured area to a high concentration of arginine (6
mM). The other iliac artery was subjected to balloon angioplasty,
but not treated with a local infusion. After four weeks, the
vessels were harvested, and segments of the arteries processed for
histomorphometry. Initial thickening in the arginine-treated
vessels was significantly reduced (FIG. 8). This study indicates
that the local intraluminal application of high doses of arginine
can reduce myointimal hyperplasia after vascular injury.
EXAMPLE 13
Exclusion of the Effect of Enhanced Nitrogen or Caloric Balance as
Causing the Observed Results
[0109] To exclude an effect of L-arginine on nitrogen or caloric
balance as the cause of these results, six animals received 1%
cholesterol diet supplemented by additional methionine to increase
the dietary methionine six-fold. At ten weeks animals were
sacrificed for studies of platelet and vascular reactivity, and
histomorphometry. Endothelium-dependent relaxation, platelet
aggregation and intimal thickness were not different from those of
animals fed 1% cholesterol diet alone. These results reveal that
another amino acid, methionine (which is not a precursor of NO)
does not mimic the effect of the amino acid L-arginine. Therefore
it seems likely that the effect of L-arginine is due to its
metabolism to nitric oxide, rather than some other effect of amino
acid administration (i.e. change in nitrogen or caloric
balance).
EXAMPLE 14
L-lysine Enhances Vascular NO Activity and Inhibits
Atherogenesis
[0110] L-lysine is a basic amino acid like L-arginine, but is not
known to be metabolized by NO synthase to NO. Therefore, the
following results were unexpected. New Zealand white rabbits were
fed a normal or high cholesterol chow (n=18). Half of the animals
on the cholesterol diet also received oral L-lysine. After ten
weeks, the thoracic aortae were harvested and bioassayed for
vascular NO synthesis, and histomorphometry to assess lesion
formation was performed as described above. The administration of
L-lysine was just as effective as L-arginine to increase vascular
NO activity in the hypercholesterolemic animals as assessed by
endothelium-dependent vasorelaxation. (FIG. 9) The improvement in
vascular NO activity was associated with a marked reduction in
vascular lesion formation.
[0111] This study revealed the unexpected result that L-lysine can
enhance vascular NO activity and inhibit atherosclerosis.
EXAMPLE 15
Oral L-arginine Normalizes Monocyte Adhesiveness in
Hypercholesterolemic Humans
[0112] Adherence of monocytes to the endothelium is the first
observable event in the development of atherosclerosis. We
hypothesized that chronic oral administration of L-arginine to
hypercholesterolemic humans would enhance the generation of
endothelium-derived NO, and thereby inhibit the interaction of
monocytes with the endothelium. In this investigation we have
developed a reproducible assay for the binding of human monocytes
to cultured endothelial cells, and we have examined the effect of
hypercholesterolemia and L-arginine treatment on this
interaction.
[0113] The control subject population in this study included 12
normal volunteers, (10 males and 2 females), with an average age of
37.+-.2 yrs. Normalcy was determined by a careful history, physical
examination, and laboratory analysis to exclude individuals with
hematologic, renal, or hepatic dysfunction or clinically evident
atherosclerosis. There were 20 patients (10 males and 10 females)
with hypercholesterolemia as defined by a total plasma cholesterol
greater than 240 mg/dl and a LDL cholesterol level greater than 160
mg/dl. These individuals had an average age of 51.+-.2 yrs. None of
the subjects were taking diuretics, vasoactive medications,
antiplatelet or hypolipidemic medications. This study was approved
by the Stanford University Administrative Panel on Human Subjects
in Medical Research and each subject gave written informed consent
before entry into the study. Blood was drawn from each subject in
the postabsorptive state.
[0114] We isolated human monocytes from citrated venous blood. The
blood was centrifuged and the buffy coat removed and resuspended
with HBSS. The suspension was then carefully layered onto a cushion
of 1.068-d Histopaque, and centrifuged. After centrifugation, the
monocytes were aspirated.
[0115] We used the transformed endothelial cell (EC) line, bEnd3 to
examine monocyte-endothelial binding ex vivo. The bEnd3 cells
express endothelial adhesion molecules and bind monocytes in a
cytokine-inducible fashion with kinetics similar to those observed
with human umbilical vein endothelium. Monocytes were added to the
wells containing the endothelial monolayers to reach a final cell
number of 3.times.10.sup.6/ml. In some studies, monocytes were
exposed in vitro for 30 minutes to sodium nitroprusside (an NO
donor) prior to the binding assay.
[0116] The six-well plates were transferred to a rocking platform
and rocked for 30 minutes at room temperature. After 30 minutes,
the cell suspension was aspirated from each well and wells were
then rinsed with binding buffer to remove nonadherent monocytes.
Videomicroscopic counting of adherent cells was performed using a
computer aided image analysis system.
[0117] Results. Oral administration of L-arginine (7 g daily for 2
weeks) to hypercholesterolemic humans increased plasma arginine
values by 60% (from 79.+-.10 to 128.+-.12 mM; n=7), whereas
L-arginine values in the placebo-treated (n=3) and
normocholesterolemic (n=6) groups remained unchanged. The
administration of oral L-arginine had no effect on any of the
biochemical or hematologic parameters and was well tolerated. Oral
L-arginine did not lower total cholesterol or LDL cholesterol. Two
patients dropped out of the study; one because he did not want to
take the pills, and one because of reactivation of oral herpes
during the study.
[0118] The results of the adhesion assays were highly reproducible.
Monocytes derived from hypercholesterolemic individuals
demonstrated a 50.+-.8% increase in bound cells/hpf in comparison
to cells from normal individuals (p<0.0001). The degree of
adhesiveness was correlated to the plasma levels of LDL cholesterol
(R=0.7, n=33; p<0.0001; FIG. 10).
[0119] In an open-label study, 3 hypercholesterolemic individuals
were treated with oral L-arginine supplementation for 2 weeks.
Arginine treatment resulted in a 38% decrease in monocyte
adhesiveness.
[0120] To confirm this effect of L-arginine treatment and to
control for any experimental bias, a double-blinded,
placebo-controlled, randomized study was performed. Ten
hypercholesterolemic subjects were randomized (1:2) to placebo or
L-arginine treatment; 6 normocholesterolemic individuals were
studied in parallel to control for variation over time in the
binding assay. At baseline, the adhesion of monocytes from both
hypercholesterolemic groups was increased in comparison to the
normocholesterolemic individuals (p<0.001). After 2 weeks of
L-arginine administration, there was an absolute reduction of 53%
in monocyte binding (n=7, p<0.005, baseline vs 2 weeks) (FIG.
11). By contrast, there was no significant change in the
adhesiveness of monocytes isolated from hypercholesterolemic
individuals treated with placebo. Two weeks after discontinuation
of the L-arginine treatment, the adhesiveness of the monocytes
isolated from hypercholesterolemic individuals had significantly
increased compared to the normocholesterolemic individuals
(34.+-.9% increase in bound cells/hpf; p<0.05), and was also
significantly increased in comparison to the binding obtained after
2 weeks of L-arginine therapy (an increase of 30.+-.9%, p<0.05).
The adhesiveness of monocytes from placebo-treated
hypercholesterolemic individuals did not change significantly
during the washout period.
[0121] In some studies monocytes were exposed to sodium
nitroprusside or vehicle control for 30 minutes in vitro.
Pre-incubation of the cells from hypercholesterolemic individuals
with the NO donor sodium nitroprusside (10.sup.-5 M) markedly
reduced binding(164.+-.9% vs 98.+-.7% vehicle vs sodium
nitroprusside; n=7, p<0.0005; values expressed as a percent of
the normocholesterolemic control exposed to vehicle; FIG. 12).
[0122] To conclude, the salient findings of this investigation are
that: 1) Hypercholesterolemia enhances the adhesiveness of
monocytes for endothelial cells, 2) oral arginine supplementation
reverses the increase in adhesiveness of monocytes from
hypercholesterolemic individuals, and 3) the effect of oral
arginine is mimicked in vitro by exposure of the monocytes from
hypercholesterolemic individuals to sodium nitroprusside, an NO
donor.
EXAMPLE 16
Platelet Hyperaggregability in Hypercholesterolemic Humans:
Reversal by Oral L-Arginine
[0123] In this study we tested the hypothesis that chronic
L-arginine supplementation would inhibit platelet reactivity in
hypercholesterolemic humans. Venous blood was collected from normal
(NC; n=11) and hypercholesterolemic (HC; n=22) volunteers for
isolation of platelet-rich plasma and aggregometry. Half the HC
group received L-arginine (7 g/d) for 2 weeks; aggregometry was
performed using collagen (5 mg/ml) before and after two weeks of
treatment.
[0124] Results: HC platelets were hyperaggregable. After two weeks
of L-arginine, the aggregability of HC platelets was reduced (FIG.
13). These studies are consistent with our previous observations in
animals that oral administration of L-arginine inhibits platelet
reactivity.
EXAMPLE 17
Intravenous Administration of L-Arginine Improves
Endothelium-Dependent Vasodilation in Hypercholesterolemic
Humans
[0125] Hyperlipoproteinemia impairs endothelium-dependent
vasodilation, even before the development of atherosclerosis. We
hypothesized that administration of L-arginine may increase
synthesis of NO and thereby improve endothelium-dependent
vasodilation in hypercholesterolemia. Indeed, our earlier studies
conducted in cholesterol-fed rabbits support this notion. The
following data demonstrates that L-arginine augments
endothelium-dependent vasodilation in forearm resistance vessels of
hypercholesterolemic humans.
[0126] The control subject population in this study included 11
normal volunteers comprising (10 males and 1 female). Their ages
ranged from 31 to 49 and averaged 39.+-.2 yr. There were 14
patients with hypercholesterolemia. Hypercholesterolemia was
defined as a serum LDL cholesterol level greater than the 75th
percentile adjusted for age and sex. These individuals included 11
males and 3 females whose ages ranged from 22 to 48 and averaged
38.+-.2 years.
[0127] Under local anesthesia and sterile conditions, a
polyethylene catheter was inserted into a brachial artery of each
subject for determination of blood pressure and for infusion of
drugs. A separate polyethylene catheter was inserted into the
antecubital vein for infusion of L-arginine. Bilateral forearm
blood flow was determined by venous occlusion strain gauge
plethysmography, using calibrated mercury-in-silastic strain
gauges, and expressed as ml/100 ml tissue per min.
[0128] To assess NO-dependent vasodilation, methacholine chloride
(which induces the endothelium to release NO) was administered via
the brachial artery. Forearm blood flow was measured during
infusion of methacholine chloride at concentrations of 0.3, 3, and
10 .mu.g/min each for 3 min.
[0129] After completion of the methacholine chloride infusions, all
normal subjects and 10 individuals with hypercholesterolemia were
given L-arginine intravenously over 30 minutes and then the
methacholine infusions were repeated. D-arginine, the enantiomer of
L-arginine, is not a precursor of NO. Thus, to ensure that any
observed effects of L-arginine were due to its contribution to the
synthesis of NO and not just secondary to its physiochemical
properties, five individuals with hypercholesterolemia received
D-arginine intravenously.
[0130] Results. Baseline blood pressure, heart rate, and forearm
blood flow did not differ between normal and hypercholesterolemic
subjects. Intraarterial infusion of methacholine chloride caused a
dose-dependent increase in forearm blood flow. In the
hypercholesterolemic subjects, however, cholinergic vasodilation
was less than that of normal subjects (p<0.05). The maximal
forearm blood flow response to methacholine in normal subjects is
19.0.+-.1.9 ml/100 ml of tissue per min, and in
hypercholesterolemic subjects, it was 13.7.+-.1.7 ml/100 ml of
tissue per min (p<0.05).
[0131] In the normal subjects, L-arginine did not potentiate the
vasodilation that occurred during the administration of
methacholine chloride. In the hypercholesterolemic subjects,
however, the L-arginine infusion augmented the vasodilation to
methacholine chloride by 25% (p<0.05). There were no
complications or side-effects of the L-arginine infusions.
[0132] The important findings in this study are: (a)
endothelium-dependent vasodilation (due to the release of NO) is
reduced in forearm resistance vessels of hypercholesterolemic
humans; and (b) intravenous administration of L-arginine improves
endothelium-dependent vasodilation in these individuals. NO not
only causes vasodilation, but it also inhibits platelet aggregation
and suppresses monocyte adhesion in hypercholesterolemic
humans.
EXAMPLE 18
Administration of Intravenous L-Arginine Improves Coronary
Endothelial Function in Cardiac Transplant Recipients
[0133] A reduction in coronary NO-dependent vasodilation occurs in
cardiac transplant recipients and may represent an early marker for
the development of graft atherosclerosis. Reduced NO-dependent
vasodilation in response to acetylcholine is an indicator of
endothelial dysfunction and has been attributed to reduced
synthesis or accelerated degradation of endothelium-derived nitric
oxide. We hypothesized that endothelial dysfunction of epicardial
coronary arteries at an early stage of coronary allograft
atherosclerosis might be reversed by L-arginine. The present study
tested the hypothesis that administration of L-arginine, the
precursor of endothelium-derived NO, improves endothelial
vasodilator function of coronary conduit and resistance
vessels.
[0134] Cardiac transplant recipients scheduled for elective annual
coronary angiography at Stanford University hospital were screened
for possible participation in the study. The study protocol was
approved by the Stanford University Committee on Human Subjects in
Medical Research. All patients gave written informed consent.
Eighteen patients who had cardiac transplantation 1 to 13 years
previously were studied.
[0135] Vasoactive medications were discontinued at least 12 hours
before the study. After diagnostic angiography revealed no visually
apparent coronary stenosis, a guiding catheter was used to
cannulate the left main coronary artery. An infusion catheter was
then advanced over a Doppler flow velocity guide wire into a
nonbranching segment of the coronary artery for infusion of
acetylcholine (which stimulates the endothelium to release NO).
After baseline angiography was performed, increasing concentrations
of acetylcholine were serially infused over 3 minutes. Infusion of
acetylcholine continued until the maximum dose (10.sup.-4 mol/L)
was reached or until total coronary occlusion occurred. Then an
intravenous infusion of L-arginine (30 g over 15 minutes) was
performed. Thereafter, the intracoronary infusion of acetylcholine
was repeated. Coronary angiography and Doppler flow velocity
recording was performed at the end of the L-arginine infusion and
after the infusion of each concentration of acetylcholine.
[0136] Results. In epicardial coronary arteries of these transplant
recipients, acetylcholine caused vasoconstriction. Epicardial
coronary vasoconstriction caused by acetylcholine was attenuated by
infusion of L-arginine (10.sup.-4 mol /L, -6.8% versus -2.8%;
p<0.01). In coronary resistance vessels, acetylcholine induced
vasodilation, reflected by increases in blood flow. The increase in
coronary blood flow was significantly enhanced with L-arginine
(p<0.002; FIG. 14). There were no complications or side-effects
of the L-arginine infusion.
[0137] The coronary vasculature of cardiac transplant recipients
exhibits a generalized reduction of NO-dependent vasodilation.
L-arginine improves endothelial-derived NO dependent vasodilation
of both coronary microvasculature and epicardial coronary
arteries.
[0138] It is evident from the above results, that by enhancing the
nitric oxide levels, by means of nitric oxide precursor compounds
or other compounds in the nitric oxide pathway, substantial
benefits will ensue to patients with vascular degenerative
diseases. This treatment will restore normal vascular tone
(preventing excessive vasoconstriction and elevation of blood
pressure; and will improve blood flow to the heart, brain, and
other critical tissues thereby enhancing exercise tolerance and
relieving symptoms such as angina or cerebral ischemia); and will
diminish the formation of atherosclerotic plaque and restenosis (by
inhibiting adhesion of monocytes and platelets, and by reducing the
proliferation of vascular smooth muscle cells). Benefits may also
ensue to normal individuals, ecause NO is critically involved in
exercise-mediated vasodilation, an enhancement of NO synthesis
could improve blood flow and exercise capacity even in normal
individuals.
[0139] By virtue of administering to the host, based on a
predetermined regimen, or providing in the host a supply of a
component in the synthetic pathway for production of nitric oxide,
so as to maintain a mildly elevated level of nitric oxide in the
host, particularly at the site to be treated, the incidence of
plaque formation can be substantially diminished. This can be
achieved in a variety of ways: by oral administration in accordance
with a predetermined regimen of various compounds associated with
nitric oxide formation, e.g. L-arginine and/or L-lysine; by
administration at the site, in a predetermined regimen of compounds
which can produce nitric oxide, either directly or as a result of
physiologic action of endogenous compounds, e.g. enzymes; by
employing combinations of compounds, which by their action result
in the production of nitric oxide; or the like. These individual
administrations, can be done independently or in conjunction with a
regimen of other compounds associated with the production of nitric
oxide.
[0140] Alternatively, one may use genetic engineering to introduce
a gene associated with a component in the synthetic pathway for
production of nitric oxide, e.g. nitric oxide synthase, where the
enhanced production of such compounds will have the effect of
driving the equilibrium to an enhanced production of nitric oxide.
Thus, the subject invention provides a plurality of pathways to
enhance the synthesis or action of nitric oxide, or reduce the
degradation of nitric oxide, thereby increasing the effect of
endogenous nitric oxide to prevent the formation of vascular
lesions and to inhibit restenosis.
[0141] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0142] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
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