United States Patent (19)
Cooke et al.
US 005428070A
(11) Patent Number: 5,428,070
(45) Date of Patent: Jun. 27, 1995
(54) TREATMENT OF VASCULAR
DEGENERATIVE DISEASES BY MODULATION OF ENDOGENOUS NITRIC OXIDE PRODUCTION OF ACTIVITY
(75) Inventors: John P. Cooke, Palo Alto; Victor J. Dzau, Los Altos Hills; Gary H. Gibbons, Palo Alto, all of Calif.
(73) Assignee: The Board of Trustees of the Leland Stanford Jr. Univ., Stanford Calif.
(21) Appl. No.: 76,312
(22) Filed: Jun. 11, 1993
(51) Int. Cl.6 ………A01N 37/00; A61K 31/19
(52) U.S. Cl. ……………….514/557; 514/310
(58) Field of Search ……………514/310, 557
(56) References Cited
PUBLICATIONS
Andrews et al., “Low-density Lipoproteins inhibit endothelium-dependent Relaxation in Rabbit Aorta”, Nature, 327:231-239 (1987).
Bath et al., “Nitric Oxide and Prostacyclin: Divergence of Inhibitory Effects on Monocyte Chemotaxis and Adhesion to Endothelium in Vitro”, Arteriosclerosis and Thrombosis, 11(2):254-260 (1991).
Cooke, “Endothelial Dysfunction in Disease States”, Current Opinion in Cardiology, 5:637-644 (1990).
Drexler et al., “Correction of Endothelial Dysfunction in Coronary Microcirculation of Hypercholesterolemic Patients”, The Lancet, 338:1546-1550 (1991).
Garg and Hassid, “Niitric Oxide-generating Vasodilators and 8-Bromo-Cyclic Guanosine Monophosphate Inhibit Mitogenesis and Proliferation of Cultured Rat Vascular Smooth Muscle Cells”, J. Clin. Invest., 83:1774-1777 (1989).
Girerd et al., “L-Arginine Augments Endothelium-Dependent Vascodilation in Cholesterol-Fed Rabbits”, Circulation Research, 67(6):1301-1308 (1990).
Heistad et al., “Augmented Responses to Vasoconstrictor Stimuli in Hypercholesterolemic and Atherosclerotic Monkeys”, Circulation Research, 54(6)711-718 (1984).
Kubes et al., “Nitric Oxide: An Endogenous Modulator of Leukocyte Adhesion”, Proc. Natl. Acad. Sci. USA, 88:4651-4655 (1991).
Kugiyama et al., “Impairment of Endothelium-dependent Arterial Relaxation by Lysolectithin in Modified Low-density Lipoproteins”, Nature, 344:160-162 (1990).
Kuo et al., “Pathophysiological Consequences of Atherosclerosis Extend Into the Coronary Microcirculation: Restoration of Endothelium-dependent Responses by L-Arginine”, Circulation Research, 70(3):465-476 (1992).
Lefer et al., “Role of Endothelium-derived Relaxing Factor as a Cardioprotective Agent in Myocardial Ishemia”, Basil Karger, pp. 190-197 (1990).
Minor et al., “Diet-induced Atherosclerosis Increases the Release of Nitrogen Oxides from Rabbit Aorta”, J. Clin. Invest., 86:2109-2116 (1990).
(List continued on next page.)
Primary Examiner – Werren B. Lone
Attorney, Agent, or Firm – Bectram I. Rowland
(57) ABSTRACT
Atherogenesis and restenosis are treated by long term administration of physiologically acceptable compounds which enhance the level of endogenous nitric oxide 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. In addition, cells may be genetically engineered to provide a component in the synthetic pathway to nitric oxide, so as drive the process to enhance nitric oxide concentration, particularly in conjunction with the administration of a nitric oxide precursor.
5 Claims, No Drawings
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Page 2
PUBLICATIONS
Mitchell et al., “Native LDL Inhibits the Release of Endothelial Derived Relaxing Factor by Reducing the Activity of Endothelial Nitric Oxide Synthase”, (Abstract), J. Vasc. Res., 29:169 (1992).
Pohl and Busse, “EDRF Increases Cyclic GMP in Platelets During Passage Through the Coronary Vascular Bed”, Circulation Research, 65(6):1798-1803 (1989).
Radomski et al., “Comparative Pharmacology of Endothelium-derived Relaxing Factor, Nitric Oxide and Prostacyclin in Platelets”, Br. J. PHarmacol., 92:181-187 (1987).
Ross, “The Pathogenesis of Atherosclerosis – an Update”, The New England Journal of Medicine, 314(8):488-500 (1986).
Rossitch, Jr. et al., “L-Arginine Normalizes Endothelial Function in Cerebral Vessels from Hypercholesterolemic Rabbits”, J. Clin. Invest., 87:1295-1299 (1991).
Stamler et al., “N-Acetylcysteine Potentiates Platelet Inhibition by Endothelium-derived Relaxing Factor”, Circulation Research, 65(3):789-795 (1989).
Tanner et al., “Oxidized Low Density Lipoproteins Inhibit Relaxations of Porcine Coronary Arteries: Role of Scavenger Receptor and Endothelium-derived Nitric Oxide”, Circulation, 83(6):2109-2116 (1991).
Tomita et al., “Rapid and Reversible Inhibition by Low Density Lipoprotein of the Endothelium-dependent Relaxation to Hemostatic Substances in Porcine Coronary Arteries”, Circulation Research, 66(1):18-27 (1990).
Weidinger et al., “Persistent Dysfunction of Regenerated Endothelium After Balloon Angioplasty of Rabbit Iliac Artery”, Circulation, 81(5):1667-1679 (1990).
Yamamoto et al., “Videomicroscopic Demonstration of Defective Cholinergic Arteriolar Vascodilation in Atherosclerotic Rabbit”, J. Clin. Invest., 81:1752-1758 (1988).
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1
TREATMENT OF VASCULAR DEGENERATIVE DISEASES BY MODULATION OF ENDOGENOUS NITRIC OXIDE PRODUCTION OF ACTIVITY
INTRODUCTION
This invention was supported in part by the United States Government under Grant 1KO7HCO266((NH-LBI). The U.S. Government may have an interest in this application.
TECHNICAL FIELD
The field of this invention is the treatment of vascular degenerative diseases, particularly atherosclerosis and restenosis.
BACKGROUND
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 form cells distort and eventually may even rupture through the endothelium.
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.
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 with three to six months after the angioplasty in a significant proportion of patients (30-40%).
Because of their great prevalence and serious consequences, it is critically important to find therapies which can diminish the incidence of atherosclerosis, vascular thrombosis and restenosis. Ideally, such therapies would inhibit the pathological processes associated with atherosclerosis, thereby providing prophylaxis or retarding the progression of the degenerative process.
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 up- 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 a 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 precursor.) This substance has been reported to relax vascular smooth muscle, inhibit platelet aggregations, inhibit mitogenesis and proliferation of cultured vascular smooth muscle,
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and leukocyte adherence. NO may have other
effects, either direct or indirect, on the various cells associated with vascular walls and degenerative diseases of the vessels.
Relevant Literature
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. Rositch, 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.
Creager, at al. (1992) J. Clin. Invest. 90, 1248-1253, report that intravenous administration of L-arginine improves endothelium-derived NO-dependent vasodilation in hypercholesterolemic patients.
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.”
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.
Cooke (1992) J. Clin. Invest. 90, 1168-1172, describe the effect of chronic administration of 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.
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.
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.
Other references which refer to activities attributed to NO or its precursor include: Pohl and Busse (1989) Circ. Res. 65:1798-1803; Radomski et al. (1987) Br. J. Pharmacol. 92:181-1187; and Stamler et al. (1989) Circ. Res. 65:795; anti-platelet activity); Garg and Hassid (1989) J. Clin Invest. 83:1774-1777; and 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 adhesions and migration); Heistad et al. (1984) Circ. Res. 43: 711-718; Rossitch et al. (1991) J. Clin Invest. 87:1296-1299; 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) 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; and Nakanishi et. Al. (1991) Lancet 338:1546-1550; and Nakanishi et al. (1992) In: Scientific Conference on Functional and Structural Mechanisms of Vascular Control, Snowbird, ET, p. 86 (abstsr.); relation of L-arginine to NO-dependent vasodilation.
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SUMMARY OF THE INVENTION
Atherosclerosis and restenosis are treated with agents that enhance nitric oxide formation. The enhancement of endogenous nitric oxide formation inhibits the progression of restenosis and atherosclerosis. As a prophylaxis or treatment for 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 as a dietary supplement with increase NO elaboration and thereby diminish the effects of atherogenesis. Other techniques to enhance NO production 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.
DESCRIPTION OF SPECIFIC EMBODIMENTS
In accordance with the subject invention, common vascular degenerative diseases such as atherosclerosis, vascular thrombosis, and restenosis, are treated prophylactically and/or therapeutically by maintaining an enhanced level of nitric oxide or its precursor in the vessel wall in accordance with a predetermined regimen over an extended period of time. The enhanced level 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. The enhanced level of nitric oxide may be a result of administration to the patient of an intermediate in the metabolic pathway to the production of nitric oxide (or its physiological equivalent), the enhanced levels of an enzyme associated with the production of nitric oxide, or a physiologically acceptable precursor, which may lead directly or indirectly, to formation of nitric oxide.
One approach is to employ L-arginine as a dietary supplement. This amino acid may be administered as any physiologically acceptable salt, such as the hydrochloride sale, glutamate sale, etc. It may also be administered as a peptide (i.e. poly-L-arginine) so as to increase plasma levels of the NO precursor. Naturally occurring sources include protamine. The administration of L-arginine or other convenient NO precursor 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, as a chronic treatment, and could last 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 or biologically equivalent compound which is used would generally provide a plasma level in the range of about 0.2 mM to 30 mM. The oral administration of L-arginine can be achieved by providing L-arginine 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 for a human host will be about 1 to 12 g per day.
The administration of L-arginine may be administered prophylactically, so as to inhibit atherogenesis or restenosis, or therapeutically after atherogenesis has been initiated. Thus, for example, a patient who is to undergo balloon angioplasty may have a regimen of L-arginine administered substantially prior to the balloon angioplasty, preferably at least about a week or substantially longer. Alternatively, in a patient where atherogenesis is suspected, the administration of L-arginine may begin at any time. Of particular interest is the incorporation of L-arginine as a supplement in a food, such as a health bar, e.t. granola, other grains, fruit bars, such as a date bar, fig bar, apricot bar, or the like. The amount of L-arginine or the equivalent would be about 2-25 g per dosage or bar, preferably about 3-15 g.
Instead of oral administration, intravascular administration may 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
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combinations of oral and parenteral administrations may be employed in accordance with the needs of the patient. Furthermore, parenteral administration may allow for the administration of compounds which would not readily be transported across the mucosa from the gastrointestinal tract into the vascular system.
For intravascular administration, a wide variety of individual or combinations of physiologically acceptable compositions may be employed, which may be provided systemically or in proximity to a lesion site. Thus, one may provide for combinations of peroxy compounds or other oxygen containing oxidants, where the odxidant is physiologically acceptable, in conjunction with a nitrogen source, such as substituted guanidines, e.g. L-arginine, amidines, or the like. Alternatively, one may reduce the degradation of endogenous nitric oxide using antioxidants (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. Thiol compounds may also find application, as well as their derivatives, such as disulfides, sulfonic acids, thiol esters, thiono thiol esters, and the like.
Other compounds which may find use include partially oxidized nitrogen compounds, such as hydroxylamines, oxazoles, oxazines, nitroso compounds, or the like. Physiologically acceptable stable free radical compounds, such as nitroxyl compounds, may find use, where the unpaired electron may be nitrogen or oxygen, analogous to NO. These compounds will be, for the most part, synthetic organic compounds generally having a molecular weight of at least about 100 and usually not more than about 2000 D.
Other compositions which may find use include nitrites, including nitrite esters, e.g. esters of carbonate, thiocarbonate, etc. These compositions may be administered at the site of a lesion to provide for rapid enhancement of nitric oxide concentration, so as to imitiate or inhibit the various physiological processes affected by the level of nitric oxide present and associated with plaque formation or restenosis. Particularly, processes associated with the vascular smooth muscle (“VSM”) cell proliferation and invasion of the endothelial layer can be modulated.
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 may enhance the amount of nitric oxide synthetase 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 may 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.
Alternatively, cells may be genetically engineered to provide for constitutive or inducible expression of the synthase. Thus, expression vectors (viral or plasmid) may be prepared which contain the NO synthase gene and which can be introduced into hose cells which will then produce high concentrations of nitric oxide. These cells may be introduced at the lesion site or at another site in the host, where the increased NO synthase activity will maintain an elevated level of NO in the vascular system.
Cultured cells can be transfected with expression vectors containing the NO synthase 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 can be employed to transfect vascular cells within the intact vessel in vivo. The NO synthase gene can be expressed at high constitutive levels or it can be linked to an inducible promoter (which may have tissue specificity) to allow for regulation of NO synthase expression.
DNA constructs are prepared, where a NO synthase gene is joined to an appropriate promoter, either with its native termination region or a different termination region, which may 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 may include the B-actin promoter, or cell-type specific promoters.
A construct is prepared in accordance with conventional techniques, the various DNA
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fragments being introduced into an appropriate plasmid or viral vector, normally a vector capable of replication in a bacterial and/or eucaryotic host. Alternatively, the vector will normally 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 may also be present in the vector.
Once the vector has been prepared and replicated, it may then be used for introduction into host cells. The plasmid vector construct may be further modified by being joined to viral elements which allow for ease of transfection, may provide a marker for selection, e.g. antibiotic resistance, or other functional elements. Introduction of the plasmid vector construct into the host cells may be achieved by calcium phosphate precipitated DNA, transfection, electroporation, fusion, lipofection, viral capsid-mediated transfer, or the like. Alternatively, the NO synthase construct within viral vectors may be introduced by standard infection techniques. For somatic cell gene therapy, autologous cells will generally be employed, although it some instances allogeneic cells or recombinantly modified cells may be employed. Usually the cells employed for genetic modification will be mature endothelial or vascular smooth muscle cells. For example, myoblasts may be employed for muscle cells or hematopoietic stem cells or high proliferative potential cells may be employed for lymphoid and/or myelomonocytic cells.
Depending upon the nature of the cells, they may be injected into tissue of the same or different cellular nature, they may be injected into the vascular system, where they may remain as mobile cells or home to a particular site (i.e. the lesion). The number of cells which are administered will depend upon the nature of the cells, the level of production of the synthase, the desired level of NO synthase in the host vascular system, at the lesion site, or the like, whether the enhanced level of 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.