A Unified Theory of Human Cardiovasc... 
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A Unified Theory of Human Cardiovascular Disease Leading the Way to the Abolition of This Disease as a Cause for Human Mortality
Matthias Rath M.D. and Linus Pauling Ph.D
"An important scientific innovation rarely, makes its way by gradually winning over and converting its opponents. What does happen is that its opponents gradually die out and that the growing generation is familiar with the idea from the beginning."
-Max Planck
This paper is dedicated to the young physicians and the medical students of this world
Abstract
Until now therapeutic concepts for human cardiovascular disease (CVD) were targeting individual pathomechanisms or specific risk factor,. On the basis of genetic, metabolic, evolutionary, and clinical evidence we present here a unified pathogenetic and therapeutic approach. Ascorbate deficiency is the precondition and common denominator of human CVD. Ascorbate deficiency is the result of the inability of man to synthesize ascorbate endogenously in combination with insufficient dietary intake. The invariable morphological consequences of chronic ascorbate deficiency in the vascular wall are the loosening of the connective tissue and the loss of the endothelial barrier function. Thus human CVD is a form of pre-scurvy. The multitude of pathomechanisms that lead to the clinical manifestation of CVD are primarily defense mechanisms aiming at the stabilization of the vascular wall. After the loss of endogenous ascorbate production during the evolution of man these defense mechanisms became life-saving. They counteracted the fatal consequences of scurvy and particularly of blood loss through the scorbutic vascular wall. These countermeasures constitute a genetic and a metabolic level. The genetic level is characterized by the evolutionary advantage of inherited features that lead to a thickening of the vascular wall, including a multitude of inherited diseases.
The metabolic level is characterized by the close connection of ascorbate with metabolic regulatory systems that determine the risk profile for CVD in clinical cardiology today. The most frequent mechanism is the deposition of lipoproteins, particularly lipoprotein (a) [Lp(a)], in the vascular wall. With sustained ascorbate deficiency, the result of insufficient ascorbate uptake, these defense mechanisms overshoot and lead to the development of CVD. Premature CVD is essentially unknown in all animal species that produce high amounts of ascorbate endogenously. In humans, unable to produce endogenous ascorbate, CVD became one of the most frequent diseases. The genetic mutation that rendered all human beings today dependent on dietary ascorbate is the universal underlying cause of CVD- Optimum dietary ascorbate intake will correct this common genetic defect and prevent its deleterious consequences. Clinical confirmation of this theory should largely abolish CVD as a cause for mortality in this generation and future generations of mankind.
Key words
Ascorbate, vitamin C, cardiovascular disease, lipoprotein (a), hype rcholestero le in i a. hypertriglyceridemia, hypoalphalipoproteinemia, diabetes, homocystinuria.
Introduction
We have recently presented ascorbate deficiency as the primary cause of human CVD. We proposed that the most frequent pathomechanism leading to the development of atherosclerotic plaques is the deposition of Lp(a) and fibrinogen/fibrin in the ascorbate-deficient vascular wall. In the course of this work we discovered that virtually every pathomechanism for human CVD known today can be induced by ascorbate deficiency. Beside the deposition of Lp(a) this includes such seemingly unrelated processes as foam cell formation and decreased reverse-cholesterol
transfer, and also peripheral angiopathies in diabetic or homocystinuric patients. We did not accept this observation as a coincidence. Consequently we proposed that ascorbate deficiency is the precondition as well as a common denominator of human CVD. This farreaching conclusion deserves an explanation; it is presented in this paper. We suggest that the direct connection of ascorbate deficiency with the development of CVD is the result of extraordinary pressure during the evolution of man. After the loss of the endogenous ascorbate production in our ancestors, severe bloodloss through the scorbutic vascular wall became a life-threatening condition. The resulting evolutionary pressure favored genetic and metabolic mechanisms predisposing to CVD.
The Loss of Endogenous Ascorbate Production in the Ancestor of Man
With few exceptions all animals synthesize their own ascorbate by conversion from glucose. In this way they manufacture a daily amount of ascorbate that varies between about 1 gram and 20 grams, when compared to the human body weight. About 40 million years ago the ancestor of man lost the ability for endogenous ascorbate production. This was the result of a mutation of the gene encoding for the enzyme L-gulono-g-lactone oxidase (GLO), a key enzyme in the conversion of glucose to ascorbate. As a result of this mutation all descendants became dependent on dietary ascorbate intake.
The precondition for the mutation of the GLO gene was a sufficient supply of dietary ascorbate. Our ancestors at that time lived in tropical regions. Their diet consisted primarily of fruits and other forms of plant nutrition that provided a daily dietary ascorbate supply in the range of several hundred milligrams to several grams per day. When our ancestors left this habitat to settle in other regions of the world the availability of dietary ascorbate dropped considerably and they became prone to scurvy.
Fatal Blood Loss Through the Scorbutic Vascular Wall - An Extraordinary Challenge to the Evolutionary Survival of Man
Scurvy is a fatal disease. It is characterized by structural and metabolic impairment of the human body, particularly by the destabilization of the connective tissue. Ascorbate is essential for an optimum production and hydroxylation of collagen and elastin, key constituents of the extracellular matrix. Ascorbate depletion thus leads to a destabilization of the connective tissue throughout the body. One of the first clinical signs of scurvy is perivascular bleeding. The explanation is obvious: Nowhere in the body does there exist a higher pressure difference than in the circulatory system, particularly across the vascular wall. The vascular system is the first site where the underlying destabilization of the connective tissue induced by ascorbate deficiency is unmasked, leading to the penetration of blood through the permeable vascular wall. The most vulnerable sites are the proximal arteries, where the systolic blood pressure is particularly high. The increasing permeability of the vascular wall in scurvy leads to petechiae and ultimately hemorrhagic blood loss.
Scurvy and scorbutic blood loss decimated the ship crews in earlier centuries within months. It is thus conceivable that during the evolution of man periods of prolonged ascorbate deficiency led to a great death toll. The mortality from scurvy must have been particularly high during the thousands of years the ice ages lasted and in other extreme conditions, when the dietary ascorbate supply approximated zero. We therefore propose that after the loss of endogenous ascorbate production in our ancestors, scurvy became one of the greatest threats to the evolutionary survival of man. By hemorrhagic blood loss through the scorbutic vascular wall our ancestors in many regions may have virtually been brought close to extinction.
The morphologic changes in the vascular wall induced by ascorbate deficiency are well characterized: the loosening of the connective tissue and the loss of the endothelial barrier function. The extraordinary pressure by fatal blood loss through the scorbutic vascular wall favored genetic and metabolic countermeasures attenuating increased vascular permeability.
Ascorbate Deficiency and Genetic Countermeasures
The genetic countermeasures are characterized by an evolutionary advantage of genetic features and include inherited disorders that
are associated with atherosclerosis and CVD. With sufficient ascorbate supply these disorders stay latent. In ascorbate deficiency, however, they become unmasked, leading to an increased deposition of plasma constituents in the vascular wall and other mechanisms that thicken the vascular wall. This thickening of the vascular wall is a defense measure compensating for the impaired vascular wall that had become destabilized by ascorbate deficiency. With prolonged insufficient ascorbate intake in the diet these defense mechanisms overshoot and CVD develops.
The most frequent mechanism to counteract the increased permeability of the ascorbate-deficient vascular wall became the deposition of lipoproteins and lipids in the vessel wall. Another group of proteins that generally accumulate at sites of tissue transformation and repair are adhesive proteins such as fibronectin, fibrinogen, and particularly apo(a). It is therefore no surprise that Lp(a), a combination of the adhesive protein apo(a) with a low density lipoprotein (LDL) particle, became the most frequent genetic feature counteracting ascorbate deficiency.' Beside lipoproteins, certain metabolic disorders, such as diabetes and homocystinuria, are also associated with the development of CVD. Despite differences in the underlying pathomechanism, all these mechanisms share a common feature: they lead to a thickening of the vascular wall and thereby can counteract the increased permeability in ascorbate deficiency. In addition to these genetic disorders, the evolutionary pressure from scurvy also favored certain metabolic countermeasures.
Ascorbate Deficiency and Metabolic Countermeasures
The metabolic countermeasures are characterized by the regulatory role of ascorbate for metabolic systems determining the clinical risk profile for CVD. The common aim of these metabolic regulations is to decrease the vascular permeability in ascorbate deficiency. Low ascorbate concentrations therefore induce vasoconstriction and hemostasis and affect vascular wall metabolism in favor of atherosclerogenesis. Towards this end ascorbate interacts with lipoproteins. coagulation factors, prostaglandins, nitric oxide, and second messenger systems such as cyclic monophosphates. It should be noted that ascorbate can affect these regulatory levels in a multiple way- In lipoprotein metabolism low density lipoproteins (LDL), Lp(a), and very low density lipoproteins (VLDL) are inversely correlated with ascorbate concentrations, whereas ascorbate and HDL levels are positively correlated. Similarly, in prostaglandin metabolism ascorbate increases prostacyclin and prostaglandin E levels and decreases the thromboxane level. In general, ascorbate deficiency induces vascular constriction and hemostatis, as well as cellular and extracellular defense measures in the vascular wall.
In the following sections we shall discuss the role of ascorbate for frequent and well established pathomechanisms of human CVD. In general, the inherited disorders described below are polygenic. Their separate description, however, will allow the characterization of the role of ascorbate on the different genetic and metabolic levels.
Apo(a) and Lp(a), the Most Effective and Most Frequent Countermeasure
After the loss of endogenous ascorbate production, apo(a) and Lp(a) were greatly favored by evolution. The frequency of occurrence of elevated Lp(a) plasma levels in species that had lost the ability to synthesize ascorbate is so great that we formulated the theory that apo(a) functions as a surrogate for ascorbate.' There are several genetically determined isoforms of apo(a). They differ in the number of kringle repeats and in their molecular size. An inverse relation between the molecular size of apo(a) and the synthesis rate of Lp(a) particles has been established. Individuals with the high molecular weight apo(a) isoform produce fewer Lp(a) particles than those with the low apo(a) isoform. In most population studies the genetic pattern of high apo(a) isoform/low Lp(a) plasma level was found to be the most advantageous and therefore most frequent pattern. In ascorbate deficiency Lp(a) is selectively retained in the vascular wall. Apo(a) counteracts increased permeability by compensating for collagen, by its binding to fibrin, as a proteinthiol antioxidant, and as an inhibitor of plasmin-induced proteolysis. Moreover, as an adhesive protein apo(a) is effective in tissue-repair processes (8). Chronic ascorbate deficiency leads to a sustained accumulation of Lp(a) in the vascular wall. This leads to the development of atherosclerotic plaques and premature CVD, particularly in individuals with genetically determined high plasma Lp(a) levels. Because of its association with apo(a), Lp(a) is the most specific repair particle among all lipoproteins. Lp(a) is predominantly deposited at predisposition sites and it is therefore found to be significantly correlated with coronary, cervical, and cerebral atherosclerosis but not with peripheral vascular disease.
The mechanism by which ascorbate resupplementation prevents CVD in any condition is by maintaining the integrity and stability of the vascular wall. In addition, ascorbate exerts in the individual a multitude of metabolic effects that prevent the exacerbation of a possible genetic predisposition and the development of CVD. If the predisposition is a genetic elevation of Lp(a) plasma levels the specific regulatory role of ascorbate is the decrease of apo(a) synthesis in the liver and thereby the decrease of Lp(a) plasma levels. Moreover, ascorbate decreases the retention of Lp(a) in the vascular wall by lowering fibrinogen synthesis and by increasing the hydroxylation of lysine residues in vascular wall constituents, thereby reducing the affinity for Lp(a) binding.
In about half of the CVD patients the mechanism of Lp(a) deposition contributes significantly to the development of atherosclerotic plaques. Other lipoprotein disorders are also frequently part of the polygenic pattern predisposing the individual patient to CVD in the individual.
Other Lipoprotein Disorders Associated with CVD
In a large population study Goldstein et al. discussed three frequent lipid disorders, familial hypercholesterolemia, familial hypertriglyceridemia, and familial combined hyperlipidemia. Ascorbate deficiency unmasks these underlying genetic defects and leads to an increased plasma concentration of lipids (e.g. cholesterol, triglycerides) and lipoproteins (e.g. LDL, VLDL) as well as to their deposition in the impaired vascular wall. As with Lp(a), this deposition is a defense measure counteracting the increased permeability. It should, however, be noted that the deposition of lipoproteins other than Lp(a) is a less specific defense mechanism and frequently follows Lp(a) deposition. Again, these mechanisms function as a defense only for a limited time. With sustained ascorbate deficiency the continued deposition of lipids and lipoproteins leads to atherosclerotic plaque development and CVD. Some mechanisms will now be described in more detail.
Hypercholesterolemia, LDL-receptor defect
A multitude of genetic defects lead to an increased synthesis and/or a decreased catabolism of cholesterol or LDL. A well characterized although rare defect is the LDL receptor defect. Ascorbate deficiency unmasks these inherited metabolic defects and leads to an increased plasma concentration of cholesterol-rich lipoproteins, e.g. LDL, and their deposition in the vascular wall. Hypercholesterolemia increases the risk for premature CVD primarily when combined with elevated plasma levels of Lp(a) or triglycerides.
The mechanisms by which ascorbate supplementation prevents the exacerbation of hypercholesterolemia and related CVD include an increased catabolism of cholesterol. In particular, ascorbate is known to stimulate 7-a-hydroxylase, a key enzyme in the conversion of cholesterol to bile acids and to increase the expression of LDL receptors on the cell surface. Moreover, ascorbate is known to inhibit endogenous cholesterol synthesis as well as oxidative modification of LDL.
Hypertriglyceridemia, Type III hyperlipidemia
A variety of genetic disorders lead to the accumulation of triglycerides in the form of chylomicron remnants, VLDL, and intermediate density lipoproteins (IDL) in plasma. Ascorbate deficiency unmasks these underlying genetic defects and the continued deposition of triglyceride-rich lipoproteins in the vascular wall leads to CVD development. These triglyceride-rich lipoproteins are particularly subject to oxidative modification, cellular lipoprotein uptake, and foam cell formation. In hypertriglyceridemia nonspecific foam-cell formation has been observed in a variety of organs." Ascorbate-deficient foam cell formation, although a less specific repair mechanism than the extracellular deposition of Lp(a), may have also conferred stability .
Ascorbate supplementation prevents the exacerbation of CVD associated with hypertriglyceridemia, Type III hyperlipidemia, and related disorders by stimulating lipoprotein lipases and thereby enabling a normal catabolism of triglyceride-rich lipoproteins. Ascorbate prevents the oxidative modification of these lipoproteins, their uptake by scavenger cells and foam cell formation. Moreover, we propose here that, analogous to the LDL receptor, ascorbate also increases the expression of the receptors involved in the metabolic clearance of triglyceride-rich lipoproteins, such as the chylomicron remnant receptor.
The degree of build-up of atherosclerotic plaques in patients with lipoprotein disorders is determined by the rate of deposition of lipoproteins and by the rate of the removal of deposited lipids from the vascular wall. It is therefore not surprising that ascorbate is also closely connected with this reverse pathway.
Hypoalphalipoproteinemia
Hypoalphalipoproteinemia is a frequent lipoprotein disorder characterized by a decreased synthesis of HDL particles. HDL is part of the 'reverse-cholesterol-transport' pathway and is critical for the transport of cholesterol and also other lipids from the body periphery to the liver. In ascorbate deficiency this genetic defect is unmasked, resulting in decreased HDL levels and a decreased reverse transport of lipids from the vascular wall to the liver. This mechanism is highly effective and the genetic disorder hypoalphalipoproteinemia was greatly favored during evolution. With ascorbate supplementation HDL production increases, leading to an increased uptake of lipids deposited in the vascular wall and to a decrease of the atherosclerotic lesion. A look back in evolution underlines the importance of this mechanism. During the winter seasons, with low ascorbate intake, our ancestors became dependent on protecting their vascular wall by the deposition of lipoproteins and other constituents. During spring and summer seasons the ascorbate content in the diet increased significantly and mechanisms were favored that decreased the vascular deposits under the protection of increased ascorbate concentration in the vascular tissue. It is not unreasonable for us to propose that ascorbate can reduce fatty deposits in the vascular wall within a relatively short time. In an earlier clinical study it was shown that 500 mg of dietary ascorbate per day can lead to a reduction of atherosclerotic deposits within 2 to 6 months."
This concept, of course, also explains why heart attack and stroke occur today with a much higher frequency in winter than during spring and summer, the seasons with increased ascorbate intake.
Other Inherited Metabolic Disorders Associated with CVD
Beside lipoprotein disorders many other inherited metabolic diseases are associated with CVD. Generally these disorders lead to an increased concentration of plasma constituents that directly or indirectly damage the integrity of the vascular wall. Consequently these diseases lead to peripheral angiopathies as observed in diabetes, homocystinuria, sickle-cell anemia (the first molecular disease described," and many other genetic disorders. Similar to lipoproteins the deposition of various plasma constituents as well as proliferative thickening provided a certain stability for the ascorbatedeficient vascular wall. We illustrate this principle for diabetic and homocystinuric angiopathy.
Diabetic Angiopathy
The pathomechanism in this case involves the structural similarity between glucose and ascorbate and the competition of these two molecules for specific cell surface receptors." Elevated glucose levels prevent many cellular systems in the human body, including endothelial cells, from optimum ascorbate uptake- Ascorbate deficiency unmasks the underlying genetic disease, aggravates the imbalance between glucose and ascorbate, decreases vascular ascorbate concentration, and thereby triggers diabetic angiopathy.
Ascorbate supplementation prevents diabetic angiopathy by optimizing the ascorbate concentration in the vascular wall and also by lowering insulin requirement-"
Homocystinuric angiopathy
Homocystinuria is characterized by the accumulation of homocyst(e)ine and a variety of its metabolic derivatives in the plasma, the tissues and the urine as the result of decreased homocysteine catabolism." Elevated plasma concentrations of homocyst(e)ine and its derivatives damage the endothelial cells throughout the arterial and venous system. Thus homocystinuria is characterized by peripheral vascular disease and thromboembolism. These clinical manifestations have been estimated to occur in 30 per cent of the patients before the age of 20 and in 60 per cent of the patients before the age of 40.
Ascorbate supplementation prevents homocystinuric angiopathy and other clinical complications of this disease by increasing the rate of homocysteine catabolism.
Thus, ascorbate deficiency unmasks a variety of individual genetic predispositions that lead to CVD in different ways. These genetic disorders were conserved during evolution largely because of their association with mechanisms that lead to the thickening of the vascular wall. Moreover, since ascorbate deficiency is the underlying cause of these diseases, ascorbate supplementation is the universal therapy.
The Determining Principles of This Theory
The determining principles of this comprehensive theory are schematically summarized in Figures I to 3 (pages 13 to 15).
1. CVD is the direct consequence of the inability for endogenous ascorbate production in man in combination with low dietary ascorbate intake.
2. Ascorbate deficiency leads to increased permeability of the vascular wall by the loss of the endothelial barrier function and the loosening of the vascular connective tissue.
3. After the loss of endogenous ascorbate production scurvy and fatal blood loss through the scorbutic vascular wall rendered our ancestors in danger of extinction. Under this evolutionary pressure over millions of years genetic and metabolic countermeasures were favored that counteract the increased permeability of the vascular wall.
4. The genetic level is characterized by the fact that inherited disorders associated with CVD became the most frequent among all genetic predispositions. Among those predispositions lipid and lipoprotein disorders occur particularly often.
5. The metabolic level is characterized by the direct relation between ascorbate and virtually all risk factors of clinical cardiology today. Ascorbate deficiency leads to vasoconstriction and hemostasis and affects the vascular wall metabolism in favor of atherosclerogenesis.
6. The genetic level can be further characterized. The more effective and specific a certain genetic feature counteracted the increasing vascular permeability in scurvy, the more advantageous it became during evolution and, generally, the more frequently this genetic feature occurs today
7. The deposition of Lp(a) is the most effective, most specific, and therefore most frequent of these mechanisms. Lp(a) is preferentially deposited at predisposition sites. In chronic ascorbate deficiency the accumulation of Lp(a) leads to the localized development of atherosclerotic plaques and to myocardial infarction and stroke.
8. Another frequent inherited lipoprotein disorder is hypoalphalipoproteinemia. The frequency of this disorder again reflects its usefulness during evolution. The metabolic upregulation of HDL synthesis by ascorbate became an important mechanism to reverse and decrease existing lipid deposits in the vascular wall.
9. The vascular defense mechanisms associated with most genetic disorders are nonspecific. These mechanisms can aggravate the development of atherosclerotic plaques at predisposition sites. Other nonspecific mechanisms lead to peripheral forms of atherosclerosis by causing a thickening of the vascular wall throughout the arterial system. This peripheral form of vascular disease is characteristic for angiopathics associated with Type III hyperlipidemia, diabetes, and many other inherited metabolic diseases.
10. Of particular advantage during evolution and therefore particularly frequent today are those genetic features that protect the ascorbate-deficient vascular wall until the end of the reproduction age. By favoring these disorders nature decided for the lesser of two evils: the death from CVD after the reproduction age rather than death from scurvy at a much earlier age. This also explains the rapid increase of the CVD mortality today from the 4th decade onwards.
11. After the loss of endogenous ascorbate production the genetic mutation rate in our ancestors increased significantly- This was an additional precondition favoring the advantage not only of apo(a) and Lp(a) but also of many other genetic countermeasures associated with CVD.
12. Genetic predispositions are characterized by the rate of ascorbate depiction in a multitude of metabolic reactions specific for the genetic disorder." The overall rate of ascorbate depletion in an individual is largely determined by the polygenic pattern of disorders. The earlier the ascorbate reserves in the body are depleted without being resupplemented, the earlier CVD develops.
13. The genetic predispositions with the highest probability for early clinical manifestation require the highest amount of ascorbate supplementation in the diet to prevent CVD development. The amount of ascorbate for patients at high risk should be comparable to the amount of ascorbate our ancestors synthesized in their body before they lost this ability: between 10,000 and 20,000 milligrams per day.
14. Optimum ascorbate supplementation prevents the development of CVD independently of the individual predisposition or pathomechanism. Ascorbate reduces existing atherosclerotic deposits and thereby decreases the risk for myocardial infarction and stroke. Moreover, ascorbate can prevent blindness and organ failure in diabetic patients, thromboembolism in homocystinuric patients, and many other manifestations of CVD.
Conclusion
In this paper we present a unified theory of human CVD. This disease is the direct consequence of the inability of man to synthesize ascorbate in combination with insufficient intake of ascorbate in the modem diet. Since ascorbate deficiency is the common cause of human CVD, ascorbate supplementation is the universal treatment for this disease. The available epidemiological and clinical evidence is reasonably convincing. Further clinical confirmation of this theory should lead to the abolition of CVD as a cause of human mortality for the present generation and future generations of mankind.
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- Re: Interesting! asdfgh_is_me
20 y
2,697
This is a reply to # 13,335Yet if this theory holds true how is it people who get high doses of vitamin C still have heart disease/heart attacks? Genetics aside (though technically vit. C should prevent exacerbation of genetic predisposition according to the paper) what about the people without predisposition who still get the disease?
This is obviously one small part of the total heart disease system.- Only U.S. Patent for the cure for He... Burnt Ice
20 y
2,963
This is a reply to # 13,336How much where high doses of vitamin C were they taking, and there diet and life style, He holds the only U.S Patent for the cure for Heart Disease, I don't think they patent things that don't work...I would love to see your documentation on those who took HIGH does and still had heart attacks, that would be great...
check out vitamincfoundation if your looking the the exact does used in these studys...
RATH/PAULING U.S. PATENT # 5278189
Prevention and treatment of occlusive cardiovascular disease with ascorbate and substances that inhibit the binding of lipoprotein (A)
--------------------------------------------------------------------------------
PATN Patent Bibliographic Information
WKU Patent Number: 05278189
SRC Series Code: 7
APN Application Number: 5575168
APT Application Type: 1
ART Art Unit: 125
APD Application Filing Date: 19900724
TTL Title of Invention: Prevention and treatment of occ
lusive cardiovascular disease with
ascorbate and substances that inhibit the binding of lipoprotein (A)
ISD Issue Date: 19940111
NCL Number of Claims: 15
ECL Exemplary Claim Number: 1
EXA Assistant Examiner: Henley, III; Raymond J.
EXP Primary Examiner: Waddell; Frederick E.
NDR Number of Drawings Sheets: 5
NFG Number of Figures: 6
INVT Inventor Information
NAM Inventor Name: Rath; Matthias W.
STR Inventor Street: Eberhardstrasse 12
CTY Inventor City: 7141 Kirchberg/Murr
CNT Inventor Country: DEX
INVT Inventor Information
NAM Inventor Name: Pauling; Linus C.
STR Inventor Street: 15 Salmon Creek
CTY Inventor City: Big Sur
STA Inventor State: CA
ZIP Inventor Zip Code: 93920
RLAP Related U.S. Application Data
COD Parent Code: 72
APN Application Number: 533129
APD Application Filing Date: 19900604
PSC Parent Status Code: 03
CLAS Classification
OCL Original U.S. Classification: 514561
XCL Cross Reference Classification: 514356
XCL Cross Reference Classification: 514474
XCL Cross Reference Classification: 514824
EDF International Classification Edition Field: 5
ICL International Classification: A61K 31195
ICL International Classification: A61K 3134
ICL International Classification: A61K 3144
FSC Field of Search Class: 514
FSS Field of Search Subclass: 474;561;562;564;567;824
;356
UREF U.S. Patent Reference
PNO Patent Number: 3956504
ISD Issue Date: 19760500
NAM Patentee Name: Sawyer
OCL Original U.S. Classification: 514567
U.S. Patent References
UREF U.S. Patent Reference
PNO Patent Number: 4424232
ISD Issue Date: 19840100
NAM Patentee Name: Parkinson
OCL Original U.S. Classification: 514474
UREF U.S. Patent Reference
PNO Patent Number: 4600582
ISD Issue Date: 19860700
NAM Patentee Name: Stevens et al.
OCL Original U.S. Classification: 514561
UREF U.S. Patent Reference
PNO Patent Number: 4954521
ISD Issue Date: 19900900
NAM Patentee Name: Sawyer et al.
OCL Original U.S. Classification: 514474
FREF Foreign Reference
PNO Patent Number: 60-4611
ISD Issue Date: 19850000
CNT Foreign Reference Country Code: JPX
FREF Foreign Reference
PNO Patent Number: 60-78560
ISD Issue Date: 19850000
CNT Foreign Reference Country Code: JPX
FREF Foreign Reference
PNO Patent Number: 60-87221
ISD Issue Date: 19850000
CNT Foreign Reference Country Code: JPX
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LREP Legal Information
FRM Legal Firm: Limbach & Limbach
ABST Abstract
A method is provided for prevention and treatment of cardiovascular disease, such as atherosclerosis, by administering therapeutically effective dosages of a drug comprised of ascorbate, lipoprotein(a) binding inhibitors, and antioxidants.
PARN Parent Case Text
This application is a continuation-in-part of application Ser. No. 07/533,129, filed Jun. 4, 1990.
BSUM Brief Summary
TECHNICAL FIELD
The present invention relates generally to the prevention and treatment of cardiovascular disease and more particularly to methods and compounds that inhibit the binding of lipoprotein (a) to components of the arterial wall.
BACKGROUND OF THE INVENTION
Lipoprotein(a) ("Lp(a)") was first identified by Blumberg, B. S., et al. (1962) J. Clin. Invest. 41: 1936-1944 , and Berg, K. (1963) Acta Pathol. 59: 369-382. The structure of Lp(a) resembles that of low-density lipoprotein ("LDL") in that both share a lipid apoprotein composition, mainly apolipoprotein B-100 ("apo B"), the ligand by which LDL binds to the LDL receptors present on the interior surfaces of arterial walls. The unique feature of Lp(a) is an additional glycoprotein, designated apoprotein(a), apo(a), which is linked to apo B by disulfide groups. The cDNA sequence of apo(a) shows a striking homology to plasminogen, with multiple repeats of kringle 4, one kringle 5, and a protease domain. The isoforms of apo(a) vary in the range of 300 to 800 kDa and differ mainly in their genetically determined number of kringle 4 structures. McLean, J. W., et al. (1987) Nature 300: 132-137. Apo(a) has no plasmin-like protease activity. Eaton, D. L., et al., (1987) Proc. Natl Acad. Sci. USA, 84: 3224-3228. Serine protease activity, however, has been demonstrated. Salonen, E., et al. (1989) EMBO J. 8: 4035-4040. Like plasminogen, Lp(a) has been shown to bind to lysine-sepharose, immobilized fibrin and fibrinogen, and the plasminogen receptor on endothelial cells. Harpel, P.C., et al. (1989) Proc. Natl. Acad. Sci. USA 86:3847-3851; Gonzalez-Gronow, M., et al. (1989) Biochemistry 28: 2374-2377; Miles, L. et al. (1989) Nature 339: 301-302; Hajjar, K. A., et al. (1989) Nature 339: 303-305. Furthermore, Lp(a) has been demonstrated to bind to other components of the arterial wall like fibronectin and glycosaminoglycans. The nature of these bindings, however, is poorly understood.
Essentially all human blood contains lipoprotein(a); however, there can a thousand-fold range in its plasma concentration between individuals. High levels of Lp(a) are associated with a high incidence of cardiovascular disease. Armstrong, V. W., et al. (1986) Atherosclerosis 62: 249-257; Dahlen, G., et al. (1986) Circulation 74: 758-765; Miles, L. A., et al. (1989) Nature 339: 301-302; Zenker, G., et al. (1986). Stroke 17: 942-945 (The term occlusive cardiovascular disease will be used hereafter as including all pathological states leading to a narrowing and/or occlusion of blood vessels throughout the body, but particularly atherosclerosis, thrombosis and other related pathological states, especially as occurs in the arteries of the heart muscle and the brain.)
For some time, general medical practice has focused on the role of LDL, th e so called "bad cholesterol," in occlusive cardiovascular disease. A great many studies have been published ostensibly linking occlusive cardiovascular disease with elevated levels of LDL. As a result, most therapies for the treatment and prevention of arteriosclerosis rely on drugs and methods for the reduction of serum levels of LDL's. Such therapies have had mixed results. The efficacy of such approaches to the problem of occlusive cardiovascular disease continues to be major source of debate.
There exists therefore a need for a drug therapy for reducing the binding of Lp(a) to vessel walls, for reducing the overall level of Lp(a) in the circulatory system and for promoting the release of existing deposits of Lp(a) on vessel walls.
SUMMARY OF THE INVENTION
The foregoing needs in the treatment and prevention of cardiovascular disease are met by the methods and compositions of the present invention.
A method is provided for the treatment of occlusive cardiovascular disease , comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors, as a mixture or as a compound comprising ascorbate covalently linked with binding inhibitors, which inhibit the binding of Lp(a) to blood vessel walls, such as arterial walls. This effect may also be obtained by administering an effective amount of one or more inhibitors, without ascorbate. The term binding inhibitor throughout the specification and claims is intended to include all substances that have an affinity for the lysine binding site present on the interior walls of blood vessels, particularly arteries, the site of Lp(a) binding. Most of these substances compete with plasmin for the lysine binding site and some of these compounds, in high doses, are in clinical use for the treatment of hyperfibrinolytic states.
A method is further provided for the prevention of atherosclerosis comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors as previously discussed but further comprising one or more antioxidants. The term antioxidant throughout the specification and the claims is intended to exclude ascorbate which has as one of its chemical properties a potent antioxidant effect.
It is thus an object of the invention to provide a method for treatment of occlusive cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or a mixture of binding inhibitors.
It is another object of the invention to provide a method for preventing o f occlusive cardiovascular disease, by administering to a subject an amount of ascorbate effective to lower the amount of Lp(a) in the plasma of the subject.
Yet another object of the present invention is to provide a method for prevention of cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or more binding inhibitors.
A further object of the present invention is to provide a pharmaceutically acceptable agent for the treatment of occlusive cardiovascular disease.
Still another object of the present invention is to provide a pharmaceutically acceptable agent for the prevention of cardiovascular disease.
These and other objects will be more readily understood upon consideration of the following detailed descriptions of embodiments of the invention and the drawings.
DRWD Drawing Description
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an immunoblot of the plasma of guinea pigs form from the test described in Example 1.
FIG. 2A a photograph of the aorta of a guinea pig receiving an adequate amount of ascorbate from the test diet in Example 1.
FIG. 2B is a photograph of an aorta of a guinea pig receiving a hypoascorbic diet after three weeks from the test diet in Example 1.
FIG. 3 is an immunoblot of plasma and tissue of guinea pigs from the test shown in Example 2.
FIG. 4 shows the potential mechanism of binding inhibitors in the therapy for atherosclerosis.
FIG. 5 show the potential mechanism of ascorbate in the binding of Lp(a) t o the arterial wall.
DETD Detail Description
DETAILED DESCRIPTION OF THE INVENTION
Our invention is based in part on our discovery that animals which have lost the ability to produce ascorbate, such as higher primates and guinea pigs, uniformly produce Lp(a). Most animals which possess the ability to synthesize ascorbate generally do not produce Lp(a). Further, we have found that ascorbate deficiency in humans and guinea pigs tends to raise Lp(a) levels and causes atherosclerosis by the deposition of Lp(a) in the arterial wall, from which we conclude that ascorbate administration lowers plasma Lp(a) levels.
We have also discovered that substances that inhibit the binding of Lp(a) to components of the arterial wall, particularly to fibrinogen, fibrin and fibrin degradation products herein identified as binding inhibitors, such as lysine or .epsilon.-aminocaproic acid used alone or in combination with ascorbate, cause release of Lp(a) from the arterial wall. Thus, ascorbate and such binding inhibitors are not only useful for the prevention of occlusive cardiovascular disease, but also for the treatment of such disease. The present invention, then, provides methods and pharmaceutical agents for the both the treatment and prevention of occlusive cardiovascular disease in vivo.
GENERAL APPLICATIONS
The present invention provides a method and pharmaceutical agent for the treatment and prevention of occlusive cardiovascular disease generally, by administering to a subject an effective amount of ascorbate and one or more binding inhibitors. Throughout the specification and claims, the term binding inhibitor is intended to cover any substance which has as at least one of its chemical properties the ability to inhibit the binding of Lp(a) to blood vessel wall components, particularly to fibrin or fibrinogen. As used herein, the term "ascorbate" includes any pharmaceutically acceptable salt of ascorbate, including sodium ascorbate, as well as ascorbic acid itself. Binding inhibitors include, but are not limited to .epsilon.-aminocaproic acid, lysine, tranexamic acid (4-aminomethylcyclohexane carboxylic acid), p-aminomethyl benzoic acid , p-benzylamine sulfuric acid, o-N-acetyl-lysine-methyl ester, PROBUCOL (a compound comprised of 2 butyl hydroxy tocopherol groups linked together by a disulphide group), Aprotinin, trans-4-aminomethylcyclohexanecarboxylic acid (AMCA), and benzamidine derivatives such as amidinophenylpyruvic acid (APPA) and 1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl (NANP). An effective amount of a binding inhibitor or a mixture of binding inhibitors may also be used, without ascorbate. Other substances used in the treatment of occlusive cardiovascular disease may be co-administered, including antioxidants, such as tocopherol, carotene and related substances; vitamins; provitamins; trace elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile acid sequestrants; and mixtures of any two or more of these substances.
Although ascorbate can be used alone or in varying combinations with one o r more representative constituents of the above classes of compounds, we prefer when treating a pre-existing cardiovascular condition to combine ascorbate with at least one each of the binding inhibitors, antioxidants and lipid lowering drugs elements in the dosages (per kilogram of body weight per day (Kg BW/d)) provided in Table 1. It should be noted that Table 1 provides differing concentration ranges of each constituent, depending upon whether the agent is to be administered orally or parenterally. The variance in dosages is reflective of variation in disease severity. It will be realized therefore that if the subject has been diagnosed for advanced stages of atherosclerosis,
dosages at the higher end of this range can be utilized. However, if only prevention of an atherosclerosis condition is the object, dosages at the lower end of this range can be utilized.
As an alternative, a pharmaceutical agent identical to the one just described, but omitting ascorbate, may be employed.
Where ascorbate and binding inhibitors are utilized in the same agent, the y may simply be mixed or may be chemically combined using synthesis methods well known in the art, such as compounds in which ascorbate and the inhibitor are covalently linked, or form ionically bound salts. For example, ascorbate may be bound covalently to lysine, other amino acids, or .epsilon.-aminocaproic acid by ester linkages. Ascorbyl .epsilon.-aminocaproate is such an example. In this form the ascorbate moiety may be particularly effective in preventing undesirable lipid peroxidation.
In the case of oral administration, a pharmaceutically acceptable and otherwise inert carrier may be employed. Thus, when administered orally, the active ingredients may be administered in tablet form. The tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid, and/or a lubricant such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring agents may be used. If administration is by parenteral injection, in isotonic saline, a phosphate buffered solution or the like, may be used as a pharmaceutically acceptable carrier.
The advisability of using binding inhibitors in treating occlusive cardiovascular disease will depend to some extent on the subject's general health, particularly with regard to hyperfibrinolytic conditions. Most binding inhibitors (except lysine) are used clinically to treat such conditions. As a result, monitoring of the subject's coagulation and fibrinolytic sysem is recommended before and during treatment for occlusive cardiovascular disease. Long-term administration of binding inhibitors will require formulations in which the dosages of binding inhibitors are in the lower ranges of the dosages given in Table 1.
Prevention, as contrasted with treatment, of cardiovascular disease may be accomplished by oral or parenteral administration of ascorbate alone. Table 1 gives a range of ascorbate concentrations sufficient to lower the serum Lp(a) concentration.
Preferably the prevention of the occlusive cardiovascular disease accordin g to the invention is accomplished by use of a physical mixture of ascorbate and one or more binding inhibitors, or by use of a compound comprising covalently linked ascorbate with one or more of the binding inhibitors, which inhibit binding of Lp(a) to the arterial wall. A binding inhibitor or mixture of binding inhibitors may also be administered without ascorbate to prevent Lp(a)-associated occlusive cardiovascular disease.
To optimize the therapeutic effect of the release of Lp(a) from the blood vessel walls, the ascorbate and the binding inhibitors described above may be separately administered. Further optimization of therapeutic effect can be gained by using a time release composition to achieve relatively constant serum concentrations of the agent through time.
TBL TABLE 1
______________________________________
DOSAGES OF COMPONENTS IN THE DRUG
COMPOSITIONS OF THE PRESENT INVENTION
Oral Parenteral
Administration
Administration
______________________________________
Ascorbate: 5 mg-2500 mg/kg
25 mg-2500 mg/kg
bw/d bw/d
Binding inhibitors:
EACA 5 mg-500 mg/kg
same
bw/d
Tranexamic Acid
1 mg-100 mg/kg
same
bw/d
Para-aminomethyl
1 mg-30 mg/kg same
benzoic acid bw/d
Lysine 5-500 mg/kg bw/d
same
Antioxidants:
Tocopherol 0,1 IU-20 IU/kg
same
bw/d
Carotene 100 IU-1000 IU/kg
same
bw/d
Lipid
Lowering Drugs:
Nicotinic Acid
1 mg-300 mg/kg
bw/d
HMG-CoA 0.1-10 mg/kg bw/d
Fibrates 0.1-20 mg/kg bw/d
Probucol 0.1-20 mg/kg bw/d
Bile Acid Sequestrants
10-400 mg/kg bw/d
______________________________________
TBL TABLE 2
______________________________________
CONCENTRATION OF COMPONENTS IN THE
SOLUTION OF THE PRESENT INVENTION
______________________________________
Ascorbate 50-5000 mg/l
Binding inhibitors
EACA 2-2000 mg/l
Tranexamic Acid 1-300 mg/l
Para-aminomethyl 1-200 mg/l
benzoic acid
Lysine 10-5000 mg/l
Antioxidants
Tocopherol 1-1000 mg/l
Carotene 0.1-100 mg/l
______________________________________
EXPERIMENTAL
Having disclosed the preferred embodiment of the present invention, the following examples are provided by way of illustration only and are not intended to limit the invention in any way.
EXAMPLE 1
Because of its metabolic similarity to man, with resepct to the metabolism of ascorbate and Lp(a), the guinea pig was used in this example.
No study has been previously reported in the guinea pig to identify the lopoprotein involved as risk factors in plasma and as constituents of the atherosclerotic plaque.
Three female Gartly guinea pigs with an average weight of 800 g and an approximate age of 1 year wer stuided. One animal received an extreme hypoascorbic diet with 1 mg ascorbate/kg body weight/d. Another animal received 4 mg/kg BW/d. The third animal served as a control receiving 40 mg ascorbate/kg/BW/d)
Blood was drawn by heart puncture from the anesthetized animals and collected into EDTA containing tubes at the beginning, after 10 days, and after 3 weeks, when the animals were sacrificed. Plasma was stored at - -80.degree. C. until analyzed. Lp(a) was detected in the plasma of the guinea pigs by use of SDS-polyacrylamide gels according to Neville (J. Biol. Chem., 246, 6328-6334 (1971)) followed by Western blotting (Beisiegel, et al., J. Biol. Chem., 257, 13150-13156 (1982)). 40 .mu.l of plasma and 20 mg of arterial wall homogenate were applied in delipidated form per lane of the gel. The immunodetection of apo(a) was performed using a polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria) followed by a rabbit anti-sheep antibody (Sigma) and the gold-conjugated goat anti-rabbit anti-body with subsequent silver enhancement (Bio-Rad). The determinations of cholesterol and triglycerides were done at California Veterinary Diagnostics (Sacramento) using the enzyme assay of Boehringer Mannheim. Plasma ascorbate was determined by the dinitrophenylhydrazine method (Schaffer, et al., J. Biol. Chem., 212, 59 (1955)).
Vitamin C deficiency in the diet led to an increase of Lp(a) in the plasma of the guinea pig indicated by a clear band in the immunoblot of the plasma after 10 and 20 days of a hypoascorbic diet (FIG. 1). At necropsy the animals were anesthetized with metophase and were exsanguinated. Aorta, heart and various other organs were taken for biochemical and histological analysis. The aorta was excised, the adventitial fat was carefully removed, and the vessel was opened longitudinally. Subsequently the aorta was placed on a dark metric paper and a color slide was taken. The picture was projected and thereby magnified by an approximate factor 10. The circumference of the ascending aorta, the aortic arch and thoracic aorta as well as the atherosclerotic lesions in this area were marked and measured with a digitalized planimetry system. The degree of atherosclerosis was expressed by the ratio of plaque area to the total aortic area defined. The difference in the 3 one-year old animals of the experiment was significant and pronounced lesions were observed in the ascending aorta and the arch of the vitamin C deficient animal (FIG. 2B).
EXAMPLE 2
To confirm the data obtained in Example 1, a second guinea pig experiment was conducted, using 33 male animals with a mean weight of 550 g and an approximate age of 5 months. One group of 8 animals served as a control and received 40 mg ascorbate/kg BW/d (group A). To induce hypoascorbemia 16 animals were fed 2 mg ascorbate/kd/d (group B). Group A and half of the animals of group B (progression sub-group) were sacrificed after 5 weeks as described above. Half of group B was kept for 2 more weeks, receiving daily intraperitoneal injection of 1.3 g sodium ascorbate/Kg BW/d as a daily intra peritoneal injection with the intention to reduce the extent of atherosclerosis lesions. After this period these animals also were sacrificed.
Plasma ascorbate levels were negatively correlated with the degree of the atherosclerotic lesion. Total cholesterol levels increased significantly during ascorbic acid deficiency (Table 3).
The aortas of the guinea pigs receiving a sufficient amount of ascorbate were essentially plaque free, with minimal thickening of the intima in the ascending region. In contrast, the ascorbate-deficient animals exhibited fatty streak-like lesions, covering most parts of the ascending aorta and the aortic arch. In most cases the branching regions of the intercostal arteries of the aorta exhibited similar lipid deposits. The difference in the precentage of lesion area between the control animals and the hypoascorbic diet animals was 25% deposition of lipids and liporpoteins in the arterial wall.
TBL TABLE 3
______________________________________
MEAN PLASMA
PARAMETERS OF THE DIFFERENT GROUPS
IN RELATION TO THE AREA OF AORTIC LESIONS
Regression
Scurvy (after
Control (progress)
Scurvy)
______________________________________
Plasma 39 54 33
Chloesterol
(mg/dl)
Total Plasma
5.03 3.01 20.64
Ascorbic
Acid .mu.g/ml
Atheroscl. -- 25 19
Lesion
(Percent of
Aorta Thorac.
Surface)
______________________________________
EXAMPLE 3
Human arterial wall was obtained post mortem from the aorta ascendens. The tissue showed homogeneous intimal thickening (early atherosclerotic lesion). It was cut into pieces, with 100 mg of the cut up tissue homogenized in a glass potter for a 1 minute in 2.5 ml of the following solutions:
TBL ______________________________________
PBS (Dulbeco) + 50 mg/ml
NaAscorbate
PBS + EACS 50 mg/ml
PBS + Tranexamic Acid
50 mg/ml
PBS + NaAscorbate +
50 mg/ml
Tranexamic Acid
______________________________________
Results of this treatment are given in Table 4 and show that, compared to the control solution, a considerable amount of Lp(a) was released from the interior arterial wall.
TBL TABLE 4
__________________________________________________________________________
Lp(a) RELEASED FROM HUMAN AORTA
IN RELATION TO SPECIFIC BINDING INHIBITORS
##STR1##
__________________________________________________________________________
By now it is apparent that the methods and compositions of the present invention meet longstanding meeds in the field of prevention and treatment of occlusive cardiovascular disease. Although preferred embodiments and examples have been disclosed, it is understood that the invention is in no way limited by them, but rather is defined by the claims that follow and equivalents thereof.
CLMS Claims
STM Claim Statement: What is claimed is:
NUM Claim Number: 1.
1. A pharmaceutical composition consisting essentially of ascorbate, traexamic acid, lysine and nicotinic acid said ingredients in an amount effective to treat Lp (a)-associated occlusive cardiovascular disease.
NUM Claim Number: 2. 2. A method of treatment of occlusive cardiovascular disease comprising th e step of administering to a subject a therapeutic composition comprising ascorbate and tranexamic acid in an amount sufficient to decrease the binding of liproprotein (a) to blood vessel walls.
NUM Claim Number: 3. 3. A method according to claim 2 wherein said ascorbate is selected from the group consisting of pharmaceutically acceptable ascorbate salts, ascorbic acid and mixtures thereof.
NUM Claim Number: 4. 4. A method for treatment of occlusive cardiovascular disease comprising the step of administering to a subject a therapeutic composition comprising tranexamic acid in an amount sufficient to decrease the binding of lipoprotein(a) to blood vessel walls.
NUM Claim Number: 5. 5. A method according to any one of the claims 2, 3 or 4 wherein occlusive cardiovascular disease comprises atherosclerosis and thrombosis and said vessel walls comprise arterial walls.
NUM Claim Number: 6. 6. A method according to any one of the claims 2, 3 or 4 wherein said therapeutic composition is administered to a subject at risk of developing and in need of preventing for Lp (a)-associated occlusive cardiovascular disease in an amount of effective to inhibit binding of liproprotein(a) to blood vessel walls.
NUM Claim Number: 7. 7. A method according to any one of the claims 2, 3 or 4 wherein said therapeutic composition is administered in an amount effective to release at least some lipoprotein(a) bound to blood vessels.
NUM Claim Number: 8. 8. A method according to any one of the claims 2, 3 or 4 wherein said therapeutic composition is administered in an amount effective to reduce concentrations of lipoprotein(a) in blood serum.
NUM Claim Number: 9. 9. A method for prevention of cardiovascular disease comprising the step o f administering to a subject at risk of developing and in need of prevention for Lp (a)-associated occlusive cardiovascular disease a therapeutic composition comprising ascorbate and tranexamic acid in an amount sufficient to decrease binding of liproprotein(a) to blood vessel walls.
NUM Claim Number: 10. 10. A method according to claim 9 wherein said ascorbate is selected from the group consisting of pharmaceutically acceptable salts of ascorbate, ascorbic acid and mixtures thereof.
NUM Claim Number: 11. 11. A method of prevention of occlusive cardiovascular disease comprising the step of administering to a subject at risk of developing and in need of prevention for Lp (a)-associated occlusive cardiovascular disease a therapeutic composition comprising tranexamic acid in an amount sufficient to decrease binding of lipoprotein(a) to blood vessel walls.
NUM Claim Number: 12. 12. A method according to any one of claims 9, 10 or 11 wherein said therapeutic composition is administered in an amount effective to inhibit binding of lipoprotein(a) to the blood vessel walls.
NUM Claim Number: 13. 13. A method according to any one of claims 9, 10, 11, or 12 wherein said therapeutic composition is administered in an amount effective to reduce concentrations of lipoprotein(a) in blood serum.
NUM Claim Number: 14. 14. A method according to any one of claims 2, 3, 4 or 9 wherein the administration is oral.
NUM Claim Number: 15. 15. A method according to any one of claims 2, 3, 4 or 9 wherein administration is by parenteral application.
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United States Patent 5,230,996
Rath , et al. July 27, 1993
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Use of ascorbate and tranexamic acid solution for organ and blood vessel
treatment prior to transplantation
Abstract
A method and pharmaceutical agent are provided for the prevention and
treatment of cardiovascular disease, particularly cardiovascular disease in
the context of diabetic angiopathy, by-pass surgery, organ transplantation,
and hemodialysis, by administering ascorbate and substances that inhibit the
binding of lipoprotein (a) to blood vessel walls. The use of ascorbate and
lipoprotein (a) binding inhibitors such as tranexamic acid in a temporary
storage solution for blood vessels and organs prior to transplantation is
also demonstrated.
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Inventors: Rath; Matthias W. (Kirchberg/Murr, DE); Pauling; Linus C. (Big
Sur, CA)
Assignee: Therapy 2000 (Palo Alto, CA)
Appl. No.: 556968
Filed: July 24, 1990
Current U.S. Class: 435/1.1; 435/1.2; 514/474; 514/659
Intern'l Class: A01N 001/02; A01N 043/08; A01N 033/02
Field of Search: 435/1,2
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References Cited [Referenced By]
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U.S. Patent Documents
4443546 Apr., 1984 Stemerman 435/240.
Other References
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associated with Lipoprotein (a)," Nature 339:301-302 (1989).
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cerebrovascular disease," Stroke 17(5)942-945 (1986).
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the arterial wall of 107 coronary bypass patients,"
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Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Saucier; S.
Attorney, Agent or Firm: Limbach; George C.
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Parent Case Text
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This application is a continuation-in-part of application Ser. No.
07/533,129, filed Jun. 4, 1990, now abandoned.
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Claims
----------------------------------------------------------------------------
What is claimed is:
1. A method for reducing lipoprotein(a) binding to vessel explants prior to
implantation comprising the step of storing the vessel explants in an
aqueous composition comprising ascorbate and tranexamic acid in
concentrations sufficient to decrease binding of lipoprotein(a) to interior
walls of the vessel explants.
2. A method according to claim 1 wherein said ascorbate is selected from the
group consisting of pharmaceutically acceptable ascorbate salts, ascorbic
acid and mixtures thereof.
3. A method according to claim 2 wherein said ascorbate is covalently linked
to said tranexamic acid.
4. A method for reducing injury to organ explants prior to implantation
comprising the step of storing the organ explants in an aqueous composition
comprising ascorbate and tranexamic acid in concentrations sufficient to
decrease binding of lipoprotein(a) to interior walls of vessels within the
organ explants.
5. A method according to claim 4 wherein said ascorbate is selected from the
group consisting of pharmaceutically acceptable ascorbate salts, ascorbic
acid and mixtures thereof.
6. A method according to claim 5 wherein said ascorbate is covalently linked
to said tranexamic acid.
7. A method according to either claim 4 or 5 wherein said composition is a
pharmaceutical composition administered in an amount effective to release at
least some of the vessel-bound lipoprotein(a).
8. A method for reducing lipoprotein(a) binding to organ explants prior to
implantation comprising the step of storing the organ explants in an aqueous
composition comprising ascorbate and tranexamic acid in concentrations
sufficient to decrease lipoprotein(a) binding to interior walls of vessels
within the organ explants.
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Description
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TECHNICAL FIELD
The present invention relates generally to the prevention and treatment of
cardiovascular disease arising as a complication from surgery or a
pre-existing, unrelated disease, and more particularly to methods and
compounds for that inhibit the binding of lipoprotein (a) to components of
the arterial wall.
BACKGROUND OF THE INVENTION
Lipoprotein (a) ("Lp(a)") was first identified by Blumberg, B. S., et al.
(1962) J. Clin. Invest. 41: 1936-1944 and Berg, K. (1963) Acta Pathol. 59:
369-382. The structure of Lp(a) resembles that of low-density lipoprotein
("LDL") in that both share a lipid/apoprotein composition, mainly
apolipoprotein B-100 ("apo B"), the ligand by which LDL binds to the LDL
receptors present on the interior surfaces of arterial walls. The unique
feature of Lp(a) is an additional glycoprotein, designated apoprotein(a),
apo(a), which is linked to apo B by disulfide groups. The cDNA sequence of
apo(a) shows a striking homology to plasminogen, with multiple repeats of
kringle 4, one kringle 5, and a protease domain. The isoforms of apo(a) vary
in the range of 300 to 800 kDa and differ mainly in their genetically
determined number of kringle 4 structures. McLean, J. W., et al. (1987)
Nature 300: 132-137. Apo(a) has no plasmin-like protease activity. Eaton, D.
L., et al., (1987) Proc. Natl Acad. Sci. USA, 84: 3224-3228. Serine protease
activity, however, has been demonstrated. Salonen, E., et al. (1989) EMBO J.
8: 4035-4040. Like plasminogen, Lp(a) has been shown to bind to
lysine-sepharose, immobilized fibrin and fibrinogen, and the plasminogen
receptor on endothelial cells. Harpel, P. C., et al. (1989) Proc. Natl.
Acad. Sci. USA 86:3847-3851; Gonzalez-Gronow, M., et al. (1989) Biochemistry
28: 2374-2377; Miles, L. et al. (1989) Nature 339: 301-302; Hajjar, K. A.,
et al. (1989) Nature 339: 303-305. Furthermore, Lp(a) has been demonstrated
to bind to other components of the arterial wall like fibronectin and
glycosaminoglycans. The nature of these bindings, however, is poorly
understood.
Essentially all human blood contains lipoprotein (a); however, there can a
thousand-fold range in its plasma concentration between individuals. High
levels of Lp(a) are associated with a high incidence of cardiovascular
disease. Armstrong, V. W., et al. (1986) Atherosclerosis 62: 249-257;Dahlen,
G., et al. (1986) Circulation 74: 758-765; Miles, L. A., et al. (1989)
Nature 339: 301-302; Zenker, G., et al. (1986) Stroke 17: 942-945 (The term
cardiovascular disease will be used hereafter as including all pathological
states leading to a narrowing and/or occlusion of blood vessels throughout
the body, but particularly atherosclerosis, thrombosis and other related
pathological states, especially as occurs in the arteries of the heart
muscle and the brain.)
It has also been suggested that Lp(a), the concentration of which increases
markedly in the blood during pregnancy, may be linked to cardiovascular
disease in woman. Zechner, R., et al. (1986) Metabolism 35: 333-336. It has
also been observed that diabetics, many of whom suffer in some degree from
atherosclerotic diseases, display greatly elevated serum levels of Lp(a).
Bruckert, E., et al. (1990) JAMA 263: 35-36.
Low levels of ascorbate have also been associated with high incidences of
cancer (Wright, L. C. et al. (1989) Int. J. Cancer 43: 241-244) and
atherosclerosis in diabetes mellitus patients (Som, S. et al. (1981)
Metabolism 30: 572-577). In all instance, serum concentrations of Lp(a) were
elevated.
In addition to atherosclerosis and thrombosis in arteries Lp(a) has also
been linked to stenosis of vein grafts after coronary bypass surgery. Hoff,
H., et al. (1988) Circulation 77: 1238-1244. Similar problems of rapid
occlusion of vessels have been observed in heart transplant patients.
For some time, general medical practice has focused on the role of LDL, the
so called "bad cholesterol," in cardiovascular disease. A great many studies
have been published ostensibly linking cardiovascular disease with elevated
levels of LDL. As a result, most therapies for the treatment and prevention
of arteriosclerosis rely on drugs and methods for the reduction of serum
levels of LDLS. Such therapies have had mixed results. The efficacy of such
approaches to the problem of cardiovascular disease continues to be major
source of debate.
There exists a need, therefore, for a drug and therapy for reducing the
binding of Lp(a) to vessel walls, for reducing the overall level of Lp(a) in
the circulatory system and for promoting the deposition of existing deposits
of Lp(a) on vessel walls.
SUMMARY OF THE INVENTION
The foregoing needs in the treatment and prevention of cardiovascular
disease are met by the methods and compositions of the present invention.
A method is provided for the treatment of cardiovascular disease,
particularly atherosclerosis, induced or promoted by kidney failure,
diabetes, transplant surgery and the like, comprising the step of
administering to a subject an effective amount of ascorbate and one or more
binding inhibitors, as a mixture or as a compound comprising ascorbate
covalently linked with binding inhibitors, which inhibit the binding of
Lp(a) to blood vessel walls, such as arterial walls and vein grafts used in
by-pass surgery. This effect may also be obtained by administering an
effective amount of one or more inhibitors, without ascorbate. The term
binding inhibitor throughout the specification and claims is intended to
include all substances that have an affinity for the lysine binding site
present on the interior walls of blood vessels, particularly arteries, the
site of Lp(a) binding. Most of these substances compete with plasmin for the
lysine binding site and some of these compounds, in high doses, are in
clinical use for the treatment of hyperfibrinolytic states.
A method is further provided for the prevention of atherosclerosis related
to or as a complication of surgery, a preexisting disease or a therapy such
as hemodialysis. comprising the step of administering to a subject an
effective amount of ascorbate and one or more binding inhibitors as
previously discussed but further comprising one or more antioxidants. The
term antioxidant throughout the specification and the claims is intended to
exclude ascorbate, which itself is a powerful antioxidant.
It is thus an object of the invention to provide a method for treatment of
induced cardiovascular disease by administering to a subject an effective
amount of ascorbate and one or more binding inhibitors, or an effective
amount of one or a mixture of binding inhibitors.
It is another object of the invention to provide a method for prevention of
induced cardiovascular disease, by administering to a subject an amount of
ascorbate effective to lower the amount of Lp(a) in the plasma of the
subject.
Yet another object of the present invention is to provide a method for
prevention of induced cardiovascular disease by administering to a subject
an effective amount of ascorbate and one or more binding inhibitors, or an
effective amount of one or more binding inhibitors.
A further object of the present invention is to provide a pharmaceutically
acceptable agent for the treatment of induced cardiovascular disease.
Still another object of the present invention is to provide a
pharmaceutically acceptable agent for the prevention of induced
cardiovascular disease.
Yet another object of the present invention is to provide a method for
preservation of explanted tissues and organs that reduces the risk of
occurrence of cardiovascular disease in the tissues and organs after
implantation.
It is also an object of the present invention to provide a pharmaceutically
acceptable agent to assist in the preservation of explanted tissues and
organs prior to implantation.
Still another object of the present invention is to provide a pharmaceutical
compound and method for treating cardiovascular disease arising from a
preexisting condition of diabetes mellitus.
These and other objects will be more readily understood upon consideration
of the following detailed descriptions of embodiments of the invention and
the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an immunoblot of the plasma of guinea pigs from the test described
in Example 1. Increase of Lp(a) in plasma of guinea pigs with a hypoascorbic
diet. Immuunoblot with anti apo(a) antibodies. Lane 1: human control plasma.
Lane 2: guinea pig plasma at the start of experiment. Lane 3: guinea pig
plasma after 10 days of hypoascorbic diet. Lane 4; guinea pig plasma after
20 days. HMW: high molecular weight standard.
FIG. 2A is a photograph of the aorta of a guinea pig receiving an adequate
amount of ascorbate from the test diet in Example 1.
FIG. 2B is a photograph of an aorta of a guinea pig receiving a hypoascorbic
diet after three weeks from the test diet in Example 1.
FIG. 3 is an immunoblot of plasma and tissue of guinea pigs from the test
shown in Example 2. Plasma and tissue of guinea pigs. Immunoblot with anti
apo(a) antibody. HC: human control plasma; L: liver tissue; B: brain tissue;
A: aortic tissue, homogenate of plaque area from FIG. 2B.
FIG. 4 is a diagrammatic representation of the action of Lp(a) binding
inhibitors on Lp(a) to cause release of Lp(a) from fibrin fibers of an
arterial wall.
FIG. 5 is a diagrammatic representation of the action of ascorbate to
prevent association and reassociation of Lp(a) to an arterial wall.
DETAILED DESCRIPTION OF THE INVENTION
Our invention is based in part on our discovery that animals which have lost
the ability to produce ascorbate, such as higher primates and guinea pigs,
uniformly produce Lp(a), whereas most animals which possess the ability to
synthesize ascorbate generally do not produce Lp(a). Further, we have found
that ascorbate deficiency in humans and guinea pigs tends to raise Lp(a)
levels and causes atherosclerosis by the deposition of Lp(a) in the arterial
wall, from which we conclude that ascorbate administration lowers plasma
Lp(a) levels.
We have also discovered that substances that inhibit the binding of Lp(a) to
components of the arterial wall, particularly to fibrinogen, fibrin and
fibrin degradation products herein identified as binding inhibitors, such as
lysine or .epsilon.-aminocaproic acid. Thus, ascorbate and such binding
inhibitors are not only useful for the prevention of cardiovascular disease,
but also for the treatment of such disease.
Some beneficial effects of ascorbate in the prevention and treatment of
cardiovascular disease have been established (see FIG. 2). Our invention
reveals the relation to and therapeutic use of ascorbate for Lp(a), one of
the most atherogenic lipoproteins, directly related to the development of
atherosclerotic plaques. The beneficial effects of ascorbate suggest that
ascorbate therapies would be useful in a variety of situations where
occlusion of blood vessels by Lp(a) deposition is a problem. For instance,
ascorbate may be useful in transplantation of blood vessels and whole
organs, where a combination of tissue damage to the transplant, such as by
oxidation, and high serum Lp(a) in the transplant recipient results in rapid
occlusion of blood vessels in the transplant. Ascorbate may also be useful
in the area of hemodialysis, where loss of ascorbate and other vitamins and
trace elements from the blood of hemodialysis patients can result in
increased serum levels of Lp(a) and thus increased risk of cardiovascular
disease. Finally, it appears that ascorbate alone and in combination with
binding inhibitors, specifically with plasmin competitors, may be
therapeutically useful for treatment of the pathogenic effects of diabetes
which is associated with elevated serum concentrations of Lp(a).
The present invention provides methods and pharmaceutical agents for both
the treatment and prevention of cardiovascular disease in vivo; methods and
agents for the preservation of damage linked vessel occlusion in explanted
tissues and organs, as well as methods and agents for the prevention of
hemodialysis-linked cardiovascular disease. Each of these embodiments is
discussed in turn below.
GENERAL APPLICATIONS
The present invention provides a method and pharmaceutical agent for the
treatment and prevention of cardiovascular disease generally, particularly
atherosclerosis, by administering to a subject an effective amount of
ascorbate and one or more binding inhibitors which inhibit the binding of
Lp(a) to blood vessel wall components, particularly to fibrin or fibrinogen.
As used herein, the term "ascorbate" includes any pharmaceutically
acceptable salt of ascorbate, including sodium ascorbate, as well as
ascorbic acid itself. Binding inhibitors include, but are not limited to
.epsilon.-aminocaproic acid (EACA), lysine, tranexamic acid
(4-aminomethylcyclohexane carboxylic acid), p-aminomethylbenzoic acid,
p-benzylamine sulfuric acid, and .alpha.-N-acetyl-lysine-methyl ester and
PROBUCOL (a compound comprised of 2 butyl hydroxy tocopherol groups linked
together by a disulphide group), Aprotinin,
trans-4-aminomethylcyclohexanecarboxylic acid (AMCA), and benzamidine
derivatives such as amidinophenylpyruvic acid (APPA) and
1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl (NANP). An
effective amount of a binding inhibitor or a mixture of binding inhibitors
may also be used, without ascorbate. Other substances used in the treatment
of cardiovascular disease may be co-administered, including: antioxidants,
such as tocopherol, carotene and related substances; vitamins; provitamins;
trace elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl
coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile acid
sequestrants; and mixtures of any two or more of these substances.
Although ascorbate can be used alone or in varying combinations with one or
more representative constituents of the above classes of compounds, we
prefer when treating a pre-existing cardiovascular condition to combine
ascorbate with at least one each of the binding inhibitors, antioxidants and
lipid lowering drugs elements in the dosages (per kilogram of body weight
per day (kg BW/d)) provided in Table 1. It should be noted that Table 1
provides differing concentration ranges of each constituent, depending upon
whether the agent is to be administered orally or parenterally. The variance
in dosages is reflective of variation in disease severity. It will be
realized therefore that if the subject has been diagnosed for advanced
stages of atherosclerosis, dosages at the higher end of this range can be
utilized. However, if only prevention of an atherosclerosis condition is the
object, dosages at the lower end of this range can be utilized.
As an alternative, a pharmaceutical agent identical to the one just
described, but omitting ascorbate, may be employed.
Where ascorbate and binding inhibitors are utilized in the same agent, they
may simply be mixed or may be chemically combined using synthesis methods
well known in the art, such as compounds in which ascorbate and the
inhibitor are covalently linked, or form ionically bound salts. For example,
ascorbate may be bound covalently to lysine, other amino acids, or
.epsilon.-aminocaproic acid by ester linkages. Ascorbyl
.epsilon.-aminocaproate is such an example. In this form the ascorbate
moiety may be particularly effective in preventing undesirable lipid
peroxidation.
In the case of oral administration, a pharmaceutically acceptable and
otherwise inert carrier may be employed. Thus, when administered orally, the
active ingredients may be administered in tablet form. The tablet may
contain a binder such as tragacanth, corn starch or gelatin; a
disintegrating agent, such as alginic acid, and/or a lubricant such as
magnesium stearate. If administration in liquid form is desired, sweetening
and/or flavoring agents may be used. If administration is by parenteral
injection, in isotonic saline, a phosphate buffered solution or the like,
may be used as pharmaceutically acceptable carrier.
The advisability of using binding inhibitors in treating cardiovascular
disease will depend to some extent on the subject's general health,
particularly with regard to hyperfibrinolytic conditions. Most binding
inhibitors (except lysine) are used clinically to treat such conditions. As
a result, monitoring of the subject's coagulation and fibrinolytic system is
recommended before and during treatment for cardiovascular disease.
Long-term administration of binding inhibitors will require formulations in
which the dosages of binding inhibitors are in the lower ranges of the
dosages given in Table 1.
Prevention, as contrasted with treatment, of cardiovascular disease may be
accomplished by oral or parenteral administration of ascorbate alone. Table
1 gives a range of ascorbate concentrations sufficient to lower the serum
Lp(a) concentration. Preferably the prevention of the cardiovascular disease
according to the invention is accomplished by use of a physical mixture of
ascorbate and one or more binding inhibitors, or by use of a compound
comprising covalently linked ascorbate with one or more of the binding
inhibitors, which inhibit binding of Lp(a) to the arterial wall. A binding
inhibitor or mixture of binding inhibitors may also be administered without
ascorbate to prevent Lp(a)-associated cardiovascular disease.
To optimize the therapeutic effect of the release of Lp(a) from the blood
vessel walls, the ascorbate and the binding inhibitors described above may
be separately administered. Further optimization of therapeutic effect can
be gained by using a time release composition to achieve relatively constant
serum concentrations of the agent through time.
CORONARY BYPASS APPLICATIONS
As discussed above, recurrence of cardiovascular disease after bypass
surgery is a frequent problem. Physicians often observe that the veins used
to replace occluded arteries become rapidly occluded themselves after
implantation, often requiring the patient to undergo successive surgical
episodes to replace clogged bypasses. While not wishing to be bound to any
theory, we believe that the rapid occlusion observed in many individual's
results from a combination of the patient's pre-existing elevated levels of
Lp(a) and injury to the bypass veins, during transplantation, particularly
as a result of oxidative damage during explantation. This damage makes
binding of Lp(a) to the vessel interior easier. Further, Lp(a) has been
detected in abundance in reoccluded by-pass veins after coronary bypass
surgery. See, Cushing, et al. (1989) Atherosclerosis 9:593-603. Lp(a) is now
known to be the most significant factor for reocclusion of bypass veins.
See, Hoff, H, et al. (1988) Circulation 77:1238-1244. Thus, a further
embodiment of this invention includes using the pharmaceutical agent of the
present invention to lower the bypass patient's Lp(a) before, during and
after surgery while at the same using a solution containing the agent to
rinse and store the bypass veins until such time as the veins are implanted
into the recipient, thereby reducing oxidative damage that can make Lp(a)
binding more likely after implantation.
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