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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
Krystal, G, Arthritis Rheum. 25:318-25 (1982).
Kurokawa, Y, Tohoku J. Ex. Med. 134:183-93 (1981).
Feng, J., In Vitro 13:91-99 (1977).
Malemud, C, Connect Tissue Res 6:171-9 (1978).
Hinrichs U, Arzneimittelforschung 33:143-9 (1983).
Risley, M, Biol Reprod. 36:985-97 (1987).
Popov I, Z. Exp. Chir. Transplant Kunstliche Organe 22:22-6 (1989).
Schiff, L, In Vitro 16:893-906 (1980).
Vinograd-Finkel, F, Otkrytiya, Izobret., Prom. Obraztsy, Tovarnye
Znaki 49:264 (1972).
Rath, M. & L. Pauling, "Solution of the puzzle of human
cardiovascular disease: Its primary cause is ascorbate deficiency,
leading to the deposition of lipoprotein(a) and fibrinogen/fibrin
in the vascular wall," J. Orthomolecular Med. (In Press 1991).
Markwardt, F. & H. P. Klocking, "Chemical control of
hyperfibrinolytic states by synthetic inhibitors of fibrinolytic
enzymes," Biomed. Biochim. Acta 42:725-730 (1983).
Werb, Z. et al., "Endogenous activation of latent collagenase by
rheumatoid synovial cells," New England J. Med 296(18):1017-1023
(1977).
Knox, E. G., "Ischaemic-heart-disease mortality and dietary intake
of calcium," Lancet, i, pp. 1465-1467, Jun. 30, 1973.
Berg, K. "A new serum type system in man--The Lp system," Acta
Path. 59:369-382 (1963).
McLean, J. et al., "cDNA sequence of human apolipoprotein(a) is
homologous to plasminogen," Nature 300:132-137 (1987).
Salonen, E-M, et al., "Lipoprotein(a) binds to fibronectin and has
serine proteinase activity capable of cleaving it," EMBO J.
8(13):4035-4040 (1989).
Harpel, P. C. et al., "Plasmin catalyzes binding of lipoprotein(a)
to immobilized fibrinogen and fibrin," Proc. Natl. Acad. Sci. USA
86:3847-3851 (1989).
Gonzalez-Gronow, M. et al., "Further characterization of the
cellular plasminogen biding site: Evidence that Plasminogen 2 and
Lipoprotein a compete for the same site," Biochemistry 28:2374-2377
(1989).
Hajjar, K. A. et al., "Lipoprotein(a) modulation of endothelial
cell surface fibrinolysis and its potential role in
atherosclerosis" Nature 339:303-305 (1989).
Armstrong, V. W. et al., "The association between serum Lp(a)
concentrations and angiographically assessed coronary
atherosclerosis"; Atherosclerosis 62:249-257 (1986).
Dahlen, G. H., et al., "Association of levels of lipoprotein Lp(a),
plasma lipids, and other lipoproteins with coronary artery disease
documented by angiography," Circulation 74(4):758-765 (1986).
Miles, L. A. et al., "A potential basis for the thrombotic risks
associated with Lipoprotein (a)," Nature 339:301-302 (1989).
Zenker, G. et al., "Lipoprotein(a) as a strong indicator for
cerebrovascular disease," Stroke 17(5)942-945 (1986).
Zechner, R. et al., "Fluctuations of plasma Lipoprotein-A
concentrations during pregnancy and post partum," Metabolism
35(4):333-336 (1986).
Hoff, H. et al., "Serum Lp(a) level as a predictor of vein graft
stenosis after coronary artery bypass surgery in patients,"
Circulation 77(6):1238-1244 (1988).
Rath, M. et al., "Detection and quantification of Lipoprotein(a) in
the arterial wall of 107 coronary bypass patients,"
Arteriosclerosis 9(5):579-592 (1989).
Cushing, G. L. et al., "Quantitation and localization of
Apolipoproteins [a] and B in Coronary artery bypass vein grafts
resected at re-operation," Arteriosclerosis 9(5):593-603 (1989).
Bruckert, E. et al., "Increased serum levels of Lipoprotein(a) in
diabetes mellitus and their reduction with glycemic control," JAMA
263(1):35-36 (1990).
Blumberg, B., et al., "A human lipoprotein polymorphism," J. Clin.
Invest. 41:1936-1944 (1962).
Eaton, D. L., et al., "Partial amoni acid sequence of
apolipoprotein(a) shows that it is homologous to plasminogen,"
Proc. Natl. Acad. Sci. USA, 84:3224-3228 (1987).
Wright, L. C. et al., "Elevated apolipoprotein(a) levels in cancer
patients," Int. J. Cancer 43:241-244 (1989).
Som, S. et al., "Ascorbic acid metabolism in diabetes mellitus,"
Metabolism 30:572-577 (1981).
Maeda, S. et al., "Transient changes in serum liproprotein(a) as an
acute phase protein," Atherosclerosis 78:145-150 (1989).
Kapeghian, J. C. et al., "The effects of glucose on ascorbic acid
uptake in heart endothelial cells: Possible pathogenesis of
diabetic angiopathies," Life Sci. 34:577 (1984).
Tomlinson, J. E. et al., "Rhesus monkey apolipoprotein(a," J. Biol.
Chem. 264:5957-5965 (1989).
Ginter, E. et al., "The effect of chronic hypovitaminosis C on the
metabolism of cholesterol and athergenesis in guinea pigs," J.
Atherosclerosis Res. 10:341-352 (1969).
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
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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.
The treatment protocols for the bypass patient generally follow those
described above for the treatment of pre-existing cardiovascular disease.
The composition of the pharmaceutical agent will generally include
ascorbate, one or more binding inhibitors, one or more antioxidants and one
or more lipid lowering drugs as enumerated and in the dosages given in Table
1. Of course, the level of dosage will depend on disease severity. Further,
the constituents of the agent can be combined just as described above, can
be administered either orally or parenterally and can be combined with a
pharmaceutically acceptable carrier.
TABLE 1
______________________________________
DOSAGES OF COMPONENTS IN THE DRUG
COMPOSITIONS OF THE PRESENT INVENTION
Parenteral
Oral Admin-
Administration istration
______________________________________
Ascorbate: 5 mg-2500 mg/kg bw/d
25 mg-2500
mg/kg
bw/d
Binding inhibitors:
EACA 5 mg-500 mg/kg bw/d
same
Tranexamic Acid
1 mg-100 mg/kg bw/d
same
Para-aminomethyl
1 mg-30 mg/kg bw/d
same
benzoic acid
Lysine 5-500 mg/kg bw/d same
Antioxidants:
Tocopherol 0,1 IU-20 IU/kg bw/d
same
Carotene 100 IU-1000 IU/kg bw/d
same
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
______________________________________
Turning now to vessel treatment and storage, it is important to provide an
in vitro environment which minimizes vessel injury. We conclude that vessel
injury can be reduced by the addition of a combination of ascorbate, binding
inhibitors and antioxidants to the solution in which the vessels are
normally stored. A range of effective concentrations of these constituents
in solution is given in Table 2. The general aspects of live vessel
preservation and storage prior to implantation are well known in the art.
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
______________________________________
APPLICATIONS IN ORGAN TRANSPLANTS
We have also found that the solution and method of the present invention are
effective in preventing cardiovascular disease from occurring in
transplanted organs that have been otherwise successfully implanted in an
organ recipient, particularly in the case of the heart.
As with occlusion of transplanted veins after bypass surgery, a transplanted
heart free of any substantial arterial occlusion may suffer accelerated
atherosclerosis after implantation. We believe that the mechanism described
for occlusion of transplanted vessels applies equally to the heart itself as
a whole, namely that the heart muscle itself, as well as the interiors of
the arterial walls become damaged, making the arteries of the heart more
prone to binding with Lp(a). Because the organ recipient often presents
elevated serum concentration of Lp(a), particularly after surgery (see,
Maeda, S. et al. (1989) Atherosclerosis 78: 145-150), atherosclerosis can
proceed at an accelerated rate.
Treatment follows along the same line as that described for bypass surgery.
Damage to the organ itself is minimized by placing the organ in a solution
containing a mixture of ascorbate, binding inhibitors, and antioxidants in
an otherwise standard storage solution. Concentration ranges for the various
components in the final solution are given in Table 2. Because of the
oxidative cellular damage during extended periods of explantation, the
concentration of antioxidants should be in the higher range of dosages
disclosed in Table 1. The standard storage solution itself is well known in
the art. Storage of the organ in this solution will tend to minimize damage
to arterial walls, thereby providing fewer places for Lp(a) to bind.
Of course, patient treatment is also desireable. If the organ recipient
suffers from some degree of atherosclerosis at the time of organ transplant,
the protocol and drug described above generally for the treatment of
atherosclerosis should be employed. If, however, the patient does not suffer
from atherosclerosis, use of the drug and protocol described above for
prevention of atherosclerosis is desired. In all cases, the lowest dosages
of ascorbate should be employed in the drug composition since ascorbate has
an immune stimulatory effect.
APPLICATIONS IN HEMODIALYSIS TREATMENT
It is well known that patients who suffered renal failure and require
regular dialysis treatment to cleanse the blood of metabolic waste products
are also at an increased risk for cardiovascular disease. We believe that
the reason for this may be a depletion of ascorbate, vitamins in general and
other essential substances from the blood supply during the hemodialytic
process. As described more fully above, the loss of ascorbate would result
in greater injury to the interior of the artery walls over time and may also
result in the production of elevated Lp(a) levels in the blood serum.
As can appreciated, the solution and method of the present invention can be
applied both to the patient and the hemodialysis solution to prevent and
control hemodialysis-related cardiovascular disease. Turning first to the
hemodialysis solution, it is desired to add a combination of ascorbate,
binding inhibitors and antioxidants to the solution to produce
concentrations of these compounds in solution in the range of concentrations
provided in Table 2.
In order to achieve the best results, treatment of the dialysis patient
should be carried out in addition to modification of the hemodialysis
solution. Treatment should follow the drug and protocols set forth in detail
above for the treatment of a preexisting atherosclerotic condition.
APPLICATIONS IN TREATMENT OF DIABETES
The composition and method of the present invention are also useful in the
treatment of the pathological effects of diabetes mellitus. In diabetes
mellitus, pathological charges in the arteries frequently lead to clinical
symptoms or complete failure in various organs such as the kidney, eye and
peripheral circulation system. Therefore, one therapeutic focus in diabetes
mellitus is the treatment of diabetic angiopathy.
It appears that glucose competitively inhibits the physiologic uptake of
ascorbate in different cell systems of the body, including the arterial
wall. Kapeghian, et al. (1984) Life Sci. 34: 577. Such damage to arterial
walls creates binding sites for Lp(a). Further, Lp(a) has been found to be
elevated in the blood serum of diabetic patients. The atherogenic process is
perhaps therefore accelerated by the combination of damaged arteries and
elevated Lp(a). Therefore, we propose that ascorbate alone or in combination
with at least one binding inhibitor has therapeutic value in treating
diabetes-related atherosclerosis.
Thus, another embodiment of the present invention is the use of a
composition and method in treating the pathogenic effects of diabetes
mellitus, particularly with regard to atherosclerotic conditions.
The treatment protocol involves the oral or parenteral administration of a
pharmaceutical composition comprised of ascorbate, one or more binding
inhibitors and one or more antioxidants. Dosages for a course of treatment
are provided in Table 1. The dosage of ascorbate should preferably fall
within the higher range, thereby increasing its chance of cellular uptake in
the presence of high serum levels of glucose.
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 respect 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
lipoprotein involved as risk factors in plasma and as constituents of the
atherosclerotic plaque.
Three female Hartly guinea pigs with an average weight of 800 g and an
approximate age of 1 year were studied. One animal received an extreme
hypoascorbic diet with 1 mg ascorbate/kg body weight/d. Another animal
received 4 mg/k 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
antibody 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 Na-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 percentage of lesion area between the control animals and the
hypoascorbic diet animals was 25% deposition of lipids and lipoproteins in
the arterial wall.
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
Cholesterol
(mg/dl)
Total Plasma
5.03 3.01 20.64
Ascorbic
Acid .mu.g/ml
Atheroscl. -- 25 19
Lesion
(Percent of
Aorta Thorac.
Surface)
______________________________________
A possible inhibitor may be identified first by adding molar amounts of the
possible inhibitor at a little larger, by approximately 5 times, the amount
of .epsilon.-aminocaproic acid found in the earlier study. If, at this
concentration, a possible inhibitor is found to inhibit the agglutination,
studies are made at lower concentrations, to determine the concentration
that has a 50% initiatory effect.
EXAMPLE 3
Human arterial wall was obtained post mortem from the aorta ascendens. The
tissue showed homogenous intimal thickening (early atherosclerotic lesion).
It was cut into pieces, with 100 mg of the cut up tissue homogenized in a
glass potter for 1 minute in 2.5 ml of the following solutions:
______________________________________
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.
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 needs in the field of prevention and treatment
of induced cardiovascular disease. Although preferred embodiments and
examples have been disclosed, it is understood that the invention is in no
way limited thereby, but rather is defined by the claims that follow and the
equivalents thereof.
* * * * *
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