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Description  |
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TECHNICAL FIELD
The present invention relates to a method and composition for treating
pathological hydrophobic interactions in which there is acute impairment
of the circulation, especially the microcirculation. More particularly,
the present invention relates to compositions and methods for treating
circulatory diseases comprising using certain ethylene oxide-propylene
oxide condensation copolymers either alone or in combination with other
compounds, including but not limited to, fibrinolytic enzymes,
anticoagulants, free radical scavengers, antiinflammatory agents,
antibiotics, membrane stabilizers and/or perfusion media.
BACKGROUND OF THE INVENTION
The term "pathological hydrophobic interactions" means detrimental adhesion
of components, including but not limited to, cells and molecules in blood
or other biological fluids thereby slowing or stopping the flow of blood
or other biological fluid. The term "fibrinolytic enzyme" means any enzyme
that is capable of cleaving fibrin or capable of causing fibrin to be
cleaved. Enzymes that are capable of cleaving fibrin or causing fibrin to
be cleaved include, but are not limited to, streptokinase, urokinase,
tissue plasminogen activator (t-PA) produced from cell cultures, tissue
plasminogen activator produced by recombinant DNA technology and
plasminogen activator produced from prourokinase. The terms "isotonic" or
"isoosmotic" solution are defined as solutions having the same osmotic
pressure as blood. The term "SOD" means superoxide dismutase and refers to
any enzyme capable of neutralizing oxygen radicals. The terms clot, fibrin
clot and thrombus are used interchangeably. The term "microcirculation"
means blood circulation through blood vessels that are about 50 microns in
diameter or less. The term "soluble fibrin" means soluble high molecular
weight polymers of fibrinogen and fibrin. The term "biological fluids"
means blood, lymph, or other fluids found in animals or humans. The term
"platelet suspension" means a suspension of platelets that has a higher
concentration of platelets than that found in blood. The term "plasma
extender" means any substance that can be added to animal or human blood
to maintain or increase coloid osmotic pressure. The term "cytoprotective"
as used herein, means an increased ability of myocardial, endothelial and
other cells to withstand ischemia or recover from ischemia, or other
noxious insults including but not limited to burns. The term "ischemic
tissue" is any tissue that is damaged from reduced blood flow. The term
"anticoagulant" is any compound or agent that inhibits the blood
coagulation process. The term "reperfusion injury" means injury to tissue
or cells which occurs during reperfusion of damaged tissue with blood. The
term "damaged tissue" means tissue damaged by ischemia, burns, toxins or
other noxious insult. The term "angioplasty" means any invasive procedure
that is used to reduce or eliminate a blockage in a blood vessel and
includes, but is not limited to, percutaneous transluminal angioplasty, or
balloon angioplasty, laser angioplasty, and endarectomy. The term
"antiplatelet drugs", as used herein, means any drug that inhibits the
proliferation of a thrombus and includes, but is not limited to drugs that
have a direct effect on platelets as well as certain nonsteroidal
antiinflammatory drugs and anticoagulants.
It is to be understood that the citation of art contained herein is in no
way to be construed as an admission that said art is suitable reference
against the present patent application nor should this citation act as a
waiver of any rights to overcome said art which may be available to the
applicant.
A number of reports have described high amounts of fibrinogen and/or
soluble fibrin in the blood of patients with thrombosis, impending
thrombosis and many other diseases. These conditions include acute or
chronic infection, severe trauma, burns, sickle cell crisis, malaria,
leukemia, myocardial infarction, sepsis, shock, and almost any serious
illness which produces tissue damage or surgical maneuvers. Evidence
indicates that the high concentrations of fibrinogen and/or soluble fibrin
may play an important role in the pathology of the conditions.
Furthermore, much of the pathology that is encountered in disease may be
due to pathological hydrophobic interactions which may be at least
partially mediated by high concentration of fibrinogen and/or soluble
fibrin.
What is needed is a means of decreasing the adverse effects of soluble
fibrin. This would involve blocking the adhesion of soluble fibrin to
cells in the circulation thereby blocking the aggregation of such cells
and their adhesion or friction to vessel walls in the microvasculature.
This would also decrease the risk of thrombosis.
Each year about 550,000 Americans die from heart attacks. Even more--close
to 700,000--have heart attacks and live. While a heart attack victim may
survive, part of his or her heart will almost certainly die. The death of
heart muscle, called myocardial infarction, is due to coronary artery
thrombosis in 70-90% of the cases. When a thrombosis, or blood clot,
occludes one of the arteries of the heart, it stops the flow of blood to
the surrounding muscle which deprives it of oxygen and other nutrients. In
the past, nothing could be done to reverse this process. The high
technology devices in intensive care units mostly support patients so they
can live while a portion of their heart dies.
Similar situations occur in many other tissues when the blood supply to the
tissue is affected by a thrombus or embolus. Stroke, deep vein thrombosis
and pulmonary embolus are examples. Typically, the clot forms and is not
treated for a relatively long period of time. Blood flow distal to the
clot is greatly diminished or is stopped completely. The tissue that is
normally fed by that vessel will be severely damaged unless blood flow is
reestablished in a short period of time.
It has been found that certain enzymes are able to degrade, initiate or
activate other enzymes that can degrade fibrin deposits to open clogged
arteries. The enzymes which have been used successfully include
streptokinase, urokinase, prourokinase, tissue plasminogen activator
produced from cell cultures and tissue plasminogen activator produced by
recombinant DNA technology. These enzymes are most successful if
administered shortly after the occlusion of the blood vessels before the
heart tissue has sustained irreversible damage. In one study of 11,806
patients treated with intravenous or intracoronary artery streptokinase,
an 18% improvement of survival was demonstrated. If the treatment was
begun within one hour after the initial pain onset of the heart attack,
the in-hospital mortality was reduced by 47%. (See The Lancet, Vol. 8478,
p. 397-401, Feb. 22, 1986). It was demonstrated that early lysis of the
thrombus resulted in salvage of a portion of heart tissue which would have
otherwise have died. In studies using angiography to assess the patency of
blood vessels, it was found that tissue plasminogen activator could
completely open the vessels of 61% of the 129 patients versus 29% of
controls who were not treated with the enzyme. (See Verstraete, et al.,
The Lancet, Vol. 8462, p. 965-969, Nov. 2, 1985). Tissue plasminogen
activator requires the addition of approximately 100 .mu.l of Tween 80 per
liter of solution to promote dispersion of the enzyme. (See Korninger, et
al., Thrombos, Haemostas, (Stuttgart) Vol. 46(2), p. 561-565 (1981)).
The natural enzymes that lyse thrombi in vessels do so by activating
fibrinolysis. Fibrin is the protein produced by polymerization of
fibrinogen. It forms a gel which holds the thrombus together. The fibrin
molecules which form clots gradually become cross-linked to make a more
stable clot. All three enzymes, urokinase, streptokinase and tissue
plasminogen activator, are effective because of their ability to activate
an enzyme, plasmin, which degrades fibrin. Thus, they have similar effects
on fibrin but they have different toxicities. If the fibrinolytic
mechanisms (i.e., plasmin) are activated in the vicinity of a clot, the
clot is lysed. If, however, they are activated systemically throughout the
circulation, the body's capacity to stop bleeding or hemorrhage is
markedly reduced. Streptokinase and urokinase tend to activate systemic
fibrinolysis. Consequently, they have been most effective when injected
directly into the affected blood vessel.
Tissue plasminogen activator or t-PA, in contrast, becomes effective only
when it is actually attached to fibrin. This means its activity is largely
localized to the immediate area of a clot and does not produce systemic
fibrinolysis. For this reason, tissue plasminogen activator is thought to
produce less risk of hemorrhage than the other enzymes. If high doses are
used in an effort to increase the rate of clot lysis or to lyse refractory
clots, then the amount of systemic fibrinolysis and risk of hemorrhage can
become significant. t-PA can be injected intravenously into the general
circulation. It circulates harmlessly until it contacts the fibrin in a
blood clot where it becomes activated and causes the lyses of the clot.
Tissue plasminogen activator is able to cause the lysis of a clot which is
extensively cross-linked. This means it is possible to lyse clots which
have been present for many hours.
Remarkable as the new enzyme therapies are, they are subject to serious
complications and are not effective in all patients. Clots in the anterior
descending branch of the left coronary artery are much more readily lysed
than those in other arteries. If the enzyme is not delivered by the blood
stream directly to the thrombus, it has no effect. For various reasons,
more blood passes by or trickles around thrombi in the left anterior
descending coronary artery than in the other major arteries. In addition,
the presence of collateral circulation which forms in response to
compromised blood flow in the major arteries adversely affects the rate of
reopening or recanalization of the thrombosed major arteries. It is
thought the presence of many collateral vessels which allow blood to
bypass the clot reduces the pressure gradient across the clot. This in
turn reduces the blood flow through the tiny openings which may persist in
the clot, impedes the delivery of enzymes to the clot, and prevents the
clot from being lysed.
Even after the clot has been lysed, the factors which led to the formation
of the thrombus persist. This produces a high incidence of re-thrombosis
and further infarction in the hours and days following lysis of the clot.
Rethrombosis has been reported in between 3% and 30% of cases in which the
initial treatment successfully lysed the clot. Anticoagulants are
currently used to prevent the formation of new thrombi, but they tend to
induce hemorrhage. There is a delicate balance between the amount of
anticoagulation necessary to prevent re-thrombosis of the vessels and that
which will produce serious hemorrhage.
A reported advantage of t-PA is its short half-life of less than 10
minutes, which may allow rapid reversal of bleeding problems should they
occur. However, the clinical value of this consideration has not yet been
demonstrated. Moreover, the short half-life may lead to an increased
reocclusion rate following discontinuation of thrombolytic therapy, (See
Williams, D. O., et al., "Intravenous recombinant tissue-type plasminogen
activator in patients with acute myocardial infarction: a report from the
NHLBI Thrombolysis in Myocardial Infarction Trial.", Circulation 1986;
73:338-46). To counter this problem, t-PA infusions have been continued
for up to 6 hours in phase II of the TIMI (Thrombolysis in Myocardial
Infarction Trial). Whether this will effectively reduce the incidence of
reocclusion without increased bleeding remains to be proven. Although
active thrombolysis ceases shortly after discontinuing administration of
t-PA, it takes several hours to replace fibrinogen, so that the risk of
continued bleeding does not terminate when t-PA is stopped. (See Rich, M.
W., "tPA: Is it worth the price?", American Heart Journal, 1987, Vol
114:1259-1261.
Finally, dissolving the clot after irreversible damage has taken place has
little effect. The irreversible damage could be either to the heart muscle
or vascular bed of the tissue supplied by the blood vessel. Once a cell is
dead, the change is irreversible. However, the term irreversible damage is
frequently applied to tissue in which a chain of events leading to cell
death has been initiated, even though most cells are not yet dead. If this
chain of events were broken, for example by restoring the microvasculature
blood supply or stabilizing fragile membranes, then many cells could be
saved. A major problem in widespread implementation of this new enzyme
therapy is to find ways of identifying and treating the patients earlier
in their disease and to find ways to make the treatment effective for a
longer period of time after the initiation of thrombosis.
Animal studies have provided a better understanding of the events which
control blood flow and tissue death following coronary artery thrombosis.
Much of the heart muscle receives blood from more than one vessel. For
this and other reasons, the tissue changes following a coronary thrombosis
are divided into distinct zones. The central zone of tissue, i.e., usually
that zone of tissue closest to the thrombus, becomes almost completely
necrotic. This is surrounded by an area of severe ischemia. Outside this
is an area of lesser ischemia called the marginal zone. Finally, there is
a jeopardized zone which surrounds the entire area.
In studies with baboons, the central necrotic area was not affected by
recanalization of the vessel after several hours. However, muscle in the
other zones which had undergone less severe damage during the ischemic
period could be salvaged. A surprising finding was that lysing of the
thrombus to produce a perfect arteriograph was insufficient to restore
normal flow in the majority of animals. (See Flameng, et al, J. Clin.
Invest., Vol. 75, p. 84-90, 1985). Some further impediment to flow had
developed in the area supplied by the vessel during the time that it was
occluded. In further studies, it was demonstrated that immediately after
removing the obstruction to the vessel, the flow through the damaged
tissue began at a high rate. However, within a short time the blood flow
through the ischemic zone decreased and the tissue died.
Consequently, the regional blood flow immediately after reperfusion is a
poor predictor of the salvage of myocardial tissue. If the blood flow
through the damaged tissue remained near the normal levels, the success of
tissue salvage was much greater. Hemorrhage occurred almost exclusively in
the severely ischemic zone reflecting damage to the small blood vessels.
The hemorrhage, however, remained limited to the severely ischemic tissue
and did not cause extension of the infarction or other serious
complication. Therapies which could preserve the blood flow through the
small blood vessels distal to the major area of thrombus after reperfusion
could be expected to markedly increase the salvage of myocardial tissue.
The damage to heart muscle cells which occurs after lysing the thrombus is
due to other factors as well as ischemia. Contact of fresh blood with
damaged or dead cells induces the influx of neutrophils, or pus cells,
which can damage or kill heart cells which would otherwise have recovered.
Much of the damage caused by neutrophils has been attributed to superoxide
ions. (For a general review, please see "Oxygen Radicals and Tissue
Injury" Proceedings of a Brook Lodge Symposium, Augusta Michigan, Barry
Halliwell, Ed.) The superoxide anion can damage tissue in several ways.
The interaction of the superoxide anion with hydrogen peroxide leads to
the production of hydroxyl radicals which are highly toxic and react
rapidly with most organic molecules. Mannitol is a selective scavenger of
hydroxyl radicals. The enzyme, superoxide dismutase, catalyzes the
decomposition of the superoxide anion. Enzymes such as superoxide
dismutase, free radical scavengers or agents which prevent the influx on
neutrophils are able to increase the salvage of heart muscle cells.
Continuing therapy is needed even after restoration of blood flow and
salvage of damaged tissue. The arteriosclerosis that caused the original
heart attack remains. American and European researchers have found that
arteriosclerosis still narrows the arteries in 70-80% of patients whose
clots were lysed by thrombolytic therapy. Many physicians believe this
obstruction must be opened for long term benefits.
Balloon angioplasty is a procedure whereby a catheter with a small balloon
is inserted into the narrowed artery. The balloon is inflated, compresses
the atherosclerotic plaque against the vessel wall and dilates the artery.
The effectiveness of this procedure is limited by the effects of ischemia
produced by the balloon, by embolization of atheromatous material which
lodges in distal vessels and by an increased tendency for immediate or
delayed thrombosis in the area damaged by the balloon. The balloon tears
the tissue exposing underlying collagen and lipid substances which induce
formation of thrombi. The thrombus may occlude the vessel immediately or
set up a sequence of events which leads to occlusion many days or weeks
later. In addition, there is an interruption of blood flow to the heart
tissue when the balloon is inflated. When the blood flow is interrupted,
tissue downstream from the balloon is deprived of blood and can be
damaged. Balloon angioplasty is representative of numerous clinical and
experimental procedures for repairing the lumen of diseased arteries and
vessels.
In other forms of angioplasty, means other than a balloon are used to clear
the blockage from the blood vessel. For example, lasers are being used to
actually burn away the offending blockage. In addition, wire stents are
being implanted in the vessel to hold the vessel open.
What is needed is a means of rendering the surface of the dilated vessel
less thrombogenic, improving the blood flow through the distal tissue and
breaking the embolized material into smaller pieces which are less likely
to produce embolic damage. A means of restoring blood flow through the
microcapillaries downstream from the site of balloon inflation is also
required.
Another area where fibrinogen/fibrin plays a role is tumors. There is now
strong evidence that fibrinogen-related proteins are localized in solid
tumors. The anatomical distribution of fibrin in tumors varies depending
on the tumor type. In carcinomas, fibrin is deposited in the tumor stroma
and around tumor nests and may be particularly abundant toward the tumor
periphery and at the tumor host interface. By contrast, fibrin is often
less prominent in older, more central tumor stroma characterized by
sclerotic collagen deposits. Fibrin may also be found between individual
carcinoma cells. In some, but not all such cases, interepithelial fibrin
deposits are related to zones of tumor necrosis; however, zones of tumor
necrosis are not necessarily sites of fibrin deposition. Fibrin deposition
in sarcomas has been less carefully studied than that in carcinomas. In
lymphomas, fibrin deposits may be observed between individual malignant
tumor cells as well as between adjacent, apparently reactive benign
lymphoid elements. Fibrin has been reported to appear in zones of tumor
sclerosis, as in Hodgkin's disease. Research has indicated that the
pattern and extent of fibrin deposition are characteristic for a given
tumor. (See Hemostasis and Thrombosis, Basic Principles and Clinical
Practice, " Abnormalities of Hemostasis in Malignancy", pp. 1145-1157, ed.
by R. W. Colman, et al., J. B. Lippincott Company, 1987).
The lack of a uniform vascular supply to tumors can impede diagnostic and
therapeutic procedures. For example, hypoxic tumors are less susceptible
to many drugs and to radiation. Conventional drugs and new drugs, such as
monoclonal antibody conjugates, are not effective unless they are
delivered to tumor cells. Fibrin deposits that surround some types of
tumors inhibit delivery of the drugs to the tumor. The blood supply of
tumors is further compromised by other factors as well. Blood vessels in
tumors are frequently small and tortuous. The hydrodynamic resistance of
such channels further impedes the flow of blood to tumors.
Finally, lipid material on the atherosclerotic wall contributes to the bulk
of the plaque which narrows the lumen of the artery and produces a highly
thrombogenic surface. What is needed is a method of extracting or covering
lipids from atherosclerotic plaques which leaves their surfaces less
thrombogenic and reduces their bulk.
Use of copolymers prepared by the condensation of ethylene oxide and
propylene oxide to treat an embolus or a thrombus has been described (See
U.S. Pat. No. 3,641,240). However, the effect is limited to recently
formed, small (preferably microscopic) thrombi and emboli which are
composed primarily of platelets. To be effective, the compound must be
used within 20 minutes after the initiation of thrombosis.
The use of the ethylene oxide and propylene oxide copolymer has little or
no effect on a clot in a patient who has suffered a severe coronary
infarction because such patients almost never receive treatment within 20
minutes following initiation of thrombosis. It is likely that many persons
do not develop symptoms until the thrombus reaches considerable size. The
clots that are occluding the blood vessel in these patients are large and
stable clots. Stable clots are clots in which the fibrin has undergone
cross linking. Fibrin which has undergone crosslinking is not effected by
presence of the ethylene oxide-propylene oxide copolymers. The copolymers
only affect new clots composed primarily of platelets in which the newly
formed fibrin has not crosslinked.
Another problem that commonly occurs in damaged tissue where blood flow is
interrupted is a phenomenon called "no reflow" phenomenon. This is a
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