U.S. patent application number 10/629515 was filed with the patent office on 2005-04-14 for antithrombotic materials and methods.
Invention is credited to Ammons, William S., White, Mark L..
Application Number | 20050080008 10/629515 |
Document ID | / |
Family ID | 24584270 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050080008 |
Kind Code |
A1 |
White, Mark L. ; et
al. |
April 14, 2005 |
Antithrombotic materials and methods
Abstract
Antithrombotic materials and methods are provided for the
treatment of thrombotic disorders, in which therapeutically
effective amounts of BPI protein products are administered.
Inventors: |
White, Mark L.; (Sonoma,
CA) ; Ammons, William S.; (Pinole, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
24584270 |
Appl. No.: |
10/629515 |
Filed: |
July 29, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10629515 |
Jul 29, 2003 |
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09610785 |
Jul 6, 2000 |
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6599881 |
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09610785 |
Jul 6, 2000 |
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09299319 |
Apr 26, 1999 |
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6107280 |
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09299319 |
Apr 26, 1999 |
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09063465 |
Apr 20, 1998 |
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5935930 |
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09063465 |
Apr 20, 1998 |
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08644290 |
May 10, 1996 |
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5741779 |
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Current U.S.
Class: |
514/2.2 ;
514/14.9 |
Current CPC
Class: |
A61K 38/1751 20130101;
A61K 2300/00 20130101; Y10S 514/822 20130101; A61P 7/02 20180101;
Y10S 530/829 20130101; A61K 38/49 20130101; A61K 38/1751 20130101;
A61K 38/49 20130101; A61P 7/00 20180101; A61K 38/49 20130101; A61P
9/00 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/37 |
Claims
What is claimed is:
1. A method for slowing clot formation in blood comprising
administering to a subject a BPI protein product in an amount
effective to delay or prevent clot formation in the blood.
2. A method for enhancing clot dissolution in blood comprising
administering to a subject a BPI protein product in an amount,
effective to enhance clot dissolution in the blood.
3. A method for treating a thrombotic disorder in a subject
comprising administration of a pharmaceutically effective amount of
a BPI protein product.
4. A method for treating a thrombotic disorder in a subject
comprising co-administration of a pharmaceutically effective amount
of a BPI protein product and a thrombolytic agent.
5. The method of claim 4 wherein the amount of the thrombolytic
agent is less than that required for a desired pharmaceutical
effect when the thrombolytic agent is administered as a
monotherapy.
6. A method for enhancing reperfusion or reducing reocclusion in a
subject treated with a thrombolytic agent comprising
co-administration of a pharmaceutically effective amount of a BPI
protein product and the thrombolytic agent.
7. A method for decreasing the dose of a thrombolytic agent
required to establish reperfusion or to reduce reocclusion in a
subject comprising co-administration of a BPI protein product and a
thrombolytic agent, the dosage of the thrombolytic agent being less
than that required for a desired pharmaceutical effect when the
thrombolytic agent is administered as a monotherapy.
8. A method of slowing clot formation in blood comprising
contacting the blood with an amount of BPI protein product
effective to delay or prevent clot formation in the blood.
9. A method for enhancing clot dissolution in blood comprising
contacting the blood with an amount of BPI protein product
effective to dissolve or lyse the clot.
10. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 and 9 wherein the
BPI protein product is an amino-terminal fragment of BPI protein
having a molecular weight of about 21 kD to 25 kD.
11. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 and 9 wherein the
BPI protein product is rBPI.sub.23 or a dimeric form thereof.
12. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 and 9 wherein the
BPI protein product is rBPI.sub.21.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to therapeutic
compositions and treatment methods utilizing
bactericidal/permeability-increasing protein (BPI) protein products
for the treatment of thrombotic disorders.
[0002] The coagulation, or blood clotting process is involved both
in normal hemostasis, in which the clot stops blood loss from a
damaged blood vessel, and in abnormal thrombosis, in which the clot
blocks circulation through a blood vessel. During normal
hemostasis, the platelets adhere to the injured blood vessel and
aggregate to form the primary hemostatic plug. The platelets then
stimulate local activation of plasma coagulation factors, leading
to generation of a fibrin clot that reinforces the platelet
aggregate. Later, as wound healing occurs, the platelet aggregate
and fibrin clot are degraded by specifically activated proteinases.
During the pathological process of thrombosis, the same mechanisms
create a platelet/fibrin clot that occludes a blood vessel.
Arterial thrombosis may produce ischemic necrosis of the tissue
supplied by the artery, e.g., myocardial infarction due to
thrombosis of a coronary artery, or stroke due to thrombosis of a
cerebral artery. Venous thrombosis may cause the tissues drained by
the vein to become edematous and inflamed, and thrombosis of a deep
vein may result in a pulmonary embolism.
[0003] An increased tendency toward thrombosis accompanies surgery,
trauma, many inflammatory disorders, malignancy, pregnancy,
obesity, vascular disorders and prolonged immobilization. Inherited
thrombotic tendencies, which are much rarer, are being increasingly
recognized and include deficiencies of the protein C-protein S
system, deficiencies of antithrombin III (ATIII),
dysfibrinogenemias, and other disorders of the fibrinolytic system.
The evaluation of hypercoagulable risk involves checking for a
family history of thromboembolism, and for other systemic
predisposing diseases or conditions that favor localized vascular
stasis (such as prolonged immobilization, pregnancy, or malignancy)
and evaluating possible laboratory abnormalities, such as
thrombocytosis, elevated blood or plasma viscosity, and elevated
plasma levels of coagulation factors or fibrin degradation
products. Levels of ATIII, protein C, or protein S levels, may also
be measured, although hypercoagulability due to such abnormalities
is uncommon compared to factors such as stasis or localized
injury.
[0004] Severe derangements of the coagulation process are seen in
disseminated intravascular coagulation (DIC), a syndrome
characterized by the slow formation of fibrin microthrombi in the
microcirculation and the development of concomitant fibrinolysis.
The net result of these processes is the consumption of platelets
and clotting factors in the thrombotic process, and the proteolytic
digestion of several clotting factors by the fibrinolytic process,
leading to decreased coagulability of the patient's blood. DIC
never occurs as a primary disorder; it is always secondary to
another disorder. These primary disorders fall into three general
categories: (1) release of procoagulant substances into the blood,
as may occur in amniotic fluid embolism, abruptio placentae,
certain snake bites, and various malignancies, (2) contact of blood
with an injured or abnormal surface, as may occur in extensive
burns, infections, heat stroke, organ grafts, and during
extracorporal circulation, and (3) generation of
procoagulant-active substances within the blood, as may occur if
red or white blood cell or platelet membranes become damaged and
release thromboplastic substances, e.g., during leukemia treatment,
hemolytic transfusion reactions and microangiopathic hemolytic
anemia. Bacterial endotoxins on, associated with or released from
gram-negative bacteria also have thromboplastin-like properties
that initiate clotting.
[0005] Intravascular clotting occurs most frequently with shock,
sepsis, cancer, obstetric complications, burns, and liver disease.
There are no specific symptoms or signs unique to DIC. Bleeding,
however, is much more evident than thrombosis. The rate and extent
of clotting factor activation and consumption, the concentration of
naturally occurring inhibitors, and the level of fibrinolytic
activity determine the severity of the bleeding tendency. In some
patients there is no clinical evidence of bleeding or thrombosis,
and the syndrome becomes apparent only as a consequence of abnormal
blood coagulation tests. Many patients develop only a few petechiae
and ecchymotic areas and bleed a little more than usual from
venipuncture sites. More pronounced forms of diffuse intravascular
clotting may become evident as a result of severe gastrointestinal
hemorrhage or genitourinary bleeding. In some instances bleeding
may cause death. Hemorrhage caused by the DIC syndrome can be
especially life threatening in association with obstetric
complications or in conjunction with surgery.
[0006] The endpoint of the coagulation process is the generation of
a powerful serine protease, thrombin, which cleaves the soluble
plasma protein fibrinogen so that an insoluble meshwork of fibrin
strands develops, enmeshing red cells and platelets to form a
stable clot. This coagulation process can be triggered by injury to
the-blood vessels and involves the rapid, highly controlled
interaction of more than 20 different coagulation factors and other
proteins to amplify the initial activation of a few molecules to an
appropriately sized, fully developed clot. Most of the coagulation
proteins are serine proteases that show a high degree of homology
(Factors II, VII, IX, and X); others are cofactors without enzyme
activity (Factors V and VIII). These proteins circulate as inactive
zymogens in amounts far greater than are required for blood
clotting. Both the injured vessel wall and platelet aggregates
provide specialized surfaces that localize and catalyze the
coagulation reactions.
[0007] The coagulation cascade can be initiated via two different
activation pathways: the intrinsic pathway, involving contact with
injured tissue or other surfaces, and the extrinsic pathway,
involving tissue factor expressed on injured or inflamed tissue.
Both pathways converge into a common pathway when Factor X is
activated at the platelet surface. [See, e.g., Cecil's Essentials
of Medicine, 3rd ed., W B Saunders Co., Pennsylvania (1983);
Goodman & Gilman, The Pharmacological Basis of Therapeutics,
9th ed., McGraw-Hill, NY (1996).] The intrinsic pathway begins when
Factor XII is activated to XIIa by contact with the altered or
injured blood vessel surface or with another negatively charged
surface, such as a glass tube. Cofactors or promoters of Factor XII
activation include prekallikrein, high molecular weight kininogen,
and Factor XI. These proteins form a surface-localized complex
which optimally activates Factor XII. The activated Factor XIIa
then converts the complex-bound Factor XI to its active form, XIa,
and also converts prekallikrein to its active form, kallikrein,
which then cleaves high molecular weight kininogen to form
bradykinin. In turn, Factor XIa requires calcium ions (Ca.sup.2+)
to activate Factor IX to IXa. Factor XIa may also activate Factor
VII (in the extrinsic pathway) as well. Activated Factor XIa also
cleaves plasminogen to form plasmin, which is the main protease
involved in the fibrinolytic mechanisms that restrain blood
clotting. In the presence of Ca.sup.2+ and phospholipid, Factor IXa
activates Factor X to Xa, which is the first step in the common
pathway. Factor X activation usually takes place at the plasma
membrane of stimulated platelets but also may occur on the vascular
endothelium.
[0008] In the extrinsic pathway, the release of tissue factor from
injured tissues directly activates Factor VII to VIIa. Tissue
factor is present in activated endothelium and monocytes as well as
in brain, vascular adventitia, skin, and mucosa. Factor VIIa then
activates Factor X to Xa in the presence of Ca.sup.2+. In addition,
the tissue factor, Factor VII, and Ca.sup.2+ form a complex that
can activate Factor IX (in the intrinsic pathway).
[0009] The activated Factor Xa (the first step in the common
pathway) then activates prothrombin (Factor II) to generate the
protease thrombin.
[0010] Assembly of the plasma prothrombinase complex on the surface
of activated platelets in the presence of Factor V, another
cofactor, enhances the efficiency of prothrombin activation to
thrombin on the platelet surface. Thrombin cleaves fibrinogen,
which is a large, asymmetric, soluble protein with a molecular
weight of about 340 kilodaltons consisting of three pairs of
polypeptide chains: A.alpha., B.beta., and .gamma.. Thrombin first
removes small peptides from the A.alpha. chain of fibrinogen to
form Fibrin I, which polymerizes end to end; further thrombin
cleavage of small peptides from the B.beta. chain leads to
formation of Fibrin II molecules, which polymerize side to side and
are then cross-linked via the .gamma. subunits by the plasma
glutaminase (Factor XIII) to form an insoluble fibrin clot.
[0011] Thrombin has multiple critical actions during coagulation in
addition to the cleavage of fibrinogen to fibrin. It activates
platelets, exposing their procoagulant activity (e.g., binding
sites for the prothrombinase complex) and induces the release of
platelet-aggregating substances such as thromboxane, Ca.sup.2+,
ADP, von Willebrand factor, fibronectin, and thrombospondin.
Thrombin cleaves Factors VIII and Va, thus augmenting the
coagulation cascade, and also cleaves plasma glutaminase, the
enzyme which cross-links fibrin and stabilizes the fibrin clot.
Thrombin acts on the endothelium by binding to the surface protein
thrombomodulin to activate protein C, which is a potent inactivator
of Factors Va and VIIIa and also stimulates fibrinolysis. Thrombin
also causes endothelial cell contraction. Conversely, endothelium
can bind and inactivate thrombin, and in some cases can generate
the vasodilatory substance prostacyclin in response to thrombin.
Thus, thrombin activation contributes to the limitation as well as
the initiation of clotting.
[0012] There are two commonly used tests for measuring the
coagulability of blood: the activated partial thromboplastin time
(APTT or PTT) and the prothrombin time (PT). Blood generally clots
in vitro in four to eight minutes when placed in a glass tube.
Clotting is prevented if a chelating agent such as
ethylenediaminetetraacetic acid (EDTA) or citrate is added to bind
Ca.sup.2+. Recalcified plasma, i.e., plasma in which Ca.sup.2+ has
been replenished, clots in two to four minutes. The clotting time
after recalcification is shortened to 26 to 33 seconds by the
addition of negatively charged phospholipids and a particulate
substance such as kaolin (aluminum silicate); this
post-recalcification clotting time is the APTT. Alternatively,
recalcified plasma will clot in 12 to 14 seconds after addition of
"thromboplastin," a saline extract of brain that contains tissue
factor and phospholipids; this post-recalcification clotting time
is the PT.
[0013] An individual with a prolonged APTT and a normal PT is
considered to have a defect in the intrinsic coagulation pathway,
because all of the components of the APTT test (except kaolin) are
intrinsic to the plasma. A patient with a prolonged PT and a normal
APTT has a defect in the extrinsic coagulation pathway, since
thromboplastin is extrinsic to the plasma. Prolongation of both the
APTT and the PT suggests a defect in a common pathway.
[0014] Whereas the blood coagulation pathways involve a series of
enzymatic activations of serine protease zymogens, downregulation
of blood clotting is influenced by a variety of natural
anticoagulant mechanisms, including antithrombin III (ATIII), the
protein C-protein S system, and fibrinolysis. Normal vascular
endothelium promotes the activation of these anticoagulant
mechanisms by acting as a source of heparin-like substances that
enhance ATIII activation, a source of thrombomodulin, a cofactor in
protein C activation, and a source of the tissue plasminogen
activators that initiate fibrinolysis.
[0015] The anticoagulant ATIII is a plasma protease inhibitor that
is specific for plasmin, the enzyme that dissolves clots. ATIII
also binds all the serine protease procoagulant proteins (Factor Xa
as well as thrombin). Complexes of ATIII and protease are rapidly
cleared by the liver and the reticuloendothelial system. The
activity of ATIII is enhanced by heparin or heparin-like
substances. Other enzymes that play a role in limiting the
coagulation process include the nonspecific plasma protease
inhibitors .alpha..sub.1-antitrypsin, .alpha..sub.2-plasmin
inhibitor, and .alpha..sub.2-macroglobulin, which rapidly
inactivate any circulating serine proteases including thrombin and
plasmin.
[0016] The final stage of the coagulation process is fibrinolysis,
or clot dissolution. The endpoint of the fibrinolytic system is the
generation of the enzyme plasmin, which dissolves intravascular
clots by digesting fibrin. Fibrinolysis is initiated during
clotting by the action of thrombin. When complexed to
thrombomodulin in the endothelium, thrombin activates protein C,
which initiates the release of tissue plasminogen activator (tPA)
from the blood vessel wall. Protein C, together with its cofactor
protein S, also inactivates Factors Va and VIIIa, thus dampening
the coagulation cascade. The tPA then cleaves a circulating
proenzyme, plasminogen, to form the active protease, plasmin, which
digests fibrin. Plasmin is a relatively nonspecific protease; it
not only digests fibrin clots but also digests other plasma
proteins, including several coagulation factors.
[0017] The fibrinolytic system is regulated in a manner so that
unwanted fibrin thrombi are removed, while fibrin in wounds
persists to maintain hemostasis. The tPA is released from
endothelial cells in response to various signals, including stasis
produced by occlusion of the blood vessel. This released tPA exerts
little effect on circulating plasminogen because tPA is rapidly
cleared from blood or inhibited by circulating inhibitors,
plasminogen activator inhibitor-1 and plasminogen activator
inhibitor-2. Both plasminogen and its activator tPA bind to fibrin.
The activity of tPA is actually enhanced by this binding to fibrin,
so that the generation of plasmin is localized to the vicinity of
the blood clot. In addition, fibrin-bound plasmin is protected from
inhibition.
[0018] Four main types of therapies are used to prevent or treat
thrombosis: antiplatelet agents, anticoagulant agents (heparin),
vitamin K antagonists (coumarin derivatives) and thrombolytic
agents. Each type of agent interferes with clotting at a different
site in the coagulation pathway [See, generally, Goodman &
Gilman, The Pharmacological Basis of Therapeutics, 9th ed.,
McGraw-Hill, NY (1996).] Dipyridamole is another agent sometimes
used to prevent or treat thrombosis; it is a vasodilator that, in
combination with warfarin (a coumarin derivative), inhibits
embolization from prosthetic heart valves and, in combination with
aspirin, reduces thrombosis in patients with thrombotic
disorders.
[0019] The antiplatelet agents include aspirin and other
non-steroidal anti-inflammatory agents such as ibuprofen, which are
all administered orally. Aspirin acts by irreversibly inhibiting
platelet cyclooxygenase and thus blocking production of thromboxane
A.sub.2, an inducer of platelet aggregation and potent
vasoconstrictor. In general, antiplatelet agents are used as
prophylaxis against arterial thrombosis, because platelets are more
important in initiating arterial than venous thrombi. Antiplatelet
therapy also reduces the risk of occlusion of saphenous vein bypass
grafts.
[0020] The anticoagulant agents include heparin and its
derivatives, which act by accelerating the activities of ATIII in
inhibiting thrombin generation and in antagonizing thrombin's
action. Low molecular weight preparations of heparin such as
dalteparin and enoxaparin may also be effective for
anticoagulation. Heparin increases the rate of the
thrombin-antithrombin reaction at least a thousandfold by serving
as a catalytic template. Heparin can only be administered
parenterally and has an immediate anticoagulant effect. It is used
to prevent and treat arterial and venous thrombosis, as well as to
keep blood fluid during extracorporeal circulation, such as with
renal hemodialysis or during cardiopulmonary bypass, and to keep
vascular access catheters patent. Heparin therapy is also standard
in patients undergoing percutaneous transluminal coronary
angioplasty.
[0021] Bleeding is the primary adverse effect of heparin. Major
bleeding occurs in 1% to 33% of patients who receive various forms
of heparin therapy. Purpura, ecchymoses, hematomas,
gastrointestinal hemorrhage, hematuria, and retroperitoneal
bleeding are regularly encountered complications of heparin
therapy. Frequently bleeding is most pronounced at sites of
invasive procedures. If bleeding is severe, the effects of heparin
can be counteracted by giving 1 mg of protamine sulfate for each
100 units of heparin. Another side effect, thrombocytopenia, also
occurs in 1% to 5% of patients receiving heparin, but subsides when
heparin is discontinued.
[0022] The vitamin K antagonists (coumarin derivatives) are
sometimes referred to as oral anticoagulants although they do not
actually directly inhibit the coagulation cascade. These agents
include 4-hydroxycoumarin, warfarin sodium, dicumarol,
phenprocoumon, indan-1,3-dione, acenocoumarol, and anisindione.
They interfere with the hepatic synthesis of Factors II, VII, IX,
and X and proteins C and S, which are all involved in the
coagulation process, and therefore have a slow onset of
anticoagulant effect that spans several days. They are given
orally; once the dose is established for an individual patient,
they can provide a steady level of anticoagulation. Vitamin K
antagonists are used for both the prevention and treatment of
arterial and venous thrombosis.
[0023] Bleeding is the major adverse effect of vitamin K
antagonists. Especially serious episodes involve sites where
irreversible damage may result from compression of vital structures
(e.g., intracranial, pericardial, nerve sheath, or spinal cord) or
from massive internal blood loss that may not be diagnosed rapidly
(e.g., gastrointestinal, intraperitoneal, retroperitoneal). The
risk of intracerebral or subdural hematoma in patients over 50
years of age taking an oral anticoagulant over a long term may be
increased ten-fold. For continued or serious bleeding, vitamin
K.sub.1 (phytonadione) is an effective antidote. Since reversal of
anticoagulation by vitamin K.sub.1 requires the synthesis of fully
carboxylated coagulation proteins, significant improvement in
hemostasis does not occur for several hours, regardless of the
route of administration, and 24 hours or longer may be needed for
maximal effect. Warfarin is contraindicated in women who are or may
become pregnant because the drug passes through placental barrier
and may cause fatal hemorrhage in the fetus. Warfarin treatment
during pregnancy may also cause spontaneous abortion, still birth
and birth defects.
[0024] The thrombolytic agents include tPA, streptokinase,
urokinase prourokinase, anisolylated plasminogen streptokinase
activation complex (APSAC), and animal salivary gland plasminogen
activators, all of which act by accelerating fibrinolysis. The
thrombolytic drugs are used to lyse freshly formed arterial and
venous thrombi; they are not efficacious in dissolving thrombi that
have been present for more than a few hours. The intravenous
administration of these agents is now accepted as useful therapy in
the management of deep vein thrombosis, pulmonary embolism, acute
myocardial infarction, and peripheral arterial thromboembolism.
[0025] The major toxicity of all thrombolytic agents is hemorrhage,
which results from two factors. Therapy with thrombolytic drugs
tends to dissolve both pathological thrombi and fibrin deposits at
sites of vascular injury. In addition, a systemic lytic state
results from systemic formation of plasmin, which produces
fibrinogenolysis and destruction of other coagulation factors.
Massive fibrinolysis is initiated, and the inhibitory controls of
the process are overwhelmed. The systemic loss of fibrinogen and
platelet dysfunction caused by the thrombolytic agents also
produces a hemorrhagic tendency. Thus, the use of thrombolytic
agents is contraindicated in situations where there is active
bleeding or a risk of major hemorrhage.
[0026] If heparin is used concurrently with either streptokinase or
t-PA, serious hemorrhage will occur in 2% to 4% of patients.
Intracranial hemorrhage is by far the most serious problem; it
occurs in approximately 1% of cases, and the frequency is the same
with all three thrombolytic agents. Retroperitoneal hemorrhage is
also a serious complication. The frequency of hemorrhage is less
when thrombolytic agents are utilized to treat myocardial
infarction compared with pulmonary embolism or venous thrombosis;
this difference may be due to the duration of therapy (1 to 3 hours
for myocardial infarction, compared to 12 to 72 hours for pulmonary
embolism and venous thrombosis).
[0027] In general, venous thrombosis and its potential for
life-threatening pulmonary embolism are prevented and treated with
heparin or warfarin. Low-dose subcutaneous heparin is frequently
used as prophylaxis against venous thrombosis in surgical patients
but is ineffective in those at highest risk, for example, after hip
fracture. Warfarin reduces mortality from pulmonary embolism and
can be given more safely to immobilized or post-surgical patients
in low-dose or stepwise regimens. Once a venous thrombosis has
developed, however, full-dose heparin treatment for 5 to 10 days
overlapping with full-dose warfarin treatment for 4 to 5 days is
necessary to prevent clot progression and/or pulmonary embolism.
Thrombolytic agents have been used to treat pulmonary embolism and
deep venous thrombosis, but their efficacy in reducing mortality
remains to be established. Aspirin offers little value in treating
venous thromboembolism.
[0028] For acute arterial thrombosis, thrombolytic therapy is
generally the treatment of choice. The goals of thrombolytic
therapy are to achieve rapid reperfusion of the thrombosed vessel
and maintain patency of the vessel; these objectives are based on
the premise that rapid and sustained restoration of blood flow
reduces associated complications. However, multiple episodes of
vessel reocclusion typically follow thrombolytic therapy. Although
widely used as an adjunct to thrombolytic therapy, heparin does not
accelerate thrombolysis or prevent reocclusion of the vessel.
[Klement et al., Thrombosis Haemostasis, 68:64-68 (1992).] In
patients with a fresh coronary thrombosis, intravenous thrombolytic
therapy can permit rapid reperfusion of the thrombosed coronary
artery, thus preserving cardiac function and reducing mortality, if
administered within a few hours of the onset of symptoms.
Thrombolytic agents can also re-establish the patency of thrombosed
peripheral arteries if administered within a few hours after acute
thrombosis. In some instances, e.g., for coronary artery
thrombosis, the thrombolytic agent is administered locally by
selective catheterization of the involved vessel. When given
systemically rather than locally, a therapeutic effect is evident
if the thrombin time is greater than twice normal. Such treatment
should generally be followed by heparin and then oral
anticoagulants to prevent further clot promulgation or recurrence.
Following thrombolytic therapy and before the thrombin time has
returned to its normal range, heparin is generally given to fully
anticoagulate the patient for five to ten days. Warfarin may be
started before the heparin is stopped, depending on whether
prolonged anticoagulation will be required in the management of the
patient's disorder. Aspirin is ineffective in the immediate
setting, but is useful for long-term prophylaxis against arterial
thrombosis. Recent studies suggest that the concurrent
administration of low doses of aspirin improves the efficacy of
thrombolytic therapy of myocardial infarction. Patients with
symptomatic strokes are acutely anticoagulated with heparin and
followed indefinitely with warfarin. Aspirin is recommended for
prophylaxis of stroke in patients with cervical bruits,
asymptomatic carotid stenosis, or a history of transient ischemic
attacks and minor stroke.
[0029] Considerable controversy continues to surround the use of
heparin in DIC. Heparin is usually reserved for fulminant,
explosive forms of diffuse intravascular clotting, in which massive
defibrination is accompanied by fibrinogen levels of less than 100
mg/dL and replacement therapy is not controlling the hemorrhage. In
these cases, heparin is given as a continuous intravenous infusion
at a rate of 10 to 15 units/kg/hour. If the patient is in immediate
danger of dying from hemorrhage, 5000 to 10,000 units of heparin
are given intravenously as a bolus and heparin is then continued at
an infusion rate of 1000 units per hour.
[0030] Heparin can also be useful in treating unstable angina and
patients undergoing elective cardioversion for atrial fibrillation
of greater than 2 days duration. Warfarin and aspirin are useful
for prophylaxis of cerebral embolism, particularly in patients at
risk because of atrial fibrillation. More than 50% of patients with
cerebral embolism have atrial fibrillation. Warfarin is also
recommended for treating patients with mechanical heart valves, for
whom the associated risk of embolism is 2% to 6% per patient per
year despite anticoagulation, patients with rheumatic mitral valve
disease, in whom the rate of associated thromboembolic
complications is 1.5% to 4.7% per year, and patients with a history
of thromboembolism. Aspirin is recommended for patients with mitral
valve prolapse.
[0031] Anti-thrombotic agents are also used routinely to prevent
the occlusion of extracorporeal devices: intravascular cannulas
(heparin), vascular access shunts in hemodialysis patients
(aspirin), hemodialysis machines (heparin), and cardiopulmonary
bypass machines (heparin). In addition, they have been utilized in
the treatment of certain renal diseases (heparin/warfarin) and
small-cell lung cancer (warfarin).
[0032] BPI is a protein isolated from the granules of mammalian
polymorphonuclear leukocytes (PMNs or neutrophils), which are blood
cells essential in the defense against invading microorganisms.
Human BPI protein has been isolated from PMNs by acid extraction
combined with either ion exchange chromatography [Elsbach, J. Biol.
Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss,
et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is
referred to herein as natural BPI and has been shown to have potent
bactericidal activity against a broad spectrum of gram-negative
bacteria. The molecular weight of human BPI is approximately 55,000
daltons (55 kD). The amino acid sequence of the entire human BPI
protein and the nucleic acid sequence of DNA encoding the protein
have been reported in FIG. 1 of Gray et al., J. Biol. Chem.,
264:9505 (1989), incorporated herein by reference. The Gray et al.
amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat.
No. 5,198,541 discloses recombinant genes encoding and methods for
expression of BPI proteins, including BPI holoprotein and fragments
of BPI.
[0033] BPI is a strongly cationic protein. The N-terminal half of
BPI accounts for the high net positive charge; the C-terminal half
of the molecule has a net charge of -3. [Elsbach and Weiss (1981),
supra.] A proteolytic N-terminal fragment of BPI having a molecular
weight of about 25 kD possesses essentially all the anti-bacterial
efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi
et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the
N-terminal portion, the C-terminal region of the isolated human BPI
protein displays only slightly detectable anti-bacterial activity
against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649
(1991).] An N-terminal BPI fragment of approximately 23 kD,
referred to as "rBPI.sub.23," has been produced by recombinant
means and also retains anti-bacterial activity against
gram-negative organisms as well as endotoxin-neutralizing activity.
[Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).]
[0034] The bactericidal effect of BPI has been reported to be
highly specific to gram-negative species, e.g., in Elsbach and
Weiss, Inflammation: Basic Principles and Clinical Correlates, eds.
Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise
mechanism by which BPI kills gram-negative bacteria is not yet
completely elucidated, but it is believed that BPI must first bind
to the surface of the bacteria through electrostatic and
hydrophobic interactions between the cationic BPI protein and
negatively charged sites on LPS. In susceptible gram-negative
bacteria, BPI binding is thought to disrupt LPS structure, leading
to activation of bacterial enzymes that degrade phospholipids and
peptidoglycans, altering the permeability of the cell's outer
membrane, and initiating events that ultimately lead to cell death.
[Elsbach and Weiss (1992), supra]. LPS has been referred to as
"endotoxin" because of the potent inflammatory response that it
stimulates, i.e., the release of mediators by host inflammatory
cells which may ultimately result in irreversible endotoxic shock.
BPI binds to and neutralizes lipid A, reported to be the most toxic
and most biologically active component of LPS.
[0035] In addition to BPI's bactericidal and endotoxin
binding/neutralizing activities, BPI has been shown to bind and
neutralize heparin. Co-owned U.S. Pat. No. 5,348,942 was issued
Sep. 20, 1994 with claims directed to methods of neutralizing the
anticoagulant effects of heparin with BPI protein products (i.e.,
their procoagulant activity). There has been no suggestion or use
of BPI as an anticoagulant or thrombolytic agent, nor any
suggestion of its use for the prophylaxis or treatment of
thrombotic disorders.
[0036] There exists a need in the art for methods and compositions
capable of exerting anticoagulant or thrombolytic effects without
severe adverse side effects, and methods and compositions capable
of improving the therapeutic effectiveness of existing
anticoagulant or thrombolytic agents, which ideally could reduce
the required dosages of such existing agents.
SUMMARY OF THE INVENTION
[0037] The present invention provides novel methods for slowing
clot formation and for enhancing clot dissolution using BPI protein
products, and further provides methods for treatment of thrombotic
disorders by administration of BPI protein products in
therapeutically effective amounts.
[0038] According to the invention, a BPI protein product such as
rBPI.sub.21 is administered to a subject suffering from thrombotic
disorder in an amount effective to treat such disorder, including
prophylactic and therapeutic treatment. BPI protein products reduce
the adverse effects of thrombotic disorder by activities that
include slowing or delaying clot formation (i.e., anticoagulant
activity) or by enhancing, accelerating or increasing clot
dissolution (i.e., thrombolytic activity).
[0039] In another aspect of the invention, methods are provided for
the treatment of thrombotic disorder by concurrent administration
of a BPI protein product with a thrombolytic agent, including tPA,
streptokinase, urokinase, prourokinase, APSAC, animal salivary
gland plasminogen activators, other plasminogen activators, and
derivatives of such plasminogen activators. According to this
aspect of the invention, the thrombolytic agent dissolves the clot,
while the BPI protein product enhances the dissolution activity of
the thrombolytic agent and/or delays clot formation, thus delaying,
decreasing or preventing rethrombosis. The BPI protein products are
effective with both endogenous levels and therapeutic levels of
thrombolytic agents such as plasminogen activators.
[0040] This aspect of the invention also provides methods for
decreasing the dose of a thrombolytic agent required for a desired
therapeutic or prophylactic effect in a patient, such as for
dissolving a blood clot, by concurrent administration of BPI
protein product and the thrombolytic agent.
[0041] The invention further provides methods for accelerating
reperfusion and/or delaying or preventing reocclusions in a subject
treated with a thrombolytic agent, e.g., tPA, by concurrent
administration of BPI protein product with the thrombolytic
agent.
[0042] As will be appreciated from the following detailed
description, methods of the present invention provide safer and
more effective treatment of thrombotic disorders than conventional
therapies. By reducing the dosage of antithrombotic agent required
to achieve a desired therapeutic effect, BPI protein products can
reduce or eliminate the potential side effects often associated
with conventional antithrombotic agent therapies, while not
interfering with the antithrombotic activity of those agents.
Importantly, use of such BPI protein products can enhance the
antithrombotic activity of such agents by slowing clot formation or
enhancing clot dissolution.
[0043] Numerous additional aspects and advantages of the invention
will become apparent to those skilled in the art upon consideration
of the following detailed description of the invention which
describes presently prepared embodiments thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Thrombotic disorders, including acute vascular diseases,
such as myocardial infarction, stroke, peripheral arterial
occlusion, deep vein thrombosis, pulmonary embolism, and other
blood system thromboses, constitute major health risks. Such
disorders are caused by either partial or total occlusion of a
blood vessel by a blood clot, which consists of fibrin and platelet
aggregates. Therapeutic intervention with agents that prevent or
delay clot formation (i.e., anticoagulants) or with agents that
dissolve blood clots (i.e., thrombolytics) is associated with
numerous limitations, complications, risks and side effects. Most
significant are the bleeding side effects associated with
therapeutic doses of such agents and the complications associated
with rethrombosis and reocclusion following reperfusion. It has now
been unexpectedly discovered that administration of BPI protein
products effectively slows clot formation and enhances clot
dissolution in blood. The administered BPI protein product present
in the blood during clot formation delays clotting time and/or may
change the character of the clot that is formed to a looser, less
stable clot.
[0045] It is contemplated that BPI protein products may be
administered alone or concurrently with other antithrombotic
(anticoagulant or thrombolytic) agents. Anticoagulant agents are
agents with the pharmacological effect of slowing clot formation,
such as dalteparin and enoxaparin, the coumarin derivative oral
anticoagulants such as warfarin, and aspirin. Thrombolytic agents
are agents with the pharmacological effect of enhancing clot
dissolution, and include plasminogen activators such as t-PA,
streptokinase, urokinase, proutokinase, APSAC, animal salivary
gland plasminogen activators and derivatives thereof.
[0046] BPI protein products used according to methods of the
invention unexpectedly have the property of making blood clots more
susceptible to dissolution or lysis either at endogenous levels or
added levels of plasminogen activators, such as tPA. Whether the
tPA is present prior to clot formation or after clot formation, the
BPI protein product enhances clot dissolution, e.g., accelerates
clot dissolution or lysis, or provides more complete clot
dissolution or lysis. Thus, BPI protein products are useful in
methods for the treatment of thrombotic disorders, for dissolving
or lysing clots in thrombotic patients, for delaying or inhibiting
hard clot formation or supplementing thrombolytic therapy in the
patients.
[0047] The previously described biological activities of BPI
protein products, including bactericidal, endotoxin binding and
neutralizing, heparin binding and neutralizing activities, do not
suggest or even hint at the anticoagulant or thrombolytic
activities of BPI protein products and the therapeutic uses that
arise from these unexpected and previously undiscovered activities.
In particular, the activity of BPI protein products as agents for
treatment of thrombotic disorders is particularly surprising in
view of the previously discovered activity of BPI protein products
to bind and neutralize heparin (see, co-assigned U.S. Pat. No.
5,348,942).
[0048] The term "treatment" as used herein encompasses both
prophylactic and therapeutic treatment of thrombotic disorders.
[0049] The term "thrombotic disorder" as used herein encompasses
conditions associated with or resulting from thrombosis or a
tendency towards thrombosis. These conditions include conditions
associated with arterial thrombosis, such as coronary artery
thrombosis and resulting myocardial infarction, cerebral artery
thrombosis or intracardiac thrombosis (due to, e.g., atrial
fibrillation) and resulting stroke, and other peripheral arterial
thrombosis and occlusion; conditions associated with venous
thrombosis, such as deep venous thrombosis and pulmonary embolism;
conditions associated with exposure of the patient's blood to a
foreign or injured tissue surface, including diseased heart valves,
mechanical heart valves, vascular grafts, and other extracorporeal
devices such as intravascular cannulas, vascular access shunts in
hemodialysis patients, hemodialysis machines and cardiopulmonary
bypass machines; and conditions associated with coagulapathies,
such as hypercoagulability and disseminated intravascular
coagulopathy that are not the result of an endotoxin-initiated
coagulation cascade.
[0050] "Concurrent administration," or "co-administration" or
"co-treatment," as used herein includes administration of the
agents, in conjunction or combination, together, or before or after
each other. The BPI protein product and other antithrombotic
(including anticoagulant or thrombolytic) agents may be
administered by different routes. For example, the BPI protein
product may be administered intravenously while the antithrombotic
agent is administered intramuscularly, intravenously,
subcutaneously or orally. Alternatively, the BPI protein product
may be administered, e.g., in an aerosolized or nebulized form
while the antithrombotic agent is administered, e.g.,
intravenously. The BPI protein product and antithrombotic agent are
preferably both administered intravenously, in which case they may
be given sequentially in the same intravenous line, or after an
intermediate flush, or in different intravenous lines. The BPI
protein product and antithrombotic agent may be administered
simultaneously or sequentially, as long as they are given in a
manner sufficient to allow both agents to achieve effective
concentrations at the site of thrombosis. During sequential
administration of BPI protein product and antithrombotic agent, it
is also contemplated that a time period varying from minutes to
hours may intervene between the administration of the agents.
[0051] Conventional antithrombotic agents are expected to be
administered in dosages and by routes consistent with the usual
clinical practice. The typical dosages and administration regimens
for some of these anticoagulant and thrombolytic agents, when
administered as monotherapy, are discussed below. Naturally, these
dosages vary as determined by good medical practice and the
clinical condition of the individual patient.
[0052] The dosing of warfarin must be individualized according to
the patient's sensitivity to the drug as indicated by its effect on
the prothrombin time (PT) ratio. The loading dose is typically 2 to
5 mg/day and most patients are satisfactorily maintained at a dose
of 2 to 10 mg/day. Warfarin is generally given orally but may be
administered intravenously if the patient cannot take the drug
orally.
[0053] Urokinase is indicated for lysis of acute pulmonary emboli
and coronary artery emboli, and is also used to restore patency to
intravenous cannulae and catheters. The drug is typically
administered in an initial dose of 2,000 units/lb over a period of
10 minutes followed by a continuous infusion of 2,000 units/lb/hr
for 12 hours. The total dose of urokinase given will range from
2.25 million to 6.25 million units, depending on the weight of the
patient. When it is used to clear intravenous cannulae or
catheters, urokinase is given as a single injection of 5,000 units
in a volume of 1 mL.
[0054] Streptokinase is indicated for use in the management of
acute myocardial infection, lysis of intracoronary thrombi,
arterial thrombosis or embolism, deep vein thrombosis, pulmonary
embolism, and for clearing blocked cannulae or catheters. For
treatment of acute myocardial infarction, 1.5 million units may be
given by intravenous infusion over 60 minutes. Alternatively, it
may be given by intracoronary infusion of a 20,000 unit bolus
followed by 2,000 units/min. over 60 minutes. For other
non-myocardial infarction indications, a dosage of 250,000 units by
intravenous infusion over 30 minutes is appropriate for the great
majority of patients.
[0055] Anistreplase (also known as ASPAC) is generally administered
in a single dose of 30 units by intravenous injection over 2 to 5
minutes. Its use is indicated in the management of acute myocardial
infection and for the lysis of coronary artery thrombi.
[0056] The drug tPA is dosed based upon patient weight, with the
total dose not exceeding 100 milligrams. For patients weighing more
than 67 kg, the recommended dose is a 15 mg initial intravenous
bolus followed by a continuous infusion of 15 mg over 30 minutes,
and further followed by 35 mg infused over the next 60 minutes. For
patients weighing less than or equal to 67 kg, the recommended dose
is a 15 mg initial intravenous bolus followed by 0.75 mg/kg (not to
exceed 50 mg) over 30 minutes, and further followed by 0.5 mg/kg
(not to exceed 35 mg) over the following 60 minutes.
[0057] Therapeutic compositions comprising BPI protein product may
be administered systemically or locally into the involved vessel.
Systemic routes of administration include oral, intravenous,
intramuscular or subcutaneous injection (including into a depot for
long-term release), intraocular and retrobulbar, intrathecal,
intraperitoneal (e.g., by intraperitoneal lavage), intrapulmonary
using aerosolized or nebulized drug, or transdermal. The preferred
systemic route is intravenous administration. In some instances,
e.g., for coronary artery or peripheral artery thrombosis, it is
advantageous to administer the BPI protein product regionally by
selective catheterization of the involved vessel. When given
parenterally, BPI protein product compositions are generally
injected in doses ranging from 1 .mu.g/kg to 100 mg/kg per day,
preferably at doses ranging from 0.1 mg/kg to 20 mg/kg per day, and
more preferably at doses ranging from 1 to 20 mg/kg/day. The
treatment may continue by continuous infusion or intermittent
injection or infusion, at the same, reduced or increased dose per
day for as long as determined by the treating physician. Those
skilled in the art can readily optimize effective dosages and
monotherapeutic or concurrent administration regimens for BPI
protein product and/or other antithrombotic agents, as determined
by good medical practice and the clinical condition of the
individual patient.
[0058] When BPI protein product is concurrently administered with
antithrombotic agents, the BPI protein product and the
antithrombotic agents may each be administered in amounts that
would be sufficient for monotherapeutic effectiveness, or they may
be administered in less than monotherapeutic amounts. It is
expected that BPI protein products are capable of improving the
therapeutic effectiveness of existing anticoagulant or thrombolytic
agents, which would reduce the dosages needed to exert their
desired anticoagulant or thrombolytic effects. This, in turn,
decreases the risk of adverse side effects associated with the use
of thrombolytic agents, including, for example, undesirable
internal or external bleeding.
[0059] BPI protein products may improve the therapeutic
effectiveness of other antithrombotic agents in a variety of ways.
For example, lowering the dosage of the antithrombotic agent
required for therapeutic effectiveness reduces toxicity and/or cost
of treatment, and thus allows wider use of the agent.
Alternatively, concurrent administration may produce an increased,
more rapid or more complete anticoagulant or thrombolytic effect
than could be achieved with either agent alone.
[0060] It is further contemplated that BPI protein product
compositions are useful, in vitro or in vivo in restoring or
maintaining patency of cannulae, catheters and tubing obstructed by
clotted blood or fibrin, in maintaining the anticoagulation of
blood, e.g., in blood bags, and in maintaining blood fluidity in,
e.g., hemodialysis and extracorporeal circulation, and around
foreign implants, e.g., heart valves or prosthetics.
[0061] As used herein, "BPI protein product" includes naturally and
recombinantly produced BPI protein; natural, synthetic, and
recombinant biologically active polypeptide fragments of BPI
protein; biologically active polypeptide variants of BPI protein or
fragments thereof, including hybrid fusion proteins and dimers;
biologically active polypeptide analogs of BPI protein or fragments
or variants thereof, including cysteine-substituted analogs; and
BPI-derived peptides. The BPI protein products administered
according to this invention may be generated and/or isolated by any
means known in the art. U.S. Pat. No. 5,198,541, the disclosure of
which is incorporated herein by reference, discloses recombinant
genes encoding and methods for expression of BPI proteins including
recombinant BPI holoprotein, referred to as rBPI.sub.50 and
recombinant fragments of BPI. Co-owned, copending U.S. patent
application Ser. No. 07/885,501 and a continuation-in-part thereof,
U.S. patent application Ser. No. 08/072,063 filed May 19, 1993 and
corresponding PCT Application No. 93/04752 filed May 19, 1993,
which are all incorporated herein by reference, disclose novel
methods for the purification of recombinant BPI protein products
expressed in and secreted from genetically transformed mammalian
host cells in culture and discloses how one may produce large
quantities of recombinant BPI products suitable for incorporation
into stable, homogeneous pharmaceutical preparations.
[0062] Biologically active fragments of BPI (BPI fragments) include
biologically active molecules that have the same or similar amino
acid sequence as a natural human BPI holoprotein, except that the
fragment molecule lacks amino-terminal amino acids, internal amino
acids, and/or carboxy-terminal amino acids of the holoprotein.
Nonlimiting examples of such fragments include a N-terminal
fragment of natural human BPI of approximately 25 kD, described in
Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant
expression product of DNA encoding N-terminal amino acids from 1 to
about 193 or 199 of natural human BPI, described in Gazzano-Santoro
et al., Infect. Immun. 60:4754-4761 (1992), and referred to as
rBPI.sub.23. In that publication, an expression vector was used as
a source of DNA encoding a recombinant expression product
(rBPI.sub.23) having the 31-residue signal sequence and the first
199 amino acids of the N-terminus of the mature human BPI, as set
out in FIG. 1 of Gray et al., supra, except that valine at position
151 is specified by GTG rather than GTC and residue 185 is glutamic
acid (specified by GAG) rather than lysine (specified by AAG).
Recombinant holoprotein (rBPI) has also been produced having the
sequence (SEQ ID NOS: 1 and 2) set out in FIG. 1 of Gray et al.,
supra, with the exceptions noted for rBPI.sub.23 and with the
exception that residue 417 is alanine (specified by GCT) rather
than valine (specified by GTT). Other examples include dimeric
forms of BPI fragments, as described in co-owned and co-pending
U.S. patent application Ser. No. 08/212,132, filed Mar. 11, 1994,
and corresponding PCT Application No. PCT/US95/03125, the
disclosures of which are incorporated herein by reference.
Preferred dimeric products include dimeric BPI protein products
wherein the monomers are amino-terminal BPI fragments having the
N-terminal residues from about 1 to 175 to about 1 to 199 of BPI
holoprotein. A particularly preferred dimeric product is the
dimeric form of the BPI fragment having N-terminal residues 1
through 193, designated rBPI.sub.42 dimer.
[0063] Biologically active variants of BPI (BPI variants) include
but are not limited to recombinant hybrid fusion proteins,
comprising BPI holoprotein or biologically active fragment thereof
and at least a portion of at least one other polypeptide, and
dimeric forms of BPI variants. Examples of such hybrid fusion
proteins and dimeric forms are described by Theofan et al. in
co-owned, copending U.S. patent application Ser. No. 07/885,911,
and a continuation-in-part application thereof, U.S. patent
application Ser. No. 08/064,693 filed May 19, 1993 and
corresponding PCT Application No. US93/04754 filed May 19, 1993,
which are all incorporated herein by reference and include hybrid
fusion proteins comprising, at the amino-terminal end, a BPI
protein or a biologically active fragment thereof and, at the
carboxy-terminal end, at least one constant domain of an
immunoglobulin heavy chain or allelic variant thereof. Similarly
configured hybrid fusion proteins involving part or all
Lipopolysaccharide Binding Protein (LBP) are also contemplated for
use in the present invention.
[0064] Biologically active analogs of BPI (BPI analogs) include but
are not limited to BPI protein products wherein one or more amino
acid residues have been replaced by a different amino acid. For
example, co-owned, copending U.S. patent application Ser. No.
08/013,801 filed Feb. 2, 1993 and corresponding PCT Application No.
US94/01235 filed Feb. 2, 1994, the disclosures of which are
incorporated herein by reference, discloses polypeptide analogs of
BPI and BPI fragments wherein a cysteine residue is replaced by a
different amino acid. A preferred BPI protein product described by
this application is the expression product of DNA encoding from
amino acid 1 to approximately 193 or 199 of the N-terminal amino
acids of BPI holoprotein, but wherein the cysteine at residue
number 132 is substituted with alanine and is designated
rBPI.sub.21.DELTA.cys or rBPI.sub.21. Other examples include
dimeric forms of BPI analogs; e.g. co-owned and co-pending U.S.
patent application Ser. No. 08/212,132 filed Mar. 11, 1994, and
corresponding PCT Application No. PCT/US95/03125, the disclosures
of which are incorporated herein by reference.
[0065] Other BPI protein products useful according to the methods
of the invention are peptides derived from or based on BPI produced
by recombinant or synthetic means (BPI-derived peptides), such as
those described in co-owned and co-pending U.S. patent application
Ser. No. 08/504,841 filed Jul. 20, 1995 and in co-owned and
copending PCT Application No. PCT/US94/10427 filed Sep. 15, 1994,
which corresponds to U.S. patent application Ser. No. 08/306,473
filed Sep. 15, 1994, and PCT Application No. US94/02465 filed Mar.
11, 1994, which corresponds to U.S. patent application Ser. No.
08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of
U.S. patent application Ser. No. 08/183,222, filed Jan. 14, 1994,
which is a continuation-in-part of U.S. patent application Ser. No.
08/093,202 filed Jul. 15, 1993 (for which the corresponding
international application is PCT Application No. US94/02401 filed
Mar. 11, 1994), which is a continuation-in-part of U.S. patent
application Ser. No. 08/030,644 filed Mar. 12, 1993, the
disclosures of all of which are incorporated herein by
reference.
[0066] Presently preferred BPI protein products include
recombinantly-produced N-terminal fragments of BPI, especially
those having a molecular weight of approximately between 21 to 25
kD such as rBPI.sub.21 or rBPI.sub.23, or dimeric forms of these
N-terminal fragments (e.g., rBPI.sub.42 dimer). Additionally,
preferred BPI protein products include rBPI.sub.50 and BPI-derived
peptides.
[0067] The administration of BPI protein products is preferably
accomplished with a pharmaceutical composition comprising a BPI
protein product and a pharmaceutically acceptable diluent,
adjuvant, or carrier. The BPI protein product may be administered
without or in conjunction with known surfactants, other
chemotherapeutic agents or additional known anti-microbial agents.
One pharmaceutical composition containing BPI protein products
(e.g., rBPI.sub.50, rBPI.sub.23) comprises the BPI protein product
at a concentration of 1 mg/ml in citrate buffered saline (5 or 20
mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of
poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and
0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc.,
Wilmington, Del.). Another pharmaceutical composition containing
BPI protein products (e.g., rBPI.sub.21) comprises the BPI protein
product at a concentration of 2 mg/mL in 5 mM citrate, 150 mM NaCl,
0.2% poloxamer 188 and 0.002% polysorbate 80. Such combinations are
described in co-owned, co-pending PCT Application No. US94/01239
filed Feb. 2, 1994, which corresponds to U.S. patent application
Ser. No. 08/190,869 filed Feb. 2, 1994 and U.S. patent application
Ser. No. 08/012,360 filed Feb. 2, 1993, the disclosures of all of
which are incorporated herein by reference.
[0068] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples. Example 1 addresses the effects of BPI protein product on
clot formation and clot lysis/dissolution under varying conditions
in tube assays. Example 2 addresses the effects of BPI protein
products on clot formation and clot lysis/dissolution, as monitored
by turbidity measurements, under varying conditions in microtiter
plate assays. Example 3 addresses the effects of BPI protein
product in vivo in a rat thrombus model with concurrent
administration of tPA.
EXAMPLE 1
Effects of BPI Protein Product on Clot Formation and Clot
Lysis/Dissolution
[0069] A tube assay was used to determine the effects of a BPI
protein product on clot formation and on clot lysis or dissolution
under a variety of conditions using human plasma samples. Unless
otherwise noted the human plasma used in these assays was prepared
from human blood drawn from a variety of donors into ACD
Vacutainer.RTM. tubes (Becton Dickinson, Mountainview, Calif.)
containing citrate as an anticoagulant, and was stored frozen at
-70.degree. C. For the preparation of platelet rich plasma (PRP),
the anticoagulated blood was centrifuged at approximately
180.times.g for 5 minutes and the plasma removed following this
low-speed centrifugation. For the preparation of platelet poor
plasma (PPP), the anticoagulated blood was centrifuged at
approximately 460.times.g for 10 minutes and the plasma removed
following this higher speed centrifugation.
[0070] In an initial experiment to determine whether BPI protein
products interacted with plasma proteins, ACD plasma pooled from
two human donors was passed over a column containing rBPI.sub.23
that was conjugated to Sepharose via cyanogen bromide.
Approximately 40 mL of plasma was passed through the
rBPI.sub.23-Sepharose column to allow binding of plasma components.
The column was washed with phosphate buffered saline (10 mM
phosphate, 0.15 M NaCl, pH 7.2) until the OD.sub.280 of the wash
was <0.02. The bound plasma components were eluted with high
salt (1.5 M NaCl) and the protein eluate was analyzed by SDS-PAGE.
Amino acid sequence analysis of several protein bands showed that
prothrombin and fibrinogen were bound by rBPI.sub.23.
[0071] The ability of exemplary BPI protein products to delay clot
formation (i.e., anticoagulant activity) and/or to enhance the
dissolution or lysis of a clot once formed (i.e., thrombolytic
activity) was evaluated. Such anticoagulant and/or thrombolytic
activity demonstrates the utility of BPI protein products for the
treatment of thrombotic disorder in a subject suffering from such
disorder. The effects of BPI protein products were evaluated in PPP
and PRP under a variety of conditions as follows.
[0072] A. Effect of Different Surface Environments
[0073] Clot formation and lysis were evaluated in the presence and
absence of rBPI.sub.21 in polypropylene or glass test tubes. For
all experiments in this and subsequent examples, the rBPI.sub.21
used was formulated at 2 mg/mL in 5 mM citrate, 150 mM NaCl, 0.2%
poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.),
and 0.002% polysorbate 80 (TWEEN 80, ICI Americas Inc., Wilmington,
Del.). Other BPI protein products used herein were similarly
formulated at 1 mg/mL. The 0.1% HSA-TBS used was 0.1% human serum
albumin (HSA) [Alpha Therapeutics, Los Angeles, Calif.] in
Tris-buffered saline (TBS) [0.02M Tris, 0.15 M NaCl, pH 7.4]. For
the clot formation part of this experiment, the following reagents
were mixed together in the following order: (1) 160 .mu.L of PPP
(Donor RL); (2) 40 .mu.L of rBPI.sub.21 (either 10 or 250 .mu.g/mL
in 0.1% HSA-TBS); and (3) 200 .mu.L of 40 mM CaCl.sub.2 in TBS, pH
7.8.
[0074] The tubes were allowed to stand at room temperature, and
every 1 minute the tubes were checked by gently tipping the tube
and visually inspecting it for clot formation. The time in minutes
to clot formation after CaCl.sub.2 addition was measured.
1 Minutes to Clot Formation (After CaCl.sub.2 Addition) Control
rBPI.sub.21 rBPI.sub.21 Tube Type (0.1% HSA-TBS) 1 .mu.g/mL 25
.mu.g/mL Polypropylene 12 14 24 Glass 6 6 9
[0075] Clot formation was faster in glass tubes than in
polypropylene tubes. However, rBPI.sub.21 prolonged clotting times
in both glass and polypropylene tubes. The higher rBPI.sub.21
concentration tested (25 .mu.g/mL) produced the longest delay in
clotting time.
[0076] For the clot dissolution part of this experiment, 33 minutes
after CaCl.sub.2 addition, 44 .mu.L of tPA [Calbiochem, San Diego,
Calif.] (600 units/mL, or 1 .mu.g/mL) were added to each tube to
provide a final tPA concentration of 60 units/mL (or 100 ng/mL).
This is in the range of elevated endogenous concentrations observed
for tPA in certain physiologic states/conditions [See, e.g., von
der Mohlen et al., Blood, 85:3437-3443 (1995); Suffradini, et al.,
New Engl. J. Med., 320:1165-1172 (1989)]. The tubes were incubated
for 10 minutes at room temperature, then placed in a 37.degree. C.
water bath. Each tube was checked for clot dissolution/lysis by
visual inspection.
[0077] After 5 minutes, the rBPI.sub.21-treated clots in glass
tubes had detached from the sides of the tube while all other clots
remained adhered to the side of the tube. At 3.5 hours the clot in
the 25 .mu.g/mL rBPI.sub.21 polypropylene tube was smaller,
approximately {fraction (1/3)} the size of the clots in the 1
.mu.g/mL rBPI.sub.21 and control polypropylene tubes. Clot
dissolution/lysis times were faster in glass than in polypropylene
tubes. The presence of 25 .mu.g/mL rBPI.sub.21 accelerated the
dissolution of the clot. In all subsequent experiments,
polypropylene tubes were utilized to minimize the protein
adsorption effects of the glass.
[0078] B. Effect of Calcium Ion Concentration
[0079] Clot formation and lysis were evaluated in the presence or
absence of rBPI.sub.21 with varying concentrations of calcium (10,
15, 20 mM) to determine optimum calcium concentration. For the clot
formation part of this experiment, the following reagents were
mixed together in a polypropylene test tube in the following order:
(1) 60 .mu.L of 0.1% HSA-TBS; (2) 100 .mu.L of PPP (Donor PC); (3)
40 .mu.L of rBPI.sub.21 (either 10 or 250 .mu.g/mL in 0.1%
HSA-TBS); and (4) 200 .mu.L of 20, 30, or 40 mM CaCl.sub.2 in TBS,
pH 7.8.
[0080] The tubes were allowed to stand at room temperature and were
checked every 1 minute for gel clot formation by visual inspection.
At all calcium concentrations tested, increased rBPI.sub.21
concentrations correlated with increased clotting time. The higher
rBPI.sub.21 concentration produced the longest delay in clotting
time.
[0081] For the clot dissolution part of this experiment, 35 minutes
after CaCl.sub.2 addition, 44 .mu.L of tPA (600 units/mL, or 1
.mu.g/mL) were added to each tube to provide a final tPA
concentration of 60 units/mL or 100 ng/mL. The tubes were incubated
in a 37.degree. C. water bath and checked approximately every
twenty minutes for clot dissolution/lysis by visual inspection.
[0082] rBPI.sub.21 accelerated the dissolution of the clot. Again,
this was most apparent at the 25 .mu.g/mL rBPI.sub.21
concentration. Optimal clot formation and clot lysis was observed
using 10 mM calcium. In all subsequent experiments, 10 mM
CaCl.sub.2 was used for initiation of clotting and lysis.
[0083] C. Effect of Pre-Clot and Post-Clot Addition of tPA
[0084] Clot formation and lysis were evaluated in the presence or
absence of rBPI.sub.21 with pre-clot or post-clot addition of tPA.
For the clot formation part of this experiment, the following
reagents were mixed together in a polypropylene test tube in the
following order: (1) 60 .mu.L of 0.1% HSA-TBS; (2) 100 .mu.L of PPP
(Donor PC); (3) 40 .mu.L of rBPI.sub.21 (either 10 or 250 .mu.g/mL
in 0.1% HSA-TBS); (4) for pre-clot only, 44 .mu.L of tPA (600
units/mL, or 100 ng/mL); and (5) 200 .mu.L of 20 mM CaCl.sub.2 in
TBS, pH 7.8.
[0085] The tubes were allowed to stand at room temperature and were
checked every 1 minute for gel clot formation by visual inspection.
The time in minutes to clot formation after CaCl.sub.2 addition
were measured. A delay in clot formation was observed only for 25
.mu.g/mL rBPI.sub.21 with pre-clot tPA addition.
[0086] For the clot dissolution part of this experiment, 35 minutes
after CaCl.sub.2 addition, 44 .mu.L of tPA (600 units/mL, or 1
.mu.g/mL) were added to the post-clot tubes. The final tPA
concentration in all tubes (pre- and post-clot) was 60 units/mL or
100 ng/mL. All tubes were incubated in a 37.degree. C. water bath
and checked approximately every twenty minutes for clot
dissolution/lysis by visual inspection. Time to clot dissolution in
hours (for post-clot addition of tPA) or in minutes (for pre-clot
addition of tPA) was evaluated. In this experiment, clot lysis when
the tPA was added pre-clot was at least 6 times faster than
post-clot. The rBPI.sub.21 at 25 .mu.g/mL appeared to accelerate
clot lysis time. Because of the unexpectedly rapid dissolution of
the clot under conditions of pre-clot tPA addition, precise times
for clot dissolution were not assessed in this experiment. These
results demonstrated that clot lysis was dramatically accelerated
by the addition of tPA prior to clot formation rather than after
clot formation.
[0087] D. Effect of Pre-Clot Addition of Varying Concentrations of
tPA
[0088] Clot formation and lysis were evaluated in the presence or
absence of rBPI.sub.21 with pre-clot addition of tPA to a final
concentration of 0, 6, or 60 units/mL. For the clot formation final
part of this experiment, the following reagents were mixed together
in a polypropylene test tube in the following order: (1) 60 .mu.L
of 0.1% HSA-TBS; (2) 100 .mu.L of PPP (Donor RL); (3) 40 .mu.L of
rBPI.sub.22 (either 10 or 250 .mu.g/mL in 0.1% HSA-TBS; (4) 44
.mu.L of tPA (600 units/mL, or 100 ng/mL) or buffer control (0.1%
HSA-TBS); and (5) 200 .mu.L of 20 mM CaCl.sub.2 in TBS, pH 7.8.
[0089] The tubes were allowed to stand at room temperature, and
were checked every 1 minute for gel clot formation by visual
inspection. The minutes to clot formation after CaCl.sub.2 addition
were measured. Adding increasing amounts of tPA (0, 6, 60 units/mL)
did not significantly alter the time to clot formation for the
conditions tested.
2 Minutes to Clot Formation tPA (After CaCl.sub.2 Addition)
Concentration Control rBPI.sub.21 .about.1 rBPI.sub.21 .about.25
(units/mL) (0.1% HSA-TBS) .mu.g/mL .mu.g/mL 0 17 19 44 6 18 19 45
60 15 20 45
[0090] For the clot dissolution part of this experiment, 53 minutes
after CaCl.sub.2 addition, all tubes were placed in a 37.degree. C.
water bath and checked for clot dissolution/lysis by visual
inspection. Time to clot dissolution in hours or minutes was
evaluated. With no tPA, no clot lysis was observed. A tPA dose
response effect was observed, with the higher tPA concentration (60
units/mL) producing the most rapid clot dissolution. Under these
conditions with this donor's plasma, rBPI.sub.21 had a much greater
effect on the delay of clot formation while having minimal effect
on clot dissolution. It is apparent that individual plasma donors
can have significantly different clotting and dissolution times
(compare results in parts A-C above). These differences could be
due to different concentrations of crucial clotting factors in
individual donor plasma.
[0091] E. Effect of rBPI.sub.21 and tPA when Added Pre-Activation
and Post-Activation
[0092] Clot formation and lysis were evaluated when both
rBPI.sub.21 and tPA were added prior to calcium addition
(pre-activation or pre-calcium) and 4 minutes after calcium
addition (post-activation or post-calcium). The following reagents
were mixed together in a polypropylene test tube in the following
order: (1) 60 .mu.L of 0.1% HSA-TBS; (2) 100 .mu.L of PPP (Donor
PC); (3) 84 .mu.L of rBPI.sub.21 (either 5, 25 or 125 .mu.g/mL)
with tPA (300 units/ml, 50 ng/mL), or buffer control (0.1%
HSA-TBS); and (4) 200 .mu.L of 20 mM CaCl.sub.2 in TBS, pH 7.8.
[0093] The tubes were allowed to stand at room temperature, and
were checked every 1 minute for gel clot formation by visual
inspection. The minutes to clot formation after CaCl.sub.2 addition
were measured.
3 Minutes to Clot Formation (After CaCl.sub.2 Addition) Timing of
Control rBPI.sub.21 and (0.1% rBPI.sub.21 .about.1 rBPI.sub.21
.about.5 rBPI.sub.21 .about.25 tPA addition HSA-TBS) .mu.g/mL
.mu.g/mL .mu.g/mL Pre-Calcium 7 7 8 11 4 min Post- 7 7 8 8
Calcium
[0094] In this experiment, 25 .mu.g/mL rBPI.sub.21 slowed clot
formation only when present prior to calcium addition. No
significant effect was observed on rate of clot formation when the
rBPI.sub.21 was added 4 minutes after calcium addition.
[0095] For the clot dissolution part of this experiment, 15 minutes
after CaCl.sub.2 addition, all tubes were placed in a 37.degree. C.
water bath and checked approximately every minute for clot
dissolution/lysis by visual inspection. The minutes to clot
dissolution were measured.
4 Minutes to Clot Lysis (After Placement in Water Bath) Timing of
Control rBPI.sub.21 and (0.1% rBPI.sub.21 .about.1 rBPI.sub.21
.about.5 rBPI.sub.21 .about.25 tPA addition HSA-TBS) .mu.g/mL
.mu.g/ML .mu.g/mL Pre-Calcium 58 54 53 51 4 min Post- 50 45 44 40
Calcium
[0096] Clot dissolution appeared to be faster when rBPI.sub.21 and
tPA were added post-calcium compared to pre-calcium. A small
rBPI.sub.21 dose-response effect on clot dissolution was detected
for both pre- and post-calcium groups. These results indicated that
the timing of rBPI.sub.21 and tPA addition relative to calcium
addition (clot activation) influenced clot formation and
dissolution.
[0097] In an additional experiment using higher plasma
concentrations, clot formation and lysis were evaluated when
rBPI.sub.21 and tPA were added pre-calcium and 3 minutes
post-calcium. The following reagents were mixed together in a
polypropylene test tube in the following order: (1) 160 .mu.L of
PPP (Donor PC); (2) 84 .mu.L of rBPI.sub.21 (either 5, 25 or 125
.mu.g/mL) with tPA (300 units/mL, or 50 ng/mL) or buffer control
(0.1% HSA-TBS); and (3) 200 .mu.L of 20 mM CaCl.sub.2 in TBS, pH
7.8.
[0098] The tubes were allowed to stand at room temperature and were
checked every 1 minute for gel clot formation by visual inspection.
The minutes to clot formation after CaCl.sub.2 addition were
measured. In this experiment, 25 .mu.g/mL rBPI.sub.21 slowed clot
formation (.about.50% prolongation) when present prior to calcium
addition. A slight effect (.about.22% prolongation) was observed on
clot formation when 25 .mu.g/mL rBPI.sub.21 was added 3 minutes
after calcium addition.
[0099] For the clot dissolution part of this experiment, 15 minutes
after CaCl.sub.2 addition, all tubes were placed in a 37.degree. C.
water bath and checked for clot dissolution/lysis by visual
inspection. The minutes to clot dissolution were measured. A small
rBPI.sub.21 dose-response effect on clot dissolution was again
detected for both pre- and post-calcium addition groups. Only
minimal differences in clot dissolution were observed between pre-
and post-calcium addition of rBPI.sub.2, and tPA. Although a larger
difference in clot dissolution between pre- and post-calcium
addition groups was observed in a previous experiment, that result
may have been due to the different plasma concentration utilized.
These results confirmed those of the previous experiments that the
timing of rBPI.sub.21 and tPA addition relative to calcium addition
(clot activation), as well as plasma concentration, influenced clot
formation and dissolution.
[0100] F. Effect of Clotting Temperature
[0101] Clot formation and lysis were evaluated at several clotting
temperatures. For the clot formation part of this experiment, the
following reagents were mixed together in a polypropylene test tube
in the following order: (1) 60 .mu.L of 0.1% HSA-TBS; (2) 100 .mu.L
of PPP (Donor PC); (3) 40 .mu.L of rBPI.sub.21 (either 10, 50 or
250 .mu.g/mL) or buffer control; (4) 44 .mu.L of tPA (600 units/mL,
or 100 ng/mL) or buffer control (0.1% HSA-TBS); and (5) 200 .mu.L
of 20 mM CaCl.sub.2 in TBS, pH 7.8.
[0102] The tubes were incubated at room temperature (R.T.) or
37.degree. C. and were checked every 1 minute for gel clot
formation by visual inspection. The minutes to clot formation after
CaCl.sub.2 addition were measured.
5 Minutes to Clot Formation Temperature (After CaCl.sub.2 Addition)
for clotting Control (Timing of tPA (0.1% rBPI.sub.21 .about.1
rBPI.sub.21 .about.5 rBPI.sub.21 .about.25 Addition) HSA-TBS)
.mu.g/mL .mu.g/mL .mu.g/mL R.T. 7 7 8 10 (Pre-clot tPA) 37.degree.
C. 5 5 6 8 (Pre-clot tPA) 37.degree. C. 5 5 6 7 (Post-clot tPA)
[0103] Clot formation proceeded more rapidly at 37.degree. C. than
at R.T. However, 25 .mu.g/mL rBPI.sub.21 delayed clot formation at
both temperatures. Under the conditions tested in this experiment,
the lower concentrations of rBPI.sub.21 did not appear to have an
effect on rate of clot formation.
[0104] For the clot dissolution part of this experiment, 15 minutes
after CaCl.sub.2 addition, 44 .mu.L of tPA (600 units/mL, or 1
.mu.g/mL) were added to post-clot tubes to provide a final
concentration of 60 units/mL (or 100 ng/mL). The tubes were placed
in a 37.degree. C. water bath and checked for clot
dissolution/lysis by visual inspection. Time to clot dissolution in
hours (for post-clot addition of tPA) or in minutes (for pre-clot
addition of tPA) was measured.
6 Minutes/Hours to Clot Lysis Temperature (After CaCl.sub.2
Addition) for clotting Control (Timing of tPA (0.1% rBPI.sub.21
.about.1 rBPI.sub.21 .about.5 rBPI.sub.21 .about.25 Addition)
HSA-TBS) .mu.g/mL .mu.g/mL .mu.g/mL R.T. 38 min. 33 min. 37 min. 38
min. (Pre-clot tPA) 37.degree. C. 45 min. 30 min. 31 min. 29 min.
(Pre-clot tPA) 37.degree. C. 10-12 hours.sup.# 7-8 hours.sup.# 7-8
hours.sup.# 6 hours.sup.# (Post-clot tPA) .sup.#Time estimated
based on clot size at the 7 hour time point.
[0105] Clot lysis was greatly accelerated when tPA was added
pre-clot formation compared to post-clot formation. For pre-clot
tPA addition, rBPI.sub.21 provided a greater acceleration of clot
lysis time when the clot was formed at 37.degree. C. compared to at
R.T. (.about.35% versus .about.3%). All subsequent experiments were
performed at 37.degree. C.
[0106] G. Effect of rBPI.sub.21, rBPI.sub.50, and rLBP
[0107] Clot formation and lysis were evaluated with rBPI.sub.21,
rBPI.sub.50, and rLBP. The following reagents were mixed together
in a polypropylene test tube in the following order: (1) 60 .mu.L
of 0.1% HSA-TBS; (2) 100 .mu.L of PPP (Donor PC); (3) 40 .mu.L of
rBPI.sub.21, rBPI.sub.50, or rLBP (either 10, 50 or 250 .mu.g/mL)
or buffer control; and (4) 200 .mu.L of 20 mM CaCl.sub.2 in TBS, pH
7.8.
[0108] The tubes were incubated in a 37.degree. C. water bath and
were checked every 1 minute for gel clot formation by visual
inspection. The minutes to clot formation after CaCl.sub.2 addition
were measured.
7 Minutes to Clot Formation (After CaCl.sub.2 Addition) Control 1
.mu.g/mL 5 .mu.g/mL 25 .mu.g/mL (0.1% of tested of tested of tested
Tested Protein HSA-TBS) protein protein protein rBPI.sub.21 6 5 7 8
rBPI.sub.50 6 4 6 6 rLBP 6 5 5 5
[0109] 25 .mu.g/mL rBPI.sub.21 slowed the rate of clot formation,
while rBPI.sub.50 and rLBP clotting times were comparable to the
controls.
[0110] For the clot dissolution part of this experiment, 15 minutes
after CaCl.sub.2 addition, 44 .mu.L of tPA (600 units/mL, 1
.mu.g/mL) were added to all tubes to provide a final concentration
of 60 units/mL (100 ng/mL). Then tubes were placed in a 37.degree.
C. water bath and checked for clot dissolution/lysis by visual
inspection. The minutes to clot dissolution were measured.
8 Hours to Clot Lysis (After Placement in Water Bath) Control 1
.mu.g/mL 5 .mu.g/mL 25 .mu.g/mL Tested Protein (0.1% of tested of
tested of tested Added HSA-TBS) protein protein protein rBPI.sub.21
13-14* 7 7 7 rBPI.sub.50 13-14* 10 11 7 rLBP 13-14* 13-14* 8 11
*Time estimated based on clot size at 11 hours.
[0111] At all concentrations tested, rBPI.sub.21 decreased clot
lysis time by approximately 50%. rBPI.sub.50 decreased clot lysis
by approximately 29% at 1 .mu.g/mL, 19% at 5 .mu.g/mL and 50% at 25
.mu.g/mL. rLBP decreased clot lysis time by approximately 57% at 5
.mu.g/mL, approximately 29% at 25 .mu.g/mL, and had no discernable
effect at 1 .mu.g/mL. These results show that, on a mass basis,
rBPI.sub.21 was more potent than rBPI.sub.50 and rLBP in enhancing
the effects of tPA on clot dissolution.
[0112] H. Effect of Fresh Platelet Rich Plasma (PRP) and Platelet
Poor Plasma (PPP)
[0113] Clot formation and lysis were evaluated in the presence and
absence of rBPI.sub.21 with freshly collected platelet rich plasma
(PRP) and platelet poor plasma (PPP) from the same donor. The
following reagents were mixed together in a polypropylene test tube
in the following order: (1) 120 .mu.L of fresh PRP or PPP (Donor
EL); (2) 80 .mu.L of rBPI.sub.21 (either 5, 25 or 125 .mu.g/mL) or
control (0.1% HSA-TBS) and tPA (300 units/mL); and (3) 200 .mu.L of
20 mM CaCl.sub.2 in TBS, pH 7.8.
[0114] The tubes were incubated in a 37.degree. C. water bath, and
were checked every 1 minute for gel clot formation by visual
inspection. The minutes to clot formation after CaCl.sub.2 addition
were measured.
9 Minutes to Clot Formation (After CaCl.sub.2 Addition) Control
(0.1% rBPI.sub.21 rBPI.sub.21 rBPI.sub.21 Type of Plasma HSA-TBS) 1
.mu.g/mL 5 .mu.g/mL 25 .mu.g/mL Platelet Poor 21 24 36 55 Platelet
Rich 15 16 21 37
[0115] A direct rBPI.sub.21 dose-response effect on rate of clot
formation was observed for both PPP and PRP; higher concentrations
of rBPI.sub.21 produced greater prolongation of clot formation. For
all conditions tested in this experiment, PRP exhibited faster
clotting times than PPP.
[0116] For the clot dissolution part of this experiment, incubation
was continued at 37.degree. C. without further addition of
reagents. The minutes to clot dissolution were measured.
10 Minutes to Clot Lysis (After CaCl.sub.2 Addition) Control (0.1%
rBPI.sub.21 rBPI.sub.21 rBPI.sub.21 Type of Plasma HSA-TBS) 1
.mu.g/mL 5 .mu.g/mL 25 .mu.g/mL Platelet Poor 71 59 66 86 Platelet
Rich 81 65 66 81
[0117] For all conditions tested, the fresh PRP had longer clot
dissolution times than fresh PPP. For each plasma type (PRP or
PPP), clot dissolution was fastest at the 1 .mu.g/mL concentration
of rBPI.sub.21. The time from clot formation to clot dissolution is
calculated and shown below.
11 Duration of Clot (Time of Clot Formation Subtracted From Time of
Clot Lysis Control (0.1% rBPI.sub.21 rBPI.sub.21 rBPI.sub.21 Type
of Plasma HSA-TBS) 1 .mu.g/mL 5 .mu.g/mL 25 .mu.g/mL Platelet Poor
50 35 30 31 Platelet Rich 66 49 45 44
[0118] In this experiment, a dose response effect was observed for
clot formation; increasing rBPI.sub.21 concentration resulted in
increasing clotting times in both PRP and PPP. However, a
rBPI.sub.21 dose response effect was not clearly observed for clot
dissolution. Overall, faster clot dissolution times were observed
for rBPI.sub.21 at 1 .mu.g/mL. However, if the time between clot
formation to clot dissolution is measured, then there are only
modest differences in clot dissolution times. From these results,
it appeared that at lower rBPI.sub.21 concentrations, greater
effects were observed on clot dissolution rather than clot
formation. As rBPI.sub.21 concentration was increased, greater
effects were observed on clot formation rather than clot
dissolution.
[0119] I. Effect of rBPI.sub.21 on Freshly Collected Blood
[0120] Clot formation was evaluated with rBPI.sub.21 and freshly
collected blood. For this experiment, blood was collected into four
siliconized 3 mL Vacutainer.RTM. tubes containing either 50, 100,
200 .mu.g/ml rBPI.sub.21 or control formulation buffer. Each tube
was inverted several times after blood collection and placed on ice
until blood had been collected for all tubes. (Collection time of
each tube was less than thirty seconds.)
[0121] All tubes were placed in a 37.degree. C. water bath and
checked every one minute by gently tipping the tube and visually
inspecting it for clot formation.
12 Minutes to Clot Formation (After Placement in 37.degree. C.
Water Bath) Control (0.1% rBPI.sub.21 rBPI.sub.21 rBPI.sub.21 Type
of Plasma HSA-TBS) 50 .mu.g/mL 100 .mu.g/mL 200 .mu.g/mL Whole
Blood 6 11 16 30 (Donor PM)
[0122] Increasing rBPI.sub.21 concentration slowed the rate of clot
formation in a dose dependent manner, but did not completely
prevent clot formation.
EXAMPLE 2
Effects of BPI Protein Products on Clot Formation and Clot Lysis in
Microtiter Plate Assays
[0123] A 96-well microtiter plate assay was used to evaluate the
effects of a BPI protein product on clot formation under a variety
of conditions using human plasma samples. These assays confirmed
the results of tube assays described in Example 1. For these
assays, human plasma, either PRP or PPP, was prepared as described
in Example 1.
[0124] For the plate-based assay, all experiments were conducted at
37.degree. C. and total volume per well was .about.200 .mu.L in the
presence or absence of tPA as a pre-clot addition or .about.250
.mu.L where tPA (50 .mu.L) was added post-clot formation to the 200
.mu.L containing well.
[0125] A. Effect of BPI Protein Product and Pre-Clot Addition of
tPA: Fresh PRP and PPP
[0126] Clot formation was evaluated when rBPI.sub.21 and tPA were
added to fresh PRP or PPP prior to calcium addition. The following
reagents were added to each well of a 96 well microtiter plate
(e.g., Dynatec, Chantilly, Va., CoStar, Cambridge, Mass.) in the
following order: (1) 60 .mu.L of 0.1% HSA-TBS; (2) 50 .mu.L of
fresh PRP or PPP (Donor PM); (3) 20 .mu.L of rBPI.sub.21 (1000,
250, 50, 10 or 2 .mu.g/mL); (4) 20 .mu.L of tPA (600 units/mL, 100
ng/mL); and (5) 50 .mu.L of 20 mM CaCl.sub.2 in TBS, pH 7.4.
[0127] Immediately after CaCl.sub.2 addition, the turbidity of the
wells was measured as the optical density at 405 nm at various
times (in this experiment at 2 minute intervals for 2 hours) by
using an automatic plate reader (Vmax Plate Reader, Molecular
Devices, Menlo Park, Calif.). The entire plate was scanned within 5
seconds. The OD.sub.405 versus time data at 2 minute intervals over
a 2 hour period was plotted. The rate of clot formation was
measured as a change in OD.sub.405 over time, i.e., as the clot
formed the OD.sub.405 increased. After the peak OD.sub.405 was
achieved, the pre-clot addition of tPA allowed dissolution of the
clot as measured by a decrease in OD.sub.405 over time.
[0128] In this experiment, rBPI.sub.21 completely prevented clot
formation of both PRP and PPP at concentrations of 5, 25 and 100
.mu.g/ml. At 1 .mu.g/mL, rBPI.sub.21 effectively delayed time to
clot as measured by a delay in time to peak OD.sub.405 levels [clot
time] and a decrease in peak OD.sub.405 intensity [clot density].
At 0.2 .mu.g/mL, rBPI.sub.21 clot formation time was comparable to
the control.
[0129] B. Effect of Various BPI Protein Products and Control
Protein with Pre-Clot Addition of tPA: Frozen PPP
[0130] Clot formation was evaluated when rBPI.sub.21, rBPI.sub.50,
rBPI.sub.42, LBP and thaumatin (a control cationic protein having a
similar size and charge as rBPI.sub.21) and tPA were added to PPP
prior to calcium addition. The following reagents were added to
each well of a 96 well microtiter plate in the following order: (1)
60 .mu.L of 0.1% HSA-TBS; (2) 50 .mu.L of PPP that had been frozen
at -70.degree. C. (Donor PM); (3) 20 .mu.L of protein product
(rBPI.sub.21, rBPI.sub.50, rBPI.sub.42, LBP and thaumatin each at
1000, 250, 50, 10 or 2 .mu.g/mL); (4) 20 .mu.L of tPA (600
units/mL, 100 ng/mL); and (5) 50 .mu.L of 20 mM CaCl.sub.2 in TBS,
pH 7.4. Immediately after CaCl.sub.2 addition, the turbidity of the
wells was measured as the OD.sub.405 at various times as described
in part A above. In this experiment, the time to clot formation was
determined as the minutes to clot formation as measured by the time
at which the OD.sub.405 had reached its peak value.
13 Minutes to Clot Formation Protein 0.2 1.0 5.0 25.0 100.0 Product
Control .mu.g/mL .mu.g/mL .mu.g/mL .mu.g/mL .mu.g/mL rBPI.sub.21 34
32 34 50 50.dagger. No clot rBPI.sub.50 34 34 34 40 46.dagger. No
clot rBPI.sub.42 40* 28 24 20 30.dagger. No clot LBP 34 30 30 32 34
38 Thaumatin 40* 32 34 32 34 34 .dagger.peak height substantially
decreased (e.g., 3-4 fold) from control *higher effective
concentration of citrate in formulation buffer control
[0131] At 100 .mu.g/mL, rBPI.sub.21, rBPI.sub.50, rBPI.sub.42
completely prevented clot formation (as measured by no detectable
increase in OD.sub.405 over the 2 hour kinetic plate reader
analysis), and at 25 .mu.g/mL, these BPI protein products
substantially prevented clotting (as measured by a slight
OD.sub.405 increase over the 2 hours). At 5 .mu.g/mL, rBPI.sub.21
and rBPI.sub.50 effectively delayed time to clot as measured by a
delay in time to peak OD.sub.405 levels. LBP and thaumatin did not
affect time to clot formation in this experiment. The lack of
effect by the cationic control protein thaumatin indicated that the
delay to clot formation by BPI protein products was not simply a
charge effect due to their cationic properties.
[0132] C. Effect of BPI Protein Product and Pre-Clot Addition of
Various Plasminogen Activators: Frozen PPP
[0133] Clot formation was evaluated when rBPI.sub.21 and a
plasminogen activator (tPA, urokinase or streptokinase) were added
to PPP prior to calcium addition. The following reagents were added
to each well of a 96 well microtiter plate in the following order:
(1) 60 .mu.L of 0.1% HSA-TBS; (2) 50 .mu.L of PPP that had been
frozen at -70.degree. C. (Donor PM); (3) 20 .mu.L of rBPI.sub.21
(1000, 250, 50, 10 or 2 .mu.g/mL); (4) 20 .mu.L of PA (tPA, 1000
ng/mL; for urokinase, 100 and 1000 ng/mL; for streptokinase, 100
and 1000 ng/mL); and (5) 50 .mu.L of 20 mM CaCl.sub.2 in TBS, pH
7.4.
[0134] Immediately after CaCl.sub.2 addition, the turbidity of the
wells was measured as the OD.sub.405 as described in part A above.
In this experiment, rBPI.sub.21 at 100 .mu.g/mL completely
prevented clot formation (as measured by no detectable increase in
OD.sub.405 over the 2 hour kinetic plate reader analysis), and at
25 .mu.g/mL, it substantially prevented clotting under conditions
where tPA, urokinase or streptokinase was present in the pre-clot
mixture. Effects on decreasing clot formation were observed with 5
.mu.g/mL rBPI.sub.21. At 1 and 0.2 .mu.g/mL, rBPI.sub.21 clot
formation time was comparable to the control. At the concentrations
of urokinase tested (10 and 100 ng/mL), clot dissolution did not
occur following clot formation, as it did with 100 ng/mL tPA and
100 ng/mL (but not 10 ng/mL) streptokinase.
[0135] D. Effect of BPI Protein Product on Plasma from Various
Donors: Fresh PPP
[0136] Clot formation was evaluated when rBPI.sub.21 (but not tPA)
was added to fresh PPP prior to calcium addition. The following
reagents were added to each well of a 96 well microtiter plate in
the following order: (1) 80 .mu.L of 0.1% HSA-TBS; (2) 50 .mu.L of
fresh PPP (Donors MW, RD and RL); (3) 20 .mu.L of rBPI.sub.21
(1000, 250, 50, 10 or 2 .mu.g/mL); and (4) 50 .mu.L of 20 mM
CaCl.sub.2 in TBS, pH 7.4.
[0137] Immediately after CaCl.sub.2 addition, the turbidity of the
wells was measured as the OD.sub.405 as described in part A above.
In this experiment, rBPI.sub.21 at various concentrations slowed or
prevented clot formation. Some individual variation was observed in
the response among the three donor plasma samples simultaneously
tested. For all three donors, rBPI.sub.21 at 25 and 100 .mu.g/mL
completely prevented clot formation of fresh PPP. At 5 .mu.g/mL,
rBPI.sub.21 also completely prevented clot formation of the PPP
from one donor (RL) and substantially prevented clotting of the PPP
from the other two donors (MW and RD). At 1 .mu.g/mL, rBPI.sub.21
substantially prevented clotting of the PPP from one donor (RL),
and reduced clotting was observed in the PPP of the other two
donors (MW and RD). A slight effect was observed even at 0.2
.mu.g/mL rBPI.sub.21 with one donor; generally at 0.2 .mu.g/mL
rBPI.sub.21, clot formation was comparable to control.
[0138] E. Effect of Multiple BPI Protein Products After
Thrombin-Driven Clot Formation and Post-Clot tPA Addition: Fresh
PPP
[0139] Clot lysis was evaluated when rBPI.sub.21 and tPA were added
to fresh PPP with 0.125 units/mL thrombin and calcium. The
following reagents were added to each well of a 96 well microtiter
plate in the following order: (1) 80 .mu.L of 0.1% HSA-TBS; (2) 50
.mu.L of PPP that had been frozen at -70.degree. C. (Donor PM); (3)
20 .mu.L of rBPI.sub.21 or rBPI.sub.42 (50, 10 or 2 .mu.g/mL); and
(4) 50 .mu.L of 20 mM CaCl.sub.2 in TBS, pH 7.4 with 0.5 units/mL
thrombin.
[0140] After thrombin and CaCl.sub.2 addition, clot formation was
allowed to occur and 50 .mu.L of tPA (300 units/mL, 500 ng/mL) with
0.5 units/mL of heparin in TBS, pH 7.4 were added. After clot
formation and tPA addition (.about.15 minutes), the turbidity of
the wells was measured as the OD.sub.405. In this initial
experiment with thrombin-driven clot formation, rBPI.sub.21 or
rBPI.sub.42 with post-clot addition of tPA allowed dissolution of
the clot as measured by a decrease in OD.sub.405 over time.
Increasing the concentration of rBPI.sub.21 or rBPI.sub.42 (0.2, 1
and 5 .mu.g/mL) with a constant concentration of tPA (100 ng/mL)
resulted in enhanced clot dissolution (i.e., the rate at which the
OD.sub.405 decreased was .about.2-4 fold faster).
EXAMPLE 3
Effects of BPI Protein Product on tPA-Induced Clot Lysis in a Rat
Thrombosis Model
[0141] A rat thrombosis model was used to determine the effects of
a BPI protein product (rBPI.sub.21) with a thrombolytic agent (tPA)
on clot lysis and reocclusion after thrombolytic therapy. The
methods of Klement et al., Thrombosis Haemostasis, 68:64-68 (1992),
were modified as described herein to determine the effects of
rBPI.sub.21 in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188,
0.002% polysorbate 80 on tPA-induced clot lysis in vivo. In this
experiment, two groups of rats (5 for the vehicle-treated group and
5 for the rBPI.sub.21-treated group) were anesthetized with
ketamine/Rompum, and catheters were placed in both jugular veins
for administration of therapeutic agents. The right common carotid
artery was cannulated to measure blood pressure and heart rate. A
midline incision was made in the lower abdomen to expose the
terminal aorta and iliac vessels, which were dissected free of
connective tissue and separated from the vena cava, where possible,
and the great veins. All of the numerous small branches from the
aorta that were observed were ligated in order to isolate a section
of the vessel approximately 1 cm in length. The left iliac artery
was cannulated with a 20 gauge blunt needle sealed with a rubber
septum. An ultrasonic flow transducer cuff was placed around the
right iliac artery in order to measure blood flow with a flow meter
[Crystal Biotech, Holliston, Mass.]. The flow signal was recorded
on a chart recorder.
[0142] The following procedures were carried out in order to injure
the aorta and thus provide a surface for clot formation. The 20
gauge needle was advanced into the aorta and slowly moved against
the vessel wall along a 1 to 1.5 cm length. This was repeated 8
times. The needle was removed and the same area of the aorta was
pinched 4 times with smooth forceps. Subsequently, an area of
stenosis was produced in the terminal aorta by gently tightening
silk thread at two locations. Right iliac blood flow was measured
at this time and designated as the "pre-occlusion" level.
[0143] At this point, the rBPI.sub.21-treated rats were
administered 20 mg/kg rBPI.sub.21 as a 2 mg/mL solution in 5 mM
citrate 0.2% poloxamer 188, 150 mM NaCl (Pluronic F-68, BASF
Wyandotte, Parsippany, N.J.) and 0.002% polysorbate 80 (Tween 80,
ICI Americas Inc., Wilmington, Del.), pH 5 as a bolus over 30
seconds (4 mL/kg) intravenously into one of the jugular veins,
followed by a constant infusion of rBPI.sub.21 at 20 mg/kg/hr that
was continued until the end of the experiment. The vehicle-treated
rats received the same volumes; the vehicle solution was 5 mM
citrate 150 mM NaCl, 0.2% poloxamer 188, (Pluronic F-68, BASF
Wyandotte, Parsippany, N.J.) and 0.002% polysorbate 80 (Tween 80,
ICI Americas Inc., Wilmington, Del.), pH 5. Next, the right iliac
artery was occluded with a small metal clip placed just above the
blood flow cuff. Since the left iliac artery was already sealed by
the needle catheter, blood was then trapped above the occlusion.
Finally, the aorta was occluded above the injured site, producing a
stenotic area with stasis. The occlusions were maintained for 30
minutes and then removed. Confirmation that a clot had formed was
provided by the failure to record flow through the right iliac
artery.
[0144] Five minutes after removal of the occlusions, t-PA was
administered into the other jugular vein (in which rBPI.sub.21 had
not been administered) as a bolus of 1 mg/kg followed by an
infusion of 1 mg/kg over 1 hour. After 1 hour, both the tPA and the
rBPI.sub.21 (or vehicle) infusions were discontinued and the
experiment was terminated; Clot lysis was defined as a return of
blood flow through the right iliac artery to 50% of the
pre-occlusion level. The length of time until clot lysis was
recorded, as well as the time until the first reocclusion after the
initial clot lysis. The number of times that the vessel became
reoccluded over the 1 hour period of t-PA treatment was also
recorded. Reocclusion was defined as a reduction in blood flow to
10% of the pre-occlusion level. Statistical comparisons between the
buffer- and rBPI.sub.21-treated groups were done with Student's T
test. Neither t-PA nor rBPI.sub.21 produced any adverse systemic
effects, other than a rise in blood pressure due solely to the
aortic occlusion.
[0145] The results revealed no significant difference in the mean
time to clot lysis, which was 14.4.+-.5.1 minutes for the
buffer-treated group and 16.1.+-.4.2 minutes for the
rBPI.sub.2-treated group. Multiple reocclusion episodes occurred in
all rats. The time to the first reocclusion was not significantly
different between the two groups. However, the number of
reocclusions was statistically significantly reduced in
rBPI.sub.21-treated rats (p<0.05); the rBPI.sub.21-treated rats
averaged 4.+-.1.6 reocclusion episodes, while the buffer-treated
rats averaged 9.+-.1.7 reocclusion episodes.
[0146] Numerous modifications and variations of the above-described
invention are expected to occur to those of skill in the art.
Accordingly, only such limitations as appear in the appended claims
should be placed thereon.
Sequence CWU 1
1
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