U.S. patent application number 11/352720 was filed with the patent office on 2006-06-29 for compositions and methods for selective dissolution of nascent intravascular blood clots.
This patent application is currently assigned to Trustees of the University of Pennsylvania. Invention is credited to Douglas Cines, Juan Carlos Murciano, Vladimir R. Muzykantov.
Application Number | 20060140917 11/352720 |
Document ID | / |
Family ID | 34062346 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060140917 |
Kind Code |
A1 |
Muzykantov; Vladimir R. ; et
al. |
June 29, 2006 |
Compositions and methods for selective dissolution of nascent
intravascular blood clots
Abstract
Compositions and methods for prevention and treatment of
uncontrolled formation of intravascular fibrin clots, which are
capable of selective dissolution of pathological nascent clots
formed intravascularly, with minimal risk of unwanted dissolution
of pre-existing hemostatic clots, are provided wherein fibrinolytic
or anticoagulant drugs are biocompatibly coupled to red blood cell
carriers.
Inventors: |
Muzykantov; Vladimir R.;
(Warwick, PA) ; Murciano; Juan Carlos; (Madrid,
ES) ; Cines; Douglas; (Wynnewood, PA) |
Correspondence
Address: |
LICATLA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Assignee: |
Trustees of the University of
Pennsylvania
|
Family ID: |
34062346 |
Appl. No.: |
11/352720 |
Filed: |
February 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10611723 |
Jul 1, 2003 |
7041287 |
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11352720 |
Feb 13, 2006 |
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10253518 |
Sep 23, 2002 |
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10611723 |
Jul 1, 2003 |
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09454666 |
Dec 3, 1999 |
6488927 |
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10253518 |
Sep 23, 2002 |
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PCT/US99/10547 |
May 12, 1999 |
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09454666 |
Dec 3, 1999 |
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60086262 |
May 21, 1998 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 38/49 20130101;
A61K 35/18 20130101; A61P 7/02 20180101; A61K 9/0019 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
A61K 35/14 20060101
A61K035/14 |
Claims
1. A composition comprising a therapeutic agent biocompatibly
coupled to a carrier similar in size to a red blood cell.
2. The composition of claim 1 wherein the carrier is a red blood
cell.
3. The composition of claim 1 wherein the therapeutic agent is an
anti-thrombotic agent.
4. (canceled)
5. A thromboprophylactic agent comprising the composition of claim
3 and a pharmaceutically acceptable vehicle.
6. A method for preventing formation of nascent intravascular blood
clots in a subject comprising administering to the subject the
thromboprophylactic agent of claim 5.
Description
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 10/611,723 filed Jul. 1, 2003, which is a
continuation-in-part of U.S. patent application Ser. No. 10/253,518
filed Sep. 23, 2002, which is a divisional application of U.S.
patent application Ser. No. 09/454,666 filed Dec. 3, 1999, now
issued as U.S. Pat. No. 6,488,927, which is a continuation-in-part
application of PCT Application PCT/US1999/10547 filed May 12, 1999,
which claims the benefit of priority to U.S. Provisional
Application Ser. No. 60/086,262 filed May 21, 1998, each of which
are herein incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Occlusions of blood vessels by intravascular clots cause
or/and contribute to the pathogenesis of a variety of disease
conditions including myocardial infarction, stroke and pulmonary
embolism and thus represent a significant medical problem. Although
fibrinolytics, such as plasminogen activators, have recently been
used in the treatment of some of these diseases or conditions,
their effectiveness and safety are still of great concern,
especially under specific prothrombotic conditions such as deep
vein thrombosis and pulmonary embolism.
[0003] Pulmonary thromboembolism, a leading cause of mortality, is
most often a complication of deep venous thrombosis. Statistics
show that more than 95% of pulmonary emboli result from thrombi in
the deep venous system of the lower extremities. Despite advances
in medicine, the incidence and/or recognition of embolism and deep
vein thrombosis appears to be increasing. This increase has been
attributed to higher survival of trauma patients, an increase in
orthopedic surgeries for joint replacement, and the widespread use
of indwelling catheters, as well as the overall increase in medical
and surgical procedures, particularly in older patients. As a
result, methods of preventing and treating deep vein thrombosis are
required to reduce the incidence of pulmonary embolisms.
[0004] Factors which promote deep vein thrombosis were defined as
early as the nineteenth century and include stasis, abnormalities
of the blood vessel wall, and alterations in the blood coagulation
system. The highest risk groups for deep vein thrombosis are
surgical patients requiring 30 minutes or more of general
anesthesia, postpartum patients, patients with right and left
ventricular failure, patients with fractures or injuries involving
the lower extremities, patients with chronic deep venous
insufficiency of the legs, patients on prolonged bed rest, cancer
patients, obese individuals, and patients using estrogens.
Treatment of deep vein thrombosis most often involves use of an
anticoagulant such as heparin. Even with this well-known drug,
however, there is no consensus regarding the optimum regimen of
anticoagulant therapy that affords both safety and efficacy. In
addition to anticoagulant therapy, thrombolytic agents, such as
streptokinase and urokinase, have been used in the management of
acute deep vein thrombosis.
[0005] Acute vascular occlusion by fibrin thrombi is also a common
and dangerous complication of surgery. Development of venous (e.g.,
pulmonary embolism) and arterial (e.g., arterial re-occlusion)
thrombi that may embolize vitally important blood vessels increases
peri-operative morbidity and mortality. Cardiopulmonary bypass
surgery, transcutaneous coronary angioplasty and carotid
endarterectomy and other interventions may be complicated by
formation of small blood clots, which embolize to cerebral vessels
and cause serious persistent neurological disorders.
[0006] In contrast to settings where ischemia is the sole threat to
health (e.g., acute myocardial infarction, AMI), the management of
thrombosis in the surgical settings is complicated by the added
risk of bleeding at and around the wound site imparted by
anti-thrombotic therapy itself. Also, in contrast with AMI, where
lysis of the mature clot occluding a single vessel is a primary
goal for the fibrinolytic intervention (therapy), the goal of the
post-surgical anti-thrombotic management is to maintain an adequate
blood flow in diverse vascular areas at risk of occlusion by
post-surgical clots (prophylaxis). In the surgical settings, the
ideal thromboprophylactic agent effectively dissolves nascent
pathological thrombi developed in a post-operative and recovery
periods, while not dissolving hemostatic fibrin clots formed during
and immediately after the operation.
[0007] In addition to the objective inadequacies of existing agents
(see below), widespread use of thromboprophylaxis in surgical
settings continues to receive limited enthusiasm for additional
reasons. The clinical significance of silent thrombotic events
(pulmonary and brain micro-embolism) is under-appreciated because
the symptoms may be delayed in onset and attributed to other
causes, while significance of bleeding is acutely evident in the
surgical practice and frequently blamed on thromboprophylaxis.
[0008] Current approaches afford some measure of protection, but
all have a limited benefit/risk ratio in the surgical setting.
Mechanical filters trap large clots, but cannot be used to prevent
arterial re-occlusion or cerebral micro-emboli. Anticoagulants
(e.g. low molecular weight heparin) reduce the incidence of
thrombosis in some vascular beds, but dosing is limited by the risk
of post-operative bleeding. Heparin has been reported to increase
the incidence of wound hematomas, which in turn may predispose to
wound infection and dehiscence. Aspirin and anti-GPIIb/IIIa agents
suppress platelet aggregation, but cannot prevent formation of
fibrin monomers and clot propagation. In addition to serious side
effects (in part discussed above), each particular anticoagulant
agent inhibits one specific mechanism for formation of clots (e.g.,
activation of platelets or fibrin formation), but none affords 100%
fidelity of anti-thrombotic protection. A modality to dissolve
nascent pathological thrombi, formed even despite anti-coagulation
or due to its inapplicability, is acutely needed.
[0009] Plasminogen activators (PA's) are effective thrombolytics,
which could be theoretically used for this purpose, but they have
fundamental inadequacies for thromboprophylactic applications
including rapid blood clearance and inactivation, non-selective
degradation of the extracellular matrix and hemostatic fibrin
plugs. The risk of hemorrhage practically precludes their use in
the surgical settings. No existing anti-thrombotic strategy permits
selective dissolution of the nascent clots without affecting
pre-existing hemostatic fibrin plugs.
[0010] Streptokinase, staphylokinase, tissue-type plasminogen
activator or tPA, and urokinase are members of a family of agents
known as plasminogen activators. These compounds act to dissolve
intravascular clots by activating plasmin, a protease that digests
fibrin. Plasminogen, the inactive precursor of plasmin, is
converted to plasmin by cleavage of a single peptide bond. Plasmin
itself is a nonspecific protease that digests fibrin clots as well
as other plasma proteins, including several coagulation
factors.
[0011] Fibrinolytic therapy with plasminogen activators have been
shown to be useful in the treatment of myocardial infarction and
stroke. However, application of these agents to dissolution of
clots formed or lodged in other vascular areas such as deep venous
areas is limited by extremely rapid elimination and inactivation
after bolus dosing (Plow, E. et al. 1995. FASEB J. 9:939-945;
Narita, M. et al. 1995. J. Clin. Invest. 96:1164-1168). Both tPA
and urokinase undergo rapid inactivation by a circulating
plasminogen activator inhibitor and plasmin itself is inactivated
by a circulating glycoprotein, .alpha.-2-antiplasmin (Collen, D.
1996. Circulation 93:857-865; Reilly, C. et al. 1991. Arterioscl.
Thromb. 11:1276-1286). .alpha.-2-antiplasmin inactivates
staphylokinase, while streptokinase is more resistant to this
endogenous glycoprotein inhibitor (Collen, B. et al. 1993. Eur. J.
Biochem. 216:307-314). Although therapeutic doses of plasminogen
activators can overwhelm the potential inhibitory activity of
plasminogen activator inhibitor and .alpha.-2-antiplasmin, other
inhibitors of plasminogen activators also are present
(C1-inhibitor, .alpha.-2-macroglobulin, anti-trypsin) and
contribute to the decrease over time in the fibrinolytic response
upon treatment with plasminogen activators (Collen, D. 1996.
Circulation 93:857-865). Such inactivation, or degradation of
plasminogen activators and plasmin reduce the effectiveness of
thrombolytic therapy and thus fail to prevent re-occlusion of blood
vessels.
[0012] To overcome this problem, attempts have been made to infuse
plasminogen activators intravenously for prolonged periods of time
with little success; failure was attributed to the harmful side
effects such as bleeding, dissolution of hemostatic clots as well
as nascent clots causing deleterious consequences, and uncontrolled
tissue proteolysis that occurred after extravascular deposition of
plasminogen activators.
[0013] Accordingly, several different approaches have been
attempted to improve efficacy of these agents in deep vein
thrombosis including: prolongation of the half-life of plasminogen
activators in blood; protection of plasminogen activators from
inactivation by inhibitors; and targeting plasminogen activators to
fibrin and thrombi. For example, chemical modifications and
incorporation of plasminogen activators into liposomes have been
used to prolong the half-life of plasminogen activators in the
circulation (Kajihara, J. et al. 1994. Biochim. Biophys. Acta
1199:202-208; Heeremans, J. et al. 1995. Thromb. Haemost.
73:488-494). However, these studies have shown that the activity of
liposome-encapsulated plasminogen activators is strongly
compromised by steric limitations. Genetically engineered tPA
compounds have also been produced which possess altered
pharmacokinetic properties, enhanced resistance to inhibitors, and
higher fibrinolytic potency (Collen, D. 1996. Circulation
93:857-865; Collen, D. 1993. Lancet 342:34-36; Krishnamurti, C. et
al. 1996. Blood 87:14-19; Lijnen, R. and D. Collen. 1992. Ann. NY
Acad. Sci. 667:357-364). Several laboratories have explored
conjugation of plasminogen activators with antibodies recognizing
fibrin or activated platelets in order to localize plasmin
generation to the clot (Holvoet, P. et al. 1993. Circulation
87:1007-1016; Runge, M. et al. 1996. Circulation 94:1412-1422;
Fears, R. and G. Poste. 1994. Fibrinolysis 8:203-213). However,
such conjugated plasminogen activators with affinity for clot
components only bind to the superficial layer of the clot and do
not enter into the clot interior (Sakharov, D. and D. Rijken. 1995.
Circulation 92:1883-1890). In addition, clots bind only a small
fraction of injected "fibrin-specific" plasminogen activator
because of limited surface area of the formed clots. Such targeting
also fails to distinguish nascent clots with deleterious
consequences from pre-existing desired hemostatis clots.
[0014] Further, to date, none of these methods for modifying
plasminogen activators prevent deposition of plasminogen activators
in tissues, which can lead to an increase in harmful side effects;
they all represent molecules or molecular complexes with sizes that
do not exceed that of blood proteins. Such deposition leads to
plasmin activation in tissues. Activated plasmin degrades the
extracellular matrix, thus causing vascular remodeling, abnormal
elevation of vascular permeability and even partial denudation of
subendothelium (Plow et al. 1995. FASEB J. 9:939-945; Shreiber et
al. 1995. J. Cell. Physiol. 165:107-118).
[0015] Accordingly, there is a need for methods of modifying
plasminogen activators which not only decrease the rate of
elimination and degradation of the plasminogen activators, but also
prevent deposition of the plasminogen activator in the
extrasvascular tissues. Further, compositions and methods are
needed which selectively target nascent clots from within, while
having minimal effects on pre-existing hemostatic clots.
[0016] Red blood cells (RBCs) normally have a life span of 120 days
and thus can serve as natural carriers for drugs and biomolecules.
Autologous RBCs can be easily obtained from the patient's blood,
loaded with drug, and re-injected. RBCs have been used as carriers
for drugs loaded into the inner volume of RBCs (Poznansky, M. and
R. Juliano. 1984. Pharmacol. Rev. 36:277-324; Kirch, M. et al.
1994. Biotechnol. App. Biochem. 19:331-363; Kinoshita, K. and T.
Tsong. 1978. Nature 272:258-260). In addition, methods for
conjugation of proteins to RBCs have been developed, including
methods using a streptavidin-biotin pair as a cross-linker.
[0017] Streptavidin is a 60 kDa protein that possesses four high
affinity biotin binding sites and the streptavidin-biotin pair is
widely used in biomedicine as a cross-linking agent (Wilchek, M.
and E. Bayer. 1988. Anal. Biochem. 171:1-32). Several groups have
reported application of streptavidin-biotin technology in vivo for
gamma-immunoscintigraphy (Kalofonos, H. et al. 1990. J. Nucl. Med.
31:1791-1796) and drug targeting (Pardridge, W. et al. 1995. Proc.
Natl. Acad. Sci. USA 92:5592-5596; Muzykantov, V. et al. 1996.
Proc. Natl. Acad. Sci. USA 93:5213-5218). Moreover, streptavidin
induces no known harmful reactions in animals or humans (Kalofonos,
H. et al. 1990. J. Nucl. Med. 31:1791-1796). Biotinylation of RBCs
can be accomplished in manner which has no effect on the life span
and biocompatibility of these cells in vivo in animals (Susuki, T.
and G. Dale. 1987. Blood 70:791-795; Muzykantov, V. et al. 1991.
Blood 78:2611-2618).
[0018] Biotinylation of proteins, including plasminogen activators,
without significant reduction of functional activity of the
plasminogen activator has been described (Muzykantov, V. et al.
1986. Biochem. Biophys. Acta, 884:355-363; Muzykantov, V. et al.
1995. Anal. Biochem., 226:279-287; Muzykantov, V. et al. 1996. J.
Pharm. Exp. Ther., 279:1026-1034). In in vitro studies, polyvalent
conjugation of various biotinylated proteins such as antibodies,
enzyme peroxidase and fibrinolytic streptokinase with streptavidin
conjugated-biotinylated RBCs (SA/b-RBC) was performed and high
functional activity of these proteins bound to SA/b-RBC in vitro
was reported (Muzykantov, V. et al. 1985. FEBS Lett. 182:62-66;
Muzykantov, V. et al. 1986. Biochim. Biophys. Acta 884:355-363;
Muzykantov, V. et al. 1987. Am. J. Pathol. 128:226-234).
[0019] However, polyvalent conjugation of biotinylated proteins to
b-RBCs via streptavidin cross-linker profoundly compromises the
biocompatibility of the carrier RBC. Binding of streptavidin to
b-RBC leads to elimination of homologous restriction of both
classical and alternative pathways of the complement thereby
causing lysis of SA/b-RBC in the plasma (Muzykantov, V. et al.
1991. Blood 78:2611-2618; Muzykantov, V. et al. 1992. Int. J.
Artif. Organs 15:620-627; Muzykantov, V. et al. 1993. FEBS Lett.
318:108-112). Streptavidin-induced cross-linking and membrane
redistribution of the complement inhibitors, DAF and CD59, in
biotinylated RBC membrane represents the likely mechanism for
complement activation and lysis (Muzykantov, V. et al. 1992.
Biochim. Biophys. Acta 1107:119-125; Zaltzman, A. et al. 1995.
Biochem. J. 305:651-656). In addition, fixation of C3b complement
component has been shown to lead to an increased rate of
elimination of SA/b-RBC from the bloodstream via hepatic and
splenic uptake (Muzykantov, V. et al. 1992. Int. J. Artif. Organs
15:620-627; Muzykantov, V. et al. 1996. Anal. Biochem.
214:109-119). Accordingly, drugs polyvalently conjugated to an RBC
carrier via streptavidin can not be delivered to their targets in
vivo.
[0020] This lack of biocompatibility of SA/b-RBC carrier can be
overcome through modifications of the conjugation method. For
example, monovalent coupling of streptavidin to b-RBCs has been
demonstrated to produce a serum-stable carrier SA/b-RBC capable of
binding up to 10.sup.5 molecules of a biotinylated model protein
per RBC (Muzykantov, V. et al. 1991. Biochem. J. 273:393-397;
Muzykantov, V. et al. 1992. Biochim. Biophys. Acta 1107:119-125;
Muzykantov, V. et al. 1993. Anal. Biochem. 208:338-342; Muzykantov,
V. and R. Taylor. 1994. Anal. Biochem. 223:142-148; Muzykantov, V.
et al. 1996. Anal. Biochem. 214:109-119;). B-RBC carrier,
monovalently conjugated with a model biotinylated protein (b-IgG)
via streptavidin, circulated for at least a day as a stable complex
after intravenous injection in animals, with no evidence of lysis
or hepatic uptake (Muzykantov, V. et al. 1996. Anal. Biochem.
214:109-119). In these studies, it was also found that the
half-life b-IgG monovalently conjugated with SA/b-RBCs
significantly exceeded that of non-conjugated b-IgG (Muzykantov, V.
et al. 1996. Anal. Biochem. 214:109-119).
[0021] As shown herein, monovalent conjugation of a biotinylated
plasminogen activator to a SA/b-RBC carrier results in prolonged
circulation of the plasminogen activator in the bloodstream and
decreased deposition of the plasminogen activator in the tissues.
Further, as shown herein, monovalent conjugation of biotinylated
soluble receptor for urokinase plasminogen activator, (b-suPAr),
and monovalent conjugation of biotinylated tissue plasminogen
activator, (tPA), to a SA/b-RBC carrier results in prolonged
circulation of these biotinylated plasminogen activators in the
bloodstream in a form of the receptor/RBC complex (b-suPAr/SA/b-RBC
complex) and the tPA/RBC complex, respectively. Moreover, as shown
herein b-suPAr/SA/b-RBC complex retains its ability to bind single
chain urokinase plasminogen activator (scuPA) even after prolonged
circulation in the bloodstream and non-covalent binding of this
fibrinolytic precursor (scuPA) to the receptor conjugated to RBC
carrier (i.e., to b-suPAr/SA/b-RBC complex) leads to scuPA
activation and greater resistance to plasma inhibitors and thus
provides increased fibrinolytic activity on the clot itself. As
also shown herein pulmonary vascular uptake of tPA is increased by
crosslinking tPA to biotinylated RBC to form a tPA/RBC complex,
with the increase seen to a level that exceeds the level of tPA/RBC
complex in larger blood vessels. In addition, conjugation of tPA
and suPAr with a monoclonal antibody against human CR1 is shown
herein to promote specific coupling of fibrinolytically active
anti-thrombotic agents to red blood cells, limiting the
non-specific binding and tissue uptake of the complexes.
[0022] Further, as shown herein coupling of an anti-thrombotic
agent to a carrier such as an RBC alters is fibrinolytic profile
rendering the anti-thrombotic agent relatively impermeable to
pre-formed (hemostatic) clots and extravascular tissues, thereby
reducing the risk of bleeding, while prolonging its circulation and
ability to be incorporated inside nascent intravascular clots,
providing their fast lysis from within.
[0023] Accordingly, the present invention relates to compositions
comprising a therapeutic agent, such as an anti-thrombotic agent,
biocompatibly coupled to a carrier sized to inhibit penetration
into existing clots, such as a red blood cell, and methods of using
these compositions in the selective dissolution of nascent
intravascular blood clots while avoiding lysis of pre-existing
(hemostatic) clots.
SUMMARY OF THE INVENTION
[0024] An object of the present invention is to provide
compositions comprising a therapeutic agent, preferably an
anti-thrombotic agent, more preferably a fibrinolytic,
biocompatibly coupled to the surface of a carrier similar in size
to a red blood cell, which circulate for a prolonged time and
selectively dissolve nascent intravascular blood clots. In a
preferred embodiment, the carrier comprises a red blood cell.
Coupling is preferably accomplished by cross-linking a biotinylated
therapeutic agent to a biotinylated carrier such as a red blood
cell via streptavidin. Alternatively, a therapeutic agent is
conjugated with molecules with high affinity to a specific red
blood cell surface protein such as monoclonal antibodies which then
bind specifically with red blood cells upon administration. An
example of a monoclonal antibody specific for a red blood cell
carrier is a monoclonal antibody against human CR1.
[0025] Another object of the present invention is to provide a
method of preventing and treating uncontrolled formation of nascent
intravascular clots while avoiding lysis of pre-existing
(hemostatic) clots in a patient which comprises administering to a
patient suffering from uncontrolled formation of intravascular
clots a composition comprising a therapeutic agent such as an
anti-thrombotic agent biocompatibly coupled to a carrier similar or
greater in size to a red blood cell. In a preferred embodiment the
carrier comprises a red blood cell.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides compositions and methods for
prevention and treatment of deep vein thrombosis, pulmonary
embolism and other diseases or syndromes characterized by
uncontrolled formation of intravascular fibrin clots. The invention
is based on the biocompatible coupling of a therapeutic agent,
preferably an anti-thrombotic agent, more preferably a plasminogen
activator, to a carrier similar in size to a red blood cell (RBC).
Preferably the carrier is a RBC. RBC carriers or carriers similar
in size to RBCs (3-5 microns) provide for prolonged circulation,
restricted tissue uptake and minimal permeation into clots of the
drug. The monovalent conjugation of plasminogen activators to
biotinylated RBCs via streptavidin (referred to herein as SA/b-RBC)
serves as an example in the present invention of a biocompatible
coupling method. Biotinylated plasminogen activators are referred
to herein as b-PAs.
[0027] Another example of biocompatible coupling is the
crosslinking of plasminogen activators to biotinylated molecules
such as monoclonal antibodies which then couple specifically with a
carrier such as red blood cells in whole blood. An example of an
antibody useful in this coupling method is a monoclonal antibody
against human CR1.
[0028] Experiments were performed to examine the conjugation of
b-PAs to biotinylated RBCs via streptavidin. Results of in vitro
tests showed that streptavidin provided specific and effective
conjugation of the b-PAs to the biotinylated RBC (Table 1).
TABLE-US-00001 TABLE 1 Number of .sup.125I-coupled Plasminogen
Activator Molecules Per Red Blood Cell Plasminogen # Coupled to #
Coupled to SA/ Activator Biotinylated RBCs biotinylated RBCs
biotinylated- <400 79,000 scuPa biotinylated- <400 30,000 tPA
biotinylated- <1200 42,000 urokinase biotinylated- <300
12,000 streptokinase
[0029] When tested in a fibrin plate lysis assay and in an assay of
release of radiolabeled iodine from a fibrin clot (formed from
.sup.125I-fibrinogen), the b-PA/SA/b-RBC conjugates were shown to
be stable and to display high fibrinolytic activity. When the
b-PA/SA/b-RBC conjugates were added to solution of
.sup.125I-fibrinogen before formation of fibrin clot, the
subsequent fibrinolysis attained 71.+-.12%. Non-conjugated PAs
induced 96.+-.5.5% lysis of fibrin clot. Thus, conjugation of b-PA
to SA/b-RBC does not compromise significantly fibrinolytic activity
of PA.
[0030] The half-life (i.e., rate of degradation and elimination
from blood) of RBC-conjugated plasminogen activators was also
examined in vivo in rats. The kinetics of blood clearance of
.sup.125I-scuPA, .sup.125I-urokinase, .sup.125I-streptokinase, or
.sup.125I-tPA were determined following intravenous injection of
scuPA or tPA at a dose of 5 .mu.g/kg. Within one hour of injection,
the blood level of scuPA dropped to less than 5% of the injected
dose. In contrast, the blood level of SA/b-RBC-conjugated scuPA was
10-fold higher at the one hour time point, and remained at high
levels (20% of injected dose) for up to 24 hours. Similar results
were seen with tPA, urokinase and streptokinase. Pharmacokinetic
analysis of the areas below the curves of blood level of
plasminogen activators versus those biocompatibly coupled to RBCs
during 24 hours after intravenous injection revealed that the
half-life of RBC-conjugated PAs exceeded that of non-conjugated PAs
by several orders of magnitude.
[0031] Further, conjugation of the biotinylated plasminogen
activators with SA/b-RBC carrier did not cause intravascular lysis
of the carrier RBC labeled with .sup.51Cr and did not change the
biodistribution of RBC in any tissues except the spleen. Splenic
uptake of the b-PA/SA/b-RBC was elevated about 4-fold. However,
such increases in splenic uptake of chemically modified RBCs are
well known in the literature. Importantly, after injection of
b-PA/SA/b-RBC conjugates, tissue uptake of plasminogen activators
was markedly reduced. For example, level of .sup.125I-tPA in the
brain tissue (expressed as percentage of that in the blood) was
18.3.+-.1.1%, whereas that parameter for .sup.125I-b-tPA/SA/b-RBC
was 1.8.+-.0.5%. Thus conjugation with an RBC carrier provides ten
fold reduction of the uptake in the brain.
[0032] In the case of tPA, the tissue distribution data revealed
that conjugation of tPA to RBC not only prolonged its
bioavailability in the circulation, but also dramatically increased
its pulmonary vascular uptake. One hour after injection, 20% of the
radiolabeled tPA was found in the lungs of rats injected with
.sup.125I-tPA/RBC, a value that is 100-fold is higher than what is
seen following injection of soluble .sup.125I-tPA. Yet, the
pulmonary uptake of tPA/.sup.51Cr-RBC was only marginally higher
than that of non-modified RBC (3.8% versus 1.2%). Thus, the lungs
of rats injected with tPA/RBC contained five-fold more radiolabeled
tPA than radiolabeled RBC, whereas the ratio of radiolabeled tPA to
radiolabeled RBC equaled one in all other tissues, including blood.
This indicates that there was a significant transfer of tPA to the
lung, the pulmonary vasculature, a result that was not seen in
other organs (an organ-specific effect). This transfer of tPA from
the carrier RBC occurred rapidly and lasted for several hours. In
addition, the lung to blood ratio of .sup.125I-tPA/RBC was 20 times
higher than that of radiolabeled tPA whereas in the kidney, brain
and heart, these organ to blood ratios were ten times lower, data
indicating that coupling of tPA to RBC strongly restricted uptake
by organs other than the lung.
[0033] Experiments were also performed to determine strategies for
activating inactive plasminogen activators in blood. The soluble
form of urokinase receptor (suPAr) has been reported to bind scuPA
and convert inactive scuPA to an active uPA, as well as protect it
from inhibition by plasma inhibitors (Higazi, A. et al. 1995. J.
Biol. Chem. 270:17375-17380; Higazi, A. et al. 1996. Blood
87:3545-3549). The half-life of suPAr in blood, however, is short,
in the range of minutes following intravenous injection.
Accordingly biotinylated suPAr was coupled to SA/biotinylated RBCs
to prolong the half-life of suPAr in vivo. This coupling yielded a
complex designated as b-suPAr/SA/b-RBC that possessed up to
1.3.times.10.sup.5 molecules of suPAr per SA/b-RBC molecule.
Without streptavidin conjugated to the biotinylated RBCs, the
binding of suPAr to the RBC carrier was an order of magnitude lower
(1.2.times.10.sup.4 suPAr molecules/RBC).
[0034] The ability of b-suPAr/SA/b-RBC complex to bind single chain
urokinase plasminogen activator (scuPA) was examined in vitro by
incubating .sup.125I-labeled scuPA with b-suPAr/SA/b-RBC or intact
RBCs (used as a control of non-specific binding of scuPA). It was
found that 1.5+0.1.times.10.sup.4 molecules of .sup.125I-scuPA per
b-suPAr/SA/b-RBC bound while only 0.06+0.01.times.10.sup.4
125I-scuPA per RBC bound. Thus, the b-suPAr/SA/b-RBC complex binds
scuPA effectively and specifically.
[0035] The fibrinolytic activity of the scuPA associated with
b-suPAr/SA/b-RBC complex (scuPA/b-suPAr/SA/b-RBC) was then
examined. Fibrin clots were prepared that contained
.sup.125I-fibrin and either phosphate-buffered saline (control
clots), RBC carrier alone, b-scuPA/SA/b-RBC complexes,
scuPA/b-suPAr/SA/b-RBC complexes, or scuPA alone. In control clots,
less than 5% of the radiolabel was released to the supernatant
after 120 minutes incubation at 37.degree. C. Neither RBC carrier
alone nor b-scuPA/SA/b-RBC complexes (b-scuPA conjugated to
SA/b-RBC directly) caused detectable fibrinolysis. Free scuPA
caused about 10% fibrinolysis. However, similar doses of
scuPA/b-suPAr/SA/b-RBC complexes caused 95.+-.4% fibrinolysis.
These results indicate that binding of scuPA to b-suPAr conjugated
with SA/b-RBC indeed stimulates fibrinolytic activity of scuPA and
that b-suPAr/SA/b-RBC complex is active as a fibrinolytic
agent.
[0036] To determine the fate of the b-suPAr/SA/b-RBC complex in
vivo, rats were injected intravenously with
.sup.125I-suPAr/SA/.sup.51Cr-b-RBC and the blood levels and
biodistribution of the complex were studied. Conjugation of suPAr
to the carrier RBC significantly increased the half-life of suPAr
in the bloodstream. The blood level of non-conjugated suPAr was
undetectable within 3 hours, while 20% of the b-suPAr/SA/b-RBC
complex was measurable out to 24 hours after injection.
[0037] Biodistribution studies showed that conjugation of suPAr did
not lead to hemolysis or to reticuloendothelial uptake of the
carrier RBC. In fact, distribution of the conjugated
b-suPAr/SA/.sup.51Cr-b-RBC was similar to that of the control
.sup.51Cr-RBC distribution. One hour after injection of rats with
b-suPAr/SA/.sup.51Cr-b-RBC, blood samples were obtained for
determination of binding of .sup.125I-scuPA to blood cells. Blood
obtained from rats injected with the b-suPAr/SA/b-RBC complexes
bound three times more scuPA than blood obtained from control
animals. These data indicate that b-suPAr/SA/b-RBC is functionally
active in the circulation and is capable of binding scuPA.
Quantitation of the binding revealed that, after circulating for
one hour in the bloodstream, each b-suPAr/SA/b-RBC complex binds
approximately 10.sup.4 molecules of scuPA. This value is similar to
the initial levels of scuPA binding of b-suPAr/SA/RBC before
injection in rats. Therefore, circulation in the bloodstream did
not alter the binding ability of this complex.
[0038] The fibrinolytic activity of crosslinked plasminogen
activators was examined in vivo in rats. Rats were injected
intravenously with either 250 .mu.g of unconjugated tPA or 50 .mu.g
of tPA conjugated to carrier RBC and blood was collected after 10,
60 and 180 minutes. .sup.125I-fibrinogen was added to all samples
and blood was allowed to coagulate at room temperature. Neither tPA
nor tPA/RBC suppressed clot formation at the test concentrations,
an important consideration in terms of safety of the present
invention. The clots were then incubated at 37.degree. C. and the
rate of clot lysis was examined. Spontaneous fibrinolysis did not
exceed 10% at either 10 minutes or 60 minutes in rats injected with
saline. Fibrinolysis was augmented 10 minutes after injection of
unconjugated tPA. However, the rate of fibrinolysis in blood
obtained 60 minutes after tPA injection only marginally exceeded
that of the control animals, as was expected in light of the rapid
elimination of tPA from blood. Remarkably, blood obtained even 3
hours after tPA/RBC injection retained significant fibrinolytic
activity (40% clot lysis), even though the injected dose of tPA was
5-fold lower. These data show that conjugation of tPA to the
carrier RBC significantly increased the circulation of
enzymatically active plasminogen activator in vivo and prolonged
the in vivo half-life of the plasminogen activator in blood to a
level that would overcome any steric limitations that might be
imposed by conjugation to RBC.
[0039] Experiments were also performed with mouse RBCs. In these
experiments, tPA was conjugated to washed murine RBC in
Krebs-Ringer buffer containing glucose and 3% bovine serum albumin
to minimize energy depletion and mechanical damage. Using methods
as described for rat RBCs, 3 to 4.times.10.sup.4 molecules of
biotinylated tPA or suPAr per biotinylated murine RBC were coupled
without detectable hemolysis as shown below in Table 2. In these
tests, the murine RBC were biotinylated with 10 .mu.M BxNHS in
Krebs-Ringer glucose (pH 7.4). The radiolabeled activators were
coupled to b-RBC via streptavidin in BSA-containing Krebs-Ringer
glucose. Therefore, murine RBC were conjugated in the same way as
human and rat RBCs. TABLE-US-00002 TABLE 2 Coupling Efficiency of
Murine RBC % Added Added Bound % Added Bound (per (of SA/B-
Activator (per RBC) (per RBC) (of RBC) SA/b-RBC) RBC)
.sup.125I-b-tPA 3 .times. 10.sup.5 4.4 .times. 10.sup.3 1.5% 3.2
.times. 10.sup.4 11% .sup.125I-b- 2 .times. 10.sup.5 3.5 .times.
10.sup.2 0.2% 4.1 .times. 10.sup.4 20% suPAr
[0040] Studies were then performed to examine the transfer of tPA
from its carrier RBC to tissue, specifically vascular tissue. As a
model for pulmonary vasculature transfer, human umbilical vein
endothelial cells (HUVEC) cells were used in culture. RBC carriers
were constructed by the method of the present invention. The RBCs
carried either no protein (naive RBC), tPA (tPA/RBC, suPAr
(suPAr/RBC), scuPA conjugated to suPAr molecules (scuPA-suPAr/RBC),
scuPA (scuPA/RBC), or anti-TM/RBC. RBC carrying the protein
molecules (3.times.10.sup.4 molecules/RBC) were incubated with
HUVEC (2.times.10.sup.7 RBC per well, 160 minutes at 37.degree.
C.). Non-bound cells were eliminated by gentle washing with a
phenol-free medium and 1 ml of water was added to lyse bound RBC.
The number of HUVEC-bound RBC was determined by measuring the
absorbance at 405 nm emitted by hemoglobin released into the cell
lysates and expressed as 1,000 RBC/well. The results are shown in
Table 3. TABLE-US-00003 TABLE 3 Adhesiveness of complexes to
endothelium in culture Naive suPAr/ scuPA- scuPA/ anti- RBC tPA/RBC
RBC suPAr/RBC RBC TM/RBC RBC 2.6 1.7 2.5 2.3 22 11 bound
Neither tPA/RBC, suPAr/RBC, nor scuPA-suPAr/RBC were adhesive to
HUVEC, as evidenced by binding levels comparable to naive RBC. In
contrast, scuPA/RBC complex or RBC conjugated with antibody against
endothelial surface glycoprotein, thrombomodulin (used as a
positive control) were highly adhesive. Accordingly, complexes
comprising a soluble form of urokinase receptor biocompatibly
coupled to a red blood cell carrier minimize the potential
hazardous interactions of urokinase with endothelial cells.
[0041] Next, .sup.125I-tPA/RBC were incubated with HUVEC, the
unbound RBC removed by washing and .sup.125I in the wells measured.
HUVEC bound 8.1+0.2.times.10.sup.9 molecules tPA/well after
incubation with .sup.125I-tPA/RBC. The results shown in Table 3
indicate that only 5% of this amount of cell-bound .sup.125I-tPA
was attributable to RBC-associated tPA in the wells
(3.times.10.sup.4 molecules
.sup.125I-tPA/RBC.times.1.7.times.10.sup.3
RBC/well=5.1.times.10.sup.8 molecules RBC/.sup.125-tPA/well).
Therefore, tPA transfers from RBC to HUVEC, demonstrating the
successful use of the RBC carrier of the instant invention.
[0042] Accordingly, the present invention provides novel
compositions for prolonging the half-life of therapeutic agents
including anti-thrombotic agents such as plasminogen activators and
anticoagulants in the bloodstream of animals including humans by
decreasing the degradation and elimination of the therapeutic
agents in the bloodstream.
[0043] The present invention also provides a method for increasing
selective dissolution of blood clots by therapeutic agents such as
anti-thrombotics in blood of a patient which comprises
administering to a patient a composition comprising a therapeutic
agent, preferably an anti-thrombotic agent, more preferably a
plasminogen activator, biocompatibly coupled to a red blood cell
carrier or a carrier similar in size to a red blood cell.
[0044] Further, as demonstrated herein, coupling of an
anti-thrombotic agent such as a fibrinolytic or anticoagulant drug
to a carrier similar in size to a red blood cell, i.e., a carrier
approximately 3-5 microns also results in a composition that
selectively dissolves pathological, nascent blood clots such as
formed after surgical interventions, while minimizing the risk of
untoward dissolution of hemostatic pre-existing clots.
[0045] Fibrin clots undergo a rapid maturation process, which
includes mechanical retraction, incorporation of pro-thrombotic
moieties and cross-linking by Factor XIII transglutaminase, all
markedly limiting its porosity. Thus, established surgical clots
and extravascular hemostatic fibrin in the wound are likely to be
far less permeable to blood components than are nascent
intravascular clots.
[0046] Once surgical hemostasis has been attained, the risk of
post-operative bleeding would be lessened if a plasminogen
activator (PA) could be delivered in a manner that limits its
penetration into existing clots, but permits inclusion into the
nascent clots. One means to achieve this goal is to link a
plasminogen activator to a large carrier of, for example 3-5
microns, such as red blood cells (RBC). Coupling to RBC would
restrict the physical penetration of PA into physiological clots
and prolong its survival. This mechanism of delivering PA may
permit prophylactic administration once surgical hemostasis has
been secured.
[0047] In these experiments, the anti-thrombotic agent tissue
plasminogen activator (tPA) was coupled to RBCs using
strepatividin(SA) biotin as a cross linker. The amount of
biotinylated tPA (.sub.btPA) that can be coupled to biotinylated
rat, mouse or human RBCs (.sub.bRBC) ranges from 10.sup.5
molecules/RBC to less than 200 molecules of non-biotinylated tPA.
Attachment to .sub.bRBC using polyvalent streptavidin inactivated
the complement controlling proteins DAF and CD59, leading to
hemolysis of .sub.bRBC/SA in autologous serum. However, RBC
biotinylated in biotin ester levels below 100 .mu.M (.sub.bxRBC
activity, wherein X.ltoreq.100) do not activate complement and are
stable in fresh homologous serum.
[0048] The activity of .sub.b10RBC/SA/.sub.btPA complexes
possessing 6.5-7.5.times.10.sup.5 tPA molecules per RBC was tested
in fibrin clots formed by adding thrombin to .sup.125I-labeled
fibrinogen as described by Murciano et al. (Am. J. Physiol. Lung
Cell Mol. Physiol 2002 282:L529-539). Clots invested with RBC/tPA
were dissolved releasing .sup.125Iodine into the supernatant.
Similar results were observed with clots formed from rat, mouse and
human plasma using tPA coupled to homologous RBCs. During a 24 hour
incubation period in fresh serum at 37.degree. C., hemolysis of
RBC/tPA by complement was comparable to control RBCs and only a
minor fraction of tPA detached from the RBCs. Dissolution of
pre-existing versus nascent fibrin clots by free tPC and RBC/tPA
was first compared in vitro. Free tPA caused 50% dissolution of
pre-existing versus 90% lysis of nascent clots (1.9 fold
difference). In contrast, tPA when coupled to a large carrier of,
for example 3-5 microns such as an RBC, caused less than 5%
dissolution of pre-existing clots versus 80% dissolution of nascent
clots (17-fold difference). Thus, compositions of the present
invention comprising an anti-thrombotic agent coupled to a large
carrier of, for example 3-5 microns such as an RBC, exhibit ten
times greater selectivity toward nascent clots than anti-thrombotic
agent alone.
[0049] Fibrinolytic efficacy of compositions of the present
invention was also tested in a mouse model of venous thrombosis.
First, experiments were performed to confirm that RBC carriage
prolongs the circulation of tPA in mice. In these experiments, free
.sup.125I-tPA disappeared from the blood stream within 20 minutes
while 50-60% of injected RBC/.sup.125I-tPA remained in the blood
stream 60 minutes post injection. Next, findings in rats that the
duration of fibrinolytic activity in the blood after bolus
injection of tPA was prolonged by RBC carriage were confirmed.
Fibrinolysis of clots formed from blood taken 2 minutes after free
tPA injection was facilitated, but this enhancement disappeared
within 20 minutes after injection. In contrast, clots formed from
blood taken 60 minutes post injection of RBC/tPA dissolved almost
completely. Therefore, like rats, active RBC/tPA, but not free tPA,
circulates for a prolonged time after injection in mice.
[0050] A model of pulmonary embolism induced by injecting
radiolabeled fibrin microemboli 3-5 microns in diameter
(.sup.125I-ME) was used to examine fibrinolysis of intravascular
clots in vivo. Momentarily after intravenous injection .sup.125I-ME
form aggregates invested with blood cells which lodge in the lungs
of mice and rats (Murciano et al. Am. J. Physiol. Lung Cell Mol.
Physiol. 2002 282:L529-539; Bdeir et al. Blood 200 96:1820-26).
Measuring radioactivity in the lungs 1 hour after .sup.125I-ME
injection provides quantitative measurement of intravascular
fibrinolysis of pulmonary emboli (Murciano et al. Am. J. Physiol.
Lung Cell Mol. Physiol. 2002 282:L529-539).
[0051] Injection of free tPA 10 minutes after .sup.125I-ME
dissolved the clots almost completely. In contrast, less that 20%
fibrinolysis was seen when tPA was injected even 5 minutes prior to
the emboli, consistent with its rapid clearance from the
bloodstream. In contrast, RBC/tPA injected after .sup.125I-ME
produced modest (less than 40%) fibrinolysis of pulmonary emboli,
while RBC/tPA injected 20 minutes before .sup.125I-ME caused 80%
fibrinolysis. Thus, the ratio of fibrinolysis of nascent versus
pre-existing clots was 0.2 and 2.2 for free tPA and RBC/tPA,
respectively.
[0052] Large, life-threatening occlusive clots are less permeable
and susceptible to fibrinolysis than microemboli due to a lower
surface to mass ratio (Murciano et al. Am. J. Physiol. Lung Cell
Mol. Physiol. 2002 282:L529-539). Accordingly, the
thromboprophylactic ability of RBC/tPA to effectively dissolve
occlusive thrombi in a large vessel was examined using the
FeCl.sub.3 model of carotid arterial injury. In these experiments,
thrombus formation and fibrinolysis were monitored by Doppler
ultrasound. Shortly after application of the FeCl.sub.3 solution to
the tunica adventitia, blood flow in the carotid artery was
practically undetectable and the vessel remained occluded for the
entirety of the experiment (60 minutes). Injection of free tPA
prior to thrombus formation did not cause clot lysis or facilitate
reperfusion. Neither did injection of free tPA or RBC/tPA 10
minutes after thrombus formation. However, RBC/tPA injected 10
minutes prior to thrombosis, though not affecting the rate of clot
formation, began to dissolve the clots within 10-20 minutes and
restored the blood flow reaching a mean of approximately 80% of
baseline by 20-30 minutes. The ratio of fibrinolysis of nascent
versus pre-existing clots in this model was 0.2 and 3.3 for free
tPA and RBC/tPA, respectively.
[0053] Thus, these experiments are demonstrative of the
thromboprophylaxic utility of compositions of the present
invention. As shown herein complexing a therapeutic agent,
particularly an anti-thrombotic agent such as a plasminogen
activator to a large carrier of, for example 3-5 microns, such as
an RBC minimizes diffusion of the anti-thrombotic agent from the
vasculature and fibrinolysis of pre-existing clots, while markedly
prolonging its life-span and permitting dissolution of nascent
clots soon after their formation, thereby converting a
anti-thrombotic agent such as a fibrinolytic into a
thromboprophylactic agent. Specifically, RBC/tPA showed a 17-fold
enhancement in the lysis of nascent versus pre-existing clots in
vitro, likely by restricting the diffusion of tPA into fibrin.
Biocompatible carriage by RBC also prolonged tPA circulation,
markedly extending duration of its fibrinolytic activity in the
blood providing the complex with the ability to dissolve pulmonary
emboli and occlusive arterial thrombi, when infected prior, but not
post thrombosis. RBC/tPA was ten times more selective than free tPA
towards lysing nascent versus pre-existing pulmonary emboli and 20
times more selective towards nascent versus pre-existing arterial
clots. Furthermore, prophylactic injection of RBC/tPA dissolved
occlusive thrombi in the carotid artery soon after their formation,
despite the fact that they proved resistant to dissolution by free
tPA injected before or after thrombosis. Therefore, complexing a
therapeutic agent such as an anti-thrombotic agent such as tPA to a
carrier such as a RBC or a carrier similar or greater in size than
a RBC converts the therapeutic agent into a potent
thromboprophylactic agent.
[0054] Preparation of compositions comprising an anti-thrombotic
agent and a biocompatible carrier such as red blood cell carriers
is performed in accordance with known methods of conjugation and is
exemplified by monovalent crosslinkage via biotin, streptavidin,
and monoclonal antibodies. However, as will be obvious to those of
skill in the art upon this disclosure, other methods of
biocompatible coupling, i.e., chemical conjugation, non-covalent
binding via a conjugated receptor, or other means of attachment
which does not lead to poor biocompatibility of the carrier
exemplified by lysis and increased phagocytosis could also be
used.
[0055] Autologous transfusion is standard practice in elective
surgery. Results from human studies exploring RBC carriage of other
drugs indicate that it is technically feasible to conjugate
therapeutic agents such as anti-thrombotic agents (e.g.
fibrinolytic agents such as tPA) to auto-donated RBC and to
re-inject the complex after surgery. Alternative bioconjugation
techniques such as coupling to the RBC via activated polyethylene
glycol (PEG) may enhance biocompatibility and minimize the immune
response to non-autologous RBC. Coupling a therapeutic agent to
RBCs via PEG may extend the applicability of the proposed strategy
by generating universal donor RBC/tPA with a reasonable shelf-life.
Furthermore, it is possible to couple therapeutic agents to RBCs
directly in the bloodstream. Human RBC possess the complement
receptor I (CRI) that binds immune complexes and transfers them to
macrophages without opsonization of RBC themselves. CRI monoclonal
antibodies and anti-CRI immunoconjugates injected in animals bind
to RBC and circulate for a prolonged time without causing cell
damage. Thus, it is expected that anti-CR1/anti-thrombotic agent
conjugates can also be used for prophylaxis without the need for
RBC extraction, modification or transfusion.
[0056] As will be understood by those of skill in the art upon
reading this disclosure, other antibodies selective to RBCs can be
used in similar fashion to anti-CRI as well. The compositions of
the present invention are also expected to be safer in cerebral
fibrinolysis. Free tPA improves reperfusion in ischemic stroke, but
causes collateral damage in the brain in hemorrhagic stroke. In
addition to unintended dissolution of hemostatic clots in the
cerebral vasculature, diffusion of anti-thrombotic agents such as
plasminogen activators into the brain aggravates cerebral edema and
matrix remodeling, and causes direct cytotoxicity to neurons. It is
believed that compositions of the present invention such as RBC/tPA
will be safer due to better retention within the vascular
compartment.
[0057] In a preferred embodiment, the compositions of the present
invention further comprise pharmaceutically acceptable vehicles for
intravenous administration to patients with a disease or condition
characterized by uncontrolled intravascular fibrin clot formation
including deep venous thrombosis. Such pharmaceutically acceptable
vehicles include saline, phosphate buffered saline, or other liquid
sterile vehicles accepted for intravenous injections in clinical
practice.
[0058] As will be understood by the skilled artisan upon reading
this patent application, the model anti-thrombotic agent used in
these experiments, tPA, represents just one of a large number of
therapeutic agents whose therapeutic profile will be enhanced or
favorably altered by complexing with a large carrier of, for
example 3-5 microns such as RBC carriage. Examples of such
therapeutic agents include, but are not limited to
anti-inflammatory agents, anti-coagulant agents such as protein C,
and enzymes that inactivate toxins in the bloodstream. Compositions
of the present invention are preferably administered systemically
as a bolus intravenous injection of a single therapeutic dose of
the drug (for example, 0.1-1.0 mg/kg for plasminogen activators).
When used prophylactically, the compositions of the present
invention is preferably administered to provide safe and effective
thromboprophylaxis in specific surgical settings that are
accompanied by a high incidence of arterial thrombosis.
[0059] For example, a few hours after angioplasty, when initial
hemostatic fibrin formation is relatively mature, a patient is
administered an injection of preformed tPA/RBC complex, or
scuPA-suPAR/anti-CR1 conjugate which binds to the circulating RBC.
The life span of RBC-bound complex in the circulation may be varied
from several hours to several weeks, depending on the number of tPA
or scuPA molecules on RBC. The complex has little or no access to
the tissues or mature fibrin clots and circulates the bloodstream
in inactive-form due to physiological suppression by plasminogen.
However, if nascent fibrin clots begin form intravascularly, the
entire complex is incorporated, resulting in fibrinolysis in a
geographically constrained area.
[0060] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Preparation of Conjugated Plasminogen Activators-Biotinylation,
Radiolabeling of Proteins, Conjugation of Proteins to RBC and
Assessment of the Fibrinolytic Activity
[0061] Biotin ester, 6-biotinylaminocaproic acid
N-hydroxysuccinimide ester (BxNHS) was dissolved in 100%
dimethylformamide to a final concentration of 10 mM or 1 mM.
Tissue-type plasminogen activator (tPA), urokinase, streptokinase
and soluble urokinase plasminogen activator receptor (suPAr) were
biotinylated at ten-fold molar excess of BxNHS. Eight microliters
of fresh 1 mM BxNHS were added to 100 .mu.l of a protein solution
(1 mg/ml in borate buffered saline, BBS, pH 8.1). After a 1 hour
incubation on ice, excess of non-reacted BxNHS was eliminated by
overnight dialysis. Biotinylated proteins were radiolabeled with
.sup.125Iodide using Iodogen-coated tubes according to the
manufacturer's recommendations (Pierce). Incubation of 100 .mu.g of
a biotinylated protein and 100 .mu.Ci of sodium .sup.125Iodide in a
tube coated with 100 .mu.g of Iodogen for 20 minutes on ice yielded
streptavidin with a specific radioactivity of approximately 500 cpm
per ng. Excess iodine was eliminated by dialysis. More than 95% of
radiolabeled proteins were precipitable by TCA.
[0062] Two milliliters of fresh heparinized blood was then
centrifuged at 1,500 rpm for 5 minutes and supernatant (i.e.,
plasma) was eliminated. The pellet was then resuspended and washed
with PBS by standard centrifugation (10 ml of PBS per 1 ml of
pellet, 1,500 rpm, 5 minutes, four times) to make a 100% suspension
of washed RBC. PBS (0.9 ml) was then added to 0.1 ml of RBC pellet
(i.e., make 10% suspension of washed RBC). One hundred microliters
of 300 mM boric acid (pH 9.0) was added to 1.0 ml of 10% RBC. BxNHS
in DMFA was then added to this suspension to obtain a final BxNHS
concentration in the reaction mixture equal to 10 .mu.M and to
obtain b.sub.10-RBC. At first, 1 .mu.l of stock solution of 0.1 M
BxNHS/DMFA was added to 99 .mu.l DMFA. Then 10 .mu.l of this fresh
1 mM BxNHS/DMFA was added to 1 ml 10% RBC and mixed well. After a
30 minute incubation with periodic gentle shaking, at 20.degree.
C., excess non-reacted BxNHS was eliminated from the reaction
mixture by standard centrifugation with PBS containing 2 mg/ml BSA
(BSA-PBS). A 10% suspension of biotinylated RBC in BSA-PBS was
prepared.
[0063] To attach streptavidin to b-RBC, 20 .mu.l of SA stock
solution (1 mg/ml in PBS) was added to 100 .mu.l 10% suspension of
b-RBC and mixed well. This provides addition of 1 .mu.g SA per
5.times.10.sup.6 b-RBC (about 2.times.10.sup.6 molecules per
b-RBC). After a 30 minute incubation with periodic gentle shaking
at 20.degree. C., non-bound SA was removed by standard
centrifugation in BSA-PBS.
[0064] To attach biotinylated plasminogen activator or suPAr to
SA/b-RBC, 5 .mu.l of stock solution b-PA or b-suPAr (1 mg/ml in
PBS) was added to 100 .mu.l 10% suspension of SA/b-RBC and mixed
well. This provides addition of 1 .mu.g b-PA or b-suPAr per
2.times.10.sup.7 SA/b-RBC (about 3.5.times.10.sup.5 molecules per
SA/b-RBC). b-PA or b-suPAr was then incubated with the 10%
suspension of SA/b-RBC for 1 hour (periodic gentle shaking,
20.degree. C.). Non-bound proteins were removed by standard
centrifugation with BSA-PBS. To quantitate binding of b-PA or
b-suPAr to SA/b-RBC, radiolabeled b-PA or b-suPAr was used as a
tracer.
[0065] Proteins were radiolabeled with .sup.125I-Na (Perkin Elmer,
Boston, Mass.) using IODOGEN (Pierce, Rockford, Ill.) and RBC were
radiolabeled with .sup.51Cr as described by Muzykantov et al.
(Anal. Biochem. 1996 241:109-119).
[0066] The suspension of .sup.51Cr-RBC/.sup.125I-tPA in phosphate
buffered saline (PBS)/3% bovine serum albumin (BSA) was stable for
at least 4 days at 4.degree. C. To test hemolysis by complement,
control RBC, .sub.bRBC/SA or .sub.bRBC/SA/.sub.btPA were incubated
in fresh serum for 1 hour at 37.degree. C., centrifuged (5 minute,
1200 g) and the released hemoglobin was determined
spectrophotometrically at 405 nm.
Example 2
In Vivo Administration of Conjugated Plasminogen Activators
[0067] To study biodistribution of radiolabeled preparations in
rats, injection of 0.5 ml of saline containing 1 .mu.g of
radiolabeled PA or suPAr, or these proteins coupled to the carrier
RBC, was made into the tail vein under anesthesia. To trace
RBC-coupled plasminogen activators after in vivo administration,
20-50 .mu.l of 10% suspension of .sup.125I-b-PA/SA/b-RBC was
injected via the tail vein in anesthetized rats. At indicated times
after injection (5 minutes-24 hours), anesthetized rats were
sacrificed by exsanguination. Blood and internal organs were
collected. Organs were rinsed with saline until free of blood and
weighed. Radioactivity of .sup.125I in aliquots of blood and
internal organs was then determined using a gamma-counter. Plasma
was then separated from the blood by centrifugation of blood and
radioactivity in the plasma was determined. Results were calculated
as cpm per gram of tissue, blood or plasma, as mean.+-.standard
error (M.+-.SE). Statistical comparisons were made using one-way
analysis of equal variance (ANOVA) followed by Student-Newman-Keuls
Method at a level of statistical significance of p<0.05.
Example 3
In Vitro Clot Lysis
[0068] Fibrin clots were formed by adding CaCl.sub.2 and thrombin
(20 mM and 0.2 units/ml final concentrations) to fibrinogen (3
mg/mL) trace-labeled with .sup.125I-fibrinogen. To simulate lysis
of pre-existing clots, clots were overlaid with each fibrinolytic
agent or saline (control) for 20 minutes at 20.degree. C. To lyse
nascent clots, fibrinolytics were added directly to
.sup.125I-fibrinogen prior to adding CaCl.sub.2 and thrombin. To
initiate fibrinolysis, clots were placed at 37.degree. C. and the
radioactivity in the supernatants was measured in a gamma-counter.
(Perkin Elmer, Boston, Mass.)
Example 4
RBC, tPA and RBC/tPA Circulation In Vivo
[0069] .sup.51Cr-RBC, .sup.125I-tPA or .sup.51Cr-RBC/.sup.125I-tPA
were injected into the tail veins of anesthetized rats or mice. At
selected times 100-200 .mu.l of blood were taken from animals into
heparin, centrifuged at 1200 g and the radioactivity was measured
in plasma supernatants and RBC pellets. The animals were sacrificed
1-3 hours after injection and radioactivity in the organs was
analyzed in a gamma-counter.
Example 5
Dissolution of Clots Formed from Blood Obtained After Injections of
tPA or RBC/tPA
[0070] Control RBC, free tPA, RBC/tPA or saline (control) were
injected intravenously into rats or mice. At indicated times,
100-200 .mu.l aliquots of blood were drawn in the absence of
anticoagulant, rapidly mixed with trace amounts of
.sup.125I-Fibrinogen and were allowed to clot in borositicate tubes
at 20.degree. C. After 20 min maturation, clots were overlaid with
saline, incubated at 37.degree. C. and the release of 125 Iodine
was measured.
Example 6
Dissolution of Pulmonary Emboli by tPA or RBC/tPA
[0071] Suspensions of .sup.125I-fibrin microemboli (.sup.125I-ME)
were prepared as described (Murciano, J. C. et al. 2002. Am J
Physiol Lung Cell Mol Physiol 282:L529-539 and Bdeir, K. et al.
2000. Blood 96:1820-1826) and injected into anesthetized mice. A
350 .mu.il sample containing a 50% suspension of RBC, RBC mixed
with saline or tPA (0.5 mg/Kg) or RBC/tPA after injection of
.sup.125I-ME. Animals were sacrificed 1 hr after injection of
.sup.125I-ME, the lungs were isolated, rinsed in saline and the
residual radioactivity was measured in the gamma-counter.
Example 7
Dissolution on the Carotid Arterial Thrombi by RBC/tPA
[0072] We used a described murine model of acute carotid
embolization (Farrehi, P. M. et al., 1998. Circulation
97:1002-1008). Occlusion of the vessel was determined by Doppler
ultra-sound using a 0.5 VB flow probe connected to a recording
system (Transonic, Ithaca, N.Y.). Complete occlusion occurred
within 8-10 min of the vessel injury by FeCl3, and in control
animals vessels remain occluded until the experiment was terminated
at 60 min. Either before FeCl3 application or 10 n-tin after
complete occlusion, 350 .mu.il solution of PBS, RBC, tPA 0.7 mg/Kg
alone or mixed with RBC, or RBC/tPA (containing 0.5 mg/Kg tPA) was
injected through the opposite jugular vein.
* * * * *