Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Stabilized enzymes or enzymes complexed with nonenzyme
Reexamination Certificate
1999-12-03
2002-12-03
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Stabilized enzymes or enzymes complexed with nonenzyme
C435S188000, C435S215000, C435S216000, C436S519000, C514S002600
Reexamination Certificate
active
06488927
ABSTRACT:
BACKGROUND OF THE INVENTION
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 a great concern, especially under specific prothrombotic conditions such as deep vein thrombosis and pulmonary embolism.
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.
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.
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.
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, &agr;-2-antiplasmin (Collen, D. 1996.
Circulation
93:857-865; Reilly, C. et al. 1991.
Arterioscl. Thromb.
11:1276-1286). &agr;-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 &agr;-2-antiplasmin, other inhibitors of plasminogen activators also are present (C1-inhibitor, &agr;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.
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 and uncontrolled tissue proteolysis that occurred, likely after extra vascular deposition of plasminogen activators.
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.
J. Drug Targeting.
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. N.Y. 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.
Further, to date, none of these methods for modifying plasminogen activators prevents 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).
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 tissues.
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-biot
Cines Douglas
Higazi Abd Al-Roof
Murciano Juan Carlos
Muzykantov Vladimir R.
Borin Michael
Licata & Tyrrell P.C.
Trustees of the University of Pennsylvania
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