Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing
Reexamination Certificate
1998-01-26
2001-04-10
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
C424S093200, C424S093600, C424S093210, C435S320100, C435S325000, C435S069600, C435S455000, C435S456000, C435S366000, C435S372000, C435S372100, C435S091400, C435S091410
Reexamination Certificate
active
06214333
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to agents that effect vasoprotection in mammals. More particularly, the invention pertains to anti-thrombotic agents that are adapted for localized rather than systemic administration. Still more particularly, the invention relates to recombinant adenovirus vectors containing a DNA sequence encoding a human tissue factor pathway inhibitor (TFPI) gene and to methods of making and using such vectors to effect local expression of TFPI in vascular smooth muscle cells at a specific blood vessel site.
2. Description of the Related Art
Pharmacological anticoagulation therapies are widely employed to deter thrombus formation in injured or atherosclerotic arteries. These therapeutic approaches typically employ physiological inhibitors of thrombin and require systemic administration of multiple drugs. However, the presence of anticoagulants in the circulating (systemic) blood is generally associated with increased bleeding risk. For instance, in clinical trials studying heparin and the thrombin inhibitor desirudin, most patients with acute coronary syndromes who developed intracranial bleeds had received aspirin, heparin or desirudin, and a thrombolytic agent (
1
-
3
). Those trials indicated that systemic blockade of multiple platelet/coagulation pathways is not without risk. Similarly, it is known that high doses of heparin are poorly tolerated in conjunction with potent platelet inhibition with c7E3 Fab (ReoPro) (
4
).
Short Term Local Administration of Antithrombotics
Since most conventional methods aimed at deterring thrombosis deposition act systemically and typically cause bleeding, some recent research efforts have focused on determining the feasibility of local anticoagulant treatment of predetermined “at risk” arterial sites, as opposed to treating the entire circulatory system. Local delivery of anticoagulant drugs has been attempted.
For example, the isolation of a portion of a vessel with a pair of angioplasty balloons and instillation of hirudin or heparin has been reported (
4
a
). However, these methods are limited by uncertain drug delivery (given the systemic escape) and the short persistence of the antithrombotic drug in the vessel wall, given the diffusion gradient towards the vessel lumen. However, even in these localized treatments, no locally delivered antithrombotic drug has been reported to be present at the target site 48 hours after delivery. Points within the human circulatory system that are subject to injury, inflammation or atherosclerosis are especially likely targets for the local application of therapeutic anticoagulant agents, and include such specific sites as those subjected to angioplasty, stent or graft placement, or arteriovenous shunt.
For the purposes of this disclosure, “local” treatment, as distinguished from “systemic” treatment, means that a specific region, site or area within the blood circulatory system (especially a blood vessel) is the focus or target of the treatment and therefore receives the significant part of the treating agent, while the rest of the vessel and/or the circulatory system receive none or only an insignificant exposure to the treating agent. Short-term administration of antithrombins does little to passivate the injured artery, and allows thrombin generation to relentlessly proceed. In prior studies, for instance, it was found that short-term administration of the direct antithrombins failed to reduce restenosis rates after percutaneous coronary balloon angioplasty (
5
,
6
). This may be explained in part by experimental and clinical evidence suggesting that the thrombin inhibitors are not capable of inhibiting thrombin generation in the course of arterial thrombosis or in systemic procoagulant states (
7
,
8
). Furthermore, after withdrawal of short-term thrombin inhibitor therapy at 3-5 days, thrombin activity soon recurs (
9
,
10
).
Similar conclusions can be drawn from trials of short-term administration of synthetic inhibitors of GP IIb/IIIa integrin receptor (
11
). The administration of platelet IIb/IIIa integrin receptor blocker, c7E3 Fab (ReoPro), was effective in reducing early ischemic events in an early trial (
12
) and reduced the need for recurrent revascularization at 6-months. However, this was associated with increased bleeding at the time of the initial intervention (
4
). In a later trial, however, ReoPro failed to reduce the need for repeated revascularization at 6 months (
13
).
In summary, while effective during their administration, systemically given antithrombotic drugs are associated with increased hemorrhagic risk and require hospitalization associated with high cost, inconvenience, and additional risk of (hospital-acquired) infection; and, finally, do not passivate the thrombogenic lesion after the drug infusion is stopped (typically 3-5 days).
Anti-thrombotic Gene Therapy
Recent trials of systemic antithrombins to prevent restenosis after percutaneous revascularization suggest that there may be advantages to local antithrombotic gene therapy, which conventional drug therapy cannot presently match (
5
,
6
). Gene therapy potentially ensures the continuous in situ production of the foreign antithrombotic protein. Lee et al., in 1993, demonstrated that a replication-defective recombinant adenovirus can serve as an efficient vector for direct in vivo arterial gene transfer (
14
). Zoldhelyi et al. have previously described the adenovirus-mediated transfer of the cyclooxygenase gene (Ad.COX-1) as a localized anti-thrombotic agent (
15
). Cyclooxygenase is the rate-limiting enzyme in the synthesis of prostacyclin, an important vasoprotective molecule that inhibits platelet aggregation and vasoconstriction. Delivery of recombinant adenovirus to the artery at the doses used in the Ad.COX-1 study was associated with only minimal inflammation (
15
). Ad.COX-1 is a reasonable antithrombotic agent but has no direct influence on the thrombin-coagulation pathway involved in fibrin formation and smooth muscle cell proliferation contributing to restenosis after percutaneous balloon angioplasty. Also, platelet aggregation plays little role in venous thrombosis where thrombin inhibition is a highly effective approach (
16
).
Tissue Factor Pathway Inhibitor
Another approach to blockading platelet/coagulation pathways involves inhibiting thrombin activation via the tissue factor metabolic pathway. Tissue factor (TF), the cellular initiator of blood coagulation, is a transmembrane protein receptor exposed after vessel injury or after cytokine activation of endothelial cells and monocytes. Blood coagulation in the extrinsic pathway begins when the serine protease, activated factor VII (factor VIIa), which binds to its cofactor, TF, and the factor VIIa/TF enzyme complex activates by limited proteolysis of coagulation factors X and IX (
17
-
19
). On the membranes of activated platelets and endothelium, factor Xa then binds to factor Va, forming the prothrombinase complex, which in the presence of Ca
2+
proteolytically converts prothrombin to thrombin (
20
). Factor IXa, also activated by the factor VIIa/TF complex, combines with factor VIIIa to activate in a second (intrinsic) pathway factor X. Thus, TF plays an initiating role for both the extrinsic and intrinsic pathway of thrombin generation (
21
).
Thrombin, the final product of the converging coagulation pathways, activates platelets and converts fibrinogen to fibrin, thereby stimulating formation of the fibrin-platelet clot. Thrombin not only activates platelets, converts fibrinogen to fibrin, and via factor XIII activation, stabilizes the fibrin clot, but also positively feeds back on its generation by activating platelets, factors V, VIII and XI (
22
-
24
). In addition, thrombin promotes release of P-selectin from storage granules of platelets and endothelial cells, contributing to platelet-leukocyte interaction and leukocyte rolling and migration into the vessel wall (
25
).
Willerson James T.
Zoldhelyi Pierre
Guzo David
Hall Elizabeth R.
McDaniel C. Steven
McDaniel & Associates, P.C.
Texas Heart Institute
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