Modified annexin proteins and methods for their use in organ transplantation and in prevention of thrombosis and reperfusion injury

Abstract

Modified annexin proteins, including a homodimer of human annexin V, are provided. Methods for their use, such as to prevent thrombosis without increasing hemorrhage and to attenuate ischemia-reperfusion injury (IPI), are also provided. The modified annexins bind phosphatidylserine (PS) on cell surfaces, thereby preventing the assembly of the prothromkinase complex. The modified annexin decreases the binding of leukocytes and platelets during post-ischemic reperfusion, thereby restoring microvascular blood flow and decreasing organ damage.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 11 / 267,837, “Modified Annexin Proteins and Methods for Use in Organ Transplantation,” filed Nov. 3, 2005, which is a continuation in part of U.S. application Ser. No. 11 / 078,231, “Modified Annexin Proteins and Methods for Preventing Thrombosis,” filed Mar. 10, 2005, which is a is a continuation in part of U.S. application Ser. No. 10 / 080,370, “Modified Annexin Proteins and Methods for Preventing Thrombosis,” filed Feb. 21, 2002, which claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 60 / 270,402, “Optimizing the Annexin Molecule for Preventing Thrombosis,” filed Feb. 21, 2001, and U.S. Provisional Application No. 60 / 332,582, “Modified Annexin Molecule for Preventing Thrombosis and Reperfusion Injury,” filed Nov. 21, 2001. U.S. application Ser. No. 11 / 078,231 also claims the benefit, under 35 U.S.C. § 119 of U.S. Provisional application No. 60 / 552,4...

AI Technical Summary

Benefits of technology

[0018] The present invention provides compounds and methods for preventing arterial or venous thrombosis. Recombinant human annexins are modified in such a way that its half-life in the vascular compartment is prolonged. This can be achieved in a variety of ways; three embodiments are an annexin coupled to polyethylene glycol, a homopolymer or heteropolymer of annexin, and a fusion protein of annexin with another protein (e.g., the Fc portion of immunoglobulin). The modified annexin binds with high affinity to phosphatidylserine on the surface of activated platelets or injured cells, thereby preventing the binding of Gas6 as well as procoagulant proteins and the formation of a prothrombinase complex. Modified annexin therefore inhibits both the cellular and humoral mechanisms by which platelet aggregation is amplified, thereby preventing thrombosis.

[0025] The present invention also provides a method of inhibiting the attachment of leukocytes to endothelial cells comprising administering an effective amount of an isolated modified annexin protein comprising an annexin dimer to a patient in need thereof. In some embodiments, the method further comprises reducing endothelial cell damage.

[0031] The present invention also provides a method of protecting organ or tissue transplants against reperfusion injury and dysfunction, comprising contacting said organ or tissue with a preservation fluid for perfusion and storage or rinsing of the organ, or tissue transplants prior to the implantation of said organ or tissue in a patient requiring such implantation, wherein said preservation fluid contains an isolated modified annexin protein comprising an annexin dimer in a concentration sufficient to prevent IRI after ischemia.

[0038] In still further embodiments, the present invention provides a method of increasing the duration of survival of blood platelets, comprising adding an isolated modified annexin protein comprising an annexin dimer to stored platelets. The addition may be in a platelet storage medium. The addition may also be in a patient to whom platelets were administered, including the case where the patient is the recipient of a liver graft, including a thrombocytopenic liver graft patient.

Problems solved by technology

However, bolus injection (as opposed to a more gradual intravenous infusion) significantly increases the risk of cerebral hemorrhage (Mehta et al., 2000).

However, the same antibody has shown no benefit in unstable angina without angioplasty, and a better method for preventing coronary occlusion in these patients is needed.

However, intravenous rtPA administration is associated with increased risk of intracerebral hemorrhage.

Venous thrombosis is a frequent complication of surgical procedures such as hip and knee arthroplasties.

Despite such promising results for preventing thrombosis, a major problem associated with the therapeutic use of annexins is their short half-life in the circulation, estimated in experimental animals to be 5 to 15 minutes (Römisch et al., 1991; Stratton et al., 1995; Thiagarajan and Benedict, 1997); annexin V also has a short half-life in the circulation of humans (Strauss et al., J.

The outcome of liver transplants is less successful if the donor organs are steatotic (Amersi et al.

Despite advances in surgical technique, patient management and immunosuppression, ischemia-reperfusion injury (IRI) remains an important clinical problem.

In addition, ischemia of longer than 12 hours is highly correlated with primary nonfunction of transplanted livers, as well as an increase incidence of both acute and chronic rejection (Fellstrom et al.

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