Modulators of coagulation factors with enhanced stability

Abstract

The invention provides improved nucleic acid ligands with enhanced stability that inhibit coagulation and improved modulators of the nucleic acids to provide ideal modulators of coagulation. These improved nucleic acids and modulators are particularly useful for inhibiting coagulation in a host undergoing a therapeutic regime such as surgery or coronary artery bypass.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. application Ser. No. 11 / 113,378, filed Apr. 22, 2005.TECHNICAL FIELD [0002] An improved agent, composition and method to regulate the pharmacological activity of a coagulation factor with nucleic acid ligands (e.g., aptamers) that have improved stability is disclosed. BACKGROUND [0003] Despite substantial efforts to treat and prevent thrombotic events, arterial thrombosis continues to be the major cause of death in adult populations of developed nations. Although numerous medical strategies exist for treating thrombosis, no available agent meets the therapeutic endpoints of both bioavailability and efficacy, while also having a reasonable safety profile (see Feuerstein et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19:2554-2562). [0004] Under normal circumstances, an injury to vascular endothelial cells lining a blood vessel triggers a hemostatic response through a sequence of events c...

AI Technical Summary

Benefits of technology

[0023] Improved nucleic acid ligands for anticoagulant therapy are disclosed as well as improved nucleic acid ligands in combination with an antidote that changes the binding of the nucleic acid ligand for its target or that degrades or otherwise cleaves, metabolizes or breaks down the nucleic acid ligand while the ligand is still exerting its effect. These improved aptamers provide favorable anticoagulant properties for in vivo applications, including during human or veterinary surgery. The anticoagulant function of the improved aptamer is conveniently neutralized on administration of its antidote when desired by the surgeon or orther medical care specialist. In particular, the aptamers of the invention include additional agents to increase bioavailability and / or decrease degradation of the active agent.

[0024] In one aspect of the invention, improved nucleic acid ligands or aptamers to a factor in the blood coagulation cascade are provided that include at least one moiety that increases bioavailability of the agent. In some embodiments, the factors include Factor IX (FIX) or the cleavage product Factor IXa (FIXa). In some embodiments, the aptamers are ligands to the complex formed by FIXa with Factor VIIIa (FVIIIa), also known as the “intrinsic tenase complex.” In some embodiments, the aptamers are ligands that inhibit the complex formation between FIXa and FVIIIa. In a subembodiment, the aptamers of the present invention bind to the complex of FIX and FVIIIa and inhibit activation of Factor X (FX). The aptamers can interact with FIX, FIXa or a complex formed with FVIIIa in the presence or absence of additional calcium. The aptamers can also interact with the factors of the complex at a cell membrane. In one embodiment, the aptamers bind to the intrinsic tenase complex at the membrane surface.

[0025] In particular, the aptamers, such as aptamers to FIXa, are linked to a stabilizing moiety on at least one terminus. In one embodiment, the aptamers are linked to a polymeric agent. In one embodiment, the aptamers are linked to one or more polyethylene glycol molecules. The aptamer and stabilizing agent can be linked at, for example, a 5′ terminus of the nucleic acid sequence. In certain embodiments, the aptamers are linked to multiple polyethylene glycol molecules. In other embodiments, a single polyethylene glycol molecule can be linked to more than one aptamer to provide improved delivery of the agent.

[0027] The aptamers can include a “suicide position.” In one embodiment, this position becomes single stranded and labile upon binding of the antidote to the improved aptamer and allows for cleavage of the improved aptamer upon binding of the antidote by enzymes in the circulation, such as blood or liver endonucleases, thereby effectively eliminating the active aptamer from circulation. The suicide position can be at a guanine in stem 2 that is hydroxylated. In one embodiment, this nucleotide is in a double stranded configuration until bound with an antidote and becomes single stranded and available for cleavage upon binding of the antidote.

[0040] In one embodiment, antidotes to the improved aptamers of the invention are provided to a mammal in need thereof to reverse the anticoagulant effects of the improved aptamers. Improved aptamers and aptamer-antidote pairs can be administered in real time as needed based on various factors, including the progress of the patient, as well as the physician's discretion in how to achieve optimal therapy. Thus, this invention discloses an improved regulatable therapeutic regime in the course of nucleic acid ligand therapy for blood coagulation. In one example, an antidote is provide that neutralizes the effect of the improved aptamer to turn off anticoagulant activity when desired by the physician or other health care provider. In another embodiment, the improved aptamers and antidotes to blood coagulation factors are administered in sequential steps, in which the aptamers are administered, the antidotes are used to limit the activity of the improved aptamers, and subsequently the aptamers are re-administered to a patient in need thereof. In one embodiment, the antidote achieves this neutralization effect by binding to or hybridizing to the improved aptamer.

Problems solved by technology

Despite substantial efforts to treat and prevent thrombotic events, arterial thrombosis continues to be the major cause of death in adult populations of developed nations.

Although numerous medical strategies exist for treating thrombosis, no available agent meets the therapeutic endpoints of both bioavailability and efficacy, while also having a reasonable safety profile (see Feuerstein et al.

Unmodified RNA is not realistically used as a therapeutic agent since blood is rich in ribonucleases.

However, there is no way to predict how a particular modification changes aptamers.

In particular, when additional limitations are required, as is the case with modulatable aptamers, no techniques exist to predict how one or more modifications can affect the capacity of the aptamer to regulate its ligands and at the same time continue to be regulated by antidote binding.

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