SEQUENCE LISTING

The instant application contains a Sequence Listing, which is submitted concomitantly with this application via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 11, 2014 is named “Sequence Listing” and is 3 kilobytes in size.

FIELD OF THE INVENTION

Provided herein are compounds, compositions comprising same and methods useful for reversibly stabilizing thrombin activity and extending thrombin's shelf-life. In particular, disclosed herein are reversible thrombin binding oligonucleotides capable of interacting with thrombin and inhibiting thrombin activity and an antisense oligonucleotide thereto, and compositions and methods of use therefore to stabilize thrombin activity in a liquid thrombin formulation.

BACKGROUND

Thrombin is a serine protease which serves as an active component in several hemostasis products. For example, fibrin sealants typically comprise a fibrinogen component and a thrombin component. When both components are mixed (e.g. when applied to a bleeding wound or surgical incision) thrombin cleaves fibrinogen and a fibrin polymer is formed. Concentrated purified thrombin in liquid form displays a reduction in activity during prolonged storage, primarily as a result of autolysis.

Hemostatic formulations containing liquid thrombin have special handling requirements in order to maintain thrombin's biologic activity and prevent autolytic degradation. For example, liquid thrombin requires freezing or the addition of protease inhibitors to maintain shelf-life stability. The shortcoming of the liquid thrombin solutions in use today are manifold: In the clinic and operating room, freezing is expensive and not always feasible, and promiscuous protease inhibitors may adversely affect activity of thrombin and other proteases in the hemostasis pathway once the fibrin sealant or the thrombin is applied.

Liquid thrombin preparations may be made into a lyophilized medical preparation, which is used after dissolving at the time of use. However, liquid preparations are advantageous compared with the lyophilized preparations in that they can be easily administered without the additional step of dissolving in a solvent prior to use. Also, the lyophilization step is costly and time consuming and may result in loss of yield.

Known compositions and methods for stabilizing thrombin are unsatisfactory and include the following: inclusion of various non-specific components (e.g. bulk carrier proteins such as albumins, different stabilizing sugars, general protease inhibitors etc.); formulation of the thrombin with inhibitors of thrombin activity, which although may be efficient, also inactivate or inhibit the thrombin, thereby reducing its effectiveness; and formulation of a low thrombin concentration solution, thereby necessitating administration of larger amounts of the formulation.

Several publications relate to stabilization of thrombin: for example International Patent Application Publication No. WO2008157304; U.S. Pat. Nos. 4,409,334; 7,351,561 and 8,394,372; U.S. Patent Application No. 20080311104; and European Patent Nos. EP0277096 B1 and EP 0478827 B1.

There remains a need for thrombin specific compounds useful to stabilize thrombin from autolytic degradation. Preferably, the compounds are reversible thrombin inhibitors which may be used with a concentrated liquid thrombin formulation.

SUMMARY OF THE INVENTION

Provided herein is a method for the use of thrombin binding oligonucleotides that have the exceptional ability to reversibly inhibit thrombin and stabilize its activity in an aqueous solution.

The thrombin binding oligonucleotides are such that they can bind to thrombin in a thrombin aqueous solution and inhibit, at least partially, the activity of thrombin. This inhibition of the thrombin activity by the thrombin binding oligonucleotide can be reversed by contacting the thrombin solution comprising the thrombin binding oligonucleotide with an antisense oligonucleotide. Thus, an aqueous thrombin solution can be stabilized by contacting it with the thrombin binding oligonucleotide in the absence of the antisense oligonucleotide. When the use of thrombin is desired, the activity of thrombin can be restored by contacting the stabilized thrombin solution with the antisense oligonucleotide. The reversibility of the binding of the thrombin binding oligonucleotide to thrombin by adding an antisense oligonucleotide provides an unequivocal advantage in the clinic and precludes the need to freeze and thaw a liquid thrombin solution or to reconstitute a lyophilized thrombin component prior to use.

The present inventors have shown for the first time that thrombin binding oligonucleotides are capable of reversibly inhibiting and stabilizing activity of liquid thrombin and that such molecules are useful in extending the shelf-life of thrombin.

Without wishing to be bound to theory, the thrombin binding molecules bind thrombin to inhibit, fully or partially, thrombin autolysis. Once the thrombin-binding oligonucleotide bound to thrombin comes in contact with an antisense oligonucleotide, the thrombin is released from inhibition and is capable of cleaving its heterologous substrates, including fibrinogen. The methods are further beneficial in that they are easily carried out in the clinic.

In one aspect, provided herein is a method for stabilizing thrombin activity in a solution (e.g. aqueous solution), the method includes inhibiting thrombin activity by contacting the thrombin with a thrombin binding oligonucleotide, wherein the thrombin binding oligonucleotide is capable of binding a second oligonucleotide; and wherein the inhibition of thrombin activity can be reversed by contacting the thrombin binding oligonucleotide with the second oligonucleotide. In some embodiments, the second oligonucleotide is an antisense oligonucleotide to the thrombin binding oligonucleotide.

In one aspect, provided herein is a method for stabilizing thrombin activity in a solution, the method includes inhibiting thrombin activity by contacting the thrombin with a thrombin binding oligonucleotide, wherein the thrombin binding oligonucleotide is capable of binding an antisense oligonucleotide; and wherein the inhibition of thrombin activity can be reversed by contacting the thrombin binding oligonucleotide bound to thrombin with the antisense oligonucleotide.

In some embodiments of the method, the solution comprises a thrombin concentration equal to or higher than 4 IU/ml and up to 15,000 IU/ml thrombin. In some embodiments, the thrombin concentration in solution is 10 IU/ml-1,000 IU/ml; 20 IU/ml-15,000 IU/ml; 100 IU/ml-5,000 IU/ml; 200 IU/ml-1000 IU/ml or 300 IU/ml-1000 IU/ml.

The thrombin binding oligonucleotide is selected to bind to any region of the thrombin to which binding would inhibit thrombin activity in a reversible manner; for example to inhibit thrombin activity from autolysis. In some embodiments, the thrombin binding oligonucleotide binds to exosite I or exosite II of thrombin or to both. In specific embodiments of the method, the thrombin binding oligonucleotide binds to exosite I. In some embodiments, the thrombin binding oligonucleotide is an aptamer comprising a DNA and/or RNA nucleotide sequence of about 10 to about 60 nucleotides in length, or about 12 to about 40 nucleotides in length, about 14 to about 35 nucleotides in length, or about 14 to about 25 nucleotides in length. The aptamer may bind to a consecutive linear sequence of amino acids in thrombin or may bind to a three dimensional region of thrombin. In some embodiments, the thrombin binding aptamer is a DNA oligonucleotide comprising a nucleotide sequence set forth in SEQ ID NO:1 5′ GGTTGGTGTGGTTGG 3′, a variant of SEQ ID NO:1 comprising a nucleotide sequence set forth in SEQ ID NO:2 5′ GGGTTGGGTGTGGGTTGGG 3′ or a DNA oligonucleotide comprising a nucleotide sequence set forth in SEQ ID NO:3 5′ AGTCCGTGGTAGGGCAGGTTGGGGTGACT 3′. In specific embodiments, the nucleotide sequence of the thrombin binding aptamer is set forth in SEQ ID NO:1. In some embodiments, the nucleotide sequence of the thrombin binding aptamer is set forth in SEQ ID NO:2. In some embodiments, the nucleotide sequence of the thrombin binding aptamer is set forth in SEQ ID NO:3.

In some embodiments, the thrombin binding aptamer is an RNA oligonucleotide comprising a nucleotide sequence set forth in SEQ ID NO:4 5′ GGUUGGUGUGGUUGG 3′, a variant of SEQ ID NO: 4 comprising a nucleotide sequence set forth in SEQ ID NO:5 5′GGGUUGGGUGUGGGUUGGG 3′ or an RNA oligonucleotide comprising a nucleotide sequence set forth in SEQ ID NO:6 5′ AGUCCGUGGUAGGGCAGGUUGGGGUGACU 3′.

The antisense oligonucleotide is selected to bind to the thrombin binding oligonucleotide. The antisense oligonucleotide includes a nucleotide sequence that binds to the thrombin binding oligonucleotide and may bind to a consecutive linear sequence or to a three dimensional structure of the thrombin binding oligonucleotide. In some embodiments of the method, the antisense oligonucleotide comprises a DNA and/or RNA nucleotide sequence of about 8 to about 60 nucleotides in length, or about 10 to about 40 nucleotides in length, about 12 to about 35 nucleotides in length, or about 12 to about 25 nucleotides in length. The antisense oligonucleotide may bind part of or the entire thrombin binding oligonucleotide. In some embodiments, the antisense oligonucleotide is a DNA oligonucleotide having a nucleotide sequence set forth in SEQ ID NO:7 5′ CCAACCACACCAACC 3′, SEQ ID NO:8 5′ CCCAACCCACACCCAACCC, or SEQ ID NO:9 5′ AGTCACCCCAACCTGCCCTACCACGGACT 3′. In some embodiments, the antisense oligonucleotide is an RNA oligonucleotide having a nucleotide sequence set forth in SEQ ID NO:10 5′ CCAACCACACCAACC 3′, SEQ ID NO:11 5′ CCCAACCCACACCCAACCC 3′ or SEQ ID NO:12 5′ AGUCACCCCAACCUGCCCUACCACGGACU 3′.

An antisense oligonucleotide can further be covalently or non-covalently attached to or associated with a molecule which may include one or more of a nucleotide or non-nucleotide moiety, e.g. a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide, a lipid moiety, a carbohydrate moiety, a marker, a matrix, beads or a tag, directly or using a linker

In some embodiments of the method, the solution comprises a molar ratio of less than about 10:1 to about 1:1 thrombin binding oligonucleotide:thrombin. In some embodiments, the ratio of the thrombin binding oligonucleotide:thrombin in the solution is about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about 2.5:1; or about 2:1 or about 1:1.

In some embodiments of the method, dissociation constant (Kd) between the thrombin binding oligonucleotide and the antisense oligonucleotide in solution is equal to or less than 0.2 μM (microM).

In another aspect, provided herein is a thrombin formulation for use in applications requiring conversion of fibrinogen to fibrin, the formulation comprising: thrombin and a thrombin binding oligonucleotide, wherein the thrombin binding oligonucleotide inhibits thrombin activity and is capable of binding an antisense oligonucleotide and wherein the inhibition of thrombin activity can be reversed by contacting the formulation with the antisense oligonucleotide.

In some embodiments of the formulation, thrombin is present in the formulation at a concentration equal to or higher than 4 IU/ml and up to 15,000 IU/ml thrombin. In some embodiments, the thrombin concentration in solution is 10 IU/ml-1,000 IU/ml; 20 IU/ml-15,000 IU/ml; 100 IU/ml-5,000 IU/ml; 200 IU/ml-1000 IU/ml or 300 IU/ml-1000 IU/ml.

The thrombin binding oligonucleotide is selected to bind to any region of the thrombin to which binding would reversibly inhibit and stabilize thrombin activity; for example fully or partially inhibit thrombin activity from autolysis. In some embodiments of the formulation, the thrombin binding oligonucleotide binds to exosite I or exosite II of thrombin. In specific embodiments, the thrombin binding oligonucleotide binds to exosite I. In some embodiments, the thrombin binding oligonucleotide is an aptamer comprising a nucleic acid sequence set forth in any of SEQ ID NOS:1-6. In one embodiment the thrombin binding oligonucleotide is an aptamer comprising a nucleic acid sequence set forth in SEQ ID NO:1.

In some embodiments of the formulation, the thrombin binding oligonucleotide and thrombin are present at a molar ratio of less than about 10:1 to about 1:1 thrombin binding oligonucleotide:thrombin. In some embodiments, the ratio of the thrombin binding oligonucleotide:thrombin in the formulation is about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about 2.5:1; or about 2:1 or about 1:1.

In some embodiments of the formulation, the dissociation constant (Kd) between the thrombin binding oligonucleotide and the antisense oligonucleotide in solution is equal to or less than 0.2 μM (microM).

In yet another aspect, provided herein is a kit comprising a container comprising thrombin and a thrombin binding oligonucleotide; a container comprising an antisense oligonucleotide to the thrombin binding oligonucleotide; and optionally instructions for use.

The containers can be a monolithic piece including at least two chambers separated by a septum.

In one embodiment, the chambers are divided by a septum, which is at least partially breakable, braking the septum allows mixing the oligonucleotide inhibited thrombin and the antisense oligonucleotide.

In some embodiments, the container and/or chambers are flexible and breaking the septum can be achieved by applying pressure (e.g. manual pressure) onto the container and/or chambers.

The size of each chamber and fill volumes are dependent e.g. on the intended use, suitable concentration ratios between the antisense oligonucleotides, and the thrombin binding oligonucleotide, and/or desired volume.

The container and/or chamber can comprise an opening e.g. including Male or Female Luer Lock. The opening can be resealable.

The antisense oligonucleotide can be in powder or liquid form. The kit can contain a liquid for reconstitution.

The kit can comprise a multi—e.g. dual chamber prefilled syringe, one chamber containing thrombin and a thrombin binding oligonucleotide and the other chamber containing an antisense oligonucleotide e.g. a dual chamber as described in PCT Patent Publication No. WO 97/46202A1.

In some embodiments of the kit, the thrombin binding oligonucleotide is capable of binding the antisense oligonucleotide and the binding of the thrombin binding oligonucleotide and the thrombin is reversed by contacting the thrombin binding oligonucleotide with the antisense oligonucleotide. In some embodiments, the antisense oligonucleotide is in solution. In alternative embodiments, the antisense oligonucleotide is solid phase or is linked to a solid phase. The kit may further comprise a container comprising fibrinogen. In various embodiments of the kit, the antisense oligonucleotide is excluded from the fibrinogen component.

In some embodiments of the kit, the thrombin binding oligonucleotide and thrombin are present at a molar ratio of less than about 10:1 to about 1:1 thrombin binding oligonucleotide:thrombin. In some embodiments, the ratio of the thrombin binding oligonucleotide:thrombin in the formulation is about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about 2.5:1; or about 2:1 or about 1:1.

Within one embodiment, the containers are sealed containers having a label affixed to an exterior surface thereof. In some embodiments, the formulation is prepared for use as a fibrin sealant component. The kit may further comprise one or more devices for the delivery of the thrombin-thrombin binding oligonucleotide; antisense oligonucleotide and/or the fibrinogen components.

In another aspect, provided is a method for converting fibrinogen to fibrin comprising:

mixing a formulation comprising: thrombin and a thrombin binding oligonucleotide, wherein the thrombin binding oligonucleotide is capable of inhibiting thrombin activity and of binding an antisense oligonucleotide, and wherein the inhibition of thrombin activity is reversed by the addition of the antisense oligonucleotide; the antisense oligonucleotide and fibrinogen.

In some embodiments of the method, the ratio between the antisense oligonucleotide and the thrombin binding oligonucleotide is in the range of about 1:1 to 2:1.

In some embodiments of the method, the thrombin binding oligonucleotide and thrombin are present at a molar ratio of less than about 10:1 to about 1:1 thrombin binding oligonucleotide:thrombin. In some embodiments, the ratio of the thrombin binding oligonucleotide:thrombin in the formulation is about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about 2.5:1; or about 2:1 or about 1:1. Fibrinogen component can be prepared as described in the art, for example, in PCT Patent Publication No. WO 93/05822 or as in the fibrin kit described in the European pharmacopeia. In some embodiments, the fibrinogen component is free of or is depleted of plasmin(ogen) as disclosed in U.S. Pat. No. 7,641,918 or in PCT Patent Publication No. WO 02/095019.

In some embodiments of the method, the nucleic acid sequence of the thrombin binding oligonucleotide is set forth in any of SEQ ID NOS:1-6, preferably SEQ ID NO:1.

In yet another aspect, provided herein is an antisense oligonucleotide of a thrombin binding oligonucleotide for use in reversing a binding between the thrombin binding oligonucleotide and thrombin in applications requiring converting fibrinogen to fibrin.

In some embodiments, the antisense oligonucleotide comprises any of SEQ ID NOS:7-12. Further provided is a composition comprising the antisense oligonucleotide disclosed herein; and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of screening for oligonucleotides capable of reversibly binding and stabilizing thrombin activity and of binding an antisense oligonucleotide. Accordingly, provided is a method for screening for an oligonucleotide capable of reversibly binding and stabilizing the activity of thrombin in an aqueous liquid thrombin formulation, comprisinga. contacting thrombin or a fragment thereof, each exhibiting an initial activity of 4 to 15,000 IU/ml, with a set of test thrombin binding oligonucleotides; and identifying one or more thrombin binding oligonucleotides which inhibit, at least partially, the initial activity; andb. contacting the thrombin bound oligonucleotide of step a) with a set of test antisense oligonucleotides;
whereby restoration of at least 4 IU/ml thrombin activity following step (b) indicates 1) a potential thrombin binding oligonucleotide for thrombin stabilization and 2) a potential antisense oligonucleotide to reverse the inhibitory effect of the thrombin binding oligonucleotide.

In some embodiments of the screening method, the thrombin has an initial activity of 4 to 15,000 IU/ml, about 20 IU/ml to 15,000 IU/ml, or 100 IU/ml to 5,000 IU/ml, 200 IU/ml to about 1000 IU/ml or about 300 IU/ml to about 1000 IU/ml.

In some embodiments the method provides an antisense oligonucleotide that restores thrombin activity to at least 4 IU/ml, at least about 20 IU/ml, at least about 100 IU/ml or at least about 300 IU/ml, at least about 1000 IU/ml and up to 1500 IU/ml of the initial activity of thrombin.

In an alternative, provided is a method for screening for an oligonucleotide capable of reversibly binding and stabilizing the activity of thrombin in an aqueous liquid thrombin formulation, comprisinga. contacting thrombin or a fragment thereof, each exhibiting an initial activity, with a set of test thrombin binding oligonucleotides; and identifying one or more thrombin binding oligonucleotides which inhibit, at least 60% of the initial activity; andb. contacting a thrombin bound oligonucleotide of step a) with a set of test antisense oligonucleotides;
whereby restoration of more than 40% of the initial activity following step (b) indicates 1) a potential thrombin binding oligonucleotide for thrombin stabilization and 2) a potential antisense oligonucleotide to reverse the inhibitory effect of the thrombin binding oligonucleotide.

In some embodiments, the method provides stabilizing thrombin activity and restoring at least, 40%, 50% and up to 100% of the initial activity of thrombin.

In another aspect, provided herein is an antisense oligonucleotide of a thrombin binding oligonucleotide for use in reversing thrombin inhibition by thrombin binding oligonucleotide in applications requiring converting fibrinogen to fibrin.

In some embodiments, the antisense oligonucleotide is bound to a solid phase.

In another aspect, provided herein is a method for reversing an oligonucleotide inhibited thrombin, the method comprising the steps of contacting the oligonucleotide inhibited thrombin with an antisense to the oligonucleotide. In one embodiment, the antisense to the oligonucleotide is immobilized on a solid phase.

In some embodiments, the antisense is bound to the solid phase directly or indirectly.

In some embodiments, the solid phase is selected from the group consisting of chromatographic media beads and filters.

In all aspects herein, the thrombin binding oligonucleotides are not used as anticoagulants.

In another aspect, the invention relates to a delivery applicator comprising:

a barrel holding an oligonucleotide inhibited thrombin; and

a vessel having a delivery opening and holding an antisense oligonucleotide linked to a solid phase;

wherein the barrel and the vessel are capable of being in fluid communication, so that after fluid communication and contact of the inhibited thrombin with the solid phase, thrombin activity is increased before delivering thrombin through the delivery opening.

In some embodiments, the fluid communication is via a re-sealable opening positioned between the barrel and the vessel.

In some embodiments, the delivery applicator further comprises a barrel having a delivery opening and holding fibrinogen.

In another aspect, the invention relates to a delivery applicator comprising:

a barrel holding an oligonucleotide inhibited thrombin; and

a vessel having a delivery opening and holding an antisense oligonucleotide;

wherein the barrel and the vessel are capable of being in fluid communication, so that after fluid communication and contact of the inhibited thrombin with the antisense, thrombin activity is increased before delivering thrombin through the delivery opening.

In another aspect, the invention relates to a delivery applicator comprising:

a barrel holding an oligonucleotide inhibited thrombin; and an antisense oligonucleotide, wherein the oligonucleotide inhibited thrombin and the antisense oligonucleotide are held in separate chambers that are capable of being in fluid communication, so that allowing fluid communication between the chambers and contact between the inhibited thrombin and the antisense results in increased thrombin activity before delivery, and wherein the barrel has an opening for delivery therethrough of the thrombin.

In another aspect, the invention relates to a delivery applicator comprising:

a container holding an oligonucleotide inhibited thrombin; and an antisense oligonucleotide linked to a solid phase, wherein the oligonucleotide inhibited thrombin and the antisense oligonucleotide are held in separate chambers that are capable of being in fluid communication, so that fluid communication between the chambers and contact between the inhibited thrombin and the solid phase results in increased thrombin activity before delivery, and wherein the container has a re-sealable opening for delivery therethrough of the thrombin.

Re-sealable opening is an opening sealed by e.g. a membrane, cap, needle, rubber cap, stent, delivery tube and/or tip.

In one embodiment, a re-sealable opening prevents or minimizes leakage of liquid.

In some embodiments, the delivery applicator further comprises a container having a delivery opening and holding fibrinogen.

In some embodiments, the inhibited thrombin, fibrinogen and/or the formulations can be in solid, dry, aqueous, and/or frozen form. The solid phase can be dry or suspended in liquid, e.g. buffers.

In some embodiments the kits, device, containers, chambers, vessel, delivery application, barrel and/or syringe etc. may comprise an aqueous liquid for reconstitution.

An oligonucleotide inhibited thrombin is a thrombin combined with an oligonucleotide, said oligonucleotide can bind to thrombin in a thrombin aqueous solution, and thrombin activity is inhibited by the oligonucleotide, at least partially.

These and other aspects and embodiments of the invention will become evident upon reference to the following detailed description of the invention and the figures.

