Aptamers that bind thrombin with high affinity

Abstract

The invention provides aptamers capable of binding to thrombin useful as therapeutics for and diagnostics of coagulation related disorders and/or other diseases or disorders in which thrombin has been implicated. The invention further provides materials and methods for the administration of aptamers capable of binding to thrombin.

1 . An aptamer that binds to a thrombin target, wherein the aptamer decreases or inhibits thrombin mediated coagulation and the aptamer is ARC2172 (SEQ ID NO 294) or an aptamer that has substantially the same ability as ARC2172 (SEQ ID NO 294) to decrease or inhibit thrombin mediated coagulation, wherein the aptamer binds to human thrombin with a K D  of less than 1 nM and wherein the aptamer is 55 nucleotides or less in length. 
     
     
         2 . (canceled) 
     
     
         3 . The aptamer of  claim 1  wherein the aptamer's ability to decrease or inhibit thrombin mediated coagulation is assessed by measuring the aptamer's ability to decrease or inhibit activated clotting time or prothrombin time. 
     
     
         4 . The aptamer of  claim 1 , wherein the aptamer decreases or inhibits thrombin mediated coagulation in vivo. 
     
     
         5 - 8 . (canceled) 
     
     
         9 . An aptamer comprising the following nucleic acid sequence
 N 1 N 2 N 3 TAGGTTGGGTAGGGTGGTN′ 3 N′ 2 N′ 1  (SEQ ID NO 442)   Wherein N 1 , N 2, or  N 3  is any nucleotide that forms a base pairs with N′ 1 , N′ 2 or  N′ 3  respectively, wherein N 1 , N 2 , and N 3  may each be the same nucleotide or different nucleotides and the aptamer decreases or inhibits thrombin mediated coagulation.   
     
     
         10 . The aptamer of  claim 9 , wherein N 1 , N 2, or  N 3  are deoxy nucleotides. 
     
     
         11 . The aptamer of  claim 9 , wherein at least two of N 1 , N 2, or  N 3  comprise a 2′ OMe modification. 
     
     
         12 . The aptamer of  claim 9  further comprising the sequence N 1 N 2 N 3 N 4 N 5 N 6 TAGGTTGGGTAGGGTGGTN′ 6 N′ 5 N′ 4  N′ 3 N′ 2 N′ 1  (SEQ ID NO 445)
 Wherein N 1 , N 2 , N 3 , N 4 , N 5 , or N 6  is any nucleotide that forms a base pairs with N′ 1 , N′ 2 , N′ 3 , N′ 4 , N′ 5 , or N′ 6  respectively, wherein N 1 , N 2 , N 3 , N 4 , N 5 , or N 6  may each be the same nucleotide or different nucleotides and the aptamer decreases or inhibits thrombin mediated coagulation. 
 
     
     
         13 . The aptamer of  claim 9  wherein N is a guanosine or cytidine nucleotide residue. 
     
     
         14 . The aptamer of  claim 9 , wherein the aptamer binds to thrombin with a K D  of less than 1 nM. 
     
     
         15 . The aptamer of  claim 9 , wherein the aptamer has at least substantially the same ability as ARC2172 (SEQ ID NO 294) to decrease or inhibit thrombin mediated coagulation. 
     
     
         16 . (canceled) 
     
     
         17 . The aptamer of  claim 9 , wherein the aptamer is deoxyribonucleic acid. 
     
     
         18 . The aptamer of  claim 9 , wherein the aptamer is single stranded deoxyribonucleic acid. 
     
     
         19 . The aptamer of  claim 9 , wherein the aptamer comprises at least one chemical modification. 
     
     
         20 . The aptamer of  claim 19 , wherein the modification is selected from the group consisting: of a chemical substitution at a sugar position; a chemical substitution at a phosphate position, and a chemical substitution at a base position, of the nucleic acid. 
     
     
         21 . The aptamer of  claim 20 , wherein the modification is selected from the group consisting of: incorporation of a modified nucleotide, 3′ capping, and conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound. 
     
     
         22 . The aptamer of  claim 19 , wherein the modification is conjugation to a non-immunogenic, high molecular weight compound and wherein the compound is polyalkylene glycol. 
     
     
         23 . The aptamer of  claim 22  wherein the polyalkylene glycol is polyethylene glycol. 
     
     
         24 . (canceled) 
     
     
         25 . A method comprising administering the aptamer of  claim 9  to a subject or an extracorporeal circuit in an amount effective to decrease or inhibit thrombin mediated coagulation in the subject. 
     
     
         26 . The method of  claim 25 , wherein the subject is human. 
     
     
         27 - 29 . (canceled) 
     
     
         30 . The method according to  claim 25 , wherein the human subject is renally impaired and wherein the aptamer for use in the method is not conjugated to a PEG. 
     
     
         31 . The method according to  claim 25 , wherein the human subject has heparin induced thrombocytopenia. 
     
     
         32 . The method according to  claim 25 , wherein the human subject is heparin resistant. 
     
     
         33 . The method according to  claim 25 , wherein the subject has impaired hepatic function. 
     
     
         34 . The method according to  claim 25 , wherein the aptamer is administered to the subject before, during, after or any combination thereof, a surgical procedure on the subject. 
     
     
         35 - 39 . (canceled)