Nucleic acid modulators of glycoprotein vi

Abstract

The present invention relates, in general, to a pharmacologic system to modulate the biology of platelets based upon a nucleic acid ligand that can interact with and modulate the activity of platelet glycoprotein GPVI to regulate platelet function. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize this pharmacologic effect and thereby restore GPVI function, including collagen binding, platelet adhesion, collagen-induced platelet activation, and collagen-induced platelet aggregation. The invention further relates to compositions comprising the nucleic acid ligand, the ligand and a modulator, methods to generate the nucleic acid ligand and its modulator, as well as methods of using these agents and compositions in medical therapeutic and diagnostic procedures.

1 . A GPVI ligand comprising a first isolated nucleic acid sequence, wherein said GPVI ligand binds glycoprotein VI (GPVI) and wherein said GPVI ligand comprises a secondary structure comprising in a 5′ to 3′ direction a first stem, a first loop, a second stem, a second loop, a third loop, a third stem and a fourth loop; and wherein said fourth loop comprises UAA. 
     
     
         2 . The GPVI ligand of  claim 1 , wherein the second loop is substituted with a spacer. 
     
     
         3 . The GPVI ligand of  claim 1 , wherein the first nucleic acid comprises at least one modified nucleotide. 
     
     
         4 . The GPVI ligand of  claim 1 , conjugated to a carrier. 
     
     
         5 . The GPVI ligand of  claim 4 , wherein the carrier is a hydrophilic moiety. 
     
     
         6 . The GPVI ligand of  claim 5 , wherein the hydrophilic moiety is a polyethylene glycol (PEG) molecule. 
     
     
         7 . The GPVI ligand of  claim 1 , comprising a modified phosphate backbone. 
     
     
         8 . The GPVI ligand of  claim 1 , comprising SEQ ID NO:69, wherein a spacer is incorporated between the 2′O-Methyl G at position 11 and the 2′O-Methyl C at position 12 of SEQ ID NO:69. 
     
     
         9 . The GPVI ligand of  claim 8 , comprising a modified phosphate backbone. 
     
     
         10 . The GPVI ligand of  claim 8 , further comprising a polyethylene glycol moiety. 
     
     
         11 . The GPVI ligand of  claim 8 , wherein said GPVI ligand is selected from the group consisting of RB569, RB570, and RB571. 
     
     
         12 . A pharmaceutical composition comprising the GPVI ligand of  claim 1 . 
     
     
         13 . A modulator which binds specifically to a GPVI ligand,
 wherein said modulator comprises a second nucleic acid sequence; and   wherein said GPVI ligand binds glycoprotein VI (GPVI); and   wherein said GPVI ligand comprises a secondary structure comprising in a 5′ to 3′ direction a first stem, a first loop, a second stem, a second loop, a third loop, a third stem and a fourth loop; and wherein said fourth loop comprises UAA.   
     
     
         14 . The modulator of  claim 13 , wherein the second nucleic acid sequence consists of about 10 nt to about 50 nt. 
     
     
         15 . A pharmaceutical composition comprising the modulator of  claim 13 . 
     
     
         16 . A modulator comprising SEQ ID NO:84. 
     
     
         17 . The modulator of  claim 16 , wherein said modulator is RB515. 
     
     
         18 . A pharmaceutical composition comprising the modulator of  claim 13 . 
     
     
         19 . A method for treating a platelet-mediated disorder comprising, administering to a host in need thereof a therapeutically effective amount of a GPVI ligand. 
     
     
         20 . The method of  claim 19 , wherein the platelet-mediated disorder is selected from the group consisting of a vascular disorder, a cerebrovascular disorder, a platelet-mediated inflammatory disorder, a diabetes-related disorder, or a cancer. 
     
     
         21 . The method of  claim 20 , wherein the vascular disorder is selected from the group consisting of acute coronary syndromes, thrombosis, thromboembolism, peripheral vascular disease, and transient ischemic attack 
     
     
         22 . The method of  claim 20 , wherein the cerebrovascular disorder is selected from the group consisting of transient ischemic attack, ischemic stroke, and embolism. 
     
     
         23 . The method of  claim 20 , wherein the platelet-mediated inflammatory disorder selected from the group consisting of arthritis, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, inflammatory bowed disease, ankylosing spondylitis, and scleroderma. 
     
     
         24 . The method of  claim 20 , wherein the diabetes-related disorder is selected from the group consisting of diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injury and chronic renal failure. 
     
     
         25 . The method of  claim 20 , wherein the cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, bone cancer and liver cancer. 
     
     
         26 . A method for modulating platelet function in a host in need thereof comprising administering to the host an effective amount of a GPVI ligand. 
     
     
         27 . The method of  claim 26 , wherein the host is undergoing a cardiac intervention. 
     
     
         28 . The method of  claim 19 , further comprising administering to the host a modulator, wherein said modulator comprises a second nucleic acid sequence, and wherein said modulated specifically binds the GPVI ligand. 
     
     
         29 . The method of  claim 26 , further comprising administering to the host a modulator, wherein said modulator comprises a second nucleic acid sequence, and wherein said modulated specifically binds the GPVI ligand.