WO2010091396A2 - Aptamères dirigés contre le facteur de von willerbrand et leur utilisation en tant que produits thérapeutiques pour des maladies thrombotiques, hématologiques et cardiovasculaires - Google Patents

Aptamères dirigés contre le facteur de von willerbrand et leur utilisation en tant que produits thérapeutiques pour des maladies thrombotiques, hématologiques et cardiovasculaires Download PDF

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WO2010091396A2
WO2010091396A2 PCT/US2010/023599 US2010023599W WO2010091396A2 WO 2010091396 A2 WO2010091396 A2 WO 2010091396A2 US 2010023599 W US2010023599 W US 2010023599W WO 2010091396 A2 WO2010091396 A2 WO 2010091396A2
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vwf
aptamer
aptamers
ome
seq
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WO2010091396A3 (fr
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Kathleen Mcginness
John L. Diener
Robert G. Schaub
Kristin Thompson
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Archemix Corp
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Archemix Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • the invention relates generally to the field of nucleic acids and more particularly to aptamers that bind to von Willebrand Factor (vWF), which are useful as therapeutics in and diagnostics of cardiovascular diseases, thrombotic diseases, hematologic diseases and/or other diseases or disorders in which vWF has been implicated.
  • vWF von Willebrand Factor
  • the aptamers may be used before, during and/or after medical procedures in order to reduce complications or side effects thereof.
  • the invention also relates to materials and methods for the administration of aptamers that bind to vWF.
  • the invention further relates to agents that reverse the effects of such aptamers.
  • An aptamer is an isolated or otherwise purified nucleic acid molecule that binds with high specificity and affinity to a target through interactions other than Watson- Crick base pairing. Unlike nucleic acid molecules, such as genes and mRNA, aptamer function, which is based upon the specific binding to a target molecule, is not dependent upon a conserved linear base sequence, but rather a particular secondary or tertiary structure. That is, aptamers are non-coding sequences. Any coding potential that an aptamer may possess is entirely fortuitous and plays no role whatsoever in the binding of an aptamer to its cognate target. Thus, while it may be that aptamers that bind to the same target, and even to the same site on that target, share a similar linear base sequence, most do not.
  • An aptamer has a three dimensional structure held in a fixed conformation that provides chemical contacts to specifically bind its given target.
  • Aptamers are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g., through binding, aptamers may inhibit or stimulate their target's ability to function.
  • This specific binding to a target is an inherent property of aptamers.
  • Functional activity i.e., inhibiting or stimulating a target's function, is not an inherent property of aptamers.
  • an aptamer may bind to a target and have little or no effect on the desired function or any other function of the target.
  • a typical aptamer is 10-15 kDa in size (20-45 nucleotides), binds to its target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family).
  • Aptamers have been generated to many targets, such as small molecules, carbohydrates, peptides and proteins, including growth factors, transcription factors, enzymes, immunoglobulins and receptors.
  • Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics, including high specificity and affinity, biological efficacy and excellent pharmacokinetic properties.
  • Cardiovascular disease is the leading cause of death in the United States.
  • Cardiovascular disease is a broad class of diseases that includes many different subsets of diseases.
  • One such subset of cardiovascular diseases is thrombotic diseases.
  • Thrombotic diseases or disorders are characterized by abnormal thrombus formation, which may lead to life-threatening events, such as heart attacks and strokes. Examples of diseases or disorders that involve abnormal thrombus formation include acute coronary syndrome (ACS); thrombotic microangiopathies (TMA); thrombotic thrombocytopenic purpura (TTP); von Willebrand's Disease, such as von Willebrand's Disease - type 2b (vWD-2b); and atherothrombosis, such as transient ischemic attack (TIA).
  • Another such subset of cardiovascular diseases is hematologic diseases. Hematologic diseases or disorders are characterized by abnormal blood or blood producing organs. Examples of hematologic diseases or disorders include anemia, deep vein thrombosis, thrombotic thrombocytopenic purpura and von Willebrand's Disease.
  • Acute Coronary Syndrome affects approximately 2 million people in the United States and includes the two forms of heart attack, Non-ST Segment Elevation Myocardial Infarction, or NSTEMI, and ST Segment Elevation Myocardial Infarction, or STEMI.
  • a heart attack is caused by an arterial blockage or thrombus that reduces blood flow to the heart muscle.
  • NSTEMI and STEMI patients are often managed with percutaneous coronary intervention (PCI), or angioplasty, a medical procedure that mechanically opens narrowed or clogged arteries to restore normal blood flow in the arteries.
  • PCI percutaneous coronary intervention
  • angioplasty a medical procedure that mechanically opens narrowed or clogged arteries to restore normal blood flow in the arteries.
  • Heart attack patients undergoing PCI receive a regimen of drugs known as anti-thrombotics, which prevent the formation of additional harmful blood clots in their arteries during the procedure.
  • This combination of anti-thrombotic drugs generally includes an anti-coagulant agent and an anti-platelet agent.
  • GPIIb/IIIa antagonists which target and bind to a site on platelets known as the GPIIb/IIIa receptor. By binding to the GPIIb/IIIa receptor, these drugs prevent platelets from aggregating and forming a thrombus.
  • GPIIb/IIIa antagonists include the approved drugs REOPRO ® and INTEGRILIN ® .
  • GPIIb/IIIa antagonists have the following limitations:
  • GPIIb/IIIa antagonists suppress platelet function independent of shear force, these drugs are active in the veins and arteries throughout the body, which is beyond the region of the primary blood clot. Accordingly, there is an increased risk of significant bleeding in the systemic circulation in patients receiving GPIIb/IIIa antagonists.
  • Thrombotic microangiopathy describes syndromes of microangiopathic hemolytic anemia, thrombocytopenia and variable signs of organ impairment due to platelet aggregation in the microcirculation.
  • TMA include thrombotic thrombocytopenic purpura and hemolytic uremic syndrome.
  • Hemolytic uremic syndrome HUS
  • HUS Hemolytic uremic syndrome
  • TTP thrombotic thrombocytopenic purpura
  • TTP refers to adult cases of TMA with predominant neurological abnormalities.
  • Examples of TTP include familial TTP, secondary TTP, idiopathic TTP, and congenital TTP (which is also known as hereditary TTP or Upshaw-Schulman syndrome). There is no approved drug treatment for patients with TMA or TPP.
  • Type 2b von Willebrand's Disease (vWD-2b) is an inherited disorder that is characterized by defective von Willebrand Factor (vWF).
  • the disease category atherothrombosis includes transient ischemic attack, stroke and myocardial infarction.
  • High risk atherothrombosis patients are those who have suffered a transient ischemic attack, or TIA, which is a temporary blockage of a cranial artery.
  • TIA transient ischemic attack
  • mini-strokes TIA's are transient and usually do not inflict permanent damage, but are often a pre-cursor for a stroke.
  • vWF plays a role in thrombosis, hemostasis and disease.
  • platelets do not adhere to healthy vessels, rather platelets typically adhere to the subendothelium of injured vessels. Platelet adhesion triggers a series of platelet activation processes that ultimately result in thrombus formation and cessation of bleeding, von Willebrand Factor is a mediator of platelet adhesion at sites of vascular damage.
  • vWF is a large multi-subunit, multimeric soluble factor mainly produced by vascular endothelial cells. The von Willebrand Factor becomes immobilized on the blood vessel wall via interactions between von Willebrand Factor domain A3 and exposed collagen.
  • GPIb platelet-receptor glycoprotein Ib
  • the Al domain of the immobilized von Willebrand Factor facilitates the adhesion, activation and aggregation of platelets at sites of vascular injury.
  • GPIb platelet-receptor glycoprotein Ib
  • von Willebrand factor is pro-thrombotic, playing an important role during hemostasis in facilitating thrombus formation at sites of vascular injury.
  • von Willebrand Factor by the same mechanism, also plays a key role in pathological conditions, such as cardiovascular diseases, involving platelet adhesion, activation and aggregation, and thrombosis formation.
  • cardiovascular diseases involving platelet adhesion, activation and aggregation, and thrombosis formation.
  • antithrombotic therapies are currently available, there is still a large unmet need for additional therapies.
  • the American Heart Association estimates that more than 60 million people in the United States alone have one or more forms of cardiovascular disease, and that a high proportion of people with cardiovascular disease are at higher risk for arterial thrombosis. S.P. Jackson and S.M. Schoenwaelder, Nature Reviews, 2, 1-12 (2003).
  • vWF is involved in platelet adhesion, activation and aggregation, and plays a pivotal role in hemostasis and in the formation of blood clots.
  • vWF is activated by means of a physical deformation that exposes its Al domain and enables binding to the platelet GPIb receptor (Siedlecki et al, Blood, 88 (8):2939-50 (1996)).
  • Activated vWF binds to cellular elements in the blood known as platelets, which play a key role in the normal process of blood clotting.
  • vWF captures platelets from the flowing bloodstream, causing the platelets to adhere to the blood vessel wall.
  • vWF This adhesive interaction between vWF and platelets activates the bound platelets and causes them to recruit additional platelets from the bloodstream. These recruited platelets aggregate on the blood vessel wall and form the beginning of a blood clot. As the primary blood clot grows and shear force within the artery is further increased, more vWF is activated, enabling the formation of new clots. These new clots break off and lodge in the smaller, distal vessels of the heart known as the micro vasculature, where they may join other clots that have formed in response to local activation of vWF. Together with the primary clot, these smaller clots restrict the normal process of delivering, or perfusing, blood to the working heart muscle, or myocardium, causing a heart attack.
  • vWF-dependent platelet adhesion, activation and aggregation can also occur at sites of vascular injury and endothelial denudation, where exposed and activated vWF can promote thrombogenesis (Blann, Thromb Haemost, 95 (l):49-55 (2006)).
  • Shear stress in conduit arteries can be elevated at the sites of stenosis (Mailhac A, et al, Circulation, 90 (2):988-96 (1994); Siegel JM, et al, JBiomech Eng, 116 (4):446-51 (1994); and Strony J, et al, Am J Physiol, 265 (5 Pt 2):H1787-96 (1993)), leading to activation of vWF, triggering vWF-platelet binding, and generating pro-coagulant platelet-derived microparticles (Jackson SP, et al., Blood, 107 (9):3418-9 (2006); Reininger AJ, et al, Blood, 107 (9):3537-45 (2006)).
  • Circulating plasma levels of vWF are chronically elevated in the clinical setting of endothelial dysfunction and atherosclerosis (Blann AD, Pathophysiol Haemost Thromb, 33 (5-6):256-61 (2003); Paramo JA, et al, J Thromb Haemost, 3 (4):662-4 (2005)), and acute elevations are observed in acute coronary syndromes (Collet JP, et al, Circulation, 108 (4):391-4 (2003); Lee et al, Blood, 105 (2):526-32 (2005); Montalescot G, et al, Circulation, 98 (4):294-9 (1998); and Ray KK, et al, Eur Heart J, 26 (5):440-6 (2005)).
  • Elevated vWF is considered to be both a prognostic marker and a pathophysiologic mediator of adverse outcomes in heart disease (Becker, Eur Heart J, 26 (5):421-2 (2005)), and normalization of vWF activity levels may represent a new therapeutic paradigm in cardiovascular medicine.
  • vWF antagonism offers an improved risk-to-benefit ratio in comparison to GPIIb/IIIa receptor antagonism for use in the management of ACS and in conjunction with percutaneous coronary intervention (PCI) (De Meyer et ah, Cardiovasc Hematol Disord Drug Targets, 6 (3): 191-207 (2006); Vanhoorelbeke K, et ah, Curr Drug Targets Cardiovasc Haematol Disord, 3 (2): 125-40 (2003)).
  • PCI percutaneous coronary intervention
  • a monoclonal antibody antagonist of vWF was found to inhibit thrombosis without inducing bleeding in pre-clinical studies (Eto K, et al., Arterioscler Thromb Vase Biol, 19 (4):877-82 (1999); Kageyama S, et al, Br J Pharmacol, 122 (1): 165-71 (1997); Kageyama et ah, Arterioscler Thromb Vase Biol, 22 (1): 187-92 (2002); Kageyama et ah, Thromb Res, 101 (5):395-404(2001); Kageyama et ah, Arterioscler Thromb Vase Biol, 20 (10):2303-8 (2000); and Yamamoto, Thromb Haemost, 79 (l):202-10 (1998)) and in a healthy volunteer human study (Machin SJ., J Thrombosis Haemostasis, Supplement 1:OC328 (2006)).
  • Thrombotic thrombocytopenic purpura is a rare blood disorder caused by elevated levels of activated von Willebrand Factor (vWF) in the blood that is due to a deficiency of the enzyme responsible for vWF degradation.
  • This enzyme known as ADAMTS 13
  • ADAMTS 13 is responsible for vWF degradation, which is necessary to maintain the normal balance between bleeding and clotting.
  • the inherited form is caused by mutations in the ADAMTS 13 gene that impair the normal function of the enzyme. Patients with the acquired form of TTP do not have mutations in this gene, but instead produce antibodies that block the activity of the ADAMTS 13 enzyme.
  • a deficiency of ADAMTS 13 or the absence of this enzyme results in excessive levels of activated vWF that cause platelet aggregation, resulting in widespread blood clotting, which can lead to life-threatening events, such as heart attack and stroke.
  • TTP ulcerative colitis
  • platelets bind together abnormally and adhere to the walls of blood vessels, forming clots throughout the body. As these clots grow in size and multiply, they restrict blood flow to critical organs, such as the brain, kidneys and heart, potentially causing stroke, seizure, kidney failure or heart attack. These events trigger acute episodes of disease resulting in hospitalization.
  • TTP is a syndrome that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, fever and renal dysfunction.
  • Type 2b von Willebrand's Disease is characterized by excessive platelet binding, which is caused by constitutively active vWF.
  • vWF constitutively active vWF.
  • Atherothrombosis is a result of elevated and activated vWF. This is due to the fact that endothelial injury and shear forces in atherosclerotic arteries lead to vWF secretion and activation, which promotes platelet adhesion, activation and aggregation. Medical Procedures
  • the aptamers may be administered in conjunction (before, during and/or after) with medical procedures in order to reduce complications or side effects of the procedures.
  • medical procedures are carotid endarterectomy, carotid stenting and dialysis.
  • Carotid endarterectomy is an operation or surgical procedure during which a vascular surgeon removes the inner lining of the carotid artery. This procedure removes plaque from the artery and restores blood flow. Blocking vWF is thought to reduce ischemia with a decreased risk of bleeding.
  • the present invention provides aptamers that bind to von Willebrand Factor
  • vWF vWF
  • vWF aptamers vWF aptamers
  • methods for using such aptamers in the treatment of cardiovascular diseases, thrombotic diseases, hematologic diseases and other vWF-mediated diseases or disorders may be used before, during and/or after medical procedures in order to reduce complications or side effects thereof.
  • the vWF aptamers modulate vWF-mediated platelet adhesion, activation and/or aggregation.
  • vWF aptamers include, but are not limited to, aptamers that comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, which is ARC13090; SEQ ID NO: 2, which is ARC14303; SEQ ID NO: 3, which is ARC15103; SEQ ID NO: 4, which is ARC15104; and SEQ ID NO: 5, which is ARC15105.
  • the vWF aptamers bind specifically to vWF or a fragment thereof.
  • the vWF is human vWF.
  • the fragment is the Al domain.
  • the vWF aptamer has a dissociation constant for vWF of 100 nM or less.
  • the vWF aptamers may comprise at least one chemical modification.
  • 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.
  • the modification is selected from the group consisting of: incorporation of a modified nucleotide; 3' capping; 5' capping; conjugation to a high molecular weight, non-immunogenic compound; and conjugation to a lipophilic compound.
  • the high molecular weight, non-immunogenic, compound is preferably polyethylene glycol.
  • the 3' cap is preferably an inverted deoxythymidine cap.
  • the vWF aptamer is an aptamer or a salt thereof comprising the structure set forth below: mGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmC-3T (SEQ ID NO: 1
  • the vWF aptamer is an aptamer or a salt thereof comprising the structure set forth below: biotin-mGmGmGmAmCmCmUmAmAmGmAmCmAmUmGmUmCmCmC-3T
  • the vWF aptamer is an aptamer or a salt thereof comprising the structure set forth below:
  • the vWF aptamer is an aptamer or a salt thereof comprising the structure set forth below: PEG20K-NH- mGmGmGmAmCmCmUmAmAmGmAmCmAmUmGmUmCmCmC-3T (SEQ ID NO: 4) (ARCl 5104), where "NH” is a 5'-hexylamine linker phosphoramidite, "3T” is an inverted deoxythymidine, "mN” is a 2'-0 Methyl containing residue and "PEG” is a polyethylene glycol.
  • the vWF aptamer is an aptamer or a salt thereof comprising the structure set forth below: PEG40K-NH- mGmGmGmAmCmCmUrriArriAmGrriAmCrriAmCrriAmUmGmUmCmC-3T (SEQ ID NO: 5) (ARCl 5105), where "NH” is a 5'-hexylamine linker phosphoramidite, "3T” is an inverted deoxythymidine, "mN” is a 2'-0 Methyl containing residue and "PEG” is a polyethylene glycol.
  • the invention also provides pharmaceutical compositions comprising a therapeutically effective amount of a vWF aptamer or a salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • the invention further provides a method for treating, preventing or ameliorating a disease or disorder mediated by vWF by administering to a subject the above pharmaceutical compositions.
  • the subject is a mammal. More preferably, the subject is a human.
  • the disease or disorder is selected from the group consisting of: essential thrombocytopenia, thrombotic microangiopathies (TMA), hemolytic uremic syndrome, thrombotic thrombocytopenic purpura (TTP), familial TTP, secondary TTP, idiopathic TTP, congenital TTP, Type 2b von Willebrand's Disease, pseudo type 2b von Willebrand's Disease, peripheral artery disease, peripheral arterial occlusive disease, unstable angina, angina pectoris, arterial thrombosis, atherosclerosis, ischemia, myocardial infarction, acute coronary syndrome (ACS), atrial fibrillation, carotid stenosis, unstable carotid disease, unstable carotid lesions, cerebral infarction, cerebral thrombosis, stroke, ischemic stroke, and transient cerebral ischemic attack.
  • TMA thrombotic microangiopathies
  • TTP thrombotic thrombocytopenic purpura
  • TTP thrombo
  • the pharmaceutical compositions may also be administered prior to, during and/or after a medical procedure.
  • the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as carotid endarterectomy, carotid stenting, carotid angioplasty (with or without stenting), medical management of a patient with symptomatic or asymptomatic carotid stenosis, CABG surgery, percutaneous coronary intervention and heart valve replacement.
  • the pharmaceutical compositions may also be administered in conjunction (before, during and/or after) with other medical procedures, such as dialysis or hemodialysis.
  • the pharmaceutical compositions may be administered in combination with another drug.
  • the pharmaceutical compositions may be administered in combination with another therapy.
  • the pharmaceutical compositions may be administered simultaneously, concurrently or sequentially with the other drug and/or therapy.
  • the vWF aptamers may be used for identification of the vWF protein.
  • the aptamers of the invention specifically bind the vWF protein or a portion thereof.
  • K D equilibrium binding constant
  • the invention also provides aptamers that have substantially the same ability to bind vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • aptamers according to the invention have substantially the same structure as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • aptamers according to the invention have substantially the same ability to bind vWF and substantially the same structure as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention also provides aptamers that have substantially the same ability to bind and modulate a biological function of vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention further provides aptamers that bind to vWF, wherein the aptamer modulates platelet adhesion, activation and/or aggregation as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention further provides for the use of a vWF aptamer in the manufacture of a pharmaceutical composition or medicament for use in the treatment, prevention and/or amelioration of a cardiovascular, thrombotic or hematologic disease.
  • ARC13090, ARC14303, ARC15103, ARC15104 and ARC15105 are used in the manufacture of a pharmaceutical composition or medicament for treating, preventing or otherwise ameliorating a cardiovascular, thrombotic or hematologic disease.
  • vWF reversal agents agents that reverse the effects of the vWF aptamers
  • a vWF reversal agent is 10-15 nucleotides in length.
  • a vWF reversal agent binds to a vWF aptamer.
  • binding is via complementary base pairing.
  • a vWF reversal agent acts by hybridizing to a vWF aptamer, thereby disrupting the vWF aptamer' s structure and preventing the binding of the vWF aptamer to vWF.
  • a vWF reversal agent comprises all 2'-0 Methyl residues and a 3 '-inverted deoxythymidine.
  • vWF reversal agents include, but are not limited to: SEQ ID NO: 1
  • ARC22743 SEQ ID NO: 9, which is ARC22742; SEQ ID NO: 10, which is ARC22741; and SEQ ID NO: 11, which is ARC22740.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mTmGmTmGmTmCmTmTmAmGmGmTmCmCmC-3T (SEQ ID NO: 6), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mCmTmTmAmGmGmTmCmCmC-3T (SEQ ID NO: 7), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mAmTmGmTmGmTmCmTmTmA-3T (SEQ ID NO: 8), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mAmCmAmTmGmTmGmTmCmTmTmAmGmGmT-3T (SEQ ID NO: 9), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mGmGmGmAmCmAmTmGmTmG-3T (SEQ ID NO: 10), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mGmGmGmAmCmAmTmGmTmGmTmCmTmTmA-3T (SEQ ID NO: 11), where "3 T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the invention also provides a kit that includes at least a first container having a quantity of one or more vWF aptamers described herein together with instructions for use of the aptamer or aptamers in the treatment, prevention, and/or amelioration of a cardiovascular, thrombotic or hematologic disease.
  • the kit includes ARC13090, ARC14303, ARC15103, ARC15104 or ARC15105, and combinations thereof.
  • the vWF aptamers in the kit are formulated as a pharmaceutical composition.
  • the kit may also comprise a second container having a quantity of one or more vWF reversal agents described herein together with instructions for use in reversing the effect of a vWF aptamer.
  • the kit may include ARC22745, ARC22744, ARC22743, ARC22743, ARC22741 or ARC22740, and combinations thereof.
  • the vWF reversal agents in the kit are formulated as a pharmaceutical composition.
  • the invention also includes methods for preparing a vWF aptamer for use in the treatment, prevention or amelioration of cardiovascular, thrombotic or hematologic diseases by synthesizing a nucleic acid set forth in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the vWF aptamer is formulated as a pharmaceutical composition.
  • the invention further includes methods for preparing a vWF reversal agent for use in reversing the effect of a vWF aptamer by synthesizing a nucleic acid set forth in SEQ ID NOs: 6, 7, 8, 9, 10 or 11.
  • the vWF reversal agents are formulated as a pharmaceutical composition.
  • the invention also provides aptamers that have been identified by the
  • SELEX process which comprises the steps of (a) contacting a mixture of nucleic acids with a target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids that have bound the target; (c) dissociating the nucleic acid-target pairs; (d) amplifying the nucleic acids dissociated from the nucleic acid-target pairs to yield a ligand-enriched mixture of nucleic acids; and optionally (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired, to obtain the aptamer(s) which bind(s) specifically to the target.
  • Figure 1 is a schematic representation of the in vitro aptamer selection
  • Figure 2 depicts the amino acid sequences of various von Willebrand Factor domain Al proteins.
  • Figure 3 depicts the amino acid sequence of the full length human von
  • Figure 4 is an illustration depicting the proposed secondary structure of
  • Figure 5 is an illustration depicting various PEGylation strategies representing standard mono-PEGylation, multiple PEGylation and oligomerization via
  • Figure 6 is an illustration of a 40 kDa branched PEG.
  • Figure 7 is an illustration of a 40 kDa branched PEG attached to the 5' end of an aptamer.
  • Figure 8A is a graph showing the direct binding of various vWF aptamers to vWF.
  • Figure 8B is a table showing the apparent Kd for each vWF aptamer in Figure 8A.
  • Figure 9 is a graph showing the results of a competition assay with
  • Figure 10 is a graph showing the results of an ELISA assay with ARC 15104
  • Figure 11 is a graph comparing ARCl 5105 and ARC15104 to ARC1779 in a
  • Figure 12 is a graph showing the results of a PFA-100 assay with
  • ARC15103 ARC15103, ARC15104 and ARC15105.
  • Figure 13 are graphs and pictures showing the results of in vitro perfusion chamber experiments for ARC15105 and ARC 1779.
  • Figure 14 is an illustration showing the design of various reversal agents binding to ARC15105. The highlighted area is the hybridization region of the reversal agent.
  • Figure 15 is a graph showing that various reversal agents block the anti-vWF activity of ARC15105 in PFA-100 assays.
  • Figure 16 is a graph showing that various reversal agents block the anti-vWF activity of ARC15105 in PFA-100 assays.
  • vWF vWF
  • vWF aptamers modulate vWF-mediated platelet adhesion, activation and/or aggregation.
  • the vWF aptamers bind specifically to v WF or a fragment thereof.
  • the vWF is human vWF.
  • the vWF aptamer has a dissociation constant for vWF of 100 nM or less.
  • the vWF aptamers are, for example, vWF antagonists or inhibitors that modulate at least one biological activity of vWF.
  • Biological activities of vWF include, for example, binding platelets and inhibiting platelet adhesion, activation and/or aggregation.
  • the vWF aptamers completely or partially inhibit vWF activity by partially or completely blocking the ability of vWF to bind to platelets.
  • the vWF aptamers are considered to completely inhibit vWF activity when the level of vWF activity in the presence of the vWF aptamer is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of vWF activity in the absence of binding with a vWF aptamer.
  • the vWF aptamers are considered to partially inhibit vWF activity when the level of vWF activity in the presence of the vWF aptamer is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of vWF activity in the absence of binding with a vWF aptamer.
  • the aptamers described herein are identified through a method known in the art as Systematic Evolution of Ligands by Exponential Enrichment or SELEXTM, which is shown generally in Figure 1. More specifically, starting with a mixture containing a starting pool of nucleic acids, the SELEXTM method includes steps of: (a) contacting the mixture with a target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to the target; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and optionally (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target.
  • SELEXTM Systematic Evolution of Ligands by Exponential Enrichment
  • the SELEX TM method further includes the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.
  • the starting pool of nucleic acids can be modified or unmodified DNA, RNA, or DNA/RNA hybrids. Additionally, selections can be performed with nucleic acid sequences incorporating modified nucleotides to stabilize the aptamer molecules against degradation in vivo. For example, resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2 '-position.
  • the present invention provides aptamers including combinations of 2'-OH, 2'-F, 2'-deoxy and 2'-0Me modifications of the ATP, GTP, CTP, TTP and UTP nucleotides.
  • the present invention provides aptamers including combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-0Me, 2'-NH 2 and 2'-methoxyethyl modifications of the ATP, GTP, CTP, TTP and UTP nucleotides.
  • the present invention provides aptamers including all or substantially all 2'-0Me modified ATP, GTP, CTP, TTP, and/or UTP nucleotides.
  • 2'-modified aptamers of the invention are created using modified polymerases, e.g., a modified RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2 '-position that is higher than that of wild-type polymerases.
  • the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to phenylalanine (Y639F).
  • the modified RNA polymerase is a mutant T7 polymerase in which the histidine at position 784 changed to an alanine residue (H784A).
  • the modified RNA polymerase is a mutant T7 polymerase in which the histidine at position 784 is changed to an alanine residue, in addition to the Y639F mutation (Y639F/H784A).
  • the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to phenylalanine, the histidine residue at position 784 has been changed to an alanine, and the lysine residue at position 378 has been changed to arginine (Y639F/H784A/K378R), and the transcription reaction mixture requires a spike of 2'-OH GTP for transcription.
  • RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2 '-position that is higher than that of wild-type polymerases is, for example, a mutant T3 RNA polymerase.
  • the mutant T3 RNA polymerase has a mutation at position 640 wherein the tyrosine residue at position 640 is replaced with a phenylalanine residue.
  • the mutant T3 RNA polymerase has mutations at position 640 and position 785 wherein the tyrosine residue at position 640 is replaced with a leucine residue and the histidine residue at position 785 is replaced with an alanine residue.
  • 2'-modified oligonucleotides may be synthesized entirely of modified nucleotides, or with a subset of modified nucleotides.
  • the modifications can be the same or different.
  • Some or all nucleotides may be modified, and those that are modified may contain the same modification.
  • all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modification.
  • All purine nucleotides may have one type of modification (or are unmodified), while all pyrimidine nucleotides have another, different type of modification (or are unmodified).
  • transcripts, or pools of transcripts are generated using any combination of modifications, including for example, ribonucleotides (2'-OH), deoxyribonucleotides (2'-deoxy), 2'-amine nucleotides (2'-NH 2 ), 2'-fluoro nucleotides (T- F) and 2'-O-methyl (2'-OMe) nucleotides.
  • modifications including for example, ribonucleotides (2'-OH), deoxyribonucleotides (2'-deoxy), 2'-amine nucleotides (2'-NH 2 ), 2'-fluoro nucleotides (T- F) and 2'-O-methyl (2'-OMe) nucleotides.
  • U triphosphates (2'-0Me ATP, 2'-0Me UTP, 2'-0Me CTP, and 2'-0Me GTP) is referred to as an MNA or mRmY mixture, and aptamers selected therefrom are referred to as MNA aptamers or mRmY aptamers and contain only 2'-O-methyl nucleotides.
  • a transcription mixture containing 2'-0Me C and U, and 2'-OH A and G is referred to as an "rRmY" mixture, and aptamers selected therefrom are referred to as "rRmY” aptamers.
  • a transcription mixture containing deoxy A and G, and 2'-0Me U and C is referred to as a "dRmY” mixture, and aptamers selected therefrom are referred to as “dRmY” aptamers.
  • a transcription mixture containing 2'-0Me A, C and U, and 2'-OH G is referred to as a "rGmH” mixture, and aptamers selected therefrom are referred to as "rGmH” aptamers.
  • a transcription mixture alternately containing 2'-0Me A, C, U and G, and 2'-0Me A, U and C, and 2'-F G is referred to as an "alternating mixture", and aptamers selected therefrom are referred to as "alternating mixture” aptamers.
  • a transcription mixture containing 2'-OH A and G, and 2'-F C and U is referred to as an "rRfY” mixture, and aptamers selected therefrom are referred to as "rRiY” aptamers.
  • a transcription mixture containing 2'-0Me A, U, and C, and 2'-F G is referred to as a "fGmH” mixture, and aptamers selected therefrom are referred to as “fGmH” aptamers.
  • a transcription mixture containing 2'-0Me A, U, C and G, where up to 10% of the G's are ribonucleotides is referred to as a "r/mGmH” mixture, and aptamers selected therefrom are referred to as "r/mGmH” aptamers.
  • a transcription mixture containing 2'-0Me A, U and C, and deoxy G is referred to as a "dGmH” mixture, and aptamers selected therefrom are referred to as “dGmH” aptamers.
  • a transcription mixture containing deoxy A, and 2'-0Me C, G and U is referred to as a "dAmB” mixture, and aptamers selected therefrom are referred to as "dAmB” aptamers.
  • a transcription mixture containing 2'-OH A, and 2'-0Me C, G and U is referred to as a "rAmB” mixture, and aptamers selected therefrom are referred to as "rAmB” aptamers.
  • a transcription mixture containing 2'-OH adenosine triphosphate and guanosine triphosphate, and deoxy cytidine triphosphate and thymidine triphosphate is referred to as an rRdY mixture, and aptamers selected therefrom are referred to as "rRdY' aptamers.
  • a transcription mixture containing 2'-0Me A, U or T, and G, and deoxy C is referred to as a "dCmD" mixture, and aptamers selected there from are referred to as "dCmD" aptamers.
  • a transcription mixture containing 2'-0Me A, G and C, and deoxy T is referred to as a "dTmV” mixture, and aptamers selected there from are referred to as “dTmV” aptamers.
  • a transcription mixture containing 2'-0Me A, C and G, and 2'-OH U is referred to as a "rUmV” mixture, and aptamers selected there from are referred to as “rUmV” aptamers.
  • a transcription mixture containing 2'-0Me A, C and G, and T- deoxy U is referred to as a "dUmV” mixture, and aptamers selected there from are referred to as "dUmV” aptamers.
  • a transcription mixture containing all 2'-OH nucleotides is referred to as a "rN” mixture, and aptamers selected therefrom are referred to as “rN", “rRrY” or RNA aptamers.
  • a transcription mixture containing all deoxy nucleotides is referred to as a “dN” mixture, and aptamers selected therefrom are referred to as “dN” or “dRdY” or DNA aptamers.
  • a leader sequence can be incorporated into the fixed sequence at the 5' end of a DNA transcription template.
  • the leader sequence is typically 6-15 nucleotides long, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long, and may be composed of all purines, or a mixture of purine and pyrimidine nucleotides.
  • Another useful factor can be the presence or concentration of 2'-OH guanosine (e.g., GMP, GTP, or another non-2 '-0Me non-triphosphate).
  • Transcription can be divided into two phases: the first phase is initiation, during which an NTP is added to the 3'-hydroxyl end of GTP (or GMP, or another non-2'-OMe non-triphosphate) to yield a dinucleotide which is then extended by about 10-12 nucleotides; the second phase is elongation, during which transcription proceeds beyond the addition of the first about 10-12 nucleotides.
  • Priming transcription with 2'-OH guanosine is useful due to the specificity of the polymerase for the initiating nucleotide.
  • the 5 '-terminal nucleotide of any transcript generated in this fashion is likely to be 2'-OH G.
  • the preferred concentration of GMP is 0.5 mM and even more preferably 1 mM.
  • HEPES buffer concentration can range from 0 to 1
  • the present invention also contemplates the use of other buffering agents having a pKa between 5 and 10 including, for example, Tris-hydroxymethyl-aminomethane.
  • the DTT concentration can range from 0 to 400 mM.
  • the methods of the present invention also provide for the use of other reducing agents including, for example, mercaptoethanol.
  • the spermidine and/or spermine concentration can range from 0 to 20 mM.
  • the PEG-8000 concentration can range from 0 to 50% (w/v).
  • the methods of the present invention also provide for the use of other hydrophilic polymers including, for example, other molecular weight PEGs or other polyalkylene glycols.
  • the Triton X-100 concentration can range from 0 to 0.1% (w/v).
  • the methods of the present invention also provide for the use of other non-ionic detergents including, for example, other detergents, including other Triton- X detergents.
  • the MgCl 2 concentration can range from 0.5 mM to 50 mM.
  • the MnCl 2 concentration can range from 0.15 mM to 15 mM.
  • the 2'-OMe NTP concentration (each NTP) can range from 5 ⁇ M to 5 mM.
  • the 2'-OH GTP concentration can range from 0 ⁇ M to 300 ⁇ M.
  • the 2'-OH GMP concentration can range from 0 to 5 mM.
  • the pH can range from pH 6 to pH 9.
  • ARC254 and ARC256 transcribe under all 2'-0Me conditions and ARC255 transcribes under rRmY conditions.
  • Variants of the SELEX process may also be used to identify aptamers. For example, one may use agonist SELEX, toggle SELEX, 2 '-Modified SELEX or Counter SELEX. Each of these variations of the SELEX process are known in the art.
  • the materials of the present invention include a series of nucleic acid aptamers of 16-26 nucleotides in length that bind specifically to vWF and functionally modulate the biological activity of vWF.
  • the vWF aptamers bind to vWF or a variant or a fragment thereof.
  • a vWF variant as used herein encompasses variants that perform essentially the same function as vWF functions, preferably includes substantially the same structure and in some embodiments includes at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of vWF.
  • the vWF aptamers bind full length vWF.
  • a vWF aptamer binds a fragment of vWF
  • it is preferable that the vWF aptamer binds the Al domain of vWF.
  • a vWF aptamer specifically binds to a von Willebrand Factor full length target and a von Willebrand Factor domain Al target, and more preferably, a vWF aptamer specifically binds to a human vWF full length target and a human vWF domain A 1 target.
  • the vWF protein may be from any species, but is preferably human.
  • a vWF aptamer preferably comprises a dissociation constant for human von Willebrand Factor domain Al, or a variant thereof, of less than 100 ⁇ M, less than 1 ⁇ M, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, and more preferably 500 pM or less.
  • the dissociation constant is determined by dot blot titration. Examples of amino acid sequences of various von Willebrand Factor domain Al proteins are illustrated in Figure 2. The amino acid sequence of the full length human von Willebrand Factor protein is illustrated in Figure 3.
  • the vWF aptamers may be ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
  • the vWF aptamers may be single stranded ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
  • the vWF aptamers comprise at least one chemical modification.
  • the chemical modification is selected from a chemical substitution of the nucleic acid at a sugar position, a chemical substitution at a phosphate position and a chemical substitution at a base position.
  • the chemical modification is selected from incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of phosphorothioate or phosphorodithioate into the phosphate back bone.
  • the non- immunogenic, high molecular weight compound is polyalkylene glycol, and more preferably is polyethylene glycol (PEG).
  • the 3' cap is an inverted deoxythymidine cap.
  • the modifications described herein may affect aptamer stability, e.g., incorporation of a capping moiety may stabilize the aptamer against endonuclease degradation. Additionally, the modifications described herein may affect the binding affinity of an aptamer to its target, e.g., site specific incorporation of a modified nucleotide or conjugation to PEG may affect binding affinity. The effect of such modifications on aptamer binding affinity can be determined using dot blot titration and comparing the binding affinities pre- and post-incorporation of modifications.
  • the vWF aptamer binds to vWF or a variant or a fragment thereof and acts as an antagonist to inhibit the function of vWF.
  • vWF When vWF is activated, it is responsible for the adhesion, activation and aggregation of platelets, which are involved in the formation of blood clots.
  • the vWF aptamers bind the Al domain of human vWF and prevent the interaction of the vWF Al domain with the platelet GPIb receptor. Therefore, the vWF aptamers are competitive antagonists of the vWF/platelet interaction. [0097]
  • the vWF aptamers prevent vWF-mediated platelet adhesion, activation and/or aggregation, preferably while not significantly increasing bleeding time in a subject. That is, the vWF aptamers do not increase bleeding time in a subject in a clinically significant (i.e., clinically meaningful) manner.
  • an increase in bleeding time is less than 15 minutes, preferably less than 10 minutes, more preferably less than 5 minutes, and in some embodiments, less than 3 minutes relative to the bleeding time of a subject not treated with the vWF aptamer.
  • the bleeding time is determined by cutaneous (or template) bleeding time.
  • the vWF aptamer helps to rapidly restore platelet count to normal, near normal or functionally safe levels.
  • Examples of aptamers that specifically bind and modulate the function of vWF for use as therapeutics and/or diagnostics include, but are not limited to, ARC 13090, ARC14030, ARC15103, ARC15104 and ARC15105.
  • the vWF aptamers comprise the following sequences:
  • ARC13090 is 2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe- guanylyl-(3' ⁇ 5')-2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe-cytidylyl-
  • ARC14303 is biotin-2'-OMe-guanylyl-(3' ⁇ 5')-2'-
  • ARC15103 6-aminohexylyl-(l ' ⁇ 5')-2'-OMe- guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-adenylyl-
  • ARCl 5104 is N-(methoxy-polyethyleneglycol)-6- aminohexylyl-( 1 ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ')-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe- guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-cytidylyl-(3 ' ⁇ 5 ')-2 ' -OMe- cytidy Iy l-(3 ' ⁇ 5 ')-2 ' -OMe-uracylyl-(3 ' ⁇ 5 ')-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-uracylyl-(
  • ARCl 5105 is N-(methoxy-polyethyleneglycol)-6- aminohexylyl-( 1 ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ')-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ')-2 ' -OMe- guanylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe-cytidylyl-(3' ⁇ 5')-2'-OMe- cytidy Iy l-(3 ' ⁇ 5 ')-2 ' -OMe-uracylyl-(3 ' ⁇ 5 ')-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe
  • ARCl 5105 The proposed secondary structure of ARCl 5105 is depicted in Figure 4, which comprises a stem and a loop motif.
  • the invention also provides aptamers that have substantially the same ability to bind vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • aptamers according to the invention have substantially the same structure as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • aptamers according to the invention have substantially the same ability to bind vWF and substantially the same structure as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention also provides aptamers that have substantially the same ability to bind and modulate a biological function of vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention further provides aptamers that bind to vWF, wherein the aptamer modulates platelet adhesion, activation and/or aggregation as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention also provides aptamers that have the same, substantially the same, and/or better ability to bind vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the invention also provides aptamers that have the same, substantially the same, and/or better ability to bind and modulate a biological function of vWF as SEQ ID NOs: 1, 2, 3, 4 or 5.
  • aptamers have substantially the same structure and the same, substantially the same, and/or better ability to bind vWF as the nucleic acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 or 5.
  • substantially the same ability to bind vWF means that the affinity is within one or two orders of magnitude of the affinity of the nucleic acid sequences and/or aptamers described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence has substantially the same ability to bind vWF.
  • the invention further provides aptamers that bind to vWF, wherein the aptamer modulates platelet adhesion, activation and/or aggregation and the aptamer is SEQ ID NO: 1, 2, 3, 4 or 5, or an aptamer that has the same ability, substantially the same, or better ability to modulate platelet adhesion, activation and/or aggregation as the nucleic acid sequences shown in SEQ ID NO: 1, 2, 3, 4 or 5 and wherein the aptamer includes a K D less than 100 ⁇ M.
  • the aptamer having the same, substantially the same, or better ability to modulate platelet adhesion, activation and/or aggregation is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 or 5.
  • the aptamer that binds to vWF has a nucleic acid sequence at least 95% identical to SEQ ID NO: 1, 2, 3, 4 or 5.
  • an aptamer of the invention modulates a function of vWF and/or modulates the interaction between vWF and platelets.
  • an aptamer of the invention inhibits a vWF function, while in another embodiment the aptamer stimulates a function of the target.
  • the aptamer binds and/or modulates a function of a vWF variant.
  • a function of vWF that is modulated by the aptamer of the invention is platelet adhesion, activation and/or aggregation.
  • the function of vWF that is modulated by the aptamer of the present invention is selected from the group consisting of: platelet adhesion, activation and/or aggregation.
  • sequence identity or “% identity” in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol.
  • BLAST basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • aptamer therapeutics of the present invention have great affinity and specificity to their targets while reducing the deleterious side effects from non-naturally occurring nucleotide substitutions if the aptamer therapeutics break down in the body of patients or subjects.
  • the therapeutic compositions containing the aptamer therapeutics of the present invention are free of or have a reduced amount of fluorinated nucleotides.
  • Aptamers of the present invention including, but not limited to, aptamers identified by the SELEX method, 2'-Modified SELEX , minimized aptamers, optimized aptamers and chemically substituted aptamers, can be manufactured using any oligonucleotide synthesis technique that is well known in the art, such as solid phase oligonucleotide synthesis techniques (see, e.g., Froehler et al, Nucl. Acid Res. 14:5399- 5467 (1986) and Froehler et al, Tet. Lett. 27:5575-5578 (1986)).
  • aptamers using solid phase oligonucleotide synthesis techniques can also be done at commercial scale.
  • Solution phase methods such as triester synthesis methods (see, e.g., Sood et al, Nucl. Acid Res. 4:2557 (1977) and Hirose et al, Tet. Lett, 28:2449 (1978)), may also be used to manufacture aptamer of the present invention, as well as recombinant means.
  • aptamers that bind to a desired target are identified, several techniques may be optionally performed in order to further increase binding and/or functional characteristics of the identified aptamer sequences.
  • Aptamers that bind to a desired target may be truncated in order to obtain the minimal aptamer sequence (also referred to herein as a "minimized construct” or “minimized aptamer”) having the desired binding and/or functional characteristics.
  • a minimal aptamer sequence also referred to herein as a "minimized construct” or “minimized aptamer”
  • One method of accomplishing this is by using folding programs and sequence analysis, e.g., aligning clone sequences resulting from a selection to look for conserved motifs and/or covariation to inform the design of minimized constructs.
  • Biochemical probing experiments can also be performed to determine the 5' and 3' boundaries of an aptamer sequence to inform the design of minimized constructs.
  • Minimized constructs can then be chemically synthesized and tested for binding and functional characteristics as compared to the non-minimized sequence from which they were derived. Variants of an aptamer sequence containing a series of 5', 3' and/or internal deletions may also be directly chemically synthesized and tested for binding and/or functional characteristics as compared to the non-minimized aptamer sequence from which they were derived. [00116] Additionally, doped reselections may be used to explore the sequence requirements within a single active aptamer sequence or a single minimized aptamer sequence. Doped reselections are performed using a synthetic, degenerate pool that has been designed based on the single sequence of interest.
  • the level of degeneracy usually varies 70% to 85% from the wild type nucleotide, i.e., the single sequence of interest.
  • sequences with neutral mutations are identified through the doped reselection process, but in some cases sequence changes can result in improvements in affinity.
  • the composite sequence information from clones identified using doped reselections can then be used to identify the minimal binding motif and aid in optimization efforts.
  • Aptamer sequences and/or minimized aptamer sequences may also be optimized post-SELEX TM using Aptamer Medicinal Chemistry to perform random or directed mutagenesis of the sequence to increase binding affinity and/or functional characteristics, or alternatively to determine which positions in the sequence are essential for binding activity and/or functional characteristics.
  • Aptamer Medicinal Chemistry is an aptamer improvement technique in which sets of variant aptamers are chemically synthesized. These sets of variants typically differ from the parent aptamer by the introduction of a single substituent, and differ from each other by the location of this substituent. These variants are then compared to each other and to the parent. Improvements in characteristics may be profound enough that the inclusion of a single substituent may be all that is necessary to achieve a particular therapeutic criterion.
  • the information gleaned from the set of single variants may be used to design further sets of variants in which more than one substituent is introduced simultaneously.
  • all of the single substituent variants are ranked, the top 4 are chosen and all possible double (6), triple (4) and quadruple (1) combinations of these 4 single substituent variants are synthesized and assayed.
  • the best single substituent variant is considered to be the new parent and all possible double substituent variants that include this highest-ranked single substituent variant are synthesized and assayed.
  • Other strategies may be used, and these strategies may be applied repeatedly such that the number of substituents is gradually increased while continuing to identify further- improved variants.
  • Aptamer Medicinal Chemistry may be used particularly as a method to explore the local, rather than the global, introduction of substituents. Because aptamers are discovered within libraries that are generated by transcription, any substituents that are introduced during the SELEX TM process must be introduced globally. For example, if it is desired to introduce phosphorothioate linkages between nucleotides then they can only be introduced at every A (or every G, C, T, U etc.) if globally substituted. Aptamers that require phosphorothioates at some As (or some G, C, T, U etc.) (locally substituted) but cannot tolerate it at other As (or some G, C, T, U etc.) cannot be readily discovered by this process. [00121] The kinds of substituents that can be utilized by the Aptamer Medicinal
  • Aptamer Medicinal Chemistry schemes may include substituents that introduce steric bulk, hydrophobicity, hydrophilicity, lipophilicity, lipophobicity, positive charge, negative charge, neutral charge, zwitterions, polarizability, nuclease-resistance, conformational rigidity, conformational flexibility, protein-binding characteristics, mass, etc.
  • Aptamer Medicinal Chemistry schemes may include base- modifications, sugar-modifications or phosphodiester linkage-modifications. [00122] When considering the kinds of substituents that are likely to be beneficial within the context of a therapeutic aptamer, it may be desirable to introduce substitutions that fall into one or more of the following categories:
  • Substituents already present in the body e.g., 2'-deoxy, 2'-ribo, 2'-O-methyl purines or pyrimidines, or 5-methyl cytosine;
  • the aptamers of the present invention include aptamers developed through Aptamer Medicinal Chemistry as described herein.
  • Target binding affinity of the aptamers of the invention can be assessed through a series of binding reactions between the aptamer and the target (e.g., a protein) in which trace 32 P-labeled aptamer is incubated with a dilution series of the target in a buffered medium and then analyzed by nitrocellulose filtration using a vacuum filtration manifold.
  • the dot blot binding assay uses a three layer filtration medium consisting (from top to bottom) of nitrocellulose, nylon filter and gel blot paper. RNA that is bound to the target is captured on the nitrocellulose filter whereas the non- target bound RNA is captured on the nylon filter.
  • the gel blot paper is included as a supporting medium for the other filters. Following filtration, the filter layers are separated, dried and exposed on a phosphor screen and quantified using a phosphorimaging system. The quantified results can be used to generate aptamer binding curves from which dissociation constants (K D ) can be calculated.
  • the buffered medium used to perform the binding reactions is IX Dulbecco's PBS (with Ca + and Mg + ) plus 0.1 mg/mL BSA.
  • the ability of an aptamer to modulate the functional activity of a target can be assessed using in vitro and in vivo models, which will vary depending on the biological function of the target.
  • the aptamers of the invention may inhibit a known biological function of the target.
  • the aptamers of the invention may stimulate a known biological function of the target.
  • the functional activity of aptamers of the invention can be assessed using in vitro and in vivo models designed to measure a known function of vWF.
  • Aptamer sequences and/or minimized aptamer sequences may also be optimized post-SELEX TM using Metabolic Profile Directed Aptamer Medicinal Chemistry to perform random or directed mutagenesis of the sequence to increase binding affinity and/or functional characteristics, or alternatively to determine which positions in the sequence are essential for binding activity and/or functional characteristics.
  • Metabolic Profile Directed Aptamer Medicinal Chemistry involves incubating a parent aptamer with a test fluid to result in a mixture. Then, the mixture is analyzed in order to determine the rate of disappearance of the parent aptamer, the specific aptamer metabolic profile and the specific aptamer metabolite sequences.
  • the method involves introducing chemical substitutions or modifications at or near the cleavage sites that are designed to block nuclease cleavage.
  • an aptamer is identified and modified by a) incubating a parent aptamer with a test fluid to result in a mixture; b) analyzing the mixture to identify metabolites of the parent aptamer, thereby detecting at least one aptamer cleavage site in the parent aptamer; and c) introducing a chemical substitution at a position proximal to the at least one aptamer cleavage site to result in a modified aptamer.
  • the test fluid is a biological matrix, particularly a biological matrix selected from the group consisting of one or more of: serum; plasma; cerebral spinal fluid; tissue extracts, including cytosolic fraction, S9 fraction and microsomal fraction; aqueous humour; vitreous humour and tissue homogenates.
  • the biological matrix is derived from a species selected from the group consisting of one or more of: mouse, rat, monkey, pig, human, dog, guinea pig and rabbit.
  • the test fluid comprises at least one purified enzyme, particularly at least one purified enzyme selected from the group consisting of: snake venom phosphodiesterase and DNAse 1.
  • the analyzing step includes analyzing the resulting aptamer using liquid chromatography and mass spectrometry, particularly electron spray ionization liquid chromatography mass spectrometry, polyacrylamide gel electrophoresis or capillary electrophoresis to determine a position of at least one aptamer cleavage site.
  • liquid chromatography and mass spectrometry particularly electron spray ionization liquid chromatography mass spectrometry, polyacrylamide gel electrophoresis or capillary electrophoresis to determine a position of at least one aptamer cleavage site.
  • the analyzing step includes analyzing the resulting aptamer using a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS, and more particularly ESI-LC/MS, ESI- LC/MS/MS and ESI-LC/MS/MS/MS.
  • a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS, and more particularly ESI-LC/MS, ESI- LC/MS/MS and ESI-LC/MS/MS/MS.
  • the proximal position includes a position selected from the group consisting of: a position immediately 5' to the aptamer cleavage site, a 5'- position at or within three nucleotides of the aptamer cleavage site, a position immediately 3' to the aptamer cleavage site, a 3 '-position at or within three nucleotides of the aptamer cleavage site, and at the cleaved internucleotide linkage.
  • the chemical substitution is selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a base position and a chemical substitution at a phosphate position. More particularly, a substitution is selected from the group consisting of: a purine substitution for a pyrimidine; a 2'-deoxy dihydrouridine substitution for a uridine; a 2'-deoxy-5-methyl cytidine for a cytidine; a 2-amino purine substitution for a purine; a phosphorothioate substituted for a phosphodiester; a phosphorodithioate substituted for a phosphodiester; a deoxynucleotide substituted for a 2'-OH nucleotide; a 2'-0Me nucleotide, a 2'-fluoro nucleotide or a 2'-O- methoxyethyl nucleotide substituted for a 2
  • the introducing step of these methods further includes introducing more than one chemical substitution at one or more cleavage sites or at a single cleavage site or both.
  • the introducing step of these methods further includes introducing at least one chemical substitution at the associated proximal position of the aptamer cleavage site determined to occur first in time during the incubating step or at any other cleavage site(s) that provides the desired properties upon introduction of a chemical substitution.
  • these methods further include the step of testing the stability of the modified aptamer in the test fluid.
  • aptamer stability is assessed by determining the percent of modified aptamer that remains intact in the test fluid as compared to the percent of the parent aptamer that remains intact in the test fluid.
  • the percent of intact aptamer is assessed by a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS, and more particularly ESI-LC/MS, ESI-LC/MS/MS and ESI-LC/MS/MS/MS.
  • the modified aptamer is more stable in the test fluid than the parent aptamer, preferably at least 2 fold, more preferably at least 5 fold and most preferably at least 10 fold more stable.
  • these methods further include determining a dissociation constant of the modified aptamer for its target.
  • chemical substitutions are introduced singly at each position or in various combinations in the aptamer, and the dissociation constant for each resulting aptamer is determined.
  • Chemical substitutions are introduced at a position proximal to the aptamer cleavage site such that a single chemical modification results in a dissociation constant for the modified aptamer that is the same or less than that of the parent aptamer.
  • the method includes selecting a modified aptamer having a dissociation constant for its target that is the same or less than that for the parent aptamer.
  • the modified aptamer binds to a target having a biological activity
  • the method further includes testing the biological activity of the target in the presence and absence of modified aptamer.
  • the method further includes selecting a modified aptamer that binds to a target having a biological activity that is the same or better than that of the parent aptamer.
  • the biological activity may be measured in any relevant assay, such as an ELISA assay or a cell-based assay.
  • the incubating, analyzing, introducing and testing steps are repeated iteratively until the desired stability is achieved.
  • the aptamers of the invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic utilization may include both in vivo or in vitro diagnostic applications. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any aptamer by procedures known in the art to incorporate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures.
  • oligonucleotide- based therapeutics including aptamers
  • Aptamers must be able to be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen.
  • the present invention provides materials and methods to affect the pharmacokinetics of aptamer compositions, and, in particular, the ability to tune aptamer pharmacokinetics.
  • the tunability oi(i.e., the ability to modulate) aptamer pharmacokinetics is achieved through conjugation of modifying moieties (e.g., PEG polymers) to the aptamer and/or the incorporation of modified nucleotides (e.g., 2'-fluoro or 2'-O-methyl) to alter the chemical composition of the nucleic acid.
  • modifying moieties e.g., PEG polymers
  • modified nucleotides e.g., 2'-fluoro or 2'-O-methyl
  • aptamers in the circulation it is desirable to decrease the residence times of aptamers in the circulation.
  • maintenance therapies where systemic circulation of a therapeutic is desired, it may be desirable to increase the residence times of aptamers in circulation.
  • the tunability of aptamer pharmacokinetics is used to modify the biodistribution of an aptamer therapeutic in a subject.
  • the aptamer therapeutic preferentially accumulates in a specific tissue or organ(s).
  • PEGylation of an aptamer therapeutic e.g., PEGylation with a 20 kDa PEG polymer
  • PEGylation of an aptamer therapeutic is used to target inflamed tissues, such that the PEGylated aptamer therapeutic preferentially accumulates in inflamed tissue.
  • aptamer therapeutics e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides
  • parameters include, for example, the half-life (ti/ 2 ), the plasma clearance (Cl), the volume of distribution (Vss), the area under the concentration-time curve (AUC), maximum observed serum or plasma concentration (C max ), and the mean residence time (MRT) of an aptamer composition.
  • ti/ 2 the half-life
  • Cl plasma clearance
  • Vss volume of distribution
  • AUC area under the concentration-time curve
  • C max maximum observed serum or plasma concentration
  • MRT mean residence time
  • the term "AUC" refers to the area under the plot of the plasma concentration of an aptamer therapeutic versus the time after aptamer administration.
  • the AUC value is used to estimate the bioavailability (i.e., the percentage of administered aptamer therapeutic in the circulation after aptamer administration) and/or total clearance (Cl) (i.e., the rate at which the aptamer therapeutic is removed from circulation) of a given aptamer therapeutic.
  • the volume of distribution relates the plasma concentration of an aptamer therapeutic to the amount of aptamer present in the body. The larger the Vss, the more an aptamer is found outside of the plasma (i.e., the more extravasation).
  • an aptamer described herein such as a stabilized aptamer
  • a modulating moiety such as a small molecule, peptide, or polymer terminal group
  • modified nucleotides into an aptamer.
  • the conjugation of a modifying moiety and/or altering nucleotide(s) chemical composition alters fundamental aspects of aptamer residence time in circulation and distribution to tissues.
  • oligonucleotide therapeutics are subject to elimination via renal filtration.
  • a nuclease-resistant oligonucleotide administered intravenously typically exhibits an in vivo half-life of ⁇ 10 min, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood stream into tissues or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus. Conjugation of small therapeutics to a PEG polymer (PEGylation), described below, can dramatically lengthen residence times of aptamers in circulation, thereby decreasing dosing frequency and enhancing effectiveness against vascular targets.
  • PEGylation PEGylation
  • Modified nucleotides can also be used to modulate the plasma clearance of aptamers.
  • an unconjugated aptamer which incorporates for example, T- fluoro, 2'-0Me, and/or phosphorothioate stabilizing chemistries, which is typical of current generation aptamers as it exhibits a high degree of nuclease stability in vitro and in vivo, displays rapid loss from plasma (i.e., rapid plasma clearance) and a rapid distribution into tissues, primarily into the kidney, when compared to unmodified aptamer.
  • aptamer compositions of the invention may be derivatized with one or more polyalkylene glycol (“PAG”) moieties.
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • polypropylene glycol including poly isopropylene glycol
  • a polyalkylene glycol such as PEG
  • PEG polyalkylene glycol
  • This polymer, alpha-, omega-dihydroxylpolyethylene glycol, can also be represented as HO-PEG-OH, where it is understood that the — PEG- symbol represents the following structural unit: - CH 2 CH 2 O-(CH 2 CH 2 O) n -CH 2 CH 2 - where n typically ranges from about 4 to about 10,000.
  • PAG polymers suitable for therapeutic indications typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity.
  • One use of PAGs is to covalently attach the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble.
  • the water-insoluble drug paclitaxel when coupled to PEG, becomes water- soluble. Greenwald, et ah, J. Org. Chem., 60:331-336 (1995).
  • PAG conjugates are often used not only to enhance solubility and stability but also to prolong the blood circulation half-life of molecules.
  • the PAG derivatized compounds conjugated to the aptamers of the invention are typically between 5 and 80 kDa in size however any size can be used, the choice dependent on the aptamer and application.
  • Other PAG derivatized compounds of the invention are between 10 and 80 kDa in size.
  • Still other PAG derivatized compounds of the invention are between 10 and 60 kDa in size.
  • the PAG moieties derivatized to compositions of the present invention are PEG ranging from 10, 20, 30, 40, 50, 60 or 80 kDa in size.
  • the PEG is linear PEG, while in other embodiments, the PEG is branched PEG.
  • the PEG is a 4OkDa branched PEG as depicted in Figure 6.
  • the 40 kDa branched PEG is attached to the 5' end of the aptamer as depicted in Figure 7.
  • Branched activated PEGs will have more than two termini, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are herein referred to as, multi-activated PEGs. In some cases, not all termini in a branch PEG molecule are activated. In cases where any two termini of a branch PEG molecule are activated, such PEG molecules are referred to as bi- activated PEGs. In some cases where only one terminus in a branch PEG molecule is activated, such PEG molecules are referred to as mono-activated.
  • the linear PEG molecule is di-functional and is sometimes referred to as "PEG diol.”
  • the terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the -OH groups, that can be activated, or converted to functional moieties, for attachment of the PEG to other compounds at reactive sites on the compound.
  • Such activated PEG diols are referred to herein as bi-activated PEGs.
  • the molecules are generated using any of a variety of art-recognized techniques.
  • one or both of the terminal alcohol functionalities of the PEG molecule can be modified to allow for different types of conjugation to a nucleic acid. For example, converting one of the terminal alcohol functionalities to an amine, or a thiol, allows access to urea and thiourethane conjugates.
  • PEG molecule on one end it is desirable to cap the PEG molecule on one end with an essentially non-reactive moiety so that the PEG molecule is mono-functional (or mono-activated).
  • bi-functional activated PEGs lead to extensive cross- linking, yielding poorly functional aggregates.
  • mono-activated PEGs one hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with non- reactive methoxy end moiety, -OCH3.
  • the other, un-capped terminus of the PEG molecule typically is converted to a reactive end moiety that can be activated for attachment at a reactive site on a surface or a molecule such as a protein.
  • the invention also includes pharmaceutical compositions containing aptamers that bind to vWF.
  • the compositions include an effective amount of a pharmacologically active vWF aptamer or a pharmaceutically acceptable salt thereof, alone or in combination, with one or more pharmaceutically acceptable carriers.
  • the compositions may contain one or more different vWF aptamers.
  • the compositions may contain ARC15105.
  • the compositions may contain ARCl 5104 and ARCl 5105.
  • the compositions may contain ARC15105 and another vWF aptamer.
  • the compositions contain ARC15105, either alone or in combination with another vWF aptamer.
  • the term "pharmaceutically acceptable salt” refers to salt forms of the active compound that are prepared with counter ions that are non-toxic under the conditions of use and are compatible with a stable formulation.
  • pharmaceutically acceptable salts of vWF aptamers include, but are not limited to, hydrochlorides, sulfates, phosphates, acetates, fumarates, maleates and tartrates.
  • pharmaceutically acceptable carrier means being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers are well known in the art. Examples of pharmaceutically acceptable carriers can be found, for example, in Goodman and Gillmans, The Pharmacological Basis of Therapeutics, latest edition.
  • Therapeutic or pharmacological compositions of the present invention will generally include a therapeutically effective amount of the active component(s) of the therapy, e.g., a vWF aptamer of the invention, dissolved or dispersed in a pharmaceutically acceptable carrier or medium.
  • a pharmaceutically acceptable carrier or medium examples include, but are not limited to, physiological saline solution and glucose solution.
  • other pharmaceutically acceptable carriers may also be used.
  • other pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the pharmaceutical compositions may contain excipients such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts, or buffers for modifying or maintaining pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or absorption of the formulation.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • compositions of the invention are prepared according to conventional mixing, granulating, or coating methods, and typically contain about 0.1% to 75%, preferably about 1% to 50%, of the active component.
  • formulation of pharmaceutical or pharmacological compositions will be known to one of skill in the art in light of the present disclosure. Typically, such compositions may be formulated as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules for slow release formulations; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like.
  • the active compound defined above may be also formulated as suppositories, using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • sterile formulations such as saline-based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful.
  • compositions may be formulated as oral dosage forms, such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
  • oral dosage forms such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture.
  • Suitable binders include, without limitation, starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta- lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • compositions can also be formulated in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Patent No. 5,262,564.
  • the aptamer molecules described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art.
  • liposomes may bear aptamers on their surface for targeting and carrying cytotoxic agents internally to mediate cell killing.
  • An example of nucleic-acid associated complexes is provided in U.S. Patent No. 6,011,020.
  • the therapeutic compositions of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the therapeutic compositions of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual.
  • a minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
  • compositions may be administered to a vertebrate, preferably a mammal, and more preferably a human.
  • patient and “subject” are used interchangeably throughout the specification, and these terms include both human and veterinary subjects.
  • vWF aptamer compositions provided herein are administered to subjects in an amount effective to inhibit, reduce, block or otherwise modulate vWF-mediated platelet adhesion, activation and/or aggregation.
  • compositions may be administered by numerous routes of administration.
  • routes of administration include, but are not limited to, oral routes; topical routes, such as intranasally, vaginally or rectally; and parenteral routes, such as intravenous, subcutaneous, intradermal, intramuscular, intraarticular and intrathecal administration.
  • Suitable routes of administration may also be used in combination, such as intravenous administration followed by subcutaneous administration.
  • the route of administration is determined by the attending physician.
  • the formulations are administered subcutaneously. Most preferably, the formulations are administered intravenously.
  • Oral dosage forms may be administered as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups or emulsions.
  • Topical dosage forms include creams, ointments, lotions, aerosol sprays and gels for intranasal vehicles, inhalants or transdermal patches.
  • Parenteral dosage forms include solutions and lyophilized powders that are reconstituted prior to administration.
  • the dosage regimen utilizing the vWF aptamers is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptamer or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • compositions are used to treat, prevent or ameliorate vWF-mediated diseases and disorders, including the treatment of cardiovascular, thrombotic and hematologic diseases and disorders involving vWF-mediated platelet adhesion, activation and/or aggregation.
  • the diseases and disorders to be treated, prevented or ameliorated are selected from the group consisting of: essential thrombocytopenia, thrombotic microangiopathies (TMA), hemolytic uremic syndrome, thrombotic thrombocytopenic purpura (TTP), familial TTP, secondary TTP, idiopathic TTP, congenital TTP, Type 2b von Willebrand's Disease, pseudo type 2b von Willebrand's Disease, peripheral artery disease, peripheral arterial occlusive disease, unstable angina, angina pectoris, arterial thrombosis, atherosclerosis, ischemia, myocardial infarction, acute coronary syndrome (ACS), atrial fibrillation, carotid stenosis, unstable carotid disease, unstable carotid lesions, cerebral infarction, cerebral thrombosis, stroke, ischemic stroke, and transient cerebral ischemic attack.
  • TMA thrombotic microangiopathies
  • TTP thrombotic thrombocytopenic purpur
  • compositions may also be administered prior to, during and/or after a medical procedure.
  • the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as carotid endarterectomy, carotid stenting, carotid angioplasty with or without stenting, medical management of a patient with symptomatic or asymptomatic carotid stenosis, CABG surgery, percutaneous coronary intervention and heart valve replacement.
  • the pharmaceutical compositions may also be administered in conjunction (before, during and/or after) with other medical procedures, such as dialysis or hemodialysis.
  • the anti-vWF aptamer compositions provided herein are used to inhibit activated vWF in order to improve the outcome for ACS patients.
  • the aptamer compositions provided herein address significant, unmet medical needs in the treatment of patients who are suffering from ACS, or heart attack, and who are undergoing a procedure called angioplasty, or PCI. These unmet needs include the improvement of blood flow to the heart, reduction of bleeding risk and improved therapeutic administration.
  • ARC 1779 has this duration of action.
  • ARC 1779 binds with high affinity and specificity to a region of activated vWF known as the Al domain. When exposed to high shear forces, the Al domain binds to its receptor on a platelet. Once bound, the platelet adheres to the blood vessel wall and then recruits and activates additional platelets. As these platelets aggregate, a thrombus is formed.
  • aptamers provided herein are agents that inhibit the three steps of platelet activity - adhesion, activation and aggregation - and, therefore, participate in all aspects of platelet-mediated thrombosis in order to provide a more precise and effective method for preventing platelet-induced thrombus formation than currently approved drugs.
  • ARC 1779 improves myocardial perfusion in patients suffering from ACS.
  • ARC 1779 inhibits the local activation of vWF and prevents clot formation in the microvasculature.
  • ARC 1779 also reduces bleeding risk during PCI.
  • ARC 1779 targets and binds to only activated vWF, the anti-platelet effect of ARC 1779 should only be present in regions subject to high shear forces. These shear forces are only present in the arteries, including those leading into and within the heart. These shear forces would be especially high in areas of vessels partially occluded by the presence of atherosclerotic lesions. In these areas, it has been estimated that shear force can be as high as 40 times that of the normal artery. Therefore, ARC 1779 can locally suppress platelet function and thrombus formation in the coronary arteries, while not disrupting normal platelet function and blood clotting in the remainder of the body.
  • the vWF aptamer compositions are used in the treatment of ACS patients who are undergoing percutaneous coronary intervention (PCI).
  • the compositions and dosages are designed to improve the risk-to-benefit profile of adjuvant pharmacotherapy of PCI for high risk patients by introducing a novel antithrombotic therapeutic principle, namely antagonism of the binding of vWF to the GPIb receptor on platelets.
  • vWF antagonism using the aptamer compositions provided herein, provides anti-thrombotic efficacy comparable to GPIIb/IIIa antagonism, while reducing the relative risk of bleeding complications.
  • the vWF aptamers help restore platelet count to within the normal range.
  • Thrombotic microangiopathies are a result of impaired cleavage of vWF. This is due to a deficiency or defect of the vWF protease, ADAMTS- 13, which leads to the formation of ultra-large vWF multimers that act as nidus for platelet-rich thrombi.
  • vWF The rationale for blocking vWF is to inhibit the formation of diffuse microvascular thrombi and improve end-organ perfusion, thereby reducing ischemia.
  • vWD von Willebrand's Disease
  • type 2 vWD is characterized by defective vWF.
  • the rationale for treating vWD-type 2b is to reduce thrombocytopenia and enable the use of concomitant procoagulant therapy. Atherothrombosis
  • Atherothrombosis is a result of elevated and activated vWF. This is due to the fact that endothelial injury and shear forces in atherosclerotic arteries lead to vWF secretion and activation, which promotes platelet adhesion, activation and aggregation.
  • the rationale for blocking vWF is to inhibit shear-dependent arterial thrombosis and improve end-organ perfusion, thereby reducing ischemia.
  • One embodiment of the invention comprises a vWF aptamer or a salt thereof or a pharmaceutical composition used in combination with one or more other treatments for cardiovascular, thrombotic or hematologic diseases or disorders.
  • a vWF aptamer is administered in combination with another useful compositions or drug, such as an anti-inflammatory agent, an immunosuppressant, an antiviral agent, or the like.
  • a vWF aptamer is used in combination with non-drug therapies or treatments, such as plasma exchange and PCI.
  • non-drug therapies or treatments such as plasma exchange and PCI.
  • the currently available dosage forms of the known therapeutic agents and the uses of non-drug therapies for use in such combinations will be suitable.
  • Combination therapy includes the administration of a vWF aptamer and at least a second agent or treatment as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents or treatments.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agent or treatments.
  • Administration of these therapeutic agents or treatments in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • Combination therapy may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents or treatments as part of separate monotherapy regimens that incidentally and arbitrarily results in the combinations of the present invention.
  • Combination therapy is intended to embrace administration of the therapeutic agents or treatments in a sequential manner. That is, wherein each therapeutic agent or treatment is administered at a different time, as well as administration of these therapeutic agents or treatments, or at least two of the therapeutic agents or treatments, in a substantially simultaneous manner.
  • Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single injection having a fixed ratio of each therapeutic agent or in multiple, single injections for each of the therapeutic agents.
  • each therapeutic agent or treatment can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, subcutaneous, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents or treatments can be administered by the same route or by different routes.
  • a first therapeutic agent or treatment of the combination selected may be administered by injection while the other therapeutic agents or treatments of the combination may be administered subcutaneously.
  • all therapeutic agents or treatments may be administered subcutaneously or all therapeutic agents or treatments may be administered by injection.
  • the sequence in which the therapeutic agents or treatments are administered is not critical unless noted otherwise.
  • Combination therapy also can embrace the administration of the therapeutic agent or treatments as described above in further combination with other biologically active ingredients.
  • the combination therapy comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co- action of the combination of the therapeutic agent and non-drug treatment is achieved.
  • the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agent, perhaps by days or even weeks.
  • compositions may be administered in combination with other drugs or therapies.
  • the compositions of the invention may be used in combination with plasma exchange, corticosteroids, immunosuppressives, aspirin, clopidogrel, or aspirin and clopidogrel for use in treating TMAs.
  • the compositions may be administered in combination with aspirin, clopidogrel, or aspirin and clopidogrel for use in treating ACS and TMAs.
  • the compositions may be administered in combination with antibiotics for use in treating HUS.
  • the compositions are also compatible with other standard hypersensitivity regimens, such as corticosteroids and antihistamines. REVERSAL AGENTS
  • the invention further relates to agents that reverse the effects of the vWF aptamers, referred to herein as "vWF reversal agents".
  • a vWF reversal agent is 10-15 nucleotides in length. However, there are no limits to the length of the reversal agent.
  • a vWF reversal agent binds to a vWF aptamer.
  • a vWF reversal agent may bind to a full length vWF aptamer or a fragment thereof. Such binding may be through ionic interactions, covalent bonding, complementary base pairing, hydrogen bonding, or any other type of chemical bond. Preferably, such binding is via complementary base pairing.
  • a vWF reversal agent acts by hybridizing to a vWF aptamer, thereby disrupting the vWF aptamer' s structure and preventing the binding of the vWF aptamer to vWF.
  • a vWF reversal agent may be a ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic and deoxyribonucleic acid.
  • a vWF reversal agent is single stranded.
  • a vWF reversal agent comprises all 2'-0 Methyl residues and a 3'- inverted deoxythymidine.
  • a vWF reversal agent may contain any nucleotides, modified or unmodified, along with any other 3 ' or 5 ' modifications that may be found on aptamers.
  • vWF reversal agents include, but are not limited to: SEQ ID NO: 1
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mTmGmTmGmTmCmTmTmAmGmGmTmCmCmC-3T (SEQ ID NO: 6), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mCmTmTmAmGmGmTmCmCmC-3T (SEQ ID NO: 7), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mAmTmGmTmGmTmCmTmTmA-3T (SEQ ID NO: 8), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below:
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mGmGmGmAmCmAmTmGmTmG-3T (SEQ ID NO: 10), where "3T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • the vWF reversal agent is a nucleic acid comprising the structure set forth below: mGmGmGmAmCmAmTmGmTmGmTmCmTmTmA-3T (SEQ ID NO: 11), where "3 T” is an inverted deoxythymidine and "mN” is a 2'-0 Methyl containing residue.
  • ARC22745 is 2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe- guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ')-2 ' -OMe- thymidylyl-(3' ⁇ 5')-2'-OMe-cytidylyl-(3' ⁇ 5')-2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe- thymidylyl-(3 ' ⁇ 5 ' )-2 '-OMe- thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3
  • ARC22744 is 2'-OMe-cytidylyl-(3' ⁇ 5')-2'-OMe- thymidylyl-(3' ⁇ 5')-2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe- guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ')-2 ' -OMe- cytidylyl-(3' ⁇ 5')-2'-OMe-cytidylyl-(3' ⁇ 5')-2'-OMe-cytidylyl-(3' ⁇ 5')-2
  • ARC22743 The chemical name of ARC22743 is 2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe- thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe- guanylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-cytidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe- thymidylyl-(3' ⁇ 5')-2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-(3'
  • ARC22742 The chemical name of ARC22742 is 2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe- cytidy Iy l-(3 ' ⁇ 5 ')-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ')-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe- guanylyl-(3' ⁇ 5')-2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe- thymidylyl-(3' ⁇ 5 ⁇ )-2'-OMe- thymidylyl-(3' ⁇ 5 ⁇ )-2'-OMe- thymidylyl-(3' ⁇ 5 ⁇ )-2'-OMe- thymidylyl-(3' ⁇ 5 ⁇
  • ARC22741 is 2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe- guanylyl-(3' ⁇ 5')-2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe-cytidylyl- (3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe-thymidylyl-(3' ⁇ 5')-2'-OMe-guanylyl- (3 ' ⁇ 5 ')-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl- (3 ' ⁇ 5 ')-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl-(3 ' ⁇ 5
  • ARC22740 is 2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe- guanylyl-(3' ⁇ 5')-2'-OMe-guanylyl-(3' ⁇ 5')-2'-OMe-adenylyl-(3' ⁇ 5')-2'-OMe-cytidylyl- (3 ' ⁇ 5 ')-2 ' -OMe-adenylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ')-2 ' -OMe-guanylyl- (3 ' ⁇ 5 ')-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 ' -OMe-guanylyl- (3 ' ⁇ 5 ')-2 ' -OMe-thymidylyl-(3 ' ⁇ 5 ' )-2 '
  • the invention also includes vWF reversal agents that have 70% identity or more to SEQ ID NOs: 6, 7, 8, 9, 10 or 11.
  • the vWF reversal agents may have 70, 75, 80, 85, 90, 95 or 100% identity to one of SEQ ID NOs: 6, 7, 8, 9, 10 or 11.
  • the invention also includes pharmaceutical compositions containing vWF reversal agents that bind to vWF aptamers.
  • the compositions include an effective amount of a pharmacologically active vWF reversal agent or a pharmaceutically acceptable salt thereof, alone or in combination, with one or more pharmaceutically acceptable carriers.
  • the compositions may contain one or more different v WF reversal agents.
  • the vWF reversal agents are administered to subjects in an amount effective to reverse the therapeutic effect of the vWF aptamer.
  • the compositions may be administered by numerous routes of administration, such as, for example, topically, intranasally or parenterally.
  • the dosage regimen for a vWF reversal agent will depend on a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration, the renal and hepatic function of the patient; the amount of vWF aptamer used to treat a patient; and the particular vWF reversal agent or salt thereof employed.
  • An ordinary skilled physician or veterinarian can readily determine and prescribe the effective amount of the vWF reversal agent required to reverse the therapeutic effect of a vWF aptamer.
  • the pharmaceutical compositions may also be packaged in a kit.
  • the kit will contain the composition, along with instructions regarding administration of the vWF aptamer.
  • the kit may also contain one or more of the following: a syringe, an intravenous bag or bottle, the same vWF aptamer in a different dosage form or another vWF aptamer.
  • the kit may contain both an intravenous formulation and a subcutaneous formulation of a vWF aptamer of the present invention.
  • the kit may contain lyophilized vWF aptamer and an intravenous bag of solution.
  • the kit form is particularly advantageous when the separate components must be administered in different dosage forms (i.e., parenteral and oral) or are administered at different dosage intervals.
  • the kit may further comprise a vWF reversal agent, along with instructions regarding administration of the reversal agent.
  • the kit may contain both an intravenous formulation and a subcutaneous formulation of the vWF reversal agent.
  • the kit may contain lyophilized vWF reversal agent and an intravenous bag of solution.
  • the kits are stored at 5 ⁇ 3°C.
  • the kits can also be stored at room temperature or frozen at -20 0 C.
  • ARC13090 is the aptamer described in SEQ ID NO: 1.
  • ARC15104 is the aptamer described in SEQ ID NO: 4.
  • ARC15105 is the aptamer described in SEQ ID NO: 5.
  • ARC14303 which is the aptamer described in SEQ ID NO: 2, has the same nucleotide sequence as ARC13090, but it also has a biotin derivative conjugated to its 5' end.
  • ARCl 5103 which is the aptamer described in SEQ ID NO: 3, has the same nucleotide sequence as ARC 13090, but it also has an amine group conjugated to its 5' end.
  • ARC13090 is the core aptamer sequence for each of ARC14303, ARC15103, ARC15104 and ARC15105.
  • ARC1779 which is described in U.S. Application Serial No. 12/156,212, filed May 30, 2008 and published as US 20090203766, is an anti-vWF aptamer that has a different nucleotide sequence than any of the above aptamers.
  • ARCl 5104, ARC15105 and ARC 1779 were each incubated with the plate-bound vWF. Aptamer binding to vWF was determined with either streptavidin or an anti-PEG antibody.
  • purified vWF (EMD BIOSCIENCES, Philadelphia, PA) protein was dissolved in Dulbecco's PBS (dPBS) buffer at a concentration of 10 ⁇ g/ml and 100 ⁇ l solution was added to a Nunc Maxisorp (NUNC #446612) 96-well plate. The plate was incubated overnight at 4°C.
  • the plate was then washed three times, each with 200 ⁇ l dPBS, followed by blocking with 5% BSA in dPBS at room temperature for 30 minutes.
  • the blocked plate was then washed three times with 200 ⁇ l dPBS each before serially diluted aptamers were added into the plate and incubated at 37°C for 2 hours.
  • the plate was then again washed three times with 200 ⁇ l dPBS each.
  • the plate was then washed three times with 200 ⁇ l dPBS before adding 100 ⁇ l of anti-Rabbit HRP (Cell Signaling Technology, #101 A) in dPBS with 1% BSA for 45 minutes at room temperature.
  • HRP Cell Signaling Technology, #101 A
  • lOO ⁇ L of StreptAvidin-HRP diluted 1 :200 in dPBS was added and incubated at room temperature for 1 hour.
  • 100 ⁇ l of Ultra-TMB (Pierce, #34028) solution was added to each well and incubated at room temperature for 5 to 10 minutes.
  • vWF EMD BIOSCIENCES, Philadelphia, PA
  • dPBS Dulbecco's PBS
  • Nunc Maxisorp NUNC #446612
  • the plate was incubated overnight at 4°C. The plate was then washed three times, each with 200 ⁇ l dPBS, followed by blocking with 5% BSA in dPBS at room temperature for 30 minutes.
  • the plate was then washed three times with 200 ⁇ l dPBS before adding 100 ⁇ l of anti-Rabbit HRP (Cell Signaling Technology, #7074) in dPBS with 1% BSA for 45 minutes at room temperature.
  • HRP Cell Signaling Technology, #7074
  • 100 ⁇ l of Ultra-TMB (Pierce, #34028) solution was added to each well and incubated at room temperature for 5 to 10 minutes.
  • 100 ⁇ L of Stop solution (2 N H 2 SO 4 ) was added to each well and the plate was then read at 450 nm for absorption.
  • Figure 9 illustrates that ARC14303 competes with ARC1779 for binding to vWF, suggesting that the two aptamers have similar or overlapping binding sites.
  • ARC1779, ARC15104 and ARC15105 were tested in a commercially available quantitative direct ELISA kit (READDS ® vWF Activity ELISA test kit, Corgenix, Inc., Riverside, CO) that is designed for detection of vWF activity, defined as the amount of active ("free") vWF with functional Al domain present in human citrated plasma.
  • RTDDS ® vWF Activity ELISA test kit Corgenix, Inc., Riverside, CO
  • Citrated plasma was mixed with ARC15105, ARC15104 and 37°C pre- treated ARC1779 at various concentrations. The test samples were added to the ELISA plate, which was coated with a mouse monoclonal anti-human vWF antibody that recognizes the functional Al domain.
  • HRP horse radish peroxidase
  • TMB 5'-tetramethylbenzidine
  • Figure 10 illustrates that ARCl 5104 and ARC 15105 are equally active as
  • ARC15105 and ARC15104 were compared to ARC1779 in the platelet functional analyzer assay (PFA-100, Dade Behring, Deerfield, IL).
  • the PFA- 100 assay is a functional assay where platelets are exposed to high sheer flow conditions.
  • the PFA-100 aspirates blood through a capillary from a sample reservoir in a test cartridge with a microscopic aperture cut into a biologically active membrane; the membrane used for these experiments were coated with collagen and adenosine diphosphate (ADP), which induces a platelet plug and closure of the aperture. Aggregation is quantified by measuring the time of aperture occlusion, with a maximum occlusion time of 300 seconds.
  • ARC15103, ARC15104 and ARC15105 were tested in the platelet functional analyzer assay (PFA-100, Dade Behring, Deerfield, IL).
  • the PFA- 100 assay is a functional assay where platelets are exposed to high sheer flow conditions.
  • the PFA-100 aspirates blood through a capillary from a sample reservoir in a test cartridge with a microscopic aperture cut into a biologically active membrane; the membrane used for these experiments were coated with collagen and adenosine diphosphate (ADP), which induces a platelet plug and closure of the aperture. Aggregation is quantified by measuring the time of aperture occlusion, with a maximum occlusion time of 300 seconds.
  • Citrated whole blood was mixed with ARC15103, ARC15104 and
  • ARC 15105 at various concentrations prior to initiation of the aggregation reaction. The occlusion times were recorded and graphed. As shown in Figure 12, both ARC 15104 and ARC 15105 were as potent as ARCl 5103 (the unPEGylated molecule) in this assay.
  • Venous blood was taken from volunteers and anti-coagulated so that the platelets could be isolated and radiolabeled.
  • Four plexiglass perfusion chambers that mimic the tube-like cylindrical shape of blood vessels were used. Each chamber contained a window (1 mm ID), allowing the direct exposure of arterial segments to the blood containing In-111 radio-labeled platelets.
  • the arterial segments were prepared from porcine arteries, which were dissected free of surrounding tissues, cut into rings, and longitudinally opened. Injured arterial segments were prepared by lifting and peeling off the intima in order to expose the subjacent media. The thrombogenic properties of these damaged arterial surfaces, which are related to platelet activation and adhesion, were demonstrated previously.
  • the flow within the chambers was adjusted to 40 ml/min with a peristaltic pump.
  • the chambers were placed in parallel (two per side) in a thermostatically controlled water bath at 37 0 C, thus permitting simultaneous parallel, pair wise perfusion over arterial tissues of treated or untreated blood at high shear (6974/sec in 1 mm ID chambers).
  • the control groups were not exposed to drug, while the experimental groups were exposed to either ARCl 5105 or ARC 1779 at various concentrations.
  • Blood was perfused and re-circulated over the arterial segments for 15 minutes in the flow chambers. Blood was pre-treated for 10 minutes with aptamer before the perfusion.
  • the surfaces were immediately fixed in tissue fix and counted in a gamma counter to calculate the level of platelet adhesion, followed by scanning electron microscope for determination of thrombus mass.
  • ARC22744, and ARC22745 were tested using the platelet functional analyzer assay (PFA-100).
  • the PFA-100 assay is a functional assay where platelets are exposed to high sheer flow conditions. Aggregation is quantified by measuring the time of aperture occlusion, with a maximum occlusion time of 300 seconds.
  • Reversal agents were added to this mixture at various concentrations prior to initiation of the aggregation reaction, and occlusion times were recorded and graphed. As shown in Figure 15, five reversal agents reduced the occlusion time for at least two of the concentrations tested; and one reversal agent, ARC22740, did not reduce the occlusion time with 100 nM ARC15105, but did reduce the occlusion time with 50 nM ARC15105 as noted in Figure 15. Of the six reversal agents, ARC22742, ARC22744 and ARC22745 required the least amount of added reversal agent to completely reverse ARC15105.

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Abstract

L'invention concerne d'une manière générale le domaine des acides nucléiques et, plus particulièrement, les aptamères qui se lient au facteur de von Willerbrand (vWF), qui sont utiles en tant que produits thérapeutiques et en tant que produits de diagnostic de maladies cardiovasculaires, de maladies thrombotiques et de maladies hématologiques et/ou autres maladies ou troubles dans lesquels vWF a été impliqué. De plus, les aptamères peuvent être utilisés avant, pendant et/ou après des actes médicaux afin de réduire les complications ou les effets secondaires de ceux-ci. L'invention concerne également des matières et des procédés pour l'administration d'aptamères qui se lient à vWF. L'invention concerne en outre des agents qui inversent les effets des aptamères.
PCT/US2010/023599 2009-02-09 2010-02-09 Aptamères dirigés contre le facteur de von willerbrand et leur utilisation en tant que produits thérapeutiques pour des maladies thrombotiques, hématologiques et cardiovasculaires Ceased WO2010091396A2 (fr)

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WO2017073536A1 (fr) * 2015-10-30 2017-05-04 タグシクス・バイオ株式会社 Aptamère d'adn capable de se lier au facteur de von willebrand (vwf)
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JPWO2017073536A1 (ja) * 2015-10-30 2018-08-16 タグシクス・バイオ株式会社 ｖＷＦに結合するＤＮＡアプタマー
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JP2020522566A (ja) * 2017-05-19 2020-07-30 バンド セラピューティクス エルエルシーＢａｎｄ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｌｌｃ フォン・ヴィレブランド因子に関連する合併症及び障害の処置のための組成物及び方法
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EP3624801A4 (fr) * 2017-05-19 2021-02-24 Band Therapeutics, LLC Compositions et méthodes pour le traitement de complications et de troubles liés au facteur de von willebrand
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CN110520128A (zh) * 2017-05-19 2019-11-29 邦德治疗有限公司 用于治疗与血管性血友病因子有关的并发症和疾病的组合物和方法
KR20200008122A (ko) * 2017-05-19 2020-01-23 밴드 쎄라퓨틱스, 엘엘씨 폰 빌레브란트 인자와 관련된 합병증 및 장애의 치료를 위한 조성물 및 방법
JP2023093535A (ja) * 2017-05-19 2023-07-04 バンド セラピューティクス エルエルシー フォン・ヴィレブランド因子に関連する合併症及び障害の処置のための組成物及び方法
JP7317805B2 (ja) 2017-05-19 2023-07-31 バンド セラピューティクス エルエルシー フォン・ヴィレブランド因子に関連する合併症及び障害の処置のための組成物及び方法
CN110520128B (zh) * 2017-05-19 2023-11-14 邦德治疗有限公司 用于治疗与血管性血友病因子有关的并发症和疾病的组合物和方法
AU2018269935B2 (en) * 2017-05-19 2024-05-16 Band Therapeutics, Llc Compositions and methods for the treatment of complications and disorders relating to von Willebrand factor
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EP4100121A4 (fr) * 2020-02-04 2024-04-10 Band Therapeutics, LLC Régulation du facteur de von willebrand (vwf)
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EP4251272A4 (fr) * 2020-11-24 2024-10-09 Band Therapeutics, LLC Compositions et méthodes de traitement de troubles hémostatiques

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