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Definitions

• the present invention relates to the field of therapeutic modalities useful in the treatment of complications and disorders relating to von Willebrand factor.
• VWF Von Willebrand Factor
• VWF is essential for the first step in maintaining hemo stasis, i.e., arresting hemorrhage at sites where there has been a break in the integrity of the vasculature.
• VWF makes a molecular bridge between the collagen structural matrix which underlies the vascular endothelium and circulating blood platelets, enabling platelets to adhere to sites of vessel damage and initiate formation of thrombus in order to arrest hemorrhage.
• This essential function of VWF is performed by high molecular weight multimers of individual VWF molecules, and these multimers are activated to bind platelets when they are exposed to hemodynamic shear forces (in a manner analogous to uncoiling a rope).
• VAD ventricular assist device
• the hemodynamic shear forces produced by the continuous flow pump design can cause a defect in the function of the coagulation system known as Acquired Von Willebrand Syndrome (aVWS) and, as a result, a substantial incidence of major bleeding, especially from the gastrointestinal (GI) tract. Abnormal blood vessel formation is also observed in these patients and contributes to the bleeding (Suarez J, et al., Mechanisms of bleeding and approach to patients with axial-flow left ventricular assist devices. Circ. Heart Fail. 201 1 4:779-84 and references therein).
• aVWS Acquired Von Willebrand Syndrome
• LVADs left ventricular assist devices
• Starling et al. who have observed an increase in the rate of thrombosis in the VAD instrument among patients who received the HeartMate IITM LVAD.
• Starling RC, et al. Unexpected Abrupt Increase in Left Ventricular Assist Device Thrombosis, 2014, N. Engl. J Med., 370:33-40.
• Others have reported similar findings with LVADs. Capoccia M, et al., Recurrent Early Thrombus Formation in Heartmate II Left Ventricular Assist Device, 2013, J. Inv. Med.,
• Thrombus development in the coronary and cerebral arteries is the most common cause of mortality and morbidity worldwide, precipitating diseases such as myocardial infarction and ischemic stroke, respectively.
• Current thrombolytic treatments target only one constituent of the thrombus, fibrin, and are therefore only partially effective in dissolving arterial and platelet-rich thrombi.
• platelet GpIIb/IIIa inhibitors treatment that seeks to disrupt the platelet - fibrin interaction
• platelet GpIIb/IIIa inhibitors is effective for preventing initial thrombus formation and dissolving fresh platelet aggregates
• disruption does not significantly prevent thrombus formation in pre-occluded arteries having very high shear rates or have a significant impact on the patency of pre-occluded arteries (vessels that are at least 50% occluded).
• Another disorder associated with VWF interactions is sickle cell disease.
• Sickle cell disease results from inheritance of a mutant ⁇ -globin allele (Glu6Val), either as 2 copies or with an allele specifying deficient or defective ⁇ -globin, yielding rigid, adhesive, lysis-prone erythrocytes.
• Glu6Val mutant ⁇ -globin allele
• These properties account for the clinical manifestations of sickle cell disease, including chronic hemolytic anemia and episodic vaso-occlusion, affecting many organs. Patients with the highest hemolytic rates are at risk for a syndrome composed of pulmonary artery hypertension, priapism, and leg ulcers.
• Several adhesive molecules have been implicated in sickle vaso-occlusion, including VWF. Acutely activated endothelial cells can release massive quantities of very large and hyperadhesive VWF molecules which are capable of spontaneously binding erythrocytes, especially sickle cells.
• VAD-associated acquired Von Willebrand Syndrome (aVWS)
• no known cure is currently available (See: Suarez J, et al., Mechanisms of Bleeding and Approach to Patients with Axial-Flow Left Ventricular Assist Devices . Circulation: Heart Failure 201 1; 4(6):779-784).
• Current treatments involve administration of additional clotting factors or concentrated VWF, each of which carries significant risk.
• VAD-associated pump thrombosis See, for example, Fig. 2 in Starling RC, et al., Unexpected Abrupt Increase in Left Ventricular Assist Device Thrombosis, 2014, N. Engl. J Med., 370:33-40, which shows thrombus formation in LVAD.
• no therapies have been proposed or adopted to prevent the incidence of thrombosis, other than anticoagulant therapies.
• agents that antagonize platelet aggregation or cross- linking during closure (occlusion) of a vessel lumen i.e., under very high shear rates, such as those found in VAD patients and/or subjects with pre-occluded vessels
• agents that are able to disaggregate or disrupt already-formed occlusive thrombi in a vessel may antagonize the interaction between VWF and platelets, thereby restoring vessel patency after occlusive thrombosis by disaggregating existing thrombi.
• agents are useful for the treatment of cardiovascular and coronary diseases, such as myocardial infarction and ischemic stroke.
• Such agents may also be useful to discourage the development of occlusive thrombi during VAD treatment.
• VAD vascular assist device
• cardiovascular and/or coronary diseases such as stroke.
• the present disclosure provides, among other things, antagonists of the VWF- platelet interaction and of the VWF-erythrocyte interaction which may occur in circumstances such as sickle cell disease either independently or in combination with VAD treatment, and which may occur in circumstances of platelet aggregation during occlusive thrombus formation in VAD patients or during closure of the lumen of a vessel such as that which may occur in any number of conditions, such as, for example ischemic stroke.
• Such antagonists may serve as VWF protective agents.
• Such antagonists may also serve as therapeutic agents in the regulation and modulation of hemostasis, therapeutic agents for the treatment of vascular assist device (VAD) associated complications and disorders, such as aVWS and occlusive thrombosis, and therapeutic agents for the treatment of disorders arising from interactions of VWF multimers with erythrocytes, such as in sickle cell disease.
• VAD vascular assist device
• agents may serve as therapeutic agents in the treatment of cardiovascular and cerebrovascular disorders, such as myocardial infarction and stroke.
• VWF protective agents comprising synthetic polynucleotides comprising at least 21 contiguous nucleotides of SEQ ID NO: 1, which exhibit a double stranded region having at least 6 base pairs.
• VWF protective agents having a double stranded region of at least 6 base pairs may have greater thermostability at higher temperatures and thereby greater affinity for VWF and increased functionality as compared to protective agents with a shorter double stranded region of 5 base pairs or less. Shorter double stranded regions may lead to unraveling of the stem-loop structure at higher temperatures and associated loss of affinity and functionality.
• the synthetic polynucleotide of the present invention may be 25-30 nucleotides in length.
• the double stranded region may be from about 6 to about 9 base pairs.
• the double stranded region is formed by 6 or more of the 3 ' and 5' nucleotides at or near the termini.
• the synthetic polynucleotides of the present invention may be chemically modified.
• Each nucleotide may contain at least one chemical modification.
• Such chemical modification may be at the sugar, nucleobase or internucleoside linker of the synthetic polynucleotide.
• Modifications to the sugar may consist of a 2' O-methyl modification.
• Terminal cap structures may also be incorporated to the 3 ' and/or 5' termini. Such structures include, but are not limited to at least one inverted deoxythymidine or amino group (Nth). In one aspect, the 3 ' terminal cap comprises an inverted deoxythymidine and the 5' terminal cap comprises an amino group (Nth). In one aspect, the synthetic polynucleotide is SEQ ID NO: 2.
• the VWF protective agents comprising nucleic acid aptamers may be modified by any number of conjugates.
• One such conjugate is a PEG (polyethylene glycol) moiety.
• Such moieties may be of any size or branch configuration.
• the PEG moiety may be conjugated to the 5' terminus of the nucleic acid aptamer.
• the PEG moiety may be conjugated to the 3' terminus of the nucleic acid aptamer.
• the synthetic polynucleotide is SEQ ID NO: 3.
• complementary synthetic polynucleotides which are capable of hybridizing with, or binds to, in whole or in part any of the VWF protective agents described above.
• the complementary synthetic polynucleotide may comprise at least 12 contiguous nucleotides of SEQ ID NO: 4.
• the complementary synthetic polynucleotide may comprise at least 15 contiguous nucleotides of SEQ ID NO: 4.
• the complementary synthetic polynucleotide may comprise at least 18-22 contiguous nucleotides of SEQ ID NO: 4.
• the complementary synthetic polynucleotides may be chemically modified.
• each of the nucleotides contains at least one chemical modification.
• the chemical modification may be a 2'-O-methyl modification to a nucleotide sugar.
• the complementary synthetic polynucleotides may further comprise a 3' terminal cap. In one aspect, the 3' terminal cap may be an inverted deoxythymidine.
• the complementary synthetic polynucleotide comprises SEQ ID NO: 5.
• VWF protective agents may function to mitigate this loss in or reduction of function.
• the synthetic polynucleotides or complementary synthetic polynucleotides described herein in preparation of a medicament for the treatment of a disease, disorder or complication relating to VWF.
• the disorder is a thrombotic disorder.
• the thrombotic disorder is ischemic stroke.
• the synthetic polynucleotides or complementary synthetic polynucleotides described herein in preparation of a medicament for the treatment of a VAD associated complication or disorder.
• the complication or disorder may be acquired Von Willebrand Syndrome (aVWS), angiodysplasia, occlusive thrombosis, or VAD-associated pump thrombosis.
• complication or disorder which comprises aberrant binding of VWF to erythrocytes.
• the complication or disorder may be sickle cell disease.
• GI gastrointestinal
• Such method includes contacting the subject with a VWF protective agent such that the one or more occlusive thrombi are disaggregated.
• the agent disaggregates the external layer of the occlusive thrombi.
• the occlusive thrombi are formed under conditions of shear rates that are 10,000 s "1 or greater.
• the occlusive thrombi are resistant to fibrinolysis and/or antithrombotic agents. Diseases or disorders arising from the formation of occlusive thrombi may include an acute coronary syndrome, acute occlusion thrombosis, and ischemic stroke.
• the synthetic polynucleotide may be administered via intravenous injection or subcutaneous injection.
• the bioavailability of subcutaneous injection is at least 85% relative to intravenous injection.
• the bioavailability of subcutaneous injection is at least 95% relative to intravenous injection.
• the synthetic polynucleotide may be administered at a dose of from about 0.01 mg/kg to about 0.5 mg/kg of the subject body weight.
• the synthetic polynucleotide may have a plasma half-life of from about 70 hours to about 100 hours. In some embodiments, the synthetic polynucleotide may not function as an anticoagulant. The synthetic polynucleotide may not prolong clotting time and/or alter thrombin generation. In one aspect, the thrombotic disorder or complication is myocardial infarction. In other aspect, the thrombotic disorder or complication is ischemic stroke.
• thrombotic disorders and complications thereof comprising contacting a patient diagnosed with said disorder or complication with any of the VWF protective agents of the invention.
• the thrombotic disorder is ischemic stroke.
• a VAD associated complication or disorder such as, for example, aVWS and occlusive thrombosis
• methods of treating a VAD associated complication or disorder comprising contacting a patient diagnosed with said complication or disorder with any of the VWF protective agents of the invention.
• the thrombosis occurs in the VAD instrument.
• occlusive thrombosis occurs in the lumen of a
• the present invention provides certain reversal agents.
• erythrocytes are sickle cells.
• the treatment of such diseases or disorders may be in combination with a VAD treatment or independent of a VAD treatment.
• a complication or disorder which comprises gastrointestinal bleeding associated with angiodysplasia with a VWF protective agent in combination with a VAD treatment or independent of a VAD treatment.
• occlusive thrombus formation can occur in one or more of the patient's vessels. In another embodiment, thrombus formation can occur in the VAD instrument itself. Additionally, provided are methods for disaggregating one or more thrombi in a VAD patient comprising contacting a VAD patient with a VWF protective agent. Further provided are methods for decreasing the rate of thrombosis in a VAD patient comprising contacting a VAD patient with a VWF protective agent such that the rate of thrombosis is decreased relative to the rate of thrombosis in an untreated VAD patient.
• a complication or disorder which comprises occlusive thrombus formation associated with very high shear rate (>10,000 s "1 ), such as the shear rate found in an occluded vessel, with a VWF protective agent.
• the occlusive thrombus formation can occur in one or more of the subject's vessels.
• restoring vessel patency in a subject having one or more occluded vessels comprising contacting said subject with a VWF protective agent.
• methods for disaggregating occlusive thrombi in one or more occluded vascular vessels comprising contacting a subject having at least one occluded vessel with a VWF protective agent.
• methods for decreasing the rate of vessel occlusion in a subject comprising contacting said subject with a VWF protective agent such that the rate of vessel occlusion is decreased relative to the rate of vessel occlusion in an untreated subject.
• the one or more vessels are at least 50% occluded.
• the agent disaggregates the external layer of the occlusive thrombi.
• the occlusive thrombi are formed under conditions of shear rates that are 10,000 s "1 or greater.
• the occlusive thrombi are resistant to fibrinolysis and/or antithrombotic agents.
• the disease or disorder is acute occlusion thrombosis or an acute coronary syndrome, including, for example, myocardial infarction and unstable angina.
• the disease or disorder is ischemic stroke.
• CNS central nervous system
• GI gastrointestinal
• Fig. 1 is a diagram illustrating three aptamer constructs.
• BT-100 SEQ ID NO: 2 of the present invention is shown for comparison to PRIOR ART compounds ARC15105 (SEQ ID NO: 6) and ARC1779 (SEQ ID NO: 7).
• Fig. 2 is a diagram illustrating a BT-100 reversal agent.
• Fig. 2 discloses SEQ ID NOs 3 and 12, respectively, in order of appearance.
• Fig. 3 shows a gel of Von Willebrand Factor multimers and the effects of BT-100 on protection of multimers in human plasma from proteolytic degradation induced by high shear conditions.
• Fig. 4 is a schematic drawing depicting the sequential steps of thrombus formation.
• Fig. 4A shows the initial stage of thrombus formation at ⁇ 50% occlusion under physiological shear stress with platelet-platelet cross-linking involving mainly GpIIb/IIIa interaction.
• Fig. 4B shows the next step of thrombus formation at >50% occlusion under very high shear stress with platelet-platelet cross-linking involving mainly Gplba-VWF interaction.
• Fig. 4C shows the next step of fully occlusive thrombosis (100% occluded) which in the early stages is resistant to GpIIb/IIIa inhibitors and sensitive to inhibitors of the Gplba-VWF interaction.
• a VWF antagonist should prevent and/or ameliorate VAD-associated complications and/or disorders or side effects, such as aVWS and occlusive thrombosis.
• VTP Thrombotic Thrombocytopenic Purpura
• TTP Thrombotic Thrombocytopenic Purpura
• Administration of a VWF antagonist to patients with TTP has been demonstrated to correct thrombocytopenia, a hallmark of this disease, and clinical experiments to demonstrate improvement of clinical outcome by this means are underway.
• VWF antagonism is in treatment of a rare, inherited disorder known as Von Willebrand Disease Type 2b (VWD Type 2b).
• VWD Type 2b a rare, inherited disorder known as Von Willebrand Disease Type 2b
• Patients with this disease have a mutation in the Al domain of VWF that renders it constitutively active, eliminating the normal requirement for activation of VWF by hemodynamic shear force.
• VWD Type 2b a rare, inherited disorder known as Von Willebrand Disease Type 2b
• thrombocytopenic purpura thrombocytopenic purpura.
• Thromb Haemost 2011 105(3):545-552; US Publication 2009/0203766; International Publication WO2010/091396 and Jilma-Stohlawetz P, et al.
• the anti-von Willebrand factor aptamer ARC 1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease.
• VWS Von Willebrand Syndrome
• exogenous VWF from donor plasma concentrates that contain VWF or from recombinant sources (not yet commercially available) could transiently restore high molecular weight VWF multimer levels.
• administration of exogenous VWF would not correct the underlying problem resulting from the causal chain starting with mechanically-induced elevated shear force causing inappropriate activation of VWF, in turn causing excessive platelet binding to VWF in turn causing exposure of VWF to proteolysis which results in a deficiency of functional VWF multimers.
• the next best way to interrupt this causal chain would be to preserve the multimers themselves as proposed herein, by any of several ways including, but not limited to, blocking the binding of platelets to the shear-force-activated and partially uncoiled VWF multimers.
• VWD Type 2b Thrombotic Thrombocytopenic Purpura or Von Willebrand Disease Type 2b
• VWD Type 2b Von Willebrand Disease Type 2b
• VWF represents a therapeutic target to ameliorate hemolysis in sickle cell disease, either in combination with VAD treatment or independently of VAD treatment.
• VWF has been implicated in a number of processes in the vasculature, including the vascular inflammation and smooth muscle cell proliferation.
• VWF is also known to be a negative regulator of angiogenesis.
• angiogenesis is defined as the formation of new blood vessels. VWF has been found to act as a "brake” by constraining angiogenesis, through control of VEGFR2 signaling (Starke RD, et al., Endothelial von Willebrand factor regulates angiogenesis. Blood, 2011, 1 17: 1071-80; the contents of which are each incorporated herein by reference in their entirety).
• Angiodysplasia as used herein, is defined as vascular malformations in the GI tract which are associated with dysregulated angiogenesis.
• VWF systemic depletion of VWF through proteolysis of VWF multimers caused by exposure to the hemodynamic shear forces may lead to a reduction of VWF in the vascular endothelium of the GI tract and result in angiodysplasia in VAD patients.
• functional VWF may be critical to maintaining healthy vascular endothelium in the GI tract.
• the present invention provides VWF agents compositions and methods which prevent depletion of VWF in the vascular endothelium.
• these compositions prevent formation of vascular malformations in the GI tract and GI bleeding in VAD patients.
• head-to-head studies described herein the inventors demonstrate that protection of VWF multimers is provided by an aptamer distinct from those in the art.
• GpIIb/IIIa platelet glycoprotein Ilb/IIIa
• plasma proteins mediate platelet cross-linking in arterial thrombi.
• GpIIb/IIIa inhibitors do not significantly impact the patency of pre-occluded arteries, failing to disperse platelet aggregates after myocardial infarction or ischemic stroke (Mehilli J, et al., Abciximab in patients with acute ST-segment- elevation myocardial infarction undergoing primary percutaneous coronary intervention after clopidogrel loading: a randomized double-blind trial . Circulation.
• glycoprotein-receptor-Ib in focal cerebral ischemia an MRI study at 17.6 Tesla.
• occlusive thrombus formation occurs in three distinct stages of development, in which each stage is subjected to different local shear rates and regulated by different mechanisms, resulting in three different regions of occlusive thrombi as follows: (1) the base of the thrombus that is exposed to shear rates below 1,500 s "1 throughout the entire thrombotic process (fibrin); (2) the core of the thrombus, corresponding to the portion of the thrombus formed during the first steps of development (0% to ⁇ 50% occlusive thrombus formation) at shear rates between about 1500 s "1 and about 10,000 s "1 (see Fig.
• the core of the thrombus is primarily made up of platelets cross-linked by GpIIb/IIIa- dependent interactions, whereas formation of the external layer of the thrombus is dependent upon Gplba-VWF interactions.
• the present disclosure provides VWF agents, compositions and methods which disaggregate occlusive thrombi formed under very high shear rates, such as shear rates found in pre-occluded vessels or in VAD treatment.
• the VWF agents dissolve occlusive thrombi containing platelet aggregates formed under shear rates of 10,000 s "1 or greater, i.e., those found in the external layer of the occlusive thrombus.
• These agents and compositions restore vessel patency in patients having acute occlusion thrombosis or acute coronary syndrome, including, for example, myocardial infarction and unstable angina.
• the agents and compositions restore vessel patency in patients who have suffered an ischemic stroke.
• the agents and compositions restore vessel patency in VAD patients.
• the agents and compositions disaggregate one or more thrombi in the VAD instrument.
• the present disclosure also provides VWF agents, compositions and methods which slow the development of occlusive thrombi formed under very high shear rates, such as shear rates found in pre-occluded vessels or in VAD treatment.
• the VWF agents slow the development of occlusive thrombi containing platelet aggregates formed under shear rates of 10,000 s "1 or greater, i.e., those found in the external layer of occlusive thrombi.
• the agents and compositions slow the development of thrombi in the VAD instrument.
• the present disclosure also provides VWF agents, compositions and methods for decreasing the rate of vessel occlusion in a subject having one or more vessel(s) that are at least 50% occluded with one or more occlusive thrombi, the method comprising contacting the subject with a VWF protective agent such that the rate of vessel occlusion is decreased relative to the rate of vessel occlusion in an untreated vessel that is at least 50% occluded.
• the VWF agents, compositions and methods of the present disclosure may be useful for treating or preventing an orphan disease or indication associated with VWF, such as, but not limited to, severe and/or cerebral malaria, and Heyde's Syndrome.
• Severe malaria is a life-threatening condition of Plasmodium falciparum infection where the infection is complicated with serious failure of the body's major organs. Sometimes severe malaria is associated with coma and is known as cerebral malaria. Elevated levels of VWF, VWF propeptide (markers of chronic and acute endothelial cell activation,
• VWF plays an active role in modulating malaria pathogenesis, possibly by sequestering platelets, recruiting malaria-infected red blood cells, inducing increased endothelial cell permeability, and eventually causing central nervous system (CNS) thrombosis (O'Regan N, et al. A novel role for von Willebrand factor in the pathogenesis of experimental cerebral malaria. Blood. 2016;127(9): 1192-201; Montgomery, R.R., The heads and the tails of malaria and VWF. Blood, 2016. 127(9): p. 1081-2; the contents of which each are incorporated herein by reference in their entirety).
• CNS central nervous system
• Heyde's Syndrome is a syndrome of gastrointestinal bleeding from angiodysplasia in the presence of aortic stenosis.
• Loss of high-molecular- weight multimers of VWF has been identified as a link between GI bleeding and aortic stenosis (Warkentin TE, et al. Aortic stenosis and bleeding gastrointestinal angiodysplasia: is acquired von Willebrand's disease the link? Lancet. 1992;340(8810):35-7), and has been reported to be present in 67-92% of patients with severe aortic stenosis (Vincentelli A, et al. Acquired von Willebrand syndrome in aortic stenosis. N Engl J Med. 2003 Jul 24;349(4):343-9; the contents of which are
• VWF activity is significantly correlated with the severity of stenosis and the rate of bleeding (Blackshear JL, et al. Indexes of von Willebrand factor as biomarkers of aortic stenosis severity (from the Biomarkers of Aortic Stenosis Severity [BASS] study) . Am J Cardiol. 2013;11 1(3):374-81 ; the contents of which are incorporated herein by reference in their entirety). It is thought that high shear stress caused by valve obstruction leads to changes in the structure of VWF multimers, increasing their proteolysis by the protease ADAMTS13 (Vaz A, et al. Heyde syndrome— the link between aortic stenosis and gastrointestinal bleeding. Rev Port Cardiol. 2010 Feb;29(2):309-14;
• VWF agents described herein can protect the loss of VWF multimers and/or antagonizes the interaction of VWF with platelets, thereby preventing GI bleeding in patients with Heyde's Syndrome.
• VADs ventricular assist devices
• LVADs left VAD or LVAD
• aVWS acquired Von Willebrand Syndrome
• aVWS acquired Von Willebrand Syndrome
• aVWS acquired Von Willebrand Syndrome
• aVWS acquired Von Willebrand Syndrome
• ischemic events as well as sickle cell disease, severe and/or cerebral malaria, and Heyde's Syndrome, either in combination with VAD treatment or independently of VAD treatment.
• the VWF protective agent may be used to treat GI bleeding associated with angiodysplasia. In some embodiments, the VWF protective agent may be used to treat a VAD patient with GI bleeding.
• the VWF protective agent may interact with or bind to VWF monomers or multimers in a way that preserves, protects or prevents the loss, reduction or destruction of VWF multimers to restore VWF mediated constraint on angiogenesis in vascular endothelial cells and prevent neovascularization in a VAD patient.
• the VWF protective agent can be used to restore vessel patency in a subject having one or more pre-occluded vessels, such as subjects with acute occlusion thrombosis or acute coronary syndrome, including, for example, myocardial infarction and unstable angina, or ischemic stroke.
• the compounds and/or compositions interact with or bind to VWF in a way that antagonizes the interaction of VWF with platelets such that occlusive thrombi in pre-occluded vessels (vessels that are at least 50% occluded) are disaggregated.
• the VWF protective agent disaggregates the external layer of the occlusive thrombi.
• the occlusive thrombi that are disaggregated are formed under conditions of shear rates of 10,000 s "1 or greater (that is, the occlusive thrombi contain platelet aggregates formed under very high shear rates of 10,000 s "1 or greater).
• the occlusive thrombi are resistant to fibrinolysis and/or antithrombotic agents.
• the VWF protective agents can be used to restore vessel patency in a subject undergoing VAD treatment.
• the VWF protective agent can be used to decrease the rate of vessel occlusion in a subject comprising contacting said subject with a VWF protective agent such that the rate of vessel occlusion is decreased relative to the rate of vessel occlusion in an untreated vessel.
• the VWF protective agents can be used to decrease the rate of vessel occlusion in a subject undergoing VAD treatment.
• occlusive thrombus refers to a thrombus or thrombi containing platelet aggregates formed under very high shear rates (10,000 s "1 or greater). Such occlusive thrombus or thrombi are formed during the closure of the vessel lumen, i.e., when the lumen is at least 50% occluded.
• pre-occluded vessel(s) or “occluded vessel(s)” refers to a vessel(s) that is at least 50% occluded and can include fully occluded vessel(s).
• the term “fully occluded vessel(s)” refers to a vessel(s) that is 100%) occluded or, in other words, a vessel with a closed lumen.
• thrombotic disorder refers to any disease or disorder characterized by abnormal thrombus formation.
• the abnormal thrombus formation characterizing the disorder is the formation of one or more occlusive thrombi.
• Non- limiting examples of thrombotic disorders include von Willebrand's Disease, ischemic stroke, acute coronary syndrome, myocardial infarction, transient ischemic attack, thrombotic thrombocytopenic purpura, and thrombotic microangiopathies.
• a thrombotic disorder may occur either in combination with VAD treatment or independently of VAD treatment.
• the thrombotic disorder is ischemic stroke.
• the VWF protective agent can be used to treat an orphan disease associated with VWF abnormality, such as, but not limited to, severe and/or cerebral malaria, and Heyde's Syndrome.
• the VWF protective agent can be used to antagonize the interaction of VWF with platelets in a subject with severe and/or cerebral malaria such that the CNS thrombus formation is prevented or reduced.
• the VWF protective agent can be used to treat GI bleeding associated with Heyde's Syndrome.
• the VWF protective agent may prevent or reduce the loss of VWF multimers and/or antagonize the interaction of VWF with platelets, thereby preventing the GI bleeding in the subject with Heyde's Syndrome.
• the VWF protective agents of the invention comprise an aptamer.
• an "aptamer” is a biomolecule that binds to a specific target molecule and modulates the target's activity, structure, or function.
• An aptamer of the present invention may be nucleic acid or amino acid based. Nucleic acid aptamers have specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Nucleic acid aptamers, like peptides generated by phage display or monoclonal antibodies (mAbs), are capable of specifically binding to selected targets and, through binding, block their targets' ability to function. In some cases, aptamers may also be peptide aptamers. As used herein, an "aptamer” specifically refers to either a nucleic acid aptamer or peptide aptamer.
• Aptamers often called “chemical antibodies,” have similar characteristics as antibodies.
• a typical nucleic acid aptamer is approximately 10-15 kDa in size (20-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets.
• Aptamers may be either monovalent or multivalent. Aptamers may be monomeric, dimeric, trimeric, tetrameric or other higher multimeric. Individual aptamer monomers may be linked to form multimeric aptamer fusion molecules.
• a linking oligonucleotide i.e., linker
• a small trimeric or tetrameric (i.e., a Holliday junction-like) DNA nanostructure will be engineered to include sequences complementary to the 3 '-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization.
• 3 to 5 or 5 to 10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors.
• multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
• multimeric aptamer or “multivalent aptamer” refers to an aptamer that comprises multiple monomeric units, wherein each of the monomeric units can be an aptamer on its own. Multivalent aptamers have multivalent binding characteristics. A multimeric aptamer can be a homomultimer or a heteromultimer.
• homomultimer refers to a multimeric aptamer that comprises multiple binding units of the same kind, i.e., each unit binds to the same binding site of the same target molecule.
• heteromultimer that binds to different target molecules can also be referred to as a multi- specific multimer.
• Nucleic acid aptamers comprise a series of linked nucleosides or nucleotides.
• nucleic acid in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.
• Exemplary nucleic acid molecules or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2 '-amino- a-LNA having a 2 '-amino functionalization) or hybrids thereof.
• RNAs ribonucleic acids
• RNA molecule or "ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a
• ribonucleoside includes a nucleoside base and a ribose sugar
• ribonucleotide is a ribonucleoside with one, two or three phosphate moieties.
• ribonucleoside and “ribonucleotide” can be considered to be equivalent as used herein.
• the RNA can be modified in the nucleobase structure, the ribofuranosyl ring or in the ribose- phosphate backbone.
• Nucleic acid aptamers may be ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid. Aptamers may be single stranded ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
• the VWF protective agent may be an aptamer or salt thereof.
• the VWF protective agent may be an aptamer or salt thereof which may comprise synthetic polynucleotides.
• the synthetic polynucleotides may comprise at least 21 contiguous nucleotides of SEQ ID NO: 1. Additionally, the synthetic
• polynucleotides may exhibit a double stranded region having at least 6 base pairs. While not wishing to be bound by theory, shorter double stranded regions of 5 base pairs or less may lead to the unraveling of the stem-loop structure at higher temperatures and the associated loss of affinity and functionality of the protective agents.
• the VWF protective agent may be an aptamer or salt thereof which may comprise synthetic polynucleotides, where the synthetic polynucleotides comprise at least 21 contiguous nucleotides of SEQ ID NO: 1, and where the synthetic polynucleotides comprise a double stranded region having at least 6 base pairs.
• VWF protective agents having a double stranded region of at least 6 base pairs may have greater thermostability at higher temperatures. This increased thermostability at higher temperatures may provide a greater affinity for VWF and increased functionality as compared to protective agents which a shorter double stranded region of 5 base pairs or less.
• the double stranded region of 6 or more base pairs is at or near (e.g., within 1-10 nucleotides) the termini of the synthetic polynucleotide.
• nucleic acid aptamers comprise at least one chemical modification. Modifications may be used to increase the stability of an aptamer, e.g., by increasing its resistance to degradation by ribonucleases (RNases) present in a cell, thereby causing its half-life in the cell to be increased. Modifications may also or alternatively be used to decrease the likelihood or degree to which aptamer introduced into cells elicit innate immune responses.
• RNases ribonucleases
• RNA interference including small-interfering RNAs (siRNAs), as described in the art, tend to be associated with reduced half-life of the RNA and/or the elicitation of cytokines or other factors associated with immune responses.
• Potential modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, (c) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
• end modifications e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.
• sugar modifications e.g., at the 2' position or 4' position
• modified aptamer compositions useful with the methods described herein include, but are not limited to, nucleic acid molecules containing modified or non-natural internucleoside linkages.
• Modified aptamer having modified internucleoside linkages include, among others, those that do not have a phosphorous atom in the internucleoside linkage.
• modified aptamer has a phosphorus atom in its internucleoside linkage(s).
• 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; 3 ' capping; 5' capping; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; and incorporation of phosphorothioate into the phosphate backbone.
• each nucleotide of the nucleic acid aptamers may contain at least one chemical modification.
• Such chemical modification may be at the sugar, nucleobase or internucleoside linker of the synthetic polynucleotide. Modifications to the sugar may consist of a 2' O-methyl modification.
• terminal cap structures may also be incorporated to the 3' and/or 5' termini.
• Such structures include, but are not limited to, at least one inverted deoxy thymidine or amino group (NH 2 ).
• the 3' cap is an inverted deoxythymidine cap.
• the 3' cap is an amino group (NH 2 ).
• the 5' cap is an inverted deoxythymidine cap.
• the 5' cap is an amino group (NH 2 ).
• VWF protective agents of the present invention may be modified by any number of conjugates.
• the conjugates may be high molecular weight non-immunogenic compounds.
• the high molecular weight, non-immunogenic compound is polyalkylene glycol, and more preferably is polyethylene glycol (PEG).
• a PEG moiety is conjugated to the 5' terminus of the nucleic acid aptamer.
• a PEG moiety is conjugated to the 3' terminus of the nucleic acid aptamer.
• a nucleic acid aptamer can also include at least one modified ribonucleoside including but not limited to a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside (e.g., ⁇ -D- ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'- amino-LNA having a 2 '-amino functionalization, and 2'-amino-a-LNA having a 2 '-amino functionalization), an abasic nucleoside, an inverted deoxynucleoside or in
• nucleoside ribonucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, a 2'-alkyl-modified nucleoside, a 2'-0-alkyl-modified nucleoside, a 2'-0-alkyl-0-alkyl-modified nucleoside, a 2'-fluoro-modified nucleoside, a 2'-fluoro-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any
• a nucleic acid aptamer can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the molecule.
• the modifications need not be the same for each of such a plurality of modified deoxy- or ribonucleosides in a nucleic acid molecule.
• Nucleic acid aptamers described herein may also contain one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted CI to CIO alkyl or C2 to CIO alkenyl and alkynyl.
• Exemplary modifications include O[(CH 2 )nO] m CH3, O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 )nON[(CH 2 )nCH 3 )] 2 , where n and m are from 1 to about 10.
• nucleic acid aptamers include one of the following at the 2' position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an aptamer, or a group for improving the pharmacodynamic properties of a nucleic acid aptamer, and other substituents having similar properties.
• the modification includes a 2' methoxyethoxy (2'-O- CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxy ethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
• Another exemplary modification is 2'- dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxy ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 2 ) 2 .
• Similar modifications may also be made at other positions on the aptamer, particularly the 3 ' position of the sugar on the 3 ' terminal nucleotide and the 5' position of 5' terminal nucleotide.
• Aptamers may also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.
• Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
• VWF protective agents which are nucleic acid based may include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
• base often referred to in the art simply as “base”
• nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8
• modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
• oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2-NH-O-CH2, CH2-N(CH 3 )- O-CH2 (known as a methylene(methylimino) or MMI backbone), CH2-O-N (CH 3 )-CH2, CH2 - N(CH 3 )-N(CH 3 )-CH 2 and 0-N(CH 3 )-CH 2 -CH 2 backbones (wherein the native phosphodiester backbone is represented as O-P-O-CH2-), amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995); which is incorporated by reference in its entirety];
• PNA peptide nucleic acid
• oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497; which is incorporated by reference in its entirety).
• Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5'-3 ' or 2'-5' to 5'-2'; see US Patent Nos.
• Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001);
• Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 122: 8595-8602 (2000), which is hereby incorporated by reference in its entirety.
• Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing
• nucleic acid aptamers are provided in which the P(0)0 group is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), P(0)NR 2 ("amidate”), P(0)R, P(0)OR', CO or CH 2 ("formacetal”) or 3 '-amine (— NH— CH 2 — CH 2 — ), wherein each R or R' is independently H or substituted or unsubstituted alkyl.
• Linkage groups can be attached to adjacent nucleotide through an— O— ,— N— , or— S— linkage. Not all linkages in the nucleic acid aptamers are required to be identical.
• nucleic acid aptamers of the invention include those taught in, for example, International Publication PCT/US2012/058519, the contents of which are incorporated herein by reference in their entirety.
• a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other preferred embodiments, 15-30 nt, 20-25 nt, 30-100 nt, 30- 60 nt, 25-70 nt, 25-60 nt, 40-60 nt, 25-40 nt, 30-40 nt, any of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nt or 40-70 nt in length.
• the sequence can be designed with sufficient flexibility such that it can accommodate interactions of aptamers with two targets at the distances described herein.
• the nucleic acid aptamer comprises one or more regions of double stranded character. Such double stranded regions may arise from internal self- complementarity or complementarity with a second or further aptamer molecule.
• the double stranded region may be from 4-12, 4-10, 4-8 base pairs in length.
• the double stranded region may be 5, 6, 7, 8, 9, 10, 1 1 or 12 base pairs.
• the double stranded region may form a stem region. Such extended stem regions having double stranded character can serve to stabilize the nucleic acid aptamer.
• Extended stem regions of at least 6 base pairs may contribute to greater thermostability at higher temperatures and thereby, greater affinity for VWF and increased overall functionality as compared to shorter stem regions of 5 base pairs or less. Shorter stem regions may lead to unraveling of the stem-loop structure at higher temperatures and associated loss of affinity and overall functionality.
• double stranded character means that over any length of two nucleic acid molecules, their sequences form base pairings (standard or nonstandard) of more than 50 percent of the length.
• base pairings standard or nonstandard
• the secondary structure of aptamers described herein may be confirmed by computational models known in the art that predict nucleic acid folding based on free energy calculation, such as Mfold.
• composition of an oligonucleotide can influence complement activation. For example, the number of
• the aptamers used in the pharmaceutical compositions and formulations and methods provided herein has a nucleotide sequence that includes no more than four, no more than three, no more than two or no more than one phosphorothioate backbone modification.
• the nucleic acid aptamer contains no phosphorothioate modifications.
• Aptamers of the present invention may be modified to increase thermostability.
• Thermostability of nucleic acid aptamers is an important factor that controls the structure, hybridization, and functions of aptamers.
• modifications that may be used to enhancer aptamer thermostability include 2'-0-Methyl nucleosides, 2' Fluoro nucleosides, locked nucleic acids (LNAs), 2,6-Diaminopurine (2-Amino-dA), 5-Methyl dC, and Super T (5-hydroxybutynl-2'-deoxyuridine).
• RNA duplexes The presence of 2'-0-Methyl nucleosides in DNA or RNA strands enhances thermostability of the duplexes and the effect is more prominent in RNA: RNA duplexes than in RNA: DNA duplexes.
• Modification of the 2'- deoxyribose with 2 '-fluoro enhances the thermostability of the DNA-DNA duplexes by 1.3 °C per insertion.
• LNA bases have a modification to the ribose backbone that locks the base in the C3 '-endo position, which favors RNA A-type helix duplex geometry. LNA modification significantly increases Tm and is also very nuclease resistant.
• thermostability of the nucleic acid aptamers is characterized by melting temperature (Tm) at which half of the DNA or RNA molecules are in a double stranded form, while the other half of the molecules are represented by single strand form.
• Tm may be determined by any method known in the art, such as UV absorbance measurement, circular dichroism spectroscopy, and thermal measurement.
• T m may be influenced by a number of factors such as aptamer concentration, pH, monovalent cation (Na + , K + ) concentration, or divalent cation (Mg 2+ ) concentration.
• aptamers have T m greater than 37 °C and maintain intact stem-loop structure in vivo.
• Aptamers may be further modified to protect the aptamers from nuclease and other enzymatic activities.
• the aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5 '-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamers.
• modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or 2'-fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants.
• 2'-0-Methyl nucleosides and LNAs can also contribute to the resistance to nucleases.
• the resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
• such modified nucleic acid aptamers may be synthesized entirely of modified nucleotides, or with a subset of modified nucleotides.
• the modifications can be the same or different. All nucleotides may be modified, and all may contain the same modification. All nucleotides may be modified, but contain different modifications, e.g., all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modification. For example, 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). In this way, oligonucleotides, or libraries of oligonucleotides are generated using any combination of modifications as disclosed herein.
• the VWF aptamer is an aptamer or a salt thereof comprising the sequence, 5'GCCAGGGACCUAAGACACAUGUCCCUGGC-3 ' (SEQ ID NO: 1).
• the VWF aptamer is an aptamer or a salt thereof comprising the structure: NH 2 - mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmUmGmUmCmCmC mUmGmGmC-idT (SEQ ID NO: 2) (BT-100), where "NH” is a 5'-hexylamine linker phosphoramidite, "idT” is an inverted deoxythymidine, "mN” is a 2'-O-Methyl containing residue.
• the VWF aptamer is an aptamer or a salt thereof comprising the structure: PEG40K-NH- mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmUmGmUmCmCmC mUmGmGmC-idT (SEQ ID NO: 3) (BT-200), where "NH” is a 5'-hexylamine linker phosphoramidite, "idT” is an inverted deoxythymidine, "mN” is a 2'-O-Methyl containing residue and "PEG” is a polyethylene glycol and PEG40K is a pegylation moiety having a molecular weight of approximately 40KDa. It should be understood that the presence of a PEG moiety is optional but when present, may be of varied size.
• reversal agents are provided.
• a "reversal agent” is one which alters, mitigates or reverses the effect of a VWF protective agent.
• the reversal agents are nucleic acid based.
• reversal agents are competitive binding molecules.
• reversal agents bind VWF protective agents.
• reversal agents hybridize with a portion of a VWF protective agent.
• nucleic acid based VWF protective agents such as aptamers
• reversal agents may hybridize with all, a portion, a region of a protective aptamers.
• Reversal agents may comprise any or all of the modifications described herein. An example is illustrated in Fig. 2.
• the reversal agent comprises at least 15 contiguous nucleotides of the sequence 5'-ACAUGUGUCUUAGGUCCCUGGC-3 ' (SEQ ID NO: 4). In some embodiments, the reversal agent comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides of SEQ ID NO: 4.
• the reversal agent comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides which are complementary to or bind to SEQ ID NO: 1.
• the least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides which are complementary to or bind to SEQ ID NO: 1 may be chemically modified. In some embodiments, at least one of the nucleotides contains at least one chemical modification. In some embodiments, each of the nucleotides contains at least one chemical modification. As a non-limiting example, the chemical modification may be a 2'-O-methyl modification to a nucleotide sugar. Additionally, least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides which are complementary to or bind to SEQ ID NO: 1 may further comprise a 3 ' terminal cap. In one embodiment, the 3 ' terminal cap may be an inverted deoxythymidine
• the reversal agent is BT-201 having the sequence
• the VWF aptamer may comprise the sequence PEG40K-NH- mGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmC-idT
• ARC15105 SEQ ID NO: 6
• PEG20K-NH- mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmGsdTmGdCdGdGdTm GmCdCmUdCdCmGmUdCmAmCmGmCidT (ARC1779)(SEQ ID NO:7), where "NET is a 5'-hexylamine linker phosphoramidite, "idT” is an inverted deoxythymidine, "mN” is a 2'-O- Methyl containing residue, “dN” is a deoxynucleotide residue, “sdT” is a phosphorothioate deoxythymidine residue and "PEG” is a polyethylene glycol and PEG20K is a pegylation moiety having a molecular weight of approximately 20KDa.
• the VWF aptamer when used in methods of treating VAD disorders, may comprise the sequence PEG40K-NH- mGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmC-idT
• ARC15105 SEQ ID NO: 6
• PEG20K-NH- mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmGsdTmGdCdGdGdTm GmCdCmUdCdCmGmUdCmAmCmGmCidT (ARC1779)(SEQ ID NO:7), where "NH” is a 5'-hexylamine linker phosphoramidite, "idT” is an inverted deoxythymidine, "mN” is a 2'-O- Methyl containing residue, “dN” is a deoxynucleotide residue, “sdT” is a phosphorothioate deoxythymidine residue and "PEG” is a polyethylene glycol and PEG20K is a pegylation moiety having a molecular weight of approximately 20KDa.
• BT-200 preserves one or more characteristics and/or properties of BT-100. In other embodiments, BT-200 has one or more improved or altered characteristics and/or properties compared to BT-100. In one aspect, BT-200 has a longer plasma half-life than BT-100. In another aspect, BT-200 has a longer duration of action than BT-100.
• BT-200 preserves one or more characteristics and/or properties of ARC15105 or ARC 1779. In some embodiments, BT-200 has one or more improved or altered characteristics and/or properties compared to ARC15105 or ARC1779. In one aspect, BT-200 has a longer plasma half-life than ARC15105 or ARC1779. In another aspect, BT-200 has a longer duration of action than ARC15105 or ARC1779. In another aspect, BT-200 has higher subcutaneous bioavailability. In another aspect, BT-200 has greater potency compared to ARC15105 or ARC 1779 as a VWF protective agent.
• the present invention contemplates several types of VWF protective agents which are nucleic acid based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. As such, included within the scope of this invention are VWF protective agents containing substitutions, insertions and/or additions, deletions and covalently modifications.
• variants refers to molecules which differ in their nucleic acid sequence from a reference sequence.
• the nucleic acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the nucleic acid sequence, as compared to a reference sequence.
• variants will possess at least about 50% identity (homology) to a reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a reference sequence.
• variable mimics are provided.
• the term “variant mimic” is one which contains one or more nucleic acids which would mimic a reference sequence.
• the nucleic acid sequences of the VWF protective agents of the invention may comprise naturally occurring nucleic acids.
• the VWF protective agents may comprise both naturally and non-naturally occurring nucleic acids.
• variants will possess at least about 70% homology to a reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a reference sequence.
• Homology as it applies to nucleic acid sequences is defined as the percentage of residues in the candidate sequence that are identical with the residues in the sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
• analogs is meant to include polynucleotide variants which differ by one or more nucleic acid alterations, respectively, e.g., substitutions, additions or deletions of residues that still maintain the properties of the parent polypeptide or polynucleotide.
• substitutional variants are those that have at least one nucleoside (or nucleotide) in a starting sequence removed and a different nucleoside (or nucleotide) inserted in its place at the same position.
• the substitutions may be single, where only one nucleoside (or nucleotide) in the molecule has been substituted, or they may be multiple, where two or more nucleosides (or nucleotides) have been substituted in the same molecule.
• “Insertional variants” are those variants with one or more nucleosides (or
• nucleotides inserted immediately adjacent to a nucleoside (or nucleotide) at a particular position in a starting sequence. "Immediately adjacent" to a nucleoside (or nucleotide) means directly to the 5' or 3 ' of the instant nucleoside or nucleotide of the polynucleotide.
• deletion variants are those with one or more nucleosides (or nucleotides) in the starting sequence removed. Ordinarily, deletional variants will have one or more nucleosides (or nucleotides) deleted in a particular region of the molecule.
• Covalent modifications are traditionally introduced by reacting targeted nucleoside (or nucleotide) residues of the molecule with an organic derivatizing agent that is capable of reacting with selected atoms or residues. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
• Covalent derivatives specifically include fusion molecules in which nucleic acids of the invention are covalently bonded to a non-proteinaceous polymer.
• the non-proteinaceous polymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymer not otherwise found in nature.
• hydrophilic polyvinyl polymers fall within the scope of this invention, e.g. polyvinylalcohol and
• polyvinylpyrrolidone Particularly useful are polyvinylalkylene ethers such a polyethylene glycol, polypropylene glycol.
• the VWF protective agents may be linked to various non- proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or
• Features are defined as distinct nucleoside (or nucleotide) sequence-based components of a molecule.
• Features of the VWF protective agents of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
• surface manifestation refers to a component of a polynucleotide appearing on an outermost surface.
• fold means the resultant conformation of a nucleoside (or nucleotide) sequence upon energy minimization.
• a fold may occur at the secondary or tertiary level of the folding process.
• turn means a bend which alters the direction of the backbone of a polynucleotide and may involve one, two, three or more nucleoside (or nucleotide) residues.
• loop refers to a structural feature of a polynucleotide which reverses the direction of the backbone of the sequence and comprises four or more nucleoside (or nucleotide) residues.
• domain refers to a motif of polynucleotide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity), serving as a site for molecular interactions.
• site as it pertains to nucleoside (or nucleotide) based embodiments is used synonymous with "nucleic acid residue".
• a site represents a position within a polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polynucleotide based molecules of the present invention.
• termini or terminus refers to an extremity of a
• polynucleotide Such extremity is not limited only to the first or final site of the polynucleotide but may include additional nucleosides (or nucleotides) in the terminal regions.
• the polynucleotide based molecules of the present invention may be characterized as having both a 5' terminus and a 3' terminus.
• any of the features have been identified or defined as a component of a molecule of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.
• Modifications and manipulations can be accomplished by methods known in the art such as site directed mutagenesis.
• the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
• the VWF protective agents of the present invention may contain one or more atoms that are isotopes.
• isotope refers to a chemical element that has one or more additional neutron.
• compounds of the present invention may be deuterated.
• deuterated refers to a substance that has had one or more hydrogen atoms replaced by deuterium isotopes.
• Deuterium isotopes are isotopes of hydrogen.
• the nucleus of hydrogen contains one proton while deuterium nuclei contain both a proton and a neutron.
• the VWF protective agents may be deuterated in order to change a physical property of the compound, such as stability, or to allow the compounds to be used in diagnostic and experimental applications.
• the VWF protective agents of the present invention may be complexed, conjugated or combined with one or more homologous or heterologous molecules.
• homologous molecule means a molecule which is similar in at least one of structure or function relative to a starting molecule while a
• heterologous molecule is one that differs in at least one of structure or function relative to a starting molecule.
• Structural homologs are therefore molecules which are substantially structurally similar. They can be identical.
• Functional homologs are molecules which are substantially functionally similar. They can be identical.
• VWF protective agents of the invention may comprise conjugates.
• conjugates of the invention may include a naturally occurring substance or ligand, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
• a protein e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin
• a carbohydrate e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid
• lipid e.g., a lipid,
• the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer).
• polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co- glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
• PLL polylysine
• poly L-aspartic acid poly L-gluta
• polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
• VWF protective agents of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), reporter molecules, polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG,
• intercalating agents e.g. acridines
• cross-linkers e.g. psoralene, mitomycin C
• porphyrins TPPC4, texaphyrin, Sapphyrin
• reporter molecules e.g., polycyclic aromatic hydrocarbons (e.g., phenazine, di
• [MPEG] 2 polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.
• haptens e.g. biotin
• transport/absorption facilitators e.g., aspirin, vitamin E, folic acid
• synthetic ribonucleases proteins, e.g., glycoproteins, or
• the conjugates can also include targeting groups, e.g., a cell or tissue targeting agent or group, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
• targeting groups e.g., a cell or tissue targeting agent or group, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
• a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, poly aspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
• Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
• Targeting groups may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamer s.
• the targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.
• the targeting group is an aptamer.
• the aptamer can be unmodified or have any combination of modifications disclosed herein.
• the VWF protective agent is covalently conjugated to a cell penetrating polypeptide.
• the cell-penetrating peptide may also include a signal sequence.
• the conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).
• VWF protective agents of the present invention may be designed to be conjugated to any number of conjugates.
• the conjugates may be high molecular weight non-immunogenic compounds.
• Conjugating moieties may be added to the VWF protective agents such that they allow labeling or flagging the VWF for clearance.
• tagging/flagging molecules include, but are not limited to ubiquitin, fluorescent molecules, human influenza hemaglutinin (HA), c- myc (a 10 amino acid segment of the human protooncogene myc with sequence
• EQKLISEEDL SEQ ID NO: 10
• His histidine
• flag a short peptide of sequence
• DYKDDDDK (SEQ ID NO: 11)
• GST glutathione S-transferase
• V5 a paramyxovirus of simian virus 5 epitope
• biotin avidin, streptavidin, horse radish peroxidase (HRP) and digoxigenin.
• VWF protective agents may be combined with other VWF protective agents, or other molecules in the treatment of a disease or condition.
• the VWF protective agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
• combination with it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
• Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
• the present disclosure encompasses the delivery of pharmaceutical,
• prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
• the combination may include a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent.
• a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent.
• a cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells.
• Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.
• Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.
• iodine e.g., iodine 125 or iodine 131
• strontium 89 phosphorous
• palladium cesium
• iridium phosphate
• cobalt yttrium 90
• samarium 153 samarium 153
• praseodymium praseodymium
• therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU),
• antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine
• alkylating agents e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU)
• cyclophosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).
• anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
• antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
• anti-mitotic agents e.g
• the combination may include a detectable agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, m In, 123 I, 133 Xe, 201 T1, 125 1, 35 S, 14 C, 3 H, or 99m Tc (e.g., as pertechnetate (technetate(VII), TcO 4 " )), and contrast agents (e.g., gold (e.g., gold), gold
• nanoparticles examples include gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons).
• Gd e.g., chelated Gd
• iron oxides e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)
• manganese chelates e.g., Mn-DPDP
• barium sulfate iodinated contrast media (iohexol), microbubbles, or perfluorocarbons.
• optically-detectable labels include for example, without limitation, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-naphthyl)maleimide;
• anthranilamide BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5' 5"- dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]-naphthalene-l-sulfon
• sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), ⁇ , ⁇ , ⁇ ', ⁇ 'tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, and
• tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
• TRITC tetramethyl rhodamine isothiocyanate
• the detectable agent may be a non-detectable pre-cursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical)).
• fluorogenic tetrazine-fluorophore constructs e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X
• enzyme activatable fluorogenic agents e.g., PROSENSE® (VisEn Medical)
• enzyme labeled compositions include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
• ELISAs enzyme linked immunosorbent assays
• IA enzyme immunoassays
• RIA radioimmunoassays
• the present invention provides aptamers (themselves monomers or multimers) that bind to von Willebrand Factor (VWF) monomers or multimers. These aptamers are referred to herein as "VWF aptamers.” As stated above, VWF aptamers may be nucleic acid based or amino acid based.
• the target of the VWF protective agents is the VWF protein or protein multimers.
• VWF protective agents may be designed to bind or associate with proteins or other biomolecules which themselves associate with the VWF protein.
• Homo sapiens Von Willebrand Factor mRNA is provided herein as SEQ ID NO: 8 (NM_000552; 8833 nt) and the protein as SEQ ID NO: 9 (NP_000543; 2813 amino acids).
• VWF von Willebrand factor
• Uridine is represented by Thymidine.
• the VWF protective agents may completely or partially inhibit VWF monomer or multimers degradation by partially or completely blocking the ability of VWF to bind to platelets or erythrocytes.
• the VWF aptamers are considered to completely inhibit VWF degradation when the level of protection of a monomer or multimers of at least 2 monomers is at least 95%, e.g., by 96%o, 97%), 98%, 99% or 100% as compared to the level of intact monomer or multimers in the absence of the VWF protective agent.
• the VWF aptamers are considered to partially inhibit VWF degradation when the level of protection of a monomer or multimers of at least 2 monomers is at least 10%>, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of intact monomer or multimers in the absence of the VWF protective agent.
• the VWF protective agents may completely or partially disaggregate occlusive thrombi by partially or completely blocking the ability of VWF to bind to platelets.
• the VWF aptamers are considered to completely disaggregate occlusive thrombi when the occlusive thrombi are at least 95% disaggregated, e.g., 95%), 96%o, 97%), 98%, 99% or 100%, disaggregated, as compared to the level of occlusive thrombi in the absence of the VWF protective agent.
• the VWF aptamers are considered to partially disaggregate occlusive thrombi when the occlusive thrombi are at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%) disaggregated as compared to the level of occlusive thrombi in the absence of the VWF protective agent.
• a VWF protective agent which is a nucleic acid aptamer may comprise a dissociation constant for human von Willebrand Factor domain Al or a variant thereof, of less than 100 uM, less than 1 uM, 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.
• Aptamers have a number of desirable characteristics for use as therapeutics including high specificity and affinity to molecular targets, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over other biologies, for example:
• Aptamers may be produced by an entirely in vitro process. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads against both toxic and non-immunogenic targets. Aptamers may also be produced synthetically, whether by chemical synthesis such as solid phase synthesis or enzymatic synthesis using polymerase enzymes such as T7 RNA polymerase.
• Toxicity and Immunogenicity .
• Aptamers as a class have demonstrated little or no toxicity or immunogenicity.
• Such immunogenicity may be further controlled by incorporating chemical modifications which mute or ameliorate such immune responses. Such modifications are taught in International Publication PCT/US2012/058519, the contents of which are incorporated herein by reference in their entirety.
• aptamers can be administered by subcutaneous injection or by other routes.
• Aptamer bioavailability via subcutaneous administration has been shown to be >80% in monkey studies.
• subcutaneous administration of VWF protective agents to a subject may yield a bioavailability of at least about 50%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 98%, or about 99% relative to that of an intravenous administration.
• Aptamers may be adapted to regain activity following exposure to heat, denaturants, etc. and can be stored for extended periods (>1 year).
• a VWF protective agent formulation of the invention used in combination with one or more other treatments for coagulation, bleeding or VAD-associated disorders.
• a VWF protective agent formulation can be used in combination with one or more other treatments for acute coronary syndrome or acute occlusion thrombosis, including for example, but not limited to, aspirin, thrombolytics (i.e., tissue plasminogen activator,reteplase), nitroglycerin, beta blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), Ca 2+ channel blockers, cholesterol-lowering drugs, (i.e., tissue plasminogen activator (tPA),reteplase), and clot preventing drugs (i.e., clopidogrel and prasugrel).
• thrombolytics i.e., tissue plasminogen activator, alteplase
• ACE angiotensin-converting enzyme
• ARBs angiotensin receptor block
• a VWF protective agent formulation can be used in combination with one or more other treatments used to disaggregate platelet aggregates, such as, for example, thrombolytic treatments targeting fibrin, and treatments that seeks to disrupt the platelet - fibrin interaction, i.e., platelet GpIIb/IIIa inhibitors, such as abciximab, eptifibatide, tirofiban).
• one or more other treatments used to disaggregate platelet aggregates such as, for example, thrombolytic treatments targeting fibrin, and treatments that seeks to disrupt the platelet - fibrin interaction, i.e., platelet GpIIb/IIIa inhibitors, such as abciximab, eptifibatide, tirofiban).
• a VWF protective agent formulation can be used in combination with one or more other treatments used to treat ischemic stroke, such as, for example, anti-coagulants, thrombolytics (tPA, alteplase), anti-platelet therapy (e.g., aspirin, dabigairan, clopidogrel, dipyridamole), and warfarin.
• the VWF protective agent formulation of the invention may contain, for example, more than one aptamer.
• a VWF protective agent formulation of the invention, containing one or more aptamers is administered in combination with another useful formulation or drug.
• the aptamer formulations are used in combination with non-drug therapies or treatments, such as surgical procedures. In general, 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 protective agent formulation of the invention 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 these 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 affected 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 agents
• 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. For example, in appropriate cases, 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.
• pharmacokinetic or pharmacodynamic properties may be modified to improve pharmacokinetic or pharmacodynamic properties such as half-life and/or duration of action.
• half-life or “plasma half-life” refers to the amount of time by which half of the administered amount of aptamer is cleared from the blood stream.
• Duration of action refers to the length of time that an aptamer is effective. The duration of action of aptamers may depend on several parameters such as plasma half-life, the administered dose, the pharmaceutical preparation, and the influence of disease on drug elimination (Carruthers SG.
• Modification of the aptamers such as altering the stem region, or attaching the aptamers to polymers (e.g., PEG), may alter the half-life and/or duration of action.
• aptamers of the present invention may have a plasma half-life of from about 20 hours to about 40 hours, from about 30 hours to about 50 hours, from about 40 hours to about 60 hours, from about 50 hours to about 70 hours, from about 55 hours to about 75 hours, from about 60 hours to about 80 hours, from about 70 hours to about 85 hours, from about 75 hours to about 90 hours, from about 85 to about 100 hours, or at least 100 hours after administered to a subject.
• aptamers of the present invention may have a duration of action of at least 8 hours, at least 16 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, from about 72 hours to about 96 hours, from about 90 hours to 120 hours, from about 110 hours to about 135 hours, from about 120 hours to 150 hours, from about 135 hours to 168 hours, from about 150 hours to 180 hours, from about 180 hours to about 210 hours, from about 210 hours to about 240 hours, from about 240 hours to 300 hours, from about 300 hours to 360 hours, or at least 360 hours.
• the aptamers of the present invention do not function as anticoagulants and/or alter clotting function.
• anticoagulant refers to a chemical substance that prevent or reduce coagulation of blood, prolonging the clotting time. Coagulation of blood may be assayed via the techniques and methods described herein and those known in the art, such as activated partial
• thromboplastin time (aPTT) assays thromboplastin time (PT) assays
• PT prothrombin time
• thrombin time test thrombin time test
• fibrinogen test anti-factor Xa assays
• rotational thrombelastometry e.g., calibrated automated thrombogram
• aptamers of the present invention do not prolong clotting time. In some embodiments, aptamers of the present invention do not prolong activated partial thromboplastin time. In some embodiments, aptamers of the present invention do not prolong prothrombin time. In some embodiments, aptamers of the present invention do not prolong thrombin time. In some embodiments, aptamers of the present invention do not alter fibrinogen concentration. In some embodiments, aptamers of the present invention do not alter thrombin generation.
• compositions of the present invention comprise at least one VWF protective agent as the active ingredient.
• the compositions are suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers.
• the compounds are especially useful in that they have very low, if any toxicity.
• the formulations may comprise any amount of aptamers of the present invention or a pharmaceutically acceptable salt thereof.
• 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. Examples of pharmaceutically acceptable salts of aptamers include sodium salts,
• the formulations may comprise any aptamer or a combination of aptamers that bind to the full length of the polypeptide or a variant or a fragment thereof.
• the polypeptide may be from any species, but is preferably from human.
• the formulations also comprise a pharmaceutically acceptable solvent.
• pharmaceutically acceptable solvent refers to being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• compositions are well known in the art. Examples of pharmaceutically acceptable solvents can be found, for example, in Goodman and Gillmans, The
• the pharmaceutically acceptable solvent is selected from the group consisting of 0.9% saline (also known as physiological saline or sterile isotonic saline solution) or phosphate buffered saline. Most preferably, the pharmaceutically acceptable solvent is 0.9% saline.
• the formulations may comprise any amount of pharmaceutically acceptable solvent.
• formulations may, optionally, include one or more of the following: buffer, pH adjuster, tonicity agent, cosolvent or pharmaceutically acceptable carrier.
• the formulations may further comprise a buffer.
• a buffer is any substance that, when added to a solution, is capable of neutralizing both acids and bases without appreciably changing acidity or alkalinity of the solution.
• buffers include, but are not limited to, pharmaceutically acceptable salts and acids of acetate, glutamate, citrate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, phosphate, malate, succinate, formate, propionate and carbonate.
• the formulations may further comprise a pH adjuster.
• a pH adjuster is used to adjust the pH of the formulation.
• Suitable pH adjusters typically include at least an acid or a salt thereof and/or a base or a salt thereof. Acids and bases can be added on an as needed basis in order to achieve a desired pH. For example, if the pH is greater than the desired pH, an acid may be used to lower the pH to the desired pH. Examples of acids include, but are not limited to, hydrochloric acid, phosphoric acid, citric acid, ascorbic acid, acetic acid, sulphuric acid, carbonic acid and nitric acid.
• a base can be used to adjust the pH to the desired pH.
• bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium citrate, sodium acetate and magnesium hydroxide.
• the formulations may further comprise a tonicity agent.
• Tonicity agents are used to adjust the osmolality of the formulations in order to bring them closer to the osmotic pressure of body fluids, such as blood or plasma.
• Examples of tonicity agents include, but are not limited to, anhydrous or hydrous forms of sodium chloride, dextrose, sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride, magnesium chloride and other inorganic salts.
• the formulations may further comprise a cosolvent.
• a cosolvent is a solvent that is added to the aqueous formulation in a weight amount that is less than that of water and assists in the solubilization of the aptamer.
• cosolvents include, but are not limited to, glycols, ethanol and polyhydric alcohols.
• the formulations may further comprise a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier refers to 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.
• stable means remaining in a state or condition that is suitable for administration to a patient.
• the formulations are, preferably, substantially pure.
• substantially pure means that the active ingredient (aptamer) is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the active ingredient comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
• a substantially pure composition will comprise more than 80% of all
• the active ingredient is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
• compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
• compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/ml to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
• the desired dosage may be delivered three times a day, two times a day,
• the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
• the total dose of aptamers is administered so as to achieve and then maintain a steady state blood concentration equal to at least the ECs>o, which is the concentration of drug that leads to 90% maximal response.
• the total dose of aptamers is administered so as to achieve and then maintain a steady state blood concentration equal to at least the ECso, which is the concentration of drug that leads to 80% maximal response.
• the total dosage may be administered in a single dose, multiple doses, repeated doses, as a continual dose or a combination thereof.
• the formulations are administered with a loading dose, a
• the dosage regimen utilizing the compounds 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 compound 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.
• Oral dosages of the present invention when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally.
• the compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient.
• Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.
• Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
• the formulations and dosages described herein are designed to maximize clinical efficacy in the treatment of VAD-mediated diseases and for disorders arising from interactions of VWF multimers with erythrocytes, such as the interaction that occurs between VWF multimers and erythrocytes in sickle cell disease, as well as the treatment of diseases and disorders arising from interactions of VWF monomers or multimers with platelets during the formation of occlusive thrombi that occurs during closing of the lumen of a vessel and/or under very high shear rates (10,000 s "1 or greater) (such as the disorders of acute coronary syndromes, acute occlusion thrombosis, and ischemic stroke), while simultaneously decreasing or minimizing adverse side effects, without the need for an antidote.
• a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal,
• injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal,
• the aptamer formulations provided herein are administered to subjects, preferably human subjects, in an amount effective to prevent, ameliorate or treat thrombotic disorders such as ischemic stroke, diseases or disorders associated with ventricular assist devices and disorders arising from interactions of VWF multimers with erythrocytes, such as the interaction that occurs between VWF multimers and erythrocytes in sickle cell disease.
• thrombotic disorders such as ischemic stroke, diseases or disorders associated with ventricular assist devices and disorders arising from interactions of VWF multimers with erythrocytes, such as the interaction that occurs between VWF multimers and erythrocytes in sickle cell disease.
• the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
• 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 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 sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or poly ethylenegly col 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.
• the compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
• compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
• the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
• the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
• Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
• the active compound is dissolved in or mixed with a
• injectable compositions are preferably aqueous isotonic solutions or suspensions.
• the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
• the compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
• injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
• the materials of the present invention can be delivered to the ocular cavity with the methods described below.
• the materials of the present invention can be administered to subjects in the modalities known in the art as described below.
• Parenteral injectable administration is generally used for subcutaneous,
• preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
• the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
• Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.
• excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
• 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.
• the compounds of the present invention can also be administered 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.
• the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
• soluble polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
• the compounds 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.
• 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 pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
• non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
• formulations may be administered in combination with other drugs or therapies.
• bioavailability refers to the systemic availability of a given amount of a VWF protective agent administered to a subject. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (Cmax) of the unchanged form of a compound following administration of the compound to a subject. AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v.
• the Cmax value is the maximum concentration of the compound achieved in the serum or plasma of a subject following administration of the compound to the subject.
• the Cmax value of a particular compound can be measured using methods known to those of ordinary skill in the art.
• VWF protective agents of the present invention are formulated into a composition with a delivery /formulation agent or vehicle as described herein to increase bioavailability.
• the phrases "increasing bioavailability" or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a VWF protective agent, measured as AUC, Cmax, or Cmin in a subject is greater, when co-administered with a delivery agent as described herein, than when such co-administration does not take place.
• the bioavailability of the VWF protective agent can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%), at least about 30%>, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
• VWF protective agent compounds and compositions of the present invention may be combined with other ingredients or reagents or prepared as components of kits or other retail products for commercial sale or distribution.
• the kit will contain the compound or composition, along with instructions regarding administration and/or use of the kit.
• the kit may also contain one or more of the following: a syringe, an intravenous bag or bottle, the same drug in a different dosage form, or another drug.
• the kit may contain both an intravenous formulation and a subcutaneous formulation of the present invention.
• the kit may contain lyophilized anti- thrombin aptamer and an intravenous bag of solution.
• the kit form is particularly
• kits are stored at 5 ⁇ 3 °C.
• the kits can also be stored at room temperature or frozen at -20 °C.
• compositions of the present invention which are VWF protective agents may be used in conjunction with one or more therapeutic devices.
• Such devices include vascular assist devices (VADs), stents, or any device useful in the regulation or management of VAVs.
• the VWF protective agents may be used as coatings on solid surfaces or as adjunct therapy where the
• VWF aptamers are formulated to be added to the blood or plasma flow during surgical procedures such as transplants, angioplasty, and stent insertion or during long-term care such as those patients having internal VAD or LVAD devices implanted.
• Formulations and/or compositions of VWF protective agents of the present invention can be packaged for use in a variety of pharmaceutically acceptable containers using any pharmaceutically acceptable container closure, as the formulations are compatible with PVC-containing and PVC-free containers and container closures. Examples of
• pharmaceutically acceptable containers include, but are not limited to, ampules, pre-filled syringes, intravenous bags, intravenous bottles and admix bags.
• the formulation may be an aqueous formulation containing both anti-thrombin aptamer and pharmaceutically acceptable solvent.
• the formulation may contain lyophilized aptamer in one compartment of an admix bag and a pharmaceutically acceptable solvent in a separate compartment of the admix bag such that the two compartments may be mixed together prior to administration to a patient.
• Pharmaceutically acceptable containers are well known in the art and commercially available.
• the formulations are stored in a Type 1 glass vial with a butyl rubber stopper. The formulations in liquid form must be stored in a refrigerated environment.
• the liquid formulations are stored at 2-8 °C.
• the lyophilized formulations may be stored at room temperature, or refrigerated or frozen.
• the formulations are sterile.
• a "sterile" formulation means a formulation that has been brought to a state of sterility and has not been subsequently exposed to microbiological contamination, i.e., the container holding the sterile composition has not been compromised.
• Sterile compositions are generally prepared by pharmaceutical manufacturers in accordance with current Good Manufacturing Practice ("cGMP") regulations of the U.S. Food and Drug Administration.
• cGMP Good Manufacturing Practice
• Procedures for filling pharmaceutical formulations in pharmaceutically acceptable containers, and their subsequent processing are known in the art. These procedures can be used to produce sterile pharmaceutical drug products often required for health care.
• Suitable procedures for producing sterile pharmaceutical drug products include, but are not limited to, terminal moist heat sterilization, ethylene oxide, radiation (i.e., gamma and electron beam) and aseptic processing techniques. Any one of these sterilization procedures can be used to produce the sterile pharmaceutical formulations described herein.
• sterile pharmaceutical formulations can be prepared using aseptic processing techniques. Sterility is maintained by using sterile materials and a controlled working environment. All containers and apparatus are sterilized, preferably by heat sterilization, prior to filling. Then, the container is filled under aseptic conditions, such as by passing the composition through a filter and filling the units. Therefore, the formulations can be sterile filled into a container to avoid the heat stress of terminal sterilization.
• the formulations are terminally sterilized using moist heat. Terminal sterilization can be used to destroy all viable microorganisms within the final, sealed container containing the pharmaceutical formulation.
• An autoclave is typically used to accomplish terminal heat-sterilization of drug products in their final packaging. Typical autoclave cycles in the pharmaceutical industry to achieve terminal sterilization of the final product are 121 °C for at least 10 minutes.
• articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
• the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
• the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
• any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
• BT-100 SEQ ID NO: 2 was synthesized with a free amine group attached to the 5 '-end through a six carbon-linker.
• BT-200 SEQ ID NO: 3
• BT-100 was first purified through anion- exchange and ultrafiltration before dissolved in 100 mM sodium bicarbonate buffer (pH 8.0) at 10 mg/mL concentration.
• the purified BT-100 was then mixed with NHS activated branched 40K-PEG (Catalog number Y-NHS-40K, JenKem Technology USA Inc, Piano, TX) at the molar ratio of BT-100 to NHS activated branched 40K-PEG to 1 :2.
• the reaction proceeded overnight at the room temperature.
• the resultant was then purified through anion-exchange and ultrafiltration to get rid of unconjugated 40K-PEG and BT-100, and BT-200 was obtained.
• ARC15105 (PEGylated) (SEQ ID NO: 6) was prepared as described previously in Siller-Matula JM, Merhi Y, Tanguay JF et al. ARC15105 Is a Potent Antagonist of Von Willebrand Factor Mediated Platelet Activation and Adhesion. Arteriosclerosis, Thrombosis, and Vascular Biology 2012; 32(4): 902-909, the contents of which are incorporated herein by reference in its entirety.
• BT-100 or BT-200 were predicted to naturally form a stem-loop structure (as shown in Fig. 1) when dissolved in aqueous based buffer solution. This was confirmed by several secondary structural prediction models such as Mfold.
• VFD Vascular assist device
• In vitro extracorporeal circuits were established using left ventricular assist devices (HEAPvTWARE®) and silicone tubing with blood inlets and outlets.
• the pump speed was varied between 1800 and 3500 rounds per minute (rpm), and the circulating blood volume was adjusted to approximately 5 liters per minute, to correspond to normal physiologic settings for humans.
• Anticoagulated blood was left circulating for 1-4 hours in the extracorporeal circuits.
• lanes 1 and 10 are normal plasma (George King Bio-Medical Inc.).
• the Von Willebrand Ristocetin Cofactor [vWF:RCo] assay measures the ability of a patient's plasma to agglutinate platelets in the presence of the antibiotic Ristocetin.
• the rate of Ristocetin induced agglutination is related to the concentration and functional activity of the plasma Von Willebrand Factor.
• VWF VWF ristocetin cofactor activity
• Plasma was pre-incubated with BT-100 or ARC15105 (pegylated) at 37°C approximately 10 minutes prior to the addition of activating reagent, ristocetin.
• the assay was conducted (BC von Willebrand reagent; Dade Behring/Siemens, Marburg, Germany) (Reiter RA, et al., Desmopressin antagonizes the in vitro platelet dysfunction induced by GPIIb/IIIa inhibitors and aspirin. Blood 2003; 102: 4594-4599, the contents of which are incorporated herein by reference in their entirety).
• the system detects the electrical impedance change due to the adhesion and aggregation of platelets on two independent electrode-set surfaces in the test cuvette.
• the area under the aggregation curve (AUC) expresses the aggregation response over the measured time (AU ⁇ min) which can predict adverse outcome of patients and even bleeding (Sibbing D, et al., Antiplatelet effects of clopidogrel and bleeding in patients undergoing coronary stent placement. J Thromb Haemost 2010; 8: 250-256; and Siller-Matula JM et al., Cross validation of the Multiple Electrode Aggregometry. A prospective trial in healthy volunteers. Thromb Haemost 2009; 102: 397—403, the contents of which each are incorporated herein by reference in their entirety).
• a number of specific Multiplate test reagents (ASPItest, COLtest, TRAPtest, ADPtest, RISTOtest) are available, which permit detection of the role of different
• AA aminoethylcholine receptor
• ADP adenosine hydrophosphate
• collagen receptor adenosine glycoprotein Ib-IX
• ARC15105 pegylated at 37°C approximately 10 minutes prior to the addition of activating reagent, ristocetin. Thereafter, ristocetin (0.77 mg/ml) was added to separate samples to establish concentration-effect curves. The increase in electrical impedance was recorded continuously for 6 min. The mean values of the two independent determinations are expressed as the AUC of the aggregation tracing.
• the MEA instrument allows two ways to express the AUC: as arbitrary aggregation units (AU x min) or as units (U). Ten AU x min correspond to 1 U. AUC was measured in U, following the current recommendation of the manufacturer and many recent studies and expressed platelet aggregation as relative increase or decrease as compared to baseline values.
• ARC15105 (pegylated) were necessary to reach 90% inhibition (p ⁇ 0.05). These data clearly demonstrate, surprisingly, that BT-100 outperforms ARC15105.
• Comparisons between BT-100 and ARC15105 show that BT-100 is up to 25-fold more potent, with an overall average of 10-fold (Compare treatment columns A to C and B to D).
• CT closure time
• the membrane is coated with collagen/adenosine diphosphate (CADP), which are very sensitive to von Willebrand factor levels. This applies to a deficiency in VWF, its blockade by anti-VWF aptamers as well as VWF release, e.g. by desmopressin or endotoxin (Jilma B. Platelet function analyzer (PFA-100): a tool to quantify congenital or acquired platelet dysfunction. J Lab Clin Med 2001 ; 138: 152-163; Jilma-Stohlawetz et al. 2012; Reiter et al. 2003; Homoncik M, Blann AD, Hollenstein U, Pernerstorfer T, Eichler HG, Jilma B.
• CRIP collagen/adenosine diphosphate
• closure time in seconds
• Aggregation is quantified by measuring the time of aperture closure, with a maximum occlusion time of 300 seconds.
• “Native” refers to normal donor samples that have not been treated, which establishes the "normal” reference level of assay result; "ND" means not tested.
• VWF VANCE® Gplb and VWF binding assay
• the VWF: Ac assay does not require the use of ristocetin, since the recombinant Gplb has two gain-of-function mutations.
• An increase in extinction by turbidometric measurements is related to increased VWF binding and agglutination.
• Lawrie AS, et al. A comparative evaluation of a new automated assay for von Willebrand factor activity. Haemophilia 2013 Mar;19(2):338-342; de Maistre E, et al., Performance of two new automated assays for measuring von Willebrand activity: HemosIL AcuStar and Innovance. Throm Haemost. 2014(4), the contents of each of which are herein incorporated by reference in their entirety.
• VWF Ac was measured using an INNOVANCE® VWF Activity kit on Sy smex
• a VWF aptamer is investigated for the ability to block the interaction of platelets, leukocytes, and erythrocytes to von Willebrand factor (VWF) strands that are secreted from activated endothelial cells.
• VWF von Willebrand factor
• the experiment makes use of human components of a recently described in vitro microvessel system ⁇ In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl. Acad. Sci. USA 2012, 109, 9342-9347, the contents of which are herein incorporated by reference in their entirety), which demonstrates the formation of very long, thick VWF strands and cables from activated endothelial cells.
• VWF strands are capable of binding platelets and other blood cells, depending on the shear stresses applied.
• the experiment provides a model of microvascular occlusion in thrombotic microangiopathies such as thrombotic thrombocytopenic purpura and sickle cell disease and tests the ability of the anti-VWF aptamer to interfere with these processes.
• Example 7 Prevention of GI Bleeding Caused by Angiodysplasia
• a VWF aptamer is investigated in in vitro and in vivo models for the ability to prevent or inhibit an increase in angiogenesis that causes angiodysplasia and results in bleeding in the GI tract.
• a VWF aptamer is contacted with Human Umbilical Vein Endothelial Cells
• VECs cardiovascular disease .
• VECs vessel network formation, cell migration, cell proliferation and adhesion. Methods for these evaluations are taught in, for example, Starke RD, et al., Endothelial von Willebrand factor regulates angiogenesis. Blood, 201 1, 1 17: 1071-80, the contents of which are incorporated herein by reference in their entirety.
• HUVECs contacted with VWF aptamer are subjected to in vitro tube formation in a Matrigel assay, which demonstrates the ability of the VWF aptamer to interfere with network formation.
• Angiogenesis is cell migration. A Scatch wound assay combined with time lapse microscopy is used to assess the ability of the aptamer to inhibit migration. In this assay, HUVEC cells contacted with the aptamer are evaluated for reduced velocity and directionality of migration. Another aspect of angiogenesis is proliferation. Reduced proliferation of
• HUVECs contacted with one or more VWF aptamers is measured as a decrease in the number of cells as compared to the untreated control.
• Yet another aspect of angiogenesis is adhesion of endothelial cells, which is measured in an adhesion assay. Using a commercially available kit (Invitrogen), the inhibitory effect of the VWF aptamer on cell migration can be measured by reduced fluorescence of bound cells in the wells.
• ECs derived from circulating progenitor cells called blood outgrowth endothelial cells (BOECs), from healthy individuals and Von Willebrand's Disease (VWD) patients are isolated according to the methods described in Starke RD, et al., Endothelial von Willebrand factor regulates angiogenesis. Blood, 201 1, 1 17: 1071-80 and Ingram DA, et al., Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004 104:2752-60, the contents of each of which are incorporated herein by reference in their entirety.
• BOECs are used to evaluate the ability of the VWF aptamer to affect a reduction in or rescue from the increased angiogenesis observed in BOECs from VWD patients, as measured by matrix assay, migration, proliferation, and adhesion assays, observed in the BOECs from VWD patients compared to BOECs from healthy controls.
• Mouse models of angiogenesis include ischemic models, such as hindlimb ischemia, heart ischemia and skin models, and non-ischemic models, such as matrigel plug assays, disc assays, eye retina and corneal assays, wound healing and ovarian models. These models are used to evaluate the effect of VWF aptamer delivery on angiogenesis in vivo.
• mice are injected with matrigel alone or containing VWF aptamer and the model can be used as described in Couffinhal et al, 2009 and Starke et al, 2011, the contents of each of which are incorporated herein by reference in their entirety, to evaluate the effect of the aptamer on blood vessel formation in the matrigel plug.
• the mice are housed in a temperature-controlled room on a 12-hour light/12-hour dark cycle with food and water ad libitum. Animals are deeply anesthetized with isoflurane 5% and, thereafter, maintained with 2.5% isoflurane in a 70%/30% mixture of NO2/O2.
• a catheter is inserted into the tail vein to allow the intravenous administration (200 ⁇ .) of saline or aptamer.
• a micropipette is filled with 1 ⁇ _, of purified murine alpha-thrombin (approximately 1000 NIH units/mg; Sigma- Aldrich). Mice are placed in a stereotaxic device, the skin between the right eye and the right ear is incised, and the temporal muscle is retracted. A small craniotomy is performed, the dura is excised, and the middle cerebral artery (MCA) is exposed. 1 ⁇ _, of purified murine alpha-thrombin (0.75 UI) is injected into the lumen of the MCA to induce the formation of a clot in situ. The pipette is removed 10 minutes after the injection of alpha thrombin to allow the clot to stabilize. To induce thrombolysis, the aptamer is intravenously injected into the tail vein 20 minutes after the injection of alpha-thrombin. The control group receives the same volume of saline under identical conditions.
• Cerebral blood flow is determined by laser Doppler flowmetry using a fiberoptic probe (Oxford Optronix) glued to the skull in the MCA territory. CBF is measured before the injection of alpha-thrombin (100% baseline) and throughout the duration of the experiment. CBF in the MCA is measured in the presence or absence of aptamer at 1 min, 5 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, and 60 mins after administration of aptamer or saline. Showing that full occlusion occurs at a later time in the presence of aptamer versus saline control demonstrates that the aptamer slows the rate of occlusion in the MCA. Showing that full occlusion does not occur within 60 minutes indicates that the aptamer prevents occlusive thrombi formation.
• ischemic brain damage in the MCA area can be assessed by magnetic resonance imaging (MRI) at 6 hours and 24 hours following full occlusion.
• MRI magnetic resonance imaging
• MRI is carried out on a Pharmascan 7 T/12 cm system using surface coils to determine lesion size, which is quantified on T2 weighted images using ImageJ software.
• a reduction in lesion size in mice treated with aptamer compared with the lesion size of saline-treated mice indicates that the aptamer prevents or decreases occlusive thrombi formation.
• mice are anesthetized with 5% isoflurane (Baxter, Paris, France), placed in a stereotaxic device and maintained under anesthesia for up to 2 hours with 2% isoflurane in a 70%/30% mixture of N 2 O/O 2 .
• the masseter muscle is excised through a skin incision between the right eye and ear, and a small craniotomy (diameter, 1 mm) is performed on the parietal bone to expose the right middle cerebral artery (MCA).
• MCA right middle cerebral artery
• Thrombus induction is achieved by placing a piece of WHATMANTM filter paper strip soaked in freshly prepared FeCl 3 (5% to 20%, Sigma Aldrich, Lisle d'Abeau, France) on the intact dura mater, on top of the MCA for 4 minutes. Previous studies in mice have shown that 5% FeCb fails to induce thrombosis, while 10% FeCb induces partial thrombosis (forming a thrombus "core” formed at shear rates below 10,000 s "1 ) and 20% FeCb results in a stable and fully occlusive thrombus (formed at shear rates 10,000 s "1 or greater).
• Cerebral blood flow (CBF) in the MCA territory is determined by laser Doppler flowmetry (Oxford Optronix) which measures blood movements in the probed tissue.
• CBF is continuously measured before MCA occlusion and throughout the duration of occlusion, ischemia and thrombolysis.
• Occlusion time is defined as the time between FeCb application and CBF diminution below 20% of baseline.
• mice are administered 20% FeCb to induce thrombosis. After approximately 4-5 minutes (when cerebral blood flow drops below 50% of basal value, i.e., after partial occlusion), either aptamer or saline (control) is administered to the mice.
• CBF in the MCA is used to measure the degree of occlusion over time in the presence or absence of aptamer (i.e., occlusion is measured 1 min, 5 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, and 60 mins after administration of aptamer or saline).
• ischemic brain damage in the MCA is assessed using magnetic resonance imaging (MRI) at 6 hours and 24 hours following full occlusion as described in Example 8.
• MRI magnetic resonance imaging
• Examples of useful positive controls include aurintricarboxylic acid ("ATA", 20 mg/kg; Sigma- Aldrich), a polycarboxylated compound which binds to the Al domain of VWF, thus preventing binding of VWF to platelet Gplba and a monoclonal antibody or antibody fragment to the Al domain of VWF which prevents binding to platelet Gplba, i.e., Fab fragments of the monoclonal antibody Xia.B2 (2.5 mg/kg, Emfret Analytics, Wiirzburg, Germany).
• ATA aurintricarboxylic acid
• a polycarboxylated compound which binds to the Al domain of VWF
• Examples of useful negative controls include GR144053 trihydrochloride ("GR", 10 mg/kg; R&D Systems, Lille, France), a non-peptidic GpIIb/IIIa inhibitor, and a monoclonal antibody or antibody fragment to GpIIb/IIIa, i.e., Fab fragments of the monoclonal antibody Leo. H4 (2.5 mg/kg, Emfret Analytics, Wiirzburg, Germany).
• ATA and GR solutions are passed through 0.22 ⁇ filters prior to administration and subsequently injected as a rapid bolus (200 ⁇ .).
• Control IgG (rat) can be purchased from Jackson Immunoresearch.
• Fab fragments are prepared using Pierce Fab preparation kit (Fisher scientific, Illkirch, France) according to the manufacturer instructions
• Immunohistological analyses of thrombi from mice euthanized 10 minutes after administration of aptamer is performed using fluorescently-labeled antibodies against VWF and CD41 (platelet marker) and DAPI nuclear staining of MCA.
• VWF and CD41 platelet marker
• DAPI nuclear staining of MCA The observation of less VWF and CD41 in the MCA (as measured by fluorescence staining) in aptamer-treated mice compared with control mice indicates disaggregation of the thrombi in the presence of aptamer.
• the MCA can be re-opened and examined to determine the size of the thrombus in aptamer-treated versus saline-treated mice.
• useful positive controls include ATA and Xia.B2.
• useful negative controls include GR144053 trihydrochloride and a monoclonal antibody or antibody fragment to GpIIb/IIIa.
• mice with fully occlusive thrombi are generated using 20% FeCb as described in Example 9.
• Saline (control) or aptamer is administered to the mice intravenously or subcutaneously at 20 minutes following full occlusion.
• Doppler flowmetry is used to determine the cerebral blood flow (CBF) in the MCA, with an observed increase in CBF in the aptamer-treated mice compared with the saline-treated mice demonstrating the restoration of vessel patency.
• CBF cerebral blood flow
• MRI magnetic resonance imaging
• MRI magnetic resonance imaging
• a reduction in lesion size in mice treated with aptamer compared with the lesion size of saline- treated mice indicates the restoration of vessel patency.
• Similar methods can be used to determine restoration of patency in larger arteries, such as the carotid artery, using the FeCb model of thrombosis and monitoring via Doppler flowmetry MRI, and immunohistological analyses, as previously described.
• NHP non-human primates
• the NHP are housed in a temperature-controlled room on a 12-hour light/12-hour dark cycle with food and water ad libitum. Animals are lightly anesthetized with an intramuscular injection of lOmg/kg ketamine hydrochloride, intubated and anesthesia maintained with 0.5% to 1.5% isoflurane in 30% O2. Body temperature is monitored continually and maintained at 37 to 39°C with a heating pad.
• a catheter is inserted into the femoral vein to allow the intravenous administration of rose bengal, saline or BT-100 aptamer.
• the transorbital approach to the right MCA is performed as described in Maeda M, Moriguchi A, Mihara K, Aoki T, Takamatsu H et al. FK419, a nonpeptide platelet glycoprotein Ilb/IIIa antagonist, ameliorates brain infarction associated with thrombotic focal cerebral ischemia in monkeys: comparison with tissue plasminogen activator. J Cereb Blood Flow Metab. 2005 Jan;25 : 108- 1 18, the contents of which are herein incorporated by reference in their entirety.
• An orbital craniotomy is performed, permitting visualization of the right MCA while keeping the dura mater intact.
• the region is then irradiated with green light (wavelength 540nm) for 10 minutes coincidentally with 6 minutes of intravenous rose Bengal administration (20 mg/kg).
• the treatment (saline or aptamer) is applied at the end of the photoirradiation.
• Cerebral blood flow is determined by laser Doppler flowmetry using a fiberoptic probe (Oxford Optronix) placed on the MCA. CBF is measured before the photoirradiation and for up to 3hrs following aptamer administration. CBF in the MCA is measured in the presence or absence of BT-100 aptamer at 1 min, 5 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 60 mins, 120 mins and 180 mins after administration of BT-100 aptamer or saline.
• Aptamers are optimized to improve one or more desired characteristics or properties of the aptamer. Such characteristics or properties include improvements in binding affinity, pharmacokinetic or pharmacodynamic properties, thermostability, potency, or any other characteristic or property known to one with skill in the art.
• aptamers are optimized by altering the stem.
• the stem length may either be increased or decreased.
• the stem length is extended and made longer.
• the duplexed region of the stem may be altered to be made shorter, longer or to alter the symmetry or asymmetry.
• Branches may be added to the stem of the aptamer.
• aptamers are optimized by adding or altering one or more conjugates and/or terminal cap structures.
• the conjugate may be a polyethelyene glycol (PEG) moiety or any other conjugate or terminal cap known in the art.
• PEG polyethelyene glycol
• One or more conjugates are added to the aptamer to improve the desired characteristics or properties.
• conjugate(s) may be at the 5' or the 3 ' end of the aptamer.
• aptamers are optimized to increase thermostability, by increasing the double stranded region of the stem to exceed 6 base pairs.
• Optimized aptamers may be tested by any of the methods known in the art for assessing the desired characteristic and/or property, including those described herewithin. The optimized aptamers should exceed the performance of the equivalent, but unoptimized aptamer in at least one characteristic and/or property.
• ARC15105 is optimized to generate BT-200.
• the optimization is to improve thermostability and pharmacokinetic and pharmacodynamic properties such as half-life and/or duration of action.
• the duplexed stem region of ARC15105 is elongated from a 5-base pair duplex to a 9-base pair duplex. The extension of the stem region increases the thermostability of BT-200 and stabilizes the integrity of the aptamer, thus making it less susceptible to nuclease cleavage and prolonging the in vivo half-life and/or duration of action of the aptamer.
• the 40kD PEG is retained for BT-200.
• the characteristics and/or properties of BT-200 are tested by any of the methods known in the art and/or described herewithin. In a direct comparison, optimized aptamer BT-200 will outperform original aptamer ARC15105.
• BT-100 is optimized to generate BT-200.
• the 40kD PEG is conjugated to the 5' terminus of the BT-100.
• pegylation improves pharmacokinetics and pharmacodynamics properties such as half-life and/or duration of action.
• the characteristics and/or properties of BT-200 are tested by any of the methods known in the art and/or described herewithin.
• BT-200 was evaluated for in vivo inhibition of the VWF activity.
• the REAADS ® von Willebrand Factor Activity Test Kit (Diapharma, Catalog #: K10826, West Chester, Ohio) was utilized to measure functional VWF in plasma samples.
• the assay was adapted to evaluate inhibition of VWF activity by BT-200.
• the test kit is an enzyme-linked immunosorbent assay (ELISA) that uses a purified murine anti-VWF monoclonal antibody to detect the Al domain of VWF.
• BT-200 stock solution was prepared as 1 mg/ml in phosphate-buffered saline (PBS).
• PBS phosphate-buffered saline
• BT-200 was spiked into plasma samples to final concentrations of 10, 30, 100, 300, 1000, 3000, 10000, and 30000 ng/ml.
• the REAADS assay was performed according to manufacturer's instruction at room temperature.
• VWF activity was reported in percent (%) of normal, relative to a calibrator that has been standardized against the third International Standard for Factor VIII and von Willebrand Factor in Plasma (91/666). VWF activity was plotted against BT-200 concentration to determine ICso values.
• Results of the in vitro REAADS assay are presented in Table 8.
• the data shows a dose-dependent inhibition of VWF Al domain activity by BT-200 in monkey plasma.
• the study confirms that BT-200 inhibits VWF activity by blocking the Al domain of VWF.
• Results of the REAADS assay are presented in Table 9.
• the inhibition of BT-200 on VWF activity lasted more than 336 hours with a dose of 3 mg/kg administered either intravenously or subcutaneously.
• BT-200 blocked the VWF activity for more than 168 hours with a dose of 1 mg/kg by S.C. injection.
• BT-200 concentrations of BT-200 were adjusted to 0.01, 0.1, 0.4, 0.6, 0.8, 1.0, 3.0 and 10.0 ug/mL.
• platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time (CADP-CT) with a platelet function analyzer, PFA-200 (Siemens, Marburg, Germany). Normal saline was used as negative control. Maximal CT measured by PFA-200 is 5 min and the instrument gives a result >300 s if this time is exceeded.
• Data was plotted against BT-200 concentration and fitted in GraphPad Prism 5 (San Diego, CA) to obtain median effective dose (ED50) values.
• ED50 concentration-dependent prolongation of CADP-CT.
• ED50 concentration-dependent prolongation of CADP-CT.
• BT-200 produced concentration-dependent inhibition of VWF activity.
• the affinity of BT-200 binding to human VWF was 3.72 times higher than monkey VWF.
• BT-200 was evaluated in cynomolgus monkeys for in vivo inhibition of VWF activity. Sixteen adult monkeys were selected and distributed into four treatment groups according to sex and weight. The monkeys weighed between 2.6 to 3.9 kg, and aged 3 to 5 years old. Each group contained four animals, two males and two females, and the average weight of each group was about 3.2 kg. BT-200 dosing solutions were prepared in 0.9% saline to a final concentration of 10 mg/ml. Groups 1-4 were administered 3 mg/kg BT-200 by intravenous injection (I.V.), 3.0 mg/kg, 1.0 mg/kg, and 5.0 mg/kg BT-200 by subcutaneous injection (S.C.), respectively.
• I.V. intravenous injection
• S.C. subcutaneous injection
• Plasma samples were collected at 30 min before injection (-30 min), 1 h, 4 h, 12 h, 24 h, 48 h, 96 h, 168 h, and 240 h after injection. Plasma samples were prepared as described previously. CADP-CT measurement was performed with PFA-200 immediately upon sample collection.
• Closure time of cynomolgus monkey pre- or post- BT-200 injection was expressed as mean ⁇ standard deviation (SD) (see Table 12), respectively.
• SD standard deviation
• the inhibition of BT-200 on VWF in vivo maintained at least to 240 h.
• Coagulation parameters including activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were assayed by an automatic coagulometer (Stago, France) at 30 min before injection (pre-dose), 15 min, 1 h, 4 h, 8 h, 24 h, 48 h, 96 h, 168 h and 240 h after injection from the same 16 monkeys as in Example 19.
• aPTT activated partial thromboplastin time
• PT prothrombin time
• TT thrombin time
• FIB fibrinogen
• Coagulation parameters of each group at different time points are presented in Tables 13-16.
• BT-200 was not previously shown to be an anticoagulant, it was not expected to prolong clotting time. The observed prolonged aPTT in Example 20 was further investigated. The aPTT assay was repeated using aPTT reagents Actin-FS (Siemens, Marburg, Germany) and aPTT-lupus anticoagulant (LA) (Stago, France) with either silica or ellagic acid as a clot activator. Actin-FS is the most sensitive reagent to mild reductions of factors VIII, IX, and XI. The results revealed that the spurious aPTT effect is reagent specific (see Table 17). While aPTT reagents with silica as activator showed increased aPTT values in the presence of BT- 200, aPTT Actin-FS with ellagic acid exhibited the same aPTT value at all BT-200
• ROTEM Rotational thrombelastometry
• coagulation assays such as PT and aPTT are widely performed, they are known to have intrinsic limitations. For example, these assays evaluate the clotting cascade in isolation and are thus non-physiological. As a result, they usually show a poor correlation with the clinical phenotype, i.e., the PT or aPTT may be prolonged but this does not necessarily predict the bleeding phenotype. Moreover, these assays are in general insensitive to prothrombotic states. Furthermore, the PT and aPTT assays use the formation of a fibrin clot as the endpoint of the test and this is known to occur when only less than 5% of the total thrombin has been generated.
• ROTEM Rotational thrombelastometry
• Thrombin generation reflects the plasma's total ability to generate thrombin.
• the calibrated automated thrombogram (CAT) assay is a routine test used to measure the generation of thrombin. This assay displays the concentration of thrombin in clotting plasma as a function of time by monitoring the splitting of a fluorogenic substrate and comparing it with a constant known thrombin activity in a parallel non-clotting sample.
• the parameters of the thrombogram include lag time, peak height, endogenous thrombin potential (ETP) (calculated as the area under the curve), maximal rising slop, and time to peak (Hemker HC, et al., Thrombin generation, a function test of the haemostatic-thrombotic system. Thromb Haemost. 2006 Nov;96(5):553-61, the contents of which are incorporated herein by reference in their entirety).
• Coagulation inhibitors such as rivaroxaban decrease the ETP, and/or the peak and lengthen the time to peak.
• BT-200 The effect of BT-200 on thrombin generation was assessed via the CAT assay using platelet-poor plasma (PPP) reagents low or high (Thrombinoscope BV, Netherlands).
• PPP platelet-poor plasma
• the PPP reagent is used as a trigger that is added to platelet-poor plasma to initiate thrombin generation. Readout was measured on a Fluoroskan ASCENTTM microplate fluorimeter (Thermo
• Hematology parameters including hemoglobin concentration, hematocrit, white blood cell (WBC) and platelet counts were analyzed at pre-dose, 1 week and 2 weeks after BT-200 injection from the same 16 monkeys as in Example 19.
• the hematology tests were performed with a Sysmex XP-100 3 -Part Differential hematology analyzer (Sysmex, Japan). Compared with pre-dose, hemoglobin and hematocrit values decreased slightly, without significant difference. The WBC and platelet counts did not change after injection of BT-200.
• Willebrand Factor to the normal host would lead to minimal risk of a bleeding phenotype, unless that inhibitor was able to effect complete, sustained inhibition.
• This prediction was confirmed with the experience of a first-generation aptamer inhibitor of von Willebrand Factor, ARC 1779, administered to non-human primates, normal human volunteers, and even patients with thrombocytopenia or patients on anti-coagulants, yet no bleeding manifestations were observed (Gilbert JC, et al., First-in-human evaluation ofanti von Willebrand factor therapeutic aptamer ARC 1779 in healthy volunteers. Circulation.
• BT-200 was engineered to be more potent, longer-acting and to have better subcutaneous bioavailability than predecessor aptamer inhibitors of von Willebrand Factor, such as ARC 1779 and ARC15105. These goals appear to have been fully met based upon the findings in PK/PD studies of BT-200 in cynomolgus monkeys. Most of the dosed animals in this study were observed to have mucocutaneous bleeding mostly occurred at the limbs and/or thoracic area beneath the skin.
• PK analysis of BT-200 was performed in plasma samples from the same 16 monkeys as described in Example 19. Blood was sampled pre-dose (-30 min), 1 h, 4 h, 8 h, 24 h, 48 h, 96 h, 168 h, 240 h, and 336 h post-dose. Approximately 0.5 ml whole blood was collected at each time point into tubes containing 1.5% K2-EDTA. Samples were centrifuged at 3000 rpm to obtain plasma. All plasma samples were stored at -80°C until analysis.
• Plasma concentration of BT-200 was determined by high-performance liquid chromatography (HPLC) using Agilent 1260 Infinity System (Agilent, Santa Clara, CA) with a DNA PAC200 Column, 4*250mm.
• PK parameters including half-life (Ti /2 ), time of maximum concentration observed (Tmax), initial concentration (Co), maximum concentration (Cmax), AUC up to the last measurable concentration (AUCiast), AUC from 0 to infinity

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