WO2009140383A2

WO2009140383A2 - Aptamers that bind to p-selectin and their use as coagulation, thrombotic, inflammatory, and metastatic disease therapeutics

Info

Publication number: WO2009140383A2
Application number: PCT/US2009/043787
Authority: WO
Inventors: Robert Schaub; Jeffrey Kurz
Original assignee: Archemix Corp.
Priority date: 2008-05-13
Filing date: 2009-05-13
Publication date: 2009-11-19
Also published as: WO2009140383A3

Abstract

Materials and methods are provided to prepare targeting nucleic acids and more particularly to aptamers that bind to P-selectin, which are useful as therapeutics in and diagnostics of coagulation/thrombotic, inflammatory, metastatic and/or other pathologies, diseases or disorders in which P-selectin has been implicated. In addition, the aptamers may be used before, during and/or after medical procedures to reduce complications or side effects thereof. Materials and methods for the administration of aptamers that bind to P-selectin are also provided.

Description

APTAMERS THAT BIND TO P-SELECTIN AND THEIR USE AS COAGULATION, THROMBOTIC, INFLAMMATORY AND METASTATIC DISEASE THERAPEUTICS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional patent application claims the benefit of and priority to U.S. Provisional Application Serial Nos. 61/127,629, filed May 13, 2008; 61/132,182, filed June 16, 2008; 61/122,642, filed December 15, 2008; and 61/167,225, filed April 7, 2009; each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to the field of nucleic acids and more particularly to aptamers that bind to P-selectin, which are useful as therapeutics in and diagnostics of coagulation/thrombotic, inflammatory, metastatic and/or other pathologies, diseases or disorders in which P-selectin has been implicated. The invention further relates to materials and methods for the administration of aptamers that bind to P-selectin.

BACKGROUND OF THE INVENTION

[0003] An aptamer is an isolated or purified nucleic acid that binds with high specificity and affinity to a target through interactions other than Watson-Crick base pairing. An aptamer has a three dimensional structure that provides chemical contacts to specifically bind to a target. Unlike nucleic acid binding, aptamer binding is not dependent upon a conserved linear base sequence, but rather a conserved base sequence within the context of a particular secondary or tertiary structure. That is, aptamers are non-coding sequences. Any coding potential that an aptamer may possess is entirely fortuitous and plays no role whatsoever in the binding of an aptamer to its target. Thus, while it may be that aptamers that bind to the same target, and even to the same site on that target, share a similar linear base sequence, most do not. A typical minimized aptamer is 5-15 kDa in size (15-45 nucleotides), binds to its target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind to other proteins from the same gene family). [0004] Aptamers have been generated to many targets, such as small molecules, carbohydrates, peptides and proteins, including growth factors, transcription factors, enzymes, immunoglobulins and receptors. [0005] Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics, including high specificity and affinity, biological activity, low immunogenicity, tunable pharmacokinetic properties, stability, and good scalability and cost.

[0006] Aptamers are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g., through binding, aptamers may inhibit or stimulate the target's ability to function. Specific binding to a target is an inherent property of an aptamer. Functional activity, i.e., inhibiting or stimulating a target's function, is not.

An aptamer may bind to a target and have little or no effect on the desired or any other function of the target.

[0007] P-selectin, which is also known as CD62P, Granule Membrane Protein 140

(GMP- 140) and Platelet Activation-Dependent Granule to External Membrane Protein

(PADGEM), belongs to a family of cell adhesion molecules called selectins. P-selectin is the largest of the known selectins at 140 kDa. P-selectin is expressed in α-granules of activated platelets and granules of endothelial cells.

[0008] P-selectin plays an essential role in the initial recruitment of leukocytes to the site of injury during inflammation. When endothelial cells are activated during inflammation, P- selectin moves from the Weibel-Palade bodies to the endothelial surface. The primary ligand for P-selectin is PSGL-I (P-Selectin Glycoprotein Ligand 1), which is constitutively found on all leukocytes. The transient interactions between P-selectin and PSGL-I allow leukocytes to roll along the vascular endothelium. Accordingly, P-selectin is largely responsible for the rolling phase of the leukocyte adhesion cascade.

[0009] Other ligands for P-selectin include CD24 and uncharacterized ligands on eosinophils and neutrophils. As such, P-selectin has been implicated in coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases and/or disorders.

[0010] Accordingly, it would be beneficial to identify novel therapies for antagonizing P- selectin in the treatment of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorder, or that are used in conjunction with medical procedures.

The present invention provides materials and methods to meet these and other needs.

SUMMARY OF THE INVENTION

[0011] The present invention provides aptamers that bind to P-selectin, referred to herein as "P-selectin aptamers", and methods for using such aptamers in the treatment of pathologies, diseases or disorders that are characterized by abnormal adhesion between any combination of erythrocytes, leukocytes, platelets and the vascular endothelium, such as, for example, coagulation/thrombotic, inflammatory, metastatic and other P-selectin-mediated pathologies, diseases or disorders. In addition, the aptamers may be used before, during and/or after medical procedures in order to reduce the complications or side effects thereof. [0012] The P-selectin aptamers bind to P-selectin or a fragment thereof. Preferably, the P-selectin is human P-selectin. Preferably, the P-selectin aptamer has a dissociation constant for P-selectin of 100 nM or less.

[0013] Examples of P-selectin aptamers include, but are not limited to, aptamers that comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, which is referred to herein as ARC5665; SEQ ID NO: 2, which is referred to herein as ARC5685; SEQ ID NO: 3, which is referred to herein as ARC5691; SEQ ID NO: 4, which is referred to herein as ARC5692; SEQ ID NO: 5, which is referred to herein as ARC5690. Other examples of P-selectin aptamers include ARC6337 and ARC5134. [0014] For example, the P-selectin aptamer is an aptamer comprising the following nucleic acid sequence set forth below: fC-fU-fC-rA-rA-fC-rG-rA-rG-fC-fC-rA-rG-rG-rA-rA- fC-rA-fU-fC-rG-rA-fC-rG-fU-fC-rA-rG-fC-rA-rA-rA-fC-rG-fC-rG-rA-rG (SEQ ID NO: 1) (ARC5665), wherein "rN" is a ribonucleotide and "fN" is a 2'-fluorinated nucleotide. ARC5665 corresponds to ligand PF377sl, which is disclosed in U.S. Patent Publication No. 2004/0072234 Al, and which is incorporated herein by reference in its entirety. In some embodiments, the P-selectin aptamer is an aptamer or salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 1.

[0015] Preferably, the P-selectin aptamer is an aptamer comprising the following nucleic acid sequence set forth below: fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA- fC-mA-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 2) (ARC5685), wherein "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide and "mN" is a 2'-0 Methyl modified nucleotide. In some embodiments, the P-selectin aptamer is an aptamer or salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 2.

[0016] More preferably, the P-selectin aptamer is an aptamer comprising the following nucleic acid sequence set forth below: NH2-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG- mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA- rG-idT (SEQ ID NO: 3) (ARC5691), wherein "NH2" is a 5'-hexylamine linker phosphoramidite, "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'- fluorinated nucleotide and "mN" is a 2'-0 Methyl containing nucleotide. In some embodiments, the P-selectin aptamer is an aptamer or salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 3.

[0017] Most preferably, the P-selectin aptamer is an aptamer comprising the following nucleic acid sequence set forth below: PEG40K-nh-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC- rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC- rG-rA-rG-idT (SEQ ID NO: 4) (ARC5692), wherein "nh" is an amine linker, "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide, "mN" is a 2'-0 Methyl containing nucleotide and "PEG40K" is a 40 kDa polyethylene glycol moiety. In some embodiments, the P-selectin aptamer is an aptamer or salt thereof that consists of the nucleic acid sequence of SEQ ID NO: 4.

[0018] The P-selectin aptamers may comprise at least one chemical modification. Preferably, the modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage and a chemical substitution at a base position. Alternatively, the modification is selected from the group consisting of: incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone. The high molecular weight, non-immunogenic, compound is preferably polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is methoxypoly ethylene glycol (mPEG). The 3' cap is preferably an inverted deoxythymidine cap.

[0019] The invention also provides aptamers that have substantially the same ability to bind to P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. In some embodiments, the aptamers have substantially the same structure as the aptamers shown in SEQ ID NOs: 2, 3 or 4. In some embodiments, the aptamers have substantially the same ability to bind to P- selectin and substantially the same structure as the aptamers shown in SEQ ID NOs: 2, 3 or 4. The invention also provides aptamers that have substantially the same ability to bind to P- selectin and modulate a biological function of P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4.

[0020] The invention also provides aptamers that bind to P-selectin and have one or more of the following characteristics: (i) includes the primary nucleic acid sequence of SEQ ID NO: 2; (ii) includes a primary sequence that has at least 70% sequence identity to the primary nucleic acid sequence shown in SEQ ID NO: 2; and/or (iii) has substantially the same, or better, ability to bind to P-selectin as that of an aptamer that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 4. As used herein, the term primary sequence refers to the 5' to 3' linear sequence of nucleotide bases of the nucleic acid sequence that forms an aptamer. For example, the primary sequence of ARC5685, ARC5691 and ARC5692 is shown in SEQ ID NO: 2.

[0021] The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a P-selectin aptamer or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

[0022] The invention further provides a method for treating, preventing or ameliorating a pathology, disease or disorder mediated by P-selectin by administering to a subject the above pharmaceutical composition. Preferably, the subject is a mammal. More preferably, the subject is a human. Preferably, the pathology, disease or disorder is selected from the group consisting of: a coagulation/thrombotic, inflammatory, metastatic or other pathology, disease or disorder. Specifically, the pathology, disease or disorder is selected from the group consisting of: sickle cell disease, sickle cell disease sequelae (pain, acute chest syndrome, vasoocclusive crisis, acute vasoocclusive syndrome, acute non-occlusive syndrome, chronic syndrome, vascular inflammation, hypoxia of tissues, vasoocclusion of organs and tissues, organ failure, thrombogenesis, cerebrovascular accident, dactylitis, priapism, hemolytic anemia, aplastic crisis, pulmonary hypertension, retinopathy, osteonecrosis and skin ulcers), sickle cell anemia, vascular diseases, cardiovascular diseases, thrombotic diseases, hemostasis diseases, myocardial infarction, stroke, transient ischemic attack, revascularization, stent restenosis, atherosclerosis, deep vein thrombosis, venous thromboembolism, hypereosinophilia, ischemia/reperfusion injury, inflammatory diseases, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, juvenile idiopathic arthritis, organ transplant, graft rejection, ocular inflammation, retinal inflammation, colitis, conjunctivitis, scleritis, tumor metastasis, renal failure, epilepsy, malaria, cerebral malaria, asthma, psoriasis, allergic diseases, allergic conjunctivitis, immune diseases, shock and hemorrhagic shock.

[0023] The pharmaceutical compositions may also be administered prior to, during and/or after a medical procedure. For example, the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as surgery, cardiopulmonary bypass, percutaneous coronary intervention (PCI), transfusion, organ transplant, dialysis, intravitreal injection, photocoagulation, photodynamic therapy, and radiation treatment. [0024] The pharmaceutical compositions may also be administered in combination with another drug. Alternatively, the pharmaceutical compositions may be administered in combination with another therapy. For example, the P-selectin aptamers may be administered in combination with any one or more of: anticoagulants, statins, vasodilators, anti-angiogenics (for AMD (acute macular degeneration) and/or metastatic disease), analgesics (e.g., opiates), corticosteroids, hydroxyurea, iron chelators, NSAIDs (non-steroidal anti-inflammatory drugs) and other anti-inflammatory agents, antibiotics, fibrinolytics, antimalarials (e.g., quinine, chloroquine), antihistamines, cytotoxics, cytostatics, chemotherapeutics, radioisotopes, immunosuppressants, antivirals, and vitamins. Specifically, the P-selectin aptamers may be administered with fibrinolytics to resolve clots, or with immunosuppressants for organ transplantation.

[0025] The P-selectin aptamers may also be used for identification of the P-selectin protein. For example, the P-selectin aptamers may be used to identify, quantify or otherwise detect the presence of the P-selectin protein in a sample, such as a biological sample or other subject-derived sample. For example, the P-selectin aptamers are used in in vitro assays, e.g., ELISA, to detect P-selectin levels in a patient sample.

[0026] The invention further provides for the use of a P-selectin aptamer in the manufacture of a medicament in the treatment, prevention and/or amelioration of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders. For example, ARC5685, ARC5691 and ARC5692 are used in the manufacture of a medicament for treating, preventing or otherwise ameliorating coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders.

[0027] In one embodiment, the invention provides an aptamer described herein for use in a method of treatment, prevention and/or amelioration of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders. [0028] In one embodiment, the invention provides for the use of an aptamer described herein in the manufacture of a diagnostic composition or product for use in a method of diagnosis practiced on the human or animal body. In some embodiments, the method of diagnosis is for the diagnosis of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders.

[0029] In one embodiment, the invention provides an aptamer described herein for use in a method of diagnosis practiced on the human or animal body. In some embodiments, the method of diagnosis is for the diagnosis of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders. [0030] In one embodiment, the invention provides the use of an aptamer described herein for diagnosis in vitro. In some embodiments, the in vitro use is for the diagnosis of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders.

[0031] The invention also provides a kit comprising at least one container comprising a quantity of one or more P-selectin aptamers along with instructions for using the P-selectin aptamer or aptamers in the treatment, prevention and/or amelioration of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders. For example, the kit includes ARC5685, ARC5691 and ARC5692 and combinations thereof. In some embodiments, the aptamers are formulated as a pharmaceutical composition.

[0032] The invention also provides aptamers that have been identified by the SELEX™ process, which comprises the steps of (a) contacting a mixture of nucleic acids with P- selectin under conditions in which binding occurs; (b) partitioning unbound nucleic acids from those nucleic acids that have bound to P-selectin; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of binding, partitioning, and amplifying through as many cycles as desired, to obtain aptamer(s) that bind specifically to P-selectin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic representation of the in vitro aptamer selection (SELEX™) process from pools of random sequence oligonucleotides. [0034] Figure 2 is an illustration depicting various PEGylation strategies representing standard mono-PEGylation, multiple PEGylation and oligomerization via PEGylation. [0035] Figure 3 is an illustration of a 40 kDa branched PEG.

[0036] Figure 4 is an illustration of a 40 kDa branched PEG that is attached to the 5 'end of an aptamer.

[0037] Figure 5 is a schematic showing the relationship between vasoocclusion, vascular inflammation and hypoxia in sickle cell disease (SCD).

[0038] Figure 6 is a cartoon illustrating vascular occlusion secondary to erythrocyte and leukocyte adhesion.

[0039] Figure 7 presents the mean concentration-time profile of and pharmacokinetic (PK) parameters for ARC5692 following intravenous administration in mice. [0040] Figure 8 presents the mean concentration-time profile of and pharmacokinetic parameters for ARC5692 following intravenous and subcutaneous administration in rats. [0041] Figure 9 presents the mean concentration-time profile of and pharmacokinetic parameters for ARC5692 following intravenous and subcutaneous administration in cynomolgus monkeys.

[0042] Figure 10 presents binding data for ARC5685 using a nitrocellulose filter binding assay to recombinant human P-selectin (circles), E-selectin (triangles) and L-selectin

(diamonds).

[0043] Figure 11 presents data for an anti-human P-selectin aptamer (ARC6337) and an anti-mouse P-selectin aptamer (ARC5134), as quantitated by surface plasmon resonance for binding to recombinant human P-selectin (left panels) and mouse P-selectin (right panels).

[0044] Figure 12 shows inhibition of recombinant human PSGL-I to immobilized, recombinant human P-selectin.

[0045] Figure 13 shows the results of ARC5692 in a FACS assay with ADP-activated platelets and THP-I cells.

[0046] Figure 14 shows a graph of the percentage of bound platelets as a function of

ARC5692 concentration for THP-I cells positive for platelets, both in the presence (solid symbols) and absence (open symbols) of ADP.

[0047] Figure 15 top shows a graph of the percentage of bound platelets as a function of

ARC5692 concentration for THP-I cells positive for binding of thrombin activated platelets from 4 donors. Figure 15 bottom shows a graph of the percentage of bound platelets as a function of ARC5692 concentration for THP-I cells for binding of platelets without thrombin activation.

[0048] Figure 16 shows the average percent inhibition of THP-I cells binding to platelets with ARC5692, with or without thrombin.

[0049] Figure 17 shows data from ARC5692 mediated rolling of monocytes on activated platelets. Panel A is a fluorescence micrograph showing adhered and rolling leukocytes in the absence of ARC5692. Panel B illustrates the loss of leukocyte rolling and adhesion in the presence of 1 μg/mL ARC5692. Panel C shows the average number or rolling or adhered leukocytes across several visual fields on platelets from two donors in the presence of increasing concentrations of ARC5692. Panel D is a plot of percent inhibition of rolling by

ARC5692 derived from the data shown in Panel C.

[0050] Figure 18 presents photomicrographs from a leukocyte rolling experiment showing that leukocyte rolling was completely inhibited in histamine stimulated veins after intravenous (IV) infusion of anti-mouse P-selectin aptamer ARC5690. [0051] Figure 19 shows two tables of leukocyte rolling data. Table 1 shows leukocyte rolling data in histamine treated mice prior to administration of anti-P-selectin aptamer.

Table 2 shows leukocyte rolling data in mice treated with anti-P-selectin aptamer ARC5690.

[0052] Figure 20 top presents photomicrographs from a leukocyte rolling experiment showing that leukocyte rolling was not inhibited in histamine stimulated veins after IV infusion of ARC5694 (a scrambled control aptamer). Figure 20 bottom shows a table containing leukocyte rolling data in mice treated with scrambled control aptamer ARC5694.

[0053] Figure 21 is an illustration depicting the proposed secondary structure of

ARC5692.

[0054] Figure 22 shows human pharmacokinetic (PK) parameter estimates based upon allometric scaling.

[0055] Figure 23 is a table showing the effect of ARC5690 on hemodynamic parameters in AS mice after hypoxia/reoxygenation (RBC adhesion experiments).

[0056] Figure 24 shows the effect of ARC5690 on red blood cell (RBC) velocity and adhesion in AS mice after hypoxia/reoxygenation. Figures 24A (left) & B (right) are graphs showing the effect of ARC5690 on RBC velocity (Figure 24A) and adhesion (Figure 24B) in

AS mice after hypoxia/reoxygenation compared to vehicle (saline) control.

[0057] Figure 25 is a table showing the effect of ARC5690 on hemodynamic parameters in AS mice after hypoxia/reoxygenation (WBC adhesion experiments).

[0058] Figure 26 shows the effect of ARC5690 on leukocyte rolling flux and adhesion in

AS mice after hypoxia/reoxygenation. Figures 26A (left) & B (right) are graphs showing the effect of ARC5690 on leukocyte rolling flux (Figure 26A) and adhesion (Figure 26B) in AS mice after hypoxia/reoxygenation.

[0059] Figure 27 is a series of graphs showing the kinetics of ARC6337 binding to pre- activated platelets. The graphs show that ARC6337 binds to TRAP-activated, but not to unactivated platelets, and this binding reaches steady state in less than two minutes and is partially abrogated by EDTA. Further increases in ARC6337 over time correspond to increased exposure of platelet surface P-selectin.

[0060] Figure 28 is a graph showing comparable platelet activation and ARC6337 binding with TRAP v. thrombin/GPRP and in citrate v. PPACK.

[0061] Figure 29 is a series of graphs showing the direct evaluation of the affinity of the

P-selectin aptamer ARC6337. The graphs show that ARC6337 binds saturably to thrombin- activated platelets with an apparent affinity of 190 nM (95% confidence interval 130-251 nM). Results are mean ± SEM, n = 3. [0062] Figure 30 shows that a two binding site model is a significantly better fit to the data than a one binding site model for ARC6337.

[0063] Figure 31 is a series of graphs showing that ARC6337 inhibits rhuPSGLl-Ig binding to activated platelets. These graphs show that ARC6337 competitively inhibits (IC₅₀

= 191 nM, 95% CI = 114-321 nM, IC₉O estimate = 1,723 nM) binding of recombinant human

PSGLl-Ig chimera to activated human platelets. Results are mean ± SEM, n = 3.

[0064] Figure 32 is a series of graphs showing that ARC5692 inhibits rhuPSGLl-Ig binding to activated platelets. These graphs show that ARC5692 competitively inhibits (IC₅₀

= 393 nM, 95% CI = 208-741 nM, IC₉₀ estimate = 3,534 nM) binding of recombinant human

PSGLl-Ig chimera to activated human platelets. Results are mean ± SEM, n = 3.

[0065] Figure 33 is a series of graphs showing aptamer inhibition of thrombin- stimulated human monocyte-platelet (top panels) and neutrophil-platelet (bottom panels) aggregate formation. Data are quantified in terms of the percentage of monocytes or neutrophils with at least one platelet bound (left panels) or in terms of fluorescence intensity of monocytes or neutrophils due to binding of labeled platelets (right panels). All data shown are mean ±

SEM, n = 6.

[0066] Figure 34 is a series of graphs showing aptamer inhibition of ADP-stimulated human monocyte-platelet and neutrophil-platelet aggregate formation. All data shown are mean ± SEM, n = 6.

[0067] Figure 35 is a series of tables showing the comparison of aptamer apparent binding affinity and IC₅₀S for inhibition of monovalent ligand binding and inhibition of multivalent cell adhesion.

[0068] Figure 36 is a series of graphs showing that ARC5692 reduces platelet forward light scatter (suggestive of platelet-platelet aggregates) of agonist- stimulated, eptifibatide- treated whole blood samples.

[0069] Figure 37 is a series of graphs showing the partial blockade of whole blood platelet aggregation by ARC5692.

[0070] Figure 38 is a series of graphs showing that ARC5692 partially blocks whole blood platelet aggregation regardless of anticoagulant or agonist.

[0071] Figure 39 is a scatter plot illustrating the range of thrombus weights between groups, n is the number of animals per group.

[0072] Figure 40 is a graph showing thrombus weight analysis 72 hours post-IVC ligation. The mean thrombus weight of the NL group was significantly different from all groups (Psel-conapt, Psel-apt and Psel-Ab, P<0.001, P<0.001, P<0.05, respectively). Aptamer (Psel-apt) and antibody (Psel-Ab) group weights were significantly lower than the control group (Psel-conapt) (P<0.05 and p<0.01, respectively), n is the number of animals per group.

[0073] Figure 41 is a graph showing a morphometric evaluation of inflammatory cells in vein wall at 72 hours post-IVC ligation, n is the number of mice per group. [0074] Figure 42 is a graph showing morphometric scoring for thrombosis surface organization, intimal thickness and intimal fibrosis at 72 hours post-IVC ligation, n is the number of mice per group.

[0075] Figure 43 is a graph showing soluble P-selectin plasma levels at 72 hours post- IVC ligation, n is the number of mice per group.

[0076] Figure 44 is a graph showing thrombus weight versus soluble P-selectin plasma levels at 72 hours post-IVC ligation, n is the number of mice per group.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular form also includes the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.

[0078] The invention provides aptamers that bind to P-selectin, which are referred to herein as "P-selectin aptamers", and methods for using such aptamers in the treatment of pathologies, diseases or disorders that are characterized by abnormal adhesion between any combination of erythrocytes, leukocytes, platelets and the vascular endothelium, including, but not limited to, coagulation/thrombotic, inflammatory, metastatic and other P-selectin- mediated pathologies, diseases and disorders. In addition, the aptamers may be used before, during and/or after medical procedures in order to reduce the complications or side effects thereof.

[0079] Leukocyte extravasation, a precursor to inflammation, represents a multi-step process initiated by leukocyte tethering and rolling along the vessel wall of post-capillary venules. The tethering and rolling steps are mediated, in part, by P-selectin and its ligands.

Rolling on P-selectin and its ligands allows leukocytes to interact with chemokines on the surface of the activated endothelium. In addition to its role in leukocyte rolling and extravasation in inflammation, P-selectin also mediates platelet-leukocyte adhesion within thrombi, and increases tissue factor expression on monocytes, thereby promoting fibrin deposition and thrombogenesis. Figure 6 illustrates vascular occlusion secondary to erythrocyte and leukocyte rolling and/or adhesion.

IDENTIFICATION OF APTAMERS

[0080] The aptamers described herein are identified through a method known in the art as Systematic Evolution of Ligands by Exponential Enrichment, or SELEX™, which is shown generally in Figure 1. More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX™ method includes steps of: (a) contacting the mixture with a target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) amplifying the bound nucleic acids to yield a ligand-enriched mixture of nucleic acids; and, optionally, (d) reiterating the steps of binding, partitioning, and amplifying through as many cycles as desired to yield highly specific, high affinity aptamers to the target molecule. In those instances where transcribed aptamers are being selected, the amplification step of SELEX method includes the steps of: (i) reverse transcribing, or otherwise transmitting the sequence information into a corresponding DNA sequence, the nucleic acids dissociated from the nucleic acid-target complexes; (ii) PCR amplification; and (iii) transcribing, or otherwise transmitting the sequence information into a corresponding DNA sequence, the PCR amplified nucleic acids) before restarting the process. The starting pool of nucleic acids can be modified or unmodified DNA, RNA, or DNA/RNA hybrids, and acceptable modifications include modifications at the base, sugar and/or at the internucleotide linkages. The composition of the pool is dependent on the desired properties of the final aptamer. Selections elections can be performed with nucleic acid sequences incorporating modified nucleotides to e.g., stabilize the aptamer molecules against degradation in vivo. For example, resistance to nuclease degradation can be greatly increased by the incorporation of modifying groups at the 2 '-position.

[0081] In one embodiment, the present invention provides aptamers including single 2' substitutions at all bases or combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-NH₂ and 2'-0Me modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, the present invention provides aptamers including combinations of 2'-OH, 2'-F, 2'-deoxy, T- OMe, 2'-NH₂, and 2'-methoxyethyl modifications of the ATP, GTP, CTP, TTP and UTP nucleotides. In some embodiments, the present invention provides aptamers including all or substantially all 2'-OMe modified ATP, GTP, CTP, TTP, and/or UTP nucleotides. [0082] In some embodiments, 2 '-modified aptamers of the invention are created using modified polymerases, e.g., a modified RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2' position that is higher than that of wild-type polymerases. In one embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to phenylalanine (Y639F). In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to phenylalanine and the lysine residue at position 378 has been changed to arginine (Y639F/K378R). In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to phenylalanine, the histidine residue at position 784 has been changed to an alanine, and the lysine residue at position 378 has been changed to arginine (Y639F/H784A/K378R), and the transcription reaction mixture requires a spike of 2'-OH GTP for transcription.

[0083] In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to leucine (Y639L). In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to leucine (Y639L) and the histidine at position 784 has been changed to an alanine residue (Y639L/H784A). In another embodiment, the modified RNA polymerase is a mutant T7 polymerase in which the tyrosine residue at position 639 has been changed to leucine, the histidine residue at position 784 has been changed to an alanine, and the lysine residue at position 378 has been changed to arginine (Y639L/H784A/K378R).

[0084] Another suitable RNA polymerase having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2' position that is higher than that of wild-type polymerases is, for example, a mutant T3 RNA polymerase. In one embodiment, the mutant T3 RNA polymerase has a mutation at position 640, wherein the tyrosine residue at position 640 is replaced with a phenylalanine residue (Y640F). In another embodiment, the mutant T3 RNA polymerase has mutations at position 640 and position 785, wherein the tyrosine residue at position 640 is replaced with a leucine residue and the histidine residue at position 785 is replaced with an alanine residue (Y640L/H785A). [0085] 2 '-modified oligonucleotides may be synthesized entirely of modified nucleotides, or with a subset of modified nucleotides. The modifications can be the same or different. Some or all nucleotides may be modified, and those that are modified may contain the same modification. For example, all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modification. All purine nucleotides may have one type of modification (or are unmodified), while all pyrimidine nucleotides have another, different type of modification (or are unmodified). In this way, transcripts, or pools of transcripts are generated using any combination of modifications, including for example, ribonucleotides (2'-OH), deoxyribonucleotides (2'-deoxy), 2'-amino nucleotides (2'-NH₂), 2'-fluoro nucleotides (2'-F) and 2'-O-methyl (2'-0Me) nucleotides.

[0086] As used herein, a transcription mixture containing only 2'-0Me A, G, C, and U and/or T triphosphates (2'-0Me ATP, 2'-0Me UTP and/or 2'-0Me TTP, 2'-0Me CTP, and 2'-0Me GTP) is referred to as an MNA or mRmY mixture, and aptamers selected therefrom are referred to as MNA aptamers or mRmY aptamers and contain only 2'-O-methyl nucleotides. A transcription mixture containing 2'-0Me C and U and/or T and 2'-OH A and G is referred to as an "rRmY" mixture and aptamers selected therefrom are referred to as "rRmY" aptamers. A transcription mixture containing deoxy A and G and 2'-0Me U and/or T, and C is referred to as a "dRmY" mixture and aptamers selected therefrom are referred to as "dRmY" aptamers. A transcription mixture containing 2'-0Me A, C, and U and/or T, and 2'-OH G is referred to as a "rGmH" mixture and aptamers selected therefrom are referred to as "rGmH" aptamers. A transcription mixture alternately containing 2'-0Me A, C, U and/or T and G and 2'-0Me A, U and/or T, and C and 2'-F G is referred to as an "alternating mixture" and aptamers selected therefrom are referred to as "alternating mixture" aptamers. A transcription mixture containing 2'-OH A and G and 2'-F C and U and/or T is referred to as an "rRfY" mixture and aptamers selected therefrom are referred to as "rRfY" aptamers. A transcription mixture containing 2'-0Me A and G and 2'-F C, and U and/or T is referred to as an "mRfY" mixture and aptamers selected therefrom are referred to as "mRfY" aptamers. A transcription mixture containing 2'-0Me A, U and/or T, and C, and 2'-F G is referred to as a "fGmH" mixture and aptamers selected therefrom are referred to as "fGmH" aptamers. A transcription mixture containing 2'-0Me A, U and/or T, C, and G, where up to 10% of the G's are ribonucleotides is referred to as a "r/mGmH" mixture and aptamers selected therefrom are referred to as "r/mGmH" aptamers. A transcription mixture containing T- OMe A, U and/or T, and C, and deoxy G is referred to as a "dGmH" mixture and aptamers selected therefrom are referred to as "dGmH" aptamers. A transcription mixture containing deoxy A, and 2'-0Me C, G and U and/or T is referred to as a "dAmB" mixture and aptamers selected therefrom are referred to as "dAmB" aptamers. A transcription mixture containing 2'-OH A and 2'-0Me C, G and U and/or T is referred to as a "rAmB" mixture and aptamers selected therefrom are referred to as "rAmB" aptamers. A transcription mixture containing 2'-OH adenosine triphosphate and guanosine triphosphate and deoxy cytidine triphosphate and thymidine triphosphate is referred to as an rRdY mixture and aptamers selected therefrom are referred to as "rRdY' aptamers. A transcription mixture containing 2'-0Me A, U and/or T, and G, and deoxy C is referred to as a "dCmD" mixture and aptamers selected there from are referred to as "dCmD" aptamers. A transcription mixture containing 2'-0Me A, G, and C, and deoxy T is referred to as a "dTmV" mixture and aptamers selected there from are referred to as "dTmV" aptamers. A transcription mixture containing 2'-0Me A, C, and G, and 2'-OH U is referred to as a "rUmV" mixture and aptamers selected there from are referred to as "rUmV" aptamers. A transcription mixture containing 2'-0Me A, C, and G, and T- deoxy U is referred to as a "dUmV" mixture and aptamers selected there from are referred to as "dUmV" aptamers. A transcription mixture containing all 2'-OH nucleotides is referred to as a "rN" mixture and aptamers selected therefrom are referred to as "rN," "rRrY" or RNA aptamers, and a transcription mixture containing all deoxy nucleotides is referred to as a "dN" mixture and aptamers selected therefrom are referred to as "dN" or "dRdY" or DNA aptamers.

[0087] A number of factors have been determined to be useful for optimizing the transcription conditions used to produce the aptamers disclosed herein. For example, a leader sequence can be incorporated into the fixed sequence at the 5 ' end of a DNA transcription template. The leader sequence is typically 6-15 nucleotides long, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long, and may be composed of all purines, or a mixture of purine and pyrimidine nucleotides.

[0088] For compositions that contain 2'-0Me GTP, another useful factor can be the presence or concentration of 2'-OH guanosine or GMP. Transcription can be divided into two phases: the first phase is initiation, during which the RNA is extended by about 10-12 nucleotides; the second phase is elongation, during which transcription proceeds beyond the addition of the first about 10-12 nucleotides. It has been found that 2'-OH GMP or guanosine added to a transcription mixture containing an excess of 2'-0Me GTP are sufficient to enable the polymerase to initiate transcription. Priming transcription with 2'-OH guanosine e.g., or GMP is useful due to the specificity of the polymerase for the initiating nucleotide. The preferred concentration of GMP is 0.5 mM and even more preferably 1 mM. [0089] Another useful factor in optimizing the incorporation of 2'-OMe substituted nucleotides into transcripts is the use of both divalent magnesium and manganese in the transcription mixture. Different combinations of concentrations of magnesium chloride and manganese chloride have been found to affect yields of 2'-0 modified transcripts, the optimum concentration of the magnesium and manganese chloride being dependent on the concentration in the transcription reaction mixture of NTPs which complex divalent metal ions.

[0090] Other reagents can be included in the transcription reaction, include buffers such as HEPES buffer, a redox reagent such as dithiothreitol (DTT), a polycation such as spermidine, spermine, a surfactant such as Triton XlOO, and any combinations thereof. [0091] In one embodiment, the HEPES buffer concentration can range from 0 to 1 M. The present invention also contemplates the use of other buffering agents having a pKa between 5 and 10 including, for example, Tris-hydroxymethyl-aminomethane. In some embodiments, the DTT concentration can range from 0 to 400 mM. The methods of the present invention also provide for the use of other reducing agents including, for example, mercaptoethanol. In some embodiments, the spermidine and/or spermine concentration can range from 0 to 20 mM. In some embodiments, the PEG-8000 concentration can range from 0 to 50 % (w/v). The methods of the present invention also provide for the use of other hydrophilic polymer including, for example, other molecular weight PEG or other polyalkylene glycols. In some embodiments, the Triton X-100 concentration can range from 0 to 0.1% (w/v). The methods of the present invention also provide for the use of other non- ionic detergents including, for example, other detergents, including other Triton-X detergents. In some embodiments, the MgCl₂ concentration can range from 0.5 mM to 50 mM. The MnCl₂ concentration can range from 0.15 mM to 15 mM. In some embodiments, the 2'-OMe NTP concentration (each NTP) can range from 5 μM to 5 mM. In some embodiments, the 2'-OH GTP concentration can range from 0 μM to 300 μM. In some embodiments, the T- OH GMP concentration can range from 0 to 5 mM. The pH can range from pH 6 to pH 9. [0092] Variants of the SELEX process may also be used to identify aptamers. For example, one may use agonist SELEX, toggle SELEX, 2 '-Modified SELEX or Counter SELEX. Each of these variations of the SELEX process is known in the art.

P-SELECTIN APTAMERS

[0093] The invention includes nucleic acid aptamers, preferably of 33-43 nucleotides in length, that bind specifically to P-selectin and which, in some embodiments, functionally modulate, e.g., stimulate, block, or otherwise inhibit or stimulate, the activity of P-selectin. The term "specifically", as used herein, means specified, precisely or particularly. [0094] The P-selectin aptamers bind to P-selectin or a variant or a fragment thereof. A P- selectin variant, as used herein, encompasses variants that perform essentially the same function as P-selectin functions, preferably includes substantially the same structure and in some embodiments includes at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and even more preferably at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of P-selectin. [0095] Preferably, the P-selectin aptamers bind to full length P-selectin. If the aptamer binds to a fragment of P-selectin, it is preferable that the aptamer bind to the lectin domain of P-selectin.

[0096] The P-selectin may be from any species, but is preferably human. [0097] The P-selectin aptamer preferably comprises a dissociation constant for human P- selectin, or a variant thereof, of less than 100 μM, less than 1 μM, less than 500 nM, less than 100 nM, preferably 50 nM or less, preferably less than 25 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. In some embodiments, the dissociation constant is determined by dot blot titration. [0098] The P-selectin aptamers may be ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid aptamers. The aptamers may be single stranded ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid aptamers.

[0099] In some embodiments, the P-selectin aptamers comprise at least one chemical modification. In some embodiments, the chemical modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at an internucleotide linkage and a chemical substitution at a base position. In other embodiments, the chemical modification is selected from the group consisting of: incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjugation to a high molecular weight, non- immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone. In a preferred embodiment, the non-immunogenic, high molecular weight compound is polyalkylene glycol, and more preferably is polyethylene glycol (PEG). In some embodiments, the polyethylene glycol is methoxypolyethylene glycol (mPEG). In another preferred embodiment, the 3' cap is an inverted deoxythymidine cap. [00100] The modifications described herein may affect aptamer stability, e.g., incorporation of a capping moiety may stabilize the aptamer against endonuclease degradation. Additionally, the modifications described herein may affect the binding affinity of an aptamer to its target, e.g., site specific incorporation of a modified nucleotide or conjugation to PEG may affect binding affinity. The effect of such modifications on thermodynamic affinity can be determined using any of a variety of art-recognized techniques, such as, e.g., binding assays, such as a dot blot assay, in which labeled trace aptamer is incubated with varying target concentrations and complexes are captured on nitrocellulose and quantitated to compare the binding affinities pre- and post-incorporation of a modification.

[00101] Preferably, the P-selectin aptamer binds to P-selectin or a variant or a fragment thereof and acts as an antagonist to inhibit the function of P-selectin.

[00102] The P-selectin aptamers bind to P-selectin and prevent the interaction of P-selectin with PSGL-I and other ligands. For example, the P-selectin aptamers prevent P-selectin- mediated adhesion between any combination of erythrocytes, leukocytes, platelets and the vascular endothelium.

[00103] Examples of aptamers that specifically bind and modulate the function of P- selectin for use as therapeutics and/or diagnostics include ARC5685, ARC5691, ARC5692,

ARC5690, ARC6337 and ARC5134.

[00104] Preferably, the P-selectin aptamers comprise one of the following nucleic acid sequences:

(ARC5685) fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG- fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: T), wherein "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide and

"mN" is a 2'-0 Methyl modified nucleotide;

(ARC5691)

NH₂-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC- mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 3), wherein "NH2" is a 5'-hexylamine linker phosphoramidite, "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide and "mN" is a 2'-0 Methyl containing nucleotide; and

(ARC5692) PEG40K-nh-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG- mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 4), wherein

"nh" is an amine linker, "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide, "mN" is a 2'-0 Methyl containing nucleotide and "PEG40K" is a 40 kDa polyethylene glycol moiety.

[00105] The proposed secondary structure of ARC5692 is depicted in Figure 21, which comprises a stem and four loops.

[00106] Some of the aptamers of the invention were generated by optimizing ARC5665.

ARC5665 comprises the following nucleic acid sequence (reading from the 5' end to the 3' end): fC-fU-fC-rA-rA-fC-rG-rA-rG-fC-fC-rA-rG-rG-rA-rA-fC-rA-fU-fC-rG-rA-fC-rG-fU- fC-rA-rG-fC-rA-rA-rA-fC-rG-fC-rG-rA-rG (SEQ ID NO: 1), where "rN" is a ribonucleotide and "fN" is a 2'-fluorinated nucleotide. ARC5665 corresponds to ligand PF377sl, which is disclosed in U.S. Patent Publication No. 2004/0072234 Al, and which is incorporated herein by reference in its entirety.

[00107] ARC5665 was further optimized with: i) selected 2'-OMe purine substitutions and ii) the addition of an inverted deoxythymidine at the 3' end to generate ARC5685, which has the following nucleotide sequence (reading from the 5' end to the 3' end): fC-fU-fC-rA-rA- fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG- fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 2).

[00108] The PK profile of ARC5685 was optimized with the addition of a 40 kDa branched PEG moiety to its 5 ' end. In order to facilitate this PEGylation, an amine group was added to the 5' end of ARC5685 to generate ARC5691, which has the following nucleotide sequence (reading from the 5' end to the 3' end): NH2-fC-fU-fC-rA-rA-fC-mG- mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG-fC-rA- mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 3).

[00109] The conjugation of a 40 kDa branched PEG moiety to the amine at the 5' end of

ARC5691 generated ARC5692, which has the following nucleotide sequence (reading from the 5' end to the 3' end): PEG40K-nh-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG- mA-rA-fC-mA.-fU-fC-mG-mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG- idT (SEQ ID NO: 4).

[00110] The invention also provides aptamers that have substantially the same ability to bind to P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. In some embodiments, the aptamers have substantially the same structure as the aptamers shown in SEQ ID NOs: 2,

3 or 4. In some embodiments, the aptamers have substantially the same ability to bind to P- selectin and substantially the same structure as the aptamers shown in SEQ ID NOs: 2, 3 or 4. The invention also provides aptamers that have substantially the same ability to bind to and modulate a biological function of P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. The invention further provides aptamers that bind to P-selectin, wherein the aptamer modulates adhesion of any combination of erythrocytes, leukocytes, platelets and the vascular endothelium as SEQ ID NOs: 2, 3 or 4.

[00111] The invention also provides aptamers that have the same, substantially the same, and/or better ability to bind to P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. The invention also provides aptamers that have the same, substantially the same, and/or better ability to bind and modulate a biological function of P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. In other embodiments, the aptamers have substantially the same structure and the same, substantially the same, and/or better ability to bind to P-selectin as the aptamers shown in SEQ ID NOs: 2, 3 or 4. As used herein, substantially the same ability to bind to P-selectin means that the affinity is within one or two orders of magnitude of the affinity of the nucleic acid sequences and/or aptamers described herein. It is well within the skill of those having ordinary skill in the art to determine whether a given sequence has substantially the same ability to bind P-selectin.

[00112] The invention further provides aptamers that bind to P-selectin, wherein the aptamer modulates adhesion and the aptamer is SEQ ID NO: 2, 3 or 4, or an aptamer that has the same, substantially the same, or better ability to modulate adhesion as the aptamers shown in SEQ ID NO: 2, 3 or 4. In some embodiments, the aptamer that binds to P-selectin has a nucleic acid sequence at least 70%, 80%, 90% or 95% identical to SEQ ID NO: 2, 3 or 4. In some embodiments, the aptamer that binds to P-selectin has a nucleic acid sequence at least 95% identical to SEQ ID NO: 2, 3 or 4.

[00113] The terms "sequence identity" or "% identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol. 48: 443 (1970); by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988); by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by visual inspection (see generally, Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc, and Wiley- Interscience (1987).

[00114] One example of an algorithm that is suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter "BLAST"), see, e.g. Altschul et al, J MoI. Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15: 3389-3402 (1997), which is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI").

[00115] In some embodiments, aptamers of the invention have great affinity and specificity for their targets while reducing the deleterious side effects from non-naturally occurring nucleotide substitutions, such as if the aptamer therapeutics break down in the body of patients or subjects. In some embodiments, the compositions containing the aptamers of the invention are free of or have a reduced amount of fluorinated nucleotides. [00116] Aptamers of the invention including, but not limited to, aptamers identified by the SELEX^™ method, 2'-Modifϊed SELEX^™, minimized aptamers, optimized aptamers and chemically substituted aptamers, can be manufactured using any oligonucleotide synthesis technique that is well known in the art, such as solid phase oligonucleotide synthesis techniques (see, e.g., Gualtiere, F. Ed., New Trends in Synthetic Medicinal Chemistry, Ch. 9, Chemistry of Antisense Oligonucleotides, p. 261-335, 2000, Wiley-VCH, New York). The manufacturing of aptamers using solid phase oligonucleotide synthesis techniques can also be done at commercial scale. Solution phase methods, such as triester synthesis methods (see, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449 (1978)), may also be used to manufacture aptamers of the invention, as well as recombinant means. [00117] In addition, a variety of functional groups can be introduced during solid phase synthesis. The functionality can be a simple linker that results in a functional group such as amine or thiol, or may be a more complex construct such as biotin or a fluorescent dye. Typically, functional group linkers or more complex moieties are introduced via a phosphoramidite, or they can be introduced post-synthetically (i.e., after solid phase synthesis). Alternatively, by utilizing a modified solid support, a variety of functionalities can be introduced at the 3 '-end of the oligonucleotide, thereby enabling a wider variety of conjugation techniques.

APTAMER MEDICINAL CHEMISTRY [00118] Once aptamers that bind to a desired target are identified, several techniques may be optionally performed to further increase binding and/or functional characteristics of the identified aptamer sequences.

[00119] Aptamers that bind to a desired target may be truncated to obtain the minimal aptamer sequence (also referred to herein as "minimized construct" or "minimized aptamer") having the desired binding and/or functional characteristics. One method of accomplishing this is by using folding programs and sequence analysis, e.g., aligning clone sequences resulting from a selection to look for conserved motifs and/or covariation to inform the design of minimized constructs. Suitable folding programs include, for example, the RNAstructure program. (Mathews, D.H.; Disney, M.D.; Childs, JX. ; Schroeder, S. J.; Zuker, M.; and Turner, D. H., "Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure," 2004. Proceedings of the National Academy of Sciences, US, 101, 7287-7292). Biochemical probing experiments can also be performed to determine the 5 ' and 3 ' boundaries of an aptamer sequence to inform the design of minimized constructs. Minimized constructs can then be chemically synthesized and tested for binding and functional characteristics as compared to the non- minimized sequence from which they were derived. Variants of an aptamer sequence containing a series of 5', 3' and/or internal deletions may also be directly chemically synthesized and tested for binding and/or functional characteristics as compared to the non- minimized aptamer sequence from which they were derived. [00120] Additionally, doped reselections may be used to explore the sequence requirements within a single active aptamer sequence, or a single minimized aptamer sequence. Doped reselections are performed using a synthetic, degenerate pool that has been designed based on the single sequence of interest. The level of degeneracy usually varies 70% to 85% from the wild type nucleotide, i.e., the single sequence of interest. In general, sequences with neutral mutations are identified through the doped reselection process, but in some cases sequence changes can result in improvements in affinity. The composite sequence information from clones identified using doped reselections can then be used to identify the minimal binding motif and aid in optimization efforts.

[00121] Aptamer sequences and/or minimized aptamer sequences may also be optimized post-SELEX using Aptamer Medicinal Chemistry to perform random or directed mutagenesis of the sequence to increase binding affinity and/or functional characteristics, or alternatively to determine which positions in the sequence are essential for binding activity and/or functional characteristics. [00122] Aptamer Medicinal Chemistry is an aptamer improvement technique in which sets of variant aptamers are chemically synthesized. These sets of variants typically differ from the parent aptamer by the introduction of a single substituent, and differ from each other by the location of this substituent. These variants are then compared to each other and to the parent. Improvements in characteristics may be profound enough that the inclusion of a single substituent may be all that is necessary to achieve a particular therapeutic criterion. [00123] Alternatively the information gleaned from the set of single variants may be used to design further sets of variants in which more than one substituent is introduced simultaneously. In one design strategy, all of the single substituent variants are ranked, the top 4 are chosen and all possible double (6), triple (4) and quadruple (1) combinations of these 4 single substituent variants are synthesized and assayed. In a second design strategy, the best single substituent variant is considered to be the new parent and all possible double substituent variants that include this highest-ranked single substituent variant are synthesized and assayed. Other strategies may be used, and these strategies may be applied repeatedly such that the number of substituents is gradually increased while continuing to identify further-improved variants.

[00124] Aptamer Medicinal Chemistry may be used particularly as a method to explore the local, rather than the global, introduction of substituents. Because aptamers are discovered within libraries that are generated by transcription, any substituents that are introduced during the SELEX^™ process must be introduced globally. For example, if it is desired to introduce phosphorothioate linkages between nucleotides then they can only be introduced at every A (or every G, C, T, U etc.) if globally substituted. Aptamers that require phosphorothioates at some As (or some G, C, T, U etc.) (locally substituted) but cannot tolerate it at other As (or some G, C, T, U, etc.) can not be readily discovered by this process.

[00125] The kinds of substituents that can be utilized by the Aptamer Medicinal Chemistry process are only limited by the ability to introduce them into an oligomer synthesis scheme. The process is certainly not limited to nucleotides alone. Aptamer Medicinal Chemistry schemes may include substituents that introduce steric bulk, hydrophobicity, hydrophilicity, lipophilicity, lipophobicity, positive charge, negative charge, neutral charge, zwitterions, polarizability, nuclease-resistance, conformational rigidity, conformational flexibility, protein-binding characteristics, mass, etc. Aptamer Medicinal Chemistry schemes may include base-modifications, sugar-modifications or phosphodiester linkage-modifications. [00126] When considering the kinds of substituents that are likely to be beneficial within the context of a therapeutic aptamer, it may be desirable to introduce substitutions that fall into one or more of the following categories:

(1) Substituents already present in the body, e.g., 2'-deoxy, 2'-ribo, 2'-O-methyl nucleotides, inosine, or 5-methyl cytosine.

(2) Substituents already part of an approved therapeutic, e.g., 2'-fluoro nucleotides.

(3) Substituents that hydro lyze, degrade or metabolize to one of the above two categories, e.g., methylphosphonate-linked oligonucleotides or phosphorothioate-linked oligonucleotides.

[00127] The aptamers of the present invention include aptamers developed through Aptamer Medicinal Chemistry as described herein.

[00128] Target binding affinity of the aptamers of the invention can be assessed through a series of binding reactions between the aptamer and the target {e.g., a protein) in which trace ³²P-labeled aptamer is incubated with a dilution series of the target in a buffered medium and then analyzed by nitrocellulose filtration using a vacuum filtration manifold. Referred to herein as the dot blot binding assay, this method uses a three layer filtration medium consisting (from top to bottom) of nitrocellulose, nylon filter and gel blot paper. RNA that is bound to the target is captured on the nitrocellulose filter whereas the non-target bound RNA is captured on the nylon filter. The gel blot paper is included as a supporting medium for the other filters. Following filtration, the filter layers are separated, dried and exposed on a phosphor screen and quantified using a phosphorimaging system. The quantified results can be used to generate aptamer binding curves from which dissociation constants (K_D) can be calculated. In a preferred embodiment, the buffered medium used to perform the binding reactions is IX Dulbecco's PBS (with Ca ⁺⁺ and Mg⁺⁺) plus 0.1 mg/mL BSA. [00129] Generally, the ability of an aptamer to modulate the functional activity of a target can be assessed using in vitro and in vivo models, which will vary depending on the biological function of the target. In some embodiments, the aptamers of the invention may inhibit a known biological function of the target. In other embodiments, the aptamers of the invention may stimulate a known biological function of the target. The functional activity of aptamers of the invention can be assessed using in vitro and in vivo models designed to measure a known function of P-selectin.

[00130] Aptamer sequences and/or minimized aptamer sequences may also be optimized using Metabolic Profile Directed Aptamer Medicinal Chemistry for site-specific identification of cleavage sites and modifications to optimize stability of the aptamer sequences and/or minimized aptamer sequences.

[00131] Metabolic Profile Directed Aptamer Medicinal Chemistry involves incubating a parent aptamer with a test fluid to result in a mixture. Then, the mixture is analyzed to determine the rate of disappearance of the parent aptamer or the amount or percentage of aptamer remaining after incubation, the specific aptamer metabolic profile and the specific aptamer metabolite sequences. Knowledge of the sequences of the specific metabolites formed allows one to identify the sites of nuclease cleavage based on the mass of the metabolite(s). After systematically conducting metabolic profiling and identifying specific aptamer cleavage sites, the method involves introducing chemical substitutions or modifications at or near the cleavage sites that are designed to optimize the stability of the aptamer sequences and/or minimized aptamer sequences.

[00132] In one embodiment, an aptamer is identified and modified by a) incubating a parent aptamer with a test fluid to result in a mixture; b) analyzing the mixture to identify metabolites of the parent aptamer, thereby detecting at least one aptamer cleavage site in the parent aptamer; and c) introducing a chemical substitution at a position proximal to the at least one aptamer cleavage site to result in a modified aptamer. This enhances the stability of the aptamer, and, in particular, the stability of the aptamer to endonucleases and exonucleases.

[00133] In some embodiments, the test fluid is a biological matrix, particularly a biological matrix selected from the group consisting of one or more of: serum; plasma; cerebral spinal fluid; tissue extracts, including cytosolic fraction, S9 fraction and microsomal fraction; aqueous humour; vitreous humour and tissue homogenates. In some embodiments, the biological matrix is derived from a species selected from the group consisting of one or more of: mouse, rat, monkey, pig, human, dog, guinea pig and rabbit. In some embodiments, the test fluid comprises at least one purified enzyme, particularly at least one purified enzyme selected from the group consisting of: snake venom phosphodiesterase and DNAse 1. [00134] In some embodiments, the analyzing step includes analyzing the resulting aptamer using liquid chromatography and mass spectrometry, particularly electron spray ionization liquid chromatography mass spectrometry, polyacrylamide gel electrophoresis or capillary electrophoresis to determine a position of at least one aptamer cleavage site. In some embodiments, the analyzing step includes analyzing the resulting aptamer using a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS, and more particularly ESI-LC/MS, ESI- LC/MS/MS and ESI-LC/MS/MS/MS.

[00135] In some embodiments, the proximal position includes a position selected from the group consisting of: a position immediately 5' to the aptamer cleavage site, a 5' position at or within three nucleotides of the aptamer cleavage site, a position immediately 3' to the aptamer cleavage site, a 3' position at or within three nucleotides of the aptamer cleavage site, and at the cleaved internucleotide linkage.

[00136] In some embodiments, the chemical substitution is selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a base position and a chemical substitution at an internucleotide linkage. More particularly, a substitution is selected from the group consisting of: a nucleotide substituted for a different nucleotide; a purine substitution for a pyrimidine; a 2'-deoxy dihydrouridine substitution for a uridine; a 2 '-deoxy-5 -methyl cytidine for a cytidine; a 2-amino purine substitution for a purine; a phosphorothioate substituted for a phosphodiester; a phosphorodithioate substituted for a phosphodiester; a 2'-deoxy nucleotide substituted for a 2'-OH nucleotide, a 2'-0Me nucleotide or a 2'-fluoro nucleotide; a 2'-0Me nucleotide substituted for a 2'-OH nucleotide, a 2'-deoxy nucleotide, or a 2'-fluoro nucleotide; a 2'-fluoro nucleotide substituted for a T- OH nucleotide, a 2'-deoxy nucleotide or a 2'-0Me nucleotide; or a 2'-O-methoxyethyl nucleotide substituted for a 2'-OH, 2'-fluoro or 2'-deoxy nucleotide; a 2'-O-methoxyethyl nucleotide or deoxy nucleotide for a 2'-fluoro nucleotide; and the addition of one or more PEG or other polymers or other PK or distribution-influencing entity.

[00137] In additional embodiments, the introducing step of these methods further includes introducing more than one chemical substitution at one or more cleavage sites or at a single cleavage site or both.

[00138] In another embodiment, wherein more than one aptamer cleavage site is detected, the introducing step of these methods further includes introducing at least one chemical substitution at the associated proximal position of the aptamer cleavage site determined to occur first in time during the incubating step or at any other cleavage site(s) that provides the desired properties upon introduction of a chemical substitution. [00139] In other embodiments, these methods further include the step of testing the stability of the modified aptamer in the test fluid. In some embodiments, aptamer stability is assessed by determining the percent of modified aptamer that remains intact in the test fluid as compared to the percent of the parent aptamer that remains intact in the test fluid. In some embodiments, the percent of intact aptamer is assessed by a bioanalytical method selected from the group consisting of one or more of: denaturing polyacrylamide gel electrophoresis (PAGE); capillary electrophoresis; HPLC and LC/MS, particularly LC/MS/MS or LC/MS/MS/MS, and more particularly ESI-LC/MS, ESI-LC/MS/MS and ESI- LC/MS/MS/MS. In other embodiments, the modified aptamer is more stable in the test fluid than the parent aptamer, preferably at least 2 fold, more preferably at least 5 fold and most preferably at least 10 fold more stable.

[00140] In additional embodiments, these methods further include determining a dissociation constant or IC₅₀ of the modified aptamer for its target. In some embodiments, chemical substitutions are introduced singly at each position or in various combinations in the aptamer, and the dissociation constant or IC₅₀ for each resulting aptamer is determined. Chemical substitutions are introduced at a position proximal to the aptamer cleavage site such that a single chemical modification results in a dissociation constant for the modified aptamer that is the same or less than that of the parent aptamer. In another embodiment of the invention, the method includes selecting a modified aptamer having a dissociation constant or IC50 for its target that is the same or less than that for the parent aptamer. [00141] In other embodiments, the modified aptamer binds to a target having a biological activity, and the method further includes testing the biological activity of the target in the presence and absence of modified aptamer. In another embodiment, the method further includes selecting a modified aptamer that binds to a target having a biological activity that is the same or better than that of the parent aptamer. The biological activity may be measured in any relevant assay, such as an ELISA assay or a cell-based assay.

[00142] In some embodiments, the incubating, analyzing, introducing and testing steps are repeated iteratively until the desired stability is achieved.

[00143] The aptamers of the invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic utilization may include either in vivo or in vitro diagnostic applications. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any aptamer by procedures known in the art to incorporate a labeling tag to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures.

APTAMERS HAVING IMMUNOSTIMULATORY MOTIFS [00144] Recognition of bacterial DNA by the vertebrate immune system is based upon the recognition of unmethylated CG dinucleotides in particular sequence contexts ("CpG motifs"). One receptor that recognizes such a motif is Toll-like receptor 9 ("TLR 9"), a member of a family of Toll-like receptors (~10 members) that participate in the innate immune response by recognizing distinct microbial components. TLR 9 is activated by unmethylated oligodeoxynucleotide ("ODN") CpG sequences in a sequence-specific manner. The recognition of CpG motifs triggers defense mechanisms leading to innate and ultimately acquired immune responses. For example, activation of TLR 9 in mice induces activation of antigen presenting cells, up-regulation of MHC class I and II molecules, and expression of important co-stimulatory molecules and cytokines including IL- 12 and IL-23. This activation both directly and indirectly enhances B and T cell responses, including a robust up-regulation of the THl cytokine IFN-gamma. Collectively, the response to CpG sequences leads to: protection against infectious diseases, improved immune response to vaccines, an effective response against asthma, and improved antibody-dependent cell-mediated cytotoxicity. Thus, CpG ODNs can provide protection against infectious diseases, function as immuno-adjuvants or cancer therapeutics (monotherapy or in combination with a mAb or other therapies), and can decrease asthma and allergic response.

[00145] Aptamers of the invention, including one or more CpG or other immunostimulatory sequences, can be identified or generated by a variety of strategies using, e.g., the SELEX^™ process described herein. The incorporated immunostimulatory sequences can be DNA, RNA, substituted DNA or RNA and/or a combination of substituted or unsubstituted DNA/RNA. In general, the strategies can be divided into two groups. In group one, the strategies are directed to identifying or generating aptamers including both a CpG motif or other immunostimulatory sequence as well as a binding site for a target, where the target (hereinafter "non-CpG target") is a target other than one known to recognize CpG motifs or other immunostimulatory sequences and known to stimulate an immune response upon binding to a CpG motif. In some embodiments of the invention the non-CpG target is a P-selectin target. The first strategy of this group includes performing SELEX^™ to obtain an aptamer to a specific non-CpG target, preferably a target, e.g., P-selectin, where a repressed immune response is relevant to disease development, using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region of the pool members includes a fixed region having a CpG motif incorporated therein, and identifying an aptamer including a

CpG motif. The second strategy of this group includes performing SELEX^™ to obtain an aptamer to a specific non-CpG target, preferably a target, e.g., P-selectin, where a repressed immune response is relevant to disease development, and following selection, appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a nonessential region, of the aptamer. The third strategy of this group includes performing SELEX to obtain an aptamer to a specific non-CpG target, preferably a target, e.g., P- selectin, where a repressed immune response is relevant to disease development, wherein during synthesis of the pool the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer including a CpG motif. The fourth strategy of this group includes performing SELEX^™ to obtain an aptamer to a specific non- CpG target, preferably a target, e.g., P-selectin, where a repressed immune response is relevant to disease development, and identifying an aptamer including a CpG motif. The fifth strategy of this group includes performing SELEX to obtain an aptamer to a specific non- CpG target, preferably a target, e.g., P-selectin, where a repressed immune response is relevant to disease development, and identifying an aptamer which, upon binding, stimulates an immune response but which does not include a CpG motif.

[00146] In group two, the strategies are directed to identifying or generating aptamers including a CpG motif and/or other sequences that are bound by the receptors for the CpG motifs (e.g., TLR9 or the other toll-like receptors) and upon binding stimulate an immune response. The first strategy of this group includes performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response using an oligonucleotide pool wherein a CpG motif has been incorporated into each member of the pool as, or as part of, a fixed region, e.g., in some embodiments the randomized region of the pool members include a fixed region having a CpG motif incorporated therein, and identifying an aptamer including a CpG motif. The second strategy of this group includes performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response and then appending a CpG motif to the 5' and/or 3' end or engineering a CpG motif into a region, preferably a non-essential region, of the aptamer. The third strategy of this group includes performing SELEX to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response wherein during synthesis of the pool, the molar ratio of the various nucleotides is biased in one or more nucleotide addition steps so that the randomized region of each member of the pool is enriched in CpG motifs, and identifying an aptamer including a CpG motif. The fourth strategy of this group includes performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences and upon binding stimulate an immune response and identifying an aptamer including a CpG motif. The fifth strategy of this group includes performing SELEX^™ to obtain an aptamer to a target known to bind to CpG motifs or other immunostimulatory sequences, and identifying an aptamer which upon binding, stimulate an immune response but which does not include a CpG motif.

[00147] A variety of different classes of CpG motifs have been identified, each resulting upon recognition in a different cascade of events, release of cytokines and other molecules, and activation of certain cell types. See, e.g., CpG Motifs in Bacterial DNA and Their Immune Effects, Annu. Rev. Immunol. 2002, 20:709-760, incorporated herein by reference. Additional immunostimulatory motifs are disclosed in the following U.S. Patents, each of which is incorporated herein by reference: U.S. Patent No. 6,207,646; U.S. Patent No. 6,239,116; U.S. Patent No. 6,429,199; U.S. Patent No. 6,214,806; U.S. Patent No. 6,653,292; U.S. Patent No. 6,426,334; U.S. Patent No. 6,514,948 and U.S. Patent No. 6,498,148. Any of these CpG or other immunostimulatory motifs can be incorporated into an aptamer. The choice of aptamers is dependent on the disease or disorder to be treated. Preferred immunostimulatory motifs are as follows (shown 5' to 3' left to right) wherein "r" designates a purine, "y" designates a pyrimidine, and "X" designates any nucleotide: AACGTTCGAG (SEQ ID NO: 9); AACGTT; ACGT, rCGy; rrCGyy, XCGX, XXCGXX, and XiX₂CGYiY₂ wherein Xi is G or A, X₂ is not C, Yi is not G and Y₂ is preferably T.

[00148] In those instances where a CpG motif is incorporated into an aptamer that binds to a specific target other than a target known to bind to CpG motifs and upon binding stimulate an immune response (a "non-CpG target"), the CpG is preferably located in a non-essential region of the aptamer. Non-essential regions of aptamers can be identified by site-directed mutagenesis, deletion analyses and/or substitution analyses. However, any location that does not significantly interfere with the ability of the aptamer to bind to the non-CpG target may be used. In addition to being embedded within the aptamer sequence, the CpG motif may be appended to either or both of the 5' and 3' ends or otherwise attached to the aptamer. Any location or means of attachment may be used so long as the ability of the aptamer to bind to the non-CpG target is not significantly interfered with.

[00149] As used herein, "stimulation of an immune response" can mean either (1) the induction of a specific response {e.g., induction of a ThI response) or of the production of certain molecules or (2) the inhibition or suppression of a specific response (e.g., inhibition or suppression of the Th2 response) or of certain molecules.

MODULATION OF PHARMACOKINETICS AND BIODISTRIBUTION

OF APTAMER THERAPEUTICS

[00150] It is important that the pharmacokinetic properties for all oligonucleotide-based therapeutics, including aptamers, be tailored to match the desired pharmaceutical application. Aptamers must be able to be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen. [00151] Thus, the present invention provides materials and methods to affect the pharmacokinetics of aptamer compositions, and, in particular, the ability to tune aptamer pharmacokinetics. The tunability of (i.e., the ability to modulate) aptamer pharmacokinetics is achieved through conjugation of modifying moieties (e.g., PEG polymers) to the aptamer and/or the incorporation of modified nucleotides (e.g., 2'-fluoro or 2'-O-methyl) or modified internucleotide linkages to alter the chemical composition of the nucleic acid. The ability to tune aptamer pharmacokinetics is used in the improvement of existing therapeutic applications, or alternatively, in the development of new therapeutic applications. For example, in some therapeutic applications, e.g., in anti-neoplastic or acute care settings where rapid drug clearance or turn-off may be desired, it is desirable to decrease the residence times of aptamers in the circulation. Alternatively, in other therapeutic applications, e.g., maintenance therapies where systemic circulation of a therapeutic is desired, it may be desirable to increase the residence times of aptamers in circulation. [00152] In addition, the tunability of aptamer pharmacokinetics is used to modify the disposition, for example the absorption, distribution, metabolism and elimination (ADME) of an aptamer to fit its therapeutic objective in a subject. Tunability of the pharmacokinetics of an aptamer can affect the manner and extent of absorption of the aptamer, the distribution of an aptamer throughout the fluids and tissue of the body, the successive metabolic transformations of the aptamer and its daughter metabolite(s) and finally, the elimination of the aptamer and its metabolite(s). For example, in some therapeutic applications, it may be desirable to alter the biodistribution of an aptamer therapeutic in an effort to target a particular type of tissue or a specific organ (or set of organs) or to increase the propensity to enter specific cell types. In these applications, the aptamer therapeutic preferentially distribute into specific tissues and/or organs and accumulate therein to cause a therapeutic effect. In other therapeutic applications, it may be desirable to target tissues displaying a cellular marker or a symptom associated with a given disease, cellular injury or other abnormal pathology, such that the aptamer therapeutic preferentially accumulates in the affected tissue. For example, PEGylation of an aptamer therapeutic (e.g., PEGylation with a 20 kDa PEG polymer or other polymer or conjugation entity) is used to target inflamed tissues, such that the PEGylated aptamer therapeutic preferentially accumulates in inflamed tissue.

[00153] To determine the pharmacokinetic profiles of aptamer therapeutics (e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides) a variety of parameters are determined during pharmacokinetic study in normal subjects, e.g., test animals or humans, or in diseased subjects, e.g., P-selectin-specifϊc animal models or diseased humans. Such parameters include, for example, the distribution or elimination half-life (tm), the plasma clearance (CL), the volume of distribution (Vss), the area under the concentration- time curve (AUC), maximum observed serum or plasma concentration (C_max), and the mean residence time (MRT) of an aptamer composition. As used herein, the term "AUC" refers to the area under the plasma concentration of an aptamer therapeutic versus the time after aptamer administration. The AUC value is used to estimate the exposure of the aptamer and also used to determine bioavailability of an aptamer after extravascular route of administration such as, e.g., subcutaneous administration. Bioavailability is determined by taking the ratio of the AUC obtained after subcutaneous administration to the AUC obtained after intravenous administration and normalize them to the doses used after each administration (i.e., the percent ratio of aptamer administered after subcutaneous administration as compared to the same aptamer administered by intravenous administration at the same dose or normalized dose). The CL value is the measurement of the removal of the parent aptamer therapeutic is removed from the systemic circulation. The volume of distribution (Vd) is a term that relates the amount of aptamer in the body at one time to its plasma concentration. The Vd is used to determine how well a drug is removed from the plasma and distributed to tissues and/or organs. A larger Vd implies wide distribution, extensive tissue binding or both a wide distribution and extensive tissue binding. There are three basics volume of distribution: (i) the apparent or initial volume of distribution at time zero obtained from back extrapolation of the concentration-time curve; (ii) the volume calculated once distribution is complete, approximating to Vdss, where the area volume is dependent upon the elimination kinetics; and (iii) the volume of distribution calculated once distribution is complete. The parameter that should ideally be measured is the Vdss, since this parameter is independent of the elimination kinetics. If the Vss for the aptamer is larger the blood volume, the data suggest that the aptamer is distributed outside of the systemic system and is likely to be found outside of the systemic compartment, the tissues or organs. Pharmacodynamic parameters may also be used to assess drug characteristics. [00154] To determine the distributions of aptamer therapeutics (e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides), a tissue distribution study or a quantitative whole body autoradiography using radiolabeled aptamer administered to normal animal or diseased target specific animal models is used. The accumulation of the radiolabeled-aptamer at specific site can be quantified.

[00155] The pharmacokinetics and biodistribution of an aptamer described herein, such as a stabilized aptamer, can be modulated in a controlled manner by conjugating an aptamer to a modulating moiety such as, but not limited to, a small molecule, peptide, or polymer, or by incorporating modified nucleotides into an aptamer. The conjugation of a modifying moiety and/or altering nucleotide(s) chemical composition alters fundamental aspects of aptamer residence time in circulation and distribution within and to tissues and cells. [00156] In addition to metabolism by nucleases, oligonucleotide therapeutics are subject to elimination via renal filtration. As such, a nuclease-resistant oligonucleotide administered intravenously typically exhibits an in vivo half- life of <10 min, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood stream into tissues or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus. Conjugation of small molecular weight therapeutics to a PEG polymer (PEGylation), described below, can dramatically lengthen residence times of aptamers in circulation, thereby decreasing dosing frequency and enhancing effectiveness against vascular targets.

[00157] Modified nucleotides can also be used to modulate the plasma clearance of aptamers. For example, an unconjugated aptamer which incorporates for example, 2'-fluoro, 2'-OMe, and/or phosphorothioate stabilizing chemistries, which is typical of current generation aptamers as it exhibits a high degree of nuclease stability in vitro and in vivo, displays rapid distribution into tissues, primarily into the liver and kidney, when compared to unmodified aptamer.

PAG-DERIVATIZED NUCLEIC ACIDS

[00158] As described above and as shown in Figure 2, derivatization of nucleic acids with high molecular weight non-immunogenic polymers has the potential to alter the pharmacokinetic and pharmacodynamic properties of nucleic acids making them more effective and/or safer therapeutic agents. Favorable changes in activity can include increased resistance to degradation by nucleases, decreased filtration through the kidneys, decreased exposure to the immune system, and altered distribution of the therapeutic through the body. [00159] The aptamer compositions of the invention may be derivatized with one or more polyalkylene glycol ("PAG") moieties. Typical polymers used in the invention include polyethylene glycol ("PEG"), also known as polyethylene oxide ("PEO") and polypropylene glycol (including poly isopropylene glycol). Additionally, random or block copolymers of different alkylene oxides can be used in many applications. In a common form, a polyalkylene glycol, such as PEG, is a linear polymer terminated at each end with hydroxyl groups: HO-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-OH. This polymer, alpha-, omega- dihydroxylpolyethylene glycol, can also be represented as HO-PEG-OH, where it is understood that the — PEG- symbol represents the following structural unit: -CH₂CH₂O- (CH₂CH₂O)_n-CH₂CH₂- where n typically ranges from about 4 to about 10,000. [00160] PAG polymers suitable for therapeutic indications typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PAGs is to covalently attach the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et ah, J. Org. Chem., 60:331-336 (1995). PAG conjugates are often used not only to enhance solubility and stability but also to prolong the blood circulation half-life of molecules and later distribution within the body.

[00161] The PAG derivatized compounds conjugated to the aptamers of the invention are typically between 5 and 80 kDa in size however any size can be used, the choice dependent on the aptamer and application. Other PAG derivatized compounds of the invention are between 10 and 80 kDa in size. Still other PAG derivatized compounds of the invention are between 10 and 60 kDa in size. In some embodiments, the PAG moieties derivatized to compositions of the present invention are PEG moieties having a molecular weight ranging from 10, 20, 30, 40, 50, 60 or 80 kDa in size. In some embodiments, the PEG is linear PEG, while in other embodiments, the PEG is branched PEG. In still other embodiments the PEG is a 4OkDa branched PEG as depicted in Figure 3. In some embodiments the 40 kDa branched PEG is attached to the 5' end of the aptamer as depicted in Figure 4. [00162] Production of high molecular weight PEGs (>10 kDa) can be difficult, inefficient, and expensive. To synthesize high molecular weight PEG-nucleic acid conjugates higher molecular weight activated PEGs are generated. Method for generating such molecules involve the formation of a linear activated PEG, or a branched activated PEG in which case two or more PEGs are attached to a central core carrying the activated group. The terminal portions of these higher molecular weight PEG molecules, i.e., the relatively non-reactive hydroxyl (-OH) moieties, can be activated, or converted to functional moieties, for attachment of one or more of the PEGs to other compounds at reactive sites on the compound. Branched activated PEGs will have more than two termini, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are herein referred to as, multi-activated PEGs. In some cases, not all termini in a branch PEG molecule are activated. In cases where any two termini of a branch PEG molecule are activated, such PEG molecules are referred to as bi-activated PEGs. In some cases where only one terminus in a branch PEG molecule is activated, such PEG molecules are referred to as mono-activated. In other cases, the linear PEG molecule is di-functional and is sometimes referred to as "PEG diol." The terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the -OH groups, that can be activated, or converted to functional moieties, for attachment of the PEG to other compounds at reactive sites on the compound. Such activated PEG diols are referred to herein as homo bi-activated PEGs. The molecules are generated using any of a variety of art-recognized techniques. In addition to activating PEG using one of the previously described methods, one or both of the terminal alcohol functionalities of the PEG molecule can be modified to allow for different types of conjugation to a nucleic acid. For example, converting one of the terminal alcohol functionalities to an amine, or a thiol, allows access to urea and thiourethane conjugates. Other functionalities include, e.g., maleimides and aldehydes.

[00163] In many applications, it is desirable to cap the PEG molecule on one end with an essentially non-reactive moiety so that the PEG molecule is mono-functional (or mono- activated). In the case of protein therapeutics which generally display multiple reaction sites for activated PEGs, homo bi-functional activated PEGs lead to extensive cross-linking, yielding poorly functional aggregates. To generate mono-activated PEGs, one hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with non-reactive methoxy end moiety, -OCH3. In this embodiment, the polymer can be represented by MeO- CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-OH and is commonly referred to as "mPEG," where n typically ranges from about 4 to about 10,000.

[00164] The other, un-capped terminus of the PEG molecule typically is converted to a reactive end moiety that can be activated for attachment at a reactive site on a surface or a molecule such as a protein, peptide or oligonucleotide. [00165] In some cases, it is desirable to produce a hetero bi-functional PEG reagent, where one end of the PEG molecule has a reactive group such as an N-hydroxysuccinimide or nitrophenyl carbonate, while the opposite end contains a maleimide or other activating group. In these embodiments, two different functionalities, for example, amine and thiol, may be conjugated to the activated PEG reagent at different times.

PHARMACEUTICAL COMPOSITIONS

[00166] The invention also includes pharmaceutical compositions comprising an aptamer that binds to P-selectin. In some embodiments, the compositions include a therapeutically effective amount of a pharmacologically active P-selectin aptamer or a pharmaceutically acceptable salt thereof, alone or in combination, with one or more pharmaceutically acceptable carriers or diluents.

[00167] The compositions may comprise one or more P-selectin aptamers. For example, the compositions may contain ARC5692. Alternatively, the compositions may contain ARC5685. Alternatively, the compositions may contain ARC5692 and another P-selectin aptamer. In embodiments where the composition includes at least two aptamers that can be the same aptamer or two different aptamers, the aptamers can optionally be tethered or otherwise coupled together. Preferably, the compositions contain ARC5692, either alone or in combination with another P-selectin aptamer.

[00168] As used herein, the term "therapeutically effective amount" refers to an amount of a P-selectin aptamer, by itself or in combination with another drug or therapy, sufficient to treat or prevent coagulation/thrombotic, inflammatory, metastatic and/or other pathologies, diseases or disorders. A therapeutically effective amount will vary depending upon the severity of the disease or disorder, age, general health condition, and weight of the subject to be treated.

[00169] As used herein, the term "pharmaceutically acceptable salt" refers to salt forms of the active compound that are prepared with counter ions that are non-toxic under the conditions of use and are compatible with a stable formulation. Examples of pharmaceutically acceptable salts of P-selectin aptamers include hydrochlorides, sulfates, phosphates, acetates, fumarates, maleates and tartrates.

[00170] The term "pharmaceutically acceptable carrier," as used herein, means being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers are well known in the art. Examples of pharmaceutically acceptable carriers can be found, for example, in Goodman and Gillmans,

The Pharmacological Basis of Therapeutics, latest edition. [00171] The pharmaceutical compositions will generally include a therapeutically effective amount of the active component(s) of the therapy, e.g., a P-selectin aptamer of the invention that is dissolved or dispersed in a pharmaceutically acceptable carrier or medium. Examples of preferred carriers include, but are not limited to, physiological saline solution and glucose solution. However it is contemplated that other pharmaceutically acceptable carriers may also be used. Examples of other pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol, and wool fat. The use of such media and agents for pharmaceutically active substances is well known in the art. [00172] Supplementary ingredients can also be incorporated into the pharmaceutical compositions. For example, the pharmaceutical compositions may contain excipients such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts, or buffers for modifying or maintaining pH, osmolality, viscosity, clarity, color, sterility, stability, rate of dissolution, or absorption of the formulation. For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. [00173] The pharmaceutical compositions are prepared according to conventional mixing, granulating or coating methods, and typically contain about 0.1% to 99.9%, for example, about 0.1% to 75%, about 0.1% to 50 %, about 0.1% to 25%, about 0.1% to 10%, about 0.1 to 5%, preferably about 1% to 50%, of the active component.

[00174] The formulation of pharmaceutical compositions is known to one of skill in the art. Typically, such compositions may be formulated as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules for slow release formulations; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like. The active compound defined above, may be also formulated as suppositories, using for example, polyalkylene glycols, as the carrier. In some embodiments, suppositories are advantageously prepared from fatty emulsions or suspensions. The use of sterile formulations, such as saline-based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful.

[00175] The compositions may be formulated as oral dosage forms, such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. For instance, for oral administration in the form of a tablet or capsule (e.g. , a gelatin capsule), the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, 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 polyethylene glycol, 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. [00176] Pharmaceutical compositions can also be formulated in 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. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564. For example, the aptamers described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art. Additionally, liposomes may bear aptamers on their surface for targeting and carrying cytotoxic agents internally to mediate cell killing. An example of nucleic-acid associated complexes is provided in U.S. Patent No. 6,011,020. [00177] The therapeutic compositions of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the therapeutic compositions of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[00178] The therapeutic compositions of the present invention may also be used in conjunction with medical devices, e.g., as a coating on a medical device such as, but not, limited to a stent.

[00179] The quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual. [00180] A minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.

ADMINISTRATION

[00181] The compositions may be administered to a vertebrate, preferably a mammal, and more preferably a human. The terms "patient" and "subject" are used interchangeably throughout the application, and these terms include both human and veterinary subjects. [00182] In embodiments where the P-selectin aptamers are antagonist aptamers, the P- selectin aptamer compositions provided herein are administered to subjects in an amount effective to inhibit, reduce, block or otherwise modulate P-selectin-mediated adhesion. The P-selectin aptamer compositions may completely or partially inhibit, reduce, block or otherwise modulate P-selectin-mediated adhesion. The P-selectin aptamers are considered to completely inhibit or otherwise modulate P-selectin activity when the level of P-selectin activity in the presence of the P-selectin aptamer is decreased by at least 50%, e.g., by 50, 60, 70, 80, 90 and 100% as compared to the level of P-selectin activity in the absence of binding with a P-selectin aptamer. The P-selectin aptamers are considered to partially inhibit or otherwise modulate P-selectin activity when the level of P-selectin activity in the presence of the P-selectin aptamer is decreased by less than 50%, e.g., by 50, 60, 70, 80, 90 or 100% as compared to the level of P-selectin activity in the absence of binding with a P-selectin aptamer. [00183] The compositions may be administered by numerous routes of administration.

Such routes of administration include, but are not limited to, oral routes; topical routes, such as intranasally, vaginally or rectally; and parenteral routes, such as intravenous, subcutaneous, intradermal, intramuscular, intraarticular and intrathecal administration.

Suitable routes of administration may also be used in combination, such as intravenous administration followed by subcutaneous administration. The route of administration, however, is determined by the attending physician. Preferably, the formulations are administered subcutaneously. Most preferably, the formulations are administered intravenously.

[00184] Oral dosage forms may be administered as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups or emulsions.

[00185] Topical dosage forms include creams, ointments, lotions, aerosol sprays and gels for intranasal vehicles, inhalants or transdermal patches.

[00186] Parenteral dosage forms include solutions and lyophilized powders that are reconstituted prior to administration.

[00187] The dosage regimen utilizing the aptamers is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptamer or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

INDICATIONS

[00188] The compositions are used to treat, prevent or ameliorate P-selectin-mediated pathologies, diseases or disorders, including the treatment of coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders involving P-selectin- mediated adhesion. The pathologies, diseases or disorders to be treated, prevented or ameliorated are selected from the group consisting of: sickle cell disease, sickle cell disease sequelae (pain, acute chest syndrome, vasoocclusive crisis, acute vasoocclusive syndrome, acute non-occlusive syndrome, chronic syndrome, vascular inflammation, hypoxia of tissues, vasoocclusion of organs and tissues, organ failure, thrombogenesis, cerebrovascular accident, dactylitis, priapism, hemolytic anemia, aplastic crisis, pulmonary hypertension, retinopathy, osteonecrosis and skin ulcers), sickle cell anemia, vascular diseases, cardiovascular diseases, thrombotic diseases, hemostasis diseases, myocardial infarction, stroke, transient ischemic attack, revascularization, stent restenosis, atherosclerosis, deep vein thrombosis, venous thromboembolism, hypereosinophilia, ischemia/reperfusion injury, inflammatory diseases, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, juvenile idiopathic arthritis, organ transplant, graft rejection, ocular inflammation, retinal inflammation, colitis, conjunctivitis, scleritis, tumor metastasis, renal failure, epilepsy, malaria, cerebral malaria, asthma, psoriasis, allergic diseases, allergic conjunctivitis, immune diseases, shock and hemorrhagic shock.

[00189] The compositions may also be administered prior to, during and/or after a medical procedure. For example, the pharmaceutical compositions may be administered in conjunction (before, during and/or after) with medical procedures, such as surgery, cardiopulmonary bypass, percutaneous coronary intervention (PCI), transfusion, organ transplant, dialysis, intra vitreal injection, photocoagulation, photodynamic therapy, and radiation treatment.

THERAPEUTIC RATIONALE

[00190] Without wishing to be bound by theory regarding mechanism of action, the following therapeutic rationale is offered by way of example only. Sickle Cell Disease (SCD)

[00191] SCD results from a single nucleotide mutation in hemoglobin that causes red cell sickling. SCD includes homozygous sickle cell disease (hemoglobin SS disease), doubly heterozygous sickle hemoglobin C disease (hemoglobin SC disease) and the sickle β- thalassemias. SCD is prevalent in over 72,000 individuals in the United States and over 2 million individuals world-wide. Over 2 million Americans are believed to carry the sickle cell allele. SCD presents clinically with any one or combination of sequelae. [00192] Pain associated with a vasoocclusive crisis among patients with SCD is a common reason for emergency department visits and hospitalization. Acute pain in patients with SCD is ischemic in nature and results from the occlusion of microvascular beds. Clinical data indicate that more than five percent of patients with SCD have from three to ten episodes of painful vasoocclusive crises per year. In many patients, a vasoocclusive episode will typically be resolved in about a week. In some cases, severe episodes may persist for several weeks or even months. An ischemia-reperfusion injury can also contribute to cumulative organ damage in SCD. In addition, irreversible organ damage can result from recurrent ischemic insults and may lead to acute chest syndrome, renal hypertrophy and isosthenuria (inability to concentrate urine), autoinfarction of the spleen, chronic skin ulcers, osteonecrosis, priapism and cerebrovascular accident. [00193] SCD has historically been viewed as a disease of red cell abnormalities. Recently, however, it has been suggested that the wide spectrum of clinical manifestations of this disease result in part from chronic inflammation due to hypoxia and direct endothelial activation by adhesion of sickle erythrocytes and leukocytes among other causes. This concept is supported by evidence that SCD patients demonstrate many clinical symptoms of chronic inflammation, such as increased cytokine levels, the presence of circulating endothelial cells, increased white blood cell counts and an increase in cellular markers of leukocyte and endothelial activation. The multifactorial nature of sickle cell disease is depicted in Figure 5.

[00194] Currently, acute sickle crises are managed primarily with analgesics. Standard treatment is palliative and consists primarily of opioids, hydration, rest and behavioral therapies. However, the pain associated with vasoocclusive crisis is often under-treated due to concerns of the physician with respect to narcotic addiction, tolerance, respiratory depression and excessive sedation.

[00195] Hydroxyurea is currently the only FDA approved drug for treating SCD. Hydroxyurea is an S-phase cytotoxic drug and is used for long-term therapy. It is believed to increase the levels of hemoglobin F, which prevents formation of S-polymers and red cell sickling. It is also believed to increase NO production. A multi-center trial of hydroxyurea in adults with SCD showed that hydroxyurea reduced the incidence of painful vasoocclusive episodes by nearly half. However, hydroxyurea is currently only administered to patients: i) who suffer severe complications of SCD and ii) who are capable of following the daily dosage regimes. The general belief is that hydroxyurea therapy is effective only if given in a structured environment with a high potential for compliance. In addition, many SCD patients are refractory to hydroxyurea.

[00196] Therefore, there is a need for new therapies for treating SCD and reducing the severity of SCD sequelae. The present invention provides materials and methods to meet these and other needs.

[00197] The treatment methods of the invention are expected to have an inhibitory effect on sickle erythrocyte/leukocyte adhesion and/or erythrocyte/endothelial interaction and, consequently, reduce the severity of SCD sequelae. For example, anti-occlusive effects are thought to result from inhibition of sRBC adhesion, anti-inflammatory effects are thought to result from inhibition of leukocyte rolling and activation of the endothelium; and antithrombotic effects are thought to result from prevention of platelet adhesion. [00198] The invention also contemplates administering P-selectin aptamers in an amount sufficient to decrease sickle red blood cell retention in the pulmonary circulation. In a preferred embodiment, the invention provides a method for the administration of a P-selectin aptamer in the treatment of acute chest syndrome in a patient with SCD. As used herein, "acute chest syndrome" refers to a pathology characterized by vasoocclusion of the pulmonary vasculature that is often, but not always, triggered by an infection in the lung that causes a decrease in oxygen tension in the pulmonary tissues, which then leads to sickling of red blood cells and causes vasoocclusion.

[00199] In another embodiment, the invention provides a method for treating SCD comprising administering to a subject presenting at least one SCD sequela a therapeutic amount of an aptamer that binds to P-selectin wherein administration of the aptamer reduces the severity of the SCD sequelae. As used herein, "SCD sequelae" include, but are not limited to, pain, acute chest syndrome, vasoocclusive crisis (which occurs when abnormally shaped or sickled red blood cells block the flow of blood through small vessels and deprive the tissues of oxygen), acute vasoocclusive syndrome, acute non-occlusive syndrome, chronic syndrome, vascular inflammation, hypoxia (i.e., oxygen deprivation) of tissues (including peripheral tissues), vasoocclusion of organs and tissues, organ failure (including, but not limited to, functional asplenia), thrombogenesis, cerebrovascular accident, dactylitis, priapism, hemolytic anemia, aplastic crisis, pulmonary hypertension, retinopathy, osteonecrosis and skin ulcers. It should be noted these sequelae can interact pathologically. For example, the relationship between vasoocclusion, vascular inflammation and hypoxia is illustrated by the schematics in Figures 5 and 6.

[00200] As used herein, "reduce the severity of SCD sequelae" or "reducing the severity of SCD sequelae" means decreasing the number, frequency and/or intensity of any SCD sequelae. While it is not intended that the invention be limited to the reduction in severity of any one SCD sequelae, in one example a reduction in the severity of vasoocclusion is evidenced by a percentage decrease in the accumulation of sickle red blood cells in any organ or tissue. In another embodiment, a reduction of pain is an example of reducing the severity of an SCD sequelae. While is not intended that the invention be limited to any pain measurement index, examples of validated indexes for the measurement of pain include, but are not limited to, the Wong-Baker faces pain scale, visual analog scale, descriptor differential scale and the Walid-Robinson pain index. [00201] It is also thought that the administration of aptamers that bind to P-selectin will decrease the vasoocclusion associated with SCD by inhibiting sickle cell RBC/leukocyte/endothelial adhesion along any one or several steps in the adhesion process. Example 3 provides experimental validation that (using a murine anti P-selectin aptamer in a mouse model) anti P-selectin aptamers can substantially prevent leukocyte rolling that is proximal to vasoocclusion in patients with SCD. Example 9 provides experimental validation that (using a murine anti-P-selectin aptamer in a transgenic mouse model of sickle trait) anti-P-selectin aptamers can substantially prevent erythrocyte and leukocyte adhesion that is proximal to vasoocclusion in patients with SCD. Organ Transplant

[00202] Amersi et al. have shown that rPSGL-Ig blockade of CD62-mediated adhesive interactions protect against severe ischemia/reperfusion injury suffered otherwise by steatotic rat livers. Pretreatment of fatty livers with rPSGL-Ig prior to transplantation extended the survival of lean Zucker rat recipients from 40 to 90%. This effect correlated with significantly improved liver function, depressed neutrophil activity and decreased histological features of hepatocyte injury. rPSGL-Ig treatment decreased intragraft infiltration by CD3/CD25 cells, diminished expression of pro-inflammatory TNF_α, IL-6, iNOS, IL-2 and IFN-γ, without significantly affecting mRNA levels coding for anti-inflammatory IL-4. Amersi et al., Am. J. Transplantation, vol. 2, pp. 600-608 (2002).

[00203] Dong and Tilney state that the selectins, a group of adhesion molecules initially responsible for leukocyte-endothelial cell interactions, appear to mediate the rolling effect, the first step in the process of leukocyte slowing, adherence to the vascular endothelium and subsequent infiltration into the injured organ. Blockade of selectins prevents this early phase of leukocyte recruitment. Inhibitors of P-selectin binding protect against ischemia/reperfusion injury in organ transplants. Dong and Tilney, Current Opinions in Organ Transplantation, vol. 6, pp. 69-74 (2001).

[00204] Farmer et al. show that treatment with recombinant P-selectin glycoprotein ligand-immunoglobulin (rPSGL-Ig) resulted in significantly improved survival after intestinal transplantation. The mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration leading to a decreased inflammatory response that is possibly driven by T_h2 cytokines. Farmer et al, Transplantation, vol. 79, pp. 44-51 (2005). [00205] Gasser et al. analyzed the effects of selectin blockade with rPSGL-Ig, with or without maintenance immunosuppression with cyclosporine A and sirolimus, in a rat model of brain death-induced renal allograft dysfunction. Gasser et al. found that rPSGL-Ig decreased inflammation in the early post-transplant period such that lower doses of maintenance immunosuppression were sufficient to maintain long-term graft function. Gasser et al., Am. J. Transplantation, vol. 5, pp. 662-670 (2005). [00206] Langer et al. show that the selectin inhibitor bimosiamose, a pan-selectin inhibitor, reduced intragraft expression of P-selectin glycoprotein ligand-1, CX₃CLl, CCL 19, CCL20 and CCL2. Thus, bimosiamose blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines. Langer et al., J. Am. Soc. Nephrol., vol. 15, pp. 2893-2901 (2004).

[00207] Tsuchihashi et al. show that blocking PSGL-I protects from ischemia/reperfusion injury in liver transplantation. They examined the effects of early PSGL-I blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 antibody with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pre-treatment with anti-PSGL-1 antibody improved portal venous flow, increased bile production and decreased hepatocellular damage. Rat pre-treatment with anti-PSGL-1 antibody prevented hepatic insult in a model of cold ischemia, followed by orthotopic liver transplantation, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-I, CD3 and OX-62 cells; 3) inhibited expression of pro-inflammatory cytokine genes (TNFα, IL-I β, IL-6, IFN-γ and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic BC1-2/BC1-X_L protective genes. Tsuchihashi et al., J. Immunology, vol. 176, pp. 616-624 (2006). Hemostasis

[00208] Andre et al. propose that soluble P-selectin should no longer be considered only as a marker of inflammation or platelet activation, but also as a direct inducer of pro-coagulant activity associated with vascular and thrombotic diseases. Andre et al. showed that plasma from mice genetically engineered to express P-selectin without the cytoplasmic tail, which constitutively show a 3-4 fold increase of soluble P-selectin in plasma, or mice infused with P-selectin-Ig contained higher concentration of pro-coagulant microparticles and clotted one minute faster than wild-type mice. This pro-coagulant phenotype of genetically engineered mice could be reversed by a 4-day treatment with PSGL-Ig, a P-selectin inhibitor. Therefore, increased levels of soluble P-selectin accelerate hemostasis in these mice. Andre et al.,

PNAS, vol. 97, no. 25, pp. 13835-13840 (2000). [00209] Kyrle et al followed 544 patients with first unprovoked venous thromboembolism for an average of 35 months and studied the relationship between P-selectin and recurrence. Their study shows that high circulating P-selectin is a risk factor of recurrent venous thromboembolism. Kyrle et al, Thromb Haemost, vol. 97, pp. 880-883 (2007). [00210] McCarty et al show that eosinophil recruitment to surface-bound platelets in shear flow follows a cascade of events that shares common features with that outlined for neutrophils. In particular, PSGL-I predominantly binds to platelet P-selectin to initiate primary tethering and rolling of free-flowing eosinophils, which assist in the secondary eosinophil recruitment mediated by L-selectin-PSGL-1 interactions. McCarty et al, Am. J. Physiol Cell Physiol, vol, 284, pp. C1223-C1234 (2003).

[00211] Tanguay et al show that pre-treatment with rPSGL-Ig reduces thrombo- inflammatory responses, neointimal proliferation and in-stent restenosis. Specifically, they demonstrate that P-selectin antagonism using recombinant PSGL-Ig is effective in reducing platelet-leukocyte reactions and in-stent restenosis in double-injured porcine coronary arteries. Tanguay et al, Thromb Haemost., vol. 91, pp. 1186-1193 (2004). [00212] Wakefield et al discuss mechanisms of venous thrombosis and the role of P- selectin and other selectins. Wakefield et al, Arterioscler. Thromb. Vase. Biol., vol. 28, pp. 387-391 (2008).

[00213] Example 18 provides experimental validation that (using a murine anti-P-selectin aptamer in a mouse ligation model of DVT) anti-P-selectin aptamers can significantly mitigate thrombus growth. Rheumatoid Arthritis

[00214] Benedetti et al demonstrate a role for P-selectin in patients with juvenile idiopathic arthritis (JIA). In patients with JIA, the mRNA levels of α(l,3)- Fucosyltransferase-VII (FucT-VII) , as well as of IFN-γ and IL-12Rβ2, were up-regulated in SF T cells compared to paired PB T cells. A higher expression of FucT-VII mRNA in SF T cells was associated with increased binding of T cells to P-selectin. Moreover, FucT-VII expression and increased P-selectin binding capacity of T cells were associated with a polyarticular course of oligoarticular JIA. Expression of FucT-VII in Jurkat T cells resulted in an increased accumulation of these cells in human rheumatoid synovial tissue grafted into SCID mice. Therefore, FucT-VII plays an important role in the enhanced homing of T cells to the inflamed synovium. Benedetti et al, J. Rheumatology, vol. 30, no. 7, pp. 1611-1615 (2003). [00215] Littler et al found that patients with rheumatoid arthritis had significant elevations of serum sICAM-1, sICAM-3, sVCAM-1, sL-selectin and sP-selectin, but not sE-selectin.

However, only sP-selectin was found to correlate with disease activity in the patients. Littler et al, British J. Rheumatology, vol. 36, pp. 164-169 (1997).

Metastasis

[00216] Borsig et al. show that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin mediated interactions of platelets with carcinoma cell surface mucin ligands. This is accomplished by interfering with formation of the platelet "cloak" around tumor cells while permitting an increased interaction of monocytes with the malignant cells.

Borsig et al, PNAS, vol. 98, no. 6, pp. 3352-3357 (2001).

[00217] Borsig et al show that metastatic spread can be facilitated by tumor cell selectin ligands other than mucins, and also that P-selectin and L-selectin work synergistically in facilitating tumor metastasis. Borsig et al also state that P-selectin mediates early interactions between platelets and tumor cells. Borsig et al, PNAS, vol. 99, no. 4, pp. 2193-

2198 (2002).

[00218] Reyes and Akiyama show that P-selectin binding to specific receptors on cancer cells can activate signals that regulate tumor cell adhesion and possibly proliferation. Reyes and Akiyama, Exp. Cell Research, vol. 314, pp. 2212-2223 (2008).

Malaria

[00219] Combes et al show that endothelial P-selectin plays an important role in the pathogenesis of cerebral malaria. Mice deficient only in endothelial P-selectin did not show any sign of cerebral malaria (vascular plugging, hemorrhages or edema), while mice lacking only platelet P-selectin showed signs of cerebral malaria similar to that seen in wild-type mice. Combes et al, Am. J. Pathology, vol. 164, no. 3 (2004).

Atherosclerosis

[00220] Dong et al show that P-selectin appears to be a key adhesion receptor mediating leukocyte recruitment into atherosclerotic lesions and promoting advance atherosclerosis in apoE-deficient mice. Dong et al, Circulation, vol. 101, pp. 2290-2295 (2000).

[00221] Galkina and Ley state that increase of P-selectin expression may be the earliest and primary event in the initiation of atherosclerosis. Galkina and Ley, Arterioscler Thromb

Vase Biol, vol. 27, pp. 1-10 (2007).

[00222] Woollard and Dusting discuss the role of P-selectin in cardiovascular disease and atherosclerosis. They also state that P-selection compounds are currently being developed to treat deep vein thrombosis, sickle cell anemia, kidney transplant, rheumatoid arthritis, atherothrombosis, asthma, psoriasis, myocardial infarction and stroke. Woollard and

Dusting, Inflammation & Allergy - Drug Targets, vol. 6, pp. 69-74 (2006).

Stroke

[00223] Htun et al show that patients with a completed stroke or transient ischemic attack had significantly increased circulating platelet-leukocyte aggregates, increased P-selectin expression on platelets and decreased L-selectin expression in the acute state compared with the control group of healthy volunteers. Therefore, platelet and leukocyte activation is substantially enhanced in the acute phase of transient ischemic attack and completed stroke.

Htun et al, Stroke, vol. 37, pp. 2283-2287 (2006).

Epilepsy

[00224] Fabene et al. show, using a mouse model of epilepsy, that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-I) and leukocyte integrins α₄βi and (X_LP₂- Inhibition of leukocyte-vascular interactions, either with blocking antibodies or by genetically interfering with PSGL-I function in mice, markedly reduced seizures. Treatment with blocking antibodies after acute seizures prevented the development of epilepsy. Fabene et al, Nature Medicine, vol. 14, no. 12, pp.

1377-1383 (2008).

Inflammation

[00225] Edwards et al show that expression of P-selectin at low site density promotes the selective attachment of eosinophils over neutrophils to the vascular endothelium, which may account for the increase in eosinophil accumulation in allergic diseases. Edwards et al, J.

Immunology, vol. 165, pp. 404-410 (2000).

[00226] Haddad et al show a substantial reduction in recruitment of in vzVo-activated cells to the lamina propria in anti-IL-12 or anti-P-selectin-treated mice, and a virtual absence of lamina propria recruitment in anti-P-selectin-treated mice given differentiated ThI cells.

These data assign important roles for IL- 12 and functional PSGL-I in the migration of activated T cells to nonlymphoid tissue in the intestine, and suggest that P-selectin and functional PSGL-I are major determinants of ThI recruitment to the lamina propria. Haddad et al, J. Experimental Medicine, vol. 198, no. 3, pp. 369-377 (2003).

[00227] Ley shows that P-selectin has a role in inflammation and other diseases, such as atherosclerosis, immune responses and coagulation. Ley, Trends in Molecular Medicine, vol.

9, no. 6, pp. 263-268 (2003). [00228] Lim et al show that patients with scleritis have a significantly increased number of rolling and arrested leukocytes in superficial ocular vessels in comparison to patients with mild allergic conjunctivitis and controls. Lim et al, Molecular Vision, vol. 12, pp. 1302-

1305 (2006).

[00229] Jayle et al. show that selectin ligand inhibition attenuates ischemia/reperfusion injury in both warm and cold ischemia in large mammals. Jayle et. al., Kidney International, vol. 69, pp. 1749-1755 (2006).

[00230] Scalia et al. show that activation of P-selectin on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Using intravital microscopy, they found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti-P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-I immunoglobulin, exhibited markedly attenuated leukocyte- endothelium interaction after hemorrhagic shock. Moreover, endogenous levels of PSGL-I mRNA were significantly increased in the lung, liver and small intestine of wild-type mice subjected to hemorrhagic shock. Scalia et al., J. Exp. Med., vol. 189, no. 6, pp. 931-938

(1999).

[00231] Singbartl et. al. show that blocking P-selectin protects mice from ischemia/reperfusion induced acute renal failure, even after the onset of reperfusion.

Singbartl et. al., FASEB J., vol. 14, pp. 48-54 (2000).

[00232] Singbartl et al. show that platelet P-selectin, but not endothelial P-selectin, is critical for neutrophil-mediated acute postischemic renal failure. Singbartl et al, FASEB J., vol. 15, pp. 2337-2344 (2001).

[00233] Spencer et al discuss how the cellular interactions in the retinal vasculature are mediated by cell adhesion molecules, such as P-selectin, similar to those seen at the site of inflammation in other organs. Spencer et al, Semin. Immunopathol, vol. 30, pp. 179-190

(2008).

[00234] Strauss et al show that soluble P-selectin Glycoprotein Ligand 1 (sPSGL-1) inhibits ocular inflammation in a murine model of allergy. Mice sensitized and challenged with pollen developed clinical signs consistent with human allergic conjunctivitis. However, sensitized and challenged mice concurrently treated with sPSGL-1 displayed no inflammatory ocular changes associated with a ragweed-induced type-1 hypersensitivity reaction. Strauss et al, Invest. Ophthalmol. Vis. Sd., vol. 40, pp. 1336-1342 (1999). [00235] Vowinkel et al. show that the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes in experimental colitis. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-I). Colitis was also associated with an increased expression of PSGL-I in the colonic vasculature. Vowinkel et al., Am J. Physiol. Gastrointest. Liver Physiol, vol. 293, pp. G1054-1060 (2007).

COMBINATION THERAPY

[00236] An embodiment of the invention comprises a P-selectin aptamer or a salt thereof or a pharmaceutical composition used in combination with one or more other treatments for coagulation/thrombotic, inflammatory, metastatic and other pathologies, diseases or disorders. In some embodiments, a P-selectin aptamer is administered in combination with another useful formulation or drug, such as: anticoagulants, statins, vasodilators, anti- angiogenics (for AMD (acute macular degeneration) and/or metastatic disease), analgesics {e.g., opiates), corticosteroids, hydroxyurea, iron chelators, NSAIDs (non-steroidal antiinflammatory drugs) and other anti-inflammatory agents, antibiotics, fibrinolytics, antimalarials {e.g., quinine, chloroquine), antihistamines, cytotoxics, cytostatics, chemotherapeutics, radioisotopes, immunosuppressants, antivirals, and vitamins. Specifically, the P-selectin aptamers may be administered with fibrinolytics to resolve clots, or with immunosuppressants for organ transplantation. In another embodiment, a P-selectin aptamer is used in combination with a non-drug therapy or treatment, such as surgery, cardiopulmonary bypass, percutaneous coronary intervention (PCI), transfusion, organ transplant, dialysis, intra vitreal injection, photocoagulation, photodynamic therapy, and radiation treatment. 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.

[00237] "Combination therapy" (or "co-therapy") includes the administration of a P- selectin aptamer and at least a second agent or treatment as part of a specific treatment regimen that is intended to provide a 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 is typically carried out over a defined time period (usually minutes, hours, days or weeks, depending upon the combination selected). [00238] 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 result in the combinations of the invention. Combination therapy is intended to embrace administration of the therapeutic agents or treatments in a sequential manner. That is, wherein each therapeutic agent or treatment is administered at a different time, as well as administration of these therapeutic agents or treatments, or at least two of the therapeutic agents or treatments, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single injection having a fixed ratio of each therapeutic agent or multiple, single injections for each of the therapeutic agents. [00239] Sequential or substantially simultaneous administration of each therapeutic agent or treatment can be effected by any appropriate route including, but not limited to, topically, orally, intravenously, subcutaneously, intramuscularly and direct absorption through mucous membrane tissues. The therapeutic agents or treatments can be administered by the same route or by different routes. For example, a first therapeutic agent or treatment of the combination may be administered by injection while the other therapeutic agents or treatments of the combination may be administered subcutaneously. Alternatively, for example, 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. [00240] Combination therapy can also embrace the administration of the therapeutic agent or treatments as described above in further combination with other biologically active ingredients. Where the combination therapy comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co- action of the combination of the therapeutic agent and non-drug treatment is achieved. 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.

KITS

[00241] The compositions may also be packaged in a kit. The kit will contain the composition, along with instructions regarding administration of the composition. The kit may also contain one or more of the following: a syringe, an intravenous bag or bottle, the same composition in a different dosage form or another drug. For example, the kit may contain both an intravenous formulation and a subcutaneous formulation of the invention. Alternatively, the kit may contain lyophilized P-selectin aptamer and an intravenous bag of physiological saline solution. The kit form is particularly advantageous when the separate components must be administered in different dosage forms (i.e., parenteral and oral) or are administered at different dosage intervals.

[00242] Preferably, the kits are stored at 5±3°C. The kits can also be stored at room temperature or frozen at -20⁰C.

[00243] Many modifications and variations of the invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[00244] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

EXAMPLES

[00245] In the examples, one or more of the following aptamers were used to conduct the various experiments. ARC5665 is the aptamer described in SEQ ID NO: 1. ARC5685 is the aptamer described in SEQ ID NO: 2. ARC5691 is the aptamer described in SEQ ID NO: 3. ARC5692 is the aptamer described in SEQ ID NO: 4. ARC5691 has the same nucleotide sequence as ARC5685, except that it also has an amine derivative conjugated to its 5' end. ARC5692 has the same nucleotide sequence as ARC5691, except that it also has a 40 kDa PEG moiety conjugated to its 5' end. ARC5685 is the core aptamer sequence for each of ARC5691 and ARC5692. ARC5690 is a murine P-selectin aptamer, which is described in SEQ ID NO: 5. ARC5694 is a scrambled control aptamer, which is described in SEQ ID NO: 6. ARC6337 is a biotinylated human P-selectin aptamer that has the same sequence as ARC5692, except that it has a biotin moiety at its 5' end rather than a PEG moiety. ARC5134 is a biotinylated mouse P-selectin aptamer that has the same sequence as ARC5690, except that it has a biotin moiety at its 5 'end rather than a PEG moiety.

EXAMPLE 1: Synthesis of ARC5691

[00246] ARC5691 was synthesized by means of solid phase synthesis on an AKTA OligoPilot 100 synthesizer (GE Healthcare, Uppsala, Sweden). The synthesis was performed by standard phosphoramidite chemistry using commercially available 2'-OMe RNA, T- Fluoro RNA and DNA phosphoramidites (Glen Research, Sterling, VA), as well as an inverted deoxythymidine CPG support (Prime Synthesis, Aston, PA), and a 5' terminal C6 TFA linker phosphoramidite (American International Chemicals, Framingham, MA). ARC5691 was then deprotected with ammonium hydroxide for 16 hours at 45°C. Following deprotection, the oligonucleotide was ethanol precipitated, purified by ion exchange chromatography on Super Q 5PW resin (Tosoh Biosciences) and ethanol precipitated again to desalt.

EXAMPLE 2: PEGylation of ARC5691

[00247] To commence the PEGylation reaction, ARC5691 was dissolved in 100% water, yielding an oligonucleotide concentration of approximately 50 mg/mL. A volume of 500 mM sodium bicarbonate (pH 8.5) equivalent to 25% of the aqueous aptamer solution was then added to the mixture. Next, a dimethylsulfoxide (DMSO) volume equivalent to the volume of the ARC5691 -containing solution was added (1 :1 DMSO: [H₂O+ NaHCO₃]). In a separate container, 2.5 molar equivalents of 40 kDa branched PEG (NOF Sunbright GL2- 400NP p-nitrophenyl carbonate ester) were dissolved in acetonitrile, yielding an effective concentration of 200 mg/mL. This PEG-containing solution was then added to the aptamer- based solution and the reaction mixture was heated to 45°C for 8-12 hours. Reaction progress was monitored by analytical SAX chromatography. Upon completion, the reaction was diluted 2.5 fold with 100% water to quench the reaction by hydrolyzing any unreacted PEG. The resulting 40 kDa PEGylated product, ARC5692, was purified by ion exchange chromatography on Super Q 5PW resin (Tosoh Biosciences), desalted using reverse phase chromatography performed on Amberchrom HPRlO resin (Rohm and Haas), and lyophilized.

EXAMPLE 3: Leukocyte Rolling

[00248] In order to evaluate the effect of aptamers that bind to P-selectin on leukocyte rolling in an animal model for inflammation, the murine anti-P-selectin aptamer designated ARC5690 was compared against its scrambled control aptamer ARC5694. These aptamers have the following nucleotide sequences (reading from the 5' end to the 3' end), wherein "nh" is an amine linker, "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide, "mN" is a 2'-0 Methyl containing nucleotide and "PEG40K" is a 40 kDa polyethylene glycol moiety: ARC5690

PEG40K-nh-fC-fU-fC-mG-fC-mA-mG-mA-fC-mA-mA-fC-fC-mG-mG-mA-fU-mG- mA-mA-mA-fU-fC-fC-mG-mA-fC-fC-mG-mG-mA-mG-idT (SEQ ID NO: 5)

ARC5694

PEG40K-nh-fC-fU-fC-fC-mA-mG-mA-fC-mA-fC-mA-mG-fC-mG-mG-mA-fU-mG- mA-mA-mA-fU-fC-fC-mG-mG-fC-fC-mA-mG-mA-mG-idT (SEQ ID NO: 6) [00249] More specifically, the rolling of rhodamine labeled leukocytes in mice was monitored in the surgically-exposed, mesenteric vasculature, stimulated with histamine in the presence and absence of ARC5690 or ARC5694. Animals

[00250] Wild-type (WT) mice on a C57BL/6J background were purchased from the Jackson Laboratory. The mice, both male and female, used for intravital microscopy, were approximately 4 weeks old and weighed between 12-20 grams. Mice were selected as a relevant species for nonclinical evaluation of ARC5690 because assessing leukocyte rolling by intravital microscopy is well established in the mouse. This mouse model has been used to test anti-P-selectin antibodies and antagonists, and P-selectin knockout mice. Intravital Microscopy

[00251] Mice were anesthetized with 2.5% tribromoethanol (0.15 ml/10 g) and a midline incision was made through the abdominal wall to expose the mesentery and mesenteric veins of 200-300 μm in diameter. The exposed mesentery was kept moist throughout the experiment with periodic perfusion of warmed (37⁰C) bicarbonate buffered saline (131.9 mM NaCl, 18 mM NaHCO₃, 4.7 mM KCl, 2.0 mM CaCl₂ and 1.2 mM MgCl₂), which was equilibrated with 5% CO₂ in N₂. The mesentery was trans luminated with a 12 V, 100 W, DC stabilized source. The shear rate was calculated using an optical Doppler velocity meter. [00252] Histamine was injected intraperitoneally at a dose of 200 μL of 1 mM histamine per 15 grams of body weight. Histamine is known to induce leukocyte rolling for at least two hours. Endogenous platelets and leukocytes were labeled with Rhodamine 6G by injecting 50 μL of 1 mg/mL dye per 20 g mouse by intravenous injection in the retro-orbital plexus. Cell fluorescence is known to last for two to three hours. After thirty minutes, aptamer (ARC5690 or ARC5694) was injected into mice intravenously in the retro-orbital plexus. For the 24 hour timepoint measurements, aptamer was pre-injected, and then the mice were prepared for surgery, injected with histamine and Rhodamine thirty minutes before observation for rolling. Next, stimulated veins, with a shear rate of 150-200 s^"1, were observed by intravital microscopy. Up to four veins per mouse were studied, and leukocyte interactions with the endothelium vessel wall was recorded on video tape for 1-10 minutes in each vein. The video tapes were subsequently analyzed for counts of the number of rolling leukocytes. For each vein in which rolling was observed, the number of rolling cells to cross an arbitrary line was determined for a 1-6 minute segment of video and the data recorded as counts/minute.

[00253] Veins were visualized using a Zeiss (Germany) Axiovert 135 inverted microscope (Objective 32X) equipped with a 100 W HBO fluorescent lamp source (Optic Quip, Highland Mills, NY) and connected to both an SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan) and a CCD video camera (Hamamatsu Photonic Systems, Hamamatsu City, Japan). [00254] Videomicrographs were analyzed by an observer blinded to the treatment of the animals. To ensure more accurate counting, the image of the vein was divided in half laterally, and rolling was counted in one half of the vein at one time. For each half vein, a line perpendicular to the direction of flow was drawn. Rolling leukocytes were defined as fluorescent cells that traveled along the vein with a velocity distinctly slower than the bulk flow, and which migrated past the perpendicular line. The video tape was then rewound, the other half of the vein was revealed, and rolling leukocytes were counted in the same manner on the other side of the vein. The start and stop time for each vein segment was noted, and the total duration of observation was recorded. To calculate the number of rolling leukocytes per minute (counts/min), the total number of rolling leukocytes (counts) in each half of the vein was added and then divided by the duration in minutes. For each mouse, the counts for each vein were averaged and the standard deviation (SD) and standard error of the mean (SEM) was reported. Results

[00255] Fluorescent, intravital microscopy was used to visualize the in vivo rolling of leukocytes in the mesenteric vasculature of wild-type C57BL/6J mice. To stimulate leukocyte rolling, the mice were treated with histamine, which induces the secretion of Weibel-Palade bodies containing P-selectin. All mice were administered fluorescent rhodamine dye, which nonspecifically labels leukocytes in situ for better visualization under the microscope. Representative images captured from the venules of mice treated in this manner are included in Figures 18 and 20. White "dots" in these images are leukocytes that are rolling on or adhered to the endothelial surface of the venule. [00256] Figure 18 shows images of leukocyte rolling prior to and after injection of 1 mg/mL of anti-mouse P-selectin aptamer ARC5690. The left hand panel displays a vein from a mouse after histamine treatment and rhodamine labeling. Several leukocytes can be visualized contacting the endothelium prior to ARC5690 administration. The average number of rolling leukocytes quantified in 13 veins from 4 mice was 204 ± 27.7 counts/min (Table 1 in Figure 19). Shown in the right hand panel of Figure 18 is a vein from a mouse infused with 1 mg/kg of ARC5690 one minute after infusion. Leukocytes were no longer captured by the inflamed endothelium. Mice 1-3 all received ARC5690 and were analyzed within 30 minutes of aptamer administration. Mice 1 and 2 received 1 mg/kg and mouse 3 received 0.1 mg/kg. Four veins were analyzed in each mouse (Table 2 in Figure 19) and no leukocyte rolling was observed in any of them. A second group of 3 mice (mice 5-7 in Table 2 in Figure 19) received 1 mg/kg of ARC5690 and were left alone overnight. After approximately 24 hours, these mice were given histamine and rhodamine, and 3 veins per mouse were analyzed for rolling. A few rolling cells were visible in vein 1 of mouse 5, but no rolling was observed in the other eight veins.

[00257] Figure 20 shows images of leukocyte rolling prior to and after injection of 1 mg/mL of scrambled control aptamer ARC5694 (left and right hand panels, respectively). ARC5694 is identical in nucleotide composition to ARC5690, and also contains a 40 kDa PEG. However, the sequence of the nucleotides was rearranged to abrogate P-selectin binding activity while preserving the predicted secondary structure. As shown in the right hand panel of Figure 20, leukocyte rolling was not blocked immediately after injection of ARC5694. Only a limited number of observations were made with ARC5694 at this timepoint (not shown), which were suggestive of a lack of blockade. Mice 8-10 were dosed with 1 mg/kg of ARC5694 and observed at 24 hours (Table 3 in Figure 20). The average number of rolling leukocytes in nine veins from these three mice (221 ± 20.6 counts/min) was indistinguishable from the pre-dose average. This suggests that the inhibition of rolling from ARC5690 was associated with the specific targeting of P-selectin by this aptamer. [00258] Based upon these data, we concluded that ARC5690 completely abolished leukocyte rolling in histamine activated mesenteric venules of wildtype mice while control aptamer ARC5694 did not. Inhibition of rolling extended to 24 hours at a dose of 1 mg/kg.

EXAMPLE 4: Binding Affinity of ARC5692 [00259] ARC5685 was ³²P-labeled at the 5 ' end by incubation with γ-³²P-ATP and polynucleotide kinase (New England Biolabs, Beverly, MA). Radiolabeled aptamer was purified away from free ATP by gel filtration. To measure anti-P-selectin aptamer affinity, radiolabeled aptamer (< 100 pM) was incubated with increasing concentrations of recombinant protein (0.002-100 nM P-selectin or 0.2-1000 nM E- or L-selectin; R&D Systems, Minneapolis, MN) in Dulbecco's phosphate buffered saline (DPBS; Invitrogen) containing 0.1 g/L MgCl₂ (492 μM) and 0.13 g/L CaCl₂ (905 μM) at 37⁰C for 30 minutes. [00260] The binding reactions were analyzed by nitrocellulose filtration using a Minifold I dot-blot, 96-well vacuum filtration manifold (Schleicher & Schuell, Keene, NH). A three- layer filtration medium was used, consisting (from top to bottom) of Protran nitrocellulose (Schleicher & Schuell), Hybond-P nylon (Amersham Biosciences, Piscataway, NJ) and GB002 gel blot paper (Schleicher & Schuell). The nitrocellulose layer, which selectively binds protein over nucleic acid, preferentially retained the anti-P-selectin aptamer in complex with a protein ligand, while non-complexed aptamer passed through the nitrocellulose and adhered to the nylon. The gel blot paper was included simply as a supporting medium for the other filters. Following filtration, the filter layers were separated, dried and exposed on a phosphor screen (Amersham Biosciences) and quantified using a Storm 860 Phosphorimager^® blot imaging system (Amersham Biosciences).

[00261] As shown in Figure 10, the percentage of ARC5685 bound to P-selectin protein increased with increasing P-selectin concentrations. The dependence of bound ARC5685 on increasing P-selectin concentration is well-described by a single-site binding model (P- selectin + ARC5685 ^ P-selectin^» ARC5685; percent bound = Cmax / (1 + K_D / [P- selectin]); Cmax is the maximum percent bound at saturating [P-selectin]; K_D is the dissociation constant). The K_D of ARC5685 for P-selectin determined by this experiment was 1.7 ± 0.1 nM. In contrast, minimal binding was observed to recombinant E-selectin, even at high protein concentrations. Binding was observed to recombinant L-selectin, but only at comparatively high protein concentrations relative to P-selectin (K_D > 200 nM).

EXAMPLE 5: Binding Affinities as Measured by Biacore

[00262] The binding affinities of P-selectin aptamers were further characterized by surface plasmon resonance (SPR). Modified constructs of ARC5692 (anti-human P-selectin aptamer) and ARC5690 (anti-mouse P-selectin aptamer) were synthesized with biotin functional groups conjugated to the 5 '-terminus in place of PEG. The biotin group was used to immobilize the aptamers on a streptavidin-coated surface for analysis by Biacore. The biotinylated anti-human and anti-mouse aptamers were designated ARC6337 and ARC5134, respectively.

[00263] Biotinylated aptamers were immobilized on a research-grade streptavidin (SA) biosensor chip (BIACORE Inc., Piscataway, NJ) to measure binding specificity by purified recombinant human P-selectin (R&D Systems) or mouse P-selectin (R&D Systems). Biotinylated aptamers were diluted to 25 nM in DPBS buffer containing 0.1 g/L MgCl₂ and 0.13 g/L CaCl₂, and injected manually over cell surfaces at a flow rate of 10 μL/min, and stopped when net increase of surface resonance units (RU) gained 500. All surfaces, including no-aptamer negative control, were blocked by injecting 50 μM biotin at 10 μL/min for 2 minutes. Recombinant human P-selectin and mouse P-selectin were serially diluted into DPBS and run through an aptamer coated SA chip. Various concentrations of P-selectin (ranging from 0.1 nM to 100 nM) samples were injected for binding at 20 μL/min for 5 minutes to monitor association kinetics, followed by 5 minutes of buffer only to monitor protein dissociation. To regenerate the non-derivatized surface, IN NaCl was injected for 30 seconds at 20 μL/min. Rate constants and dissociation constants were calculated using BIAevaluation software.

[00264] Immobilized, biotinylated anti-human P-selectin (ARC6337) and biotinylated anti-mouse P-selectin (ARC5134) aptamers bound with high affinity to their cognate proteins, as determined by this method (Figure 11). The human-specific aptamer bound to human P-selectin with an association rate constant (k_on) of 1.4 x 10⁵ M ¹S ¹ and a dissociation rate constant (k_o/j) of 2.2 x 10^"5 s^"1. This k_of/, which corresponds to a half-life (ty₂) of > 2 hr, is a rough estimate of the actual value oϊk_ojf, since dissociation kinetics are measured, optimally, over several half-lives. Nevertheless, the thermodynamic dissociation constant calculated from the kinetic measurements (K_D = k_o/f/ k_on) was 1.6 nM, consistent with the results of the nitrocellulose filter binding assay described in Example 4. Similarly, the mouse-specific aptamer bound to mouse P-selectin with an association rate constant (k_on) of 2.0 x 10⁵ M ¹S ¹ and a dissociation rate constant {k_ojj) of 7.8 x 10^"5 s^"1, corresponding to a K_D of 0.4 nM. Neither aptamer bound with significant affinity to protein of the other species.

EXAMPLE 6: Performance of ARC5692 in a PSGL-I Assay

[00265] The ability of ARC5692 to compete for binding to P-selectin with its cognate ligand, the membrane-associated protein P-selectin Glycoprotein Ligand-1 (PSGL-I), was measured using a microplate assay with purified components, including a soluble, recombinant version of PSGL-I hybridized to the Fc domain of IgG (PSGLl-Fc). PSGLl-Fc was biotinylated for the purpose of detection, then mixed with aptamer prior to addition to immobilized P-selectin. Bound PSGLl-Fc was then measured using streptavidin conjugated to horseradish peroxidase (HRP).

[00266] To immobilize human recombinant P-selectin, 500 ng of recombinant P-selectin (R&D Systems) in 100 μL of PBS (pH 7.4) was pipetted into the wells of a 96-well Maxisorb plate (Nunc) and incubated overnight at 4⁰C. On a separate plate, various concentrations of aptamers were diluted in DPBS containing 0.2% bovine serum albumin (BSA). 50 μL of aptamer solution was mixed with 50 μL of 100 nM biotinylated PSGLl-Fc (R&D Systems) in the same buffer and incubated at room temperature for 5 minutes. The plate containing immobilized P-selectin protein was washed three times with 200 μL of TBST (25 mM Tris- HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). This plate was blocked with 200 μL TBST containing 5% nonfat dry milk for 30 minutes at room temperature and washed again three times with 200 μL of DPBS. The mixture of aptamer and biotinylated-PSGLl-Fc was added to the P-selectin plate and incubated at room temperature for 1.5 hours. The plate was washed three times with 200 μL of DPBS. 100 μL of streptavidin-HRP (R&D Systems), diluted 1 :200 in DPBS containing 0.2% BSA, was added and incubated for 1 hour at room temperature. The plate was washed again with DPBS followed by the addition of 100 μL TMB (3,3',5,5'-tetramethylbenzidine) substrate solution (Pierce). The plate was incubated with TMB for 10-15 minutes in the dark at room temperature. Development of the substrate was stopped by the addition of 100 μL 2N H₂SO₄ and the plate was read on a UV/vis spectrophotometer at 450 nm.

[00267] As shown in Figure 12, increasing concentrations of ARC5692 inhibited the interaction between immobilized P-selectin and soluble PSGLl-Fc. The midpoint, or EC50, of the inhibition curve was 1.5 nM, which is consistent with measures of aptamer/P-selectin affinity described above. At the highest concentrations of ARC5692, this interaction approached 100% blocked.

EXAMPLE 7: Inhibition of Platelet/Leukocyte Interactions with ARC5692 Reagents

[00268] THP-I cells were obtained from ATCC (Manassas, VA) and were cultured in Complete Growth Medium (CGM), containing 500 mL RPMI medium, 10 mM Hepes, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol (all from Sigma, St. Louis, MO) penicillin/streptomycin (cat. no. 10378-016, Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (cat. no. 30-2020, ATCC). Phosphate buffered saline (PBS) (cat. no. MT- 21-040-CV), was obtained from Mediatech (Manassas, VA). Cells were maintained in incubators at 37⁰C with 5% CO₂.

[00269] Tyrode's buffer was prepared containing the following ingredients: 137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.2% Glucose, 5 mM Hepes, pH 7.3, and 0.36% BSA dissolved in deionized water. Hank's balanced saline solution (HBSS) without MgCl₂ or CaCl₂ (cat. no. H6648, Sigma) was used with added 0.5% BSA (Sigma). Calcium chloride was obtained from Sigma. Tyrode's buffer and HBSS were refrigerated and calcium chloride was stored at room temperature.

[00270] D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) was obtained from EMD (cat. no. 520222, Darmstadt, Germany). It was dissolved in a solution of 5% acetic acid (made with deionized water) to a stock solution of 10 mM. 90 μL of stock was placed in a syringe able to hold a 10 mL volume and used in the blood draw discussed below. The stock solution was aliquoted into Eppendorf tubes and stored at -8O⁰C for up to 12 months. Individual aliquots were thawed at room temperature or on ice and used only once. [00271] Adenosine diphosphate (ADP) was obtained from Diamed (Cressier, Switzerland) purchased as DiaAdin (cat. no. 308161). A stock solution of 110 μM was made by dissolving the lyophilized ADP powder into 1 mL of deionized water. ADP solution was refrigerated at 4⁰C and used for up to one month once reconstituted. Lyophilized powder was stored up to one year at 4⁰C.

[00272] Human thrombin (cat. no. T7009) was purchased from Sigma. Thrombin stock solution was made by dissolving lyophilized powder into phosphate buffered saline (PBS) at a concentration of 100 LVmL. This solution was aliquoted into Eppendorf tubes and stored at -80°C for up to 12 months. Individual aliquots were thawed at room temperature or on ice and used only once.

[00273] Blood was drawn from healthy human donors and anticoagulated with either the thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) obtained from EMD (cat. no. 520222, Darmstadt, Germany) or anticoagulant citrate dextrose (ACD) obtained from BD Biosciences (cat. no. 364606). For the PPACK draw, blood was collected directly into a syringe containing 90 μL of stock anticoagulant. The concentration of PPACK was 90 μM in a volume of about 10 mL. For the ACD anticoagulated blood, a standard blood draw into vacutainer tubes was done collecting 8.5 mL of blood per draw. Platelets were isolated from whole blood by centrifugation and then washed and resuspended in Tyrode's buffer. [00274] For flow cytometry, a monoclonal antibody raised against the platelet marker CD41a (clone HIP8) labeled with fluorescein isothiocyanate (FITC) was purchased from BD Pharmingen (San Jose, CA) and refrigerated. Analysis of THP-1/ADP-activated Platelet Complex Formation

[00275] Platelets were isolated from human blood that had been anticoagulated using the thrombin inhibitor PPACK. Platelets were washed and then resuspended in Tyrode's buffer. THP-I monocytes, cultured in CGM, were concentrated by centrifugation, washed and resuspended in HBSS. Isolated platelets (5 μL) were mixed with THP-I cells (45 μL) to a final ratio of 4 X 10⁶ platelets to 45,000 cells, along with 2.5 μM ADP, 2.5 mM CaCl₂ and various concentrations of aptamer (or blocking anti-P-selectin monoclonal antibody) for 20 minutes at 37°C. Fluorescein isothiocyanate (FITC) labeled anti-CD41a antibody (5 μL) was then added to label the platelets. Cells plus stain were incubated at 37°C for 5 minutes. 400 μL of PBS was added and then the reaction was transferred to a FACS tube. Samples were analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA) to determine the percent of THP-I cells that had platelets bound (denoted as "% Pit (+)"). Data was analyzed using CellQuest (Version 3.3).

Analysis of THP-1/Thrombin-activated Platelet Complex Formation [00276] Platelets were isolated from human blood that had been anti-coagulated using ACD. Platelets were washed and resuspended in Tyrode's buffer. THP-I monocytes, cultured in CGM, were concentrated by centrifugation, washed and resuspended in HBSS. Isolated platelets (5 μL) were mixed with THP-I cells (45 μL) to a final ratio of 4 X 10⁶ platelets to 45,000 cells, along with 2.5 μL of 110 mM CaCl₂ (5 mM final concentration), 2.5 μL of 4.4 LVmL thrombin (0.2 LVmL final concentration) and various concentrations of aptamer for 20 minutes at 37°C. Additionally, a control containing no additional calcium chloride and also the calcium chelator EDTA to 10 mM was included to account for non-P- selectin/PSGL-1 dependent, or alternatively, non-specific platelet adherence. Fluorescein isothiocyanate (FITC) labeled anti-CD41a antibody (5 μL) was then added to label the platelets. Cells plus stain were incubated at 37°C for 5 minutes. 400 μL of PBS was added and the reaction was transferred to a FACS tube. Samples were analyzed by flow cytometry using FACSCalibur to determine the percent of THP-I cells that had platelets bound. Data was analyzed using CellQuest (Version 3.3). The data was plotted, and IC50 and IC90 values were determined using the XLfit 4.1 plug-in for Excel^®. Results [00277] The graphs in Figure 13 show FACS results from ADP-activated platelets combined with THP-I cells in the presence and absence of aptamer. The y (ordinate) axis, labeled FL-I, reports the degree to which a cell is stained with an anti-platelet fluorescein labeled antibody. Each dot represents THP-I cell(s). Dots with a high level of fluorescence are bound to one or more platelets. In the left-hand panel, the population above the line denotes THP-I cells positive for platelet fluorescence, with the population below the line denoting THP-I cells negative for platelet fluorescence. In the right-hand panel, ARC5692 added to a concentration of 1 μg/ml (77.2 nM) knocks down binding of platelets to THP-I cells. There is always a residual amount of THP-I cells that appear positive for the fluorescent dye. EDTA is a divalent cation chelator that disrupts binding of P-selectin to PSGL-I . THP-I cells appear positive for dye even when EDTA is added to the mixture (data not shown), representing either non-specific binding of the antibody to THP-I cells, or interaction of platelets and THP-I in a P-selectin/PSGL-1 independent mechanism. [00278] Significant platelet activation was observed both in the presence (closed symbols) and absence (open symbols) of ADP, and results are shown for both treatments (Figure 14). Replicate experiments, performed with different donors on two separate days, are shown in Figure 14. In the first experiment (Donor 1), the platelet/THP-1 interaction is maximally inhibited at the lowest concentration of aptamer tested of 1.00 μg/ml (77.2 nM). Other concentrations of aptamer tested were 10.0 and 100 μg/ml. In the second experiment (Donor 2), the experiment was repeated at 0.100, 1.00, 5.00 and 10.0 μg/ml (7.72 nM, 77.2 nM, 386 nM and 772 nM), and the interaction is maximally inhibited at 1.00 μg/ml. [00279] Although ADP was used as an agonist to stimulate platelet P-selectin expression and platelet/leukocyte complex formation, ADP addition did not greatly enhance complex formation in these experiments. The percentage of platelet positive THP-I cells in the absence of P-selectin blocking aptamer increased less than 2-fold from 40-50% to 60-80%. However, this observation concurs with published reports of ADP resistance of washed platelets. Walkowiak et al. found that P-selectin expression on washed platelets did not significantly increase with ADP treatment (Walkowiak, B., Kralsiz, U., Michalec, L., Majewska, E., Koziolkiewicz, W., Ligocka, A., Cierniewski, CS. Comparison of Platelet Aggregability and P-selectin Surface Expression on Platelets Isolated by Different Methods. Thrombosis Research. 2000; vol 99: 495-502). Cazenave et al. observed that washed platelets are resistant to ADP-mediated degranulation in the absence of fibrinogen (Cazenave, J.P., Ohlmann, P., Cassel, D., Eckly, A., Hechler, B., Gachet, C, Preparation of Washed

Platelet Suspensions From Human and Rodent Blood. Methods in Molecular Biology, vol. 272: Platelets and Megakaryocytes, vol. 1 : Functional Assays. Ed. J. M. Gibbons and M. P. Mahaut-Smith. Humana Press Inc.: Totowa, 2004: 1064-3745). For the experiments described in this report, plasma was excluded as fibrinogen addition would have caused aggregation and particle formation making flow cytometric sample analysis difficult. [00280] In a second set of experiments, incubations were performed using platelets activated with thrombin (0.2 U/mL), which is a much stronger activator/inducer of P-selectin expression than ADP regardless of the preparative method used to isolate platelets. In this case, the blood could not be drawn into the thrombin inhibitor PPACK, which would have blocked the work of the agonist, so anticoagulant citrate dextrose (ACD) was used instead. Platelet and THP-I cell washes were performed as described for the THP-I /ADP activated platelet experiment, as were the incubations and flow cytometry. In this case, 4 replicates were performed on different days with platelets from 4 donors. The individual data, expressed as the percentage of THP-I cells with platelets bound [%Plt(+)] is plotted in Figure 15. As observed above, significant platelet activation, reflected in their binding to THP-I cells, was seen with or without thrombin treatment (Figure 15). The average level of THP-I cells with platelets bound was 84.8 ± 10.1% in the absence of thrombin, and 91.9 ± 4.2% in the presence of thrombin. As in the ±ADP experiment, (Figure 14) increasing concentrations of ARC5692 inhibited platelet/THP-1 interactions, although higher aptamer concentrations were required here for complete inhibition. The % Inhibition for the binding of THP-I cells to platelets in the presence or absence of thrombin was calculated for each sample and the average % Inhibition across donors was plotted versus [ARC5692] and fit to a standard IC₅₀ curve (Figure 16). IC50 values of 44.6 ± 30.4 nM and 43.3 ± 35.8 nM were determined for measurements in the presence and absence thrombin, respectively, and error values are the standard errors derived from the curve-fit. For both conditions, the IC90 values were approximately 400 nM.

EXAMPLE 8: Rolling of Monocytes as Mediated by ARC5692

[00281] The P-selectin mediated rolling of THP-I monocytes on activated, human donor platelets under flow was measured by microscopy using fluorescently-labeled cells and an in vitro flow chamber.

Cell line and Tissue Culture Reagents

[00282] THP-I cells were obtained from ATCC (Manassas, VA) and were cultured in Complete Growth Media (500 mL RPMI, 10 mM Hepes, 1 mM sodium pyruvate, 2 mM L- Glutamine, 0.05 mM 2-mercaptoethanol (all from Sigma, St. Louis, MO) penicillin/streptomycin (10378-016, Invitrogen, Carlsbad, CA), 10% fetal bovine serum (30- 2020, ATCC). Phosphate buffered saline (PBS) part number MT-21-040-CV, was obtained from Mediatech (Manassas, VA). Cells were maintained in incubators at 37⁰C with 5% CO₂.

Chemicals

[00283] Tyrode's buffer is comprised of the following ingredients: 137 mM NaCl, 2.7 mM

KCl, 12 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.2% Glucose, 5 mM Hepes, pH 7.3 and 0.36 %

BSA, all purchased from Sigma (St. Louis, MO). Tyrodes buffer was aliquoted and stored at

-2O⁰C. Just prior to use, the pH of the thawed buffer was adjusted to 7.3 at room temperature by adding HCl, and the buffer was placed in a 37°C water bath.

[00284] Hank's balanced saline solution (cat. no. H6648) was purchased from Sigma.

Hank's balanced saline solutions (HBSS) used throughout the study had calcium chloride and magnesium chloride (Sigma) added to 1.5 mM, and bovine serum albumin (Sigma) added to

0.5%. HBSS was pre -warmed to 37⁰C just prior to use.

[00285] Human thrombin (cat. no. T7009) was purchased from Sigma. Thrombin stock solution was made by dissolving lyophilized powder into phosphate buffered saline (PBS) at a concentration of 100 LVmL. This solution was aliquoted into Eppendorf tubes and stored at

-80°C for up to 12 months. Individual aliquots were thawed at room temperature or on ice and used only once.

[00286] Prostaglandin El (PGEl) (cat. no. P5515-1MG, Sigma) was resuspended in ethanol at 1 mg/mL and frozen in 10 μL aliquots at -80⁰C for up to 6 months. Individual aliquots were thawed at room temperature or on ice and used only once.

[00287] Apyrase (cat. no. A6535-100U, Sigma) was resuspended in deionized water at

300 LVmL and frozen in 10 μL aliquots at -80°C for up to 6 months. Individual aliquots were thawed at room temperature or on ice and used only once.

Platelet Isolation and Labeling

[00288] Blood was drawn from healthy human donors and anticoagulated with low molecular weight heparin enoxaparin (Lovenox, cat. no. 0075-0626-03) obtained from

Aventis Pharmaceuticals, Inc. (Frankfurt, Germany) to a final concentration of 100 μg/mL.

The stock solution, at 100 mg/mL, was diluted in PBS to make a solution of 300 μg/mL by adding 30 μL of Lovenox in 10 mL of PBS. Then, 3.3 mL of this solution was added to syringes into which approximately 6.6 mL of blood was drawn. Blood was kept on the bench-top for 15 minutes at room temperature, then centrifuged for 10 minutes at 800 rpm

(-13 Og) to make 5-6 mL platelet-rich plasma (PRP). 300 μL PRP was diluted into 700 μL

Tyrodes buffer containing Prostaglandin El (PGEl) at a final concentration of 1 μg/ml and apyrase at a final concentration of 1 LVmL in a 1.5 mL tube. This mixture was centrifuged at 2400 rpm (50Og) for 3.5 minutes to pellet the platelets, which were then resuspended in 150 μL Tyrodes buffer. CellTrace Calcein Red-Orange dye (cat. no. C34851, Invitrogen, Carlsbad, CA) was added to a 1 :200 final dilution for 5 minutes at room temperature to label platelets. Platelets were labeled so that when they were used to coat covers lips visual inspection could be used to verify uniform coverage. THP-I Cell Preparation

[00289] THP-I monocytes were grown in Complete Growth Medium to a concentration of -500,000 cells/mL. Just prior to use, 20 mL of cell suspension was pelleted by centrifugation for 5 minutes at 200 g. Cells were resuspended in 20 mL PBS (no MgCl₂ or CaCl₂), re- centrifuged for 5 minutes at 200 g, and resuspended in 10 mL PBS. To fluorescently label the cells, 5 μL carboxyfluorescein diacetate, succinimidyl ester (CellTrace CFSE, cat. no. C34554, Invitrogen, Carlsbad, CA) was added and cells were incubated for 5 minutes at 37°C. Cells were centrifuged for 5 minutes at 200 g and resuspended to 500,000 cells/mL in HBSS. Cells were kept in a 37°C water bath until use. Just prior to use, aliquots of cells (~3 mL) were prepared +/- ARC5692 (0.03-1 μg/mL) in standard glass test tubes. Coverslip Preparation

[00290] Circular, glass coverslips (35 mm diameter, cat. no. 31-0008; Glycotech, Gaithersburg, MD) were washed in acetone and incubated in 4% 3- aminopropyltriethoxysilane (APTES) (cat. no. 440140, Sigma) in acetone for 30 minutes at 37°C to create an adhesive surface for the immobilization of labeled platelets. APTES- treated coverslips were rinsed once with acetone and four times with deionized water, then dried at 37°C. 200 μL labeled platelet solution was pipetted onto dry coverslips and incubated for 30 minutes at 37°C in the dark, leaving a dense monolayer of platelets on the coverslip surface. Platelet-coated coverslips were washed three times with PBS containing 0.5% BSA. Regions of the coverslip left uncoated were "blocked" by the addition of 1% BSA in 200 μL Tyrodes buffer, followed by incubation for 30 minutes at 37°C. In order to activate the platelets, the Tyrodes/1% BSA solution was aspirated and replaced with 200 μL thrombin (1 LVmL) for 2 minutes at room temperature. Thrombin was removed and replaced with 200 μL Tyrodes/1% BSA for 10 minutes at room temperature. Flow Chamber

[00291] Flow chamber apparatus and gasket (Glycotech) were cleaned with isopropanol and assembled according to the manufacturer's instructions. The Tyrodes/1% BSA solution was aspirated from the platelet-coated coverslip. The flow chamber assembly was pressed onto the coverslip, which was held on via a vacuum line attached to an external vacuum pump. PBS was pulled through manually, using a syringe attached to the outlet line, to fill the flow chamber inlet and outlet lines with buffer. Experiments were run by passing THP-I cell suspensions, +/- ARC5692, through the inlet line into the flow chamber at a constant shear rate of 200 s^"1 (~80 mL/min) using a syringe pump (Harvard Apparatus, Holliston, MA). A separate coverslip was prepared for each concentration of aptamer tested. For Donor 1, images were collected but rolling was not quantified. For Donor 2, two coverslips were used for the zero aptamer data, and one coverslip each was used for 0.100 μg/mL, 0.300 μg/mL and 1.00 μg/mL ARC5692. For Donor 3, one coverslip each was used for no aptamer control, 0.0300 μg/mL, 0.100 μg/mL, 0.300 μg/mL and 1.00 μg/mL of ARC5692. Methods of Analysis

[00292] Images were visualized using a Zeiss inverted microscope connected to an SVHS Panasonic AG-6720A video recorder (Matsushita Electric, Kadoma City, Japan) using a charge-coupled device video camera (Hamamatsu Photonics Systems, Hamamatsu City, Japan). Discreet THP-I cells immobilized in, or in slow migration across the field of view were counted as adhered or rolling, respectively. Leukocyte rolling/adhesion was quantified as the mean number of rolling or adhered THP-I cells per field, based on observations across several fields of view (n = 6-9). The percentage inhibition was calculated from the ratio of rolling/adhered cells observed in the absence of aptamer (C_{no ARC5692}) versus in the presence of aptamer (C+_ARC5692): %inh = 100% x (1 - C+_ARC5692 / C_no ARC5692). Results

[00293] Experiments to measure THP-I monocyte rolling on activated platelets were performed on three separate occasions using platelets isolated from the blood of three different donors. In Figure 17, single-frame images are shown of fluorescently labeled leukocytes under flow, using platelets obtained from a single donor (Donor 1). Bright dots represent rolling or adhered cells, while streaks represent cells that flow unimpeded through the chamber. In the absence of ARC5692 (Panel A), several monocytes were observed as adhered or rolling on the platelet-covered surface. The addition of 1.00 μg/mL ARC5692 (Panel B) blocked interactions with the platelet surface, as no rolling cells were observed. [00294] Adhered/rolling monocytes were quantified in experiments performed with platelets from two additional donors. Graphical representations of the cell number and %Inhibition data are shown in Figure 17, Panels C and D, respectively. For Donor 2, the number of rolling/adhered THP-I cells in the absence of aptamer was measured on two separate coverslips, and the counts from both were averaged in calculating the %Inhibition

(Panel D). Adhesion and rolling were quantified for 0.100, 0.300 and 1.00 μg/mL ARC5692 for Donor 2, and 0.0300, 0.100, 0.300 and 1.00 μg/mL ARC5692 for Donor 3. In these experiments, the number of adhered/rolling platelets decreased with increasing concentrations of ARC5692 (0.0300 - 1.00 μg/mL). As observed for Donor 1, leukocyte rolling/adhesion was nearly 100% inhibited at a concentration of 1.00 μg/mL (77.2 nM) for both Donors 2 and 3. Substantial inhibition of rolling/adhesion was observed at all concentrations tested: 50- 60% at 0.0300 μg/mL (2.32 nM), 70-90% at 0.100 μg/mL (7.72 nM), and 80-90% at 0.300 μg/mL (23.2 nM).

Example 9: KO-TG Mouse Model of Sickle Cell Disease

[00295] This was a preclinical efficacy study of the anti-P-selectin aptamer ARC5690 in a knockout-transgenic (KO-TG) mouse model of sickle cell disease (SCD). The mouse model used in this study was originally developed at the University of Alabama, Birmingham, AL (Ryan, T.M., Ciavatta, D. J., and Townes, T. M. 1997. Knockout-transgenic mouse model of sickle cell disease. Science 278:873-876). Homozygous sickle animals carry deletions of the murine α- and β-globin alleles and possess a complex transgene of the human α-, β- and γ- globin gene sequences. Consequently, animals homozygous for the knockout alleles do not produce any murine hemoglobin and express high levels of human β-sickle hemoglobin. The SCD phenotype is mature by the age of eight to ten weeks, consisting of the characteristic hemolytic anemia, vessel congestion, and changes in the structure and function of multiple organs.

[00296] This study utilized intravital microscopy to study both sickle red blood cell (RBC) adhesion and white blood cell (WBC) adhesion in knockout-transgenic SCD mice that were heterozygous for the human sickle gene (AS). RBCs were withdrawn from an AS donor mouse and fluorescently labeled ex vivo using 2',7'-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein (BCECF), then injected into study mice for visualization by intravital microscopy. Microscopy was performed of the vasculature in the bone marrow of the exposed front-parietal skull. In this location, the skull is thin and provides sufficient optical transparency to allow for the recording of microvascular events without the need for surgery (Mazo, Gutierrez-Ramos, Frenette, Hynes, Wagner, von Andrian (1998) J Exp Med 188, 465- 474). Fluorescent labeling of leukocytes was performed in vivo by injecting a fluorescein isothiocyanate labeled antibody to CD45. Cell adhesion was stimulated by exposure of subject mice to one hour of hypoxia (12% O₂, balanced N₂) followed by one hour of reoxygenation at ambient conditions (21% O₂). Anesthetized animals were ventilated with a small animal ventilator, body temperature maintained at 37⁰C with a heater plate, and blood pressure continuously monitored with a carotid artery pressure transducer. Digital recordings of the microcirculation were obtained from ten randomly chosen windows and analyzed offline for RBC adhesion, leukocyte rolling and adhesion, RBC velocity and shear stress. RBC adhesion (events/min), RBC velocity (μm/sec), wall shear rate (WSR, s^"1), leukocyte adhesion (cells/100 μm), leukocyte rolling velocity (μm/sec) and leukocyte rolling flux (cells/min) were measured essentially as described (Kaul, DK and Hebbel, RP (2000) Hypoxia/reoxygenation causes inflammatory response in transgenic sickle mice but not in normal mice, J Clin Invest 106, 411-420).

[00297] Experimental mice were given an intraperitoneal injection of ARC5690 (20 mg/kg) 2.5 hours before hypoxia/reoxygenation. Control mice were given an intraperitoneal injection of saline (0.9% NaCl) 2.5 hours before hypoxia/reoxygenation. The hemodynamic parameters determined by visualization of fluorescently-labeled red blood cells are shown in Figures 23 and 24. Red blood cell velocity was increased by 100% in ARC5690-treated mice (230.6 ± 34.88 μm/sec; n = 4) versus the saline control (116.7 ± 17.7 μm/sec; n = 6) (Figure 24, left panel). The wall shear rate was increased by a similar amount from 43.6 ± 7.35 s^"1 in saline -treated mice to 90.2 ± 14.58 s^"1 in ARC5690-treated mice. The number of adhered RBCs decreased by >75% (Figure 24, right panel). The hemodynamic parameters determined by visualization of fluorescently-labeled leukocytes are shown in Figures 25 and 26. Leukocyte velocity was increased by 40% in ARC5690-treated mice (197.9 ± 23.82 μm/sec; n = 5) versus the saline control (142.3 ± 6.04 μm/sec; n = 4) (Figure 25). The wall shear rate was increased by a similar amount from 53.3 ± 6.20 s^"1 in saline-treated mice to 73.6 ± 7.55 s^"1 in ARC5690-treated mice. Both the number of adhered leukocytes and the leukocyte rolling flux decreased by >50% (Figure 26, right and left panels, respectively). All ARC5690-dependent changes in hemodynamic parameters were statistically significant, with p values noted in the pertinent figures.

[00298] P-selectin blockade using a specific anti-P-selectin aptamer significantly decreased adhesion of both red and white blood cells in a mouse model of sickle cell trait, and increased hemodynamic flow. These results suggest that anti-P-selectin therapy should prevent the adhesive events that lead to vasoocclusion in SCD-affected individuals. Example 10: Binding of P-selectin Specific Aptamer to Activated Platelets in Whole

Blood

[00299] The ability of ARC6337, which comprises ARC5685 modified at the 5 '-end with biotin instead of PEG, to bind to activated platelets was measured in human whole blood by flow cytometry. [00300] The time-dependence of ARC6337 binding was investigated first in PPACK- anticoagulated whole blood treated with TRAP. PPACK (D-phenylalanyl-L-pro IyI-L- arginine chloromethyl ketone) is a tripeptide, active-site inhibitor of thrombin, and TRAP (thrombin receptor activating peptide) is a peptide having the sequence SFLLRNP (SEQ ID NO: 7) that binds to and activates protease-activated receptor type 1 (PAR-I). In the experiment shown in Figure 27, whole blood was activated for 5 minutes with TRAP (20 μM) in the presence of eptifibatide (a heptapeptide that blocks the platelet glycoprotein Ilb/IIIa receptor to prevent platelet-platelet aggregation), then combined with 1000 nM ARC6337, with or without 10 mM EDTA. At selected times, samples were removed and fixed by the addition of formaldehyde (1% FC). The fixed samples were then stained for 15 minutes with the fluorescently-labeled streptavidin (PE-streptavidin) and CD41-PECy5 (as a platelet identifier). To confirm P-selectin expression on the platelet surface, TRAP activated blood was incubated with vehicle only (no ARC6337) and fixed at the indicated times, and then stained for 15 minutes with PE-conjugated P-selectin antibody and CD41-PECy5. Results (Figure 27, left-hand panel) show that addition of ARC6337 to the TRAP-activated platelets for as little as 1 minute, resulted in significant binding of PE-streptavidin to the platelets and that this binding was partially blocked by 10 mM EDTA. Binding of PE- streptavidin was minimal when ARC6337-biotin was omitted. Longer incubation of ARC6337-biotin with TRAP-activated blood resulted in a slight time-dependent increase in the amount of PE-streptavidin staining. This increase paralleled a slight increase in platelet surface expression of P-selectin over time (Figure 27, right-hand panel). These results suggest that binding of ARC6337-biotin to TRAP-activated platelets is very rapid, reaching steady state within seconds to only a few minutes. The residual binding of ARC6337 in the presence of EDTA most likely represents non-specific interactions with the target, as both the aptamer and the target P-selectin are thought to be non-functional in the absence of divalent cations (Jenison, RD, Jennings, SD, Walker, DW, Bargatze, RF and Parma, D (1998) Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion. Antisense & Nucleic Acid Drug Development 8, 265-279).

[00301] The binding of ARC6337 to platelets was next investigated to compare the effects of different anticoagulants and different activating methods. In Figure 28, the binding of ARC6337 at 0, 200 and 2000 nM to platelets was measured in blood containing citrate and no platelet agonist, citrate and 20 μM TRAP, citrate and 1 LVmL thrombin with GPRP, or PPACK with 20 μM TRAP. The tetratpeptide GPRP (SEQ ID NO: 8)) was used in experiments containing thrombin because it inhibits thrombin-induced fibrin clot formation and platelet aggregation, but not thrombin-induced platelet activation. As shown in Figure 28, ARC6337 at 200 nM or 2000 nM bound equivalently to platelets regardless of anticoagulant (citrate or PPACK) or platelet agonist (TRAP or thrombin). In the absence of agonist, no binding was observed, which is consistent with the lack of P-selectin expression on the platelet surface. Although citrate is a chelator of divalent metal ions, it is a weaker chelator than EDTA, so the binding activity of ARC6337 appears to be preserved. [00302] The affinity of ARC6337 was investigated in citrate-anticoagulated blood using thrombin as the agonist to activate platelet P-selectin expression. Varying concentrations of ARC6337 up to a maximum of 3 μM were added to whole blood in the presence or absence of 2 U/mL thrombin, and binding measured by flow cytometry using PE-streptavidin as the detection reagent. As shown in the left-hand panel of Figure 29, the fluorescence due to ARC6337 binding increased with aptamer concentration in the presence of activating thrombin. In the absence of thrombin, a small amount of background binding was observed only at high ARC6337 concentrations, suggesting that the majority of the binding in the presence of thrombin reflected interactions with P-selectin. In the right-hand panel, the fluorescence due to binding in the absence of thrombin was subtracted out, and the background-corrected data was fit to a single-site binding model with a K_D of 190 nM. In Figure 30, the same data was fit to a two-site binding model. The fit of the data to this model was superior to the single-site model, suggesting that ARC6337 may bind to two independent sites on P-selectin under the conditions of the assay, with a K_D for the first site (K_D1) of 24 nM and for the second site (K_D2) of 533 nM. This second site could reflect a specific binding site, or a non-specific mode of binding consistent with that observed in the presence of EDTA described above. Example 11: ARC5692 Inhibition of PSGL-I Binding to Activated Platelets in Whole

Blood

[00303] The ability of ARC6337 (biotinylated ARC5685) and ARC5692 to inhibit the binding of platelet P-selectin to its native ligand, PSGL-I, was investigated in whole blood using flow cytometry. A recombinant version of PSGL-I, which is normally a membrane protein, was used containing the PSGL extracellular domain fused to the Fc domain of human IgG (R&D Systems). The human recombinant PSGL-I fusion protein (rhuPSGLl-Ig) is expressed as a homodimer. The protein used in the following experiments was labeled with fluorescein isothiocyanate (FITC) for detection by flow cytometry. [00304] In Figures 31 and 32, respectively, increasing concentrations of ARC6337 and

ARC5692 (0-3 μM) were incubated with rhuPSGLl-Ig (20 μg/mL) in whole blood treated with 2 LVmL thrombin and GPRP (SEQ ID NO: 8). In the absence of thrombin activation, a low level of fluorescent labeling of platelets was observed, presumably due to some basal level of P-selectin independent rhuPSGLl-Ig binding. When thrombin was added to stimulate P-selectin expression, the fluorescent signal increased 4-5 fold due to interactions between platelet P-selectin and rhuPSGLl-Ig. Increasing concentrations of ARC6337 (Figure 31) or ARC5692 (Figure 32) inhibited the binding of rhuPSGLl-Ig. At the highest aptamer concentration (3 μM), the fluorescent signal due to rhuPSGLl-Ig binding to P- selectin dropped to nearly the basal level. In the case of ARC6337, the data may be fit to a standard inhibition curve with an IC₅₀ of 191 nM. The inhibitory activity of ARC5692 was only slightly weaker with an IC50 of 393 nM. Example 12: ARC5692 Inhibition of Human Leukocyte-Platelet Interactions in Whole

Blood

[00305] Leukocytes constitutively express the cognate receptor to P-selectin, P-Selectin Glycoprotein Ligand (PSGL-I). Leukocyte recruitment from the circulation to areas of inflammation involves endothelial contact and rolling, mediated largely by P-selectin, followed by firm adhesion, transendothelial migration and subendothelial migration. Upon activation by an agonist like TRAP or thrombin, platelets expressing P-selectin can bind to PSGL-I -expressing leukocytes, often with several platelets bound to a single leukocyte to form an aggregate. Although their significance is unclear, elevated levels of platelet- leukocyte aggregates have been observed in myocardial infarction, unstable angina, peripheral vascular disease, hypertension, stroke and diabetes. The following experiments describe the ability of ARC5692 to inhibit the formation of platelet-leukocyte aggregates. [00306] Experiments to measure the effects of ARC5692 on platelet-leukocyte aggregates were performed by flow cytometry in citrated whole blood. Blood samples spiked with increasing concentrations of ARC5692 (0-3 μM) were treated with thrombin and GPRP (SEQ ID NO: 8). During flow cytometry, monocytes and neutrophils were distinguished by their characteristic forward and side scattering properties. Platelet bound monocytes and neutrophils, in turn, were identified by staining with the platelet specific anti-CD41-PECy5. Increasing concentrations of ARC5692 inhibited the binding of activated platelets to both monocytes (Figure 33, top panels) and neutrophils (Figure 33, bottom panels). The left-hand panels in Figure 33 record the percentage of monocytes (top) or neutrophils (bottom) with one or more platelets bound. In the absence of ARC5692, -90% of monocytes and -80% of neutrophils were bound by platelets. At the highest concentration of ARC5692 tested, approximately 20% of monocytes still had platelets bound, but nearly all of the neutrophils were free of platelets. The right-hand panels in Figure 33 indicate platelet binding in terms of overall fluorescence, which takes into account the number of platelets bound to each leukocyte. The baseline fluorescence following activation of platelets with thrombin was approximately 3 -fold higher for monocytes than for neutrophils. The addition of ARC5692 up to 3 μM decreased the fluorescence nearly to zero for both monocytes and neutrophils, although approximately twice as much aptamer was required for monocytes than for neutrophils (Figure 33, right-hand panels). The data for all data-sets were fit with standard inhibition curves, yielding IC₅₀ and IC₉₀ values that are indicated in with their associated plots in Figure 33 and summarized in the table in Figure 35.

[00307] In a second set of experiments, the effects of ARC5692 on platelet-leukocyte aggregates were measured in blood treated with ADP instead of thrombin. ADP is a much weaker agonist for platelets than thrombin and stimulates less P-selectin expression. Under the conditions of this assay, -80% of monocytes and -30% of neutrophils were bound by one or more platelets (Figure 34, left-hand panels). As observed for thrombin activation, increasing concentrations of ARC5692 inhibited platelet-leukocyte aggregate formation in the presence of ADP; however, nearly complete inhibition was observed for both monocytes and neutrophils in these assays, consistent with the lower levels of P-selectin expression. The right-hand panels, again, show aggregate formation quantified in terms of overall fluorescence, which was lower for both monocytes and neutrophils in blood treated with ADP than in blood treated with thrombin. As previously observed, ARC5692 inhibited aggregation essentially to completion by this measure. IC₅₀ and IC₉₀ values derived from inhibition curves were indicated with the associated plots in Figure 34. All were lower by 2- 5 fold than the equivalent values calculated for curves derived in the presence of thrombin. Again, this presumably reflects the lower P-selectin expression induced by ADP versus thrombin.

[00308] IC50 and IC90 values determined for ARC5692 inhibition of platelet-monocyte and platelet-neutrophil aggregate formation are summarized in the table in Figure 35, along with measures of binding to platelets and inhibition of rhuPSGL-1-Ig binding.

Example 13: ARC5692 Inhibition of Human Platelet-Platelet Interactions in Whole

Blood

[00309] Upon treatment with thrombin, a small number of platelets can form platelet- platelet aggregates in citrated blood, even in the presence of eptifibatide and GPRP (SEQ ID NO: 8). These aggregates may be detected by the scattering properties of platelets by flow cytometry (Figure 36). Treatment with high concentrations of ARC5692 blocked the formation of these aggregates (IC₅₀ = 861 nM). When assessed by a standard platelet- aggregation assay in hirudin-anticoagulated whole blood using ADP as an agonist, partial blockade of platelet aggregation was observed with ARC5692 with an IC50 of 40-50 nM (Figure 37). This observation may be recapitulated in hirudin-anticoagulated whole blood treated with TRAP or citrated whole blood treated with ADP or collagen (Figure 38).

Example 14: Pharmacokinetic Analysis of ARC5692 in Mice

[00310] The pharmacokinetics of ARC5692 were evaluated in CD-I mice. The study design, mean concentration-time profile and PK parameters are all summarized in Figure 7. Each animal was dosed separately via a lateral tail vein injection with 20 mg/kg of ARC5692. Blood samples were obtained by terminal cardiac puncture at pre-dose, and at 0.083, 0.5, 1, 4, 8, 16, 24, 32, 48, 72, 96, 120 and 144 hours post-dose. Blood samples (-500 μL) were transferred into dipotassium (K2) EDTA tubes, placed on wet ice, and centrifuged within 30 minutes of collection at approximately 37⁰C. The plasma was transferred and stored frozen at -8O⁰C prior to analysis for ARC5692 concentration.

[00311] The concentrations of ARC5692 in mouse plasma were determined by a validated HPLC method with UV detection by Archemix Corp. Mean plasma concentration data were used in a model-independent PK analysis using WinNonlin, version 5.1 (Pharsight Corporation, Mountainview, CA). Following intravenous administration in mice, the mean Cmax value was 424.8 μg/mL and occurred at 5 minutes post-dose (the first sampling time). The AUCo-iast and AUCo-∞ values were 2073.5 and 2074.4 μg-hr/mL, respectively. The tYi, CL, MRTo-oo and Vss values for ARC5692 were 2.72 hour, 9.64 mL/hr/kg, 5.14 hour and 49.58 mL/kg, respectively.

Example 15: Pharmacokinetic Analysis of ARC5692 in Rats

[00312] Six male Sprague-Dawley rats were assigned to two dosing groups receiving intravenous bolus or subcutaneous doses of 20 mg/kg of ARC5692. At the time of dosing and throughout sample collection, each animal was observed for any clinically relevant abnormalities and none were noted. Blood samples (approximately 0.3 mL) were collected from the jugular vein at specified time points (pre-dose and 0.083, 0.25, 0.5, 1, 4, 8, 10, 24, 48, 72, 96, 120 and 144 hours post-dose. Collected blood samples were placed into K2EDTA-coated tubes and kept on wet ice until centrifuged at 2-5⁰C within 1 hour of collection to obtain plasma samples.

[00313] Plasma samples were stored frozen at -8O⁰C prior to analysis for ARC5692 concentration by a validated high performance liquid chromatography (HPLC) assay. All ARC5692 concentrations reported are based on oligonucleotides mass, excluding the mass of PEG. Individual plasma concentration-time data were used in a model-independent PK analysis using WinNonlin, version 5.1 (Pharsight Corporation, Mountainview, CA). Mean concentration-time profiles and PK parameters are shown in Figure 8. The mean Cmax value following IV administration was 653.18 μg/mL and occurred at 15 minutes post-dose. The AUCo-kst and AUCo-∞ values were 3300 and 3306 μg-hr/mL, respectively. The tV≥, CL, MRTo-oo and Vss values for ARC5692 were 2.62 hour, 6.05 mL/hr/kg, 4.20 hour and 25.66 mL/kg, respectively. The mean Cmax value following subcutaneous administration was 6.08 μg/mL and occurred at 25 hours post-dose. The AUCo-kst and MRTo_∞ values were 180 μg-hr/mL and 24.39 hr, respectively.

Example 16: Pharmacokinetic Analysis of ARC 5692 in Cynomolgus Macaques [00314] Three male cynomolgus monkeys were administered 20 mg/kg of ARC5692 by a single bolus intravenous (IV) infusion, followed after a washout period by 20 mg/kg subcutaneous administration. The dosage of ARC5692 was based on the oligonucleotide mass, excluding the mass of the PEG moiety. Blood (~1 mL) samples were obtained from a peripheral vein at pre-dose, and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120 and 144 hours post-dose and were placed into a dipotassium (K2) EDTA tube. Blood samples were placed immediately on wet ice and centrifuged within 30 minutes of collection at approximately 4°C. The plasma samples obtained were frozen at -80° C prior to bioanalysis to quantify ARC5692 concentration.

[00315] Concentrations of ARC5692 in cynomolgus monkey plasma were determined by high performance liquid chromatography (HPLC) method with UV detection. Individual and mean plasma concentration-time profiles are shown in Figure 9. Pharmacokinetic analyses were performed on concentration-time values using WinNonlin version 5.1 (Pharsight Corp., Mountainview, CA). The PK parameter estimates for ARC5692 are shown in tabular form in Figure 9. The mean Cmax value of ARC5692 following IV administration was 694 μg/mL and occurred at a mean Tmax of 0.14 hours post-dose. The AUCo-iast and AUCo-∞ values were 4480 and 4711 μg-hr/mL, respectively. The tV≥, CL, MRTo_∞ and Vss values for ARC5692 were 38.78 hour, 4.32 mL/hr/kg, 7.22 hour and 32.89 mL/kg, respectively. The mean Cmax value following subcutaneous administration was 46 μg/mL and occurred at 32.00 hours post-dose. The AUCo-kst and AUCo-∞ values were 2683 and 2688 μg-hr/mL, respectively. The tV≥, CL, and MRT₀__∞ values were 17.50 hour, 7.66 mL/hr/kg and 46.98 hour, respectively. The subcutaneous bioavailability (Fsc) was calculated to be 60%. Example 17: Estimation of Human Dose by Allometric Scaling

[00316] Allometric scaling was conducted to predict human PK parameter estimates for ARC5692 for clearance (CL), volume of distribution at steady state (Vss) and mean residence time (MRT). The PK parameter estimates CL, Vss and MRT from IV bolus PK studies in CD-I mice, Sprague-Dawley rats, and cynomolgus monkeys were subjected to allometric scaling using the allometric equation (Y = a W b or log Y= b logW+log a) and used to predict the PK parameter estimates for humans. The allometric exponent "b" values were then used to extrapolate to the human equivalent dose (HED) comparable to a 20 mg/kg dose in monkeys using the equation, Dh=Da (Wh/Wa) b (where D and W represent dose and weight, respectively). Allometric scaling of CL, Vss and MRT of ARC5692 across species produced the allometric exponent "b" values for CL, Vss and MRT values of 0.8, 1.0 and 0.22, respectively. These allometric exponent "b" values for CL and Vss fell within or very near the typical ranges of 0.6-0.9, 0.8-1.0 and 0.2-0.25 respectively. The predicted ARC5692 human (70 kg) values for CL, Vss and tl/2β by allometric scaling are -188 mL/hr, -2810 mL and 14 hours, respectively. Predicted values are summarized in Figure 22. Example 18: Mouse Model of Deep Vein Thrombosis

[00317] By blocking the interactions between P-selectin and its ligand PSGL-I with either anti P-selectin antibodies, soluble recombinant PSGL-I ligands, or P-selectin small molecule antagonists, multiple animal model studies have demonstrated decreased thrombus formation, thus elucidating the importance of P-selectin and inflammation in thrombosis. The goal of this study was to evaluate the prophylactic effects of the anti-mouse P-selectin aptamer ARC5690 and its ability to alter thrombosis formation in a murine in-vivo model of primary venous stasis. Reagents

[00318] Anti-mouse P-selectin aptamer ARC5690 (PEG40K-nh-fC-fU-fC-mG-fC-mA- mG-mA-fC-mA-mA-fC-fC-mG-mG-mA-fU-mG-mA-mA-mA-fU-fC-fC-mG-mA-fC-fC- mG-mG-mA-mG-idT) (SEQ ID NO: 5) and "scrambled" anti-P-selectin control aptamer ARC5694 (PEG40K-nh-fC-fU-fC-fC-mA-mG-mA-fC-mA-fC-mA-mG-fC-mG-mG-mA-fU- mG-mA-mA-mA-fU-fC-fC-mG-mG-fC-fC-mA-mG-mA-mG-idT) (SEQ ID NO: 6) used in these experiments was synthesized and purified by Archemix Corp. Anti-mouse P-selectin antibody RB40.34 (CD62P) (BD Pharmingen, San Jose CA) is a parenterally administered systemic inhibitor of P-selectin. The following antibodies were used for flow cytometry: rat anti-murine phycoerythrin (red; MAC-I) (Millipore and BD Biosciences) and rat anti-murine fluorescein isothiocyanate green (CD41) (BD Pharmingen), antibody binding beads (anti-rat) from (BD Pharmingen), and SPHERO Rainbow calibration 3μm beads. Chemicals/Surgical Methods

[00319] The following chemicals were used: isoflurane, oxygen, nosecone, 7-0 Prolene (Ethicon, Inc, Somerville, NJ.), 5-0 Vicryl (Ethicon, Inc, Somerville, NJ), Nexaband surgical glue (Veterinary Product Laboratories, Phoenix, AZ, ), EDTA Vacutainers (Becton Dickinson), 10% acid citrate dextrose, hematoxylin, eosin, and paraffin. Animals

[00320] Six to 10 week old male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) weighing 18-25g were utilized in this study. All work was approved by the University of Michigan, University Committee on Use and Care of Animals and was performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Inferior Vena Cava (IVC) Ligation Model

[00321] Inferior vena cava ligation was used to stimulate thrombosis in mice. Mice were anesthetized via inhalation of 1-2% isoflurane with 100% oxygen as a carrier gas delivered by a tight fitting nosecone. A midline laparotomy was performed with retraction and exteriorization of the intestinal contents to expose the inferior vena cava (IVC). Blunt dissection was performed to locate dorsal branches which were cauterized and occasional side branches which were ligated using 7-0 Prolene (Ethicon, Inc, Somerville, NJ). The IVC was ligated just distal to the renal veins with 7-0 Prolene. The abdominal musculature was closed with 5-0 Vicryl (Ethicon, Inc, Somerville, NJ) using a simple continuous pattern. The skin was closed using Nexaband surgical glue (Veterinary Product Laboratories, Phoenix, AZ). Mice were evaluated daily until harvesting 72h post-IVC ligation. At harvest, mice were anesthetized with isoflurane as previously described and blood was collected by direct cardiac puncture, placed into EDTA Vacutainers (Becton Dickinson) or syringes filled with 10% acid citrate dextrose, depending upon the assay.

[00322] Study compounds (P-selectin control aptamer ARC5694, anti-P-selectin aptamer ARC5690, and anti-P-selectin antibody) were administered 48 hours pre-IVC ligation. Additional doses of ARC5690 and ARC5694 were administered 24 hours pre-IVC ligation, concomitantly with IVC ligation and 24, 48 and 68 hours post-IVC ligation. Euthanasia, blood collection, and IVC harvesting were performed 72h post-IVC ligation. Histology, thrombus mass, soluble P-selectin, and microparticles analysis were performed on collected tissues. Study Groups and Design

[00323] Mice were assigned to one of the following four groups: [00324] 1) Psel-conapt: P-selectin control aptamer "scrambled" ARC5694. (n=15) lmg/kg IP at 48h pre-IVC ligation, 24h pre-IVC ligation, immediately post-IVC ligation, 24h post-ligation, and 48h post-ligation. Total volume <250μl;

[00325] 2) Psel-apt: P-selectin aptamer ARC5690. (n=21) 2mg/kg IP at 48h pre-IVC ligation, 24h pre-IVC ligation, immediately post-IVC ligation, 24h post-ligation, 48h post- ligation, and 68h or 4h prior to harvesting. Total volume <250ul. There were more mice in this group due to the addition of data from the pilot study;

[00326] 3) Psel-Ab: anti-P-selectin aptamer. (n=15) 0.2 mg IP at 48h pre-IVC ligation. Total volume = 200 μl; and

[00327] 4) NL: Non-ligated or no thrombosis control. (n=10) No compounds were administered to mice in this group. Mice were euthanized for IVC harvesting and blood collection.

[00328] Depending upon their designated group, mice were dosed either with P-selectin aptamer, P-selectin control aptamer, or anti-P-selectin antibody. IVC ligations were performed on all mice except those mice in the non-ligated control group. At 72 h post-IVC ligation, mice were euthanized via exsanguination while anesthetized with inhalation of isoflurane 1-2% in a mixture of 100% oxygen. The IVC of each mouse was harvested, weighed, and measured for all groups excluding those mice utilized for histology where the descending abdominal aorta was preserved. IVCs for morphometric analysis were prepared using standard methods for tissue fixation with paraffin-embedded sections. Blood was collected via cardiac puncture for plasma soluble P-selectin by ELISA and plasma leukocyte/platelet microparticles (MPs) via double-stained fluorescence-activated cell scanning (FACS) analysis. Methods of Analysis

[00329] Thrombus Weight. This technique was used as an indirect measure of thrombus content. At sacrifice, the IVC was removed, weighed, and measured for length. The major component of weight is the thrombus as opposed to the vein wall tissue itself and in comparing one animal to another or one group to another, the vein wall contribution to thrombus weight cancels out. A 0.3 cm section was removed for analysis from mice in the Psel-Ab group that did not develop a clot or show visual evidence of thrombosis. A 0.5 cm section of IVC was removed for each mouse in the NL group. [00330] Vein Wall Morphometries . In a blinded fashion, veins were examined under high- power oil immersion light microscopy by a board-certified pathologist. Sections were stained with hematoxylin and eosin from paraffin-embedded tissues. Five representative high-power fields (HPFs, oil immersion XlOOO) were examined around the vein wall and the cell counts of the vein wall were analyzed. Cells were identified as neutrophils, monocytes/macrophages, or lymphocytes based on standard histologic criteria. Results from the five high-power fields were added together and the mean SE calculated for each group. [00331] Morphometric Scoring. Sections were scored for thrombus surface organization, intimal thickness and intimal fibrosis according to the following criteria:

Thrombus Surface Organization Scoring Criteria

1 : A few scattered fibroblasts and/or white blood cells are present in superficial thrombus.

2: There are at least 5 fibroblasts per high power field (HPF) which are aligned parallel to the surface of the thrombus.

3. At least two layers of fibroblasts are aligned parallel to the surface of the thrombus, usually accompanied by clumps of white blood cells.

4: At least four layers of fibroblasts are aligned parallel to the surface of the thrombus with or without collagen formation, or the thrombus has recanalized close to the surface. This stage is usually accompanied by some early evidence of recanalization of blood flow.

5 : Highly organized thrombus that has altered the lumen. Original endothelial surface is either hard or impossible (therefore no counts can be made) to identify due to the adherent thrombus which is highly organized with recanalization, fibrosis, mineralization, necrosis, etc. at that site.

Intimal Thickness Scoring Criteria

0: Intima appears as just a potential space with space occupied only by endothelial cells.

1 : Very small spaces present between endothelial cells and the internal elastic lamina.

Intima still appears generally as thick as the nuclei of normal spindle-shaped endothelial cells.

2: Intima is at least twice as thick as an endothelial nucleus (about same as a red blood cell diameter) at its widest point in the HPF. 3. Intima is at least 5 times the thickness of a red blood cell diameter at its widest point in the HPF. Intimal thickness tends to be highly variable and may contain cells other than endothelial cells.

4. Intima is greatly thickened and contains either fibroblasts, white blood cells and/or hemorrhage at its widest point.

Intimal Fibrosis Scoring Criteria

0: No fibrosis evident.

1 : Intima contains a small amount of dense eosinophilic or amphiphilic material.

Fibroblasts may or may not be evident.

2: Intima contains fibroblasts and some small dense bundle of eosinophilic or amphiphilic collagenous connective tissue.

3. Intima contains numerous fibroblasts and is irregularly thickened by large amounts of collagenous connective tissue. This is usually accompanied by white blood cells and/or red blood cells.

[00332] Soluble P-selectin. Blood samples were collected (300 μl) in 0.1 μl of EDTA at time of harvest (72 h post IVC ligation) and centrifuged for 20 minutes at 200Og. Circulating P-selectin plasma levels were measured with a mouse soluble P-selectin ELISA. Data was reported as ng/mg total protein. Results

[00333] Thrombus Weight. The Psel-Ab group had significantly smaller venous thrombi versus the Psel-conapt group (0.0194 ± 0.004 vs. 0.0370 ± 0.002 grams, P<0.005, mean weight ± SEM ) (Figure 39 and Figure 40).

[00334] The Psel-apt group had significantly smaller venous thrombi versus the Psel- conapt group (0.0290 ± 0.003 vs. 0.0370 ± 0.002 grams, P<0.030, mean weight ± SEM) (Figure 39 and Figure 40).

[00335] There were no significant differences between the Psel-Ab compared to the Psel- apt group (0.0194 ± 0.004 vs. 0.0290 ± 0.003 grams, P>0.1, mean weight ± SEM) (Figure 39 and Figure 40).

[00336] Vein Wall Morphometries . As expected the NL groups showed minimal inflammatory infiltration due to the absence of thrombosis in this group. There were no significant differences with respect to white blood cell types and total cell counts when Psel- conapt, Psel-apt, and Psel Ab were compared to each other (Figure 41). There was a tendency of neutrophil cell populations to decrease in the order of P-selectin control aptamer, P-selectin aptamer, and anti-P-selectin antibody (Figure 41).

[00337] Morphometric Scoring. The NL group was not included in the statistical analysis since no thrombosis was evident in each sample within the NL group. There were no significant differences between Psel-conapt, Psel-apt, and Psel-Ab groups with respect to scoring parameters (Figure 42).

[00338] Soluble P-selectin. The mean ± SEM soluble P-selectin level of the Psel-Ab group is significantly smaller versus the Psel-conapt and the Psel-apt group (134 ± 21.8 vs. 344.4 ± 22.3 and 272 ± 19.9 ng/mg, P<0.0001 and P<0.001 respectively) (Figure 43). [00339] The mean ± SEM soluble P-selectin level of the Psel-apt group is significantly smaller versus the Psel-conapt (272 ± 19.9 vs. 344.4 ± 22.3 ng/mg, P<0.05) (Figure 43). [00340] The mean ± SEM soluble P-selectin level of the NL group is significantly smaller than the Psel-conapt group (130.6 ± 66.9 vs. 344.4 ± 22.3 ng/mg, P<0.05) (Figure 43). [00341] The graph shows a correlation between thrombus weight and soluble P-selectin. (r=0.75, Pearson correlation coefficient, p<0.001) with a regression r²=0.56. (Figure 44). [00342] Conclusion. Dose escalation studies performed with the P selectin aptamer may show a correlation between higher P-selectin aptamer doses and decreases in vein wall neutrophil counts. On the contrary, there was a tendency for monocyte populations to increase in the order of P-selectin control, P-selectin aptamer, and anti-P-selectin antibody. [00343] The P-selectin aptamer and anti-P-selectin antibody groups showed significant decreases in thrombus weights, showed a decreasing trend for neutrophil extravasation, but showed an increase in monocyte influx which has been shown to play a role in thrombus resolution.

[00344] Significant differences were not expected to be present between groups for morphometric scoring with respect to thrombosis surface organization, intimal thickness, and intimal fibrosis within a relatively short time span of 72h post-IVC ligation. [00345] The correlation between thrombus weight and soluble P-selectin provides increased evidence for the use of soluble P-selectin as a clinical bio-marker of venous thrombosis. The Psel-apt and Psel-Ab groups trended towards decreasing plasma levels of soluble P-selectin.

[00346] Clots within the IVCs of some mice in the Psel-apt and Psel-Ab groups appeared to move freely within the vein compared the Psel-conapt mice. Multiple Psel-conapt had clots extending from the IVC ligation to iliac bifurcation. There were no gross bleeding abnormalities visualized. [00347] Those of skill in the art will recognize that the invention, having now been described by way of written description and example, can be practiced in a variety of embodiments and that the description and examples above are for purposes of illustration and not for limitation of the following claims.

Claims

What is claimed is:

1) An aptamer that binds to P-selectin, wherein the aptamer comprises the nucleic acid sequence of SEQ ID NO: 2.

2) The aptamer of claim 1 , wherein the P-selectin is human P-selectin.

3) The aptamer of claim 1, wherein the aptamer has a dissociation constant for human P- selectin of 100 nM or less.

4) The aptamer of claim 1 , wherein the aptamer further comprises at least one chemical modification.

5) The aptamer of claim 4, wherein the modification is selected from the group consisting of: a chemical substitution at a sugar position, a chemical substitution at a phosphate position, and a chemical substitution at a base position.

6) The aptamer of claim 5, wherein the modification is selected from the group consisting of: incorporation of a modified nucleotide; a 3' cap; a 5' cap; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; incorporation of a CpG motif; and incorporation of a phosphorothioate or phosphorodithioate into the phosphate backbone.

7) The aptamer of claim 6, wherein the high molecular weight, non-immunogenic, compound is polyethylene glycol.

8) The aptamer of claim 6, wherein the 3' cap is an inverted deoxythymidine cap.

9) The aptamer of claim 1 comprising the nucleic acid sequence set forth below: fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC-mG- fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 2), wherein "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide and

"mN" is a 2'-0 Methyl modified nucleotide. 10) The aptamer of claim 6 comprising the nucleic acid sequence set forth below: NH2-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG-mA-fC- mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 3), wherein "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide, "mN" is a 2'-0 Methyl modified nucleotide and "NH₂" is an amine group.

11) The aptamer of claim 7 comprising the nucleic acid sequence set forth below: PEG40K-nh-fC-fU-fC-rA-rA-fC-mG-mA-mG-fC-fC-rA-mG-mG-mA-rA-fC-mA-fU-fC-mG- mA-fC-mG-fU-fC-mA-mG-fC-rA-mA-rA-fC-rG-fC-rG-rA-rG-idT (SEQ ID NO: 4), wherein "idT" is an inverted deoxythymidine, "rN" is a ribonucleotide, "fN" is a 2'-fluorinated nucleotide, "mN" is a 2'-0 Methyl modified nucleotide, "nh" is an amine linker and "PEG40K" is a 40 kDa PEG moiety.

12) A pharmaceutical composition comprising a therapeutically effective amount of the aptamer of claim 11 or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

13) A method for treating, preventing or ameliorating a disease or disorder mediated by P- selectin comprising administering to a subject the composition of claim 12.

14) The method of claim 13, wherein the subject is a mammal.

15) The method of claim 14, wherein the mammal is a human.

16) The method of claim 13, wherein the disease or disorder is selected from the group consisting of: a coagulation or thrombotic disease or disorder, an inflammatory disease or disorder, and a metastatic disease or disorder.

17) The method of claim 16, wherein the disease or disorder is selected from the group consisting of: sickle cell disease, sickle cell disease sequelae, pain, acute chest syndrome, vasoocclusive crisis, acute vasoocclusive syndrome, acute non-occlusive syndrome, chronic syndrome, vascular inflammation, hypoxia of tissues, vasoocclusion of organs and tissues, organ failure, thrombogenesis, cerebrovascular accident, dactylitis, priapism, hemolytic anemia, aplastic crisis, pulmonary hypertension, retinopathy, osteonecrosis and skin ulcers, sickle cell anemia, vascular diseases, cardiovascular diseases, thrombotic diseases, hemostasis diseases, myocardial infarction, stroke, transient ischemic attack, revascularization, stent restenosis, atherosclerosis, deep vein thrombosis, venous thromboembolism, hypereosinophilia, ischemia/reperfusion injury, inflammatory diseases, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, juvenile idiopathic arthritis, organ transplant, graft rejection, ocular inflammation, retinal inflammation, colitis, conjunctivitis, scleritis, tumor metastasis, renal failure, epilepsy, malaria, cerebral malaria, asthma, psoriasis, allergic diseases, allergic conjunctivitis, immune diseases, shock and hemorrhagic shock.

18) The method of claim 13, wherein the composition is administered prior to, during and/or after a medical procedure.

19) The method of claim 13, wherein the composition is administered in combination with another drug.

20) The method of claim 13, wherein the composition is administered in combination with another therapy.