Attorney Docket: 0245.90/02PCT

INHIBITION OF THE ACTIVATION OF COAGULATION FACTOR XII BY LIGANDS OF PHOSPHATIDLYSERINE

TECHNICAL FIELD

[0001] The invention provides compositions and methods for treatment in medicine and surgery.

BACKGROUND

[0002] Blood coagulation factor XII (FXII) (Hageman factor) is an 80-kD glycoprotein synthesized by the liver and circulated in plasma as an inactive serine protease zymogen. FXII readily binds to and is autoactivated by anionic surfaces such as silicates, dextran sulfate, sulfatides, kaolin, glass, ellagic acid, articular cartilage, skin, fatty acids, endotoxin, amyloid protein, and heparins. There are two pathways for FXII activation: autoactivation upon exposure to negatively charged surfaces and proteolytic activation on cell membranes. Autoactivation results in a change in shape of FXII. Once activated, FXII is converted to enzyme Factor Xlla (a-FXIIa), a serine protease that activates FXI, prekallikrein (PK), and CI esterase (a subunit of the complement cascade). The consequence of this activity is the initiation of the intrinsic blood clotting cascade and the production of kinins. Activation of PK by a-FXIIa forms plasma kallikrein, which reciprocally activates more FXII and liberates bradykinin and other kinins from high molecular weight kininogen (HK). Kinins are mediators of increased vascular permeability. More a-FXIIa is cleaved by plasma kallikrein forming β-FXIIa which then activates the CI complement complex of the classic complement system. Plasma kallikrein also directly activates C3 and C5 of the complement cascade.

[0003] FXII activation is involved in a variety of diseases and conditions. Therefore, there is a need in the art to provide new methods and compositions for inhibiting activation of FXII.

[0004] Provided herein are methods and compositions directed toward overcoming one or more of the problems discussed above.

SUMMARY OF THE EMBODIMENTS

[0005] Provided herein are compositions and methods of treatment in medicine and in surgery. Attorney Docket: 0245.90/02PCT

[0006] The present invention provides a method of treating a disease or condition associated with FXII activation, the method comprising administering to a patient in need thereof a phosphatidylserine (PS) binding agent.

[0007] In some aspects, embodiments herein include methods for treating acute angioedema attacks, pain, sickle cell crisis, acute renal failure, and vasculitis.

[0008] In other aspects, embodiments herein include methods for treating systemic inflammatory response syndrome, disseminated intravascular coagulation, hypotension, septic shock, cardiogenic shock, hypovolemic shock, obstructive shock, neurogenic shock, and anaphylactic shock.

[0009] Aspects of the present invention also provide a method of treating a disease or condition associated with FXII activation, the method comprising administering to a patient in need thereof a PS binding agent along with an antibiotic or an antiviral agent.

[0010] Aspects of the present invention also provide a method of treating a disease or condition associated with FXII activation, the method comprising administering to a patient in need thereof an annexin.

[0011] In other embodiments, compositions are provided that are useful in treatment of the FXII related diseases or conditions.

[0012] These and various features and advantages of the invention will be apparent from a reading of the following detailed description and a review of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 demonstrates that treatment with Diannexin prolonged clot initiation time following exposure to Kaolin.

[0014] Figure 2 demonstrates that treatment with Diannexin increased time to reach a specific clot strength following exposure to Kaolin.

[0015] Figure 3 demonstrates that treatment with Diannexin attenuated the maximum rate of thrombin generation following exposure to Kaolin.

[0016] Figure 4 demonstrates that treatment with Diannexin prolonged the time to reach the maximum rate of thrombin generation after exposure to Kaolin.

[0017] Figure 5 demonstrates that Diannexin decreases edema in the mouse brain 36 hours after commencing post-ischemic reperfusion. Extension of brain damage during reperfusion is also reduced. Attorney Docket: 0245.90/02PCT

[0018] The Figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

DETAILED DESCRIPTION

[0019] Representative embodiments are provided below. While the invention will be described in conjunction with such embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the disclosure and any appended claims.

[0020] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein can be used in and are within the scope of the practice of the present disclosure.

[0021] Unless described otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0022] As used herein, the singular forms "a", "an", and "the" include plural references, unless the content clearly dictates otherwise, and are used interchangeably with "at least one" and "one or more".

[0023] As used herein, the term "about" represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.

[0024] As used herein, the terms "comprises", "comprising", "includes", "including",

"contains", "containing", and any variations thereof, are intended to cover a non-exclusive inclusion, such that a system, device, process, method, etc. that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such system, composition, process, method, etc.

Phosphatidylserine (PS) and Factor XII

[0025] It has been determined herein that the negatively charged aminophospholipid phosphatidylserine (PS), which is translocated to the outer leaflet of the plasma membrane Attorney Docket: 0245.90/02PCT bilayer in activated and anoxic cells and in microparticles shed from those cells, is an activator of FXII. Surprisingly, this relationship provides a superior target for mitigating or treating a plethora of disease states associated with activation of FXII. Targeting FXII inhibition at the level of PS translocation has beneficial therapeutic effects additional to those achieved by targeting FXII directly, using an enzyme inhibitor or antibody. For example, binding PS on cell surfaces inhibits the docking and activity of sPLA2, thereby decreasing the production of proinflammatory and procoagulant lipid mediators (Kuypers et al. Thromb Hemost. 2007; 97:478). Additionally, masking PS suppresses the recruitment of leukocytes and platelets into sites of post-ischemic reperfusion (Teoh et al. Gastroenterology 2007; 133: 632).

[0026] PS is normally confined to the inner leaflet of the plasma membrane bilayer of healthy cells. This asymmetry is maintained by the action of an ATP-dependent

aminophospholipd transporter (flipase) which transports PS from the outer to the inner leaflet of the bilayer (Soupene and Kuypers, Identification of an erythroid ATP-dependent

aminophospholipid transporter. Brit J Hematol 2006; 135:436-438). As a result of blood platelet activation, PS is translocated to the cell surface (Thiagarajan and Tait, Binding of

annexin V/placental anticoagulant protein I to platelets. J Biol Chem 1990; 265: 17420-17423). Likewise, under anoxic conditions following, for example, thrombosis or organ storage before transplantation, ATP is depleted, the flipase cannot function, and PS is translocated to cell surfaces. This is demonstrable in cultures of vascular endothelial cells (ECs) (Ran S et al.

Increased exposure of anionic phospholipids on the surface of tumor blood vessels. Cancer Res 2002; 62: 6132-6140). Anoxia in the human forearm produced by a tourniquet accompanied by muscular exercise results in PS exposure on vascular ECs as measured by local accumulation of annexin V (Rongen GA et al. Annexin A5 scintigraphy of forearm as a novel in vivo model of ischemic preconditioning in humans. Circulation 2005; 111: 173-178). PS extemalization of this type is reversible upon reoxygenation in contrast to PS eversion accompanying apoptosis.

Ischemia followed by reperfusion results in the attachment of leukocytes and platelet aggregates to ECs and obstruction of microvascular blood flow in mouse liver (Teoh NC et al., Diannexin, a novel annexin V homodimer, provides prolonged protection against hepatic ischemia- reperfusion injury in mice. Gastroenterology 2007; 133: 632-646). These authors showed that Diannexin, a ligand of PS, suppresses the attachment of leukocytes and platelets to endothelial cells and preserves microvascular blood flow. Attorney Docket: 0245.90/02PCT

[0027] PS eversion has several consequences. First, serine proteases of the

prothrombinase complex use PS on the cell surface as a docking and activation site leading to thrombosis (Kuypers FA et al. Interaction of an annexin V homodimer (Diannexin) with phosphatidylserine on cell surfaces and consequent antithrombotic activity. Thromb Hemost 2007; 97:478-486). Secondly, secreted isoforms of phospholipase A ₂ (sPLA ₂ ) also use PS on cell surfaces as a docking site (Lambeau G, Gelb MH. Biochemistry and physiology of mammalian secreted phospholipase A ₂ . Annu Rev Biochem 2008; 77:495-520). Such enzymatic activity generates lipid products that are metabolized into prothrombotic and proinflammatory mediators such as prostaglandins, leukotrienes, lysophospholipids, platelet activating factor, and others.

[0028] FXII is activated by negatively charged molecules including PS translocated to the surface of cells. Activation of FXII can result in initiation of the blood clotting cascade, the production of kinins, and complement activation.

[0029] Humans deficient in FXII do not bleed excessively, and this is also true of mice in which FXII has been genetically inactivated (Renne et al. Defective thrombus formation in mice lacking coagulation FXII. J Exp Med 2005; 202:271-281). However, the formation and stabilization of platelet-rich occlusive thrombi was significantly reduced following collagen and epinephrine injection in FXII-deficient mice. Further, brain reperfusion injury following middle cerebral artery occlusion was attenuated without excessive bleeding in FXII-deficient mice (Kleinschnintz et al. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering in hemostasis. J Exp Med 2006; 203:513- 518). Inhibiting FXII activation therefore has the potential to prevent thrombosis without increasing hemorrhage, a desirable property. As mentioned by Nieswandt and Renne (U.S.

Publication No. 20080254039), this can be achieved by inhibitors of FXII serine esterase activity or antibodies against FXII.

[0030] Thus, provided herein are compositions and methods of treatment in medicine and in surgery.

Inhibition of FXII to Treat FXII related Diseases and Conditions.

[0031] An alternative strategy for suppressing FXII activation, and thereby exerting anticoagulant activity without increasing hemorrhage, is provided herein. Inhibition can occur upstream of FXII activation or can occur at the stage where FXII is autoactivated, cleaved, or Attorney Docket: 0245.90/02PCT changed in shape. In some embodiments, inhibition occurs upstream by blocking negatively charged PS on the surface of activated cells and microparticles shed from such cells to prevent exposure of FXII to PS and consequent FXII activation. Blocking PS on cell and microparticle surfaces has additional desirable effects, for example preventing activation of sPLA ₂ , thereby suppressing the production of proinflammatory and procoagulant lipid mediators.

[0032] As used herein, the phrase "a disease or condition associated with FXII activation" and other similarly worded phrases are understood to include any disease or condition in which activated FXII is implicated, regardless of the pathway to activation. The inventors determined that, surprisingly, inhibition of FXII is a key linking all these diseases.

[0033] Inhibition of FXII activation can be used to treat acute angioedema attacks and to prevent/treat systemic inflammatory response syndrome (SIRS) which plays a major role in the pathogenesis of septic, traumatic, or hemorrhagic shock.

[0034] Thus, in some embodiments, a disease or condition associated with FXII activation can be treated by administering to a patient in need thereof a PS binding agent.

[0035] In other embodiments, a disease or condition associated with FXII activation can be treated by administering to a patient in need thereof a PS binding agent in combination with an antibiotic or an antiviral agent.

[0036] In still other embodiments, a disease or condition associated with FXII activation can be treated by administering to a patient in need thereof an annexin.

[0037] In further embodiments, a method of inhibiting FXII activation comprises administering to a patient in need thereof another PS binding agent.

SIRS, DIC, Hypotension, and Shock

[0038] In some embodiments, compositions and methods are provided for prevention and/or treatment of the systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulation (DIC), hypotension, and other pathogenic mechanisms involved in septic, traumatic, or hemorrhagic shock.

Systemic Inflammatory Response Syndrome (SIRS)

[0039] SIRS is the clinical response to a nonspecific insult of either infection or noninfectious origin and can be caused by ischemia, inflammation, trauma, infection, or a combination of several insults. Bacteremia refers to the presence of bacteria within the blood stream; however bacteremia does not always lead to SIRS or sepsis. Sepsis is the systemic Attorney Docket: 0245.90/02PCT response to infection and is defined as the presence of SIRS in addition to infection. Severe sepsis is further associated with organ dysfunction, hypoperfusion, or hypotension. Septic shock refers to persistent hypotension and perfusion abnormalities despite adequate fluid resuscitation. Multi-organ dysfunction syndrome (MODS) is a state of physiological derangements in which organ function is not capable of maintaining homeostasis.

[0040] Regardless of the etiology, SIRS has shared pathophysiologic events with minor differences in inciting cascades. Inflammation is the body's response to nonspecific insults that arise from chemical, traumatic, or infectious stimuli. The inflammatory cascade involves humoral and cellular responses, complement, and cytokine cascades. Following an initial insult, locally produced cytokines incite an inflammatory response to recruit and activate leukocytes, thereby limiting infections, and to promote wound repair. A decrease in proinflammatory mediators and the release of endogenous antagonists controls the initial inflammatory response in an attempt to restore homeostasis. If homeostasis is not restored, cytokine release leads to aggravation of the disorder rather than protection. As a consequence, the numerous cascades and the reticuloendothelial system are activated with subsequent loss of circulatory integrity.

Ultimately, this results in end-organ dysfunction.

[0041] Trauma, inflammation, or infection lead to the activation of the inflammatory cascade. When SIRS is mediated by an infectious insult, the inflammatory cascade is often initiated by an endotoxin or exotoxin. Cytokines released by tissue macrophages, monocytes, neutrophils, mast cells, platelets, and endothelial cells include TNF-a and IL-1. TNF-a and IL-1 are responsible for fever and the release of stress hormones including norepinephrine and vasopressin, and activate the renin-angiotensin-aldosterone pathway. The cytokines also initiate several cascades, leading to cleavage of the NF-κΒ inhibitor, activation of NF-κΒ, and subsequent production of mRNA for other proinflammatory cytokines (including IL-6, IL-8, and interferon gamma). IL-6 and other cytokines stimulate the release of acute-phase reactants such as C-reactive protein (CRP).

[0042] The proinflammatory interleukins either function directly on tissue or exert their effects through secondary mediators to activate the coagulation cascade, complement cascade, and the release of nitric oxide, platelet- activating factor, prostaglandins, and leukotrienes.

Proinflammatory polypeptides within the complement cascade such as C3a and C5a contribute to the release of additional cytokines, induce vasodilatation and increase vascular permeability. Attorney Docket: 0245.90/02PCT

[0043] The relationship between inflammation and coagulation underlies the progression of SIRS. Unchecked, the coagulation cascade leads to complications of microvascular thrombosis and organ dysfunction. The complement system also plays a role in the coagulation cascade; infection-related procoagulant activity is generally more severe than that produced by trauma.

[0044] The cumulative effect of the SIRS inflammatory cascade is an unbalanced state with inflammation and coagulation dominating.

[0045] Causes of SIRS include but are not limited to bacterial sepsis, burn wound infections, candidiasis, cellulitis, cholecystitis, community-acquired pneumonia, diabetic foot infection, erysipelas, infective endocarditis, influenza, intraabdominal infections (e.g., diverticulitis, appendicitis), gas gangrene, meningitis, nosocomial pneumonia,

pseudomembranous colitis, pyelonephritis, septic arthritis, toxic shock syndrome, urinary tract infections (both male and female), acute mesenteric ischemia, autoimmune disorders, burns, chemical aspiration, cirrhosis, dehydration, drug reaction, electrical injuries, erythema multiforme, hemorrhagic shock, intestinal perforation, medication side effect (e.g. theophylline), myocardial infarction, pancreatitis, substance abuse (stimulants such as cocaine and

amphetamines), surgical procedures, toxic epidermal necrolysis, transfusion reactions, upper gastrointestinal bleeding, and vasculitis.

[0046] Inhibition of FXII activation as described herein is useful in preventing SIRS, treating SIRS, and mitigating the effects of SIRS.

Disseminated Intravascular Coagulation (DIC)

[0047] DIC is a complex systemic thrombohemorrhagic disorder involving the generation of intravascular fibrin and the consumption of procoagulants and platelets. The resultant clinical condition is characterized by intravascular coagulation and hemorrhage.

[0048] DIC exists both acutely and chronically. Acute DIC develops upon sudden exposure of blood to procoagulants (including tissue thromboplastin) inducing intravascular coagulation. Compensatory hemostatic mechanisms are quickly overwhelmed resulting in severe consumptive coagulopathy leading to hemorrhage. Chronic DIC develops when blood is continuously or intermittently exposed to small amounts of tissue factor but compensatory mechanisms in the liver and bone marrow are not overwhelmed. There can be little obvious Attorney Docket: 0245.90/02PCT clinical or laboratory indication of the presence of chronic DIC, though it is frequently associated with solid tumors and large aortic aneurysms.

[0049] DIC is caused by widespread and ongoing activation of coagulation that leads to intravascular or microvascular fibrin deposition, ultimately compromising adequate blood supply to various organs. Four mechanisms are responsible for the hematologic derangements seen in DIC: increased thrombin generation; suppressed anticoagulant pathways; impaired fibrinolysis; and inflammatory activation. Activation of FXII, with consequent activation of the intrinsic clotting pathway, is an important mechanism leading to intravascular coagulation.

[0050] Multiple hemostatic mechanisms regulate thrombin generation, but once intravascular coagulation commences, these compensatory mechanisms are overwhelmed or incapacitated. For example, antithrombin is reduced in patients with sepsis as it is continuously consumed by ongoing coagulation activation, is degraded by elastase produced by activated neutrophils, is lost through capillary leakage, and its production is secondarily impaired by the damaged liver resulting from under-perfusion and microvascular coagulation.

[0051] Proteins C and S are also part of the anticoagulant compensatory mechanism.

Normally, protein C is activated by thrombin and complexed on endothelial cell surfaces with thrombomodulin. Activated protein C inhibits coagulation via proteolytic cleavage of factors Va and Villa. However, the cytokines produced in sepsis and other generalized inflammatory states disable the protein C pathway and down-regulate the expression of thrombomodulin on the endothelial cell surface. Protein C levels are further reduced by consumption, extravascular leakage, reduced hepatic production, and by reduction in freely circulating protein S.

[0052] Another anticoagulant mechanism disabled in DIC is tissue factor pathway inhibitor (TFPI) - TFPI inhibits the tissue factor- VII complex and blocks the procoagulant effect of endotoxin. TFPI depletion predisposes patients to DIC. Intravascular fibrin produced by thrombin is normally eliminated by fibrinolysis. The initial response to inflammation is augmentation of fibrinolytic action. This response is quickly reversed when inhibitors of fibrinolysis, including plasminogen activator inhibitor- 1 (PAI-1) are released. High levels of PAI-1 precede DIC and predict poor outcomes. Fibrinolysis cannot keep pace with increased fibrin formation and eventually results in under-opposed vasculature fibrin deposition.

[0053] Inflammatory and coagulation pathways interact in substantial ways. Activated coagulation factors produced in DIC contribute to the propagation of inflammation by Attorney Docket: 0245.90/02PCT stimulating endothelial cell release of proinflammatory cytokines. Factor Xa, thrombin, and the FXIIa complex each elicit proinflammatory actions. Inhibition of the anti-inflammatory action of activated protein C and AT contributes to additional dysregulation of inflammation.

[0054] As mentioned above, DIC is typically a complication or an effect of progression of other clinical conditions generally involving activation of systemic inflammation:

sepsis/severe infection, trauma (including neurotrauma), organ destruction, malignancy (solid and myeloproliferative malignancies), severe transfusion reactions, rheumatologic illness (including adult Stills disease and lupus), obstetric complications (including amniotic fluid embolism, abruptio placentae, hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome/eclampsia, and retained dead fetus syndrome), vascular abnormalities (Kasabach- Merritt syndrome and large vascular aneurysms), severe hepatic failure, and severe toxic reactions (envenomations, transfusion reactions and transplant rejection).

[0055] DIC is most commonly observed in severe sepsis and septic shock; the development and severity of DIC correlates with mortality in severe sepsis. Gram positive or gram negative bacteria, viruses, fungi, parasites, and other organisms are associated with DIC.

[0056] Neurotrauma patients, and particularly those who develop the systemic inflammatory response syndrome, are prone to developing DIC. Inflammatory cytokines are central to DIC in both trauma and septic patients.

[0057] Inhibition of FXII activation as described herein is useful in preventing DIC, treating DIC, and mitigating the effects of DIC.

Septic Shock

[0058] Sepsis is a systemic inflammatory response to a documented infection with clinical conditions like those of SIRS but further including tachycardia and tachypnea as well as some manifestation of inadequate organ function/perfusion. Severe sepsis is associated with organ dysfunction, hypoperfusion, or hypotension, and may include lactic acidosis, oliguria, or an acute alteration in mental status. Sepsis-induced hypotension (systolic blood pressure of <90 mm Hg or a reduction of >40 mm Hg from baseline) can develop despite adequate fluid resuscitation. Septic shock from hypotension develops in a subset of patients with severe sepsis despite adequate fluid resuscitation, along with the presence of perfusion abnormalities including lactic acidosis, oliguria, or acute alteration in mental status. Attorney Docket: 0245.90/02PCT

[0059] The predominant hemodynamic feature of septic shock is arterial vasodilation.

Diminished peripheral arterial vascular tone results in dependency of blood pressure on cardiac output, causing vasodilation to result in hypotension and shock if insufficiently compensated by a rise in cardiac output. Early in septic shock, the rise in cardiac output is limited by

hypovolemia and a fall in preload due to low cardiac filling pressures. When intravascular volume is augmented, the cardiac output is elevated (the hyperdynamic phase of sepsis and shock). Even though the cardiac output is elevated, the performance of the heart is typically depressed.

[0060] Sepsis is an autodestructive process where a normal pathophysiologic response to infection results in multiple organ dysfunction syndrome. Organ dysfunction or organ failure can be the first clinical sign of sepsis, and no organ or system is immune to the consequences of the inflammatory excesses of sepsis.

[0061] Inflammatory mediators are key players in the pathogenesis of sepsis. Bacteria induce a variety of proinflammatory mediators including cytokines, which play a role in initiating sepsis and shock. Components of the bacterial cell wall, including lipopolysaccharide (gram-negative bacteria), peptidoglycan (gram-positive and gram-negative bacteria), and lipoteichoic acid (gram-positive bacteria), as well as other bacterial products, induce the release of cytokines (including IL-1 and TNF). Both IL-1 and TNF initially help keep an infection localized, but once the infection becomes systemic, the effects of these cytokines can be detrimental. The complement system is activated, contributing to the clearance of infecting microorganisms but enhancing tissue damage. Kinins are generated, nitrous oxide is induced, and hypotension results.

[0062] The imbalance of homeostatic mechanisms leads to DIC and microvascular thrombosis causing organ dysfunction and death. Inflammatory mediators such as TNF induce endothelial cells (ECs) to release microparticles (MPs) with PS on their surfaces, which have the capacity to activate FXII, ultimately triggering the intrinsic coagulation cascade and accelerating production of thrombin and deposition of fibrin.

[0063] Activation of coagulation in sepsis is demonstrated by increases in thrombin- antithrombin complex and the presence of D-dimer in plasma, indicating activation of the clotting system and fibrinolysis. Tissue plasminogen activator (t-PA) facilitates conversion of plasminogen to plasmin, a natural fibrinolytic. Attorney Docket: 0245.90/02PCT

[0064] Endotoxins impair fibrinolysis by increasing the activity of fibrinolysis inhibitors including plasminogen activator inhibitor (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI). The levels of protein C and endogenous activated protein C are also decreased in sepsis. Protein C is activated by thrombin via thrombomodulin and is an important proteolytic inhibitor of coagulation cofactors Va and Vila. Endogenous activated protein C also enhances fibrinolysis by neutralizing PAI-1 and by accelerating t-PA-dependent clot lysis.

[0065] This imbalance among inflammation, coagulation, and fibrinolysis results in widespread coagulopathy, microvascular thrombosis, and suppressed fibrinolysis, ultimately leading to multiple organ dysfunction and death.

[0066] Inhibition of FXII activation as described herein is useful in preventing septic shock, treating septic shock, and mitigating the effects of septic shock.

Cardiogenic Shock, Hypovolemic Shock, Obstructive Shock, Neurogenic Shock, and Anaphylactic Shock

[0067] The state of shock, irrespective of etiology, is initiated by acute systemic hypoperfusion that leads to tissue hypoxia and vital organ dysfunction. All forms of shock are characterized by inadequate perfusion to meet tissue metabolic demands.

[0068] Hypovolemic shock is characterized by a greater than 15% decrease in

intravascular volume and typically occurs with other types of shock. Hypovolemic shock can be caused by hemorrhage, burns, and severe dehydration.

[0069] Cardiogenic shock relates to the compromised pumping ability of the heart such that it cannot maintain cardiac output and adequate tissue perfusion. Typical causes include myocardial infarction or cardiac arrest. The patient ultimately develops left and right sided heart failure.

[0070] Obstructive shock is caused by obstruction of the heart or great vessels, impeding venous return or cardiac pumping action and resulting in widespread vasodilation and decreased peripheral resistance. Typical causes include pulmonary embolism or pneumothorax.

[0071] Neurogenic shock is an imbalance between parasympathetic and sympathetic nervous stimulation of vascular smooth muscle, resulting in sustained vasodilatation. Head injury, spinal cord trauma, insulin reactions, and anesthesia are common causes of neurogenic shock. Attorney Docket: 0245.90/02PCT

[0072] Anaphylactic shock is a result of widespread hypersensitivity, i.e. an allergic reaction with release of large amounts of histamine leading to increased permeability and massive vasodilatation. Vasodilatation leads to hypovolemia and altered cellular metabolism. The patient develops respiratory distress with bronchospasm and laryngospasm.

[0073] Inhibition of FXII activation as described herein is useful in preventing shock, treating shock, and mitigating the effects of shock.

Kinins and Pain

[0074] Pain can occur in healthy tissues as an arousing protective reflex or defensive alert or can be associated with inflammation. Strong pain is still one of the most debilitating and significant symptoms in patients suffering from most diseases. Pain receptors are typically divided into three types: nociceptive pain activated by heat, acid, and mechanical force, mainly mediated through various types of ion-channels including vanilloid receptor, capsaicin-sensitive ion channel, and TRY- VI; inflammatory pain, mediated by chemical substances produced in inflammatory exudates including bradykinin, lysophosphatidic acid, and prostaglandins; and neuropathic pain, such as caused by spinal cord injury, thalamic stroke, etc.

[0075] The kinin-kallikrein system or kinin system involves blood proteins that play a role in inflammation, blood pressure control, coagulation, and pain. High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) are precursors of the blood proteins but have no activity themselves. HMWK is produced by the liver with prekallikrein while LMWK is produced locally in several tissues.

[0076] Kinins are among the most potent autacoids involved in inflammatory, vascular, and pain processes. Autacoids are biological factors with short half-lives that act like local hormones near the site of synthesis. Kinins are generated during tissue injury and noxious stimulation. Prekallikrein is the precursor of plasma kallikrein and can only activate kinins after being activated itself by FXII or other stimuli. Exemplary kinins include bradykinin, kallidin, and T-kinin. Bradykinin is a potent endothelium-dependent vasodilator, causes contraction of non-vascular smooth muscle, increases vascular permeability, and is involved in regulation of pain. Bradykinin also causes natriuresis, contributing to a drop in blood pressure. Kallidin is a bioactive kinin formed in response to injury from kininogen precursors through the action of kallikreins. Kallidin is identical to bradykinin with an additional lysine residue at the N-terminal and can be converted to bradykinin by an aminopeptidase. Attorney Docket: 0245.90/02PCT

[0077] Kinins exert their biological effects through the activation of two transmembrane

G-protein-coupled receptors, bradykinin Bi and B ₂ . Whereas the B ₂ receptor is constitutive and activated by the parent molecules, the Bi receptor is generally underexpressed in normal tissues and is activated by kinins deprived of the C-terminal Arg (des-Arg ⁹ -kinins). The induction and increased expression of Bi receptor occurs following tissue injury or after exposure to bacterial endotoxins or cytokines such as IL-Ι β and/or TNF-a.

[0078] B ₂ receptors play a role in the acute phase of the inflammatory and pain response, while Bi receptors are involved in the chronic phase of the response. The Bi receptors also play a role in inflammatory diseases with an immune component (for example, diabetes, asthma, rheumatoid arthritis, and multiple sclerosis).

[0079] Kinin receptor stimulation induces increased vascular permeability, relaxation of venular smooth muscle, hypotension, contraction of intestinal smooth muscle, increased airway resistance, stimulation of sensory neurons (pain), alteration of ion secretion by epithelial cells, production of nitric oxide, release of cytokines by leukocytes, and release of eicosanoids from various cell types.

[0080] Illustratively, inhibition of FXII activation can be used to treat sickle-cell crises.

Sickle-cell crises are characterized by vascular inflammation and intractable pain, major mediators of which are kinins, prostacyclin, and lysophosphatidic acid (LP A). In sickle-cell crises, PS is found on the surface of erythrocytes and other cell types (Setty BNY et al. Role of phosphatidylserine in sickle red cell-endothelial adhesion. Blood 2002; 99 : 1564- 1571). This externalized PS has the capacity to activate FXII with consequent kinin generation. PS on cell surfaces also provides a docking site for secretory phospholipase A2, activity of which increases the formation of lysophosphatidic acid (LPA) and prostaglandins. It follows that a ligand of PS can inhibit FXII and sPLA2 activation, thereby preventing the formation of kinins, LPA, and prostaglandins, and exerting beneficial effects in sickle cell crises.

Acute Renal Failure

[0081] In further embodiments, methods are provided to treat or prevent acute renal failure (ARF). ARF is typically caused by an underlying clinical condition, for example, hemorrhage or a complication of cardiac surgery, which reduces blood volume and renal perfusion. A decrease in renal perfusion results in increased reabsorption of sodium and water secondary to renal arteriolar vasoconstriction, increased secretion of ADH, and activation of the Attorney Docket: 0245.90/02PCT renin-angiotensin-aldosterone system. A persistent decrease in glomerular filtration rate (GFR) and tubular necrosis leads to tubular obstruction and increased tubular permeability. Decreased kidney output leads to inefficient elimination of metabolic waste, water, electrolytes, and acids from the body, ultimately resulting in azotemia (retention of excessive amounts of nitrogenous compounds in the blood), fluid retention, electrolyte imbalance, and metabolic acidosis. Due to the high salt and water retention, the patient is at risk for heart failure and pulmonary edema.

[0082] Acute renal failure can be caused by any condition that significantly reduces renal perfusion pressure and causes decreased GFR and azotemia. Exemplary clinical conditions include, but are not limited to, extracellular fluid losses secondary to burns, prolonged vasoconstriction (hypertension), and reduced cardiac output as seen in patients with shock or congestive heart failure.

[0083] Acute renal failure can also be caused by actual damage to nephrons and renal parenchyma, i.e. intrarenal damage. The use of nephrotoxic drugs such as streptomycin, penicillin, or amphotericin in older patients or patients with underlying renal insufficiency may lead to ischemic damage to the nephron and increase the risk of developing acute tubular necrosis.

[0084] Other causes of acute renal failure include clinical conditions which obstruct urine flow, for example, tumors, benign prostatic hypertrophy, kidney stones, and bladder neck obstruction.

[0085] Illustratively, inhibition of FXII activation can be used to address renal and other complications common following cardiac operations with cardiopulmonary bypass. The incidence of ARF is as high as 7.5% after valvular operations (Grayson et al. Ann Thor Surg 2003; 75: 1829). The pathogenesis of ARF following cardiac surgery is complex; however, during cardiopulmonary bypass cell-derived, highly procoagulant, microparticles are released into the circulation (Nieuwland R et al. Cell-derived microparticles generated in patients during cardiopulmonary bypass and are highly procoagulant. Circulation 1997; 96: 3534-3541).

Inhibition of FXII activation will prevent thrombosis as well as complement activation and the generation of kinins and lipid mediators of inflammation, thus decreasing ARF and

complications of cardiac surgery.

[0086] Inhibition of FXII activation as described herein is useful in preventing acute renal failure, treating acute renal failure, and mitigating the effects of acute renal failure. Attorney Docket: 0245.90/02PCT

Vascular Inflammation/V asculitis Increased Vascular Permeability

[0087] Vasculitis is inflammation of blood vessels, often with ischemia, necrosis, and occlusive changes. It can affect arteries, veins, venules, or capillaries. Most damage results when inflammation narrows vessels and obstructs the blood supply, thereby causing tissue necrosis. Clinical manifestations of specific vasculitic disorders are diverse and depend on the size of the vessels involved and the organs affected by ischemia.

[0088] Vasculitis resulting from an inflammatory response targeting the vessel walls and having no known cause is considered primary vasculitis. Vasculitis triggered by an infection, a drug, or a toxin or occurring as part of another inflammatory disorder or cancer is considered secondary vasculitis.

[0089] Inhibition of FXII as described herein is useful in preventing vascular

inflammation and vasculitis, treating vascular inflammation and vasculitis, and mitigating the effects of vascular inflammation and vasculitis, including, for example, vascular permeability.

Cerebral edema and the Compartment Syndromes

[0090] In some embodiments, methods are provided for suppressing cerebral edema following reperfusion (see Example 3) or head injury and other disorders leading to this complication. Increased leakage of fluid from the vascular into the extravascular compartment can exert pressure on blood vessels, decrease blood flow and impair organ function. This can occur in the abdomen during shock (abdominal compartment syndrome) or following peripheral vascular surgery (peripheral compartment syndrome). It is therefore desirable to prevent excessive leakage of fluids from the vascular compartment by suppressing FXII activation and other procedures.

Angioedema

[0091] In other embodiments, methods are provided for treatment of angioedema, which is due to unopposed complement activation when the natural inhibitor (Cl-INH) is congentially lacking. Mutations of FXII can also result in angioedema, showing the importance of this cascade in complement activation, kinin generation, and edema (Bork, Hereditary angioedema with normal CI inhibition. Curr. Allergy Asthma Reports 2009, 9: 280-285). Thus, an agent having the capacity to suppress FXII activation is a useful therapeutic agent in angioedema. Attorney Docket: 0245.90/02PCT

Illustrative Embodiments

[0092] Embodiments herein include compositions and methods useful in the prevention and/or treatment of FXII related diseases and conditions. These diseases and conditions include SIRS, DIC, hypotension, septic shock, cardiogenic shock, hypovolemic shock, obstructive shock, neurogenic shock, anaphylactic shock, kinin formation, somatic and/or visceral inflammation, pain, acute renal failure, vascular permeability, vascular inflammation, vasculitis, thrombosis, sickle cell crisis, and angioedema. Novel compositions and methods described herein can be administered alone or in combination with other known prevention and/or treatment regimens, for example, in combination with antibiotics for the treatment of septic shock. As more fully described below, compositions and methods herein provide a surprising and unexpected improvement over conventional therapies for the above-mentioned disease states and conditions.

[0093] The terms "treat" or "treatment" and the like refer to the relief or alleviation of at least one symptom associated with the FXII related diseases and conditions described above. "Treat" or "treatment" may also refer to a slowing or reversing of the progression of one of the above mentioned disease states or conditions as determined by an ordinary health care provider.

Pharmaceutical Agents for Inhibiting FXII Activation

[0094] Compositions are provided herein having activity which prevents activation of

FXII. In some embodiments, the composition prevents activation of FXII by exposure to negatively charged surfaces of cells and of micropapticles shed from them. Exemplary agents that inhibit FXII activation include agents that bind PS or "PS binding agents". The term

"inhibits" refers to any decrease in FXIIa activity relative to FXIIa activity in the absence of the agents(s), including partial decrease and complete inhibition. Thus, provided herein are pharmaceutical compositions comprising one or more PS binding agents and a pharmaceutically acceptable carrier (see below for a description of such carriers). Such pharmaceutical compositions can be added to cells, groups of cells, tissues, or organs, and or administered to patients.

[0095] As used herein, a "PS binding agent" is any molecule that binds to PS

externalized on cell and microparticle surfaces and inhibits interaction thereby, for example, interaction between FXII and PS. In some embodiments, inhibition can occur because the binding agent is bound to PS. In other embodiments, the binding agent becomes attached to a Attorney Docket: 0245.90/02PCT molecule associated with PS, for example a lipid raft constituent. In some aspects, this inhibition restrains or retards physiologic, chemical, or enzymatic action between PS and PS interacting molecules. In other aspects, a binding agent blocks, restricts, or interferes with a particular chemical reaction or other biologic activity. In still other aspects, a binding agent prevents recognition of PS by cells such as leukocytes, monocytes and platelets, thereby preventing interaction between a cell expressing PS and the monocytes, leukocytes and platelets.

[0096] According to the compositions and methods herein, a PS binding agent is a protein or other agent that binds to PS exposed on cell surfaces. Such an agent can be any molecule that binds or interacts with PS or binds some structure on cell surfaces associated with PS, such as a component of lipid rafts. The PS -binding agent can bind PS translocated to the surface of ECs as a result of anoxia, or to PS externalized to the surface of platelets or other cells during their activation. By binding PS on cell surfaces, such an agent can inhibit the attachment to them of other cell types or of some enzymes. An example is the attachment of leukocytes and platelets to ECs during IRI. A second example is the docking and activity of secretory isoforms of PLA ₂ . A third example is the assembly and activity of the prothrombinase complex on PS translocated to the surface of platelets, ECs and other cell types.

[0097] Additional exemplary agents that inhibit FXII activation include agents that directly act on FXII to prevent conformation change, cleavage, etc., for example, polyclonal and/or monoclonal antibodies prepared by Abeam, Pic. or Abgent, Inc. against human FXII. It is also envisioned that PS binding agents can be combined with agents that act directly on FXII to inhibit FXII activation.

Antibodies

[0098] In some aspects, the PS binding agent is an antibody capable of recognizing PS on a cell surface. Isolated antibodies are antibodies that have been removed from their natural environment, but the term "isolated" does not refer to the state of purity of such antibodies. The phrase "recognizing" refers to the ability of such antibodies to preferentially bind PS. Binding affinities, commonly expressed as equilibrium association constants, typically range from about l(r 3 M 1 to about 101 ¹ 2" M 1. Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays, immunofluorescent antibody assays, immunoelectron microscopy and binding to cells or liposomes with PS on their surfaces. Attorney Docket: 0245.90/02PCT

[0099] The term "antibody" refers to a Y-shaped molecule having a pair of antigen binding sites, a hinge region, and a constant region. PS antibodies used according to the methods described herein include polyclonal and monoclonal antibodies. Functional equivalents are also contemplated, including, for example, antibody fragments, genetically-engineered antibodies, single chain antibodies, and chimeric antibodies. Useful antibodies include those generated in an animal to which PS has been administered, then serum or plasma recovered using techniques known to those skilled in the art. Other useful antibodies include those produced by recombinant methods. Antibodies produced against defined antigens can be especially useful as they are not substantially contaminated with antibodies against other substances.

[00100] An illustrative monoclonal antibody that can be useful according to the method described herein was generated by Ran et al. to detect cell surface phospholipids on tumor vasculature (Cancer Research, 2002; 62:6132, incorporated herein by reference). The 9D2 antibody bound with specificity to PS, as well as to other anionic phospholipids, without requiring the presence of Ca ²⁺ . Similarly, Ran et al. developed a murine monoclonal antibody, 3G4, to target PS on tumor vasculature which also may be useful according to the method herein (Clin. Cancer Res. 2005; 11: 1551, incorporated herein by reference). Thus, the 9D2 antibody and the 3G4 antibody are exemplary PS-binding agents. Other antibodies considered useful herein include the naturally occurring antiphospholipid antibodies and PS-binding fragments.

[00101] It is further contemplated that antibodies that prevent FXII activation or prevent the downstream effects of FXII activation are useful according to the methods described herein. Exemplary antibodies include antibodies against lactoferrin, Tim4, and BAIL Derivatives of these proteins including homodimers and heterodimers are also contemplated herein as useful inhibitors of FXII activation.

Protein Ligands

[00102] In some embodiments, the PS binding agent is a ligand having an affinity for PS, for example, an affinity that is at least about 10% of the affinity of annexin V for PS. Such ligands include, for example, proteins, polypeptides, receptors, and peptides which interact with PS. Exemplary ligands include those described in U.S. Publication No. 2006/0228299 (Thorpe et al.), for example, Beta 2-glycoportein I, Mer, α ₅ β ₃ integrin and other integrins, CD3, CD4, CD14, CD93, SRB (CD36), SRC, PSOC and PSr, as well as the proteins, polypeptides, and peptides thereof. Attorney Docket: 0245.90/02PCT

[00103] Protein ligands preventing activation of FXII including the milk fat globule protein MFG-E8 (lactadherin), GAS-6, Tim4, and Ptdsr (Boese et al. J Biol 2004;3: 15) and the brain- specific angiogenesis inhibitor BAI1 (Park et al Nature 2007; 450 :430-435), as well as dimers or other constructs and/or fragments of these proteins.

[00104] The ligand can, in some embodiments, be a construct where one or more proteins, polypeptides, receptors, or peptides are coupled to an Fc portion of an antibody. The Fc regions used herein are derived from an antibody or immunoglobulin. It is necessary that the ligand retains the PS -binding property or FXII binding property when attached to the Fc portion of an antibody. The Fc portion and the ligand can be operatively attached such that each functions sufficiently as intended. In some embodiments, two ligands are coupled to an Fc portion such that they form a dimer. As used herein, "Fc" refers to both native and mutant forms of the Fc region of an antibody that contain one or more of the Fc region's CH domains, including truncated forms of Fc polypeptides containing the dimerization-promoting hinge region.

[00105] Other protein ligands can bind directly to FXII to prevent activation.

Annexins

[00106] Annexins are exemplary agents which prevent activation of FXII upon exposure to negatively charged surfaces. Annexin V is a 34 kD protein having antithrombotic activity in several experimental animal models (Romisch J et al. In vivo antithrombotic activity of placental anticoagulant placenta protein 4 (annexin V). Thromb Res 1991; 61:93-104). The molecular weight of monomeric annexin V is below the renal filtration threshold having a circulating half- life in non-human primates of less than 15 minutes. These properties make the annexin V monomer less suitable as a pharmaceutical agent, however some embodiments described herein include annexin monomers. As used herein, the term "annexin" refers to any annexin including, for example, an annexin modified to increase its circulating half life, an annexin fragment, and an annexin multimer.

[00107] Annexin homodimers and heterodimers or annexin monomers coupled with a 30+ kD peptide have a much longer half-life in circulation than the annexin monomers (Kuypers F et al. Thromb Hemost 2007; 97: 478-486). An exemplary annexin dimer, annexin V - annexin V, at concentrations therapeutically attainable in the human circulation (1 to 20 nM/mL) inhibits assembly and activity of the prothrombinase complex and the activity of sPLA ₂ . At 200 μg/kg, the annexin V - annexin V dimer (or Diannexin) was shown to inhibit venous thrombosis in rats. Attorney Docket: 0245.90/02PCT

[00108] Thus, in some embodiments, the PS binding agent is an annexin monomer or

PEGylated annexin, or an annexin multimer such as, for example, an annexin heterodimer, an annexin homodimer, an annexin trimer, annexin tetramer, or a combination and/or fragment thereof.

[00109] In treating sickle cell crises, annexin homodimers, heterodimers, trimers, tetramers, and otherwise modified annexins have the potential to inhibit FXII activation as well as the activity of secretory phospholipases A2, which leads to generation of LPA and

prostacyclin. As such, annexin dimers can block the formation of several important mediators of pain in sickle cell crises. Further, by inhibiting FXII activation, annexin dimers can also prevent thrombosis and the formation of inflammatory mediators contributing to the pathogenesis of the acute chest syndrome in sickle-cell crises.

[00110] Thus, in some aspects, the PS binding agent is a modified annexin. As used herein, the phrase "modified annexin" refers to any annexin protein that has been modified in such a way that its half-life in a recipient is prolonged. Modified annexin refers to the subject matter disclosed in U.S. Patent Application No. 11/267,837, which is incorporated by reference in its entirety.

[00111] Annexins include proteins of the annexin family, such as Annexin I, Annexin II

(lipocortin 2, calpactin 1, protein I, p36, chromobindin 8), Annexin III (lipocortin 3, PAP-III), Annexin IV (lipocortin 4, endonexin I, protein II, chromobindin 4), Annexin V (Lipocortin 5, Endonexin 2, VAC-alpha, Anchorin CII, PAP-I), Annexin VI (Lipocortin 6, Protein III,

Chromobindin 20, p68, p70), Annexin VII (Synexin), Annexin VIII (VAC-beta), Annexin XI (CAP-50), and Annexin XIII (ISA).

[00112] An annexin gene includes all nucleic acid sequences related to a natural annexin gene such as regulatory regions that control production of the annexin protein encoded by the gene (such as, but not limited to, transcription, translation, or post-translation control regions) as well as the coding region itself. An annexin gene in accordance to the disclosure herein includes allelic variants. An allelic variant is a gene that occurs at essentially the same locus in the genome, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants are well known to those skilled in the art and would be expected to be found within a Attorney Docket: 0245.90/02PCT given human since the genome is diploid and/or among a population comprising two or more humans.

[00113] In more detail, Annexin I (SEQ ID NO: 1) is a 37 kDa member of the annexin superfamily of proteins. The protein is predominantly expressed within gelatinase granules of neutrophils and is externalized onto the cell membrane after cell adhesion to endothelial cells.

[00114] Annexin II (SEQ ID NO: 2) is involved in diverse cellular processes such as cell motility (especially that of the epithelial cells), linkage of membrane-associated protein complexes to the actin cytoskeleton, endocytosis, fibrinolysis, ion channel formation, and cell matrix interactions. It is a calcium-dependent phospholipid-binding protein whose function is to help organize exocytosis of intracellular proteins to the extracellular domain.

[00115] Annexin III (SEQ ID NO: 3) is also called "lipocortin 3" or "placental anticoagulant protein 3" and is a member of the lipocortin/annexin family. Annexin III binds to phospholipids and membranes in a Ca ²⁺ dependent manner and has been shown to have anticoagulant and anti-phospholipase A ₂ properties. Suppression of Annexin III expression has been shown to inhibit DNA synthesis in rat hepatocytes (Nimmi, et al., Biol. Pharm. Bull. 2005; 28:424).

[00116] Annexin IV (SEQ ID NO: 4) (endonexin) is a 32 kDa, Ca ²⁺ -dependent membrane-binding protein which shares many of the properties of Annexin V. The translated amino acid sequence of Annexin IV shows the four domain structure characteristic of proteins in this class. Annexin IV is a close structural homologue of Annexin V and has 45-59% identity with other members of its family, sharing a similar size and exon-intron organization. The sequence of Annexin IV is shown in Hamman et al., Biochem. Biophys. Res. Comm., 156:660- 667. (1988). Isolated from human placenta, Annexin IV encodes a protein that has in vitro anticoagulant activity, binds acidic phospholipid membranes in the presence of calcium, and inhibits phospholipase A ₂ activity. Annexin IV is almost exclusively expressed in epithelial cells.

[00117] Annexin V (SEQ ID NO: 5) is a member of the Ca ²⁺ -dependent phospholipid- binding proteins. It binds to PS with high affinity. The core domain is a concave discoid structure that can be closely apposed to phospholipid membranes. It contains 4 subdomains, each consisting of a 70 amino-acid annexin repeat made up of five alpha-helices. The sequence of annexin V is well known (See Funakoshi et al., 1987; 26:8087). Attorney Docket: 0245.90/02PCT

[00118] Annexin VI (SEQ ID NO: 6) is 68 kd protein whose actin binding is positively regulated by calcium (other actin binding proteins are typically negatively regulated by calcium). Annexin VI can bind G- as well as actin filaments and also binds lipids. Annexin VI has been localized to stress fibers, membrane ruffles, microspikes and focal contacts. On stress fibers annexin VI is periodic and coincident with a-actinin. Annexin VI contains 8 annexin repeats.

[00119] Annexin VII (SEQ ID NO: 7) is a member of the annexin family of calcium- dependent phospholipid binding proteins. The Annexin VII gene contains 14 exons and spans approximately 34 kb of DNA. An alternatively spliced cassette exon results in two mRNA transcripts of 2.0 and 2.4 kb which generate two protein isoforms differing in their N-terminal domain. The alternative splicing event is tissue specific and the mRNA containing the cassette exon is prevalent in brain, heart and skeletal muscle. The transcripts also differ in their 3'-non coding regions by the use of two alternative poly(A) signals. Annexin VII encodes a protein with a molecular weight of approximately 51 kDa with a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Structural analysis of the protein indicates that Annexin VII is a membrane binding protein with diverse properties, including voltage-sensitive calcium channel activity, ion selectivity, and membrane fusion.

Transcript Variant: This variant (1) lacks an alternate in-frame exon, compared to variant 2, resulting in a shorter protein (isoform 1) that lacks an internal segment, compared to isoform 2.

[00120] Annexin VIII (SEQ ID NO: 8) belongs to the family of Ca ²⁺ -dependent phospholipid binding proteins (annexins) having high sequence identity to Annexin V (56%) (Hauptmann, et al., Eur. J. Biochem. 1989; 185(1):63-71). Initially isolated as a 2.2 kb vascular anticoagulant-beta, annexin VIII is neither an extracellular protein nor associated with the cell surface. Annexin VIII is expressed at low levels in human placenta and shows restricted expression in lung, vascular ECs, skin, liver, and kidney.

[00121] Annexin XI (SEQ ID NO: 9) is a 56-kD antigen recognized by sera from patients with various autoimmune diseases. Transcript variants encoding the same isoform have been identified.

[00122] Annexin XIII (SEQ ID NO: 10) is associated with the plasma membrane of undifferentiated, proliferating endothelial cells and differentiated villus enterocytes.

Alternatively spliced transcript variants encoding different isoforms have been identified. Attorney Docket: 0245.90/02PCT

[00123] In some aspects, annexin proteins are modified to increase their half-life in humans or other mammals. In some embodiments, the annexin protein is annexin V, annexin IV or annexin VIII. One suitable modification of annexin is an increase in its effective size, which inhibits loss of the modified annexin, i.e., from the vascular compartment, into the extravascular compartment and urine, thereby prolonging the annexin activity in the vascular compartment. Any increase in effective size of the annexin protein that maintains a sufficient binding affinity with PS is contemplated herein.

[00124] In one embodiment, an annexin protein is coupled to one or more annexin proteins (homodimers, heterodimers, etc., for example an annexin V homodimer contemplated herein is referred to as Diannexin) or to one or more non-annexin proteins. Modification can be accomplished through a fusion segment, or by the Fc portion of an immunoglobulin. An alternative method for increasing the effective size of proteins is coupling to polyethylene glycol (PEG) or another molecule. For example, coupling by pegylation is achieved by coupling one or more PEG chains to one or more annexin proteins. A PEG chain can have a molecular weight of at least about 10 kDa, or at least about 20 kDa, or at least about 35 kDa. The annexin is coupled to PEG in such a way that the modified annexin is capable of performing the function of annexin binding to PS on cell surfaces.

[00125] According to some embodiments, modified annexin proteins and mixtures thereof are used in methods for preparing pharmaceutical compositions intended for use in any of the therapeutic methods of treatments described above.

[00126] In one embodiment, a modified annexin contains a recombinant human annexin protein coupled to PEG in such a way that the modified annexin is capable of performing the function of annexin in a phosphatidylserine (PS)-binding assay. The activity of the intravenously administered annexin-PEG conjugate is prolonged as compared with that of the free or non- modified annexin. The recombinant annexin protein coupled to PEG can be annexin V protein or another annexin protein. In one embodiment, the annexin protein is annexin V, annexin IV or annexin VIII.

[00127] PEG consists of repeating units of ethylene oxide that terminate in hydroxyl groups on either end of a linear or, in some cases, branched chain. The size and molecular weight of the coupled PEG chain depend upon the number of ethylene oxide units it contains, which can be selected. Any size of PEG and number of PEG chains per annexin molecule can be Attorney Docket: 0245.90/02PCT used such that the half-life of the modified annexin is increased, relative to annexin, while preserving the function of binding of the modified molecule to PS. The optimal molecular weight of the conjugated PEG varies with the number of PEG chains. In one embodiment, two PEG molecules of molecular weight of at least about 15 kDa, are each coupled to an annexin molecule. The PEG molecules can be linear or branched. The Ca ²⁺ -dependent binding of annexins to PS is affected not only by the size of the coupled PEG molecules, but also the sites on the protein to which PEG is bound. Optimal selection ensures that desirable properties are retained. Selection of PEG attachment sites is facilitated by knowledge of the three-dimensional structure of the molecule and by mutational and crystallographic analyses of the interaction of the molecule with phospholipid membranes (Campos et al., Biochemistry 37:8004-8008 (1998), incorporated herein by reference).

[00128] PEG derivatives have been widely used in covalent attachment (referred to as pegylation) to proteins to enhance solubility, as well as to reduce immunogenicity, proteolysis, and kidney clearance. The superior clinical efficacy of recombinant products coupled to PEG is established. For example, PEG-interferon alpha-2a administered once weekly is significantly more effective against hepatitis C virus than three weekly doses of the free interferon (Heathcote et al., N. Engl. J. Med. 343: 1673-1680 (2000), incorporated herein by reference). Coupling to PEG has been used to prolong the half-life of recombinant proteins in vivo (Knauf et al., J. Biol. Chem. 266:2796-2804 (1988), incorporated herein by reference), as well as to prevent the enzymatic degradation of recombinant proteins and to decrease the immunogenicity sometimes observed with homologous products (references in Hermanson, Bioconjugate techniques. New York, Academic Press (1996), pp. 173-176, incorporated herein by reference).

[00129] In another embodiment, the modified annexin protein is a polymer of annexin proteins that has an increased effective size. It is believed that the increase in effective size results in prolonged half-life in the vascular compartment and prolonged activity. One such modified annexin is a dimer of annexin proteins. In one embodiment, the dimer of annexin is a homodimer of annexin V, annexin IV or annexin VIII. In another embodiment, the dimer of annexin is a heterodimer of annexin V and other annexin protein (e.g., annexin TV or annexin VIII), annexin IV and another annexin protein (e.g., annexin V or annexin VIII) or annexin VIII and another annexin protein (e.g., annexin V or annexin IV). The annexin homodimer or heterodimer can be produced by bioconjugate methods or recombinant methods, and be Attorney Docket: 0245.90/02PCT administered by itself or in a PEG-conjugated form. Table 1 provides possible annexin combinations with their SEQ ID NOs.

Table 1

Attorney Docket: 0245.90/02PCT

[00130] In some embodiments, two or more annexins are linked directly to each other. In other embodiments, two or more annexins are linked together by a fusion segment.

[00131] One or more fusion segments or linkers can be used to couple one or more annexin proteins, typically referred to as "fusion proteins". A "fusion protein" refers to a first protein having attached one or more additional proteins. The protein can be fused using recombinant DNA techniques, such that the first and second proteins are expressed in frame.

[00132] Inclusion of a fusion sequence as part of a modified annexin nucleic acid molecule can enhance stability during production, storage, and/or use of the protein encoded by the nucleic acid molecule. The fusion segment can be a domain of any size that has the desired function. Fusion segments can be constructed to contain restriction sites to enable cleavage for recovery of desired proteins.

[00133] Illustratively, a flexible linker contains a sequence of amino acids flanked by a glycine and a serine residue at either end to serve as swivels. Such swivels allow rotation of each annexin monomer around the long axis of the linker. The linker can comprise one or more such "swivels." In some aspects, the linker comprises 2 swivels which can be separated by at least 2 amino acids, at least 4 amino acids, at least 6 amino acids, at least 8 amino acids, or at least 10 amino acids. The overall length of the linker can be 5 to 30 amino acids, 5 to 20 amino acids, 5 to 10 amino acids, 10 to 15 amino acids, or 10 to 20 amino acids. The dimer can fold in such a way that the convex surfaces of the monomer, which bind Ca ²⁺ and PS, can both gain access to externalized PS. Flexible linkers are known in the art, for example, (GGGGS) _(n) (SEQ ID NO: 115) (n=3-4), as well as helical linkers with less flexibility, (EAAAK) _(n) (SEQ ID NO: 116) (n=2-5), described in Arai, et al., Proteins. 2004 Dec. 1; 57(4):829-38.

[00134] An illustrative linker represented by SEQ ID NO: 111 comprises 12 amino acids with a Gly-Ser sequence on both ends. The linker was designed to have no secondary structure and to allow flexibility and rotation around its length. The particular amino acids of the linker were also chosen for their low immunogenicity. Various linker amino acid sequences and lengths are described in U. S. Patent Application Serial No. 11/613,125, filed December 19, 2006 which is incorporated by reference herein in its entirety. Attorney Docket: 0245.90/02PCT

[00135] Exemplary embodiments of two annexin monomers joined by a linker include

SEQ ID NOs 112, 113, and 114, annexin IV homodimer, annexin V homodimer, and annexin VIII homodimer, respectively.

Therapeutic Applications

[00136] Compositions described herein can be administered in the form of a

pharmaceutical composition comprising the composition and a pharmaceutically acceptable carrier. Such a composition is sometimes referred to as a "therapeutic composition". Aspects of potential therapeutic compositions are described herein.

[00137] A patient can be a human or non-human. In some embodiments, the patient suffers from or is at risk of developing a disease or condition associated with activation of FXII (see above).

[00138] Provided herein are therapeutic compositions that also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier. For example, a composition can be formulated in an excipient that the patient can tolerate. Illustrative excipients include water, saline, Ringer's solution, dextrose solution, mannitol, Hanks' solution, the University of Wisconsin Belzer solution and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as triglycerides may also be used. Excipients can contain minor amounts of additives, including substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include thimerosal, m- or o- cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. The composition can be further combined with or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.

[00139] A therapeutically effective amount includes an amount sufficient to prevent, attenuate, or partially reverse a condition or disease associated with FXII activation. A therapeutically effective amount can be any amount or dose sufficient to bring about the desired amount of protection from the disease or condition, or the desired attenuation of the disease, Attorney Docket: 0245.90/02PCT condition, or symptoms. This amount can depend, in part, on the composition used in treatment, the frequency and duration of administration, the condition of the patient, the duration of the disease or condition, etc. Other factors such as the size and health of the patient are known to those skilled in the art and taken into account at the time of administration. It will be understood that recitation herein of a "therapeutically effective" amount herein does not necessarily require that the drug be therapeutically effective if only a single such dose is administered; in some situations repeated administration may be needed to provide effective treatment.

[00140] Illustratively, the composition is a modified annexin protein which can be administered in a range of about 50 to about 500 μg/kg, for example, about 200, or about 300, or about 400 μg/kg ^g PS-binding agent/kg of patient's weight).

[00141] The compositions described herein, including for example, PS binding agents, can be administered by any method known in the art. The composition can be administered in a single dose, or as several doses, for example, twice a day or in a dosing regimen that covers two or three days or one or more weeks.

[00142] Administration of an agent or therapeutic composition can be by any suitable route, including without limitation parenteral (e.g., intravenous, subcutaneous, intrasternal, intramuscular, or infusion techniques), oral, sublingual, buccal, intranasal, pulmonary, topical, transdermal, intradermal, mucosal, ocular, otic, rectal, vaginal, intragastric, intrasynovial, and intra- articular routes. A route such as parenteral that provides systemic delivery is generally desirable. In some aspects, the method comprises intravenous administration of the agent or composition. In other aspects, the method comprises administration by bolus injection. In still other aspects, the method comprises administration by injection or introduction into an intravenous drip.

[00143] Pharmaceutical compositions can be in the form of sterile injectable preparations or aerosol sprays allowing absorption through the nasal mucosa or lungs.

[00144] For administration by inhalation or aerosol, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulations. The

compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art. Attorney Docket: 0245.90/02PCT

[00145] For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides and fatty acids (including oleic acid). Solutions or suspensions of the inhibiting agents can be prepared in water or isotonic saline (for example, phosphate buffered saline), optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin, and mixtures thereof. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[00146] The pharmaceutical dosage for injection or infusion can include sterile aqueous solutions, sterile dispersions, or sterile powders, comprising an active ingredient adapted for the extemporaneous preparation of sterile injectable solutions, sterile infusible solutions, or sterile dispersions. In all cases, the ultimate dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (including, for example, but not limited to, glycerol, propylene glycol, or liquid polyethylene glycol), vegetable oil, nontoxic glyceryl ester, or suitable mixture thereof. Desired fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size (in the case of dispersion), or by the use of nontoxic surfactants. Prevention of microbial action can be achieved by using various antibacterial and antifungal agents. Illustrative antimicrobial or antifungal agents include parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In some aspects, isotonic agents are desirable, and include sugar, buffer, or sodium chloride.

[00147] One embodiment is a controlled release formulation that is capable of slowly releasing a composition into a patient. As used herein, a controlled release formulation comprises a composition as described herein in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations include liquids that, upon administration to a patient, form a solid or a gel in situ. In some embodiments, the controlled release formulations are biodegradable (i.e., bioerodible). Prolonged absorption of injectable compositions can be brought about by the inclusion in the Attorney Docket: 0245.90/02PCT composition of agents delaying absorption, for example, aluminum monostearate hydrogels and gelatin.

[00148] Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various other ingredients as enumerated above. Such solutions are subsequently sterilized, typically using a filter. Sterile powders used in the preparation of sterile injectable solutions are vacuum dried or freeze dried, yielding a powder of the active ingredient plus any additional desired ingredient present in the previously sterile- filtered solutions.

[00149] Where annexin monomers (or other PS binding agents having a short half life) are utilized, administration may be continuous or periodic based on maintaining a sufficient level of annexin monomer in the patient's system. Suitable administration methods include intravenous infusion, pump, or multiple administrations over the course of a predetermined time period.

[00150] In some embodiments, the PS binding agent is an annexin monomer such as an annexin V monomer. In some aspects, the monomer can be administered continuously, about every fifteen minutes, about every thirty minutes, about every hour, about every two hours, about every three hours, about every four hours, about every 6 hours, about every 8 hours, about every 12 hours, or about every 24 hours. In other aspects, the monomer can be administered every day for at least about two days, at least about 3 days, at least about 4 days, or at least about 1 week.

[00151] Where administration of a PS binding agent and direct FXII inhibitor are contemplated, the materials can be administered simultaneously or alternating as determined by the health care professional. For example, a modified annexin V composition can be combined with an anti-FXII antibody and administered on the same regimen or at different times. In addition, each material, including multiple PS binding agents, can be administered by different routes, for example, Diannexin via injection once a day and annexin monomer via intravenous infusion over the course of a day.

EXAMPLES

Example 1. Effect of PS ligand on kaolin-induced blood coagulation

[00152] Tracking measurements of fibrin formation, clot strength, thrombin generation, platelet function, and fibrinolysis using Thrombelastograph (TEG, Haemoscope Corp.) mapping provided a comprehensive evaluation of thrombosis and hemostasis under various treatment Attorney Docket: 0245.90/02PCT conditions. Four parameters were examined: initial clotting time (R), time from R to a specific clot strength (K), maximum rate of thrombin generation (MRTG), and time to reach the maximum rate of thrombin generation (TMRTG).

[00153] Whole-blood specimens were drawn from normal volunteers (n = 9). Each 4-8 mL sample was collected into sodium citrate buffer (4.5 mL BD Vacutainer®, 0.105 M ~ 3.2%), pooled immediately and left at room temperature for 30 minutes with intermittent mixing.

Diannexin was the PS ligand used in the experiment. Aliquots of Diannexin (lmg/Ml) were previously prepared and frozen from a 7 mg/mL Diannexin stock. Dilutions of Diannexin were made using saline. After 30 minutes, specimen preparation continued according to coagulation initiation. Kaolin initiation was used in the experiments because it is dependent on FXII activation.

[00154] At 30 minutes, a whole-blood specimen was pipetted in lmL aliquots into manufacturer-prepared kaolin tubes (TEG Hemostasis Systems). Diannexin was added to achieve final concentrations of 1 μg/mL (13 nM), 0.5 μg/mL (6.5 nM), 0.25 μg/mL (3.3 nM), 1.25 μg/mL (1.6 nM). A control sample was also prepared without Diannexin. 340 μΐ _^ of each prepared sample was immediately loaded into a TEG assay cup with 20μL· CaCl ₂ and

coagulation was mapped and evaluated using TEG parameters R, K, MRTG, and TMRTG.

[00155] Diannexin treatment prolonged clot initiation time (R), (see Figure 1) and the time to reach a specific clot strength (K) (see Figure 2). Diannexin also attenuated the maximum rate of thrombin generation (MRTG) (see Figure 3) and prolonged the time to reach the maximum rate of thrombin generation (TRMTG) (see Figure 4). These effects were dose-dependent over the range of Diannexin studied (0-13.7 nm). Corn trypsin inhibitor was used to block intrinsic pathway activation as a control. Thus, Diannexin in clinically attainable concentrations can inhibit FXII-dependent blood coagulation.

Example 2. Effect of vascular permeability during post-ischemic reperfusion(IRI)

[00156] Example 1 demonstrates that Diannexin inhibits FXII-dependent blood coagulation. This example shows that a PS ligand such as Diannexin can also inhibit FXII- dependent kinin generation, and consequent edema, in vivo. An experimental animal model using a flap containing the cremaster muscle of the rat which can be studied by intravital microscopy allows quantification of increased vascular permeability at the level of single blood vessels. A fluorescent protein was injected into the blood, most of which was retained within the Attorney Docket: 0245.90/02PCT vasculature, but some protein passed into the extravascular space. Sequential measurements of fluorescent light intensity (pixels) within and around venules provided a ratio or Mean

Permeability Index (MPI). When vascular permeability increased, the ratio of extravascular to intravascular fluorescence (MPI) rose.

[00157] A detailed description of the model used in the experiments described in this example was published (Molski M. et al., Diannexin treatment decreases ischemia-reperfusion injury at the endothelial cell level of the microvascular bed in muscle flaps. Ann. Plast. Surg. (2009) 63: 564-571). Briefly, male Lewis Rtl rats weighing about 150 grams were anesthetized and the cremaster muscles prepared for microscopy. In 20 animals the arterial supply to the muscle was clamped off for 5 hours and then restored. In another 10 control animals the arterial supply was maintained (no ischemia). In 10 of the rats undergoing post-ischemic reperfusion, Diannexin (100 micrograms per kg) was injected intravenously. In the other 10 rats undergoing reperfusion the same volume of saline was injected as a placebo. All animals received 3 mg of fluorescein isothiocyanate-conjugated albumin intravenously.

[00158] The ratio of extravascular to intravascular fluorescence intensity (pixels) was measured at the commencement of reperfusion, and after 15 minutes, 30 minutes, and

60 minutes. Results of a representative set of experiments are summarized in Table 2. The observations show that in the cremaster muscle, as in other tissues, vascular permeability is increased during post-ischemic reperfusion. Diannexin treatment inhibits this increase in vascular permeability during reperfusion, in contrast to the saline placebo.

[00159] Thus, a PS ligand such as Diannexin counteracts the increase in vascular permeability, ultimately preventing edema that occurs during post-ischemic reperfusion. These findings made at the single- vessel level are complementary to the findings in the following Example 3 and show that Diannexin opposes reperfusion-related edema in several vascular beds. This effect is attributable, at least in part, to inhibition of FXII activation by inhibition of PS activation.

[00160] Table 2. Comparison of the mean ratios of extravascular to intravascular fluorescence at different time periods after commencing reperfusion, and at all time periods (All). P values comparing all groups are from F-tests, whereas paired-comparison p values are from t- tests that assume equal variance across groups. Differences that are statistically significant (p<0.05) are starred. Attorney Docket: 0245.90/02PCT

Example 3 : Effect on Hemorrhage in Mouse Brain

[00161] This example shows the efficacy of Diannexin in attenuating post-ischemic reperfusion injury (IRI) in a mouse brain model, and in particular the hemorrhage associated with that condition. The mouse stroke model on which the experiment was performed was developed by Maier et al. (Ann. Neurol. (2006) 59: 929-938). Knock-out (KO) mice with targeted disruption of the gene encoding inducible mitochondrial manganese-containing superoxide dismutase (SOD2) were subjected to a mild stroke followed by early reperfusion and 3 day survival. Heterozygous SOD2-KO mice (SOD2 -/+) are more susceptible to ischemic damage than their wild- type (SOD +/+) counterparts and exhibit a significant increase in matrix metalloproteinase-9 (MMP9) expression in blood vessels during IRI. The tight-junction transmembrane protein occludin is highly susceptible to degradation by MMP9, and depletion of occludin is one factor leading to loss of vascular integrity, and consequent hemorrhage, during IRI. This model was developed to evaluate targets for therapies designed to attenuate cerebral IRI.

[00162] A detailed description of the mouse cerebral artery occlusion (MCRO) model has been published by Maier et al. (id). Briefly, 35 mice heterozygous for SOD2 knockout (on a CD1/SW129 background), 32-35 gm, and 34 of their wild-type (WT) littermates were used. Under isoflurane anesthesia, a middle cerebral artery was occluded by intraluminal suture for 30 min after which arterial circulation was re-established. Reperfusion was allowed to continue Attorney Docket: 0245.90/02PCT for 24 or 72 hr, after which the animals were killed for histological and other studies. To quantify vascular permeability, 2.5 ml/kg of 4% Evans blue dye in 0.9% saline was injected intravenously. Sections were examined by fluorescence microscopy to evaluate Evans Blue extravasation (edema). In one half of the MCAO mice, Diannexin (200 micro grams/kg) was injected intravenously a few minutes after the commencement of reperfusion, and in the other MCAO mice normal saline was similarly administered as a placebo control.

[00163] The main experimental findings are shown in Figure 5 and Table 3. Not shown are observations 24 hours after commencing reperfusion. Diannexin did not affect the primary infarct area resulting from the effects of 30 minutes anoxia on the mouse brain. However, in mice receiving the placebo treatment (saline), the percentage of infarcted area markedly increased; presumably this represents failure of recovery of blood flow in the hypoperfused areas surrounding the primarily infarcted tissues. In contrast, when the mice received Diannexin the infarcted area did not increase between 24 and 72 hours, and was less at 72 hours than in the controls that had received saline (see Figure 5).

[00164] The edema, as measured by Evans blue extravasation, also increased between 24 and 72 hours in the saline-treated animals, whereas the increase in edema was minimal in Diannexin-treated animals (see Figure 5). At 72 hours edema was lower in Diannexin-treated animals than in controls that had received saline. This inhibition is associated with a significant decrease of FXII activation and binding in perfused brains, quantified by Western blots, in Dinnexin-treated animals.

[00165] The most remarkable effect of Diannexin was the reduction in rates of

hemorrhage following reperfusion in the heterozygous SOD2-KO mice (Table 3). Areas of hemorrhage were easily identified, and the results are unambiguous. In the saline treated controls, 68% showed hemorrhage, whereas of the Diannexin-treated animals, only 20% showed hemorrhage. Since Diannexin exerts potent antithrombotic activity, it might be expected to increase hemorrhage, especially in mice genetically engineered to have high hemorrhage rates during brain reperfusion. The observed reduction in hemorrhage rates in Diannexin-treated mice shows that the protein has minimal effects on hemostatic mechanisms, as expected of an inhibitor of FXII, and as observed in clinical trials in humans. By preserving vascular integrity during reperfusion, Diannexin actually decreased hemorrhage, a major complication of reperfusion in stroke patients. All these observations indicate that Diannexin is therapeutically Attorney Docket: 0245.90/02PCT efficacious in attenuating the complications of thrombotic strokes, in part because of the inhibition of FXII activation.

[00166] Table 3. Hemorrhage rates in the brains of heterozygous SOD2-KO mice following 30 minutes middle cerebral artery occlusion and 72 hours reperfusion.

* p <0.0001

[00167] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

[00168] The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the invention to the form disclosed. The scope of the present invention is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments described and shown in the figures were chosen and described in order to best explain the principles of the invention, the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. Attorney Docket: 0245.90/02PCT

SEQUENCES

SEQ ID NO: 1 ANNEXIN I MONOMER

MAMVSEFLKQ AWFIENEEQE YVQTVKSSKG GPGSAVSPYP TFNPSSDVAA LHKAIMVKGVDEATIIDILT KRNNAQRQQI KAAYLQETGK PLDETLKKAL TGHLEEVVLA LLKTPAQFDADELRAAMKGL GTDEDTLIEI LASRTNKEIR DINRVYREEL KRDLAKDITS DTS GDFRN ALLS LAKGDRS E DFGVNEDLAD SDARALYEAG ERRKGTDVNV FNTILTTRSY PQLRRVFQKYTKYSKHDMNK VLDLELKGDI EKCLTAIVKC ATSKPAFFAE KLHQAMKGVG TRHKALIRIM VSRSEIDMND IKAFYQKMYG ISLCQAILDE TKGDYEKILV ALCGGN

SEQ ID NO: 2 ANNEXIN II MONOMER

MSTVHEILCK LSLEGDHSTP PSAYGSVKAY TNFDAERDAL NIETAIKTKG VDEVTIVNILTNRS N AQRQD IAFAYQRRTK KELASALKSA LSGHLETVIL GLLKTPAQYD ASELKASMKGLGTDEDSLIE IICSRTNQEL QEINRVYKEM YKTDLEKDII SDTSGDFRKL MVALAKGRRAEDGSVIDYEL IDQDARDLYD AGVKRKGTDV PKWISIMTER SVPHLQKVFD RYKS YS P YDMLES IRKEVKG DLENAFLNLV QCIQNKPLYF ADRLYDSMKG KGTRDKVLIR IMVSRSEVDM LKIRSEFKRK YGKSLYYYIQ QDTKGDYQKA LLYLCGGDD

SEQ ID NO: 3 ANNEXIN III MONOMER

MAS IW VGHRG TVRDYPDFSP SVDAEAIQKA IRGIGTDEKM LISILTERSN AQRQLIVKEYQAAYGKELKD DLKGDLSGHF EHLMVALVTP PAVFDAKQLK KSMKGAGTNE DALIEILTTRTSRQMKDISQ AYYTVYKKSL GDDISSETSG DFRKALLTLA DGRRDES LKV DEHLAKQDAQILYKAGENRW GTDEDKFTEI LCLRSFPQLK LTFDEYRNIS QKDIVDSIKG ELSGHFEDLLLAIVNCVRNT PAFLAERLHR ALKGIGTDEF TLNRIMVSRS EIDLLDIRTE FKKHYGYSLYSAIKSDTSGD YEITLLKICG GDD Attorney Docket: 0245.90/02PCT

SEQ ID NO: 4 ANNEXIN IV MONOMER

M AM ATKGGT VKA AS GFN AMED AQTLRKAMKGLGTDED AIIS VLA YRNTAQ RQEIRTAYKSTIGRDLIDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRA MKG AGTDEGCLIEILAS RTPEEIRRIS QT YQQQ YGRRLEDDIRS DTS FMF QRVLVS LS AGGRDEGN YLDD ALVRQD AQDLYE AGEKKWGTDE VKFLT VLC S RNRNHLLH VFDE YKRIS QKDIEQS IKS ETS GS FED ALLAIVKCMRNKS A YFAEKLYKS MKGLGTDDNTLIR VM VS R AEIDMLDIR AHFKRLYGKS LYS F IKGDTS GD YRKVLLVLCGGDD

SEQ ID NO: 5 ANNEXIN V MONOMER

MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQ EISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALK G AGTNEKVLTEIIAS RTPEELR AIKQ V YEEE YGS S LEDD V VGDTS G Y YQR MLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTR S VS HLRKVFDKYMTIS GFQIEETIDRETS GNLEQLLLA V VKS IRS IP A YL AETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIK GDTS GD YKKALLLLCGEDD

SEQ ID NO: 6 ANNEXIN VI MONOMER

MAKPAQGAKY RGSIHDFPGF DPNQDAEALY TAMKGFGSDK EAILDIITSR SNRQRQEVCQSYKSLYGKDL IADLKYELTG KFERLIVGLM RPPAYCDAKE IKDAISGIGT DEKCLIEILAS RTNEQMHQL VAAYKDAYER DLEADIIGDT SGHFQKMLVV LLQGTREEDD VVSEDLVQQDVQDLYEAGEL KWGTDEAQFI YILGNRSKQH LRLVFDEYLK TTGKPIEASI RGELSGDFEKLMLAVVKCIR STPEYFAERL FKAMKGLGTR DNTLIRIMVS RSELDMLDIR EIFRTKYEKS LYS MIKNDTS GEYKKTLLKL SGGDDDAAGQ FFPEAAQVAY QM WELS AVAR VELKGTVRPANDFNPDADAK ALRKAMKGLG TDEDTIIDII THRSNVQRQQ IRQTFKSHFG RDLMTDLKSEISGDLARLIL GLMMPPAHYD AKQLKKAMEG AGTDEKALIE ILATRTNAEI RAINEA YKED YHKS LED ALS SDTSGHFRRI LISLATGHRE EGGENLDQAR EDAQVAAEIL EIADTPSGDKTSLETRFMTI Attorney Docket: 0245.90/02PCT

LCTRSYPHLR RVFQEFIKMT N YD VEHTIKKEMS GD VRD AFV AIVQS VKNKPLF FADKLYKSMKGAGTDEKTLTRIMVSRS EIDLLNIRRE FIEKYDKSLH QAIEGDTSGDFLKALLALCG GED

SEQ ID NO: 7 ANNEXIN VII MONOMER

MSYPGYPPTG YPPFPGYPPA GQESSFPPSG QYPYPSGFPP MGGGAYPQVP SSGYPGAGGY PAPGGYPAPG GYPGAPQPGG APSYPGVPPG QGFGVPPGGA GFSGYPQPPS QSYGGGPAQV PLPGGFPGGQ MPSQYPGGQP TYPSQPATVT QVTQGTIRPA ANFDAIRDAE ILRKAMKGFG TDEQAIVDVV ANRSNDQRQK IKAAFKTSYG KDLIKDLKSE LSGNMEELIL ALFMPPTYYD AWS LRKAMQG AGTQERVLIE ILCTRTNQEI REIVRCYQSE FGRDLEKDIR SDTSGHFERL LVSMCQGNRD ENQSINHQMA QEDAQRLYQA GEGRLGTDES CFNMILATRS FPQLRATMEA YSRMANRDLL SSVSREFSGY VESGLKTILQ CALNRPAFFA ERLYYAMKGA GTDDSTLVRI VVTRSEIDLV QIKQMFAQMY QKTLGTMIAG DTSGDYRRLL LAIVGQ

SEQ ID NO: 8 ANNEXIN VIII MONOMER

MAWWKAWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTK RS NTQRQQIAKS FKAQFGKDLTETLKS ELS GKFERLIV ALM YPP YRYE AK ELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQAD TSGYLERILVCLLQGSRDDVSSFVDPALALQDAQDLYAAGEKIRGTDEMK FITILCTRS ATHLLR VFEEYEKIANKS IEDS IKS ETHGS LEEAMLT V VKC TQNLHS YF AERLY Y AMKG AGTRDGTLIRNI VS RS EIDLNLIKCHFKKM YG KTLS S MIMEDTS GD YKN ALLS LVGS DP

SEQ ID NO: 9 ANNEXIN XI MONOMER

MSYPGYPPPP GGYPPAAPGG GPWGGAAYPP PPSMPPIGLD NVATYAGQFN QDYLSGMAANMSGTFGGANM PNLYPGAPGA GYPPVPPGGF GQPPSAQQPV PPYGMYPPPG GNPPSRMPSYPPYPGAPVPG QPMPPPGQQP PGAYPGQPPV TYPGQPPVPL PGQQQPVPSY PG YPGS GTVTP A VPPTQFGS RGTITDAPGF DPLRDAEVLR KAMKGFGTDE QAIIDCLGSR SNKQRQQILLSFKTAYGKDL Attorney Docket: 0245.90/02PCT

IKDLKSELSG NFEKTILALM KTPVLFDIYE IKEAIKGVGT DEACLIEILASRSNEHIREL NRAYKAEFKK TLEEAIRSDT SGHFQRLLIS LSQGNRDEST NVDMSLAQRD AQELYAAGEN RLGTDESKFN AVLCSRSRAH LVAVFNEYQR MTGRDIEKSI CREMSGDLEEGMLAVVKCLK NTPAFFAERL NKAMRGAGTK DRTLIRIMVS RSETDLLDIR S E YKRM YGKS LYHDIS GDTS GDYRKILLKI CGGND

SEQ ID NO: 10 ANNEXIN XIII MONOMER

MGNRHAKASS PQGFDVDRDA KKLNKACKGM GTNEAAIIEI LSGRTSDERQ QIKQKYKATYGKELEEVLKS ELSGNFEKTA LALLDRPSEY AARQLQKAMK GLGTDESVLI EVLCTRTNKEIIAIKEAYQR LFDRSLESDV KGDTS GNLKK ILVSLLQANR NEGDDVDKDL AGQDAKDLYDAGEGRWGTDE LAFNEVLAKR SYKQLRATFQ AYQILIGKDI EEAIEEETSG DLQKAYLTLVRCAQDCEDYF AERLYKSMKG AGTDEETLIR IVVTRAEVDL QGIKAKFQEK YQKS LS DM VRS DTS GDFRKL LVALLH

SEQ ID NO: 111 ILLUSTRATIVE LINKER GSLEVLFQGPSG

SEQ ID NO: 112 ANNEXIN IV HOMODIMER WITH LINKER

M AM ATKGGT VKA AS GFN AMED AQTLRKAMKGLGTDED AIIS VLA YRNTAQ RQEIRTAYKSTIGRDLIDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRA MKG AGTDEGCLIEILAS RTPEEIRRIS QTYQQQ YGRRLEDDIRS DTS FMF QRVLVS LS AGGRDEGN YLDD ALVRQD AQDLYE AGEKKWGTDE VKFLT VLC S RNRNHLLH VFDE YKRIS QKDIEQS IKS ETS GS FED ALLAIVKCMRNKS A YFAEKLYKS MKGLGTDDNTLIR VM VS R AEIDMLDIR AHFKRLYGKS LYS F IKGDTSGDYRKVLLVLCGGDDGSlevlfqgpSOfiTLAMATKGGTVKAASGF

NAMED AQTLRKAMKGLGTDED AIISVLAYRNTAQRQEIRTAYKSTIGRDL IDDLKSELSGNFEQVIVGMMTPTVLYDVQELRRAMKGAGTDEGCLIEILA S RTPEEIRRIS QTYQQQ YGRRLEDDIRS DTS FMFQRVLVS LS AGGRDEGN YLDD ALVRQD AQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVFDEYKR Attorney Docket: 0245.90/02PCT

IS QKDIEQS IKS ETS GS FED ALLAIVKCMRNKS A YF AEKLYKS MKGLGTD DNTLIRVMVSRAEIDMLDIRAHFKRLYGKSLYSFIKGDTSGDYRKVLLVLCGGDD

SEQ ID NO: 113 ANNEXIN V HOMODIMER WITH LINKER

MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQR QEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHAL KG AGTNEKVLTEIIAS RTPEELR AIKQ V YEEE YGS S LEDD V VGDTS G Y YQ RMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGT RS VS HLRK VFD KYMTIS GFQIEETIDRETS GNLEQLLLA V VKS IRS IP AY LAETLY Y AMKG AGTDDHTLIR VM VS RS EIDLFNIRKEFRKNFATS L YS MI KGDTSGDYKKALLLLCGEDDGSLEVLFOGPSGKLAQVLRGTVTDFPGFDE RAD AETLRKAMKGLGTDEES ILTLLTS RS N AQRQEIS A AFKTLFGRDLLD DLKS ELTGKFEKLIV ALMKPS RLYD A YELKHALKG AGTNEKVLTEIIAS R TPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGI DEAQ VEQD AQ ALFQ AGELKWGTDEEKFITIFGTRS VS HLRK VFD KYMTIS GFQIEETIDRETS GNLEQLLLA V VKS IRS IP A YLAETLY Y AMKG AGTDDH TLIRVM VS RS EIDLFNIRKEFRKNFATS LYS MIKGDTS GD YKK ALLLLCG EDD

SEQ ID NO: 114 ANNEXIN VIII HOMODIMER WrfH LINKER

MAWWKAWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTK RS NTQRQQIAKS FKAQFGKDLTETLKS ELS GKFERLIV ALM YPP YRYE AK ELHDAMKGLGTKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQAD TSGYLERILVCLLQGSRDDVSSFVDPALALQDAQDLYAAGEKIRGTDEMK FITILCTRS ATHLLR VFEEYEKIANKS IEDS IKS ETHGS LEEAMLT V VKC TQNLHS YF AERLY Y AMKG AGTRDGTLIRNI VS RS EIDLNLIKCHFKKM YG KTLS S MIMEDTS GD YKN ALLS LVGS DPGSLE VLFQGPSGfiTLA WWKA WIEQ

EGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTKRSNTQRQQIA KSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLG TKEGVIIEILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILV CLLQGS RDD VS S F VDP ALALQD AQDLY A AGEKIRGTDEMKFITILCTRS A Attorney Docket: 0245.90/02PCT

THLLRVFEEYEKIANKS IEDS IKS ETHGS LEE AMLT V VKCTQNLHS YF AE

RLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKMYGKTLSSMIMED

TSGDYKNALLSLVGSDP

SEQIDNO: 115 LINKER

GGGGS

SEQIDNO: 116 LINKER EAAAK