WO2011069090A1 - Inhibition de l'activation du facteur xii de la coagulation par des ligands de phosphatidylsérine - Google Patents

Inhibition de l'activation du facteur xii de la coagulation par des ligands de phosphatidylsérine Download PDF

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WO2011069090A1
WO2011069090A1 PCT/US2010/058937 US2010058937W WO2011069090A1 WO 2011069090 A1 WO2011069090 A1 WO 2011069090A1 US 2010058937 W US2010058937 W US 2010058937W WO 2011069090 A1 WO2011069090 A1 WO 2011069090A1
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annexin
shock
binding agent
fxii
activation
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PCT/US2010/058937
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English (en)
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Anthony Allison
Gordon Ringold
James L. Zehnder
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Alavita Pharmaceuticals, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention provides compositions and methods for treatment in medicine and surgery.
  • Blood coagulation factor XII (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.
  • anionic surfaces such as silicates, dextran sulfate, sulfatides, kaolin, glass, ellagic acid, articular cartilage, skin, fatty acids, endotoxin, amyloid protein, and heparins.
  • 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).
  • a-FXIIa Factor Xlla
  • PK prekallikrein
  • CI esterase a subunit of the complement cascade
  • a-FXIIa is cleaved by plasma kallikrein forming ⁇ -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.
  • 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.
  • compositions and methods of treatment in medicine and in surgery are compositions and methods of treatment in medicine and in surgery.
  • 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.
  • PS phosphatidylserine
  • embodiments herein include methods for treating acute angioedema attacks, pain, sickle cell crisis, acute renal failure, and vasculitis.
  • 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.
  • 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.
  • 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.
  • compositions are provided that are useful in treatment of the FXII related diseases or conditions.
  • Figure 1 demonstrates that treatment with Diannexin prolonged clot initiation time following exposure to Kaolin.
  • Figure 2 demonstrates that treatment with Diannexin increased time to reach a specific clot strength following exposure to Kaolin.
  • Figure 3 demonstrates that treatment with Diannexin attenuated the maximum rate of thrombin generation following exposure to Kaolin.
  • Figure 4 demonstrates that treatment with Diannexin prolonged the time to reach the maximum rate of thrombin generation after exposure to Kaolin.
  • 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.
  • 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.
  • PS Phosphatidylserine
  • Factor XII Factor XI
  • 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).
  • 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
  • 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).
  • secreted isoforms of phospholipase A 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.
  • 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.
  • 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.
  • Nieswandt and Renne U.S.
  • compositions and methods of treatment in medicine and in surgery are 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.
  • 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.
  • 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.
  • 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.
  • SIRS systemic inflammatory response syndrome
  • a disease or condition associated with FXII activation can be treated by administering to a patient in need thereof a PS binding agent.
  • 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.
  • a disease or condition associated with FXII activation can be treated by administering to a patient in need thereof an annexin.
  • a method of inhibiting FXII activation comprises administering to a patient in need thereof another PS binding agent.
  • 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.
  • SIRS systemic inflammatory response syndrome
  • DIC disseminated intravascular coagulation
  • hypotension hypotension
  • other pathogenic mechanisms involved in septic, traumatic, or hemorrhagic shock are provided.
  • SIRS Systemic Inflammatory Response Syndrome
  • 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 is a state of physiological derangements in which organ function is not capable of maintaining homeostasis.
  • 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.
  • cytokines 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.
  • the numerous cascades and the reticuloendothelial system are activated with subsequent loss of circulatory integrity.
  • SIRS 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- ⁇ inhibitor, activation of NF- ⁇ , 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).
  • CRP C-reactive protein
  • 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.
  • 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.
  • Inhibition of FXII activation as described herein is useful in preventing SIRS, treating SIRS, and mitigating the effects of SIRS.
  • 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.
  • 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.
  • 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.
  • Proteins C and S are also part of the anticoagulant compensatory mechanism.
  • 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.
  • 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.
  • tissue factor pathway inhibitor TFPI
  • TFPI tissue factor pathway inhibitor
  • 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.
  • PAI-1 plasminogen activator inhibitor- 1
  • 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.
  • 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.
  • DIC is typically a complication or an effect of progression of other clinical conditions generally involving activation of systemic inflammation:
  • 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.
  • Inhibition of FXII activation as described herein is useful in preventing DIC, treating DIC, and mitigating the effects of DIC.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ECs endothelial cells
  • MPs microparticles
  • Tissue plasminogen activator facilitates conversion of plasminogen to plasmin, a natural fibrinolytic.
  • Endotoxins impair fibrinolysis by increasing the activity of fibrinolysis inhibitors including plasminogen activator inhibitor (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI).
  • PAI-1 plasminogen activator inhibitor
  • TAFI thrombin activatable fibrinolysis inhibitor
  • 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.
  • 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
  • Hypovolemic shock is characterized by a greater than 15% decrease in
  • Hypovolemic shock can be caused by hemorrhage, burns, and severe dehydration.
  • 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.
  • 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.
  • 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
  • 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.
  • Inhibition of FXII activation as described herein is useful in preventing shock, treating shock, and mitigating the effects of shock.
  • 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.
  • 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.
  • 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
  • Bi receptor G-protein-coupled receptors, bradykinin Bi and B 2 .
  • 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- ⁇ ⁇ and/or TNF-a.
  • 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).
  • 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.
  • 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).
  • kinins major mediators of which are kinins, prostacyclin, and lysophosphatidic acid (LP A).
  • LP A lysophosphatidic acid
  • ARF acute renal failure
  • 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.
  • GFR glomerular filtration rate
  • tubular necrosis leads to tubular obstruction and increased tubular permeability.
  • 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.
  • 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.
  • Acute renal failure can also be caused by actual damage to nephrons and renal parenchyma, i.e. intrarenal damage.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • inflammation and vasculitis treating vascular inflammation and vasculitis, and mitigating the effects of vascular inflammation and vasculitis, including, for example, vascular permeability.
  • 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 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
  • 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.
  • 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.
  • 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.
  • compositions are provided herein having activity which prevents activation of
  • the composition prevents activation of FXII by exposure to negatively charged surfaces of cells and of micropapticles shed from them.
  • agents that inhibit FXII activation include agents that bind PS or "PS binding agents".
  • inhibitors refers to any decrease in FXIIa activity relative to FXIIa activity in the absence of the agents(s), including partial decrease and complete inhibition.
  • 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.
  • PS binding agent is any molecule that binds to PS
  • inhibition can occur because the binding agent is bound to PS.
  • the binding agent becomes attached to a Attorney Docket: 0245.90/02PCT molecule associated with PS, for example a lipid raft constituent.
  • this inhibition restrains or retards physiologic, chemical, or enzymatic action between PS and PS interacting molecules.
  • a binding agent blocks, restricts, or interferes with a particular chemical reaction or other biologic activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • immunoblot assays immunoprecipitation assays, radioimmunoassays, enzyme immunoassays, immunofluorescent antibody assays, immunoelectron microscopy and binding to cells or liposomes with PS on their surfaces.
  • 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.
  • 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+ .
  • 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).
  • 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.
  • 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.
  • 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.
  • 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, ⁇ 5 ⁇ 3 integrin and other integrins, CD3, CD4, CD14, CD93, SRB (CD36), SRC, PSOC and PSr, as well as the proteins, polypeptides, and peptides thereof.
  • 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.
  • 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.
  • two ligands are coupled to an Fc portion such that they form a dimer.
  • 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.
  • 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.
  • 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.
  • 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 .
  • the annexin V - annexin V dimer was shown to inhibit venous thrombosis in rats.
  • 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.
  • 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
  • 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.
  • the PS binding agent is a modified annexin.
  • 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.
  • Annexins include proteins of the annexin family, such as Annexin I, Annexin II
  • Chromobindin 20 p68, p70
  • Annexin VII Synexin
  • Annexin VIII VAC-beta
  • Annexin XI CAP-50
  • Annexin XIII ISA
  • 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.
  • Annexin I 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • Annexin XIII (SEQ ID NO: 10) is associated with the plasma membrane of undifferentiated, proliferating endothelial cells and differentiated villus enterocytes.
  • annexin proteins are modified to increase their half-life in humans or other mammals.
  • 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.
  • 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.
  • 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.
  • 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.
  • the annexin protein is annexin V, annexin IV or annexin VIII.
  • 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.
  • 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).
  • 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.
  • pegylation covalent attachment
  • the superior clinical efficacy of recombinant products coupled to PEG is established.
  • 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.
  • 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.
  • the dimer of annexin is a homodimer of annexin V, annexin IV or annexin VIII.
  • 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).
  • 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.
  • two or more annexins are linked directly to each other. In other embodiments, two or more annexins are linked together by a fusion segment.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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.
  • Exemplary embodiments of two annexin monomers joined by a linker include
  • compositions described herein can be administered in the form of a
  • composition comprising the composition and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Such a composition is sometimes referred to as a "therapeutic composition”. Aspects of potential therapeutic compositions are described herein.
  • a patient can be a human or non-human.
  • the patient suffers from or is at risk of developing a disease or condition associated with activation of FXII (see above).
  • compositions that also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
  • 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.
  • buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof
  • 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.
  • 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.
  • 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.
  • the composition is a modified annexin protein which can be administered in a range of about 50 to about 500 ⁇ g/kg, for example, about 200, or about 300, or about 400 ⁇ g/kg ⁇ g PS-binding agent/kg of patient's weight).
  • compositions described herein 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.
  • 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.
  • 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.
  • the method comprises intravenous administration of the agent or composition.
  • the method comprises administration by bolus injection.
  • compositions can be in the form of sterile injectable preparations or aerosol sprays allowing absorption through the nasal mucosa or lungs.
  • compositions for administration by inhalation or aerosol, can be prepared according to techniques well-known in the art of pharmaceutical formulations.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • isotonic agents are desirable, and include sugar, buffer, or sodium chloride.
  • a controlled release formulation that is capable of slowly releasing a composition into a patient.
  • 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.
  • 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.
  • 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.
  • 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.
  • the PS binding agent is an annexin monomer such as an annexin V monomer.
  • 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.
  • 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.
  • the materials can be administered simultaneously or alternating as determined by the health care professional.
  • a modified annexin V composition can be combined with an anti-FXII antibody and administered on the same regimen or at different times.
  • 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.
  • 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.
  • 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 ⁇ g/mL (13 nM), 0.5 ⁇ g/mL (6.5 nM), 0.25 ⁇ g/mL (3.3 nM), 1.25 ⁇ g/mL (1.6 nM).
  • a control sample was also prepared without Diannexin. 340 ⁇ ⁇ of each prepared sample was immediately loaded into a TEG assay cup with 20 ⁇ L ⁇ CaCl 2 and
  • 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 1 demonstrates that Diannexin inhibits FXII-dependent blood coagulation.
  • 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
  • MPI Permeability Index
  • a PS ligand such as Diannexin counteracts the increase in vascular permeability, ultimately preventing edema that occurs during post-ischemic reperfusion.
  • 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.
  • SOD2-KO mice 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.
  • MMP9 matrix metalloproteinase-9
  • 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.
  • 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.
  • Diannexin 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.

Abstract

L'invention porte sur des compositions et des procédés pour le traitement de maladies et états associées au Facteur XII. Des compositions à titre d'exemples contiennent un dimère de l'annexine ou autre agent de liaison à la phosphatidylsérine (PS).
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