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US 20140227350A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0227350 A1 Wang et al. (43) Pub. Date: Aug. 14, 2014

(54) ANNEXIN A2 AND TISSUE PLASMINOGEN Publication Classification ACTIVATOR FORTREATING VASCULAR DISEASE (51) Int. Cl. A638/49 (2006.01) (71) Applicant: The General Hospital Corporation, A638/17 (2006.01) Boston, MA (US) (52) U.S. Cl. CPC ...... A61K 38/49 (2013.01); A61K 38/17 (72) Inventors: Xiaoying Wang, West Roxbury, MA (2013.01) (US); Eng Lo, Newton, MA (US) USPC ...... 424/450; 424/94.64; 424/94.3 (21) Appl. No.: 14/197.988 (57) ABSTRACT The use of thA to treat hemorrhagic transformation, neuro toxicity has been limited to short treatment time windows (22) Filed: Mar. 5, 2014 because a high dose of t?A required to generate Sufficient amounts of the enzyme plasmin for clot lysis. The present Related U.S. Application Data invention combines tRA with recombinant Annexin A2 result ing in without hemorrhagic transformation at (63) Continuation of application No. 12/918,726, filed on delayed times after stroke. This embodiment allows the Oct. 20, 2010, now abandoned, filed as application No. administration of a lower, non-neurotoxic, tRA dose. Our PCT/US09/01057 on Feb. 19, 2009. results suggest this novel combination for stroke therapy may (60) Provisional application No. 61/030,033, filed on Feb. greatly improve both efficacy and safety, and prolong tPA 20, 2008. therapeutic time window. Patent Application Publication Aug. 14, 2014 Sheet 1 of 6 US 2014/0227350 A1

A.

a 14 Ces 12 10 E E

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Figure 1 Patent Application Publication Aug. 14, 2014 Sheet 2 of 6 US 2014/0227350 A1

m L-tpA - H-tRPA - L-toA -- A2 : 4. O 2

O 3O 6O 90 12O 5 LS 30 60 lschemia (min) After Treatment (min)

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FIGURE 2 Patent Application Publication Aug. 14, 2014 Sheet 3 of 6 US 2014/0227350 A1

Safe HA -it k r.

FIGURE 3 Patent Application Publication Aug. 14, 2014 Sheet 4 of 6 US 2014/0227350 A1

-ArcA.

Figure 3 (cont'd) Patent Application Publication Aug. 14, 2014 Sheet 5 of 6 US 2014/0227350 A1

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Pre-ischemia 5 Ischemia (JThrombolysis

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FIGURE 4

US 2014/0227350 A1 Aug. 14, 2014

ANNEXIN A2 AND TISSUE PLASMINOGEN medium comprising Annexin A2 and tissue plasminogen acti ACTIVATOR FORTREATING VASCULAR vator (tPA); and b) administering said medium to said patient DISEASE under conditions such that said symptoms are reduced. In one embodiment, the vascular disorder is selected from the group FIELD OF INVENTION comprising stroke, , pulmonary embo 0001. The present invention is related to the field of vas lism, deep vein or intracerebral hematoma. In one cular disorders. In particular, the present invention is related embodiment, the Annexin A2 and the tRA have a dose ratio of to the treatment and management of diseases including, but 2:1. In one embodiment, the medium comprises a carrier. In not limited to, stroke, myocardial infarction, deep vein throm one embodiment, the Annexin A2 and the tBA are attached to bosis, or pulmonary embolism. For example, a patient having the carrier. In one embodiment, the carrier is selected from the Suffered a vascular disorder may be administered a composi group comprising a liposome or a microparticle. In one tion comprising tPA and Annexin A2. In Such cases, the tBA embodiment, the medium comprises a liquid. In one embodi dose may be reduced Such that the risk of hemorrhagic side ment, the administering is intravenous. In one embodiment, effects are minimal. the patient is a human. In one embodiment, the patient is a non-human. BACKGROUND 0007. In one embodiment, the present invention contem plates a method comprising: a) providing: i) a patient exhib 0002. Each year, about 600,000 American suffer from iting symptoms associated with a recently incurred stroke, ii) stroke. Thrombolytic therapy with tissue plasminogen acti a medium comprising Annexin A2 and tissue plasminogen vator (tPA) is the only FDA-approved medicine for achieving activator (tPA), wherein Annexin A2 and tRA have a dose ratio both vascular reperfusion and clinical benefit, but only 2-5% of 2:1; and, b) administering said medium to said human of stroke patients receive tea in the US. In part, this because Subject under conditions such that said symptoms are tPA therapy unfortunately increases the risk of intracerebral reduced. In one embodiment, the administering occurred less hemorrhage by approximately 10-fold. Perhaps even more than three hours after said stroke. In one embodiment, the importantly, there is accumulating evidence from experimen administering occurred less than six hours after said stroke. In tal models and clinical studies that tRA can have neurotoxic one embodiment, the administering occurred less than twelve actions separate from its beneficial clot lysis properties, tRA hours after said stroke. In one embodiment, the tBA dose is at neurotoxicity may further exacerbate ischemic brain damage, least two-fold lower than the currently recommended dose. In particularly in the 50% of patients who have no perfusion one embodiment, the tRA dose is at least three-fold lower than improvement after receiving intravenous tFA. the currently recommended dose. In one embodiment, the tRA 0003. Many clinical trials attempting to provide neuropro dose is at least four-fold lower than the currently recom tection following stroke have failed. While, to date, tRA mended dose. based thrombolytic therapy is the only FDA-approved treat 0008. In one embodiment, the present invention contem ment for achieving vascular reperfusion and clinical benefit, plates a medium comprising Annexin A2 and tissue plasmi this agent is given to only about 2-5% of stroke patients (25. nogen activator (tPA). In one embodiment, the Annexin A2 26). This may be related, in part, to the elevated risks of and the tBA have a dose ratio of 2:1. In one embodiment, the symptomatic intracranial hemorrhage, and a short therapeutic medium further comprises a carrier. In one embodiment, the time window in order to decrease the clinical risk of tRA's Annexin A2 and the tBA are attached to the carrier. In one limitations. Specifically, tRA therapy limitations include: (1) embodiment, the carrier comprises a liposome population. In short 3 hr treatment time window, (2) risk of intracerebral hemorrhage, and (3) neurotoxicity. Others have tried to find one embodiment, the carrier comprises a microparticle popu other lytics with equal thrombolysis properties for safe and lation. In one embodiment, the medium comprises a liquid. effective reperfusion at longer times after stroke onset. One 0009. In one embodiment, the present invention contem example is the vampire bat saliva molecule desmoteplase. plates a kit comprising a medium comprising Annexin A2 and However, the recent completion of a desmoteplase (DIAS-2) tissue (tPA). In one embodiment, the clinical trial failed. So the problem of a safe and effective use medium further comprises a carrier. In one embodiment, the oftBA in the treatment of stroke remains unsolved. tPA and the Annexin A2 are attached to said carrier. In one 0004 What is needed is a composition and method that embodiment, the kit further comprises a sheet of instructions increases the thrombolytic efficacy of tA, while reducing regarding administration of said medium following a vascular neurotoxicity and the risk of hemorrhagic transformation. disorder. In one embodiment, the vascular disorder is selected from the group comprising stroke, myocardial infarction, SUMMARY pulmonary embolism, deep vein thrombosis or intracerebral hematoma. In one embodiment, the kit further comprises a 0005. The present invention is related to the field of vas Syringe. In one embodiment, the kit further comprises an cular disorders. In particular, the present invention is related intravenous catheter. In one embodiment, the kit further com to the treatment and management of diseases including, but prises an intravenous drip bag capable of fluid communica not limited to, stroke, myocardial infarction, deep vein throm tion with said intravenous catheter. bosis, or pulmonary embolism. For example, a patient having Suffered a vascular disorder may be administered a composi DEFINITIONS tion comprising Annexin A2 and tRA. In Such cases, the tBA dose may be reduced Such that the risk of hemorrhagic side 0010. The term “attached as used herein, refers to any effects are minimal. interaction between a medium (or carrier) and a drug. Attach 0006. In one embodiment, the present invention contem ment may be reversible or irreversible. Such attachment plates a method comprising: a) providing: i) a patient exhib includes, but is not limited to, covalent bonding, ionic bond iting symptoms associated with a vascular disorder, and ii) a ing, Van der Waals forces or friction, and the like. A drug is US 2014/0227350 A1 Aug. 14, 2014

attached to a medium (or carrier) if it is impregnated, incor compound has its intended effect on the patient. For example, porated, coated, in Suspension with, in Solution with, mixed one method of administering is by an indirect mechanism with, etc. using a medical device Such as, but not limited to a Syringe, an 0011. The term “medium' as used herein, refers to any intravenous catheter, etc. A second exemplary method of material, or combination of materials, which may serve as administering is by a direct mechanism Such as, local tissue vehicle for delivering of a drug, or carrier, to a treatment point administration (i.e., for example, extravascular placement), (e.g., a thrombosis, a stenosis etc.). Preferably, a medium is oral ingestion, transdermal patch, topical, inhalation, Sup selected from the group including, but not limited to, liquids, pository etc. foams, or gels (including, but not limited to, hydrogels). In 0018. The term “biocompatible’, as used herein, refers to Some cases a medium constitutes a drug delivery system that any material does not elicita Substantial detrimental response provides a controlled and Sustained release of drugs over a in the host. There is always concern, when a foreign object is period of time lasting approximately from 1 day to 6 months. introduced into a living body, that the object will induce an Alternatively, the controlled and Sustained drug release is immune reaction, such as an inflammatory response that will from a carrier mixed within the medium. have negative effects on the host. In the context of this inven 0012. The term “carrier as used herein, refers to any tion, biocompatibility is evaluated according to the applica material capable of attaching a drug or composition wherein tion for which it was designed: for example; a bandage is a medium facilitates delivery of the carrier to a treatment regarded a biocompatible with the skin, whereas an implanted point. Preferably, a carrier is selected from the group includ medical device is regarded as biocompatible with the internal ing, but not limited to, liposomes, Xerogels, or microparticles tissues of the body. Preferably, biocompatible materials (i.e., microspheres, liposomes, microcapsules etc.). Any car include, but are not limited to, biodegradable and biostable rier contemplated by this invention may comprise a con materials. trolled release formulation. (0019. The term “local delivery” as used herein, refers to 0013 The term “xerogel as used herein, refers to any any drug or compound that is placed on or near a tissue device comprising a combination of silicone and oxygen surface without systemic distribution. The tissue surface having a plurality of air bubbles and an entrapped drug. The includes, but is not limited to, the external skin or any internal resultant glassy matrix is capable of a controlled release of an tissue (i.e., for example, the periadvential blood vessel) and/ entrapped drug during the dissolution of the matrix. or organ Surface. 0014. The term “foam’ as used herein, refers to a disper 0020. The term “antiplatelets' or “ as sion in which a large proportion of gas, by volume, is in the used herein, refers to any drug that prevents aggregation of form ofgas bubbles and dispersed within a liquid, Solid or gel. platelets or fibrin formation (i.e., for example as a prior event The diameter of the bubbles are usually relatively larger than to adhesion formation). For example, an antiplatelet drug the thickness of the lamellae between the bubbles. comprises an inhibitor of glycoprotein IIb/IIIa (GPIb/IIIa). 0015 The term “gel” as used herein, refers to any material Further a GPIIb/IIIa inhibitor includes, but is not limited to, forming, to various degrees, a medium Viscosity liquid or a Xemilofiban, (ReoProR) cromafiban, elarofiban, jelly-like product when Suspended in a solvent. A gel may orbofiban, roxifiban, sibrafiban, RPR 1098.91, (Ag also encompass a solid or semisolid colloid containing a grastat(R), (IntegrilinR), UR-4033, UR-3216 or certain amount of water. These colloid solutions are often UR-2922. referred to in the art as hydrosols. One specific type of gel is 0021. The term, “' or “ drug’ a hydrogel. The term “hydrogel' as used herein, refers to any as used herein, refers to any drug that inhibits or reduces material forming, to various degrees, a jelly-like product thrombi formation and include, but are not limited to, biva when Suspended in a solvent, typically water or polar solvents lirudin, , , hirulog, , , comprising such as, but not limited to, gelatin and pectin and efegatran, or thrombomodulin. fractions and derivatives thereof. Typically, a hydrogel is 0022. The term, “' or “ drug’ capable of Swelling in water and retains a significant portion as used herein, refers to any drug that inhibits the blood of water within its structure without dissolution. In one coagulation cascade. A typical anticoagulant comprises hep embodiment, the present invention contemplates a gel that is arin, including but not limited to, low molecular weight hep liquid at lower than body temperature and forms a firm gel arin (LMWH) or unfractionated (UFH). Other anti when at body temperature. coagulants include, but are not limited to, tinzaparin, 0016. The term “drug or “compound as used herein, certoparin, parnaparin, nadroparin, ardeparin, enoxaparin, refers to any pharmacologically active Substance capable of reviparin ordalteparin. Specific inhibitors of the blood coagul being administered which achieves a desired effect. Drugs or lation cascade include, but are not limited to, Factor Xa (FXa) compounds can be synthetic or naturally occurring, non inhibitors (i.e., for example, ), Factor IXa peptide, proteins or peptides, oligonucleotides or , (FIXa) inhibitors, Factor XIIIa (FXIIIa) inhibitors, and Fac polysaccharides or Sugars. Drugs or compounds may have tor VIIa (FVIIa) inhibitors. any of a variety of activities, which may be stimulatory or 0023 The term “patient’, as used herein, is a human or inhibitory, such as antibiotic activity, antiviral activity, anti animal (i.e., for example, a dog, cat, horse, cow, pig etc.) and fungal activity, Steroidal activity, cytotoxic, cytostatic, anti need not be hospitalized. For example, out-patients, persons proliferative, anti-inflammatory, analgesic or anesthetic in nursing homes are “patients.” A patient may comprise any activity, or can be useful as contrast or other diagnostic age of a human or non-human animal and therefore includes agents. Drugs or compounds may be capable of reducing both adult and juveniles (i.e., children and/or offspring). It is thromboses and/or adhesions. not intended that the term “patient connote a need for medi 0017. The term “administered’ or “administering a drug cal treatment, therefore, a patient may Voluntarily or involun or compound, as used herein, refers to any method of provid tarily be part of experimentation whether clinical or in Sup ing a drug or compound to a patient such that the drug or port of basic Science studies. US 2014/0227350 A1 Aug. 14, 2014

0024. The term “medical device', as used herein, refers brane. Special solutions that facilitate removal of toxins may broadly to any apparatus used in relation to a medical proce be infused in, remain in the abdomen for a time, and then dure. Specifically, any apparatus that contacts a patient during drained out. a medical procedure or therapy is contemplated herein as a 0028. The term “fixed split-tip dialysis catheter as used medical device. Similarly, any apparatus that administers a herein, refers to any catheter having at least two distinct drug or compound to a patient during a medical procedure or elongated end portions that extend Substantially parallel to therapy is contemplated herein as a medical device. “Direct the longitudinal axis of the catheter and are flexible to the medical implants' include, but are not limited to, urinary and lateral displacement of an infused fluid. It is believed that this intravascular catheters, dialysis catheters, wound drain tubes, flexibility prevents a permanent catheter tip splay that is skin Sutures, vascular grafts and implantable meshes, known to injure tissue. Usually a fixed-tip dialysis catheter intraocular devices, implantable drug delivery systems and provides indwelling vascular access for patients undergoing heart valves, and the like. “Wound care devices' include, but long-term renal dialysis care (i.e., for example, end-stage are not limited to, general wound dressings, non-adherent renal disease). dressings, burn dressings, biological graft materials, tape clo 0029. The term “femoral catheter as used herein, refers to Sures and dressings, Surgical drapes, sponges and absorbable any catheter that is inserted into the femoral vein. Femoral hemostats. “Surgical devices” include, but are not limited to, catheters are typically provided for intermediate term blood Surgical instruments, endoscope systems (i.e., catheters, vas access because the Superior Vena cava is relatively close to the cular catheters, Surgical tools such as scalpels, retractors, and right atrium of the heart, the minimal range of shape changes the like) and temporary drug delivery devices such as drug of these veins with natural movements of the patient (to ports, injection needles etc. to administer the medium. A minimize the damage to the vessel intima), and because of medical device is "coated when a medium comprising a tRA good acceptance by the patients of the skin exit on the tho and Annexin A2 becomes attached to the surface of the medi racic wall. Further, the femoral veins are easy to cannulate, so cal device (either directly or indirectly). Such an indirect that catheters of this invention may be inserted into the femo attachment may result from coating a medical device with a ral veins at the bed side. polymer comprising the medium. This attachment may be 0030 The term “endoscope” refers to any medical device permanent or temporary. When temporary, the attachment that is capable of being inserted into a living body and used for may result in a controlled release of a tRA/rA2 combination. tasks including, but not limited to, observing Surgical proce 0025. The term “dialysis/apheresis catheter as used dures, performing Surgical procedures, or applying medium herein, refers to any multi-lumen catheter (i.e., for example, a to a surgical site. An endoscope is illustrated by instruments triple lumen catheter) capable of providing a simultaneous including, but not limited to, an arthroscope, a laparoscope, withdrawal and return of blood to a patient undergoing a hysteroscope, cytoscope, etc. It is not intended to limit the use blood treatment process. Apheresis (called also pheresis) of an endoscope to hollow organs. It is specifically contem comprises a blood treatment process involving separation of plated that endoscopes, such as an arthroscope or a laparo blood elements that can remove soluble drugs or cellular Scope is inserted through the skin and courses to a closed elements from the circulation. Deisseroth et al., “Use Of Surgical site. Blood And Blood Products, Cancer: Principles And Practice 0031. The term, “microparticle' as used herein, refers to Of Oncology, Devita, V.T. Jr. etal. Editors, Philadelphia: J. B. any microscopic carrier to which a drug or compound may be Lippincott Company 1989, p. 2045-2059. For example, blood attached. Preferably, microparticles contemplated by this is withdrawn from a donor, some blood elements (i.e., for invention are capable of formulations having controlled example, plasma, leukocytes, platelets, etc.) are separated release properties. and retained. The unretained blood elements are then retrans 0032. The term “PLGA as used herein, refers to mixtures fused into the donor. of polymers or copolymers of lactic acid and glycolic acid. As used herein, lactide polymers are chemically equivalent to 0026. The term “dialysis catheter as used herein, refers to lactic acid polymer and glycolide polymers are chemically any device capable of removing toxic Substances (impurities equivalent to glycolic acid polymers. In one embodiment, or wastes) from the body when the kidneys are unable to do PLGA contemplates an alternating mixture of lactide and so. A dialysis catheter may comprise a single catheter having glycolide polymers, and is referred to as a poly(lactide-co at least a dual lumen (i.e., one lumen withdraws arterial blood glycolide) polymer. and a second lumen returns the dialyzed blood to the venous 0033. The term “stenosis” is defined herein as referring to system) or involve placing two catheters-one that is placed in any narrowing of the internal diameter of a biological tissue, an artery, and one in an adjacent vein. Dialysis catheters are Such as a vessel. In particular, Such narrowing is caused by most frequently used for patients who have kidney failure, but phenomenon including, but not limited to, arteriosclerosis, may also be used to quickly remove drugs or poisons in acute scar tissue and/or adhesions. situations. 0034. The term “restenosis” is defined herein as referring 0027. The term “peritoneal dialysis catheter as used to any condition wherein “stenosis', having been treated and herein, refers to any continuous flow catheters with at least at least partially reversed, recurs. two lumens, one of which is a short lumen (used to infuse a 0035. The term “vascular access site' is defined herein as dialysis solution into the peritoneum), and the other of which referring to any percutaneous insertion of a medical device is a long coiled lumen having a plurality of openings, gener into the vasculature. For example, a hemodialysis catheter ally located on the inside of the coil. It is believed that peri placement comprises a vascular access site. Such sites may be toneal Solutes enter into the coiled lumen openings and are temporary (i.e., placed for a matter of hours) or permanent thereby removed from the peritoneum. One hypothesis sug (i.e., placed for days, months or years). gests that peritoneal dialysis works by using the peritoneal 0036. The term "syringe' or “catheter as used herein, membrane inside the abdomen as the semipermeable mem refers to any device or apparatus designed for liquid admin US 2014/0227350 A1 Aug. 14, 2014

istration, as defined herein. A Syringe or catheter may com aggregation. It also may act as a "plasmin activator Such that prise at least one storage vessel (i.e., for example, a barrel) when in contact with a plasminogen activator, the production wherein a single medium resides prior to administration. A of plasmin is increased. Syringe or catheter comprising two or more barrels, each 0046. The term "plasminogen activator” as used herein, containing a separate medium, may mix the media from each refers to any compound (i.e., usually a protein) that is capable barrel prior to administration or the media of each barrel may of converting plasminogen into plasmin (i.e., for example, be administered separately. One of skill in the art will recog tissue plasminogen activator (tPA) or -type plasmi nize that any catheter designed to perform dialysis, as defined nogen activator (uPA)). herein, may also administer liquids. 0047. The term “population' as used herein, refers to any 0037. The term “vascular graft' as used herein, refers to mixture of particles (i.e., for example, liposomes or micro any conduit or portion thereof intended as a prosthetic device particles) having a distribution in diameter size. For example, for conveying blood and, therefore, having a blood contacting a population of microparticles may range is particle diameter surface (i.e., “luminal). While usually in a tubular form, the from between approximately 1-5000 um, preferably between graft may also be a sheet of material useful for patching approximately 350-3500 um, more preferably between portions of the circumference of living blood vessels (these approximately 1000-2000 um. materials are generally referred to as Surgical wraps). Like 0048. The term “symptoms are reduced as used herein, wise, the term Vascular graft includes intraluminal grafts for refers to a qualitative or quantitative reduction in detectable use within living blood vessels. The inventive grafts as such symptoms, including, but not limited to, a detectable impact may also be used as a stent covering on the exterior, luminal on the rate of recovery from disease (e.g. rate of thrombus or both surfaces of an implantable vascular stent. regression) or a detectable impact on the rate of development 0038. The term “anti-thrombotic drug combination' as of disease (e.g., rate of thrombus growth). used herein, refers to any composition comprising at least one 0049. The term “recently incurred as used herein, refers plasminogen activator (i.e., for example, tRA) and at least one to the onset of a particular vascular disorder within the past Annexin protein (i.e., for example, recombinant Annexin A2; twelve hours. Preferably onset within the past six hours, but rA2). Other drugs including, but not limited to, antithrombin more preferably onset within the past four hours. drugs, anticoagulant drugs or antiinflammatory drugs may 0050. The term “a sheet of instructions” as used herein, also be in this combination. refers to any method of means of storing and retrieving writ 0039. The term “controlled release drug elution” as used ten or spoken information. Such instructions are preferably herein, refers to any stable and quantifiable drug release from related to the use of a kit containing a therapeutic method a polymer-based medium as contemplated herein. contemplated herein. Such instructions may also provide 0040. The term “synthetic vascular graft' as used herein, information regarding interpretation of the results of the refers to any artificial tube or cannula designed for insertion therapeutic method such that a clinical diagnosis can be into a blood vessel. Such grafts may be constructed from reached. polytetrafluoroethylene (PTFE). 0041. The term “fibrin sheath' as used herein, refers to any BRIEF DESCRIPTION OF THE FIGURES encapsulation of a medical device Subsequent to implanta 0051 FIG. 1 presents exemplary data showing an effect of tion. One hypothesis suggests that platelets and white blood rA2 on tPA-dependent plasmain generation in vitro. cells respond to foreign Substances in much the same way as 0.052 FIG. 1A demonstrates plasmin activity expressed as an injured tissue (i.e., for example, a blood vessel) and that a ratio that generated by either 2.5 mg/ml oftBA alone or 100 platelet adherence, followed by fibrin encapsulation, is Units/ml of uPA alone. Data were expressed as mean+S.e.m, involved in fibrin sheath formation. *P<0.001, n=4 per group. 0042. The term “non-adhesive luminal surface' as used 0053 FIG. 1B demonstrates a range of concentrations of herein, refers to any vascular graft having been constructed, tPA (1, 2.5, 5, 10 mg/ml) plus/without the indicated concen or treated, that prevents platelet attachment and Subsequent trations of ra2 (0, 0.5, 1.2, 2.5, 5 mg/ml). Plasmin activity is thrombosis formation. represented as fold of plasmin activity related to 1 mg/ml of 0043. The term “currently recommended dose” as used tPA alone. Data were expressed as mean+S.e.m., n 4 per herein, refers to the approved dosage established by a recog group. nized regulatory body approving the administration of com 0054 FIG. 2 presents exemplary data showing an effect of pounds to living being. For example, the Food & Drug treating rats at 2 hrs after initiation of cerebral ischemia. Administration (FDA) approves dosages for human and non 0055 FIG. 2A demonstrates that two hours after initiation human administration. For example, the FDA has approved of ischemia, animals were treated intravenously with either the administration to humans of no more than 100 mg of saline, high dose tRA (10 mg/kg, H-tpA), intermediate dose tissue plasminogen activator (tPA: Activase R, Genentech) tPA (5 mg/kg, M-tPA), low dose tRA (2.5 mg/kg, L-tPA), ra2 over the period of one hour. alone (5 mg/kg), or a combination of low dose tRA (2.5 0044. The term “vascular disorder” as used herein, refers mg/kg) plus ra2 (5 mg/kg). Laser doppler flowmetry was to any biochemical, physiological, structural, or anatomical used to monitor regional cerebral blood flow (rCBF) for up to abnormality occurring within the cardiovascular system. For 1 h after treatment. example, such vascular disorders include, but are not limited 0056 FIG. 2B demonstrates that at 24 hrs after stroke, to, stroke, myocardial infarction, deep vein thrombosis, pull brain infarction was stained by TTC, and the volume was monary embolism, thrombophlebitis, or intracerebral quantified using computer-assisted image analysis. Data hematoma. expressed as mean+s.e.m., *P<0.05 for L-tRA plus raN, 0045. The term “annexin' as used herein, refers to a family #P-0.05 for H-tRA, respectively, n=10 per group. of highly homologous proteins believed to 0057 FIG.3 presents exemplary data showing an effect of prevent both cellular and humoral amplification of platelet treating rats at 4 hrs after initiation of ischemia. US 2014/0227350 A1 Aug. 14, 2014

0058 FIG. 3A. At 4 h after stroke onset, three groups of in in Vivo thrombus formation. Although it is not necessary to rats were treated intravenously with either saline, high dose understand the mechanism of an invention it is believed that a tPA (10 mg/kg, H-tRA), or low dose tRA (2.5 mg/kg, L-tRA) combination of Annexin A2 with thA can significantly plus r A2 (5 mg/kg). Shown here are representative images of enhance thrombolysis efficacy, such that lower doses oftBA brain sections after TTC staining at 24 h after initiating can be administered that avoid neurotoxic and hemorrhagic ischemia. Ischemic infarctions (white color area) were complications. detected in all three groups, however large visible hemor 0069. For example, after synthesis, purified recombinant rhage appeared only on the brain sections of H-tpA treated human Annexin A2 protein was confirmed to provide rats pointed by arrows. Annexin A2-tPA induced in vitro amplification of plasmin 0059 FIG. 3B. At 24 h after stroke, brain infarction was generation. Using a rat embolic stroke model, a tFA-recom quantified using computer-assisted image analysis. binant Annexin A2 protein combination was administered 2 0060 FIG.3C. Volumes of intracerebral hemorrhage ware hours after the ischemia-induced embolic stroke. The effec quantified with hemoglobin assay at 24 h after stroke. tive dose required for tRA to restore cerebral blood flow was 0061 FIG. 3D. Hemispheric swelling rates were calcu reduced by 4-fold, also resulting in reduced brain infarctions. lated from TITC stained brain slices. Data expressed as Further, the Annexin A2-tPA combination also prolonged the mean+s.e.m., *P<0.05, n=11 per group. effective treatment time window to prevent thrombolysis (the 0062 FIG. 4 present exemplary data showing an effect of currently accepted therapeutic window is limited to three tPA along or in combination with ra2 on , and hours post stroke event). For example, when compared to tA reperfusion improvement. (10 mg/kg) alone, the Annexin A2 (5 mg/kg)/tPA (2.5 mg/kg) 0063 FIG. 4A. Effects of ra2 plus low dose tRA in fibrin (i.e., a 2:1 Annexin A2-tPA ratio) combination significantly olysis. Plasma samples were collected before ischemia (Pre enhanced fibrinolysis and reperfusion; attenuated mortality, ischemia), just before thrombolytic therapy (Ischemia), and 1 brain infarctions, and hemorrhagic transformations, when h after treatment (Thrombolysis). Concentrations of fibrin administered at 4 hours post stroke event. These data show degradation product D-dimer in plasma were quantified by that when used in combination with recombinant Annexin ELISA analysis. Data expressed as mean+s.e.m., *P<0.01 A2, the effective thrombolytic dose ofteA can be decreased. versus ischemia, #P-0.01, n=6 per group. As a result, brain hemorrhage and infarction can be reduced, 0064 FIG. 4B. Representative MR angiograms (MRA). and the therapeutic time window for stroke reperfusion pro rA2 (5 mg/kg) plus low dose tRA (2.5 mg/kg) was IV injected longed. at 4 hr after stroke onset in embolic stroke rats. Time of flight 0070 Although it is not necessary to understand the (TOF) technique was used to assess cerebral vessel MRA mechanism of an invention it is believed that the molecular imaging. It showed clear MCA occlusion examined at 3 hr complex of tRA-Annexin A2-plasminogen amplifies t?A- after stroke (Before Thrombolysis), and clear recanalization converted plasmin generation in vitro and inhibits clotforma at 1 hr after thrombolysis by the combination. tion in vivo. For example, in the rat embolic model, a combi 0065 FIG. 4C. Representative cerebral blood flow (CBF) nation treatment at 2 hrs after stroke onset with low dose (2.5 map (perfusion image). The map was obtained using a 2-coil mg/kg) plus Annexin A2 (5 mg/kg) showed similar therapeu continuous arterial spin labeling technique. On the same rat, tic benefits as treatment with high dose (10 mg/kg) tA alone CBF was significantly reduced at 3 hr after stroke (before in terms of restoring cerebral perfusion and reducing infarct thrombolysis) in the ipsilateral region, and a clear improve Volumes at 24 hrs. It is further shown that when treatment was ment in the same region at 1 hr after thrombolysis by given the delayed to four hrs after stroke onset, a tRA plus Annexin A2 combination. The CBF improving rate was 62.3% for ra2 combination treatment improved cerebral perfusion and plus low dose tRA versus 28.3% for high dose tRA alone reduced infarction without hemorrhagic transformation. (n=2). 0071. With the advantages of this invention, more 0066 FIG. 5 presents one embodiment of a human ischemic stroke patients may be able to receive tRA treat Annexin II protein sequence (A) and corresponding nucle ments beyond the currently recommended three hour post otide sequence (B). stroke event time window. As a result, it is expected that patients receiving a tpA/Annexin A2 combination treatment DETAILED DESCRIPTION will have lower risk for intracerebral hemorrhage, and better 0067. The present invention is related to the field of vas clinic outcomes. cular disorders. In particular, the present invention is related I. Tissue Plasminogen Activator (tPA) to the treatment and management of diseases including, but 0072 Tissue plasminogen activator (tPA) is a fibrin-spe not limited to, stroke, myocardial infarction, deep vein throm cific activator that converts plasminogen to plasmin. tRA bosis, or pulmonary embolism. For example, a patient having stimulates thrombolysis and rescues the ischemic brain by Suffered a vascular disorder may be administered a composi restoring blood flow. However, emerging data Suggest that tion comprising tPA and Annexin A2. In Such cases, the tBA besides clot lysis per se, exogenous tEPA may have: i) pleio dose may be reduced Such that the risk of hemorrhagic side tropic actions in the brain (14); ii) direct vasoactivity (27); iii) effects are minimal. cleavage of the N-methyl-D-aspartate (NMDA) NR1 subunit 0068. Hemorrhagic transformations, neurotoxicities and a (28); and iv) activation of other extracellular proteases such as short treatment time window include, but not limited to, some matrix metalloproteinases (MMPs) (3, 13, 28, 29). Further, limitations for effective tea stroke therapy. These limitations these effects may increase neuronal excitotoxicity, further are related to the high dose of tA required to generate the damage the blood brain barrier, and/or worsen edema and necessary amounts of plasmin Sufficient for clot lysis. In one cerebral hemorrhage (14). Because of these shortcomings, embodiment, the present invention contemplates that soluble there is a clinical need to develop new approaches that might Annexin A2 unexpectedly potentiates tA-mediated plasmin increase thrombolytic efficacy with longer therapeutic time generation in vitro, thereby resulting in improved reductions windows, reduce tRA-neurotoxicity and diminish the risk of US 2014/0227350 A1 Aug. 14, 2014

hemorrhagic transformation. Combination therapies that can binant human tRA. This combination resulted in reduced improve the benefit/risk ratio and lengthen treatment win blood brain barrier leakage, brain edema and ischemic lesion dows would make more patients eligible for tRA stroke Volume as compared to treatment with tA alone. Zhanget al., therapy (30). “Adjuvant Treatment with Neuroserpin Increases the Thera 0073. The above described shortcomings of using only peutic Window for Tissue-Type Plasminogen Activator tPA to treat stroke underscores that it is clinically and timely Administration in a Rat Model of Embolic Stroke' Circula needed to identify new thrombolytics that might increase tion 106:740–745 (2002). thrombolytic efficacy. Such an advance should also reduce 0077 Finally, another recent study suggested that stabili tPA-neurotoxicity and the risk of hemorrhagic transforma Zation of mast cells with cromoglycate results in a significant tion, thereby improving the benefit/risk ratio for tRA stroke reduction in tA-mediated hemorrhage and reperfusion therapy. injury following focal cerebral ischemia in rats (40). Such a 0074. In one embodiment, the present invention contem wide variety of experimental data only show that many plates a composition capable of being administered using an mechanisms may contribute to the deleterious effects oftBA. effective tea dose that is four times lower than currently All above experimental findings Support the novelty of some recommended. Although it is not necessary to understand the embodiments of this invention contemplating that a lower tRA mechanism of an invention it is believed that such a reduced dose will reduce dose-related side effects, and enhance dose reduces tha-mediated side effects resulting from intrac thrombolytic efficacy and/or safety. erebral hemorrhage and/or direct neurotoxicity. In one embodiment, the present invention contemplates a composi II. Annexin A2 tion comprising a combination of thA and recombinant 0078 Annexin A2 (also known as Annexin II) is a cal human Annexin A2, wherein plasmin generation is amplified. cium- and phospholipid-binding protein that serves as a pro Although it is not necessary to understand the mechanism of fibrinolytic co-receptor for tRA and plasminogen on endothe an invention it is believed that such plasmin generation will lial cells (16, 17). Annexin A2 accelerates the activation of the improve perfusion and provide greater clinical benefits. In clot-dissolving protease plasmin by complexing with thA and one embodiment, the present invention contemplates a with the plasmin precursor plasminogen which binds on method comprising administering a composition comprising endothelial cell surface and exist in the clot (18, 19). Although tPA and Annexin A2 to a patient more than three hours after a initially identified as an intracellular protein, Annexin A2 is stroke event, and under conditions such that effective treat also transported to the extracellular environment, and exists ment is provided. Although it is not necessary to understand as both soluble and membrane-bound protein. Annexin A2 the mechanism of an invention it is believed that because can be detected in human plasma and can be transported to the Annexin A2 is an endogenous human protein a combined cell surface in response to cellular stress (33). Recent reports treatment with thA may reduce the risk of an immune have demonstrated that soluble Annexin A2 not only dramati response. cally increases t?A-dependent plasmin generation in vitro, 0075. The present embodiments identify new strategies but also reduces thrombus formation in rat carotidarteries and that can increase the thrombolytic efficacy of tRA, while middle cerebral arteries in vivo (20, 21, respectively). reducing its associated neurotoxicity and hemorrhagic trans 0079. In one embodiment, the present invention contem formation (1-6). Successful clot lysis for stroke therapy has plates a recombinant human Annexin A2 having a tRA bind required high doses of thA, but it is well accepted that this ing site. In one embodiment, the present invention contem practice increases the risk of intracerebral hemorrhage (7-9). plates a tRA peptide is bound to the Annexin A2 peptide. In Emerging evidence from experimental models and clinical one embodiment, the binding site is modified to provide a studies demonstrates that t?A administered systemically can high affinity tPA binding site. enter both normal and injured brain parenchyma (10, 11). 0080. In one embodiment, the present invention contem Moreover, exogenous tA can have neurotoxic actions that plates a recombinant human Annexin A2 having a plasmin are not related to any clot lysis properties (12-15). Also, it has binding site. In one embodiment, the present invention con been reported that tha mediated neurotoxicity may exacer templates a plasmin peptide is bound to the Annexin A2 bate ischemic damage, particularly to the about 50% of peptide. In one embodiment, the binding site is modified to patients who have poor improvement in perfusion after provide a high affinity plasmin binding site. receiving intravenous tA (14). I0081. The use of annexin proteins, including Annexin A2, 0076. Other studies have attempted to develop new for the treatment of thrombosis without increasing the risk of approaches for tRA combination therapy in animal stroke hemorrhaging has been reported. For example, Annexin A2 models. For instance, the EEIIMD hexapeptide correspond may be linked with the Fc portion of an immunoglobulin. ing to amino acids 350-355 of plasminogen activator inhibitor However, this administration of Annexin A2 is advocated for type 1 abolished the tRA-induced increases in infarct size and administration in a "heterotetramer' form. Modified Annexin intracranial bleeding in both mechanical and embolic models A2 proteins, including recombinant proteins, were suggested ofstroke in rats (4). Co-treatment with thA plus MMP inhibi to prevent transientischemic attacks from developing into full tors ameliorated reperfusion injury in a focal stroke model in blown strokes (i.e., a preventative therapy). Combinations of rats (34), and also reduced the incidence of teA-induced Annexin A2 with thA were not suggested, and in fact the hemorrhage in rabbits (35). Reduced brain infarction and/or disadvantages of tRA (i.e., increased bleeding risk) were hemorrhage and thrombolytic window extension have been pointed out tending to teach away from the presently contem reported using combinations of tRA with: i) bortezomib (a plated embodiment. Allison, “Modified Annexin Proteins and potent and selective inhibitor of the proteasome) (36); ii) a Methods for Preventing Thrombosis.” U.S. Pat. No. 6,962, glycoprotein IIb/m a receptor antagonist (37); iii) an antioxi 903 (herein incorporated by reference). Annexin A2 has also dant (2, 38); and iv) activated (39). Further, a rat been proposed as an effective therapeutic to reduce hemor model was used to test treatment with neuroserpin and recom rhaging when administered following retinal trauma and US 2014/0227350 A1 Aug. 14, 2014

other ophthalmological disorders. Rich, “Methods of Stabi tPA plus ra2 significantly increased D-dimer levels by about lizing Hyaluronidase with Annexin A2 U.S. Pat. No. 7,115, 2.9 fold compared to either saline or tRA alone. 408 (herein incorporated by reference). I0086 A. Annexin A2-tPA Relationships I0087 Annexin A2 has been reported to be the putative III. Annexin A2 and tRA Combination Treatments human endothelial tRA cell surface receptor. This relationship 0082 In one embodiment, the present invention contem has resulted in broad speculation that the Annexin A2-tPA plates that Annexin A2 may be therapeutically administered relationship may play a role in hemostasis and thrombosis. with tissue plasminogen activator (tPA) for treatment follow Hajjar et al., “An Endothelial Cell Receptor for Plasminogen/ ing a stroke. The present invention addresses current prob Tissue Plasminogen Activator” Journal of Biological Chem lems associated with the fact that tRA is currently the only istry 269:21191-21197 (1994). Other, in vitro studies, have FDA approved medicine for the generation of vascular rep shown that Annexin A2 is capable of binding both plasmin erfusion in stroke patients but relatively few patients actually and plasminogen in human endothelial cells and Suggest that receive tRA due to the increased risk of intracerebral hemor Annexin A2 may act as a catalyst for the activation of plas rhaging. In one embodiment, the present invention contem minogen by tPA. Haijaret al., “Annexin A2: A Mediator of the plates that Annexin A2, an endogenous protein of human Plasmin/Plasminogen Activator System, Trends in Cardio plasma, may be combined in treatment regimens with tRA vascular Medicine” 9:128-138 (1999). However, until the Such that lower, non-neurotoxic dosages of tRA may be presently disclosed invention, the therapeutic usefulness of administered. Although it is not necessary to understand the providing a combination therapy of Annexin A2 and tRA has mechanism of an invention it is believed that the dose oftBA not been proposed. may be reduced at least four-fold from currently recom I0088 Annexin A2-plasminogen activator combinations mended dosages when administered in combination with have been used for the treatment of other diseases. For Annexin A2 a still achieve similar therapeutic effectiveness. example, an anti-angiogenic plasmin fragment and an Since tea is broadly used in treating a range of thrombotic Annexin A2 heterotetramer was reported to affect angiogen disorders, besides acute ischemic stroke, this new combina esis. In one combination used to effect angiogenesis, an tion approach for lowering the minimum effective tRA dose Annexin A2 heterotetramer was combined with a urokinase may potentially improve tRA treatments for other conditions type plasminogen activator; an enzyme that interacts with Such as acute myocardial infarction, pulmonary embolism, tPA. Novokhatny et al., “Tissue-type plasminogen activator deep venous thrombosis, thrombosed medical devices (i.e., (tPA) interacts with urokinase-type plasminogen activator for example, intravenous catheters), and lysis of intracerebral (uPA) via tRA's lysine binding site. An explanation of the hematomas. poor fibrin affinity of recombinant t?A/uPA chimeric mol 0083 Specific advantages of a combination of tRA and ecules” Journal of Biological Chemistry 270:8680-8685 Annexin A2 is that, tRA's associated risks when administered (1995). The urokinase-type plasminogen activator-tPA (uPA) alone and at a high dose (i.e., for example, rat brain hemor interaction in regards to angiogenesis, however, was not rhaging after a 10 mg/kg dose) may be alleviated and/or linked to Annexin A2 to provide an effective combination for avoided. Other embodiments contemplated by the present the treatment of stroke. Waisman et al. "Anti-Angiogenesis invention are that tha/Annexin A2 combination therapies Methods, Compositions and Uses Therefor U.S. Pat. No. may further improve treatment outcomes for other medical 7.250.271 (herein incorporated be reference). Further, while conditions including, but not limited to, myocardial infarc tPA is believed to stimulate plasmin production, there are tion, pulmonary embolism, deep vein thrombosis and lysis those in the art that believe glycation of Annexin A2 may for intracerebral hematoma. impair the appropriate formation of the plasminogen/tPA/ 0084. Although it is not necessary to understand the Annexin A2 complex. Ribo et al. Acute Hyperglycemia mechanism of an invention, it is believed that a combination State is Associated with Lower tRA-Induced Recanalization of Annexin A2 and tRA can significantly enhance throm Rates in Stroke Patients' Stroke 36:1705-1709 (2005). This bolytic efficacy so that lower doses of tRA can be applied in evidence Suggests that Annexin A2 and tRA have counterbal ischemic stroke to avoid neurotoxic and hemorrhagic com ancing physiological roles, thereby arguing against an effec plications. The data presented herein demonstrate that this tive combination therapy. novel approach Successfully achieved improvements in per I0089. B. ra2 Amplifies tA-Mediated Plasmin Genera fusion and brain tissue protection. Further, the risk of intrac tion. In Vitro erebral hemorrhage was also significantly reduced. It is fur 0090 Recombinant human Annexin A2 protein (ra2) was ther believed that these beneficial effects on blood flow, synthesized in accordance with Example I and purified by infarct Volume, hemorrhage and mortality Suggest that ra2 SDS-PAGE and confirmed using a Western Blot (data not might safely augment the efficacy and lengthen the treatment shown). In vitro plasmin activity assays showed a combina window oftBA stroke therapy. tion of ra2 (5ug/ml) and tRA (2.5ug/ml) significantly ampli 0085 Administered doses for a tRA and ra2 in vivo com fied tRA-converted plasmin generation. This amplification bination was based on in vitroplasmin generation data. The in was Annexin A2-tPA specific because a combination of BSA vitro data indicated that an ra2/tBA combinationata 2:1 ratio protein (5ug/ml) and tRA or a combination of ra2 and uPA increases the plasmin-generating capability oftBA by almost did not increase plasmin activity. See, FIG. 1A. Further, a four-fold. The thrombolytic profiles may be evaluated by combination of ra2 and tRA significantly amplified tRA-con examining plasma levels of a fibrin degradation product, Verted plasmin generation in a dose-dependent manner. The D-dimer. The assay is based upon degradation of cross-lined data demonstrate that equal plasmin activity levels can be fibrin (enriched in fibrin thrombi) that produces a number of reached by either: i) a high dose tRA alone (clinically associ fragments containing the D-dimer epitope. The released ated with cerebral hemorrhage); orii) different ra2/teA com D-dimer epitope is then utilized as an indicator the extent of binations having lower doses ofteA, such as: i) tha 5 g/ml fibrin clot lysis by ELISA. ELISA data showed that low dose (alone) versus a tRA (1 lug/ml)/rA2 (2.5ug/ml) combination; US 2014/0227350 A1 Aug. 14, 2014

or ii) tea 10 ug/ml (alone) versus a tpA (2.5 g/ml)/rA2 (5 high dose tRA (10 mg/kg), or a combination of low dose tpA ug/ml) combination. These data indicate that by combining (2.5 mg/kg) and ra2 (5 mg/kg). At 24 h after initiation of rA2 with teA at approximately a 2:1 w/v ratio (or alterna stroke, this combination significantly reduced infarction Vol tively, 4:1 molar ratio) increased the in vitro plasmin-gener ume, compared with either saline controls or rats treated with ating capability oftBA by almost four-fold in vitro. See, FIG. high-dose-tpA alone. See FIG. 3A, 3B. As expected, high 1B. dose tRA administered at the delayed 4 h time point induced 0091 C. ra2 Decreases the Dose oftBA for Focal Embolic significant hemorrhagic transformation at 24 h. This combi Stroke nation significantly ameliorated the severity of hemorrhagic 0092. It has been suggested that species differences may transformation and hemispheric swelling. See FIG. 3C, 3D. exist in fibrin specificities (22). Consequently, the equivalent effective dose of human recombinant tBA used in rat embolic (0099 Finally, in concert with these beneficial effects on stroke model studies is usually about 10 mg/kg, whereas an cerebral perfusion, infarction and hemorrhage, combination approximate 10 times higher dose is used in human clinical therapy of low-dose tRA plus ra2 also reduced mortality. treatment. Mortality rates were 29% in untreated rats and 41% in the 0093. Therefore, two hours after initiation of ischemia high dose tRA group, whereas combination therapy with low (i.e., for example, by clot infusion) rats were intravenously dose tRA plus ra2 significantly reduced mortality to 15%. administered with either i) saline; ii) a high dose tpA (10 See, Table 1. mg/kg); iii) an intermediate dose tpA (5 mg/kg); iv) a low dose tRA (2.5 mg/kg); V) ra2 alone (5 mg/kg); or vi) a com TABLE 1 bination of low dose tRA (2.5 mg/kg) plus ra2 (5 mg/kg). 0094 Laser doppler flow metry was then used to monitor Mortality Rates regional cerebral blood flow (rCBF) for up to 1 h after treat Groups Saline H-tPA L-tRA+ ra2 ment. Brain infarction volume was quantified at 24 h after stroke. As expected, high dose tRA (10 mg/kg) induced Tested Animals 14 17 13 almost complete reperfusion, and also decreased infarct Vol Dead Animals 4 7 2 umes by about 45%. It should be noted that neither the inter Mortality% 29 41 15 For the animals treated at 4h after the initiation of stroke, the mortality rate of each group for mediate dose tRA or the low dose tRA (alone), nor the rA2 the first 24 h are shown. dose (alone) were effective in improving reperfusion and Combination treatment significantly eliminated the mortality related to H-tPA treatment, reducing infarction. Surprisingly, the combination of low dose tpA and ra2 dose successfully achieved similar reper 0100. In addition, physiological parameters measured fusion and reduced infarct size as observed with high dose tRA (alone). See, FIG. 2A and FIG. 2A. before ischemia, after ischemia, and 30 min following throm 0095. These data suggest that this combination is pharma bolysis remained within normal range in all groups. See, cologically more effective and specific for fibrin clot lysis Table 2. than currently recommended treatments (24). Further, the data Supports embodiments of the present invention by pro TABLE 2 viding improved therapy based upon plasmin-dependent Measurements of Physiological Parameters fibrinolysis. Consequently, intravenous injection of a combi nation of 2.5 mg/kg tPA and 5 mg/kg ra2 may generate Saline H-tPA L-tRA+ ra2 plasmin in equal or even greater amounts than 10 mg/kg tPA Weight (g) 291 7.91 287 6.17 295 8.22 alone. This conclusion is consistent with the almost 100% Rectal Temperature (C.) 37.1 - 0.23 37.O. O.13 36.8 O.28 rCBF recovery in the 2 h treatment protocols (supra), and the MABP (mmHg) 123 4.23 12O3.05 121 - 4.21 pH 7.36 0.04 741 - 0.04 7.38, O.OS significant improvement in reperfusion even in rats treated at PCO, (mmHg) 39.5 2.87 39.83.66 40.1 + 4.24 the delayed 4 h time point (infra). Although it is not necessary PO, (mmHg) 125 4.7 1274.8 1264.6 to understand the mechanism of an invention, it is believed that such increased efficiency of fibrinolysis by the A2-tPA Physiological parameters were measured at 30 min after thrombolysis at 4h after onset of combination might even generate more plasmin locally in the stroke, All measurements remained within normal range in three groups. Mean + S.E.M. clot site. Previous findings have demonstrated that Annexin 0101. In some embodiments, the presently contemplated A2 accelerates the activation of plasmin by complexing with invention demonstrates that addition of a “plasmin amplifier” tPA and with the plasmin precursor plasminogen, which such as ra2 can decrease the effective thrombolytic dose of binds the endothelial cell surface and is enriched in the clot tPA, thereby reducing hemorrhage and brain infarction, and (19, 31). Therefore, a combination of tRA and recombinant prolonging the reperfusion time window for stroke. Annexin A2 might locally bind plasminogen and conse quently amplify plasmin generation in the vicinity of the clot, 01.02 E. Fibrin Degradation Product D-Dimer thereby resulting in more locally effective fibrinolysis. 0103 Plasma levels were examined of the fibrin degrada 0096. D. Extension of the Thrombolytic Time Window tion product D-dimer, a biomarker for fibrinolysis. ELISA 0097. Previous studies using a rat model of embolic focal data showed both high dose tRA and low dose tRA plus ra2 cerebral ischemia have shown that tRA treatment is effective significantly increased D-dimer after thrombolysis for 2.2- only when given within 2-3 h after a stroke event (23). In one fold and 3.2-fold, respectively, and the increase in low dose embodiment, the present invention contemplates a method tPA plus ra2 combination was significantly greater compared wherein a combination therapy with tA plus ra2 prolongs to high dose tpA-only treatment. See, FIG. 4A. These data the conventional thrombolytic time window. indicate that this combination thrombolytic therapy was more 0098. For example, at 4 hours after stroke onset, three effective and specific for fibrinolysis than tA along in groups of rats were treated intravenously with either saline, delayed 4 hrs treatment (24). US 2014/0227350 A1 Aug. 14, 2014

0104 F. MRI Reperfusion Imaging orrhagic stroke occurs when a blood vessel bursts inside the 0105. Our initial MRI analysis also indicated the reperfu brain. The brain is very sensitive to bleeding and damage can sion improvement by the combination in the delayed 4 hrs occur very rapidly, either because of the presence of the blood treatments. See, FIG. 4B, 4C. itself, or because the fluid increases pressure on the brain and harms it by pressing it against the skull. Bleeding irritates the IV. Clinical Applications brain tissue, causing Swelling. The Surrounding tissues of the 0106. In one embodiment, the present invention contem brain resist the expansion of the bleeding, which is finally plates administering a ra2/tPA composition for the treatment contained by forming a mass (i.e., for example, an intracere of a vascular disorder. In one embodiment, the disorder may bral hematoma). Both Swelling and hematoma will compress comprise a stroke. In one embodiment, the disorder may and displace normal brain tissue. Most often, hemorrhagic comprise a thrombosis. In one embodiment, the thrombosis stroke is associated with high blood pressure, which stresses may be attached to a medical device. In one embodiment, the the artery walls until they break. disorder may comprise an embolism. 0114. One cause of hemorrhagic stroke is an aneurysm. 01.07 A. Stroke This is a weak spot in an artery wall, which balloons out 0108. Each year, about 600,000 Americans suffer from because of the pressure of the blood circulating inside the stroke. Thrombolytic therapy with tA is the only FDA-ap affected artery. Eventually, it can burst and cause serious proved medicine for achieving both vascular reperfusion and harm. The larger the aneurysm is, the more likely it is to burst. clinical benefit, but only 2-5% of stroke patients receive teA 0115 3. Symptomology in the US. In part, this because tRA therapy unfortunately 0116 Stroke symptoms are typically of sudden onset and increases the risk of intracerebral hemorrhage by approxi may quickly become worse. Stroke symptoms may include, mately 10-fold. Perhaps even more importantly, there is accu but are not limited to: i) Weakness or inability to moveabody mulating evidence from experimental models and clinical part; ii) Numbness or loss of sensation; iii) Decreased or lost studies that tRA can have neurotoxic actions separate from its vision (may be partial); iv) Speech difficulties; v) Inability to beneficial clot lysis properties. tea neurotoxicity may further recognize or identify familiar things; vi) Sudden headache; exacerbate ischemic brain damage, particularly in the 50% of vii) Vertigo; viii) Dizziness; xi) Loss of coordination; x) patients who have no perfusion improvement after receiving Swallowing difficulties; and Xi) Sleepy, stuporous, lethargic, intravenous tRA. The present invention contemplates increas comatose, and/or unconscious. ing the thrombolytic efficacy of tRA, while reducing neuro 0117. A stroke event may be detected by using a neuro toxicity and the risk of hemorrhagic transformation. logic exam, which would be expected to show abnormal Although it is not necessary to understand the mechanism of results. Further, a patient may look drowsy and confused. An an invention, it is believed that Annexin A2 will lower the eye examination may show abnormal eye movements, and dose oftPA required to generate plasmin thereby allowing the changes may be seen upon retinal examination (examination use of lower non-neurotoxic doses oftBA and also extending of the back of the eye with an instrument called ophthalmo the treatment time window without incurring risks of brain Scope). The patient may also have abnormal reflexes. A com hemorrhage. puterized tomography scan will confirm the presence of a 0109 1. Ischemic Strokes brain hemorrhage by providing pictures of the brain. A brain 0110 Approximately eighty percent of strokes may be magnetic resonance imaging (MRI) scan can also be obtained caused by too little blood reaching an area of the brain, which later to better understand what caused the bleeding. A con is usually due to a clot that has blocked a blood vessel (i.e., for ventional angiography (i.e., for example, an X-ray of the example, a cerebral thrombosis). This is called “ischemic arteries using dye) may be required to identify aneurysms or stroke.” This type of stroke can sometimes lead to a brain AVM. Other tests may include, but are not limited to: com hemorrhage because the affected brain tissue softens and this plete blood count, bleeding time, prothrombin/partial throm can lead to breaking down of small blood vessels. In addition, boplastin time (PT/PTT), and CSF (cerebrospinal fluid) brain hemorrhage can occur when people have problems examination. forming blood clots. Clots, which are the body's way of 0118 B. Thromboses stopping any bleeding, are formed by proteins called coagul 0119 Thrombosis may be defined as the formation, devel lation factors and by sticky blood cells called platelets. When opment, or presence of a blood clot (i.e. for example, a throm ever the coagulation factors or platelets do not work well or bus) in a blood vessel and is believed to be a common severe are insufficient in quantity, people may develop a tendency to medical disorder. Thromboses may be involved in the gen bleed excessively. eration of a variety of vascular disorders including, but not 0111 Ischemic strokes may be preceded by transient limited to, myocardial infarctions, cardiac ischemia, and/or ischemic attacks (TIA), and it is estimated that about 300,000 deep vein thrombosis. persons suffer a TIA every year in the United States. It would I0120) 1.. Myocardial Infarction be desirable to have a safe and effective agent that could be I0121 The most frequent example of arterial thrombosis is administered as a bolus and would for several days prevent coronary thrombosis, which leads to occlusion of the coro recurrence of cerebral thrombosis without increasing the risk nary arteries and often to myocardial infarction (heart attack). of cerebral hemorrhage. Thrombosis also contributes to More than 1.3 million patients are admitted to the hospital for peripheral arterial occlusion in diabetics and other patients, myocardial infarction each year in North America. The stan and an efficacious and safe antithrombotic agent for use in dard therapy is administration of a thrombolytic protein by Such patients is needed. infusion. Thrombolytic treatment of acute myocardial infarc 0112 2. Hemorrhagic Strokes tion is estimated to save 30 lives per 1000 patients treated: 0113 Approximately twenty percent of strokes may nevertheless the 30-day mortality for this disorder remains involve bleeding within the brain, which damages nearby substantial (Mehta et al., Lancet 356:449-454 (2000), incor brain tissue (i.e., for example, a hemorrhagic stroke). Hem porated herein by reference). It would be convenient to US 2014/0227350 A1 Aug. 14, 2014

administer antithrombotic and thrombolytic agents by bolus 0128. Evidence has accumulated that the formation of injection, since they might be used before admission to hos tight macroaggregates of platelets is facilitated by a cellular pital with additional benefit (Rawles, J. Am. Coll. Cardiol. and a humoral amplification mechanism, which reinforce 30:1 181-1186 (1997), incorporated herein by reference). each other. In the cellular mechanism, the formation of rela However, bolus injection (as opposed to a more gradual intra tively loose microaggregates of platelets, induced by moder venous infusion) significantly increases the risk of cerebral ate concentrations of agonists such as ADP, A, hemorrhage (Mehta et al., 2000). The development of an or collagen, is accompanied by the release from platelet-O- agent able to prevent thrombosis and/or increase thromboly granules of the 85-kD protein Gas6 (Angelillo-Scherreretal. sis, without augmenting the risk of bleeding, would be desir Nature Medicine 7:215-221 (2001), incorporated herein by able. reference). Binding of released Gasó to receptor tyrosine 0122 2. Cardiac Ischemia kinases (Axl, Sky, Mer) expressed on the surface of platelets 0123. Unstable , caused by inadequate oxygen induces complete degranulation and the formation of tight delivery to the heart due to coronary occlusion, is the most macroaggregates of these cells. In the humoral amplification common cause of admission to hospital, with 1.5 million mechanism, a prothrombinase complex is formed on the Sur cases a year in the United States alone. When patients with face of activated platelets and microvesicles. This generates occlusion of coronary arteries are treated with angioplasty and fibrin. Thrombin is itself a potent platelet acti and stenting, the use of an antibody against platelet GP IIb/ vator and inducer of the release of Gas6 (Ishimoto and IIIa decreases the likelihood of restenosis. However, the same Nakano, FEBS Lett. 446:197-199 (2000), incorporated antibody has shown no benefit in without herein by reference). Fully activated platelets bind tightly to angioplasty, and a better method for preventing coronary the fibrin network deposited around them. Histological obser occlusion in these patients is needed. vations show that both platelets and fibrin are necessary for (0.124 3. Deep Vein Thrombosis the formation of a stable coronary thrombus in humans (Falk 0.125 Deep venous thrombosis is a frequent complication et al. Interrelationship between atherosclerosis and thrombo of Surgical procedures Such as hip and knee arthroplasties. It sis. In Vanstraete et al. (editors), Cardiovascular Thrombosis would be desirable to prevent thrombosis without increasing Thrombocardiology and Thromboneurology. Philadelphia: hemorrhage into the field of operation. Similar considerations Lipincott-Raven Publishers (1998), pp. 45-58, incorporated apply to venous thrombosis associated with pregnancy and herein by reference). Another platelet adhesion molecule, parturition. Some persons are prone to repeated venous amphoterin, is translocated to the platelet Surface during acti thrombotic events and are currently treated by antithrombotic Vation, and binds anionic phospholipid (Rouhainen et al., agents such as -type drugs. The dose of such drugs Thromb. Hemost. 84:1087-1094 (2000), incorporated herein must be titrated in each patient, and the margin between by reference). Like Gas6, amphoterin could form a bridge effective antithrombotic doses and those increasing hemor during platelet aggregation. rhage is Small. Having a treatment with better separation of I0129. The question arises whether it is possible to inhibit antithrombotic activity from increased risk of bleeding is these amplification mechanisms but not the initial platelet desirable. All of the recently introduced antithrombotic thera aggregation step, thereby preventing thrombosis without pies, including ligands of platelet GP IIb/IIIa, low molecular increasing hemorrhage. The importance of cellular amplifi weight , and a pentasaccharide inhibitor of factor Xa, cation has recently been established by studies of mice with carry an increased risk of bleeding (Levine et al., Chest 119: targeted inactivation of Gas6 (Angelillo-Scherrer et al., 108 S-121S (2001), incorporated herein by reference). 2001). The Gas6 mice were found to be protected against 0126 Hence there is a need to explore alternative strate thrombosis and embolism induced by collagen and epineph gies for preventing arterial and venous thrombosis without rine. However, the Gas6 mice did not suffer from sponta augmenting the risk of hemorrhage. Deep vein thrombosis neous hemorrhage and had normal bleeding after tail clip may be detected by tests including, but not limited to: i) ping. Furthermore, antibodies against Gas6 inhibited platelet Doppler ultrasound exam of an extremity blood flow studies: aggregation in vitro as well as thrombosis induced in vivo by ii) Venography of the legs; oriii) Plethysmography of the legs. collagen and epinephrine. In principle, such antibodies, or 0127. To inhibit the extension of arterial or venous ligands competing for Gas6 binding to receptor tyrosine thrombi without increasing hemorrhage, it is necessary to kinases, might be used to inhibit thrombosis. However, in exploit potential differences between mechanisms involved view of the potency of humoral amplification, it might be in hemostasis and those involved in thrombosis in large blood preferable to inhibit that step. Ideally such an inhibitor would vessels. Primary hemostatic mechanisms include the forma also have additional Suppressive activity on the Gas6-medi tion of platelet microaggregates, which plug capillaries and ated cellular amplification mechanism. accumulate over damaged or activated endothelial cells in 0.130. A strategy for preventing both cellular and humoral Small blood vessels. Inhibitors of platelet aggregation, amplification of platelet aggregation is provided by the including agents Suppressing the formation or action of annexins, a family of highly homologous antithrombotic pro thromboxane A, ligands of GPIIa/IIIb, and drugs acting on teins of which ten are expressed in several human tissues ADP receptors such as (Hallopeter, Nature 409: (Benz and Hofmann, Biol. Chem. 378:177-183 (1997), incor 202-207 (2001), incorporated herein by reference), interfere porated herein be reference). Annexins share the property of with this process and therefore increase the risk of bleeding binding calcium and negatively charged phospholipids, both (Levine et al., 2001). In contrast to microaggregate formation, of which are required for blood coagulation. Under physi occlusion by an arterial or venous thrombus requires the ological conditions, negatively charged phospholipid is continued recruitment and incorporation of platelets into the mainly supplied by phosphatidylserine (PS) in activated or thrombus. To overcome detachment by shear forces in large damaged cell membranes. In intact cells, PS is confined to the blood vessels, platelets must be bound tightly to one another inner leaflet of the plasma membrane bilayer and is not acces and to the fibrin network deposited around them. sible on the surface. When platelets are activated, the amounts US 2014/0227350 A1 Aug. 14, 2014

of PS accessible on their surface, and therefore the extent of 0.134 Platelet-mediated thrombosis is also known to com annexin binding, are greatly increased (Sun et al., Thrombo plicate Successful native and synthetic graft implantation. sis Res. 69:289-296 (1993), incorporated herein by refer Hemodialysis vascular access sites or an obstructed arterial ence). During activation of platelets, microvesicles are vasculature (i.e., for example, in the vascular periphery or the released from their surfaces, greatly increasing the Surface heart) bypass utilize these grafts. Vascular neointimal forma area expressing anionic phospholipids with procoagulant tions are known to occur in native and synthetic grafts, par activity (Merten et al., Circulation 99:2577-2582 (1999); ticularly in the venous outflow tracts. Wales et al., “Func Chow et al., J. Lab. Clin. Med. 135:66-72 (2000), both incor tional Neointima Characterization Of Vascular Prostheses. In porated herein by reference). These may play an important Human” Ann Thorac Surg. 77:864-868 (2004). Vascular role in the propagation of platelet-mediated arterial thrombi. neointimal formations (i.e., for example, lesions) are com Proteins involved in the blood coagulation cascade (factors X, posed primarily of Smooth muscle cells, and ultimately lead Xa, and Va) bind to membranes bearing PS on their surfaces, to a decreased blood flow within the grafts. Platelet-released and to one another, forming a stable, tightly bound prothrom growth factors may, in part, stimulate vascular neointimal binase complex. Several annexins, including II, V, and VIII, formations. As a neointimal lesion develops, blood flow bind PS with high affinity, thereby preventing the formation becomes more turbulent and further injury occurs, resulting of a prothrombinase complex and exerting antithrombotic in additional platelet recruitment. With additional platelet activity. recruitment, fibrin deposition may result with complete graft 0131 4. Medical Devices failure as a probable consequence. Thus, a drug combination 0132 Platelets and white blood cells respond to foreign comprising tPA and Annexin A2 may have distinct advan Substances in much the same way as an injured tissue (i.e., for tages over currently recommended antiproliferative and/or example, a blood vessel). Although it is not necessary to anticoagulant therapies. In one embodiment, the present understand the mechanism of an invention, it is believed that invention contemplates devices and methods to administer a platelet adherence, followed by fibrin deposition and subse drug combination to a graft venous outflow tract. In one quent encapsulation, is involved in fibrin sheath formation. embodiment, a drug combination is administered comprising Fibrin sheaths are known to be responsible for intravascular tPA and Annexin A2. In one embodiment, the medium or catheter medical complications, in particular, when using carrier may be wrapped or draped around the exterior graft central venous and intraperitoneal dialysis catheters. Santilli, Surface Such that the drug combination diffuses to an intralu J., “Fibrin Sheaths And Central Venous Catheter Occlusions: minal blood vessel Surface (i.e., for example, the vaso Diagnosis And Management Tech. in Vascular and Interven vasorum). tional Radiology 5:89-94 (2002). In one embodiment, the 0.135 Another embodiment of the present invention con present invention contemplates a method to prolong catheter templates coating a medical device with a medium or carrier function comprising coating the outside Surface of an intra comprising thA and Annexin A2. A medical device is vascular catheter with a drug combination comprising tPA “coated when a medium comprising tea and Annexin A2 and Annexin A2. becomes attached to the surface of the medical device. For example, Such attachment includes, but is not limited to, 0.133 Platelets are also known to release growth factors, in Surface adsorption, impregnation into the material of manu particular, platelet-derived growth factor (PGDF) which pro facture, covalent or ionic bonding and simple friction adher mote smooth muscle cell proliferation. Schwartz et al., ence to the surface of the medical device. “Common Mechanisms Of Proliferation Of Smooth Muscle In Atherosclerosis And Hypertension’ Hum Pathol. 18:240 0.136 Carriers or mediums contemplated by this invention 247 (1987). For example, following stent placement in may comprise a polymer including, but not limited to, gelatin, patients with coronary lesions, platelets adhere to the injured collagen, cellulose esters, dextran Sulfate, pentosan polysul blood vessels intraluminal surface. Subsequently, the bound fate, chitin, Saccharides, albumin, fibrin Sealants, synthetic platelets release growth factors that result in restenosis. Res polyvinyl pyrrolidone, polyethylene oxide, polypropylene tenosis is a condition where Smooth muscle cells accumulate oxide, block polymers of polyethylene oxide and polypropy within an injured blood vessel such that vessel blockage lene oxide, polyethylene glycol, acrylates, acrylamides, occurs within 3-6 months (i.e., such as following an intravas methacrylates including, but not limited to, 2-hydroxyethyl cular stent placement). Restenosis may be reduced with the methacrylate, poly(ortho esters), cyanoacrylates, gelatin-re use of drug-eluting stents, in particular with drugs such as Sorcin-aldehyde type bioadhesives, polyacrylic acid and rapamycin. Falotico et al., “Drug/Drug Delivery Systems For copolymers and block copolymers thereof. The Prevention And Treatment Of Vascular Disease' United I0137 C. Pulmonary Embolisms States Patent Application Publ. No: 2002/0016625A1 Filed: 0.138 A pulmonary embolus is a blockage of an artery in May 7, 2001. Published: Feb. 7, 2002. However, thrombus the lungs by fat, air, blood clot, or tumor cells. Pulmonary formation followingstent placement is a problem. Jeremias et emboli are most often caused by blood clots in the veins, al., “Stent Thrombosis After Successful Sirolimus-Eluting especially veins in the legs or in the pelvis (hips). More rarely, Stent Implantation' Circulation 109(16):1930-1932 Epub air bubbles, fat droplets, amniotic fluid, or clumps of parasites Apr. 12 (2004). Stent technology is attempting to solve this or tumor cells may obstruct the pulmonary vessels. problem using antiplatelet drug-eluting stents or grafts, but its 0.139. One cause of a pulmonary embolism is a blood clot efficacy is as yet unknown. Falotico, R., “Coated Medical in the veins of the legs, called a deep vein thrombosis (DVT) Devices For The Prevention And Treatment Of Vascular Dis (Supra). Many clear up on their own, though some may cause ease” United States Patent Application 2003/0216699 A1. severe illness or even death. Filed: May 7, 2003. Published; Nov. 20, 2003. The present 0140 Risk factors for a pulmonary embolus may include, invention contemplates administering a drug combination but are not limited to: i) Prolonged bed rest or inactivity comprising tPA and Annexin A2 at, or near, an intravascular (including long trips in planes, cars, or trains); ii) Oral con stent placement. traceptive use; iii) Surgery (especially pelvic Surgery); iv) US 2014/0227350 A1 Aug. 14, 2014

Childbirth; v) Massive trauma; vi) Burns; vii) Cancer; viii) ents and other suitable additives such as, but not limited to, Stroke: ix) Heart attack; x) Heart surgery; or xi) Fractures of penetration enhancers, carrier compounds and other pharma the hips or femur. Further, persons with certain clotting dis ceutically acceptable carriers or excipients. orders and/or autoimmune diseases (i.e., for example, anti 0150 Pharmaceutical compositions of the present inven cardiolipin antibody syndrome) may also have a higher risk. tion include, but are not limited to, Solutions, emulsions, and 0141 Symptoms of pulmonary embolism may be vague, liposome-containing formulations. These compositions may or they may resemble symptoms associated with other dis be generated from a variety of components that include, but eases. Symptoms can include, but are not limited to: i) Sudden are not limited to, preformed liquids, self-emulsifying Solids cough; ii) Bloody sputum (significant amounts of visible and self-emulsifying semisolids. blood or lightly blood streaked sputum); iii) Sudden onset of 0151. The pharmaceutical formulations of the present shortness of breath at rest or with exertion; iv) splinting of ribs invention, which may conveniently be presented in unit dos with breathing (bending over or holding the chest); v) chest age form, may be prepared according to conventional tech pain; vi) rapid breathing; or vii) rapid heart rate (tachycardia) niques well known in the pharmaceutical industry. Such tech 0142 Pulmonary emboli may be identified using tests niques include the step of bringing into association the active including, but not limited to: i) Arterial blood gases; ii) Pulse ingredients with the pharmaceutical carrier(s) or excipient(s). oximetry; iii) Chest X-ray; iv) Pulmonary ventilation/perfu In general the formulations are prepared by uniformly and sion scan; v) Pulmonary angiogram; vi) electrocardiogram; intimately bringing into association the active ingredients and V) computerized tomographic chest angiogram. with liquid carriers or finely divided solid carriers or both, and 0143 D. Superficial Thrombophlebitis then, if necessary, shaping the product. 0144. Thrombophlebitis is swelling (inflammation) of a 0152 The compositions of the present invention may be vein caused by a blood clot. Such conditions are usually a formulated into any of many possible dosage forms such as, result of sitting for a long period of time (such as on a long but not limited to, tablets, capsules, liquid syrups, soft gels, airplane trip). Disorders that increase a person's chance for Suppositories, and enemas. The compositions of the present blood clots also lead to thrombophlebitis. Superficial throm invention may also be formulated as Suspensions in aqueous, bophlebitis affects veins near the skin surface. non-aqueous or mixed media. Aqueous Suspensions may fur 0145 Symptoms often associated with superficial throm ther contain Substances that increase the Viscosity of the Sus bophlebitis may include but are not limited to: i) Warmth and pension including, for example, sodium carboxymethylcel tenderness over the vein; ii) Pain in the part of the body lulose, Sorbitol and/or dextran. The Suspension may also affected; iii) Skin redness (not always present); or iv) Inflam contain stabilizers. mation (swelling) in the part of the body affected. Objective 0153. In one embodiment of the present invention the tests may be performed to detect thrombophlebitis including, pharmaceutical compositions may be formulated and used as but not limited to: i) Doppler ultrasound; ii) Venography; and foams. Pharmaceutical foams include formulations such as, iii) Blood coagulation studies. but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formu V. Pharmaceutical Formulations lations vary in the components and the consistency of the final 0146 The present invention further provides pharmaceu product. tical compositions (e.g., comprising the Annexin A2/tPA 0154 The compositions of the present invention may combinations described above). The pharmaceutical compo additionally contain other adjunct components convention sitions of the present invention may be administered in a ally found in pharmaceutical compositions. Thus, for number of ways depending upon whether local or systemic example, the compositions may contain additional, compat treatment is desired and upon the area to be treated. Admin ible, pharmaceutically-active materials such as, for example, istration may be topical (including ophthalmic and to mucous antipruritics, astringents, local anesthetics or anti-inflamma membranes including vaginal and rectal delivery), pulmo tory agents, or may contain additional materials useful in nary (e.g., by inhalation or insufflation of powders or aero physically formulating various dosage forms of the compo Sols, including by nebulizer, intratracheal, intranasal, epider sitions of the present invention, such as dyes, flavoring agents, mal and transdermal), oral or parenteral. Parenteral preservatives, antioxidants, opacifiers, thickening agents and administration includes intravenous, intraarterial, Subcutane stabilizers. However, such materials, when added, should not ous, intraperitoneal or intramuscular injection or infusion; or unduly interfere with the biological activities of the compo intracranial, e.g., intrathecal or intraventricular, administra nents of the compositions of the present invention. The for tion. mulations can be sterilized and, if desired, mixed with auxil 0147 Pharmaceutical compositions and formulations for iary agents, e.g., lubricants, preservatives, stabilizers, wetting topical administration may include transdermal patches, oint agents, emulsifiers, salts for influencing osmotic pressure, ments, lotions, creams, gels, drops, suppositories, sprays, buffers, colorings, flavorings and/or aromatic Substances and liquids and powders. Conventional pharmaceutical carriers, the like which do not deleteriously interact with the nucleic aqueous, powder or oily bases, thickeners and the like may be acid(s) of the formulation. necessary or desirable. 0155 Dosing is dependent on severity and responsiveness 0148 Compositions and formulations for oral administra of the disease state to be treated, with the course of treatment tion include powders or granules, Suspensions or solutions in lasting from several days to several months, or until a cure is water or non-aqueous media, capsules, Sachets or tablets. effected or a diminution of the disease state is achieved. Thickeners, flavoring agents, diluents, emulsifiers, dispersing Optimal dosing schedules can be calculated from measure aids or binders may be desirable. ments of drug accumulation in the body of the patient. The 0149 Compositions and formulations for parenteral, administering physician can easily determine optimum dos intrathecal or intraventricular administration may include ages, dosing methodologies and repetition rates. Optimum sterile aqueous Solutions that may also contain buffers, dilu dosages may vary depending on the relative potency of indi US 2014/0227350 A1 Aug. 14, 2014

vidual peptide combinations, and can generally be estimated Annexin A2. Liposomes are microscopic spherical lipid based on ECsos found to be effective in in vitro and in vivo bilayers Surrounding an aqueous core that are made from animal models or based on the examples described herein. In amphiphilic molecules such as phospholipids. For example, a general, dosage is from 0.01 ug to 100 g per kg of body liposome may comprise tpA and Annexin A2 trapped weight, and may be given once or more daily, weekly, between hydrophobic tails of the phospholipid micelle. In monthly or yearly. The treating physician can estimate rep addition to these peptides, water soluble drugs can be etition rates for dosing based on measured residence times entrapped in the core and lipid-soluble drugs can be dissolved and concentrations of the drug in bodily fluids or tissues. in the shell-like bilayer. Liposomes have a special character Following successful treatment, it may be desirable to have istic in that they enable water soluble and water insoluble the Subject undergo maintenance therapy to prevent the recur chemicals to be used together in a medium without the use of rence of the disease state, wherein the peptide combination is Surfactants or other emulsifiers. administered in maintenance doses, ranging from 0.01 ug to 0162 Liposomes form spontaneously by forcefully mix 100g per kg of body weight, once or more daily, to once every ing phospholipids in aqueous media. Water Soluble com 20 years. pounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, VI. Drug Delivery Systems therefore, these compounds are trapped within the aqueous 0156 The present invention contemplates several drug liposomal center. The liposome wall, being a phospholipid delivery systems that provide for the administration of a membrane, holds fat soluble materials such as oils. Lipo roughly uniform distribution, having controllable rates of Somes provide controlled release of incorporated com release, of a combination of Annexin A2 and tRA. A variety of pounds. In addition, liposomes can be coated with water different media are described below that are useful in creating soluble polymers, such as polyethylene glycol to increase the Such drug delivery systems. It is not intended that any one pharmacokinetic half-life. One embodiment of the present medium or carrier is limiting to the present invention. Note invention contemplates an ultra high-shear technology to that any medium or carrier may be combined with another refine liposome production, resulting in stable, unilamellar medium or carrier; for example, in one embodiment a poly (single layer) liposomes having specifically designed struc mer microparticle carrier attached to a compound may be tural characteristics. These unique properties of liposomes, combined with a gel medium. allow the simultaneous storage of normally immiscible com 0157 Carriers or mediums contemplated by this invention pounds and the capability of their controlled release. comprise a material selected from the group comprising gela 0163 The present invention contemplates cationic and tin, collagen, cellulose esters, dextran Sulfate, pentosan anionic liposomes, as well as liposomes having neutral lipids polysulfate, chitin, Saccharides, albumin, fibrin Sealants, Syn comprising tPA and Annexin A2. Preferably, cationic lipo thetic polyvinyl pyrrolidone, polyethylene oxide, polypropy Somes comprise negatively-charged materials by mixing the lene oxide, block polymers of polyethylene oxide and materials and fatty acid liposomal components and allowing polypropylene oxide, polyethylene glycol, acrylates, acryla them to charge-associate. Clearly, the choice of a cationic or mides, methacrylates including, but not limited to, 2-hy anionic liposome depends upon the desired pH of the final droxyethyl methacrylate, poly(ortho esters), cyanoacrylates, liposome mixture. Examples of cationic liposomes include gelatin-resorcin-aldehyde type bioadhesives, polyacrylic lipofectin, lipofectamine, and lipofectace. acid and copolymers and block copolymers thereof. 0164. One embodiment of the present invention contem 0158. One embodiment of the present invention contem plates a medium comprising liposomes that provide con plates a medical device comprising several components trolled release oftBA and Annexin A2. Preferably, liposomes including, but not limited to, a reservoir comprising thA/ that are capable of controlled release: i) are biodegradable and Annexin A2, a catheter, a sprayer or a tube. In one embodi non-toxic; ii) carry both water and oil soluble compounds; iii) ment, said medical device administers either an internal or solubilize recalcitrant compounds; iv) prevent compound external spray to a patient. In another embodiment, said medi oxidation; v) promote proteinstabilization; vi) control hydra cal device administers either an internal or external gel to a tion; vii) control compound release by variations in bilayer patient. composition Such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of satu Microparticles rated and unsaturated fatty acids, and physical configuration; 0159. One embodiment of the present invention contem viii) have solvent dependency; iv) have pH-dependency and plates a medium comprising a microparticle. Preferably, V) have temperature dependency. microparticles comprise liposomes, nanoparticles, micro 0.165. The compositions of liposomes are broadly catego spheres, nanospheres, microcapsules, and nanocapsules. rized into two classifications. Conventional liposomes are Preferably, some microparticles contemplated by the present generally mixtures of stabilized natural lecithin (PC) that may invention comprise poly(lactide-co-glycolide), aliphatic comprise synthetic identical-chain phospholipids that may or polyesters including, but not limited to, poly-glycolic acid may not contain glycolipids. Special liposomes may com and poly-lactic acid, hyaluronic acid, modified polysaccha prise: i) bipolar fatty acids; ii) the ability to attach antibodies rides, chitosan, cellulose, dextran, polyurethanes, polyacrylic for tissue-targeted therapies; iii) coated with materials such acids, pseudo-poly(amino acids), polyhydroxybutrate-re as, but not limited to lipoprotein and carbohydrate; iv) mul lated copolymers, polyanhydrides, polymethylmethacrylate, tiple encapsulation and V) emulsion compatibility. poly(ethylene oxide), lecithin and phospholipids. 0166 Liposomes may be easily made in the laboratory by 0160 Liposomes methods such as, but not limited to, Sonication and vibration. 0161. One embodiment of the present invention contem Alternatively, compound-delivery liposomes are commer plates liposomes capable of attaching and releasing tPA and cially available. For example, Collaborative Laboratories, US 2014/0227350 A1 Aug. 14, 2014

Inc. are known to manufacture custom designed liposomes centrifugal extrusion, pan coating and air Suspension. Micro for specific delivery requirements. spheres/microcapsules can be engineered to achieve particu lar release rates. For example, Oliosphere R (Macromed) is a Microspheres, Microparticles and Microcapsules controlled release microsphere system. These particular 0167 Microspheres and microcapsules are useful due to microsphere’s are available in uniform sizes ranging between their ability to maintain a generally uniform distribution, 5-500 um and composed of biocompatible and biodegradable provide stable controlled compound release and are economi polymers. Specific polymer compositions of a microsphere cal to produce and dispense. Preferably, an associated deliv may control the drug release rate Such that custom-designed ery gel or the compound-impregnated gel is clear or, alterna microspheres are possible, including effective management tively, said gel is colored for easy visualization by medical of the burst effect. ProMaxXR (Epic Therapeutics, Inc.) is a personnel. One of skill in the art should recognize that the protein-matrix drug delivery system. The system is aqueous terms “microspheres, microcapsules and microparticles' in nature and is adaptable to standard pharmaceutical drug (i.e., measured in terms of micrometers) are synonymous delivery models. In particular, ProMaxx(R) are bioerodible with their respective counterparts "nanospheres, nanocap protein microspheres that deliver both Small and macromo Sules and nanoparticles' (i.e., measured in terms of nanom lecular drugs, and may be customized regarding both micro eters). It is also clear that the art uses the terms “micro/ sphere size and desired drug release characteristics. nanosphere, micro?nanocapsule and micro?nanoparticle' 0173. In one embodiment, a microsphere or microparticle interchangeably, as will the discussion herein. comprises a pH sensitive encapsulation material that is stable 0168 Microspheres can be obtainable commercially (Pro at a pH less than the pH of the internal mesentery. The typical leasee, Alkermes: Cambridge, Mass.) wherein thA and range in the internal mesentery is pH 7.6 to pH 7.2. Conse Annexin A2 may be homogenized in a Suitable solvent and quently, the microcapsules should be maintained at a pH of sprayed to manufacture microspheres in the range of 20 to 90 less than 7. However, if pH variability is expected, the pH um. Techniques are then followed that maintain Sustained sensitive material can be selected based on the different pH release integrity during phases of purification, encapsulation criteria needed for the dissolution of the microcapsules. The and storage. Scott et al., Improving Protein Therapeutics With encapsulated compound, therefore, will be selected for the Sustained Release Formulations, Nature Biotechnology, Vol pH environment in which dissolution is desired and stored in ume 16:153-157 (1998). a pH preselected to maintain stability. Examples of pH sen 0169 Modification of the microsphere composition by the sitive material useful as encapsulants are Eudragit(R) L-100 or use of biodegradable polymers can provide an ability to con S-100 (Rohm GMBH), hydroxypropyl methylcellulose trol the rate of tRA/Annexin A2 release. Miller et al., Degra phthalate, hydroxypropyl methylcellulose acetate Succinate, dation Rates of Oral Resorbable Implants {Polylactates and polyvinyl acetate phthalate, cellulose acetate phthalate, and Polyglycolates: Rate Modification and Changes in PLA/PGA cellulose acetate trimellitate. In one embodiment, lipids com Copolymer Ratios, J. Biomed. Mater. Res., Vol. 1:711–719 prise the inner coating of the microcapsules. In these compo (1977). sitions, these lipids may be, but are not limited to, partial 0170 Alternatively, a sustained or controlled release esters of fatty acids and hexitiol anhydrides, and edible fats microsphere preparation is prepared using an in-water drying such as triglycerides. Lew C. W. Controlled-Release pH Sen method, where an organic solvent solution of a biodegradable sitive Capsule And Adhesive System And Method. U.S. Pat. polymer metal salt is first prepared. Subsequently, a dissolved No. 5,364,634 (herein incorporated by reference). or dispersed medium of tRA/Annexin A2 is added to the biodegradable polymer metal salt solution. The weight ratio 0.174. In one embodiment, a microparticle contemplated of tRA/Annexin A2 to the biodegradable polymer metal salt by this invention comprises a gelatin, or other polymeric may for example be about 1:100000 to about 1:1, preferably cation having a similar charge density to gelatin (i.e., poly about 1:20000 to about 1:500 and more preferably about L-lysine) and is used as a complex to form a primary micro 1:10000 to about 1:500. Next, the organic solvent solution particle. A primary microparticle is produced as a mixture of containing the biodegradable polymer metal salt and tRA/ the following composition: i) Gelatin (60 bloom, type A from Annexin A2 is poured into an aqueous phase to prepare an porcine skin), ii) chondroitin 4-sulfate (0.005%-0.1%), iii) oil/water emulsion. The solvent in the oil phase is then evapo glutaraldehyde (25%, grade 1), and iv) 1-ethyl-3-(3-dimethy rated off to provide microspheres. Finally, these microspheres laminopropyl)-carbodiimide hydrochloride (EDC hydro are then recovered, washed and lyophilized. Thereafter, the chloride), and ultra-pure Sucrose (Sigma Chemical Co., St. microspheres may be heated under reduced pressure to Louis, Mo.). The source of gelatin is not thought to be critical; remove the residual water and organic solvent. it can be from bovine, porcine, human, or other animal source. 0171 Other methods useful in producing microspheres Typically, the polymeric cation is between 19,000-30,000 that are compatible with a biodegradable polymer metal salt daltons. Chondroitin sulfate is then added to the complex with and atPA/Annexin A2 mixture are: i) phase separation during Sodium sulfate, or ethanol as a coacervation agent. a gradual addition of a coacervating agent; ii) an in-water 0.175. Following the formation of a microparticle, a tRA/ drying method or phase separation method, where an anti Annexin A2 composition is directly bound to the surface of flocculant is added to prevent particle agglomeration and iii) the microparticle or is indirectly attached using a “bridge' or by a spray-drying method. 'spacer. The amino groups of the gelatin lysine groups are 0172. In one embodiment the present invention contem easily derivatized to provide sites for direct coupling of a plates a medium comprising a microsphere or microcapsule composition. Alternatively, spacers (i.e., linking molecules capable of delivering a controlled release of a tRA/Annexin and derivatizing moieties on targeting ligands) Such as avidin A2 composition for a duration of approximately between 1 biotin are also useful to indirectly couple targeting ligands to day and 6 months. Controlled release microcapsules may be the microparticles. Stability of the microparticle is controlled produced by using known encapsulation techniques such as by the amount of glutaraldehyde-spacer crosslinking induced US 2014/0227350 A1 Aug. 14, 2014

by the EDC hydrochloride. A controlled release medium is NaCl (pH8), and sonicated for 2 minutes. The lysate was also empirically determined by the final density of glutaral purified using a nickel-affinity column (His-Trap, purchased dehyde-spacer crosslinks. from Amersham Pharmacia). Protein purity of ra2 protein was confirmed by SDS-PAGE followed by Coomassie blue VII. Kits staining, and its identity was verified by Western Blot analy 0176). In another embodiment, the present invention con S1S. templates kits for the practice of the methods of this inven tion. The kits preferably include one or more containers con Example II taining a vascular disorder treatment method of this invention. The kit can include a medium comprising thA and Plasmin Activity Assay Annexin A2. The kit can include a container comprising the 0180 Individual or combined concentrations of ra2, tRA, medium. The medium can optionally be a liquid. The kit can uPA (100 units/ml), BSA protein were added directly to wells optionally include medical devices including, but not limited of 96-well culture plate preloaded with N-terminal lysine to, injection syringes, intravenous drip bags, intravenous plasminogen (2.5ug/ml) and a fluorogenic plasmin Substrate catheters, tubing comprising connector (i.e., for example, (D-Val-Leu-Lys-AMC (200 nM)) in a final volume of 100 ul Leur Lock connectors) capable of attaching a drip bag to a PBS. After incubation at 37°C. for 30 min, plasmin genera catheter. The kit can optionally include a pharmaceutically tion or activity was read on a fluorescent plate reader at acceptable excipient and/or a drug delivery vehicle (e.g., a excitation 360 nm and emission 460 nm (20). The plate read liposome). The medium may be provided Suspended in the ings were expressed as relative fluorescent units for each well, excipient and/or delivery vehicle or may be provided as a and the final result was represented as fold of plasminactivity separate component which can be later combined with the generated by tPA or uPA alone. excipient and/or delivery vehicle. 0177. The kits may also optionally include appropriate Example III systems (e.g. opaque containers) or stabilizers (e.g. antioxi dants) to prevent degradation of the reagents by light or other Animal Models of Focal Embolic Cerebral Ischemia adverse conditions. 0.178 The kits may optionally include instructional mate 0181 All experiments were performed following an insti rials containing directions (i.e., protocols) providing for the tutionally approved protocol in accordance with the National use of the mediums for treatment of vascular disorders. In Institutes of Health Guide for the Care and Use of Laboratory particular the disorders can include, but are not limited to, Animals. stroke, myocardial infarction, deep vein thrombosis, or pull 0182 Male Wistar rats (270-300g) were anesthetized with monary embolism. While the instructional materials typically 1-2% isoflurane under spontaneous respiration in a 30% oxy comprise written or printed materials they are not limited to gen/70% nitrous oxide mixture. Rectal temperatures were Such. Any material capable of storing Such instructions and maintained between 37° C. and 38° C. with a thermostat communicating them to an end user is contemplated by this controlled heating pad. The right femoral artery was cannu invention. Such material include, but are not limited to elec lated, and physiologic parameters including rectal tempera tronic storage material (e.g., magnetic discs, tapes, cartridges, ture, mean arterial blood pressure, pH, PCO, and PO, were chips), optical material (e.g., CD ROM), and the like. Such monitored throughout all experiments. The right femoral vein material may include addresses to internet sites that provide was cannulated for drug administration. Such instructional materials. 0183 Focal embolic strokes were induced as previously described (2), except that two blood clots, 20-mm in length, EXPERIMENTAL were infused. In the appropriate groups, human recombinant tPA (Activase?R), Genentech Inc, San Francisco, Calif., U.S. Example I A.) and/or ra2 protein were administered intravenously. An initial 10% bolus dose was followed by continuous infusion Preparation of Recombinant Human Annexin A2 of the remaining drug over a 30 minute period. The relatively high dose of tRA was chosen based on the approximately 0179. Histidine-tagged recombinant Annexin A2 (ra2) ten-fold difference in fibrin-specific enzyme activity between was produced in E. coli. using a known bacterial expression human and rodent systems (22). Only animals surviving 24h vector containing full-length human Annexin A2 cl DNA (20). after stroke were included in the study. Numbers of dead Briefly. A full-length human Annexin II cDNA (See, FIG.5B) animals were counted in calculating the overall mortality rate was inserted into a histidine-tagged bacterial expression vec for each group of animals. All drug treatments were per tor, pCRE (purchased from Qiagen), between the KpnI and Sall formed by an investigator blinded to the Surgical groups. sites (pCE-A2). A mutant A2 (mA2) expression vector that lacks 224 amino acids of the C-terminal was created as fol Example IV lows. A stop codon was introduced at 114 amino acids using an oligonucleotide-based mutagenesis kit (Quick Change, Laser-Doppler Flowmetry Analysis Clontech) and two complement oligonucleotides 5'-GCT TCTGAGCTATA-GGCITCCATGAAG-3' and 5'-CT 0.184 Regional cerebral blood flow (rCBF) was monitored TCATGGAAGCCTATAGCT-CAGAAGC-3' according to continuously by laser-doppler flow metry (LDF). In brief an the manufacturers instructions. The mutated sequence was LDF probe was positioned 2 mm posterior and 5 mm lateral to confirmed by DNA sequencing. A cell lysate prepared from the bregma, thus monitoring the rCBF of the middle cerebral overnight cultures of pGE-AN II-transformed JM109 was artery (MCA). Initial stable records of pre-ischemic baseline pelleted (8000 rpm, 15 minutes), resuspended in a buffer were set as 100% for each rat. Following clot infusion, the containing 50 mmol/L sodium phosphate and 300 mmol/L MCA was considered to have been successfully occluded if US 2014/0227350 A1 Aug. 14, 2014

the rCBF dropped to less than 30% of the pre-ischemic base (0192 4. Armstead W M, Nassar T, Akkawi S, et al. Neu line. Animals with rCBF levels of more than 40% of pre tralizing the neurotoxic effects of exogenous and endog ischemic baseline were excluded from the study. In the enous tA. Nat. Neurosci. September 2006: 9(9): 1150 delayed 4 hr treatment study, rCBF was monitored for 2 h 1155. after induction of ischemia, and then reprobed in the same (0193 5. Lapchak PA, Araujo DM, Pakola S, et al. Micro position and continuously monitored for 1 h after treatment. plasmin: a novel thrombolytic that improves behavioral outcome after embolic strokes in rabbits. Stroke. Septem Example V ber 2002; 33(9):2279-2284. 0194 6. Lapchak PA, Song D. Wei J, et al. Coadministra Analysis of Infarct Volumes tion of NXY-059 and six hours following 0185. At 24 hours after induction of ischemia, rats were embolic strokes in rabbits improves clinical rating scores. sacrificed with a lethal dose of sodium pentobarbital and Exp Neurol. August 2004; 188(2):279-285. transcardially perfused to remove all intravascular blood. (0195 7. Haley EC, Jr., Levy D. E. Brott TG, et al. Urgent Coronal brain sections (2 mm thick) were stained with 2.3.5- therapy for stroke. Part II. Pilot study of tissue plasminogen triphenyltetrazolium chloride (TTC, Sigma, St. Louis, Mo., activator administered 91-180 minutes from onset. Stroke. U.S.A.). Infarct Volumes were quantified using computer May 1992; 23(5):641-645. assisted image analysis (2). To eliminate confounding effects (0196) 8. Brott TG, Haley EC, Jr., Levy DE, et al. Urgent of edema and Swelling, the indirect method was used (con therapy for stroke. Part I. Pilot study of tissue plasminogen tralateral Volume minus un-infarcted ipsilateral Volume). activator administered within 90 minutes. Stroke. May 1992; 23(5):632-640. Example VI 0197) 9. Zivin J. A. Thrombolytic stroke therapy: past, present, and future. Neurology. Jul. 13 1999; 53(1):14-19. Spectrophotometric Assay of Intracerebral 0198 10. Benchenane K, Berezowski V. Ali C, et al. Tis Hemorrhage Sue-type plasminogen activator crosses the intact blood brain barrier by low-density lipoprotein receptor-related 0186 The volume of intracerebral hemorrhage was quan protein-mediated transcytosis. Circulation. May 3, 2005; tified using a previously described spectrophotometric hemo 111(17):2241-2249. globin assay (2). 0199 11. Harada T. Kano T. Katayama Y, et al. Tissue Example VII plasminogen activator extravasated through the cerebral vessels: evaluation using a rat thromboembolic stroke Measurement of Plasma D-Dimer Levels model. Thromb Haemost. October 2005: 94(4):791-796. (0200 12. Tsirka SE, Rogove AD, Strickland S. Neuronal 0187 Blood samples were collected, anticoagulated upon cell death and tRA. Nature. Nov. 14 1996: 384(6605):123 addition of one-ninth volume of 3.2% (0.109M) trisodium 124. citrate, and centrifuged at 400 g for 15 min. Plasma samples 0201 13. Benchenane K, Lopez-Atalaya JP. Fernandez were stored at -80°C. until assayed. D-dimer was assayed on Monreal M. et al. Equivocal roles of tissue-type plasmino plasma samples diluted 1:4 in an ELISA kit (American Diag gen activator in stroke-induced injury. Trends Neurosci. nostica Inc., Stamford, Conn., USA) according to the manu March 2004; 27(3):155-160. facturers instructions. 0202 14. Kaur J, Zhao Z, Klein GM, et al. The neurotox icity of tissue plasminogen activator?J Cereb Blood Flow Example VIII Metab. September 2004; 24(9):945-963. Statistical Analysis 0203 15. Tsirka SE. Clinical implications of the involve ment ofteA in neuronal cell death. Mol. Med. May 1997: 0188 Data were expressed as mean+standard error of the 75(5):341-347. mean (S.E.M.). The Chi-square test was used to evaluate dif 0204 16. Kim J. Hajjar K.A. Annexin II: a plasminogen ferences in mortality. rCBF levels, infarct volumes, hemor plasminogen activator co-receptor. Front Biosci. Feb. 1 rhage volumes and D-dimer levels were assessed with 2002; 7:d341-348. ANOVA analysis followed by Tukey-Kramer tests. Differ (0205 17. Hajjar KA, Menell J S. Annexin II: a novel ences with P<0.05 were considered statistically significant. mediator of cell surface plasmin generation. Ann NY Acad. Sci. Apr. 15 1997; 811:337-349. REFERENCES 0206. 18. Hajjar KA, Acharya SS. Annexin II and regu 0189 1. Ding G, Jiang Q, Zhang L, et al. Analysis of lation of cell surface fibrinolysis. Ann NY Acad. Sci. May combined treatment of embolic stroke in rat with r-tRA and 2000:902:265-271. a GPIb/IIIa inhibitor. J Cereb Blood Flow Metab. January 0207. 19. Hajjar K.A. Krishnan S. Annexin II: a mediator 2005; 25(1): 87-97. of the plasmin/plasminogen activator system. Trends Car 0190. 2. Asahi M., Asahi K. Wang X, et al. Reduction of diovasc Med. July 1999; 9(5):128-138. tissue plasminogen activator induced hemorrhage and 0208. 20. Ishii H, Yoshida M, Hiraoka M, et al. Recombi brain injury by free radical spin trapping after embolic nant annexin II modulates impaired fibrinolytic activity in focal cerebral ischemia in rats. JCereb Blood Flow Metab. vitro and in rat carotid artery. Circ Res. Dec. 7. 2001; March 2000: 2003):452-457. 89(12): 1240-1245. 0191 3. Wang X, Tsuji K, Lee S R, et al. Mechanisms of (0209. 21. Tanaka Y. Ishii H, Hiraoka M, et al. Efficacy of hemorrhagic transformation after tissue plasminogen acti recombinant annexin 2 for fibrinolytic therapy in a rat vator reperfusion therapy for ischemic stroke. Stroke. embolic stroke model: A magnetic resonance imaging November 2004; 35(11 Suppl 1):2726-2730. study. Brain Res. Aug. 24 2007: 1165:135-143. US 2014/0227350 A1 Aug. 14, 2014 17

0210 22. Koringer C. Collen D. Studies on the specific 0221 33. Aarli A, Skeie Jensen T. Kristoffersen E K, et al. fibrinolytic effect of human extrinsic (tissue-type) plasmi Inhibition of phytohaemagglutinin-induced lymphoprolif nogen activator in human blood and in various animal eration by soluble annexin II in sera from patients with species in vitro. Thromb Haemost. Aug. 28 1981: 46(2): renal cell carcinoma. Apmis. September 1997: 105(9):699 561-565. 704. 0211 23. Morris DC, Zhang L, Zhang ZG, et al. Exten 0222 34. Pfefferkom T. Rosenberg G. A. Closure of the sion of the therapeutic window for recombinant tissue plas blood-brain barrier by matrix metalloproteinase inhibition minogen activator with argatroban in a rat model of embo reduces rtPA-mediated mortality in cerebral ischemia with lic stroke. Stroke. November 2001; 32(11):2635-2640. delayed reperfusion. Stroke. August 2003: 34(8): 2025 0212 24. Knecht MF, Heinrich F, Spanuth E. Evaluation 2O3O. of plasma D-dimer in the diagnosis and in the course of 0223. 35. Lapchak PA, Chapman D F. Zivin J. A. Metal fibrinolytic therapy of deep vein thrombosis and pulmo loproteinase inhibition reduces thrombolytic (tissue plas nary embolism. Thromb Res. Jul. 15 1992: 67(2):213-220. minogen activator)-induced hemorrhage after thromboem 0213 25. Marler J. R. Goldstein L. B. Medicine. Stroke bolic stroke. Stroke. December 2000; 31 (12):3034-3040. tRA and the clinic. Science. Sep.19 2003:301 (5640): 1677. 0224, 36. Zhang L, Zhang ZG, Liu X, et al. Treatment of 0214) 26. Grotta J C, Burgin W. S. El-Mitwalli A, et al. embolic stroke in rats with bortezomib and recombinant Intravenous tissue-type plasminogen activator therapy for human tissue plasminogen activator. Thromb Haemost. ischemic stroke: Houston experience 1996 to 2000. Arch January 2006:95(1):166-173. Neurol. December 2001: 58(12):2009-2013. 0215 27. Nassar T, Akkawi S, Shina A, et al. In vitro and 0225 37. Zhang L, Zhang ZG, Zhang R, et al. Adjuvant in vivo effects of tRA and PAI-1 on blood vessel tone. treatment with a glycoprotein IIb/IIIa receptor inhibitor Blood. Feb. 1 2004; 103(3):897-902. increases the therapeutic window for low-dose tissue plas 0216. 28. Nicole O. Docagne F, Ali C, et al. The pro minogen activator administration in a rat model of embolic teolytic activity of tissue-plasminogen activator enhances stroke. Circulation. Jun. 10 2003: 107(22):2837-2843. NMDA receptor-mediated signaling. Nat. Med. January 0226) 38. Lapchak PA, Araujo DM, Song D, et al. Effects 2001; 7(1):59-64. of the spin trap agent disodium-tert-butylimino)methyl 0217 29. Wang X, Lee S R, Arai K, et al. Lipoprotein benzene-1,3-disulfonate N-oxide (generic NXY-059) on receptor-mediated induction of matrix metalloproteinase intracerebral hemorrhage in a rabbit Large clot embolic by tissue plasminogen activator. Nat. Med. October 2003: stroke model: combination studies with tissue plasmino 9(10): 1313-1317. gen activator. Stroke. June 2002; 33(6):1665-1670. 0218. 30. Sena E. vander Worp HB, Howells D, et al. How 0227 39. Cheng T. Petraglia A. L. Li Z. et al. Activated can we improve the pre-clinical development of drugs for protein C inhibits tissue plasminogen activator-induced stroke? Trends Neurosci. September 2007:30(9):433-439. brain hemorrhage. Nat. Med. November 2006; 12(11): 0219. 31. Sakharov DV, Rijken DC. Superficial accumu 1278-1285. lation of plasminogen during plasma clot lysis. Circula 0228 40. Strbian D. Karjalainen-Lindsberg ML, Kovanen tion. Oct. 1 1995; 92(7): 1883-1890. PT, et al. Mast cell stabilization reduces hemorrhage for 0220 32. Siever DA, Erickson HP Extracellular annexin mation and mortality after administration of thrombolytics II. IntJ Biochem Cell Biol. November 1997: 29(11):1219 in experimental ischemic stroke. Circulation. Jul. 24 2007: 1223. 116(4):411-418.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 4

<21 Os SEQ ID NO 1 &211s LENGTH: 192 212s. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 1 Met Glu Gln His Phe Lieu. Gly Cys Val Lys Arg Ala Trp Asp Ser Ala 1. 5 1O 15

Glu Wall Ala Pro Glu Pro Glin Pro Pro Pro Asn. Ser Ser Glu Asp Arg 2O 25 3 O

Gly Pro Trp Pro Leu Pro Leu Tyr Pro Val Lieu. Gly Glu Tyr Ser Lieu. 35 4 O 45

Asp Ser Cys Asp Lieu. Gly Lieu Lleu Ser Ser Pro Cys Trp Arg Lieu Pro SO 55 60

Gly Val Tyr Trp Glin Asn Gly Lieu Ser Pro Gly Val Glin Ser Thir Lieu. 65 70 7s 8O

Glu Pro Ser Thr Ala Lys Pro Thr Glu Phe Ser Trp Pro Gly Thr Glin

US 2014/0227350 A1 Aug. 14, 2014

- Continued

<4 OOs, SEQUENCE: 3 gcttctgagc tataggct tc catgaag 27

<210s, SEQ ID NO 4 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 4

Ctt catggaa gcctatagct Cagaa.gc 27

We claim: 10. The method of claim 1, wherein said patient is a non 1. A method comprising: human. a) providing: 11. A method comprising: i) a patient exhibiting symptoms associated with a vas a) providing: cular disorder, and i) a human patient exhibiting symptoms associated with ii) a medium comprising Annexin A2 and tissue plasmi a recently incurred stroke, nogen activator (tPA); and ii) a medium comprising Annexin A2 and tissue plasmi b) administering said medium to said patient under condi nogen activator (tPA), wherein said Annexin A2 and tions such that said symptoms are reduced. said tRA have a dose ratio of 2:1; 2. The method of claim 1, wherein said vascular disorder is and, selected from the group consisting of stroke, myocardial inf b) administering said medium to said human patient under arction, pulmonary embolism, deep vein thrombosis and conditions such that said symptoms are reduced. intracerebral hematoma. 12. The method of claim 11, wherein said administering 3. The method of claim 1, wherein said Annexin A2 and occurred less than three hours after said stroke. said tRA have a dose ratio of 2:1. 13. The method of claim 11, wherein said administering 4. The method of claim 1, wherein said medium comprises occurred less than six hours after said stroke. a carrier. 14. The method of claim 11, wherein said administering 5. The method of claim 4, said Annexin A2 and said tRA are occurred less than twelve hours after said stroke. attached to said carrier. 15. The method of claim 11, wherein said tRA dose is at 6. The method of claim 1, wherein said carrier is selected least two-fold lower than the currently recommended dose. from the group consisting of a liposome and a microparticle. 16. The method of claim 11, wherein said tRA dose is at 7. The method of claim 1, wherein said medium is a liquid. least three-fold lower than the currently recommended dose. 8. The method of claim 7, wherein said administering is 17. The method of claim 11, wherein tA dose is at least intravenous. four-fold lower than the currently recommended dose. 9. The method of claim 1, wherein said patient is a human. k k k k k