TITLE

Metal Ion Chelation for Enhancing the

Effect of Tissue Plasminogen Activator (tPA) in Thrombolysis

RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application Ser. No. 62/157,808, filed under 35 U.S.C. § 111(b) on May 6, 2015, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with no government support. The government has no rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Ischemic stroke is the rapid loss of brain function because of the interruption of blood flow, and subsequently brain damage from oxygen-glucose deprivation due to thrombosis or embolism. Stroke is the second leading cause of death worldwide according to the World Health Organization, and the fifth leading cause of death in the US according to the Centers for Disease Control. Furthermore, it is a leading cause of serious long-term disability. Therefore, stroke has a significant impact and burden on individuals, the health care system, and society. Early and adequate medical intervention is critical for improving stroke outcomes. However, the only drug approved by FDA for treatment of acute thrombotic stroke so far is tissue plasminogen activator (tPA), referred to as the Gold Standard by the American Stroke Association.

[0004] The treatment of tPA, a thrombolytic agent, lyses the clot and reperfuses the occluded artery, which is important for saving neurons by improving blood flow to the part of the brain being deprived of blood flow under stroke. Unfortunately, only 1-3% of stroke patients in the US receive this therapy because of the narrow time window for, and severe side effects from, using tPA. Though tPA is effective at dissolving the blot clot which causes a stroke, tPA can dissolve other tissues as well, thereby causing hemorrhaging and other adverse effects. The most deadly and damaging side effect of tPA is the risk of intracranial bleeding or hemorrhage, which deteriorates the outcome of stroke. Since tPA dissolves walls of blood vessels, tPA at higher dosages significantly increases the risk of hemorrhage. For that reason, the dose of tPA and its overall administration are under tight control, while the effect of thrombolysis may be compromised. Thus, there are needs in the art for new and improved methods and compositions for the treatment of ischemic stroke, including methods that would increase the efficacy of tPA, improve the rate of successful thrombolysis by tPA, or reduce the side effects caused by tPA. SUMMARY OF THE INVENTION

[0005] Provided herein is a composition comprising an effective amount of a thrombolytic drug, at least one metal ion chelator capable of chelating zinc or iron, and a pharmaceutically acceptable excipient, diluent, or carrier. In certain embodiments, the thrombolytic drug comprises tissue plasminogen activator (tPA). In certain embodiments, the thrombolytic drug comprises streptokinase. In certain embodiments, the metal ion chelator is capable of chelating both zinc and iron. In certain embodiments, the metal ion chelator comprises ethylenediaminetetra-acetic acid (EDTA). In particular embodiments, the EDTA comprises calcium saturated EDTA (CaEDTA). In certain embodiments, the effective amount of the thrombolytic drug is less than an amount of the thrombolytic drug needed to be effective in the absence of the at least one metal ion chelator. In certain embodiments, the effective amount of the thrombolytic drug ranges from about 0.25 mg/kg to about 20 mg kg. In certain embodiments, the effective amount of the thrombolytic drug is about 2 mg kg. In certain embodiments, the effective amount of the thrombolytic drug is about 1 mg/kg. In certain embodiments, the effective amount of the thrombolytic drug is about 0.5 mg/kg. In certain embodiments, the at least one metal ion chelator is present at a concentration ranging from about 1 μΜ to about 100 mM . In certain embodiments, the composition further includes a second metal ion chelator capable of chelating zinc or ion. In certain embodiments, the metal ion chelator comprises N,N,N',N'-tetrakis(2-pyrdiylmethyl) ethylenediamine (TPEN). In certain embodiments, the metal ion chelator comprises EDTA and TPEN. In certain embodiments, the composition further includes an additional agent selected from the group consisting of: aspirin, heparin, coumarins (such as warfarin), clopidogrel, oxalate, citrate, hirudin, and combinations thereof.

[0006] Further provided is a method of treating a clotting event, the method comprising the steps of administering an effective amount of a metal ion chelator to a patient having a clotting event, and administering an effective amount of a thrombolytic drug to the patient to treat the clotting event. In certain embodiments, the thrombolytic drug comprises tissue plasminogen activator (tPA). In certain embodiments, the metal ion chelator and the thrombolytic drug are administered simultaneously. In particular embodiments, the metal ion chelator and the thrombolytic drug are in a single composition. In certain embodiments, the metal ion chelator and the thrombolytic drug are administering sequentially. In certain embodiments, the clotting event is selected from the group consisting of: stroke, myocardial infarction, vein thrombosis, and pulmonary embolism. In certain embodiments, the clotting event is a stroke, and the thrombolytic drug is tPA. In particular embodiments, the effective amount of the tPA is about 0.5 mg/kg or higher. In certain embodiments, the metal ion chelator comprises EDTA. In particular embodiments, the metal ion chelator comprises calcium saturated EDTA (CaEDTA). In certain embodiments, the metal ion chelator comprises TPEN. In certain embodiments, the thrombolytic drug comprises

streptokinase. In particular embodiments, the effective amount of streptokinase ranges from about 10,000 units to about 100,000,000 units. In certain embodiments, the metal ion chelator comprises EDTA and TPEN.

[0007] Further provided is a kit for preparing a composition, the kit comprising a first container housing a thrombolytic drug, and a second container housing a metal ion chelator. In certain embodiments, the metal ion chelator comprises EDTA and the thrombolytic drug comprises tPA. In certain embodiments, the kit further includes a syringe.

BRIEF DESCRIPTION OF THE DRAWING

[0008] The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees.

[0009] FIG. 1: Diagram of embolic or thrombotic stroke and stroke treatment with tPA.

[0010] FIG. 2: Diagram of embolic or thrombotic stroke and stroke treatment with tPA versus with a composition having a metal ion chelator and one -half the otherwise required dosage of tPA.

[0011] FIGS. 3A-3C: The set-up for blood clot lysis (thrombolysis) in vitro spectrophotometric measurement. FIG. 3A shows a schematic diagram of the custom-modified cuvette used. FIG. 3B shows a photograph of the cuvettes at the start of thrombolysis. Each blood clot was formed from 200 recalcified CDl mouse blood, and transferred into the cuvette. The color of lystate at this point was clear, and the absorbance was low. FIG. 3C shows a photograph of the cuvettes after 60 minutes of thrombolysis. Cuvette 1, 2, and 3 represented lysate of three doses of tPA (0.005 mg, 0.01 mg, and 0.05 mg).

[0012] FIGS. 4A-4D: The clot-lysis represented by absorbance at 1 hour in different

thrombolysis agents, measured by spectrophotometry at 580 nm wavelength. FIG. 4A shows the thrombolysis curve of tPA. FIG. 4B shows quantified thrombolysis at one hour treatment with tPA and tPA plus different metal ions. The dose of tPA was 0.01 mg. ZnCl ₂ : 200 μΜ. FeC , FeC : 100 μΜ. FIG. 4C shows the rate of thrombolysis of tPA. FIG. 4D shows the maximum rate of thrombolysis of tPA and tPA plus metal ions. Saline was used as a vehicle in these treatments. Data were shown as mean + SEM. The numbers of samples tested: 22 in tPA, 13 in (tPA + EDTA), 6-9 in tPA with Zn ²⁺ , Fe ²⁺ , and Fe ³⁺ , and 4 in saline. * p < 0.05. *** P < 0.001.

[0013] FIGS. 5A-5I: Quantified clot lysis in 60 min shown as absorbance increase measured by spectrophotometry at 580 nm. n = 22 in tPA alone. FIGS. 5A-5C show thrombolysis of tPA compared to tPA plus different doses of Fe ³⁺ . n = 6-10 per group of (tPA + Fe ³⁺ ). FIGS. 5D-5F show thrombolysis of tPA compared to tPA plus different doses of Zn ²⁺ . n=6-9 per group of (tPA + Zn ²⁺ ). FIGS. 5G-5I show thrombolysis of different doses of Fe ²⁺ with tPA. n=6-9 per group of (tPA + Fe ²⁺ ). * P< 0.05, *** P< 0.001.

[0014] FIGS. 6A-6I: The reaction velocity of thrombolysis in different treatments, n = 22 in tPA alone group. FIGS. 6A-6C show the rate of thrombolysis in different doses of Fe ⁺ plus tPA. n = 6-10 per group of (Fe ³⁺ + tPA). FIGS. 6D-6F show the rate of thrombolysis in different doses of Zn ²⁺ plus tPA. n = 6-9 per group of (Zn ²⁺ + tPA). FIGS. 6G-6I show the rate of thrombolysis in different doses of Fe ²⁺ plus tPA. n=6-9 per group of (Fe ²⁺ + tPA). * P< 0.05.

[0015] FIGS. 7A-7B: Effect of EDTA on clot-lysis of tPA. Ion chelation promotes tPA-induced thrombolysis. These graphs show the effect of EDTA on tPA-induced thrombolysis in 60 minutes. FIG. 7A shows quantified thrombolysis represented by change of optical density in 60 minutes. FIG. 7B shows the velocity of thrombolysis. Values are mean + SEM. n = 22 in tPA, n = 13 in (tPA + EDTA). * p < 0.05. The numbers of samples tested: 22 in tPA, and 13 in (tPA + EDTA).

[0016] FIGS. 8A-8C: Clot-lysis in vivo. FIG. 8A shows the exposure of the femoral artery on the CD1 mouse. The arrow shows the femoral artery. Scale bar: 1 cm. FIG. 8B shows photo thrombosis formed in the femoral artery (shown as the arrow), leaving the downstream blood-free (outlined by dash lines). FIG. 8C shows thrombolysis after tPA perfusion. Scale bar: 1 mm

[0017] FIGS. 9A-9B: Ion chelation improves reperfusion outcomes after photothrombosis of artery. These figures show real-time thrombolysis in vivo. FIG. 9A shows thrombolysis at each time point (0, 10, 20, 30, and 40 min), with different tPA treatments (tPA, ½ tPA and ½ tPA + CaEDTA). Femoral arteries were outlined by dash lines. Scale bar, 0.5mm. FIG. 9B shows the change of light transmission in each thrombosis. Empty dots represent data from reperfused cases. Black square dots represent data from non-reperfused cases. P < 0.01 between those two groups.

[0018] FIGS. 10A-10B: Table (FIG. 10A) and graph (FIG. 10B) showing the percentage of reperfusion versus non-reperfusion in vivo in different thrombolytic treatments. There were more reperfused cases in treatments of tPA and (½ tPA + CaEDTA). n = 10-12 in each group. *p<0.05 between (½ tPA) and (½ tPA + CaEDTA).

[0019] FIG. 11: Change of thrombolysis in tPA treatment and (½ tPA + CaEDTA). n = 7 in tPA group, n = 9 in (½ tPA + CaEDTA) group. *P<0.05.

[0020] FIG. 12: Graph showing the percentage of blood clot lysis from different treatments. The metal ion chelator CaEDTA or TPEN increased clot lysis when co-applied with streptokinase (SK), compared to lysis of the control groups (SK, CaEDTA, or TPEN alone). The addition of chelator without SK showed little to no lysis.

[0021] FIG. 13: Graph showing the blood clot lysis from different treatments. The addition of zinc (200 μΜ) inhibited the streptokinase (SK)-induced clot lysis when compared to the lysis of the streptokinase treatment group.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Throughout this disclosure, various publications, patents and published patent

specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this disclosure pertains.

[0023] Although tPA is the only drug approved by the FDA for treatment of acute thrombotic stroke, and the standard of care for treatment of acute thrombotic stroke, it must be administered as early as possible after the onset of stroke symptoms in order to be effective in the treatment of ischemic stroke. Protocol guidelines require its use intravenously within the first three hours of the event, after which its detrimental side effects may outweigh its benefits. Therefore, the patient needs to present in the hospital soon after the onset of stroke symptoms, which is almost impossible. For this reason, only about 1-3% of stroke patients in the United States receive this therapy.

[0024] The bloodstream contains and transports a large variety of ions, including major trace elements zinc and iron. The dyshomeostasis of these metal ions is closely correlated to the pathogenesis of cerebral ischaemia, contributing significantly to brain damage in stroke. Zinc (Zn ²⁺ ) has been correlated to cell damage in stroke. Without wishing to be bound by theory, it is believed that both zinc and iron are involved in the clotting of blood. Iron is important for red blood cells and plays role in coagulation. Similarly, zinc deficiency can lead to increased bleeding tendency and delayed wound healing by poor platelet aggregation.

[0025] The concentration of total zinc in blood plasma is around 15 μΜ or 60-120μg /100ml, which is bound predominantly to blood proteins. A substantial amount of zinc has also been found in various cellular compartments, vesicles, or vacuoles. Zn is stored in many types of cells, including platelets, which are responsible for making clots. Zn concentration is high (in the micromolar range) in platelets. In the bloodstream, the most zinc-enriched site is the a-granule of platelets, where the zinc concentration reaches 500μΜ. This is almost a 30-fold higher level of zinc in platelets than in the serum. Like zinc, iron also has intracellular and extracellular repositories. The amount of serum iron is 60-170 μg /100ml, which is slightly higher than serum zinc. The hemoglobin of erythrocytes, on the other hand, is the major site of iron storage. About 65% of the iron in the body is bound up in hemoglobin, where iron is bound with heme. Iron is strictly regulated by the binding of proteins such as transferrin in the bloodstream and ferritin in cell plasma.

[0026] The platelets and erythrocytes are physiologically important for thrombus formation. During the clot formation, a large amount of zinc releases from platelets and leads to a surge in zinc concentrations in the local microenvironment of an expanding thrombus, which promotes fibrin formation and triggers key signalling events that lead to platelet aggregation. Together with fibrin, aggregated platelets and erythrocytes are major components in thrombus composition. Because platelets and erythrocytes store a substantial amount of zinc (in platelet granules) and iron (in hemes), thrombi become a source of these metal ions as they are "trapped" in the thrombi. The precise role of these thrombus ions is not known. However, without wishing to be bound by theory, it believed that zinc enhances the production of fibrin, and free iron promotes blood coagulation and increases fibrinogen strength. Hence, the presence of increasing free zinc and iron creates resistance to thrombolysis.

[0027] Zinc is part of the processes of thrombosis and thrombolysis, and is involved in several steps of blood coagulation. For example, zinc triggers platelets aggregation. High concentrated zinc is packed in a-granules of platelets, which make platelets the highest zinc-containing cells among hematocytes. Zinc can affect hemostasis by directly affecting the function of platelets. The activated platelets secrete the contents of these granules through platelet exocytosis. During coagulation and thrombosis, the platelets undergo the cascade of activation, adhesion, and aggregation as the important part of the clot formation process. Zinc has been shown to induce platelet aggregation and affect ADP-induced aggregation in a synergistic manner. Zinc deficient rodents and humans have all displayed an increase in bleeding time and a decrease in platelet function. Zinc interacts with factor FXII and may be an essential cofactor. When local zinc concentration is 10-30 fold higher than plasma, zinc activates FXII and potentiates blood clotting. Zinc has been shown to regulate fibrinolytic pathways and to enhance the production of fibrin. When it comes to intensifying polymerization of fibrin oligomer, zinc is an introducer stronger than calcium. Furthermore, zinc prevents thrombolysis by inhibiting fibrin clearance. The presence of a substantial amount of zinc provides a positive feedback mechanism for platelet aggregation, the propagation of coagulation, and stabilizing the blood clot.

[0028] Another ion that is important for thrombolysis is iron. Generally, an adult human contains 3-4 grams of iron with 2-3 grams in erythrocytes. Iron is also strictly regulated by the binding of proteins such as transferring in the bloodstream and ferritin in cell plasma. Free iron promotes blood coagulation. Like zinc, iron also has intracellular and extracellular repositories, which are able to yield a locally high level of free Fe ³⁺ /Fe ²⁺ during thrombolysis.

[0029] Platelets (storing zinc) collapse during clot formation, creating a significant amount of Zn in the clot. Without wishing to be bound by theory, it is believed that during thrombolysis, zinc and iron are dissociated from the blood clot, creating a focal high concentration of these ions. When a thrombolytic drug, such as tPA, dissolves the clot, the dissolution of the clot releases the Zn from the clot. Without wishing to be bound by theory, it is believed that this released Zn interacts with the thrombolytic drug. Similarly, a high concentration of Fe is present in blood clots, such that when a thrombolytic drug dissolves a blood clot, the dissolution of the clot releases Fe from the clot. Without wishing to be bound by theory, it is believed that this released Fe interacts with the thrombolytic drug. More generally, it is believed that there are interactions between ions, such as zinc and iron, and thrombolysis mediated by a thrombolytic drug such as tPA.

[0030] The zinc increase triggers platelets' activation and blood coagulation. The examples herein describe the effect of zinc and iron on the tPA-induced thrombolysis of a whole blood clot measured in vitro, which is a physiologically relevant model. The results show that the application of zinc or iron significantly inhibits tPA-induced thrombolysis. The therapeutic effect of metal ion chelation on tPA-induced thrombolysis in vivo was also evaluated. As seen from the examples, because zinc and iron inhibit tPA-induced thrombolysis, the chelation of these ions enhance its action. Removing zinc, removing iron, or removing both zinc and iron, with metal ion chelators accelerates tPA-mediated thrombolysis. Removing zinc, removing iron, or removing both zinc and iron, with metal ion chelators also reduces the necessary dose of tPA. The chelation removes not only chelatable ions in the serum but also free zinc and iron dissociated from the blood clots where tPA is inducing thrombolysis. As described in the examples, the co- application of a metal ion chelator facilitates the action of tPA on the thrombolysis of a femoral artery thrombus. The chelation accelerates the rate of tPA-induced thrombolysis, enhances overall tPA-induced thrombolysis, and reduces tPA doses necessary for effective thrombolysis. Thus, described herein are compositions and methods that utilize the co-application of a chelator and a thrombolytic drug (such as tPA) to improve the efficacy, potency, and safety of the thrombolytic drug in the treatment of a clotting event such as a stroke.

[0031] In accordance with the above, provided herein are compositions that include a

thrombolytic drug and at least one metal ion chelator, as well as methods that involve the co- application (either simultaneously or sequentially) or a thrombolytic drug and at least one metal ion chelator. The presence of the metal ion chelator can enhance the potency and efficacy of the thrombolytic drug, and can reduce the necessary concentration of the thrombolytic drug. In some embodiments of the compositions, the composition can have the same efficacy as a certain concentration of the thrombolytic drug alone when it contains one-half the concentration of the thrombolytic drug in combination with a metal ion chelator.

[0032] Both zinc and iron inhibit tPA-induced thrombolysis by postponing the maximum effect or decreasing tPA efficacy. The examples herein show further that the removal of zinc and/or iron, by the co-application of a metal ion chelator with a thrombolytic drug, enhances the effect of the thrombolytic drug-induced thrombolysis in vivo. Though tPA is described for exemplary purposes, it is to be understood that the present disclosure is not limited to tPA, and is not limited to applications involving clotting events in the brain. In fact, a metal ion chelator may be combined and/or co-applied with any other thrombolytic drugs to improve their potency and efficacy in treating, for example, myocardial infarction, vein thrombosis, or pulmonary embolism. To illustrate this broad effect of metal ion chelators, the examples herein include an evaluation of a metal ion chelator with streptokinase. Streptokinase is a thrombolytic drug used for treating myocardial infarction and pulmonary embolism. As the examples show, co-application of a metal ion chelator was able to increase clot lysis caused by streptokinase (FIG. 12) as well as tPA

(FIGS. 7A-7B, FIGS. 10A-10B, FIG. 11). [0033] The compositions and methods herein can include any thrombolytic drugs (or combinations thereof), and are not limited to the treatment of strokes. Suitable thrombolytic drugs include, but are not limited to: tPA, streptokinase (Kabiknase, Streptase) or recombinant streptokinase, reteplase (Retavase), tenecteplase (TNKase), anistreplase (Eminase), urokinase (Abbokinase), single-chain urokinase-type plasminogen activator, plasminogen acetylation- streptokinase activate complex, alteplase, PPA, Batroxobin, Sak, venom thrombolytic enzymes, or combinations thereof. Further, it is understood that reference to "tissue plasminogen activator" and "tPA" can include recombinant tissue plasminogen activator (rtPA).

[0034] The thrombolytic drug can be present in the composition at a concentration lower than would be necessary for effective thrombolysis in the absence of a metal ion chelator. Generally, the concentration of the thrombolytic drug in the compositions ranging from about 0.1 mg kg to about 30 mg/kg, or from about 0.25 mg/kg to about 20 mg/kg. In one non-limiting example, the thrombolytic drug is present at a concentration of only about 0.5 mg/kg or higher. In another non- limiting example, the thrombolytic drug is present at a concentration of about 10 mg/kg. When the thrombolytic drug is streptokinase, the effective amount of the streptokinase present in the composition may be expressed as units or international units. As used herein, a "unit" refers to an amount of streptokinase which will liquefy a standard clot of fibrinogen, plasminogen, and thrombin at pH 7.5 at 37 °C in 10 minutes. An "international unit" of streptokinase, on the other hand, is defined as the activity contained in 0.002090 mg of the international standard for streptokinase-streptodornase. In some embodiments, the effective amount of streptokinase in accordance with the present disclosure can range from about 10,000 units to about 100,000,000 units.

[0035] The metal ion chelator can be a chelator capable of chelating zinc (a "zinc chelator"), a chelator capable of chelating iron (an "iron chelator"), or a chelator capable of chelating both zinc and iron. In some embodiments, the composition includes multiple metal ion chelators. In such embodiments, the multiple metal ion chelators can each be zinc chelators, iron chelators, or combinations thereof.

[0036] Suitable zinc chelators include, but are not limited to: ethylenediaminetetra-acetic acid (EDTA); l,3-diaminopropane-N,N,N',N'-tetraacetic acid (DTP A); N,N,N',N'-tetrakis(2- pyrdiylmethyl) ethylenediamine (TPEN); 1,10-phenanthroline; clioquinol; diethyldithiocarbamate (DEDTC), 2,3-dimercapto-l-propanesulfonic acid (DMPS); ethylenediamine-N,N'-diacetic-N,N'- di-B-propionic acid (EDPA); l,2-dimethyl-3-hydroxy-4-pyridinone (DMHP); l,2-diethyl-3- hydroxy-4-pyridinone (DEHP); ethylmaltol (EM), 4-(6-methoxy-8-quinaldinyl- aminosulfonyl)benzoic acid potassium salt (TFLZn); dithiozone; N-(6-methoxy-8-quinolyl)-para- toluenesulfonamide (TSQ); carnosine; deferasirox; trans- l,2-cyclohexane-diamine-N,N,N',N'- tetraacetic acid (CyDTA); dihydroxyethylglycine (DHEG); l,3-diamino-2-hydroxypropane- Ν,Ν,Ν',Ν'-tetraacetic (DTPA-OH); ethylenediamine-N,N'-diacetic acid (EDDA); ethylenediamine- Ν,Ν'-dipropionic acid (EDDP); ethylenediarnine-N,N'-bis(methylpliosplioriic) acid (EDDPO); N- hydroxy-ethylenediamine-N,N',N'-triacetic acid (EDTA-OH); aminophenol triacetic acid

(APTRA); ethylenediarninetetra(methylenephosplioriic) acid (EDTPO); N,N'-bis(2- hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED); hexamethylene-1,6- diaminetetraacetic acid (HDTA); hydroxyethyliminodiacetic acid (HIDA); iminodiacetic acid (IDA); methyl-EDTA, nitrilotriacetic acid (NTA); nitrilotripropionic acid (NTP),

nitrilotrimethylenphosphonic acid (NTPO); 7,19,30-trioxa-l,4,10,13,16,22,27,33- octaazabicyclo[l 1,11,11] pentatriacontane (O-Bistren); triethylenetetraaminehexaacetic acid (TTHA); ethyleneglycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); dimercaptosuccinic acid (DMSA); deferoxamine; dimercaprol; zinc citrate; combinations of bismuth and citrate; penicilamine;

succimer; etidronate; ethylenediamine-di (O-hydroxyphenylacetic acid) (EDDHA); trans- 1,2- cyclohexanediaminetetraacetic acid (CDTA); N-(2 -hydro xyethyl) ethylenedinitrilotriacetic acid (HEDTA); N-(2-hydroxyethyl) iminodiacetic acid (HEIDA); 9-(0-carboxyphenyl)-2,7-dichloro- 4,5,-bis[bis(2-pyridylmethyl)-aminomethyl]-6-hydroxy-3-xanth one; 9-(0-carboxyphenyl)-4,5- bis[bis(2-pyridylmethyl)-aminomethyl]-6-hydroxy-3-xanthanone ; 9-(0-carboxyphenyl-2-chloro-5- [2-{bis(2-pyridylmethyl)aminomethyl}-N-methylaniline]-6-hydr oxy-3-xanthanone; calprotectin; zinc fingers; lactoferrin; ovotransferrin; conalbumin; salts thereof; and combinations thereof. In other embodiments, the zinc chelator is a wild-type apoenzyme or genetically engineered mutant thereof. Examples of the apoenzyme include, but are not limited to, apo-carbonic anhydrase, a S166C mutant, a H36C mutant, or a E117A mutant.

[0037] Suitable iron chelators include, but are not limited to: ethylenediaminetetraacetic acid (EDTA); nitriloacetic acid; diethylenetriaminepentaacetic acid; Ν,Ν'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED); dimercaptosuccinic acid; 2,3-dimercato-l- propanesulfonic acid (DMPS); alpha lipoic acid (ALA); 8 -hydroxy quinoline derivatives, such as 5- [4-(2-hydroxyethyl)piperazin-l-ylmethyl]-8-hydroxyquinoline; hydro xypyridinones, such as deferiprone; hydroxamates, such as desferrioxamine (DFO); amine carboxylates; catechols;

hydroxpyridinones; pyridoxal isonicotinoly hydrazone (PIH) and derivatives thereof; siderophores, such as enterobactin, parabactin, agrobactin, fluviabactin, vulnibactin, vibriobactin, ferrioxamine B, ferrioxamine E, aerobactin, ferrichrome, and desferrithiocin and its analogues and derivatives; diketones, such as dibenzoylmethane (l,3-dephenyl-l,3-propanedione); beta-diketones, such as hydroxydibenzoyl methane or hydroxymethyldibenzoylmethane; salts thereof; and combinations thereof.

[0038] In certain embodiments, the metal ion chelator is EDTA or a salt thereof, such as

CaEDTA. EDTA is a cell-impermeable metal ion chelator that has extremely high affinity to zinc and iron compared to other biologically relevant ions. Thus, EDTA is both a zinc chelator and an iron chelator. The EDTA stability constants of Zn ²⁺ , Fe ²⁺ , and Fe ³⁺ are 16.5, 14.3, and 25.1, respectively. CaEDTA is particularly useful as the metal ion chelator for several reasons. For instance, CaEDTA is more specific for zinc than other ions, such as calcium. Also, CaEDTA is an extracellular, membrane-impermeable chelator, meaning it generally does not cross the cellular membrane and remain inside cells.

[0039] The metal ion chelator can be present in the composition at a concentration ranging from about 1 μΜ to about 500 mM, or from about 10 μΜ to about 100 mM. The metal ion chelator is typically present in the composition at a concentration ranging from about 1 mM to about 20 mM. For example, the concentration of the metal ion chelator can be about 5 mM. In other embodiments, the concentration of the metal ion chelator in the composition ranges from about 10 μΜ to about 1000 μΜ. The concentration of the metal ion chelator can be selected based on the desired use. For instance, the optimal concentration of metal ion chelator present in a composition with tPA, to be used to treat a stroke, may be different from the optimal concentration of metal ion chelator present in a composition with streptokinase, to be used to treat myocardial infarction. The metal ion chelator can increase the efficacy and potency of tPA-mediated thrombolysis, and can expand the time window allotted for tPA treatment without the risk of its side effects.

[0040] Changes in ionic concentration have a vital effect on numerous physiological processes related to blood coagulation and fibrinolysis. Though the compositions and methods described herein can effectively remove some Zn ²⁺ ions, zinc is nonetheless an integral component of thousands of different proteins, none of which are functional if the zinc is removed. Zinc metalloproteins include transcription factors containing zinc fingers, structural proteins, and enzymes from all six enzyme classes. Zinc metalloproteins are present in virtually every cellular organelle of every cell type in the brain and body. Zinc is also important for DNA stabilization and gene expression. Thus, zinc deficiency can be harmful, or even fatal in some situations. Zinc deficiency can lead to increased bleeding tendency. Without wishing to be bound by theory, it is believed that chelating zinc to too low a level can induce seizures or facilitate apoptosis.

Therefore, the compositions and methods herein should be practiced with an understanding of, and respect for, the importance of zinc in the overall body. Similarly, iron is essential for red blood cells, and plays a role in coagulation. Iron deficiency is the most common cause of anemia. Iron deficiency may also cause reduced immunity. Accordingly, certain embodiments of the methods herein involve either monitoring free zinc and/or iron levels in a subject undergoing the treatment, and/or supplementing zinc and/or iron levels during or following the treatment.

[0041] As one non-limiting example, free zinc levels can be monitored through the use of zintrodes. Zintrodes are chemical sensors/probes based on optical fibers which can be used to transmit excitation light from an illumination source to an analyte captured within an area of the probe, and to collect the return signal, either emission or scattering, onto a detector. The fiber optic probe is small and enables in vitro or in vivo real-time monitoring of biological fluids and in vivo implantation. Typically, the zintrode body is made of an inert, biocompatible material, such as a fluorocarbon, with a port or an area in the wall that is fitted with a material permeable to the analyte (Zn ²⁺ ), which reacts with a reagent contained within the body of the sensor. Similarly, serum iron levels can be monitored through a standard blood test.

[0042] As another non-limiting example, zinc levels can be supplemented through the

administration of any suitable zinc supplement. Zinc supplements include, but are not limited to, one or more forms of inorganic or organic zinc, or combinations thereof, for oral or parenteral administration, including solid, gel, liposomal, and liquid forms. Non-limiting examples include zinc chloride (ZnC ); tetrabasic zinc chloride (ZnsCkfOEOs); zinc oxide (ZnO); zinc sulfate (ZnS04); zinc proteinates; zinc chelates; zinc amino acid complexes, such as zinc histidine, zinc methionine, or zinc lysine complexes; zinc acetate; zinc ascorbate; zinc aspartate; zinc butyrate; zinc carbonate; zinc citrate; zinc gluconate; zinc glycinate; zinc histidinate; zinc lactate; zinc maleate; zinc picolinate; zinc propanoate; zinc stearate; and zinc succinate. In certain embodiments, the zinc supplements are administered to deliver from about 10 mg to about 400 mg of elemental zinc per day for a desired period of time.

[0043] Similarly, iron levels can be supplemented through the administration of any suitable iron supplement. Commonly available iron supplements generally include a single form of iron.

Examples of common single forms of iron used in iron supplements include iron salt, that is, a salt containing divalent or ferrous iron salt, such as a salt containing trivalent or ferric iron and iron (0) powder, such as carbonyl iron. Iron supplements are available commercially in rapid release dosage forms and in controlled release dosage forms. Rapid release iron supplement dosage forms typically contain a "rapidly dissolving" iron salt. Certain iron salts are significantly more soluble in water and gastrointestinal fluids than other salts and metallic forms of iron. Hence, these more soluble iron salts or "rapidly dissolving" iron salts are incorporated into rapid release iron supplement dosage forms. In certain embodiments, the iron supplements are administered to deliver from about 1 mg to about 45 mg of iron per day for a desired period of time. As another method, consuming meat and fish can help supplement iron levels, as meat and fish generally contain a significant amount of heme iron, which is readily absorbed by humans.

[0044] Furthermore, there is increased free zinc levels following hypoxic stress, which can be a precursor for cell death. Abnormal high zinc levels can cause mitochondrial dysfunction and reinforce apoptotic signaling cascades. Zinc chelation has been shown to be neuroprotective in cellular and animal models. Subarachnoid hemorrhage (SAH) causes excess quantities of hemoglobin and its degradation production product, iron, in the perivascular space. Treatment with an iron chelator has been proven to reduce brain edema, cerebralvasospasm, and neuronal cell death in a rat model of SAH. The effective zinc and iron chelation facilitates tPA-induced thrombolysis and reduces the necessary dose of tPA treatment. Therefore, by co-applying a metal ion chelator and a thrombolytic drug, a smaller dose of the thrombolytic drug (such as 50% than an otherwise effective dose) is needed to achieve a higher rate of reperfusion. Accordingly, the co- application of a thrombolytic drug and a metal ion chelator (within the same or a separate, co- applied composition) can reduce the adverse effects of tPA, such as hemorrhage. This co- application can also offer additional benefits. For instance, the presence of the metal ion chelator may remove zinc or iron accumulated inside of cells caused by hypoxic stress. Accumulated free iron injures endothelial cells by the production of free radicals. Therefore, the presence of the metal ion chelator can improve neuronal survival by reducing the secondary brain injury or reperfusion brain injury. For example, in the unwanted event of tPA-induced hemorrhage, having a metal ion chelator can reduce the brain damage caused by hemorrhage. Therefore, metal ion chelation can also improve the outcome of thrombolysis by further reducing the adverse effect of a thrombolytic drug such as tPA, as well as the toxicities associated with dissociated zinc and iron. Diverse serial and parallel events contribute to ischemic neuronal cell death. As such, combined treatments that target multiple events for better thrombolytic results are encompassed herein.

[0045] A pharmaceutical composition as described herein may be formulated with any

thrombolytic drug and a metal ion chelator, plus any pharmaceutically acceptable excipients, diluents, or carriers. A composition disclosed herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for such routes of administration as injection. Compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically (i.e., transdermal), intramuscularly, subcutaneously, mucosally (i.e., intranasally, vaginal, etc.), in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), parenterally (i.e., routes that bypass the alimentary tract, such as by injection or by continuous or discontinuous intra- arterial infusion), compressed into tablets or formulated as elixirs or solutions for convenient oral administration or administration by intramuscular or intravenous routes, formulated as sustained release dosage forms, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington' s Pharmaceutical Sciences, 2003, incorporated herein by reference).

[0046] The compositions provided herein are useful for treating animals, such as humans, for various embolic or thrombotic conditions and clotting events. A method of treating a human patient according to the present disclosure includes the administration of an effective amount of a thrombolytic drug and a metal ion chelator, or pharmaceutical composition comprising the combination of a thrombolytic drug and a metal ion chelator. In other embodiments, multiple pharmaceutical compositions - a first composition including a thrombolytic drug, and a second composition including a metal ion chelator - are administered. The metal ion chelator and thrombolytic drug can be administered sequentially or simultaneously. When the metal ion chelator and thrombolytic drug are administered sequentially, there can be a delay of a desired period of time between the first administration and the second administration. Similarly, multiple cycles of sequential administration can be practiced, with or without a significant time delay between cycles.

[0047] When formulated into compositions which may be administered by the oral or rectal routes, or parentarelly, the thrombolytic drug and metal ion chelator can be in the form of, for example, tablets, lozenges, sublingual tablets, sachets, cachets, elixirs, gels, suspensions, aerosols, ointments, for example, containing from 1 to 10% by weight of the active compound in a suitable base, soft and hard gelatin capsules, suppositories, injectable solutions, and suspensions in physiologically acceptable media, or sterile packaged powders adsorbed onto a support material for making injectable solutions.

[0048] The formulations useful for separate administration of the thrombolytic drug and metal ion chelator normally contain at least one compound selected from thrombolytic drugs and metal ion chelators (which may, individually or together, be referred to herein as the active ingredient or active substance) mixed with a pharmaceutically acceptable carrier, or diluted by a carrier, or enclosed or encapsulated by an ingestible carrier in the form of a capsule, sachet, cachet, paper, or other container, or by a disposable container such as an ampoule. The phrases "pharmaceutical" or "pharmacologically acceptable" refer to molecular entities and compositions that produce no adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human. A carrier or diluent may be a solid, semi-solid, or liquid material which serves as a vehicle, excipient, or medium for the active therapeutic substance. Some examples of the diluents or carriers which may be employed in the pharmaceutical compositions of the present disclosure are lactose, dextrose, sucrose, sorbitol, mannitol, propylene glycol, liquid paraffin, white soft paraffin, kaolin, fumed silicon dioxide, microcrystalline cellulose, calcium silicate, silica, polyvinylpyrrolidone, cetostearyl alcohol, starch, modified starches, gum acacia, calcium phosphate, cocoa butter, ethoxylated esters, oil of theobroma, arachis oil, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate, methyl and propyl hydro xybenzoate, sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol, and propellants such as trichloromonofluoromethane, dichlorodifluoromethane, and dichlorotetrafluoroethane. In the case of tablets, a lubricant may be incorporated to prevent sticking and binding of the powdered ingredients in the dies and on the punch of the tableting machine. For such purposes there may be employed, for instance, aluminum, magnesium, or calcium stearates, talc, or mineral oil.

[0049] In certain embodiments, a composition and/or additional agent is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0050] Solutions of the compositions disclosed herein as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as

hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In certain cases the form should be sterile and should be fluid to the extent that easy injectability exists. It should be stable under the conditions of manufacture and storage and may optionally be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, such as, but not limited to, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate, or gelatin.

[0051] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0052] Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

[0053] Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder. Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum as well as any other suitable absorption, emulsion, or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture. Transdermal administration of the compositions may also comprise the use of a "patch." For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.

[0054] In certain embodiments, the compositions are suitable for delivery by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays have been described in U.S. Patents 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in their entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts and could be employed to deliver the compositions described herein. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Patent 5,780,045 (specifically incorporated herein by reference in its entirety), and could be employed to deliver the

compositions described herein.

[0055] It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight, and the severity and response of the symptoms.

[0056] The actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient, and the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The compounds of the present disclosure are generally effective over a wide dosage range. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0057] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by those preparing such

pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0058] In other non-limiting examples, a dose may also comprise from about 1

microgram kg/body weight, about 5 micro gram/kg/body weight, about 10 microgram kg/body weight, about 50 microgram kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. The dosages will depend on many factors, and will in any event be determined by a suitable practitioner. Therefore, the dosages described herein are not intended to be limiting.

[0059] In some embodiments, the compositions further include an additional active ingredient, in addition to the thrombolytic drug and metal ion chelator. The preparation of a pharmaceutical composition that contains at least one compound or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington' s

Pharmaceutical Sciences, 2003, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA Office of Biological Standards. In certain embodiments, pharmaceutical compositions of the present disclosure comprise an effective amount of a thrombolytic drug and an effective amount of a metal ion chelator, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. Suitable additional agents include, but are not limited to, anti-coagulants or blood thinners. In some non-limiting examples, the composition includes an additional agent selected from the group consisting of: aspirin, heparin, coumarins (such as warfarin), clopidogrel, oxalate, citrate, hirudin, and combinations thereof.

[0060] KITS

[0061] The compositions and methods described herein can be embodied as parts of a kit or kits. A non-limiting example of such a kit comprises the ingredients for preparing a composition, namely a thrombolytic drug and a metal ion chelator, in separate (or at least two or more) containers, where the containers may or may not be present in a combined configuration. Many other kits are possible, such as kits comprising a second metal ion chelator or pharmaceutically acceptable additive in a separate container. In certain embodiments, the kits further comprise a means for administering the composition, such as a syringe. The kits may further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, such as a flash drive, CD-ROM, or diskette. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, such as via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0062] EXAMPLES

[0063] Example 1 - tPA

[0064] An in vitro clot model and an animal model were used to evaluate the effects of Zn and Fe on tPA-induced thrombolysis, as well as the effects of co-applying a metal ion chelator with the tPA. The results show that zinc and iron inhibit thrombolysis by reducing the efficacy of tPA. Chelating extracellular free zinc and iron by EDTA increased tPA efficacy and promoted tPA- induced thrombolysis in vitro. Furthermore, in vivo application of CaEDTA increased the efficacy and potency of tPA.

[0065] Recombinant tPA (Genentech, South San Fransisco, CA) was used for the tests in this example. The thrombus in vitro was made of recalcified mice whole blood at 37 °C for 3h incubation. Thrombolysis was quantified by spectrophotometry at 580 nm wavelength.

Alteplase/Activase (tPA) was used for thrombolysis. Zn ²⁺ , Ca ²⁺ , Fe ³⁺ , and Fe ²⁺ were added together with tPA accordingly. EDTA was used for ion chelation. In vivo, Rose Bengal (4,5,6,7- tetrachloro-2 ^* ,4',5',7'-tetraiodofluorescein) and local light exposure were used to introduce photo- thrombosis at the femoral artery. Thrombolysis was monitored and quantified by microscopy with software. The results show that Zn ²⁺ , Fe ³⁺ , and Fe ²⁺ inhibited thrombolysis of tPA, with Zn ²⁺ and Fe ²⁺ being the most potent. Ion chelation of EDTA markedly increased the efficacy of thrombolysis. Data in vivo show that adding calcium saturated EDTA (CaEDTA) improved the thrombolysis rate to 66.7% compared with 20% in tPA alone. Analysis of the thrombolysis images show that EDTA accelerated thrombolysis compared with that of tPA alone. These result indicate that zinc and iron inhibit the tPA reaction. Chelation of zinc and iron improves efficacy and potency of tPA in thrombolysis.

[0066] Citrated blood of male CD1 mice was purchased from Innovative research (46430 Peary Court Novi, MI 48377) and BioChemed services (172 Linden Drive, SuitelOl, Winchester, VA 22601). Blood arrived on the next day of harvest and was stored in 4 °C. Citrated blood was used in 3 days due to its decreased clotting ability. An aliquot of 200 μΐ blood was placed in 0.5 ml tubes and recalcified with 3 μΐ of 1M CaC . The blood samples were incubated in 0.5 ml centrifuge tubes at 37 °C for 3 hours. Blood clots were gently rinsed by saline 3 times and then transferred to custom-modified cuvettes for measurement of absorbance.

[0067] Custom-modified cuvettes were designed for the absorbance measurement. A schematic diagram of these modified cuvettes is shown in FIG. 3A. A nylon web was placed at the narrowed part of the cuvette to separate the cuvette into an upper chamber and a lower chamber. The lower chamber was where the light passed through during spectrophotometric measurement. The optical intensity change of the light was detected and shown as absorbance value. The upper chamber was to hold the blood clot from interfering with the measurement. Cuvettes were filled with tPA in 1.5 ml saline solution. The lysis treatments were tPA, tPA plus EDTA, and tPA plus metal ions (Zn ²⁺ , Ca ²⁺ , Fe ³⁺ , or Fe ²⁺ ). At the start of blood clot- lysis, the color of lysate was clear and the absorbance (optical density) was low. As the blood clots were being lysed, more hemoglobin was released into the vehicle, which gave samples a red color and increased absorbance (FIGS. 3A- 3B).

[0068] A spectrophotometer (Biomate3) from Thermo Spectronic (Waltham, MA, USA) was used to test the most sensitive wavelength for hemoglobin at 500 nm to 600 nm. The peaks of absorbance were between 540 nm and 580 nm, which was consistent with previous hemoglobin studies. The wavelength of 580 nm was chosen to measure the absorbance as the optimal wavelength. Samples (cuvettes with blood clots and lysis treatments dissolved in saline) were placed in the spectrophotometer. Saline was used to set up the absorbance baseline. The absorbance of each sample was measured every 5 minutes, for up to 1 hour. The absorbance change between two time points divided by the time duration was considered the rate of lysis.

[0069] Adult CD1 mice (male) were kept under 12-h light/12-h dark cycles and acclimatized for at least 24 h before surgery. All animal work in this example was conducted according to Ohio University Institutional Animal Care and Use Committee (IACUC) guidelines.

[0070] The CD1 mouse was anesthetized by intraperitoneal (IP) injection with a cocktail of ketamine, xylazine, and aceptromazine. The anesthesia cocktail was made by Ketamine (100 mg/ml) 1 ml, Xylazine (20 mg/ml) 1 ml, Aceptromazine (10 mg/ml) 0.3 ml, and sterile water or saline 7.7 ml. According to the body weight, 6.5 μΐ/g anesthesia cocktail was given through IP injection. The mouse (in surgical plane anesthesia) was placed on dorsal recumbency on a heat plate connected with ATC 1000 animal temperature controller (World Precision Instruments, 175 Sarasota Center Boulevard, Sarasota, FL 34240-9258 USA). The femoral artery of the mouse was exposed, followed by the intravenous injection of Rose Bengal (30 mg kg). Cold white light that was provided by a Fiber-Lite Illuminator through a probe in 2.16 mm diameter (Dolan-Janner Industries) was placed on the femoral artery. The light intensity was 1300 μW/cm ² . The duration of light exposure was 20 minutes. Occlusion of the femoral artery was observed at the location of light exposure, as a dark blood clot and emptiness observed downstream. 2 mg/kg tPA was given to the mouse with 0.01 mg as initial bolus, then 30-minute perfusion of the rest via Syringe Infusion Pump 22 (Harvard Apparatus, Holliston, MA). Half dose of tPA was lmg/kg, with 0.01 mg as initial bolus and 30-minute perfusion of the rest.

[0071] Images of the femoral arteries were taken under dissection microscope connected with a Moticam 2500 camera and Motic Images Plus 2.0 software. Images from each trial were made into sequenced images and analyzed by ImagePro software to quantify the thrombolysis process. The rest of the images were taken by a Nikon 300s.

[0072] Ethylenedinitrilo-tetraacetic acid (EDTA) was purchased from Eastman (Kingsport, Tennessee). Rose Bengal and Ethylenediaminetetraacetic acid disodium-calcium salt (CaEDTA) were purchased from Sigma Aldrich (St Louis, MO). Alteplase/Activase (tPA) was purchased from Genentech (South San Francisco, CA). All chemicals were dissolved in saline unless stated otherwise.

[0073] Statistical analysis of the percentage of reperfusion under different treatment of lysis was performed using Fisher's exact test under R program. Statistical analysis of absorbance increase of clot lysis and the rate change were performed using two-tailed Student t test under Microsoft Excel. P < 0.05 was considered significant.

[0074] The amount of hemoglobin in a solution can be measured by a spectrophotometer and shown as absorbance. The more hemoglobin contained in a sample, the more opaque the sample becomes. Therefore, a sample yields increasing absorbance value when hemoglobin keeps being released. In this example, the blood clot lysis (thrombolysis) was monitored and quantified by measuring the absorbance change at 580 nm wavelength. The amplitudes of absorbance were depicted in arbitrary units.

[0075] When the blood clots were first transferred into cuvettes, the samples were clear with low absorbance value (FIG. 3B). As tPA was lysing the clots, hemoglobin was released into clear solution, which gives the sample an increased absorbance value (FIG. 3C). Additionally, different doses of tPA (from 0.005 mg to 0.05 mg, as shown in FIG. 3C, cuvettes 1-3) resulted in different amounts of hemoglobin release. Therefore, the higher absorbance value indicated a greater amount of thrombolysis. A steady increase of absorbance value started 10 minutes after the initiation of thrombolysis, and the absorbance value kept increasing within the one-hour observation (FIG. 4A). The thrombolysis of vehicle (saline) was also measured. The vehicle showed an extremely low level of thrombolysis (FIG. 4B). The measurement of absorbance was performed every 5 minutes. The absorbance change per minute was calculated as the velocity of the thrombolysis reaction. The amplitudes of rate were depicted in arbitrary units. The rate of thrombolysis started to increase after 10 minutes and reached the maximum at 20-25 minutes (FIG. 4C).

[0076] To investigate the effect of metal ions on thrombolysis, Zn ²⁺ , Ca ²⁺ , Fe ²⁺ , and Fe ³⁺ were added accordingly together with tPA during thrombolysis. Spectrophoto metric measurement showed a decrease of overall absorbance in Zn ²⁺ , Fe ²⁺ , and Fe ³⁺ treated groups, indicating that Zn ²⁺ , Fe ²⁺ , and Fe ³⁺ attenuated thrombolysis of tPA. Both Zn ²⁺ and Fe ²⁺ yielded potent inhibition in overall thrombolysis and decreased the efficacy (maximum rate) of thrombolysis. Compared to the two divalent cations, the inhibiton caused by Fe ³⁺ is much milder (FIGS. 4B, 4D). Fe ³⁺ is not as stable as Fe ²⁺ . Without wishing to be bound by theory, it is believed this is why Fe ²⁺ was seen to be more effective at inhibition of thrombolysis than Fe ³⁺ was. Ca ²⁺ did not affect thrombolysis of tPA.

[0077] The effect of each ion (Zn ²⁺ , Fe ²⁺ , Fe ³⁺ ) was further tested in different doses. At low dose (10 μΜ), only Zn ²⁺ yielded mild inhibition in thrombolysis. Fe ²⁺ did not affect tPA, and Fe ³⁺ slightly increased thrombolysis (FIGS. 5A, 5D, 5G). At medium dose (100 μΜ), all three ions steadily inhibited thrombolysis throughout the one-hour observation. At high concentration (200 μΜ), Zn ²⁺ and Fe ²⁺ markedly decreased thrombolysis. Fe ²⁺ yielded significant inhibition from 30 minutes to 55 minutes of thrombolysis. Zn ²⁺ inhibited thrombolysis from 10 minutes onward. Of the three ions, Zn ²⁺ inhibited thrombolysis most consistently in a dose-dependent manner. The inhibition effect of Fe ²⁺ is significant and saturated at 100 μΜ, with no further inhibition at 200 μΜ. Fe ³⁺ yielded mild but significant inhibition only at 100 μΜ (FIGS. 5A-5I).

[0078] The rate of tPA reaction gradually went up and reached the maximum at 20-25 minutes (FIG. 2B, FIGS. 6A-6I). Zn ²⁺ started to show significant inhibition effect at 10 μΜ, during the first 30 minutes of thrombolysis, but slightly increased the rate after 40 minutes of the reaction. In a dose-dependent manner, the inhibition effect of Zn ²⁺ lasted longer through the thrombolysis as the Zn ²⁺ concentration increased (FIGS. 6D-6F). Fe ²⁺ did not affect the reaction rate at 10 μΜ (FIG. 6G). The inhibition of Fe ²⁺ on the reaction rate was significant and saturated at 100 μΜ. Increasing Fe ²⁺ concentration over 100 μΜ did not enhance the inhibition. Fe ³⁺ showed a slight effect of promotion on tPA, and only showed consistent inhibition at ΙΟΟμΜ. These results indicate that Zn ²⁺ acts as a reliable inhibitor in thrombolysis, by reducing the rate of the tPA reaction. Locally high dose (100 μΜ-200 μΜ) of Fe ³⁺ and Fe ²⁺ as well contributed to reducing tPA-induced thrombolysis (FIGS. 6A-6I).

[0079] EDTA was used as a reliable and high-affinity zinc and iron chelator. Blood clots were assigned into two groups. One group was treated with tPA alone. The other group was treated with EDTA (5 mM) together with tPA. The EDTA group significantly augmented the amount of thrombolysis from 20 minutes of the reaction (FIG. 7A).

[0080] In the trend of reaction rate, the treatment of (tPA + EDTA) started to separate itself as being significant higher than tPA-only treatment at 15 minutes (FIG. 7B). Both treatments had a similar trend of speed change, with higher amplitude in (tPA + EDTA) group. However, with EDTA treatment, the maximum speed of thrombolysis was dramatically higher when EDTA was co-treated with tPA. EDTA yielded a significant higher velocity of thrombolysis from 15 minutes to 40 minutes. After 40 minutes, the curves of reaction rate in EDTA group and tPA alone group merged together.

[0081] Photothrombosis is to introduce in vivo blood clotting by light exposure. Rose Bengal is a photosensitive chemical producing singlet oxygen under light exposure. Singlet oxygen breaks down endothelial cells of the blood vessel and initiates thrombosis. The femoral artery runs superficially in the hind limb of mice (FIG. 8A). The occlusion of the femoral artery was achieved by a photothrombotic method. Rose Bengal was systematically delivered through the tail vein followed by 20 minutes of light exposure (intensity: 1300 on top of the femoral artery. The successful occlusion was shown after light exposure as a dark blood clot in exposed location and emptiness of the artery downstream (FIG. 8B).

[0082] The dose of tPA was given by body weight through 30 minutes of continuous intravenous perfusion which was preceded by a tPA bolus. Images of the femoral artery were acquired every 10 minutes under a dissection microscope. The blood clot in the femoral artery was initially dark, and gradually became lighter during the tPA treatment. The reperfusion was observed as blood refilling in the previous empty downstream region in the femoral artery (FIG. 8C), which was later confirmed by cutting the artery downstream of the occlusion. Bleeding was considered as successful reperfusion of the artery. In some cases, reperfusion was not achieved. Unsuccessful reperfusion was shown as a dark blood clot still occluding the vessel with emptiness remained in the artery (FIG. 8B) and no bleeding when the femoral artery was cut open. [0083] ImagePro was used to quantify the sequenced images of each trial. Change of light transmission in the blood clot was measured as light intensity change, which was shown as (F- Fo)/Fo in arbitrary units. In the reperfused cases, light transmission of the blood clot was gradually increased which was shown as a positive change of light transmission. In non-reperfused cases, in contrast, light transmission was decreased, which caused change of light transmission being negative. The two groups had less than 1 % overlap at all time points, so the positive change of light transmission was used as an indicator of successful thrombolysis (FIG. 9B).

[0084] EDTA, the high affinity chelator of zinc and iron, also combines with calcium. Given the fact that plasma contains high level of calcium, in order to maintain calcium homeostasis in blood, calcium saturated EDTA (CaEDTA) was used in in vivo experiments instead of EDTA to minimize calcium disturbance. CaEDTA was applied so that normal calcium concentration was maintained. Calcium is known to play an important role in physiological functions and is an important factor contributing to coagulation.

[0085] After photothrombotic treatment, thrombolysis agents were given through intravenous perfusion. Three different treatments, which were tPA (20 mg kg), half dose of tPA (10 mg kg), and half dose of tPA plus CaEDTA (30 mg/kg), were given accordingly. Images of the occluded femoral artery were taken every 10 minutes since the intravenous perfusion started.

Representative images of each treatment were shown in FIG. 8. In the tPA (20 mg/kg) group, most of the occlusion cases (7/10) were successfully perfused, accounting for 70%. This reperfusion percentage was decreased to 20% when the dose of tPA was reduced into half (10 mg/kg). However, the poor reperfusion outcome was significantly improved in the presence of CaEDTA. Half dose of tPA combined with CaEDTA yielded 66.7% perfused rate (Table 1, FIG. 10A). The results indicate that ion chelation improves the potency of tPA-induced thrombolysis, and with the help of chelation, the necessary dose of tPA can be reduced.

[0086] ImagePro was used to analyze the speed of thrombolysis of tPA treatment and half dose of tPA (½tPA) co-administrated with CaEDTA, which is indicated as the change of light transmission. Although two treatments yielded a similar rate of reperfusion (FIG. 10A, Table 1), ½ tPA together with CaEDTA showed faster thrombolysis during the first 20 minutes (FIG. 11). The data revealed that ion chelation not only improved thrombolysis outcomes of tPA, but also accelerated the thrombolysis process.

[0087] This example demonstrates that zinc and iron inhibit tPA-induced thrombolysis by reducing the overall effectiveness of tPA. The kinetics of thrombolysis was quantified by using spectrophotometry in vitro. The removal of zinc and iron with metal ion chelation increased tPA efficacy and promoted tPA-induced thrombolysis of the femoral artery thrombus in vivo.

Application of the chelator alone did not cause a noticeable clot lysis. The co- application of metal ion chelator CaEDTA and tPA showed significant increases in the rate and the amplitude of thrombolysis. In other words, significantly higher thrombolysis was achieved by the co- application of the metal ion chelator and tPA than that by tPA alone, and this higher thrombolysis was even higher than that achieved by double the amount of tPA alone. Importantly, these results indicate that the application of metal ion chelator, removing zinc and iron, enhances overall tPA- induced thrombolysis. Furthermore, this regiment can improve the safety of tPA by reducing the tPA dose.

[0088] This example further demonstrates that the application of zinc significantly inhibits tPA- induced thrombolysis by reducing tPA' s potency and efficacy (maximum rate of thrombolysis) of the reaction in a dose-dependent manner. Zn ²⁺ started to show inhibition at 10 μΜ. When Zn ²⁺ concentration was as high as 100 μΜ or 200 μΜ, the amount of thrombolysis was significantly reduced (FIGS. 5E-5F, 6E-6F). Application of zinc itself (without tPA) did not have an effect on the blood clot. This indicates that there is direct interaction between zinc and tPA. Without wishing to be bound by theory, it is believed that zinc binds to tPA and suppresses the conversion of plasminogen to plasmin in a dose-dependent manner. However, the sites of the zinc binding domain in tPA remain unknown.

[0089] The tests in the lysis of the whole blood clot, which is a more physiologically and pathophysiologically relevant model, indicate further the direct inhibitory action of zinc on tPA. Zinc also acts on others: zinc binds heparin and neutralizes its effect of anticoagulation, and zinc abolishes the enhancement effect of decorin on tPA. Because zinc inhibits tPA lysis, the chelation of zinc enhances its effect. The metal ion chelator not only removes cirlulation zinc but also zinc dissociated from thrombi where tPA directly interact in thrombus. Removal of zinc or iron with metal ion chelation facilitates tPA mediated thrombolysis by removing the inhibitory effect of zinc or iron. The aggregation and accumulation of platelets produce a high concentration of zinc in the clot.

[0090] Erythrocytes contain 2-3 g iron, or about 2/3 of the total iron contained in the body, with hemoglobin as one of the major sites of storage. Iron is an important factor in blood coagulation. The erythrocyte is another major composite in the clot. During thrombolysis, the release of hemoglobin contributes to locally high concentration of Fe ³⁺ and Fe ²⁺ . In this example, Fe ³⁺ was observed to postpone the maximum velocity of thrombolysis at 100 μΜ dose (FIG. 6B). At 200 μΜ, Fe ³⁺ significantly reduced the efficacy of tPA-induced thrombolysis (FIG. 6C). Fe ²⁺ showed more severe inhibition by reducing velocity of the thrombolysis reaction than Fe ³⁺ at 100 μΜ and 200 μΜ (FIGS. 6H, 61). Without wishing to be bound by theory, it is believed that when iron is binding to proteins, the bond distance generated by trivalent iron (Fe ³⁺ ) is longer than that generated by divalent iron (Fe ²⁺ ). Therefore, Fe ²⁺ binding with proteins is more stable.

[0091] Increased circulating ferritin and free iron have been found in a variety of disease states associated with thrombophilia. When blood or plasma is exposed to iron addition, characteristic changes in thrombus formation are observed by scanning electron microscopy, which include fusion of fibrin polymers, matting, and even sheeting of fibrin. Fe ³⁺ generates hydroxyl radicals by the following reaction: Fe ³⁺ + HCT→ Fe ²⁺ + HO. Whole blood studies have revealed that fibrinogen can be converted to a fibrin-like polymer in the presence of hydro xyl radicals. Fe ³⁺ also strengthens fibrin and makes it more resistant to proteolysis. Without wishing to be bound by theory, it is believed that the mechanism of Fe ²⁺ is that iron blocks the fibrinolysis pathway by either binding and inhibiting tPA like zinc, or by blocking the pathway of fibrin degradation. The in vitro results of this example showed no effect of inhibition on thrombolysis when adding 10 μΜ of Fe ³⁺ or Fe ²⁺ (FIGS. 5A, 5G). The explanation is that increase of iron may promote plasmin activity on the cell surface. When increasing iron concentration, the inhibition effect of iron overcomes the promotion effect by suppressing fibrinolytic enzymes and accelerating thrombosis. Thus, reversible iron binding to fibrinogen mechanistically explains a significant portion of coagulation kinetic and ultrastructural hypercoagulability.

[0092] As seen in this example, CaEDTA promotes thrombolysis by augmenting efficacy of tPA-mediated thrombolysis. The efficacy of thrombosis, which is represented by the maximum velocity, is increased by 55% by EDTA (FIG. 7B). Among Zn ²⁺ , Fe ²⁺ and Fe ³⁺ , the enhancement of thrombolysis by EDTA is because of Zn ²⁺ and Fe ³⁺ chelation, not Fe ³⁺ , drop. This is because the application of Fe ³⁺ has little effect on tPA-induced thrombolysis. Interestingly, even though Fe ³⁺ has a much higher binding affinity to EDTA than Zn ²⁺ , Zn ²⁺ is able to displace Fe ³⁺ -EDTA. Without wishing to be bound by theory, it is believed that Zn ²⁺ reduces FeEDTA-mediated oxidation by 90% when Zn ²⁺ is added in 1 : 1 molar ratio to Fe ³⁺ . The mechanism of displacement of Fe ³⁺ from EDTA by Zn ²⁺ is not settled. It may involve hydrolysis-aided dissociation of Fe ³⁺ , or the reduction of Fe ³⁺ (as a redox-active ion) to Fe ²⁺ which is outcompeted by Zn ²⁺ at binding EDTA.

[0093] As seen from FIGS. 9A-9B, ion chelation improves reperfusion outcomes in vivo after photothrombosis of the artery. FIG. 9A shows representative sequenced images of tPA-induced thrombolysis in the femoral artery (outlined by dash lines). (Scale bar: 0.5 mm.) FIG. 9B shows sequenced images of thrombolysis analyzed by ImagePro and presented as the change of light transmission every 5 minutes during tPA perfusion. Reperfused cases generate a positive change in light transmission, indicating the blood clot being lysed gradually. Thrombosis from non- perfused cases generate a negative change of light transmission due to unsuccessful lysis. The two groups have less than 1% overlap (p < 0.01).

[0094] A significant superior effect of thrombolysis can be seen when chelation is combined with tPA, as compared to tPA being used alone (FIGS. 9A-9B). A half dose of tPA plus CaEDTA achieved a similar reperfusion rate to a full dose of tPA alone (Table 1, FIG. 10A), demonstrating that CaEDTA increases the potency of tP A- mediated thrombolysis. The measurement of light transmission of the thrombosis showed that the half dose of tPA together with CaEDTA lysed the blood clot faster than the full dose of tPA (FIG. 11). Thus, metal ion chelators such as EDTA increase the efficacy of thrombolysis in vitro. The data indicates that by co-applying CaEDTA with tPA, the therapeutic dose of tPA is reduced with improved efficacy. This combination can therefore lead to fewer side effects and better thrombolytic outcomes. Reducing the tPA dose (from 1 mg kg to 0.5 mg kg) has been shown to shorten the primary bleeding time by 60%.

Secondly, the application of tPA during stroke increases the permeability of the blood-brain barrier, thereby causing brain edema. tPA induces brain edema without plasminogen or matrix metallopeptidases,and tPA-deficient mice have reduced edema size. Additionally, the cytotoxicity of tPA contributes to neuronal death. Therefore, by reducing the therapeutic dose of tPA, co- application of ion chelation can reduce side effects of thrombolysis, and can extend the window of its clinical application.

[0095] Example 2 - Streptokinase

[0096] The effect of metal ion chelators on streptokinase-induced thrombolysis was investigated using procedures similar to those described in Example 1. The co- application of a metal ion chelator, CaEDTA or TPEN, with the thrombolytic drug streptokinase also increased clot lysis when compared to lysis of the control groups streptokinase, CaEDTA, or TPEN alone (FIG. 12). As seen from FIG. 12, the addition of a metal ion chelator without streptokinase showed little to no lysis. As seen from FIG. 13, the addition of zinc (200 μΜ) inhibited the streptokinase (SK)- induced clot lysis when compared to the lysis of the streptokinase treatment group.

[0097] Certain embodiments of the compositions and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.